Lactobacillus plantarum and application thereof

Abstract

The invention discloses a strain of lactobacillus plantarum and application thereof, the lactobacillus plantarum is named as lactobacillus plantarum SMU5928 and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M2024731, and the preservation date is April 19, 2024. The lactobacillus plantarum has the advantages that the lactobacillus plantarum is named as the lactobacillus plantarum SMU5928, the lactobacillus plantarum is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M2024731; the strain is separated from yak feces. Protease generated by the strain can decompose a complex protein composition into amino acid and peptide, the strain has good acid resistance and cholate resistance, the acid resistance is high, and the survival rate is 64 + / -4.06% after the strain is placed in artificial gastric juice with the pH value of 3.0 for 3 h; the strain can slowly grow in cholate with the concentration of 1.0%, and the growth efficiency reaches 11.36 + / -1.19% of that of cholate-free culture. The strain has high enzyme production capacity, and the activity is 198.5 U / mL. Protease secreted by the bacterium can help the host digest macromolecular protein in the feed.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a plant lactobacillus strain and application thereof. Background Art

[0002] Protein is an important factor for the growth, development and milk production of yaks. Therefore, yaks have a great demand for protein during supplementary feeding, especially for pregnant and lactating yaks. However, due to the limited secretion of protease by yaks themselves, they may not be able to completely decompose the protein in the feed, resulting in reduced protein utilization, affecting the growth and development of yaks and reducing their immunity. In particular, supplementing protease during winter supplementary feeding can help yaks better decompose the protein in the feed and improve the utilization of protein, thereby meeting their growth and production needs.

[0003] Proteases are a class of enzymes that can catalyze the hydrolysis of protein peptide bonds to produce short peptides or amino acids. Proteases have a wide range of sources, among which microbial proteases have great potential for industrial production due to their low production cost, good stability, and strong specificity.

[0004] As a probiotic, Lactobacillus plantarum secretes a variety of enzymes, including proteases, during its growth and metabolism. These enzymes help Lactobacillus plantarum decompose and utilize nutrients in the environment, but the ability of Lactobacillus plantarum to secrete proteases found so far is limited.

[0005] Among Lactobacillus plantarum, Lactobacillus plantarum ST-Ⅲ is considered to be a strain rich in protease. This strain can produce high protease activity during the fermentation process, which helps to decompose protein and generate beneficial substances such as peptides and amino acids. However, due to species specificity, Lactobacillus plantarum ST-Ⅲ cannot adapt to the harsh geographical environment of the Qinghai-Tibet Plateau. It is difficult to colonize and survive in the intestines of yaks, and cannot play the role of secreting proteases, so it is not suitable for yaks. Summary of the invention

[0006] In view of this, the present invention aims at the above-mentioned problems and provides a strain of Lactobacillus plantarum and its application. The Lactobacillus plantarum can adapt to the severe environment of the Qinghai-Tibet Plateau and the internal environment of the yak and can produce high protease.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: In a first aspect, the present invention discloses a strain of Lactobacillus plantarum SMU5928, which is named Lactobacillus plantarum SMU5928 ( Lactobacillus plantarum SMU5928), deposited in China Center for Type Culture Collection (CCTCC), with the deposit number being CCTCC NO: M2024731, and the deposit date being April 19, 2024.

[0008] Furthermore, the gene sequence of the Lactobacillus plantarum SMU5928 is shown in SEQ ID NO.3.

[0009] In a second aspect, the present invention provides use of the Lactobacillus plantarum SMU5928 described in the first aspect in preparing a probiotic.

[0010] In a third aspect, the present invention provides a probiotic, wherein the probiotic contains the Lactobacillus plantarum SMU5928 described in the first aspect, or a fermentation broth containing Lactobacillus plantarum SMU5928, or a freeze-dried powder containing Lactobacillus plantarum SMU5928.

[0011] Furthermore, in the probiotic agent, the viable count of Lactobacillus plantarum SMU5928 is 10 8 above.

[0012] In a fourth aspect, the present invention provides a fermented product rich in protease, wherein the fermented product is prepared according to the following method: The Lactobacillus plantarum SMU5928 described in the first aspect was inoculated into an enzyme-producing liquid culture medium at an inoculation rate of 2% (v / v) to obtain a Lactobacillus plantarum fermentation broth, wherein the number of viable bacteria in the fermentation broth was 10 8 above.

[0013] Furthermore, the components of the enzyme-producing liquid culture medium are as follows: 5 g / L peptone, 10 g / L yeast extract, 40.0 g / L glucose, 1.5 g / L dipotassium hydrogen phosphate, 10 g / L cellulose, 2.5 g / L sodium citrate, and 0.5 g / L magnesium sulfate, and the pH value is adjusted to 6.7.

[0014] In a fifth aspect, the present invention provides use of the Lactobacillus plantarum SMU5928 described in the first aspect or the probiotic agent described in the third aspect in the preparation of a product for promoting the growth and development of yaks and regulating the body's immunity.

[0015] In a sixth aspect, the present invention provides a product comprising the Lactobacillus plantarum SMU5928 described in the first aspect, or the probiotic agent described in the third aspect, wherein the product is an animal health product.

[0016] Compared with the prior art, the present invention can achieve the following technical effects: The present invention provides a plant lactobacillus SMU5928, which is isolated from yak feces. The protease produced by the strain can decompose complex protein compositions into amino acids and peptides, and the strain has good acid and bile salt resistance, and has strong acid resistance, and the survival rate is 64±4.06% after 3 hours in artificial gastric juice with a pH of 3.0; it can grow slowly under a bile salt concentration of 1.0%, and the growth efficiency reaches 11.36±1.19% of that of culture without bile salt.

[0017] The plant lactobacillus has stronger adaptability to the severe environment of the Qinghai-Tibet Plateau and the intestinal environment of yaks, and the secreted protease can help yaks digest and decompose a large amount of macromolecular proteins in feed during supplementary feeding, improve the absorption rate of feed, and thus play a role in promoting the growth and development of yaks and regulating the body's immunity. The plant lactobacillus of the present invention has high protease production, and the activity of the produced protease is high, the ACE inhibition rate is 54%, and the enzyme activity is 198.5U / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 It is the hydrolysis circle of the protease production ability of Lactobacillus plantarum of the present invention; Figure 2 It is the electrophoresis diagram of the PCR product of 16SrDNA gene of Lactobacillus plantarum SMU5928 of the present invention; M represents DNA marker; 1-4 are samples. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application belong to the scope of protection of this application.

[0020] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0021] Sodium chloride (0.2 g / 100 mL), NaCL eluent, sodium carbonate solution, trichloroacetic acid, ammonium sulfate, hydrochloric acid solution, and ethyl acetate involved in the examples were purchased from ** Group Chemical Reagent Co., Ltd.; Pepsin (0.35 g / 100 mL), casein, and borate buffer (pH 8.3) were purchased from Chengdu ** Biotechnology Co., Ltd.; Ox bile salt, PBS, Folin reagent, Hip-His-Leu, and ACE solution were purchased from Chengdu ** Chemicals Co., Ltd.

[0022] The components of the culture medium involved in the embodiment are as follows: Enzyme-producing liquid culture medium: 5 g / L peptone, 10 g / L yeast extract, 40.0 g / L glucose, 1.5 g / L dipotassium hydrogen phosphate, 10 g / L cellulose, 2.5 g / L sodium citrate, 0.5 g / L magnesium sulfate, and the pH value was adjusted to 6.7.

[0023] Enzyme-producing solid culture medium: 5 g / L peptone, 10 g / L yeast extract, 40.0 g / L glucose, 1.5 g / L potassium dihydrogen phosphate, 10 g / L cellulose, 2.5 g / L sodium citrate, 0.5 g / L magnesium sulfate, 10 g / L agar powder, adjust the pH to 6.7.

[0024] Enzyme-producing (containing 0.5% calcium carbonate) solid culture medium: Add 0.5% (W / V) calcium carbonate to the enzyme-producing solid culture medium.

[0025] Enzyme-producing THIO medium: Add 0.2% (W / V) sodium thioglycolate to the enzyme-producing liquid medium.

[0026] Example 1: Isolation and identification of Lactobacillus plantarum SMU5928 1. Isolation of bacteria Weigh 2 g of fresh yak feces sample collected from Hongyuan County, Aba Prefecture, in 20 ml sterile water, shake and mix, then dilute in 10-fold series, take different dilutions, inoculate on enzyme-producing solid culture medium plates (containing 0.5% calcium carbonate) under sterile conditions, and culture at 37°C for 48 h. Pick single colonies with calcium-dissolving rings and different shapes for Gram staining, select Gram-positive bacteria, inoculate in enzyme-producing liquid culture medium, culture at 37°C, number the screened lactic acid bacteria, draw lines on enzyme-producing solid culture medium, purify and store for later use; Thirteen strains of lactic acid bacteria were screened from 45 strains of lactic acid bacteria that were acid-resistant and bile-resistant and had a significant inhibitory effect on the indicator strain. After eliminating the influence of acid, three strains of lactic acid bacteria still had a significant ability to produce protease. The lactic acid bacteria with the best protease production ability were named Lactobacillus plantarum SMU5928. Figure 1 As shown, 2. Molecular Biological Identification 1. Identification of morphological characteristics, culture characteristics, and physiological and biochemical characteristics: According to the methods described in "Berge's Manual of Bacterial Identification" (8th edition) and "Handbook of Common Bacterial System Identification" (Dong Xiuzhu, Cai Miaoying, etc., Beijing: Science Press, 2001.2), the morphological characteristics and culture characteristics of strain SMU5928 were analyzed. The specific results are as follows: Bacterial characteristics: Gram-positive, rod-shaped cells, about 0.5-1.0µm wide, 1.5-4µm long, in single, paired or chain forms, do not form spores, and have round ends.

[0027] Colony characteristics: Obvious colonies are formed on the enzyme-producing solid culture medium, with a diameter between 0.5-2.0 mm, round, with neat edges, milky white, sometimes light yellow or dark yellow, with a moist and smooth surface and no pigmentation.

[0028] The strain of Lactobacillus plantarum has strong acid resistance, with a survival rate of 64±4.06% after 3 hours in artificial gastric juice at pH 3.0; it can grow slowly at a bile salt concentration of 1.0%, and the growth efficiency reaches 11.36±1.19% of that in culture without bile salt.

[0029] 16S rDNA test A single colony of the frozen test strain was inoculated into 50 ml of enzyme-producing liquid culture medium, and cultured at 30°C for 24 hours. 2 mL of the bacterial culture at the end of logarithmic growth was taken and placed in a sterile EP tube for centrifugation. After centrifugation at 8000×g for 3 minutes (4°C), the bacterial cells were collected, the supernatant was discarded, and the genomic DNA of the strain was extracted using the CTAB freeze-thaw method specifically for lactic acid bacteria. Universal primers were used, the forward primer was F: 5′-AAGTCGTAACAAGGTAACC-3′ (the nucleotide sequence is shown in SEQ ID NO.1); the reverse primer was R: 5′-GGATAACACCTGGAAACAG-3′ (the nucleotide sequence is shown in SEQ ID NO.2), and the 16S rRNA gene region was amplified by PCR using bacterial DNA as an amplification template. The amplified product was purified and subjected to gel electrophoresis, as shown in FIG. Figure 2 As shown, the 16S rRNA gene sequence was determined, and the sequence was shown in SEQ ID No. 3, and then the gene sequence was compared. According to the Gen-Bank sequence homology comparison, strain SMU5928 and Lactiplantibacillus plantarum strain 3335 (GenBank accession number MT613628.1) had a homology of 99%; the obtained strain showed the highest molecular systematic relationship with Lactobacillus plantarum, and was preliminarily determined to be Lactobacillus plantarum.

[0030] Based on the above characteristics, strain SMUN5928 was named Lactobacillus plantarum SMU5928 ( Lactobacillus plantarumSMU5928). The strain was deposited in China Center for Type Culture Collection (CCTCC) on April 19, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024731.

[0031] SEQ ID No.3

[0032] Example 2: Determination of probiotic properties Activation of Lactobacillus plantarum SMU5928: An inoculation loop of freeze-dried bacteria was selected and streaked continuously on an enzyme-producing solid culture medium. After culturing at 37°C for 24 hours, a single colony was selected and streaked continuously on an enzyme-producing solid culture medium. This was repeated three times to obtain activated Lactobacillus plantarum SMU5928 bacteria, which were stored at 4°C for later use.

[0033] Preparation of bacterial suspension: Take 5 mL of activated bacterial solution and pour it into a sterilized 10 mL centrifuge tube on a sterile operating table. Collect the bacteria by centrifugation at 3000 r / min for 10 min, add 5 mL of sterile saline and mix well to make a bacterial suspension.

[0034] (2) Screening of probiotics tolerant to artificial gastric juice at pH 3.0.

[0035] Preparation of artificial gastric juice: Dissolve sodium chloride (0.2 g / 100 mL) and pepsin (0.35 g / 100 mL) in ultrapure water, adjust the pH value to 3.0 with 1 mol / L hydrochloric acid, and filter and sterilize with a vacuum pump in a sterile operating table for later use.

[0036] Determination of probiotic tolerance to artificial gastric juice: Take 1mL of bacterial suspension and mix it with 9mL of artificial gastric juice with pH 3.0, shake it well, and culture it in a constant temperature oscillator (37℃, 300r / min). Samples are taken at 0h and 3h respectively, and 15ml of enzyme-producing solid culture medium is poured and cultured at 37℃ for 48h. The number of viable bacteria is determined by the plate count method, and the survival rate (%) is calculated: survival rate (%) = number of viable bacteria at 3h / number of viable bacteria at 0h × 100%.

[0037] (3) Determination of probiotics’ tolerance to bile salts.

[0038] Take 5mL of activated bacterial solution and inoculate it with a 2% (v / v) inoculation volume (100μL) using a pipette into the enzyme-producing THIO liquid culture medium containing 0.0% ox bile salt (W / V) (blank), 0.3% ox bile salt (W / V), 0.5% ox bile salt (W / V), and 1.0% ox bile salt (W / V). After culturing at 37°C in a constant temperature oscillator for 24 hours, use the blank culture medium as the control (uninoculated enzyme-producing THIO culture medium) to measure the OD of the above different concentrations of culture medium. 600nm The value was used to calculate the tolerance of the strain to bile salts. Bile salt tolerance = OD of the culture medium containing bile salts 600nm Value / OD of blank culture medium 600nm value × 100%.

[0039] Table 1 Results of acid and bile resistance of Lactobacillus plantarum SMU5928

[0040] As shown in Table 1, the survival rate of strain SMU5928 is very high in the environment of pH 3.0, and it has strong tolerance to acid. After passing through the stomach, the surviving bacteria will come into contact with bile salts in the small intestine. In this experiment, the resistance of lactic acid bacteria to bile salts is used as a selection criterion for potential probiotics. The results of the acid-resistant strain SMU5928 tolerating different concentrations of bile salts show that it has a certain tolerance to 0.3% bile salts. When the bile salt concentration increases to 1.0%, the tolerance of SMU5928 to bile salts decreases.

[0041] The strain has good acid and bile salt resistance, and has strong acid resistance. The survival rate is 64±4.06% after 3 hours in artificial gastric juice with pH 3.0. It can grow slowly under 1.0% bile salt concentration, and the growth efficiency reaches 11.36±1.19% of that in culture without bile salt. Example 3: Method for producing protease by Lactobacillus plantarum (1) Preparation of crude protease The prepared SMU5928 bacterial suspension was inoculated into 5 mL of enzyme-producing liquid culture medium at an inoculation volume of 2% (v / v), and the mixture was shaken at 170 rpm / min, and the mixture was cultured at a constant temperature of 37°C for 24-36 hours to obtain Lactobacillus plantarum fermentation liquid. The Lactobacillus plantarum fermentation liquid was inoculated into 5 mL of enzyme-producing liquid culture medium at an inoculation volume of 2% (v / v), and the mixture was shaken at 170 rpm / min, and the mixture was cultured at a constant temperature of 37°C for 24-36 hours. This operation was performed for three consecutive generations of activation. The fermentation liquid of the third generation was centrifuged at 10000×g for 10 minutes, the bacteria were collected, the bacteria were washed three times with sterilized PBS, and the bacteria were resuspended. The resuspended bacterial liquid was collected, the bacteria were broken with an ultrasonic disruptor, and the supernatant was collected by centrifugation at 4°C and 12000×g for 20 minutes. The supernatant was filtered with a 0.45 μm filter membrane to remove residual cell debris to obtain a crude enzyme liquid.

[0042] (2) Purification of crude protease Ammonium sulfate with a final concentration of 45% (mass concentration) was added to the crude enzyme solution to precipitate the protease, which was then purified by DEAE-Sephadex A-25 chromatography and then eluted with 0.1 mol / L NaCL eluent. The purified enzyme solution was collected and the purified protein was detected by SDS-PAGE electrophoresis, with a molecular weight of 50 kD.

[0043] (3) Protease activity assay Take 1mL of the above purified enzyme solution, add 1mL of casein with a concentration of 20g / L as a substrate, and react for 10min at 40℃. After the reaction is completed, immediately add 3mL of trichloroacetic acid with a concentration of 0.4mol / L to terminate the reaction, and let it stand for 15min. Let it stand until the protein is completely precipitated, filter it with filter paper, and get the supernatant filtrate. Take 1mL of the supernatant filtrate, add 5mL of sodium carbonate solution with a concentration of 0.4mol / L, 1mL of Folin reagent, and perform a color reaction in a constant temperature water bath at 40℃ for 20min. After the color reaction is completed, the absorbance value at a wavelength of 680nm is measured by a spectrophotometer, and the boiled inactivated enzyme solution is used as a blank control. The results show that the enzyme activity of the purified enzyme is 198.5U / mL.

[0044] (4) Determination of protease ACE inhibitory activity Hip-His-Leu was prepared into a 5 mol / L solution using 0.1 mol / L borate buffer (pH 8.3) containing 0.3 mol / L NaCL. 200 μL of 5 mol / L Hip-His-Leu solution and 80 μL of purified sample were added to a 10 mL test tube, and the mixture was kept at 37°C for 3 min. 20 μL of ACE solution was added, and the mixture was mixed and kept at 37°C for 30 min. 250 μL of 1 mol / L hydrochloric acid solution was added to terminate the reaction, and 1.7 mL of ethyl acetate was added. After 15 s of oscillation and mixing, the mixture was allowed to stand for 5 min. 1 mL of ethyl acetate layer was taken out with a pipette, and dried in an oven at 120°C. 1 mL of distilled water was added. After mixing, the absorbance was measured at 228 nm.

[0045] ACE inhibition rate = [(BA) / B] × 100% IC 50 The concentration of inhibitor required to inhibit ACE activity by 50%.

[0046] Wherein, A represents the absorbance of the reaction between ACE and Hip-His-Leu when ACE inhibitory peptide is added; B represents the absorbance of the reaction between ACE and Hip-His-Leu when ACE inhibitory peptide is not added.

[0047] Results The ACE inhibition rate of the purified protease hydrolyzed whey protein was 54%.

[0048] Example 4 Preparation of a fermented product rich in protease This embodiment provides a fermented product rich in protease, and the preparation method thereof comprises the following steps: (1) Inoculate the activated SMU5928 bacterial solution into 5 mL of enzyme-producing liquid culture medium at a 2% (v / v) inoculation rate, shake and mix at 170 rpm / min, and culture at 37°C for 24-36 h to obtain Lactobacillus plantarum fermentation liquid.

[0049] (2) Count the number of live bacteria in the Lactobacillus plantarum fermentation broth, and then dilute the fermentation broth to 10 with enzyme-producing liquid culture medium. 8 Then repack.

[0050] Example 5 Preparation of probiotics This embodiment provides a probiotic agent, wherein the probiotic agent contains the fermentation liquid of Lactobacillus plantarum SMU5928 prepared in Example 4, and the number of viable bacteria of Lactobacillus plantarum SMU5928 in the probiotic agent is 10 8 above.

[0051] Example 6 This embodiment provides a probiotic agent, wherein the probiotic agent contains Lactobacillus plantarum SMU5928, and the number of viable bacteria of Lactobacillus plantarum SMU5928 is 10 8 above.

[0052] Example 7 This embodiment provides a probiotic agent, wherein the probiotic agent contains freeze-dried powder of Lactobacillus plantarum SMU5928, and the number of viable bacteria of Lactobacillus plantarum SMU5928 is 10 8 above.

[0053] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A plant lactobacillus, characterized in that: The plant lactobacillus was named as plant lactobacillus SMU5928 ( Lactobacillus plantarum SMU5928), deposited in China Center for Type Culture Collection (CCTCC), with the deposit number being CCTCC NO: M2024731, and the deposit date being April 19, 2024.

2. The Lactobacillus plantarum SMU5928 according to claim 1, characterized in that Its gene sequence is shown in SEQ ID NO.

3.

3. Use of Lactobacillus plantarum SMU5928 according to claim 1 or 2 in the preparation of probiotics.

4. A probiotic, characterized in that: The probiotic contains the Lactobacillus plantarum SMU5928 according to claim 1 or 2, or a fermentation liquid containing Lactobacillus plantarum SMU5928, or a freeze-dried powder containing Lactobacillus plantarum SMU5928.

5. The probiotic according to claim 4, characterized in that In the probiotic agent, the viable count of the plant lactobacillus SMU5928 is 10 8 above.

6. A fermented product rich in protease, characterized in that: The fermented product is prepared according to the following method: The Lactobacillus plantarum SMU5928 according to claim 1 or 2 is inoculated into an enzyme-producing liquid culture medium at an inoculation rate of 2-5% (v / v), and fermented to obtain a Lactobacillus plantarum fermentation liquid, wherein the number of viable bacteria in the fermentation liquid is 10 8 above.

7. The fermented product according to claim 6, characterized in that The components of the enzyme-producing liquid culture medium are as follows: 5 g / L peptone, 10 g / L yeast extract, 40.0 g / L glucose, 1.5 g / L dipotassium hydrogen phosphate, 10 g / L cellulose, 2.5 g / L sodium citrate, 0.5 g / L magnesium sulfate, 1 L deionized water, and the pH value is adjusted to 6.

7.

8. Use of the Lactobacillus plantarum SMU5928 according to any one of claims 1 to 2, or the probiotic according to any one of claims 4 to 5, in the preparation of a product for promoting the growth and development of yaks and regulating the body's immunity.

9. A product, characterized in that: The product contains the Lactobacillus plantarum SMU5928 according to any one of claims 1 to 2, or the probiotic according to any one of claims 4 to 5, and is an animal health product.

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