阅读说明：本技术 一种发酵法高效制备β-葡聚糖的方法、剑菌及其筛选方法 (Method for efficiently preparing beta-glucan by fermentation method, Jianjun and screening method thereof ) 是由 程慧君 李舒宇 费颖 樊启磊 于 2021-04-15 设计创作，主要内容包括：本发明公开了一种发酵法高效制备β-葡聚糖的方法、剑菌及其筛选方法,包括以下步骤：剑菌菌种的活化：将剑菌菌种接种到活化培养基上静置恒温培养,取得单菌落；种子活化：从所述活化培养基上刮取单菌落接种到种子培养液中,进行摇瓶培养,至培养液OD600值在0.6-0.9之间时结束培养,得到种子液；发酵培养：将所述种子液接种到与种子活化步骤中种子培养液相同的培养基中,进行扩大培养发酵,得到发酵液；分离干燥：将所述发酵液加入乙醇,静置,离心后取沉淀物,干燥后得到β-葡聚糖。本发明还公开了剑菌的筛选和验证方法,通过优化发酵条件的控制,实现利用微生物对蔗糖的高效转化制备β-葡聚糖,进而实现发酵制备β-葡聚糖的工业化生产。(The invention discloses a method for efficiently preparing beta-glucan by a fermentation method, Jianjun and a screening method thereof, which comprises the following steps: activation of the sword fungus strain: inoculating the sword fungus strain to an activation culture medium, standing and culturing at constant temperature to obtain a single colony; seed activation: scraping a single colony from the activation culture medium, inoculating the single colony into a seed culture solution, performing shake flask culture, and ending the culture when the OD600 value of the culture solution is between 0.6 and 0.9 to obtain a seed solution; fermentation culture: inoculating the seed solution into a culture medium which is the same as the seed culture solution in the seed activation step, and performing expanded culture fermentation to obtain a fermentation solution; separation and drying: adding ethanol into the fermentation liquor, standing, centrifuging, taking a precipitate, and drying to obtain the beta-glucan. The invention also discloses a screening and verifying method of the sword mushroom, which realizes the high-efficiency conversion of the saccharose by utilizing the microorganism to prepare the beta-glucan by optimizing the control of the fermentation condition, thereby realizing the industrial production of preparing the beta-glucan by fermentation.)

1. A method for preparing beta-glucan by a fermentation method is characterized by comprising the following steps:

A. activation of Jianjun (Ensifer) species: inoculating the sword fungus strain to an activation culture medium, standing and culturing at constant temperature to obtain a single colony;

B. seed activation: scraping a single colony from the activation culture medium, inoculating the single colony into a seed culture solution, performing shake flask culture, and ending the culture when the OD600 value of the culture solution is between 0.6 and 0.9 to obtain a seed solution;

C. fermentation culture: b, inoculating the seed solution into a culture medium which is the same as the seed culture solution in the step B, and performing expanded culture fermentation to obtain a fermentation solution;

D. separation and drying: adding ethanol into the fermentation liquor, standing, centrifuging, taking a precipitate, and drying to obtain the beta-glucan.

2. The method for preparing beta-glucan by fermentation according to claim 1, wherein the activation medium in step a comprises 0.8-1.2g/L of yeast powder, 5.0-10g/L of glucose or 8-13.0g/L of sucrose, 30-50g/L of soil, 12-18.0g/L of agar, and 5-11 of pH;

the conditions of constant temperature culture in the step A are as follows: the culture temperature is 25-30 ℃; the culture time is 2-3 days.

3. The method of claim 1, wherein the seed culture solution in step B is a mixed aqueous solution of peptone 8.0-12.0g/L, yeast powder 4.0-6.0g/L and sodium chloride 8.0-12.0g/L, and has a pH of 5-11.

4. The method for preparing beta-glucan by fermentation according to claim 1, wherein the volume ratio of the seed culture solution to the seed solution in the step C is 1:300-1: 800; the fermentation conditions were: the fermentation temperature is 25-30 deg.C, and the fermentation time is 3-5 days.

5. The method for preparing beta-glucan by fermentation according to claim 1, wherein the volume ratio of the fermentation liquor to the ethanol in the step D is 1: (2-4), the standing time is 2-4h, the centrifugation speed is 6000-8000rpm, and the drying temperature is 45 ℃.

6. The Ensifer used in the method for preparing beta-glucan according to any one of claims 1 to 5, wherein the nucleotide sequence of the Ensifer (Ensifer) is shown as SEQ ID No. 1.

7. The method for screening for Jianjun according to claim 6, comprising the steps of preparing a screening medium, shake-flask culture of enriched β -glucan producing species and screening of single colony with clear mucus secretion for scale-up culture.

8. The screening method according to claim 7,

the step of preparing the screening medium comprises the following steps:

weighing the substances according to the concentration of 0.8-1.2g/L yeast powder, 8-13.0g/L sucrose, 30-50g/L soil and 12-18.0g/L agar, dissolving the substances in distilled water to a constant volume, adjusting the pH value to 5-11, sterilizing at 121 ℃ for 20-30 minutes to prepare a screening culture medium; the method comprises the following steps of (1) shaking culture and enrichment of strains producing beta-glucan:

adding 0.5-1.5g of the collected saline-alkali soil sample for screening into 5ml of liquid culture medium, and carrying out shake culture in a shaking table at 28 ℃ for 2-3 days to obtain a strain culture for enriching and producing beta-glucan;

the screening of the single colony with the secretion of the transparent mucus for scale-up culture comprises the following steps:

coating the culture on the screening culture medium by an inoculum size of 5 percent of the total weight, carrying out inverted culture at 25-30 ℃ for 2-3 days, selecting a single bacterial colony with secretion of transparent mucus by utilizing the special morphology of the bacterial colony, transferring the single bacterial colony into 5-8ml of the screening culture medium for amplification culture, and carrying out shake culture at 25-30 ℃ and 300rpm for 2-3 days to obtain the sword fungus strain.

9. Beta-glucan produced by the process according to any one of claims 1 to 5.

10. Use of the beta-glucan according to claim 9 in fields including general foods, health foods, foods formulated for special medical use, and pharmaceuticals.

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for efficiently preparing beta-glucan by a fermentation method, a sword fungus and a screening method thereof.

Background

Beta-glucan is a glucose polymer with various biological activities, is extracted by special steps and is a substance which is considered to be safe by the FDA in the United states, can be added to general foods, and reports show that blood fat and blood sugar of mice are reduced after the beta-glucan is orally taken for a period of time. In addition, the glucan has the functions of removing free radicals, resisting radiation and resisting infection caused by filtering viruses, fungi, bacteria and the like, so that the glucan can be widely applied to the industries of medicines, foods, cosmetics and the like.

Traditionally, beta-glucan is produced by extracting from Lentinus edodes, yeast, oat, and highland barley. Due to the limitation of raw material scale and production period, the process for producing the beta-glucan by the extraction method is complex, the yield is low, and the market demand can not be met.

Beta-glucan not only exerts various biological activities in various organisms, but also has various functions in the process of interaction between various organisms, and is a highly effective biological response regulator (BRM). However, the exertion of the efficacy of β -glucan requires some solubility, steric structure and proper molecular weight support. Many beta-glucans are less water soluble, for example curdlan is a water insoluble gel polysaccharide glucan produced by agrobacterium or alcaligenes; the fungus pall polysaccharide has the effect of inhibiting the tumor, but the solubility of the fungus pall polysaccharide in water is not high. The water-insoluble glucan is difficult to be directly absorbed after entering the human body, so that the application range of the glucan is greatly limited.

In conclusion, research and development of beta-glucan with high water solubility, avoidance of limiting factors such as raw material scale and production period and the like are urgent needs in the field of food nutrition and health.

Disclosure of Invention

Aiming at the problems and the defects in the prior art, the invention firstly provides a method for preparing beta-glucan by a fermentation method, a sword fungus and a screening method thereof, and solves the problems of low water solubility, long production period and the like of the beta-glucan in the prior art.

Firstly, one technical scheme provided by the invention is a method for preparing beta-glucan by a fermentation method, which comprises the following steps:

A. activation of the sword fungus strain: inoculating the sword fungus strain to an activation culture medium, standing and culturing at constant temperature to obtain a single colony;

B. seed activation: scraping a single colony from the activation culture medium, inoculating the single colony into a seed culture solution, performing shake flask culture, and ending the culture when the OD600 value of the culture solution is between 0.6 and 0.9 to obtain a seed solution;

C. fermentation culture: b, inoculating the seed solution into a culture medium which is the same as the seed culture solution in the step B, and performing expanded culture fermentation to obtain a fermentation solution;

D. separation and drying: adding ethanol into the fermentation liquor, standing, centrifuging, taking a precipitate, and drying to obtain the beta-glucan.

In one embodiment, the activation medium in step A contains 0.8-1.2g/L of yeast powder, 5.0-10g/L of glucose or 8-13.0g/L of sucrose, 30-50g/L of soil, 12-18.0g/L of agar, and the pH value is 5-11.

In one embodiment, the conditions for the isothermal cultivation in step a are: the culture temperature is 25-30 ℃; the culture time is 2-3 days.

In one embodiment, the seed culture solution in step B is a mixed aqueous solution of 8.0-12.0g/L peptone, 4.0-6.0g/L yeast powder and 8.0-12.0g/L sodium chloride, and has a pH of 5-11.

In one embodiment, the volume ratio of the seed culture solution to the seed solution in the step C is 1:300-1: 800; the fermentation conditions were: the fermentation temperature is 25-30 deg.C, and the fermentation time is 3-5 days.

In one embodiment, the volume ratio of the fermentation liquid to ethanol in the step D is 1: (2-4), the standing time is 2-4h, the centrifugation speed is 6000-8000rpm, and the drying temperature is 45 ℃.

As a second aspect of the present invention, there is provided a sword-shaped bacterium used in a method for producing β -glucan, the nucleotide sequence of the sword-shaped bacterium (Ensifer) being represented by SEQ ID No. 1.

As a third aspect of the invention, a screening method of sword-shaped bacteria is provided, which comprises a step of preparing a screening culture medium, a step of carrying out shake flask culture on enriched beta-glucan-producing strains and a step of screening single colony with secretion of transparent mucus for scale-up culture.

In one embodiment, the step of preparing the screening medium comprises:

weighing the substances according to the concentration of 0.8-1.2g/L yeast powder, 8-13.0g/L sucrose, 30-50g/L soil and 12-18.0g/L agar, dissolving the substances in distilled water to a constant volume, adjusting the pH value to 5-11, sterilizing at 121 ℃ for 20-30 minutes to prepare a screening culture medium;

in one embodiment, the shake flask culture is enriched for β -glucan-producing species:

adding 0.5-1.5g of the collected saline-alkali soil sample for screening into 5ml of liquid culture medium, and carrying out shake culture in a shaking table at 28 ℃ for 2-3 days to obtain a strain culture for enriching and producing beta-glucan;

in one embodiment, the step of screening for single colony expansion with secreted clear mucus comprises:

coating the culture on the screening culture medium by an inoculum size of 5 percent of the total weight, carrying out inverted culture at 25-30 ℃ for 2-3 days, selecting a single bacterial colony with secretion of transparent mucus by utilizing the special morphology of the bacterial colony, transferring the single bacterial colony into 5-8ml of the screening culture medium for amplification culture, and carrying out shake culture at 25-30 ℃ and 300rpm for 2-3 days to obtain the sword fungus strain.

As a third aspect of the present invention, there is provided a β -glucan produced by the above method.

As a fourth aspect of the invention, the invention provides an application of the beta-glucan prepared by the method in the fields of common food, health food, special medical application formula food and medicines.

Compared with the prior art, the invention has the beneficial effects that:

the invention uses a sword fungus strain, realizes the high-efficiency conversion of the microorganism to cane sugar to prepare the beta-glucan with high water solubility by optimizing the control of fermentation conditions, and further realizes the industrial production of preparing the beta-glucan by fermentation; by the aid of the sword fungus and the screening method thereof, the yield and the purity of the sword fungus strain are high, and a foundation is laid for preparing high-purity beta-glucan; the beta-glucan prepared by the invention is food grade, can be applied to the food fields of common food, health food, special medical formula food and the like, can control the rise of blood sugar, remove free radicals, resist radiation and resist virus and fungus infection, and provides a new solution for health food.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

The sequence table is a 16S rDNA sequence obtained by sequencing after purifying the PCR product of the strain of the invention.

FIG. 1 shows the colony morphology of strain ZB-1 on a plate.

FIG. 2 shows the content of beta-glucan prepared in examples 1 to 3 and comparative examples 1 to 2 in a different manner.

FIG. 3 shows the yields of beta-glucan prepared in different manners in examples 1 to 3 and comparative examples 1 to 2.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The invention is further illustrated by the following examples and figures. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The method for preparing the beta-glucan by the fermentation method in the embodiment of the invention comprises the following steps:

A. activation of the sword fungus strain: inoculating the sword fungus strain to an activation culture medium, standing and culturing at constant temperature to obtain a single colony;

B. seed activation: scraping a single colony from the activation culture medium, inoculating the single colony into a seed culture solution, performing shake flask culture, and ending the culture when the OD600 value of the culture solution is between 0.6 and 0.9 to obtain a seed solution;

C. fermentation culture: b, inoculating the seed solution into a culture medium which is the same as the seed culture solution in the step B, and performing expanded culture fermentation to obtain a fermentation solution;

D. separation and drying: adding ethanol into the fermentation liquor, standing, centrifuging, taking a precipitate, and drying to obtain the beta-glucan.

The screening method of the sword mushroom used in the embodiment of the invention comprises the steps of preparing a screening culture medium, carrying out shake flask culture on a strain enriched and producing beta-glucan and screening single colony with secretion transparent mucus for amplification culture:

wherein, the steps for preparing the screening culture medium are as follows:

weighing the substances according to the concentration of 0.8-1.2g/L yeast powder, 8-13.0g/L sucrose, 30-50g/L soil and 12-18.0g/L agar, dissolving the substances in distilled water to a constant volume, adjusting the pH value to 5-11, sterilizing at 121 ℃ for 20-30 minutes to prepare a screening culture medium;

the steps of culturing enriched beta-glucan producing strains by shaking the flask are as follows:

adding 0.5-1.5g of the collected saline-alkali soil sample for screening into 5ml of liquid culture medium, and carrying out shake culture in a shaking table at 28 ℃ for 2-3 days to obtain a strain culture for enriching and producing beta-glucan;

the steps of screening the single colony with the secretion of the transparent mucus for expansion culture are as follows: :

coating the culture on the screening culture medium by an inoculum size of 5 percent of the total weight, carrying out inverted culture at 25-30 ℃ for 2-3 days, selecting a single bacterial colony with secretion of transparent mucus by utilizing the special morphology of the bacterial colony, transferring the single bacterial colony into 5-8ml of the screening culture medium for amplification culture, and carrying out shake culture at 25-30 ℃ and 300rpm for 2-3 days to obtain the sword fungus strain.

The method for verifying the sword fungus used in the embodiment of the invention comprises the following steps of 16S rDNA sequence analysis: the slant culture of the strain is picked and added into 5-10ml of liquid seed culture medium, the culture is carried out overnight at 25-30 ℃, 2ml of bacterial liquid is taken, centrifugation is carried out for 3-5min at 6000rpm with 5000-. Heating in 100 deg.C boiling water bath for 10-15min, centrifuging at 10000-; the primer sequences are as follows:

an upstream primer: 5'-CGGCTCCCTTGTTACGACTT-3'

A downstream primer: 5'-AGAGTTTGATACTGGCTCAG-3'

The 16S rDNA sequence of the strain obtained by sequencing after the purification of the PCR product is shown in SEQ ID No.1, and the sequence comparison result shows that the strain is Jianjun (Ensifer).

In embodiments 1 to 3, the screening and verification methods of the sword fungi are the same as those described above, and specifically include the following steps:

example 1

A method for preparing beta-glucan by using the sword fungus obtained by the screening method comprises the following specific steps:

(1) activation of the sword fungus strain: firstly, preparing an activation culture medium of the sword mushroom according to the proportion, namely 1.2g/L of yeast powder, 10.0g/L of cane sugar, 50g/L of soil and 18.0g/L of agar, and adjusting the pH value to 7.0 after heating and dissolving; the constant temperature culture temperature is 30 ℃; inoculating the strain of the sword fungus to an activated plate culture medium, standing and culturing for 3 days at constant temperature to obtain a plate single colony.

(2) Seed activation: scraping single colony from the strain activating culture medium and inoculating the single colony into liquid seed culture liquid for shake flask culture to obtain seed liquid. The method for preparing the seed liquid by using the sword fungi comprises the following steps: dissolving peptone 12.0g/L, yeast powder 6.0g/L and sodium chloride 12.0g/L with distilled water to a constant volume, adjusting pH to 7.0, sterilizing at 121 ℃ for 20 minutes under high temperature and high pressure, cooling, and slightly scraping off the single colony of the sword fungus obtained in the step (1) to be added; fermenting at 30 deg.C for 48h, shaking table at 300rpm for 3 days, and finishing culturing until OD600 value of culture solution is 0.8 to obtain seed culture solution.

(3) And (3) performing fermentation culture, namely preparing the culture solution again according to the culture solution preparation method in the step (2), wherein the preparation amount is determined according to the amount of the seed culture solution obtained in the step (2). The volume ratio of the seed culture solution to the newly prepared culture solution is 1:800, and then the fermentation is continued for 3 days at 30 ℃.

(4) Separation and drying: adding ethanol into the fermentation liquor obtained in the step (3), wherein the volume ratio of the fermentation liquor to the ethanol is 1: 2, standing for 4 hours, centrifuging at 8000rpm, and drying the solid obtained by alcohol precipitation at 45 ℃ by adopting a low-temperature drying method. To obtain beta-glucan powder prepared by using the sword fungus.

Example 2

A method for preparing beta-glucan by using the sword fungus. The preparation method is the same as that of example 1, except that glucose is used as the carbon source for the activation medium. The method comprises the following specific steps:

(1) activation of the sword fungus strain: firstly, preparing an activation culture medium of the sword mushroom according to the proportion, namely 1.2g/L of yeast powder, 10.0g/L of glucose, 50g/L of soil and 18.0g/L of agar, and adjusting the pH value to 7.0 after heating and dissolving; the constant temperature culture temperature is 30 ℃; inoculating the strain of the sword fungus to an activated plate culture medium, standing and culturing for 3 days at constant temperature to obtain a plate single colony.

(2) Seed activation: scraping single colony from the strain activating culture medium and inoculating the single colony into liquid seed culture liquid for shake flask culture to obtain seed liquid. The method for preparing the seed liquid by using the sword fungi comprises the following steps: dissolving peptone 12.0g/L, yeast powder 6.0g/L and sodium chloride 12.0g/L with distilled water to a constant volume, adjusting pH to 7.0, sterilizing at 121 ℃ for 20 minutes under high temperature and high pressure, cooling, and slightly scraping off the single colony of the sword fungus obtained in the step (1) to be added; fermenting at 30 deg.C for 48h, shaking table at 300rpm for 3 days, and finishing culturing until OD600 value of culture solution is 0.8 to obtain seed culture solution.

(3) And (3) performing fermentation culture, namely preparing the culture solution again according to the culture solution preparation method in the step (2), wherein the preparation amount is determined according to the amount of the seed culture solution obtained in the step (2). The volume ratio of the seed culture solution to the newly prepared culture solution is 1:800, and then the fermentation is continued for 3 days at 30 ℃.

(4) Separation and drying: adding ethanol into the fermentation liquor obtained in the step (3), wherein the volume ratio of the fermentation liquor to the ethanol is 1: 2, standing for 4 hours, centrifuging at 8000rpm, and drying the solid obtained by alcohol precipitation at 45 ℃ by adopting a low-temperature drying method. To obtain beta-glucan powder prepared by using the sword fungus.

Example 3

A method for preparing beta-glucan by using the sword fungus. The preparation method is the same as example 1, except that the process parameters are different. The method comprises the following specific steps:

(1) activation of the sword fungus strain: firstly, preparing an activation culture medium of the sword mushroom according to the proportion, namely 1.2g/L of yeast powder, 13.0g/L of cane sugar, 50g/L of soil and 18.0g/L of agar, and adjusting the pH value to 5.0 after heating and dissolving; the constant temperature culture temperature is 25 ℃; inoculating the strain of the sword fungus to an activated plate culture medium, standing and culturing for 2 days at constant temperature to obtain a plate single colony.

(2) Seed activation: scraping single colony from the strain activating culture medium and inoculating the single colony into liquid seed culture liquid for shake flask culture to obtain seed liquid. The method for preparing the seed liquid by using the sword fungi comprises the following steps: dissolving peptone 8.0g/L, yeast powder 4.0g/L and sodium chloride 8.0g/L in distilled water to a constant volume, adjusting the pH to 7.0, sterilizing at 115 ℃ for 20 minutes under high temperature and high pressure, and slightly scraping the single colony of the sword fungus obtained in the step (1) after cooling; fermenting at 25 deg.C for 24h, shaking table at 250rpm for 2 days, and finishing culturing until OD600 value of culture solution is 0.6 to obtain seed culture solution.

(3) And (3) performing fermentation culture, namely preparing the culture solution again according to the culture solution preparation method in the step (2), wherein the preparation amount is determined according to the amount of the seed culture solution obtained in the step (2). The volume ratio of the seed culture solution to the newly prepared culture solution is 1:300, and then the fermentation is continued for 5 days at 25 ℃.

(4) Separation and drying: adding ethanol into the fermentation liquor obtained in the step (3), wherein the volume ratio of the fermentation liquor to the ethanol is 1: and 4, standing for 4 hours, centrifuging at 6000rpm, and drying the solid obtained by alcohol precipitation at a low temperature of 45 ℃ by adopting a low-temperature drying method. To obtain beta-glucan powder prepared by using the sword fungus.

To better explain the remarkable effects of the present invention, the comparative examples were added as follows:

comparative example 1

A method for extracting and preparing beta-glucan from barley. The method comprises the following specific steps:

(1) 500g of barley is ground into powder and sieved by a 40-mesh sieve. Then baking for 1h at 85 ℃ to kill endogenous enzyme activity.

(2) Adding 250g of the powder obtained in the step (1) into absolute ethyl alcohol according to the weight-volume ratio of 1:4, heating and refluxing for 20min, centrifuging (3500r/min) for 10min when the powder is hot, and removing the supernatant.

(3) Dissolving the residue obtained in the step (2) in water according to the weight-volume ratio of 1:6, adding 1.5ml of high-temperature-resistant alpha-amylase at 90 ℃, gelatinizing the starch after 30min, then adding efficient saccharifying enzyme after the temperature is reduced to 60 ℃, adjusting the pH value to 4.0, and treating for 2 h; when the temperature is reduced to 32 ℃, adding trypsin (the adding amount of the enzyme is 0.05 percent), adjusting the pH value to 8.0, and processing for 2 hours. This process is to remove lipids, starch, and proteins contained in barley. Then keeping the temperature at 90 ℃ for 10min, and inactivating the enzyme.

(4) And (4) centrifuging (8000r/min, 10min) the enzymolysis liquid obtained in the step (3) for separation to obtain a supernatant rich in beta-glucan, amino acid, oligopeptide, monosaccharide and oligosaccharide.

(5) Concentrating the supernatant obtained in step (4) with a membrane with molecular weight cutoff of 10000 under 0.21MPa at 30 deg.C. Then adding equal volume of absolute ethyl alcohol to wash the trapped solid phase for multiple times, wherein the washing condition is (8000r/min, 10 min).

(6) And (4) discarding the supernatant obtained in the step (5), and drying the solid phase at a low temperature of 45 ℃ to obtain beta-glucan powder.

Comparative example 2

A method for preparing beta-glucan by using Saccharomyces cerevisiae. The method comprises the following specific steps:

(1) the saccharomyces cerevisiae is washed by deionized water for multiple times to prepare bacterial suspension with the mass fraction of 15%, then NaCl with the mass fraction of 3% is added, the pH value is adjusted to 5.0, autolysis is induced in a constant-temperature water bath oscillator with the temperature of 55 ℃ for 24 hours, and then the temperature is raised to 85 ℃ for 20min to inactivate enzyme.

(2) And (2) after the bacterial suspension obtained in the step (1) is cooled to room temperature, centrifugally washing the bacterial suspension for multiple times by using deionized water, preparing the bacterial suspension with the mass fraction wet weight of 20% by using a phosphate buffer solution with the concentration of 0.02mol/L and the pH of 7.5, oscillating the bacterial suspension in a constant-temperature water bath at the temperature of 95 ℃ for 130r/min, leaching the bacterial suspension for 4 hours by using hot water, cooling the bacterial suspension to the room temperature, centrifugally washing the bacterial suspension for multiple times by using the deionized water, and preserving the precipitate at the temperature of 4 ℃.

(3) Homogenizing the yeast cell sap after hot water extraction in the step (2) by using high-pressure microjet under the following treatment conditions: the mass concentration is 20 percent of wet weight, the pressure is 105MPa, and the cycle number is 6.

(4) Mannase and yeast drawer are selected to not destroy protein components in yeast cell wall, the ratio of feed to liquid is 1:4, the enzyme addition amount is 0.5% and 0.5%, the temperature is 55 ℃, the pH value is 7.0, and the time is 50 min.

(5) After the composite enzymolysis in the step (4), homogenizing the enzymolysis liquid by high-pressure micro-jet. The conditions are as follows: the mass concentration is 20 percent of wet weight, the pressure is 210MPa, and the cycle time is 3 times.

(6) Centrifuging the sample after the microjet treatment, and drying the sample in a constant-temperature air-blast drying oven at 65 ℃ to obtain the yeast beta-glucan product.

Characterization and comparison of results for examples and comparative examples:

1. determination of beta-glucan content in samples obtained by different preparation methods

The content of β -glucan in each of examples 1 to 3 and comparative examples 1 to 2 was measured by congo red method by first plotting a standard curve of β -glucan and determining the microgram number (i.e., K value) of β -glucan equivalent to 1 in absorbance on the curve. Respectively taking 2ml of undried liquid of a sample to be detected, respectively adding 4ml of Congo red, accurately timing to react in a water bath at 20 ℃ for 10min, replacing the sample with 2ml of distilled water to perform blank zeroing, and measuring the absorbance A of the reaction solution. The content of β -glucan in the sample (mg/L) ═ K/2 × absorbance a × dilution factor n.

The results are shown in Table 1:

TABLE 1 content of beta-glucan prepared in different ways

As can be seen from table 1 and fig. 2, the content of β -glucan is significantly increased in the microbial fermentation methods of examples 1 to 3 and comparative example 2 compared to the chemical extraction method of comparative example 1; compared with the method for preparing the beta-glucan by using the saccharomyces cerevisiae through the microbial fermentation method in the comparative example 2, the method for preparing the beta-glucan by using the sword fungus adopted in the examples 1 to 3 has obvious improvement on the purity of the beta-glucan, and fully shows that the sword fungus is more beneficial to the conversion of the beta-glucan than the saccharomyces cerevisiae.

2. Yield of beta-glucan in samples from different preparation methods

The results of the yield (%) of β -glucan prepared by different methods ═ mass (g) of β -glucan obtained/mass (g) of substrate x 100 are shown in table 2:

TABLE 2 yield of beta-glucan prepared in different ways

As can be seen from table 2 and fig. 3, the yield of β -glucan is slightly improved and the effect is not significant in comparative example 2, compared to the chemical extraction method of comparative example 1, in which β -glucan is prepared by the microbial fermentation method using saccharomyces cerevisiae. In examples 1-3, the beta-glucan is prepared by the microorganism fermentation method using the sword fungus, the yield of the beta-glucan is obviously improved, and the improvement effect is 5-6 times as high as that of the beta-glucan prepared by the chemical method. Fully shows that the method for preparing the beta-glucan by using the sword fungus adopted in the embodiment has great improvement on the yield of the beta-glucan, and has very important promotion significance on production and application.

In conclusion, the invention uses the high-yield beta-glucan strain screened from the saline-alkali soil to realize the efficient conversion of the sucrose by the microorganism to prepare the beta-glucan by optimizing the control of the fermentation condition. The method is expected to realize the industrial production of preparing the beta-glucan by fermentation, avoid or reduce the defects that the traditional beta-glucan extraction and preparation method is highly dependent on raw materials and has long and complex production period, and simultaneously expand the high-value processing and utilization of sucrose resources. Based on the market demands of the fields of current food, medicine, cultivation and the like on beta-glucan, the invention has good application prospect.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Suzhou Langpang Nutrition technology Co., Ltd

<120> method for efficiently preparing beta-glucan by fermentation method, Jianjun and screening method thereof

<141> 2021-04-15

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1321

<212> DNA

<213> Jianjun (Ensifer)

<400> 1

tacgagcggc agacgggtga gtaacgcgtg ggaatctacc cttttctacg gaataacgca 60

gggaaacttg tgctaatacc gtatgagccc ttcgggggaa agatttatcg ggaaaggatg 120

agcccgcgtt ggattagcta gttggtgggg taaaggccta ccaaggcgac gatccatagc 180

tggtctgaga ggatgatcag ccacattggg actgagacac ggcccaaact cctacgggag 240

gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagccatgc cgcgtgagtg 300

atgaaggccc tagggttgta aagctctttc accggtgaag ataatgacgg taaccggaga 360

agaagccccg gctaacttcg tgccagcagc cgcggtaata cgaagggggc tagcgttgtt 420

cggaattact gggcgtaaag cgcacgtagg cggacattta agtcaggggt gaaatcccgg 480

ggctcaaccc cggaactgcc tttgatactg ggtgtctaga gtccagaaga ggtgagtgga 540

attccgagtg tagaggtgaa attcgtagat attcggagga acaccagtgg cgaaggcggc 600

tcactggtct ggtactgacg ctgaggtgcg aaagcgtggg gagcaaacag gattagatac 660

cctggtagtc cacgccgtaa acgatgaatg ttagccgtcg ggcagtttac tgttcggtgg 720

cgcagctaac gcattaaaca ttccgcctgg ggagtacggt cgcaagatta aaactcaaag 780

gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgcag 840

aaccttacca gcccttgaca tcccggtcgc gggaacgaga gatcgtttcc ttcagttcgg 900

ctggaccgga gacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 960

agtcccgcaa cgagcgcaac cctcgccctt agttgccagc atttagttgg gcactctaag 1020

gggactgccg gtgataagcc gagaggaagg tggggatgac gtcaagtcct catggccctt 1080

acgggctggg ctacacacgt gctacaatgg tggtgacagt gggcagcgag accgcgaggt 1140

cgagctaatc tccaaaagcc atctcagttc ggattgcact ctgcaactcg ggtgcatgaa 1200

gttggaatcg ctagtaatcg cagatcagca tgctgcggtg aatacgttcc cgggccttgt 1260

acacaccgcc cgtcacacca tgggagttgg ttctacccga aggtagtgcg ctaaccgcaa 1320

g 1321