Bacterial agent for fermenting soybean milk, its application, soybean cheese stick and preparation method

Abstract

The present invention belongs to the technical fields of biological bacterium agents and functional food processing. Specifically, it relates to a bacterium agent for soybean milk fermentation and its application, a soy cheese stick and a preparation method thereof. The bacterium agent for soybean milk fermentation provided by the present invention uses Lactobacillus helveticus HL88 and Lactobacillus casei BL25 in combination. It can not only quickly acidify the soy milk, shortening the coagulation time of soy cheese, but also more fully hydrolyze soy proteins, releasing more short peptides and amino acids. In particular, it can highly produce γ-aminobutyric acid (GABA), enhancing the fermentation effect and nutritional value of soybean milk. Further, the soy cheese food prepared by fermenting with the above bacterium agent not only has a strong soy milk fragrance and a smooth texture, but is also easily digested and absorbed, improving the digestibility and reducing the soybean allergenicity, especially helping to improve exercise fatigue and enhance sleep quality. Therefore, the above bacterium agent provided by the present invention has excellent application prospects in the field of preparing functional foods for anti-fatigue and promoting sleep.

Description

Technical Field

[0001] The invention belongs to the technical field of biological microbial agents and functional food processing, and in particular, 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] To overcome the deficiencies in the prior art, the purpose of the present invention is to provide a bacterial agent for fermenting soybean milk, which has a strong ability to hydrolyze soybean protein and can simultaneously produce a high yield of γ-aminobutyric acid (GABA), and is very suitable for fermenting soybean milk to prepare functional soybean cheese products.

[0007] Meanwhile, the purpose of the present invention is also to provide the application of the above-mentioned bacterial agent for fermenting soybean milk.

[0008] Meanwhile, the purpose of the present invention is also to provide a soybean cheese stick.

[0009] Meanwhile, the purpose of the present invention is also to provide a preparation method of the above-mentioned soybean cheese stick.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is:

[0011] A bacterial agent for fermenting soybean milk, the bacterial agent for fermenting soybean milk includes: Lactobacillus helveticus HL88 and Lactobacillus casei BL25;

[0012] Among them, Lactobacillus helveticus HL88 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC NO. 28126;

[0013] Lactobacillus casei BL25 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M20241210.

[0014] The bacterial agent for fermenting soybean milk provided by the present invention simultaneously includes Lactobacillus helveticus HL88 and Lactobacillus casei BL25. Through the synergistic effect of the two strains, on the one hand, it has a strong ability to hydrolyze soybean protein, and on the other hand, it can produce a high yield of γ-aminobutyric acid (GABA), and is very suitable for fermenting soybean milk to prepare functional products such as soybean cheese.

[0015] As a preferred scheme, in the bacterial agent for fermenting soybean milk, the viable bacteria number ratio of Lactobacillus helveticus HL88 to Lactobacillus casei BL25 is 1∶(1.8 - 2.2), and more preferably 1∶2.

[0016] The application of the above-mentioned bacterial agent for fermenting soybean milk in the preparation of functional foods with anti-fatigue and / or sleep-promoting effects.

[0017] As a preferred scheme, the functional food is a soybean cheese stick.

[0018] A soybean cheese stick is prepared by fermenting soybean milk with the above-mentioned bacterial agent for fermenting soybean milk.

[0019] The soy cheese product prepared by using the bacterial agent for fermenting soy milk of the present invention not only has good flavor, 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 exercise fatigue and improve sleep quality. Therefore, this bacterial agent is suitable for application in functional foods for anti-fatigue and promoting sleep.

[0020] A preparation method of a soy cheese stick, comprising the following steps:

[0021] (1) Inoculate the bacterial agent for fermenting soy milk into soy milk for fermentation treatment, then add a coagulant to solidify to obtain curd;

[0022] (2) Cut the curd, then drain the soy whey to obtain semi-hard soy cheese;

[0023] (3) Add auxiliary materials to the semi-hard soy cheese for chopping and mixing, then shape and refrigerate to obtain the soy cheese stick.

[0024] As a preferred scheme, in step (1), the dosage ratio of soy milk, the bacterial agent for fermenting soy milk, 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.

[0025] As a preferred scheme, the viable bacteria count in the bacterial agent for fermenting soy milk is (1-2)×10 9 CFU / mL; the temperature of the fermentation treatment is 40-45°C, the fermentation time is 1-3 h; the coagulant is calcium chloride; the solidification time is 1-5 h.

[0026] As a preferred scheme, in step (3), based on 100 parts by mass of the semi-hard soy cheese, the auxiliary materials include: 8-12 parts of corn oil, 1.2-3.0 parts of emulsifying salt, and 0.1-0.5 parts of edible salt.

[0027] As a further preferred scheme, the emulsifying salt is a combination of sodium citrate, sodium hexametaphosphate, and sodium tripolyphosphate with a mass ratio of 1:(1.5-2.5):(1.5-2.5). As a more preferred scheme, the emulsifying salt is a combination of sodium citrate, sodium hexametaphosphate, and sodium tripolyphosphate with a mass ratio of 1:2:2.

[0028] As a preferred scheme, in step (3), the process conditions for chopping and mixing are: temperature 70-90°C, rotation speed 1500-2200 r / min, chopping and mixing for 20-60 min; the refrigeration temperature is 2-8°C.

[0029] The beneficial effects of the above technical solutions of the present invention are as follows:

[0030] (i) The bacterial agent for fermenting soy milk provided by the present invention uses Lactobacillus helveticus HL88 and Lactobacillus casei BL25 in combination, which can play a synergistic role. It can not only quickly acidify soy milk, shortening the curdling time of soy cheese, but also more fully hydrolyze soy protein, releasing more short peptides and amino acids. In particular, it can produce a high yield of γ-aminobutyric acid (GABA), improving the fermentation effect and nutritional value of soy milk.

[0031] (ii) The soy cheese stick prepared by fermenting with the above bacterial agent has a strong soy milk flavor and a smooth texture. It is easy to be digested and absorbed, improving the digestibility and reducing the soy allergenicity.

[0032] (iii) Experiments have confirmed that ingesting the soy cheese stick prepared by fermenting with the above bacterial agent after exercise can reduce the fatigue time. And due to the use of the compound starter, the soy cheese stick contains a relatively high content of GABA, which can effectively improve the sleep quality. Therefore, the soy cheese stick prepared by fermenting with the above bacterial agent has the functions of anti-fatigue and promoting sleep, and has good application prospects in the field of functional food preparation. Description of the Drawings

[0033] Figure 1 It is the comparison result of the curdling time of each strain in the present invention;

[0034] Figure 2 It is the comparison result of the activity of hydrolyzing soy protein of each strain in the present invention;

[0035] Figure 3 It is the comparison result of the GABA concentration of the soy milk fermented by each strain in the present invention;

[0036] Figure 4 It is the comparison result of the antigenicity of the soy milk fermented by each strain in the present invention;

[0037] Figure 5 It is the flavor determination result of the soy cheese sticks of different test groups in the present invention;

[0038] Figure 6 It is the GABA determination result of the soy cheese sticks of different test groups in the present invention;

[0039] Figure 7 It is the allergenicity determination result of the soy cheese sticks of different test groups in the present invention;

[0040] Figure 8 It is the determination result of the digestion characteristics of the soy cheese sticks of different test groups in the present invention;

[0041] Figure 9Results of the effects of soy cheese sticks in different experimental groups on the motor performance of rats in the present invention; among them, Figures A - D are the measurement results of swimming time, serum urea, liver glycogen, and blood lactic acid in sequence. Detailed implementation manners

[0042] The technical solutions and technical effects of the present invention will be clearly and completely described below in combination with specific examples and test examples. However, those skilled in the art should understand that the examples are only used to illustrate the technical solutions of the present invention and should not be regarded as limiting the protection scope of the present invention. Unless otherwise specified, the test methods used in the following examples are all conventional methods; unless otherwise specified, the raw materials used are all commonly used in the art, publicly available, or items that can be obtained through commercial channels.

[0043] Among them, in the following examples, the preservation information involved is as follows:

[0044] Preservation information 1

[0045] Preservation name: Lactobacillus helveticus HL88, Latin name Lactobacillus helveticus HL88;

[0046] Preservation number: CGMCC NO.28126;

[0047] Preservation unit: China General Microbiological Culture Collection Center (CGMCC);

[0048] Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0049] Preservation date: August 9, 2023.

[0050] Preservation information 2

[0051] Preservation name: Lactobacillus casei BL25, Latin name Lactobacillus casei BL25;

[0052] Preservation number: CCTCC NO: M 20241210;

[0053] Preservation unit: China Center for Type Culture Collection (CCTCC);

[0054] Preservation address: Wuhan University;

[0055] Preservation date: June 7, 2024.

[0056] In the following embodiments of the present invention, the composition and preparation method of the MRS medium are as follows: Take 12 g of peptone, 6 g of yeast extract, 6 g of beef extract, 18 g of glucose, 5 g of sodium acetate, 2.15 g of ammonium citrate, 1 g of Tween, 0.58 g of magnesium sulfate, 0.05 g of manganese sulfate, and 2 g of dipotassium hydrogen phosphate. Make up the volume to 1 L with pure water, adjust the pH to 6.5, and sterilize it at 121 °C for 15 min using an autoclave for later use. In other embodiments, the MRS medium can be obtained from commercial channels, and the present invention does not make any special limitations on it.

[0057] In the following embodiments of the present invention, the preparation process of the soybean milk adopted is as follows:

[0058] ① Soybean pretreatment: After washing and removing impurities from the soybeans, soak them in water at a material-liquid ratio of 1:5 to 1:10 for 12 to 24 h to fully swell the soybeans. Then boil the soybeans at 100 °C for 20 to 30 min, take them out and cool for later use.

[0059] ② Preparation of soybean milk: Mix the cooked soybeans with water at a material-liquid ratio of 1:4 to 1:6. First, coarsely crush them with a blender for 1 to 3 min, then wet-crush them with a colloid mill for 5 to 10 min. Adjust the grinding gap of the colloid mill to 5 to 20 μm and the rotational speed of the colloid mill to 2,800 to 3,000 r / min. After crushing, filter through a 120-mesh filter cloth 2 to 3 times, sterilize the filtered emulsion at 108 °C for 15 min, and then cool it to 37 °C to obtain soybean milk for later use.

[0060] In other embodiments, the soybean milk can also be prepared by other preparation processes well-known in the art. The present invention does not make any special limitations on it, and it will not affect the fermentation effect of the bacterial agent of the present invention.

[0061] Example 1

[0062] This example provides a bacterial agent for fermenting soybean milk, including: Lactobacillus helveticus HL88 and Lactobacillus casei BL25; Lactobacillus helveticus HL88 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO. 28126; Lactobacillus casei BL25 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20241210. In this bacterial agent, the quantity ratio of Lactobacillus helveticus HL88 to Lactobacillus casei BL25 is 1:2.

[0063] The separation, screening and identification process of Lactobacillus helveticus HL88, Lactobacillus casei BL25 involved in this embodiment and other comparative strains (Lactobacillus plantarum LP11, Lactobacillus plantarum LP12, Lactobacillus plantarum LP14, Lactobacillus helveticus HL01, Lactobacillus helveticus HL03, Lactobacillus helveticus HL13, Lactobacillus helveticus HL14, Lactobacillus helveticus HL101) involved in the following test example 1 are as follows:

[0064] (1) Strain source

[0065] 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.

[0066] 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, Right Middle Banner, Xing'an League, Inner Mongolia, Left 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.

[0067] Table 1. Strain isolation information

[0068] Number Strain Name Isolation Source Isolation Location LP11 Lactiplantibacillus plantarum LP11 Yoghurt Xiwu Banner, Xilingol League, Inner Mongolia LP12 Lactiplantibacillus plantarum LP12 Yoghurt Xiwu Banner, Xilingol League, Inner Mongolia BL14 Lactobacillus casei BL14 Yoghurt Left Middle Banner, Tongliao City, Inner Mongolia BL25 Lactobacillus casei BL25 Yoghurt Xiahe County, Gannan Tibetan Autonomous Prefecture HL01 Lactobacillus helveticus HL01 Yoghurt Right Middle Banner, Xing'an League, Inner Mongolia HL03 Lactobacillus helveticus HL03 Yoghurt Right Middle Banner, Xing'an League, Inner Mongolia HL13 Lactobacillus helveticus HL13 Yoghurt Xinyuan County, Ili Kazakh Autonomous Prefecture, Xinjiang HL14 Lactobacillus helveticus HL14 Yoghurt Xinyuan County, Ili Kazakh Autonomous Prefecture, Xinjiang HL88 Lactobacillus helveticus HL88 Yoghurt Xinyuan County, Ili Kazakh Autonomous Prefecture, Xinjiang HL101 Lactobacillus helveticus HL101 Yoghurt Left Middle Banner, Tongliao City, Inner Mongolia

[0069] (2) Isolation, culture and routine identification of lactic acid bacteria

[0070] ① Isolation, purification and preservation of lactic acid bacteria in samples

[0071] 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.

[0072] 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.

[0073] Among them, Lactobacillus helveticus HL88 is Gram-positive, with rod-shaped cells, round ends, arranged in short chains, without spores and metachromatic granules. The colonies are irregular in shape, rough on the surface, flat, with uneven edges, light yellow and opaque. Lactobacillus casei BL25 is Gram-positive, with short rod-shaped cells, arranged singly, and the colonies are round, small, relatively moist and white.

[0074] ② Molecular identification of lactic acid bacteria isolates

[0075] The genomic DNA of lactic acid bacteria isolates was extracted and the 16S rDNA gene was amplified by PCR. The sequencing results were subjected to homology alignment by BLAST in the NCBI database. If the homology of the 16S DNA sequence of the sequenced strain and the type strain is greater than 97%, then the strain can be identified as belonging to the same species as the type strain.

[0076] Among them, the homology of the 16S DNA sequence of Lactobacillus helveticus HL88 and the type strain is 99.45%, so it is confirmed as Lactobacillus helveticus. The homology of the 16S DNA sequence of Lactobacillus casei BL25 and the type strain is 99.37%, so it is confirmed as Lactobacillus casei. The isolation and identification processes of other strains are similar to those of Lactobacillus helveticus HL88 and Lactobacillus casei BL25, and the identification results are shown in Table 1, which will not be elaborated in this invention.

[0077] ③ Microbial activation and preparation of bacterial suspension

[0078] The experimental strains were all stored in a glycerol aqueous solution at -80 °C and 20% (v / v). Before the experiment, the strains were inoculated into sterile MRS medium at 18 °C and incubated in a constant temperature incubator at 37 °C for 24 h. After incubation, Lactobacillus helveticus HL88 and Lactobacillus casei BL25 were mixed, centrifuged at 2000 rpm for 10 min, the medium was removed, and the precipitate was harvested. The precipitate was washed twice with sterile phosphate buffer (PBS). The washed precipitate was diluted with PBS to a suspension with an effective bacterial concentration of 1.8×10 9 CFU / mL, which is the bacterial agent for fermenting soy milk in Example 1. In the bacterial agent, the number ratio of Lactobacillus helveticus and Lactobacillus casei is 1:2.

[0079] Example 2

[0080] This example provides a soy cheese stick, which is prepared by fermenting soy milk with the bacterial agent for fermenting soy milk in Example 1.

[0081] The preparation method of this soy cheese stick specifically includes the following steps: ·

[0082] (1) Take the sterilized soy milk, pour it into a small cheese vat, inoculate the bacterial agent for soy milk fermentation in Example 1, ferment at 42 °C for 2 hours, then add a coagulant (CaCl2) and coagulate at 48 °C for 2 hours to obtain curd; wherein, the dosage ratio of soy milk, bacterial agent, and coagulant is 100 g∶3 mL∶0.3 g; the viable count of the bacterial agent for soy milk fermentation is 1.8×10 9 CFU / mL.

[0083] (2) Cut the curd into 1-cm pieces, transfer it to a perforated mold with a layer of gauze to drain, and press overnight to drain the soy whey to obtain semi-hard soy cheese;

[0084] (3) Pour the semi-hard soy cheese into a chopping mixer, add auxiliary materials (based on 100 parts by mass of the semi-hard soy cheese, add 10 parts of corn oil, 2 parts of emulsifying salt, and 0.3 part of edible salt), chop and emulsify at 80 °C and 1800 r / min for 30 min, pour it into a mold for shaping, and then refrigerate it in a 4 °C refrigerator for 24 h to obtain the soy cheese stick of this example. Among them, the emulsifying salt is a combination of sodium citrate, sodium hexametaphosphate, and sodium tripolyphosphate with a mass ratio of 1∶2∶2.

[0085] The following is the determination of the curdling time, hydrolyzed soy protein activity, GABA content, and allergenicity of the above-mentioned bacterial agent involved in the present invention, and the flavor texture, GABA content, allergenicity, digestion characteristics, and animal tests of the prepared soy cheese stick are carried out to confirm the technical effects 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, one-way ANOVA is used for the data between different groups by SPSS18.0 software, and GraphPad Prism5.0 and Origin 9.0 software are used for plotting, and P<0.05 is considered statistically significantly different. The test process and test results are as follows.

[0086] Test Example 1. Evaluation of the Fermentation Effect of Strains

[0087] 1.1. Determination of the Curdling Time of Strains

[0088] Curdling time: It 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 curdling time determination results of each strain (LP11, LP12, BL14, BL25, HL01, HL03, HL13, HL14, HL88, HL101) are as Figure 1 shown.

[0089] From Figure 1It can be seen that there are significant differences in the curdling time of each strain in soy milk. Among them, strains BL25 and BL14 have the fastest curdling time, and the curdling time of strain BL25 is only 7.3 h. A shorter curdling time indicates that the strain can grow rapidly in the soy milk environment, produce acid and curdle the milk by utilizing carbohydrates.

[0090] 1.2 Determination of the activity of strains in hydrolyzing soy protein

[0091] The strain types are the same as those in Section 1.1. The method for determining the activity of strains in hydrolyzing soy protein is as follows: Take 100 g of sterilized soy milk, and add 3 mL of different bacterial agents (the viable count is 1.8×10 9 CFU / mL) and ferment at 42 °C for 24 h to obtain fermented milk; Take 10 mL of the fermented milk and add an equal volume of 10% trichloroacetic acid for precipitation. After standing at room temperature for 1 h, centrifuge (15000 r / min, 30 min), take the supernatant of the fermented sample, and store it at low temperature for later use. The supernatant of the fermented sample is determined by o-phthalaldehyde (OPA). The reagent solution is prepared by mixing three different solutions; Solution 1 consists of 3.81 g of sodium tetraborate and 100 mg of sodium dodecyl sulfate (SDS), and is prepared by dissolving in 75 mL of distilled water; Solution 2 is prepared by dissolving 80 mg of o-phthalaldehyde in 2 mL of 96% ethanol; Solution 3 is prepared by dissolving 88 mg of dithiothreitol (DTT) in 50 mL of MilliQ water. The reagent solution is prepared on the day of analysis, and Solutions 1-3 are mixed shortly before use. When testing, thaw the supernatant samples in triplicate, add 30 μL to a 96-well plate, then add 270 μL of the reagent solution, incubate the plate in the dark for 2 minutes, and read the absorbance at 340 nm using a microplate reader to determine the free amino group content, and then evaluate the activity of each strain in hydrolyzing soy protein. The results are as Figure 2 shown.

[0092] It can be Figure 2 seen that there are significant differences in the protein hydrolysis ability of each strain in soy milk. Compared with other strains, strains HL88 and HL101 have the highest soy protein hydrolysis activity. A higher protein hydrolysis activity indicates that the strain can hydrolyze soy protein more fully during the fermentation of soy milk, obtaining more short peptides and amino acids, which is beneficial to improving the flavor and increasing the digestibility.

[0093] 1.3 Determination of GABA

[0094] The method for determining the concentration of γ-aminobutyric acid (GABA) in the fermented milk obtained by fermenting soy milk with the strain is as follows: Take 10 mL of the fermented milk obtained in Section 1.2 and stir well, add an equal volume of 10% trichloroacetic acid for dilution, filter after standing at room temperature for 1 h, and centrifuge the obtained solution (15000 r / min, 30 min), then take the supernatant for standby. The content of GABA in the supernatant of the fermented broth of the strain to be tested is determined by high performance liquid chromatography (HPLC). HPLC chromatographic conditions: Use a Luna-C18 chromatographic column (250 mm × 4.6 mm), column temperature 30 °C; injection volume is 5 μL; the two mobile phases are 20 mmol / L sodium acetate aqueous solution (A) and methanol (B), and after optimization, the volume ratio A:B = 55:45 is determined; the flow rate is set at 1 mL / min; wavelength 334 nm.

[0095] Take 0.10 g of GABA standard product, dissolve it with ultrapure water and make up the volume to a 50 mL volumetric flask, shake well to prepare a 2 mg / mL GABA standard solution. Then dilute this standard solution to standard solutions of 0.5, 0.8, 1.0, 1.2, 1.5 mg / mL respectively for standby. Add 200 μL of the fermented milk supernatant or GABA standard product solution to a 5 mL EP tube, add 600 μL of OPA derivatizing agent, shake well, add 800 μL of potassium dihydrogen phosphate buffer solution, react in the dark for 90 s, filter through a 0.22 μm filter membrane, and then inject the sample immediately. The time for the whole derivatization process is controlled within 3 min. The GABA test results are as Figure 3 shown.

[0096] As Figure 3 can be seen, compared with other strains, the strain BL25 produces the highest content of GABA. And GABA is crucial for relieving fatigue and improving sleep quality.

[0097] 1.4. Determination of allergenicity

[0098] The method for determining the antigenicity of the soy milk obtained by fermenting the strain is as follows: The antigenicity of soy milk is determined by ELISA. During the test, add 100 μL of the fermented milk sample (1 μg / mL) diluted with PBS solution to a 96-well plate, place it overnight at 4 °C, then block it with skim milk (50 mL / L, pH 7.2) at 37 °C for 1 h. After washing with PBS, incubate each well with the serum for 1 h, wash, and incubate with the serum of soy allergy patients for 1 h, and wash 3 times with PBST washing solution. Add 100 μL of TMB chromogenic solution to each well, react in the dark at 37 °C for 15 min, add 50 μL of sulfuric acid termination solution to each well to terminate the reaction, and measure the absorbance value of the sample at 450 nm, which reflects the IgE binding ability and characterizes the antigenicity of soy milk. The antigenicity test results are as Figure 4 shown.

[0099] It can be seen from Figure 4 that compared with other strains, the antigenicity of the fermented soy milk by strain HL88 is the lowest, which may be due to the higher proteolytic activity of strain HL88, which hydrolyzes and destroys the soybean protein allergen during the fermentation process.

[0100] Based on the above screening results of strains, strain BL25 has a shorter curdling time and the ability to produce high levels of GABA; strain HL88 has a higher ability to hydrolyze soybean protein and lower antigenicity. Therefore, in the present invention, strain BL25 and strain HL88 are used for the subsequent fermentation preparation of soy cheese.

[0101] Experimental Example 2: Performance evaluation of soy cheese sticks

[0102] Soy cheese sticks fermented with a single Lactobacillus casei BL25 (Group A) and soy cheese sticks fermented with a single Lactobacillus helveticus HL88 (Group B) were respectively prepared. During the preparation process, only the type of inoculant was changed, and other conditions were the same as in Example 2. The soy cheese sticks fermented with a single inoculant were compared with the soy cheese sticks (Group C) fermented with the compound inoculant of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in Example 2 of the present invention.

[0103] 2.1 Determination of the flavor of soy cheese sticks

[0104] The flavor of the soy cheese sticks prepared in each group was determined as follows:

[0105] Electronic nose: Accurately weigh 10 g of the soy cheese stick sample, transfer it into a 40 mL sample bottle (with a lid), and use the electronic nose to test at room temperature. The sensor was cleaned with air. The cleaning time was 120 s. The gas in the sample was inhaled into the electronic nose using a vacuum pump. The inlet speed was 1 L / min, and the detection time was 120 s.

[0106] Electronic tongue: 5 g of the evenly crushed soy cheese sample was mixed in 100 mL of deionized water and centrifuged at 6000×g and 20 °C for 20 minutes. The supernatant was filtered with filter paper, and the clear liquid was used for analysis. The acidic, bitter, astringent, salty, umami, sweet sensors and the reference electrode of the electronic tongue device were activated in advance, and the activation time was 24±2 h. The liquid sample without precipitation was poured into the sample cup for taste determination.

[0107] The flavor test results are as Figure 5 shown. Among them, Figure 5Among them, A: the group of soy cheese sticks prepared by fermenting Lactobacillus casei BL25; B: the group of soy cheese sticks prepared by fermenting Lactobacillus helveticus HL88; C: the group of soy cheese sticks prepared by co-fermenting Lactobacillus casei BL25 and Lactobacillus helveticus HL88, the same hereinafter. The meanings of each number are as follows: Electronic nose: 1 - Aromatic compounds; 2 - Nitrogen oxides; 3 - Ammonia, aromatic molecules; 4 - Hydrides; 5 - Olefins, aromatics, polar molecules; 6 - Alkanes; 7 - Sulfur compounds; 8 - Detection of alcohols, some aromatic compounds; 9 - Aromatic compounds, organic compounds of sulfur; 10 - Alkanes and aliphatics. Electronic tongue: 1 - Sour taste; 2 - Bitter taste; 3 - Astringent taste; 4 - Bitter aftertaste; 5 - Astringent aftertaste; 6 - Umami taste; 7 - Rich taste; 8 - Salty taste.

[0108] From Figure 5 it can be seen that compared with the cheese fermented by single bacteria, the rich taste of the cheese fermented by the compound bacterium agent of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention is more obvious, the sour taste is less, and the overall flavor and taste are more acceptable.

[0109] 2.2 Determination of GABA in soy cheese sticks

[0110] The method for determining the GABA content of the soy cheese sticks prepared in each group is as follows: Take 10 g of soy cheese sample and grind it into powder, add an equal volume of 10% trichloroacetic acid for dilution, filter after standing at room temperature for 1 h, and take the supernatant after centrifuging the obtained solution (15000 r / min, 30 min) for standby. The GABA determination method is shown in Section 1.3. The results are as Figure 6 shown.

[0111] From Figure 6 it can be seen that compared with the other two groups of single-bacteria fermented cheese, the GABA content in the cheese of group C is the highest. It shows that the mixed fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention can increase the GABA content in soy cheese. GABA is mainly produced by Lactobacillus casei BL25. The possible reason for the production of more GABA by mixed fermentation is that Lactobacillus helveticus HL88 hydrolyzes soy protein to generate more short peptides and amino acids, and these nutrients promote the growth of Lactobacillus casei BL25, and then Lactobacillus casei BL25 produces a higher content of GABA.

[0112] 2.3 Determination of the allergenicity of soy cheese sticks

[0113] The method for determining the allergenicity of the soy cheese sticks prepared in each group is as follows: First, grind and crush the soy cheese sample, and dilute the sample with PBS to 1 μg / mL for determination. The specific determination method is shown in Section 1.4. The results are as Figure 7 shown.

[0114] From Figure 7It can be seen that, compared with the other two groups of single-strain fermented cheeses, the C-group cheese has the lowest allergenicity. This shows that the mixed-strain fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention can reduce the allergenicity of soy cheese. The soy protein hydrolysis effect of the mixed-strain fermentation is better, and it can hydrolyze more soy protein allergens.

[0115] 2.4 Determination of the Digestibility of Soy Cheese Sticks

[0116] The determination method for the digestibility of the soy cheese sticks prepared in each group is as follows:

[0117] In vitro digestion process: Prepare the simulated gastric juice and simulated intestinal juice solutions as 1.25× concentrated solutions. According to the previously measured protein concentration, disperse the soy cheese sample equivalent to 150 mg of protein into 9.6 mL of SGF, and homogenize it in an ice bath using a biological homogenizer (5500 rpm, 2×30 s, cooling interval 60 s). To initiate gastric digestion, change the pH of the mixed solution to 3.0 with hydrochloric acid (1 M), and add pepsin to reach a final enzyme activity of 2000 U / mL. Incubate the sample at 37 °C for 2 h, and terminate the digestion by adjusting the pH to 7.5 with 1 mol / L NaOH. For the intestinal stage of digestion, mix the gastric chyme with 10 mL of SIF, then add trypsin and hydrolyze at 37 °C for 2 h, and stop the digestion by heating the sample at 100 °C for 5 minutes to obtain the digestion solution.

[0118] Determination of the digestibility: Mix the digestion solution with 3 volumes of ethanol respectively, and let it stand at 4 °C for 12 h. Then centrifuge the mixture at 8000 g and 4 °C for 20 minutes to remove undigested proteins and high-molecular-weight peptides. Determine the protein content of the precipitate using a BCA protein assay kit according to the instructions. The protein digestibility is calculated according to the following formula: Digestibility % = (W0 - W1) / W × 100; where W0 and W1 represent the protein content of the blank and the sample after digestion, and W represents the protein content of the sample before digestion.

[0119] Determination of peptide content: Dissolve 160 mg of OPA 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. Then add deionized water to a final volume of 200 mL. Before the experiment, mix the digest with 10% trichloroacetic acid (TCA) in a 1:1 ratio and centrifuge at 10,000 g for 10 minutes at 4 °C to collect the supernatant. Peptides with a molecular weight of <10 kDa were collected through a membrane. Then, mix 50 μL of the filtered sample into 2 mL of the OPA solution and react the mixture in the dark for 2 min. Measure the absorbance at 340 nm using a multi-functional microplate reader and use casein as a standard. The test results of the digestion characteristics (digestion rate and peptide content) are as Figure 8 shown.

[0120] As Figure 8 can be seen, the digestion rate of the cheese in group C is higher than that of the cheeses in groups A and B, indicating that the mixed fermentation of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention improves the digestibility of soy cheese (left figure). At the same time, the present invention measured the polypeptide content in the digests of the three groups of cheeses (right figure), and the results showed that the polypeptides in the digest of the cheese in group C were significantly higher than those in the cheeses in groups A and B, indicating that the soy cheese fermented by the mixed bacteria of Lactobacillus casei BL25 and Lactobacillus helveticus HL88 in the present invention can produce more peptides after simulated digestion and is easy to digest.

[0121] 2.5. Investigation of the effects of cheese sticks on animal fatigue and sleep in animal experiments

[0122] Animal experiment design: Healthy female Sprague-Dawley (SD) rats (60.0 ± 10.0 g, 3 w) were purchased from Harbin Medical University, China. The rats were raised in accordance with the guidelines for animal management and use, with three rats in each cage, and the standard cage allowed the rats to freely access food and water. The environmental temperature was maintained at 22 ± 2 °C and the humidity at 45 ± 5%, and the light-off / dark cycle was 12 hours.

[0123] Exercise performance test: All rats were first allowed to freely consume standard feed for one week for adaptation and observation. After the adaptation period, the rats were divided into four groups: a control group (N), a group fed with cheese sticks prepared with single-strain Lactobacillus casei BL25 (Group A), a group fed with cheese sticks prepared with single-strain Lactobacillus helveticus HL88 (Group B), and a group fed with cheese sticks prepared with the compound starter of Lactobacillus casei BL25 + Lactobacillus helveticus HL88 of Example 2 (Group C). There were 6 rats in each group. After the adaptation period, each group was fed with a standard diet (the standard diet was purchased from Beijing Keao Xieli Feed Co., Ltd.) for 4 weeks. Group A was intragastrically administered 1 g of soy cheese prepared by single-strain fermentation of the corresponding Lactobacillus helveticus HL88 (dissolved in PBS) every day. Group B was intragastrically administered 1 g of soy cheese prepared by single-strain fermentation of the corresponding Lactobacillus casei BL25 (dissolved in PBS) every day. Group C was intragastrically administered 1 g of soy cheese prepared by compound fermentation of the corresponding Lactobacillus helveticus HL88 and Lactobacillus casei BL25 (dissolved in PBS) every day. The control group (Group N) was infused with an equal volume of sterile PBS as a solvent. Intragastric administration was carried out continuously for 4 weeks. 40 minutes after the last intragastric administration, a rat negative gravity exhaustion swimming experiment was carried out. The rats were placed in a plastic container with a temperature of 25°C and a water surface height of about 25 cm, and an iron block with a mass of 10% of the body weight was loaded on the rat's tail for the swimming experiment. The time when the rat sank into the water surface and could not float out for 10 s was used as the negative gravity exhaustion swimming time, and the serum urea, liver glycogen, and blood lactic acid contents of the rats in each group were tested. The effects of soy cheese sticks on the exercise performance of rats are as Figure 9 shown.

[0124] Figure 9 shown. Among them, A to D are the measurement results of swimming time, serum urea, liver glycogen, and blood lactic acid in sequence. As Figure 9 can be seen, after the cheese sticks prepared by compound fermentation of Lactobacillus helveticus HL88 and Lactobacillus casei BL25 act on rats, compared with the control group, the swimming time of rats is significantly increased (P < 0.01) ( Figure 9 A); the serum urea content of rats is significantly decreased (P < 0.01) ( Figure 9 B); the liver glycogen content of rats is significantly increased (P < 0.01) ( Figure 9 C); the blood lactic acid content of rats is significantly decreased (P < 0.01) ( Figure 9 D). It shows that the compound fermentation of Lactobacillus helveticus HL88 and Lactobacillus casei BL25 used in the present invention has a significant anti-fatigue effect on rats.

[0125] Direct sleep experiment: According to the experimental grouping in the above-mentioned motor performance section, rats in each group were intragastrically administered with soy cheese solutions of different groups (1 g of soy cheese sample was dissolved in PBS in each case), and the control group was infused with an equal volume of PBS solvent. Then, the sleep state of the rats was observed. The disappearance of the righting reflex was used as an indicator of sleep. When the rat was in the dorsal recumbent position, immediately righting its body position indicated that it had not entered sleep. If it could not right itself within 30 - 60 s, it was considered that the righting reflex had disappeared and it had entered sleep. The recovery of the righting reflex was the sign of the animal's awakening, and the sleep time of the animal could be considered as the time from the disappearance of the righting reflex to its recovery. The number of sleeping animals and the corresponding sleep times in the blank control group and the sample intake group were recorded. The results are shown in Table 2.

[0126] Table 2. Effects of soy cheese sticks on the sleep time of rats treated with sodium pentobarbital

[0127] Group Soy Cheese Treatment Sleep Time / min Prolongation Rate / % Control Group Sterilized PBS <![CDATA[26.85±7.41 d > <![CDATA[0 d > Group A BL25 Cheese <![CDATA[35.12+8.97 b > <![CDATA[30.80 b > Group B HL88 Cheese <![CDATA[30.66+9.13 c > <![CDATA[23.43 c <!-- 9 -->]]> Group C BL25 + HL88 Cheese <![CDATA[42.97+9.06 a > <![CDATA[60.04 a >

[0128] As can be seen from Table 2, compared with the other three groups, the sleep time of the rats in group C was significantly prolonged, indicating that the soy cheese co-fermented with Lactobacillus helveticus HL88 and Lactobacillus casei BL25 in the present invention had a better sleep effect on rats after exercise.

[0129] In summary, the bacterial agent provided by the present invention for soy milk fermentation, using Lactobacillus helveticus HL88 and Lactobacillus casei BL25 in combination, can not only quickly acidify soy milk and shorten the curdling time of soy cheese, but also more fully hydrolyze soy protein and release more short peptides and amino acids. In particular, it can produce a high yield of γ-aminobutyric acid (GABA), improving the fermentation effect and nutritional value of soy milk. In particular, the soy cheese stick prepared by fermenting with the above-mentioned compound bacterial agent has a good flavor, low allergenicity, a high content of GABA and good digestive characteristics. More importantly, through animal experiments, the present invention found that the soy cheese stick obtained by co-fermenting Lactobacillus casei BL25 and Lactobacillus helveticus HL88 can significantly improve exercise fatigue and enhance sleep quality. Therefore, the above-mentioned bacterial agent provided by the present invention has good application prospects in the field of preparing functional foods with anti-fatigue and / or sleep-promoting effects.

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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 1×10 9 ~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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