A strain of Lactobacillus plantarum derived from sourdough and its application

Abstract

The invention discloses a plant lactobacillus derived from sour dough and an application thereof, belonging to the technical field of microorganisms. The invention screens a plant lactobacillus L23104 from sour dough, which is deposited in the China General Microbiology Center with a deposit number of CGMCC No. 29668. The strain has the ability to efficiently produce extracellular polysaccharides, and the extracellular polysaccharides have the effect of inhibiting free radical activity, which can enhance the quality of fermented food and improve the safety of fermented food, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to a plant lactobacillus derived from sour dough and application thereof, belonging to the fields of fermentation engineering and biotechnology. Background Art

[0002] Sourdough is a traditional starter obtained by mixing wheat flour and other cereal powder with water and fermenting it naturally with microorganisms such as yeast and lactic acid bacteria. The sourdough system is complex and consists of a rich microbial flora structure. During the fermentation process, lactic acid bacteria is one of the most important fermenting microorganisms. The lactic acid bacteria in sourdough are mainly composed of a large number of Lactobacillus and a small amount of Weissella, Pediococcus, Streptococcus, Leuconostoc and Enterococcus. As probiotics, lactic acid bacteria are widely present in the natural environment and the human intestine. They are recognized as safe food-grade microorganisms with multiple probiotic effects such as lowering cholesterol, anti-oxidation, and inhibiting the growth of harmful bacteria. In addition, lactic acid bacteria can also decompose fermentable sugars to produce organic acids such as lactic acid. While giving food a sour taste, they can also react with fatty acids and alcohol to produce esters, giving food a unique flavor. There are more than 60 species of lactic acid bacteria reported in sourdough, among which the most representative ones are mainly heterofermentative Lactobacillus. Lactobacillus plantarum is a type of lactic acid bacteria. Compared with other lactic acid bacteria, it has strong advantages. It is a normal probiotic flora in the intestines of humans and animals, and is also a safe food-grade microorganism. The important position of Lactobacillus plantarum in the food industry is receiving more and more attention and attention.

[0003] Microbial exopolysaccharides (EPS) are a type of mucus-like compounds secreted into the extracellular matrix by microorganisms during their growth. Compared with plant polysaccharides, microbial polysaccharides have the advantages of short production cycle and no restrictions on geographical and seasonal conditions, and have broad development prospects. At present, there are many studies on the exopolysaccharide of Lactobacillus plantarum. Patent CN102453689B discloses a strain of Lactobacillus plantarum producing exopolysaccharide and its application, and its exopolysaccharide has the effect of improving immunity; Patent CN110106121B discloses that the exopolysaccharide of Lactobacillus plantarum JNULCC001 is mainly composed of glucose, mannose, galactose and galacturonic acid, and can be used for whey fermentation; Patent CN111100810B discloses that Lactobacillus plantarum DNB1 and its exopolysaccharide have significant antioxidant capacity, and the exopolysaccharide of 0.4mg / mL reaches 57.66% for the scavenging rate of DPPH free radical, and reaches 32.65% and 28.64% for the scavenging capacity of hydroxyl free radical and ABTS free radical, respectively. The above-mentioned prior art is mostly focused on the research of the property of Lactobacillus plantarum exopolysaccharide itself, ignoring the functionality of exopolysaccharide as an additive.

[0004] Lactic acid bacteria exopolysaccharides are usually used as natural substitutes for commercial stabilizers, thereby increasing the viscosity of the fermentation broth, enhancing stability, improving physical and chemical properties such as emulsification and gel properties, and improving the rheological properties, texture and taste of the fermentation broth. Therefore, it can be predicted that Lactobacillus plantarum exopolysaccharides, as a new, efficient and safe physiological function regulator, will have theoretical research significance and practical application value. Summary of the invention

[0005] In order to solve the above problems, the present invention screens out a plant lactobacillus L23104 from sourdough, and preserves it in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.29668. The strain can produce high extracellular polysaccharides and has excellent fermentation performance. The extracellular polysaccharides have the effect of inhibiting the activity of free radicals, and can enhance the quality of fermented food and improve the safety of fermented food. The strain has broad application prospects.

[0006] The first object of the present invention is to provide a plant lactobacillus (Lactiplantibacillus plantarum) L23104, which has been deposited in the Institute of Microbiology, Chinese Academy of Sciences on January 18, 2024, with a deposit number of CGMCC No.29668.

[0007] The plant lactobacillus L23104 of the present invention has the following functions:

[0008] (1) The present invention provides a Lactobacillus plantarum strain L23104, which has a highest OD value of a bacterial liquid at 20°C to 40°C, indicating that it grows well within this temperature range and is suitable for different temperature requirements of fermented products.

[0009] (2) The present invention provides a strain of Lactobacillus plantarum L23104 that can produce a high amount of extracellular polysaccharides. After culturing for 24 hours, the extracellular polysaccharide content reaches a maximum of 2460.12±1.46 mg / L, which can be used as a stabilizer to enhance the stability of the fermented product, thereby achieving the purpose of improving the texture and taste of the product.

[0010] (3) The polysaccharide solution produced by the high-yield extracellular polysaccharide Lactobacillus plantarum L23104 provided by the present invention was used for antioxidant experiments. The clearance rates of HO·, DPPH· and ABTS+ by Lactobacillus plantarum L23104 were higher than those of the control group VC, which were (88.55±0.28)%, (82.15±0.14)% and (85.60±0.26)%, respectively. This shows that the extracellular polysaccharide produced by Lactobacillus plantarum L23104 has good antioxidant capacity.

[0011] (4) When the high-yield extracellular polysaccharide Lactobacillus plantarum L23104 provided by the present invention is used to ferment foods such as yogurt, bread, cheese, kimchi, fermented black beans and chili sauce, the polysaccharide content of Lactobacillus plantarum L23104 after fermentation is significantly increased, increasing by more than 15% and up to 25%, compared with commercially available starter cultures.

[0012] (5) When the high-yield extracellular polysaccharide plant lactobacillus L23104 provided by the present invention is used to ferment animal feed such as alfalfa silage, the polysaccharide content increases by 21.5%, which can provide nutrients for alfalfa silage and promote the fermentation process.

[0013] In one embodiment, the colony morphology of the Lactobacillus plantarum is characterized by raised colonies, milky white, round and smooth, translucent, and neat edges.

[0014] The present invention also provides a microbial preparation, which contains the above-mentioned Lactobacillus plantarum L23104 and / or its metabolites.

[0015] In one embodiment, the microbial preparation is in the form of a solid preparation or a liquid preparation.

[0016] In one embodiment, the number of Lactobacillus plantarum per gram or per milliliter of the microbial preparation is ≥ 1×10 8 CFU.

[0017] In one embodiment, the microbial preparation contains living cells or freeze-dried dry cells or immobilized cells of the above-mentioned Lactobacillus plantarum L23104, or a strain of Lactobacillus plantarum L23104 containing extracellular polysaccharides in any other form.

[0018] The second object of the present invention is to provide a microbial agent containing Lactobacillus plantarum L23104.

[0019] In one embodiment, the microbial agent is prepared as follows: the activated Lactobacillus plantarum L23104 is inoculated into a triangular flask containing 50 mL of liquid MRS culture medium, and cultured at 37° C. for 24 hours to obtain a primary seed solution of the strain; then inoculated into liquid MRS culture medium at a volume ratio of 5.0%, and cultured at 37° C. for 24 hours to obtain a bacterial concentration of 10 8 CFU / mL of bacterial agent.

[0020] The third object of the present invention is to provide an extracellular polysaccharide prepared from the above-mentioned Lactobacillus plantarum L23104 or any of the above-mentioned microbial preparations.

[0021] In one embodiment, the exopolysaccharide is prepared as follows:

[0022] Activate two generations of Lactobacillus plantarum L23104 and inoculate them into mMRS (MRS with 5% w / w sucrose) liquid culture medium and culture them at 37°C for 24h. Place the test tube in a boiling water bath for 10min and then cool it to room temperature. Centrifuge at high speed for 30min at 4°C to remove the precipitate. Take the supernatant and add trichloroacetic acid (TCA) solution to a final concentration of 4% and keep it at 4°C overnight. Centrifuge at high speed for 30min to take the supernatant and add 95% (w / v) pre-cooled ethanol and let it stand overnight. Centrifuge at high speed for 30min at 4°C and repeat the operation twice. Redissolve the precipitate in deionized water. After fully dissolving, dialyze it at 4°C using a dialysis bag with a molecular weight cutoff of 8000-14000Da for 48h. After dialysis, freeze-dry it to obtain extracellular polysaccharides.

[0023] In one embodiment, the exopolysaccharide prepared by the above-mentioned Lactobacillus plantarum L23104 or any of the above-mentioned microbial preparations has an antioxidant capacity of scavenging free radicals.

[0024] The fourth object of the present invention is to provide the use of the above-mentioned Lactobacillus plantarum L23104 or any of the above-mentioned microbial preparations in the preparation of extracellular polysaccharides or fermented products or antioxidant products or feed additives.

[0025] In one embodiment, the fermented product includes dairy products, pasta products, fermented vegetables, condiments or animal feed, wherein the dairy product includes yogurt or cheese; the pasta product includes bread; the fermented vegetable includes kimchi; the condiment includes fermented black beans or chili sauce; and the animal feed includes alfalfa.

[0026] In one embodiment, the fermented food includes but is not limited to yogurt, bread, cheese, kimchi, fermented black beans and chili sauce.

[0027] In one embodiment, the application is for yogurt fermentation, using the high-exopolysaccharide-producing Lactobacillus plantarum L23104 as a starter.

[0028] In one embodiment, the yogurt fermentation is obtained by inoculating the high-exopolysaccharide-producing Lactobacillus plantarum L23104 into milk at an inoculation rate of 0.5% to 10.0% w / w, and fermenting at 30° C. to 42° C. for 8 h to 24 h.

[0029] In one embodiment, the application is for dough fermentation, using the high-exopolysaccharide-producing Lactobacillus plantarum L23104 as a starter.

[0030] In one embodiment, the dough fermentation is performed by using the high-yield extracellular polysaccharide plant lactobacillus L23104 as a starter, adding it to the dough in amounts of 0.25 mL to 3.0 mL and 0.05 g to 0.25 g, respectively, using high-gluten flour, sucrose, sodium chloride and drinking water as main raw materials, mixing them in a mass ratio of (10.0 to 25.0): (0.5 to 1.5): (1.0 to 3.0): (5.0 to 10), and then fermenting and baking to obtain the dough.

[0031] In one embodiment, the application is for cheese fermentation.

[0032] In one embodiment, the dough fermentation is obtained by inoculating the high-exopolysaccharide plant lactobacillus L23104 starter into pasteurized milk at a ratio of 2% to 8% (v / v), and then fermenting, filtering, cutting, hanging filtering, secondary fermentation, adding salt, compression molding and packaging.

[0033] In one embodiment, the application is for kimchi fermentation, using the high-exopolysaccharide-producing Lactobacillus plantarum L23104 as a starter.

[0034] In one embodiment, the kimchi fermentation is obtained by inoculating the high-yield extracellular polysaccharide plant lactobacillus L23104 starter into mustard at an inoculation rate of 1.0% to 2.0% v / v, adding salt in an amount of 1.0% to 3.0% w / w, and performing processes such as filling, pressing, sealing and fermentation.

[0035] In one embodiment, the application is for fermentation of fermented black beans.

[0036] In one embodiment, the fermented black beans are obtained by spraying the high-yield extracellular polysaccharide plant lactobacillus L23104 starter at an inoculation rate of 1.0% to 3.0% v / v evenly on soybeans that have been steamed at high temperature, and fermenting at a temperature of 38° C. and a humidity of 70% for 12 hours to 18 hours.

[0037] In one embodiment, the application is for chili sauce fermentation.

[0038] In one embodiment, the chili sauce is fermented by inoculating the high-yield extracellular polysaccharide plant lactobacillus L23104 starter at an inoculation rate of 1.0% to 3.0% v / v into chili peppers boiled in boiling water, adding 1.0% to 5.0% w / w salt, and fermenting at 30° C. for 2 to 5 days to obtain the sauce.

[0039] The sixth object of the present invention is to provide the use of the above-mentioned Lactobacillus plantarum L23104 or any of the above-mentioned microbial preparations in fermenting animal feed.

[0040] In one embodiment, the animal feed includes but is not limited to silage alfalfa feed and the like.

[0041] In one embodiment, the alfalfa silage fermentation is obtained by inoculating the high-exopolysaccharide-producing Lactobacillus plantarum L23104 starter into alfalfa at gradient concentrations, and then vacuum-sealing and fermenting at room temperature for 20 to 45 days.

[0042] The seventh object of the present invention is to provide a method for reducing capsaicin in chili sauce, wherein the above-mentioned Lactobacillus plantarum L23104 or any of the above-mentioned microbial preparations or the above-mentioned exopolysaccharide is added during the preparation of the chili sauce.

[0043] In one embodiment, the amount of Lactobacillus plantarum L23104 or Lactobacillus plantarum L23104 inoculant added to the chili sauce is 0.5% to 5% (inoculation amount is 10 5 ~10 7 CFU / g).

[0044] The eighth object of the present invention is to provide a method for increasing the crude protein content, dry matter and carbohydrates in alfalfa silage feed, by adding the above-mentioned Lactobacillus plantarum L23104 or any of the above-mentioned microbial preparations or the above-mentioned extracellular polysaccharides when preparing the alfalfa silage feed.

[0045] In one embodiment, the amount of Lactobacillus plantarum L23104 or Lactobacillus plantarum L23104 inoculant added to alfalfa silage is 10 5 ~10 7 CFU / g wet weight.

[0046] Beneficial Effects

[0047] The invention screens a plant lactobacillus L23104 from sourdough and preserves it in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.29668. The strain can produce high amounts of extracellular polysaccharide and has excellent fermentation performance. The extracellular polysaccharide has the function of inhibiting free radical activity, can enhance the quality of fermented food and improve the safety of fermented food, and has broad application prospects.

[0048] Specific:

[0049] (1) The present invention provides a strain of Lactobacillus plantarum L23104, which has a highest OD value of a bacterial liquid at 20°C to 40°C, indicating that it grows well within this temperature range and is suitable for different temperature requirements of fermented products.

[0050] (2) The present invention provides a strain of Lactobacillus plantarum L23104 that can produce a high amount of extracellular polysaccharides. After culturing for 24 hours, the extracellular polysaccharide content reaches a maximum of 2460.12±1.46 mg / L, which can be used as a stabilizer to enhance the stability of the fermented product, thereby achieving the purpose of improving the texture and taste of the product.

[0051] (3) The polysaccharide solution produced by the high-yield extracellular polysaccharide Lactobacillus plantarum L23104 provided by the present invention was used for antioxidant experiments. The clearance rates of HO·, DPPH· and ABTS+ by Lactobacillus plantarum L23104 were higher than those of the control group, reaching (88.55±0.28)%, (82.15±0.14)% and (85.60±0.26)%, respectively, indicating that the extracellular polysaccharide produced by Lactobacillus plantarum L23104 has good antioxidant capacity.

[0052] (4) When the high-yield extracellular polysaccharide Lactobacillus plantarum L23104 provided by the present invention is used to ferment foods such as yogurt, bread, cheese, kimchi, fermented black beans and chili sauce, the polysaccharide content of Lactobacillus plantarum L23104 after fermentation is significantly increased, increasing by more than 15% and up to 25%, compared with commercially available starter cultures.

[0053] (5) When the high-yield extracellular polysaccharide plant lactobacillus L23104 provided by the present invention is used to ferment animal feed such as alfalfa silage, the polysaccharide content increases by 21.5%, which can provide nutrients for alfalfa silage and promote the fermentation process.

[0054] Biological deposit materials

[0055] The plant lactobacillus (Lactiplantibacillus plantarum) L23104 of the present invention is classified and named as plant lactobacillus Lactiplantibacillus plantarum, and has been deposited in the China General Microbiological Culture Collection Center on January 18, 2024, with a deposit number of CGMCC No.29668, and a deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is the Gram staining picture of Lactobacillus plantarum L23104 of the present invention.

[0057] Figure 2 It is a phylogenetic tree diagram of Lactobacillus plantarum L23104 of the present invention.

[0058] Figure 3 This is a 500-fold magnification scanning electron microscope image of the extracellular polysaccharide of Lactobacillus plantarum L23104 of the present invention.

[0059] Figure 4 This is a 2000-fold magnification scanning electron microscope image of the extracellular polysaccharide of Lactobacillus plantarum L23104 of the present invention.

[0060] Figure 5 It is a Fourier infrared analysis diagram of the extracellular polysaccharide of Lactobacillus plantarum L23104 of the present invention. DETAILED DESCRIPTION

[0061] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0062] Detection of physical and chemical indicators of fermented foods: pH value determination is determined by the method of GB 5009.237-2016. The total colony count is determined by the method of GB 4789.2-2022 National Food Safety Standard for Food Microbiological Examination for the Determination of Total Colony Count. The organic acid content is determined by high performance liquid chromatography (HPLC). The sample texture is determined by a texture analyzer. The nitrite content is determined by the spectrophotometric method in GB 5009.33-2016 National Food Safety Standard for the Determination of Nitrite and Nitrous Acid in Food. The dry matter (DM) content is determined by the oven drying method, and the crude protein (CP) content is determined by the Kjeldahl method; the water-soluble carbohydrates (WSC) content is determined by the anthrone colorimetric method; neutral detergent fiber and acid detergent fiber are determined by the Van soest method.

[0063] Dough sample source: Taken from Bozhou, Anhui;

[0064] Lactobacillus niger NC516.11, with the deposit number: CGMCC No.23107, was disclosed in patent CN113736713A on December 3, 2021.

[0065] Example 1: Screening and identification of Lactobacillus plantarum

[0066] 1. Sample preparation and strain isolation

[0067] Traditional fermented sourdough samples from different regions of my country were collected and stored in sealed sterile bags at -20°C. 2.0-5.0 g of sourdough sample was weighed and added to 45.0 mL of sterile saline (0.85%, m / v). Vortexed until the sample was thoroughly and evenly suspended. 1.0 mL of the suspension was diluted 10-fold to 10 -1 ~10 -7Use a sterile pipette tip to draw 100 μL of the dilution solution and apply it on the MRS plate culture medium. After culturing in an anaerobic incubator at 37°C for 24 hours, carefully observe the colony morphology and pick out a single colony with good growth.

[0068] The single colony was inoculated into liquid MRS medium, cultured at 37°C for 24 hours, and then streaked on solid MRS medium to observe the purity of the colony. This process was repeated three times until no foreign bacteria were found under the microscope. The purified colonies were subjected to catalase and Gram staining, and the catalase-negative, Gram-positive strain was preliminarily identified as lactic acid bacteria and named L23104. The single colony obtained was cultured in MRS liquid medium for 48 hours, then packaged and added to a sterilized 25% v / v glycerol protective agent and stored at -80°C.

[0069] 2. Morphological and physiological and biochemical characteristics of Lactobacillus plantarum L23104

[0070] The strain L23104 isolated in 1 was streaked on an MRS solid plate and cultured at 37°C for 24 hours to observe its colony morphology. A single colony was selected for Gram staining and its cell morphology was observed under a microscope. The colony of this strain was convex, milky white, round and smooth, translucent, with neat edges; the bacteria were rod-shaped and Gram-positive, such as Figure 1 The physiological and biochemical characteristics of lactic acid bacteria were determined by a lactic acid bacteria biochemical identification kit (purchased from Qingdao Haibo Biological, model HBIG11) as shown in Table 1, and it can metabolize most sugars except esculin and sodium hippurate.

[0071] Table 1 Physiological and biochemical characteristics of Lactiplantibacillus plantarum L23104

[0072]

[0073] Note: "+" indicates that the strain reacts positively and can metabolize; "-" indicates that the strain reacts negatively and cannot metabolize;

[0074] 3. Strain identification

[0075] The genomic DNA of lactic acid bacteria L23104 was extracted using a DNA extraction kit, and its OD value (A260 / A280) and concentration were determined. The qualified purity sample was diluted to about 100 ng / μL for PCR amplification.

[0076] The universal primers for 16S rDNA were: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′; SEQ ID NO. 1), 1492R (5′-GGTTACCTTGTTACGACTT-3′; SEQ ID NO. 2);

[0077] The PCR amplification system (50 μL) was: 25 μL 2×Phanta Max Buffer, 0.5 μL 10 mM dNTP Mix, 0.5 μL upstream primer (10 μM) and downstream primer (10 μM), 0.4 μL high-fidelity DNA polymerase, 5 μL template DNA, and sterile water to a total volume of 50 μL; the PCR amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 90 s, 35 cycles, and complete extension at 72°C for 5 min.

[0078] After 1% w / v agarose gel electrophoresis, the product was purified using a product purification kit and sent to the company for sequencing. The sequencing results were compared with the NCBI database for homology analysis to find the sequence of the closest known species. The homology of 99% or more can directly identify the strain to be tested. By constructing a phylogenetic tree, such as Figure 2 As shown, the lactobacillus was identified as Lactobacillus plantarum and was named Lactiplantibacillus plantarum L23104.

[0079] 4. Wire drawing experiment of Lactobacillus plantarum L23104

[0080] Take 1 drop of freshly prepared 4% v / v KOH aqueous solution on a clean glass slide, use an inoculation loop to take a small amount of freshly prepared L23104 colonies (3 mg) on ​​the solid culture medium, stir and mix in the KOH solution, and lift the inoculation loop every few seconds to observe whether a thread can be drawn. Repeat the operation for 4 to 5 colonies. If a thread can be drawn with the inoculation loop, it is positive, and if it is still a suspension, it is negative. Measure the maximum length (cm) of the colony thread, and the results are expressed as the average value. The initial screening obtained lactic acid bacteria with thread-drawing characteristics. The results showed that the longest thread drawn by L23104 was 5.5 cm.

[0081] 5. Strain preservation

[0082] The Lactobacillus plantarum L23104 strain was deposited in the China General Microbiological Culture Collection Center on January 18, 2024, with a deposit number of CGMCC No. 29668, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The Lactobacillus plantarum 16SrDNA sequence provided by the present invention is shown in SEQ ID NO.3.

[0083] Example 2: Growth characteristics detection of Lactobacillus plantarum L23104

[0084] 1. Growth curve

[0085] The glycerol-preserved plant lactobacillus L23104 strain prepared in Example 1 was inoculated on MRS solid medium for activation, and a single colony was picked and inoculated into 25.0 mL of liquid MRS medium for cultivation, and finally inoculated into 300.0 mL of MRS broth medium with a 2% (v / v) inoculum, and placed in a 37°C constant temperature incubator for static cultivation for 24 h, with three groups of biological replicates. Samples were taken every 2 h to measure the pH value and the number of colonies was calculated by gradient dilution.

[0086] Lactobacillus plantarum L23104 has a short growth cycle and enters the logarithmic growth phase after 10 hours. 600 The value increases rapidly. After 20 hours, it enters the stable period, and the number of viable bacteria reaches 10 9 CFU / mL and maintained a dynamic balance for a long time. Its pH showed a linear downward trend from 0 to 8 hours, dropped to 4.12 after 20 hours, and then gradually stabilized.

[0087] 2. Temperature curve

[0088] The activated Lactobacillus plantarum L23104 was inoculated into MRS liquid culture medium at a ratio of 2% (v / v), and cultured at 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C for 12 hours. The blank MRS liquid culture medium was used as a control to measure the OD value of the culture medium after fermentation. 600 value.

[0089] The results showed that Lactobacillus plantarum L23104 could grow in all temperature ranges, and the OD value of the bacterial solution was high at 20℃~40℃, indicating that the strain grew well in this temperature range, and its optimal growth temperature was 35℃.

[0090] 3. Sugar production curve

[0091] The glycerol-preserved Lactobacillus plantarum L23104 strain prepared in Example 1 was inoculated on MRS solid medium for activation, and a single colony was picked and inoculated into 25.0 mL of liquid MRS medium for cultivation, and finally inoculated into 300.0 mL of MRS broth medium at a 2% (v / v) inoculum, and placed in a 37°C constant temperature incubator for static culture for 24 h.

[0092] The activated Lactobacillus plantarum L23104 was inoculated into 150 mL mMRS (MRS medium with 5% w / v sucrose) medium at a ratio of 2% (v / v) (bacterial concentration was 10 5 CFU / mL), cultured at 37°C for 48 h, sampled every 6 h, and the phenol-sulfuric acid method was used to determine the extracellular polysaccharide content.

[0093] The results showed that Lactobacillus plantarum L23104 produced the most extracellular polysaccharide during the growth period of 18 to 24 hours, reaching the maximum at 24 hours, which was 2460.12±1.46 mg / L.

[0094] Example 3: Preparation of Lactobacillus plantarum L23104 inoculant

[0095] The preparation method of Lactobacillus plantarum L23104 bacterial agent is as follows:

[0096] The activated Lactobacillus plantarum L23104 was inoculated into a triangular flask containing 50 mL of liquid MRS medium and cultured at 37°C for 24 h to obtain the primary seed solution of the strain; then inoculated into liquid MRS medium at a ratio of 5.0% (v / v) and cultured at 37°C for 24 h to obtain a bacterial concentration of 10 8 According to different fermented food requirements, the prepared Lactobacillus plantarum L23104 bacterial agent is used for fermentation production.

[0097] Example 4: Extraction and determination of extracellular polysaccharides of Lactobacillus plantarum L23104

[0098] 1. Extraction and determination of extracellular polysaccharide of Lactobacillus plantarum L23104, the method is as follows:

[0099] After activating two generations of Lactobacillus plantarum L23104 prepared in Example 1, 2% v / v was inoculated into mMRS (MRS medium was supplemented with 5% w / v sucrose) liquid culture medium and cultured at 37° C. for 24 h.

[0100] Place the test tube in a boiling water bath for 10 minutes, then cool to room temperature, centrifuge at 4°C for 30 minutes to remove the precipitate, take the supernatant and add trichloroacetic acid (TCA) solution to a final concentration of 4% w / w, and keep at 4°C overnight. Centrifuge at high speed for 30 minutes, take the supernatant and add 95% (w / v) precooled ethanol, let stand overnight, centrifuge at 4°C for 30 minutes, and repeat the operation twice.

[0101] The precipitate was redissolved in deionized water. After being fully dissolved, it was dialyzed at 4°C for 48 h using a dialysis bag with a molecular weight cutoff of 8000-14000 Da. After dialysis was completed, it was freeze-dried.

[0102] Determination of the purity of extracellular polysaccharides: The extracted extracellular polysaccharides were prepared into a 0.1 mg / mL aqueous solution, and the absorption peaks of the samples in the range of 190 to 350 nm were scanned by a UV-visible spectrophotometer. No absorption peaks appeared at 260 and 280 nm, indicating that the sample did not contain nucleic acids and proteins, and the polysaccharide purity was high.

[0103] 2. Scanning electron microscopy (SEM) of extracellular polysaccharides of Lactobacillus plantarum L23104

[0104] The prepared extracellular polysaccharide was freeze-dried and then attached to a copper platform containing conductive glue using double-sided tape. Conductive gold was plated in a vacuum jet forging device. The microstructure of the extracellular polysaccharide and dough was observed at different magnifications. The results are shown in Figure 2. Figure 3 and Figure 4 As shown, the results show that the surface of extracellular polysaccharide is rough and presents a raised granular structure, which can bind more water molecules and give the extracellular polysaccharide a higher water holding capacity.

[0105] 3. Fourier transform infrared analysis of extracellular polysaccharides of Lactobacillus plantarum L23104

[0106] The functional groups in the extracellular polysaccharide were analyzed by Fourier transform infrared (FT-IR). 1.0-5.0 mg of freeze-dried extracellular polysaccharide was mixed with 100.0 mg of KBr, pressed into thin slices by a tablet press, and then heated at 400-4000 cm -1 Scan within the range and record the infrared spectrum.

[0107] The results are as follows Figure 5 As shown, 3430cm -1 The strong absorption peak at 2930 cm is caused by the stretching vibration of -OH. -1 The strong absorption peaks at 1640cm are caused by CH stretching. These two absorption peaks are characteristic peaks of polysaccharides. -1 The peak at 800cm is the characteristic peak of bound water, indicating that there is a strong interaction between polysaccharides and water molecules. -1 ~1000cm -1 The absorption peaks at , indicating the presence of α and β configuration glycosidic bonds.

[0108] Example 5: Analysis of the antioxidant capacity of Lactobacillus plantarum L23104 and its extracellular polysaccharides

[0109] The extracellular polysaccharide of Lactobacillus plantarum L23104 prepared in Example 4 was prepared into a 1.5 mg / mL solution and the OD was measured. 517 The value was used to detect its antioxidant capacity, and Vc and Lactobacillus cirrhosae NC516.11 were used as controls to prepare a 1.5 mg / mL Lactobacillus cirrhosae NC516.11 exopolysaccharide solution. The preparation method of the Lactobacillus cirrhosae NC516.11 inoculum and exopolysaccharide was consistent with that of Lactobacillus plantarum L23104.

[0110] 1. Hydroxyl radical (HO·) scavenging assay of extracellular polysaccharide of Lactobacillus plantarum L23104

[0111] 1.0 mL of Lactobacillus plantarum L23104 extracellular polysaccharide solution, 1.0 mL of 9 mmol / L H2O2 solution, 0.6 mL of 9 mmol / L salicylic acid solution, and 1.0 mL of 9 mmol / L ferrous sulfate solution were added to the test tube in sequence, and the tube was kept in a constant temperature water bath at 37°C for 1 hour, and the absorbance was measured at a wavelength of 562 nm. The scavenging ability of VC with an initial concentration of 1.5 mg / mL for HO· was determined by the same method.

[0112] 2. DPPH scavenging test of extracellular polysaccharide of Lactobacillus plantarum L23104

[0113] Add 1.0 mL of Lactobacillus plantarum L23104 extracellular polysaccharide solution, 1.0 mL of DPPH-ethanol solution, and 1.0 mL of deionized water to the test tube in turn, protect from light for 30 minutes, and measure the absorbance at a wavelength of 517 nm. The same method was used to measure the scavenging ability of VC with an initial concentration of 1.5 mg / mL for DPPH· in the positive control.

[0114] 3. ABTS+ removal test of extracellular polysaccharide of Lactobacillus plantarum L23104

[0115] 1.0 mL of Lactobacillus plantarum L23104 extracellular polysaccharide solution and 1.0 mL of ABTS+ test solution were added to the test tube in turn, the reactants were allowed to stand at room temperature for 20 minutes, and the absorbance was measured at a wavelength of 734 nm. The scavenging ability of VC with an initial concentration of 1.5 mg / mL of the positive control on ABTS+ was measured in the same way.

[0116] Clearance (%) = 1-(A1-A0) / A2×100%

[0117] Where: A1 is the absorbance of the sample, A2 is the absorbance of ABTS+, and A0 is the absorbance of distilled water.

[0118] The test results of hydroxyl free radicals, DPPH and ABTS are shown in Table 2. Lactobacillus plantarum L23104 was significantly better than the control group Lactobacillus niger NC516.11, and the free radical scavenging ability of the extracellular polysaccharide of Lactobacillus plantarum L23104 was close to that of VC.

[0119] Table 2 Antioxidant capacity of Lactobacillus plantarum L23104 and its extracellular polysaccharides

[0120]

[0121] Example 6: Application of Lactobacillus plantarum L23104 in fermented yogurt

[0122] The plant lactobacillus L23104 inoculum prepared in implementation case 3 and the commercially available yogurt starter (purchased from the official website of Ubit yogurt starter, model number is new product 99) were inoculated into milk at an inoculum amount of 1.0% v / v, and the analysis was performed after fermentation at 37°C for 24 hours. The crude polysaccharide determination method was referred to implementation case 4, with Lactobacillus cirrhosae NC516.11 and commercially available yogurt starter (purchased from the official website of Ubit yogurt starter, model number is new product 99) as the control group. The fermentation capacity of plant lactobacillus L23104 fermented yogurt was evaluated using pH, titratable acid (TTA) and total lactic acid bacteria count (TLC) as indicators.

[0123] The results showed that Lactobacillus plantarum L23104 could reach a lower pH and a significantly increased TTA value compared with the control group. The TLC and polysaccharide contents were significantly increased after fermentation, reaching 1.0×10 8 CFU / mL and 89.5mg / mL. This shows that Lactobacillus plantarum L23104 has good fermentation performance and exopolysaccharide production ability, and has great application potential in yogurt development.

[0124] Table 3 Fermentation characteristics of Lactobacillus plantarum L23104

[0125] index Control group Lactobacillus plantarum L23104 pH 4.3 3.5 TTA(mL) 5.3 7.5 TLC (CFU / mL) <![CDATA[10 7 ]]> <![CDATA[10 8 ]]> Polysaccharide (mg / mL) 58.1 89.5

[0126] Example 7: Application of Lactobacillus plantarum L23104 in fermented flour products

[0127] Bread preparation method: According to the mass, high-gluten flour, sucrose, sodium chloride and drinking water are used as the main raw materials, and the ratio of 10: 0.6: 0.2: 0.6 is mixed, 0.25 mL of the plant lactobacillus L23104 bacterial agent prepared in Example 3 and 0.25 g of commercial fermentation agent (purchased from Angel Yeast Company, high sugar tolerant type) are taken, and the mass ratio of the bacterial agent to the flour is 1: 100, and the dough is made and placed in a 35°C proofing box for 3 hours. After the fermentation is completed, the dough is baked in an oven at 190°C and 200°C for 25 minutes. After the baking is completed, the bread is placed at 25°C for balance and sealed for further analysis, and the same proportion of commercial food sinus Weiss lactic acid bacteria NC516.11 and commercial yeast are used as fermentation agents as a control group.

[0128] The fermentation power was determined by the CO2 weight loss method; the extracellular polysaccharide content in the bread was determined with reference to Example 4; the texture characteristics of the bread were determined by a texture analyzer, including hardness, elasticity, resilience, cohesion, viscosity, chewiness and other indicators, and each sample was measured at least 6 times.

[0129] The results of texture determination are shown in Table 4. Compared with the control group, the elasticity, resilience and cohesion of the bread fermented with Lactobacillus plantarum L23104 were not significantly different. The hardness, stickiness and chewiness of commercial yeast were greater. Compared with commercial yeast, the bread fermented with Lactobacillus plantarum L23104 had lower hardness and less stickiness, was not easy to stick together, and was relatively fluffy and soft. On this basis, the polysaccharide content in the bread fermented with Lactobacillus plantarum L23104 was 20.40% higher than that in the control group.

[0130] Table 4 Texture characteristics of sourdough fermented by Lactobacillus plantarum L23104

[0131]

[0132]

[0133] Example 8: Application of Lactobacillus plantarum L23104 in fermented cheese

[0134] 5.0L raw milk (purchased from Yili Industrial Group Co., Ltd.) was pasteurized at 63°C for 30min. 2% v / v of the plant lactobacillus inoculum prepared in Example 3 and 2% v / v of a traditional commercial cheese starter (purchased from the official website of Ubit yogurt starter, model number is new product 99) were inoculated into the pasteurized milk, and 2% v / v of a traditional commercial cheese starter (purchased from the official website of Ubit yogurt starter, model number is new product 99) and 2% v / v of Lactobacillus niger NC516.11 and commercial yeast were inoculated into the control group, stirred evenly, and fermented at 37°C for 8h to form curd.

[0135] The container is sealed with plastic wrap and the fermentation process is carried out under sealed conditions. When the pH of the curd drops to about 4.6 and can be separated from the container wall, cut it into 1 cm pieces with a cheese knife. 3 Small pieces. Filter the curd through four layers of gauze for about 5 hours until no whey is filtered out. Add 1% w / w salt to the curd and mix well, put it into a mold and press it for 24 hours until the moisture content is about 50%. Cut the formed curd into small pieces and vacuum pack it, then mature it in an incubator at 4°C and 90% humidity for 40 days. Collect cheese samples every 10 days, quick-freeze them in liquid nitrogen, and then store them at -80°C until analysis.

[0136] The samples were named and tested separately, where M represents cheese fermented with L23104, S represents cheese fermented with the control group starter, and 1 to 40 represent different maturation days.

[0137] 1. Determination of viable bacteria count in cheese

[0138] The cheese sample was diluted 10 times with 0.85% (w / v) sterile saline and homogenized in a sterile bag for 10 min. After continuous dilution to the required gradient, the plate count was performed. The lactic acid bacteria in the fermented cheese were counted using MRS agar medium at 37°C for 36 h, and the commercial starter culture fermented cheese was used as a control.

[0139] 2. Determination of basic physical and chemical indicators of cheese

[0140] 5.0 mL of sterile water and 1.0 g of grated cheese were thoroughly mixed and then measured using a pH meter; the sodium chloride content was determined by silver nitrate precipitation titration (GB 5009.44-2016); the water content was determined by direct drying method (GB 5009.3-2016); the protein content was determined by Kjeldahl nitrogen determination method (GB / T 5009.5-2016).

[0141] 3. Determination of polysaccharide content in cheese

[0142] The polysaccharide content in cheese was determined by reference to Example 4.

[0143] Table 5 Physical and chemical indexes of cheese

[0144]

[0145]

[0146] Note: S is commercial yeast, M is sample; 10, 20, 30 and 40 are fermentation days respectively.

[0147] It is generally believed that the number of probiotics in cheese should be greater than 10 6 CFU / g, which can have a positive impact on human health. The results are shown in Table 5. The number of probiotics in cheese made with Lactobacillus plantarum L23104 tends to be stable after 30 days of maturation, reaching 10 8 CFU / g. Compared with cheese made from traditional commercial cheese culture, the results of moisture, pH value, protein content and salt content of the test group with added Lactobacillus plantarum L23104 were slightly higher, but there was no significant difference; on this basis, the polysaccharide content of cheese made with added Lactobacillus plantarum L23104 was 17.5% higher.

[0148] Example 9: Application of Lactobacillus plantarum L23104 in fermented kimchi

[0149] After washing the fresh mustard greens, put them into boiling water for about 10 seconds. When the vegetables are blanched into emerald green, remove them, spread them out to cool and drain the water. The amount of salt added is 1.0% w / w, and 1.0% v / v of the plant lactobacillus L23104 prepared in Example 3 and the commercial Lactobacillus edodes NC516.11 are inoculated respectively and then put into jars, sealed tightly and fermented at room temperature. The fermentation end point is when the pickled vegetable sour aroma is obvious and the total acid content reaches 0.4%, and the natural fermentation group is used as the control.

[0150] pH value determination: Determine according to the method of GB 5009.237-2016;

[0151] Determination of nitrite: refer to the spectrophotometric method in GB 5009.33-2016 National Food Safety Standard Determination of Nitrite and Nitrous Acid in Foods;

[0152] Determination of organic acids: Chromatographic column: Agilent XDB-C18 (5μ, 250×4.6mm), mobile phase: 100% acetonitrile, 1% phosphoric acid aqueous solution (pH 2.8), flow rate 1 mL / min, injection volume 10 μL, UV detection wavelength 210 nm, column temperature 35°C.

[0153] The results showed that the physical and chemical indicators of kimchi fermented with Lactobacillus plantarum were normal, the nitrite content in kimchi added with Lactobacillus plantarum L23104 was 3.03 mg / kg lower than that in the control group, and significantly lower than the 20 mg / kg stipulated by the national food safety standard, and the polysaccharide content in kimchi added with Lactobacillus plantarum L23104 was 94.76 mg, 21.33% higher than that in the control group. That is, the Lactobacillus plantarum L23104 method improved the nutritional value and safety of kimchi.

[0154] Example 10: Application of Lactobacillus plantarum L23104 in fermentation of fermented black beans

[0155] After weighing and screening soybeans, add enough water to soak at 10 ℃ of water temperature until most of the beans have no wrinkles, and the soaking water volume is set to 3 times the mass of soybeans for 8 hours. The soaked soybeans are steamed with 0.1Mpa high pressure steam for 20 minutes to soften the soybean tissue and denature the protein, which is conducive to the growth, metabolism and secretion of microorganisms. The surface color of the soybeans after steaming is yellow-brown, and the beans are soft, with bean fragrance and no raw taste. The steamed soybeans are cooled to room temperature and artificially inoculated under a clean and tidy aseptic environment. The bacterial liquid of plant lactobacillus L23104 prepared by inoculation embodiment 3 of 1.0% v / v inoculation amount and the bacterial liquid of lactobacillus NC516.11 of food sinus Weiss are evenly sprayed on the surface of the beans. After inoculation, they are placed under the conditions of temperature 38 ℃ and humidity 70% and fermented for 12 hours. The fermentation is completed and is a bacterial type fermented black bean product. Compared with the control group, the polysaccharide content in the fermented black beans inoculated with Lactobacillus plantarum L23104 was 74.43 mg, which was 14.8% higher than that of the control group. The texture of the fermented black beans was appropriate, and other physical and chemical indicators were normal.

[0156] Example 11: Application of Lactobacillus plantarum L23104 in chili sauce fermentation

[0157] Select fresh and intact peppers, wash them, remove the stems and dry them, crush them and weigh them for later use. Boil the fermenter in boiling water for 30 minutes and then dry them for later use. Add 5.0% w / w salt, inoculate 1.0% v / v of the plant lactobacillus L23104 prepared in Example 3, and use an equal amount of Lactobacillus niger NC516.11 as a control group, and ferment at 30°C for 2 to 5 days.

[0158] Testing the physical and chemical indicators of chili sauce:

[0159] pH value determination: Determine according to the method of GB 5009.237-2016;

[0160] Determination of organic acid content: Determine with reference to the method in GB / T 21266-2007 "Determination of capsaicinoids in peppers and pepper products and expression of spiciness". Weigh 5.0g of chili sauce sample and dry it in an oven at 60℃ until the moisture is less than 15%, add 25mL of methanol and tetrahydrofuran (1:1) solution, seal with plastic wrap and extract in a 60℃ water bath with ultrasonic vibration for 1h, filter and repeat the above operation twice, combine the filtrate and evaporate it to 20mL at 70℃ and make it to 50mL. Filter through a 0.45μm filter membrane for chromatographic analysis. Chromatographic conditions: chromatographic column: Thermo Fisher C18 (4.6×250mm, 5μm); mobile phase: methanol: water = 80:20 (V:V); flow rate is 0.8mL / min; injection volume: 20μL; column oven temperature: 30℃; detection wavelength: 280nm.

[0161] The results showed that the pH changes between the experimental group and the control group were not obvious; before fermentation, the capsaicin content was as high as 0.68 mg / g. After fermentation with Lactobacillus plantarum L23104, the capsaicin content was significantly reduced to 0.28 mg / g, which softened the spicy taste of the chili sauce and made it more acceptable.

[0162] Example 12: Application of Lactobacillus plantarum L23104 in silage alfalfa feed

[0163] The Lactobacillus plantarum L23104 bacterial agent prepared in Example 3 was added in an amount of 1.0×10 5 CFU / gFM, 1.0×10 6 CFU / g FM and 1.0×10 7 CFU / g FM; 1.0×10 7 CFU / g FM concentration of Lactobacillus niger NC516.11 was used as the control group, and an equal amount of clean water was added. Three replicates were performed for each group and each gradient concentration. After being fully mixed with the alfalfa raw material, the bags were quickly evacuated and sealed using a vacuum sealer. The vacuum bags were stored at room temperature, and the sealing status was checked regularly. After storage at room temperature for 30 days, the bags were opened and samples were taken to measure various indicators.

[0164] Take 5.0g of alfalfa raw material and add 50.0mL of deionized water, extract at 4℃ for 12h, filter through 4-6 layers of gauze and measure the pH value. Determine the dry matter (DM) content by oven drying method, the crude protein (CP) content by Kjeldahl method; the water soluble carbohydrate (WSC) content by anthrone colorimetry; neutral detergent fiber and acid detergent fiber by Van soest method.

[0165] Table 6 Chemical composition of alfalfa before silage

[0166] project Numeric pH 6.12 <![CDATA[Dry matter / g·kg －1 FM]]> 146.69 <![CDATA[Crude protein / g·kg －1 DM]]> 45.76 <![CDATA[Carbohydrates / g·kg －1 DM]]> 98.90 <![CDATA[Neutral detergent fiber / g·kg －1 DM]]> 232.40 <![CDATA[Acid detergent fiber / g·kg －1 DM]]> 120.03

[0167] Note: FM, fresh weight; DM, dry matter.

[0168] Table 7 Effects of adding different concentrations of Lactobacillus plantarum L23014 on the chemical composition and fermentation performance of alfalfa silage

[0169]

[0170] The results are shown in Tables 6 and 7. The pH value of silage with plant acid bacteria was significantly lower than that of the control group at all concentrations (P<0.05), and decreased with the increase of the added concentration. The addition of lactic acid bacteria directly increased the number of lactic acid bacteria in the silage raw materials. At the same time, compared with the control group, the dry matter content and crude protein content increased by 23.1%, providing nutrients for alfalfa silage and promoting the fermentation process; on this basis, the polysaccharide content of plant acid bacteria Lactobacillus L23014 increased by 21.5% compared with that of Lactobacillus niger NC516.11.

[0171] 16S rDNA sequence SEQ ID NO.3:

[0172]

[0173] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A Lactobacillus plantarum ( Lactiplantibacillus plantarum ) L23104, characterized in that It was deposited in the China General Microbiological Culture Collection Center on January 18, 2024, with the deposit number CGMCC No.29668.

2. A microbial preparation, characterized in that: The microbial preparation contains the Lactobacillus plantarum L23104 or its fermentation liquid according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The number of Lactobacillus plantarum L23104 per gram or milliliter of the microbial preparation is not less than 1×10 8 CFU.

4. The microbial preparation according to claim 2 or 3, characterized in that: The microbial preparation contains living cells, freeze-dried dry cells or immobilized cells of Lactobacillus plantarum L23104 according to claim 1.

5. Use of the plant lactobacillus L23104 according to claim 1 or the microbial preparation according to any one of claims 2 to 4 in the preparation of extracellular polysaccharides or fermented products or feed additives.

6. The use according to claim 5, characterized in that: The fermented products include dairy products, flour products, fermented vegetables, condiments or animal feed.

7. The use according to claim 6, characterized in that: The dairy products include yogurt or cheese; the flour products include bread and steamed buns; the fermented vegetables include kimchi; the condiments include fermented black beans or chili sauce; and the animal feed includes alfalfa.

8. A product, characterized in that The product contains the Lactobacillus plantarum L23104 described in claim 1 or the microbial agent described in any one of claims 2 to 4.

9. The product according to claim 8, characterized in that The product is food, health product or feed.

10. A method for reducing capsaicin in chili sauce, characterized in that: When preparing the chili sauce, add the plant lactobacillus L23104 described in claim 1 or the microbial preparation described in any one of claims 2 to 4.

11. A method for increasing the crude protein content, dry matter and carbohydrate content in alfalfa silage, characterized in that: When preparing silage alfalfa feed, the plant lactobacillus L23104 according to claim 1 is added.

12. A method for increasing the crude protein content, dry matter and carbohydrate content in alfalfa silage, characterized in that: When preparing alfalfa silage feed, the microbial preparation according to any one of claims 2 to 4 is added.

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