WO2025059363A1 - Biodegradable microcapsules made from enzymes - Google Patents

Abstract

This disclosure relates to a biodegradable core-shell microcapsule that includes (a) a microcapsule core comprising an active material; and (b) a microcapsule shell comprising a reaction product of an unfolded enzyme with a polyisocyanate; wherein the microcapsule shell is substantially free of or free of a self-condensed polyisocyanate, and the microcapsule shell has a biodegradation rate of at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, based on the weight of the microcapsule shell, within 60 days according to OECD301F. This disclosure also relates to a liquid fragrance composition containing such biodegradable core-shell microcapsule. This disclosure also relates to a consumer product containing such biodegradable core-shell microcapsule. This disclosure also relates to a core-shell microcapsule slurry containing such biodegradable core-shell microcapsule. This disclosure also relates to a process for producing a core-shell microcapsule slurry.

Description

TITLE

Biodegradable Microcapsules Made from Enzymes

BACKGROUND

Field of the Disclosure

The present disclosure relates to biodegradable microcapsules made from enzymes. Particularly, the microcapsule shell comprises a reaction product of an unfolded enzyme with a polyisocyanate. The present disclosure also relates to a process of producing a core-shell microcapsule slurry comprising such biodegradable microcapsules. The present disclosure also relates to a liquid fragrance composition comprising such biodegradable microcapsules. The present disclosure also relates to a consumer product comprising such biodegradable microcapsules.

Description of Related Art

Microcapsules are useful in a variety of applications where there is a need to deliver, apply, or release a fragrance or other active material in a time-delayed and controlled manner.

Current commercial fragrance capsules including melamine-formaldehyde and polyurea are made from synthetic materials, which are not readily biodegradable. This is an issue for several reasons. Firstly, consumers are demanding more environmentally friendly products. Secondly, new regulation from the European Chemicals Agency (ECHA) has banned the use of microplastics in a variety of consumer goods products (e.g., cosmetics, detergents, etc.). As a result, there is an ever-increasing demand for fragrance delivery technologies where the capsule wall material is more biodegradable and/or sustainable, and will satisfy the requirements set out in any currently existing and newly proposed regulations banning the use of microplastics.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure provides a biodegradable core-shell microcapsule comprising: (a) a microcapsule core comprising an active material; and (b) a microcapsule shell comprising a reaction product of an unfolded enzyme with a polyisocyanate; wherein the microcapsule shell is substantially free of or free of a self-condensed polyisocyanate, and the microcapsule shell has a biodegradation rate of at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, based on the weight of the microcapsule shell, within 60 days according to OECD301 F.

The present disclosure also provides a process for producing a core-shell microcapsule slurry. The process comprises: (a) providing an aqueous phase comprising an unfolded enzyme, wherein the pH of the aqueous phase is equal to or greater than the isoelectric point of the enzyme; (b) providing an oil phase comprising an active material and a polyisocyanate; (c) emulsifying the oil phase with the aqueous phase to form an emulsion; (d) allowing the unfolded enzyme to react with the polyisocyanate to form a microcapsule shell encapsulating a microcapsule core, thereby forming a core-shell microcapsule slurry comprising a biodegradable core-shell microcapsule, wherein the microcapsule core comprises the active material, and the microcapsule shell comprises a reaction product of the unfolded enzyme with the polyisocyanate; and (e) curing the microcapsule shell at an elevated temperature.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments are illustrated in the accompanying figures to improve understanding of concepts as presented herein.

FIG. 1 shows the fluorescence measurement results of Samples “Water”, “A”, “B” and “C” prepared at room temperature (RT), 30 °C, 40 °C, and 50 °C respectively. FIG. 1 also shows the fluorescence measurement result of Sample “PBS” prepared at room temperature.

FIG. 2 shows the appearance of Comparative Microcapsules 21 -27.

DETAILED DESCRIPTION

The foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other features and benefits of any one or more of the embodiments will be apparent from the following detailed description, and from the claims.

As used herein, the terms “comprises”, “comprising”, “includes”, “including”, “has”, “having”, “contains”, “containing” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, use of “a” or “an” are employed to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

When an amount, concentration, or other value or parameter is given as either a range, preferred range or a list of upper preferable values and/or lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. For example, when a range of "1 to 10" is recited, the recited range should be construed as including ranges "1 to 8", “3 to 10”, "2 to 7", "1 .5 to 6", “3.4 to 7.8”, "1 to 2 and 7-10", “2 to 4 and 6 to 9”, “1 to 3.6 and 7.2 to 8.9”, "1 -5 and 10", “2 and 8 to 10”, “1 .5-4 and 8”, and the like.

The present disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. While compositions and methods are described herein in terms of “comprising” various components or steps, the compositions and methods also can “consist essentially of” or “consist of” the various components or steps, unless stated otherwise. All parts, percentages and proportions referred to herein and in the claims are by weight unless otherwise indicated.

Before addressing details of embodiments described below, some terms are defined or clarified.

The term “elevated temperature”, as used herein, means a temperature higher than the room temperature (e.g., 22 °C).

The terms “obtainable” and “obtained” can be used interchangeably in this disclosure and do not mean to indicate that, e.g., a product must be obtained by, e.g., the sequence of steps following the term “obtained” though such a limited understanding is always included by the terms as a preferred aspect of the present disclosure.

The term “microcapsule slurry”, as used herein, means an aqueous suspension of the biodegradable core-shell microcapsule. In some embodiments, the biodegradable core-shell microcapsule product produced in accordance with the methods and examples described in the present disclosure is in the form of a microcapsule slurry. The microcapsule slurry may be used directly in a consumer product. The microcapsule slurry may also be washed, coated, dried (e.g., spray-dried) and/or combined with one or more other microcapsules, active materials, and/or carrier materials.

The term “self-condensed polyisocyanate”, as used herein, means the polyurea formed by the self-polymerization of polyisocyanate in the presence of water. A person skilled in the art appreciates that isocyanate can react with water to form amine which can further react with isocyanate to form urea linkage. Accordingly, polyisocyanate can selfpolymerize in the presence of water to form polyurea. The self-condensed polyisocyanate is non-biodegradable.

The term “residual enzyme”, as used herein, means unreacted enzyme present in the core-shell microcapsule slurry. During the process of this disclosure for producing a core-shell microcapsule slurry, a small amount of the enzyme starting material does not react with polyisocyanate or a crosslinker, and remains in the slurry as a residual enzyme. The residual enzyme is a free enzyme, that is, the residual enzyme is not agglomerated and is free from extraneous matter (e.g., unbound from other compounds). The residual enzyme has about the same molecular weight (Mw) as the enzyme starting material used in the process of this disclosure for producing a core-shell microcapsule slurry. By “about the same molecular weight”, it is meant that the molecular weight difference between the residual enzyme and the enzyme starting material (e.g., enzyme used in step (a) of the process) is within a range of ± 2%, ± 5%, ± 10% or ± 15% of the molecular weight of the enzyme starting material. In some embodiments, the enzyme comprises or is glucoamylase.

As used herein, the term “w/w” means weight by weight, and the term “w/v” means weight per volume. As used herein, the term “wt%” means percentage by weight.

As used herein, the terms “kg”, “g”, “mg” and “pg” refer to “kilogram”, “gram”, “milligram” and “microgram” respectively. The terms “L”, “mL” and “pL” refer to “liter”, “milliliter” and “microliter” respectively. The terms “cm”, “mm”, “pm” and “nm” refer to “centimeter”, “millimeter”, “micrometer” and “nanometer” respectively. The terms “mM” and “M” refer to molar concentration units “millimolar” (mmol/L) and “molar” (mol/L) respectively. Polyisocyanate

The term “polyisocyanate”, as used herein, means a chemical compound having two or more isocyanate (-NCO) groups. Polyisocyanates can be aromatic, aliphatic, linear, branched, or cyclic. In some embodiments, the polyisocyanate contains, on average, 2 to 4 isocyanate groups. In some embodiments, the polyisocyanate contains at least three isocyanate functional groups. In some embodiments, the polyisocyanate is water insoluble. In certain aspects, the polyisocyanate is an oligomeric polyisocyanate obtained from hexamethylene diisocyanate (HDI), which is a monomeric diisocyanate. In certain aspects, the polyisocyanate is an oligomeric polyisocyanate having a biuret, isocyanurate, allophanate, uretdione and/or oligomeric HDI structure. Exemplary polyisocyanates are sold under the tradenames TAKENATE® (e.g., TAKENATE® D-1 10N; Mitsui Chemicals), DESMODUR® (Covestro), BAYHYDUR® (Covestro), and LUPRANATE® (BASF).

In some embodiments, the polyisocyanate comprises or is an aromatic polyisocyanate. In some embodiments, the polyisocyanate is an aromatic polyisocyanate or a combination of aromatic polyisocyanates, substantially free of or free of an aliphatic polyisocyanate. In some embodiments, the amount of aliphatic polyisocyanate (in the polyisocyanate) is no more than 20 wt%, or no more than 15 wt%, or no more than 10 wt%, or no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, based on the total weight of the polyisocyanate. The term “aromatic polyisocyanate”, as used herein, means a polyisocyanate containing one or more aromatic moiety. For example, an aromatic polyisocyanate can contain phenyl, tolyl, xylyl, naphthyl or diphenyl moiety as the aromatic component. In some embodiments, the aromatic polyisocyanate is selected from the group of polyisocyanurate of toluene diisocyanate, trimethylol propane-adduct of toluene diisocyanate, trimethylol propane-adduct of xylylene diisocyanate, and mixtures thereof. In some embodiments, the polyisocyanate comprises or is trimethylol propane-adduct of xylylene diisocyanate.

Trimethylol propane-adduct of xylylene diisocyanate has the structural formula shown below:

In some embodiments, the aromatic polyisocyanate has the structural formula shown below, and includes structural isomers thereof

wherein n can vary from zero to a desired integral number (e.g., 0-50, 0-20, 0-10, or 0-6). Preferably, n is no more than 6. The polyisocyanate may also be a mixture of polyisocyanates where the value of n can vary from 0 to 6. In the case where the polyisocyanate is a mixture of various polyisocyanates, the average value of n preferably is from 0.5 to 1 .5.

In some embodiments, the aromatic polyisocyanate has the structural formula shown below, and includes structural isomers thereof

wherein R can be a C1-C10 alkyl, C1-C10 ester, or an isocyanurate. Representative polyisocyanates having this structure are sold under the trademarks TAKENATE® D-110N (Mitsui), DESMODUR® L75 (Covestro), and DESMODUR® IL (Covestro).

In some embodiments, the aromatic polyisocyanate is selected from the group of 1 ,5-naphthylene diisocyanate, 4,4'-diphenylmethane diisocyanate (MDI), hydrogenated MDI (H12MDI), xylylene diisocyanate (XDI), tetramethylxylol diisocyanate (TMXDI), 4,4'- diphenyldimethylmethane diisocyanate, di- and tetraalkyldiphenylmethane diisocyanate, 4,4'-dibenzyl diisocyanate, 1 ,3-phenylene diisocyanate, 1 ,4-phenylene diisocyanate, the isomers of tolylene diisocyanate (TDI), 4,4'-diisocyanatophenylperfluoroethane, phthalic acid bisisocyanatoethyl ester, aromatic polyisocyanates with reactive halogen atoms, and mixtures thereof. In some embodiments, the aromatic polyisocyanate with reactive halogen atom is selected from the group of 1 -chloromethylphenyl 2,4-diisocyanate, 1 -bromomethyl- phenyl 2,6-diisocyanate, 3,3-bischloromethyl ether 4,4'-diphenyldiisocyanate, and mixtures thereof.

In some embodiments, the polyisocyanate comprises or is an aliphatic polyisocyanate. The term “aliphatic polyisocyanate”, as used herein, means a polyisocyanate containing no aromatic moiety. In some embodiments, the aliphatic polyisocyanate is selected from the group of trimer of hexamethylene diisocyanate, trimer of isophorone diisocyanate, biuret of hexamethylene diisocyanate, and mixtures thereof. In some embodiments, the aliphatic polyisocyanate is selected from the group of 1 -methyl-

2.4-diisocyanatocyclohexane, 1 ,6-diisocyanato-2,2,4-trimethylhexane, 1 ,6-diisocyanato-

2.4.4-trimethylhexane, 1 -isocyanatomethyl-3-isocyanato-1 ,5,5-trimethylcyclohexane, chlorinated aliphatic diisocyanates, brominated aliphatic diisocyanates, phosphorus- containing aliphatic diisocyanates, tetramethoxybutane 1 ,4-diisocyanate, butane 1 ,4- diisocyanate, hexane 1 ,6-diisocyanate (HDI), dicyclohexylmethane diisocyanate, cyclohexane 1 ,4-diisocyanate, ethylene diisocyanate, and mixtures thereof. In some embodiments, the polyisocyanate comprises a sulfur-containing polyisocyanate which can be obtained, for example, by reacting hexamethylene diisocyanate with thiodiglycol or dihydroxydihexyl sulfide. In some embodiments, the polyisocyanate is an aliphatic diisocyanate selected from the group of trimethylhexamethylene diisocyanate, 1 ,4- diisocyanatobutane, 1 ,2-diisocyanatododecane, dimer fatty acid diisocyanate, and mixtures thereof.

In some embodiments, the weight average molecular weight of the polyisocyanate ranges from 250 Da (daltons) to 1000 Da, or from 275 Da to 500 Da. In some embodiments, the polyisocyanate is a single kind of polyisocyanate. In other embodiments, the polyisocyanate is a mixture of polyisocyanates. In some embodiments, the polyisocyanate comprises or is a mixture of an aliphatic polyisocyanate and an aromatic polyisocyanate.

Enzyme

Enzyme is a single protein with a defined protein sequence, as opposed to a mixture of proteins such as soy protein or pea protein. As is conventional in the art, an enzyme is a protein that is capable of catalyzing a chemical reaction. Enzymes are made up of amino acids which are linked together via amide (peptide) bonds in a linear chain. This is the primary structure. The hydrogen in the amino group (NH2) and the oxygen in the carboxyl group (COOH) of each amino acid can bond with each other by means of hydrogen bond, this means that the amino acids in the same chain can interact with each other. As a result, the protein chain can fold up on itself, and it can fold up in two ways, resulting in two secondary structures: it can either wrap round forming the a-helix, or it can fold on top of itself forming the [3-sheet. As a consequence of the folding-up of the 2D linear chain in the secondary structure, the protein can fold up further and in doing so gains a three-dimensional structure. This is its tertiary structure. Disulfide bridges formed between thiol groups in two cysteine residues are also important components of the secondary and tertiary structure of proteins. Likewise, metals can be coordinated to a protein through the carboxylate side chains of glutamate and aspartate to facilitate protein folding. In some embodiments, the enzyme of this disclosure is selected from the group of glucoamylase, amylase, phytase, and mixtures thereof. In some embodiments, the enzyme comprises or is glucoamylase. The glucoamylase can be T. reesei glucoamylase and/or H. grisea glucoamylase. In some embodiments, the enzyme comprises or is H. grisea glucoamylase. In some embodiments, the enzyme comprises or is T. reesei glucoamylase. The enzyme of this disclosure may be obtained directly from a natural source and/or by recombinant production and fermentation of recombinant microorganisms {e.g., Trichoderma sp., Pichia sp., Neurospora sp., Saccharomyces sp.) modified to express or overexpress the enzyme. In some embodiments, the enzyme has a molecular weight (Mw) that is at least 45 kD (kilodaltons), or at least 50 kD. In some embodiments, the enzyme has an isoelectric point (pl) of at least 3.8, or at least 4.0, or at least 4.2. The isoelectric point (pl) is the pH of a solution at which the net charge of a protein becomes zero. In some embodiments, the enzyme is isolated and purified to homogeneity or near homogeneity. In some embodiments, the enzyme is purified by ultrafiltration. In some embodiments, the enzyme is substantially free of or free of other proteins (i.e., proteins different from the enzyme). In some embodiments, the amount of other proteins contained in the enzyme (e.g., enzyme fed into the process of producing the biodegradable coreshell microcapsule and the core-shell microcapsule slurry) is no more than 30 wt%, 25 wt%, 20 wt%, 15 wt%, 10 wt%, 5 wt%, 2 wt%, 1 wt%, 0.5 wt%, 0.2 wt%, or 0.1 wt%, based on the weight of the enzyme. The enzyme is glucoamylase, amylase, or phytase.

In some embodiments, the number of cysteine residues in the amino acid sequence of the enzyme is at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10. In some embodiments, the number of cysteine residues in the amino acid sequence of the enzyme is in the range of from 4 to 12, or from 6 to 12, or from 8 to 12. In some embodiments, the enzyme, before unfolded, has at least 2 disulfide bonds, or at least 3 disulfide bonds, or at least 4 disulfide bonds, or at least 5 disulfide bonds. In some embodiments, the enzyme has a tertiary structure that is created by disulfide bonding e.g., cysteine-cysteine bonding) and/or metal ion bonding e.g., via aspartic acid and/or glutamic acid residues), in particular Ca⁺².

In some embodiments, the enzyme is rich in lysine and arginine, the side-chain amine groups of which, together with the N-terminus, can react with polyisocyanate to form a crosslinked polymeric network of the microcapsule shell. In some embodiments, the combined number of lysine and arginine residues in the amino acid sequence of the enzyme is at least 20, or at least 23, or at least 26, or at least 29, or at least 32, or at least 34, or at least 36, or at least 38, or at least 40, or at least 42, or at least 44, or at least 46, or at least 48, or at least 50, or at least 52, or at least 54, or at least 56. In some embodiments, the combined number of lysine and arginine residues in the amino acid sequence of the enzyme is no more than 85, or no more than 80, or no more than 75, or no more than 72, or no more than 70, or no more than 68, or no more than 66, or no more than 64, or no more than 62, or no more than 60, or no more than 58, or no more than 56, or no more than 54. In some embodiments, the number of lysine residues in the amino acid sequence of the enzyme is at least 10, 12, 14, 16, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or 34. In some embodiments, the number of arginine residues in the amino acid sequence of the enzyme is at least 10, 12, 14, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26. In some embodiments, the combined number of lysine and arginine residues in the amino acid sequence of the enzyme is in the range of from 20 to 80, or from 25 to 70, or from 30 to 65, or from 30 to 60, or from 35 to 60, or from 40 to 65, or from 40 to 60.

In some embodiments, the combined number of aspartic acid and glutamic acid residues in the amino acid sequence of the enzyme is at least 40, or at least 42, or at least 44, or at least 46, or at least 48, or at least 50, or at least 52, or at least 54, or at least 56. In some embodiments, the number of aspartic acid residues in the amino acid sequence of the enzyme is at least 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 . In some embodiments, the number of glutamic acid residues in the amino acid sequence of the enzyme is at least 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 . In some embodiments, the combined number of aspartic acid and glutamic acid residues in the amino acid sequence of the enzyme is in the range of from 40 to 60, or from 42 to 56.

Glucoamylase (EC 3.2.1 .3) is an enzyme that can catalyze the hydrolysis of terminal (1 — >4)-linked a-D-glucose residues successively from non-reducing ends of the chains with release of p-D-glucose in a polysaccharide substrate. Glucoamylase may be isolated or otherwise obtained from (e.g., cloned) a fungal source, e.g., Aspergillus (e.g., A. nidulins or A. niger), Neurospora (e.g., N. crassa), Saccharomyces (e.g., S. cerevisiad), Schizosaccharomyces, Trichoderma (e.g., T. reesei) or Humicola (e.g., grisea). An exemplary glucoamylase is disclosed, e.g., in W02006/060062 A2.

In some embodiments, the glucoamylase has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:1 . SEQ ID N0:1

SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPSTIDPDYYYMWTRDSALVFKN LIDRFTETYDAGLQRRIEQYITAQVTLQGLSNPSGSLADGSGLGEPKFELTLKPFTGNWG RPQRDGPALRAIALIGYSKWLINNNYQSTVSNVIWPIVRNDLNYVAQYWNQTGFDLWEEV NGSSFFTVANQHRALVEGATLAATLGQSGSAYSSVAPQVLCFLQRFWVSSGGYVDSNIN TNEGRTGKDVNSVLTSIHTFDPNLGCDAGTFQPCSDKALSNLKVVVDSFRSIYGVNKGIP AGAAVAIGRYAEDVYYNGNPWYLATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVP GVTAGTYSSSSSTFTNIINAVSTYADGFLSEAAKYVPADGSLAEQFDRNSGTPLSALHLTW SYASFLTATARRAGIVPPSWANSSASTIPSTCSGASVVGSYSRPTATSFPPSQTPKPGVP SGTPYTPLPCATPTSVAVTFHELVSTQFGQTVKVAGNAAALGNWSTSAAVALDAVNYAD NHPLWIGTVNLEAGDVVEYKYINVGQDGSVTWESDPNHTYTVPAVACVTQVVKEDTWQ GA

In some embodiments, the glucoamylase has the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:2.

SEQ ID NO:2

MHTFSKLLVLGSAVQSALGRPHGSSRLQERAAVDTFINTEKPIAWNKLLANIGPNGKAAP GAAAGVVIASPSRTDPPYFFTWTRDAALVLTGIIESLGHNYNTTLQTVIQNYVASQAKLQQ VSNPSGTFADGSGLGEAKFNVDLTAFTGEWGRPQRDGPPLRAIALIQYAKWLIANGYKST AKSVVWPVVKNDLAYTAQYWNETGFDLWEEVPGSSFFTIASSHRALTEGAYLAAQLDTE CRACTTVAPQVLCFQQAFWNSKGNYVVSNINGGEYRSGKDANSILASIHNFDPEAGCDN LTFQPCSERALANHKAYVDSFRNLYAINKGIAQGKAVAVGRYSEDVYYNGNPWYLANFA AAEQLYDAIYVWNKQGSITVTSVSLPFFRDLVSSVSTGTYSKSSSTFTNIVNAVKAYADGFI EVAAKYTPSNGALAEQYDRNTGKPDSAADLTWSYSAFLSAIDRRAGLVPPSWRASVAKS QLPSTCSRIEVAGTYVAATSTSFPSKQTPNPSAAPSPSPYPTACADASEVYVTFNERVST AWGETIKVVGNVPALGNWDTSKAVTLSASGYKSNDPLWSITVPIKATGSAVQYKYIKVGT NG KITWESDPN RS ITLQTASSAG KCAAQTVNDSWR

Amylase or a-amylase (EC 3.2.1 .1 ) is an enzyme that can catalyze the hydrolysis of starch, glycogen, and related polysaccharides by cleaving internal a-1 ,4-glycosidic bonds at random. Amylase may be isolated or otherwise obtained from {e.g., cloned) a fungal source, e.g., Aspergillus e.g., A. nidulins, A. tereus, or A. niger), Neurospora e.g., N. crassa), Saccharomyces {e.g., S. cerevisiae), or Schizosaccharomyces.

In some embodiments, the a-amylase has the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:3.

SEQ ID NO:3 LTPAEWRSQSIYFLLTDRFGRTDNSTTAACDTSDRVYCGGSWQGIINQLDYIQGMGFTAI WITPVTGQFYENTGDGTSYHGYWQQDIYDLNYNYGTAQDLKNLANALHERGMYLMVDV VANHMGYDGAGNTVDYSVFNPFSSSSYFHPYCLISNYDNQTNVEDCWLGDTTVSLPDLD TTSTAVRNIWYDWVADLVANYSIDGLRVDTVKHVEKDFWPGYNSAAGVYCVGEVYSGDP AYTCPYQNYMDGVLNYPIYYQLLYAFESSSGSISDLYNMISSVASSCKDPTLLGNFIENHD NPRFASYTSDYSQAKNVITFIFLSDGIPIVYAGQEQHYSGGSDPANREATWLSGYSTSATL YTWIATTNQIRSLAISKDAGYVQAKNNPFYSDSNTIAMRKGTTAGAQVITVLSNKGASGSS YTLSLSGTGYSAGATLVETYTCTTVTVDSSGNLPVPMTSGLPRVFVPSSWVNGSALCNT ECTAATSISVLFEELVTTTYGENIYLSGSISQLGSWNTASAVALSASQYTSSNPEWYVSVT LPVGTSFQYKFIKKGSDGSVVWESDPNRSYTVPAGCEGATVTVADTWR

Phytase (EC 3.1 .3.26, EC 3.1 .3.8, or EC 3.1 .3.72) is an enzyme that can catalyze the hydrolysis of phytic acid (myo-inositol hexakisphosphate) - an indigestible, organic form of phosphorus that is found in many plant tissues, especially in grains and oil seeds, and release a usable form of inorganic phosphorus. Phytase may be isolated or otherwise obtained from e.g., cloned) a fungal source, e.g., Aspergillus {e.g., A. niger) or a bacterial source, e.g., Escherichia e.g., E. coli) or Buttiauxella sp. An exemplary phytase is sold under the tradename AXTRA® PHY, developed and manufactured by Danisco Animal Nutrition & Health (IFF).

In some embodiments, the phytase has the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:4.

SEQ ID NO:4

SEAAPSGYHLDKAVILSRHGVRAPTKMTQLMRDVTPYTWPEWPVPLGYITPRGEHLVSL MGGFYRQYFQQQGLLSRDRCPTANDVYVWTDVNQRTRKTGEAFLAGLAPECDLTIHHQ NDIKQVDPLFHPLKAGICSMDKTQVQQAVEKQAGMPIDKLNQHYRPELALMSNVLNFPKS PYCRQHSVEQPCDFANAFPSYLNISDDGNEVQLEGAVGLSSTLAEIFLLEYAQGMPVVA WGNIHSEQEWNDLLKLHNAQFDLMERTPYIAKHQGTPLLQTIVNALNSNTTESKLPDISPS VKILFLAGHDTNIANIGGMLGMTWTLPGQPDNTPPGGALLFELWSDKDGTQYVSVKMVY QTLAQLRNMTPLTLKEPAGSVPLKIPGCDDQTAEGYCPLDTFTRLVNQVVEPACQLP

The term “unfolded enzyme”, as used herein, means a partially or completely unfolded enzyme. In some embodiments, the enzyme is reversibly unfolded and can, under appropriate conditions, refold to have a secondary and optionally tertiary structure. In some embodiments, the enzyme is unfolded before the step of emulsifying the oil phase with the aqueous phase. Exemplary conditions for enzyme unfolding include, but are not limited to, exposure to heat or cold, dilution in water, changes in pH, exposure to denaturing agents such as detergents, urea, f/?/o/-containing reducing agent (e.g., cysteine, dithiothreitol, or 2-mercaptoethanol), other chaotropic agents, or mechanical stress including shear. In some embodiments, the enzyme is partially unfolded, e.g., 30%, 40%, 50%, 60%, 70%, 80%, or 85% (w/w) unfolded. In some embodiments, the enzyme is substantially or completely unfolded, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (w/w) unfolded. In some embodiments, the enzyme is unfolded so that it no longer has its native secondary and/or tertiary structure. For example, when an 8% enzyme solution (w/v) is used, the solution can be treated at a temperature of from 80 °C to 90 °C for 20 to 30 minutes (or preferably at 85 °C for 25 minutes) to yield a substantially unfolded enzyme. Depending on the degree of denaturation desired, it will be appreciated that higher or lower temperatures and shorter or longer times can also be employed.

In some embodiments, a chaotropic agent can be used to unfold the enzyme. As is conventional in the art, a chaotropic agent is a chemical compound which disrupts hydrogen bonding in aqueous solution, leading to increased entropy. Generally, this reduces hydrophobic effects which are essential for three dimensional structures of enzymes. In some embodiments, the chaotropic agent comprises or is a guanidinium salt. In some embodiments, the guanidinium salt is selected from the group of guanidinium sulphate, guanidinium carbonate, guanidinium nitrate, guanidinium chloride, and mixtures thereof. In some embodiments, the chaotropic agent comprises or is guanidinium carbonate.

In some embodiments, the enzyme is unfolded by dilution of the ultrafiltration concentrate (UFC) of enzyme or formulated enzyme product in water or an aqueous solution. In some embodiments, a thiol -containing reducing agent can be added into the aqueous solution to help unfolding the enzyme. In some embodiments, the thiol-containing reducing agent is selected from the group of cysteine, dithiothreitol, 2-mercaptoethanol, and mixtures thereof. In some embodiments, the thiol-containing reducing agent comprises or is cysteine. In some embodiments, the enzyme is diluted at least 10-fold, or at least 15- fold, or at least 25-fold in water or an aqueous solution. In some embodiments, the enzyme is unfolded in the presence of a thiol-containing reducing agent (e.g., cysteine). In some embodiments, the enzyme is unfolded in the absence of the thiol-containing reducing agent (e.g., cysteine). The temperature and amount of time needed to unfold the enzyme may vary depending on the extent of unfolding desired, while higher temperatures, e.g., 30 °C, 35 °C, 40 °C, 45 °C, or 50 °C, and longer incubation times may favor greater degree of unfolding.

Active Material

The core of the biodegradable core-shell microcapsule comprises an active material. In some embodiments, the active material is hydrophobic. In some embodiments, the active material has a logP value (partition coefficient) of less than 2. In some embodiments, the active material comprises fragrance, flavor, agricultural active, pesticide, insecticide, herbicide, fungicide, pharmaceutical active, nutraceutical active, animal nutrition active, food active, microbio active, malodor counteractant, and/or cosmetic active. In some embodiments, the active material is selected from the group of fragrance, pro-fragrance, malodor counteractive agent, and combinations thereof. In some embodiments, the active material comprises or is a fragrance.

In some embodiments, the active material comprises or is a fragrance comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten High Performance fragrance ingredients selected from the group of Ultra High-Impact fragrance ingredients as listed in Table 1 and High- Impact fragrance ingredients as listed in Table 2.

TABLE 1 - Ultra High-Impact Fragrance Ingredients

Available from International Flavors & Fragrances Inc.

TABLE 2 - High-Impact Fragrance Ingredients

¹ Available from International Flavors & Fragrances Inc (New York).

² Available from TRIPPER PTE Ltd. (Indonesia).

³ Available from TREATT & CO. Ltd. (United Kingdom).

The High Performance fragrance ingredients can deliver improved perceived intensity, perceived longevity and/or perceived fidelity of the fragrance profile at the various “touch points” (e.g., opening a fabric conditioner container, damp clothes upon opening a washing machine after washing laundry, opening a laundry dryer after drying laundry, drying clothes on drying frame and wearing laundered clothes) associated with the laundry experience. In addition to the High Performance fragrance ingredients listed in Tables 1 and 2, the fragrance may further comprise at least one additional fragrance ingredient. In some embodiments, the fragrance further comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or more additional fragrance ingredients, which are not listed in Tables 1 and 2. Non-limiting examples of such additional fragrance ingredients include those described in US2018/0325786 A1 , US4, 534, 891 , US5,1 12,688, and US5, 145,842. Other suitable active materials that can be encapsulated include those listed in WO2016/049456, pages 38-50.

In some embodiments, the additional fragrance ingredients, when combined with one or more High Performance fragrance ingredients of Tables 1 and 2, constitute the total fragrance composition. In this respect, the balance of the 100 wt% relative to the total weight of the fragrance is made up of one or more Ultra High-Impact and High-Impact fragrance ingredients of Tables 1 and 2 and one or more additional fragrance ingredients.

It has been surprisingly discovered that a fragrance that includes certain types of Ultra High-Impact and High-Impact fragrance ingredients results in High Performance fragrance ingredients for use in fabric care composition, preferably fabric conditioner, that improves fragrance profile and/or performance of the system. As used herein, the term “fragrance profile” means the description of how the fragrance is perceived by the human nose. A fragrance profile is composed of 2 characteristics: “intensity” and “character”. The “intensity” relates to the perceived strength whilst “character” refers to the odor impression or quality of the perfume, i.e., fresh, clean, etc.

In some embodiments, the fragrance of the present disclosure can be used at a dosage level of < 1 wt% relative to the total weight of the consumer product composition without significantly impacting the fragrance profile, i.e., perceived fragrance intensity, perceived fragrance longevity and/or perceived fragrance fidelity, particularly for select characters (e.g., fresh and/or clean). In some embodiments, the fragrance is used at a dosage level of no more than 1 wt%, 0.99 wt%, 0.95 wt%, 0.9 wt%, 0.8 wt%, 0.7 wt%, 0.6 wt%, 0.5 wt%, 0.4 wt%, 0.3 wt%, 0.2 wt%, 0.1 wt%, 0.05 wt% or 0.01 wt% of the total weight of the aqueous fabric conditioner composition, or any range delimited by any pair of the foregoing values.

In some embodiments, the active material comprises agricultural active, pesticide, insecticide, herbicide, and/or fungicide. Encapsulation of agricultural actives, pesticides, insecticides, herbicides, and/or fungicides can increase their efficacy, extend their effective period and protect the environment. In some embodiments, the insecticide is an organophosphate insecticide. In some embodiments, the insecticide can be an organophosphate insecticide selected from the group of acephate, azinphos-mehyl, chlorfenvinphos, chlorethoxyfos, chlorpyriphos-methyl, diazinon, dimethoate, disulfoton, ethoprophos, fenitrothion, fenthiom, fenamiphos, fosthiazate, malathion, methamidophos, methidathion, omethoate, oxydemeton-methyl, parathion, parathion-methyl, phorate, phosmet, profenofos, trichlorfon, and mixtures thereof. In some embodiments, the insecticide is selected from the group of cypermethrin, bifenthrin, A-cyhalothrin, and mixtures thereof. In some embodiments, the herbicide is selected from the group of clomazone, acetochlor, pendimethalin, and mixtures thereof. In some embodiments, the fungicide is tebuconazole.

Methods to make microcapsule shells with biopolymers have been described. The biopolymers either form coacervates or crosslink with polyisocyanate through interfacial polymerization. See, e.g., US8,663,690, WO2021/122633, WO2021/239742, and US2022/0071865. However, polyisocyanate, in the presence of water, can self-polymerize to form self-condensed polyisocyanate which is not biodegradable.

It has been surprisingly discovered that an unfolded enzyme can so effectively react with a polyisocyanate during the interfacial polymerization that the self-polymerization of polyisocyanate can be substantially minimized or eliminated. Accordingly, the present disclosure provides a biodegradable core-shell microcapsule which comprises: (a) a microcapsule core comprising an active material; and (b) a microcapsule shell comprising a reaction product of an unfolded enzyme with a polyisocyanate; wherein the microcapsule shell is substantially free of or free of a self-condensed polyisocyanate, and the microcapsule shell has a biodegradation rate of at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, based on the weight of the microcapsule shell, within 60 days according to OECD301 F.

As used herein, the terms “capsule”, “microcapsule” and “core-shell microcapsule” are used interchangeably and refer to a substantially spherical structure having a well- defined core and a well-defined envelope or wall or shell. The “core” comprises an active material or material submitted to encapsulation. The terms “wall” and “shell” are used interchangeably to denote the structure formed by the encapsulating polymer surrounding the core being encapsulated. In general, the wall of the microcapsule is made of a continuous, polymeric phase with an inner surface and outer surface. The inner surface is in contact with the microcapsule core. The outer surface is in contact with the environment in which the microcapsule resides, e.g., an aqueous phase, skin, or hair. Ideally, the wall protects the core against deterioration by oxygen, moisture, light, and effect of other compounds or other factors; limits the losses of volatile core materials; and releases the core material under desired conditions. In this respect, the core-shell microcapsule of the present disclosure provides controlled release and/or diffusional release of the active material. As used herein, “controlled release” refers to retention of the active material in the core until a specified triggering condition occurs. Such triggers include, e.g., friction, swelling, a pH change, an enzyme, a change in temperature, a change in ionic strength, or a combination thereof.

The microcapsule core comprises an active material. In some embodiments, the microcapsule core further comprises an adjunct core material such as a solvent, an emollient, and/or a core modifier material. Examples of adjunct core materials include nanoscale solid particulate materials, polymeric core modifiers, solubility modifiers, density modifiers, stabilizers, humectants, viscosity modifiers, pH modifiers, or a combination thereof. In some embodiments, the solvent is selected from the group of caprylic/capric triglyceride, benzyl benzoate, ethyl acetate, 3-methoxybutyl acetate, phenyl ethyl benzoate, and combinations thereof. In some embodiments, the adjunct core material can be present in the core in an amount of from 0.01% to 25% e.g., from 0.5% to 10%) by weight of the capsule.

In some embodiments, the amount of the active material (e.g., fragrance) present in the microcapsule core is in a range of from about 50 wt% to about 90 wt%, or from about 50 wt% to about 85 wt%, or from about 55 wt% to about 80 wt%, or from about 60 wt% to about 80 wt%, or from about 65 wt% to about 75 wt%, based on the weight of the microcapsule. In some embodiments, the amount of the active material (e.g., fragrance) present in the microcapsule core is at least 30 wt%, or at least 35 wt%, or at least 40 wt%, or at least 45 wt%, or at least 50 wt%, or at least 55 wt%, or at least 60 wt%, or at least 65 wt%, or at least 70 wt%, or at least 75 wt%, based on the weight of the microcapsule. In some embodiments, the amount of the active material (e.g., fragrance) present in the microcapsule core is no more than 95 wt%, or no more than 92 wt%, or no more than 90 wt%, or no more than 85 wt%, or no more than 80 wt%, or no more than 75 wt%, or no more than 70 wt%, based on the weight of the microcapsule. In some embodiments, the microcapsule core comprises an active material (e.g., fragrance) and a solvent. In some embodiments, the weight ratio of the active material to the solvent is from about 90:10 to about 60:40, or from about 85:15 to about 60:40, or from about 80:20 to about 65:35.

The microcapsule shell comprises, consists essentially of, or consists of a reaction product of an unfolded enzyme with a polyisocyanate. In some embodiments, the reaction product is formed via an interfacial polymerization reaction between the unfolded enzyme and the polyisocyanate. The unfolded enzyme and the polyisocyanate are covalently bonded or crosslinked to form a polymeric network, that is, in the microcapsule shell, the enzyme moiety and the polyisocyanate moiety are connected or crosslinked together via covalent bond. In some embodiments, the microcapsule shell comprises, consists essentially of, or consists of a polymeric network comprising an unfolded enzyme crosslinked with a polyisocyanate. In some embodiments, the unfolded enzyme and the polyisocyanate are crosslinked via a covalent bond such as a urea bond (-NHCONH-). In some embodiments, the microcapsule shell is a single-layered shell, that is, the microcapsule shell has only one layer or one-layered structure. In some embodiments, the microcapsule shell is substantially free of or free of a coacervate. In some embodiments, the microcapsule shell is substantially free of or free of a coacervate which at least partially covers or is coated on the reaction product of the unfolded enzyme with the polyisocyanate. In some embodiments, the microcapsule shell is substantially free of or free of a coacervate formed by one or more polyelectrolytes. In some embodiments, the amount of the coacervate present in the microcapsule shell is no more than 10 wt%, or no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, based on the total weight of the microcapsule shell.

In some embodiments, the microcapsule shell is substantially free of or free of an additional biopolymer. The term “biopolymer”, as used herein, means a polymer obtained from a natural source (e.g., plant, fungus, bacterium or animal) or modified biopolymer thereof. The biopolymer can be a polypeptide (e.g., protein) or a polysaccharide. The term “additional biopolymer”, as used herein, means a biopolymer other than the enzyme(s) used in the present disclosure. In some embodiments, the additional biopolymer is a biopolymer other than glucoamylase, amylase, and/or phytase. In some embodiments, the additional biopolymer is selected from the group of gelatin, collagen, chitosan, guar gum and modified guar gum, glucan and modified glucan, gum Arabic and modified gum Arabic, xanthan gum, albumins, plant proteins, vegetable globulins, hydrophobin, alginate, carrageenan, pectin, modified starch, modified cellulose, whey protein, pea protein, soy protein, rice protein, wheat protein, egg protein, plant storage proteins, milk proteins, and mixtures thereof. In some embodiments, the amount of the additional biopolymer present in the microcapsule shell is no more than 10 wt%, or no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, based on the total weight of the microcapsule shell.

In some embodiments, the microcapsule shell consists essentially of or consists of a reaction product of an unfolded enzyme with a polyisocyanate. In some embodiments, the microcapsule shell consists essentially of or consists of a polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate. By “consist essentially of”, it is meant herein that the microcapsule shell can also contain other chemical compounds or moieties that do not materially affect the diameter, sensory performance, and/or storage stability of the microcapsule. In some embodiments, the amount of the reaction product of the unfolded enzyme with the polyisocyanate, or the polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate, present in the microcapsule shell is at least 95 wt%, or at least 96 wt%, or at least 97 wt%, or at least 98 wt%, or at least 99 wt%, or at least 99.2 wt%, or at least 99.5 wt%, or at least 99.8 wt%, or at least 99.9 wt%, or at least 99.92 wt%, or at least 99.95 wt%, based on the total weight of the microcapsule shell. In some embodiments, the weight ratio of the enzyme moiety to the polyisocyanate moiety present in the microcapsule shell is in a range of from about 20:1 to about 1 .5:1 , or from about 15:1 to about 2:1 , or from about 10:1 to about 2.5:1 , or from about 8:1 to about 3:1 . In some embodiments, the weight ratio of the enzyme moiety to the polyisocyanate moiety present in the microcapsule shell is at least 0.7:1 , or at least 1 :1 , or at least 1 .2:1 , or at least 1 .5:1 , or at least 1 .8:1 , or at least 2:1 , or at least 2.2:1 , or at least 2.5:1 , or at least 2.8:1 , or at least 3:1 , or at least 3.2:1 , or at least 3.5:1 , or at least 3.7:1 , or at least 4:1 , or at least 4.5:1 , or at least 5:1 , or at least 5.5:1 , or at least 6:1 , or at least 6.5:1 , or at least 7:1 . In some embodiments, the weight ratio of the enzyme moiety to the polyisocyanate moiety present in the microcapsule shell is no more than 40:1 , or no more than 35:1 , or no more than 30:1 , or no more than 25:1 , or no more than 20:1 , or no more than 18:1 , or no more than 15:1 , or no more than 12:1 , or no more than 11 :1 , or no more than 10:1 , or no more than 9:1 , or no more than 8:1 , or no more than 7:1 .

In some embodiments, the microcapsule shell is substantially free of or free of a self-condensed polyisocyanate. In some embodiments, the amount of the self-condensed polyisocyanate present in the microcapsule shell is no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, based on the total weight of the microcapsule shell.

The term “biodegradable” as used herein with respect to a material, such as a microcapsule shell as a whole or a polymer (e.g., biodegradable polymer) of the microcapsule shell, means that the material has no real or perceived health and/or environmental issues, and is capable of undergoing and/or does undergo physical, chemical, thermal, microbial, biological and/or UV or photo-degradation. Ideally, a microcapsule shell and/or polymer is deemed “biodegradable” when the microcapsule shell and/or polymer passes one or more of the following tests including: a respirometry biodegradation method in aquatic media, available from Organization for Economic Cooperation and Development (OECD), International Organization for Standardization (ISO) and the American Society for Testing and Material (ASTM) tests including, but not limited to OECD 301 F or 310 (Ready biodegradation), OECD 302 (inherent biodegradation), ISO 17556 (solid stimulation studies), ISO 14851 (fresh water stimulation studies), ISO 18830 (marine sediment stimulation studies), OECD 307 (soil stimulation studies), OECD 308 (sediment stimulation studies), and OECD 309 (water stimulation studies). In some embodiments, the microcapsule shell has a biodegradation rate of at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%, based on the weight of the microcapsule shell, within 60 days according to the OECD301 F test. In some embodiments, the microcapsule shell has a biodegradation rate of at least 60%, based on the weight of the microcapsule shell, within 60 days according to the OECD301 F test.

In some embodiments, the reaction product of the unfolded enzyme with the polyisocyanate, or the polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate, can be crosslinked with a crosslinker to further improve performance. In some embodiments, the reaction product of the unfolded enzyme with the polyisocyanate, or the polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate, is not further crosslinked with a crosslinker. Crosslinker is a crosslinking agent. The term “crosslinker”, as used herein, means a crosslinking agent other than the enzyme(s), unfolded enzyme(s) and polyisocyanate(s) used in the present disclosure. In some embodiments, the crosslinker comprises or is tannic acid and/or glutaraldehyde. The tannic acid can be hydrolyzed tannic acid and/or unhydrolyzed tannic acid. In some embodiments, the crosslinker is selected from the group of polyfunctional aldehydes, polyols including polyphenols, polyamines, triethyl citrate, and mixtures thereof. In some embodiments, the crosslinker comprises or is a polyfunctional aldehyde and/or a polyphenol. The term “polyfunctional aldehyde”, as used herein, means a chemical compound having two or more formyl (-CHO) groups. The term “polyphenol”, as used herein, means a chemical compound having two or more hydroxyphenyl groups.

In some embodiments, the polyfunctional aldehyde is selected from the group of glutaraldehyde, glyoxal, genipin (e.g., polymerized genipin), di-aldehyde starch, malondialdehyde, succinic dialdehyde, 1 ,3-propane dialdehyde, 1 ,4-butane dialdehyde, 1 ,5-pentane dialdehyde, 1 ,6-hexane dialdehyde, glyoxal trimer, paraformaldehyde, bis(dimethyl) acetal, bis(diethyl) acetal, polymeric dialdehydes, and mixtures thereof. In some embodiments, the polyfunctional aldehyde is selected from the group of glutaraldehyde, glyoxal, genipin, and mixtures thereof. In some embodiments, the polyfunctional aldehyde comprises or is an oxidized sugar having two or more formyl (- CHO) groups. The term “oxidized sugar”, as used herein, means a sugar (e.g., monosaccharide, oligosaccharide, and/or polysaccharide) that has been treated with an oxidizing agent to generate reactive formyl groups. In some embodiments, the sugar has a molecular weight of less than 1000 g/mol and is soluble or dispersible in an aqueous solution. In some embodiments, the sugar is selected from the group of glucose, glucosamine, sucrose, maltose, lactose, maltodextrin, cyclodextrin, polysaccharide, hydrolyzed polysaccharide, and mixtures thereof. In some embodiments, the sugar is selected from the group of sucrose, glucosamine, maltodextrin, cyclodextrin, and mixtures thereof. In some embodiments, the oxidizing agent is selected from the group of sodium periodate, hydrogen peroxide, laccases, oxidases, and combinations thereof. In some embodiments, the oxidizing agent comprises sodium periodate. In some embodiments, the polyfunctional aldehyde comprises or is an oxidized sucrose.

In some embodiments, the polyphenol is selected from the group of flavonoid, isoflavonoid, neoflavonoid, gallotannin, ellagotannin, catechol, DL-3,4- dihydroxyphenylalaline, catecholamine, dopamine, phloroglucinol, phenolic acid, phenolic ester, phenolic heteroside, curcumin, polyhydroxylated coumarin, polyhydroxylated lignan, neolignan, poly-resorcinol, tannin, tannic acid, and mixtures thereof. Examples of suitable phenolic acid include gallic acid and tannic acid. Examples of suitable phenolic ester include methyl gallate and ethyl gallate. In some embodiments, the polyphenol comprises or is a phenolic acid having 3,4,5-trihydroxyphenyl group or 3,4-dihydroxyphenyl group. In some embodiments, the polyphenol is selected from the group of tannic acid, gallic acid, methyl gallate, ethyl gallate, and mixtures thereof.

In some embodiments, the microcapsule shell is substantially free of or free of moiety of an additional polyfunctional nucleophile and/or moiety of an additional polyfunctional electrophile. The term “polyfunctional nucleophile”, as used herein, means a chemical compound having two or more nucleophilic groups such as amine group and hydroxyl group. The term “additional polyfunctional nucleophile”, as used herein, means a polyfunctional nucleophile other than the enzyme(s) used in the present disclosure. In some embodiments, the additional polyfunctional nucleophile is a polyfunctional nucleophile other than glucoamylase, amylase, and/or phytase. In some embodiments, the additional polyfunctional nucleophile is polyamine and/or polyol (e.g., polyphenol). The term “polyol”, as used herein, means a chemical compound having two or more hydroxyl groups. The term “polyamine”, as used herein, means a chemical compound having two or more amine groups. The term “polyfunctional electrophile”, as used herein, means a chemical compound having two or more electrophilic groups such as formyl (-CHO) group and isocyanate (-NCO) group. The term “additional polyfunctional electrophile”, as used herein, means a polyfunctional electrophile other than polyisocyanate(s) used in the present disclosure. In some embodiments, the additional polyfunctional electrophile is polyfunctional aldehyde.

In some embodiments, the amount of the moiety of an additional polyfunctional nucleophile present in the microcapsule shell is no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, based on the total weight of the microcapsule shell. In some embodiments, the amount of the moiety of an additional polyfunctional electrophile present in the microcapsule shell is no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, based on the total weight of the microcapsule shell.

The microcapsules in the present disclosure do not have to be perfectly spherical. The term “diameter”, as used herein with respect to a microcapsule, means the diameter of a sphere having the same volume as the microcapsule. In some embodiments, at least 80% (by volume) of the microcapsules have diameter ranging from 1 pm to 200 pm, or from 2 pm to 150 pm, or from 3 pm to 100 pm, or from 4 pm to 80 pm, or from 5 pm to 75 pm, or from 6 pm to 75 pm, or from 7 pm to 75 pm, or from 8 pm to 75 pm. In some embodiments, at least 80% (by volume) of the microcapsules have diameter of at least 0.5 pm, or at least 1 pm, or at least 2 pm, or at least 3 pm, or at least 4 pm, or at least 5 pm, or at least 6 pm, or at least 7 pm, or at least 8 pm, or at least 10 pm, or at least 12 pm, or at least 15 pm, or at least 18 pm, or at least 20 pm. In some embodiments, at least 80% (by volume) of the microcapsules have diameter of no more than 400 pm, or no more than 300 pm, or no more than 200 pm, or no more than 150 pm, or no more than 120 pm, or no more than 110 pm, or no more than 100 pm, or no more than 90 pm, or no more than 85 pm, or no more than 80 pm, or no more than 75 pm, or no more than 70 pm, or no more than 65 pm, or no more than 60 pm, or no more than 55 pm, or no more than 50 pm. The present disclosure also provides a process for producing the biodegradable core-shell microcapsules described in this disclosure or a microcapsule slurry comprising such microcapsule. The process comprises: (a) providing an aqueous phase comprising an unfolded enzyme, wherein the pH of the aqueous phase is equal to or greater than the isoelectric point of the enzyme; (b) providing an oil phase comprising an active material and a polyisocyanate; (c) emulsifying the oil phase with the aqueous phase to form an emulsion; (d) allowing the unfolded enzyme to react with the polyisocyanate to form a microcapsule shell encapsulating a microcapsule core, thereby forming a core-shell microcapsule slurry comprising a biodegradable core-shell microcapsule, wherein the microcapsule core comprises the active material, and the microcapsule shell comprises a reaction product of the unfolded enzyme with the polyisocyanate; and (e) curing the microcapsule shell at an elevated temperature. In some embodiments, the reaction product comprises or is a polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate. The present disclosure also provides a coreshell microcapsule or a core-shell microcapsule slurry obtainable by such processes described in this disclosure.

In step (a), an aqueous phase is prepared and provided. In some embodiments, the aqueous phase is homogenous. In step (a), an enzyme is unfolded. In some embodiments, a native enzyme is unfolded in step (a). The native enzyme means an enzyme in its native state. The native enzyme is in its properly folded and/or assembled form, which is operative and functional in catalyzing a chemical reaction. In some embodiments, the unfolded enzyme is prepared by diluting the enzyme in water or an aqueous solution, optionally in the presence of a thiol-containing reducing agent (e.g., cysteine). In some embodiments, the unfolded enzyme is prepared in substantial absence or in absence of a chaotropic agent other than cysteine. In some embodiments, the unfolded enzyme is prepared in substantial absence or in absence of a thiol-containing reducing agent (e.g., cysteine). In some embodiments, the concentration of the unfolded enzyme in the aqueous phase is from about 1 wt% to about 10 wt%, or from about 2 wt% to about 9 wt%, or from about 3 wt% to about 8 wt%, based on the total weight of the aqueous phase. In some embodiments, the concentration of the unfolded enzyme in the aqueous phase is at least 1 wt%, or at least 2 wt%, or at least 3 wt%, or at least 4 wt%, or at least 5 wt%, based on the total weight of the aqueous phase. In some embodiments, the concentration of the unfolded enzyme in the aqueous phase is no more than 15 wt%, or no more than 12 wt%, or no more than 1 1 wt%, or no more than 10 wt%, or no more than 9 wt%, or no more than 8 wt%, or no more than 7 wt%, or no more than 6 wt%, based on the total weight of the aqueous phase.

In some embodiments, the aqueous phase further comprises a thiol-containing reducing agent (e.g., cysteine). In some embodiments, the weight ratio of the unfolded enzyme to the thiol-containing reducing agent is in a range of from about 150:1 to about 5:1 , or from about 120:1 to about 7:1 , or from about 100:1 to about 8:1 , or from about 80:1 to about 10:1 , or from about 60:1 to about 15:1 . In some embodiments, the aqueous phase is substantially free of or free of a chaotropic agent other than cysteine. In some embodiments, the amount of the chaotropic agent other than cysteine present in the aqueous phase is no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, or no more than 0.005 wt%, based on the total weight of the aqueous phase. In some embodiments, the aqueous phase is substantially free of or free of a thiol-containing reducing agent. In some embodiments, the amount of the thiol-containing reducing agent present in the aqueous phase is no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, or no more than 0.005 wt%, or no more than 0.002 wt%, based on the total weight of the aqueous phase.

In some embodiments, the aqueous phase further comprises an emulsifier. In some embodiments, the concentration of the emulsifier in the aqueous phase is at least 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, or 0.3 wt%, based on the total weight of the aqueous phase. In some embodiments, the concentration of the emulsifier in the aqueous phase is no more than 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, or 1 wt%, based on the total weight of the aqueous phase.

In some embodiments, the unfolded enzyme can function as an emulsifier, and the aqueous phase is substantially free of or free of an emulsifier. The term “emulsifier”, as used herein, means an emulsifier other than the enzyme(s) and unfolded enzyme(s) used in the present disclosure. In some embodiments, the emulsifier comprises or is an amphiphilic compound selected from the group of partially neutralized acid esters, polyvinyl alcohol and modified polyvinyl alcohol, polystyrene sulfonates (e.g., Flexan® II), sodium salt of alkylnaphthalene sulfonate condensate (Morwet® D-425), poloxamers, polysorbates, copolymer of vinyl pyrrolidone and quaternized dimethylaminoethyl methacrylate (Polyquaternium 11 ), tristyrylphenol ethoxylates, phosphated or sulfated tristyrylphenol ethoxylates, polyvinyl pyrrolidone, carboxymethyl cellulose (CMC), polyacrylic acid, polymethacrylic acid, copolymers of acrylic acid and acrylamide, copolymer of acrylamide and acrylamidopropyltrimonium chloride, terpolymers of (acrylic acid, acrylamide, and acrylamidopropyltrimonium chloride), co-polymers of ethylene and maleic anhydride (ZeMac), maleic anhydride copolymers and their hydrolysates, acrylic acid butyl acrylate copolymer, crotonic acid homopolymers and copolymers, vinyl benzenesulfonate homopolymers and copolymers, 2-acrylamido-2- methylpropanesulfonate homopolymers and copolymers, phospholipids, glycolipids, fatty acids, saponins, quillaia extract, surfactant salts with carboxylate, sulfate, sulfonate, phosphate, betaine, and/or linear alcohol groups, and mixtures thereof. In some embodiments, the amount of the emulsifier present in the aqueous phase is no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, or no more than 0.005 wt%, based on the total weight of the aqueous phase.

In some embodiments, the aqueous phase is substantially free of or free of an additional biopolymer. In some embodiments, the amount of the additional biopolymer present in the aqueous phase is no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, based on the total weight of the aqueous phase.

In some embodiments, the aqueous phase is substantially free of or free of an additional polyfunctional nucleophile. In some embodiments, the amount of the additional polyfunctional nucleophile present in the aqueous phase is no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, or no more than 0.005 wt%, based on the total weight of the aqueous phase.

In some embodiments, the aqueous phase is at room temperature. In some embodiments, the temperature of the aqueous phase is in a range of from 10 °C to 30 °C, or from 15 °C to 30 °C, or from 20 °C to 30 °C, or from 18 °C to 28 °C, or from 20 °C to 25 °C. The pH of the aqueous phase is equal to or greater than the isoelectric point (pl) of the enzyme. In some embodiments, the pH of the aqueous phase is at least 5.5, or at least 6.5, or at least 7.5. In some embodiments, the pH of the aqueous phase is in a range of from about 5.5 to about 10, or from about 6.5 to about 9.5, or from about 8 to about 9.

In step (b), an oil phase is prepared and provided. The oil phase comprises an active material and a polyisocyanate. In some embodiments, the polyisocyanate comprises or is an aromatic polyisocyanate. In some embodiments, the polyisocyanate comprises or is trimethylol propane-adduct of xylylene diisocyanate. In some embodiments, the amount of the polyisocyanate present in the oil phase is in a range of from 0.1 wt% to 10 wt%, or from 0.1 wt% to 8 wt%, or from 0.2 wt% to 5 wt%, or from 0.5 wt% to 4 wt%, or from 0.7 wt% to 3 wt%, based on the total weight of the oil phase. In some embodiments, the amount of the polyisocyanate present in the oil phase is at least 0.1 wt%, or at least 0.2 wt%, or at least 0.3 wt%, or at least 0.4 wt%, or at least 0.5 wt%, or at least 0.6 wt%, or at least 0.7 wt%, or at least 0.8 wt%, or at least 0.9 wt%, or at least 1 wt%, or at least 1 .1 wt%, or at least 1 .2 wt%, based on the total weight of the oil phase. In some embodiments, the amount of the polyisocyanate present in the oil phase is no more than 15 wt%, or no more than 12 wt%, or no more than 10 wt%, or no more than 9 wt%, or no more than 8 wt%, or no more than 7 wt%, or no more than 6 wt%, or no more than 5 wt%, or no more than 4 wt%, or no more than 3 wt%, or no more than 2 wt%, or no more than 1 .5 wt%, based on the total weight of the oil phase. In some embodiments, the oil phase further comprises an adjunct core material such as a solvent, an emollient, and/or a core modifier material. In some embodiments, the solvent is selected from the group of caprylic/capric triglyceride, benzyl benzoate, ethyl acetate, 3-methoxybutyl acetate, phenyl ethyl benzoate, and mixtures thereof. The oil phase can be prepared by mixing an active material and a polyisocyanate. In some embodiments, an adjunct core material (e.g., a solvent) is also mixed with the active material and the polyisocyanate. In some embodiments, the polyisocyanate is dissolved in a solution comprising a solvent and an active material. In some embodiments, the oil phase is at room temperature. In some embodiments, the temperature of the oil phase is in a range of from 10 °C to 30 °C, or from 15 °C to 30 °C, or from 20 °C to 30 °C, or from 18 °C to 28 °C, or from 20 °C to 25 °C.

In step (c), the oil phase is emulsified with the aqueous phase to form an emulsion. In some embodiments, an emulsifier is used in step (c), that is, the emulsion comprises an emulsifier (e.g., polystyrene sulfonates). In some embodiments, essentially no emulsifier is used in step (c), that is, the emulsion is substantially free of or free of an emulsifier. In some embodiments, the amount of the emulsifier present in the emulsion is no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, or no more than 0.005 wt%, or no more than 0.002 wt%, based on the total weight of the emulsion. The emulsifying process can be carried out using technologies known in the art. For example, a stirrer, an agitator or a homogenizer (e.g., high shear homogenizer) can be used for emulsification. In some embodiments, the emulsifying process is carried out at room temperature. In some embodiments, the emulsifying process is carried out at a temperature of from about 10 °C to about 30 °C, or from about 15 °C to about 30 °C, or from about 20 °C to about 30 °C, or from about 18 °C to about 28 °C, or from about 20 °C to about 25 °C.

Interfacial polymerization occurs during the emulsifying process, that is, the unfolded enzyme and the polyisocyanate react at the interface between the oil phase and the aqueous phase to form a reaction product of the unfolded enzyme with the polyisocyanate, wherein the unfolded enzyme and the polyisocyanate are covalently bonded or crosslinked to form a microcapsule shell encapsulating a microcapsule core, thereby forming a core-shell microcapsule slurry comprising a biodegradable core-shell microcapsule, wherein the microcapsule core comprises the active material, and the microcapsule shell comprises the reaction product of the unfolded enzyme with the polyisocyanate. In some embodiments, the weight ratio of the unfolded enzyme to the polyisocyanate used in the process (of producing the microcapsule and the microcapsule slurry) is in a range of from about 20:1 to about 1 .5:1 , or from about 15:1 to about 2:1 , or from about 10:1 to about 2.5:1 , or from about 8:1 to about 3:1 . In some embodiments, the weight ratio of the unfolded enzyme to the polyisocyanate used in the process is at least 0.7:1 , or at least 1 :1 , or at least 1 .2:1 , or at least 1 .5:1 , or at least 1 .8:1 , or at least 2:1 , or at least 2.2:1 , or at least 2.5:1 , or at least 2.8:1 , or at least 3:1 , or at least 3.2:1 , or at least 3.5:1 , or at least 3.7:1 , or at least 4:1 , or at least 4.5:1 , or at least 5:1 , or at least 5.5:1 , or at least 6:1 , or at least 6.5:1 , or at least 7:1 . In some embodiments, the weight ratio of the unfolded enzyme to the polyisocyanate used in the process is no more than 40:1 , or no more than 35:1 , or no more than 30:1 , or no more than 25:1 , or no more than 20:1 , or no more than 18:1 , or no more than 15:1 , or no more than 12:1 , or no more than 1 1 :1 , or no more than 10:1 , or no more than 9:1 , or no more than 8:1 , or no more than 7:1 . In some embodiments, the produced core-shell microcapsule has the composition and/or property of the biodegradable core-shell microcapsule described in this disclosure.

In step (e), the microcapsule shell is cured at an elevated temperature. The term “curing”, as used herein, means a toughening or hardening process of a polymer brought about by heat, chemical additives, and/or light radiation. In some embodiments, the microcapsule shell is cured at a temperature in a range of from about 35 °C to about 80 °C, or from about 40 °C to about 75 °C. In some embodiments, the microcapsule shell is cured at a temperature of no more than 150 °C, or no more than 120 °C, or no more than 100 °C, or no more than 90 °C, or no more than 80 °C, or no more than 70 °C. In some embodiments, the microcapsule shell is cured at a temperature of at least 35 °C, or at least 40 °C, or at least 45 °C, or at least 50 °C, or at least 55 °C, or at least 60 °C, or at least 65 °C. In some embodiments, the microcapsule shell is cured for about 30 minutes to 24 hours, or 30 minutes to 12 hours, or 1 hour to 8 hours. In some embodiments, the microcapsule shell is cured for at least 10 minutes, or at least 20 minutes, or at least 30 minutes, or at least 45 minutes, or at least 1 hour, or at least 1 .5 hours, or at least 2 hours. In some embodiments, the microcapsule shell is cured for no more than 48 hours, or no more than 36 hours, or no more than 24 hours, or no more than 20 hours, or no more than 16 hours, or no more than 12 hours, or no more than 10 hours, or no more than 8 hours, or no more than 6 hours, or no more than 4 hours.

In some embodiments, a crosslinker can be added or used in the process to crosslink with the reaction product of the unfolded enzyme with the polyisocyanate. In some embodiments, the crosslinker can be added before, after, or during the curing step (e). In some embodiments, the crosslinker is added after the emulsifying process begins. In some embodiments, the crosslinker is added into the microcapsule slurry after the curing step. In some embodiments, the crosslinker is tannic acid and/or glutaraldehyde. In some embodiments, essentially no crosslinker is added or used in the process. In some embodiments, the amount of the crosslinker added or used in the process is no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, or no more than 0.005 wt%, or no more than 0.002 wt%, based on the total weight of the microcapsule slurry.

Advantageously, essentially no self-condensed polyisocyanate is formed during the process. In some embodiments, the amount of the self-condensed polyisocyanate generated in the process is no more than 10 wt%, or no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, based on the total weight of the polyisocyanate fed or used in the process.

Using the process of the present disclosure, a high encapsulation efficiency (EE) can be achieved. The term “encapsulation efficiency”, as used herein with respect to the preparation or production of microcapsule, means the amount (in weight) of the active material being encapsulated relative to the total amount (in weight) of the active material used in the preparation or production of the microcapsule. In some embodiments, the encapsulation efficiency of the microcapsule produced in the process is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%.

In some embodiments, gum Arabic can be added into the microcapsule slurry before or during the curing step (e) so that the microcapsules can be dispersed in the slurry for a prolonged period of time. In some embodiments, the amount of gum Arabic added into the microcapsule slurry is at least 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, or 0.3 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the amount of gum Arabic added into the microcapsule slurry is no more than 10 wt%, 5 wt%, 2 wt%, or 1 wt%, based on the total weight of the microcapsule slurry.

In some embodiments, a thickener can be added into the microcapsule slurry before, after, or during the curing step (e) to improve the stability of the microcapsule slurry. In some embodiments, the thickener is selected from the group of xanthan gum, microcrystalline cellulose (MCC), coprocessed mixture of MCC and carboxymethyl cellulose (CMC), hydroxypropyl methyl cellulose (HPMC), and combinations thereof. In some embodiments, the thickener comprises or is a combination of xanthan gum and a coprocessed mixture of MCC and CMC. In some embodiments, the thickener comprises a combination of HPMC and a coprocessed mixture of MCC and CMC. In some embodiments, the amount of the thickener added into the microcapsule slurry is at least 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the amount of the thickener added into the microcapsule slurry is no more than 5 wt%, 2 wt%, 1 wt%, or 0.5 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the process further comprises step (f): reducing the amount of residual enzyme (e.g., glucoamylase) present in the core-shell microcapsule slurry. In some embodiments, the amount of residual enzyme is reduced to no more than 1 wt%, 0.8 wt%, 0.6 wt%, 0.4 wt%, 0.2 wt%, 0.1 wt%, 0.05 wt%, 0.02 wt%, or 0.01 wt%, based on the total weight of the microcapsule slurry. The reduction can be effected by lowering the pH value of the slurry. Generally, lower pH can reduce the amount of residual enzyme more effectively. In some embodiments, the amount of residual enzyme is reduced by lowering or adjusting the pH of the microcapsule slurry to no more than 4.5, 4.2, 4.0, 3.8, 3.6, 3.5, 3.4, 3.2, 3.1 , 3.0, 2.8, 2.6, 2.4, 2.2, or 2.0. The lower limit of the pH of the microcapsule slurry can be -1 , -0.5, 0, 0.5, 1 , 1 .5, or 2. The pH value can be lowered or adjusted by adding an acid into the microcapsule slurry. In some embodiments, the acid is selected from the group of citric acid, tartaric acid, acetic acid, phosphoric acid, hydrochloric acid (HCI), and mixtures thereof. In some embodiments, the reducing step (f) can be carried out during or after the curing step (e). For example, the pH of the microcapsule slurry can be lowered or adjusted to the desired value (for reducing the amount of residual enzyme) during or after the curing step (e).

In some embodiments, the reducing step (f) is carried out at an elevated temperature. Generally, higher temperature can reduce the amount of residual enzyme more quickly. In some embodiments, the reducing step (f) is carried out at a temperature of at least 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, or 60 °C. In some embodiments, the reducing step (f) is carried out at a temperature of no more than 120 °C, 100 °C, 90 °C, 80 °C, or 70 °C. In some embodiments, the reducing step (f) is carried out for at least 5 minutes, 10 minutes, 20 minutes, 30 minutes, or 45 minutes. In some embodiments, the reducing step (f) is carried out for no more than 36 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, or 2 hours.

In some embodiments, after the reducing step (f), the pH of the microcapsule slurry is adjusted to pH in a range of from about 4 to about 7.5, or from about 4 to about 6.0, or from about 5 to about 7.5, or from about 5 to about 7.0. In some embodiments, a preservative can be added to the microcapsule slurry, preferably after the curing step, to prevent inadvertent growth of microorganisms. The preservative can be any organic preservative that does not cause damage to the microcapsule slurry. Suitable preservatives include a combination of methylchloroisothiazolinone and methylisothiazolinone available under the tradename KATHON® CG; 5-bromo-5-n tro-1 ,3- dioxane available under the tradename BRONIDOX L®; 2-bromo-2-nitro-1 ,3-propanediol available under the tradename BRONOPOL®; polyaminopropyl biguanide; dehydroacetic acid; phenoxyethanol; sodium benzoate; sodium sorbate; potassium sorbate; and 1 ,2- benzisothiazolin-3-one available under the tradenames PROXEL® GXL and ROCIMA™ BT NV2.

In some embodiments, the process may further comprise a step of drying the coreshell microcapsule to remove water. The microcapsules can be dried at a temperature of from 20 °C to 250 °C, or at room temperature. In some embodiments, the microcapsules are dried by a dehumidifier configured to supply desiccated air to the microcapsules, a radiant heat source for facilitating drying of the microcapsules, or submitting the microcapsules under a gas flow to obtain dried free-flowing microcapsules. It is understood that any standard method known by a person skilled in the art to perform such drying is also applicable.

The present disclosure also provides a core-shell microcapsule slurry, comprising: (a) the biodegradable core-shell microcapsule described in this disclosure; and (b) water. In some embodiments, the amount of water in the microcapsule slurry is at least 20 wt%, 30 wt%, 40 wt%, 45 wt%, 50 wt%, or 55 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the amount of water in the microcapsule slurry is no more than 95 wt%, 90 wt%, 85 wt%, 80 wt%, 75 wt%, or 70 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the core-shell microcapsule slurry further comprises a thickener. In some embodiments, the thickener is selected from the group of xanthan gum, microcrystalline cellulose (MCC), coprocessed mixture of MCC and carboxymethyl cellulose (CMC), hydroxypropyl methyl cellulose (HPMC), and combinations thereof. In some embodiments, the thickener comprises or is a combination of xanthan gum and a coprocessed mixture of MCC and CMC. In some embodiments, the thickener comprises a combination of HPMC and a coprocessed mixture of MCC and CMC. In some embodiments, the amount of the thickener in the microcapsule slurry is at least 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the amount of the thickener in the microcapsule slurry is no more than 5 wt%, 2 wt%, 1 wt%, or 0.5 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the microcapsule slurry further comprises gum Arabic. In some embodiments, the amount of gum Arabic in the microcapsule slurry is at least 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, or 0.3 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the amount of gum Arabic in the microcapsule slurry is no more than 10 wt%, 5 wt%, 2 wt%, or 1 wt%, based on the total weight of the microcapsule slurry.

In some embodiments, the core-shell microcapsule slurry further comprises an emulsifier. In some embodiments, the concentration of the emulsifier in the slurry is at least 0.005 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the concentration of the emulsifier in the slurry is no more than 4 wt%, 3 wt%, 2 wt%, 1 wt%, or 0.5 wt%, based on the total weight of the microcapsule slurry. In some embodiments, the core-shell microcapsule slurry is substantially free of or free of an emulsifier. In some embodiments, the amount of the emulsifier present in the microcapsule slurry is no more than 0.1 wt%, 0.05 wt%, 0.02 wt%, 0.01 wt%, or 0.005 wt%, based on the total weight of the microcapsule slurry.

The core-shell microcapsule slurry of the present disclosure has reduced amount of residual enzyme. In some embodiments, the amount of residual enzyme (e.g., glucoamylase) present in the microcapsule slurry is no more than 1 .5 wt%, 1 wt%, 0.8 wt%, 0.6 wt%, 0.4 wt%, 0.2 wt%, 0.1 wt%, 0.05 wt%, 0.02 wt%, or 0.01 wt%, based on the total weight of the microcapsule slurry. The amount of residual enzyme (e.g., glucoamylase) present in the microcapsule slurry can be determined by using gel permeation chromatography (GPC).

In some embodiments, the microcapsule slurry has pH of at least 1 , 1 .5, 2.0, 2.5, 2.8, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, or 4.5. In some embodiments, the microcapsule slurry has pH of no more than 7.5, 7.2, 7.0, 6.8, 6.5, 6.2, 6.0, 5.8, 5.5, 5.2, 5.0, 4.5, 4.2, or 4.0. In some embodiments, the microcapsule slurry has pH in a range of from about 4 to about 7.5, or from about 4 to about 6.0, or from about 5 to about 7.5, or from about 5 to about 7.0.

The microcapsule slurry can be formulated into a capsule delivery system for use in consumer products. The capsule delivery system can be a microcapsule slurry suspended in an external solvent e.g., water, ethanol, or a combination thereof), wherein the microcapsule is present at a level from about 0.1% to about 80% (e.g., 70-75%, 40-55%, 50-90%, 1 -65%, or 5-45%) by weight of the capsule delivery system. In some embodiments, the microcapsule and its slurry prepared in the process of the present disclosure can be subsequently purified. In some embodiments, purification can be achieved by washing the microcapsule slurry with water until a neutral pH is obtained.

The biodegradable core-shell microcapsule of the present disclosure is well-suited for inclusion in consumer products where controlled release of active material (e.g., fragrances or flavors) is desired. The present disclosure also provides a consumer product comprising the biodegradable core-shell microcapsule of this disclosure. In some embodiments, the consumer product is selected from the group of fabric softener, fabric conditioner, detergent, scent booster, fabric refresher spray, body wash, body soap, shampoo, hair conditioner, body spray, hair refresher spray, hair dye, hair moisturizer, skin moisturizer, hair treatment, skin treatment, antiperspirant, deodorant, insect repellant, candle, surface cleaner, bathroom cleaner, bleach, cat litter, refresher spray, pesticide, insecticide, herbicide, fungicide, paint, and combinations thereof. Liquid Fragrance Composition

The present disclosure also provides a liquid fragrance composition. In some embodiments, the liquid fragrance composition comprises (i) 3 wt% to 40 wt% (e.g., 5 wt% to 30 wt%, preferably 8 wt% to 20 wt%) of a free fragrance, (ii) 0.1 wt% to 10 wt% (e.g., 0.2 wt% to 5 wt%, preferably 0.5 wt% to 2 wt%) of glyceryl ricinoleate, (iii) 0.1 wt% to 20 wt% (e.g., 0.2 wt% to 15 wt%, preferably 0.5 wt% to 10 wt%, and more preferably 1 wt% to 6 wt%) of a microcapsule, (iv) 0.02 wt% to 5 wt% (e.g., 0.05 wt% to 3 wt%, preferably 0.08 wt% to 2 wt%, and more preferably 0.1 wt% to 1 wt%) of a thickening agent, and (v) 50 wt% to 95 wt% (e.g., 60 wt% to 95 wt%, preferably 70 wt% to 90 wt%) of water. All amounts are based on the weight of the liquid fragrance composition. In some embodiments, the liquid fragrance composition has a pH of 3.5 to 8, preferably 4 to 6. The term “free fragrance”, as used herein, means a neat fragrance essentially free of a fragrance carrier. The free fragrance is not encapsulated or enclosed within a polymeric network, or otherwise immobilized in a delivery system. In some embodiments, the free fragrance is present in the liquid fragrance composition as fragrance oil droplets dispersed homogeneously in the aqueous phase to form an oil-in-water emulsion. The oil droplets can have a size in diameter of from 0.1 pm to 200 pm, preferably from 0.5 pm to 150 pm, and more preferably from 5 pm to 75 pm. In some embodiments, the liquid fragrance composition is stable for at least 8 weeks (preferably at least 26 weeks) at a temperature of 25 °C. In some embodiments, the liquid fragrance composition is suitable for a consumer product such as an air freshener, an air spray, a concentrated floor cleaner, a laundry scent booster, a shampoo, a hair conditioner, a body lotion, a fine fragrance, a facial lotion, a facial tissue, a cleansing wipe, a hand soap, or an alcohol free perfume.

In some embodiments, the microcapsule comprises or is the biodegradable coreshell microcapsule of the present disclosure. In some embodiments, the microcapsule comprises or is a microcapsule comprising a biopolymer in the microcapsule shell (such as the microcapsules described in WO2023/137121 ). In some embodiments, the microcapsule comprises or is a microcapsule with the microcapsule shell formed by the self-condensation of a polyisocyanate in the presence of a denatured pea protein (such as the microcapsules described in W02023/009514).

In some embodiments, the liquid fragrance composition has a viscosity of from 400 cP (centipoise) to 3000 cP (e.g., from 450 cP to 2500 cP). The viscosity can be measured with a viscometer, such as Brookfield DV-I 1 1 Ultra Programmable viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, Mass), at 25 °C and a shear rate of 1 to 120 second^-1 (e.g., 21 second^-1). The viscosity can be adjusted for better delivery and/or greater stability using a thickening agent. In some embodiments, the thickening agent is selected from the group of acrylate copolymers, cationic acrylamide copolymers, polysaccharides, and combinations thereof. Examples of acrylate copolymer include copolymers of acrylic acid and acrylate, acrylate/Cio-Cso alkyl acrylate cross-polymers, and combinations thereof. Examples of cationic acrylamide copolymers include copolymers of acrylamide and methacrylate cross-linked with a difunctional vinyl addition monomer such as methylene bisacrylamide. Examples of polysaccharides include starch, modified starch, pectin, alginate, guar gum, locust bean gum, gellan gum, xanthan gum, and combinations thereof. In some embodiments, the thickening agent comprises or is a combination of acrylate/C -Cso alkyl acrylate cross-polymer with gellan gum or xanthan gum.

In some embodiments, the thickening agent comprises or is a combination of acrylate/Cw-Cso alkyl acrylate cross-polymer and gellan gum, the amount of acrylate/Cio- C30 alkyl acrylate cross-polymer in the liquid fragrance composition is from 0.02 wt% to 3 wt% (e.g., 0.05 wt% to 2 wt%, preferably 0.08 wt% to 1 wt%, and more preferably 0.1 wt% to 0.5 wt%), and the amount of gellan gum in the liquid fragrance composition is from 0.001 wt% to 1 wt% (e.g., 0.005 wt% to 0.5 wt%, preferably 0.008 wt% to 0.2 wt%, and more preferably 0.01 wt% to 0.1 wt%). All amounts are based on the weight of the liquid fragrance composition.

In some embodiments, the thickening agent comprises or is a combination of acrylate/C -Cso alkyl acrylate cross-polymer and xanthan gum, the amount of acrylate/Cw- C30 alkyl acrylate cross-polymer in the liquid fragrance composition is from 0.02 wt% to 3 wt% (e.g., 0.05 wt% to 2 wt%, preferably 0.08 wt% to 1 wt%, and more preferably 0.1 wt% to 0.5 wt%), and the amount of xanthan gum in the liquid fragrance composition is from 0.02 wt% to 3 wt% (e.g., 0.05 wt% to 2 wt%, preferably 0.08 wt% to 1 wt%, and more preferably 0.1 wt% to 0.5 wt%). All amounts are based on the weight of the liquid fragrance composition.

Applications

The biodegradable core-shell microcapsule, microcapsule slurry and capsule delivery system of the present disclosure are well-suited for use, without limitation, in a laundry detergent, a liquid laundry detergent, a powder laundry detergent, a tablet laundry detergent, a laundry detergent bar, a laundry detergent cream, a hand wash laundry detergent, a fabric conditioner or softener, a fabric refresher, a scent booster, a shampoo, a hair conditioner, a bar soap, a shower gel, a body wash, an antiperspirant, a body spray, a body mist, a lotion, a candle or a textile.

More specifically, the biodegradable core-shell microcapsules of the present disclosure can be used in the following products:

A) Fabric Care Products such as Rinse Conditioners (containing 1 to 30 weight % of a fabric conditioning active), Fabric Liquid Conditioners (containing 1 to 30 weight % of a fabric conditioning active), Tumble Drier Sheets, Fabric Refreshers, Fabric Refresher Sprays, Ironing Liquids, and Fabric Softener Systems such as those described in US6,335,315, US5,674,832, US5,759,990, US5,877,145, US5,574,179, US5,562,849, US5,545,350, US5,545,340, US5,41 1 ,671 , US5,403,499, US5,288,417, US4,767,547 and US4,424,134.

Liquid fabric softeners/fresheners contain at least one fabric softening agent present, preferably at a concentration of 1 to 30% (e.g., 4% to 20%, 4% to 10%, and 8% to 15%) by weight of the liquid fabric softener/freshener. The ratio between the active material and the fabric softening agent can be 1 :500 to 1 :2 e.g., 1 :250 to 1 :4 and 1 :100 to 1 :8). As an illustration, when the fabric softening agent is 5% by weight of the fabric softener, the active material is 0.01% to 2.5%, preferably 0.02% to 1 .25% and more preferably 0.1% to 0.63%. As another example, when the fabric softening agent is 20% by weight of the fabric softener, the active material is 0.04% to 10%, preferably 0.08% to 5% and more preferably 0.4% to 2.5%. The active material is a fragrance, malodor counteractant or a combination thereof. The liquid fabric softener can have 0.15% to 15% of capsules {e.g., 0.5% to 10%, 0.7% to 5%, and 1 % to 3%). When including capsules at these levels, the neat oil equivalent (NOE) in the softener is 0.05% to 5% e.g., 0.15% to 3.2%, 0.25% to 2%, and 0.3% to 1 %).

Suitable fabric softening agents include cationic surfactants. Non-limiting examples are quaternary ammonium compounds (QAC) such as alkylated quaternary ammonium compounds, ring or cyclic quaternary ammonium compounds, aromatic quaternary ammonium compounds, diquaternary ammonium compounds, alkoxylated quaternary ammonium compounds, amidoamine quaternary ammonium compounds, ester quaternary ammonium compounds, or a combination thereof.

Fabric softening product includes an aqueous QAC which are characterized by: a) The viscosity of the final product ranges from 5 to 300 cps @ 106 s^-1, preferable 20 to 150 cps; b) The level of QAC ranges from 0.5 to 20 wt%, preferably from 1 to 16 wt%, more preferably from 6 to 12 wt% softening active. The preferred, typical cationic fabric softening components include water-insoluble quaternary-ammonium fabric softeners, the most commonly used having been di-long alkyl chain ammonium chloride or methyl sulfate. Preferred cationic softeners include but are not limited to the following: a. rapidly biodegradable quaternary ammonium compounds which contain 1 or more ester bonds situated between the quaternary-ammonium group and the long alkyl chain e.g., TEA ester quats, DEEDMAC and HEQ); b. Non-Ester quaternary ammonium compounds {e.g., ditallow dimethylammonium chloride (DTDMAC); dihydrogenated tallow dimethylammonium chloride; dihydrogenated tallow dimethylammonium methylsulfate; distearyl dimethylammonium chloride; dioleyl dimethylammonium chloride; dipalmityl hydroxyethyl methylammonium chloride; stearyl benzyl dimethylammonium chloride; tallow trimethylammonium chloride; hydrogenated tallow trimethylammonium chloride; C12-14 alkyl hydroxyethyl dimethylammonium chloride; C12-18 alkyl dihydroxyethyl methylammonium chloride; di(stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC); di(tallowoyloxyethyl) dimethylammonium chloride; ditallow imidazolinium methylsulfate; 1 -(2-tallowylamidoethyl)-2-tallowyl imidazolinium methylsulfate.

A first group of quaternary ammonium compounds (QACs) suitable for use according to the present disclosure is represented by formula (I):

[(CH₂)_n(TR)]_m

I R’-N⁺-[(CH₂)_n(OH)]_{3 m}X ) wherein each R is independently selected from a C1-C35 alkyl or alkenyl group; R¹ represents a C1-C4 alkyl, C2-C4 alkenyl or a C1-C4 hydroxyalkyl group; T is generally O-CO (/.e., an ester group bound to R via its carbon atom), but may alternatively be CO-O (i.e., an ester group bound to R via its oxygen atom); n is a number selected from 1 to 4; m is a number selected from 1 , 2, or 3; and X is an anionic counter-ion, such as a halide or alkyl sulphate, e.g., chloride or methylsulphate. Di-esters variants of formula (I) (i.e., m = 2) are preferred and typically have mono- and tri-ester analogues associated with them.

Especially preferred agents are preparations which are rich in the di-esters of triethanolammonium methylsulfate, otherwise referred to as "TEA ester quats". Commercial examples include STEPANTEX® LIL85, ex Stepan, Prapagen™ TQL, ex Clariant, and Tetranyl™ AHT-1 , ex Kao, (both di-[hardened tallow ester] of triethanolammonium methylsulphate), AT-1 (di-[tallow ester] of triethanolammonium methylsulphate), and L5/90 (di-[palm ester] of triethanolammonium methylsulphate), both ex Kao, and REWOQUAT® WE15 (a di-ester of triethanolammonium methylsulphate having fatty acyl residues deriving from CI 0-C20 and C16-C18 unsaturated fatty acids), ex Evonik.

Also suitable are soft quaternary ammonium actives such as STEPANTEX® VK90, STEPANTEX® VT90, SP88 (ex-Stepan), Prapagen™ TQ (ex-Clariant), DEHYQUART® AU-57 (ex-Cognis), REWOQUAT® WE18 (ex-Degussa) and Tetranyl™ L190 P, Tetranyl™ L190 SP and Tetranyl™ L190 S (all ex-Kao).

A second group of QACs suitable for use according to the present disclosure is represented by formula (II):

(R¹)2-N⁺-[(CH₂)n-T-R²]₂X- (II) wherein each R¹ group is independently selected from C1-C4 alkyl, or C2-C4 alkenyl groups; and wherein each R² group is independently selected from C8-C28 alkyl or alkenyl groups; and n, T, and X- are as defined above. Preferred materials of this second group include bis(2tallwoyloxyethyl)dimethyl ammonium chloride and hardened versions thereof.

A third group of QACs suitable for use according to the present disclosure is represented by formula (III):

wherein each R¹ group is independently selected from C1-C4 alkyl, hydroxyalkyl or C2-C4 alkenyl groups; and wherein each R² group is independently selected from C8-C28 alkyl or alkenyl groups; and wherein n, T, and X are as defined above. Preferred materials of this second group include 1 ,2 bis[tallowoyloxy]-3-trimethylammonium propane chloride, 1 ,2 bisfhardened tallowoyloxy]-3-trimethylammonium propane chloride, 1 ,2-bis[oleoyloxy]-3 trimethylammonium propane chloride, and 1 ,2 bis[stearoyloxy]-3-trimethylammonium propane chloride. Such materials are described in US4,137,180 (Lever Brothers).

Preferably, these materials also comprise an amount of the corresponding mono-ester.

Co-softeners. Co-softeners, also referred to as co-softeners and fatty complexing agents may be used in fabric conditioner composition of the present disclosure. When employed, they are typically present at from 0.1 to 20% and particularly at from 0.1 to 5%, based on the total weight of the composition. Preferred co-softeners include fatty alcohols, fatty esters, and fatty N-oxides. Fatty esters that may be employed include fatty monoesters, such as glycerol monostearate, fatty sugar esters, such as those disclosed WO01/46361 (Unilever).

In some embodiments, the fabric conditioner compositions of the present disclosure may comprise a co-active. Especially suitable fatty complexing agents include fatty alcohols and fatty acids. Of these, fatty alcohols are most preferred. Without being bound by theory it is believed that the fatty complexing material improves the viscosity profile of the composition by complexing with mono-ester component of the fabric conditioner material thereby providing a composition which has relatively higher levels of di-ester and tri-ester linked components. The di-ester and tri-ester linked components are more stable and do not affect initial viscosity as detrimentally as the mono-ester component. It is also believed that the higher levels of mono-ester linked component present in compositions comprising quaternary ammonium materials based on TEA may destabilize the composition through depletion flocculation. By using the co-active material to complex with the mono-ester linked component, depletion flocculation is significantly reduced. In other words, the co-active at the increased levels, as required by the present disclosure in some embodiments, “neutralizes” the mono-ester linked component of the quaternary ammonium material. This in situ di-ester generation from mono-ester and fatty alcohol also improves the softening of the composition.

Silicone. In some embodiments, the fabric conditioner compositions of the present disclosure may further contain a silicone based fabric softening agent. Preferably the fabric softening silicone is a polydimethylsiloxane. The fabric softening silicones include but are not limited to 1 ) non-functionalized silicones such as polydimethylsiloxane (PDMS) or alkyl (or alkoxy) functional silicones; 2) functionalized silicones or copolymers with one or more different types of functional groups such as amino, phenyl, polyether, acrylate, silicon hydride, carboxylic acid, quaternized nitrogen, etc. Suitable silicones may be selected from polydialkylsiloxanes, preferably polydimethylsiloxane more preferably amino functionalised silicones; anionic silicones and carboxyl functionalized silicone. An amino silicone that may also be used, for example, Arristan 64, ex CHT or Wacker CT45E, ex Wacker.

In terms of silicone emulsions, the particle size can be in the range from about 1 nm to 100 microns and preferably from about 10 nm to about 10 microns including microemulsions (< 150 nm), standard emulsions (about 200 nm to about 500 nm) and macroemulsions (about 1 micron to about 20 microns).

Non-ionic surfactants. In some embodiments, the fabric conditioner compositions may further comprise a nonionic surfactant. Typically, these can be included for the purpose of stabilizing the compositions. Suitable nonionic surfactants include addition products of ethylene oxide with fatty alcohols, fatty acids, and fatty amines. Any of the alkoxylated materials of the particular type described hereinafter can be used as the nonionic surfactant. Suitable surfactants are substantially water soluble surfactants of the general formula (V): R-Y-(C2H4O)z-CH2-CH2-OH (V) where R is selected from the group of primary, secondary and branched chain alkyl and/or acyl hydrocarbyl groups; primary, secondary and branched chain alkenyl hydrocarbyl groups; and primary, secondary and branched chain alkenyl-substituted phenolic hydrocarbyl groups; the hydrocarbyl groups having a chain length of from 8 to about 25, preferably 10 to 20, e.g., 14 to 18 carbon atoms. In the general formula for the ethoxylated nonionic surfactant, Y is typically: -O- , - C(O)O- , -C(O)N(R)- or -C(O)N(R)R in which R has the meaning given above for formula (V), or can be hydrogen; and Z is at least about 8, preferably at least about 10 or 1 1 .

Preferably the nonionic surfactant has an HLB of from about 7 to about 20, more preferably from 10 to 18, e.g., 1 to 16. GENAPOL® C200 (Clariant) based on coco chain and 20 EO groups is an example of a suitable nonionic surfactant. If present, the nonionic surfactant is present in an amount from 0.01 to 10%, more preferably 0.1 to 5 by weight, based on the total weight of the composition. LUTENSOL® AT25 (BASF) based on coco chain and 25 EO groups is an example of a suitable non-ionic surfactant. Other suitable surfactants include RENEX® 36 (Trideceth-6), ex Croda; TERGITOL® 15-S3, ex Dow Chemical Co.; Dihydrol LT7, ex Thai Ethoxylate ltd; CREMOPHOR® CO40, ex BASF and NEODOL® 91 -8, ex Shell.

Cationic Polysaccharide. In some embodiments, the fabric conditioner compositions may further comprise at least one cationic polysaccharide. The cationic polysaccharide can be obtained by chemically modifying polysaccharides, generally natural polysaccharides. By such modification, cationic side groups can be introduced into the polysaccharide backbone The cationic polysaccharides are not limited to: cationic cellulose and derivatives thereof, cationic starch and derivatives thereof, cationic callose and derivatives thereof, cationic xylan and derivatives thereof, cationic mannan and derivatives thereof, cationic galactomannan and derivatives thereof, such as cationic guar and derivatives thereof. Cationic celluloses which are suitable include cellulose ethers comprising quaternary ammonium groups, cationic cellulose copolymers or celluloses grafted with a water-soluble quaternary ammonium monomer.

The cellulose ethers comprising quaternary ammonium groups are described in French patent 1 ,492,597 and in particular include the polymers sold under the names "JR" (JR 400, JR 125, JR 30M) or "LR" (LR 400, LR 30M) by the company Dow. These polymers are also defined in the CTFA dictionary as hydroxyethylcellulose quaternary ammoniums that have reacted with an epoxide substituted with a trimethylammonium group. Suitable cationic celluloses also include LR3000 KC from Solvay. The cationic cellulose copolymers or the celluloses grafted with a water-soluble quaternary ammonium monomer are described especially in patent US4,131 ,576, such as hydroxyalkylcelluloses, for instance hydroxymethyl-, hydroxyethyl- or hydroxypropylcelluloses grafted especially with a methacryloyl-ethyltrimethylammonium, methacrylamidopropyltrimethylammonium or dimethyl-diallylammonium salt.

The commercial products corresponding to this definition are more particularly the products sold under the names CELQUAT® L 200 and CELQUAT® H 100 by Akzo Nobel. Cationic starches suitable for the present disclosure include the products sold under POLYGELO® (cationic starches from Sigma), the products sold under SOFTGEL®, AMYLOFAX® and SOLVITOSE® (cationic starches from Avebe), CATO from National Starch. Suitable cationic galactomannans can be those derived from Fenugreek Gum, Konjac Gum, Tara Gum, Cassia Gum or Guar Gum.

In some embodiments, the cationic polysaccharide of the present disclosure may have an average Molecular Weight (Mw) of between 100,000 daltons and 3,500,000 daltons, preferably between 100,000 daltons and 1 ,500,000 daltons, more preferably between 100,000 daltons and 1 ,000,000 daltons.

In some embodiments, the fabric conditioner composition of the present disclosure preferably comprises from 0.01 to 2 wt % of cationic polysaccharide based on the total weight of the composition. More preferably, 0.025 to 1 wt % of cationic polysaccharide based on the total weight of the composition. Most preferably, 0.04 to 0.8 wt % of cationic polysaccharide based on the total weight of the composition. In the context of the present application, the term "Degree of Substitution (DS)" of cationic polysaccharides, such as cationic guars, is the average number of hydroxyl groups substituted per sugar unit. DS may notably represent the number of the carboxymethyl groups per sugar unit. DS may be determined by titration.

The DS of the cationic polysaccharide is preferably in the range of 0.01 to 1 , more preferably 0.05 to 1 , most preferably 0.05 to 0.2. In the context of the present application, "Charge Density (CD)" of cationic polysaccharides, such as cationic guars, means the ratio of the number of positive charges on a monomeric unit of which a polymer is comprised to the molecular weight of said monomeric unit. CD of the cationic polysaccharide, such as the cationic guar, is preferably in the range of 0.1 to 3 (meq/gm), more preferably 0.1 to 2 (meq/gm), most preferably 0.1 to 1 (meq/gm).

Non-ionic Polysaccharide. In some embodiments, the fabric conditioner composition may further comprise at least one non-ionic polysaccharide. The nonionic polysaccharide can be a modified nonionic polysaccharide or a non-modified nonionic polysaccharide. The modified non-ionic polysaccharide may comprise hydroxyalkylation and/or esterification. In the context of the present disclosure, the level of modification of non-ionic polysaccharides can be characterized by Molar Substitution (MS), which means the average number of moles of substituents, such as hydroxypropyl groups, per mole of the monosaccharide unit. MS can be determined by the Zeisel-GC method, notably based on the following literature reference: Hodges, et al. (1979) Anal. Chem. 51 (13). Preferably, the MS of the modified nonionic polysaccharide is in the range of 0 to 3, more preferably 0.1 to 3 and most preferably 0.1 to 2.

In some embodiments, the nonionic polysaccharide of the present disclosure may be especially chosen from glucans, modified or non-modified starches (such as those derived, for example, from cereals, for instance wheat, corn or rice, from vegetables, for instance yellow pea, and tubers, for instance potato or cassava), amylose, amylopectin, glycogen, dextrans, celluloses and derivatives thereof (methylcelluloses, hydroxyalkylcelluloses, ethylhydroxyethylcelluloses), mannans, xylans, lignins, arabans, galactans, galacturonans, chitin, chitosans, glucuronoxylans, arabinoxylans, xyloglucans, glucomannans, pectic acids and pectins, arabinogalactans, carrageenans, agars, gum Arabics, gum tragacanths, ghatti gums, karaya gums, carob gums, galactomannans such as guars and nonionic derivatives thereof (hydroxypropyl guar), and mixtures thereof.

Among the celluloses that can be especially used are hydroxyethylcelluloses and hydroxypropylcelluloses. Suitable non-limiting examples include products sold under the trade names KLUCEL® EF, KLUCEL® H, KLUCEL® LHF, KLUCEL® MF and KLUCEL® G by Aquaion, and CELLOSIZE® Polymer PCG-10 by Amerchol, and HEC, HPMC K200, HPMC K35M by Ashland.

In some embodiments, the fabric conditioner composition of the present disclosure preferably comprises from 0.01 to 2 wt % of non-ionic polysaccharide based on the total weight of the composition. More preferably, 0.025 to 1 wt % of non-ionic polysaccharide based on the total weight of the composition. Most preferably, 0.04 to 0.8 wt % of non-ionic polysaccharide based on the total weight of the composition. Preferably the fabric conditioning composition comprises combined weight of the cationic polysaccharide and non-ionic polysaccharide of 0.02 to 4 wt %, more preferably 0.05 to 2 wt % and most preferably 0.08 to 1 .6 wt %. Preferably the ratio of the weight of the cationic polysaccharide in the composition and the weight of the nonionic polysaccharide in the composition is between 1 :10 and 10:1 , more preferably, between 1 :3 and 3:1 .

In a preferred embodiment, the cationic polysaccharide and non-ionic polysaccharide are mixed prior to addition to the fabric conditioner composition. Preferably the mix is prepared as a suspension in water. Preferably, the ratio of the weight of the quaternary ammonium compound in the composition and the total weight of the cationic polysaccharide and the nonionic polysaccharide in the composition is between 100:1 and 2:1 , more preferably, between 30:1 and 5:1 .

Water. In some embodiments, the fabric conditioner composition of the present disclosure comprises water. The compositions are rinse-added softening compositions suitable for use in a laundry process. The compositions are pourable liquids. The liquid compositions have a pH ranging from about 2.0 to about 7, preferably from about 2 to about 4, more preferably from about 2.5 to about 3.5. The compositions may also contain pH modifiers preferably hydrochloric acid, lactic acid or sodium hydroxide. The composition is preferably a ready-to-use liquid comprising an aqueous phase. The aqueous phase may comprise water-soluble species, such as mineral salts or short chain (C1-C4) alcohols. The composition is preferably for use in the rinse cycle of a home textile laundering operation, where, it may be added directly in an undiluted state to a washing machine, e.g., through a dispenser drawer or, for a top-loading washing machine, directly into the drum. The compositions may also be used in a domestic hand-washing laundry operation.

The fabric conditioner composition may typically be made by combining a melt comprising the fabric softening agent with an aqueous phase. The polymer may be combined with the water phase, or it may be post dosed into the composition after combination of the melt and water phase. A preferred method of preparation is as follows:

1 . Heat water to about 40 to 50°C, preferably above 45°C.

2. Add the rheology modifiers to the water slowly, preferably over about 1 minute with stirring.

3. Mix thoroughly, preferably from 1 to 10 minutes.

4. Add any minor ingredients, such as antifoams, sequestrants and preservatives.

5. Melt the softening active and optional fatty alcohol together to form a co-melt.

6. Add the co-melt to the heated water.

7. Add acid to the preferred pH, if required.

8. Add dyes and perfumes.

9. Cool. B) Liquid dish detergents such as those described in US6,069,122 and US5,990,065.

C) Automatic Dish Detergents such as those described in US6,020,294,

US6, 017,871 , US5,968,881 , US5,962,386, US5,939,373, US5, 914,307, US5,902,781 , US5,705,464, US5,703,034, US5,703,030, US5,679,630, US5,597,936, US5,581 ,005, US5,559,261 , US4,515,705, US5,169,552, and US4,714,562.

D) All-purpose cleaners including bucket dilutable cleaners and toilet cleaners, bathroom cleaners, bath tissue, rug deodorizers, candles (e.g., scented candles), room deodorizers, floor cleaners, disinfectants, window cleaners, garbage bags/ trash can liners, air fresheners (e.g., room deodorizer, car deodorizer, sprays, scent oil air freshener, automatic spray air freshener, and neutralizing gel beads), moisture absorber, household devices (e.g., paper towels and disposable wipes), and moth balls/traps/cakes.

E) Personal care products: cosmetic or pharmaceutical preparations. More specifically personal cleansers (e.g., bar soaps, body washes, and shower gels), in-shower conditioner, sunscreen (e.g., sprays, lotions and sticks), insect repellents, hand sanitizers, anti-inflammatory (e.g., balms, ointments and sprays), antibacterial (e.g., ointments and creams), sensates, deodorants and antiperspirants (including aerosol, pump spray and wax based), lotions, body powder and foot powder, body mist or body spray, shave cream and male groom products, bath soak, exfoliating scrub.

F) Hair Care products. More specifically, shampoos (liquid and dry powder), hair conditioners (rinse-out conditioners, leave-in conditioners, and cleansing conditioners), hair rinses, hair refreshers, hair perfumes, hair straightening products, hair styling products, hair fixative and styling aids, hair combing creams, hair wax, hair foam, hair gel, non-aerosol pump spray, hair bleaches, dyes and colorants, perming agents, and hair wipes.

In particular aspects, the core-shell microcapsule slurry of this disclosure is of use in improving a freshness impression to a fabric. Accordingly, in certain aspects, the biodegradable core-shell microcapsules of the present disclosure are included in a fabric conditioner or softener having a pH of from 2 to 4, preferably a pH of from 2.5 to 3.5.

Many aspects and embodiments have been described above and are merely exemplary and not limiting. After reading this specification, skilled artisans appreciate that other aspects and embodiments are possible without departing from the scope of the invention.

EXAMPLES

The concepts described herein will be further described in the following examples, which do not limit the scope of the invention described in the claims.

General

T. reesei glucoamylase was recombinantly produced and isolated from T. reesei. A. tereus a-amylase was recombinantly produced and isolated from A. tereus. Bacterial phytase was recombinantly produced and isolated from a bacterial. H. grisea glucoamylase was recombinantly produced and isolated from H. grisea. FLEXAN® II emulsifier is a sodium salt of polystyrene sulfonate. NEOBEE® oil is caprylic/capric triglyceride. Takenate® D-110N is a 75 wt% polyisocyanate (i.e., trimethylol propane-adduct of xylylene diisocyanate) solution in ethyl acetate. Takenate® BB is a 50 wt% polyisocyanate (i.e., trimethylol propaneadduct of xylylene diisocyanate) solution in benzyl benzoate. ROCIMA™ BT NV2 is 1 ,2- benzisothiazolin-3-one. Proxel® GXL is 1 ,2-benzisothiazolin-3-one. Avicel® CL-611 is a thickener comprising microcrystalline cellulose (MCG). Lattice® NTC-61 is also a thickener comprising microcrystalline cellulose (MCG).

Enzyme Selection

Four enzymes were selected to be used in the Examples for their amino acid contents, i.e., number of lysine, arginine, cysteine, aspartic acid, and glutamic acid residues in the amino acid sequence of the respective enzymes (shown in Table 3). They are: T. reesei glucoamylase (64 kDa, pl about 4.7), A. tereus a-amylase (64 kDa, pl 4.2), bacterial phytase (46 kDa, pl 5.4) and H. grisea glucoamylase (68 kDa, pl 9.1 ).

TABLE 3 - Amino Acid Contents of the Enzymes

Example 1 : Unfolding Enzyme Materials: Fluorescamine, monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), L-cysteine, and acetone (HPLC grade) were obtained from Sigma Chemical Company (St Louis, MO). An ultrafiltration concentrate (UFC) containing native T. reesei glucoamylase at the concentration of 273 g/kg was obtained from International Flavors & Fragrances Inc.

Preparation of buffer solution: 1 M (mol/L) PBS (potassium phosphate buffer solution) was prepared by combining 19.8 mL 1 M KH2PO4 and 80.2 mL 1 M K2HPO4. 0.1 M PBS was then prepared by diluting 1 part of 1 M PBS with 9 parts of water.

Preparation of samples: Sample “Water” was a 10 wt% glucoamylase solution prepared by diluting glucoamylase UFC with deionized water. Sample “A” was a 10 wt% glucoamylase solution containing about 0.2 wt% L-cysteine. It was prepared by adding 0.020 g of L- cysteine into 10 g of Sample “Water”. Sample “B” was a 10 wt% glucoamylase solution containing about 0.26 wt% L-cysteine. It was prepared by adding 0.026 g of L-cysteine into 10 g of Sample “Water”. Sample “C” was a 10 wt% glucoamylase solution containing about 0.33 wt% L-cysteine. It was prepared by adding 0.033 g of L-cysteine into 10 g of Sample “Water”. The amount of L-cysteine in Samples “A”, “B” and “C” were respectively 1 .5 folds, 2.0 folds, and 2.5 folds of the amount of cysteine residues in the amino acid sequence of the glucoamylase contained in corresponding samples. Sample “PBS” was prepared by diluting glucoamylase UFC with 0.1 M PBS until the final solution contained 10 wt% glucoamylase. Samples “Water”, “A”, “B” and “C” were respectively heated under stirring to 30 °C, 40 °C, and 50 °C sequentially and held for 10 minutes at each of the temperatures. Aliquots were taken at room temperature (RT), 30 °C, 40 °C, and 50 °C respectively for Fluorescence analysis.

Fluorescence analysis: The degree of unfolding of glucoamylase in Samples “Water”, “A”, “B”, “C” and “PBS” were measured with fluorescamine protein assay. Fluorescamine is not fluorescent itself, but reacts with free amino groups to form highly fluorescent products. Therefore, it has been used as a reagent for the detection of free amino groups in a protein structure. Aliquots were taken from Samples and diluted with 0.1 M PBS to glucoamylase concentration of 0.1 mg/mL. After dilution, 150 pL aliquots of samples were pipetted into microplate wells. The microplate was placed on a microplate shaker that was preheated at 25 °C and 50 pL of 10.8 mM (3 mg/mL) fluorescamine solution in acetone was added to each well. The microplate was then shaken for one minute, and the fluorescence was then determined using a BioTek Synergy H1 microplate reader with excitation at 392 nm and emission at 480 nm. The fluorescence measurement results of Samples “Water”, “A”, “B”, “C” and “PBS” are shown in FIG. 1 .

Based upon this fluorescence analysis, it was observed that native glucoamylase largely maintained its tertiary structure in the phosphate buffer solution. By comparison, dilution to 10% in water resulted in unfolding of the native glucoamylase, as evidenced by an increase in fluorescence. The addition of excess cysteine (Samples “B” and “C”) maintained the glucoamylase in an unfolded state with an increase in temperature.

Example 2: Microcapsules and Microcapsule Slurries Preparation Microcapsule 1 : Microcapsule Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution in the Presence of Cysteine

An aqueous phase was prepared by first adding 2.10 g FLEXAN® II emulsifier to water to form an aqueous solution and then adding 76.92 g T. reesei glucoamylase UFC (containing 21.00 g glucoamylase) into the aqueous solution. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5. Then 13.65 g of 5% cysteine solution (in water) was added to the aqueous solution and mixed well to form the aqueous phase. The total weight of the aqueous phase was 413 g and the concentration of the glucoamylase was 5.1% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 224.00 g fragrance with 56.00 g NEOBEE® oil as solvent) with 7.00 g Takenate® D-1 10N (containing 5.25 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 50 °C for 2 hours. The resulting microcapsule slurry was cooled to room temperature and adjusted to pH 5-5.5 with 5% citric acid solution. 35.00 g xanthan gum solution (containing 1 .40 g xanthan gum) and 0.70 g ROCIMA™ BT NV2 preservative were then added to form the final microcapsule slurry (Microcapsule slurry 1 comprising Microcapsule 1 ). 80% (by volume) of the microcapsules (Microcapsule 1 ) had diameter in a range of from 3.79 pm to 18.67 pm.

Microcapsule 2: Microcapsule Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution in the Presence of Cysteine, and No Emulsifier Was Used. An aqueous phase was prepared by first adding 73.26 g T. reesei glucoamylase UFC (containing 20.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5. Then 13.00 g of 5% cysteine solution (in water) was added to the aqueous solution and mixed well to form the aqueous phase. The total weight of the aqueous phase was 295 g and the concentration of the glucoamylase was 6.8% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 160.00 g fragrance with 40.00 g NEOBEE® oil as solvent) with 5.00 g Takenate® D-1 10N (containing 3.75 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 50 °C for 2 hours. The resulting microcapsule slurry was cooled to room temperature. 12.25 g xanthan gum solution (containing 0.50 g xanthan gum) and 0.50 g ROCIMA™ BT NV2 preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 2 comprising Microcapsule 2). 80% (by volume) of the microcapsules (Microcapsule 2) had diameter in a range of from 4.00 pm to 20.66 pm.

Microcapsule 3: Microcapsule Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution without Cysteine, and No Emulsifier Was Used.

An aqueous phase was prepared by first adding 73.26 g T. reesei glucoamylase UFC (containing 20.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 296.25 g and the concentration of the glucoamylase was 6.8% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 160.00 g fragrance with 40.00 g NEOBEE® oil as solvent) with 3.75 g Takenate® D-110N (containing 2.81 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 1 hour. The resulting microcapsule slurry was cooled to room temperature and adjusted to pH 5-5.5 with 5% citric acid solution. 25 g xanthan gum solution (containing 1 .00 g xanthan gum) and 0.50 g ROCIMA™ BT NV2 preservative were then added to form the final microcapsule slurry (Microcapsule slurry 3 comprising Microcapsule 3). 80% (by volume) of the microcapsules (Microcapsule 3) had diameter in a range of from 3.05 pm to 16.42 pm.

Microcapsule 4: Microcapsule Prepared with the Same Process as for Microcapsule 3, except that Takenate® BB Replaced Takenate® D-1 10N as the Polyisocyanate

Microcapsule 4 was made with the same process as for Microcapsule 3, except that 5.63 g Takenate® BB (containing 2.81 g polyisocyanate) was used in the preparation of Microcapsule 4 in place of the 3.75 g Takenate® D-110N. The formed microcapsule slurry is Microcapsule slurry 4 comprising Microcapsule 4. 80% (by volume) of the microcapsules (Microcapsule 4) had diameter in a range of from 3.21 pm to 24.70 pm.

Microcapsule 5: Microcapsule Prepared with a Similar Process as for Microcapsule 4, except with Less Glucoamylase and More Polyisocyanate

An aqueous phase was prepared by first adding 45.29 g T. reesei glucoamylase UFO (containing 12.50 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 292.50 g and the concentration of the glucoamylase was 4.3% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 160.00 g fragrance with 40.00 g NEOBEE® oil as solvent) with 7.50 g Takenate® BB (containing 3.75 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was cooled to room temperature. 25 g xanthan gum solution (containing 0.25 g xanthan gum) and 0.50 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 5 comprising Microcapsule 5). 80% (by volume) of the microcapsules (Microcapsule 5) had diameter in a range of from 7.80 pm to 41.40 pm.

Microcapsule 6: Microcapsule Prepared with a Similar Process as for Microcapsule 5, except with More Glucoamylase

An aqueous phase was prepared by first adding 54.35 g T. reesei glucoamylase UFC (containing 15.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 292.50 g and the concentration of the glucoamylase was 5.1 % in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 160.00 g fragrance with 40.00 g NEOBEE® oil as solvent) with 7.50 g Takenate® BB (containing 3.75 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 4 hours. The resulting microcapsule slurry was cooled to room temperature. 25 g xanthan gum solution (containing 0.25 g xanthan gum) and 0.50 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 6 comprising Microcapsule 6). 80% (by volume) of the microcapsules (Microcapsule 6) had diameter in a range of from 5.38 pm to 33.70 pm.

Microcapsule 7: Microcapsule Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution without Cysteine, and Final Curing Temperature > 65 °C

An aqueous phase was prepared by first adding 65.22 g T. reesei glucoamylase UFC (containing 18.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 291 g and the concentration of the glucoamylase was 6.2% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 192.00 g fragrance with 48.00 g NEOBEE® oil as solvent) with 9.00 g Takenate® BB (containing 4.50 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 1 hour. The resulting microcapsule slurry was added with 120 g gum Arabic solution (containing 6 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to room temperature. 6 g xanthan gum solution (containing 0.06 g xanthan gum) and 0.60 g Proxel® GXL preservative were then added to form the final microcapsule slurry (Microcapsule slurry 7 comprising Microcapsule 7). 80% (by volume) of the microcapsules (Microcapsule 7) had diameter in a range of from 4.69 pm to 59.0 pm.

Microcapsule 8: Microcapsule Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution without Cysteine, and Final Curing Temperature > 65 °C

An aqueous phase was prepared by first adding 72.46 g T. reesei glucoamylase UFC (containing 20.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 468 g and the concentration of the glucoamylase was 4.3% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 256.00 g fragrance with 64.00 g NEOBEE® oil as solvent) with 12.00 g Takenate® BB (containing 6.00 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was added with 16 g gum Arabic solution (containing 4 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to room temperature. 8 g xanthan gum solution (containing 0.08 g xanthan gum) and 0.80 g Proxel® GXL preservative were then added to form the final microcapsule slurry (Microcapsule slurry 8 comprising Microcapsule 8). 80% (by volume) of the microcapsules (Microcapsule 8) had diameter in a range of from 6.53 pm to 80.8 pm.

Microcapsules 9-11 : Microcapsules Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution without Cysteine, and different Neobee® oil to fragrance ratios

An aqueous phase was prepared by first adding 54.35 g T. reesei glucoamylase UFC (containing 15.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 246 g and the concentration of the glucoamylase was 6.1% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 130.00 g fragrance with 70.00 g NEOBEE® oil as solvent for Microcapsule 9, containing 140.00 g fragrance with 60.00 g NEOBEE® oil as solvent for Microcapsule 10, or containing 150.00 g fragrance with 50.00 g NEOBEE® oil as solvent for Microcapsule 11 ) with 7.50 g Takenate® BB (containing 3.75 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 1 hour. The resulting microcapsule slurry was added with 20 g gum Arabic solution (containing 5 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 65 °C for 1 hour. The resulting microcapsule slurry was cooled to room temperature. 2.50 g xanthan gum solution (containing 0.10 g xanthan gum), 40 g Avicel® CL-61 1 solution (2.5 wt%), and 0.50 g Proxel® GXL preservative were then added to form the final microcapsule slurry (Microcapsule slurries 9, 10 and 1 1 respectively comprising Microcapsules 9, 10 and 11 ). 80% (by volume) of the microcapsules had diameter in a range of from 4.96 pm to 43.4 pm for Microcapsule 9, from 6.62 pm to 55.0 pm for Microcapsule 10, and from 4.76 pm to 39.6 pm for Microcapsule 1 1 , respectively.

Microcapsule 12: Microcapsule Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution in the Presence of Cysteine, and with Crosslinker Tannic Acid

An aqueous phase was prepared by first adding 0.50 g FLEXAN® II emulsifier to water to form an aqueous solution and then adding 10.99 g T. reesei glucoamylase UFC (containing 3.00 g glucoamylase) into the aqueous solution. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5. Then 1 .95 g of 5% cysteine solution (in water) was added to the aqueous solution and mixed well to form the aqueous phase. The total weight of the aqueous phase was 59 g and the concentration of the glucoamylase was 5.1% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 32.00 g fragrance with 8.00 g NEOBEE® oil as solvent) with 1 .00 g Takenate® D-1 10N (containing 0.75 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 50 °C for 2 hours. The resulting microcapsule slurry was cooled to room temperature, added with 1 .60 g tannic acid solution (containing 0.40 g tannic acid), and subsequently adjusted to pH 5-5.5 with 5% citric acid solution. 5.00 g xanthan gum solution (containing 0.20 g xanthan gum) and 0.10 g ROCIMA™ BT NV2 preservative were then added to form the final microcapsule slurry (Microcapsule slurry 12 comprising Microcapsule 12). 80% (by volume) of the microcapsules (Microcapsule 12) had diameter in a range of from 4.84 pm to 20.53 pm.

Microcapsule 13: Microcapsule Prepared with Glucoamylase Unfolded by Dilution in an Aqueous Solution, and with Crosslinker Glutaraldehyde

An aqueous phase was prepared by first adding 54.35 g T. reesei glucoamylase UFC (containing 15.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 292.50 g and the concentration of the glucoamylase was 5.1 % in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 160.00 g fragrance with 40.00 g NEOBEE® oil as solvent) with 7.50 g Takenate® BB (containing 3.75 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was added with 125 g water and heated to 70 °C for 30 minutes. After cooling to room temperature, 20 g glutaraldehyde solution (containing 1 .00 g glutaraldehyde) was added and mixed for 1 hour. 10.00 g xanthan gum solution (containing 0.10 g xanthan gum) and 0.50 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 13 comprising Microcapsule 13). 80% (by volume) of the microcapsules (Microcapsule 13) had diameter in a range of from 7.80 pm to 41 .80 pm.

Microcapsule 14: Microcapsule Prepared with a-Amylase Unfolded by Dilution in an Aqueous Solution

An aqueous phase was prepared by first adding 98.90 g A. tereus a-amylase UFC (containing 18.00 g a-amylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 351 g and the concentration of the a-amylase was 4.6% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 192.00 g fragrance with 48.00 g NEOBEE® oil as solvent) with 9.00 g Takenate® BB (containing 4.50 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was cooled to room temperature and stirred overnight. 6 g xanthan gum solution (containing 0.06 g xanthan gum) and 0.60 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 14 comprising Microcapsule 14). 80% (by volume) of the microcapsules (Microcapsule 14) had diameter in a range of from 3.43 pm to 27.0 pm.

Microcapsule 15: Microcapsule Prepared with a-Amylase Unfolded by Dilution in an Aqueous Solution, and Final Curing Temperature > 65 °C

An aqueous phase was prepared by first adding 98.90 g A. tereus a-amylase UFC (containing 18.00 g a-amylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 351 g and the concentration of the a-amylase was 4.6% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 192.00 g fragrance with 48.00 g NEOBEE® oil as solvent) with 9.00 g Takenate® BB (containing 4.50 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was added with 24 g gum Arabic solution (containing 6 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to room temperature and stirred overnight. 6 g xanthan gum solution (containing 0.06 g xanthan gum) and 0.60 g Proxel® GXL preservative were then added to form the final microcapsule slurry (Microcapsule slurry 15 comprising Microcapsule 15). 80% (by volume) of the microcapsules (Microcapsule 15) had diameter in a range of from 2.15 pm to 24.0 pm.

Microcapsule 16: Microcapsule Prepared with the Same Process as for Microcapsule 15, except that the a-Amylase Was Unfolded in the Presence of Cysteine Microcapsule 16 was made with the same process as for Microcapsule 15, except that after adjusting the pH of the aqueous solution to 8-8.5, 15 g of L-cysteine solution (containing 0.75 g L-cysteine) was added to form the aqueous phase. The formed microcapsule slurry is Microcapsule slurry 16 comprising Microcapsule 16. 80% (by volume) of the microcapsules (Microcapsule 16) had diameter in a range of from 2.92 pm to 22.3 pm.

Microcapsule 17: Microcapsule Prepared with Bacterial Phytase Unfolded by Dilution in an Aqueous Solution

An aqueous phase was prepared by first adding 135.98 g bacterial phytase UFC (containing 24.00 g phytase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8-8.5 to form the aqueous phase. The total weight of the aqueous phase was 479.45 g and the concentration of the phytase was 5.0% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 256.00 g fragrance with 64.00 g NEOBEE® oil as solvent) with 12.00 g Takenate® BB (containing 6.00 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was cooled to room temperature and stirred overnight. 8 g xanthan gum solution (containing 0.08 g xanthan gum) and 0.80 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 17 comprising Microcapsule 17). 80% (by volume) of the microcapsules (Microcapsule 17) had diameter in a range of from 8.48 pm to 43.9 pm.

Microcapsule 18: Microcapsule Prepared with the Same Process as for Microcapsule 17, except that the Final Curing Temperature > 65 °C

Microcapsule 18 was made with the same process as for Microcapsule 17, except that after curing the microcapsule shell at 40 °C and 50 °C, the resulting microcapsule slurry was added with 32 g gum Arabic solution (containing 8 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to room temperature and stirred overnight. 8 g xanthan gum solution (containing 0.08 g xanthan gum) and 0.80 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 18 comprising Microcapsule 18). 80% (by volume) of the microcapsules (Microcapsule 18) had diameter in a range of from 7.05 pm to 42.5 pm.

Microcapsule 19: Microcapsule Prepared with H. grisea Glucoamylase Unfolded by Dilution in an Aqueous Solution

An aqueous phase was prepared by first adding 88.66 g H. grisea glucoamylase UFC (containing 24.00 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 9 to form the aqueous phase. The total weight of the aqueous phase was 478.57 g and the concentration of the glucoamylase was 5.0% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 256.00 g fragrance with 64.00 g NEOBEE® oil as solvent) with 12.00 g Takenate® BB (containing 6.00 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was cooled to room temperature and stirred overnight. 8 g xanthan gum solution (containing 0.08 g xanthan gum) and 0.80 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 19 comprising Microcapsule 19). 80% (by volume) of the microcapsules (Microcapsule 19) had diameter in a range of from 13.1 pm to 68.7 pm.

Microcapsule 20: Microcapsule Prepared with the Same Process as for Microcapsule 19, except that the Final Curing Temperature > 65 °C

Microcapsule 20 was made with the same process as for Microcapsule 19, except that after curing the microcapsule shell at 40 °C and 50 °C, the resulting microcapsule slurry was added with 32 g gum Arabic solution (containing 8 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to room temperature and stirred overnight. 8 g xanthan gum solution (containing 0.08 g xanthan gum) and 0.80 g Proxel® GXL preservative were then added to form the final microcapsule slurry having pH of 5.5-7 (Microcapsule slurry 20 comprising Microcapsule 20). 80% (by volume) of the microcapsules (Microcapsule 20) had diameter in a range of from 5.19 pm to 73.2 pm. Comparative Microcapsules 21 and 22: Microcapsules Prepared with Gelatin

An aqueous phase was prepared by first adding 24.00 g Gelita beef gelatin powder into 436.00 g water. The mixture was heated to 40 °C to dissolve the gelatin, then cooled to room temperature. The resulting aqueous solution was added with a diluted sodium hydroxide solution (in water) to adjust pH to 8-9 to form the aqueous phase. The total weight of the aqueous phase was 462.44 g and the concentration of the gelatin was 5.2% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 256.00 g fragrance with 64.00 g NEOBEE® oil as solvent) with 12.00 g Takenate® BB (containing 6.00 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was split into two portions. A 394 g portion was cooled to room temperature to form the final microcapsule slurry (Microcapsule slurry 21 comprising Microcapsule 21 ). 400 g of the other portion was added with 16 g gum Arabic solution (containing 4 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to room temperature to form the final microcapsule slurry (Microcapsule slurry 22 comprising Microcapsule 22). Both Microcapsule slurries 21 and 22 completely solidified after the process, indicating there were a lot of unreacted gelatins in the slurry (FIG. 2, Gelita Beef Gelatin).

Comparative Microcapsules 23 and 24: Microcapsules Prepared with Pea Protein

An aqueous phase was prepared by first adding 24.00 g TRUPRO® 2000 pea protein powder into 436.00 g water. The mixture was stirred for 1 hour. The resulting dispersion was added with a diluted sodium hydroxide solution (in water) to adjust pH to 8- 9 to form the aqueous phase. The total weight of the aqueous phase was 461 .71 g and the concentration of the pea protein was 5.2% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 256.00 g fragrance with 64.00 g NEOBEE® oil as solvent) with 12.00 g Takenate® BB (containing 6.00 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was split into two portions. A 393 g portion was cooled to room temperature, added with 0.75 g xanthan gum solution (containing 0.03 g xanthan gum) and 0.3 g Proxel® GXL preservative to form the final microcapsule slurry (Microcapsule slurry 23 comprising Microcapsule 23). 400 g of the other potion was added with 16 g gum Arabic solution (containing 4 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to ambient temperature, added with 0.75 g xanthan gum solution (containing 0.03 g xanthan gum) and 0.3 g Proxel® GXL preservative to form the final microcapsule slurry (Microcapsule slurry 24 comprising Microcapsule 24). Both Microcapsule slurries 23 and 24 displayed significant sedimentation, indicating there were a lot of unreacted pea protein components in the slurry (FIG. 2, Trupro 2000).

Comparative Microcapsules 25 and 26: Microcapsules Prepared with Whey Protein

An aqueous phase was prepared by first adding 24.00 g Hydrovon 195 whey protein isolate powder into 436.00 g water. The mixture was stirred for 1 hour. The resulting dispersion was added with a diluted sodium hydroxide solution (in water) to adjust pH to 8-9 to form the aqueous phase. The total weight of the aqueous phase was 465.52 g and the concentration of the whey protein was 5.2% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 256.00 g fragrance with 64.00 g NEOBEE® oil as solvent) with 12.00 g Takenate® BB (containing 6.00 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 2 hours. The resulting microcapsule slurry was split into two portions. A 397 g portion was cooled to room temperature, added with 0.75 g xanthan gum solution (containing 0.03 g xanthan gum) and 0.3 g Proxel® GXL preservative to form the final microcapsule slurry (Microcapsule slurry 25 comprising Microcapsule 25). 400 g of the other portion was added with 16 g gum Arabic solution (containing 4 g gum Arabic) and adjusted to pH 4.5-5 with 10% citric acid solution. Subsequently the slurry was heated to 70 °C for 30 minutes. The resulting microcapsule slurry was cooled to room temperature, added with 0.75 g xanthan gum solution (containing 0.03 g xanthan gum) and 0.3 g Proxel® GXL preservative to form the final microcapsule slurry (Microcapsule slurry 26 comprising Microcapsule 26). Microcapsule slurry 25 solidified in the next day, and Microcapsule slurry 26 displayed sedimentation, indicating there were a lot of unreacted whey protein components in the slurry (FIG. 2, Hydrovon 195).

Comparative Microcapsule 27: Microcapsule Prepared with Brown Rice Protein

An aqueous phase was prepared by first adding 24.00 g Oryzatein® Silk 90 brown rice protein isolate powder into 436.00 g water. The mixture was stirred for 1 hour. The resulting dispersion was added with a diluted sodium hydroxide solution (in water) to adjust pH to 8-9 to form the aqueous phase. The total weight of the aqueous phase was 468.00 g and the concentration of the brown rice protein was 5.2% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 256.00 g fragrance with 64.00 g NEOBEE® oil as solvent) with 12.00 g Takenate® BB (containing 6.00 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase at room temperature. However, a stable emulsion was not able to form, and microcapsules could not be made (FIG. 2, Oryzatein® Silk 90).

Microcapsule 28: Microcapsule Slurry with Reduced Amount of Residual Glucoamylase

An aqueous phase was prepared by first adding 67.92 g T. reesei glucoamylase UFC (containing 14.40 g glucoamylase) into water. The resulting aqueous solution was mixed well, and a diluted sodium hydroxide solution (in water) was added to adjust the pH of the aqueous solution to 8.5 to form the aqueous phase. The total weight of the aqueous phase was 226.78 g and the concentration of the glucoamylase was 6.35% in the aqueous phase before being mixed with the oil phase. Separately, an oil phase was prepared by mixing a fragrance composition (containing 134.49 g fragrance with 57.64 g NEOBEE® oil as solvent) with 7.21 g Takenate® BB (containing 3.60 g polyisocyanate). Subsequently, the oil phase was emulsified with the aqueous phase to form an oil-in-water emulsion at room temperature. Curing of the microcapsule shell was carried out at 40 °C for 1 hour and then 50 °C for 1 hour. The resulting microcapsule slurry was added with 14.6 g gum Arabic solution (containing 2.4 g gum Arabic) and adjusted to pH 3.5 with 25% citric acid solution. Subsequently the slurry was heated to 65 °C for 1 hour. The resulting microcapsule slurry was cooled to room temperature. 42.80 g xanthan gum and Lattice® NTC-61 solution (containing 0.575 g xanthan gum and 0.805 g Lattice® NTC-61 ) and 0.50 g Proxel® GXL preservative were then added to form the final microcapsule slurry (Microcapsule slurry 28 comprising Microcapsule 28). 80% (by volume) of the microcapsules (Microcapsule 28) had diameter in a range of from 5.69 pm to 45.7 pm.

Microcapsule 29: Microcapsule Slurry with Reduced Amount of Residual Glucoamylase Microcapsule 29 was made with the same process as for Microcapsule 28, except that after curing the microcapsule shell at 40 °C for 1 hour and then 50 °C for 1 hour, the resulting microcapsule slurry was added with 14.6 g gum Arabic solution (containing 2.4 g gum Arabic) and adjusted to pH 4.5 with 25% citric acid solution. Subsequently the slurry was heated to 65 °C for 1 hour. The resulting microcapsule slurry was adjusted to pH 3.5 with 25% citric acid solution and maintained at 65 °C for 30 minutes before being cooled to room temperature. 42.80 g xanthan gum and Lattice® NTC-61 solution (containing 0.575 g xanthan gum and 0.805 g Lattice® NTC-61 ) and 0.50 g Proxel® GXL preservative were then added to form the final microcapsule slurry (Microcapsule slurry 29 comprising Microcapsule 29). 80% (by volume) of the microcapsules (Microcapsule 29) had diameter in a range of from 4.67 pm to 42.2 pm.

Microcapsule 30: Microcapsule Slurry with Reduced Amount of Residual Glucoamylase Microcapsule 30 was made with the same process as for Microcapsule 28, except that after curing the microcapsule shell at 40 °C for 1 hour and then 50 °C for 1 hour, the resulting microcapsule slurry was added with 24.21 g gum Arabic solution (containing 4.8 g gum Arabic) and adjusted to pH 4.0 with 25% citric acid solution. Subsequently the slurry was heated to 75 °C for 1 hour. The resulting microcapsule slurry was cooled to room temperature. 42.80 g xanthan gum and Lattice® NTC-61 solution (containing 0.575 g xanthan gum and 0.805 g Lattice® NTC-61 ) and 0.50 g Proxel® GXL preservative were then added to form the final microcapsule slurry (Microcapsule slurry 30 comprising Microcapsule 30). 80% (by volume) of the microcapsules (Microcapsule 30) had diameter in a range of from 4.90 pm to 50.7 pm.

Example 3: Microcapsule Characterization

Microcapsules’ encapsulation efficiencies of the active material (fragrance) were measured and shown in Table 4.

TABLE 4

Example 4: Sensory Performance Evaluation of Microcapsules 1 -20 and 28-30 in Fabric Conditioner

Sensory performance of the microcapsule was evaluated in a fabric conditioner application. A testing microcapsule slurry was blended into the model fabric conditioner base to form a fabric conditioner sample having a fragrance load of 0.1 % neat oil equivalent (NOE). The fragrance intensity of the fragrance encapsulated in the testing microcapsule was evaluated by conducting a laundry experiment using accepted experimental protocols using European wash machine (Miele). Terry towels were used for the laundry experiments and were washed with the fabric conditioner samples. Washed terry towels were removed from the washing machine and line dried overnight. The fragrance intensity released from the line dried towels were evaluated by a panel of 20-24 trained judges at three different stages (pre-rub, gentle handling, and post-rub) and rated on a scale ranging from 0 to 30 (Table 5). “Pre-rub” refers to the evaluation of the towels by panelists before the folding of the towels. “Gentle handling” refers to the folding of the towels twice, followed by the evaluation of the towels by the panelists. “Post-rub” refers to vigorous application of mechanical force using both hands to rub the towels at least once to rupture the testing microcapsule and then evaluate for signs of released fragrance. A numerical value of 0 indicates that the towels produced no signs of released fragrance, 5 indicates that the towels only produced weak fragrance intensity and 30 indicates a very strong smell of released fragrance from the towels. The evaluation results shown in Table 5 demonstrated that the testing microcapsules delivered increasing fragrance intensity from “Pre-rub” to “Gentle handling” and to “Post-rub”, showing superior performance as compared with test results of neat fragrances (/.e., non-encapsulated fragrances) which showed fragrance intensity below 4 at both “Pre-rub” and “Post-rub” stages. TABLE 5

Example 5: Residual Glucoamylase Measurement Microcapsule slurries 10 and 28-30 were analyzed respectively using gel permeation chromatography (GPC) to determine the amount of residual glucoamylase present in the slurry. The results (weight percentage of the residual glucoamylase based on the total weight of slurry) of this GPC analysis are presented in Table 6. When the amount of residual glucoamylase was less than 0.01 %, the result is reported as ND (non detectable).

TABLE 6

Example 6: Biodegradation Tests with OECD301 F

Testing microcapsules were first washed three times with water to remove any residual unreacted materials. The microcapsules were then freeze dried to remove excess water and then extracted with methanol several times until standard gas chromatography (GC) analysis indicated that the residual fragrance in the microcapsule is less than 2%. The resulting microcapsule shells were then dried and their biodegradation were evaluated according to OECD301 F. The microcapsule shells of Microcapsules 1 , 3 and 7 had biodegradation rates of 68%, 84% and 72% respectively, based on the weight of the respective microcapsule shells, within 60 days according to OECD301 F.

Example 7: Validation of Highly Crosslinked Microcapsule Shells

The microcapsule shells of Microcapsules 3, 7, 28 and 29 obtained from the process described in Example 6 were analyzed respectively using size exclusion chromatography to determine the molecular weights and the polydispersity (PD). Polydispersity characterizes the distribution of the molecular weights for a given polymer sample. PD is defined as M_w/M_n which is the weight average molecular weight (Mw) divided by the number average molecular weight (Mn) of the polymer. The results of this analysis are presented in Table 7. Mz is z- average molecular weight. The data demonstrated that the microcapsule shells of Microcapsules 3, 7, 28 and 29 have significantly higher molecular weights (Mw, Mz) and significantly broader distribution of the molecular weights, compared with the molecular weight (64 KDa) and PD (1 .0) of the glucoamylase used to prepare the microcapsule shells.

TABLE 7

Note that not all of the activities described above in the general description or the examples are required, that a portion of a specific activity may not be required, and that one or more further activities may be performed in addition to those described. Still further, the order in which activities are listed are not necessarily the order in which they are performed.

In the foregoing specification, the concepts have been described with reference to specific embodiments. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of invention.

Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any feature(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature of any or all the claims.

It is to be appreciated that certain features are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

EMBODIMENTS

For further illustration, additional non-limiting embodiments of the present disclosure are set forth below.

For example, embodiment 1 is a biodegradable core-shell microcapsule. The biodegradable core-shell microcapsule comprises: (a) a microcapsule core comprising an active material; and (b) a microcapsule shell comprising a reaction product of an unfolded enzyme with a polyisocyanate; wherein the microcapsule shell is substantially free of or free of a self-condensed polyisocyanate, and the microcapsule shell has a biodegradation rate of at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, based on the weight of the microcapsule shell, within 60 days according to OECD301 F.

Embodiment 2 is a biodegradable core-shell microcapsule as set forth in embodiment 1 , wherein the polyisocyanate comprises or is an aromatic polyisocyanate, and optionally or preferably, the polyisocyanate is substantially free of or free of an aliphatic polyisocyanate.

Embodiment 3 is a biodegradable core-shell microcapsule as set forth in embodiment 2, wherein the aromatic polyisocyanate is selected from the group of polyisocyanurate of toluene diisocyanate, trimethylol propane-adduct of toluene diisocyanate, trimethylol propane-adduct of xylylene diisocyanate, and mixtures thereof, and optionally or preferably, the polyisocyanate comprises or is trimethylol propane-adduct of xylylene diisocyanate.

Embodiment 4 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the enzyme is selected from the group of glucoamylase, amylase, phytase, and mixtures thereof; optionally or preferably, the enzyme comprises or is glucoamylase; and optionally or preferably, the glucoamylase comprises or is T. reesei glucoamylase and/or H. grisea glucoamylase.

Embodiment 5 is a biodegradable core-shell microcapsule as set forth in embodiment 4, wherein the glucoamylase has the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:1 .

Embodiment 6 is a biodegradable core-shell microcapsule as set forth in embodiment 4, wherein the glucoamylase has the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:2.

Embodiment 7 is a biodegradable core-shell microcapsule as set forth in embodiment 4, wherein the amylase is an a-amylase, and the a-amylase has the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:3.

Embodiment 8 is a biodegradable core-shell microcapsule as set forth in embodiment 4, wherein the phytase has the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having less than 100% sequence identity with and at least 50%, or at least 60%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:4.

Embodiment 9 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the enzyme has a molecular weight (Mw) of at least 45 kD, or at least 50 kD.

Embodiment 10 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the enzyme is isolated and purified to homogeneity or near homogeneity; optionally or preferably, the enzyme is purified by ultrafiltration; and optionally or preferably, the amount of other proteins contained in the enzyme is no more than 30 wt%, 25 wt%, 20 wt%, 15 wt%, 10 wt%, 5 wt%, 2 wt%, 1 wt%, 0.5 wt%, 0.2 wt%, or 0.1 wt%, based on the weight of the enzyme.

Embodiment 11 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein (i) the number of cysteine residues in the amino acid sequence of the enzyme is at least 4, 5, 6, 7, 8, 9, or 10 and optionally no more than 12; (ii) the combined number of lysine and arginine residues in the amino acid sequence of the enzyme is at least 20, 23, 26, 29, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, and no more than 85, 80, 75, 72, 70, 68, 66, 64, 62, 60, 58, 56, or 54; and (iii) the combined number of aspartic acid and glutamic acid residues in the amino acid sequence of the enzyme is at least 40, 42, 44, 46, 48, 50, 52, 54, or 56, and optionally no more than 60 or 56.

Embodiment 12 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the enzyme, before unfolded, has at least 2 disulfide bonds, or at least 3 disulfide bonds, or at least 4 disulfide bonds, or at least 5 disulfide bonds.

Embodiment 13 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the enzyme is substantially or completely unfolded. Embodiment 14 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the active material comprises a fragrance, flavor, agricultural active, pesticide, insecticide, herbicide, fungicide, pharmaceutical active, nutraceutical active, animal nutrition active, food active, microbio active, malodor counteractant, and/or cosmetic active; optionally or preferably a fragrance; and optionally or preferably a fragrance comprising at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten High Performance fragrance ingredients selected from the group of Ultra High-Impact fragrance ingredients as listed in Table 1 and High-Impact fragrance ingredients as listed in Table 2, and optionally at least one additional fragrance ingredient.

Embodiment 15 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the active material is hydrophobic or has a logP value of less than 2.

Embodiment 16 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the active material is selected from the group of fragrance, pro-fragrance, malodor counteractive agent, and combinations thereof.

Embodiment 17 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the amount of the active material (e.g., fragrance) present in the microcapsule core is at least 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, or 75 wt%, and no more than 95 wt%, 92 wt%, 90 wt%, 85 wt%, 80 wt%, 75 wt%, or 70 wt%, based on the weight of the microcapsule.

Embodiment 18 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the amount of the self-condensed polyisocyanate present in the microcapsule shell is no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, based on the total weight of the microcapsule shell.

Embodiment 19 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the microcapsule shell comprises a polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate.

Embodiment 20 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the microcapsule shell is a single-layered shell. Embodiment 21 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the microcapsule shell is substantially free of or free of a coacervate, optionally or preferably a coacervate formed by one or more polyelectrolytes.

Embodiment 22 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the microcapsule shell is substantially free of or free of an additional biopolymer.

Embodiment 23 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the microcapsule shell consists essentially of or consists of a reaction product of an unfolded enzyme with a polyisocyanate, optionally or preferably a polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate.

Embodiment 24 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the weight ratio of the enzyme moiety to the polyisocyanate moiety present in the microcapsule shell is at least 0.7:1 , 1 :1 , 1 .2:1 , 1 .5:1 , 1 .8:1 , 2:1 , 2.2:1 , 2.5:1 , 2.8:1 , 3:1 , 3.2:1 , 3.5:1 , 3.7:1 , 4:1 , 4.5:1 , 5:1 , 5.5:1 , 6:1 , 6.5:1 , or 7:1 , and no more than 40:1 , 35:1 , 30:1 , 25:1 , 20:1 , 18:1 , 15:1 , 12:1 , 1 1 :1 , 10:1 , 9:1 , 8:1 , or 7:1.

Embodiment 25 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein the reaction product of the unfolded enzyme with the polyisocyanate, or the polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate, is crosslinked with a crosslinker, optionally or preferably the crosslinker is selected from the group of polyfunctional aldehydes, polyols including polyphenols, polyamines, triethyl citrate, and mixtures thereof, optionally or preferably the crosslinker comprises or is a polyfunctional aldehyde and/or a polyphenol, and optionally or preferably the crosslinker comprises or is tannic acid and/or glutaraldehyde.

Embodiment 26 is a biodegradable core-shell microcapsule as set forth in one of embodiments 1 -24, wherein the microcapsule shell is substantially free of or free of moiety of an additional polyfunctional nucleophile and/or moiety of an additional polyfunctional electrophile.

Embodiment 27 is a biodegradable core-shell microcapsule as set forth in any of the preceding embodiments, wherein at least 80% (by volume) of the microcapsules have diameter of at least 0.5 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 , 6 pm, 7 pm, 8 pm, 10 pm, 12 pm, 15 pm, 18 pm, or 20 pm, and no more than 400 pm, 300 pm, 200 pm, 150 pm, 120 pm, 110 pm, 100 pm, 90 pm, 85 pm, 80 pm, 75 pm, 70 pm, 65 pm, 60 pm, 55 pm, or 50 pm.

Embodiment 28 is a core-shell microcapsule slurry. The core-shell microcapsule slurry comprises: (a) the biodegradable core-shell microcapsule as set forth in any of the preceding embodiments; and (b) water; optionally or preferably the amount of water in the microcapsule slurry is at least 20 wt%, 30 wt%, 40 wt%, 45 wt%, 50 wt%, or 55 wt%, and no more than 95 wt%, 90 wt%, 85 wt%, 80 wt%, 75 wt%, or 70 wt%, based on the total weight of the microcapsule slurry.

Embodiment 29 is a core-shell microcapsule slurry as set forth in embodiment 28, wherein the core-shell microcapsule slurry further comprises a thickener; optionally or preferably the thickener is selected from the group of xanthan gum, microcrystalline cellulose (MCC), coprocessed mixture of MCC and carboxymethyl cellulose (CMC), hydroxypropyl methyl cellulose (HPMC), and combinations thereof; optionally or preferably the thickener comprises or is a combination of xanthan gum and a coprocessed mixture of MCC and CMC; optionally or preferably the thickener comprises a combination of HPMC and a coprocessed mixture of MCC and CMC.

Embodiment 30 is a core-shell microcapsule slurry as set forth in embodiment 29, wherein the amount of the thickener in the microcapsule slurry is at least 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt%, and no more than 5 wt%, 2 wt%, 1 wt%, or 0.5 wt%, based on the total weight of the microcapsule slurry.

Embodiment 31 is a core-shell microcapsule slurry as set forth in one of embodiments 28-30, wherein the microcapsule slurry further comprises gum Arabic; optionally or preferably the amount of gum Arabic in the microcapsule slurry is at least 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, or 0.3 wt%, and no more than 10 wt%, 5 wt%, 2 wt%, or 1 wt%, based on the total weight of the microcapsule slurry.

Embodiment 32 is a core-shell microcapsule slurry as set forth in one of embodiments 28-31 , wherein the microcapsule slurry has reduced amount of residual enzyme (e.g., glucoamylase), optionally or preferably the amount of residual enzyme (e.g., glucoamylase) present in the microcapsule slurry is no more than 1 .5 wt%, 1 wt%, 0.8 wt%, 0.6 wt%, 0.4 wt%, 0.2 wt%, 0.1 wt%, 0.05 wt%, 0.02 wt%, or 0.01 wt%, based on the total weight of the microcapsule slurry.

Embodiment 33 is a core-shell microcapsule slurry as set forth in one of embodiments 28-32, wherein the microcapsule slurry has pH of at least 1 , 1 .5, 2.0, 2.5, 2.8, 3.0, 3.2, 3.5, 3.7, 4.0, 4.2, or 4.5, and no more than 7.5, 7.2, 7.0, 6.8, 6.5, 6.2, 6.0, 5.8, 5.5, 5.2, 5.0, 4.5, 4.2, or 4.0; optionally the microcapsule slurry has pH in a range of from about 4 to about 7.5, or from about 4 to about 6.0, or from about 5 to about 7.5, or from about 5 to about 7.0.

Embodiment 34 is a process for producing a biodegradable core-shell microcapsule or a core-shell microcapsule slurry, optionally or preferably the biodegradable core-shell microcapsule is one as set forth in one of embodiments 1 -27, and optionally or preferably the core-shell microcapsule slurry is one as set forth in one of embodiments 28-33. The process comprises: (a) providing an aqueous phase comprising an unfolded enzyme, wherein the pH of the aqueous phase is equal to or greater than the isoelectric point of the enzyme; (b) providing an oil phase comprising an active material and a polyisocyanate; (c) emulsifying the oil phase with the aqueous phase to form an emulsion; (d) allowing the unfolded enzyme to react with the polyisocyanate to form a microcapsule shell encapsulating a microcapsule core, thereby forming a core-shell microcapsule slurry comprising a biodegradable core-shell microcapsule, wherein the microcapsule core comprises the active material, and the microcapsule shell comprises a reaction product of the unfolded enzyme with the polyisocyanate; and (e) curing the microcapsule shell at an elevated temperature.

Embodiment 35 is a process as set forth in embodiment 34, wherein the enzyme is selected from the group of glucoamylase, amylase, phytase, and mixtures thereof; optionally or preferably, the enzyme comprises or is glucoamylase; and optionally or preferably, the glucoamylase comprises or is T. reesei glucoamylase and/or H. grisea glucoamylase.

Embodiment 36 is a process as set forth in embodiments 34 or 35, wherein the enzyme has a molecular weight (Mw) of at least 45 kD, or at least 50 kD.

Embodiment 37 is a process as set forth in one of embodiments 34-36, wherein the enzyme is isolated and purified to homogeneity or near homogeneity; optionally or preferably, the enzyme is purified by ultrafiltration; and optionally or preferably, the amount of other proteins contained in the enzyme is no more than 30 wt%, 25 wt%, 20 wt%, 15 wt%, 10 wt%, 5 wt%, 2 wt%, 1 wt%, 0.5 wt%, 0.2 wt%, or 0.1 wt%, based on the weight of the enzyme.

Embodiment 38 is a process as set forth in one of embodiments 34-37, wherein (i) the number of cysteine residues in the amino acid sequence of the enzyme is at least 4, 5, 6, 7, 8, 9, or 10 and optionally no more than 12; (ii) the combined number of lysine and arginine residues in the amino acid sequence of the enzyme is at least 20, 23, 26, 29, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, and no more than 85, 80, 75, 72, 70, 68, 66, 64, 62, 60, 58, 56, or 54; and (iii) the combined number of aspartic acid and glutamic acid residues in the amino acid sequence of the enzyme is at least 40, 42, 44, 46, 48, 50, 52, 54, or 56, and optionally no more than 60 or 56.

Embodiment 39 is a process as set forth in one of embodiments 34-38, wherein the enzyme, before unfolded, has at least 2 disulfide bonds, or at least 3 disulfide bonds, or at least 4 disulfide bonds, or at least 5 disulfide bonds.

Embodiment 40 is a process as set forth in one of embodiments 34-39, wherein the aqueous phase is homogenous.

Embodiment 41 is a process as set forth in one of embodiments 34-40, wherein the enzyme, before unfolded, comprises or is a native enzyme.

Embodiment 42 is a process as set forth in one of embodiments 34-41 , wherein the unfolded enzyme is prepared by diluting the enzyme in water or an aqueous solution, optionally in the presence of a thiol-containing reducing agent (e.g., cysteine).

Embodiment 43 is a process as set forth in one of embodiments 34-42, wherein the aqueous phase comprises a thiol-containing reducing agent (e.g., cysteine), optionally or preferably the weight ratio of the unfolded enzyme to the thiol-containing reducing agent is in a range of from about 150:1 to about 5:1 , or from about 120:1 to about 7:1 , or from about 100:1 to about 8:1 , or from about 80:1 to about 10:1 , or from about 60:1 to about 15:1.

Embodiment 44 is a process as set forth in one of embodiments 34-42, wherein the unfolded enzyme is prepared in substantial absence or in absence of a thiol-containing reducing agent (e.g., cysteine), that is, the aqueous phase is substantially free of or free of a thiol-containing reducing agent. Embodiment 45 is a process as set forth in one of embodiments 34-44, wherein the unfolded enzyme is prepared in substantial absence or in absence of a chaotropic agent other than cysteine, that is, the aqueous phase is substantially free of or free of a chaotropic agent other than cysteine.

Embodiment 46 is a process as set forth in one of embodiments 34-45, wherein the enzyme is substantially or completely unfolded.

Embodiment 47 is a process as set forth in one of embodiments 34-46, wherein the concentration of the unfolded enzyme in the aqueous phase is at least 1 wt%, 2 wt%, 3 wt%, 4 wt%, or 5 wt%, and no more than 15 wt%, 12 wt%, 11 wt%, 10 wt%, 9 wt%, 8 wt%, 7 wt%, or 6 wt%, based on the total weight of the aqueous phase.

Embodiment 48 is a process as set forth in one of embodiments 34-47, wherein the aqueous phase further comprises an emulsifier.

Embodiment 49 is a process as set forth in one of embodiments 34-47, wherein the aqueous phase is substantially free of or free of an emulsifier.

Embodiment 50 is a process as set forth in one of embodiments 34-49, wherein the aqueous phase is substantially free of or free of an additional biopolymer.

Embodiment 51 is a process as set forth in one of embodiments 34-50, wherein the aqueous phase is substantially free of or free of an additional polyfunctional nucleophile.

Embodiment 52 is a process as set forth in one of embodiments 34-51 , wherein the temperature of the aqueous phase is in a range of from 10 °C to 30 °C, or from 15 °C to 30 °C, or from 20 °C to 30 °C, or from 18 °C to 28 °C, or from 20 °C to 25 °C.

Embodiment 53 is a process as set forth in one of embodiments 34-52, wherein the pH of the aqueous phase is at least 5.5, 6.5, or 7.5, and optionally no more than 1 1 , 10, 9.5, or 9.

Embodiment 54 is a process as set forth in one of embodiments 34-53, wherein the polyisocyanate comprises or is an aromatic polyisocyanate, and optionally or preferably, the polyisocyanate is substantially free of or free of an aliphatic polyisocyanate.

Embodiment 55 is a process as set forth in one of embodiments 34-54, wherein the aromatic polyisocyanate is selected from the group of polyisocyanurate of toluene diisocyanate, trimethylol propane-adduct of toluene diisocyanate, trimethylol propaneadduct of xylylene diisocyanate, and mixtures thereof, and optionally or preferably, the polyisocyanate comprises or is trimethylol propane-adduct of xylylene diisocyanate. Embodiment 56 is a process as set forth in one of embodiments 34-55, wherein the amount of the polyisocyanate present in the oil phase is at least 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1 .1 wt%, or 1 .2 wt%, and no more than 15 wt%, 12 wt%, 10 wt%, 9 wt%, 8 wt%, 7 wt%, 6 wt%, 5 wt%, 4 wt%, 3 wt%, 2 wt%, or 1 .5 wt%, based on the total weight of the oil phase.

Embodiment 57 is a process as set forth in one of embodiments 34-56, wherein the active material is one recited in one of embodiments 14-17.

Embodiment 58 is a process as set forth in one of embodiments 34-57, wherein the temperature of the oil phase is in a range of from 10 °C to 30 °C, or from 15 °C to 30 °C, or from 20 °C to 30 °C, or from 18 °C to 28 °C, or from 20 °C to 25 °C.

Embodiment 59 is a process as set forth in one of embodiments 34-58, wherein an emulsifier is used in step (c).

Embodiment 60 is a process as set forth in one of embodiments 34-58, wherein essentially no emulsifier is used in step (c).

Embodiment 61 is a process as set forth in one of embodiments 34-60, wherein the emulsifying process is carried out at a temperature of from about 10 °C to about 30 °C, or from about 15 °C to about 30 °C, or from about 20 °C to about 30 °C, or from about 18 °C to about 28 °C, or from about 20 °C to about 25 °C.

Embodiment 62 is a process as set forth in one of embodiments 34-61 , wherein the weight ratio of the unfolded enzyme to the polyisocyanate used in the process is at least 0.7:1 , 1 :1 , 1.2:1 , 1.5:1 , 1 .8:1 , 2:1 , 2.2:1 , 2.5:1 , 2.8:1 , 3:1 , 3.2:1 , 3.5:1 , 3.7:1 , 4:1 , 4.5:1 , 5:1 , 5.5:1 , 6:1 , 6.5:1 , or 7:1 , and no more than 40:1 , 35:1 , 30:1 , 25:1 , 20:1 , 18:1 , 15:1 , 12:1 , 1 1 :1 , 10:1 , 9:1 , 8:1 , or 7:1.

Embodiment 63 is a process as set forth in one of embodiments 34-62, wherein the microcapsule shell is cured at a temperature of no more than 150 °C, 120 °C, 100 °C, 90 °C, 80 °C, or 70 °C, and at least 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, or 65 °C.

Embodiment 64 is a process as set forth in one of embodiments 34-63, wherein the microcapsule shell is cured for at least 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, or 2 hours, and no more than 48 hours, 36 hours, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, 8 hours, 6 hours, or 4 hours. Embodiment 65 is a process as set forth in one of embodiments 34-64, further comprising: adding a crosslinker to crosslink with the reaction product of the unfolded enzyme with the polyisocyanate.

Embodiment 66 is a process as set forth in embodiment 65, wherein the crosslinker is selected from the group of polyfunctional aldehydes, polyols including polyphenols, polyamines, triethyl citrate, and mixtures thereof, optionally or preferably the crosslinker comprises or is a polyfunctional aldehyde and/or a polyphenol, and optionally or preferably the crosslinker comprises or is tannic acid and/or glutaraldehyde.

Embodiment 67 is a process as set forth in one of embodiments 34-64, wherein essentially no crosslinker is added or used in the process.

Embodiment 68 is a process as set forth in one of embodiments 34-67, wherein the reaction product comprises or is a polymeric network comprising an unfolded enzyme covalently bonded or crosslinked with a polyisocyanate.

Embodiment 69 is a process as set forth in one of embodiments 34-68, wherein essentially no self-condensed polyisocyanate is formed during the process.

Embodiment 70 is a process as set forth in one of embodiments 34-69, wherein the encapsulation efficiency of the microcapsule produced in the process is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%.

Embodiment 71 is a process as set forth in one of embodiments 34-70, wherein a gum Arabic is added into the microcapsule slurry before or during the curing step (e), optionally or preferably the amount of gum Arabic added into the microcapsule slurry is at least 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, or 0.3 wt%, and no more than 10 wt%, 5 wt% 2 wt%, or 1 wt%, based on the total weight of the microcapsule slurry.

Embodiment 72 is a process as set forth in one of embodiments 34-71 , wherein a thickener is added into the microcapsule slurry before, after, or during the curing step (e); optionally or preferably the thickener is selected from the group of xanthan gum, microcrystalline cellulose (MCC), coprocessed mixture of MCC and carboxymethyl cellulose (CMC), hydroxypropyl methyl cellulose (HPMC), and combinations thereof; optionally or preferably the thickener comprises or is a combination of xanthan gum and a coprocessed mixture of MCC and CMC; optionally or preferably the thickener comprises a combination of HPMC and a coprocessed mixture of MCC and CMC. Embodiment 73 is a process as set forth in embodiment 72, wherein the amount of the thickener added into the microcapsule slurry is at least 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt%, and no more than 5 wt%, 2 wt%, 1 wt%, or 0.5 wt%, based on the total weight of the microcapsule slurry.

Embodiment 74 is a process as set forth in one of embodiments 34-73, further comprising: (f) reducing the amount of residual enzyme (e.g., glucoamylase) present in the core-shell microcapsule slurry, optionally or preferably the amount of residual enzyme is reduced to no more than 1 wt%, 0.8 wt%, 0.6 wt%, 0.4 wt%, 0.2 wt%, 0.1 wt%, 0.05 wt%, 0.02 wt%, or 0.01 wt%, based on the total weight of the microcapsule slurry, optionally or preferably the reducing step (f) is carried out during or after the curing step (e).

Embodiment 75 is a process as set forth in embodiment 74, wherein the pH of the core-shell microcapsule slurry in step (f) is adjusted to no more than 4.5, 4.2, 4.0, 3.8, 3.6, 3.5, 3.4, 3.2, 3.1 , 3.0, 2.8, 2.6, 2.4, 2.2, or 2.0.

Embodiment 76 is a process as set forth in embodiments 74 or 75, wherein the reducing step (f) is carried out at an elevated temperature, optionally or preferably the reducing step (f) is carried out at a temperature of at least 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, or 60 °C, and no more than 120 °C, 100 °C, 90 °C, 80 °C, or 70 °C.

Embodiment 77 is a process as set forth in one of embodiments 74-76, wherein the reducing step (f) is carried out for at least 5 minutes, 10 minutes, 20 minutes, 30 minutes, or 45 minutes, and optionally no more than 36 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, or 2 hours.

Embodiment 78 is a process as set forth in one of embodiments 74-77, wherein after the reducing step (f), the pH of the microcapsule slurry is adjusted to pH in a range of from about 4 to about 7.5, or from about 4 to about 6.0, or from about 5 to about 7.5, or from about 5 to about 7.0.

Embodiment 79 is a process as set forth in one of embodiments 34-78, wherein the biodegradable core-shell microcapsule produced or formed in the process is the biodegradable core-shell microcapsule set forth in one of embodiments 1 -27, and/or the core-shell microcapsule slurry produced or formed in the process is the core-shell microcapsule slurry set forth in one of embodiments 28-33.

Embodiment 80 is a liquid fragrance composition. The liquid fragrance composition comprises: (i) 3 wt% to 40 wt% of a free fragrance, (ii) 0.1 wt% to 10 wt% of glyceryl ricinoleate, (iii) 0.1 wt% to 20 wt% of a biodegradable core-shell microcapsule set forth in one of embodiments 1 -27, (iv) 0.02 wt% to 5 wt% of a thickening agent, and (v) 50 wt% to 95 wt% of water, wherein all amounts are based on the weight of the liquid fragrance composition.

Embodiment 81 is a liquid fragrance composition as set forth in embodiment 80, wherein the liquid fragrance composition has a pH of 3.5 to 8, preferably 4 to 6.

Embodiment 82 is a liquid fragrance composition as set forth in embodiments 80 or 81 , wherein the free fragrance is present in the liquid fragrance composition as fragrance oil droplets dispersed homogeneously in the aqueous phase to form an oil-in-water emulsion.

Embodiment 83 is a liquid fragrance composition as set forth in one of embodiments 80-82, wherein the liquid fragrance composition has a viscosity of from 400 cP to 3000 cP, or from 450 cP to 2500 cP, at 25 °C.

Embodiment 84 is a liquid fragrance composition as set forth in one of embodiments 80-83, wherein the thickening agent is selected from the group of acrylate copolymers, cationic acrylamide copolymers, polysaccharides, and combinations thereof, optionally or preferably the thickening agent comprises or is a combination of acrylate/C -Cso alkyl acrylate cross-polymer with gellan gum or xanthan gum.

Embodiment 85 is a consumer product comprising a biodegradable core-shell microcapsule set forth in one of embodiments 1 -27, wherein the consumer product is a fabric softener, fabric conditioner, detergent, scent booster, fabric refresher spray, body wash, body soap, shampoo, hair conditioner, body spray, hair refresher spray, hair dye, hair moisturizer, skin moisturizer, hair treatment, skin treatment, antiperspirant, deodorant, insect repellant, candle, surface cleaner, bathroom cleaner, bleach, cat litter, refresher spray, pesticide, insecticide, herbicide, fungicide, or paint.

Claims

CLAIM(S) What is claimed is:

1 . A biodegradable core-shell microcapsule comprising:

(a) a microcapsule core comprising an active material; and

(b) a microcapsule shell comprising a reaction product of an unfolded enzyme with a polyisocyanate; wherein the microcapsule shell is substantially free of or free of a self-condensed polyisocyanate, and the microcapsule shell has a biodegradation rate of at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, based on the weight of the microcapsule shell, within 60 days according to OECD301 F.

2. The biodegradable core-shell microcapsule of claim 1 , wherein the enzyme is selected from the group of glucoamylase, amylase, phytase, and mixtures thereof.

3. The biodegradable core-shell microcapsule of claim 1 or 2, wherein

(i) the number of cysteine residues in the amino acid sequence of the enzyme is at least 4;

(ii) the combined number of lysine and arginine residues in the amino acid sequence of the enzyme is at least 20; and

(iii) the combined number of aspartic acid and glutamic acid residues in the amino acid sequence of the enzyme is at least 40.

4. The biodegradable core-shell microcapsule of any one of the preceding claims, wherein the amount of the self-condensed polyisocyanate present in the microcapsule shell is no more than 5 wt%, or no more than 2 wt%, or no more than 1 wt%, or no more than 0.5 wt%, or no more than 0.2 wt%, or no more than 0.1 wt%, or no more than 0.05 wt%, or no more than 0.02 wt%, or no more than 0.01 wt%, based on the total weight of the microcapsule shell.

5. The biodegradable core-shell microcapsule of any one of the preceding claims, wherein the microcapsule shell is a single-layered shell.

6. The biodegradable core-shell microcapsule of any one of the preceding claims, wherein the weight ratio of the enzyme moiety to the polyisocyanate moiety present in the microcapsule shell is in a range of from about 20:1 to about 1 .5:1 .

7. A core-shell microcapsule slurry, comprising:

(a) the biodegradable core-shell microcapsule of any one of the preceding claims; and

(b) water.

8. The core-shell microcapsule slurry of claim 7, wherein the amount of residual enzyme present in the core-shell microcapsule slurry is no more than 0.1 wt% based on the total weight of the core-shell microcapsule slurry.

9. The core-shell microcapsule slurry of claim 7 or 8, further comprising a thickener.

10. A process for producing a core-shell microcapsule slurry, comprising:

(a) providing an aqueous phase comprising an unfolded enzyme, wherein the pH of the aqueous phase is equal to or greater than the isoelectric point of the enzyme;

(b) providing an oil phase comprising an active material and a polyisocyanate;

(c) emulsifying the oil phase with the aqueous phase to form an emulsion;

(d) allowing the unfolded enzyme to react with the polyisocyanate to form a microcapsule shell encapsulating a microcapsule core, thereby forming a core-shell microcapsule slurry comprising a biodegradable core-shell microcapsule, wherein the microcapsule core comprises the active material, and the microcapsule shell comprises a reaction product of the unfolded enzyme with the polyisocyanate; and

(e) curing the microcapsule shell at an elevated temperature.

1 1. The process of claim 10, wherein the unfolded enzyme is prepared by diluting the enzyme in an aqueous solution.

12. The process of claim 10 or 1 1 , further comprising:

(f) reducing the amount of residual enzyme present in the core-shell microcapsule slurry, preferably the amount of residual enzyme is reduced to no more than about 0.1 wt% based on the total weight of the core-shell microcapsule slurry.

13. The process of claim 12, wherein the pH of the core-shell microcapsule slurry is adjusted to no more than about 4 in step (f).

14. A liquid fragrance composition comprising:

(i) 3 wt% to 40 wt% of a free fragrance,

(ii) 0.1 wt% to 10 wt% of glyceryl ricinoleate,

(iii) 0.1 wt% to 20 wt% of the biodegradable core-shell microcapsule of any one of claims 1 to 6,

(iv) 0.02 wt% to 5 wt% of a thickening agent, and

(v) 50 wt% to 95 wt% of water.

15. A consumer product comprising the biodegradable core-shell microcapsule of any one of claims 1 to 6, wherein the consumer product is a fabric softener, fabric conditioner, detergent, scent booster, fabric refresher spray, body wash, body soap, shampoo, hair conditioner, body spray, hair refresher spray, hair dye, hair moisturizer, skin moisturizer, hair treatment, skin treatment, antiperspirant, deodorant, insect repellant, candle, surface cleaner, bathroom cleaner, bleach, cat litter, refresher spray, pesticide, insecticide, herbicide, fungicide, or paint.