CN114891663A - Lactobacillus plantarum LP1406 and isolated culture method - Google Patents

Abstract

The invention belongs to the field of microbial culture, and particularly relates to a culture method of lactobacillus plantarum capable of fermenting and degrading kelp. The lactobacillus plantarum is preserved in China center for type culture Collection at 11 month and 29 month in 2021, and the preservation number is as follows: CCTCC NO: M20211500. The culture method of the plant milk straw provided by the invention comprises the following steps: s1: separating and culturing lactobacillus plantarum; s2: screening lactobacillus plantarum for fermenting kelp; s3: fermenting the kelp; s4 evaluation of fermentation effect. The lactobacillus plantarum provided by the invention can utilize natural kelp to grow rapidly, the fermented kelp has excellent performance, the yield of metabolites is increased, and a novel microbial treatment method is provided for improving the bioactivity of the fermented kelp; the strain has obvious effect of fermenting kelp, can improve the antioxidant and hypoglycemic activity of a fermented sample, has 82 percent of ABTS inhibition capacity and 75.40 percent of alpha-glucosidase inhibition capacity.

Description

Lactobacillus plantarum LP1406 and its isolation and culture method

technical field

The invention belongs to the field of microorganism culture, in particular to a Lactobacillus plantarum, and also to a method for culturing the above-mentioned Lactobacillus plantarum.

Background technique

Lactobacillus plantarum is a Lactobacillus genus 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 more than 5% NaCl. It has the characteristics of fast reproduction, short growth cycle, and rich 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 strong metabolism, and has the potential to use kelp components for fermentation. The hypoglycemic mechanism of lactic acid bacteria mainly reduces blood sugar levels by regulating the intestinal balance of the human body 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 Lactobacillus plantarum and its application in the preparation of low-salt fermented meat food; CN114196600A A Lactobacillus plantarum with relieving effect on irritable bowel syndrome and its application; CN114134089A A compound bacterial agent with high activity and its application in food production. The literature on the application of Lactobacillus plantarum in the degradation of kelp is seldom disclosed.

Kelp is rich in chemical components and biologically active substances, and also contains rich fucoidan in kelp, which has 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 shown that kelp has various biological functions such as lowering blood lipids, lowering blood sugar, regulating immunity, anticoagulation, antitumor, detoxification and antioxidation. However, my country's kelp industry is still in the primary state of low added value and low efficiency, mainly 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 care value meet the strategic needs of Healthy China and promote the sustainable development of the kelp industry chain. If a suitable Lactobacillus plantarum is found and cultivated, which can ferment and degrade kelp and improve the hypoglycemic and antioxidative functions of kelp by fermentation, it will inevitably provide the basis for large-scale production and application for the deep processing of kelp.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides Lactobacillus plantarum LP1406, which can utilize natural kelp to grow rapidly and has the ability to degrade alginic acid, through which Lactobacillus plantarum LP1406 can improve the hypoglycemic and antioxidant capacity of kelp fermentation samples, and is a kind of kelp. The deep processing provides a good foundation.

The present invention also provides a specific method for the application of the above strain in fermented kelp, and evaluates the hypoglycemic and antioxidative abilities of the product obtained after application.

The Lactobacillus plantarum LP1406 described in the present invention was deposited in the China Center for Type Culture Collection on November 29, 2021, the deposit address: Wuhan, China, the deposit number CCTCC NO: M20211500, and the classification name is: 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 culturing in MRS medium at 37° C. for 24 hours, the colony on the surface has a diameter of about 3 mm, a convex shape, and a Round, smooth surface, dense, white in color, occasionally pale yellow or dark yellow; colony texture is uniform, and the color of front and back, edges, and central parts are uniform; somatic cells are rod-shaped, single or in chains; Gram stain is Positive, H ₂ O ₂ test was positive, methyl red test was positive, gelatin liquefaction test was positive, VP test was negative, hydrogen sulfide test was negative, oxidase test was negative, and glucose, maltose, galactose, and mannose tests were all positive; Staphylococcus aureus, Escherichia coli and Salmonella typhimurium have obvious bacteriostatic effect; pH tolerance range is 2.0-6.0.

When separating and culturing Lactobacillus plantarum, solid MRS medium was 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 content to be protected by the present invention.

The application of Lactobacillus plantarum LP1406 in fermented kelp, its concrete steps are: firstly, screening the Lactobacillus plantarum of fermented kelp, and then fermented kelp;

When screening the Lactobacillus plantarum of fermented kelp, the single colony on the solid plate is inoculated in the seed medium and cultivated, and then inoculated in the kelp enzymatic hydrolyzate medium and cultivated; 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 hydrolysate medium is as follows: the kelp is washed, dried and ground, the powder is passed through a 40-mesh sieve, collected, 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 enzymatic hydrolysis, 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 to 8.0, add 0.3 wt% alkaline pectinase, incubated at 60 °C for another 1.5 hours, and finally, the reaction product was centrifuged, and the enzymatic hydrolysis supernatant was collected as the kelp hydrolysate;

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

When fermenting kelp, a single colony on the MRS plate was inoculated into the seed medium, and cultured at 37°C for 24 hours; then when inoculated into the kelp fermentation medium according to the inoculum amount of 5%-10%, the kelp enzymatic hydrolyzed solution was first inoculated into the kelp fermentation medium. Glucose was supplemented to 3%, sterilized at 121°C for 20 minutes, and then inserted into Lactobacillus plantarum with 3% content and placed at 37°C for static culture, and the fermentation time was 48h.

Utilize the method for fermenting kelp with Lactobacillus plantarum LP1406, comprising the following steps:

S1: Isolation and culture of Lactobacillus plantarum

When separating and culturing Lactobacillus plantarum, the specific steps are:

(1) Take 1 mL of dairy products and put them into a test tube containing 9 mL of normal saline, and vortex the vortex shaker sufficiently to mix the samples evenly;

(2) Fully suck up 1 mL of the mixed sample with a pipette, add it to a test tube containing 9 mL of normal saline, and make a suspension of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 by analogy. ;

(3) Fully absorb 0.1 mL of the mixture from the test tubes with concentrations of 10 ^-2 , 10 ^-4 , and 10 ^-6 with a pipette, dilute and spread them in sterilized solid MRS medium, and cultivate at 37°C for 24 hours. ;

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

(5) The microscopically detected rod-shaped colonies should be separated, purified and cultured. After the colonies grow well, check whether there are any miscellaneous bacteria. If there are, they need to be separated and purified again until a pure strain culture is obtained;

S2: Screening of Lactobacillus plantarum for fermenting kelp

The specific steps of screening Lactobacillus plantarum fermented kelp are as follows: inoculate a single colony on a solid plate into a seed medium, cultivate at 37° C. for 24 hours, and then inoculate it in the screening medium according to the inoculum amount of 5-10%, Incubate at 37°C for 40-60h;

S3: Fermented kelp

Fermented 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; The enzymatic hydrolyzate was supplemented with glucose to 3%, sterilized at 121 °C for 20 min, and then inserted into Lactobacillus plantarum with a content of 3%, placed at 37 °C for static culture, and the fermentation time was 48h.

The beneficial effects of the present invention are:

(1) 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 antioxidant 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 from the effect of ordinary Lactobacillus plantarum;

(3) Compared with the traditional method, the Lactobacillus plantarum fermented and degraded kelp provided by the present invention has high efficiency, energy saving, time saving and simple operation, and can realize high value-added production.

Description of drawings

Fig. 1 is the colony morphology photo of Lactobacillus plantarum;

Figure 2 is a microscopic photograph 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 with reference to the specific embodiments.

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

Example 1

The cultivation method of Lactobacillus plantarum comprises the following steps:

S1: Isolation and culture of Lactobacillus plantarum

When separating and culturing Lactobacillus plantarum, the specific steps are:

(1) Take 1 mL of dairy products and put them into a test tube containing 9 mL of normal saline, and vortex the vortex shaker sufficiently to mix the samples evenly;

(2) Fully suck up 1 mL of the mixed sample with a pipette, add it to a test tube containing 9 mL of normal saline, and make a suspension of 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 by analogy. ;

(3) Fully absorb 0.1 mL of the mixed solution from the test tubes with concentrations of 10 ^-2 , 10 ^-4 , and 10 ^-6 with a pipette, dilute and spread them 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 ignited and cooled by an 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 by pressing "Z". Then burn the seed ring to cool, rotate the plate from the end of the first scribed area to scribe in the same way on another area, 3-4 areas. After streaking, incubate upside down for 24h in a constant temperature incubator at 37°C;

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

(5) The microscopically detected rod-shaped colonies should be isolated, purified and cultured. After the colonies grow well, check whether there are any miscellaneous bacteria. If there are, they need to be separated and purified again until a pure strain culture is obtained.

(6) Qualified colonies were subjected to 16S rDNA sequencing analysis, 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 into a liquid medium and cultured to log phase, and 80% glycerol was autoclaved for use. Take 1mL of sterilized glycerol and mix it thoroughly with the bacterial solution to make the glycerol concentration about 10%-30%, and freeze it at -80℃.

The liquid medium of the above-mentioned preservation method is composed 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:

The isolated single colonies were picked with the inoculation loop after burning with alcohol in the ultra-clean bench, inoculated on the solid medium plate of Lactobacillus plantarum, and cultured at 37°C for 24h to stop the culture. Check whether there are any miscellaneous bacteria by observing the colony morphology and microscopic examination.

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

Gram stain 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, and glucose, maltose, galactose, and mannose tests were all positive. It has obvious bacteriostatic effect on Staphylococcus aureus, Escherichia coli and Salmonella typhimurium; pH tolerance range is 2.0-6.0.

Example 4.1

By adding Lactobacillus plantarum to the kelp enzymatic hydrolysate, the effect of Lactobacillus plantarum on the antioxidant and hypoglycemic effect of kelp fermentation after fermentation was verified.

The specific steps are as follows:

S2: Screening Lactobacillus plantarum for fermenting kelp

To screen Lactobacillus plantarum fermented kelp, the specific steps are as follows: inoculate a single colony on a solid plate into a seed medium, cultivate at 37°C for 24 hours, and then inoculate it into a screening medium (kelp enzyme) according to an inoculum of 5-10%. solution), incubate at 37°C for 40-60h;

The above seed medium is 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

Fermented 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; The enzymatic hydrolyzate was supplemented with glucose to 3%, sterilized at 121°C for 20min, and then inserted with 3% Lactobacillus plantarum, placed at 37°C, and cultured at rest for 48h;

In the above S2 and S3, the specific preparation process of the kelp enzymolysis solution is as follows: the kelp is washed, dried and ground, the powder is passed through a 40-mesh sieve, collected, and then dissolved in distilled water at a ratio of 1:50 (w/v) , adjust the pH to 4.8, add cellulase and pectinase for enzymatic hydrolysis, 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, 0.3 wt% alkaline pectinase was added, and incubated at 60° C. for another 1.5 hours. Finally, the reaction product was centrifuged, and the enzymatic hydrolysis supernatant was collected as the kelp enzymatic hydrolysis solution.

The fermentation effect of the fermented product obtained by the above-mentioned process was evaluated.

The fermentation effect evaluation is mainly carried out from two aspects: in vitro blood sugar lowering evaluation and in vitro antioxidation:

Regarding in vitro blood sugar lowering evaluation and in vitro antioxidant evaluation, the specific steps are as follows:

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

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

The specific steps are as follows:

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

The results showed that the antioxidative and hypoglycemic abilities of the fermented samples were significantly improved by fermenting kelp with Lactobacillus plantarum.

Example 4.2

The kelp enzymatic hydrolyzate was supplemented with glucose to 3%, sterilized at 121 °C for 20 min, and then added to Lactobacillus plantarum with a content of 3% and placed in a shaking table at 37 °C and 180 r/min for 36 h. The scavenging efficiency of fermentation samples to ABTS can reach 80.63%, and the inhibitory ability to α-glucosidase can reach 76.40%, respectively.

Example 4.3

The kelp enzymatic hydrolyzate was supplemented with glucose to 3%, sterilized at 121 °C for 20 min, and then inserted into Lactobacillus plantarum with a content of 3% and placed at 37 °C for static culture, and the fermentation time was 72 h. The scavenging efficiency of fermentation samples to ABTS can reach 77.21%, and the inhibitory ability to α-glucosidase can reach 73.25%, respectively.

Comparative ratio

It can be seen from the data in the above table that the product of the present invention is far superior to other strains in terms of 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 culture method

<141> 2022-04-13

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1455

<212> DNA

<213> Lactiplantibacillus plantarum LP1406

<400> 1

cctaatacat gcagtcgaac gaactctggt attgattggt gcttgcatca tgatttacat 60

ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc tgcccagaag cgggggataa 120

cacctggaaa cagatgctaa taccgcataa caacttggac cgcatggtcc gagcttgaaa 180

gatggcttcg gctatcactt ttggatggtc ccgcggcgta ttagctagat ggtggggtaa 240

cggctcacca tggcaatgat acgtagccga cctgagaggg taatcggcca cattgggact 300

gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca atggacgaaa 360

gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaaa ctctgttgtt 420

aaagaagaac atatctgaga gtaactgttc aggtattgac ggtatttaac cagaaagcca 480

cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggattta 540

ttgggcgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagcct tcggctcaac 600

cgaagaagtg catcggaaac tgggaaactt gagtgcagaa gaggacagtg gaactccatg 660

tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt 720

ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac aggattagat accctggtag 780

tccataccgt aaacgatgaa tgctaagtgt tggagggttt ccgcccttca gtgctgcagc 840

taacgcatta agcattccgc ctggggagta cggccgcaag gctgaaactc aaaggaattg 900

acggggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt 960

accaggtctt gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga 1020

tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080

cgagcgcaac ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg 1140

gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1200

tacacacgtg ctacaatgga tggtacaacg agttgcgaac tcgcgagagt aagctaatct 1260

cttaaagcca ttctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg 1320

ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380

cgtcacacca tgagagtttg taacacccaa agtcggtggg gtaacctttt aggaaccagc 1440

ctgctaaggt gacaa 1455

Claims (10)

1. Lactobacillus plantarum LP1406, deposited in China center for type culture Collection at 29 months 11/2021, with the deposition number CCTCC NO: m20211500, class name: lactobacillus plantarum LP1406Lactiplantibacillus plantarumLP1406。

2. The lactobacillus plantarum LP1406 according to claim 1, wherein the nucleotide sequence of the lactobacillus plantarum LP1406 is shown in SEQ ID No. 1.

3. The lactobacillus plantarum LP1406 according to claim 1, having a surface colony diameter of about 3mm, convex, rounded, smooth, fine, white, occasionally light yellow or dark yellow, after 24h of incubation in MRS medium at 37 ℃; the colony has uniform texture, and the colors of the front and back sides, the edges and the central part are uniform; the somatic cells are rod-shaped, single or chain-forming; gram staining positive, H ₂ O ₂ The test is positive, the methyl red test is positive, the gelatin liquefaction test is positive, the V.P. test is negative, the hydrogen sulfide test is negative, the oxidase test is negative, and the glucose, maltose, galactose and mannose tests are positive; has obvious bacteriostatic effect on staphylococcus aureus, escherichia coli and salmonella typhimurium; pH valueThe tolerance range is 2.0-6.0.

4. The lactobacillus plantarum LP1406 according to claim 1, wherein in isolating and culturing lactobacillus plantarum, a solid MRS medium is used, comprising: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of ammonium citrate, 0.58g/L of magnesium sulfate heptahydrate, 3.12g/L of sodium acetate, 1.63g/L of disodium hydrogen phosphate, 2.75g/L of potassium acetate, 801 mL of tween and 0.25g/L of manganese sulfate tetrahydrate.

5. Use of the Lactobacillus plantarum LP1406 according to claim 1 in a fermented seaweed preparation.

6. Use of lactobacillus plantarum LP1406 according to claim 1 for fermenting kelp.

7. The use according to claim 6, wherein, first, the lactobacillus plantarum fermenting kelp is selected, and then the kelp is fermented;

when lactobacillus plantarum for fermenting kelp is screened, a single colony on a solid plate is inoculated in a seed culture medium for culture, and then is inoculated in a kelp enzymolysis liquid culture medium for culture; the seed culture medium is a liquid MRS culture medium and contains: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of ammonium citrate, 0.58g/L of magnesium sulfate heptahydrate, 3.12g/L of sodium acetate, 1.63g/L of disodium hydrogen phosphate, 2.75g/L of potassium acetate, 801 mL of tween and 0.25g/L of manganese sulfate tetrahydrate;

the preparation method of the kelp enzymolysis liquid culture medium comprises the following steps: washing, drying and grinding the kelp, sieving the powder with a 40-mesh sieve, collecting, dissolving the powder in distilled water according to a ratio of 1:50 (w/v), adjusting the pH to 4.8, adding cellulase and pectinase for enzymolysis, wherein the addition amounts of the cellulase and the pectinase are 2.5wt% and 0.26 wt% respectively, stirring the reaction solution at 50 ℃ for 2 hours, adjusting the pH to 8.0, adding 0.3wt% of alkaline pectinase, incubating at 60 ℃ for 1.5 hours, finally, centrifuging the reaction product, and collecting the enzymolysis supernatant as kelp enzymolysis liquid;

when the kelp is fermented, inoculating a single colony on an MRS plate into a seed culture medium, and culturing at 37 ℃ for 24 hours; then inoculating the kelp into a kelp enzymolysis liquid culture medium according to the inoculation amount of 5-10%, and culturing for 48h at 37 ℃.

8. The use according to claim 7, wherein, when the kelp is fermented, a single colony on an MRS plate is inoculated into a seed culture medium and cultured at 37 ℃ for 24 hours; then inoculating the kelp into a kelp fermentation culture medium according to the inoculation amount of 5-10%, supplementing glucose to 3% in kelp enzymolysis liquid, sterilizing at 121 ℃ for 20min, inoculating lactobacillus plantarum with the content of 3%, standing and culturing at 37 ℃, and fermenting for 48 hours.

9. A method for fermenting kelp by using Lactobacillus plantarum LP1406, comprising the steps of:

s1: isolation and culture of Lactobacillus plantarum

When the lactobacillus plantarum is separated and cultured, the specific steps are as follows:

(1) taking 1mL of milk product, putting the milk product into a test tube filled with 9mL of physiological saline, and fully and evenly mixing the samples by fully and whirling an eddy oscillator;

(2) using a pipette to fully suck 1mL of the mixed sample, adding the sample into a test tube containing 9mL of physiological saline, and repeating the steps to obtain 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 The suspension of (a);

(3) using a pipette with a concentration of 10 ^-2 ，10 ^-4 ，10 ^-6 0.1mL of mixed solution is fully absorbed in the test tube, diluted and coated in a sterilized solid MRS culture medium respectively, and cultured for 24h at 37 ℃;

(4) observing the colony morphology after the culture is finished, and selecting a milky white smooth and wet single colony for microscopic examination;

(5) performing separation and purification culture on rod-shaped bacterial colonies through microscopic examination, checking whether mixed bacteria exist after the bacterial colonies grow well, and performing separation and purification again if the mixed bacteria exist, until pure bacterial strain culture is obtained;

s2: screening of Lactobacillus plantarum for fermenting Laminaria japonica

Screening lactobacillus plantarum for fermenting kelp, which comprises the following specific steps: inoculating the single colony on the solid plate into a seed culture medium, culturing at 37 ℃ for 24h, then inoculating into a screening culture medium according to the inoculation amount of 5-10%, and culturing at 37 ℃ for 40-60 h;

s3: fermented kelp

The kelp fermentation method comprises the following specific operation steps: inoculating a single colony on an MRS plate into a seed culture medium, and culturing at 37 ℃ for 24 h; then inoculating the kelp into a kelp fermentation culture medium according to the inoculation amount of 5-10%, supplementing glucose to 3% in kelp enzymolysis liquid, sterilizing at 121 ℃ for 20min, inoculating lactobacillus plantarum with the content of 3%, standing and culturing at 37 ℃, and the fermentation time is 48 hours.

10. The method of claim 9, further comprising S4: evaluation of fermentation effect:

in vitro blood sugar reduction evaluation and in vitro oxidation resistance evaluation;

the specific operation method for the in vitro blood sugar reduction evaluation comprises the following steps: respectively dissolving fermented kelp samples in PBS (phosphate buffer solution) with the pH value of 6.8, diluting the samples into different concentrations, respectively adding 0.2U/mL alpha-glucosidase solution, preheating the test tube in a water bath kettle at the temperature of 37 ℃ for 40min, adding a PNPG substrate into the test tube after preheating is finished to react at the temperature of 37 ℃ for 20min, finally adding 1mol/L sodium carbonate solution with the volume being 3 times that of the liquid into the test tube to terminate the reaction, and determining the inhibition rate of the alpha-glucosidase;

the specific operation method of the in vitro antioxidant experiment comprises the following steps: the crude was purified by mixing ABTS stock solution (7 mM) and potassium persulfate solution (4.9 mM) at a concentration of 1: 1 to prepare working solution; incubating the mixture in the dark at room temperature overnight; then, the mixture was diluted with distilled water to give an absorbance of 0.7. + -. 0.02 at 734 nm; the samples were mixed with ABTS working solution and left at room temperature in the dark for 8 minutes, then absorbance was recorded at 734nm with a spectrophotometer, and blank and ascorbic acid positive controls were included.

Images