Plant lactobacillus and application thereof in preparation of fiveleaf gynostemma herb probiotic oral liquid

Abstract

The invention discloses a plant lactobacillus and an application method thereof in preparation of a fiveleaf gynostemma herb probiotic oral liquid. The bacterial strain is lactobacillus plantarum R22, and is preserved in the China Center for Type Culture Collection on April 30, 2024, and the preservation number of the bacterial strain is CCTCC M 2024848. The fiveleaf gynostemma herb probiotic oral liquid is prepared by fermenting the fiveleaf gynostemma herb with the bacterial strain. Through determination of a plurality of indexes such as total saponins, total flavonoids, total phenols, viable count, lactic acid and the like, compared with a sample which is not inoculated and fermented, the content of the total saponins is increased by about 7.09%, the content of the total flavonoids is increased by about 7.10%, the content of the total phenols is increased by about 30.56%, and the viable count of the plant lactobacillus R22 can reach 7.1 * 10 < 8 > CFU / mL. Compared with unfermented fiveleaf gynostemma herb tea, the prepared fiveleaf gynostemma herb probiotic oral liquid has the advantages that the taste is improved, and the probiotic value of the fiveleaf gynostemma herb probiotic oral liquid is increased. Lactic acid and other antibacterial substances generated during fermentation of the plant lactobacillus R22 inhibit the growth of infectious microbes, have a certain preservative effect and can prolong the shelf life of the product.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a plant lactobacillus strain and application thereof in the preparation of gynostemma pentaphyllum probiotic oral liquid. Background Art

[0002] Gynostemma pentaphyllum is a herbaceous climbing plant of the Cucurbitaceae family and the genus Gynostemma. It has high medicinal value and health functions. It is a large variety of medicinal materials used for both medicine and food, and is known as the southern ginseng. The main chemical components of the Gynostemma plant include saponins, polysaccharides, vitamins, flavonoids, inorganic elements, etc. It has pharmacological effects such as anti-tumor, anti-cardiovascular disease, and liver protection, and has great research and development potential. [1] People call Jiaogulan the "magical" "longevity herb" that has the effects of clearing away heat and detoxifying, relieving cough and removing phlegm. Long-term drinking Jiaogulan tea can protect the human organs and endocrine organs, thereby achieving the effect of lowering blood sugar and improving sugar metabolism. [2] In recent years, a large number of studies at home and abroad have shown that Gynostemma pentaphyllum has many medicinal functions such as lowering blood lipids, lowering blood sugar, resisting arteriosclerosis, and protecting the liver. [3] .

[0003] Traditional Chinese medicine proecological preparations refer to preparations developed by combining the effects of traditional Chinese medicine by fermenting it with microorganisms. During the fermentation process, the two interact with each other, and usually the sum of the two can be greater than the sum of the two. Studies have shown that under the action of beneficial microorganisms, the dissolution of effective substances in traditional Chinese medicine can be promoted to increase the efficacy of the medicine; secondly, some microorganisms can reduce the toxic side effects of traditional Chinese medicine; in addition, some proteases will be produced during the metabolism of microorganisms, and under the action of certain enzymes, some substances in traditional Chinese medicine can be converted into effective ingredients, thereby improving the efficacy of traditional Chinese medicine. For microorganisms, traditional Chinese medicine can provide certain nutrients for the growth of microorganisms and promote the growth of microorganisms; secondly, when microorganisms play a probiotic role in the intestines, traditional Chinese medicine can assist microorganisms in regulating the balance of flora and maintaining intestinal health. Therefore, under the synergistic effect of the two, they usually have stronger probiotic effects such as resisting pathogens and improving the body's immunity than when the two are used alone. [4] .

[0004] Currently, there is a severe shortage of excellent strains that can be used for Gynostemma pentaphyllum fermentation, and there is an urgent need to screen and obtain such probiotic strains and develop Gynostemma pentaphyllum fermentation process technology.

[0005] References:

[0006] [1] Yuan Zhiying, Xie Mengzhou, Huang Huiyong. Research progress on plant resources, chemical components and pharmacology of Gynostemma pentaphyllum. Asia-Pacific Traditional Medicine, 2019, 15(07): 190-197.

[0007] [2] Ma Qian, Du Yu. Detection of sucrose content in Gynostemma pentaphyllum tea by HPLC. Modern Food, 2022, 28(16): 200-202+206.

[0008] [3] Bian Guzheng, Yu Yuhua, Yu Shujun, et al. Research progress on medicinal plant Gynostemma pentaphyllum. Agriculture and Technology, 2014, 34(02): 1-5.

[0009] [4] Huang Ye. On the development strategy and strategic significance of microecological Chinese medicine preparations. China Pharmaceutical Industry, 2012, 21(16): 23-27. Summary of the invention

[0010] In view of the above-mentioned current status, the present invention provides a plant lactobacillus and its application method in the preparation of gynostemma pentaphyllum probiotic oral liquid. The present invention utilizes plant lactobacillus to ferment gynostemma pentaphyllum for the first time, prepares the gynostemma pentaphyllum probiotic oral liquid, and gives the fermented product the comprehensive efficacy of the two. Through measuring multiple indicators such as total saponin, total flavonoids, total phenol, viable count, lactic acid, reducing sugar in the fermentation process of gynostemma pentaphyllum, and sensory evaluation is performed on the fermented product.

[0011] The Lactobacillus plantarum R22 provided by the present invention is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province), with a preservation number of CCTCC M 2024848 and a preservation date of April 30, 2024.

[0012] The preparation of the probiotic oral liquid of Gynostemma pentaphyllum is carried out by fermenting Gynostemma pentaphyllum with Lactobacillus plantarum R22 provided by the present invention, and the implementation steps involved are as follows:

[0013] The first step is to prepare Gynostemma pentaphyllum and water: prepare Gynostemma pentaphyllum and water in a material ratio (mass ratio) of 2:120;

[0014] Step 2: Enzyme hydrolysis: 0.5% (based on the dry weight of Gynostemma pentaphyllum) of cellulase was added for enzymolysis at a temperature of 50°C, a pH of 5.0, and a time of 120 min.

[0015] Step 3: Sterilization: 121℃ for 20min;

[0016] Step 4, inoculation: inoculate Lactobacillus plantarum R22 seed liquid at a 5% inoculation rate;

[0017] The fifth step is fermentation: the fermentation is carried out in a constant temperature incubator at 37°C for static culture and fermentation, and multiple indicators such as total saponins, total flavonoids, total phenols, viable bacteria count, lactic acid, reducing sugar, etc. are measured during the fermentation process.

[0018] The sixth step is to pass the mature fermented liquid through a 120-mesh sieve and fill it to obtain the Jiaogulan probiotic oral liquid.

[0019] The present invention uses plant lactobacillus to ferment Gynostemma pentaphyllum for the first time. Compared with the fermentation without inoculation, the total saponin content after fermentation is increased by about 7.09%, the total flavonoid content is increased by about 7.10%, the total phenol content is increased by about 30.56%, and the number of viable bacteria can reach 7.1×10 8 CFU / mL. The prepared Jiaogulan probiotic oral liquid has an improved taste compared to the unfermented Jiaogulan tea, and the lactic acid and other antibacterial substances produced during the fermentation of Lactobacillus plantarum R22 inhibit the growth of miscellaneous bacteria, have a certain antiseptic effect, and can extend the shelf life of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Attached Figure 1 It is the effect of different material ratios on the number of viable bacteria, where the abbreviation CFU stands for colony forming unit; Figure 2 It is the growth curve of Lactobacillus plantarum R22 strain during the fermentation of Gynostemma pentaphyllum, where the abbreviation CFU stands for colony forming unit. DETAILED DESCRIPTION

[0021] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0022] Example 1: Strain screening

[0023] 1.1 Strain Isolation

[0024] Take an appropriate amount of sample from naturally fermented sauerkraut and add it to MRS liquid culture medium, and place it in a 37℃ incubator for static culture for 24h. Dilute the culture solution in a gradient manner, take 100μL of the appropriate concentration dilution and apply it on the MRS solid culture medium. After the colony grows, select the single colony for streaking separation and purification according to the colony morphology, size and microscopic observation until a single colony is obtained. Preparation method of MRS liquid culture medium: 10g soy peptone, 5g beef extract powder, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 4g yeast extract powder, 0.2g magnesium sulfate, 2g triammonium citrate, 20g glucose, 0.05g manganese sulfate, 5g sodium acetate, 1L distilled water, adjust the pH value to 6.2±0.2, and sterilize at 121℃ for 20min. MRS solid culture medium is to add 20g agar powder to the MRS liquid culture medium formula.

[0025] 1.2 Strain screening

[0026] The individual colonies obtained in the above 1.1 experiment were numbered to obtain candidate strains, and the ability of different strains to produce acid and reduce nitrite was tested as follows:

[0027] Preparation of seed solution: Inoculate each strain into MRS liquid culture medium and culture it in a 37°C incubator for 24 hours to obtain seed culture medium. The preparation method of MRS liquid culture medium is as described in 1.1.

[0028] The seed liquid of each strain was inoculated with 2% of the inoculation amount into MRS liquid culture medium, incubated in a 37°C incubator for 24 hours, centrifuged, and the pH and acidity were determined. The acidity determination was carried out in accordance with the national standard GB5009.239-2016 "National Food Safety Standard Determination of Food Acidity"; the seed liquid of each strain was inoculated with 2% of the inoculation amount into MRS liquid culture medium containing 100 mg / mL sodium nitrite, incubated in a 37°C incubator for 24 hours, centrifuged, and 1 mL of the supernatant was placed in a test tube, and nitrite was determined in accordance with the national standard GB5009.33-2016 "National Food Safety Standard Determination of Nitrite and Nitrate in Food".

[0029] Among them, in the determination of acid production ability, the pH of R22 strain was 3.62 at 24 hours, and the acidity was 209.26±0.73°T; in the determination of nitrite reduction ability, the degradation rate of nitrite by R22 strain was 99% at 24 hours. R22 strain had the best comprehensive ability in various tests and was determined to be the strain of the present invention.

[0030] Example 2: Strain identification and preservation

[0031] The whole genome of the R22 strain of the present invention was sequenced, and the genome sequence of the strain was compared with the genome sequences of its closely related strains by ANI (average nucleotide identity). It was found that the whole genome sequence of the R22 strain was similar to that of the model strain of Lactobacillus plantarum ATCC 14917. T The genome sequence of the strain R22 has the highest homology of 99.07%, so the strain R22 is determined to be Lactobacillus plantarum and named as Lactiplantibacillus plantarum R22. The genome sequence of the strain R22 has been submitted to the NCBI genome database with the accession number JAYFSH000000000.

[0032] The strain Lactiplantibacillus plantarum R22 of the present invention was deposited in the China Center for Type Culture Collection on April 30, 2024, with the deposit number being CCTCC M 2024848.

[0033] Example 3: Determination of material ratio

[0034] Gynostemma pentaphyllum and water were prepared in a material ratio (mass ratio) of 1:120, 2:120, 3:120, and 4:120, and sterilized at 90°C for 1 hour and then cooled for use. Plant lactobacillus R22 seed liquid was inoculated with 5% of the above four groups of Gynostemma pentaphyllum aqueous solutions and placed in a 37°C constant temperature incubator for static culture. The number of viable bacteria was determined by plate counting method.

[0035] The preparation method of Lactobacillus plantarum R22 seed liquid is as follows: inoculate Lactobacillus plantarum R22 into MRS liquid culture medium, and culture it in a 37° C. incubator for 14 hours to obtain Lactobacillus plantarum R22 seed culture liquid. The preparation method of MRS liquid culture medium is as described in 1.1.

[0036] As attached Figure 1 As shown: when the material ratio is 1:120, the number of viable bacteria is only 2.8×10 8 CFU / mL; With the continuous increase of material ratio, the number of viable bacteria increased significantly. When the material ratio was 2:120, the number of viable bacteria could reach 5.03×10 8 CFU / mL, which is equivalent to the number of viable bacteria when the material ratio is 3:120 and 4:120. When the material ratio is too large, the fermentation liquid will be too concentrated and affect the taste, so the material ratio is selected as 2:120.

[0037] Example 4: Cellulase Hydrolysis

[0038] Gynostemma pentaphyllum and water were prepared at a material ratio (mass ratio) of 2:120, and cellulase from different brands (McLean, Yu Wanbang, Yinuo) was added at a ratio of 0.5% (based on the dry weight of Gynostemma pentaphyllum) for enzymolysis. The cellulase without cellulase was used as a control. The temperature was 50°C, the enzymolysis pH was 5.0, and the enzymolysis time was 120 min. The OD of the enzymolysis solution after enzymolysis was 600 The value was measured, the indirect reaction enzymatic hydrolysis effect, OD 600 The higher the value, the more substances are released into the solution, which indirectly indicates that the cellulase hydrolysis effect is better. The results show that the OD 600 The values ​​were all higher than those of the control group, indicating that the dissolution of the substance was promoted under the treatment of cellulase. Among them, the cellulase from the second brand had the best enzymatic hydrolysis effect and the cheapest price, so the cellulase from the second brand was selected for enzymatic hydrolysis.

[0039] Example 5: Preparation of Gynostemma pentaphyllum probiotic oral liquid

[0040] The specific process is:

[0041] The first step is to prepare Gynostemma pentaphyllum and water: prepare Gynostemma pentaphyllum and water in a material ratio (mass ratio) of 2:120;

[0042] Step 2: Enzyme hydrolysis: add Yu Wanbang cellulase at a ratio of 0.5% (based on the dry weight of Gynostemma pentaphyllum) for enzymolysis, the enzymolysis temperature is 50°C, the enzymolysis pH is 5.0, and the enzymolysis time is 120 min;

[0043] Step 3: Sterilization: sterilize at 121℃ for 20min, then cool for later use;

[0044] Step 4, inoculation: first prepare Lactobacillus plantarum R22 seed solution according to the method described in Example 3, and inoculate the Lactobacillus plantarum R22 seed solution at an inoculum amount of 5%;

[0045] Step 5, fermentation: the culture inoculated in step 4 is placed in a 37°C constant temperature incubator for static fermentation, and multiple indicators such as total saponins, total flavonoids, total phenols, viable bacteria count, lactic acid, reducing sugar, etc. are measured during the fermentation process;

[0046] The sixth step is to pass the mature fermented liquid through a 120-mesh sieve and fill it to obtain the Gynostemma pentaphyllum probiotic oral liquid.

[0047] Example 6: Determination of viable counts during fermentation of Gynostemma pentaphyllum

[0048] The plate count method was used to determine the number of viable bacteria during the fermentation process. The results are shown in the attached Figure 2 As shown in the figure, the number of live bacteria increased first and then decreased during the fermentation process, reaching 7.1×10 8 CFU / mL.

[0049] Example 7: Determination of total saponins during fermentation of Gynostemma pentaphyllum

[0050] The total saponin content was determined by the vanillin-perchloric acid method. The total saponin content increased slightly from 0 to 18 hours, increasing by about 7.09%.

[0051] Example 8: Determination of total flavonoids during fermentation of Gynostemma pentaphyllum

[0052] The total flavonoids were determined by sodium nitrite-aluminum nitrate colorimetric method. As the fermentation progressed, the content of total flavonoids increased. At 22h, the content of total flavonoids could reach 0.035mg / mL, which was about 7.10% higher than that at 0h.

[0053] Example 9: Determination of total phenols during fermentation of Gynostemma pentaphyllum

[0054] The total phenol content was determined by the Folin phenol method. During the fermentation process, the total phenol content increased. At 18h, the total phenol content reached 0.0117mg / mL, which was about 30.56% higher than that at 0h.

[0055] Example 10: Determination of reducing sugars during fermentation of Gynostemma pentaphyllum

[0056] The reducing sugar was determined by the dinitrosalicylic acid method. During the fermentation process, the reducing sugar consumption decreased rapidly from 0 to 10 hours, and at 14 hours, the reducing sugar was basically consumed, with only a very small amount of reducing sugar remaining unused.

[0057] Example 11: Determination of lactic acid during fermentation of Gynostemma pentaphyllum

[0058] The lactic acid was measured by biosensor analyzer. During the fermentation process, the lactic acid content first increased and then decreased slightly. At the 14th hour, the lactic acid content reached the highest level, which was 0.54 g / L.

[0059] Example 12: Sensory evaluation of Jiaogulan probiotic oral liquid

[0060] The probiotic oral liquid of Gynostemma pentaphyllum of the present invention and the commercially available Gynostemma pentaphyllum tea were compared and evaluated in terms of taste, smell and color. The results are shown in the following table: After fermentation, Gynostemma pentaphyllum tastes better than unfermented due to the presence of lactic acid bacteria and the production of lactic acid, and promotes the dissolution of some beneficial substances, making the color richer.

[0061]

[0062] In addition, according to observation, under normal temperature, Gynostemma pentaphyllum tea was contaminated on the second day, but the Gynostemma pentaphyllum probiotic oral liquid of the present invention was not contaminated after being placed for several weeks, indicating that in the presence of Lactobacillus plantarum R22 and substances such as lactic acid produced by it, the growth of miscellaneous bacteria can be inhibited, and a certain antiseptic effect can be obtained, which can extend the shelf life of the product.

[0063] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Lactiplantibacillus plantarum R22, whose deposit number in China Center for Type Culture Collection is CCTCC M 2024848.

2. Application of plant lactobacillus R22 according to claim 1 in the preparation of gynostemma pentaphyllum probiotic oral liquid.

3. The use according to claim 2, characterized in that: The lactobacillus plantarum R22 described in claim 1 is used to prepare the gynostemma pentaphyllum probiotic oral liquid, and the main raw material used is gynostemma pentaphyllum.

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