Agile combined lactobacillus and application thereof

Abstract

The invention relates to the technical field of food microorganisms, in particular to agile combined lactobacillus and application thereof. The invention provides an agile combined lactobacillus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.33722. The agile combined lactobacillus has the advantages that the preservation number is CGMCC NO.33722; according to the invention, the lactic acid bacteria capable of promoting rapid propagation and having a modification effect on soybean protein are separated from the soybean whey water, and the lactic acid bacteria can adapt to relatively high culture temperature and is high in propagation speed. The lactic acid bacterium agent prepared by utilizing the microbial strain can be applied to the production process of the soybean protein, can be used for replacing hydrochloric acid and improving the dispersity of the soybean protein, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of food microbiology, and more particularly to an agile *Lactobacillus* species and its applications. Background Technology

[0002] Soy protein isolate, a high-quality plant protein source rich in nutrients and diverse functional properties, is increasingly widely used in nutritional protein powders and instant beverages due to its excellent amino acid composition, good biocompatibility, and rich functional characteristics. However, currently available soy protein isolates face key technical challenges in their practical application in nutritional protein powders and instant beverages, including insufficient dispersibility and solubility, which affects the consumer product experience. Furthermore, the traditional alkali-dissolving and acid-precipitating process results in a high salt content in the final product due to the addition of acid.

[0003] To address the aforementioned issues, relevant improvement research has been conducted within the industry, but many shortcomings still exist. Chinese invention patent CN110250316B discloses a process for producing rapidly dissolving soy protein isolate. This process uses low-temperature defatted soybean meal and gluten as raw materials, mixes them with water, and then adds phytase, neutral protease, and transglutaminase for enzymatic hydrolysis. However, while this method improves the dispersibility of the soy protein isolate, the dispersion time is still as high as 15 seconds. Chinese invention patent CN111587947A discloses a method for preparing highly dispersible and stable soy protein isolate. This method mainly modifies soy protein through enzymatic hydrolysis with flavor proteases, but it does not evaluate the modification effect on the final product.

[0004] Besides the enzymatic hydrolysis methods mentioned above for improving the functionality of soybean protein, microbial methods for improving the functionality of soy protein isolate have also been studied in recent years. However, these studies have focused more on reducing the beany odor and anti-nutritional factors, with less research on improving dispersibility and solubility. Chinese invention patent CN108041254B discloses a soy protein isolate with no beany odor and high emulsification properties and its preparation method, which uses lactic acid bacteria fermentation mainly for removing the beany odor rather than improving functionality.

[0005] Therefore, screening out microbial strains from nature that can improve the functional properties of soybean protein through fermentation, and replacing the use of added acids such as hydrochloric acid / citric acid in traditional soybean protein production processes, has become one of the research focuses. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a lactic acid bacteria that can not only improve the functionality of soybean protein, but also replace the use of external acids such as hydrochloric acid / citric acid in the traditional soybean protein production process.

[0007] Another objective of this invention is to provide the application of the above-mentioned lactic acid bacteria in soybean protein production and related fields such as microbial agents.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution;

[0009] A strain of *Ligilactobacillus agilis*, with accession number CGMCCNO.33722.

[0010] After long-term research, this invention has screened a microbial strain from soybean whey that can improve the dispersibility of soybean protein, and named this strain R-2. Experiments have shown that this strain can be applied to soybean protein isolate production lines, effectively improving protein dispersibility, and can replace the use of external acids such as hydrochloric acid / citric acid in traditional soybean protein production processes.

[0011] The present invention isolates and obtains Ligilactobacillus agilis R-2. This strain is inoculated into soy milk and cultured at 35°C and 150 r / min for 4-24 h. The fermentation broth after culture is centrifuged to obtain a solid precipitate. The solid precipitate is then freeze-dried to obtain soy protein. The results show that the dispersibility of soy protein prepared by fermentation with Ligilactobacillus agilis is improved by more than 40%.

[0012] Meanwhile, the present invention, by combining Lactobacillus agilis R-2 with fermented soy milk, eliminates the need for additional acids such as hydrochloric acid and citric acid. The pH of the fermented soy milk is the acid precipitation isoelectric point, which greatly saves on acid used in production and thus reduces the salt content in the finished product.

[0013] In view of the excellent effects of Ligilactobacillus agilis R-2, the present invention provides the application of this bacterium in the production of soybean protein and the preparation of microbial agents for modifying soybean protein, wherein the soybean protein production includes, but is not limited to, various production lines that require improvement of soybean protein dispersibility.

[0014] This invention provides a microbial agent comprising the *Lactobacillus agileis* as described in the above technical solution.

[0015] The microbial agent described in this invention is a liquid microbial agent or a solid microbial agent, wherein the number of microbial cells in the liquid microbial agent is 1×10⁻⁶. 9 - 5×10 9 cfu / mL, solid bacterial agent cell count 2×10 9 - 3×10 10 cfu / g.

[0016] This invention also provides a method for preparing a soybean protein-modified microbial agent, which is obtained by activating and fermenting the Lactobacillus agile described in the above technical solution.

[0017] More preferably, the method involves inoculating *Lactobacillus agile* R-2 with accession number CGMCC NO.33722 into MRS liquid medium for activation, and then replacing it with modified MRS liquid medium for expanded culture to obtain liquid microbial inoculum.

[0018] The modified MRS liquid culture medium includes glucose, yeast powder, soy protein isolate, beef extract, sodium acetate, KH2PO4, ammonium citrate, MgSO4, MnSO4, and Tween 80.

[0019] Preferably, the modified MRS liquid culture medium formula is as follows: glucose 0.5%~2.5%, yeast powder 0.3%~0.8%, soy protein isolate 1%~3%, sodium acetate 0.3%~0.5%, KH2PO4 0.05%~0.2%, ammonium citrate 0.05%~0.2%, MgSO4 0.01%~0.05%, MnSO4 0.01%~0.02%, and Tween 80 0.05%~0.1%.

[0020] Preferably, the fermentation can be divided into two steps: seed fermentation tank fermentation and fermentation tank fermentation. The difference between the two lies in their fermentation specifications, and the appropriate method can be selected based on actual needs. In a specific embodiment of the present invention, the preparation process of the liquid microbial agent is as follows:

[0021] (1) Activation of bacterial strain: Take 1~5uL of frozen Lactobacillus agileensis and inoculate it into a liquid medium containing 5mL MRS, and incubate at 30~40℃ and 150r / min for 12~24h;

[0022] (2) Liquid seed preparation: The activated strains were transferred to 200 mL of MRS liquid culture medium and cultured at 30-40℃ and 150 r / min for 12-24 h.

[0023] (3) Seed fermentation tank: The prepared liquid seeds are inoculated into a 30L seed tank with a liquid volume of 60-70% at an inoculation rate of 5%-10% v / v. The stirring speed is 50-140 rpm, the culture temperature is 30-40℃, the fermentation tank pressure is 0.01-0.2 MPa, and the fermentation cycle is 6-20 h.

[0024] (4) Fermentation in a fermenter: The bacterial culture obtained from the seed fermenter was inoculated into the culture medium of a 300L fermenter with a volume of 60% to 70% at an inoculation rate of 5% to 10% v / v. The culture medium and culture conditions were the same as those for the seed fermenter fermentation. After fermentation, the number of bacteria reached 1×10⁻⁶. 9~ 5×10 9 The concentration of cfu / mL indicates that the liquid bacterial agent is obtained by removing the culture medium from the tank after fermentation.

[0025] The fermentation culture medium of the present invention comprises the following raw materials in the following mass percentages: glucose 2%, yeast powder 0.5%, soy protein isolate 2%, sodium acetate 0.5%, KH2PO4 0.2%, ammonium citrate 0.2%, MgSO4 0.05%, MnSO4 0.018%, Tween 80 0.1%, and the balance being water.

[0026] Furthermore, this invention can also prepare a solid microbial agent by adding trehalose, soy protein isolate, and inulin to the liquid microbial agent. After the above-mentioned fermented liquid agent is treated with adsorption by adding 2-3% trehalose, 2-5% soy protein isolate, and 2-3% inulin at a mass ratio, the liquid agent fermentation broth is centrifuged and separated to obtain solid microbial cells. These cells are then dried to obtain microbial powder. The moisture content of the microbial powder is controlled below 10%, and the effective viable count is tested to be 2 × 10⁻⁶. 9 ~3×10 10 cfu / g.

[0027] The present invention also provides the application of the above-described Agile Combined Lactobacillus or bacterial agent in the preparation of soy protein isolate.

[0028] Depending on the application, this invention also provides a method for modifying soybean protein, based on a traditional alkali-dissolving and acid-precipitating production line (see details for the preparation process). Figure 1 The Agile Combined Lactobacillus or the bacterial agent described in the above technical solution is inoculated into the mixed soy milk. Figure 1 (Dashed box), after fermentation, the modified soybean protein is obtained through sedimentation, flash evaporation, spray drying, and packaging.

[0029] More preferably, the following method is used: the Lactobacillus agileensis R-2 with accession number CGMCC NO.33722 or the microbial agent prepared therefrom is added to the mixing tank of soybean milk in the soybean protein production line. The reference usage of the liquid microbial agent is 0.5%-10%; specifically, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0030] The recommended usage for solid microbial inoculants is 0.2% to 1.5%. Specifically, these percentages can be 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, and 1.5%.

[0031] When using the above-mentioned Agile combined with Lactobacillus or microbial agents prepared therefrom for soybean protein modification, the temperature should be controlled at 30~50℃ and the initial pH at 6.0~8.5.

[0032] The fermentation time described in this invention is 4 to 12 hours; specifically, it can be 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0033] The modified soy protein isolate provided by this invention is prepared by the preparation method described in the above technical solution.

[0034] The specific preparation method described above has been clearly described in this invention and will not be repeated here.

[0035] When modifying soybean protein, the agile combined with Lactobacillus fermentation described in this invention can lower the pH to 4.5 or below, with the fermentation endpoint being the isoelectric point. This eliminates the need for external acids such as hydrochloric acid or citric acid to adjust the pH to the isoelectric point to precipitate soybean protein, reducing the amount of external acid used and lowering the salt content of the final product. In addition, the dispersibility of the fermented and modified soybean protein is improved by more than 40%, resulting in a highly dispersible soybean protein. This soybean protein has the advantages of good dispersibility and low beany odor compared to existing soybean protein products.

[0036] This invention provides a method for preparing soybean protein through continuous fermentation modification using a "simultaneous input and output" process, which enables continuous production and reduces the cost of adding external acid multiple times.

[0037] As can be seen from the above technical solutions, this invention isolates a strain of *Lactobacillus agileis* from soybean whey that can improve the dispersibility of soybean protein while reducing the need for added acid during soybean protein production. The microorganism described in this invention enables continuous production, reducing the cost of multiple enzyme additions. The lactic acid bacteria agent prepared using the microbial strain described in this invention can be applied to soybean protein production lines, improving functionality while reducing the use of added acid, showing broad application prospects.

[0038] Biological Preservation Instructions

[0039] Biological material R-2, classified and named: *Ligilactobacillus agilis*, was deposited on March 5, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33722. Attached Figure Description

[0040] Figure 1 The process flow for preparing soy protein isolate;

[0041] Figure 2 The pH change curves during the cultivation of different strains;

[0042] Figure 3 Comparison of the dispersibility of soybean protein modified by different microorganisms;

[0043] Figure 4 Changes in pH and isoelectric point of R-2 fermented soy milk;

[0044] Figure 5 The effect of different fermentation times on the dispersibility of R-2. Detailed Implementation

[0045] This invention discloses an agile combined Lactobacillus and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The agile combined Lactobacillus and its applications described in this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the denitrifying bacteria and their applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0046] The Agile Lactobacillus combined with the present invention can be used alone or in combination with other soybean modified microbial strains.

[0047] The *Lactobacillus agile* for modifying soybean protein provided in this invention cannot be mixed with bactericides or other products.

[0048] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0049] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and, unless otherwise stated, does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0050] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0051] To further illustrate the present invention, the following describes in detail, with reference to embodiments, an agile combined with Lactobacillus and its application.

[0052] In the following embodiments,

[0053] 1. Dispersibility detection method in solid milkshakes and nutritional protein powder applications

[0054] Weigh 100g of tap water and pour it into a glass beaker; then weigh 10g of egg white and stir with a glass rod until all the powder is dissolved in the water. Record the dispersion time.

[0055] 2. Viscosity testing methods in the application of solid milkshakes and nutritional protein powders:

[0056] Weigh 160g of room temperature water, add 20g of protein powder, stir for 5 minutes, adding 4-5 drops of defoamer during the process, transfer to a beaker, and measure the viscosity using a Bollerfeld viscometer at 15 rpm for 30 seconds. Select a suitable rotor based on the viscosity.

[0057] Example 1: Strain Screening

[0058] (1) Strain isolation and purification: The plate method was used for strain isolation. MRS medium (liquid medium with 1.5-2% agar added) was poured into solid plates, and after cooling, it was serially diluted 10 times. -1 10 -2 ...10 -5 Spread the bacteria on plates and incubate them in a 35°C constant temperature incubator. After the bacteria have grown, select single colonies and perform streak purification to obtain 5 isolated and purified strains, which were named R-1, R-2, R-3, R-4 and R-5, respectively.

[0059] (2) Activation of bacterial strains: single colonies were picked and inoculated into modified MRS liquid medium sterilized at 121℃ for 20 min, and cultured at 35℃ and 150 r / min for 24 h.

[0060] (3) Initial screening of bacterial strains: The OD of the activated bacterial solution was measured using a UV spectrophotometer. 600 Sterile water was used to adjust the OD of different strains. 600 =0.5, will adjust OD 600 The bacterial culture was inoculated into 200 ml of sterilized modified MRS liquid medium at an inoculation rate of 0.1%-0.5% v / v and cultured at 35°C and 150 rpm for 24 h. The pH of the fermentation broth was measured every 2 h during the process. The strain with a final pH below 4.2 was considered the target strain. The pH change curve during the strain culture is shown in the figure below. Figure 2 .

[0061] (4) Secondary screening of bacterial strains:

[0062] The strains R-2 and R-3 obtained from the initial screening, and the commercially available Lactobacillus plantarum GMNL-662 fermentation broth were added to the mixed soy milk at a ratio of 1%, respectively, and labeled as 2#, 3#, and 4#. The effective viable count of the fermentation broth was adjusted to 2×10⁻⁶. 9The concentration of cfu / mL was set at 35℃, the stirring speed was 1000r / min, and the fermentation time was 8h. The blank group 1# was the same as 2#~4# except that no bacterial solution was added. After fermentation, the final soybean protein product was obtained by centrifugation, neutralization, flash sterilization, spray drying, and packaging according to the soybean protein preparation method.

[0063] The dispersibility of soybean protein obtained from samples #1, #2, #3, and #4 was tested, and the results are as follows: Figure 3 As shown, Figure 2 The dispersibility of soybean protein modified by different microorganisms was compared. R-2 was ultimately selected for the subsequent preparation of modified soybean protein, while R-3 was eliminated due to poor dispersibility.

[0064] The 16S rDNA of strain R-2 was sequenced, and its nucleotide sequence is as follows:

[0065]

[0066] Table 1. Comparison of dispersibility of soy protein isolate modified by different microorganisms

[0067] Group Dispersed time 1# <![CDATA[21.81±3.17 b ]]> 2# <![CDATA[12.29±0.60 a ]]> 3# <![CDATA[19.97±0.65 b ]]> 4# <![CDATA[21.57±1.16 b ]]>

[0068] Note: Different letters between different groups indicate that there are significant differences in samples at the P < 0.05 level.

[0069] Example 2: Preparation of microbial inoculants

[0070] (1) Activation of bacterial strain: Take 1-5 uL of frozen Lactobacillus agile R-2 and inoculate it into a sterile culture medium containing 5 mL of MRS liquid, and incubate at 30-40℃ and 150 r / min for 12-24 h;

[0071] The MRS liquid culture medium formula is as follows: glucose 2%, yeast powder 0.5%, peptone 1%, beef extract 1%, sodium acetate 0.5%, KH2PO4 0.2%, ammonium citrate 0.2%, MgSO4 0.05%, MnSO4 0.018%, Tween 80 0.1%, distilled water balance, pH 6.5-8.0, sterilized at 121℃ for 20 min.

[0072] (2) Liquid seed preparation: The activated strains were transferred to 200 mL of MRS liquid medium and cultured at 30-40℃ and 150 r / min for 12-24 h.

[0073] (3) Seed fermentation tank: The prepared liquid seeds were inoculated into a 30L seed tank with a liquid volume of 60% at an inoculation rate of 10% v / v. The stirring speed was 50-140 rpm, the culture temperature was 30-40℃, the fermentation tank pressure was 0.05MPa, and the fermentation cycle was 12h.

[0074] The culture medium formula in the seed tank is as follows: glucose 2%, yeast powder 0.5%, soy protein isolate 2%, sodium acetate 0.5%, KH2PO4 0.2%, ammonium citrate 0.2%, MgSO4 0.05%, MnSO4 0.018%, Tween 80 0.1%, pH 6.5-8.0, sterilized at 121℃ for 20 min.

[0075] (4) Fermentation in a fermenter: The seed culture obtained above was inoculated into the culture medium of a 300L fermenter with a volume of 60-70% at an inoculation rate of 5%-10% v / v. The culture medium and culture conditions were the same as those for seed tank fermentation. After fermentation, the cell count reached 2×10⁻⁶. 9 The concentration of cfu / mL indicates that the liquid bacterial agent is obtained by removing the culture medium from the tank after fermentation is complete.

[0076] After obtaining the above-mentioned liquid bacterial agent, a bacterial cell agent can also be obtained based on it, and the preparation method is as follows:

[0077] (5) Preparation of solid inoculum: After fermentation, the liquid inoculum obtained after fermentation is treated with 2-3% trehalose, 2-5% soy protein isolate, and 2-3% inulin by mass ratio. The fermentation broth is then centrifuged and separated to obtain solid inoculum cells. After drying, inoculum powder is obtained. The moisture content of the inoculum powder is controlled below 10%, and the effective viable count is measured to be 2 × 10⁻⁶. 10 cfu / g.

[0078] Example 3: Effect of Lactobacillus agile combined with Lactobacillus R-2 fermentation on the isoelectric point of soy milk

[0079] (1) Activation of bacterial strain: Take 1-5 uL of frozen Lactobacillus agileensis and inoculate it into sterile culture medium containing 5 mL MRS liquid, and incubate at 30-40℃ and 150 r / min for 12-24 h;

[0080] The MRS liquid culture medium formula is as follows: glucose 2%, yeast powder 0.5%, peptone 1%, beef extract 1%, sodium acetate 0.5%, KH2PO4 0.2%, ammonium citrate 0.2%, MgSO4 0.05%, MnSO4 0.018%, Tween 80 0.1%, distilled water balance, pH 6.5-8.0, sterilized at 121℃ for 20 min.

[0081] (2) Liquid seed preparation: The activated strains were transferred to 200 mL of MRS liquid medium and cultured at 30-40℃ and 150 r / min for 12-24 h.

[0082] (3) Culture of strain: The prepared liquid seed was inoculated into 2L of modified MRS liquid medium at an inoculation rate of 10% v / v for expansion culture. The culture temperature was 30-40℃, the rotation speed was 150r / min, and the culture time was 12h.

[0083] (4) Fermented soy milk: Add Lactobacillus aggregata R-2 fermentation broth to the mixed soy milk at a ratio of 1%, and adjust the effective viable bacteria count of the fermentation broth to 2×10⁻⁶. 9 The concentration of cfu / mL was set at 35℃, the stirring speed at 1000 rpm, and the fermentation time was 14 hours. The pH and isoelectric point of the fermented soybean milk were measured every hour. The pH and isoelectric point change curves during fermentation are shown below. Figure 4As shown, the pH of the fermented soybean milk broth rapidly decreases with prolonged fermentation time, approaching the isoelectric point of unfermented soybean milk (4.5) at 6 hours. As fermentation time continues, the pH of the fermented soybean milk broth continues to decrease, and its isoelectric point also decreases synchronously. Since the pH of the fermented soybean milk broth is the isoelectric point of the fermented soybean milk, fermentation can alter the isoelectric point of the soybean milk. Therefore, there is no need to add external acids such as hydrochloric acid or citric acid to adjust the pH to the isoelectric point, saving on the use of external acids.

[0084] Example 4: Fermentation-modified soybean protein preparation process

[0085] (1) Mix non-GMO low-temperature soybean meal with process water evenly. The mass ratio of non-GMO low-temperature soybean meal to process water is 1:10. Adjust the pH to 7.0 and the temperature to 40℃. Add silicone oil defoamer with a mass concentration of 50g / L. Use a homogenizing emulsification pump to extract at low speed for 40min to obtain the first extraction liquid.

[0086] (2) Single extraction centrifugation separation: The single extraction liquid was separated using a horizontal screw discharge centrifuge with a rotation speed of 3200 rpm / min; the solid phase and liquid phase after single extraction centrifugation were obtained.

[0087] (3) Secondary extraction: The solid phase after the first extraction centrifugation is added to the process water and stirred evenly. The mass ratio of the solid phase after the first extraction centrifugation to the process water is 1:5. The pH value is adjusted to 7.0 with sodium hydroxide solution. The mass fraction of sodium hydroxide is 30%. The extraction time is 20 min and the extraction temperature is 30 min. The secondary extraction solution is obtained after the secondary extraction treatment.

[0088] (4) Secondary extraction and centrifugation: The secondary extraction liquid was separated using a horizontal screw centrifuge with a rotation speed of 3200 rpm / min; the solid phase and liquid phase after secondary extraction and centrifugation were obtained.

[0089] (5) Mixed soy milk: The liquid phase after the first extraction and centrifugation is mixed with the liquid phase after the second extraction and centrifugation to obtain mixed soy milk;

[0090] (6) Fermentation modification: Add the inoculum or agent to the mixed soy milk at a ratio of 1%, set the temperature to 35℃, ferment for 2-16 hours, and the pH at the end of fermentation is below 4.5.

[0091] (7) Centrifugal separation: The fermented soybean milk mixture is centrifuged and dehydrated in a horizontal screw discharge centrifuge at a speed of 3200 rpm / min. After dehydration, the solid phase and liquid phase after acid precipitation and centrifugation are obtained.

[0092] (8) Neutralization and preparation: Using the traditional neutralization process, process water and sodium hydroxide solution were added to the solid phase after acid precipitation and centrifugation and stirred evenly to obtain a protein solution with a pH of 7.0 and a solid mass concentration of 120 g / L.

[0093] (9) Flash sterilization: Sterilization and deodorization by steam heating is carried out. The steam heating temperature is 155℃, the protein solution temperature is controlled at 70℃, and sterilization is carried out for 10 seconds to obtain the protein slurry after flash sterilization.

[0094] (10) Spray drying: After flash sterilization, the protein slurry is transported by a material pump and spray dried at 300 bar to obtain highly emulsified soy protein isolate;

[0095] (11) Packaging: Soy protein powder is rapidly cooled using a cooling treatment device and then packaged.

[0096] Example 5: Effect of Agile Combined with Lactobacillus R-2 on Soy Protein Dispersibility

[0097] (1) Activation of bacterial strain: Take 1-5 uL of frozen Lactobacillus agileensis and inoculate it into sterile culture medium containing 5 mL MRS liquid, and incubate at 30-40℃ and 150 r / min for 12-24 h;

[0098] The MRS liquid culture medium formula is as follows: glucose 2%, yeast powder 0.5%, peptone 1%, beef extract 1%, sodium acetate 0.5%, KH2PO4 0.2%, ammonium citrate 0.2%, MgSO4 0.05%, MnSO4 0.018%, Tween 80 0.1%, distilled water balance, pH 6.5-8.0, sterilized at 121℃ for 20 min.

[0099] (2) Liquid seed preparation: The activated strains were transferred to 200 mL of MRS liquid medium and cultured at 30-40℃ and 150 r / min for 12-24 h.

[0100] (3) Culture of strain: The prepared liquid seed was inoculated into 2L of modified MRS liquid medium at an inoculation rate of 10% v / v for expansion culture. The culture temperature was 30-40℃, the rotation speed was 150r / min, and the culture time was 12h.

[0101] (4) Preparation of modified soybean protein: The fermented modified soybean protein product was prepared according to the method described in Example 4. In the fermentation modification step, the fermentation time was 12 hours, and samples were taken every 4 hours to prepare the finished soybean protein product, obtaining soybean protein products with fermentation times of 0 hours, 4 hours, 8 hours, and 12 hours.

[0102] (5) Soybean protein dispersibility test: Weigh 100g of tap water and pour it into a glass beaker; then weigh 10g of protein and stir with a glass rod until all the powder is in the water and record the dispersion time.

[0103] Dispersion time of soybean protein products prepared with different fermentation times, such as Figure 5 As shown, Figure 5 The dispersion time of soybean protein was determined for different fermentation times. As the fermentation time increased, the dispersion time of soybean protein significantly decreased, indicating that the combination of Agile and Lactobacillus R-2 can effectively improve the dispersion speed of soybean protein, facilitating its end-use applications.

[0104] Table 2. Dispersion time of soybean protein products prepared at different fermentation times

[0105]

[0106] Note: Different letters between different groups indicate that there are significant differences in samples at the P < 0.05 level.

Claims

1. *Ligilactobacillus agilis*, characterized by: Its accession number is CGMCCNO.33722.

2. A microbial agent, characterized in that, Includes the Agile Combined Lactobacillus as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, It can be a liquid or solid inoculant; the liquid inoculant has a bacterial count of 1×10⁻⁶. 9 - 5×10 9 cfu / mL, solid bacterial agent cell count 2×10 9 - 3×10 10 cfu / g.

4. The method for preparing the microbial agent according to claim 2 or 3, characterized in that, The *Lactobacillus agileus* as described in claim 1 is obtained by activation and fermentation.

5. The preparation method according to claim 4, characterized in that, The activated culture medium comprises the following raw materials in the indicated weight percentages: glucose 0.5-2.5%, yeast extract 0.3-0.8%, soy protein isolate 1-3%, sodium acetate 0.3-0.5%, KH₂PO₄ 0.05-0.2%, ammonium citrate 0.05-0.2%, MgSO₄ 0.01-0.05%, MnSO₄ 0.01-0.02%, and Tween 80 0.05-0.1%. The fermentation medium comprises the following raw materials in the following weight percentages: glucose 2%, yeast powder 0.5%, soy protein isolate 2%, sodium acetate 0.5%, KH2PO4 0.2%, ammonium citrate 0.2%, MgSO4 0.05%, MnSO4 0.018%, Tween 80 0.1%, and the balance being water.

6. The preparation method according to claim 4, characterized in that, The fermentation process also includes adding trehalose, soy protein isolate, and inulin to the bacterial solution, followed by drying.

7. The use of the Agile Lactobacillus as described in claim 1, or the bacterial agent as described in claim 2 or 3, in the preparation of modified soybean protein.

8. The method for preparing modified soybean protein using the *Lactobacillus agglutinosa* as described in claim 1, characterized in that, The *Lactobacillus agile* as described in claim 1 or the bacterial agent as described in claim 2 or 3 is inoculated into mixed soy milk, and modified soy protein is obtained by fermentation.

9. The method according to claim 8, characterized in that, The inoculum or inoculant amount is 0.5%-10%, and the fermentation conditions include 30-50℃ and fermentation time of 4-12 hours.

10. A modified soy protein isolate, characterized in that, It is prepared by the method described in any one of claims 8 to 9.

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