Lactobacillus plantarum L13 and application thereof

Abstract

The lactobacillus plantarum L13 is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on May 23, 2023, and the preservation number of the lactobacillus plantarum L13 is CGMCC (China General Microbiological Culture Collection Center) NO.27398, and the lactobacillus plantarum L13 is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on May 23, 2023. The probiotic fermented porphyra yezoensis has the advantages of salt resistance, acid resistance, high DPPH free radical scavenging rate, high ABTS < + > free radical scavenging rate and high antioxidant activity, and guidance is provided for development of probiotic fermented porphyra yezoensis products with high nutritional value and safety; the method is widely applied to the fields of biology, medicine, food and the like; human intestinal flora can be improved, the digestive ability can be improved, cholesterol can be reduced, and the like, which is beneficial to human bodies.

Description

Technical Field

[0001] The invention relates to the technical field of probiotic application, and in particular to a Lactobacillus plantarum L13 and an application thereof. Background Art

[0002] Porphyra yezoensis is an important economic algae in the northern waters of my country. It is mainly cultivated in the coastal areas of Bohai Sea, Yellow Sea and the northern East China Sea. Porphyra contains a variety of nutrients. Usually, dry porphyra contains about 38±12% crude protein, 30±10% carbohydrates, 2±1% crude fat and a large amount of minerals. Porphyra yezoensis has important application value, especially in biology and pharmacy. The biological activity of porphyra is inseparable from these nutrients. Many studies have shown that Porphyra yezoensis has antioxidant activity, anti-fatigue activity, blood sugar lowering and immunity regulation.

[0003] Because Porphyra yezoensis has good nutritional value, it is often used as an important raw material in the food industry. Porphyra yezoensis processing is an important category in our algae food processing, especially Lianyungang City, Jiangsu Province is one of the main origins and processing bases of Porphyra yezoensis in the country, and more than half of the Porphyra yezoensis in the country is produced in Lianyungang. However, with the rapid expansion of Porphyra yezoensis cultivation area, the total output of Porphyra yezoensis has increased dramatically. At the same time, the processing method of Porphyra yezoensis is relatively single, mainly based on dried laver and seaweed, which limits the development of Porphyra yezoensis processing industry and also affects the economy of Porphyra yezoensis without obtaining better economic benefits.

[0004] In recent years, people have done a lot of research on laver polysaccharides. Laver polysaccharides are a type of galactan sulfate with a structure similar to agar. This naturally formed macromolecular polymer is usually composed of multiple monosaccharides and linear glycosidic bonds. Since then, people have discovered that laver polysaccharides have many high-quality functions. With people's continuous discoveries, people have become enthusiastic about the research of laver polysaccharides.

[0005] A large number of studies have shown that polysaccharides, as a bioactive substance, have many physiological activities and can play a good health care role, such as antioxidant activity, antibacterial activity, anticancer activity, blood sugar regulation activity, etc., and have non-toxic and harmless characteristics. Ramírez, José A et al. found in their study that laver polysaccharides have a positive effect on reducing the proportion of propylene glycol, lipid peroxidation and oxidative stress. Zhou Huiping et al. found that laver polysaccharides can promote the biosynthesis of serum proteins in mice, and significantly enhance cellular immunity and humoral immunity; Mahmoud et al. found that laver polysaccharides can completely inhibit or slow down the development of cytopathic effects of cells infected with herpes simplex virus (HSV), and even at a concentration of up to 250μg / mL, it did not show any cytotoxic side effects on African green monkey kidney cells. Kazutoshi Nishizawa et al. fed rats with 1.75%±0.25% laver powder and found that it could inhibit the cancer rate by 20%. Further research on its effective ingredients showed that laver polysaccharides have a positive effect on anti-tumor. Porphyra polysaccharides can also promote glucose metabolism by controlling glycogen synthesis and gluconeogenesis, thereby delaying glucose absorption and diffusion, thereby controlling blood sugar. Therefore, the excellent properties of Porphyra polysaccharides have become a new hot research issue.

[0006] As the high-quality activities of laver polysaccharides are gradually studied and reported, people have a deeper understanding of the biological activities of laver polysaccharides. Based on this, the in-depth exploration and research of laver polysaccharides can have an important impact on many aspects such as the food industry and the pharmaceutical industry. At the same time, it plays a great role in expanding the utilization and development of polysaccharides in more fields. Summary of the invention

[0007] The invention aims to provide a strain of Lactobacillus plantarum L13 screened from Guizhou brine and growing well in Porphyra yezoensis. The strain has multiple functions such as anti-oxidation, acid resistance, salt resistance and the like and has important potential application value.

[0008] The object of the present invention is achieved as follows: a plant lactobacillus (Lactiplantibacillus plantarum) L13, characterized in that the plant lactobacillus L13 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 23, 2023, and the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.27398.

[0009] The 16S rDNA sequence of the Lactobacillus plantarum L13 is shown in SEQ ID NO: 1.

[0010] SEQ ID NO:1

[0011]

[0012] The second purpose of the present invention is to use the polysaccharide extracted from fermented Porphyra yezoensis to obtain a FRAP value of 2.79, a DPPH free radical scavenging rate of 75.1%, and an ABTS + The free radical scavenging rate reached 85.2%.

[0013] The third purpose of the present invention is the use of Lactobacillus plantarum L13 in the preparation of antioxidant products.

[0014] The fourth purpose of the present invention is to use Lactobacillus plantarum L13 as a functional probiotic in the preparation of food, food additives or medicines, wherein the medicines are used to stabilize intestinal balance and regulate intestinal health.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a plant lactobacillus L13, which is screened out from Guizhou brine, and the strain has salt tolerance, acid resistance, high DPPH free radical scavenging rate, ABTS + The free radical scavenging rate and high antioxidant activity provide guidance for the development of probiotic fermented Porphyra yezoensis products with high nutritional value and safety; they are widely used in biology, medicine, food and other fields; they help to improve the human intestinal flora, improve digestion ability, lower cholesterol and other beneficial effects on the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the technical description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is the colony morphology of Lactobacillus plantarum L13.

[0018] Figure 2 This is the phylogenetic tree of Lactobacillus plantarum L13.

[0019] Figure 3 The numbers of viable bacteria and FRAP values ​​of the extracts of different strains of Porphyra yezoensis fermented.

[0020] Figure 4 The salt tolerance of Lactobacillus plantarum L13.

[0021] Figure 5 The acid resistance of Lactobacillus plantarum L13.

[0022] Figure 6 The effect of fermentation time on the antioxidant capacity of fermented Porphyra yezoensis polysaccharides. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Main ingredients: brine from Qiandongnan Prefecture, Guizhou Province and Porphyra yezoensis from Lianyungang.

[0025] Main reagents: MRS liquid and solid culture medium, LB liquid and solid culture medium, purchased from Beijing Luqiao Technology Co., Ltd.; new bacterial biochemical identification kit, purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; genome extraction kit, purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0026] Sample collection: Use a sterile spoon to take an appropriate amount of brine sample, and filter it with sterile gauze. Put the filtrate into a sterile conical bottle, seal it, and store it in a -4℃ refrigerator for later use.

[0027] Embodiment 1:

[0028] Isolation and purification of strains: Add 10 mL of brine sample to a conical flask with 90 mL of MRS liquid culture medium, seal it with newspaper, and place it in a 37°C incubator for enrichment culture. Take the bacterial liquid after enrichment culture, draw three or four zones in MRS solid culture medium, and culture it in a 37°C incubator for 48 hours. Pick several single colonies and inoculate them into centrifuge tubes containing MRS liquid culture medium in turn, culture them in a 37°C incubator for 48 hours, draw three or four zones in MRS solid culture medium, culture them in a 37°C incubator for 48 hours, and repeat the streaking culture until there is a single colony in the plate. Pick the single colony after separation and purification, expand the culture, and store it in glycerol.

[0029] Screening of strains: Add 100.0g of Porphyra yezoensis powder to 120mL of distilled water, stir into a paste, and sterilize at 121℃ for 30min. After cooling to room temperature, add strains to the laver paste at a 3% inoculation rate, and ferment at 37℃ for 72h. Using FRAP value and viable count as indicators, uninoculated and fermented Porphyra yezoensis was used as a blank control to screen strains suitable for fermenting Porphyra yezoensis and improving the antioxidant activity of the extract. Take the fermented Porphyra yezoensis and dilute it with ultrapure water in a ratio of 1:5 (g / mL) in a beaker. After stirring with a magnetic stirrer for 30min, centrifuge at 1000r / min for 15min, centrifuge twice under the same conditions, and take the supernatant for index determination.

[0030] A total of 267 strains were isolated and purified from Guizhou brine, of which 170 strains were Gram-positive, with short rod-shaped and smooth surfaces. Five of them were further screened for fermentation of Porphyra yezoensis through acid production experiments. Figure 3 The results showed that all five strains could enhance the activity of Porphyra yezoensis extract on Fe 3+ The reducing ability of L13 increased, and the number of viable bacteria also increased. The effectiveness of improving the antioxidant capacity of Porphyra was ranked from high to low as L13, X5, YY9, T8 and C6. At the same time, the number of viable bacteria after fermentation of L13 was also high. Therefore, strain L13 was selected as the strain for fermenting Porphyra yezoensis to further identify L13.

[0031] Identification of strain L13

[0032] (1) The isolated and purified strains were preliminarily identified by colony morphology observation, Gram staining and physical and chemical identification. The colony morphology of strain L13 was convex dots, with a smooth surface, clear edges and milky white color.

[0033] Specific method: dilute the bacterial solution to 2McFarland with 0.85% saline, add 1 to 2 drops to each microvial, culture at 37°C for 24 hours, and observe the color change according to the identification kit instructions to determine whether it is Lactobacillus.

[0034] (2) The strain obtained by the above screening was inoculated into liquid MRS medium (inoculation volume 1%) and cultured at 37°C overnight. The culture medium was centrifuged for 2 min using a TGL-20MW centrifuge at 12000 r / min, and the supernatant was discarded. After extracting the genome of the target strain, PCR amplification was performed, wherein 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used as primers, and amplification was performed in a total reaction system of 50 μL. The PCR amplification product was sent to Nanjing Sipujin Company for sequence detection. The BLAST program was used for comparison, and the phylogenetic tree was constructed using MEGA 7.0 software.

[0035] The 16S rDNA sequencing results of strain L13 showed a total length of 1475 bp, and it was found that the similarity with Lactobacillus plantarum was 100%, and it was preliminarily determined that the strain screened was Lactobacillus plantarum.

[0036] Biochemical identification revealed that strain L13 did not produce gas when fermenting glucose, and the gelatin liquefaction result was negative, and it could ferment and utilize sugars such as trehalose, xylose, fructose, arabinose, salicylic acid, etc. Therefore, combined with the above results, strain L13 was determined to be Lactobacillus plantarum.

[0037] Biochemical identification table of strain L13

[0038]

[0039] Note: + indicates that more than 90% of the strains are positive, - indicates that more than 90% of the strains are negative.

[0040] Example 2: FRAP value (Fe 3+ Reducing power) determination

[0041] Accurately take 5 μL of fermented Porphyra yezoensis supernatant, add 100 μL of formaldehyde and 2.85 mL of FRAP working solution, mix well, keep it in the dark for 30 minutes, and measure the absorbance at 593 nm. Use Trolox as the standard, the standard solution concentration as the horizontal axis, and the absorbance as the vertical axis to draw a standard curve.

[0042] FRAP value (mg / mL) = A × N

[0043] Where: A is the value obtained by substituting the absorbance of the sample into the regression equation, mmol / L; N is the dilution factor.

[0044] Example 3: Determination of DPPH free radical scavenging rate

[0045] Take 2 mL of fermentation broth, add 2 mL of 0.1 mmol / mL DPPH solution, react at room temperature in the dark for 30 minutes, use anhydrous ethanol as the reference solution, measure the absorbance at 517 nm (A1), and simultaneously measure the absorbance after mixing 2 mL of fermentation broth and 2 mL of anhydrous ethanol (A2), and the absorbance after mixing 2 mL of anhydrous ethanol and 2 mL of DPPH solution (A3). Calculate the DPPH free radical scavenging rate using the following formula:

[0046]

[0047] Example 4: ABTS + Free radical scavenging rate assay

[0048] ABTS was prepared by mixing 7mmol / L ABTS and 2.45mmol / L potassium persulfate in equal volumes. + Free radical working solution, and adjust the absorbance of the working solution at 734nm to 0.7±0.02 with distilled water for later use. Take 0.5mL fermentation liquid and 5mL working solution and mix them evenly. The reaction should be protected from light for 5min. Measure its absorbance at 734nm.

[0049]

[0050] Wherein: A1 is the absorbance value of 0.5 mL fermentation liquid and 5 mL working liquid; A2 is the absorbance value of 0.5 mL fermentation liquid and 5 mL anhydrous ethanol; A3 is the absorbance value of 0.5 mL anhydrous ethanol and 5 mL working liquid.

[0051] Embodiment five:

[0052] Extraction of polysaccharides from fermented Porphyra yezoensis: The polysaccharides from the fermented broth of Porphyra were extracted by water extraction and alcohol precipitation. Weigh 10.00g of sample and place it in a 100mL beaker. Add 20mL of ultrapure water and mix well, stir with a magnetic stirrer at 90℃, and extract for 2h. After the extraction, centrifuge at 10000r / min for 15min, repeat the centrifugation twice under the same conditions, discard the precipitate and take the supernatant. After the supernatant is concentrated to 1 / 6 of the original volume under reduced pressure, add four times the volume of anhydrous ethanol to the concentrate, let it stand at 4℃ for 12h, and centrifuge at 8000r / min for 15min. The obtained precipitate is re-dissolved in water to obtain a polysaccharide solution of Porphyra yezoensis.

[0053] 1) Sample pretreatment: Add pure water to the crude polysaccharide extract solid to dissolve, add papain, and enzymolyze overnight. Then add 1 / 4 volume of chloroform and n-butanol to the enzymolysis solution, mix well, and collect the upper aqueous phase. Add petroleum ether to the upper aqueous phase, mix well, and collect the lower aqueous phase. Add macroporous resin AD-8 to the lower aqueous phase, mix well, and adsorb overnight. Collect the liquid, dialyze it with a 3000Da dialysis bag for 48 hours to remove small molecular components, and freeze-dry to obtain the polysaccharide after impurities are removed.

[0054] 2) Determination of total sugar content and purity: Accurately weigh a small amount of polysaccharide freeze-dried powder, add water to dissolve, add sulfuric acid-phenol reagent, react for 10 minutes in the dark, measure the absorbance at 490nm, and use glucose as the standard to prepare a series of standard solutions to draw a standard curve.

[0055] The total sugar content in crude polysaccharides was calculated according to the standard curve:

[0056]

[0057] Where: X is the total sugar content, mg / mg; C is the result calculated according to the standard curve; M is the actual weighed mass; V is the actual dissolved volume of the sample.

[0058] Polysaccharide purity calculation formula:

[0059]

[0060] Where: m is the sample mass.

[0061] Example 6: Salt tolerance test of strain L13

[0062] like Figure 4As shown in the figure, the growth of L13 decreased significantly with the increase of sodium chloride concentration in the culture medium, and showed a trend consistent with the generated curve. Compared with the control group, the growth of the strain was almost unaffected when the sodium chloride content in the culture medium was 4%. When the sodium chloride concentration was 8%, 12% and 16%, the growth of all strains was affected to varying degrees, and the higher the salt concentration, the greater the impact. However, when the sodium chloride concentration was 4%, 8% and 12%, the OD of the strain 600 The values ​​were all maintained above 2.0, indicating that the salt tolerance of strain L13 could reach 12%. Through comparison, it was found that the plant lactobacillus L13 screened in this study had good salt tolerance and had good application potential in fermentation.

[0063] Example 7: Acid resistance test of strain L13

[0064] like Figure 5 As shown, the OD of L13 600 The value decreased significantly with the decrease of the pH value of the culture medium, and its growth trend showed a trend of first increasing significantly and then tending to be flat as time went on. When the pH value of the culture medium was 4.0 and 5.0, the OD 600 The value should not be less than 2.0. When the pH is 6.0, the OD 600 The value is between 1.5 and 2.0. When the pH is 2.0 and 3.0, the OD 600 The values ​​were seriously affected, and their OD 600 The value is less than 0.5. 600 The value can reach 2.0 as the critical point of acid resistance, and it is concluded that the acid resistance of strain L13 can reach pH 5.0. Studies have shown that acid stress caused by the accumulation of acidic metabolites seriously affects the fermentation performance of lactic acid bacteria, so having good acid resistance is very important for the growth and application of lactic acid bacteria.

[0065] Example 8: Effect of fermentation time on the antioxidant capacity of fermented Porphyra yezoensis polysaccharides

[0066] like Figure 6 As shown in the figure, during the period of 0-48h, the antioxidant capacity of Porphyra yezoensis fermented by strain L13 increased with the extension of fermentation time. When fermented to 72h, the FRAP value reached 2.79, which was about 179% higher than the initial value; the DPPH free radical scavenging rate reached 75.1%, which was 131.79% higher; and the ABTS + The free radical scavenging rate reached 85.2%, an increase of 123.62%. This indicates that the fermentation of Porphyra yezoensis by strain L13 can enhance the antioxidant capacity of Porphyra yezoensis polysaccharides.

[0067] The above description is only a specific implementation mode of the present invention, but the protection scope of the invention is not limited thereto. Any changes or substitutions that can be easily thought of by a technician familiar with the field within the technical scope disclosed by the present invention should be covered within the protection scope of the invention.

Claims

1. A Lactobacillus plantarum ( Lactiplantibacillus plantarum ) L13, characterized in that, The plant lactobacillus L13 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 23, 2023, with the deposit number of CGMCC NO.27398.

2. according to claim 1, Lactobacillus plantarum L13, it is characterized in that: The 16S rDNA sequence of the Lactobacillus plantarum L13 is shown in SEQ ID NO:

1.

3. The use of Lactobacillus plantarum L13 according to claim 1 in extracting polysaccharides from Porphyra yezoensis, characterized in that: The FRAP value reached 2.79, the DPPH free radical scavenging rate reached 75.1%, and the ABTS + The free radical scavenging rate reached 85.2%.

4. Application of plant lactobacillus L13 according to claim 1 in the preparation of antioxidant products.

5. Use of plant lactobacillus L13 according to claim 1 as a functional probiotic in the preparation of food, food additives or medicines, wherein the medicine is used to stabilize intestinal balance and regulate intestinal health.

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