Lactobacillus brevis X4-4 and application thereof in fermented food

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus brevis X4-4 and application of the lactobacillus brevis X4-4 in fermentation. The preservation number of the lactobacillus brevis X4-4 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No. 31284. The activity of pyruvate dehydrogenase, malic acid-lactase, N-acetyltransferase and acid protease of the strain is remarkably higher than that of existing common lactobacillus, malic acid and protein in food raw materials can be efficiently utilized, and beneficial products can be generated. The lactobacillus brevis X4-4 has the specific advantages that 1) the lactobacillus brevis X4-4 can survive in a large quantity in an environment with relatively low pH (Potential of Hydrogen); (2) malic acid is converted into lactic acid which participates in pyruvic acid metabolism and arginine synthesis, so that the irritative sour feeling of the raw materials is reduced; (3) protein can be hydrolyzed into small molecule peptide under an acidic condition, so that the utilization rate of the protein is increased; and 4) the method can be used for single fruit raw materials or high-protein raw materials, and can also be used for fruit and protein composite raw materials.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus breve X4-4 and application thereof in fermentation. Background Art

[0002] Lactobacillus brevis is an important lactic acid bacteria with the functions of detoxification, anti-aging, and improving immunity, and is known as the "longevity bacteria". However, the optimal pH of Lactobacillus brevis is around 6. It is mainly found in fermented vegetables such as sauerkraut and kimchi. It shows low tolerance and metabolic efficiency to fruits with high organic acid content, especially sour fruits such as apples, sea buckthorn, and hawthorn with high malic acid content. At the same time, Lactobacillus brevis has a very limited ability to decompose protein and cannot be directly applied to high-protein meat products, dairy products, and egg products, and its application space is severely restricted.

[0003] Fruits, milk, meat, and eggs all have different nutritional elements and are indispensable foods for people. The types of processed products are constantly increasing. In order to achieve nutritional complementarity, the combination of high-vitamin fruits with high-protein milk, meat, and eggs has become a new demand for the development of the food industry. However, fruits have high acidity, and proteins are easy to condense and precipitate in a high-acid environment. These natural characteristics hinder their combination, which is also a major problem in food science.

[0004] In order to expand the application field of Lactobacillus brevis, solve the deficiencies in the processing of fruits, dairy products and meat products, reduce the amount of sucrose added and improve the utilization rate of protein, and give full play to the probiotic effect of Lactobacillus brevis, it is necessary to breed an efficient and powerful Lactobacillus brevis to meet the market demand for high-quality fermented products. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a strain of Lactobacillus brevis X4-4 and its application in fermented food. The Lactobacillus brevis X4-4 provided by the present invention can efficiently utilize malic acid and protein, can grow more effectively in an environment containing malic acid and protein and produce beneficial products, and can be used to prepare high-quality fermented products.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The invention provides a strain of Levilactobacillus brevis X4-4, whose deposit number is CGMCC No.31284.

[0008] The present invention provides a microbial preparation, the active ingredients of which include the breve growth-promoting Lactobacillus X4-4 described in the above technical solution.

[0009] Preferably, the number of viable bacteria of Lactobacillus breve X4-4 in the microbial preparation is ≥ 1×10 9 CFU / mL.

[0010] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising the following steps:

[0011] The activated Lactobacillus breve X4-4 is inoculated into MRS liquid culture medium for cultivation to obtain the microbial preparation.

[0012] Preferably, the MRS liquid culture medium is further added with 10-30 g / L DL-malic acid and 10-30 g / L protein powder; the pH value of the MRS liquid culture medium is 2-6; and the culture temperature is 28-40°C.

[0013] The present invention provides the use of the breve growth-promoting Lactobacillus X4-4 described in the above technical scheme or the microbial preparation described in the above technical scheme or the microbial preparation prepared by the preparation method described in the above technical scheme in fermented food.

[0014] Preferably, the fermented raw material is a food raw material containing malic acid and / or protein.

[0015] Preferably, the fermented raw materials include: one or more of fruits, juices, plant proteins and animal proteins.

[0016] Preferably, the fermentation temperature is 28-40°C.

[0017] Preferably, the mass ratio of the fermentation raw material to the bacterial liquid is 100:(6-9).

[0018] Beneficial effects:

[0019] The present invention provides a strain of Lactobacillus breve X4-4, the deposit number of which is CGMCC No.31284. The Lactobacillus breve X4-4 provided by the present invention is a strain of 60The mutated strain was obtained after Co-γ ray irradiation of yogurt. The activities of pyruvate dehydrogenase, malic acid-lactic acid enzyme, N-acetyltransferase and acid protease of this strain are significantly higher than those of the commonly used lactobacilli. It can efficiently utilize malic acid and protein in food raw materials and produce beneficial products. The specific advantages include: 1) Lactobacillus breve X4-4 can survive in large quantities in an environment with low pH (pH = 2-4); 2) It converts malic acid into lactic acid, participates in pyruvate metabolism and arginine synthesis, and reduces the irritating sourness of the raw materials; 3) It can hydrolyze proteins into small molecule peptides under acidic conditions to improve the utilization rate of proteins; 4) It can be used for single fruit raw materials or high-protein raw materials, as well as for raw materials with a combination of fruit and protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 The morphological diagram of Lactobacillus breve X4-4 on solid culture medium and under a microscope;

[0022] Figure 2 This is the phylogenetic tree of Lactobacillus breve X4-4.

[0023] Biological Deposit Description

[0024] Lactobacillus brevis X4-4, classified and named Levilactobacillus brevis, was deposited at the General Microbiology Center of China Culture Collection Administration on July 12, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31284. DETAILED DESCRIPTION

[0025] The invention provides a strain of Lactobacillus breve X4-4, whose preservation number is CGMCC No.31284.

[0026] The present invention provides a short growth-promoting lactobacillus X4-4, which is a strain 60 The induced strain obtained by Co-γ ray irradiation of yogurt has a large colony with regular round shape, milky white color, certain thickness, slightly raised in the middle, opaque, smooth surface without wrinkles, moist, uniform and viscous texture, easy to pick up, and short rod-shaped under microscopic examination (see Figure 1). Gram staining is positive and purple. Catalase is negative and no bubbles are produced, indicating that strain X4-4 does not produce active catalase. The short growth-promoting Lactobacillus X4-4 can efficiently utilize malic acid and protein, and can efficiently convert malic acid into other compounds, thereby effectively reducing the sourness of fruit products, improving product quality, and promoting the development of the fruit industry; in addition, the short growth-promoting Lactobacillus X4-4 has a strong protein decomposition ability, can more effectively utilize protein raw materials, improve nutritional value and flavor, and can be used to prepare high-quality fermented products. The activities of pyruvate dehydrogenase, malic acid-lactic acid enzyme, N-acetyltransferase and acid protease of this strain are significantly higher than those of the existing commonly used lactobacilli, and can efficiently utilize malic acid and protein in food raw materials and produce beneficial products. Specific advantages include: 1) Lactobacillus breve X4-4 can survive in large numbers in an environment with low pH; 2) It converts malic acid into lactic acid, participates in pyruvate metabolism and arginine synthesis, and reduces the irritating sourness of the raw materials; 3) It can hydrolyze proteins into small molecule peptides under acidic conditions to improve the utilization rate of protein; 4) It can be used for single fruit raw materials or high-protein raw materials, and can also be used for raw materials with a combination of fruit and protein.

[0027] Based on the above advantages, the present invention provides a microbial preparation, the active ingredient of which includes the breve growth-promoting Lactobacillus X4-4 described in the above technical solution. As an embodiment, the number of viable bacteria of breve growth-promoting Lactobacillus X4-4 in the microbial preparation is ≥ 1×10 9 CFU / mL.

[0028] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising the following steps:

[0029] The activated Lactobacillus breve X4-4 is inoculated into MRS liquid culture medium for cultivation to obtain the microbial preparation.

[0030] In one embodiment, the culture temperature is 28-40°C. In another embodiment, the culture temperature is 32-37°C. In another embodiment, the culture temperature is 36-37°C. In another embodiment, the culture temperature is 37°C.

[0031] As an embodiment, 10 to 30 g / L of DL-malic acid and 10 to 30 g / L of protein powder are further added to the MRS liquid culture medium. As another embodiment, 10 g / L of DL-malic acid and 10 g / L of protein powder are further added to the MRS liquid culture medium. As an embodiment, the pH value of the MRS liquid culture medium is 2 to 6. As another embodiment, the pH value of the MRS liquid culture medium is 2 to 3. The present invention can prove that the malic acid and protein degradation ability of breve growth-promoting Lactobacillus X4-4 is strong by adding malic acid and protein powder to the MRS liquid culture medium.

[0032] Based on the above advantages, the present invention provides the use of the short growth-promoting Lactobacillus X4-4 described in the above technical scheme or the microbial preparation described in the above technical scheme or the microbial preparation prepared by the preparation method described in the above technical scheme in fermentation. As an embodiment, the raw material for the fermentation contains malic acid and / or protein. As an embodiment, the raw material for the fermentation includes: one or more of fruit, fruit juice, plant protein and animal protein. As another embodiment, the fruit may be one or more of apples, grapes, pears, hawthorns and sea buckthorns. As another embodiment, the fruit juice may be one or more of apple juice, grape juice, pear juice, hawthorn juice and sea buckthorn juice. As another embodiment, the plant protein may be one or more of soy milk, soybeans, peanuts and peanut paste. As another embodiment, the animal protein may be one or more of chicken, pork, mutton, pigeon meat, milk and goat milk.

[0033] As an embodiment, the fermentation temperature is 28-40°C. As another embodiment, the fermentation temperature is 32-37°C. As another embodiment, the fermentation temperature is 36-37°C. As another embodiment, the fermentation temperature is 37°C.

[0034] As an embodiment, the mass ratio of the fermentation raw material to the microbial preparation is 100:6-9.

[0035] In order to further illustrate the present invention, a strain of Lactobacillus breve X4-4 provided by the present invention and its application in fermentation are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] The formula of the culture medium in the following examples or comparative examples is as follows:

[0037] MRS liquid culture medium: 5.0 g yeast extract, 10.0 g beef extract, 20.0 g glucose, 0.58 g magnesium sulfate, 3.0 g anhydrous sodium acetate, 10.0 g peptone, 2.0 g diammonium hydrogen citrate, and 0.25 g manganese sulfate. Dissolve in distilled water and make up to 1 L. Sterilize at 121°C for 20 min.

[0038] MRS solid medium: Add 20g / L agar to MRS liquid medium.

[0039] The measurement methods of different indicators in the following test examples are as follows:

[0040] 1) Malic acid: high performance liquid chromatography.

[0041] 2) Flavonoids: The total flavonoids content was determined by the sodium borohydride / tetrachlorobenzoquinone (SBC) method.

[0042] 3) Total phenols: determined by Folin phenol colorimetric method.

[0043] 4) Soluble solids: The total soluble solids were determined by Abbe refractometer.

[0044] 5) Total acid: titration method.

[0045] 6) Total antioxidant capacity: The total antioxidant capacity of apple samples was determined using the DPPH free radical scavenging capacity method.

[0046] 7) Crude protein content: refer to the national standard GB 5009.5-2016 Determination of crude protein in food.

[0047] 8) Free amino acid (FAA) content: The total amount of free amino acids in chicken was determined by sampling using the ninhydrin colorimetric method.

[0048] 9) pH: pH meter.

[0049] 10) Moisture content: Adopt normal pressure drying method.

[0050] 11) Juice yield: Mix the fruit and water in a mass ratio of 1:1, extract the juice with a wall breaker, and then filter through eight layers of gauze to obtain the fruit pulp. The juice yield is the percentage of the mass of the fruit pulp minus the mass of water to the mass of the fruit.

[0051] 12) Water holding capacity: Use a 10 mL centrifuge tube, weigh the empty tube, empty tube + sample mass = 10.0 g, centrifuge at 4500 rpm for 20 min, remove the supernatant, weigh, calculate the water holding capacity according to formula I, and measure 3 groups in parallel.

[0052] C = (m1-m0) / (10-m0) × 100% Formula I;

[0053] Where C is the water holding capacity; m1 is the mass of the sample after centrifugation, in g; m0 is the mass of the centrifuge tube, in g.

[0054] 13) The activity of pyruvate dehydrogenase was determined using a kit purchased from Solarbio on Taobao, and the activity of malate-lactic acid was determined using a colorimetric method.

[0055] 14) Protein hydrolysis degree: Kjeldahl method was used.

[0056] 15) Peptide mass concentration: colorimetric method was used.

[0057] 16) Acidic protease activity, N-acetyltransferase activity: using colorimetric method.

[0058] 17) Fruit firmness was determined using a texture analyzer method.

[0059] Example 1 Isolation and identification of strains

[0060] (1) Original bacterial liquid: Huishan "Shitian" plain yogurt, purchased from a supermarket, 100 g of yogurt per bottle, add 100 mL of apple juice (directly squeezed from diced apples, no water), stir evenly and set aside.

[0061] (2) Radiation mutagenesis: 60 Co-γ-ray irradiation treatment, source intensity 7.51×10 15 Bq, dose uniformity was 1.0, absorbed dose was 400 Gy, and dynamic 360-degree flipping was continuous during irradiation.

[0062] (3) Isolation of bacterial strains: Add 50 mL of sterile water to the irradiated yogurt, stir evenly, filter the solution through three layers of sterilized gauze to obtain the mutagenic bacterial solution, and then dilute it to 10 -2 , 10 -3 , 10 -4 , take 150 μL and spread it on MRS plate culture medium (pH = 5) containing 10 g / L malic acid, culture it at 37°C for 48 to 72 h, pick 15 colonies with intact strain morphology and obvious calcium dissolution circle, and carry out single bacteria slant culture.

[0063] (4) Screening of bacterial strains: The single-bacteria slant culture medium was repeatedly rinsed with sterile water, and the obtained bacterial suspension was cultured on a shaker for 48 h. It was then inoculated into a modified MRS culture medium at a rate of 10 v / v%, and cultured at 37°C for 5 days. The number of live bacteria in the culture medium was determined, and the dominant strain X4-4 was screened out by comparison. The modified MRS culture medium is an MRS culture medium in which milk powder with a final concentration of 10 g / L is added, and malic acid is used to adjust the pH value to 5.

[0064] (5) Pick a single colony of X4-4, streak and culture it three times, observe the colony morphology of the strain, and identify it through Gram staining experiment, as follows: First, smear, add a drop of distilled water on a clean slide, use a sterile inoculation loop to take a small amount of bacteria from the freshly cultured X4-4 colony, mix it with water, and evenly spread it into a thin layer. Pass the slide coated with bacteria through a flame quickly several times to kill the bacteria and make them adhere to the slide for fixation. Add ammonium oxalate crystal violet stain solution for initial staining, cover the entire surface, and keep it for 1 minute. Wash with water. Add iodine-potassium iodide solution as a mordant and let it act for 1 minute. Decolorize, tilt the slide, add 95% ethanol, and continue for 30 seconds until the outflowing liquid no longer appears purple. Finally, use safranin for counterstaining, and rinse it with water after about 30 seconds. After the slide is completely dry, observe it under a microscope.

[0065] Results Figure 1 The results showed that the X4-4 colony was large and regular round, milky white, with a certain thickness, slightly raised in the middle, opaque, smooth and wrinkle-free, moist, uniform and viscous, easy to pick up, and short rod-shaped under microscopic examination. Gram staining was positive and purple.

[0066] (6) Select a single X4-4 colony from the MRS agar plate and prepare a bacterial suspension with sterile saline. Take a drop of the bacterial suspension and place it on a clean glass slide. Add a small drop of 3% hydrogen peroxide solution to the bacteria. Immediately observe whether bubbles are formed. If a large number of bubbles are generated, it indicates the presence of catalase; if there are no obvious bubbles, it indicates a negative result, that is, the absence of catalase.

[0067] The strain X4-4 was tested and no bubbles were produced, indicating that the strain X4-4 did not produce active catalase. The strain X4-4 was preliminarily determined to be a suspected lactic acid bacteria.

[0068] (7) Biological identification

[0069] In order to further analyze and identify the lactic acid bacteria obtained in the initial screening, the 16S rDNA of strain X4-4 was amplified by PCR, purified and sequenced. After comparison with the basic local alignment search tool (BLAST) module in NCBI, the phylogenetic tree of the screened strains was drawn with the help of MEGA7.0 software. From the phylogenetic tree, strain X4-4 has the best homology with Lactobacillus brevis and has the closest relationship. Based on the morphological characteristics and physiological and biochemical experimental results of the above strains, strain X4-4 was finally identified as Levilactobacillus brevis, and it was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.31284.

[0070] The specific sequence of the primer sequences for the PCR amplification and the 16S rDNA is as follows:

[0071] Primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1);

[0072] Primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0073]

[0074] Example 2 Cultivation characteristics of strain X4-4

[0075] Test strains: strain X4-4 screened in Example 1, with commercially available Lactobacillus casei (numbered BNCC 134415), Lactobacillus breve (numbered BNCC 337373), Lactobacillus acidophilus (numbered BNCC 186447) and Lactobacillus helveticus (numbered BNCC 189793) as controls.

[0076] (1) pH tolerance

[0077] The pH values ​​of the MRS liquid culture medium were adjusted to 2, 3, 4, 5 and 6 with malic acid to obtain MRS liquid culture medium with different pH values. The bacterial suspension was prepared with distilled water and inoculated into the MRS liquid culture medium with different pH values ​​at a volume ratio of 1:9. The culture was shaken at 37°C for 24 hours, and the number of viable bacteria was determined. The results are shown in Table 1.

[0078] Table 1 Viable bacterial counts (CFU / mL) of strains at different pH values

[0079]

[0080] As shown in Table 1, compared with other strains, strain X4-4 showed stronger acid resistance at lower pH values. At pH = 4, the number of viable bacteria of strain X4-4 was 0.91×10 7 CFU / mL, while the viable counts of other strains were all lower than 0.4×10 7 CFU / mL. At pH=3, the viable count of strain X4-4 was 0.19×10 7 CFU / mL, while the viable counts of other strains were all less than 0.1×10 7 CFU / mL. At pH = 2, the viable count of strain X4-4 was 0.08×10 7 CFU / mL, while the viable counts of other strains were all less than 0.05×10 7 Therefore, the X4-4 strain showed stronger acid resistance in an environment of pH = 4 and below, especially at pH = 3 and pH = 2, where its viable bacterial count was significantly higher than that of other strains.

[0081] (2) Fermentation temperature

[0082] The bacterial suspension was prepared with distilled water and inoculated into MRS liquid medium with a pH value of 5 (pH value was adjusted with malic acid) at a volume ratio of 1:9. The suspension was cultured in a shaking incubator at 28°C, 32°C, 34°C, 36°C, 38°C, and 40°C for 24 hours, and the number of viable bacteria was determined. The results are shown in Table 2.

[0083] Table 2 The number of viable bacteria (CFU / mL) of strains at different fermentation temperatures

[0084] strains 28℃ 32℃ 34℃ 36℃ 40℃ X4-4 <![CDATA[4.57×10 6 ]]> <![CDATA[5.43×10 6 ]]> <![CDATA[7.54×10 6 ]]> <![CDATA[8.93×10 6 ]]> <![CDATA[11.58×10 6 ]]> Lactobacillus casei <![CDATA[4.15×10 6 ]]> <![CDATA[6.37×10 6 ]]> <![CDATA[7.01×10 6 ]]> <![CDATA[7.69×10 6 ]]> <![CDATA[9.72×10 6 ]]> Lactobacillus breve <![CDATA[3.33×10 6 ]]> <![CDATA[5.76×10 6 ]]> <![CDATA[6.88×10 6 ]]> <![CDATA[8.40×10 6 ]]> <![CDATA[10.13×10 6 ]]> Lactobacillus acidophilus <![CDATA[3.98×10 6 ]]> <![CDATA[5.01×10 6 ]]> <![CDATA[6.26×10 6 ]]> <![CDATA[7.42×10 6 ]]> <![CDATA[10.35×10 6 ]]> Lactobacillus helveticus <![CDATA[3.46×10 6 ]]> <![CDATA[4.74×10 6 ]]> <![CDATA[6.12×10 6 ]]> <![CDATA[7.06×10 6 ]]> <![CDATA[7.54×10 6 ]]>

[0085] As shown in Table 2, as the temperature increases, the number of viable bacteria of strain X4-4 also increases. From 28°C to 36°C, the number of viable bacteria increases from 4.57×10 6 CFU / mL increased to 9.58×10 6 CFU / mL, showing that the X4-4 strain has better growth performance at higher temperatures. Compared with other strains, the number of viable bacteria of the X4-4 strain at 34°C, 36°C and 40°C is higher than that of other strains, indicating that the adaptability and growth performance of the X4-4 strain at higher temperatures are better than those of other strains. Therefore, it can be considered that the X4-4 strain has a wider range of optimal fermentation temperatures, especially in the temperature range of 32°C to 40°C, where its number of viable bacteria is significantly higher than that of other strains.

[0086] (3) Malic acid degradation and metabolism

[0087] The bacterial suspension was prepared with distilled water and inoculated into MRS liquid medium with a pH value of 4 (the pH value was adjusted with malic acid) at a volume ratio of 1:9. The culture was shaken at 37°C for 24 h, the malic acid content was determined, and the malic acid degradation rate, pyruvate dehydrogenase and malate-lactic acid enzyme activities were calculated.

[0088] Malic acid degradation rate = (initial malic acid content - malic acid content after the end of the culture) / initial malic acid content × 100%. The results are shown in Table 3.

[0089] Table 3 Comparison of malic acid degradation ability of different strains

[0090]

[0091] As can be seen from Table 3, Lactobacillus breve X4-4 has significant advantages in the ability to degrade malic acid, which is specifically manifested in the following aspects: X4-4 has the highest number of viable bacteria and can degrade malic acid more effectively. The malic acid degradation rate is the highest, indicating that it has the strongest ability to degrade malic acid. The pyruvate dehydrogenase activity is the highest, indicating that it can more effectively carry out pyruvate dehydrogenation reactions during the malic acid degradation process. The malic acid-lactic acid enzyme activity is the highest, indicating that it can more effectively convert malic acid into lactic acid during the malic acid degradation process. These advantages enable X4-4 to significantly improve the degradation efficiency of malic acid during the fermentation process, thereby improving the texture and flavor of the fermented product.

[0092] (4) Protein degradation and metabolism

[0093] The pH value of the MRS liquid medium was adjusted to 4 with malic acid, and milk powder was added at a final concentration of 10 g / L to obtain a modified MRS liquid medium. A bacterial suspension was prepared with distilled water, and the modified MRS liquid medium was inoculated at a volume ratio of 1:9. The culture was shaken at 37°C for 24 hours, and the protein hydrolysis degree, peptide mass concentration, acid protease activity and N-acetyl transferase (NAT) activity were determined. The results are shown in Table 4.

[0094] Table 4 Comparison of protein degradation ability of different strains

[0095]

[0096]

[0097] As can be seen from Table 4, Lactobacillus breve X4-4 has significant advantages in protein degradation ability, which is specifically manifested in the following aspects: X4-4 has the highest number of viable bacteria and can degrade protein more effectively. X4-4 has the highest degree of protein hydrolysis, indicating that it has the strongest ability to degrade protein. X4-4 has the highest peptide mass concentration, indicating that it can more effectively degrade protein into peptides. X4-4 has the highest acid protease activity, indicating that it has stronger protease activity in an acidic environment. X4-4 has the highest N-acetyltransferase activity, indicating that it can more effectively transfer N-acetyl groups during protein degradation. These advantages enable X4-4 to significantly improve the efficiency of protein degradation during the fermentation of meat products, thereby improving the texture and flavor of meat products.

[0098] Example 3 Preparation of bacterial solution

[0099] (1) First-level seeds:

[0100] The strain X4-4 in Example 1 was activated and inoculated on an MRS slant medium and cultured at 37°C for 4 days, then repeatedly rinsed with distilled water 5 times to obtain a bacterial suspension, which was then inoculated into an MRS liquid medium at a ratio of 20 v / v%, pH = 3, containing 20 g / L DL-malic acid (purity 95%), and cultured at 37°C in a shaking incubator for 48 h, and then supplemented with 20% (g / 100 mL) peptone, and cultured continuously for 3 to 5 times until the number of viable cells was ≥10 9 CFU / mL is reserved.

[0101] (2) Secondary Seed

[0102] The germinated barley was ground into powder and boiled for 40 minutes to make saccharified malt juice (sugar content 18°Bx), which was mixed with apple juice (freshly squeezed, without water) at a volume ratio of 1:2. After sterilization and cooling, the first-class seed was inoculated at a ratio of 30% (v / v), and cultured in a shaking incubator at 37°C for 24 hours. 20% protein powder was supplemented every other day for 3 to 5 times until the number of viable bacteria was ≥10 12 CFU / mL.

[0103] Example 4 Application of Lactobacillus breve X4-4 in fruits

[0104] The fruits were washed, pitted, cut into pieces, and mixed with the bacterial solution prepared in Example 3 at a mass ratio of 100:9, and solid-state fermented at 36°C for 14 days. The fruits used were grapes, pears, and hawthorns. Unfermented fruits were used as controls.

[0105] The hardness, juice yield, soluble solids, malic acid, total acid and total antioxidant capacity of each fruit were measured before and after fermentation. The results are shown in Table 5.

[0106] Table 5 Effects of Lactobacillus breve X4-4 on different fruit raw materials

[0107]

[0108] From the results in Table 5, it can be seen that the hardness of the fruit is significantly reduced after fermentation with Lactobacillus brevis X4-4, which helps to improve the extraction efficiency of the juice because softer fruits are easier to squeeze. Although the juice yield decreased slightly, the overall change was not significant, indicating that fermentation had little effect on the juice yield. X4-4 can effectively reduce the organic acids in apples, especially malic acid. This is because Lactobacillus brevis X4-4 has a strong metabolic capacity and can convert some acidic substances into other products or directly use them as a source of energy for growth. The reduction in acidity may improve the taste of apple products, making them milder and more delicious. The soluble solids content increased significantly after fermentation, indicating that fermentation helps to improve the sweetness and flavor of the juice. The total acid content decreased after fermentation, further improving the taste of the juice. The total antioxidant capacity increased significantly after fermentation, indicating that fermentation helps to improve the nutritional value and health benefits of the juice. The fermentation treatment of Lactobacillus brevis X4-4 has a significant improvement on the fruit raw materials and helps to improve the processing efficiency and product quality of the juice. After fermentation, the firmness of the fruit decreases, the juice yield decreases slightly but the overall impact is not significant, the soluble solids content increases, the malic acid and total acid content decreases, and the total antioxidant capacity increases. These changes work together to make the juice taste better, have higher nutritional value, and be more suitable for processing and consumption.

[0109] Example 5 Application of Lactobacillus breve X4-4 in fruit juice

[0110] The fruit was washed and blanched, 3 times the weight of water was added, and the juice was squeezed into juice, and 9% (v / v) of the bacterial solution prepared in Example 3 was added, and the juice was fermented in liquid state at 37°C for 4 days. The juice used was apple juice, sea buckthorn juice, hawthorn juice, pear juice, and wild grape juice. The juice not inoculated with the activated bacterial solution was used as a control. The malic acid, flavonoids, total phenols, total acid, soluble solids and total antioxidant capacity in the juice before and after fermentation were determined. The results are shown in Table 6.

[0111] Table 6 Effects of Lactobacillus breve X4-4 on different fruit juices

[0112]

[0113] As shown in Table 6, Lactobacillus breve X4-4 significantly reduced the content of malic acid and total acid, which would make the taste of the juice softer and less sour. The sugar-acid ratio was significantly improved, which usually means that the sweetness and acidity of the juice are more balanced and the taste is better. The content of flavonoids and total phenols generally increased. The enzymes produced during the fermentation process can promote the release of flavonoids from the plant cell walls, increasing their concentration in the juice. Flavonoids are an important natural antioxidant that has many health benefits for the human body, such as antioxidant, anti-inflammatory, and anti-cancer. Similar to flavonoids, the increase in total phenolic content is also due to the fact that fermentation promotes the release of these compounds from the cells into the juice. Phenolic compounds also have strong antioxidant capacity and play a positive role in preventing cardiovascular diseases. During the fermentation process, microbial activity may produce some new soluble substances, such as sugars and amino acids. As the content of antioxidant substances such as flavonoids and total phenols increases, the antioxidant capacity of the entire system is also enhanced. This means that the fermented juice has a higher antioxidant potential, which helps to resist free radical damage and is beneficial to maintaining good health.

[0114] In summary, the fermentation treatment of different juices with Lactobacillus breve X4-4 significantly improved the taste of the juices, making them more balanced and softer, and significantly increased the nutritional and functional components of the juices, especially the antioxidant capacity and vitamin C content. These improvements are of great significance for improving the quality and health benefits of juices.

[0115] Example 6 Application of Lactobacillus breve X4-4 in meat products

[0116] Fresh lean beef, lean mutton, lean pork, chicken and pigeon meat were cleaned, cut into pieces and minced, and inoculated into the bacterial solution prepared by the method of Example 3, respectively, with a mass ratio of 100g:9g, and solid-state fermentation was cultured at 37°C for 7 days. Unfermented meat was used as a control (denoted as CK), and the crude protein content, free amino acid (FAA) content, pH value, texture characteristics and other indicators of the meat before and after fermentation were determined. The results are shown in Table 7.

[0117] Table 7 Effects of Lactobacillus breve X4-4 on different meat ingredients

[0118]

[0119] As shown in Table 7, the crude protein content in the meat did not change much before and after fermentation. The reason for this is that the determination of crude protein is obtained by converting the measured nitrogen content. The nitrogen volatilization in the whole process is very small, so the fluctuation of the total nitrogen content is small. The total amount of free amino acids increased significantly after X4-4 fermentation, indicating that the protein was continuously hydrolyzed to produce amino acids during the whole fermentation process, resulting in a continuous increase in amino acid nitrogen, which improved the flavor and nutritional value of the meat. This is directly related to the fact that the strain produces more proteolytic enzymes during the fermentation process. The pH value of short lactobacillus X4-4 decreased after fermentation, which can effectively inhibit the growth of harmful bacteria, especially Staphylococcus aureus. The lactic acid produced during the fermentation process has a certain destructive effect on myofibrils and connective tissue, so it can improve the tenderness of the meat. Short lactobacillus X4-4 is active, giving the mixed meat a dense structure, so the viscosity increases. At the same time, the growth and reproduction of short lactobacillus X4-4 promotes the gelation of protein, which can increase the cohesion and chewiness of the mixed meat. The physical structure of the meat is loose before fermentation. As the fermentation time increases, the moisture gradually dissipates, the meat becomes more tightly bound together, and the elasticity gradually increases.

[0120] The fermentation of Lactobacillus breve X4-4 can significantly improve the pH value, nitrite content, free amino acid content and texture characteristics of meat raw materials, thereby improving the processing and eating quality of meat products. The acidic environment and protein degradation generated during the fermentation process are the main reasons.

[0121] Example 7 Application of Lactobacillus breve X4-4 in fruity sour bean juice and fruity yogurt

[0122] The apple juice prepared in Example 5 was mixed with milk or soy milk in a volume ratio of 1:1, stirred evenly, and fermented at 37°C for 3 days to obtain liquid yogurt and sour bean juice with a fruity taste. No sucrose was added during the processing, and the taste was rich. The natural fermentation (the difference was that the activated bacterial liquid of X4-4 was not inoculated) was used as a control to measure the relevant indicators. The results are shown in Table 8.

[0123] Table 8 Effects of Lactobacillus breve X4-4 on fruit-flavored yogurt and sour soy milk

[0124]

[0125]

[0126] As shown in Table 8, Lactobacillus breve X4-4 produces proteases during the fermentation process. These enzymes can decompose protein molecules in milk and soy milk and degrade them into smaller peptides or even single amino acids. Therefore, although the total amount of protein seems to have decreased, they have actually been converted into smaller molecular forms. Partially degraded proteins may be metabolized and utilized by Lactobacillus breve X4-4 itself for growth and reproduction, which is also a reason for the decrease in protein content. Proteins are degraded into free amino acids by proteases, and the concentration of these amino acids in the fermentation broth will increase accordingly. In addition to amino acids directly derived from protein degradation, lactic acid bacteria may also produce some new amino acids or their derivatives during metabolism. Lactobacillus breve X4-4 produces lactic acid through fermentation. Lactic acid is a weak acid, and its accumulation will cause the pH value of the fermentation broth to decrease. In general, during the fermentation of milk and soy milk by Lactobacillus breve X4-4, macromolecular proteins are degraded into free amino acids through the action of proteases, and acidic metabolites such as lactic acid are produced, resulting in a decrease in pH. These changes not only affect the physical properties of soy milk, but may also have a positive impact on its nutritional components and functional properties.

[0127] The fruit yogurt and sour soy milk fermented by the X4-4 strain have obvious apple and yogurt aromas, which are rich and harmonious. Compared with unfermented products, the aroma is more obvious and harmonious. The sour-sweet ratio is soft, the taste is excellent, and the sour-sweet ratio is moderate, which is very popular. This shows that the X4-4 strain can effectively control the acidity of yogurt and sour soy milk, making them more palatable. The texture is uniform and delicate, without bubbles, stratification, and whey precipitation, which shows that the fermentation process can effectively improve the texture of the product and make it more uniform and delicate. The product has no precipitation, which shows that the fermentation process can effectively prevent the precipitation of ingredients in the product and maintain the stability of the product. For sour soy milk, the product fermented by the X4-4 strain can effectively mask the beany smell and make it more palatable.

[0128] It can be seen that the X4-4 strain has a significant effect on improving fruit yogurt and sour soy milk. It can effectively improve the flavor, acidity, texture and stability of the products, while masking the beany smell and making them more palatable.

[0129] In summary, the short growth-promoting Lactobacillus X4-4 provided by the present invention can improve the utilization rate of malic acid and protein, and can grow more effectively and produce beneficial products in an environment containing high concentrations of malic acid or protein. Specific advantages include: fruit acid reduction, the new strain can efficiently convert malic acid into other compounds, thereby effectively reducing the acidity of fruit products and improving product quality; protein utilization, through the enhanced protein decomposition ability and amino acid recovery mechanism after mutagenesis, short growth-promoting Lactobacillus X4-4 can more effectively utilize protein raw materials, improve nutritional value and flavor, and reduce waste.

[0130] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Levilactobacillus brevis X4-4, with a deposit number of CGMCC No.31284.

2. A microbial preparation, characterized in that: The active ingredient includes the breve growth-promoting Lactobacillus X4-4 described in claim 1.

3. The microbial preparation according to claim 2, characterized in that The number of viable bacteria of Lactobacillus breve X4-4 in the microbial preparation is ≥ 1×10 9 CFU / mL.

4. The method for preparing the microbial preparation according to claim 2 or 3, characterized in that: The following steps are involved: The activated Lactobacillus breve X4-4 is inoculated into MRS liquid culture medium for cultivation to obtain the microbial preparation.

5. The preparation method according to claim 4, characterized in that: The MRS liquid culture medium is further added with 10-30 g / L of DL-malic acid and 10-30 g / L of protein powder; the pH value of the MRS liquid culture medium is 2-6; and the culture temperature is 28-40°C.

6. Use of the breve growth-promoting Lactobacillus X4-4 according to claim 1, the microbial preparation according to claim 2 or 3, or the microbial preparation prepared by the preparation method according to claim 4 or 5 in fermented foods.

7. The use according to claim 6, characterized in that: The fermented raw material is a food raw material containing malic acid and / or protein.

8. The use according to claim 6 or 7, characterized in that: The fermented raw materials include: one or more of fruits, juices, plant proteins and animal proteins.

9. The use according to claim 6 or 7, characterized in that: The fermentation temperature is 28-40°C.

10. The use according to claim 6 or 7, characterized in that: The mass ratio of the fermentation raw material to the bacterial liquid is 100:(6-9).

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