Lactobacillus plantarum IMAUYHD26 and its application and method for degrading condensed tannins of red bean grass

Abstract

The invention discloses a plant lactobacillus IMAUYHD26 and an application thereof and a method for degrading condensed tannins of red bean grass. The classification name of plant lactobacillus IMAUYHD26 is: plant lactobacillus ( Lactiplantibacillus plantarum ), deposited in China Center for Type Culture Collection, the deposit time is: November 11, 2024, and the deposit number is CCTCC NO: M 20242507. The degradation rate of sainfoin condensed tannins screened by the plant lactobacillus IMAUYHD26 in the present invention reaches 76.73%, which effectively improves the utilization value of sainfoin condensed tannins or feed containing sainfoin.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and more specifically to a plant lactobacillus IMAUYHD26, an application thereof and a method for degrading sainfoin condensed tannins. Background Art

[0002] Plant tannins are a type of secondary metabolites with a polyphenolic structure that are widely present in plants. They can be divided into hydrolyzed tannins, condensed tannins and complex tannins according to their chemical structure. Condensed tannins are composed of flavan-3-ol monomers, which are oligomers (2 to 10 monomers) and polymers (greater than 10 monomers) formed by polymerization of catechins, epicatechins, epigallocatechins and gallocatechins in a certain proportion. Flavan-3-ol monomers are connected by carbon-carbon double bonds to form stable chemical bonds, making them difficult to be decomposed by acids or enzymes. Condensed tannins are an anti-nutritional factor. Excessive intake will affect the digestion and absorption of nutrients by animal bodies. However, the depolymerized condensed tannins have multiple biological activities such as antibacterial, free radical scavenging, anti-oxidation, and promotion of stem cell differentiation. Therefore, effective degradation of condensed tannins is of great significance to enhance their application value.

[0003] The degradation of condensed tannins mainly includes microbial degradation, chemical degradation, catalytic hydrogenation degradation, etc. Among them, the microbial degradation method has the advantages of environmental protection, low cost, and mild reaction conditions. The relevant reports on the degradation of condensed tannins by microbial degradation involve grape seeds, raspberry seeds, sea buckthorn, larch, etc. The relevant microorganisms are mainly molds, such as Aspergillus niger Aspergillus niger , D. Penicillium shititi Penicillium purpurogenum straj Since the structures of condensed tannins in different plants are different, for example, the condensed tannin structure in purple dalbergia (Dalbergia genus, Leguminosae) is Figure 1 As shown in Figure 2, it is mainly composed of epicatechin and epigallocatechin. The condensed tannin structure in white clover (Trifolium genus, Leguminosae) is shown in Figure 2. Figure 2 As shown in Figure 1, it is mainly composed of prodelphinidin (gallic acid + epigallocatechin), while the condensed tannin structure in sainfoin (Leguminosae) is mainly composed of proanthocyanidins (catechin + epicatechin) and prodelphinidin (gallic acid + epigallocatechin), with prodelphinidin accounting for a larger proportion. Therefore, the suitable microorganisms for degrading condensed tannins of different plants are also different. At present, no research related to the microbial degradation of condensed tannins in sainfoin has been found. Summary of the invention

[0004] The purpose of the present invention is to provide a plant lactobacillus IMAUYHD26 and its application and a method for degrading sainfoin condensed tannins, so as to at least solve the above problems.

[0005] In order to achieve the purpose and other advantages of the present invention, one aspect of the present invention provides a strain of Lactobacillus plantarum IMAUYHD26, which is classified as: Lactobacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus plantarum ( Lactiplantibacillus plantarum )IMAUYHD26 was deposited in the China Center for Type Culture Collection on November 11, 2024. The deposit number is CCTCC NO: M 20242507. The address of the depository is: Wuhan University, Wuhan, China.

[0006] Another aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum IMAUYHD26 in degrading sainfoin condensed tannins.

[0007] Another aspect of the present invention provides a method for degrading sainfoin condensed tannins, wherein the above-mentioned Lactobacillus plantarum IMAUYHD26 is inoculated in a sainfoin condensed tannin degradation medium at an inoculation rate of 8% to 12%, and cultured at 28° C. to 40° C. for 72 to 120 hours.

[0008] Preferably, the above Lactobacillus plantarum IMAUYHD26 is inoculated into a sainfoin condensed tannin degradation medium at an inoculation rate of 10%, and cultured at 37° C. for 96 hours.

[0009] Preferably, the sainfoin condensed tannin degradation medium consists of the following components: 4.0 g of sainfoin condensed tannin, 3.0 g of sucrose, 3.0 g of NaNO3, 1.0 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, and 1,000 mL of distilled water.

[0010] In yet another aspect, the present invention provides a bacterial agent for degrading condensed tannins of sainfoin, which comprises the above-mentioned Lactobacillus plantarum IMAUYHD26.

[0011] The present invention has at least the following beneficial effects:

[0012] The present invention isolated a strain IMAUYHD26 suitable for degrading condensed tannins of sainfoin from naturally fermented sainfoin. The strain was identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum ), the degradation rate of the strain on sainfoin condensed tannins reached 76.73% when the inoculation amount was 10%, the culture time was 96 h and the culture temperature was 37 ℃, which effectively improved the utilization value of sainfoin condensed tannins or feed containing sainfoin.

[0013] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a condensed tannin structure found in purple dalbergia;

[0015] Figure 2 It is a condensed tannin structure found in white clover;

[0016] Figure 3 This is a graph showing the effect of the inoculation amount of the present invention on the degradation of condensed tannins by Lactobacillus plantarum IMAUYHD26;

[0017] Figure 4 This is a graph showing the effect of the culture time on the degradation of condensed tannins by Lactobacillus plantarum IMAUYHD26 of the present invention;

[0018] Figure 5 Graph showing the effect of the culture temperature on the degradation of condensed tannins by Lactobacillus plantarum IMAUYHD26 of the present invention.

[0019] Deposit of biological materials:

[0020] Lactobacillus plantarum IMAUYHD26, whose classification name is Lactobacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus plantarum ( Lactiplantibacillus plantarum )IMAUYHD26 was deposited in the China Center for Type Culture Collection (CCTCC) on November 11, 2024. The deposit number is CCTCC NO: M 20242507. The deposit address is: Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with embodiments and drawings so that those skilled in the art can implement the invention with reference to the description.

[0022] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0023] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0024] Sainfoin condensed tannin is extracted from the stems and leaves of sainfoin, with a purity of 85.0%.

[0025] MRS liquid culture medium: peptone 10.0 g, beef extract powder 10.0 g, yeast powder 5.0 g, glucose 20.0 g, K2HPO4 2.0 g, diammonium citrate 2.0 g, sodium acetate 5.0 g, MgSO4·7H2O 0.5 g, MnSO4 0.25 g, Tween-80 1.0 mL, distilled water 1 000 mL, the final pH value was 5.7±0.2.

[0026] MRS solid medium: Add 1.5% agar powder to the MRS liquid medium, then add 0.7% sterile CaCO3 solution to the MRS solid medium, and sterilize at 121℃ for 15 min. After sterilization, shake the medium and cool it to 45℃, then pour it into the plate and set it aside for use after solidification.

[0027] Preliminary screening of solid culture medium: tannic acid 5.0 g, agar powder 25.0 g, NaNO3 3.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, FeSO4·7H2O 0.1 g, bromophenol blue 0.4 g, distilled water 1 000 mL, and finally adjust the pH value to 5.0.

[0028] Sainfoin condensed tannin degradation culture medium: Sainfoin condensed tannin 4.0 g, sucrose 3.0 g, NaNO3 3.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, and distilled water 1 000 mL.

[0029] Example 1: Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Screening and identification of IMAUYHD26.

[0030] First, 30 g of red bean grass stems and leaves were cut into pieces under sterile conditions, placed in a fermentation bag and a certain amount of water was added. Three groups were paralleled and fermented in an incubator at 37°C for about 7 days. After the fermentation was completed, 0.5 g of the sample was weighed and placed in a test tube with 4.5 mL of physiological saline. This test tube was 10 -1 , dilute to 10 -3. Use a pipette to transfer 200 μL of each gradient dilution to MRS solid medium (temperature <60°C), spread it evenly on the agar plate with a spreading rod, and culture at 37°C for 2-3 days. Select single colonies with different morphologies, milky white, and calcium-dissolving rings that meet the morphological characteristics of lactic acid bacteria colonies on each gradient, select 5 colonies from each plate, streak the plate, and culture at 37°C for 2 days. All purified strains were subjected to Gram staining and catalase tests (3% H2O2 was directly added to the colonies). All Gram-positive, catalase-negative, non-spore-forming strains (preliminarily judged to be LAB) were selected, a total of 18 strains, for subsequent experiments.

[0031] Then, the selected third-generation pure bacterial liquid was injected into the holes made in advance on the primary screening solid culture medium using a puncher (6mm). Tannic acid was not added to the blank control group and cultured at 37°C for 3-5 days. If the strain can degrade the tannic acid in the culture medium and produce gallic acid, it will cause the color of the surrounding bromophenol blue indicator to change (from blue-purple to yellow-green) and form an obvious degradation circle around the hole, which can be preliminarily judged that the strain has the ability to degrade tannin. Based on the diameter of the color change area, 6 lactic acid strains with strong tannase activity were initially screened out, including YHD1-4, YHD1-8, YHD1-9, YHD2-1, IMAUYHD26, and YHD3-1.

[0032] Finally, the strains screened out were inoculated into the degradation medium of sainfoin condensed tannins at a ratio of 8%, and a blank control was used. The culture was carried out at 37°C for 48 h. The condensed tannin content was determined by the hydrochloric acid-n-butanol method, and the degradation rate was calculated. The results are shown in Table 1. Among them, the strain IMAUYHD26 had the highest ability to degrade sainfoin condensed tannins, which was 61.36%. It was significantly different from other strains and had good stability, so this strain was selected as the target strain.

[0033] Table 1

[0034]

[0035] Note: The values ​​in the table are mean ± standard deviation. Different lowercase letters in the same column indicate significant differences (P<0.05).

[0036] The strain IMAUYHD26 was identified by 16S rRNA gene sequence analysis and was named Lactobacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus plantarum ( Lactiplantibacillus plantarum )IMAUYHD26 was deposited in the China Center for Type Culture Collection (CCTCC) on November 11, 2024. The deposit number is CCTCC NO: M 20242507. The deposit address is: Wuhan University, Wuhan, China.

[0037] Example 2: Optimization of fermentation conditions for degradation of sainfoin condensed tannins by Lactobacillus plantarum IMAUYHD26.

[0038] Lactobacillus plantarum IMAUYHD26 was inoculated into the condensed tannin degradation medium of Sainfoin at an inoculum rate of 4%, 6%, 8%, 10% and 12% respectively. After culturing at 37 ℃ for 48 h, the condensed tannin content was determined by the hydrochloric acid-n-butanol method, and its degradation rate was calculated. The results are shown in Figure 3 As shown in the figure (different lowercase letters indicate significant differences, the same below). It can be seen that when the inoculation amount of Lactobacillus plantarum IMAUYHD26 is 4%-10%, the degradation rate gradually increases, and when it is 10%-12%, the degradation rate decreases, and the highest degradation rate is reached when the inoculation amount is 10%. Therefore, the optimal inoculation amount is determined to be 10%.

[0039] Lactobacillus plantarum IMAUYHD26 was inoculated into the condensed tannin degradation medium of Sainfoin at an inoculation rate of 8%. After culturing at 37°C for 24, 48, 72, 96, and 120 h, the condensed tannin content was determined by the hydrochloric acid-n-butanol method, and its degradation rate was calculated. The results are shown in Figure 4 As shown in the figure, the degradation rate of Lactobacillus plantarum IMAUYHD26 gradually increased during the culture time of 24-96 h, and the degradation rate tended to be stable during the culture time of 96-120 h, and reached the highest degradation rate when the culture time was 96 h. Therefore, the optimal culture time was determined to be 96 h.

[0040] Lactobacillus plantarum IMAUYHD26 was inoculated with 8% of the inoculum into the condensed tannin degradation medium of Sainfoin. After culturing at 28, 32, 37, 42, and 45 ℃ for 48 h, the condensed tannin content was determined by the hydrochloric acid-n-butanol method, and its degradation rate was calculated. The results are shown in Figure 2. Figure 5 As shown. It can be seen that the degradation rate of Lactobacillus plantarum IMAUYHD26 gradually increased when the culture temperature was 28-37 ℃, and gradually decreased when the culture temperature was 37-45 ℃, and reached the highest degradation rate when the culture temperature was 37 ℃. Therefore, the optimal culture temperature was determined to be 37 ℃.

[0041] The optimal combination of the above three culture conditions was selected, that is, Lactobacillus plantarum IMAUYHD26 was cultured at 10% inoculation amount and 37 ℃ for 96 h. It was determined that the degradation rate of condensed tannins reached 76.73%.

[0042] Comparative Example 1: Comparison of the degradation of condensed tannins from plants of the same genus but different species by Lactobacillus plantarum IMAUYHD26.

[0043] The fermentation conditions optimized in Example 2 were used to degrade the condensed tannins in Sainfoin, Sainfoin and Sainfoin of Don River, respectively. The composition of the condensed tannin degradation medium of Sainfoin of Don River was: 4.0 g of condensed tannin of Sainfoin of Don River (purity of 83.7%), 3.0 g of sucrose, 3.0 g of NaNO3, 1.0 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, and 1 000 mL of distilled water; the composition of the condensed tannin degradation medium of Sainfoin of Don River was: 4.0 g of condensed tannin of Sainfoin of Don River (purity of 85.4%), 3.0 g of sucrose, 3.0 g of NaNO3, 1.0 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, and 1 000 mL of distilled water. The hydrochloric acid-n-butanol method was used to determine the condensed tannin content and calculate its degradation rate. The results are shown in Table 2.

[0044] Table 2

[0045]

[0046] Note: The values ​​in the table are mean ± standard deviation. Different lowercase letters in the same column indicate significant differences (P<0.05).

[0047] As shown in Table 2, there are significant differences in the degradation rates of sainfoin condensed tannins, sainfoin condensed tannins and sainfoin condensed tannins by Lactobacillus plantarum IMAUYHD26. Among them, the degradation rate of sainfoin condensed tannins is the highest, reaching 75.56%. This may be due to the structural differences among sainfoin condensed tannins, sainfoin condensed tannins and sainfoin condensed tannins.

[0048] The number of equipment and processing scale described here are used to simplify the description of the present invention. The application, modification and variation of the plant lactobacillus IMAUYHD26 and its application and the method for degrading condensed tannins of sainfoin of the present invention are obvious to those skilled in the art.

[0049] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. Lactobacillus plantarum IMAUYHD26, characterized in that The classification name of Lactobacillus plantarum IMAUYHD26 is: Lactobacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus plantarum ( Lactiplantibacillus plantarum )IMAUYHD26 was deposited in the China Center for Type Culture Collection on November 11, 2024. The deposit number is CCTCC NO: M 20242507. The address of the depository is: Wuhan University, Wuhan, China.

2. Use of the plant lactobacillus IMAUYHD26 as claimed in claim 1 in degrading condensed tannins of sainfoin.

3. A method for degrading sainfoin condensed tannins, characterized in that: The plant lactobacillus IMAUYHD26 as claimed in claim 1 is inoculated into a sainfoin condensed tannin degradation medium at a volume percentage of 8% to 12%, and cultured at 28° C. to 40° C. for 72 to 120 hours.

4. The method for degrading sainfoin condensed tannins according to claim 3, characterized in that: The plant lactobacillus IMAUYHD26 as claimed in claim 1 was inoculated into the sainfoin condensed tannin degradation medium at a volume percentage of 10%, and cultured at 37° C. for 96 hours.

5. The method for degrading sainfoin condensed tannins according to claim 3, characterized in that: The sainfoin condensed tannin degradation medium is composed of the following components: 4.0 g of sainfoin condensed tannin, 3.0 g of sucrose, 3.0 g of NaNO3, 1.0 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, and 1000 mL of distilled water.

6. A bacterial agent for degrading condensed tannins of sainfoin, characterized in that: It contains the Lactobacillus plantarum IMAUYHD26 described in claim 1.

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