CN111206005A - Culture medium for culturing lactobacillus helveticus strain with function of relieving lactose intolerance and culture method thereof - Google Patents
Culture medium for culturing lactobacillus helveticus strain with function of relieving lactose intolerance and culture method thereof Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 76
- 201000010538 Lactose Intolerance Diseases 0.000 title claims abstract description 46
- 240000002605 Lactobacillus helveticus Species 0.000 title claims abstract description 43
- 235000013967 Lactobacillus helveticus Nutrition 0.000 title claims abstract description 43
- 229940054346 lactobacillus helveticus Drugs 0.000 title claims abstract description 43
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- 238000012136 culture method Methods 0.000 title claims description 11
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a culture medium for culturing a lactobacillus helveticus strain with a function of relieving lactose intolerance. The basic culture medium formula is as follows: 15g/L of yeast peptone, 20g/L of yeast extract, 20g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of potassium dihydrogen phosphate, 5g/L of sodium acetate and the balance of purified water, wherein the pH value is 6.60. The optimized culture medium formula comprises: 25g/L of yeast peptone, 35g/L of yeast extract, 35g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 0.5g/L of calcium chloride, 5g/L of sodium acetate, 2g/L of monopotassium phosphate and the balance of purified water, wherein the pH value is 6.60. The Lactobacillus helveticus strain with the function of relieving lactose intolerance, which is prepared by the invention, has the advantages of high viable count, high enzyme activity and the like, wherein the viable count is as high as 109cfu/mL, and the enzyme activity reaches 0.584U/mL.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a culture medium for culturing a lactobacillus helveticus strain with a function of relieving lactose intolerance and a culture method thereof.
Background
Lactose is a disaccharide specific to female mammals and only exists in milk, lactose cannot be directly absorbed in human bodies and needs to be decomposed into glucose and galactose under the action of lactase, the lactase is also called β -galactosidase, a digestive enzyme located in human small intestine villus, the lactase can catalyze and decompose lactose and has the transferring effect of galactoside, the activity and secretion of lactase in the human bodies are influenced by primary and multiple secondary factors, and people lacking the lactase can not digest lactose in dairy products after taking the lactose, so that dyspepsia symptoms such as flatulence, borborborborygmus, diarrhea and the like occur, and the lactose intolerance is called.
Lactose intolerant people are ubiquitous worldwide, but the prevalence of lactose intolerance varies widely among people of different ethnic groups in different regions. The northern european population is the lowest lactose intolerant population (e.g., less than 10% in sweden and denmark), the southern and middle east european population is relatively high (e.g., about 50% in nomadic nationalities in spain, arabian in france), and the highest in non-nomadic nationalities in asia and africa. Epidemiological survey data show that the prevalence rate of lactose intolerance in China is as high as more than 86%. Lactose intolerance not only causes a decrease in milk and dairy product intake, but also affects calcium intake levels, decreased bone density, metabolic syndrome, and the like, and affects people's health to some extent.
At present, the treatment methods for lactose intolerance mainly comprise dietary lactose elimination therapy, bifidobacterium preparation and lactase preparation therapy. The dietary lactose elimination therapy is to reduce the intake of lactose, and lactose intolerance is avoided by eating lactose-free milk powder or milk, yoghourt and other alternative diets; the bifidobacterium preparation can show the activity of lactase by utilizing bifidobacterium, has the function of hydrolyzing lactose, can repair epithelial cells of small intestinal mucosa, promote the secretion of lactase, relieve clinical symptoms such as abdominal distension, borborborygmus and the like, and is a bifidobacterium triple viable tablet; the lactase preparation therapy can effectively improve lactose malabsorption without changing lactose-containing diet, and ensures normal dietary nutrition intake, and the therapy is widely accepted, but the domestic lactase raw material production technology is not mature, the existing production capacity cannot meet the domestic market demand, most of the lactase preparation therapy needs to be imported, and the cost is high.
Lactobacillus helveticus is a relatively common lactic acid bacterium, and has the probiotic characteristics of preventing gastrointestinal tract infection, resisting hypertension, regulating immunity and the like, and has no function of relieving lactose intolerance. However, the research finds that the lactobacillus helveticus has the proliferation effect on intestinal lactobacillus and bifidobacterium. The invention aims to provide a culture medium for culturing the lactobacillus helveticus with the function of relieving lactose intolerance, to culture the functional strain of the lactobacillus helveticus with the function of relieving lactose intolerance, to assist and strengthen the function of lactose intolerance of bifidobacterium when the functional strain is used together with the bifidobacterium, to provide a basis for obtaining the lactobacillus helveticus preparation which can effectively improve lactose malabsorption and does not need to eat alternative diet, and to provide more choices for the treatment of lactose intolerance.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a culture medium for culturing a lactobacillus helveticus strain with the function of relieving lactose intolerance and a culture method thereof.
The technical scheme adopted by the invention is as follows: a culture medium for culturing a strain with the function of relieving lactose intolerance of Lactobacillus helveticus comprises a basic culture medium and an optimized culture medium,
the basic culture medium comprises the following components in percentage by weight: 10-20 g/L of yeast peptone, 15-25 g/L of yeast extract, 15-25 g/L of lactose, 3-5 g/L of citric acid, 2-4 g/L of L-malic acid, 0.4-0.6 g/L of magnesium sulfate heptahydrate, 1-3 g/L of monopotassium phosphate, 4-6 g/L of sodium acetate and the balance of purified water, and adjusting the pH value to 6.60;
the formula of the optimized culture medium is as follows: 20-30 g/L of yeast peptone, 30-40 g/L of yeast extract, 30-40 g/L of lactose, 3-5 g/L of citric acid, 2-4 g/L of L-malic acid, 0.4-0.6 g/L of magnesium sulfate heptahydrate, 0.4-0.6 g/L of calcium chloride, 4-6 g/L of sodium acetate, 1-3 g/L of potassium dihydrogen phosphate and the balance of purified water, and the pH value is adjusted to 6.60.
Preferably, the culture medium for culturing the lactobacillus helveticus strain with the function of relieving lactose intolerance comprises a basal culture medium and an optimized culture medium,
the basic culture medium comprises the following components in percentage by weight: 15g/L of yeast peptone, 20g/L of yeast extract, 20g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of potassium dihydrogen phosphate, 5g/L of sodium acetate and the balance of purified water, and adjusting the pH value to 6.60;
the formula of the optimized culture medium is as follows: 25g/L of yeast peptone, 35g/L of yeast extract, 35g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 0.5g/L of calcium chloride, 5g/L of sodium acetate, 2g/L of monopotassium phosphate and the balance of purified water, and the pH value is adjusted to 6.60.
The preparation method of the culture medium for culturing the lactobacillus helveticus strain with the function of relieving lactose intolerance comprises the following steps: weighing according to the formula proportion, heating for dissolving, adjusting the pH value of the culture medium to 6.60 by using 1mol/L food-grade NaOH solution, and sterilizing for 30min at 115 ℃;
the preparation method of the optimized culture medium comprises the following steps: weighing the components according to the formula proportion, heating to dissolve, adjusting the pH value of the culture medium to 6.60 by using 1mol/L food-grade NaOH solution, and sterilizing for 30min at 115 ℃.
The culture method of the culture medium for culturing the lactobacillus helveticus strain with the function of relieving lactose intolerance comprises the following steps:
1) recovering the frozen strains: taking a Lactobacillus helveticus strain cryopreservation tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the cryopreservation tube are melted;
2) first-stage culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the opening of the test tube, performing static culture for 16-20 h in a 37 ℃ incubator at constant temperature, and storing in a 4 ℃ refrigerator for later use;
3) secondary culture: inoculating the bacterial suspension after the first-stage culture into a triangular flask filled with a basic culture medium, sealing, and carrying out static culture for 16-20 h at the constant temperature of an incubator at 37 ℃;
4) and (3) third-stage culture: and (3) inoculating the bacterial suspension after the second-stage culture to a triangular flask filled with an optimized culture medium, and performing static culture for 16-20 h at the constant temperature of an incubator at 37 ℃.
5) And (3) yield determination: the centrifugal equipment adopts a table centrifuge, the rotating speed is 12000rpm, 10min, supernatant is discarded after centrifugation, and the net weight of bacterial sludge is weighed according to the formula:
calculating the yield of the fermentation liquor;
6) and (4) measuring the number of the three-stage viable bacteria.
Preferably, in the above culture method, the amount of the inoculated medium in the steps 3) and 4) is 5%.
Preferably, in the culture method, steps 3) and 4), the liquid contents of the basic culture medium and the optimized culture medium in the triangular flask are 80%.
Compared with the prior art, the invention has the following beneficial effects:
1. the lactobacillus culture medium can culture lactobacillus helveticus functional strains with the function of relieving lactose intolerance.
2. Aiming at the strain for culturing the lactobacillus helveticus strain for relieving the lactose intolerance function, the invention respectively adopts a basic culture medium and an optimized culture medium which are more suitable for different growth stages of thalli, takes the viable count, the yield and the enzyme activity as investigation indexes, strengthens the lactose intolerance function of the lactobacillus helveticus and promotes the propagation culture of the lactobacillus helveticus.
3. The culture medium and the neutralizer adopted in the strain purification and fermentation processes are food-grade raw materials, so that the strain has high safety and can be applied to food fermentation or freeze-dried bacterial powder and the like.
4. The Lactobacillus helveticus strain with the function of relieving lactose intolerance, which is prepared by the technology of the invention, has the advantages of high viable count, high enzyme activity and the likeThe number of bacteria is as high as 109cfu/mL, and the enzyme activity reaches 0.584U/mL.
Drawings
FIG. 1 is a concentration standard curve of the amount of an o-nitrophenol (ONP) substance.
Detailed Description
Example 1 culture Medium and culture method of Lactobacillus helveticus strain for alleviating lactose intolerance
1. The culture medium of the lactobacillus helveticus strain with the function of relieving lactose intolerance comprises a basic culture medium and an optimized culture medium:
the basic culture medium formula and preparation: 15g/L of yeast peptone, 20g/L of yeast extract, 20g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of potassium dihydrogen phosphate, 5g/L of sodium acetate and the balance of purified water; weighing according to the formula proportion, heating to dissolve, adjusting the pH value of the culture medium to 6.60 with 1mol/L food-grade NaOH solution, and sterilizing at 115 ℃ for 30 min.
Optimizing the formula and preparation of the culture medium: 25g/L of yeast peptone, 35g/L of yeast extract, 35g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 0.5g/L of calcium chloride, 5g/L of sodium acetate, 2g/L of potassium dihydrogen phosphate and the balance of purified water; weighing according to the formula proportion, heating to dissolve, adjusting the pH value of the culture medium to 6.60 with 1mol/L food-grade NaOH solution, and sterilizing at 115 ℃ for 30 min.
2. The culture method of the lactobacillus helveticus strain with the function of relieving lactose intolerance comprises the following steps:
1) recovering the frozen strains: taking a Lactobacillus helveticus strain cryopreservation tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the cryopreservation tube are melted;
2) first-stage culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the opening of the test tube, performing static culture for 16-20 h in a 37 ℃ incubator at constant temperature, and storing in a 4 ℃ refrigerator for later use;
3) secondary culture: inoculating the bacterial suspension after the first-stage culture into a triangular flask filled with 80% of a basic culture medium according to the inoculation amount of 5%, sealing, and carrying out static culture for 16-20 h at the constant temperature of an incubator at 37 ℃;
4) and (3) third-stage culture: and (3) inoculating the bacterial suspension after the second-stage culture to a triangular flask filled with 80% of optimized culture medium according to the inoculation amount of 5%, and performing static culture for 16-20 h at the constant temperature of a 37 ℃ incubator.
5) And (3) yield determination: the centrifugal equipment adopts a table centrifuge, the rotating speed is 12000rpm, 10min, supernatant is discarded after centrifugation, and the net weight of bacterial sludge is weighed according to the formula:
calculating the yield of the fermentation liquor;
6) and (4) measuring the number of the three-stage viable bacteria.
Example 2 determination of enzymatic Activity of Lactobacillus helveticus Strain relieving lactose intolerance
The o-nitrophenyl- β -D-galactopyranoside (ONPG) is a colorless compound which is easy to dissolve in water and can be used as a β -galactosidase substrate, the glycosidic bond is broken under the catalytic action of enzyme to generate o-nitrophenol (ONP) and β -D-galactopyranose, the product ONP is a yellow substance, the color of the solution is gradually deepened along with the decomposition of the substrate, and the maximum absorption peak is formed at 420nm, so that the yield of ONP can be measured and calculated by measuring the absorbance of the solution.
1. Preparation of test strains
1) Recovering the frozen strains: taking a Lactobacillus helveticus strain cryopreservation tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the cryopreservation tube are melted;
2) first-stage culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the opening of the test tube, performing static culture for 16-20 h in a 37 ℃ incubator at constant temperature, and storing in a 4 ℃ refrigerator for later use;
3) secondary culture: inoculating the bacterial suspension after the first-stage culture into a triangular flask filled with 100mL of a basic culture medium according to the inoculation amount of 5%, sealing, and carrying out static culture for 16-20 h at the constant temperature of an incubator at 37 ℃;
4) and (3) third-stage culture: and (3) inoculating the bacterial suspension after the secondary culture is finished into a triangular flask filled with 100mL of optimized culture medium according to the inoculation amount of 5%, and performing static culture for 16-20 h at the constant temperature of an incubator at 37 ℃.
2. Drawing of o-nitrophenol (ONP) concentration standard curve
Weighing 0.1390g of o-nitrophenol (ONP) in a small beaker, adding 10ml of 96% ethanol for dissolving, transferring the solution into a 1L volumetric flask, adding water to a constant volume, and shaking up;
transferring 2mL, 4mL, 6mL, 8mL, 10mL, 12mL and 14mL solutions from the solutions to different 100mL volumetric flasks by a pipette, adding 25mL sodium carbonate solution, shaking up with buffer solution to constant volume to prepare ONP of 0.02mmol/L, 0.04mmol/L, 0.06mmol/L, 0.08mmol/L, 0.10mmol/L, 0.12mmol/L and 0.14mmol/L, and using sterile water as blank;
the quantity concentration of the o-nitrophenol substance is taken as the abscissa, and the absorbance delta OD of the diluent is taken420And drawing a standard curve for the ordinate to obtain a regression equation.
3.β determination of galactosidase Activity (ONPG method)
Centrifuging 15mL of fermentation liquor after three-stage culture at 10000r/min and 4 ℃ for 5 min. The bacterial sludge was collected and washed twice with equal volumes of 0.05mol/L phosphate buffer (pH 6.80). The cells were resuspended in 3mL of the above phosphate buffer and disrupted by sonication. The crushing conditions are as follows: the ultrasonic power is 200W, the working time/interval time is 1s/9s, 60 times and 4 ℃. Centrifuging at 4 deg.C at 10000r/min for 20min to obtain supernatant to obtain crude enzyme solution. The crude enzyme solution was diluted appropriately, 200. mu.L of the crude enzyme solution was then preheated in water bath at 30 ℃ for 5min, 1mL of ONPG solution preheated at 30 ℃ for 5min was added, shaken well, and then washed in water bath at 30 ℃ for 10min, and 400. mu.L of sodium carbonate solution was immediately added to terminate the reaction. The OD was measured at 420nm within 30 min.
Preparation of a blank: the order of addition of the ONPG substrate and sodium carbonate solution was reversed and the remaining steps were performed in the same manner as the sample treatment.
One unit of enzyme activity is defined as (U): the amount of enzyme required for the hydrolytic release of 1. mu. mol ONP per minute at 30 ℃.
The enzyme activity calculation formula is as follows:
in the formula: e is hairEnzyme activity in the fermentation liquid, U/mL; f is the dilution times of the enzyme solution; v is the total volume of the reaction system and is 1.6 mL; k is the slope of the standard curve, 3.1625; t is reaction time, 10 min; v1The volume of the enzyme solution was 0.2 mL.
The enzyme activity of the Lactobacillus helveticus lactose intolerance functional strain HCS23-015 is 0.584U/mL.
Example 3 determination of enzyme Activity of lactose intolerance relieving functional Strain of control Strain
The results in Table 1 show that the enzyme activity of Bifidobacterium lactis BB-12 in Hansen of Danish family is the highest and has a value of 0.731U/mL, and Lactobacillus reuteri LR92 in Italiaceae and has an enzyme activity of 0.499U/mL, the enzyme activity of Lactobacillus helveticus HCS23-015 is 0.584U/mL according to the determination result of example 2, and the enzyme activity is inferior to that of Bifidobacterium lactis BB-12 in Hansen of Danish family.
TABLE 1 List of control strains with lactose intolerance alleviating test data
Example 4 Lactobacillus helveticus Strain culture Condition test for lactose intolerance alleviating function
1. Strain culture condition test method
1.1) recovering the frozen strains: taking a Lactobacillus helveticus strain cryopreservation tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the cryopreservation tube are melted;
1.2) primary culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the opening of the test tube, performing static culture for 16-20 h in a 37 ℃ incubator at constant temperature, and storing in a 4 ℃ refrigerator for later use;
1.3) secondary culture: inoculating the bacterial suspension obtained by the first-stage culture into a 100mL basic culture medium, performing static culture for 17h under different conditions, measuring the pH value and the yield of the bacterial suspension, and inspecting the influence of different culture temperatures, different initial pH values, inoculation amounts and aerobic types on the growth of the thalli. The experimental protocol is shown in table 2.
TABLE 2 experiment factor level table for culture conditions
The basic culture medium formula for screening the culture conditions is as follows: 25g/L of yeast peptone, 10g/L of yeast extract, 20g/L of lactose, 2g/L of citric acid, 3g/L of L-malic acid, 1g/L of magnesium sulfate heptahydrate and the balance of purified water.
1.4) three-stage culture: inoculating the secondary bacterial suspension cultured under the optimized condition into a third-level optimized culture medium for standing culture for 16h, measuring the pH, yield and viable count of the bacterial suspension, and inspecting the influence of different inoculation amounts and liquid loading amounts on the growth of thalli. The experimental protocol is shown in table 3.
TABLE 3 experiment factor level table for culture conditions
The formula of the optimized culture medium used for screening the culture conditions is as follows: 20g/L of yeast peptone, 20g/L of yeast extract, 30g/L of lactose, 2g/L of citric acid, 3g/L of L-malic acid, 1g/L of magnesium sulfate heptahydrate, 0.5g/L of calcium chloride and the balance of purified water.
2. Test results of strain culture conditions
2.1) results of the tests of Table 2
2.1.1) Effect of culture temperature on growth of the Strain
Fixing the inoculum size of other conditions to be 4 percent, culturing at the initial pH of 6.60 by covering tin foil, observing the influence of the culture temperature on the growth of the thalli, and verifying the better conditions. The test results are shown in tables 4 and 5.
TABLE 4 Effect of culture temperature on growth of the strains
TABLE 5 verification of the Effect of culture temperature on the growth of the strains
According to the test results in Table 4, the yield of the strain is high at the temperature of 35 ℃ and 37 ℃, and the optimal culture temperature of the strain is 37 ℃ as shown in Table 5.
2.1.2) Effect of initial pH on growth of the Strain
The inoculum size is 4% under other fixed conditions, the culture temperature is 37 ℃, the culture is performed by covering tin foil, the influence of the initial pH value on the growth of the thalli is inspected, and the better conditions are verified. The test results are shown in tables 6 and 7.
TABLE 6 Effect of initial pH on growth of the strains
TABLE 7 verification of the Effect of initial pH on Strain growth
As is clear from the test results in Table 6, when the initial pH values were 6.20 and 6.40, the sludge state was loose and the growth rate of viable bacteria was low; when the initial pH value is 6.60 and 6.80, the bacterial sludge state is neat, and the yield is high. Further verification was made for initial pH values of 6.60 and 6.80, and as shown in table 7, the yield of bacterial sludge at an initial pH of 6.60 was still higher than that at an initial pH of 6.80, so that the optimal initial pH condition was determined to be 6.60.
2.1.3) Effect of aerobic type on Strain growth
The inoculum size was 4%, the culture temperature was 37 ℃ and the initial pH was 6.60 under the other conditions, and the effect of aerobic type on the growth of the cells was examined, and the test results are shown in Table 8.
TABLE 8 test results of the effect of aerobic type on the growth of the strains
According to the test results in Table 8, since the yield of the strain obtained by culturing under the tin-coated paper condition was higher than that of tin-free paper, the tin-coated paper culture was selected.
2.1.4) Effect of inoculum size on growth of the Strain
The culture temperature was set at 37 ℃ under other conditions, the initial pH was 6.60, the cells were cultured in tinfoil, the effect of the inoculum size on the growth of the cells was examined, and the test results are shown in Table 9.
TABLE 9 Effect of inoculum size on growth of the strains
According to the test results in Table 9, when the inoculation amount is 5%, the yield of the bacterial sludge is the highest.
2.2) results of the tests of Table 3
2.2.1) Effect of inoculum size on growth of the Strain
According to the result of a secondary fermentation experiment, the preferable result of the strain culture condition is that the culture temperature is 37 ℃, the initial pH value is 6.60, the strain is cultured by covering tin foil, the strain is cultured in a tertiary mode under the condition, the yield of bacterial sludge and the number of viable bacteria are measured, and the influence of the inoculation amount on the growth of the strain is further verified. The results are shown in Table 10.
TABLE 10 verification of the Effect of inoculum size on Strain growth
According to the test results in Table 10, with the increase of the inoculation amount, the pH value is lower and lower, the yield and the viable bacteria number are higher and higher, the trend of increasing is presented, when the inoculation amount is 5%, the yield of bacterial sludge and the viable bacteria number are the highest, and therefore, the optimum inoculation amount of the second-stage culture and the third-stage culture of the bacterial strain is 5%.
2.2.2) Effect of liquid Loading on growth of the Strain
The influence of different liquid contents on the growth of the strain was examined by three-stage culture, and the results are shown in Table 11.
TABLE 11 influence of liquid loading on growth of strains
As shown in Table 11, the yield of bacterial sludge and the number of viable bacteria were the highest when the liquid content was 80%, and the optimum liquid content for culturing the strain was 80%.
Example 5 Lactobacillus helveticus Strain Medium screening test for lactose intolerance alleviating function
According to the characteristics of the lactobacillus helveticus strain with the function of relieving lactose intolerance, the invention designs the following culture medium experimental scheme with specific raw materials and proportion. Wherein yeast peptone, yeast extract, glucose and lactose are used as basic nutrient substances in the culture medium to provide a carbon source, a nitrogen source and other growth factors to meet the requirement of bacterial growth; citric acid, L-malic acid, sodium acetate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride, etc. provide various growth factors required by Lactobacillus helveticus, while inhibiting the growth of bacteria other than lactic acid bacteria. Taking the yield of bacterial sludge, the number of viable bacteria in fermentation liquor and the enzyme activity of the fermentation liquor as investigation indexes, and preferably selecting a basic culture medium and optimizing a culture medium formula.
1. Screening test method for basic culture medium
1.1) recovering the frozen strains: taking a Lactobacillus helveticus strain cryopreservation tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the cryopreservation tube are melted;
1.2) primary culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the opening of the test tube, performing static culture for 16-20 h in a 37 ℃ incubator at constant temperature, and storing in a 4 ℃ refrigerator for later use;
1.3) secondary culture: according to the inoculation amount of 5%, the bacterial suspension obtained by the first-stage culture is inoculated into 80mL of basal medium, and is statically cultured for 17h at 37 ℃. And (3) measuring the pH value and bacterial sludge yield of the bacterial suspension, and investigating the influence of different basic culture medium formulas on the growth of the bacteria. The experimental protocol and results are shown in Table 12.
TABLE 12 basal Medium screening protocol and results
According to the experimental results of table 12, the lactobacillus helveticus strain for relieving lactose intolerance can utilize glucose and lactose, but hardly utilize galacto-oligosaccharide, and scheme 2 is the most suitable formula of the basic culture medium, in combination with the determination result of the activity of the tertiary fermentation enzyme.
2. Optimized culture medium screening test method
2.1) recovering the frozen strains: taking a Lactobacillus helveticus strain cryopreservation tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the cryopreservation tube are melted;
2.2) first-stage culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the opening of the test tube, and performing constant-temperature static culture in an incubator at 37 ℃ for 16-20 h;
2.3) secondary culture: inoculating the bacterial suspension obtained by the first-stage culture into 80mL of a basic culture medium according to the inoculation amount of 5%, and performing static culture at 37 ℃ for 16-20 h;
2.4) three-stage culture: and (3) inoculating the bacterial suspension obtained by the secondary culture into 200mL of optimized culture medium according to the inoculation amount of 5%, and standing and culturing for 16-20 h at 37 ℃.
And (3) selecting the optimal optimized culture medium formula by taking the pH value of the fermentation liquor, the viable count, the enzyme activity and the bacterial sludge yield as investigation indexes. The protocol is shown in Table 13.
Wherein the basic culture medium comprises the following components in percentage by weight: 15g/L of yeast peptone, 20g/L of yeast extract, 20g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of potassium dihydrogen phosphate, 5g/L of sodium acetate and pH value of 6.60.
TABLE 13 optimized Medium formulation screening protocol and results
According to the test results in Table 13, the Lactobacillus helveticus strain for relieving lactose intolerance has higher viable count and enzyme activity than glucose and the mixture of glucose and lactose when lactose is used as a carbon source; according to the lactose gradient comparison results of the schemes 8, 9 and 10, when 35g/L of lactose is added, the enzyme activity and the viable count are the highest, so the scheme 9 is the optimized culture medium formula for most relieving lactose intolerance.
Claims (6)
1. A culture medium for culturing a strain with the function of relieving lactose intolerance of Lactobacillus helveticus is characterized by comprising a basic culture medium and an optimized culture medium,
the basic culture medium comprises the following components in percentage by weight: 10-20 g/L of yeast peptone, 15-25 g/L of yeast extract, 15-25 g/L of lactose, 3-5 g/L of citric acid, 2-4 g/L of L-malic acid, 0.4-0.6 g/L of magnesium sulfate heptahydrate, 1-3 g/L of monopotassium phosphate, 4-6 g/L of sodium acetate and the balance of purified water, and adjusting the pH value to 6.60;
the formula of the optimized culture medium is as follows: 20-30 g/L of yeast peptone, 30-40 g/L of yeast extract, 30-40 g/L of lactose, 3-5 g/L of citric acid, 2-4 g/L of L-malic acid, 0.4-0.6 g/L of magnesium sulfate heptahydrate, 0.4-0.6 g/L of calcium chloride, 4-6 g/L of sodium acetate, 1-3 g/L of potassium dihydrogen phosphate and the balance of purified water, and the pH value is adjusted to 6.60.
2. The culture medium for culturing the Lactobacillus helveticus strain with the lactose intolerance relieving function according to claim 1, which comprises a basal medium and an optimized medium,
the basic culture medium comprises the following components in percentage by weight: 15g/L of yeast peptone, 20g/L of yeast extract, 20g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 2g/L of potassium dihydrogen phosphate, 5g/L of sodium acetate and the balance of purified water, and adjusting the pH value to 6.60;
the formula of the optimized culture medium is as follows: 25g/L of yeast peptone, 35g/L of yeast extract, 35g/L of lactose, 4g/L of citric acid, 3g/L of L-malic acid, 0.5g/L of magnesium sulfate heptahydrate, 0.5g/L of calcium chloride, 5g/L of sodium acetate, 2g/L of monopotassium phosphate and the balance of purified water, and the pH value is adjusted to 6.60.
3. The culture medium for the lactobacillus helveticus strain for alleviating the lactose intolerance as recited in claim 1 or 2, wherein the basic culture medium is prepared by the following method: weighing according to the formula proportion of claim 1 or 2, heating for dissolving, adjusting the pH value of the culture medium to 6.60 by using 1mol/L food-grade NaOH solution, and sterilizing at 115 ℃ for 30 min;
the preparation method of the optimized culture medium comprises the following steps: weighing according to the formula proportion of claim 1 or 2, heating for dissolving, adjusting the pH value of the culture medium to 6.60 with 1mol/L food-grade NaOH solution, and sterilizing at 115 ℃ for 30 min.
4. The method for culturing the culture medium for the lactobacillus helveticus strain for alleviating the lactose intolerance as set forth in claim 1 or 2, which comprises the following steps:
1) recovering the frozen strains: taking a Lactobacillus helveticus strain cryopreservation tube stored in a low-temperature refrigerator, immediately putting the tube into a water bath kettle at 37 ℃ for strain recovery for 15-30 s until all solids in the cryopreservation tube are melted;
2) first-stage culture: inoculating recovered 1mL of frozen tube strains into a test tube filled with 10mL of basal medium, sealing the test tube opening with tinfoil, carrying out static culture at constant temperature of an incubator at 37 ℃ for 16-20 h, and storing in a refrigerator at 4 ℃ for later use;
3) secondary culture: inoculating the bacterial suspension after the first-stage culture into a triangular flask filled with a basic culture medium, sealing with tinfoil, and performing static culture at constant temperature of an incubator of 37 ℃ for 16-20 hours;
4) and (3) third-stage culture: and (3) inoculating the bacterial suspension after the second-stage culture to a triangular flask filled with an optimized culture medium, and performing static culture for 16-20 h at the constant temperature of an incubator at 37 ℃.
5. The culture method according to claim 4, wherein the amount of the inoculated medium in step 3) and step 4) is 5%.
6. The culture method according to claim 4, wherein the amounts of the basal medium and the optimal medium in the flask in steps 3) and 4) are 80%.
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Address after: 331200 Zhangshu North Economic and Technological Development Zone, Yichun City, Jiangxi Province Applicant after: Jiangxi Renren Health Microecological Technology Co.,Ltd. Address before: 331200 Zhangshu North Economic and Technological Development Zone, Yichun City, Jiangxi Province Applicant before: Jiangxi Renren Health Industry Co.,Ltd. |
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| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200529 |