发明领域field of invention
本发明涉及外切淀粉酶在延缓包含动物蛋白和淀粉组分的食品的口感和质地弹性(柔软性)劣化方面的用途。本发明还涉及用于制备含有动物蛋白组分和淀粉组分的食品的工艺，其中使用了外切淀粉酶来增加食品的货架期。本发明还涉及根据本发明的方法所获得的食品。The present invention relates to the use of exoamylases for delaying the deterioration of mouthfeel and texture elasticity (softness) of food products comprising animal protein and starch components. The present invention also relates to a process for the preparation of a food product comprising an animal protein component and a starch component, wherein an exoamylase is used to increase the shelf life of the food product. The invention also relates to a food product obtained according to the method of the invention.
发明背景Background of the invention
肉制品(例如香肠、肉丸等)常常含有天然淀粉，例如玉米淀粉。所述淀粉组分可占整个产品的10％或更多。然而，所述淀粉组分可在烹饪之前、之中或之后胶凝。新鲜制备时，此类含有淀粉的肉制品将显示出湿润的口感和良好的弹性(柔软的)质地。然而，随着时间的延长，质地弹性(柔软性)和口感将劣化。毫无疑问，这些特征方面的改变都是因为受到淀粉老化的影响。Meat products (eg, sausages, meatballs, etc.) often contain native starches, such as cornstarch. The starch component may account for 10% or more of the entire product. However, the starch component may gel before, during or after cooking. When freshly prepared, such starch-containing meat products will exhibit a moist mouthfeel and a good springy (soft) texture. However, texture elasticity (softness) and mouthfeel will deteriorate over time. Undoubtedly, these changes in characteristics are due to the effect of starch aging.
WO8400876涉及含有热稳定的α淀粉酶的含有淀粉的食品。WO8400876 relates to starch-containing food products containing a thermostable alpha-amylase.
JP63146746和JP1055160涉及通过向切碎的鱼肉中加入用α-淀粉酶处理淀粉而产生的淀粉水解产物以及随后冷冻来防止冷冻变性和改善鱼肉质量。JP63146746 and JP1055160 relate to preventing freeze denaturation and improving fish meat quality by adding starch hydrolyzate produced by treating starch with α-amylase to minced fish meat and subsequent freezing.
发明目的purpose of invention
本发明的目的是提供制备含有动物蛋白的食品的方法，同时增加其货架期，例如通过该方法来延缓这些食品的口感和质地弹性(柔软性)劣化的方法。本发明目的还包括提供具有更长货架期和/或更好的湿润口感及良好的弹性质地的食品，例如可在较长的时期中保持柔软性的食品。The object of the present invention is to provide a method for preparing foodstuffs containing animal protein while increasing their shelf life, for example by delaying the deterioration of the mouthfeel and texture elasticity (softness) of these foodstuffs. It is also an object of the present invention to provide food products with a longer shelf life and/or better moist mouthfeel and a good springy texture, such as food products that retain their softness over a longer period of time.
发明概述Summary of the invention
本发明发现通过用外源的外切淀粉酶处理含有淀粉和动物蛋白的食品，抑制或降低了食品口感和质地(柔软性)劣化，从而延长了食品的货架期。The present invention finds that by treating food containing starch and animal protein with exogenous exoamylase, the deterioration of food taste and texture (softness) is inhibited or reduced, thereby prolonging the shelf life of food.
因此，本发明的第一个方面涉及制作具有改善的货架期的、包含动物蛋白和淀粉组分的食品的方法，所述方法包括下列步骤：Accordingly, a first aspect of the present invention relates to a method of making a food product comprising animal protein and starch components having an improved shelf life, said method comprising the steps of:
a)将外源外切淀粉酶与含淀粉的组分相混合，a) mixing an exogenous exoamylase with a starch-containing component,
b)在允许外源外切淀粉酶至少部分地水解淀粉的条件下，加工在步骤a)中获得的混合物，b) processing the mixture obtained in step a) under conditions allowing the exogenous exoamylase to at least partially hydrolyze the starch,
c)在步骤a)或b)的任一项之前、与之同时或之后，将含淀粉组分的混合物与动物蛋白组分相混合，c) mixing the mixture of starch-containing components with the animal protein component before, simultaneously with or after any of steps a) or b),
d)加工在步骤a)-c)之后获得的、包含动物蛋白和淀粉的混合物，以获得一种食品，其货架期可得到延长。d) Processing the mixture comprising animal protein and starch obtained after steps a)-c) to obtain a food product whose shelf life can be extended.
本发明的第二方面涉及抑制包含动物蛋白和淀粉的组合物的口感和质地(柔软性)劣化的方法，所述方法包含下列步骤：A second aspect of the present invention relates to a method of inhibiting deterioration of mouthfeel and texture (softness) of a composition comprising animal protein and starch, said method comprising the steps of:
a)将外源外切淀粉酶与含淀粉的组分相混合，a) mixing an exogenous exoamylase with a starch-containing component,
b)在允许外源外切淀粉酶至少部分地水解淀粉的条件下，加工在步骤a)中获得的混合物，b) processing the mixture obtained in step a) under conditions allowing the exogenous exoamylase to at least partially hydrolyze the starch,
c)在步骤a)、b)或c)的任一项之前、与之同时或之后，将含淀粉组分的混合物与动物蛋白组分相混合。c) admixing the mixture of starch-containing components with the animal protein component before, simultaneously with or after any of steps a), b) or c).
本发明的第三个方面涉及根据本发明的方法获得的食品。A third aspect of the invention relates to a food product obtained according to the method of the invention.
本发明的第四个方面涉及根据本发明的方法获得的、口感和质地(柔软性)劣化减少的、包含动物蛋白和淀粉的组合物。A fourth aspect of the present invention relates to a composition comprising animal protein and starch with reduced deterioration of mouthfeel and texture (softness), obtained according to the method of the present invention.
本发明的其它方面涉及外源外切淀粉酶用于改善含动物蛋白和淀粉组分的食品货架期的用途。Other aspects of the invention relate to the use of exogenous exoamylases for improving the shelf life of food products containing animal protein and starch components.
本发明的其它方面涉及外源外切淀粉酶用于抑制含动物蛋白和淀粉组分的食品的口感和质地(柔软性)劣化的用途。Other aspects of the invention relate to the use of exogenous exoamylases for inhibiting the deterioration of mouthfeel and texture (softness) of food products containing animal protein and starch components.
附图说明Description of drawings
图1显示了如何制备本发明的食品以及如何实施所述方法的工艺流程图。Figure 1 shows a process flow diagram of how to prepare the food product of the present invention and how to implement the method.
图2显示了在实施例1中获得的质地分析结果。FIG. 2 shows the results of texture analysis obtained in Example 1.
图3显示了在实施例4中获得的质地分析结果。FIG. 3 shows the results of texture analysis obtained in Example 4.
图4四种淀粉酶对玉米淀粉的抗老化作用；1c：对照-玉米淀粉；2c：对照-(玉米淀粉∶木薯淀粉＝1∶1)；3c：对照-木薯淀粉；4c：对照-玉米淀粉和质地改进剂；3：POWERFreshBread8100-200ppm；4：POWERFreshBread8100-400ppm；5：Novamyl10000BG-200ppm；6：Novamyl10000BG-400ppm；7：Opticake Fresh50BG-100ppm；8：OpticakeFresh50BG-200ppm；9：Opticake Fresh50BG-300ppm；10：OpticakeFresh50BG-400ppm；11：1271608-25ppm；12：1271608-50ppm；13：1271608-75ppm；14：1271608-100ppmFig. 4 four kinds of amylases are to the antiaging effect of cornstarch; 1c: contrast-cornstarch; 2c: contrast-(cornstarch: tapioca=1: 1); 3c: contrast-tapioca; 4c: contrast-cornstarch和质地改进剂；3：POWERFreshBread8100-200ppm；4：POWERFreshBread8100-400ppm；5：Novamyl10000BG-200ppm；6：Novamyl10000BG-400ppm；7：Opticake Fresh50BG-100ppm；8：OpticakeFresh50BG-200ppm；9：Opticake Fresh50BG-300ppm；10 : OpticakeFresh50BG-400ppm; 11: 1271608-25ppm; 12: 1271608-50ppm; 13: 1271608-75ppm;
发明详述Detailed description of the invention
根据此详细描述，适用下列简称和定义。应注意，如本文中所使用的，单数形式名词包括复数指代，除非上下文明确地另有指明。因此，例如，提及“酶”时包括多个此类酶，而提及“制剂”时包括指代一种或多种制剂以及本领域技术人员已知的其等同物，等等。除非另有定义，本文中所使用的所有技术和科学术语具有与本领域普通技术人员通常所理解的相同含义。下面的术语提供如下。From this detailed description, the following abbreviations and definitions apply. It should be noted that as used herein, nouns in the singular include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes and reference to "a preparation" includes reference to one or more preparations as well as equivalents thereof known to those skilled in the art, and so on. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following terms are provided below.
淀粉酶和淀粉酶来源Amylase and Amylase Sources
如本文中所使用的，术语“外源”指不是天然的(或内源性的)包含在淀粉组分中的淀粉酶。此术语包括淀粉酶，其可天然地在某些淀粉组分中起作用(例如少量的)，但是其被加入到淀粉组分中(例如以纯酶的形式或过量的添加)。在一些实施方案中，外源淀粉酶对给定的淀粉组分起作用并不为人所知。As used herein, the term "exogenous" refers to an amylase that is not natively (or endogenously) contained in a starch component. This term includes amylases which are naturally active in certain starch components (eg, in small amounts), but which are added to the starch component (eg, in pure enzyme form or added in excess). In some embodiments, the exogenous amylase is not known to act on a given starch component.
如本文中所使用的，“淀粉酶”是指能够催化淀粉降解的酶。As used herein, "amylase" refers to an enzyme capable of catalyzing the degradation of starch.
如本文中所使用的，术语“外切淀粉酶”和“外切作用淀粉酶”是指能够从底物的非还原端水解淀粉分子的任何淀粉酶。As used herein, the terms "exoamylase" and "exoamylase" refer to any amylase capable of hydrolyzing a starch molecule from the non-reducing end of a substrate.
如本文中所使用的，术语“外切α-淀粉酶”和“外切作用α-淀粉酶”是指能够从底物的非还原端水解淀粉分子中的α-1，4键的任何α-淀粉酶。As used herein, the terms "exo-α-amylase" and "exo-acting α-amylase" refer to any α-amylase capable of hydrolyzing α-1,4 linkages in starch molecules from the non-reducing end of the substrate. - Amylase.
合适的外切淀粉酶包括从多糖链(优选外部的)的非还原端水解α-1，4-糖苷键的外切淀粉酶。Suitable exoamylases include exoamylases that hydrolyze alpha-1,4-glycosidic linkages from the non-reducing ends of polysaccharide chains (preferably external).
在一个实施方案中，合适的外切淀粉酶包括β-淀粉酶(EC3.2.1.2)，其从葡聚糖的非还原端起作用，催化α-1，4糖苷键的水解，以去除连续的β-麦芽糖单元。其它合适的β-淀粉酶是释放β-构象的寡糖的那些，例如二聚体、三聚体、四聚体、五聚体或六聚体。特别地，有用的是释放二聚体和六聚体(例如β构型的麦芽糖或麦芽六糖残基)的淀粉酶。一种合适的β-淀粉酶是Diazyme BBA(DaniscoA/S)或如EP 234 858中所公开的、可由芽孢杆菌(Bacillus)菌株NCIB11608产生的生麦芽糖β-淀粉酶，所述文献在此通过引用而并入。In one embodiment, suitable exoamylases include β-amylases (EC 3.2.1.2), which act from the non-reducing end of dextran to catalyze the hydrolysis of α-1,4 glycosidic bonds to remove Consecutive β-maltose units. Other suitable β-amylases are those which release oligosaccharides in the β-conformation, eg dimers, trimers, tetramers, pentamers or hexamers. Particularly useful are amylases that release dimers and hexamers (eg maltose or maltohexaose residues in the beta configuration). A suitable beta-amylase is Diazyme BBA (Danisco A/S) or the maltogenic beta-amylase producible by Bacillus strain NCIB11608 as disclosed in EP 234 858, which is hereby incorporated by reference And incorporated.
在另一个实施方案中，合适的外切淀粉酶包括葡糖淀粉酶(EC3.2.1.3，α-淀粉酶，淀粉葡糖苷酶)，其从葡聚糖多糖的非还原端起作用，催化α-1，4糖苷键的水解，以去除连续的β-D-葡萄糖单元。In another embodiment, suitable exoamylases include glucoamylases (EC 3.2.1.3, alpha-amylases, amyloglucosidases), which act from the non-reducing end of the glucan polysaccharide to catalyze Hydrolysis of alpha-1,4 glycosidic linkages to remove consecutive beta-D-glucose units.
在另一个实施方案中，合适的外切淀粉酶包括葡聚糖1，4-α-麦芽四糖水解酶(EC3.2.1.60)，其从葡聚糖多糖的非还原末端起作用，催化α-1，4糖苷键的水解，以去除连续的麦芽四糖单元。这些外切淀粉酶也称为G4淀粉酶或麦芽四糖-形成性麦芽四糖水解酶，这些术语可互换地使用。G4淀粉酶可源自施氏假单胞茵(Pseudomonasstutzeri)，嗜糖假单胞茵(Pseudomonas saccharophila)和环状芽胞杆菌(Bacillus circulans)。来自嗜糖假单胞茵和施氏假单胞茵的外切麦芽四糖水解酶的合适的实例公开于EP0298645中，在此将其通过引用而并入。另一个具有G4外切淀粉酶活性的合适的酶制剂是POWERFreshBread8100烘烤用酶(材料编号1271729，丹尼斯克公司(Danisco A/s))。其他可用于根据本发明的方法中的合适外切淀粉酶是在以下任一专利中具体公开的任一具体外切淀粉酶：国际专利申请公开号WO2010133644、美国专利US7776576和美国专利US7833770，这些参考文献以引用方式并入本文。In another embodiment, suitable exoamylases include dextran 1,4-α-maltotetraose hydrolase (EC 3.2.1.60), which acts from the non-reducing end of the dextran polysaccharide to catalyze Hydrolysis of alpha-1,4 glycosidic linkages to remove consecutive maltotetraose units. These exoamylases are also known as G4 amylases or maltotetraose-forming maltotetraose hydrolases, the terms being used interchangeably. G4 amylases may be derived from Pseudomonas stutzeri, Pseudomonas saccharophila and Bacillus circulans. Suitable examples of exomaltotetralases from Pseudomonas saccharophila and Pseudomonas stutzeri are disclosed in EP0298645, which is hereby incorporated by reference. Another suitable enzyme preparation having G4 exoamylase activity is POWERFreshBread 8100 Baking Enzyme (Material No. 1271729, Danisco A/s). Other suitable exoamylases that can be used in the method according to the invention are any of the specific exoamylases specifically disclosed in any of the following patents: International Patent Application Publication No. WO2010133644, US Patent US7776576 and US Patent US7833770, which refer to Documents are incorporated herein by reference.
在另一个实施方案中，合适的外切淀粉酶包括葡聚糖1，4-α-麦芽糖水解酶(EC3.2.1.133)，其也可被称作生麦芽糖α-淀粉酶或1，4-α-D-葡聚糖α-麦芽糖水解酶，其从葡聚糖的非还原端起作用，催化α-1，4糖苷键的水解，以去除连续的α-麦芽糖单位。葡聚糖1，4-α-麦芽糖水解酶的合适的实例公开在WO9950399，WO1991004669，US4598048，US4604355，US6890572和WO2010133644中，将它们在此引用。葡聚糖1，4-α-麦芽糖水解酶的合适例子源于芽孢杆菌属，如脂肪嗜热芽孢杆菌(Bacillus stearothermophilus)。合适的酶制剂的一个具体例子是Novamyl 10000 BG。In another embodiment, suitable exoamylases include dextran 1,4-alpha-maltohydrolase (EC 3.2.1.133), which may also be referred to as maltogenic alpha-amylase or 1,4 - α-D-glucan α-maltohydrolase, which acts from the non-reducing end of glucan, catalyzing the hydrolysis of α-1,4 glycosidic linkages to remove consecutive α-maltose units. Suitable examples of dextran 1,4-alpha-maltose hydrolases are disclosed in WO9950399, WO1991004669, US4598048, US4604355, US6890572 and WO2010133644, which are incorporated herein by reference. Suitable examples of glucan 1,4-alpha-maltohydrolase originate from the genus Bacillus, such as Bacillus stearothermophilus. A specific example of a suitable enzyme preparation is Novamyl 10000 BG.
在另一个实施方案中，合适的外切淀粉酶包括葡聚糖1，4-α-麦芽糖己糖苷酶(EC3.2.1.98)，其从葡聚糖多糖的非还原端起作用，催化α-1，4糖苷键的水解，以去除连续的麦芽己糖单位。这些外切淀粉酶也被称为G6-淀粉酶。来自芽孢杆菌种#707的外切麦芽糖己糖水解酶的实例被公开于Tsukamoto等人的Biochem.Biophys.Res.Commun.中；来自环状芽胞杆菌F2的外切麦芽糖己糖水解酶被公开于Taniguchi，ACS Symp.，1991，Ser.458，111-124中，且来自产气杆菌(Aerobacter aerogenes)的外切麦芽糖己糖水解酶被公开于Kainuma等人，Biochim.Biophys.Acta，1975，410，333-346中，所有这些通过引用而被包括。In another embodiment, suitable exoamylases include dextran 1,4-α-maltohexosidase (EC 3.2.1.98), which acts from the non-reducing end of the dextran polysaccharide to catalyze α - Hydrolysis of 1,4 glycosidic linkages to remove consecutive maltohexose units. These exoamylases are also known as G6-amylases. An example of an exomaltose hexose hydrolase from Bacillus sp. #707 is disclosed in Tsukamoto et al., Biochem. Biophys. Res. Commun.; an exomaltose hexose hydrolase from Bacillus circulans F2 is disclosed in Taniguchi, ACS Symp., 1991, Ser.458, 111-124, and an exomaltose hexose hydrolase from Aerobacter aerogenes is disclosed in Kainuma et al., Biochim.Biophys.Acta, 1975, 410 , 333-346, all of which are included by reference.
在另一个实施方案中，合适的外切淀粉酶包括外切麦芽糖戊糖水解酶，其从葡聚糖多糖的非还原端起作用，催化α-1，4糖苷键的水解，以去除连续的麦芽糖戊糖单位。来自嗜碱性革兰氏阳性细菌的外切麦芽糖戊糖水解酶的实例公开于美国专利5,204,254中，且来自假单胞茵的种的外切麦芽糖戊糖水解酶公开于Shida等人，Biosci.Biotechnol.Biochem.，1992，56，76-80中；所有这些都通过引用而被包括。In another embodiment, suitable exoamylases include exomaltose pentose hydrolase, which acts from the non-reducing end of the dextran polysaccharide to catalyze the hydrolysis of α-1,4 glycosidic bonds to remove consecutive Maltose pentose unit. Examples of exo-maltose pentose sugar hydrolases from basophilic Gram-positive bacteria are disclosed in U.S. Patent 5,204,254, and exo-maltose pentose sugar hydrolases from Pseudomonas species are disclosed in Shida et al., Biosci. Biotechnol. Biochem., 1992, 56, 76-80; all of which are incorporated by reference.
在一些实施方案中，待根据本发明使用的外切淀粉酶不是源于曲霉属如米曲霉(Aspergillus Oryzae)或者黑曲霉(Aspergillus niger)的α-淀粉酶。In some embodiments, the exoamylase to be used according to the invention is not an alpha-amylase derived from Aspergillus, such as Aspergillus Oryzae or Aspergillus niger.
在一些实施方案中，待根据本发明使用的外切淀粉酶不是源于假单胞茵属如Pseudomonas amyloderamosa的异淀粉酶。In some embodiments, the exoamylase to be used according to the invention is not an isoamylase derived from a Pseudomonas such as Pseudomonas amyloderamosa.
根据本发明而使用的外切淀粉酶被认为也可具有一定程度的内切淀粉酶活性，只要其主要活性为外切活性。It is contemplated that exoamylases used in accordance with the invention may also have some degree of endoamylase activity, provided that their predominant activity is exo.
在一个实施方案中，根据本发明而使用的外切淀粉酶的外切活性比内切活性更高，如在实施详细描述的，例如Betamyl和Phadebas测定。In one embodiment, the exoamylase used according to the invention has a higher exo-activity than endo-activity, as described in detail in the Examples, eg Betamyl and Phadebas assays.
在另一个实施方案中，根据本发明而使用的外切淀粉酶具有的外切活性为总淀粉酶活性的至少10％，例如至少20％，例如至少30％，例如至少40％，例如至少50％，例如至少60％，例如至少70％，例如至少80％，例如至少90％，例如至少95％，例如至少97％，例如至少99％。In another embodiment, the exoamylase used according to the invention has an exoactivity of at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50% of the total amylase activity. %, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 99%.
根据本发明而使用的外切淀粉酶可以源自微生物或来自植物。The exoamylases used according to the invention may be of microbial origin or of plant origin.
产生特定聚合度(DP)的麦芽寡糖(包括麦芽己糖)的合适的淀粉酶可源自数种微生物，例如克雷伯氏肺炎杆菌(Klebsiellapneumonia)，枯草芽孢杆菌(Bacillus subtilis)，环状芽孢杆菌G-6，环状芽孢杆菌F-2和热溶芽孢杆菌(B.caldovelox)。Suitable amylases that produce maltooligosaccharides (including maltohexose) of a specific degree of polymerization (DP) can be derived from several microorganisms, such as Klebsiella pneumoniae (Klebsiellapneumonia), Bacillus subtilis (Bacillus subtilis), cyclic Bacillus G-6, Bacillus circulans F-2 and Bacillus caldovelox.
因此，产生麦芽戊糖的淀粉酶可源自地衣芽孢杆菌(B.licheniformis)584和假单胞茵的种，产生麦芽四糖的淀粉酶可来自施氏假单胞茵NRRL B-3389，芽孢杆菌的种MG-4和假单胞茵的种IMD353，以及产生麦芽三糖的淀粉酶可来自灰色链霉菌(Streptomycesgriseus)NA-468和枯草芽孢杆菌。Thus, amylases producing maltopentose can be derived from B. licheniformis (B. licheniformis) 584 and species of Pseudomonas, and amylases producing maltotetraose can be derived from Pseudomonas stutzeri NRRL B-3389, sp. Bacillus sp. MG-4 and Pseudomonas sp. IMD353, and maltotriose-producing amylases can be derived from Streptomyces griseus NA-468 and Bacillus subtilis.
EP298645描述了使用遗传工程技术制备施氏假单胞茵或嗜糖假单胞茵的外切麦芽四糖水解酶的方法。US 5204254描述了来自嗜碱性细菌(DSM5853)的天然的和经遗传修饰的外切麦芽糖戊糖水解酶。根据本发明而使用的其它合适的淀粉酶包括下列淀粉酶：来自芽孢杆菌的种H167，产生麦芽己糖；来自细菌分离株(163-26，DSM 5853)，产生麦芽戊糖；来自芽孢杆菌的种IMD370，产生麦芽四糖和更小的麦芽寡糖；以及来自芽孢杆菌的种GM 8901，其起初由淀粉产生麦芽己糖，然后所述麦芽己糖在延长的水解期间被转变为麦芽四糖。合适的β-淀粉酶也可源自植物，例如从大豆提取。EP298645 describes a method for preparing an exomaltotetraose hydrolase of Pseudomonas stutzeri or Pseudomonas saccharophila using genetic engineering techniques. US 5204254 describes native and genetically modified exomaltose pentose sugar hydrolases from basophilic bacteria (DSM5853). Other suitable amylases for use according to the invention include the following: from Bacillus species H167, which produces maltohexose; from a bacterial isolate (163-26, DSM 5853), which produces maltopentose; from Bacillus species IMD370, which produces maltotetraose and smaller maltooligosaccharides; and species GM 8901 from Bacillus, which initially produces maltohexose from starch, which is then converted to maltotetraose during prolonged hydrolysis . Suitable beta-amylases may also be of vegetable origin, for example extracted from soybeans.
根据本发明使用的非麦芽糖生成性外切淀粉酶的另一个实例是来自嗜碱性芽孢杆菌菌株GM8901的外切淀粉酶。Another example of a non-maltogenic exoamylase for use according to the invention is the exoamylase from the basophilic Bacillus strain GM8901.
本发明的方法中使用的酶制剂，可以是以颗粒或块状的粉末形式。所述酶制剂可具有窄的粒径分布，其中超过95％(按重量)的颗粒在25至500μm的范围内。可通过常规的方法制备颗粒和块状的粉末，例如通过在流化床造粒机中将外切淀粉酶喷洒到载体上。所述载体可由具有合适的粒径的颗粒核心组成。所述载体可以是可溶的或不可溶的，例如盐(如NaCl或硫酸钠)，糖(例如蔗糖或乳糖)，糖醇(例如山梨糖醇)，淀粉，稻，玉米渣或大豆。The enzyme preparation used in the method of the present invention may be in the form of granule or block powder. The enzyme preparation may have a narrow particle size distribution with more than 95% (by weight) of the particles in the range of 25 to 500 μm. Granular and agglomerated powders can be prepared by conventional methods, for example by spraying the exoamylase onto the carrier in a fluidized bed granulator. The carrier may consist of a particle core having a suitable particle size. The carrier may be soluble or insoluble, such as salts (such as NaCl or sodium sulfate), sugars (such as sucrose or lactose), sugar alcohols (such as sorbitol), starch, rice, corn grits or soybeans.
在一些实施方式中，根据本发明的方法和工艺中使用的外切淀粉酶是热稳定的。In some embodiments, the exoamylases used in the methods and processes according to the invention are thermostable.
如本申请中所使用的，术语“热稳定的”涉及酶在暴露于升高的温度后保持活性的能力。一般，外切淀粉酶能够在约55℃至约80℃或更高的温度下降解淀粉。合适的，所述酶在暴露于高达95℃的温度时还可保持其活性。As used in this application, the term "thermostable" relates to the ability of an enzyme to retain activity after exposure to elevated temperatures. Typically, exoamylases are capable of degrading starch at temperatures from about 55°C to about 80°C or higher. Suitably, the enzyme also retains its activity when exposed to temperatures up to 95°C.
通过其半衰期来测量酶(例如非麦芽糖生成性外切淀粉酶)的热稳定性。因此，用于本发明的外切淀粉酶在温度(55℃至约95℃或更高，优选约80℃)下可具有长的半衰期。Thermostability of an enzyme such as a non-maltogenic exoamylase is measured by its half-life. Thus, exoamylases useful in the present invention may have a long half-life at temperature (55°C to about 95°C or higher, preferably about 80°C).
如本申请中所使用的半衰期(t1/2)是在确定的热条件下酶活性的损失一半所需的时间(按分钟计算)。在优选的实施方案中，半衰期是在80℃下测定的。一般在80℃或更高的温度下将样品加热1-10分钟。然后通过本文所描述的任何方法，通过测量剩余淀粉酶活性来计算半衰期的值。半衰期的测定将在实施例中更详细的描述。Half-life (t1/2 ) as used in this application is the time (in minutes) required for the loss of half of the enzyme activity under defined thermal conditions. In a preferred embodiment, the half-life is measured at 80°C. Typically the sample is heated at 80°C or higher for 1-10 minutes. Half-life values are then calculated by measuring remaining amylase activity by any of the methods described herein. Determination of half-life is described in more detail in the Examples.
根据本发明所使用的外切淀粉酶在超过80℃的温度下是有活性的并且在大约55℃的温度下，在淀粉颗粒胶凝的过程中和之后水解淀粉。非麦芽糖生成性外切淀粉酶越热稳定，其保持活性的时间越长，因此其将提供更好的抗老化效果。然而，在高于约85℃的温度进行酶促保温的过程中，可导致酶失活。如果这种情况发生，可使非麦芽糖生成性外切淀粉酶逐渐灭活，从而使最终食品或含淀粉的组分中基本上没有酶活。因此，适于使用非麦芽糖生成性外切淀粉酶的最佳温度高于50℃且低于98℃。The exoamylases used according to the invention are active at temperatures above 80°C and at temperatures of about 55°C hydrolyze starch during and after gelatinization of starch granules. The more thermally stable the non-maltogenic exoamylase, the longer it will remain active and thus the better anti-aging effect it will provide. However, enzymatic incubation at temperatures above about 85°C can result in inactivation of the enzyme. If this occurs, the non-maltogenic exoamylases can be gradually inactivated such that there is essentially no enzymatic activity in the final food or starch-containing component. Therefore, the optimal temperature for the use of non-maltogenic exoamylases is above 50°C and below 98°C.
根据本发明所使用的外切淀粉酶的热稳定性可通过蛋白质工程化而被改善，以变得更为热稳定并因此更适于本文所描述的用途；我们因此包括了使用变种外切淀粉酶，其通过蛋白质工程化而被修饰以变得更为热稳定。The thermostability of the exoamylases used according to the invention can be improved by protein engineering to become more thermostable and thus more suitable for the uses described herein; we therefore include the use of variant exoamylases Enzymes that have been modified by protein engineering to become more thermostable.
根据本发明所使用的外切淀粉酶是pH稳定的；如本申请中所使用的，术语“pH稳定的”是指酶在宽范围的pH中保持活性的能力。根据本发明所使用的外切淀粉酶能够在约5至约10.5的pH下降解淀粉。在一个实施方案中，可通过测量在特定pH条件下的酶的半衰期来测定pH的稳定性。在另一个实施方案中，可通过测量在特定pH条件下的酶的活性或比活性来测定pH的稳定性。所述特定的pH条件可以是pH5至pH10.5的任意pH。The exoamylases used according to the invention are pH stable; as used in this application, the term "pH stable" refers to the ability of the enzyme to maintain activity over a wide range of pH. Exoamylases used in accordance with the present invention are capable of degrading starch at a pH of about 5 to about 10.5. In one embodiment, pH stability can be determined by measuring the half-life of the enzyme under specific pH conditions. In another embodiment, pH stability can be determined by measuring the activity or specific activity of an enzyme under specific pH conditions. The specific pH condition may be any pH from pH 5 to pH 10.5.
外切特异性Exo-specificity
已知一些外切淀粉酶可具有一定程度的内切淀粉酶活性。It is known that some exoamylases may have some degree of endoamylase activity.
通常可通过测定总淀粉酶活性与总内切淀粉酶活性的比率来测量外切特异性。在本文中将此比率称作“外切特异性指数”。在优选的实施方案中，酶被认为是外切淀粉酶，如果其具有2或更大的外切特异性指数，即其总的淀粉酶活性(包括外切淀粉酶活性)是其内切淀粉酶活性的2倍或更多。在优选的实施方案中，外切淀粉酶的外切特异性指数为5乃至100或更大。在高度优选的实施方案中，外切特异性指数为150乃至600或更大。Exo-specificity can generally be measured by determining the ratio of total amylase activity to total endo-amylase activity. This ratio is referred to herein as the "exo-specificity index". In a preferred embodiment, an enzyme is considered an exoamylase if it has an exospecificity index of 2 or greater, i.e. its total amylase activity (including exoamylase activity) is greater than that of its endoamylase 2 times or more than the enzyme activity. In preferred embodiments, the exoamylase has an exospecificity index of 5 to 100 or greater. In highly preferred embodiments, the exo-specificity index is 150 to 600 or greater.
可通过本领域已知的任何手段来测量总的淀粉酶活性和内切淀粉酶活性。例如，可通过测定从淀粉底物释放的还原端的总数目来测量总的淀粉酶活性。另外，在实施例中进一步详细描述了Betamyl测定的应用。Total and endoamylase activity can be measured by any means known in the art. For example, total amylase activity can be measured by determining the total number of reducing ends released from the starch substrate. Additionally, the use of the Betamyl assay is described in further detail in the Examples.
可通过使用Phadebas试剂盒(Pharmaciaand Upjohn)来测定内切淀粉酶活性。这利用了蓝色标记的交联淀粉(用偶氮染料标记的)；仅仅是淀粉分子中的内部剪切释放标记，而外部剪切不释放。可通过分光光度法来测量染料的释放。因此，Phadebas试剂盒测量内切淀粉酶活性，并且为了方便，此类测定的结果在本文中被称作“Phadebas单位”。Endoamylase activity can be determined by using the Phadebas kit (Pharmacia and Upjohn). This utilizes blue-labeled cross-linked starch (labeled with an azo dye); only internal shear in the starch molecule releases the label, while external shear does not. Dye release can be measured spectrophotometrically. Accordingly, the Phadebas kit measures endoamylase activity, and the results of such assays are referred to herein as "Phadebas units" for convenience.
因此，在高度优选的实施方案中，外切特异性指数表达为Betamyl单位/Phadebas单位，也称作“B/Phad”。Thus, in a highly preferred embodiment, the exo-specificity index is expressed as Betamyl units/Phadebas units, also referred to as "B/Phad".
也可根据现有技术中所描述的方法来测定外切特异性，例如，在我们的国际专利公开号WO99/50399中所描述的。其由内切淀粉酶活性与外切淀粉酶活性之间的比率来测量外切特异性。因此根据本发明所使用的外切淀粉酶将具有少于0.5内切淀粉酶单位(EAU)/外切淀粉酶活性单位。优选地，根据本发明所使用的外切淀粉酶具有少于0.05EAU/外切淀粉酶活性单位，并且更优选地，少于0.01 EAU/外切淀粉酶活性单位。Exo-specificity can also be determined according to methods described in the prior art, for example, in our International Patent Publication No. WO99/50399. It measures exo-specificity by the ratio between endo- and exo-amylase activity. The exoamylase used according to the invention will therefore have less than 0.5 endoamylase units (EAU) per exoamylase activity unit. Preferably, the exoamylase used according to the invention has less than 0.05 EAU/exoamylase activity unit, and more preferably, less than 0.01 EAU/exoamylase activity unit.
根据本发明所使用的外切淀粉酶将优选地具有外切特异性，例如如上文所述，通过外切特异性指数所测量的，这与它们是外切淀粉酶相一致。Exo-amylases used according to the invention will preferably have exo-specificity, eg as measured by the exo-specificity index, as described above, consistent with them being exo-amylases.
淀粉和淀粉来源Starches and Starch Sources
如本申请中所使用的，术语″淀粉″指包含植物(例如玉米)的复杂多糖碳水化合物的任何物质，包括具有式(C6H10O5)x的直链淀粉和支链淀粉，其中X可以是任何数字。术语″淀粉″指生的(即未烹饪的)淀粉，例如未经受胶凝的淀粉。As used in this application, the term "starch" refers to any substance comprising complex polysaccharide carbohydrates of plants such as corn, including amylose and amylopectin having the formula (C6 H10 O5 )x , wherein X can be any number. The term "starch" refers to raw (ie uncooked) starch, eg starch that has not been subjected to gelatinization.
淀粉可适当地来自谷物或伪谷物(pseudo cereal)例如玉米、小麦、大麦、黑麦、燕麦、荞麦和稻；根类植物，例如马铃薯、甘薯、木薯、竹芋、波利尼西亚竹芋；豆类，例如蚕豆、扁豆、绿豆、豌豆和鹰嘴豆。淀粉的其它来源包括西米、木薯、蜀黍、香蕉、arracacha、面包果、美人蕉、colacasia、katakuri、葛、malanga、粟、oca、芋头、栗、荸荠、山药、玉米穗轴(cob)和橡实。本领域的技术人员了解用于制备本申请中所公开的淀粉底物的方法。例如，有用的淀粉底物可以获得自根茎、根、茎、豆、谷物或全谷。更具体地，淀粉来自产生大量淀粉的植物。例如，淀粉可来自玉米、玉米稻穗、小麦、大麦、黑麦、高梁、西米、木薯、木薯淀粉、蜀黍、稻、豌豆、豆、香蕉或马铃薯。玉米含有大约60-68％的淀粉；大麦含有大约55-65％的淀粉；粟含有大约75-80％的淀粉；小麦含有大约60-65％的淀粉；以及精米含有70-72％的淀粉。特别涉及的淀粉底物为玉米淀粉、小麦淀粉以及大麦淀粉。来自谷粒的淀粉可以是经研磨的或完整的并且包括玉米固体，例如核、麸和/或穗轴。淀粉可以是高度精细的生淀粉或来自淀粉精制过程的原料。各种淀粉都是商业化的。例如，玉米淀粉可获得自Cerestar，Sigma和Katayama ChemicalIndustry Co.(日本)；小麦淀粉可获得自Sigma；甘薯淀粉可获得自Wako Pure ChemicalIndustry Co.(日本)；以及马铃薯淀粉可获得自Nakaari ChemicalPharmaceutical Co.(日本)。The starch may suitably be derived from cereals or pseudocereals such as corn, wheat, barley, rye, oats, buckwheat and rice; root vegetables such as potato, sweet potato, cassava, arrowroot, Polynesian arrowroot; legumes, Examples include beans, lentils, mung beans, peas, and chickpeas. Other sources of starch include sago, tapioca, milo, banana, arracacha, breadfruit, canna, colacasia, katakuri, kudzu, malanga, millet, oca, taro, chestnut, water chestnut, yam, corncob (cob), and acorn . Those skilled in the art are aware of methods for preparing the starch substrates disclosed in this application. For example, useful starch substrates may be obtained from rhizomes, roots, stems, beans, cereals or whole grains. More specifically, starch comes from plants that produce large amounts of starch. For example, the starch may be from corn, ear of corn, wheat, barley, rye, sorghum, sago, tapioca, tapioca, milo, rice, peas, beans, bananas or potatoes. Corn contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch; wheat contains about 60-65% starch; and polished rice contains 70-72% starch. Particularly contemplated starch substrates are corn starch, wheat starch and barley starch. Starch from the grain may be ground or whole and includes corn solids such as kernels, bran and/or cobs. The starch can be highly refined raw starch or raw material from the starch refining process. Various starches are commercially available. For example, corn starch is available from Cerestar, Sigma, and Katayama Chemical Industry Co. (Japan); wheat starch is available from Sigma; sweet potato starch is available from Wako Pure Chemical Industry Co. (Japan); and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).
术语“包含淀粉组分的组合物”意指任何合适的包含淀粉的组合物。该术语包括任何含有或者基于或者衍自淀粉的产品。通常，包含淀粉组分的组合物含有或者基于或者衍自从细粉如小麦细粉获得的淀粉。如本文所用，术语“细粉”是任何淀粉来源、具体地讲谷物如小麦、玉米和/或大米的细磨的粉的同义词。但是，优选地，该术语意指从玉米、大米、马铃薯或者小麦获得的细粉。但是，还设想到包含衍自其他类型的谷物例如黑麦、大麦和蜀黍的细粉的组合物。细粉或者淀粉还可以是来自不同来源、具体地讲来自玉米、大米、马铃薯和/或小麦的细粉或者淀粉的混合物。The term "composition comprising a starch component" means any suitable composition comprising starch. The term includes any product that contains or is based on or derived from starch. Typically, compositions comprising a starch component contain or are based on or are derived from starch obtained from a flour such as wheat flour. As used herein, the term "flour" is synonymous with finely ground flour of any starch source, in particular cereals such as wheat, corn and/or rice. However, preferably, the term means a fine flour obtained from corn, rice, potato or wheat. However, compositions comprising flours derived from other types of grains such as rye, barley and milo are also contemplated. The flour or starch may also be a mixture of flours or starches from different sources, in particular from corn, rice, potato and/or wheat.
在本发明的实施方案中，麦芽糊精可用作淀粉底物。麦芽糊精包含具有约20或更少的右旋糖(葡萄糖)单位的淀粉水解产物。通常，市售的麦芽糊精包含多糖的混合物，所述多糖包括约3至约19个连接的右旋糖单位。麦芽糊精被FDA定义为具有少于20的葡萄糖当量(DE)的产物。它们是公认为安全的(GRAS)食物成分，可用于人消耗。葡萄糖当量(DE)是与100的右旋糖(葡萄糖)标准相比的还原能力的量度。DE越高，淀粉解聚程度就越高(这导致更小的平均聚合物(多糖)大小)，和甜度就越高。特别有用的麦芽糊精是获自玉米淀粉的M040(其可获自Grain Processing Corp.(Muscatine，Iowa))：DE4.0-7.0；堆积密度0.51g/cc；测量的水含量为6.38％，按重量计算。In an embodiment of the invention, maltodextrin may be used as a starch substrate. Maltodextrins comprise starch hydrolysates having about 20 or less dextrose (glucose) units. Typically, commercially available maltodextrins contain a mixture of polysaccharides comprising from about 3 to about 19 linked dextrose units. Maltodextrins are defined by the FDA as products having a dextrose equivalent (DE) of less than 20. They are generally recognized as safe (GRAS) food ingredients for human consumption. Dextrose Equivalent (DE) is a measure of reducing power compared to a dextrose (glucose) standard of 100. The higher the DE, the higher the degree of starch depolymerization (which results in a smaller average polymer (polysaccharide) size), and the higher the sweetness. Particularly useful maltodextrins are obtained from cornstarch M040 (available from Grain Processing Corp., Muscatine, Iowa): DE 4.0-7.0; bulk density 0.51 g/cc; measured water content 6.38% by weight.
淀粉底物可以是来自研磨的全谷的粗制淀粉，其含有非淀粉组分，例如胚芽残留物和纤维。研磨可包括湿研磨或干研磨。在湿研磨中，将全谷浸泡在水中或稀释的酸中，以将谷粒分离成为其组成部分，例如淀粉、蛋白质、胚、油、核、纤维。湿研磨有效地分离胚与粉(即淀粉颗粒与蛋白质)并且特别适于生产糖浆。在干研磨中，将全谷研磨成为细粉并进行加工而不将谷粒分级成为其组成部分。因此，除了淀粉之外，干研磨的谷粒还将包含大量的非淀粉碳水化合物。大多数乙醇来自干研磨。备选地，待加工的淀粉可以是高度精细的淀粉质量，例如至少约90％、至少95％、至少97％或者至少99.5％纯。The starch substrate may be crude starch from ground whole grains, which contains non-starch components such as germ residue and fiber. Grinding can include wet grinding or dry grinding. In wet milling, whole grains are soaked in water or diluted acid to separate the grain into its component parts such as starch, protein, germ, oil, kernel, fiber. Wet milling effectively separates germ from flour (ie starch granules from protein) and is particularly suitable for the production of syrups. In dry milling, whole grains are ground into a fine powder and processed without fractionating the grains into its constituents. Therefore, in addition to starch, dry milled grains will also contain significant amounts of non-starch carbohydrates. Most ethanol comes from dry grinding. Alternatively, the starch to be processed may be of a highly refined starch quality, eg, at least about 90%, at least 95%, at least 97%, or at least 99.5% pure.
动物蛋白和来源Animal Protein and Sources
如本文中使用的，术语″动物蛋白组分″是指，包含衍生自来源于任何动物的食用肉的部分，全肉肌肉，或其部分的蛋白或含蛋白质的组合物，所述任何动物包括牛/牛肉，猪/猪肉，火鸡，鸭，鹅，猎禽，鸡，家禽，绵羊，马，山羊，野生狩猎物(wild game)，啮齿类动物，海产食物，或水生贝壳类动物，例如虾，鱼，以及其组合。在一些实施方案中，“动物蛋白”是指包含低含量的脂肪和腱的食用肉或全肉肌肉的部分。备选地，该术语可以指，从食用肉或全肉肌肉纯化的更纯的动物蛋白级分。根据本发明，尽管卵和乳以及衍生自卵和乳的组合物含有显著比例的蛋白质，但它们被称为“非动物蛋白组分”。As used herein, the term "animal protein component" refers to a protein or protein-containing composition comprising a portion of edible meat, whole meat muscle, or a portion thereof derived from any animal, including Cattle/beef, pig/pork, turkey, duck, goose, game birds, chicken, poultry, sheep, horses, goats, wild game, rodents, seafood, or shellfish such as Shrimp, fish, and combinations thereof. In some embodiments, "animal protein" refers to portions of edible meat or whole meat muscle that contain low levels of fat and tendons. Alternatively, the term may refer to a purer animal protein fraction purified from table meat or whole meat muscle. According to the invention, eggs and milk and compositions derived from eggs and milk are referred to as "non-animal protein components" although they contain a significant proportion of protein.
动物蛋白可以衍生自其中的代表性的合适的鱼包括，去骨鲽，sole，黑线鳕，鳕，鲈鱼，鲑鱼，金枪鱼，鳟等。代表性的合适的水生贝壳类动物包括，带壳的虾，蟹肉，螯虾，龙虾，扇贝，牡蛎，或贝壳中的虾等。动物蛋白可以衍生自其中的代表性的合适的肉包括火腿，牛肉，羔羊肉，猪肉，鹿肉，小牛肉，水牛等，家禽例如鸡，机械去骨的家禽肉，火鸡，鸭，猎禽或鹅等，以肉片形式或以磨碎的形式，例如碎牛肉。当骨不会负面影响肉的可食性时，所述肉可包括动物的骨，例如肋条肉，羔羊排或猪排。Representative suitable fish from which the animal protein may be derived include deboned plaice, sole, haddock, cod, perch, salmon, tuna, trout, and the like. Representative suitable aquatic shellfish include, shelled shrimp, crabmeat, crawfish, lobster, scallops, oysters, shrimp in shells, and the like. Representative suitable meats from which animal protein may be derived include ham, beef, lamb, pork, venison, veal, buffalo, etc., poultry such as chicken, mechanically deboned poultry meat, turkey, duck, game birds or goose, etc., in cutlet form or in ground form, such as ground beef. The meat may comprise the bone of an animal, such as rib meat, lamb chops or pork chops, when the bone does not negatively affect the edibility of the meat.
除非另有指出，否则动物蛋白组分的重量或重量百分比以其具有天然含水量(其可以高达65％)的新鲜食用肉或肉的总重量给出。Unless otherwise indicated, the weight or weight percent of the animal protein component is given in terms of the fresh meat or the total weight of the meat with its natural moisture content (which can be as high as 65%).
除非另有指出，否则动物蛋白组分的盐含量以其天然盐含量给出，其可以为约1.5％。在一些实施方案中，在已用外源淀粉酶处理的组合物中，盐的总量保持低于10％。Unless otherwise indicated, the salt content of the animal protein component is given at its natural salt content, which may be about 1.5%. In some embodiments, the total amount of salt is kept below 10% in the composition that has been treated with the exogenous amylase.
具有动物蛋白的食品foods with animal protein
根据本发明的方法产生的食品通常是切碎的肉产品。切碎的肉产品可以是利用切碎的肉作为原材料的任何食物，例如，碎牛肉，肉馅饼，肉丸，粗剁香肠，shishkebabs，烧卖，饺子等。特别地，本发明可用于产生食品，例如碎牛肉，肉馅饼，粗剁香肠等，其可以经历烘烤收缩，并且需要适当的硬度或柔软性以及其他感觉(基于切碎的肉)。The food product produced according to the method of the present invention is typically a minced meat product. The minced meat product may be any food that utilizes minced meat as a raw material, for example, ground beef, meat pies, meatballs, coarse chop sausages, shishkebabs, shumai, dumplings, etc. In particular, the invention can be used to create food products such as ground beef, meat patties, chop sausages, etc., which can undergo bake shrinkage and require appropriate firmness or softness and other sensations (based on minced meat).
根据本发明的其他食品包括香肠和香肠组合物，热狗组合物，可切片的肉产品和可涂抹的肉产品，包括但不限于，磨碎的牛肉，香肠，法兰克福香肠，维也纳小香肠(热狗)，大红肠，和午餐肉。Other food products according to the invention include sausages and sausage compositions, hot dog compositions, sliceable meat products and spreadable meat products including, but not limited to, ground beef, sausages, frankfurters, wieners (hot dogs) , bologna, and luncheon meat.
根据本发明方法制备的香肠是任何动物肉制备的广泛类别的肉制品，包括猪肉香肠(松散的或包装的，早餐类型或乡村类型)，热狗(维也纳小香肠)，法兰克福香肠，德国熟香肠，大香肠，bockwurst，大红肠，夏季香肠，熏肝香肠，肝脏香肠，午餐肉，蒸煮火腿，切片火腿，荷兰肉糕(dutch loaf)，意大利腊肠，波兰型香肠，肉糜型猪肉肠和牛肉肠，和肉糕。香肠的制备可基本上按照用于全脂肪肉产品的正常制备方法，其需要添加直至约10％的佐料，除了添加经外切淀粉酶处理的淀粉组分或将所述淀粉组分与动物蛋白组分混合外。The sausages prepared according to the method of the invention are a broad class of meat products prepared from any animal meat, including pork sausages (loose or packaged, breakfast type or country type), hot dogs (wieners), frankfurters, German mortadella, Bolognese, bockwurst, bologna, summer sausage, smoked liverwurst, liver sausage, luncheon meat, cooked ham, sliced ham, dutch loaf, salami, Polish type sausage, minced pork and beef sausage, and meatloaf. Sausages can be prepared essentially as normal for full-fat meat products, which require the addition of up to about 10% seasoning, except for the addition of an exoamylase-treated starch component or mixing said starch component with the animal Protein components are mixed outside.
除了具有动物蛋白和淀粉组分的食品外，食品根据其所需用途，可以以大量的最终形式进行加工。它们可以以块或片的形式制备，用于汤，酱等。它们可以是预先烹饪的，冷冻的，冻干的，罐装的，包装在袋中的，或这些的组合。它们还可以形成为部分，切卖给消费者，然后消费者可以根据其需要将它们配制成最合适的形状。In addition to foods with animal protein and starch components, foods can be processed in a number of final forms depending on their desired use. They can be prepared in chunks or flakes and used in soups, sauces and more. They can be pre-cooked, frozen, freeze-dried, canned, packaged in bags, or a combination of these. They can also be formed into parts, cut and sold to consumers who can then formulate them into the most suitable shape according to their needs.
另外的组合物成分和来源Additional Composition Ingredients and Sources
包含动物蛋白的食品，例如包含肉，例如鸡肉，牛肉或鱼肉的香肠或热狗组合物还可以包括香草例如鼠尾草，辛香料，碳水化合物/糖或甜味剂，胡椒粉，膳食纤维，盐和填充剂，例如粉产品，所述产品是本领域已知的。Food products containing animal protein, such as sausage or hot dog compositions containing meat such as chicken, beef or fish may also include herbs such as sage, spices, carbohydrates/sugar or sweeteners, pepper, dietary fiber, salt and fillers, such as powder products, which are known in the art.
还可以将各种另外的成分添加至根据本发明的任何食品。例如，可包括乳化剂，非动物蛋白，其他酶，水胶体，芳香剂，氧化剂，矿物质和维生素，抗氧化剂，抗微生物剂及其组合。可包含抗氧化剂添加剂(其包括BHA，BHT，TBHQ，维生素A，C和E及其衍生物，和各种植物提取物例如包含具有抗氧化剂性质的类胡罗卜素，生育酚或类黄酮的那些)，以增加货架期或在营养上改善动物肉组合物。抗氧化剂和抗微生物剂可以以约0.01％至约10％，优选地约0.05％至约5％，和更优选地约0.1％至约2％的水平(按将被挤出的包含蛋白的材料的重量计算)组合存在。Various additional ingredients may also be added to any food product according to the invention. For example, emulsifiers, non-animal proteins, other enzymes, hydrocolloids, fragrances, oxidizing agents, minerals and vitamins, antioxidants, antimicrobials, and combinations thereof may be included. May contain antioxidant additives (which include BHA, BHT, TBHQ, vitamins A, C and E and their derivatives, and various plant extracts such as those containing carotenoids, tocopherols or flavonoids with antioxidant properties those) to increase shelf life or nutritionally improve animal meat compositions. Antioxidants and antimicrobials can be present at levels of from about 0.01% to about 10%, preferably from about 0.05% to about 5%, and more preferably from about 0.1% to about 2% (based on the protein-containing material to be extruded The weight calculation) combination exists.
脂肪或油，例如动物脂肪或任何合适的动物或植物油，例如椰子油，玉米油，油菜油，猪油，或鱼油也可以添加至根据本发明的食品和组合物。食品中肉对脂肪的比率取决于类型，但脂肪含量通常限于30％，35％或50％的最大值(按重量计算)，取决于类型。Fats or oils, such as animal fat or any suitable animal or vegetable oil, such as coconut oil, corn oil, canola oil, lard, or fish oil may also be added to foods and compositions according to the invention. The ratio of meat to fat in a food depends on the type, but the fat content is usually limited to a maximum of 30%, 35%, or 50% by weight, depending on the type.
可以添加至根据本发明的食品和组合物的其他成分包括谷物，辛香料，例如胡椒，姜，辣椒，肉豆蔻，豆蔻香料，百里香，多香果，洋葱，大蒜，胡荽，小豆蔻，香菜，鼠尾草，月桂，墨角兰，丁香，或肉桂。辛香料可以以任何状况使用；未加工的，干燥的，粉末，提取物，浓缩的提取物，或乳化物；防腐剂，例如异抗坏血酸钠，亚硝酸钠，山梨酸或山梨酸钾)；着色剂，稳定剂包括增稠多糖例如黄原胶，胶凝糖胶，瓜尔胶，角叉菜胶，果胶，黄芪胶和蒟蒻甘露聚糖，和淀粉；和乳化剂。Other ingredients that can be added to foods and compositions according to the invention include cereals, spices such as pepper, ginger, chilli, nutmeg, cardamom spice, thyme, allspice, onion, garlic, coriander, cardamom, coriander , sage, bay, marjoram, cloves, or cinnamon. Spices may be used in any form; raw, dried, powder, extract, concentrated extract, or emulsion; preservatives such as sodium erythorbate, sodium nitrite, sorbic acid, or potassium sorbate); coloring stabilizers include thickening polysaccharides such as xanthan gum, gellan gum, guar gum, carrageenan, pectin, tragacanth and konjac mannan, and starches; and emulsifiers.
乳化剂可以适当地以0.01-5％，例如0.05-3％的量包含。乳化剂的实例包括各种非肉蛋白组分，例如卵蛋白，大豆蛋白，谷蛋白大豆，乳蛋白，从此类蛋白分离的蛋白和此类蛋白的(部分)分解的产物，蔗糖脂肪酸酯，山梨聚糖脂肪酸酯，聚氧乙烯山梨聚糖脂肪酸酯，甘油脂肪酸单酯，聚甘油脂肪酸酯，聚甘油缩合的蓖麻油酸酯，甘油有机酸脂肪酸酯，丙二醇脂肪酸酯，卵磷脂和酶促分解的卵磷脂。Emulsifiers may suitably be included in an amount of 0.01-5%, for example 0.05-3%. Examples of emulsifiers include various non-meat protein components such as egg protein, soybean protein, gluten soy, milk protein, proteins isolated from such proteins and products of (partial) breakdown of such proteins, sucrose fatty acid esters, Sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid monoester, polyglycerol fatty acid ester, polyglycerol condensed ricinoleic acid ester, glycerin organic acid fatty acid ester, propylene glycol fatty acid ester, egg Phospholipids and enzymatically broken down lecithin.
合适的乳化剂包括酪蛋白酸钠，卵磷脂，聚氧乙烯硬脂酸酯，可食用的脂肪酸的甘油单酯和二酯，可食用的脂肪酸的甘油单酯和二酯的乙酸酯，可食用的脂肪酸的甘油单酯和二酯的乳酸酯，可食用的脂肪酸的甘油单酯和二酯的柠檬酸酯，可食用的脂肪酸的甘油单酯和二酯的二乙酰基酒石酸酯，可食用的脂肪酸的蔗糖酯，硬脂酰基-2-乳酸钠，和硬脂酰基-2-乳酸钙。Suitable emulsifiers include sodium caseinate, lecithin, polyoxyethylene stearate, edible mono- and diglycerides of fatty acids, acetates of edible mono- and diglycerides of fatty acids, Lactate esters of mono- and diesters of edible fatty acids, citric acid esters of mono- and diesters of edible fatty acids, diacetyl tartrate esters of mono- and diesters of edible fatty acids, may Edible sucrose esters of fatty acids, sodium stearoyl-2-lactate, and calcium stearoyl-2-lactate.
可以0.01％至5％，例如0.05％至3％的量适当地含有非动物蛋白。非动物蛋白的实例包括各类非肉蛋白成分，例如卵蛋白、大豆蛋白、麦谷蛋白、豆乳蛋白、分离自这些蛋白的蛋白、和这些蛋白的(部分)分解产物等，以及它们的组合。The non-animal protein may suitably be included in an amount of 0.01% to 5%, for example 0.05% to 3%. Examples of non-animal proteins include various non-meat protein ingredients such as egg protein, soy protein, glutenin, soy milk protein, proteins isolated from these proteins, and (partial) breakdown products of these proteins, etc., and combinations thereof.
加工processing
用于制备根据本发明的食品的方法可以基本上按照标准方法制备食品。Methods for preparing food products according to the invention Food products can be prepared essentially according to standard methods.
因此，例如香肠制备：将绞碎的原料肉(通常猪肉，牛肉，鸡，火鸡与酶处理过的淀粉，盐，香草，和其他辛香料混合的。然后填充入和形成入管状肠衣(通常从小肠制备，但也可以是化学合成的)。香肠可以进行烹饪(作为加工的一部分)，可以去除肠衣。香肠可以通过食品加工，干燥或烟熏来进行防腐。So, for example, sausage preparation: Ground raw meat (usually pork, beef, chicken, turkey mixed with enzyme-treated starch, salt, herbs, and other spices. Then stuffed and formed into tubular casings (usually prepared from the small intestine, but can also be chemically synthesized). Sausages can be cooked (as part of processing) and casings can be removed. Sausages can be preserved by food processing, drying or smoking.
根据本发明的方法要求，允许外源外切淀粉酶对包含淀粉组分的组合物起作用。在所述方法的某些阶段，将淀粉酶处理的淀粉组分与包含动物蛋白的组分混合。在一些实施方案中，在添加外切淀粉酶之前或同时将动物蛋白组分与包含淀粉组分的组合物混合。备选地，包含淀粉组分的组合物先用外切淀粉酶进行处理，然后将处理过的淀粉与动物蛋白组分混合。The method according to the invention requires that an exogenous exoamylase is allowed to act on a composition comprising a starch component. At certain stages of the method, the amylase-treated starch component is mixed with a component comprising animal protein. In some embodiments, the animal protein component is mixed with the composition comprising the starch component prior to or simultaneously with the addition of the exoamylase. Alternatively, the composition comprising the starch component is first treated with an exoamylase and the treated starch is mixed with the animal protein component.
除非另有指明，否则根据本发明的方法中使用的特定温度是指混合物或组合物的核心温度。这通过本领域技术人员已知的标准技术来测量。因此，如果将要灭活在所述方法中使用的外切淀粉酶，那么食品组分的核心温度应优选地维持在外切淀粉酶的变性温度下，优选地超过所述变性温度至少1℃，至少3℃，至少5℃或至少10℃，进行一定量的时间。适合于大多数外切淀粉酶的温度是95℃。根据所使用的特定酶和组合物，特定的时间和温度当然可以变化，所述酶在对于升高的温度的稳定性方面有所差异。Unless otherwise indicated, the specific temperature used in the method according to the invention refers to the core temperature of the mixture or composition. This is measured by standard techniques known to those skilled in the art. Therefore, if the exoamylase used in the method is to be inactivated, the core temperature of the food component should preferably be maintained at the denaturation temperature of the exoamylase, preferably at least 1°C above said denaturation temperature, at least 3°C, at least 5°C or at least 10°C for an amount of time. A suitable temperature for most exoamylases is 95°C. The particular times and temperatures may, of course, vary depending upon the particular enzymes employed which vary in their stability to elevated temperatures and the compositions employed.
对于商用和家用食物产生而言，重要的是在包含淀粉的组合物中使用适量的外切淀粉酶。太高的活性可以导致太粘和/或生面团似的产品，从而导致产品滞销。For commercial and domestic food production, it is important to use the appropriate amount of exoamylase in starch-containing compositions. Too high an activity can result in a product that is too sticky and/or doughy, resulting in an unmarketable product.
温度控制例如冷却是控制微生物生长部分措施。Temperature control such as cooling is part of the control of microbial growth.
根据所述方法中使用的特定的外切淀粉酶，温度和pH可优化。除了温度和pH外，其他因素，例如离子强度也可以影响酶促反应。这些物理和化学参数均是本领域技术人员熟知的，可以通过优化，以使酶促反应精和复活。Depending on the particular exoamylase used in the process, temperature and pH can be optimized. In addition to temperature and pH, other factors such as ionic strength can also affect enzymatic reactions. These physical and chemical parameters are well known to those skilled in the art and can be optimized to refine and revive the enzymatic reaction.
如本文中使用的，“最佳pH”是指，在进行测量的pH范围中，本文中公开的外切淀粉酶在淀粉酶活性的标准测定中展示最高活性的pH。As used herein, "pH optimum" refers to the pH at which the exoamylases disclosed herein exhibit the highest activity in standard assays for amylase activity, within the pH range in which the measurements are made.
本发明的具体实施方案Specific embodiments of the present invention
如上所述，本发明涉及制备食品的方法，所述食品具有片延长架期，且包含动物蛋白和淀粉组分，所述方法包括下述步骤：a)将外源外切淀粉酶与包含淀粉组分的组合物混合，b)在允许外源外切淀粉酶至少部分水解淀粉组分的条件下，处理步骤a)获得的混合物，c)在步骤a)或b)的任一个之前，同时，或之后，混合包含淀粉组分的组合物与动物蛋白组分，和d)处理在步骤a)-c)之后获得的包含动物蛋白和淀粉组分的组合物以获得具有改善的货架期的食品。As stated above, the present invention relates to a method of preparing a food product having an extended shelf life and comprising animal protein and starch components, the method comprising the steps of: a) combining an exogenous exoamylase with a starch-containing The composition of the components is mixed, b) the mixture obtained in step a) is treated under conditions that allow the exogenous exoamylase to at least partially hydrolyze the starch components, c) before any of steps a) or b), while , or thereafter, mixing a composition comprising a starch component with an animal protein component, and d) treating the composition comprising animal protein and a starch component obtained after steps a)-c) to obtain a product with improved shelf life food.
应理解，在根据本发明的一些具体实施方案中，包含淀粉组分的组合物与动物蛋白组分的混合可在上述方法中的步骤a)或者b)(即用外源外切淀粉酶进行混合和加工)任一步骤之前进行，或者与所述任一步骤同时进行。It should be understood that in some embodiments according to the present invention, the mixing of the composition comprising the starch component and the animal protein component can be carried out in step a) or b) (i.e. using an exogenous exoamylase) in the above method. Mixing and processing) before any step, or simultaneously with any step.
本发明还涉及延缓组合物的口感和质地弹性(柔软性)的劣化的方法，所述组合物包含动物蛋白和淀粉，所述方法包括下述步骤：a)混合外源外切淀粉酶与包含淀粉组分的组合物，b)在允许所述外源外切淀粉酶至少部分水解所述淀粉组分的条件下，处理步骤a)获得的混合物，和c)在步骤a)，b)，或c)的任一个之前，同时，或之后，混合包含淀粉组分的组合物与动物蛋白组分。The present invention also relates to a method for delaying the deterioration of mouthfeel and texture elasticity (softness) of a composition comprising animal protein and starch, said method comprising the steps of: a) mixing exogenous exoamylase with a composition of starch components, b) treating the mixture obtained in step a) under conditions allowing said exogenous exoamylase to at least partially hydrolyze said starch components, and c) in steps a), b), or before, simultaneously with, or after any of c), mixing the composition comprising the starch component with the animal protein component.
在一些实施方案中，根据本发明的方法还包含步骤：处理在步骤b)获得的混合物以灭活所述外源外切淀粉酶。在一些实施方案中，该灭活通过热处理来进行，例如在距离所述外源外切淀粉酶的变性温度不超过10℃，例如8℃，例如6℃，例如4℃的温度下，处理超过2分钟，例如4，6，8，10，12，14，16，18，20，25，或30分钟，或可选地，在95℃的温度下进行超过2分钟，例如4，6，8，10，12，14，16，18，20，25，或30分钟。In some embodiments, the method according to the invention further comprises the step of treating the mixture obtained in step b) to inactivate said exogenous exoamylase. In some embodiments, the inactivation is carried out by heat treatment, for example, at a temperature not exceeding 10°C, such as 8°C, such as 6°C, such as 4°C, for more than 2 minutes, such as 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, or 30 minutes, or alternatively, at a temperature of 95 °C for more than 2 minutes, such as 4, 6, 8 , 10, 12, 14, 16, 18, 20, 25, or 30 minutes.
在一些实施方案中，外切淀粉酶以下述量添加：100-5000ppm，例如200-4000ppm，例如200-4000ppm，例如300-3000ppm，例如400-2000ppm，例如500-1000ppm的淀粉组分。In some embodiments, the exoamylase is added in an amount of 100-5000 ppm, such as 200-4000 ppm, such as 200-4000 ppm, such as 300-3000 ppm, such as 400-2000 ppm, such as 500-1000 ppm of the starch component.
外切淀粉酶可以不仅仅具有外切淀粉酶活性。所述酶还可以含有或多或少的内切淀粉酶活性。因此，在一些实施方案中，外切淀粉酶以下述量添加：根据本发明方法中使用的特定外切淀粉酶所展示的外切淀粉酶活性的百分比而调整的ppm。因此，如果提及外切淀粉酶以100ppm的量使用，且外切淀粉酶活性仅占淀粉酶活性的50％，那么可以使用总共200ppm的外切淀粉酶。An exoamylase may have more than just exoamylase activity. The enzyme may also contain more or less endoamylase activity. Thus, in some embodiments, the exoamylase is added in an amount of ppm adjusted according to the percentage of exoamylase activity exhibited by the particular exoamylase used in the methods of the invention. Thus, if it is mentioned that exo-amylase is used in an amount of 100 ppm, and the exo-amylase activity is only 50% of the amylase activity, then a total of 200 ppm of exo-amylase can be used.
在一些实施方案中，步骤b)中的处理温度是30℃至60℃，例如35℃至60℃，40℃至60℃，45℃至60℃，或50℃至60℃，例如温度距离所述外源外切淀粉酶的最佳温度不超过10℃，例如8℃，6℃，或4℃。In some embodiments, the treatment temperature in step b) is from 30°C to 60°C, such as from 35°C to 60°C, from 40°C to 60°C, from 45°C to 60°C, or from 50°C to 60°C, e.g. The optimum temperature of the exogenous exoamylase is not more than 10°C, such as 8°C, 6°C, or 4°C.
在一些实施方案中，步骤b)中的处理pH在4-8的范围内，例如在5-7的范围内，例如pH距离所述外源外切淀粉酶的最佳pH不超过0.5，1，1.5。In some embodiments, the pH of the treatment in step b) is in the range of 4-8, such as in the range of 5-7, for example, the pH is no more than 0.5, 1 from the optimum pH of the exogenous exoamylase. , 1.5.
在一些实施方案中，食品的货架期被改善，如通过下述测量的：在制备食品后1天，例如2天，例如3天，例如4天，例如5天，例如6天，例如7天，例如9天，例如11天，例如14天，例如16天，例如18天，例如20天，例如21天，例如24天，例如26天，例如27天，例如28天，例如30天，例如32天，例如34天，例如35天，例如6周，例如7周，例如8周，例如9周，例如10周，较低的最大破坏力。In some embodiments, the shelf life of the food product is improved, as measured by 1 day, such as 2 days, such as 3 days, such as 4 days, such as 5 days, such as 6 days, such as 7 days after preparation of the food product , such as 9 days, such as 11 days, such as 14 days, such as 16 days, such as 18 days, such as 20 days, such as 21 days, such as 24 days, such as 26 days, such as 27 days, such as 28 days, such as 30 days, such as 32 days, such as 34 days, such as 35 days, such as 6 weeks, such as 7 weeks, such as 8 weeks, such as 9 weeks, such as 10 weeks, lower maximum destructive power.
在一些实施方案中，口感和质地弹性(柔软性)的劣化的延缓测量为下述：在制备包含动物蛋白和淀粉的组合物后1天，例如2天，例如3天，例如4天，例如5天，例如6天，例如7天，例如9天，例如11天，例如14天，例如16天，例如18天，例如20天，例如21天，例如24天，例如26天，例如27天，例如28天，例如30天，例如32天，例如34天，例如35天，例如6周，例如7周，例如8周，例如9周，例如10周，最大破坏力的降低。In some embodiments, the delay in deterioration of mouthfeel and texture elasticity (softness) is measured as follows: 1 day, such as 2 days, such as 3 days, such as 4 days, such as after preparation of the composition comprising animal protein and starch 5 days, such as 6 days, such as 7 days, such as 9 days, such as 11 days, such as 14 days, such as 16 days, such as 18 days, such as 20 days, such as 21 days, such as 24 days, such as 26 days, such as 27 days , such as 28 days, such as 30 days, such as 32 days, such as 34 days, such as 35 days, such as 6 weeks, such as 7 weeks, such as 8 weeks, such as 9 weeks, such as 10 weeks, the reduction of the maximum destructive power.
在一些实施方案中，包含动物蛋白和淀粉的食品或组合物的最大破坏力，与不使用外源外切淀粉酶而制备的相同产品相比较，减少至少5％，例如至少10％，例如至少15％，例如至少20％，例如至少25％，例如至少30％，例如至少35％，例如至少40％，例如至少45％，例如至少50％，例如至少55％，例如至少60％，例如至少65％，例如至少70％，例如至少75％，例如至少80％，例如至少85％，例如至少90％，例如至少95％，例如至少100％。In some embodiments, the maximum destructive power of a food or composition comprising animal protein and starch is reduced by at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%.
在一些实施方案中，食品的货架期被改善，如通过下述测量的：与不使用外源外切淀粉酶而制备的相同食品相比较，在制备食品后至少约5天，例如10天，例如15天，例如20天，例如25天，例如30天，例如35天，例如40天，例如45天，例如50天，例如55天，例如60天测量的较低的最大破坏力。In some embodiments, the shelf life of the food product is improved, as measured by at least about 5 days, such as 10 days, after preparation of the food product, as compared to the same food product prepared without the use of an exogenous exogenous exoamylase, For example 15 days, such as 20 days, such as 25 days, such as 30 days, such as 35 days, such as 40 days, such as 45 days, such as 50 days, such as 55 days, such as 60 days The lower maximum destructive force measured.
在一些实施方案中，包含动物蛋白和淀粉的组合物的口感和质地弹性(柔软性)的劣化的延缓被改善，如通过下述测量的：与不使用外源外切淀粉酶而制备的相同产品相比较，在制备包含动物蛋白和淀粉的组合物后至少约5天，例如10天，例如15天，例如20天，例如25天，例如30天，例如35天，例如40天，例如45天，例如50天，例如55天，例如60天测量的较低的最大破坏力。In some embodiments, the delay in deterioration of mouthfeel and texture elasticity (softness) of compositions comprising animal protein and starch is improved, as measured by the same as prepared without the use of exogenous exogenous exoamylases. Product comparison, at least about 5 days, such as 10 days, such as 15 days, such as 20 days, such as 25 days, such as 30 days, such as 35 days, such as 40 days, such as 45 days after preparation of the composition comprising animal protein and starch The lower maximum destructive force measured in days, such as 50 days, such as 55 days, such as 60 days.
在一些实施方案中，淀粉组分占至少约4％，例如6％，例如8％，例如10％，例如12％，例如14％，例如16％，例如18％，例如20％，例如22％，例如24％，例如26％，例如28％，例如30％，例如32％，例如36％，例如38％，例如40％，按最终的食品的重量百分比计算。In some embodiments, the starch component comprises at least about 4%, such as 6%, such as 8%, such as 10%, such as 12%, such as 14%, such as 16%, such as 18%, such as 20%, such as 22% , such as 24%, such as 26%, such as 28%, such as 30%, such as 32%, such as 36%, such as 38%, such as 40%, calculated by weight percentage of the final food.
在一些实施方案中，淀粉组分占至少约4％，例如6％，例如8％，例如10％，例如12％，例如14％，例如16％，例如18％，例如20％，例如22％，例如24％，例如26％，例如28％，例如30％，例如32％，例如36％，例如38％，例如40％，按包含动物蛋白和淀粉的最终组合物的重量百分比计算。In some embodiments, the starch component comprises at least about 4%, such as 6%, such as 8%, such as 10%, such as 12%, such as 14%, such as 16%, such as 18%, such as 20%, such as 22% , such as 24%, such as 26%, such as 28%, such as 30%, such as 32%, such as 36%, such as 38%, such as 40%, calculated by weight percentage of the final composition comprising animal protein and starch.
在一些实施方案中，外切淀粉酶来源于芽孢杆菌属(Bacillus)的菌株，例如克劳氏芽孢杆菌(Bacillus Clausii)，来源于假单胞菌属(Pseudomonas)，例如嗜糖假单胞菌(Pseudomonas saccharophila)，例如外切淀粉酶选自G4淀粉酶，例如POWERFreshBread8100，和生麦芽糖 -淀粉酶，例如Novamyl 10000 BG。In some embodiments, the exoamylase is derived from a strain of Bacillus, such as Bacillus Clausii, from a Pseudomonas, such as Pseudomonas saccharophila (Pseudomonas saccharophila), for example an exoamylase selected from G4 amylases, such as POWERFreshBread 8100, and maltogenic-amylases, such as Novamyl 10000 BG.
在一些实施方案中，淀粉组分源于：玉米，小麦，马铃薯，甘薯，木薯，稻，例如细粉或粉，例如玉米细粉，玉蜀黍细粉，稻细粉，黑麦粉，黑麦细粉，燕麦细粉，燕麦粉，大豆细粉，高粱粉，高粱细粉，马铃薯粉或马铃薯细粉。In some embodiments, the starch component is derived from: corn, wheat, potato, sweet potato, cassava, rice, such as flour or flour, such as corn flour, maize flour, rice flour, rye flour, rye flour , oat flour, oat flour, soybean flour, sorghum flour, sorghum flour, potato flour or potato flour.
在一些实施方案中，动物蛋白源于下列之任何一种：牛/牛肉，猪/猪肉，火鸡，鸭，鹅，猎禽，鸡，家禽，绵羊，马，山羊，野生狩猎物，啮齿动物，海鲜或贝类，例如虾，鱼及其组合。In some embodiments, the animal protein is derived from any of the following: cattle/beef, pig/pork, turkey, duck, goose, game bird, chicken, poultry, sheep, horse, goat, wild game, rodent , seafood or shellfish such as shrimp, fish and combinations thereof.
在一些实施方案中，动物蛋白占最终食品重量的至少约10％，例如最终食品的至少约15％，例如至少约20％，例如至少约25％，例如至少约30％，例如至少约35％，例如至少约40％，例如至少约45％，例如至少约50％，例如至少约55％，例如至少约60％，例如至少约65％，例如至少约70％。In some embodiments, animal protein comprises at least about 10% by weight of the final food product, such as at least about 15% of the final food product, such as at least about 20%, such as at least about 25%, such as at least about 30%, such as at least about 35% , such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%.
在一些实施方案中，动物蛋白占包含动物蛋白和淀粉的最终组合物重量的至少约10％，例如包含动物蛋白和淀粉的最终组合物的例如至少约15％，例如至少约20％，例如至少约25％，例如至少约30％，例如至少约35％，例如至少约40％，例如至少约45％，例如至少约50％，例如至少约55％，例如至少约60％，例如至少约65％，例如至少约70％。In some embodiments, the animal protein comprises at least about 10% by weight of the final composition comprising animal protein and starch, such as, for example, at least about 15%, such as at least about 20%, such as at least About 25%, such as at least about 30%, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65% %, such as at least about 70%.
本发明的编号的实施方案：Numbered embodiments of the present invention:
1.一种制备具有改善的货架期的包含动物蛋白和淀粉组分的食品的方法，所述方法包括下列步骤：1. A method of preparing a food product comprising animal protein and starch components with improved shelf life, said method comprising the steps of:
a)将外源外切淀粉酶与包含淀粉组分的组合物相混合，a) mixing an exogenous exoamylase with a composition comprising a starch component,
b)在允许所述外源外切淀粉酶至少部分地水解所述淀粉组分的条件下加工在步骤(a)获得的混合物，b) processing the mixture obtained in step (a) under conditions allowing said exogenous exoamylase to at least partially hydrolyze said starch component,
c)在步骤(a)或(b)之任一项之前，与之同时或之后，将所述包含淀粉组分的组合物与动物蛋白组分相混合，以及c) mixing said composition comprising a starch component with an animal protein component before, simultaneously with, or after any of steps (a) or (b), and
d)加工在步骤(a)～(c)之后获得的包含动物蛋白和淀粉组分的所述组合物，以获得所述具有改善的货架期的食品。d) Processing said composition comprising animal protein and starch components obtained after steps (a) to (c) to obtain said food product with improved shelf life.
2.根据实施方案1的方法，还包括加工在步骤(b)获得的混合物以灭活所述外源外切淀粉酶的步骤。2. The method according to embodiment 1, further comprising the step of processing the mixture obtained in step (b) to inactivate said exogenous exoamylase.
3.根据实施方案2的方法，其中所述灭活是通过热处理进行的，例如：在距离所述外源外切淀粉酶的变性温度不超过10℃，例如8℃，例如6℃，例如4℃的温度处理多于2分钟，例如4，6，8，10，12，14，16，18，20，25或30分钟，或备选地于95℃温度处理多于2分钟，例如4，6，8，10，12，14，16，18，20，25或30分钟。3. The method according to embodiment 2, wherein said inactivation is carried out by heat treatment, for example: within 10 °C, such as 8 °C, such as 6 °C, such as 4 °C, from the denaturation temperature of said exogenous exoamylase C for more than 2 minutes, for example 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 or 30 minutes, or alternatively at 95 C for more than 2 minutes, for example 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 or 30 minutes.
4.根据实施方案1～3之任一项的方法，其中以所述淀粉组分的100～5000ppm，例如200～4000ppm，例如200～4000ppm，例如300～3000ppm，例如400～2000ppm，例如500～1000ppm的量添加所述外切淀粉酶。4. The method according to any one of embodiments 1 to 3, wherein 100 to 5000 ppm of the starch component, such as 200 to 4000 ppm, such as 200 to 4000 ppm, such as 300 to 3000 ppm, such as 400 to 2000 ppm, such as 500 to The exoamylase was added in an amount of 1000 ppm.
5.根据实施方案1～4之任一项的方法，其中在步骤(b)的加工温度是30℃～60℃，例如35℃～60℃，40℃～60℃，45℃～60℃，或50℃～60℃，例如在距离所述外源外切淀粉酶的最佳温度不超过10℃，例如8℃，6℃或4℃的温度。5. The method according to any one of embodiments 1 to 4, wherein the processing temperature in step (b) is 30°C to 60°C, for example 35°C to 60°C, 40°C to 60°C, 45°C to 60°C, Or 50°C to 60°C, eg at a temperature no more than 10°C, eg 8°C, 6°C or 4°C from the optimum temperature of the exogenous exoamylase.
6.根据实施方案1～5之任一项的方法，其中在步骤(b)的加工pH在4～8的范围内，如在5～7的范围内，例如距离所述外源外切淀粉酶的最佳pH不超过0.5，1，1.5的pH。6. The method according to any one of embodiments 1 to 5, wherein the processing pH in step (b) is in the range of 4 to 8, such as in the range of 5 to 7, for example, a distance from the exogenous exo-cut starch The optimal pH of the enzyme does not exceed the pH of 0.5, 1, 1.5.
7.根据实施方案1～6之任一项的方法，其中所述食品的货架期得到改善，如通过下述测量的：在制备食品之后1天，例如2天，例如3天，例如4天，例如5天，例如6天，例如7天，例如9天，例如11天，例如14天，例如16天，例如18天，例如20天，例如21天，例如24天，例如26天，例如27天，例如28天，例如30天，例如32天，例如34天，例如35天，例如6周，例如7周，例如8周，例如9周，例如10周，较低的最大破坏力。7. The method according to any one of embodiments 1 to 6, wherein the shelf life of the food product is improved, as measured by 1 day, such as 2 days, such as 3 days, such as 4 days after preparation of the food product , such as 5 days, such as 6 days, such as 7 days, such as 9 days, such as 11 days, such as 14 days, such as 16 days, such as 18 days, such as 20 days, such as 21 days, such as 24 days, such as 26 days, such as 27 days, such as 28 days, such as 30 days, such as 32 days, such as 34 days, such as 35 days, such as 6 weeks, such as 7 weeks, such as 8 weeks, such as 9 weeks, such as 10 weeks, lower maximum destructive power.
8.根据实施方案1～7之任一项的方法，其中所述食品的最大破坏力相比未用外源外切淀粉酶制备的相同的食品降低至少5％，例如至少10％，例如至少15％，例如至少20％，例如至少25％，例如至少30％，例如至少35％，例如至少40％，例如至少45％，例如至少50％，例如至少55％，例如至少60％，例如至少65％，例如至少70％，例如至少75％，例如至少80％，例如至少85％，例如至少90％，例如至少95％，例如至少100％。8. The method according to any one of embodiments 1 to 7, wherein the maximum destructive power of the food product is reduced by at least 5%, such as by at least 10%, such as by at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%.
9.根据实施方案1～8之任一项的方法，其中所述食品的货架期得到改善，如通过下述测量的：与未用外源外切淀粉酶制备的相同的食品相比，在制备所述食品之后至少约5天，例如10天，例如15天，例如20天，例如25天，例如30天，例如35天，例如40天，例如45天，例如50天，例如55天，例如60天，测得的较低的最大破坏力。9. The method according to any one of embodiments 1 to 8, wherein the shelf life of the food product is improved, as measured by: compared to the same food product prepared without exogenous exogenous exoamylase, at At least about 5 days, such as 10 days, such as 15 days, such as 20 days, such as 25 days, such as 30 days, such as 35 days, such as 40 days, such as 45 days, such as 50 days, such as 55 days after preparation of the food product, For example 60 days, a lower maximum destructive power was measured.
10.根据实施方案1～9之任一项的方法，其中所述淀粉组分的量以最终食品的重量百分比计达至少约4％，例如6％，例如8％，例如10％，例如12％，例如14％，例如16％，例如18％，例如20％，例如22％，例如24％，例如26％，例如28％，例如30％，例如32％，例如36％，例如38％，例如40％。10. The method according to any one of embodiments 1 to 9, wherein the amount of the starch component amounts to at least about 4%, such as 6%, such as 8%, such as 10%, such as 12% by weight of the final food product. %, such as 14%, such as 16%, such as 18%, such as 20%, such as 22%, such as 24%, such as 26%, such as 28%, such as 30%, such as 32%, such as 36%, such as 38%, For example 40%.
11.根据实施方案1～10之任一项的方法，其中所述外切淀粉酶源于芽孢杆菌属(Bacillus)的菌株，例如克劳氏芽孢杆菌(BacillusClausii)，源于假单胞茵属(Pseudomonas)的菌株，例如嗜糖假单胞茵(Pseudomonas saccharophila)；例如选自下列的外切淀粉酶：G4淀粉酶，例如POWERFreshBread8100，及生麦芽糖α-淀粉酶，例如Novamyl 10000 BG。11. The method according to any one of embodiments 1 to 10, wherein the exoamylase is derived from a strain of the genus Bacillus, for example Bacillus Clausii, derived from the genus Pseudomonas Strains of (Pseudomonas), such as Pseudomonas saccharophila; for example, an exoamylase selected from the group consisting of G4 amylases, such as POWERFreshBread 8100, and maltogenic alpha-amylases, such as Novamyl 10000 BG.
12.根据实施方案1～11之任一项的方法，其中所述淀粉组分源于：玉米，小麦，马铃薯，甘薯，木薯，稻，例如细粉或粉，例如玉米细粉，玉蜀黍细粉，稻细粉，黑麦粉，黑麦细粉，燕麦细粉，燕麦粉，大豆细粉，高粱粉，高粱细粉，马铃薯粉或马铃薯细粉。12. Process according to any one of embodiments 1 to 11, wherein said starch component originates from: corn, wheat, potato, sweet potato, cassava, rice, e.g. flour or flour, e.g. maize flour, maize flour , rice flour, rye flour, rye flour, oat flour, oat flour, soybean flour, sorghum flour, sorghum flour, potato flour or potato flour.
13.根据实施方案1～12之任一项的方法，其中所述动物蛋白源于下列之任何一种：牛/牛肉，猪/猪肉，火鸡，鸭，鹅，猎禽，鸡，家禽，绵羊，马，山羊，野生狩猎物，啮齿动物，海鲜或贝类，例如虾，鱼和其组合。13. The method according to any one of embodiments 1-12, wherein the animal protein is derived from any of the following: beef/beef, pig/pork, turkey, duck, goose, game bird, chicken, poultry, Sheep, horses, goats, wild game, rodents, seafood or shellfish such as shrimp, fish and combinations thereof.
14.根据实施方案1～12之任一项的方法，其中所述动物蛋白占最终食品重量的至少约10％，例如最终食品重量的至少约15％，例如至少约20％，例如至少约25％，例如至少约30％，例如至少约35％，例如至少约40％，例如至少约45％，例如至少约50％，例如至少约55％，例如至少约60％，例如至少约65％，例如至少约70％。14. The method according to any one of embodiments 1 to 12, wherein the animal protein comprises at least about 10% by weight of the final food product, such as at least about 15% by weight of the final food product, such as at least about 20%, such as at least about 25% by weight. %, such as at least about 30%, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as at least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, For example at least about 70%.
15.一种在包含动物蛋白和淀粉的组合物中延缓口感及质地弹性(柔软性)劣化的方法，所述方法包括下列步骤：15. A method of delaying deterioration of mouthfeel and texture elasticity (softness) in a composition comprising animal protein and starch, said method comprising the steps of:
a)将外源外切淀粉酶与包含淀粉组分的组合物相混合，a) mixing an exogenous exoamylase with a composition comprising a starch component,
b)在允许所述外源外切淀粉酶至少部分地水解所述淀粉组分的条件下处理在步骤(a)获得的混合物，b) treating the mixture obtained in step (a) under conditions allowing said exogenous exoamylase to at least partially hydrolyze said starch component,
c)在(a)，(b)或(c)之任何一个步骤之前，同时或之后，将所述包含淀粉组分的组合物与动物蛋白组分混合。c) before, simultaneously with or after any one of steps (a), (b) or (c), mixing said composition comprising a starch component with an animal protein component.
16.根据实施方案15的方法，还包括处理在步骤(b)获得的混合物以灭活所述外源外切淀粉酶的步骤。16. The method according to embodiment 15, further comprising the step of treating the mixture obtained in step (b) to inactivate said exogenous exoamylase.
17.根据实施方案16的方法，其中所述灭活是通过热处理进行的，例如在距离所述外源外切淀粉酶的变性温度不超过10℃，例如8℃，例如6℃，例如4℃的温度处理多于2分钟，例如4，6，8，10，12，14，16，18，20，25或30分钟，或备选地于95℃的温度处理多于2分钟，例如4，6，8，10，12，14，16，18，20，25或30分钟。17. The method according to embodiment 16, wherein said inactivation is performed by heat treatment, for example at no more than 10°C, such as 8°C, such as 6°C, such as 4°C, from the denaturation temperature of said exogenous exoamylase treatment at a temperature of 95° C. for more than 2 minutes, for example 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 or 30 minutes, or alternatively at a temperature of 95° C. for more than 2 minutes, for example 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 or 30 minutes.
18.根据实施方案15～17之任一项的方法，其中以所述淀粉组分的100～5000ppm，例如200～4000ppm，例如200～4000ppm，例如300～3000ppm，例如400～2000ppm，例如500～1000ppm的量添加所述外切淀粉酶。18. The method according to any one of embodiments 15 to 17, wherein 100 to 5000 ppm of the starch component, such as 200 to 4000 ppm, such as 200 to 4000 ppm, such as 300 to 3000 ppm, such as 400 to 2000 ppm, such as 500 to The exoamylase was added in an amount of 1000 ppm.
19.根据实施方案15～18之任一项的方法，其中在步骤(b)的处理温度是30℃～60℃，例如35℃～60℃，40℃～60℃，45℃～60℃，或50℃～60℃，例如距离所述外源外切淀粉酶的最佳温度不超过10℃，例如8℃，6℃或4℃的温度。19. The method according to any one of embodiments 15-18, wherein the treatment temperature in step (b) is 30°C-60°C, for example 35°C-60°C, 40°C-60°C, 45°C-60°C, Or 50°C to 60°C, such as a temperature not exceeding 10°C, such as 8°C, 6°C or 4°C, from the optimum temperature of the exogenous exoamylase.
20.根据实施方案15～19之任一项的方法，其中在步骤(b)的处理pH在4～8的范围内，如在5～7的范围内，例如距离所述外源外切淀粉酶的最佳pH不超过0.5，1，1.5的pH。20. The method according to any one of embodiments 15-19, wherein the pH of the treatment in step (b) is in the range of 4-8, such as in the range of 5-7, for example a distance from the exogenous exo-starch The optimal pH of the enzyme does not exceed the pH of 0.5, 1, 1.5.
21.根据实施方案15～20之任一项的方法，其中口感及质地弹性(柔软性)劣化的延缓通过下述测量：在制备所述包含动物蛋白和淀粉的组合物之后1天，例如2天，例如3天，例如4天，例如5天，例如6天，例如7天，例如9天，例如11天，例如14天，例如16天，例如18天，例如20天，例如21天，例如24天，例如26天，例如27天，例如28天，例如30天，例如32天，例如34天，例如35天，例如6周，例如7周，例如8周，例如9周，例如10周，最大破坏力的降低。21. The method according to any one of embodiments 15 to 20, wherein the delay in deterioration of mouthfeel and texture elasticity (softness) is measured by 1 day, for example 2 days after preparation of said composition comprising animal protein and starch. days, such as 3 days, such as 4 days, such as 5 days, such as 6 days, such as 7 days, such as 9 days, such as 11 days, such as 14 days, such as 16 days, such as 18 days, such as 20 days, such as 21 days, For example 24 days, such as 26 days, such as 27 days, such as 28 days, such as 30 days, such as 32 days, such as 34 days, such as 35 days, such as 6 weeks, such as 7 weeks, such as 8 weeks, such as 9 weeks, such as 10 days Zhou, the reduction of the maximum destructive power.
22.根据实施方案15～21之任一项的方法，其中所述包含动物蛋白及淀粉的组合物的最大破坏力相比未用外源外切淀粉酶制备的相同的产品降低至少5％，例如至少10％，例如至少15％，例如至少20％，例如至少25％，例如至少30％，例如至少35％，例如至少40％，例如至少45％，例如至少50％，例如至少55％，例如至少60％，例如至少65％，例如至少70％，例如至少75％，例如至少80％，例如至少85％，例如至少90％，例如至少95％，例如至少100％。22. The method according to any one of embodiments 15 to 21, wherein the maximum destructive power of the composition comprising animal protein and starch is reduced by at least 5% compared to the same product prepared without exogenous exoamylase, For example at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, For example at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 100%.
23.根据实施方案15～22之任一项的方法，其中所述包含动物蛋白及淀粉的组合物的口感及质地弹性(柔软性)劣化的延缓通过下述测量：与未用外源外切淀粉酶制备的相同的产品相比，在制备所述包含动物蛋白和淀粉的组合物之后至少约5天，例如10天，例如15天，例如20天，例如25天，例如30天，例如35天，例如40天，例如45天，例如50天，例如55天，例如60天，测得的较低的最大破坏力。23. The method according to any one of embodiments 15 to 22, wherein the retardation of the deterioration of the mouthfeel and texture elasticity (softness) of the composition comprising animal protein and starch is measured by: At least about 5 days, such as 10 days, such as 15 days, such as 20 days, such as 25 days, such as 30 days, such as 35 days after the preparation of the composition comprising animal protein and starch, compared to the same product prepared by amylase days, such as 40 days, such as 45 days, such as 50 days, such as 55 days, such as 60 days, the lower maximum destructive power measured.
24.根据实施方案15～23之任一项的方法，其中所述淀粉组分的量以包含动物蛋白和淀粉的最终混合物的重量百分比计达至少约4％，例如6％，例如8％，例如10％，例如12％，例如14％，例如16％，例如18％，例如20％，例如22％，例如24％，例如26％，例如28％，例如30％，例如32％，例如36％，例如38％，例如40％。24. The method according to any one of embodiments 15 to 23, wherein the amount of the starch component amounts to at least about 4%, such as 6%, such as 8%, by weight of the final mixture comprising animal protein and starch, eg 10%, eg 12%, eg 14%, eg 16%, eg 18%, eg 20%, eg 22%, eg 24%, eg 26%, eg 28%, eg 30%, eg 32%, eg 36 %, such as 38%, such as 40%.
25.根据实施方案15～24之任一项的方法，其中外切淀粉酶源于芽孢杆菌属(Bacillus)的菌株，例如克劳氏芽孢杆菌(BacillusClausii)，源于假单胞菌属(Pseudomonas)的菌株，例如嗜糖假单胞茵(Pseudomonas saccharophila)；例如选自下列的外切淀粉酶：G4淀粉酶，例如POWERFreshBread8100，及生麦芽糖α-淀粉酶，例如Novamyl 10000 BG。25. The method according to any one of embodiments 15 to 24, wherein the exoamylase is derived from strains of the genus Bacillus (Bacillus), such as Bacillus Clausii (Bacillus Clausii), derived from Pseudomonas ) strains, such as Pseudomonas saccharophila; for example, an exoamylase selected from the group consisting of G4 amylase, such as POWERFreshBread8100, and maltogenic alpha-amylase, such as Novamyl 10000 BG.
26.根据实施方案15～25之任一项的方法，其中所述淀粉组分源于：玉米，小麦，马铃薯，甘薯，木薯，稻，例如细粉或粉，例如玉米细粉，玉蜀黍细粉，稻细粉，黑麦粉，黑麦细粉，燕麦细粉，燕麦粉，大豆细粉，高粱粉，高粱细粉，马铃薯粉或马铃薯细粉。26. Process according to any one of embodiments 15 to 25, wherein said starch component originates from: corn, wheat, potato, sweet potato, cassava, rice, e.g. flour or flour, e.g. maize flour, maize flour , rice flour, rye flour, rye flour, oat flour, oat flour, soybean flour, sorghum flour, sorghum flour, potato flour or potato flour.
27.根据实施方案15～26之任一项的方法，其中所述动物蛋白源于下列之任何一种：牛/牛肉，猪/猪肉，火鸡，鸭，鹅，猎禽，鸡，家禽，绵羊，马，山羊，野生狩猎物，啮齿动物，海鲜或贝类，例如虾，鱼和其组合。27. The method according to any one of embodiments 15-26, wherein the animal protein is derived from any of the following: beef/beef, pig/pork, turkey, duck, goose, game bird, chicken, poultry, Sheep, horses, goats, wild game, rodents, seafood or shellfish such as shrimp, fish and combinations thereof.
28.根据实施方案15～27之任一项的方法，其中所述动物蛋白占包含动物蛋白和淀粉的最终组合物重量的至少约15％，例如最终食品重量的至少约10％，包含动物蛋白和淀粉的最终组合物的例如至少约15％，例如至少约20％，例如至少约25％，例如至少约30％，例如至少约35％，例如至少约40％，例如至少约45％，例如至少约50％，例如至少约55％，例如至少约60％，例如至少约65％，例如至少约70％。28. The method according to any one of embodiments 15 to 27, wherein the animal protein comprises at least about 15% by weight of the final composition comprising animal protein and starch, such as at least about 10% by weight of the final food product, comprising animal protein Such as at least about 15%, such as at least about 20%, such as at least about 25%, such as at least about 30%, such as at least about 35%, such as at least about 40%, such as at least about 45%, such as At least about 50%, such as at least about 55%, such as at least about 60%, such as at least about 65%, such as at least about 70%.
29.一种食品，其通过根据实施方案1～14之任一项的方法获得。29. A food product obtained by a method according to any one of embodiments 1-14.
30.一种口感及质地弹性(柔软性)劣化降低的包含动物蛋白和淀粉的组合物，其根据实施方案15～28之任一项的方法获得。30. A composition comprising animal protein and starch with reduced deterioration of mouthfeel and texture elasticity (softness), obtained according to the method of any one of embodiments 15-28.
31.外源外切淀粉酶用于改善包含动物蛋白和淀粉组分的食品的货架期的用途。31. Use of an exogenous exoamylase for improving the shelf life of a food product comprising animal protein and starch components.
32.外源外切淀粉酶用于延缓包含动物蛋白和淀粉组分的食品的口感及质地弹性(柔软性)劣化的用途。32. Use of exogenous exoamylase for delaying deterioration of mouthfeel and texture elasticity (softness) of food products comprising animal protein and starch components.
实施例1Example 1
配方formula
香肠配方Sausage Recipe
混和物配方Mixture recipe
着色剂配方Colorant formulation
乳化物配方Emulsion formula
流程(见图1)Process (see Figure 1)
过程：process:
a)鸡胸和鸡皮制备a) Chicken breast and chicken skin preparation
·将冷冻的鸡胸和鸡皮解冻，直到温度在-4℃左右。然后将这两种原料碾磨备用。Thaw frozen chicken breasts and chicken skin until the temperature is around -4°C. These two raw materials are then milled for later use.
b)混合物制备b) Mixture preparation
·将MSG粉、抗坏血酸钠和白胡椒充分混合在一起。· Mix MSG powder, sodium ascorbate and white pepper together well.
c)着色剂制备c) Colorant preparation
·将亚硝酸钠和红曲色素溶于水中。Dissolve sodium nitrite and monascus pigment in water.
d)乳化物制备d) Preparation of emulsion
·将大豆浓缩蛋白、大豆分离蛋白和热水(90℃)在5L切割机(DADAUX，法国制造)中斩碎3分钟。然后将预先碾磨的鸡皮加到该混合物，斩碎约2分钟，直到它变成均质的乳化物。此乳化物的最终温度为13℃。最终温度可优选在10-14℃之间。• Soy protein concentrate, soy protein isolate and hot water (90° C.) were minced in a 5 L cutter (DADAUX, made in France) for 3 minutes. The pre-milled chicken skin is then added to the mixture, chopped for about 2 minutes until it becomes a homogeneous emulsion. The final temperature of this emulsion was 13°C. The final temperature may preferably be between 10-14°C.
e)玉米淀粉和淀粉酶。e) Corn starch and amylases.
·将玉米淀粉和淀粉酶混合在一起・Mix cornstarch and amylase together
f)香肠制备f) Sausage preparation
首先，将碾磨的鸡胸放入5L切割机(DADAUX，法国制造)中，斩碎1分钟。然后，将着色剂溶液、盐和半冰(half ice)加到肉中，斩碎3分钟。然后，将乳化物放入切割机中，斩碎1分钟。最后，将玉米淀粉和淀粉酶的混合物连同其他成分一起加到切割机中，斩碎2分钟。此最终混合物的温度应不超过10℃。然后将产物放入肠衣中蒸煮。蒸煮温度为55℃40分钟，然后90℃40分钟。在香肠冷却之后，将其保存于5℃。First, put the ground chicken breast into a 5L cutter (DADAUX, made in France) and chop for 1 minute. Then, the colorant solution, salt and half ice were added to the meat and chopped for 3 minutes. Then, put the emulsion into a cutter and chop for 1 minute. Finally, add the cornstarch and amylase mixture along with the other ingredients to the cutter and chop for 2 minutes. The temperature of this final mixture should not exceed 10°C. The product is then cooked in casings. The cooking temperature was 55°C for 40 minutes, then 90°C for 40 minutes. After the sausages had cooled, they were stored at 5°C.
以基于淀粉量的约100～800ppm的量加入外切淀粉酶。最终的含淀粉酶的制备物于55℃处理40分钟。为了灭活淀粉酶，在最后步骤中，于90℃以上的温度处理制备物40分钟。质地分析方法(测试工具：物性测定仪TA.XTplus，来自Stable Micro Systems.Ltd，UK)。The exoamylase is added in an amount of about 100-800 ppm based on the amount of starch. The final amylase-containing preparation was treated at 55°C for 40 minutes. In order to inactivate the amylase, in a final step the preparation is treated at a temperature above 90° C. for 40 minutes. Texture analysis method (test tool: physical property analyzer TA.XTplus, from Stable Micro Systems.Ltd, UK).
将香肠切成20mm高度。TA-XT2设置如下：Cut the sausage into 20mm height. TA-XT2 settings are as follows:
记录肠体破坏之前探头切断香肠时所用来力(破坏力)。根据最大力(破坏力)测量硬度。The force (destructive force) applied by the probe to cut through the sausage prior to gut destruction is recorded. Hardness is measured according to the maximum force (destructive force).
质地分析结果(见图2)Texture analysis results (see Figure 2)
当将外切淀粉酶(G4淀粉酶，Powerfresh)加入含有10％玉米淀粉的香肠里，香肠的硬度随着淀粉酶的添加量增加而降低。添加越多淀粉酶，结构越软。存储期间，香肠变得越来越硬，预计是受了玉米淀粉老化的影响。外切淀粉酶的添加可延缓口感及质地弹性(柔软性)劣化。存储41天之后，添加400ppm淀粉酶的香肠与新鲜的对照保持相同的硬度。当淀粉酶的剂量增至400ppm以上时，和对照样相比，香肠可在相当长的时间保持一定程度的柔软度。当使用玉米淀粉时，淀粉酶具有延缓口感及质地弹性(柔软性)劣化的良好效应。When exoamylase (G4 amylase, Powerfresh) was added to the sausage containing 10% corn starch, the firmness of the sausage decreased as the amount of amylase added increased. The more amylase added, the softer the structure. During storage, the sausages became increasingly tough, which is expected to be affected by the aging of the cornstarch. The addition of exoamylase can delay the deterioration of mouthfeel and texture elasticity (softness). After 41 days of storage, the sausage supplemented with 400 ppm amylase maintained the same firmness as the fresh control. When the dosage of amylase was increased above 400ppm, compared with the control, the sausage could maintain a certain degree of softness for a long time. When cornstarch is used, amylase has a good effect of delaying deterioration of mouthfeel and texture elasticity (softness).
实施例2Example 2
将4种不同淀粉酶用于香肠，以测试对玉米淀粉的抗老化能力。基本上如实施例1中所述进行处理。Four different amylases were used in sausages to test their anti-aging ability against corn starch. Workup was performed essentially as described in Example 1.
与Diazyme FA和Bakezyme AN 301(DSM)对照测试G4外切淀粉酶POWERFreshBread8100(Danisco A/S)。Bakezyme AN 301和Diazyme FA主要具有内切淀粉酶活性，然而POWERFreshBread8100主要具有如本发明需要的外切淀粉酶活性。The G4 exoamylase POWERFreshBread 8100 (Danisco A/S) was tested against Diazyme FA and Bakezyme AN 301 (DSM). Bakezyme AN 301 and Diazyme FA mainly have endoamylase activity, whereas POWERFreshBread 8100 has mainly exoamylase activity as required by the present invention.
此实施例中制备的香肠含有20％玉米淀粉。基于玉米淀粉，将800ppm的各酶用于香肠。允许酶于60℃起作用50min，然后于95℃灭活50min。质地分析结果(见图3)。The sausage prepared in this example contained 20% cornstarch. 800 ppm of each enzyme was used for sausages based on cornstarch. Allow the enzyme to function at 60°C for 50min, then inactivate at 95°C for 50min. Texture analysis results (see Figure 3).
将香肠于5℃保存1个月，和用质地分析测量抗老化效应。TA结果和感官结果显示，与内切淀粉酶相比，外切淀粉酶POWERFreshBread8100具有良好的抗老化效果。The sausages were stored at 5° C. for 1 month and the anti-aging effect was measured by texture analysis. TA results and sensory results showed that compared with endo-amylase, exo-amylase POWERFreshBread8100 has good anti-aging effect.
可利用来自英国Stable Micro Systems.Ltd的物性测试仪TA.XTplus或本领域知道的类似设备进行质地分析。Texture analysis can be carried out using a property tester TA.XTplus from Stable Micro Systems. Ltd, UK, or similar equipment known in the art.
实施例3Example 3
与Diazyme FA和Bakezyme AN 301(DSM)对照测试外切淀粉酶Novamyl 10000 BG(Novozymes A/S)。Bakezyme AN 301和DiazymeFA主要具有内切淀粉酶活性，然而Novamyl 10000 BG主要含有本发明需要的外切淀粉酶活性。Exoamylase Novamyl 10000 BG (Novozymes A/S) was tested against Diazyme FA and Bakezyme AN 301 (DSM). Bakezyme AN 301 and DiazymeFA mainly have endoamylase activity, whereas Novamyl 10000 BG mainly contains exoamylase activity required by the present invention.
此实施例中制备的香肠含有20％玉米淀粉。基于玉米淀粉，将800ppm的各酶用于香肠。允许酶于60℃起作用50min，然后于95℃灭活50min。质地分析结果(见图3)。The sausage prepared in this example contained 20% cornstarch. 800 ppm of each enzyme was used for sausages based on cornstarch. Allow the enzyme to function at 60°C for 50min, then inactivate at 95°C for 50min. Texture analysis results (see Figure 3).
将香肠于5℃保存1个月，且用质地分析测量抗老化效应。TA结果和感官结果显示，与内切淀粉酶相比，外切淀粉酶Novamyl 10000BG具有良好的抗老化效应。The sausages were stored at 5°C for 1 month and the anti-aging effect was measured by texture analysis. TA results and sensory results showed that exoamylase Novamyl 10000BG had a good anti-aging effect compared with endoamylase.
可利用来自英国Stable Micro Systems.Ltd的物性测试仪TA.XTplus或本领域已知的类似设备进行质地分析。Texture analysis may be carried out using a property tester TA.XTplus from Stable Micro Systems. Ltd, UK, or similar equipment known in the art.
实施例4Example 4
备选的香肠配方Alternative Sausage Recipe
配方formula
混合物、着色剂以及乳化物的配方如实施例1中所描述的。The formulations of mixtures, colorants and emulsions are as described in Example 1.
质地分析结果显示在图3中。The texture analysis results are shown in Fig. 3.
1：Diazyme BBA1: Diazyme BBA
2：Diazyme FA2: Diazyme FA
3：POWERFreshBread81003: POWERFreshBread8100
4：Novamyl 10000 BG4: Novamyl 10000 BG
5：Bakezyme AN 3015: Bakezyme AN 301
在含有20％玉米淀粉的香肠制备物中测试了五种不同的淀粉酶。对于每种所测试的淀粉酶，测试了淀粉酶剂量与抗老化效果之间的关系。将香肠于5℃保存将近1个月，且在将香肠保存0天(烹饪后)、5天、21天和28天时测试硬度。Five different amylases were tested in sausage preparations containing 20% corn starch. For each amylase tested, the relationship between amylase dosage and anti-aging effect was tested. The sausages were stored at 5°C for approximately 1 month, and the firmness was tested when the sausages were stored for 0 days (after cooking), 5 days, 21 days and 28 days.
当在5℃保存时，对于所有所测试的酶以及对于对照，香肠的硬度随时间增加，尤其是前5天。然而，当香肠中加入了淀粉酶时，与没有淀粉酶的对照相比，硬度减少了。加入的淀粉酶越多，香肠变得越软。根据质地分析结果以及感官结果，Novamyl 10000 BG对于玉米淀粉具有最好的抗老化效果。同时，POWERFreshBread8100也具有良好的抗老化能力，虽然不如Novamyl 10000 BG好。When stored at 5°C, the firmness of the sausage increased over time, especially the first 5 days, for all tested enzymes as well as for the control. However, when amylase was added to the sausage, firmness was reduced compared to the control without amylase. The more amylase you add, the softer the sausage becomes. According to the texture analysis results and sensory results, Novamyl 10000 BG has the best anti-aging effect on corn starch. At the same time, POWERFreshBread8100 also has good anti-aging ability, although it is not as good as Novamyl 10000 BG.
实施例5Example 5
配方formula
香肠配方Sausage Recipe
乳化物、着色剂和混合物的配方同实施例1。The formula of emulsion, coloring agent and mixture is with embodiment 1.
质地分析结果(见图4)。Texture analysis results (see Figure 4).
如通常一样，玉米淀粉有老化的问题。但是，经改性的木薯淀粉以及玉米淀粉和经改性的木薯淀粉的混合物几乎没有老化问题。As usual, cornstarch has aging problems. However, modified tapioca starch and mixtures of corn starch and modified tapioca starch had little staleness problem.
这四种淀粉酶都能在一个月的保藏期中抑制硬度的增加。加入的淀粉酶越多，香肠越软。它们都对香肠中的玉米淀粉具有抗老化作用。All four amylases could inhibit the increase of firmness during the one-month storage period. The more amylase added, the softer the sausage. They both have an anti-aging effect on the cornstarch in the sausage.
在相同剂量下，这四种淀粉酶中1271608具有最佳的抗老化作用。而且，Opticake Fresh 50 BG也具有与Novamyl 10000 BG一样的良好抗老化作用。1271608、Opticake Fresh 50 BG和Novamyl 10000 BG的抗老化作用优于POWERFreshBread8100。At the same dose, 1271608 had the best anti-aging effect among the four amylases. Moreover, Opticake Fresh 50 BG also has the same good anti-aging effect as Novamyl 10000 BG. The anti-aging effect of 1271608, Opticake Fresh 50 BG and Novamyl 10000 BG is better than that of POWERFreshBread8100.
淀粉酶测定Amylase assay
Betamyl测定Betamyl assay
一个Betamyl单位定义为这样的活性：每1min降解0.0351mmol ePNP-偶联的麦芽戊糖的活性，从而每1min可由测定混合物中的过量α-葡萄糖苷酶释放0，0351mmole PNP。测定混合物含有50μl50mM柠檬酸Na，5mM CaCl 2，pH6，5，以及25μl酶样品和25μl来自Megazyme，爱尔兰的Betamyl底物(Glc5-PNP和α-葡萄糖苷酶)(溶解于10m1水的1瓶)。将测定混合物于40C温育30min，然后通过添加150μl4％Tris停止。使用ELISA-读取器测量在420nm处的吸光度，且以检定的酶样品的Betamyl单位/m1基于活性＝A420*d计算Betamyl活性。对于烘烤试验中的剂量测定，使用1BMK＝1000Betamyl单位。One Betamyl unit is defined as the activity that degrades 0.0351 mmole ePNP-coupled maltopentose per 1 min, thereby releasing 0,0351 mmole PNP per 1 min from the excess α-glucosidase in the assay mixture. Assay mix containing 50 μl 50 mM Na-citrate, 5 mM CaCl2, pH 6, 5, and 25 μl enzyme sample and 25 μl Betamyl substrates (Glc5-PNP and α-glucosidase) from Megazyme, Ireland (1 vial dissolved in 10 ml water) . The assay mixture was incubated at 4OC for 30 min and then stopped by adding 150 μl 4% Tris. The absorbance at 420 nm was measured using an ELISA-reader and the Betamyl activity was calculated as Betamyl units/ml of the assayed enzyme sample based on activity=A420*d. For dosimetry in the baking test, 1BMK = 1000 Betamyl units were used.
内切淀粉酶测定Endoamylase assay
内切淀粉酶测定与根据制造商(Pharmacia&Upjohn DiagnosticsAB)运行的Phadebas测定完全相同。The endoamylase assay was identical to the Phadebas assay run according to the manufacturer (Pharmacia & Upjohn Diagnostics AB).
外切特异性Exo-specificity
使用外切淀粉酶活性与Phadebas活性的比来评价外切特异性。Exo-specificity was assessed using the ratio of exoamylase activity to Phadebas activity.
| Application Number | Priority Date | Filing Date | Title |
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| CN201110442515.3 | 2011-12-26 | ||
| CN201210017956.3 | 2012-01-19 | ||
| US201261645786P | 2012-05-11 | 2012-05-11 | |
| EP12167691 | 2012-05-11 | ||
| EP12167691.0 | 2012-05-11 | ||
| US61/645786 | 2012-05-11 | ||
| PCT/EP2012/076969WO2013098338A1 (en) | 2011-12-26 | 2012-12-27 | Use of amylase enzyme |
| Publication Number | Publication Date |
|---|---|
| CN104023555Atrue CN104023555A (en) | 2014-09-03 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201280064246.4APendingCN104023555A (en) | 2011-12-26 | 2012-12-27 | Use of amylase enzyme |
| Country | Link |
|---|---|
| CN (1) | CN104023555A (en) |
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| WO1991004669A1 (en)* | 1989-09-27 | 1991-04-18 | Novo Nordisk A/S | Antistaling process and agent |
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| US6056980A (en)* | 1996-05-15 | 2000-05-02 | Nihon Shokuhin Kako Co., Ltd. | Process for producing starch sugar composition |
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| WO2011055690A1 (en)* | 2009-11-06 | 2011-05-12 | 理研ビタミン株式会社 | Quality-improving agent for fish paste products |
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