Lactobacillus plantarum LP198 and application thereof

Abstract

The invention relates to lactobacillus plantarum, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32642, and the preservation date is November 15, 2024. The lactobacillus plantarum LP198 is obtained through screening and purification, the 24-hour nitrite degradation rate of the lactobacillus plantarum LP198 is larger than or equal to 99%, the lactobacillus plantarum LP198 can tolerate NaCl with the mass concentration smaller than or equal to 80 g / L, the acid production capacity is high, the lactobacillus plantarum LP198 has a high inhibition effect on the activity of alpha-glucosidase and alpha-amylase, and the lactobacillus plantarum LP198 has a high inhibition effect on escherichia coli E.coli ATCC 8739 and staphylococcus aureus S.aureus ATCC 29213. The strain can be used for preparing hypoglycemic functional foods or medicines and fermented foods such as pickles and the like, and can also be used for shortening the fermentation period of the fermented foods and inhibiting the growth of harmful microorganisms in the fermented foods.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and more particularly, relates to a Lactobacillus plantarum LP198 and an application thereof. Background Art

[0002] Kimchi is a food made from fresh vegetables such as cabbage, radish, cucumber, and bell pepper, with the addition of water, salt, spices and other auxiliary ingredients. It is fermented in an anaerobic environment using the microorganisms attached to the vegetables themselves or by adding artificially cultured lactic acid bacteria starter.

[0003] The food safety of kimchi cannot be ignored, and the nitrite content is an important indicator that cannot be ignored when processing and storing fermented vegetables. In recent years, lactic acid bacteria have been used to improve the quality of kimchi because lactic acid bacteria can significantly reduce the production of nitrite. Lactic acid bacteria produce lactic acid during the fermentation process, which leads to a decrease in pH, which is beneficial to NO. 2- Reduction to NO, thereby reducing the nitrite content. Therefore, in the process of vegetable fermentation, the appropriate addition of lactic acid bacteria can reduce and degrade the accumulation of nitrite content to reduce the harm of nitrite to the human body.

[0004] If we can screen lactic acid bacteria with strong blood sugar-lowering, nitrite-lowering and antibacterial abilities, it will have great application value for the development of kimchi with relevant functionalities. Summary of the invention

[0005] The invention provides a plant lactobacillus, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.32642 and a deposit date of November 15, 2024.

[0006] The present invention also provides the use of the plant lactobacillus in preparing blood sugar lowering functional food or medicine.

[0007] The present invention also provides application of the plant lactobacillus in preparing fermented food.

[0008] The present invention also provides the use of the plant lactobacillus in preparing blood sugar lowering functional food or medicine.

[0009] The present invention also provides a method for shortening the fermentation period of fermented food, comprising the step of using the plant lactobacillus as a starter to prepare the fermented food.

[0010] The present invention also provides the above method for inhibiting the growth of harmful microorganisms in fermented food, comprising the step of using the above Lactobacillus plantarum as a starter for preparing the fermented food.

[0011] The present invention obtains a plant lactobacillus LP198 by screening and purifying existing fermented foods. The strain has a nitrite degradation rate of ≥99% in 24 hours, can tolerate NaCl with a mass concentration of ≤80g / L, has strong acid production ability, has a strong inhibitory effect on the activity of α-glucosidase and α-amylase, and has a strong inhibitory effect on Escherichia coli E.coli ATCC 8739 and Staphylococcus aureus S.aureus ATCC 29213. Therefore, the strain can be used to prepare blood sugar-lowering functional foods or medicines, as well as fermented foods such as kimchi, and can also be used to shorten the fermentation period of fermented foods and inhibit the growth of harmful microorganisms in fermented foods.

[0012] Microbial Deposit

[0013] The strain 198 of the present invention is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC NO: 32642 and a deposit date of November 15, 2024; the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; it is named Lactobacillus plantarum, and its Latin name is Lactobacillus plantarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The colony morphology and hemolysis of LP 198 on MRS plate.

[0015] Figure 2 This is the acid production curve of LP 198.

[0016] Figure 3 is the standard curve of nitrite content.

[0017] Figure 4 The growth of LP 198 in different mass concentrations of NaCl.

[0018] Figure 5 is the relative inhibition rate of LP 198 on α-glucosidase.

[0019] Figure 6 is the relative inhibition rate of LP 198 on α-amylase.

[0020] Figure 7 Phylogenetic tree of LP 198 based on 16S rRNA. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] 1. Isolation, purification and morphological identification of lactic acid bacteria

[0023] The mustard tuber stock solution was collected from the traditional fermented mustard tuber pool in Qianjiang District, Chongqing, and diluted 10-fold to 10 -8 , respectively, take 0.1 mL of the dilution solution onto MRS-2% CaCO3 solid medium plates, spread and separate, and culture at 37°C for 24 hours. Pick the colonies with obvious calcium dissolution circles onto another MRS solid medium and purify them by streaking three times continuously. Purified lactic acid bacteria strains were obtained.

[0024] The purified strains were subjected to Gram staining and catalase test, and the cell morphology was observed under an oil microscope.

[0025] Gram-positive, catalase-negative, and rod-shaped strains were selected for continuous subculture for more than 5 times, and strains with stable growth and fast growth were screened out, which were initially considered to be lactic acid bacteria. Figure 1 The colony was milky white and smooth and round in shape. It was numbered 198 and transferred to liquid MRS medium and cultured at 37°C until the stable period. It was mixed with 50% glycerol in a 1:1 ratio and stored at -80°C for subsequent experiments.

[0026] 2. Safety Determination

[0027] A single strain was selected and streaked on Columbia blood agar plates, and the plates were incubated at 37°C for 24-48 h. Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 29213 were used as controls to observe hemolysis. Each group was repeated three times.

[0028] The results are as follows Figure 1 As shown, there is no hemolysis in this bacterium.

[0029] Take the strain 198 that has grown to the logarithmic phase and inoculate it into MRS medium at 2% and culture it at 37℃ for 24h. Collect the bacteria by centrifugation and resuspend them in PBS. Adjust the concentration of the bacterial suspension to 10 7 CFU / mL, 100 μL of bacterial suspension was taken and spread on MRS solid medium, and then the antibiotic sensitivity sheet was placed on the surface of the plate covered with bacteria, and cultured in a 37°C constant temperature incubator for 48 hours, and the inhibition zone was observed and the diameter of the drug sensitivity zone was measured. The results are shown in Table 1, and the bacteria are sensitive to most antibiotics.

[0030] The above experiments show that strain 198 is safe, does not cause harm to human health, can be killed by a variety of antibiotics, and is controllable.

[0031] Table 1 Drug resistance of strain LP 198

[0032]

[0033] Note: “S” stands for sensitive; “I” stands for intermediate; “R” stands for resistant

[0034] 3. Determination of acid production capacity

[0035] Take 198 grown to the logarithmic phase and inoculate it into a centrifuge tube with 30mL MRS liquid medium at a 2% inoculum and culture at 37℃. Measure the pH value of the fermentation liquid every 1h. Repeat 3 times and take the average value of the results. The curve of pH value changing with time is shown in Figure 2 As shown, the strain grew for 19 h and the pH of the culture medium dropped to 3.63±0.01.

[0036] 4. Determination of nitrite degradation ability

[0037] (1) Take 198 cells grown to the logarithmic phase and inoculate 2% into MRS (200 μg / mL nitrite) medium, incubate at 37°C for 24 h, centrifuge and take 1 mL of the supernatant to measure the nitrite concentration. At the same time, make a blank tube without nitrite and a blank tube without bacteria. Repeat 3 times for each group.

[0038] (2) Determine the nitrite content by spectrophotometry according to GB5009.33-2016 "National Food Safety Standard for the Determination of Nitrite and Nitrate in Food" and draw a standard curve for the nitrite content. Obtain the nitrite content according to the standard curve. Take 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL, 3.00mL, 3.50mL, 4.00mL of sodium nitrite standard solution (equivalent to 0.0μg, 1.0μg, 2.0μg, 3.0μg, 4.0μg, 5.0μg, 7.5μg, 10.0μg, 12.5μg, 15μg, 17.5μg, 20μg of sodium nitrite), and take 1mL of the centrifugal supernatant into 10mL stoppered colorimetric tubes respectively. Add 2mL of 4g / L p-aminobenzenesulfonic acid solution, mix well, let stand for 3-5 minutes, then add 1mL of 2g / L naphthaleneethylenediamine hydrochloride solution, add water to 50mL, mix well, let stand for 15 minutes, adjust to zero with a zero tube, measure the absorbance at 538nm, and make a reagent blank at the same time. The standard curve of nitrite content is as follows Figure 3 As shown, the regression equation is y=0.0394x+0.0039, R2=0.9982.

[0039] (3) Calculation formula for nitrite degradation rate:

[0040] Nitrite degradation rate = (A1-A2) / A1×100%

[0041] A1 is the initial content of NaNO2 in the culture medium of the control group; A2 is the content of NaNO2 in the culture medium after 24 hours of culture. The degradation rate of nitrite by strain 198 is shown in Table 2, and the degradation rate is as high as 99.61%±0.21%.

[0042] Table 2 Nitrite degradation rate and inhibition zone diameter of LP 198

[0043]

[0044] Note: The initial concentration of nitrite in MRS was 200 μg / mL, the incubation time was 24 h, the diameter of the Oxford cup was 7.8 mm, and all values ​​are mean ± SD.

[0045] 5. Determination of salt tolerance

[0046] 198 cells grown to the logarithmic phase were inoculated with 2% of MRS liquid culture medium with NaCl concentrations of 0%, 2%, 4%, 6%, and 8%, respectively, and cultured at 37°C for 24 h. The absorbance was measured at 600 nm using MRS of various concentrations as blank. This was repeated three times and the results were averaged.

[0047] The results are as follows Figure 4 As shown, in each concentration of NaCl-MRS medium, OD600 was higher than 1.0. It can be seen that the strain has a strong salt tolerance and can be used for the preparation of salt-containing fermented foods.

[0048] 6. Determination of antibacterial ability

[0049] (1) 198 grown to the logarithmic phase was inoculated into MRS medium at 2% and cultured at 37°C for 24 h. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane to obtain the fermentation supernatant CFS.

[0050] (2) Take 0.1 mL of E. coli ATCC 8739 and Staphylococcus aureus ATCC29213 (10 7 CFU / mL), spread on TSB agar medium, and place 3 Oxford cups evenly. Add 0.1mL CFS to each, diffuse at 4℃ for 2h, and culture at 37℃ for 24h before measuring the diameter of the inhibition zone with a vernier caliper.

[0051] The results are shown in Table 2. The strain had a strong inhibitory effect on both pathogenic bacteria.

[0052] 7. Determination of blood sugar lowering ability

[0053] (1) 198 cells grown to the logarithmic phase were inoculated at 2% into MRS medium and cultured at 37°C for 24 h. The supernatant was collected by centrifugation and the inhibitory activity against α-amylase was determined by the DNS method, and the inhibitory activity against α-glucosidase was determined by the PNPG method. 0.2 g / L acarbose was used as a positive control, as follows.

[0054] (2) PNPG method: 1 mL of 20 mmol / L PNPG solution and 0.5 mL of supernatant were added to 1 mL of PBS solution in sequence, incubated at 37°C for 10 min, and then 0.6 mL of α-glucosidase solution was added. The reaction was continued for 20 min, and finally 1 mL of Na2CO3 was added to terminate the reaction. The absorbance was measured at OD405 and recorded as A3. Each group was repeated 3 times.

[0055] Calculation formula for α-glucosidase inhibition rate:

[0056]

[0057] A1: contains enzyme but no sample, A2: contains neither enzyme nor sample, and A4: contains sample but no enzyme.

[0058] The results are as follows Figure 5 As shown, the inhibitory effect of this strain on α-glucosidase is comparable to that of acarbose.

[0059] (3) DNS method: 0.2 mL of supernatant was mixed with 0.2 mL of 10 g / L soluble starch. After 10 min at 37 °C, 0.2 mL of α-amylase was added. After 10 min, 0.4 mL of DNS solution was added. After cooling in a boiling water bath for 5 min, 4 mL of distilled water was added. The absorbance was measured at OD540 and recorded as A3. Each group was repeated 3 times.

[0060] Calculation formula of α-amylase inhibition rate:

[0061]

[0062] A1: contains enzyme but no sample, A2: contains neither enzyme nor sample, and A4: contains sample but no enzyme.

[0063] The results are as follows Figure 6 Its inhibitory effect on α-amylase is comparable to that of acarbose.

[0064] The above experiments show that strain 198 has a strong ability to lower blood sugar.

[0065] 8. Molecular Biological Identification of Lactic Acid Bacteria

[0066] Strain 198 was inoculated into MRS plate culture medium for activation, and the plate was inverted and cultured at 37°C for 24 hours. Pick 4 rings of activated bacteria, inoculate them into 30mL MRS liquid culture medium for reactivation, and place them at 37°C for static culture for 24 hours. Take the cultured bacterial liquid, centrifuge it at 10,000rpm for 1min, aspirate the supernatant as much as possible, collect the bacteria, and send them to Sangon Biotechnology for whole genome sequencing. Use Blast to compare the 16s rRNA sequence predicted by the gene with the 16s database of NCBI, and set the parameter ident i fy>95. Then select the top 30 16s rRNA sequences with the highest identify ify (if insufficient, take all), and use mafft software to perform multiple sequence alignment and cutting, and use FastTree / i qtree / raxml software to construct a phylogenetic tree as shown below. Figure 7 , and identified 198 as Lactobacillus plantarum.

[0067] Strain 198 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO: 32642 and the deposit date November 15, 2024; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; named Lactobacillus plantarum, Latin name Lactobacillus plantarum.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Lactobacillus plantarum, characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.32642 and the deposit date is November 15, 2024.

2. Application of plant lactobacillus according to claim 1 in preparing blood sugar lowering functional food or medicine.

3. Application of plant lactobacillus according to claim 1 in preparing fermented food.

4. Application of the plant lactobacillus according to claim 1 in preparing blood sugar lowering functional foods or medicines.

5. A method for shortening the fermentation period of fermented food, characterized in that: The method comprises the step of using the plant lactobacillus described in claim 1 as a starter for preparing the fermented food.

6. A method for inhibiting the growth of harmful microorganisms in fermented food, characterized in that: The method comprises the step of using the plant lactobacillus described in claim 1 as a starter for preparing the fermented food.

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