CN114891663B - Lactobacillus plantarum LP1406 and its isolation and cultivation method - Google Patents

Abstract

The invention belongs to the field of microorganism cultivation, and in particular relates to a method for cultivating Lactobacillus plantarum capable of fermenting and degrading kelp. The Lactobacillus plantarum was deposited in the China Center for Type Culture Collection on November 29, 2021, with a deposit number of CCTCC NO: M20211500. The method for cultivating lactobacillus plantarum provided by the present invention comprises the following steps: S1: isolation and cultivation of lactobacillus plantarum; S2: strain screening of lactobacillus plantarum fermented with kelp; S3: fermentation of kelp; S4: evaluation of fermentation effect. The Lactobacillus plantarum provided by the present invention can utilize natural kelp to grow rapidly, and its fermented kelp has excellent performance, and the yield of metabolites increases, providing a new microbial treatment method for fermenting kelp to improve the biological activity of kelp; the bacterial strain of the present invention , the effect of fermented kelp is remarkable, which can improve the antioxidant and hypoglycemic activities in the fermented samples. The inhibitory ability of ABTS is 82%, and the inhibitory ability of α-glucosidase reaches 75.40%.

Description

Lactobacillus plantarum LP1406 and its isolation and cultivation method

technical field

The invention belongs to the field of microorganism cultivation, and in particular relates to a plant lactobacillus, and also relates to the above-mentioned cultivation method of the plant lactobacillus.

Background technique

Lactobacillus plantarum is a genus of Lactobacillus in the Lactobacillus family, which can survive in various environments and is closely related to human life. Lactobacillus plantarum has strong acid resistance, and most of them can tolerate NaCl concentration above 5%. It has the characteristics of fast reproduction, short growth cycle, and rich nutrition of the bacteria. Its physiological functions mainly include immune regulation, antagonizing pathogenic bacteria, lowering cholesterol, lowering blood sugar, preventing cardiovascular and cerebrovascular diseases, etc. It is a widely used probiotic. In addition, Lactobacillus plantarum is rich in enzymes and has a strong metabolism, which has the potential to use kelp components for fermentation. The hypoglycemic mechanism of lactic acid bacteria mainly reduces blood sugar levels by regulating the balance of the human intestinal tract and the immune function of the body, improving antioxidant capacity, regulating glucose metabolism and lipid metabolism, etc.

Regarding the application of Lactobacillus plantarum, the following patent documents have been disclosed:

CN114231473A a probiotic plant Lactobacillus and its application in the preparation of low-salt fermented meat food; CN114196600A a plant Lactobacillus with alleviating effect on irritable bowel syndrome and its application; CN114134089A a compound bacterial agent with high activity and its application in food production. There are few disclosures about the application of Lactobacillus plantarum in the degradation of kelp.

Kelp is rich in chemical components and biologically active substances, and also rich in fucoidan, which is of very high value in the processing and application of functional foods and medicines. In addition, it also contains a variety of vitamins and minerals. Studies have proved that kelp has various biological functions such as lowering blood fat, lowering blood sugar, regulating immunity, anticoagulation, anti-tumor, lead detoxification and anti-oxidation. However, my country's kelp industry is still in the primary state of low added value and low benefit based on traditional extensive processing and primary processed products, and the development space of kelp industry is far from being discovered.

Therefore, the innovative development of the kelp processing and fermentation industry and the research and development of kelp products with nutritional and health value meet the strategic needs of a healthy China and promote the sustainable development of the kelp industry chain. If we find and cultivate a suitable Lactobacillus plantarum that can ferment and degrade kelp and improve the hypoglycemic and antioxidant functions of kelp, it will certainly provide the basis for large-scale production and application of kelp's deep processing.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides Lactobacillus plantarum LP1406, which can grow rapidly by using natural kelp and has the ability to degrade alginic acid. Through this Lactobacillus plantarum LP1406, it can improve the hypoglycemic and antioxidant capabilities of kelp fermentation samples. It provides a good foundation for deep processing.

The invention also provides a specific method for the application of the above bacterial strains in fermented kelp, and evaluates the hypoglycemic and anti-oxidative abilities of the product obtained after the application.

The Lactobacillus plantarum LP1406 mentioned in the present invention was preserved in the China Typical Culture Collection Center on November 29, 2021, the preservation address: Wuhan, China, the preservation number CCTCC NO: M20211500, and the classification name is: Lactobacillus plantarum LP1406 Lactiplantibacillus plantarum LP1406.

The nucleotide sequence of Lactobacillus plantarum LP1406 (hereinafter referred to as Lactobacillus plantarum) of the present invention is shown in SEQ ID NO.1. After being cultured in MRS medium at 37°C for 24 hours, the diameter of the colony on the surface is about 3mm, raised, showing a Round, smooth surface, fine and dense, white in color, occasionally light yellow or dark yellow; the colony is uniform in texture, and the color of the front and back sides, edges, and central parts is very uniform; the cells of the bacteria are rod-shaped, single or in chains; Gram staining is Positive, H ₂ O ₂ test positive, methyl red test positive, gelatin liquefaction test positive, VP test negative, hydrogen sulfide test negative, oxidase test negative, glucose, maltose, galactose, mannose test were all positive; Staphylococcus aureus, Escherichia coli and Salmonella typhimurium have obvious antibacterial effect; the pH tolerance range is 2.0-6.0.

When isolating and cultivating Lactobacillus plantarum, solid MRS medium is used, containing: peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 20g/L, ammonium citrate 2g/L, heptahydrate Magnesium sulfate 0.58g/L, sodium acetate 3.12g/L, disodium hydrogen phosphate 1.63g/L, potassium acetate 2.75g/L, Tween 80 1mL, manganese sulfate tetrahydrate 0.25g/L.

The application of Lactobacillus plantarum LP1406 in fermented seaweed products, especially the application in fermented kelp, is the key protection content of the present invention.

The specific steps of the application of Lactobacillus plantarum LP1406 in fermented kelp are as follows: firstly, screening the Lactobacillus plantarum of fermented kelp, and then fermenting kelp;

When screening the Lactobacillus plantarum of fermented kelp, the single colony on the solid plate is inoculated in the seed medium for cultivation, and then inoculated in the kelp enzymatic solution medium for cultivation; the seed medium is a liquid MRS medium containing : Peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 20g/L, ammonium citrate 2g/L, magnesium sulfate heptahydrate 0.58g/L, sodium acetate 3.12g/L, disodium hydrogen phosphate 1.63g/L, potassium acetate 2.75g/L, Tween 80 1mL, manganese sulfate tetrahydrate 0.25g/L;

The preparation method of the kelp enzymatic hydrolysis medium is as follows: the kelp is washed with water, dried, and ground, and the powder is collected through a 40-mesh sieve, then dissolved in distilled water at a ratio of 1:50 (w/v), and the pH is adjusted to 4.8 , add cellulase and pectinase to carry out enzymolysis, the addition amount of cellulase and pectinase is 2.5wt% and 0.26wt% respectively, stir the reaction solution at 50 ℃ for 2 hours, after adjusting the pH to 8.0, add 0.3 wt% alkaline pectinase, incubate at 60°C for another 1.5 hours, finally, centrifuge the reaction product, and collect the enzymatic hydrolysis supernatant as kelp enzymatic hydrolysis solution;

When fermenting kelp, inoculate a single colony on the MRS plate into the seed medium and incubate at 37°C for 24 hours; then inoculate 5%-10% of the inoculum into the kelp enzymatic solution medium and inoculate at 37°C 48h.

When fermenting kelp, inoculate a single colony on the MRS plate into the seed medium, and incubate at 37°C for 24 hours; then inoculate the kelp fermentation medium according to the inoculation amount of 5%-10%, first inoculate the kelp enzymatic hydrolyzate Supplement glucose to 3%, sterilize at 121°C for 20 minutes, insert 3% Lactobacillus plantarum and place it at 37°C for static culture, and the fermentation time is 48h.

Utilize the method for plantaractobacillus LP1406 to ferment sea-tangle, comprise the following steps:

S1: Isolation and cultivation of Lactobacillus plantarum

When isolating and cultivating Lactobacillus plantarum, the specific steps are:

(1) Take 1mL of milk products, put them into a test tube filled with 9mL of normal saline, and vortex fully with a vortex oscillator to mix the samples evenly;

(2) Fully absorb 1mL of the mixed sample with a pipette gun, add it into a test tube containing 9mL of normal saline, and so on to make a suspension of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 ;

(3) Use a pipette gun to fully draw 0.1mL of the mixed solution from the test tubes with concentrations of 10 ^-2 , 10 ^-4 , and 10 ^-6 , respectively dilute and spread in sterilized solid MRS medium, and incubate at 37°C for 24h ;

(4) After the cultivation, observe the colony shape, and pick a milky white smooth and moist single colony for microscopic examination;

(5) Separation and purification of the rod-shaped colonies under the microscope. After the colonies grow well, check whether there are any bacteria. If there are any, they need to be separated and purified again until the pure strain culture is obtained;

S2: Screening of Lactobacillus plantarum fermented with kelp

Screening of Lactobacillus plantarum fermented with kelp, the specific steps are: inoculate a single colony on a solid plate into a seed medium, cultivate at 37°C for 24 hours, then inoculate a 5-10% inoculum into the screening medium, and inoculate Cultivate at 37°C for 40-60h;

S3: Fermented Kelp

To ferment kelp, the specific operation steps are: inoculate a single colony on the MRS plate into the seed medium, and cultivate it at 37°C for 24 hours; Supplement glucose to 3% in the enzymatic solution, sterilize at 121°C for 20 minutes, add 3% Lactobacillus plantarum and place at 37°C for static culture, and the fermentation time is 48h.

The beneficial effects of the present invention are:

(1) The Lactobacillus plantarum LP1406 provided by the present invention can quickly ferment natural kelp, and has a strong ability to degrade alginic acid during the fermentation process;

(2) The obtained fermented product has excellent hypoglycemic and antioxidative abilities. Through in vitro hypoglycemic evaluation and in vitro antioxidative experiments, the results show that the fermented product of the present invention has an inhibitory ability to α-glucosidase of 75.40%, The inhibition rate of ABTS is 82%, which is far beyond the reach of ordinary Lactobacillus plantarum;

(3) Compared with traditional methods, the fermented and degraded kelp provided by Lactobacillus plantarum provided by the present invention is highly efficient, energy-saving, time-saving, simple to operate and can realize high value-added production.

Description of drawings

Fig. 1 is the colony form photo of plantarum lactobacillus;

Figure 2 is a microscope photo of Lactobacillus plantarum.

Detailed ways

In order to enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

The Lactobacillus plantarum provided by the present invention was preserved in the China Center for Type Culture Collection on November 29, 2021, with the preservation number CCTCC NO: M20211500.

Example 1

The culture method of plant lactobacillus comprises the following steps:

S1: Isolation and cultivation of Lactobacillus plantarum

When isolating and cultivating Lactobacillus plantarum, the specific steps are:

(1) Take 1mL of milk products, put them into a test tube filled with 9mL of normal saline, and vortex fully with a vortex oscillator to mix the samples evenly;

(2) Fully absorb 1mL of the mixed sample with a pipette gun, add it into a test tube containing 9mL of normal saline, and so on to make a suspension of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 ;

(3) Use a pipette gun to fully draw 0.1mL of the mixed solution from the test tubes with concentrations of 10 ^-2 , 10 ^-4 , and 10 ^-6 , respectively dilute and spread in sterilized solid MRS medium, and incubate at 37°C for 24h ;

In solid MRS medium, peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 20g/L, ammonium citrate 2g/L, magnesium sulfate heptahydrate 0.58g/L, sodium acetate 3.12g/L , disodium hydrogen phosphate 1.63g/L, potassium acetate 2.75g/L, Tween 801mL, manganese sulfate tetrahydrate 0.25g/L;

After the inoculation loop is burned and cooled by the alcohol lamp, pick a ring of colonies, place the plate next to the alcohol lamp, and use the inoculation loop to draw 3-4 continuous straight lines according to "Z". Then burn the seed ring to cool down, turn the plate to draw a line from the end of the first marked area to another area in the same way, and draw 3-4 areas. After streaking, incubate upside down in a constant temperature incubator at 37°C for 24 hours;

(4) After the cultivation, observe the colony shape, and pick a milky white smooth and moist single colony for microscopic examination;

(5) The rod-shaped colony under the microscope is isolated and purified. After the colony grows well, check whether there are any bacteria. If there is any, it needs to be isolated and purified again until a pure strain culture is obtained.

(6) 16S rDNA sequencing analysis was performed on qualified colonies, and the sequencing results were compared with BLAST to obtain Lactobacillus plantarum.

Example 2

The preservation method of Lactobacillus plantarum is as follows:

Lactobacillus plantarum was inoculated in liquid medium and cultured to the logarithmic phase, and 80% glycerol was autoclaved for use. Take 1mL of sterilized glycerin and mix it with the bacterial solution thoroughly to make the glycerin concentration about 10%-30%, and freeze it at -80°C.

The liquid medium of the above preservation method is as follows: peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 20g/L, ammonium citrate 2g/L, magnesium sulfate heptahydrate 0.58g/L, sodium acetate 3.12g/L, disodium hydrogen phosphate 1.63g/L, potassium acetate 2.75g/L, Tween 80 1mL, manganese sulfate tetrahydrate 0.25g/L.

Example 3

The purification method of Lactobacillus plantarum is as follows:

Pick the isolated single colony with an inoculation loop burnt by alcohol in the ultra-clean bench, inoculate it on the solid medium plate of Lactobacillus plantarum, and incubate at a constant temperature of 37°C for 24 hours to stop the cultivation. Check whether there are any miscellaneous bacteria by observing the colony shape and microscopic examination. If there are any miscellaneous bacteria, continue to pick a single colony for purification until a pure culture of Lactobacillus plantarum is obtained.

The characteristics of the plantarum lactobacillus obtained by the above method are as follows:

Gram staining was positive, H2O2 test was positive, methyl red test was positive, gelatin liquefaction test was positive, V.P. test was negative, hydrogen sulfide test was negative, oxidase test was negative, glucose, maltose, galactose and mannose tests were all positive. It has obvious antibacterial effect on Staphylococcus aureus, Escherichia coli and Salmonella typhimurium; the pH tolerance range is 2.0-6.0.

Example 4.1

By adding Lactobacillus plantarum to the kelp enzymatic hydrolysis solution, the effect of Lactobacillus plantarum on the antioxidant and hypoglycemic effects of kelp fermentation was verified.

The specific steps are as follows:

S2: Screening Lactobacillus plantarum fermented with kelp

To screen Lactobacillus plantarum fermented with kelp, the specific steps are: inoculate a single colony on a solid plate into a seed medium, cultivate at 37°C for 24 hours, and then inoculate a 5-10% inoculum into a screening medium (laminase solution), cultured at 37°C for 40-60h;

The above seed medium is a liquid MRS medium, which contains: peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 20g/L, ammonium citrate 2g/L, magnesium sulfate heptahydrate 0.58g/L L, sodium acetate 3.12g/L, disodium hydrogen phosphate 1.63g/L, potassium acetate 2.75g/L, Tween 80 1mL, manganese sulfate tetrahydrate 0.25g/L;

S3: Fermented Kelp

To ferment kelp, the specific operation steps are: inoculate a single colony on the MRS plate into the seed medium, and cultivate it at 37°C for 24 hours; Supplement glucose to 3% in the enzymatic solution, sterilize at 121°C for 20 minutes, insert 3% Lactobacillus plantarum, place at 37°C, and culture statically, the fermentation time is 48h;

In the above S2 and S3, the specific preparation process of the kelp enzymatic hydrolyzate is as follows: the kelp is washed, dried, and ground, and the powder is collected through a 40-mesh sieve, and then dissolved in distilled water at a ratio of 1:50 (w/v) , adjust the pH to 4.8, add cellulase and pectinase to carry out enzymolysis, the addition amount of cellulase and pectinase is 2.5wt% and 0.26wt% respectively, stir the reaction solution at 50 ℃ for 2 hours, adjust the pH After reaching 8.0, add 0.3wt% alkaline pectinase and incubate at 60°C for another 1.5 hours. Finally, centrifuge the reaction product and collect the enzymatic hydrolysis supernatant as kelp enzymatic hydrolysis solution.

The fermented product obtained by the above process is evaluated for its fermentation effect.

The evaluation of fermentation effect is mainly carried out from two aspects: in vitro hypoglycemic evaluation and in vitro antioxidation:

Regarding the in vitro hypoglycemic evaluation and in vitro antioxidant evaluation, the specific steps are as follows:

The specific operation method for in vitro hypoglycemic evaluation is as follows: respectively dissolve the fermented kelp samples in PBS with pH 6.8, then dilute the samples to different concentrations in test tubes, add 0.2U/mL α-glucosidase solution respectively, and dilute the test tubes Preheat in a water bath at 37°C for 40 minutes. After the preheating is complete, add PNPG substrate to the test tube for reaction. The reaction temperature is 37°C and the reaction time is 20 minutes. Finally, add 3 times the liquid volume of 1mol/L carbonic acid into the test tube. Sodium solution terminates the reaction. Determination of α-glucosidase inhibition rate;

The specific operation method of the in vitro antioxidant experiment was as follows: a working solution was prepared by mixing ABTS stock solution (7 mM) and potassium persulfate solution (4.9 mM) at a ratio of 1:1; the mixture was incubated overnight at room temperature in the dark; after that, Dilute with distilled water, the absorbance at 734nm is 0.7±0.02; mix the sample with ABTS working solution, place it in the dark at room temperature for 8 minutes, then record the absorbance at 734nm with a spectrophotometer, and set the blank and ascorbic acid positive control.

The specific steps are as follows:

The removal efficiency of fermented samples to ABTS can reach 87.63%, respectively, and the inhibitory ability to α-glucosidase can reach 80.40%.

The results showed that the anti-oxidation and hypoglycemic abilities of the samples after fermentation were significantly improved by fermenting kelp with Lactobacillus plantarum.

Example 4.2

Glucose was supplemented to 3% in the kelp enzymatic hydrolysis solution, sterilized at 121°C for 20 minutes, and then 3% of Lactobacillus plantarum was inserted into it and cultured on a shaking table at 37°C and 180 r/min, and the fermentation time was 36 hours. The removal efficiency of fermented samples to ABTS can reach 80.63%, respectively, and the inhibitory ability to α-glucosidase can reach 76.40%.

Example 4.3

Glucose was supplemented to 3% in the kelp enzymatic hydrolyzate, sterilized at 121°C for 20 minutes, then added with 3% Lactobacillus plantarum and placed at 37°C for static culture, and the fermentation time was 72 hours. The removal efficiency of fermented samples to ABTS can reach 77.21%, respectively, and the inhibitory ability to α-glucosidase can reach 73.25%.

comparative example

As can be seen from the data in the above table, the product of the present invention is far superior to other strains in both the removal efficiency of ABTS and the inhibitory ability to α-glucosidase.

sequence listing

<110> Shandong Xiaoying Biotechnology Co., Ltd.

Qilu University of Technology

Shandong Zhuoran Biotechnology Co., Ltd.

Shengsheng Xiangrong Biotechnology (Shandong) Co., Ltd.

Shandong Chenzhang Biotechnology Co., Ltd.

<120> Lactobacillus plantarum LP1406 and its isolation and cultivation method

<141> 2022-04-13

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Lactobacillus plantarum LP1406 (Lactiplantibacillus plantarum LP1406)

<400> 1

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Claims (1)

1. The application of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) LP1406 in fermented kelp, characterized in that, using said Lactobacillus plantarum LP1406 to ferment kelp improves the antioxidant and hypoglycemic effects of fermented products; The Lactobacillus plantarum LP1406 was deposited in the China Center for Type Culture Collection on November 29, 2021, with the preservation number CCTCC NO: M 20211500, and the classification name is Lactobacillus plantarum ( Lactiplantibacillus plantarum ) LP1406; The specific steps of fermenting kelp are as follows: Inoculate a single colony of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) LP1406 on the MRS plate into the seed medium and culture it at 37°C for 24 hours; Glucose was added to the enzymatic solution to 3%, sterilized at 121°C for 20 minutes, then inserted into Lactobacillus plantarum LP1406 and placed at 37°C for static culture, and the fermentation time was 48 hours; The specific preparation process of the kelp enzymatic hydrolysis solution is as follows: the kelp is washed, dried, and ground, and the powder is collected through a 40-mesh sieve, and then dissolved in distilled water at a ratio of 1:50 (w/v), and the pH is adjusted. To 4.8, add cellulase and pectinase for enzymolysis, the addition amount of cellulase and pectinase is 2.5wt% and 0.26wt% respectively, stir the reaction solution at 50°C for 2 hours, adjust the pH to 8.0 , add 0.3wt% alkaline pectinase, incubate at 60°C for another 1.5 hours, finally, centrifuge the reaction product, collect the enzymatic hydrolysis supernatant as kelp enzymatic hydrolysis solution.

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