CN107267420B - High-yield β -1, 3-glucan bacillus subtilis and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield β -1, 3-glucan Bacillus subtilis strain and a fermentation culture method thereof, which are characterized in that an inventor obtains a strain ZJS-1 by screening from soil, the classification name is that the Bacillus subtilis has a preservation number of CGMCC No. 12176. the strain ZJS-1 directly converts cane sugar through fermentation culture to synthesize β -1, 3-glucan, namely β -1, 3-glucan directly takes cane sugar as a carbon source through biotransformation.

Description

High-yield β -1, 3-glucan bacillus subtilis and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield β -1, 3-glucan bacillus subtilis strain and a fermentation culture method thereof.

Background

β -1,3 glucan is a branched chain macromolecular polysaccharide with main chain structure connected by β -1, 3-glycosidic bond, usually containing β -1,3, β -1,4, β -1,6 glycosidic bond connected branches with different proportions and sizes, β -1,3 glucan has been proved to have the functions of enhancing the immune activity of mammals, resisting tumors, bacteria, viruses, reducing cholesterol and blood fat, promoting wound healing and the like in the 20 th century in 60 years, shows the potential application value in the field of pharmacy, and is widely concerned, and researches in recent years show that β -1,3 glucan can play the roles of moisturizing, resisting inflammation, resisting aging, removing wrinkles, removing dandruff, removing, accelerating repair and increasing skin elasticity when being applied to livestock and poultry breeding, can enhance the organism immunity of livestock and poultry, improve the gastrointestinal function and the like, and further expand the application field of β -1,3 glucan.

In order to improve the production mode of β 0-glucan and improve the yield of β -glucan, in recent years, screening research on microorganisms with high yield of β -glucan is carried out in domestic and overseas researches, currently screened microorganisms suitable for large-scale production of β -glucan comprise beer yeast, bacillus subtilis and the like, and attempts for large-scale production of β -glucanase from β -glucan bacteria through microbial fermentation culture are carried out abroad, but related researches on producing β -glucan by fermenting microorganisms in China are late, mainly using beer yeast and less other sources, but the production of β -glucan by using beer yeast still has the defects of low raw material conversion utilization rate, low product purity, poor quality and the like, and is difficult to adapt to the requirement of the current market for high-quality β -glucan.

Disclosure of Invention

The invention discloses a high-yield β -1, 3-glucan bacillus subtilis strain ZJS-1. the strain ZJS-1 directly converts sucrose to synthesize β -1, 3-glucan through fermentation culture, namely, the strain ZJS-1 directly takes sucrose as a carbon source to synthesize β -1, 3-glucan through biotransformation.

The strain is obtained by screening soil under the forest of the Zhongshan scenic region of Nanjing city through a colony drawing method and a method for increasing the yield of glucan in a liquid culture, is named as ZJS-1, and is white, rough, wrinkled and irregular in edge. And (3) classification and naming: bacillus subtilis, Bacillus subtilis. The preservation number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is as follows: CGMCC No. 12176. Preservation time: year 2016, 3, 4, and the storage address: the institute of scientific research microorganisms of China, No. 3, Xilu No.1, Beijing, Chaoyang, North.

Screening of high-yield β -1, 3-glucan strain:

in accordance with KH₂PO₄0.3-1.0g/L；CaCl₂0.3-1.0g/L，MgSO4 0.6-1.2g/L，FeSO₄·7H₂Weighing 0.6-1.2g/L of O, 3.0-8.0g/L of sucrose and 12.0-18.0g/L of agar, redistilling water to dissolve and fix the volume, adjusting pH to 6.5-7.0, sterilizing at 121 ℃ for 20-30 minutes to prepare a screening culture medium, adding 0.5-1.5g of collected soil sample or 1-2mL of water sample into 5mL of liquid culture medium, performing shake culture in a shaker at 30 ℃ for 48-72h to enrich glucan-producing bacteria, then coating the culture for 48-72h in 5% inoculation amount in a screening plate culture medium, performing inversion culture at 25-30 ℃ for 48-72h, detecting the fiber drawing performance of each colony by using a colony fiber drawing method, selecting the colony with obvious fiber drawing performance to be transferred into 5-8mL of screening liquid culture medium, performing shake culture at 28-32 ℃, at 150-300rpm for 24-48h, determining β -1 in each sampling culture by using a Congo-633 method, selecting strains with highest polysaccharide content, and performing ZJS strain analysis under Z633-361-363 conditions of ZJS, wherein the strain yield is optimized for high yield of glucan-1-3 strain, ZJS strain, Z633 strain, and ZJS strain is obtained by using ZJS strain, and Z β.

16S rDNA identification of β -1, 3-glucan-producing strains:

the identification of the strain is completed by 16S rDNA sequence analysis. Selecting a proper amount of the slant culture of the strain ZJS-1, adding the slant culture into 5-10mL of liquid seed culture medium, culturing overnight at 28-32 ℃, taking 2mL of bacterial liquid, centrifuging for 5min at 5000 Xg, removing supernatant, adding 300 mu L of cell lysate into the precipitate, adding 10-20 mu L of protease K, and carrying out 56 ℃ water bath for 1-2 h. Heating in 100 deg.C boiling water bath for 10-15min, centrifuging at 12000rpm for 10min, and collecting supernatant for PCR reaction. The primer sequences are as follows:

an upstream primer: 5'-CGGCTCCCTTGTTACGACTT-3'

A downstream primer: 5'-AGAGTTTGATACTGGCTCAG-3'

And (4) purifying the PCR product, and sequencing to obtain the 16S rDNA sequence of the strain ZJS-1. The sequencing result is shown as SEQ ID No: 1 is shown.

The invention also discloses a method for producing β -1, 3-glucan by fermenting, culturing and transforming the ZJS-1 strain by taking cane sugar as a raw material, wherein the preferable method is as follows:

a. inoculating the strain of claim 1 to a strain activation medium, and culturing at a constant temperature;

b. scraping the activated strain from the strain activation culture medium, transferring the strain to a liquid seed culture solution for culture, and taking the strain as a seed solution for fermentation culture;

c. inoculating the seed solution into the fermentation culture solution, and fermenting in a fermentation tank to obtain the final product.

In step a, the preferred activation medium contains KH₂PO₄0.3-1.0g/L、CaCl₂0.3-1.0g/L、MgSO₄0.6-1.2g/L、FeSO₄·7H₂0.6-1.2g/L of O, 3.0-8.0g/L of cane sugar and 12.0-18.0g/L of agar. Preferably the pH of the activation medium is 6.5-7.0; the constant temperature culture temperature is preferably 25-30 ℃; the culture time is preferably 2 to 3 days.

In the step b, the liquid seed culture solution preferably contains 8.0-10.0g/L of peptone, 3.0-5.0g/L of yeast extract powder and 3.0-5.0g/L of sodium chloride. The pH of the liquid seed culture is preferably 7.0 to 7.8.

In step b, the culture temperature in the liquid seed culture solution is preferably 28-32 ℃, and the culture solution is preferably shake-cultured in a shaker at a speed of 150-300rpm for 48-72h until the culture solution OD₆₀₀Ending the culture when the value is between 0.6 and 0.9, and using the culture as a seed solution for fermentation culture.

In the step c, the fermentation culture solution preferably contains 0-3.0g/L of peptone, 0-1.5g/L of yeast extract powder and 120g/L of sucrose, and the pH of the fermentation culture solution is preferably 7.0-7.8.

In the step c, the volume ratio of the seed liquid to the fermentation culture liquid is preferably 1: 300-1: 800. The fermentation temperature of the fermentation tank is preferably 25-35 ℃, and the fermentation time is preferably 5-9 days.

The culture medium of the present invention is a culture medium or a culture solution prepared by dissolving the above-mentioned preferred components in water.

The method has the advantages that the conversion rate of the cane sugar reaches 85-95 percent, and the yield of β -1, 3-glucan crude product can reach 60 percent.

The invention utilizes high-yield β -1, 3-glucan strains obtained by screening from soil, realizes the high-efficiency conversion of cane sugar to prepare β -1, 3-glucan bacteria by optimizing the control of fermentation conditions, can realize the industrial preparation of β -1, 3-glucan bacteria, avoids or reduces the defect that the traditional extraction and preparation method of β -1, 3-glucan bacteria is highly dependent on raw material sources, expands the high-value processing and utilization of cane sugar resources, and has good application prospect based on the market demand of current food, medicine and breeding fields on β -1, 3-glucan.

Drawings

FIG. 1 shows the results of screening for glucan-producing strains

FIG. 2 media selection of Glucan producing strains

FIG. 3 is a graph showing the relationship between the carbon source concentration and the biomass of a culture medium of Bacillus subtilis ZJS-1 for producing glucan

FIG. 4 shows the effect of the carbon source concentration of the Glucan-producing Bacillus subtilis ZJS-1 medium on product accumulation

Detailed Description

Example 1

Strain screening

In accordance with KH₂PO₄1.0g/L；CaCl₂1.0g/L，MgSO₄1.0g/L，FeSO₄·7H₂Weighing substances with the concentration of 1.0g/L of O and 5.0g/L of cane sugar, redistilling water to dissolve and fix the volume, adjusting pH to 7.0, sterilizing at 115 ℃ for 30 minutes to prepare a screening culture medium, adding 1.0g of collected soil sample for screening into 5mL of liquid culture medium, performing shaking culture in a shaker at 30 ℃ for 60 hours, coating the culture with the inoculation amount of 5 percent in a screening plate culture medium, performing inversion culture at 28 ℃ for 48 hours, detecting the wiredrawing performance of each bacterial colony by using a bacterial colony wiredrawing method, selecting bacterial colonies with obvious wiredrawing performance, transferring the bacterial colonies into 5mL of screening liquid culture medium, performing shaking culture at 28 ℃ and 200rpm for 30 hours, sampling, determining the content of β -1 and 3-glucan in each culture by using a Congo red method, selecting a bacterial strain with the highest content of β -1 and 3-glucan, naming the bacterial colony as ZJS-1, wherein the bacterial colony is white, rough and wrinkled, and irregular edge classification, and the Bacillus subtilis is a biological preservation number of a preservation management committee of the general microbiological culture collection center of CGMCC No.12176Time: year 2016, 3, 4, and the storage address: the institute of scientific research microorganisms of China, No. 3, Xilu No.1, Beijing, Chaoyang, North.

Example 2

β preparation of 1, 3-glucan

Screening of a strain activation medium: according to KH respectively₂PO₄1.0g/L；CaCl₂1.0g/L，MgSO₄1.0g/L，FeSO₄·7H₂O1.0 g/L, sucrose 5.0g/L and KH₂PO₄1.0g/L；CaCl₂1.0g/L，MgSO₄1.0g/L，FeSO₄·7H₂Weighing substances according to the mass concentration of O1.0 g/L and glucose 5.0g/L, dissolving by redistilled water to a constant volume, adjusting pH to 7.0, sterilizing at 115 ℃ for 30 minutes to respectively prepare sucrose and glucose culture media, inoculating the bacillus subtilis ZJS-1 strain in example 1 into two culture media, performing shake culture at 28 ℃, 200rpm for 30 hours, sampling and centrifuging to determine the wet weight of bacteria in each culture medium, and selecting the culture medium with the largest wet weight of the bacteria, wherein the screening result is shown in figure 2, and the culture medium taking sucrose as a carbon source is more favorable for the growth of β grape strains and the conversion of glucan 5.

Culturing and preserving the strain: weighing KH₂PO₄1.0；CaCl₂1.0，MgSO₄1.0，FeSO₄·7H₂Weighing substances with the mass concentration of 1.0g of O, 5.0g of cane sugar and 16.0g of agar, dissolving the substances in redistilled water to a constant volume of 1L, adjusting the pH value to 6.5, sterilizing the substances for 30 minutes at 115 ℃, inverting the plane in a plate, and inverting the slant culture medium in a strain storage tube; inoculating Bacillus subtilis ZJS-1 strain in planar culture medium according to S-shaped streak, standing at 28 deg.C for 3 days, selecting bacteria colony without bacteria, inoculating on slant culture medium, standing at 28 deg.C for 3 days, and preserving at 4 deg.C.

Screening fermentation culture medium, weighing 10g/L peptone, 5g/L yeast extract powder, sucrose from 40g/L to 200g/L, each 10g/L being a concentration gradient, dissolving and fixing volume to 10L in redistilled water in a 10L fermentation tank, adjusting pH7.4 of the culture solution, introducing steam for sterilization at 115 ℃ for 30 minutes, respectively inoculating 100ml seed solution into the fermentation culture solution, fermenting at 30 ℃ for 8 days, sampling and centrifuging every day to determine wet weight of thallus in each culture medium, selecting the culture medium with the largest thallus wet weight, weighing 1g/L to 10g/L peptone, 0.5g/L to 5g/L yeast extract powder and 120g/L sucrose, sampling in a 10L fermentation tank, dissolving and fixing volume to 10L redistilled water, adjusting pH7.4 of the culture solution, introducing steam for sterilization at 115 ℃ for 30 minutes, respectively inoculating 100ml seed solution into the fermentation culture solution, fermenting at 30 ℃, measuring conversion rates of sucrose and glucan, selecting the highest conversion rate, as shown in the culture medium concentration, selecting strains, the strain

Culturing and fermenting: weighing KH₂PO₄1.0；CaCl₂1.0，MgSO₄1.0，FeSO₄·7H₂Weighing substances with the mass concentrations of O1.0 g/L, sucrose 5.0g/L and agar 16.0g/L, dissolving in redistilled water to a constant volume of 1L, adjusting the pH value to 6.5, and sterilizing at 115 ℃ for 30 minutes; inoculating Bacillus subtilis ZJS-1 strain to strain activating culture medium according to S-shaped streak, standing at 28 deg.C, and culturing at constant temperature for 2 days; weighing the substances according to the mass concentrations of 10.0g of peptone, 3.0g of yeast extract powder and 5.0g of sodium chloride, dissolving by redistilled water to a constant volume of 1L, adjusting the pH value of a culture solution to 7.0-7.4, subpackaging the mixture into 500mL triangular flasks according to 100 mL/flask, and sterilizing at 115 ℃ for 30 minutes; scraping activated ZJS-1 from strain activation culture medium, transferring into 100mL liquid seed culture solution, shake culturing in 30 ℃ shaker at 200rpm for 72h until culture solution OD₆₀₀Weighing 300g of peptone, 50g of yeast extract powder and 9000g of sucrose, putting the peptone, the yeast extract powder and the sucrose into a 100L fermentation tank, dissolving the peptone with redistilled water to 100L, adjusting the pH value of a culture solution to 7.4, introducing steam to sterilize at 115 ℃ for 30 minutes, inoculating 100mL of the seed solution into the fermentation culture solution, fermenting at 30 ℃ for 8 days, measuring that the conversion rate of the sucrose is 91%, and finally obtaining the β -1, 3-glucan crude product of 4000g, wherein the concentration of β -1, 3-glucan in the fermentation solution is 65.8 g/L.

SEQUENCE LISTING

<110> Nanjing obsidian kinetic energy-saving environmental protection science and technology Limited

<120> high-yield β -1, 3-glucan bacillus subtilis and application thereof

<130>256

<160>1

<170>PatentIn version 3.5

<210>1

<211>1355

<212>DNA

<213> Bacillus subtilis

<400>1

gcagtcgaac gccccgcaag gggagtggca gacgggtgag taacgcgtgg gaatctaccc 60

atctctgcgg aatagctctg ggaaactgga attaataccg catacgccct acgggggaaa 120

gatttatcgg ggatggatga gcccgcgttg gattagctag ttggtggggt aaaggcctac 180

caaggcgacg atccatagct ggtctgagag gatgatcagc cacattggga ctgagacacg 240

gcccaaactc ctacgggagg cagcagtggg gaatattgga caatgggcgc aagcctgatc 300

cagccatgcc gcgtgagtga tgaaggcctt agggttgtaa agctctttca ccgatgaaga 360

taatgacggt agtcggagaa gaagccccgg ctaacttcgt gccagcagcc gcggtaatac 420

gaagggggct agcgttgttc ggaattactg ggcgtaaagc gcacgtaggc ggatatttaa 480

gtcaggggtg aaatcccgca gctcaactgc ggaactgcct ttgatactgg gtatcttgag 540

tatggaagag gtaagtggaa ttccgagtgt agaggtgaaa ttcgtagata ttcggaggaa 600

caccagtggc gaaggcggct tactggtcca ttactgacgc tgaggtgcga aagcgtgggg 660

agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgaatgt tagccgtcgg 720

gcagtatact gttcggtggc gcagctaacg cattaaacat tccgcctggg gagtacggtc 780

gcaagattaa aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 840

aattcgaagc aacgcgcaga accttaccag ctcttgacat tcggggtatg ggcattggag 900

acgatgtcct tcagttaggc tggccccaga acaggtgctg catggctgtc gtcagctcgt 960

gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc ctcgccctta gttgccagca 1020

tttagttggg cactctaagg ggactgccgg tgataagccg agaggaaggt ggggatgacg 1080

tcaagtcctc atggccctta cgggctgggc tacacacgtg ctacaatggt ggtgacagtg 1140

ggcagcgaga cagcgatgtc gagctaatct ccaaaagcca tctcagttcg gattgcactc 1200

tgcaactcga gtgcatgaag ttggaatcgc tagtaatcgc agatcagcat gctgcggtga 1260

atacgttccc gggccttgta cacaccgccc gtcacaccat gggagttggt tttacccgaa 1320

ggtagtgcgc taaccgcaag gaggcagcta accac 1355

Claims (8)

1. A Bacillus subtilis strain is deposited as follows: CGMCC No. 12176.

2. The bacillus subtilis strain of claim 1, having a 16S rDNA sequence as set forth in SEQ ID No: 1 is shown.

3. A method for preparing β -1, 3-glucan, comprising fermenting the strain of claim 1 in culture for direct conversion of sucrose to β -1, 3-glucan, comprising the steps of:

a. inoculating the strain of claim 1 to a strain activation medium, and culturing at a constant temperature;

b. scraping the activated strain from the strain activation culture medium, transferring the strain to a liquid seed culture solution for culture, and taking the strain as a seed solution for fermentation culture;

c. inoculating the seed solution into a fermentation culture solution, and fermenting in a fermentation tank to obtain the seed solution;

wherein in step a, the activation medium contains KH₂PO₄0.3-1.0g/L、CaCl₂0.3-1.0g/L、MgSO₄0.6-1.2g/L、FeSO₄·7H₂O0.6-1.2g/L, sucrose 3.0-8.0g/L and agar 12.0-18.0g/L, and the pH of the activation medium is 6.5-7.0; the constant temperature culture temperature is 25-30 ℃; the culture time is 2-3 days.

4. The method of claim 3, wherein in step b, the liquid seed culture medium contains 8.0-10.0g/L peptone, 3.0-5.0g/L yeast extract and 3.0-5.0g/L sodium chloride, and the liquid seed culture medium has a pH of 7.0-7.8.

5. The method as claimed in claim 3, wherein in step b, the liquid seed culture medium is cultured at 28-32 deg.C in a shaker at 150-₆₀₀The cultivation is terminated at a value between 0.6 and 0.9.

6. The method of claim 3, wherein in step c, the fermentation broth contains peptone 0-3.0g/L, yeast extract 0-1.5g/L and sucrose 120g/L, and the fermentation broth has a pH of 7.0-7.8.

7. The method of claim 3, wherein in the step c, the volume ratio of the seed solution to the fermentation culture solution is 1: 300-1: 800.

8. The method of claim 3, wherein in step c, the fermentation temperature in the fermentation tank is 25-35 ℃ and the fermentation time is 5-9 days.

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