Preparation method of lactobacillus exopolysaccharide

Abstract

The invention provides a lactic acid bacteria exopolysaccharide and a preparation method thereof, and the preparation method comprises the following steps: firstly, optimizing culture medium components and culture conditions to respectively obtain separately-cultured lactic acid bacteria and yeast seed solutions, then mixing the lactic acid bacteria and yeast seed solutions for co-culture, and further optimizing the culture medium components and the culture conditions to obtain an exopolysaccharide culture solution. The yield of the exopolysaccharide obtained by the preparation method disclosed by the invention is greatly improved compared with that of the exopolysaccharide obtained by independently culturing lactobacillus, and the content can be up to 90g / L. The invention belongs to the technical field of microbial co-culture technology and production and application of probiotic secondary metabolites. Lactobacillus plantarum and Pseudomonas stutzeri are co-cultured, expression of quorum sensing related genes of lactobacillus plantarum is up-regulated, biomass is increased, expression of genes of dextran sucrase which is a key enzyme for synthesizing exopolysaccharides is up-regulated, activity of dextran sucrase is improved, and yield of exopolysaccharides is increased. The co-culture extracellular polysaccharide is homopolysaccharide with glucose as monosaccharide, and compared with single culture extracellular polysaccharide and commercial glucan, the co-culture extracellular polysaccharide has good oxidation resistance, emulsification, heavy metal adsorption and probiotic proliferation promoting capacity, and is expected to be widely applied to the fields of food, medicine, environmental protection and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial co-cultivation, and relates to a co-cultivation method of lactic acid bacteria and yeast and application of secondary metabolites of lactic acid bacteria. Background Art

[0002] Lactic acid bacteria exopolysaccharide is a secondary metabolite produced by the growth and metabolism of lactic acid bacteria. It can be loosely attached to the cell surface, called capsular polysaccharide; it can also be directly secreted into the extracellular environment, called mucus polysaccharide. Capsular polysaccharide and mucus polysaccharide are collectively referred to as exopolysaccharide, which is the product of microorganisms adapting to environmental changes. Since lactic acid bacteria are recognized as safe food-grade microorganisms, lactic acid bacteria exopolysaccharide is also considered to have good biosafety, with a wide variety of species, a wide range of sources, and various structural and functional properties. It is widely used in food, medicine, and chemical industries. Although lactic acid bacteria exopolysaccharide has good functional properties, the yield of lactic acid bacteria exopolysaccharide is lower than that of other polysaccharide polymers, such as pectin, chitosan, and xanthan gum, which is the main reason that hinders the application of lactic acid bacteria exopolysaccharide in industry. Lactic acid bacteria strains from different sources have significant differences in the yield and structural characteristics of exopolysaccharides under different growth and fermentation conditions. These differences affect the physicochemical properties, biological activity, and application of exopolysaccharides.

[0003] There are two common production technologies for microbial extracellular polysaccharides. The first is to use wild-type microbial single bacteria culture and separate and prepare from the fermentation broth. The advantage is that the strain is widely available and easy to separate, but the disadvantage is that the sugar production is not high. The second is to use genetic engineering technology to modify the original strain to increase the sugar production by targeting the key enzyme genes for extracellular polysaccharide synthesis. The advantage is that the yield is increased, but the disadvantage is that the increase is limited, the modification technology is cumbersome, the engineered strain is prone to degeneration, and the polysaccharide production is unstable.

[0004] The microbial co-culture system includes a group of natural microorganisms composed of different species or different strains of the same species, whose members can interact with each other in a symbiotic / antagonistic manner. This can induce the synthesis of natural products. Studies have shown that co-culture can promote quorum sensing of lactic acid bacteria, increase biomass, change the metabolic characteristics of lactic acid bacteria, and dynamically regulate the changes in substrates and metabolites during fermentation, thereby promoting the synthesis of extracellular polysaccharides by lactic acid bacteria. Changes in metabolites caused by co-culture may also be a factor affecting the production of extracellular polysaccharides.

[0005] Lactic acid bacteria exopolysaccharides have good biological functions such as antioxidant properties and heavy metal adsorption, but the production of lactic acid bacteria exopolysaccharides is low, and large-scale applications are limited. In the natural environment, lactic acid bacteria often coexist with yeast. The present invention establishes a co-cultivation system, is committed to increasing the production of lactic acid bacteria exopolysaccharides, helps to analyze the exopolysaccharide synthesis mechanism in the natural environment, and expands the application range of exopolysaccharide-producing lactic acid bacteria. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of low yield and limited use of existing lactic acid bacteria extracellular polysaccharides, and to provide a method for preparing lactic acid bacteria extracellular polysaccharides by co-culturing lactic acid bacteria and yeast. The yield of lactic acid bacteria extracellular polysaccharides prepared by this method is significantly improved compared with the yield of single culture, and the adsorption capacity for metal ions, the emulsification capacity of organic solvents and the ability to promote the proliferation of probiotics are significantly enhanced.

[0007] A method for co-culturing a lactic acid bacterium and a yeast, comprising seven steps: lactic acid bacteria monoculture, yeast monoculture, lactic acid bacteria and yeast co-culture, lactic acid bacteria quorum sensing related gene detection in co-culture, lactic acid bacteria glucansucrase gene detection in co-culture, metabolite detection in co-culture and co-culture extracellular polysaccharide preparation.

[0008] The components of the lactic acid bacteria monoculture medium are: glucose 15-25 g / L, peptone 7-13 g / L, beef extract 8-12 g / L, yeast extract 3-7 g / L, K2HPO4 1.5-2.5 g / L, anhydrous sodium acetate 3-7 g / L, ammonium citrate 1.5-2.5 g / L, magnesium sulfate 0.4-0.6 g / L, manganese sulfate 0.2-0.3 g / L, Tween-80 0.8-1.2 mL / L, pH 5-6. The culture conditions are: shake flask culture at 28-32°C, 120-160 rpm for 14-18 hours. Wash the cells three times with 0.85% sodium chloride and adjust the cell concentration to 10 8 pcs / mL is the seed solution.

[0009] The components of the yeast monoculture medium are: glucose 15-25 g / L, peptone 15-25 g / L, yeast extract powder 8-12 g / L. The pH remains natural and does not need to be adjusted. The culture conditions are: shake flask culture at 27-33°C and 130-170 rpm for 10-14 hours. Wash the cells three times with 0.85% sodium chloride and adjust the cell concentration to 10 8 pcs / mL is the seed solution.

[0010] The components of the sugar-producing medium for co-culture of lactic acid bacteria and yeast are: sucrose 60-80 g / L, peptone 5-7 g / L, beef extract 8-12 g / L, yeast extract powder 5-7 g / L, K2HPO4 2.5-3.5 g / L, anhydrous sodium acetate 3-7 g / L, ammonium citrate 1.5-2.5 g / L, magnesium sulfate 0.4-0.6 g / L, manganese sulfate 0.2-0.3 g / L, Tween-80 0.8-1.2 mL / L, pH 5-6. The lactic acid bacteria seed liquid is inoculated into the sugar-producing medium at a rate of 1.5-2.5%, and the culture is shaken at 28-32°C and 120-160 rpm for 3-5 hours. After that, 1.5-2.5% yeast seed liquid is inoculated and the culture is continued for 56-64 hours. The extracellular polysaccharide content in the culture medium is 80-100 g / L.

[0011] The relative expression levels of quorum sensing-related genes luxS, prcK, and prcR of lactic acid bacteria in co-culture reached the highest level at 26-34 hours, which were 14.5-16.5 times, 3.5-5.5 times, and 3.5-5.5 times higher than those of lactic acid bacteria monoculture, respectively.

[0012] The relative expression level of the lactic acid bacteria glucansucrase gene dsr in co-culture reached the highest level at 26-34 hours, which was 8.5-10.5 times higher than that in single culture of lactic acid bacteria.

[0013] The contents of sucrose, acetic acid and lactic acid in co-culture decreased, reaching 2.8-3.2 g / L, 0.24-0.28 g / L and 3.4-3.8 g / L at 34-38 hours, respectively, which were lower than 3.6-4.0 g / L, 0.28-0.32 g / L and 7.8-8.2 g / L in single culture. The content of fructose increased, reaching 5.6-6.0 g / L at 34-38 hours, which was higher than 4.8-5.2 g / L in single culture.

[0014] Co-culture of lactic acid bacteria and yeast. Centrifuge the co-culture solution at 6000-10000 rpm for 15-25 minutes, collect the supernatant and mix it with 95% ethanol at a ratio of 1:2.5-1:3.5 overnight, centrifuge at 6000-10000 rpm for 15-25 minutes, and dissolve the precipitate in an equal amount of deionized water. Add an equal volume of 10% trichloroacetic acid, stir for 4 hours, centrifuge at 6000-10000 rpm for 15-25 minutes, and retain the supernatant. Centrifuge at 6000-10000 rpm for 15-25 minutes, collect the supernatant and mix it with 95% ethanol at a ratio of 1:2.5-1:3.5 overnight, centrifuge at 6000-10000 rpm for 15-25 minutes, and dissolve the precipitate in an equal amount of deionized water. Dialyze overnight in a 8000-12000 Da dialysis bag, and change the water every 4 hours. The dialyzate was passed through a Sephadex G-100 gel column, and the peak liquid was collected, concentrated, and then freeze-dried to obtain the co-cultured extracellular polysaccharide.

[0015] As a preferred embodiment, the lactic acid bacteria monoculture medium comprises 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L K2HPO4, 5 g / L anhydrous sodium acetate, 2 g / L ammonium citrate, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 1 mL / L Tween-80, and pH 5.5. The culture conditions are: 140 rpm shaking flask culture at 30°C for 16 hours.

[0016] As a preferred solution, the yeast monoculture medium composition is glucose 20 g / L, peptone 20 g / L, yeast extract powder 10 g / L, pH is kept at a natural state without adjustment, and the culture conditions are: 30°C, 140 rpm shake flask culture.

[0017] As a preferred embodiment, the sugar-producing medium components in the co-culture of lactic acid bacteria and yeast are sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract powder 6 g / L, K2HPO4 3 g / L, anhydrous sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The lactic acid bacteria seed solution is inoculated with a 2% inoculation amount into the sugar-producing medium, and the flask is shaken at 30°C and 140 rpm for 4 hours. Thereafter, 2% yeast seed solution is inoculated and the culture is continued for 60 hours. The extracellular polysaccharide content in the culture solution is 90 g / L.

[0018] As a preferred solution, after 30 hours of co-culture, the relative expression levels of lactic acid bacteria quorum sensing-related genes luxS, prcK, and prcR increased by 15.5 times, 4.5 times, and 4.5 times, respectively, compared with single culture of lactic acid bacteria.

[0019] As a preferred solution, after 30 hours of co-culture, the relative expression level of the lactic acid bacteria glucansucrase gene dsr increased by 9.5 times compared with the lactic acid bacteria single culture.

[0020] As a preferred solution, after 36 hours of co-culture, the contents of sucrose, acetic acid and lactic acid in the culture medium were 3.0 g / L, 0.26 g / L and 3.6 g / L, respectively, which were lower than 3.8 g / L, 0.3 g / L and 8.0 g / L in single culture. The fructose content was 5.8 g / L, which was higher than 5.0 g / L in single culture.

[0021] As a preferred embodiment, in the method for preparing extracellular polysaccharides by co-culturing lactic acid bacteria and yeast, the culture solution is centrifuged at 8000 rpm for 20 minutes, and the supernatant is collected and mixed with 95% ethanol in a ratio of 1:3 overnight.

[0022] The lactic acid bacteria in the present invention is Lactobacillus plantarum, which is disclosed in "Study on Isolation, Identification and Culture Conditions of Bacteriocin-producing Lactic Acid Bacteria" and is stored in the Key Laboratory of Microbiology of Provincial Universities of Heilongjiang University. The yeast is Candida shehatae, which is disclosed in "Construction of Engineering Strain of Saccharomyces cerevisiae for Co-fermentation of Xylose and Glucose with High Ethanol Production" and is stored in the Key Laboratory of Microbiology of Provincial Universities of Heilongjiang University.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1) The present invention discloses a culture medium, culture conditions, culture, co-cultured exopolysaccharide and uses thereof for co-culture of a strain of exopolysaccharide-producing lactic acid bacteria and yeast. The co-culture system provided by the present invention is simple to construct, the culture medium components are simple, the exopolysaccharide preparation steps are few, and the cost is low. The co-cultured exopolysaccharide obtained by the present invention is glucan, which has significantly improved antioxidant activity, emulsification, heavy metal adsorption capacity and the effect of promoting the proliferation of probiotics compared with the exopolysaccharide obtained by lactic acid bacteria monoculture and commercial glucan.

[0025] 2) There are two points to be explained about the co-cultivation method of the present invention. First, the increase in the production of extracellular polysaccharides by lactic acid bacteria requires direct contact between lactic acid bacteria and yeast cells, and yeast extracellular metabolites and intracellular metabolites cannot stimulate lactic acid bacteria to increase the production of extracellular polysaccharides. Second, yeast was added after the lactic acid bacteria were cultured alone for 4 hours, and the two were not co-cultured from the beginning of fermentation.

[0026] 3) The co-culture medium of the present invention is based on the lactic acid bacteria monoculture medium and culture conditions, and is optimized by single factor test and response surface test in turn to obtain the optimal co-culture medium components and culture conditions. After statistical test and verification test, under this component and condition, the co-culture extracellular polysaccharide yield reaches 90 g / L, which is significantly higher than that of lactic acid bacteria monoculture.

[0027] 4) Under the co-culture conditions of the present invention, the expression of the quorum sensing-related genes and the glucan sucrase gene of lactic acid bacteria is upregulated. The synthesis of extracellular polysaccharides of lactic acid bacteria is regulated by quorum sensing and is related to cell density. The up-regulated expression of quorum sensing genes indicates that the biomass of lactic acid bacteria increases, accompanied by an increase in polysaccharide production. Glucan sucrase is a key enzyme in the synthesis of extracellular polysaccharides. Sucrose in the substrate is a disaccharide composed of two monosaccharides, glucose and fructose. Glucan sucrase hydrolyzes sucrose into glucose and fructose, and glucose is used to synthesize the extracellular polysaccharides in the present invention. During co-culture, the expression of the glucan sucrase gene is upregulated, the activity of glucan sucrase is increased, and the fructose content increases accordingly. At the same time, the content of sucrose, lactic acid, and acetic acid decreases, and the fructose content increases. The above results are significantly different from the monoculture of lactic acid bacteria. It shows that the addition of yeast activates the quorum sensing regulation mechanism of lactic acid bacteria, the increase in lactic acid bacteria cell density is accompanied by an increase in extracellular polysaccharide production, and the change in metabolites also stimulates the production of lactic acid bacteria extracellular polysaccharides.

[0028] 5) Compared with the lactic acid bacteria monoculture exopolysaccharide and commercial glucan, the co-culture exopolysaccharide obtained by the present invention has better antioxidant activity, emulsification, heavy metal ion adsorption capacity and ability to promote the proliferation of probiotics. The present invention can provide guidance for the construction of lactic acid bacteria and yeast co-culture system and product application research, and also provide materials for the application of exopolysaccharides in the fields of food, medicine, environmental protection, etc. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with data results and embodiments, and the protection scope of the present invention is not limited to the following. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0030] Example 1 Effects of culture medium components on exopolysaccharide production by co-cultured lactic acid bacteria

[0031] The components and culture conditions of lactic acid bacteria monoculture medium were as follows: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The culture was carried out at 30°C and 140 rpm for 60 hours. The optimized co-culture medium was as follows: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: The lactic acid bacteria and yeast seed liquid were inoculated into the sugar-producing medium at a 2% inoculation rate, and the shake flask was cultured at 30°C and 140rpm for 60 hours. The biomass of lactic acid bacteria and the content of extracellular polysaccharides in the co-culture solution were determined, and the results are shown in Table 1. The co-culture using the optimized culture medium significantly increased the biomass of lactic acid bacteria, and the production of extracellular polysaccharides increased from 45 g / L to 60g / L. This example shows that compared with the monoculture of lactic acid bacteria, the biomass and extracellular polysaccharide production of lactic acid bacteria under co-culture conditions increased significantly. That is, the addition of yeast significantly promotes the growth of lactic acid bacteria and the synthesis of extracellular polysaccharides.

[0032] Table 1 Example 1 Results

[0033]

[0034] Example 2 Effect of yeast contact mode on exopolysaccharide production by co-cultured lactic acid bacteria

[0035] Lactic acid bacteria monoculture medium components and culture conditions: glucose 18 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, anhydrous sodium acetate 4.5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Shake flask culture at 30°C, 140 rpm for 60 hours. Co-culture medium: sucrose 72 g / L, peptone 5.8 g / L, beef extract 10.2 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, anhydrous sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: lactic acid bacteria and yeast seed liquid were inoculated with 2% inoculation amount into sugar-producing medium, and cultured at 30 ° C and 130 rpm for 58 hours. There are three co-culture conditions, one is to inoculate 2% yeast cells, the second is to inoculate 2% yeast cell contents for 36 hours, and the third is to inoculate 2% yeast monoculture cell-free culture fluid for 36 hours. The biomass and extracellular polysaccharide content of lactic acid bacteria were determined, and the results are shown in Table 2. Yeast cell contents and cell-free culture fluid had no significant effect on lactic acid bacteria growth and sugar production. When yeast cells are in direct contact with lactic acid bacteria cells, the biomass and extracellular polysaccharide production of lactic acid bacteria are significantly increased. This example shows that the co-culture method significantly affects the growth and sugar production of lactic acid bacteria. Neither yeast cell contents nor yeast cell-free culture fluid can stimulate lactic acid bacteria growth and sugar production. Only when yeast is in direct contact with lactic acid bacteria, the biomass and extracellular polysaccharide production of co-cultured lactic acid bacteria are significantly increased compared with lactic acid bacteria monoculture.

[0036] Table 2 Example 2 Results

[0037]

[0038] Example 3 Effect of yeast inoculation time on exopolysaccharide production by co-cultured lactic acid bacteria

[0039] Lactic acid bacteria monoculture medium components and culture conditions: glucose 22 g / L, peptone 12 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, anhydrous sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Shake flask culture at 30°C, 1450 rpm for 60 hours. Co-culture medium: sucrose 72 g / L, peptone 7 g / L, beef extract 11 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, anhydrous sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: The lactic acid bacteria and yeast seed liquid were inoculated into the sugar-producing medium at a 2% inoculation rate, and the flask was cultured at 30°C and 140 rpm for 60 hours. The yeast inoculation method was 2% yeast cells. The yeast inoculation time was simultaneous inoculation with lactic acid bacteria, delayed inoculation for 4 hours, and delayed inoculation for 8 hours. The biomass and extracellular polysaccharide content of lactic acid bacteria were determined, and the results are shown in Table 3. The results of this embodiment show that compared with the single culture of lactic acid bacteria and the simultaneous inoculation of yeast, the delayed inoculation of yeast significantly increased the growth and sugar production of lactic acid bacteria than the simultaneous inoculation. Delayed inoculation for 4 hours is better than 8 hours.

[0040] Table 3 Example 3 Results

[0041]

[0042] Example 4 Effect of yeast on the expression of genes related to quorum sensing of lactic acid bacteria

[0043] The components and culture conditions of lactic acid bacteria monoculture medium were as follows: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The culture was carried out at 30°C and 140 rpm for 60 hours. The optimized co-culture medium was as follows: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: The lactic acid bacteria and yeast seed liquid were inoculated with 2% inoculation amount into the sugar-producing medium, and the flask was cultured at 30°C and 140rpm for 60 hours. The yeast inoculation method was 2% yeast cells. The yeast inoculation time was delayed for 4 hours. After 30 hours of fermentation, the expression of lactic acid bacteria quorum sensing related genes luxS, prcK, and prcR was measured, and the results are shown in Table 4. The results of this embodiment show that compared with the single culture of lactic acid bacteria, the access of yeast initiated the quorum sensing response of lactic acid bacteria, and the expression of related genes luxS, prcK, and prcR was significantly upregulated. This result of the group is mutually corroborated by the result that the biomass of co-cultured lactic acid bacteria is higher than that of single cultured lactic acid bacteria.

[0044] Table 4 Example 4 Results

[0045]

[0046] Example 5 Effect of yeast on the expression and enzyme activity of the key gene for exopolysaccharide synthesis in lactic acid bacteria, glucansucrase gene dsr

[0047] The components and culture conditions of lactic acid bacteria monoculture medium were as follows: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The culture was carried out at 30°C and 140 rpm for 60 hours. The optimized co-culture medium was as follows: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: The lactic acid bacteria and yeast seed liquid were inoculated into the sugar-producing medium at a 2% inoculation rate, and the flask was cultured at 30°C and 140rpm for 60 hours. The yeast inoculation method was 2.5% yeast cells. The yeast inoculation time was delayed for 5 hours. After 30 hours of fermentation, the expression and enzyme activity of the key gene for exopolysaccharide synthesis of lactic acid bacteria, glucansucrase gene dsr, were determined, and the results are shown in Table 5. The results of this example show that, with lactic acid bacteria monoculture as the control, the inoculation of yeast significantly increased the relative expression of the glucansucrase gene dsr and the activity of glucansucrase.

[0048] Table 5 Example 5 Results

[0049]

[0050] Example 6 Effect of yeast on co-culture metabolites

[0051] Lactic acid bacteria monoculture medium components and culture conditions: glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, anhydrous sodium acetate 4.5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Shake flask culture at 30°C, 140 rpm for 62 hours. Co-culture medium: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, anhydrous sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: The lactic acid bacteria and yeast seed liquid were inoculated into the sugar-producing medium at a 2% inoculation rate, and the flask was shaken at 30°C and 140 rpm for 60 hours. The yeast inoculation method was 2% yeast cells. The yeast inoculation time was delayed for 4 hours. After 36 hours of fermentation, the contents of sucrose, acetic acid, lactic acid and fructose in the fermentation broth were determined, and the results are shown in Table 6. The results of this embodiment show that, with lactic acid bacteria monoculture as the control, the sucrose, acetic acid and lactic acid contents of the fermentation broth B were significantly reduced, and the fructose content was significantly increased during co-culture. Under the co-culture conditions, sucrose was more hydrolyzed into glucose and fructose, so the sucrose was lower and the fructose was higher. The addition of yeast consumed the acetic acid and lactic acid produced by lactic acid bacteria, so the lactic acid and acetic acid were reduced.

[0052] Table 6 Example 6 Results

[0053]

[0054] Example 7 Effect of preparation conditions on the efficiency of co-culture exopolysaccharide preparation

[0055] Lactic acid bacteria monoculture medium components and culture conditions: glucose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, K2HPO42 g / L, anhydrous sodium acetate 5g / L, ammonium citrate 2g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.25g / L, Tween-80 1mL / L, pH 5.5. Shake flask culture at 30℃, 140rpm for 60 hours. Co-culture medium: sucrose 70g / L, peptone 6g / L, beef extract 10g / L, yeast extract 6g / L, K2HPO43 g / L, anhydrous sodium acetate 5g / L, ammonium citrate 2g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.25g / L, Tween-80 1mL / L, pH 5.5. Co-culture method and conditions: lactic acid bacteria and yeast seed liquid were inoculated into sugar-producing medium at a 2% inoculation rate, and cultured at 30°C and 140rpm for 60 hours. The yeast inoculation method was 2% yeast cells. The yeast inoculation time was delayed for 4 hours. Exopolysaccharides were prepared from the fermentation broth after 60 hours. Preparation method 1) The culture solution was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected and mixed with 95% ethanol at a ratio of 1:3 overnight. Preparation method 2) The culture solution was centrifuged at 6000 rpm for 15 minutes, and the supernatant was collected and mixed with 95% ethanol at a ratio of 1:2 overnight. Preparation method 3) The culture solution was centrifuged at 10000 rpm for 25 minutes, and the supernatant was collected and mixed with 95% ethanol at a ratio of 1:4 overnight. The weight of the lyophilized powder of exopolysaccharide obtained from 1 liter of co-culture solution with an exopolysaccharide concentration of 92 g / L was determined and the preparation efficiency was calculated. The results are shown in Table 7. The results show that the preparation condition parameters proposed in the present invention, namely preparation method 1), are more conducive to the purification of co-cultured extracellular polysaccharides and have significantly higher preparation efficiency.

[0056] Table 7 Example 7 Results

[0057]

[0058] Example 8 Antioxidant activity of co-cultured exopolysaccharides

[0059] The components and culture conditions of the lactic acid bacteria monoculture medium were as follows: glucose 18 g / L, peptone 9 g / L, beef extract 9 g / L, yeast extract 4.5 g / L, K2HPO4 2 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The culture was carried out at 30°C and 140 rpm in a shaking flask for 60 hours. The optimized co-culture medium was as follows: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: Inoculate the lactic acid bacteria and yeast seed liquid into the sugar-producing medium at a 2% inoculation rate, and culture at 30°C and 140rpm for 60 hours. The yeast inoculation method is 2% yeast cells. The yeast inoculation time is delayed for 4 hours. The preparation conditions of extracellular polysaccharides are as follows: centrifuge the culture solution at 8000 rpm for 20 minutes, collect the supernatant and mix it with 95% ethanol at a ratio of 1:3 overnight. Freeze-dry the precipitated polysaccharide to obtain a powder. Adjust the concentration of extracellular polysaccharides to 5 mg / mL, and measure the effects of extracellular polysaccharides on DPPH, ABTS, hydroxyl, and O2 - The results of free radical scavenging ability are shown in Table 8. Compared with single culture exopolysaccharide and commercial dextran, the scavenging rate of co-culture exopolysaccharide on four free radicals was significantly higher, all reaching more than 60%.

[0060] Table 8 Example 8 Results

[0061]

[0062] Example 9 Anti-emulsification of co-cultured exopolysaccharides

[0063] The components and culture conditions of lactic acid bacteria monoculture medium were as follows: glucose 20 g / L, peptone 11 g / L, beef extract 8 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The culture was carried out at 29°C and 140 rpm in a shaking flask for 60 hours. The optimized co-culture medium was as follows: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: lactic acid bacteria and yeast seed liquid were inoculated into sugar-producing medium at a 2% inoculation rate, and cultured at 30°C and 135 rpm for 60 hours. The yeast inoculation method was 2% yeast cells. The yeast inoculation time was delayed for 4 hours. The parameters for the preparation of extracellular polysaccharides were centrifugation of the culture solution at 8000 rpm for 20 minutes, and the supernatant was collected and mixed with 95% ethanol at a ratio of 1:3 overnight. The precipitated polysaccharide was freeze-dried to obtain powder. The concentration of extracellular polysaccharides was adjusted to 2 mg / mL, and 2.5 mL of hydrocarbons or oils (ether, gasoline, diesel, benzene, petroleum, soybean oil, xylene) and 5 mL of 2 mg / mL extracellular polysaccharide solution were shaken and mixed evenly, and the total height of the mixed solution was measured, recorded as H0. The mixed solution was allowed to react at room temperature for 48 hours. The height of the emulsion layer between the aqueous phase and the organic phase was measured at 1, 12, 24 and 48 hours, recorded as H1. The emulsification rate was calculated by the following formula: emulsification rate (%) = H1 / H0 × 100%. The results are shown in Table 9. Compared with single culture exopolysaccharide and commercial glucan, co-culture exopolysaccharide had stronger emulsification ability for the seven substrates tested, all reaching more than 60%.

[0064] Table 9 Example 9 Results

[0065]

[0066] Example 10 Heavy metal adsorption capacity of co-cultured extracellular polysaccharides

[0067] The components and culture conditions of lactic acid bacteria monoculture medium were as follows: glucose 20 g / L, peptone 11 g / L, beef extract 8 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The culture was carried out at 29°C and 140 rpm in a shaking flask for 60 hours. The optimized co-culture medium was as follows: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: lactic acid bacteria and yeast seed liquid were inoculated into sugar-producing medium at a 2% inoculation rate, and cultured in a shaking flask at 30°C and 135 rpm for 60 hours. The yeast inoculation method was 2% yeast cells. The yeast inoculation time was delayed for 4 hours. The exopolysaccharide preparation condition parameters were that the culture solution was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected and mixed with 95% ethanol at a ratio of 1:3 overnight. The precipitated polysaccharide was freeze-dried to obtain powder. FeSO4·7H2O, ZnSO4·7H2O and CuSO4·5H2O were weighed and dissolved in an appropriate amount of deionized water to a final concentration of 1 mg / mL. The exopolysaccharide solution was mixed with the metal salt solution at a ratio of 1:1 (v / v) to make the final concentration of the exopolysaccharide 0.5 mg / mL, and reacted at room temperature for 0.5 hours. The reaction solution was mixed with pre-cooled acetone at a ratio of 1:3, centrifuged at 5000rpm for 30 minutes, and the supernatant was retained. The mixed acid solution was added to the supernatant (8 mL) in three portions, and the mixed solution was heated to become colorless and transparent. After cooling, the mixed solution was fixed to 10 mL with double distilled water. The adsorption capacity (q) of EPS for metals was calculated according to the following formula: q (mg / g) = V (C i -C f ) / W. Where V (L) is the volume of the mixture of extracellular polysaccharide and metal salt solution, C f (mg / L) is the final concentration of metal ions, C i (mg / L) is the initial concentration of metal ions, and W (g) is the mass of exopolysaccharide. The results are shown in Table 10. Compared with the single culture exopolysaccharide and commercial glucan controls, the co-culture exopolysaccharide has a stronger adsorption capacity for heavy metals.

[0068] Table 10 Example 10 Results

[0069]

[0070] Example 11 Ability of co-cultured exopolysaccharides to promote probiotic proliferation

[0071] The components and culture conditions of lactic acid bacteria monoculture medium were as follows: glucose 20 g / L, peptone 11 g / L, beef extract 8 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. The culture was carried out at 29°C and 140 rpm in a shaking flask for 60 hours. The optimized co-culture medium was as follows: sucrose 70 g / L, peptone 6 g / L, beef extract 10 g / L, yeast extract 6 g / L, K2HPO4 3 g / L, sodium acetate anhydrous 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. Co-culture method and conditions: lactic acid bacteria and yeast seed liquid were inoculated into sugar-producing medium at a 2% inoculation rate, and cultured in a shaking flask at 30°C and 135 rpm for 60 hours. The yeast inoculation method was 2% yeast cells. The yeast inoculation time was delayed for 4 hours. The parameters for the preparation of extracellular polysaccharides were centrifugation of the culture solution at 8000 rpm for 20 minutes, and the supernatant was collected and mixed with 95% ethanol at a ratio of 1:3 overnight. The precipitated polysaccharide was freeze-dried to obtain powder. The probiotic culture medium was prepared, and the components were: glucose 5 g / L, peptone 11 g / L, beef extract 8 g / L, yeast extract powder 5 g / L, K2HPO4 2 g / L, anhydrous sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.5. On this basis, 1% of lactic acid bacteria monoculture exopolysaccharide and co-culture exopolysaccharide were added, and lactic acid bacteria monoculture exopolysaccharide and commercial glucan were used as controls. Six probiotics, including Lactobacillus plantarum, Lactobacillus zeae, Lactobacillus rhamnosus, Bifidobacterium bifidum, Lactobacillus casei and Lactobacillus paracasei, were inoculated into the above culture medium at a 2% (v / v) inoculation rate, and cultured at 30°C and 140 rpm for 48 h, and the number of bacteria was determined. The results are shown in Table 11. In the basic probiotic culture medium, the cell number was always below 10 9 , and they were all lower than co-cultured exopolysaccharides, single-cultured exopolysaccharides and commercial glucans. Co-cultured exopolysaccharides promoted the proliferation of all six probiotics, and the promoting effects were better than those of single-cultured exopolysaccharides. Compared with commercial glucans, co-cultured exopolysaccharides had a stronger promoting effect on the proliferation of Lactobacillus casei. Compared with commercial glucans, co-cultured exopolysaccharides had a stronger promoting effect on the proliferation of Lactobacillus plantarum, Lactobacillus zeae, Lactobacillus rhamnosus, Bifidobacterium bifidum and Lactobacillus casei.

[0072] Table 11 Example 11 Results

[0073]

Claims

1. A lactic acid bacteria exopolysaccharide, characterized in that: The lactic acid bacteria extracellular polysaccharide is prepared by co-culturing lactic acid bacteria and yeast.

2. The method for preparing lactic acid bacteria exopolysaccharide according to claim 1, characterized in that: The steps of co-culturing lactic acid bacteria and yeast are as follows: (1) Lactobacillus monoculture: the culture medium components are: glucose 15-25 g / L, peptone 7-13 g / L, beef extract 8-12 g / L, yeast extract powder 3-7 g / L, K2HPO4 1.5-2.5 g / L, anhydrous sodium acetate 3-7 g / L, ammonium citrate 1.5-2.5 g / L, magnesium sulfate 0.4-0.6 g / L, manganese sulfate 0.2-0.3 g / L, Tween-80 0.8-1.2 mL / L, pH 5-6, culture conditions are: shake flask culture at 28-32°C, 120-160 rpm for 14-18 hours, wash cells three times with 0.85% sodium chloride, adjust the cell concentration to 10 8 / mL, which is the seed solution; (2) Yeast monoculture: The culture medium components are: glucose 15-25 g / L, peptone 15-25 g / L, yeast extract powder 8-12 g / L. The pH remains natural and does not need to be adjusted. The culture conditions are: culture at 27-33°C, 130-170 rpm, shake flask culture for 10-14 hours, wash cells three times with 0.85% sodium chloride, and adjust the cell concentration to 10 8 / mL, which is the seed solution; (3) Co-culture of lactic acid bacteria and yeast: The components of the sugar-producing culture medium are: sucrose 60-80 g / L, peptone 5-7 g / L, beef extract 8-12 g / L, yeast extract powder 5-7 g / L, K2HPO4 2.5-3.5 g / L, anhydrous sodium acetate 3-7 g / L, ammonium citrate 1.5-2.5 g / L, magnesium sulfate 0.4-0.6 g / L, manganese sulfate 0.2-0.3 g / L, Tween-80 0.8-1.2 mL / L, pH 5-6, the lactic acid bacteria seed solution is inoculated with 1.5-2.5% of the sugar-producing culture medium, and cultured in a shaking flask at 28-32°C and 120-160 rpm for 3-5 hours, and then inoculated with 1.5-2.5% of the yeast seed solution, and cultured for 56-64 hours until the lactic acid bacteria biomass reaches 8.5-9.5 × 10 8 / mL, and the extracellular polysaccharide content in the culture medium is 80-100 g / L.

3. The method for preparing lactic acid bacteria exopolysaccharide according to claim 2, characterized in that: The co-culture solution of step (3) was centrifuged at 6000-10000 rpm for 15-25 minutes, the supernatant was collected and mixed with 95% ethanol at a ratio of 1:2.5-1:3.5 overnight, and then centrifuged at 6000-10000 rpm for 15-25 minutes. The precipitate was dissolved in an equal amount of deionized water, an equal volume of 10% trichloroacetic acid was added, and the mixture was stirred for 4 hours and then centrifuged at 6000-10000 rpm for 15-25 minutes. The supernatant was retained and centrifuged at 6000-10000 rpm for 15-25 minutes. The supernatant was collected and mixed with 95% ethanol at a ratio of 1:2.5-1:3.5 overnight, and then centrifuged at 6000-10000 rpm for 15-25 minutes. The precipitate was dissolved in an equal amount of deionized water, and dialyzed in a 8000-12000Da dialysis bag overnight, with the water changed every 4 hours. The dialyzate was filtered through Sephadex G-100 gel column, collect the peak liquid, concentrate it and then freeze-dry it to obtain the co-culture extracellular polysaccharide freeze-dried powder.

4. The method for preparing the lactic acid bacteria exopolysaccharide according to claim 2, characterized in that The culture medium components in step (1) are glucose 20 g / L, peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, K2HPO4 2 g / L, anhydrous sodium acetate 5 g / L, ammonium citrate 2 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween-80 1 mL / L, pH 5.

5. The culture conditions are: 30°C, 140 rpm shaking flask culture for 16 hours.

5. The method for preparing lactic acid bacteria extracellular polysaccharide according to claim 2, characterized in that The culture medium components in step (2) are 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract powder. The pH is kept at a natural state without adjustment. The culture conditions are: shaking flask culture at 30°C and 140 rpm for 12 hours.

6. The method for preparing lactic acid bacteria extracellular polysaccharide according to claim 2, characterized in that The sugar-producing medium in step (3) comprises 70 g / L sucrose, 6 g / L peptone, 10 g / L beef extract, 6 g / L yeast extract, 3 g / L K2HPO4, 5 g / L anhydrous sodium acetate, 2 g / L ammonium citrate, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 1 mL / L Tween-80, and a pH of 5.

5. The lactic acid bacteria seed solution is inoculated with a 2% inoculation amount into the sugar-producing medium, and the culture is carried out at 30°C and 140 rpm in a shaking flask for 4 hours. Thereafter, 2% yeast seed solution is inoculated and the culture is continued for 60 hours.

7. The method for preparing the lactic acid bacteria exopolysaccharide according to claim 2, characterized in that Step (3) The biomass of lactic acid bacteria in the culture medium was 9.0×10 8 / mL, and the extracellular polysaccharide content was 90 g / L.

8. The method for preparing the lactic acid bacteria exopolysaccharide according to claim 2, characterized in that: After 36 hours of co-cultivation, the contents of sucrose, acetic acid and lactic acid in the culture medium were 3.0 g / L, 0.26 g / L and 3.6 g / L, respectively, and the fructose content was 5.8 g / L.

9. The method for preparing the lactic acid bacteria exopolysaccharide according to claim 3, characterized in that: In the method for preparing the co-cultured extracellular polysaccharide in step (3), the culture solution is centrifuged at 8000 rpm for 20 minutes, and the supernatant is collected and mixed with 95% ethanol at a ratio of 1:3 overnight.

10. The extracellular polysaccharide prepared by the method according to any one of the above claims, and its use in organic solvent emulsification, heavy metal adsorption, and probiotic proliferation.

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