Low-sensitivity soybean yogurt prepared by enzyme hydrolysis and bacterial fermentation and preparation method thereof

Abstract

The invention discloses a low-sensitivity soybean yogurt prepared by enzymolysis and synergistic bacterial fermentation and a preparation method thereof, belongs to the technical field of bean food processing, and provides a method for preparing low-sensitivity soybean yogurt by enzymolysis and synergistic bacterial fermentation, comprising: soaking soybeans in a sodium carbonate solution, then washing, preheating in a boiling water bath, mixing with water and grinding, ripening treatment, filtering, and obtaining cooked soybean milk; mixing with a flavor protease enzyme solution, enzymolysis, ice water bath to room temperature, and obtaining soybean milk enzymolysis solution; mixing with a glucose solution to obtain a fermentation substrate, inoculating plant lactobacillus, fermenting, and obtaining a fermentation product; post-ripening, and obtaining low-sensitivity soybean yogurt. The invention adopts flavor protease enzymolysis and synergistic bacterial fermentation to prepare soybean yogurt, and the obtained soybean yogurt has a reduced gel size, a delicate microstructure, a weakened syneresis shrinkage effect, and a significantly reduced allergenicity, and a scientific processing technology, a simple method, and easy operation.

Description

Technical Field

[0001] The invention belongs to the technical field of bean food processing, and in particular relates to a low-sensitivity soybean yogurt prepared by enzymatic hydrolysis and cooperative bacterial fermentation and a preparation method thereof. Background Art

[0002] Soybean yogurt is a plant protein fermented product made from soybeans, which is ground and pulped, and then sterilized and fermented. The protein content in soybeans is about 40% of the dry weight, and its amino acid score is the most beneficial among a series of plant-based yogurts, which is comparable to the protein content in traditional milk yogurt. It is a high-quality alternative protein source for vegetarians, people with cow's milk allergies and lactose intolerance. my country's plant-based fermented milk market started late. In addition to formula optimization, research on soy yogurt mainly focuses on strain screening, starter improvement and beany smell removal. There is little research on low-allergy soy yogurt.

[0003] The gel properties of plant-based yogurt are key factors affecting its quality and customer acceptance. Compared with animal-based yogurt such as cow's milk, plant-based dairy products such as soy milk have certain limitations in gel properties when forming yogurt. Plant protein aggregation easily forms large pores, resulting in an uneven gel network. This weak gel structure will lead to poor sensory properties of yogurt, which will have a certain impact on consumer acceptance. At the same time, soybeans are one of the eight major allergens recognized by the world, which can cause a series of allergic reactions ranging from mild discomfort to shock and death. There is a certain degree of sensitization risk after lactic acid bacteria fermentation. In response to the above shortcomings, improvements are mainly made through food additives and high-temperature heating. The former does not meet the requirements of clean labels and consumers' psychological expectations, and the latter will lead to the loss of food nutrients and flavor substances. There are great limitations in application. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a low-sensitivity soybean yogurt prepared by enzymatic hydrolysis and synergistic bacterial fermentation and a preparation method thereof, so as to solve the existing problems of poor gel properties, rough structure and high allergenicity of soybean yogurt. The present invention adopts flavor protease enzymatic hydrolysis and synergistic bacterial fermentation to prepare soybean yogurt, and the obtained soybean yogurt gel has a reduced size, a delicate microstructure, a weakened syneresis shrinkage effect, and significantly reduced allergenicity. The processing technology is scientific, the method is simple, and the operation is easy.

[0005] To achieve the above object, the present invention provides a method for preparing low-allergy soybean yogurt by enzymatic hydrolysis and synergistic bacterial fermentation, comprising the following steps:

[0006] (1) Soaking soybeans in a sodium carbonate solution and then washing them to obtain soaked soybeans; preheating the soaked soybeans in a boiling water bath to obtain preheated soybeans; mixing the preheated soybeans with water and grinding them to obtain raw soybean milk; and subjecting the raw soybean milk to ripening treatment and filtering to obtain cooked soybean milk;

[0007] (2) The pH of the cooked soy milk obtained in step (1) is adjusted to 7.0, mixed with a flavor protease solution, subjected to enzymatic hydrolysis, and cooled to room temperature in an ice-water bath to obtain a soy milk enzymatic hydrolyzate;

[0008] (3) The soymilk enzymatic hydrolyzate obtained in step (2) is adjusted to pH 7.0, mixed with a glucose solution to obtain a fermentation substrate, and the fermentation substrate is inoculated with Lactobacillus plantarum and fermented until the pH of the fermentation substrate is 4.5 to obtain a fermentation product;

[0009] (4) The fermentation product obtained in step (3) is post-ripened to obtain low-allergy soy yogurt.

[0010] Preferably, the soaking time in step (1) is 8 to 12 hours; the mass concentration of the sodium carbonate solution in step (1) is 0.4 to 0.6%; and the ratio of soybeans to sodium carbonate solution in step (1) is 1 g: 3 to 5 mL.

[0011] Preferably, the temperature of the boiling water bath preheating in step (1) is 100° C., and the time of the boiling water bath preheating is 5 to 15 minutes; the ratio of the preheated soybeans and water mixed in step (1) is calculated based on the ratio of soybeans to water of 1 g: 6 to 10 mL.

[0012] Preferably, the aging treatment in step (1) is carried out by keeping the temperature at 90-100° C. for 4-6 minutes; and the filtration in step (1) is carried out by using a 200-mesh filter.

[0013] Preferably, the mixed volume ratio of the cooked soy milk and the flavor protease solution in step (2) is 9:1; the enzyme activity of the flavor protease solution in the mixed system of cooked soy milk and the flavor protease solution in step (2) is 400-600 U / g; the enzymatic hydrolysis in step (2) is carried out in a water bath at 50-60° C., and the enzymatic hydrolysis time is 4-6 min.

[0014] Preferably, the volume ratio of the soymilk enzymatic hydrolyzate and the glucose solution in step (3) is 96:4; and the concentration of the glucose solution in step (3) is 0.3-0.7 g / mL.

[0015] Preferably, the inoculation amount of Lactobacillus plantarum in step (3) is 2-4% by volume, and the total effective viable count of Lactobacillus plantarum is 8.16 CFU / mL; and the fermentation temperature in step (3) is 37°C.

[0016] Further preferably, the Lactobacillus plantarum in step (3) is Lactobacillus plantarum T1.

[0017] Preferably, the ripening temperature in step (4) is 4° C., and the ripening time is 12 h.

[0018] The present invention also provides the method for preparing low-allergy soybean yogurt and the obtained low-allergy soybean yogurt.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] The present invention adopts flavor protease enzymolysis to cooperate with plant lactobacillus fermentation to prepare soy yogurt, and the obtained soy yogurt gel size is reduced, the microstructure is delicate, the syneresis shrinkage effect is weakened, and the sensitization is significantly reduced at the same time, the processing technology is scientific, the method is simple, and the operation is easy. The present invention utilizes the flavor protease enzymolysis of 500U / g enzyme activity for 5min, and adopts the soy yogurt prepared by the mode of restrictive enzymolysis and cooperative bacteria fermentation, the yogurt appearance is significantly optimized, the whey precipitation rate is significantly reduced, the protein gel particle size is reduced, the microstructure is more uniform, and the sensitization is further reduced. In addition, the soy yogurt preparation process provided by the present invention is simple, the fermentation cycle is short, the production equipment requirements are low, and it is easy to promote and apply.

[0021] (1) The preparation time of the soybean yogurt produced by the present invention is effectively shortened, and the number of viable bacteria is above 8 CFU / mL when the fermentation end point is reached, and the syneresis effect is reduced to 21.58±1.61%.

[0022] (2) The soy yogurt produced by the present invention exhibits significant uniformity in both visual and microscopic images. Specifically, the size of its gel particles is significantly reduced, thereby presenting a more delicate and soft texture structure, improving the shortcomings of the soy yogurt, which is hard and rough in texture.

[0023] (3) The antigenicity of the soybean yogurt prepared by the present invention was reduced to 1.14±1.01%, which proved that enzymatic hydrolysis and Lactobacillus plantarum fermentation had a significant effect in reducing the antigenicity of soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The pH change diagram of the low-sensitivity soybean yogurt of Example 1 and the soybean yogurt of Comparative Example 1 during the fermentation process;

[0026] Figure 2 This is a graph showing the change in the number of live bacteria during the fermentation of the low-sensitivity soybean yogurt of Example 1 and the soybean yogurt of Comparative Example 1;

[0027] Figure 3 It is a visual diagram of the change in the number of live bacteria during the fermentation of the low-sensitivity soybean yogurt of Example 1 and the soybean yogurt of Comparative Example 1, wherein A is Comparative Example 1 and B is Example 1;

[0028] Figure 4 It is a statistical graph of gel dehydration rate of low-sensitivity soybean yogurt in Example 1 and soybean yogurt in Comparative Example 1;

[0029] Figure 5 It is a statistical diagram of the particle size of the low-sensitivity soybean yogurt of Example 1 and the soybean yogurt gel particle of Comparative Example 1;

[0030] Figure 6 These are laser confocal images of the low-sensitivity soybean yogurt of Example 1 and the soybean yogurt gel of Comparative Example 1, wherein A is Comparative Example 1 and B is Example 1;

[0031] Figure 7 The antigenicity statistical graphs of cooked soy milk and low-allergy soy yogurt in Example 1 and cooked soy milk and soy yogurt in Comparative Example 1 are shown. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Sources of materials used in the present invention: Dongnong 252 soybeans were purchased from Heilongjiang Pulan Agricultural Development Co., Ltd.; flavor protease enzyme solution was purchased from Beijing Solebaugh Technology Co., Ltd.; plant lactobacillus ( Lactiplantibacillus plantarum )T1 was isolated from Tibetan Linggu and deposited in China Center for Type Culture Collection on September 7, 2023, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO:M 20231553; MRS broth medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; RIDASCREEN® FAST Soya kit was purchased from R-Biopharm, Germany. The pH adjustment treatment of the present invention is carried out using 2M hydrochloric acid or sodium hydroxide.

[0038] Example 1

[0039] Activation and preparation of strains: The plant lactobacillus T1 preserved by the -20℃ glycerol method was inoculated into MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 24h to obtain a mixture of activated bacteria and culture medium. Subsequently, the mixture of activated bacteria and culture medium was inoculated into new MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 12h to obtain seed fermentation liquid. After the seed fermentation liquid was centrifuged at 5000rpm for 4min, the precipitate was taken, and an equal volume of physiological saline (0.85%, m / v) was added and vortexed and mixed. After repeating twice, the plant lactobacillus T1 bacterial liquid was obtained. The total effective viable count of the plant lactobacillus T1 bacterial liquid was 8.16CFU / mL.

[0040] (1) Soak the Dongnong 252 soybeans with complete and full grains in a sodium carbonate solution with a mass concentration of 0.5% for 10 h (the ratio of soybeans to sodium carbonate solution is 1 g: 4 mL), then wash with clean water to obtain the soaked soybeans, preheat them in a boiling water bath at 100°C for 10 min to obtain preheated soybeans, mix the preheated soybeans with water at a ratio of 1 g: 8 mL, grind them to obtain raw soybean milk, and perform the aging treatment by keeping the temperature at 95°C for 5 min, and filter them with a 200-mesh filter to obtain cooked soybean milk;

[0041] (2) The pH of cooked soy milk was adjusted to 7.0, and the mixture was mixed with flavor protease solution (enzyme activity of 500 U / g) at a volume ratio of 9:1, and enzymolyzed in a 55°C water bath for 5 min. After the enzymolysis, the mixture was inactivated and sterilized in a 100°C boiling water bath for 10 min, and then cooled to room temperature in an ice water bath to obtain soy milk enzymolyzate.

[0042] (3) The soymilk enzymatic hydrolysate was adjusted to pH 7.0, and mixed with glucose solution (0.5 g / mL) at a volume ratio of 96:4 to obtain a fermentation substrate. The inoculation amount of Lactobacillus plantarum T1 bacterial solution was 3% by volume, and Lactobacillus plantarum T1 was inoculated and fermented at 37° C. until the pH of the fermentation substrate reached 4.5 to obtain a fermentation product.

[0043] (4) The fermented product was post-ripened at 4°C for 12 h to obtain low-allergy soy yogurt.

[0044] Example 2

[0045] Activation and preparation of strains: The plant lactobacillus T1 preserved by the -20℃ glycerol method was inoculated into MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 24h to obtain a mixture of activated bacteria and culture medium. Subsequently, the mixture of activated bacteria and culture medium was inoculated into new MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 12h to obtain seed fermentation liquid. After the seed fermentation liquid was centrifuged at 5000rpm for 4min, the precipitate was taken, and an equal volume of physiological saline (0.85%, m / v) was added and vortexed and mixed. After repeating twice, the plant lactobacillus T1 bacterial liquid was obtained. The total effective viable count of the plant lactobacillus T1 bacterial liquid was 8.16CFU / mL.

[0046] (1) Soak the Dongnong 252 soybeans with complete and full grains in a sodium carbonate solution with a mass concentration of 0.4% for 12 h (the ratio of soybeans to sodium carbonate solution is 1 g: 5 mL), then wash with clean water to obtain the soaked soybeans, preheat them in a boiling water bath at 100°C for 5 min to obtain preheated soybeans, mix the preheated soybeans with water at a ratio of 1 g: 10 mL, grind them to obtain raw soybean milk, and perform the aging treatment by keeping the temperature at 90°C for 6 min, and filter them with a 200-mesh filter to obtain cooked soybean milk;

[0047] (2) The pH of cooked soy milk was adjusted to 7.0, and the mixture was mixed with flavor protease solution (enzyme activity of 400 U / g) at a volume ratio of 9:1, and enzymolyzed in a 50°C water bath for 6 min. After the enzymolysis, the enzyme was inactivated and sterilized in a 100°C boiling water bath for 10 min, and then cooled to room temperature in an ice water bath to obtain soy milk enzymolyzate.

[0048] (3) The soymilk enzymatic hydrolysate was adjusted to pH 7.0, and mixed with glucose solution (0.3 g / mL) at a volume ratio of 96:4 to obtain a fermentation substrate. The inoculation amount of Lactobacillus plantarum T1 bacterial solution was 2% by volume, and Lactobacillus plantarum T1 was inoculated and fermented at 37° C. until the pH of the fermentation substrate reached 4.5 to obtain a fermentation product.

[0049] (4) The fermented product was post-ripened at 4°C for 12 h to obtain low-allergy soy yogurt.

[0050] Example 3

[0051] Activation and preparation of strains: The plant lactobacillus T1 preserved by the -20℃ glycerol method was inoculated into MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 24h to obtain a mixture of activated bacteria and culture medium. Subsequently, the mixture of activated bacteria and culture medium was inoculated into new MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 12h to obtain seed fermentation liquid. After the seed fermentation liquid was centrifuged at 5000rpm for 4min, the precipitate was taken, and an equal volume of physiological saline (0.85%, m / v) was added and vortexed and mixed. After repeating twice, the plant lactobacillus T1 bacterial liquid was obtained. The total effective viable count of the plant lactobacillus T1 bacterial liquid was 8.16CFU / mL.

[0052] (1) Soak the Dongnong 252 soybeans with complete and full grains in a sodium carbonate solution with a mass concentration of 0.6% for 8 h (the ratio of soybeans to sodium carbonate solution is 1 g: 3 mL), then wash with clean water to obtain the soaked soybeans, preheat them in a boiling water bath at 100°C for 15 min to obtain preheated soybeans, mix the preheated soybeans with water at a ratio of 1 g: 6 mL, grind them to obtain raw soybean milk, and perform the aging treatment by keeping the temperature at 100°C for 4 min, and filter them with a 200-mesh filter to obtain cooked soybean milk;

[0053] (2) The pH of cooked soy milk was adjusted to 7.0, and the mixture was mixed with flavor protease solution (enzyme activity of 600 U / g) at a volume ratio of 9:1, and enzymolyzed in a 60°C water bath for 4 min. After the enzymolysis, the enzyme was inactivated and sterilized in a 100°C boiling water bath for 10 min, and then cooled to room temperature in an ice water bath to obtain soy milk enzymolyzate.

[0054] (3) The soymilk enzymatic hydrolysate was adjusted to pH 7.0, and mixed with glucose solution (0.7 g / mL) at a volume ratio of 96:4 to obtain a fermentation substrate. The inoculation amount of Lactobacillus plantarum T1 bacterial solution was 4% by volume, and Lactobacillus plantarum T1 was inoculated and fermented at 37° C. until the pH of the fermentation substrate reached 4.5 to obtain a fermentation product.

[0055] (4) The fermented product was post-ripened at 4°C for 12 h to obtain low-allergy soy yogurt.

[0056] Example 4

[0057] Activation and preparation of strains: The plant lactobacillus T1 preserved by the -20℃ glycerol method was inoculated into MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 24h to obtain a mixture of activated bacteria and culture medium. Subsequently, the mixture of activated bacteria and culture medium was inoculated into new MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 12h to obtain seed fermentation liquid. After the seed fermentation liquid was centrifuged at 5000rpm for 4min, the precipitate was taken, and an equal volume of physiological saline (0.85%, m / v) was added and vortexed and mixed. After repeating twice, the plant lactobacillus T1 bacterial liquid was obtained. The total effective viable count of the plant lactobacillus T1 bacterial liquid was 8.16CFU / mL.

[0058] (1) Soak the Dongnong 252 soybeans with complete and full grains in a sodium carbonate solution with a mass concentration of 0.5% for 9 h (the ratio of soybeans to sodium carbonate solution is 1 g: 4 mL), then wash with clean water to obtain the soaked soybeans, preheat them in a boiling water bath at 100°C for 12 min to obtain preheated soybeans, mix the preheated soybeans with water at a ratio of 1 g: 7 mL, grind them to obtain raw soybean milk, and perform the aging treatment by keeping the temperature at 92°C for 5 min, and filter them with a 200-mesh filter to obtain cooked soybean milk;

[0059] (2) The pH of cooked soy milk was adjusted to 7.0, and the mixture was mixed with flavor protease solution (enzyme activity of 450 U / g) at a volume ratio of 9:1, and enzymolyzed in a 53°C water bath for 5 min. After the enzymolysis, the mixture was inactivated and sterilized in a 100°C boiling water bath for 10 min, and then cooled to room temperature in an ice water bath to obtain soy milk enzymolyzate.

[0060] (3) The soymilk enzymatic hydrolysate was adjusted to pH 7.0, and mixed with a glucose solution (0.4 g / mL) at a volume ratio of 96:4 to obtain a fermentation substrate. The inoculation amount of Lactobacillus plantarum T1 bacterial solution was 2.5% by volume, and Lactobacillus plantarum T1 was inoculated and fermented at 37° C. until the pH of the fermentation substrate reached 4.5 to obtain a fermentation product.

[0061] (4) The fermented product was post-ripened at 4°C for 12 h to obtain low-allergy soy yogurt.

[0062] Example 5

[0063] Activation and preparation of strains: The plant lactobacillus T1 preserved by the -20℃ glycerol method was inoculated into MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 24h to obtain a mixture of activated bacteria and culture medium. Subsequently, the mixture of activated bacteria and culture medium was inoculated into new MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 12h to obtain seed fermentation liquid. After the seed fermentation liquid was centrifuged at 5000rpm for 4min, the precipitate was taken, and an equal volume of physiological saline (0.85%, m / v) was added and vortexed and mixed. After repeating twice, the plant lactobacillus T1 bacterial liquid was obtained. The total effective viable count of the plant lactobacillus T1 bacterial liquid was 8.16CFU / mL.

[0064] (1) Soak the Dongnong 252 soybeans with complete and full grains in a sodium carbonate solution with a mass concentration of 0.5% for 11 h (the ratio of soybeans to sodium carbonate solution is 1 g: 4 mL), then wash with clean water to obtain the soaked soybeans, preheat them in a boiling water bath at 100°C for 8 min to obtain preheated soybeans, mix the preheated soybeans with water at a ratio of 1 g: 9 mL, grind them to obtain raw soybean milk, and perform the aging treatment by keeping the temperature at 98°C for 5 min, and filter them with a 200-mesh filter to obtain cooked soybean milk;

[0065] (2) The pH of cooked soy milk was adjusted to 7.0, and the mixture was mixed with flavor protease solution (enzyme activity of 550 U / g) at a volume ratio of 9:1, and enzymolyzed in a 58°C water bath for 5 min. After the enzymolysis, the mixture was inactivated and sterilized in a 100°C boiling water bath for 10 min, and then cooled to room temperature in an ice water bath to obtain soy milk enzymolyzate.

[0066] (3) The soymilk enzymatic hydrolysate was adjusted to pH 7.0, and mixed with glucose solution (0.6 g / mL) at a volume ratio of 96:4 to obtain a fermentation substrate. The inoculation amount of Lactobacillus plantarum T1 bacterial solution was 3.5% by volume, and Lactobacillus plantarum T1 was inoculated and fermented at 37° C. until the pH of the fermentation substrate reached 4.5 to obtain a fermentation product.

[0067] (4) The fermented product was post-ripened at 4°C for 12 h to obtain low-allergy soy yogurt.

[0068] Comparative Example 1

[0069] Activation and preparation of strains: The plant lactobacillus T1 preserved by the -20℃ glycerol method was inoculated into MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 24h to obtain a mixture of activated bacteria and culture medium. Subsequently, the mixture of activated bacteria and culture medium was inoculated into new MRS broth medium at a ratio of 3% (v / v) and cultured at 37℃ for 12h to obtain seed fermentation liquid. After the seed fermentation liquid was centrifuged at 5000rpm for 4min, the precipitate was taken, and an equal volume of physiological saline (0.85%, m / v) was added and vortexed and mixed. After repeating twice, the plant lactobacillus T1 bacterial liquid was obtained. The total effective viable count of the plant lactobacillus T1 bacterial liquid was 8.16CFU / mL.

[0070] (1) Soak the Dongnong 252 soybeans with complete and full grains in a sodium carbonate solution with a mass concentration of 0.5% for 10 h (the ratio of soybeans to sodium carbonate solution is 1 g: 4 mL), then wash with clean water to obtain the soaked soybeans, preheat them in a boiling water bath at 100°C for 10 min to obtain preheated soybeans, mix the preheated soybeans with water at a ratio of 1 g: 8 mL, grind them to obtain raw soybean milk, and perform the aging treatment by keeping the temperature at 95°C for 5 min, and filter them with a 200-mesh filter to obtain cooked soybean milk;

[0071] (2) The pH of cooked soy milk was adjusted to 7.0, and the mixture was mixed with a glucose solution (0.5 g / mL) at a volume ratio of 96:4 to obtain a fermentation substrate. The inoculation amount of Lactobacillus plantarum T1 bacterial solution was 3% by volume, and Lactobacillus plantarum T1 was inoculated and fermented at 37° C. until the pH of the fermentation substrate reached 4.5 to obtain a fermentation product.

[0072] (3) The fermented product is post-ripened at 4°C for 12 hours to obtain soy yogurt.

[0073] Experimental example

[0074] 1. Changes in pH and viable bacteria count during fermentation

[0075] The samples in the fermentation process of Example 1 and Comparative Example 1 were measured using a handheld pH meter at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h of fermentation, and the number of viable bacteria was measured at the above time points at the same time. Each measurement was repeated three times. The fermentation was stopped when the pH of the sample reached the fermentation end point of 4.5, and the time required to reach the fermentation end point was recorded.

[0076] like Figure 1 As shown, for the comparative example 1 without enzymolysis, it takes 12 hours to reach the fermentation end point after inoculation with Lactobacillus plantarum T1. The pH drop rate of the example 1 after enzymolysis is significantly accelerated, and the fermentation is completed within 8 hours. The fermentation rate is 1.5 times that of the comparative example 1.

[0077] like Figure 2 As shown, compared with the comparative example 1 without enzymatic hydrolysis, the viable bacteria growth rate of the example 1 enzymatically hydrolyzed by the flavor protease was accelerated and the number of viable bacteria at the end of the fermentation was higher, indicating that the flavor protease fermentation created more suitable growth conditions for Lactobacillus plantarum T1, which was consistent with the pH decrease rate.

[0078] 2. Intuitive diagram

[0079] The intuitive pictures of the low-sensitivity soybean yogurt prepared in Example 1 and the soybean yogurt prepared in Comparative Example 1 are as follows: Figure 3 Middle A and Figure 3 As shown in B, Figure 3 As shown in A, the soy protein yogurt prepared in Comparative Example 1 has a harder texture, a rough internal structure and larger particles, while the low-sensitivity soy yogurt prepared in Example 1 treated with flavor protease is softer, has enhanced fluidity and a delicate and smooth surface.

[0080] 3. Syneresis

[0081] Accurately weigh 20g of the low-sensitivity soy yogurt prepared in Example 1 and 20g of the soy yogurt sample prepared in Comparative Example 1, and then place it at room temperature for 2h with filter paper. Collect the filtrate after filtration and weigh it as m2, and calculate the syneresis (%) = (m2 / m1) × 100%. The syneresis statistical results are as follows: Figure 4 As shown, the syneresis shrinkage rate of the soybean yogurt of the comparative example 1 without enzymatic hydrolysis is as high as 39.92±1.50%, and the syneresis shrinkage rate of the low-sensitivity soybean yogurt of the embodiment 1 after enzymatic hydrolysis is reduced to 21.58±1.61% (p<0.05), and the water retention of the yogurt gel is significantly improved.

[0082] 4. Gel particle size and microstructure

[0083] The particle size of the gel particles was measured by a laser particle size analyzer (Mastersizer 3000). The refractive index of the water dispersant was set to 1.333, and the refractive index of the scatterer of the sample solution was set to 1.46.

[0084] Table 1 Gel particle size

[0085]

[0086] Note: a and b in the table represent significant differences.

[0087] The particle sizes of the gel particles of the low-sensitivity soybean yogurt of Example 1 and the soybean yogurt of Comparative Example 1 are as follows: Figure 5 As shown in Table 1, compared with Comparative Example 1 (22.73±2.70 μm), the particle size of the gel particles of the low-allergy soy yogurt obtained in Example 1 was significantly reduced (9.94±0.23 μm) (p<0.05).

[0088] The yogurt gel was stained with 0.005% (m / v) rhodamine B at 4°C for 12 h to ensure that the dye was completely diffused and evenly distributed. The excess dye was washed away with ultrapure water. The sample was excited with an argon laser at 543 nm to produce red rhodamine B and an image was taken.

[0089] like Figure 6 As shown, Figure 6 As shown in Figure A, the distribution of protein gel in the soybean yogurt of Comparative Example 1 shows obvious inhomogeneity, accompanied by large-scale agglomeration, such as Figure 6 As shown in B, the low-sensitivity soy yogurt protein gel of Example 1 hydrolyzed by flavor protease is evenly distributed without significant agglomeration.

[0090] 5. Antigenicity

[0091] The antigenicity changes of soybean protein before and after fermentation were determined by sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using the RIDASCREEN® FAST Soya kit. The extract, washing solution, HRP-labeled secondary antibody, substrate, and stop solution were all from the kit.

[0092] like Figure 7 As shown, the antigenicity of the cooked soy milk of Example 1 was reduced to 15.80±0.82% (p<0.05) compared with the cooked soy milk of Comparative Example 1, and the antigenicity of the low-sensitivity soy yogurt of Example 1 was reduced to 1.14±1.01% (p<0.05) compared with 46.38±5.33% of the soy yogurt of Comparative Example 1, indicating that the enzymatic hydrolysis and synergistic bacterial fermentation technology can further reduce the antigenicity of soybeans.

[0093] The preparation method of low-sensitivity soybean yogurt prepared by enzymatic hydrolysis and lactic acid bacteria fermentation of the present invention comprises the following steps: step 1: soybean pretreatment and preparation of soybean milk; step 2: restrictive enzymatic hydrolysis of soybean milk by flavor protease; step 3: fermentation and enzymatic hydrolysis of soybean milk by bacteria to prepare low-sensitivity soybean yogurt. The preparation method provided by the present invention optimizes the gel properties of soybean yogurt, and makes the appearance and texture of yogurt more delicate and soft. In addition, it can also effectively reduce the allergenicity of soybean protein and expand the audience group of the product. In terms of technical process, the method of the present invention is simple to operate and the conditions are easy to control, so that the method has broad application prospects, can bring higher added value to yogurt production enterprises, and also provide consumers with safer and healthier product choices.

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing low-sensitivity soybean yogurt by enzymatic hydrolysis and bacterial fermentation, characterized in that: The following steps are involved: (1) Soaking soybeans in a sodium carbonate solution and then washing them to obtain soaked soybeans; preheating the soaked soybeans in a boiling water bath to obtain preheated soybeans; mixing the preheated soybeans with water and grinding them to obtain raw soybean milk; and subjecting the raw soybean milk to ripening treatment and filtering to obtain cooked soybean milk; (2) The pH of the cooked soy milk obtained in step (1) is adjusted to 7.0, mixed with a flavor protease solution, subjected to enzymatic hydrolysis, and cooled to room temperature in an ice-water bath to obtain a soy milk enzymatic hydrolyzate; (3) The soymilk enzymatic hydrolyzate obtained in step (2) is adjusted to pH 7.0, mixed with a glucose solution to obtain a fermentation substrate, and the fermentation substrate is inoculated with Lactobacillus plantarum and fermented until the pH of the fermentation substrate is 4.5 to obtain a fermentation product; (4) post-ripening the fermentation product obtained in step (3) to obtain low-allergy soy yogurt; The aging treatment in step (1) is carried out at 90-100°C for 4-6 minutes; The mixed volume ratio of the cooked soy milk and the flavor protease solution in step (2) is 9:1; the enzyme activity of the flavor protease solution in the mixed system of cooked soy milk and the flavor protease solution in step (2) is 400-600 U / g; the enzymatic hydrolysis in step (2) is carried out in a water bath at 50-60° C., and the enzymatic hydrolysis time is 4-6 min; The Lactobacillus plantarum in step (3) is Lactobacillus plantarum T1, which was deposited in China Center for Type Culture Collection on September 7, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCNO: M 20231553.

2. The method for preparing low-allergy soybean yogurt according to claim 1, characterized in that: The soaking time in step (1) is 8 to 12 hours; the mass concentration of the sodium carbonate solution in step (1) is 0.4 to 0.6%; and the ratio of soybean to sodium carbonate solution in step (1) is 1 g: 3 to 5 mL.

3. The method for preparing low-allergy soybean yogurt according to claim 1, characterized in that: The temperature of the boiling water bath preheating in step (1) is 100° C., and the time of the boiling water bath preheating is 5 to 15 minutes. The ratio of the preheated soybeans and water mixed in step (1) is calculated based on the ratio of soybeans to water of 1 g: 6 to 10 mL.

4. The method for preparing low-allergy soybean yogurt according to claim 1, characterized in that: The filtration in step (1) uses a 200-mesh filter.

5. The method for preparing low-allergy soy yogurt according to claim 1, characterized in that: The volume ratio of the soymilk enzymatic hydrolyzate and the glucose solution in step (3) is 96:4; the concentration of the glucose solution in step (3) is 0.3-0.7 g / mL.

6. The method for preparing low-allergy soybean yogurt according to claim 1, characterized in that: The inoculation amount of Lactobacillus plantarum in step (3) is 2-4% by volume, and the total effective viable count of Lactobacillus plantarum is 8.16 CFU / mL; the fermentation temperature in step (3) is 37°C.

7. The method for preparing low-allergy soybean yogurt according to claim 1, characterized in that: The after-ripening temperature in step (4) is 4° C., and the after-ripening time is 12 hours.

8. The low-allergy soy yogurt prepared by the method for preparing low-allergy soy yogurt according to any one of claims 1 to 7.

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