High-yield linear-chain glucan strain and glucan fermentation and production method thereof

Abstract

The invention discloses a high-yield linear-chain glucan strain and a glucan fermentation and production method thereof. The high-yield linear-chain glucan strain is named Leuconostoc citreum N21. Thehigh-yield linear-chain glucan strain is separated from dried milk curds made by indigenous herdsmen in Xilin Gol League of Xilinhot City of Inner Mongolia, and an experiment discovers that the high-yield linear-chain glucan strain is capable of generating EPS (Exopolysaccharides) having good characteristics. The glucan fermentation and production method comprises the following steps: inoculatingthe Leuconostoc citreum N21 to an MRS basic culture medium containing 5 percent of sucrose, and culturing for 36 to 72 hours under the conditions that the pH (Potential of Hydrogen) value of an initial culture medium is 6 to 8, the temperature is 25 to 37 DEG C and the shaking speed is 0 to 170 rpm (Revolutions Per Minute), thus obtaining a fermentation solution; purifying crude polysaccharide, thus obtaining the EPS. The high-yield linear-chain glucan strain and the glucan fermentation and production method, disclosed by the invention, have the advantages of short EPS production period, highEPS yield, safety and no toxic and side effects; the EPS is higher in solubility, emulsibility and heat stability, theoretical foundation and basis are provided for industrial application, and a widecommercial application prospect is obtained.

Description

technical field

[0001] The invention relates to a high-yield linear glucan strain and a glucan fermentation production method thereof. Background technique

[0002] Exopolysaccharide (EPS) is a mucus polysaccharide or capsular polysaccharide secreted by microorganisms (such as bacteria, yeast, fungi) during their growth and metabolism. Microbial exopolysaccharides have been the main source of theoretical and practical research on exopolysaccharides because of their wide range of sources, mild reaction conditions, and easy separation and purification. Lactic acid bacteria (LAB) is a general term for a class of non-spore-forming, catalase-negative, Gram-positive bacteria that are widely distributed in nature, can ferment carbohydrates, and produce a large amount of lactic acid during the fermentation process. As a kind of green, safe and non-toxic food-grade microorganisms (GRAS), lactic acid bacteria have the advantages of short fermentation cycle and simple nutritional requ...

Examples

Embodiment 1

[0036] Example 1: Isolation and screening of a high-production exopolysaccharide strain

[0037] (1) culture medium

[0038] MRS basal medium: glucose 20g, tryptone 10g, beef extract 10g, yeast extract 5g, ammonium citrate 2g, K 2 HPO 4 2g, CH 3 COONa 5g, MgSO 4 ·7H 2 O 0.58g, MnSO 4 ·H 2 O 0.25g, Tween 80 1mL, distilled water 1000mL, pH 6.5, sterilized at 115°C for 20min.

[0039] MRS sugar production fermentation medium: 50g sucrose, 10g tryptone, 10g beef extract, 5g yeast extract, K 2 HPO 4 2g, CH 3 COONa 5g, ammonium citrate 2g, MgSO 4 ·7H 2 O 0.58g, MnSO 4 ·H 2 O 0.25g, Tween 80 1mL, distilled water 1000mL, pH 6.5, sterilized at 115°C for 20min.

[0040] (2) Sample pretreatment

[0041] Weigh 1g of milk tofu and add it into 9mL sterile saline to make 10 -1 Concentration of the sample solution, after shaking and mixing, gradually dilute to the appropriate concentration, and set aside for later use.

[0042] (3) Primary screening of slime-producing coloni...

Embodiment 2

[0046] Example 2: Identification of a high-production exopolysaccharide strain

[0047] (1) Morphological identification

[0048] Inoculate the strain N21 on the MRS agar plate with a three-section line, and after culturing at 30°C for 24-48 hours, observe and record the single colony characteristics of the strain on the plate. Use an inoculation needle to pick a small amount of fresh bacteria and spread it on a clean glass slide for Gram staining, and then observe the individual shape and arrangement of the cells under a microscope.

[0049] (2) Physiological and biochemical identification

[0050] ① Contact enzyme test: Select the strains with positive Gram staining results for contact enzyme test.

[0051] ②Glucose acid and gas production test: Add 1.6% bromocresol violet 2mL / L and inverted Duchenne tubules to ordinary MRS liquid medium. The color of the indicator in the medium changes from purple to yellow, indicating acid production, and if it is still purple, it indic...

Embodiment 3

[0066] Example 3: A fermentation process for producing exopolysaccharides by fermentation of a high-yield exopolysaccharide strain

[0067] In this embodiment, a fermentation process for producing exopolysaccharide by fermentation of a high-yield exopolysaccharide strain is carried out according to the following steps:

[0068] (1) Preparation of seed solution

[0069] Leuconostoc citreum N21 was inoculated in the basic MRS medium, so that the initial cell concentration in the medium was 1.0×10 8 unit / mL is the seed solution.

[0070] (2) Fermentation conditions for sugar production

[0071] Inoculate Leuconostoc citreum N21 with an inoculation amount of 2.0% (V / V) in a 100 / 250mL medium with an initial pH value of 6-8, and culture it with constant temperature shaking at a temperature of 25-37°C 36-72h, use the phenol-sulfuric acid method to measure the polysaccharide content of the fermentation supernatant.

[0072] Under the conditions of an initial pH value of 6.5, a tem...

technical field

[0001] The invention relates to a high-yield linear glucan strain and a glucan fermentation production method thereof. Background technique

[0002] Exopolysaccharide (EPS) is a mucus polysaccharide or capsular polysaccharide secreted by microorganisms (such as bacteria, yeast, fungi) during their growth and metabolism. Microbial exopolysaccharides have been the main source of theoretical and practical research on exopolysaccharides because of their wide range of sources, mild reaction conditions, and easy separation and purification. Lactic acid bacteria (LAB) is a general term for a class of non-spore-forming, catalase-negative, Gram-positive bacteria that are widely distributed in nature, can ferment carbohydrates, and produce a large amount of lactic acid during the fermentation process. As a kind of green, safe and non-toxic food-grade microorganisms (GRAS), lactic acid bacteria have the advantages of short fermentation cycle and simple nutritional requ...

Examples

Embodiment 1

[0036] Example 1: Isolation and screening of a high-production exopolysaccharide strain

[0037] (1) culture medium

[0038] MRS basal medium: glucose 20g, tryptone 10g, beef extract 10g, yeast extract 5g, ammonium citrate 2g, K 2 HPO 4 2g, CH 3 COONa 5g, MgSO 4 ·7H 2 O 0.58g, MnSO 4 ·H 2 O 0.25g, Tween 80 1mL, distilled water 1000mL, pH 6.5, sterilized at 115°C for 20min.

[0039] MRS sugar production fermentation medium: 50g sucrose, 10g tryptone, 10g beef extract, 5g yeast extract, K 2 HPO 4 2g, CH 3 COONa 5g, ammonium citrate 2g, MgSO 4 ·7H 2 O 0.58g, MnSO 4 ·H 2 O 0.25g, Tween 80 1mL, distilled water 1000mL, pH 6.5, sterilized at 115°C for 20min.

[0040] (2) Sample pretreatment

[0041] Weigh 1g of milk tofu and add it into 9mL sterile saline to make 10 -1 Concentration of the sample solution, after shaking and mixing, gradually dilute to the appropriate concentration, and set aside for later use.

[0042] (3) Primary screening of slime-producing coloni...

Embodiment 2

[0046] Example 2: Identification of a high-production exopolysaccharide strain

[0047] (1) Morphological identification

[0048] Inoculate the strain N21 on the MRS agar plate with a three-section line, and after culturing at 30°C for 24-48 hours, observe and record the single colony characteristics of the strain on the plate. Use an inoculation needle to pick a small amount of fresh bacteria and spread it on a clean glass slide for Gram staining, and then observe the individual shape and arrangement of the cells under a microscope.

[0049] (2) Physiological and biochemical identification

[0050] ① Contact enzyme test: Select the strains with positive Gram staining results for contact enzyme test.

[0051] ②Glucose acid and gas production test: Add 1.6% bromocresol violet 2mL / L and inverted Duchenne tubules to ordinary MRS liquid medium. The color of the indicator in the medium changes from purple to yellow, indicating acid production, and if it is still purple, it indic...

Embodiment 3

[0066] Example 3: A fermentation process for producing exopolysaccharides by fermentation of a high-yield exopolysaccharide strain

[0067] In this embodiment, a fermentation process for producing exopolysaccharide by fermentation of a high-yield exopolysaccharide strain is carried out according to the following steps:

[0068] (1) Preparation of seed solution

[0069] Leuconostoc citreum N21 was inoculated in the basic MRS medium, so that the initial cell concentration in the medium was 1.0×10 8 unit / mL is the seed solution.

[0070] (2) Fermentation conditions for sugar production

[0071] Inoculate Leuconostoc citreum N21 with an inoculation amount of 2.0% (V / V) in a 100 / 250mL medium with an initial pH value of 6-8, and culture it with constant temperature shaking at a temperature of 25-37°C 36-72h, use the phenol-sulfuric acid method to measure the polysaccharide content of the fermentation supernatant.

[0072] Under the conditions of an initial pH value of 6.5, a tem...

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