A kind of probiotic microcapsule and preparation method thereof based on whole water phase complex coacervationTechnical field
The present invention relates to food biotechnology and pharmaceutical technology fields more particularly to a kind of based on whole water phase complex coacervationProbiotic microcapsule and preparation method thereof.
Background technique
Probiotics is most important functional food component, has and maintains intestinal flora balance, enhancing immunity of organism, controlThe functions such as cholesterol, blood lipid and blood pressure, the extensive utilization in the food such as fermented product, dairy products and milk beverage.Only with enoughAfter the viable bacteria of quantity reaches human body intestinal canal, probiotics can just play its prebiotic function, but probiotics to temperature, humidity, oxygen,The environmental factors such as acid are highly sensitive, easily dead during processing, storage, digestion etc., and microencapsulation is often by as improvement, this is askedThe important means of topic.The probiotics microencapsulation method generallyd use in food industry has spray drying process, fluidized bed process, emulsificationMethod, extrusion etc..However the preparation condition of these methods is stringent, unfriendly to environment, the microcapsules of preparation have poor ringBorder tolerance and controlled release capability.
Complex coacervation is a concept in Polymer Physics, is referred under certain pH, between the polyelectrolyte of oppositely chargedElectrostatic interaction occurs, last desolvation forms the very big enrichment phase of polyelectrolyte content difference and dilution phase.It is compound solidifyingPoly- method is usually used in the microencapsulation of the liposoluble active ingredients such as grease, essential oil, liposoluble vitamin, lipid fractions, formerReason is to prepare oil/water lotion by emulsification, then cause oppositely charged by external condition according to water phase and an oil phase incompatibilityPolyelectrolyte forms compound on oil-water interfaces, to stabilize the liposoluble constituent inside oil droplet.Complex coacervation, which is given out a contract for a project, buriesThe research of lipophilic bioactive ingredients extensively and profoundly, and has had preferable industrial application.However complex coacervation embedding parentAqueous biological study of active components is less, and having quite a few embedding is based on water/oil/water emulsion system, and preparation process is multipleMiscellaneous, load capacity is low.
Chinese patent CN102580638A disclose it is a kind of by complex coacervation prepare hydroaropic substance be core material it is micro-Encapsulation process, this method are that addition oil phase substance and emulsifier, high speed dispersion form cream into hydrophily core material firstThen emulsion is added in the wall material solution that specific protein gelatin and polysaccharide are mixed in a certain proportion by shape liquid, adjust pHComplex coacervation reaction is carried out to 3.9~4.2, the addition glutamine transaminage of pH to 6.0 is adjusted after cooling again and is solidified to obtainMicrocapsule solution, it is dry to filter resulting hygrometric state microcapsules to get powdered microcapsule product is arrived.Complex coacervation in the patentContain emulsifying step during preparing microcapsules, and its embedding is to embed activity by oil/water or water/oil/water systemIngredient, preparation process is complicated, and embedding rate is low.Traditional complex coacervation generally requires the oily phase of addition, forms oil/water by emulsificationOr water/oil/water lotion, to realize the embedding to active constituent, this method not only needs additionally to add oily phase, and increases operationTriviality, microcapsules preparation time is long, and not applicable probiotics etc. should not be exposed to the sensitive core material in air for a long time.
Application of the complex coacervation in terms of probiotics embedding is very deficient.Coacervate microcapsules method is as a kind of newEmerging embedding method, about being rarely reported using the country in probiotics embedding field.How a kind of suitable probiotics is developedComplex coacervation becomes urgent problem to be solved to prepare probiotic microcapsule.
Summary of the invention
It is an object of the invention to overcome the defect of the prior art, provide a kind of based on the prebiotic of whole water phase complex coacervationBacteria microcapsule and preparation method thereof, this method are not need emulsification step based on the complex coacervation of type A gelatin/acidic protein systemSuddenly, method is simplified, material has been saved, has shortened microcapsules preparation time；The probiotic microcapsule survival rate of method preparation is high,With good storage, digestion, heat treatment equistability.
The present invention is implemented as follows:
It is an object of the present invention to provide the preparation methods of the probiotic microcapsule based on whole water phase complex coacervation, specialSign is that described method includes following steps:
Step 1, the probiotics for cultivating freezing, centrifugation, discard supernatant, and obtain bacterium mud, spare；
Step 2, the type A gelatin for weighing certain mass are soluble in water, are prepared into the type A gelatin that mass concentration is 0.5~5%Solution is kept the temperature spare at 30~60 DEG C；
Step 3, the na caseinate solution for weighing certain mass are soluble in water, and being prepared into mass concentration is 0.5~5%Na caseinate solution is kept the temperature spare at 30~60 DEG C；
Step 4, at 30~60 DEG C, by the bacterium mud, type A gelatin solution, na caseinate solution mix after, adjustPH, induction complex coacervation reaction, prepares hygrometric state microcapsules；The additive amount of the bacterium mud are as follows: every 1g gelatin addition 108~1012cfuBacterium mud, the mass ratio of the type A gelatin and casein sodium are as follows: 0.25~4:1, the pH range be 5.5~6.0；
The hygrometric state microcapsules that step 5, the dry step 4 obtain obtain the dry powder of probiotic microcapsule.
Preferably, temperature employed in the step 2~step 4 is 35 DEG C~45 DEG C.
The most preferred mass ratio of the type A gelatin and casein sodium is 2:1；PH is most preferably in the step 46.0。
Preferably, after type A gelatin solution, na caseinate solution described in the step 4 are mixed with bacterium mud, quality is addedThe powder for the small molecular sugar that score is 0~5%, adjusts pH again after mixing evenly.
Specifically, the small molecular sugar includes glucose, sucrose, fructose, trehalose, maltose, oligofructose, oligomeric woodOne of sugar is a variety of；The speed of agitator is 100~1000r/min, and the time is 10~60min.
Preferably, in the step 4 will bacterium mud, type A gelatin solution, na caseinate solution mix after using 100~1000r/min stirs 10~60min, then the cooling 5~30min of ice-water bath.
Preferably, dry in the step 5 is spray drying or freeze-drying；The condition of the spray drying are as follows: control100~250 DEG C of inlet temperature, outlet temperature are 50~150 DEG C；The condition of the freeze-drying are as follows: controlled at -20 DEG C~-80 DEG C, cooling time is 6~48h.
The second object of the present invention is to provide a kind of probiotic microcapsule that the method is prepared.
Compared with prior art, the invention has the advantages that and effect:
1, the preparation method of the probiotic microcapsule provided by the invention based on whole water phase complex coacervation, using type A gelatin/Casein sodium complex coacervation system makes that type A gelatin is positively charged, junket by adjusting pH in neutral pH (5.5~6.0) rangeProtein acid sodium is negatively charged, probiotics is negatively charged, and, microphase-separated compound by electrostatic forms droplet structure, cooling and solidifying, after dryPowdered microcapsule product can be obtained.The first, emulsifying step is not needed, method is simplified, has saved material, has shortened micro- glueCapsule preparation time；The second, the embedding rate and storage survival rate of probiotics are effectively raised, and the probiotic microcapsule is with goodStorage well, digestion, heat treatment equistability；It is third, easy to operate, it is easy to industrialized production.
2, in the prior art there are no using type A gelatin/casein sodium complex coacervation system successfully to probiotics intoRow embedding, this is because the complex coacervation there are many difficult points, be most difficult to be how to regulate and control microphase-separated；The present invention providesThe probiotic microcapsule based on whole water phase complex coacervation preparation method, using " 30~60 DEG C of temperature ", 5.5~6.0The concentration of pH, type A gelatin solution and na caseinate solution are the matter of 0.5~5% and type A gelatin and casein sodiumWhen amount is than being 0.25~4:1, so as to artificially regulate and control microphase-separated, and then succeeds in doing and full water is carried out to probioticsThe complex coacervation of phase, this is to need the applicant by obtained by a large amount of innovation sex exploration.
3, the preparation method of a kind of probiotic microcapsule based on whole water phase complex coacervation provided by the invention, using A typeGelatin solution and wall material of the na caseinate solution as complex coacervation, can not only improve embedding and the protecting effect of microcapsules,For edibility microcapsules, moreover it is possible to provide nutrition for human body；And type A gelatin is a kind of typical basic protein, isoelectric point is logicalIt is cheap and easy to get, without biocidal property often in pH 8.0-9.0, complex coacervation pH can be improved to neutral range, to significantly improve thallusSurvival rate durings drying, storage etc..
Detailed description of the invention
Fig. 1 is the Optical Morphology figure of the complex coacervation object of different embodiments and comparative example；
Fig. 2 is the storage survival results of probiotic microcapsule；Wherein A is 11%RH, is measured under the conditions of 25 DEG C；B is33%RH is measured under the conditions of 25 DEG C；C is 11%RH, is measured under the conditions of 30 DEG C；D is 33%RH, is measured under the conditions of 30 DEG C.
Specific embodiment
Embodiment 1
The preparation method of probiotic microcapsule based on whole water phase complex coacervation includes the following steps:
Step 1, the probiotics for cultivating freezing, centrifugation, discard supernatant, and obtain 1 × 109CFU/ml bacterium mud, it is spare；
Step 2, type A gelatin (GE) powder for weighing certain mass are dissolved in ultrapure water, are prepared into concentration 2% (w/w)GE solution, 40 DEG C of heat preservations are spare；
Step 3, casein sodium (NaCaS) powder for weighing certain mass are dissolved in ultrapure water, are prepared into concentration 2%(w/w) NaCaS solution, 40 DEG C of heat preservations are spare；
Step 4, at 40 DEG C, by 0.3ml bacteria suspension (1 × 109CFU/ml), (w/w) GE of 40g 2% solution, 20g 2%(w/w) NaCaS solution, 1.2g sucrose (final concentration 2%w/w) powder mix, and mix 20min under 400rpm；Using 10% acetic acidPH is adjusted respectively to 15min is persistently stirred under 5.5,400rpm, keeps complex coacervation complete；The cooling 15min of ice-water bath again, obtains micro-Capsule solution, then spray drying is powder to get to probiotics under conditions of 110 DEG C of inlet temperature, outlet temperature are 70 DEG CMicrocapsules.
Embodiment 2
The embodiment is in addition to the pH value of mixing cohesion is 5.6, remaining is the same as embodiment 1.
Embodiment 3
The embodiment is in addition to the pH value of mixing cohesion is 5.8, remaining is the same as embodiment 1.
Embodiment 4
The embodiment is in addition to the pH value of mixing cohesion is 6.0, remaining is the same as embodiment 1.
Embodiment 5
The pH value of embodiment mixing cohesion is 6.0, the ratio of GE/NaCaS is 0.25, remaining is the same as embodiment 4.
Embodiment 6
The pH value of embodiment mixing cohesion is 6.0, the ratio of GE/NaCaS is 0.5, remaining is the same as embodiment 4.
Embodiment 7
The pH value of embodiment mixing cohesion is 6.0, the ratio of GE/NaCaS is 1, remaining is the same as embodiment 4.
Embodiment 8
The pH value of embodiment mixing cohesion is 6.0, the ratio of GE/NaCaS is 4, remaining is the same as embodiment 4.
Comparative example 1
The comparative example is in addition to the pH value of mixing cohesion is 4.5, remaining is the same as embodiment 1.
Comparative example 2
The comparative example is in addition to the pH value of mixing cohesion is 6.5, remaining is the same as embodiment 1.
Comparative example 3
The comparative example is NaCaS, and pH value 6.0, remaining is the same as embodiment 4.
Comparative example 4
The comparative example is GE, and pH value 6.0, remaining is the same as embodiment 4.
Comparative example 5
The pH value of comparative example mixing cohesion is 6.0, the ratio of GE/NaCaS is 6, remaining is the same as embodiment 4.
Experimental example 1
1, microencapsulated forms measure: monitoring above-described embodiment 1-4 and right by BT-1600 imaged particles analysis systemThe form of ratio 1-2 microcapsules.It takes a small amount of hygrometric state microcapsules to be added dropwise on glass slide, being placed in microscopically observation and taking pictures, putsBig multiple is 10 times and 20 times.
2, embedding rate measures: taking 0.01g microcapsules dry powder respectively in above-described embodiment 1-4 and comparative example 1-2 microcapsulesIt is added in 0.99g peptonate solution (PS, 1.0g/L peptone, 8.5g/L NaCl, pH 6.8), with vortex oscillator point30s is dissipated, it is completely dispersed.0.1mL liquid is taken, after being diluted to suitable multiple with physiological saline (9g/L NaCl), is coated onOn MRS solid medium, then counted after 37 DEG C of stationary culture 48h.Meanwhile it being taken from the original bacterium solution for being added to wall material1.0mL after dilution, is spread evenly across on MRS solid medium, is counted after 37 DEG C of stationary culture 48h.
The embedding rate (EY) of microcapsules calculates as follows:
EY=N/N0 × 100%
Wherein, N indicates that the viable count released in microcapsules, N0 indicate to be used for microcapsule embedded total bacteria count.
3, the Optical Morphology of the microcapsules of embodiment 1-4 and comparative example 1-2 and embedding rate data are as shown in table 2, micro-The Optical Morphology figure of capsule is as shown in Figure 1.
Table 1
It as shown in Table 1, is preferred pH when pH value range is 5.5-6.0, experimental group 1- experimental group 4 can show preferablySpherical particles form, and embedding rate is high.When the selection of comparative example group 1 pH is 4.5 and when the selection of comparative example group 2 pH is 6.5,Microcapsules can't be spherical shape, and embedding rate is low.
Experimental example 2
Use the complex coacervation object form of the mixed proportion of the method microscopically observation difference GE/NaCaS of experimental example 1 withAnd calculating embedding rate, i.e. embodiment 4- implements the morphological feature and embedding rate data such as table of the microcapsules of 8 and comparative example 3-52。
Table 2
As shown in Table 2, when the mixed proportion of GE/NaCaS is 0.25-4:1, embodiment 4- embodiment 8 can showPreferable spherical particles form, and embedding rate is high.The microcapsules of comparative example 3- comparative example 5 can't be spherical shape, and embedding rateIt is low.
The measurement of the storage survival rate of experimental example 3
1, in complex coacervation pH6.0, complex coacervation temperature is that preparation GE/NaCaS (embodiment 4), NaCaS are (right at 40 DEG CRatio 3) and GE (comparative example 4) compound system, measure the storage survival rate of each group probiotic microcapsule.It is different by measurementMicrocapsules survival volume situation at temperature and relative humidity (RH) evaluates microcapsules storage stability.By embodiment 4, comparative example3 and the probiotic microcapsule of comparative example 4 be respectively placed under the conditions of 11%RH (25 DEG C, 30 DEG C) and 33%RH (25 DEG C, 30 DEG C)Hermetically storing, the corresponding saturated solution of difference relative humidity is respectively as follows: lithium chloride saturated solution (relative humidity at 25 DEG C11%)；Magnesium chloride saturated solution (relative humidity 33%).Sample under 33%RH storage condition, every 4d measurement thallus survivalAmount；Sample under 11%RH storage condition measures thallus survival volume every 7d, and measuring method is colony counting method, experimental periodFor 28d.With the opposite survival logarithm of thallus to storage number of days mapping, and curve linear is fitted to obtain deactivation constant.
2, inactivation of the lactic acid bacteria during storage uses opposite survival logarithm (log N/N0) to indicate that formula is expressed as follows:
LogNt=logN0+kTt
Wherein, N0 indicates original bacterium number (CFU/g), and Nt indicates thallus survival number (CFU/g) after storage a period of time, t tableShow that storage time (d), kT indicate the deactivation constant (d-1) under temperature T.
3, experimental result is as shown in Fig. 2, Fig. 2 is variation of the opposite survival logarithm of thallus with storage time, the slope of curveThallus deactivation constant is represented, thallus deactivation constant k is smaller, and thallus inactivation rate is lower, and storage stability is then better.As shown in Fig. 2,At the same temperature, relative humidity has larger impact for thallus survival, and humidity is higher, and thallus inactivation rate is bigger, and oppositeUnder lower humidity (RH11%), the influence that temperature survives for thallus is relatively small, and high temperature and humidity can accelerate the dead of lactic acid bacteriaDie rate.Known by figure, under each storage condition, can observe the deactivation constant of lactic acid bacteria, k (GE/NaCaS) < k(NaCaS) < k (GE) illustrates what casein sodium stored the protection that lactic acid bacteria room temperature is stored compared with type A gelatin to lactic acid bacteria room temperatureProtecting effect is good, and after two kinds of albumen complex coacervations, lactic acid bacteria deactivation constant k value is minimum compared with the k value of two groups of controls, illustrates AThe complex coacervation of type gelatin and casein sodium has better protecting effect to the room temperature storage of lactic acid bacteria.
Described is only presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention andWithin principle, any modification, equivalent replacement, improvement and so on should all include within protection scope of the present invention.