Microbial agent for soybean milk fermentation, application of microbial agent, soybean cheese bar and preparation method of soybean cheese bar

Abstract

The invention belongs to the technical field of biological bacterial agents and functional food processing, and particularly relates to a bacterial agent for soybean milk fermentation, application of the bacterial agent, soybean cheese bars and a preparation method of the soybean cheese bars. According to the microbial agent for soybean milk fermentation, lactobacillus helveticus HL88 and lactobacillus casei BL25 are combined for use, so that soybean milk can be quickly acidified, and the curding time of soybean cheese is shortened; the soybean protein can be more fully hydrolyzed, and more short peptides and amino acids are released; particularly, the high yield of gamma-aminobutyric acid (GABA) can be achieved, and the fermentation effect and the nutritional value of the soybean milk are improved. Furthermore, the soybean cheese food prepared by fermentation of the microbial agent has rich soybean milk flavor and smooth texture; moreover, the soybean milk is easy to digest and absorb, the digestibility is improved, the allergenicity of soybeans can be reduced, and particularly, the soybean milk is helpful for improving exercise fatigue and improving sleep quality. Therefore, the microbial agent provided by the invention has excellent application prospects in the field of preparation of functional foods for resisting fatigue and promoting sleep.

Description

Technical Field

[0001] The invention belongs to the technical field of biological microbial agents and functional food processing, and specifically relates to a microbial agent for soybean milk fermentation and its application, a soybean cheese stick and a preparation method. Background Art

[0002] With the booming development of plant-based foods around the world, plant-based dairy products such as soy cheese made from soybeans have attracted much attention. Cheese is a fermented product that is made through a series of steps such as coagulating casein with a starter and rennet, and expelling whey. Soy cheese is a product made from soybeans, imitating the traditional dairy cheese production process.

[0003] At present, soy cheese is not popular in the market, and most products are only in the experimental stage. The preparation of soy cheese is inseparable from the fermentation process and the use of starter. At present, the starter used in soy cheese fermentation is mainly lactic acid bacteria starter. The use of lactic acid bacteria to properly ferment soy cheese can reduce the beany smell and produce "pleasant" volatile flavor compounds such as ester compounds. In addition, after lactic acid bacteria ferment soy milk, soy protein is degraded into peptides and amino acids, making it easier for the body to absorb. Fermentation can also reduce anti-nutritional compounds in soy milk, such as trypsin inhibitors, tannins and phytic acid, thereby improving the digestion and absorption of nutrients. Lactic acid bacteria fermentation of soy milk can also produce many bioactive substances such as bioactive glycosides with higher bioavailability, B vitamins and other small molecules, which are beneficial to human health.

[0004] At present, although there are many types of lactic acid bacteria starters on the market, most of the commercially available cheese starters are dairy-based cheese starters, and very few are exclusive starters for soy cheese. Due to the essential differences in the protein and carbohydrate composition between animal milk and soy milk, dairy-based cheese starters cannot be directly applied to the processing of soy cheese. In addition, the soy cheese prepared by fermentation with commercially available conventional dairy-based starters is still lacking in sensory aspects such as flavor and texture, such as strong beany smell, heavy sour taste, loose texture, and is not easily accepted by consumers. In addition, the soy cheese obtained by fermentation with dairy-based starters often lacks functionality and has low market demand, which also limits its promotion and application in the field of functional foods.

[0005] Therefore, there is an urgent need to develop a starter culture suitable for the preparation of soy cheese so as to better promote its application in the industrial production of soy cheese and the development of functional foods. This will not only improve the production level of soy cheese and enhance consumers' awareness of soy cheese, but will also produce greater social benefits and application prospects. Summary of the invention

[0006] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a bacterial agent for soybean milk fermentation, which has a strong ability to hydrolyze soybean protein and can produce high levels of gamma-aminobutyric acid (GABA), and is very suitable for fermenting soybean milk to prepare functional soybean cheese products.

[0007] At the same time, the purpose of the present invention is also to provide the application of the above-mentioned bacterial agent for soybean milk fermentation.

[0008] At the same time, the object of the present invention is also to provide a soybean cheese stick.

[0009] At the same time, the object of the present invention is also to provide a method for preparing the soy cheese stick.

[0010] To achieve the above object, the technical solution adopted by the present invention is: A bacterial agent for soybean milk fermentation, the bacterial agent for soybean milk fermentation comprising: Lactobacillus helveticus HL88 ( Lactobacillus helveticus ) and Lactobacillus casei ( Lactobacillus casei )BL25; Among them, Lactobacillus helveticus HL88 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC NO. 28126; Lactobacillus casei BL25 was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20241210.

[0011] The bacterial agent for soybean milk fermentation provided by the present invention comprises Lactobacillus helveticus HL88 and Lactobacillus casei BL25. Through the synergistic effect of the two strains, the bacterial agent has a strong ability to hydrolyze soybean protein on the one hand, and can produce high gamma-aminobutyric acid (GABA) on the other hand, and is very suitable for fermenting soybean milk to prepare functional products such as soybean cheese.

[0012] As a preferred solution, in the bacterial agent used for soybean milk fermentation, the ratio of the number of live bacteria of Lactobacillus helveticus HL88 to that of Lactobacillus casei BL25 is 1:(1.8-2.2), more preferably 1:2.

[0013] The above-mentioned bacterial agent for soybean milk fermentation is used in the preparation of functional foods for resisting fatigue and / or promoting sleep.

[0014] As a preferred solution, the functional food is a soy cheese stick.

[0015] A soybean cheese stick is prepared by fermenting soybean milk with the above-mentioned bacterial agent for soybean milk fermentation.

[0016] The soy cheese product prepared by using the bacterial agent for soybean milk fermentation of the present invention not only has good flavor and low allergenicity, but also has a high content of GABA and good digestive properties. In particular, experiments have confirmed that the prepared soy cheese can effectively improve animal sports fatigue and improve sleep quality. Therefore, the bacterial agent is suitable for use in functional foods that resist fatigue and promote sleep.

[0017] A method for preparing a soybean cheese stick comprises the following steps: (1) inoculating a bacterial agent for fermentation of soybean milk into the soybean milk for fermentation, and then adding a coagulant for coagulation to obtain curd; (2) cutting the curd and then draining the soy whey to obtain soy semi-hard cheese; (3) Adding auxiliary materials to the soybean semi-hard cheese, chopping and mixing, and then shaping and refrigerating to obtain the soybean cheese stick.

[0018] As a preferred embodiment, in step (1), the ratio of the amount of soybean milk, the bacterial agent used for soybean milk fermentation, and the coagulant is (80-120) g: (2.0-4.0) mL: (0.2-0.4) g, and more preferably 100 g: 3 mL: 0.3 g.

[0019] As a preferred solution, the number of viable bacteria in the bacterial agent for soybean milk fermentation is (1-2)×10 9 CFU / mL; the fermentation temperature is 40-45°C, and the fermentation time is 1-3h; the coagulant is calcium chloride; and the coagulation time is 1-5h.

[0020] As a preferred solution, in step (3), based on 100 parts of soy semi-hard cheese, the auxiliary materials include: 8 to 12 parts of corn oil, 1.2 to 3.0 parts of emulsified salt, and 0.1 to 0.5 parts of edible salt.

[0021] As a further preferred solution, the emulsifying salt is a combination of sodium citrate, sodium hexametaphosphate, and sodium tripolyphosphate in a mass ratio of 1: (1.5-2.5): (1.5-2.5). As a more preferred solution, the emulsifying salt is a combination of sodium citrate, sodium hexametaphosphate, and sodium tripolyphosphate in a mass ratio of 1: 2: 2.

[0022] As a preferred solution, in step (3), the process conditions for chopping and mixing are: temperature 70-90°C, rotation speed 1500-2200 r / min, chopping and mixing 20-60 min; the refrigeration temperature is 2-8°C.

[0023] The beneficial effects of the above technical solution of the present invention are: (i) The bacterial agent for soybean milk fermentation provided by the present invention uses Lactobacillus helveticus HL88 and Lactobacillus casei BL25 in combination, which can play a synergistic role, not only can quickly acidify soybean milk and shorten the coagulation time of soybean cheese; but also can more fully hydrolyze soybean protein and release more short peptides and amino acids; in particular, it can produce high yield of gamma-aminobutyric acid (GABA), thereby improving the fermentation effect and nutritional value of soybean milk.

[0024] (ii) The soy cheese sticks prepared by fermentation with the above-mentioned bacterial agent have a strong soy milk flavor and a smooth texture; they are easily digested and absorbed, thus improving the digestibility and reducing the allergenicity of soy.

[0025] (iii) The experiment confirmed that consuming soybean cheese sticks prepared by fermentation with the above-mentioned bacterial agent after exercise can reduce fatigue time. In addition, due to the use of the compound fermentation agent, the soybean cheese sticks contain a higher content of GABA, which can effectively improve sleep quality. Therefore, the soybean cheese sticks prepared by fermentation with the above-mentioned bacterial agent have the effect of anti-fatigue and promoting sleep, and have good application prospects in the field of functional food preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The comparison results of the coagulation time of each strain in the present invention are shown in FIG. Figure 2 The results of the comparison of the soy protein hydrolysis activities of various strains in the present invention are shown below; Figure 3 The comparison results of GABA concentrations of soybean milk fermented by various strains in the present invention are shown in FIG. Figure 4 The antigenicity comparison results of soybean milk obtained by fermentation of various strains in the present invention are as follows; Figure 5 The flavor test results of the soy cheese sticks of different test groups in the present invention are shown; Figure 6 The GABA test results of soybean cheese sticks in different test groups in the present invention are shown; Figure 7 The allergenicity test results of soybean cheese sticks in different test groups in the present invention are shown in FIG. Figure 8 The results of the determination of the digestibility of the soy cheese sticks of different test groups in the present invention; Fig. 9 These are the results of the effects of soy cheese sticks in different test groups on the athletic performance of rats in the present invention; wherein Figures A to D are the results of the determination of swimming time, serum urea, liver glycogen, and blood lactate, respectively. DETAILED DESCRIPTION

[0027] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and test examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the raw materials used are all commonly used in the art, available to the public or commercially available, unless otherwise specified.

[0028] Among them, in the following embodiments, the preservation information involved is as follows: Collection information 1 Deposit name: Lactobacillus helveticus HL88, Latin name Lactobacillus helveticus HL88; Deposit number: CGMCC NO. 28126; Depository: China General Microbiology Center (CGMCC); Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Date of preservation: August 9, 2023.

[0029] Collection information 2 Deposit name: Lactobacillus casei BL25, Latin name Lactobacillus casei BL25; Deposit number: CCTCC NO: M 20241210; Depository: China Center for Type Culture Collection (CCTCC); Deposit address: Wuhan University; Date of preservation: June 7, 2024.

[0030] In the following embodiments of the present invention, the composition and preparation method of the MRS culture medium involved are: take 12g of peptone, 6g of yeast extract, 6g of beef extract, 18g of glucose, 5g of sodium acetate, 2.15g of ammonium citrate, 1g of Tween, 0.58g of magnesium sulfate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate, use pure water to make the volume to 1L, adjust the pH to 6.5, and use a high-pressure steam sterilizer to sterilize at 121°C for 15min for standby use. In other embodiments, the MRS culture medium can be obtained from commercial channels, and the present invention does not specifically limit it.

[0031] In the following embodiments of the present invention, the preparation process of soybean milk is as follows: ① Soybean pretreatment: After washing and removing impurities from the soybeans, soak them in clean water at a solid-liquid ratio of 1:5 to 1:10 for 12 to 24 hours to allow the soybeans to fully swell. Then boil the soybeans at 100°C for 20 to 30 minutes, remove them, cool them, and set aside.

[0032] ② Preparation of soybean milk: Mix the cooked soybeans with clean water at a material-liquid ratio of 1:4 to 1:6, first use a wall breaker to coarsely crush for 1 to 3 minutes, then use a colloid mill to wet crush for 5 to 10 minutes, adjust the colloid mill grinding interval to 5 to 20 μm, and the colloid mill speed to 2800 to 3000 r / min. After crushing, filter through a 120-mesh filter cloth for 2 to 3 times, sterilize the filtered emulsion at 108°C for 15 minutes, and then cool to 37°C to obtain soybean milk for later use.

[0033] In other embodiments, the soybean milk may also be prepared by other preparation processes well known in the art, which are not particularly limited in the present invention and will not affect the fermentation effect of the bacterial agent of the present invention.

[0034] Example 1

[0035] This embodiment provides a bacterial agent for soybean milk fermentation, including: Lactobacillus helveticus ( Lactobacillus helveticus )HL88 and Lactobacillus casei ( Lactobacillus casei ) BL25; Lactobacillus helveticus HL88 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee, with a deposit number of CGMCC NO. 28126; Lactobacillus casei BL25 is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20241210. In the bacterial agent, the number ratio of Lactobacillus helveticus HL88 to Lactobacillus casei BL25 is 1:2.

[0036] The separation, screening and identification process of Lactobacillus helveticus HL88, Lactobacillus casei BL25 involved in this embodiment and other comparative strains involved in the following test example 1 (Lactobacillus plantarum LP11, Lactobacillus plantarum LP12, Lactobacillus plantarum LP14, Lactobacillus helveticus HL01, Lactobacillus helveticus HL03, Lactobacillus helveticus HL13, Lactobacillus helveticus HL14, Lactobacillus helveticus HL101) are as follows: (1) Strain source Lactobacillus helveticus HL88 was isolated from Xinyuan County, Yili Kazakh Autonomous Prefecture, Xinjiang, and the sample was yogurt fermented by local herdsmen through traditional natural fermentation. Lactobacillus casei BL25 was isolated from Xiahe County, Gannan Tibetan Autonomous Prefecture, and the sample was yogurt fermented by local herdsmen through traditional natural fermentation.

[0037] Sample collection method: A total of 10 groups of yogurt samples were collected from Xinyuan County, Ili Kazakh Autonomous Prefecture, Xinjiang, Xiahe County, Gannan Tibetan Autonomous Prefecture, Youyi Middle Banner, Xing'an League, Inner Mongolia, Zuoyi Middle Banner, Tongliao City, Inner Mongolia, and Xiwu Banner, Xilin Gol League, Inner Mongolia. When sampling, a sterile pipette was used to draw about 20-25 mL of the sample into a 50 mL sterile sampling tube. The sample number was marked and refrigerated and transported back to the laboratory for isolation and identification tests. The isolation source and isolation location information of each strain are shown in Table 1.

[0038] Table 1. Strain isolation information

[0039] (2) Isolation, culture and routine identification of lactic acid bacteria ① Isolation, purification and preservation of lactic acid bacteria in samples Take 1mL of the above-collected yogurt sample, inoculate it in sterilized physiological saline, perform 10-fold gradient dilution in sequence, and take 100μL of the dilution solution to apply MRS on the solid culture medium by plate coating method, then put it into a constant temperature incubator and culture it at 37℃ for 48h. Observe and record the characteristics of the colonies, pick different colonies for further separation and purification 3~4 times, observe the degree of colony purification under a microscope, until a single colony is isolated and purified. The isolated lactic acid bacteria strains are stored in a -80℃ ultra-low temperature refrigerator for standby use.

[0040] Gram staining was used to observe cell morphology and determine classification characteristics such as cell shape, size, and Gram staining status, and to conduct preliminary tests on lactic acid bacteria.

[0041] Among them, Lactobacillus helveticus HL88 is Gram-positive, with long rod-shaped bacteria, round ends, arranged in short chains, no spores, no heterochromatic particles, irregular colonies, rough surface, flat, uneven edges, light yellow, and opaque. Lactobacillus casei BL25 is Gram-positive, with short rod-shaped bacteria, single arrangement, round, small, moist, and white colonies.

[0042] ②Molecular identification of lactic acid bacteria isolates The genomic DNA of the lactic acid bacteria isolates was extracted and the 16S rDNA gene was amplified by PCR. The sequencing results were compared by BLAST in the NCBI database. If the 16S DNA sequence homology between the sequenced strain and the model strain was greater than 97%, it could be identified that the strain and the model strain belonged to the same species.

[0043] Wherein, the 16S DNA sequence homology of Lactobacillus helveticus HL88 and the model strain is 99.45%, so it is confirmed as Lactobacillus helveticus. The 16S DNA sequence homology of Lactobacillus casei BL25 and the model strain is 99.37%, so it is confirmed as Lactobacillus casei. The separation and identification process of other bacterial strains is similar to Lactobacillus helveticus HL88 and Lactobacillus casei BL25, and the identification result is as shown in Table 1, and the present invention will not repeat them.

[0044] ③Microbial activation and preparation of bacterial suspension The experimental strains were stored in a 20% (v / v) glycerol aqueous solution at -80°C. Before the experiment, the strains were inoculated in sterile MRS medium at 18°C ​​and incubated in a constant temperature incubator at 37°C for 24 hours. After incubation, Lactobacillus helveticus HL88 and Lactobacillus casei BL25 were mixed and centrifuged at 2000rpm for 10 minutes. After removing the medium, the precipitate was harvested and washed twice with sterile phosphate buffered saline (PBS). The washed precipitate was diluted with PBS to an effective bacterial concentration of 1.8×10 9 The suspension of 100 CFU / mL is the bacterial agent for soybean milk fermentation in Example 1. In the bacterial agent, the number ratio of Lactobacillus helveticus to Lactobacillus casei is 1:2.

[0045] Example 2

[0046] This embodiment provides a soybean cheese stick, which is prepared by fermenting soybean milk using the bacterial agent for soybean milk fermentation of Example 1.

[0047] The method for preparing the soybean cheese stick specifically comprises the following steps: (1) Sterilized soybean milk was poured into a small cheese tank, inoculated with the bacterial agent for soybean milk fermentation of Example 1, fermented at 42°C for 2 hours, and then added with a coagulant (CaCl2) and coagulated at 48°C for 2 hours to obtain curd; wherein the ratio of soybean milk, bacterial agent, and coagulant was 100 g: 3 mL: 0.3 g; the number of viable bacteria in the bacterial agent for soybean milk fermentation was 1.8 × 10 9 CFU / mL.

[0048] (2) Cut the curd into 1 cm pieces, transfer to a perforated mold with a layer of gauze to drain, and press overnight to expel the soy whey to obtain soy semi-hard cheese; (3) Pour the semi-hard soybean cheese into a chopping pot, add auxiliary materials (based on 100 parts of the mass of the semi-hard soybean cheese, add 10 parts of corn oil, 2 parts of emulsifying salt, and 0.3 parts of edible salt), chop and emulsify for 30 minutes at 80°C and 1800 r / min, pour into a mold and shape it, and then put it in a refrigerator at 4°C for 24 hours to obtain the soybean cheese stick of this embodiment. The emulsifying salt is a combination of sodium citrate, sodium hexametaphosphate, and sodium tripolyphosphate in a mass ratio of 1:2:2.

[0049] The above-mentioned bacterial agent of the present invention is subjected to the coagulation time, hydrolyzed soybean protein activity, GABA content, and allergenicity determination, and the prepared soybean cheese stick is subjected to flavor texture, GABA content, allergenicity, digestive characteristics, and animal tests to confirm the technical effect that can be achieved by the technical solution of the present invention. The test data are all expressed as mean ± standard deviation, repeated at least 3 times, and the data between different groups are subjected to single-factor variance using SPSS18.0 software, and plotted using GraphPad Prism5.0 and Origin 9.0 software, P <0.05 is statistically significant. The test process and test results are as follows.

[0050] Test Example 1: Evaluation of strain fermentation effect 1.1. Determination of coagulation time of strains Coagulation time: refers to the time required for fermented milk to change from a liquid state to a non-flowing semi-solid state during the fermentation process. The coagulation time test results of each strain (LP11, LP12, BL14, BL25, HL01, HL03, HL13, HL14, HL88, HL101) are as follows: Figure 1 shown.

[0051] Depend on Figure 1 It can be seen that the coagulation time of each strain in soybean milk is significantly different, among which strain BL25 and strain BL14 have the fastest coagulation time, and the coagulation time of strain BL25 is only 7.3h. The short coagulation time indicates that the strain can grow rapidly in the soybean milk environment and use carbohydrates to produce acid and coagulate milk.

[0052] 1.2. Determination of the activity of the strain in hydrolyzing soybean protein The strain types were the same as those in Section 1.1. The method for determining the activity of the strain in hydrolyzing soy protein was as follows: 100 g of sterilized soy milk was taken and 3 mL of different bacterial agents (the number of viable bacteria was 1.8 × 10 9CFU / mL) was fermented at 42℃ for 24 hours to obtain fermented milk; 10mL of fermented milk was added with an equal volume of 10% trichloroacetic acid for precipitation, and then centrifuged at room temperature for 1h (15000r / min, 30min) to obtain the supernatant of the fermented sample and store it at low temperature for later use. The supernatant of the fermented sample was tested for o-phthalaldehyde (OPA). The reagent solution was prepared by mixing three different solutions; solution 1 was composed of 3.81g sodium tetraborate and 100mg sodium dodecyl sulfate (SDS), dissolved in 75mL distilled water; solution 2 was prepared by dissolving 80mg o-phthalaldehyde in 2mL 96% ethanol; solution 3 was prepared by dissolving 88mg dithiothreitol (DTT) in 50mL MilliQ water. The reagent solution was prepared on the day of analysis, and solutions 1 to 3 were mixed shortly before use. During the test, the supernatant samples were thawed in triplicate, 30 μL was added to a 96-well plate, and then 270 μL of the reagent solution was added. The plate was incubated in the dark for 2 minutes, and the absorbance was read at 340 nm using a microplate reader to determine the free amino content. The activity of each strain in hydrolyzing soy protein was then evaluated. The results are shown in Figure 2 shown.

[0053] Depend on Figure 2 It can be seen that the proteolytic ability of each strain in soy milk is significantly different. Compared with other strains, strains HL88 and HL101 have the highest soy protein hydrolysis activity. The higher proteolytic activity indicates that the strain can more fully hydrolyze soy protein during the fermentation of soy milk, obtain more short peptides and amino acids, which is beneficial to improve the flavor and increase the digestibility.

[0054] 1.3 Determination of GABA The method for determining the concentration of γ-aminobutyric acid (GABA) in fermented milk obtained by fermenting soybean milk with strains is as follows: take 10 mL of fermented milk obtained in Section 1.2 and stirred evenly, add an equal volume of 10% trichloroacetic acid to dilute, place at room temperature for 1 hour and filter, centrifuge the resulting solution (15000r / min, 30min) and take the supernatant for use. The GABA content in the fermentation supernatant of the strain to be tested was determined by high performance liquid chromatography (HPLC). HPLC chromatographic conditions: Luna-C18 column (250 mm×4.6mm) was used, column temperature was 30℃; injection volume was 5uL; two mobile phases were 20mmol / L sodium acetate aqueous solution (A) and methanol (B), and the volume ratio A:B=55:45 was determined after optimization; flow rate was set to 1mL / min; wavelength was 334nm.

[0055] Take 0.10g of GABA standard, dissolve it with ultrapure water and dilute it to a 50mL volumetric flask, shake and mix, and prepare a 2mg / mL GABA standard solution. Then dilute the standard solution to 0.5, 0.8, 1.0, 1.2, and 1.5mg / mL standard solutions for use. Add 200uL of fermented milk supernatant or GABA standard solution to a 5mL EP tube, add 600uL of OPA derivatization agent, shake and mix, add 800uL of potassium dihydrogen phosphate buffer, react in the dark for 90s, filter through a 0.22um filter membrane, and then inject immediately. The time for the entire derivatization process is controlled within 3min. GABA test results are as follows Figure 3 shown.

[0056] Depend on Figure 3 It can be seen that compared with other strains, strain BL25 produces the highest GABA content. GABA is essential for relieving fatigue and improving sleep quality.

[0057] 1.4. Determination of sensitization The method for determining the antigenicity of soybean milk obtained by strain fermentation is as follows: the antigenicity of soybean milk is determined by ELISA. During the test, 100 μL of fermented milk sample (1 μg / mL) diluted with PBS solution is added to a 96-well plate, placed at 4°C overnight, and then blocked with skim milk (50 mL / L, pH 7.2) at 37°C for 1 hour. After washing with PBS, each well is incubated with serum for 1 hour, washed, and incubated with serum from soybean allergy patients for 1 hour, and washed 3 times with PBST washing solution. 100 μL of TMB colorimetric solution is added to each well, reacted at 37°C in the dark for 15 minutes, 50 μL of sulfuric acid stop solution is added to each well to terminate the reaction, and the absorbance value of the sample at 450 nm is measured, which reflects the IgE binding ability, thereby characterizing the antigenicity of soybean milk. The antigenicity test results are as follows: Figure 4 shown.

[0058] Depend on Figure 4 It can be seen that compared with other strains, the antigenicity of fermented soy milk by strain HL88 is the lowest, which may be due to the high proteolytic activity of strain HL88, which hydrolyzes and destroys soy protein allergens during the fermentation process.

[0059] From the above screening results, it can be seen that strain BL25 has a shorter coagulation time and a high GABA production capacity, and strain HL88 has a higher soy protein hydrolysis capacity and lower antigenicity. Therefore, the present invention uses strain BL25 and strain HL88 as the subsequent fermentation preparation of soy cheese.

[0060] Test Example 2: Performance Evaluation of Soy Cheese Sticks Soybean cheese sticks fermented with a single Lactobacillus casei BL25 (Group A) and soybean cheese sticks fermented with a single Lactobacillus helveticus HL88 (Group B) were prepared respectively, and only the type of bacterial agent was changed during the preparation process, and other conditions were the same as in Example 2. The effects of the soybean cheese sticks fermented with a single bacterial agent and the soybean cheese sticks fermented with a composite bacterial agent of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 (Group C) in Example 2 of the present invention were compared.

[0061] 2.1. Determination of flavor of soy cheese sticks The flavor determination method of the soy cheese sticks prepared by each group is: Electronic nose: Accurately weigh 10g of soy cheese stick sample, transfer to a 40mL sample bottle (with cap), and test using the electronic nose at room temperature. The sensor is cleaned with air. The cleaning time is 120s. A vacuum pump is used to draw the gas in the sample into the electronic nose. The inlet speed is 1L / min and the detection time is 120s.

[0062] Electronic tongue: 5 g of uniformly crushed soy cheese sample was mixed in 100 mL of deionized water and centrifuged at 6000 × g and 20 ° C for 20 min. The supernatant was filtered with filter paper and the clear liquid was used for analysis. The acid, bitter, astringent, salty, umami, sweet sensors and reference electrode of the electronic tongue device were activated in advance for 24 ± 2 h. The liquid sample without precipitation was poured into the sample cup for taste measurement.

[0063] Flavor test results Figure 5 As shown. Among them, Figure 5 In the figure, A: soybean cheese stick group prepared by fermentation of Lactobacillus casei BL25; B: soybean cheese stick group prepared by fermentation of Lactobacillus helveticus HL88; C: soybean cheese stick group prepared by fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88, the same below. The meaning of each number is: Electronic nose: 1-aromatic compounds; 2-nitrogen oxides; 3-ammonia, aromatic molecules; 4-hydride; 5-olefins, aromatic, polar molecules; 6-alkanes; 7-sulfur compounds; 8-detection alcohols, some aromatic compounds; 9-aromatic compounds, organic compounds of sulfur; 10-alkanes and aliphatics. Electronic tongue: 1-sour; 2-bitter; 3-astringent; 4-bitter aftertaste; 5-astringent aftertaste; 6-umami; 7-rich taste; 8-salty.

[0064] Depend on Figure 5 It can be seen that compared with cheese fermented with single bacteria, the cheese fermented with the composite bacterial agent of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention has a more obvious rich taste, less sour taste, and the overall flavor and taste are more acceptable to people.

[0065] 2.2. GABA determination in soy cheese sticks The GABA content of the soy cheese sticks prepared by each group was determined as follows: 10 g of soy cheese sample was taken and ground, an equal volume of 10% trichloroacetic acid was added for dilution, the solution was allowed to stand at room temperature for 1 hour and then filtered, the resulting solution was centrifuged (15000 r / min, 30 min) and the supernatant was taken for later use. The GABA determination method is shown in Section 1.3. The results are shown in Figure 6 shown.

[0066] Depend on Figure 6 It can be seen that compared with the other two groups of single-bacteria fermented cheeses, the GABA content in the cheese of group C is the highest. This shows that the mixed fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 can increase the GABA content in soy cheese. GABA is mainly produced by Lactobacillus casei BL25. The possible reason for the mixed fermentation to produce more GABA is that Lactobacillus helveticus HL88 hydrolyzes soy protein to generate more short peptides and amino acids. These nutrients promote the growth of Lactobacillus casei BL25, and then Lactobacillus casei BL25 produces a higher content of GABA.

[0067] 2.3 Determination of the allergenicity of soy cheese sticks The allergenicity test method of the soy cheese sticks prepared by each group was as follows: first, the soy cheese samples were ground and pulverized, and the samples were diluted with PBS to 1 μg / mL for testing. The specific test method is shown in Section 1.4. Figure 7 shown.

[0068] Depend on Figure 7 It can be seen that compared with the other two groups of single-bacteria fermented cheeses, the allergenicity in group C cheese is the lowest. This shows that the mixed fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention can reduce the allergenicity in soy cheese. The soy protein hydrolysis effect of mixed fermentation is better, and more soy protein allergens can be hydrolyzed.

[0069] 2.4. Determination of digestibility of soy cheese sticks The digestibility characteristics of the soy cheese sticks prepared by each group were determined as follows: In vitro digestion process: Simulated gastric fluid and simulated intestinal fluid solutions were prepared as 1.25× concentrates. According to the pre-measured protein concentration, the soy cheese sample equivalent to 150 mg of protein was dispersed into 9.6 mL of SGF and homogenized in an ice bath using a biohomogenizer (5500 rpm, 2× 30 s, cooling interval 60 s). To initiate gastric digestion, the pH of the mixed solution was changed to 3.0 with hydrochloric acid (1 M), and pepsin was added to achieve a final enzyme activity of 2000 U / mL. The samples were incubated at 37°C for 2 h, and the digestion was terminated by adjusting the pH to 7.5 with 1 mol / L NaOH. For the intestinal stage of digestion, the gastric chyme was mixed with 10 mL of SIF, and then trypsin was added for hydrolysis at 37°C for 2 h. The digestion was stopped by heating the sample at 100°C for 5 min to obtain the digestion solution.

[0070] Determination of digestibility: The digestion solutions were mixed with 3 volumes of ethanol respectively and allowed to stand at 4 °C for 12 h. The mixture was then centrifuged at 8000 g and 4 °C for 20 min to remove undigested proteins and high molecular weight peptides. The protein content of the precipitate was determined using the BCA protein assay kit according to the instructions. The protein digestibility was calculated according to the following formula: digestibility % = (W0-W1) / W×100; where W0 and W1 represent the protein content of the blank and sample after digestion, and W represents the protein content of the sample before digestion.

[0071] Determination of peptide content: 160 mg of OPA was dissolved in 4 mL of methanol, 100 mL of sodium tetraborate (0.1 mol / L), 400 μL of β-mercaptoethanol, and 10 mL of 20% (w / w) sodium dodecyl sulfate. Deionized water was then added to a final volume of 200 mL. Prior to the experiment, the digest was mixed with 10% trichloroacetic acid (TCA) in a 1:1 ratio and centrifuged at 10,000 g for 10 min at 4 °C to collect the supernatant. Peptides with a molecular weight of <10 kDa were collected by membrane. Then, 50 μL of the filtered sample was mixed into 2 mL of OPA solution, and the mixture was reacted in the dark for 2 min. The absorbance was measured at 340 nm using a multifunctional microplate reader, and casein was used as a standard. The test results of digestion characteristics (digestibility and peptide content) are shown in Figure 2. Figure 8 shown.

[0072] Depend on Figure 8It can be seen that the digestibility of group C cheese is higher than that of group A and group B cheese, indicating that the present invention uses mixed fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 to improve the digestibility of soy cheese (left figure). At the same time, the present invention measured the polypeptide content of the three groups of cheese after digestion (right figure), and the results showed that the polypeptides in the digested products of group C cheese were significantly higher than those in group A and group B cheese, indicating that the soy cheese fermented with mixed fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention can produce more peptides after simulated digestion and is easy to digest.

[0073] 2.5 Animal experiments to investigate the effects of cheese sticks on animal fatigue and sleep Animal Experimental Design: Healthy female Sprague-Dawley (SD) rats (60.0±10.0g, 3w) were purchased from Harbin Medical University, China. The rats were housed in accordance with the Animal Care and Use Guidelines, with three rats per cage, and standard cages allowed rats to freely access food and water. The ambient temperature was maintained at 22±2°C to 45±5%, with a 12-hour light-off / dark cycle.

[0074] Motor performance test: All rats first ate standard feed freely for one week to adapt and observe. After the adaptation period, the rats were divided into three groups: control group (N), cheese stick group prepared by single bacteria Lactobacillus casei BL25 (group A), cheese stick group prepared by single bacteria Lactobacillus helveticus HL88 (group B), and cheese stick group prepared by the composite fermentation agent of Lactobacillus casei BL25 + Lactobacillus helveticus HL88 in Example 2 (group C). There were 6 rats in each group. After the adaptation period, each group ate standard diet feed (standard diet was purchased from Beijing Keao Xieli Feed Co., Ltd.) for 4 weeks. Group A was gavaged with 1g of soybean cheese prepared by single bacteria fermentation of Lactobacillus helveticus HL88 (dissolved in PBS) every day. Group B was gavaged with 1g of soybean cheese prepared by single bacteria fermentation of Lactobacillus casei BL25 (dissolved in PBS) every day. Group C was gavaged with 1g of soybean cheese prepared by composite fermentation of Lactobacillus helveticus HL88 and Lactobacillus casei BL25 (dissolved in PBS) every day. The control group (group N) was infused with an equal volume of solvent sterile PBS. The rats were placed in a plastic container at 25°C and 25 cm above the water surface. An iron block weighing 10% of their body weight was placed on their tails for swimming. The time it took for the rats to sink to the water surface for 10 seconds without being able to float to the surface was taken as the weight-exhaustive swimming time. The serum urea, liver glycogen, and blood lactate levels of the rats in each group were tested. Fig. 9 shown.

[0075] Fig. 9 In the figure, A~D are the results of swimming time, serum urea, liver glycogen, and blood lactate. Fig. 9It can be seen that the cheese sticks prepared by the combined fermentation of Lactobacillus helveticus HL88 and Lactobacillus casei BL25 acted on rats, which significantly increased the swimming time of rats compared with the control group (P < 0.01) ( Fig. 9 A); The serum urea content of rats was significantly decreased (P<0.01) ( Fig. 9 B); The content of glycogen in rat liver increased significantly (P<0.01) ( Fig. 9 C); the blood lactate content of rats was significantly reduced (P<0.01) ( Fig. 9 D). This indicates that the composite fermentation of Lactobacillus helveticus HL88 and Lactobacillus casei BL25 in the present invention has a significant anti-fatigue effect on rats.

[0076] Direct sleep experiment: According to the experimental grouping of the above motor performance part, each group of rats was gavaged with different groups of soybean cheese solutions (1g soybean cheese sample was dissolved in PBS), and the control group was infused with an equal volume of PBS solvent, and then the occurrence of sleep state in the rats was observed. The disappearance of the righting reflex is used as an indicator of sleep. When the rat is in a dorsal recumbent position, it immediately turns right, which means that it has not entered sleep. If it cannot turn right for more than 30 to 60 seconds, it is considered that the righting reflex has disappeared and it has entered sleep. The sign of animal awakening is the recovery of the righting reflex. The animal's sleep time can be considered as the time from the disappearance of the righting reflex to the recovery. The number of sleeping animals in the blank control group and the sample intake group and the corresponding sleep time are recorded. The results are shown in Table 2.

[0077] Table 2. Effect of soy cheese sticks on the sleep time of rats with sodium pentobarbital

[0078] As shown in Table 2, compared with the other three groups, the sleep time of the rats in group C increased significantly, indicating that the soybean cheese co-fermented by Lactobacillus helveticus HL88 and Lactobacillus casei BL25 of the present invention has a better sleep effect on rats after exercise.

[0079] In summary, the bacterial agent for soybean milk fermentation provided by the present invention, using Lactobacillus helveticus HL88 and Lactobacillus casei BL25 in combination, can not only quickly acidify soybean milk and shorten the coagulation time of soybean cheese; but also can more fully hydrolyze soybean protein and release more short peptides and amino acids; in particular, it can produce high-yield γ-aminobutyric acid (GABA), and improve the fermentation effect and nutritional value of soybean milk. In particular, the soybean cheese sticks prepared by fermentation of the above-mentioned composite bacterial agent in the present invention have good flavor, low allergenicity, high content of GABA and good digestive properties. More importantly, the present invention found through animal experiments that the soybean cheese sticks obtained by compound fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 can significantly improve sports fatigue and improve sleep quality. Therefore, the above-mentioned bacterial agent provided by the present invention has a good application prospect in the field of preparation of functional foods for anti-fatigue and / or sleep promotion.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bacterial agent for soybean milk fermentation, characterized in that: The bacterial agent used for soybean milk fermentation includes: Lactobacillus helveticus ( Lactobacillus helveticus )HL88 and Lactobacillus casei ( Lactobacillus casei )BL25; Among them, Lactobacillus helveticus HL88 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC NO. 28126; Lactobacillus casei BL25 was deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20241210.

2. The microbial agent for soybean milk fermentation according to claim 1, characterized in that: In the bacterial agent used for soybean milk fermentation, the ratio of the number of live bacteria of Lactobacillus helveticus HL88 to that of Lactobacillus casei BL25 is 1:(1.8-2.2).

3. The use of the bacterial agent for soybean milk fermentation according to claim 1 or 2, characterized in that: Application in the preparation of anti-fatigue and / or sleep-promoting functional foods.

4. The use of the bacterial agent for soybean milk fermentation according to claim 3, characterized in that: The functional food is a soybean cheese stick.

5. A soy cheese stick, characterized in that The soybean milk is prepared by fermenting the soybean milk using the bacterial agent for soybean milk fermentation as claimed in claim 1 or 2.

6. A method for preparing a soy cheese stick as claimed in claim 5, characterized in that: The following steps are involved: (1) inoculating a bacterial agent for fermentation of soybean milk into the soybean milk for fermentation, and then adding a coagulant for coagulation to obtain curd; (2) cutting the curd and then draining the soy whey to obtain soy semi-hard cheese; (3) Adding auxiliary materials to the soybean semi-hard cheese, chopping and mixing, and then shaping and refrigerating to obtain the soybean cheese stick.

7. The method for preparing the soy cheese stick according to claim 6, characterized in that: In step (1), the ratio of soybean milk, bacterial agent for soybean milk fermentation, and coagulant is (80-120) g: (2.0-4.0) mL: (0.2-0.4) g; the number of viable bacteria in the bacterial agent for soybean milk fermentation is (1-2) × 10 9 CFU / mL; the fermentation temperature is 40-45°C, and the fermentation time is 1-3h; the coagulant is calcium chloride; and the coagulation time is 1-5h.

8. The method for preparing the soy cheese stick according to claim 6, characterized in that: In step (3), based on 100 parts of soybean semi-hard cheese, the auxiliary materials include: 8 to 12 parts of corn oil, 1.2 to 3.0 parts of emulsified salt, and 0.1 to 0.5 parts of edible salt.

9. The method for preparing the soy cheese stick according to claim 8, characterized in that: The emulsifying salt is a combination of sodium citrate, sodium hexametaphosphate and sodium tripolyphosphate in a mass ratio of 1:(1.5-2.5):(1.5-2.5).

10. The method for preparing soybean cheese sticks according to claim 6, characterized in that: In step (3), the process conditions for chopping and mixing are: temperature 70-90°C, rotation speed 1500-2200 r / min, chopping and mixing 20-60 min; the refrigeration temperature is 2-8°C.

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