CN111849853A - Method for improving tolerance of lactic acid bacteria bile salt - Google Patents
Method for improving tolerance of lactic acid bacteria bile salt Download PDFInfo
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Abstract
The invention discloses a method for improving tolerance of bile salt of lactic acid bacteria, and belongs to the technical field of microorganisms. The method for improving the bile salt tolerance of the lactic acid bacteria is to culture the lactic acid bacteria by using a culture medium containing soybean lecithin and concentrated whey protein, wherein the mass volume ratio of the final concentration of the soybean lecithin in the culture medium is 0.6-1.0%, and the mass volume ratio of the final concentration of the concentrated whey protein in the culture medium is 2.0-3.0%. The invention has the following beneficial effects: the method is simple, low in production cost and easy for industrial production, and the survival rate of the lactobacillus strains which are not acid-resistant and bile salt-resistant can be remarkably improved by simultaneously adding the soybean lecithin and the concentrated whey protein in a certain proportion and concentration into the culture medium under the conditions of low acid and high bile salt concentration.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for improving the tolerance of lactic acid bacteria bile salt.
Background
The world health organization and the food and agriculture organization defined probiotics as "viable microorganisms that when ingested can provide a probiotic effect, particularly an improvement or restoration of the intestinal flora". Probiotics are the most effective and direct means of influencing human health by regulating intestinal microorganisms. To date, lactic acid bacteria and bifidobacteria, the most traditional and prevalent probiotics, are commonly used as additives and fermented foods and are increasingly being studied for their potential probiotic effects. Scientific research shows that the probiotics have the function of regulating related diseases caused by intestinal flora disorder, such as gastrointestinal diseases, autoimmune diseases, neurological diseases, metabolic diseases and the like. The clinical effects of modulating gut microbiology by ingestion of probiotics to treat diarrhea resulting from antibiotic use, diarrhea caused by clostridium difficile infection, irritable bowel syndrome and inflammatory bowel disease have been evaluated. In addition, the regulation of intestinal microbial abundance and composition is also associated with successful treatment of various cancers.
Despite such a wide range of probiotic effects, the importance of their use is how to ensure that their active state is maintained after ingestion of the probiotic in order to effectively exert their effect. During the production and application process, probiotics are usually stressed by gastric acid, bile salt, osmotic pressure, temperature, oxygen and the like. After the human body takes the probiotics, the first condition is how to tolerate gastric acid and bile salt before the probiotics can successfully reach and colonize the intestinal tract to exert the probiotic effect. That is, the key to the probiotic bacteria to exert their probiotic effect is how to pass the acidic environment of the gastrointestinal tract and the bile salt barrier in the form of live bacteria.
In the body of mammals, bile salts are mainly produced by the liver and then secreted into the intestinal tract to esterify and dissolve fat, thereby promoting the decomposition and absorption of fat. Meanwhile, because the bile salt has fat solubility and water solubility characteristics, the activity of protein related to fatty acid metabolism can be changed, so that the fat composition of a cell membrane is changed to kill bacteria, and cells can be killed by disturbing lipid package or proton pump power. In addition, bile salts can also kill cells by causing oxidative damage to DNA and RNA, misfolding of proteins, and an increase in intracellular acidity. Therefore, how to optimize the activity of probiotics based on the characteristics of the gastrointestinal tract is an important consideration index for screening potential probiotic strains and developing novel probiotic products.
In order to reduce the killing effect of bile salts on cells, as a long-term evolution result, lactic acid bacteria also develop a series of bile salt tolerance mechanisms, such as bile salt efflux pumps, expression of bile salt hydrolase, change of fatty acid composition, secretion of extracellular polysaccharide and the like, so as to neutralize the damage of bile salts on cell structures. Therefore, combining the mechanism of the bacteria and the way in which bile salts act on cells, researchers have also developed methods to improve the tolerance of target strains to bile salts: constructing engineering strain with high expression of bile salt hydrolase, adding lactose, soybean lecithin, isolated whey protein and other foreign matter, and microcapsule technology to prevent or relieve direct contact between bile salt and cell. However, the genetically engineered strains cannot be applied in the form of live bacteria due to the safety limitation, and the exogenous substances added alone can improve the bile salt tolerance of the related strains to a certain extent, but the survival rate is still not high.
The soybean lecithin can effectively change the fatty acid composition and hydrophobicity of cell membranes, and the addition of protein substances can effectively neutralize the influence of bile salt on cell protein active molecules. At present, no relevant literature data on improving the bile salt tolerance of the lactobacillus by treating the lactobacillus by adding lipid and protein substances in a compound way is disclosed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for improving the tolerance of the bile salt of the lactic acid bacteria, which is simple and convenient to operate, strong in practicability and low in cost.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for improving the tolerance of bile salt of lactic acid bacteria is characterized by comprising the following steps: the method comprises the step of culturing the lactic acid bacteria by using a culture medium containing soybean lecithin and concentrated whey protein, wherein the mass volume ratio of the final concentration of the soybean lecithin in the culture medium is 0.6-1.0%, and the mass volume ratio of the final concentration of the concentrated whey protein in the culture medium is 2.0-3.0%.
The mass volume ratio of the final concentration of the soybean lecithin in the culture medium is 0.74%, and the mass volume ratio of the final concentration of the concentrated whey protein in the culture medium is 2.54%.
The lactic acid bacteria comprise streptococcus thermophilus, lactobacillus bulgaricus, lactobacillus paracasei and lactobacillus plantarum.
The culture medium is a liquid culture medium or a solid culture medium.
The culture medium comprises an MRS liquid culture medium, a TJA liquid culture medium, a soybean lecithin-MRS liquid culture medium, a concentrated lactalbumin-MRS liquid culture medium, a soybean lecithin-concentrated lactalbumin-MRS liquid culture medium, an MRS solid culture medium and a TJA solid culture medium.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, by adding the soybean lecithin and the concentrated whey protein in a certain proportion and concentration into the culture medium, the survival rate of the acid-intolerant and cholate-intolerant lactobacillus strains under the conditions of low acid and high cholate concentration can be remarkably improved.
The principle that the acid and bile salt resistance of the lactobacillus strain can be improved after the simultaneous culture of the soybean lecithin and the concentrated whey protein is as follows:
since the cell wall of lactic acid bacteria is mainly composed of teichoic acid/polysaccharides and peptidoglycan composed of proteins, the cell membrane is composed of different types of protein and phospholipid bilayers. The direct damage of bile salt to cells results from the amphiphilic character of the bile salt, especially the strong fat solubility which destroys the cell structure and function and causes cell death. Therefore, after the research, the inventor believes that the addition of the main components of protein and lipid in the cell structure can form a protective film to be adsorbed on the outer surface of the cell or relieve the damage of bile salt and acidic environment to the cell structure, and can also change the fatty acid composition of the cell membrane and improve the hydrophobicity of the cell surface so as to maintain the cell integrity under the conditions of strong acid and high bile salt. At present, most of researches only add a certain substance, the survival rate is greatly improved but still is lower than 10 percent, the possible reason is that the influence of the acidic environment and bile salts on the cell structure and the function is all-round, and the damage cannot be prevented by adding a single exogenous substance. The research result shows that the isolated whey protein can improve the bile salt tolerance of Streptococcus thermophilus ST-M5(Streptococcus thermophilus ST-M5) and lactobacillus bulgaricus LB-12(lactobacillus bulgaricus LB-12) in a mode of relieving protein damage or promoting protein repair. Soybean lecithin can improve the surface hydrophobicity and membrane integrity of Lactobacillus plantarum (Lactobacillus plantarum) cells by modifying the fatty acid composition. The soybean protein can be combined with bile salt for precipitation so as to relieve the inhibition effect of the bile salt on Bifidobacterium breve (Bifidobacterium breve) in the Mylabris to a certain extent. Although there are also results indicating that lactose addition can also improve the bile salt tolerance of Lactobacillus bulgaricus and Streptococcus thermophilus, the improvement effect is not great. And bile salts mainly disrupt protein function in cell structures and alter fatty acid composition. Therefore, in our research, we focused on the effect of adding two major cellular components, namely protein and lipid, on the tolerance of lactic acid bacteria to bile salts. The result shows that the two substances are added simultaneously, and the bile salt tolerance of the target strain can be effectively improved compared with the single addition. In addition, studies show that the tolerance of the bile salt of the lactic acid bacteria is improved, and the survival rate of the lactic acid bacteria under the condition of low pH is also improved, and the conclusion is also proved in the experimental results of the lactic acid bacteria.
2. The method is simple, low in production cost and easy for industrial production. The method can obviously improve the acid and cholate resistance of the strains such as lactobacillus paracasei, streptococcus thermophilus, lactobacillus bulgaricus and the like, and is beneficial to the function of lactobacillus probiotics in intestinal tracts. According to the processing method of example 3, when the soybean lecithin and the concentrated whey protein are added at the concentrations of 0.74% and 2.54%, respectively, the logarithm value of the viable count of lactobacillus paracasei L9 is improved to 9.47 from less than 1.0 before optimization after the lactobacillus paracasei L9 acts for 2.5 hours under the condition of 0.3% bile salt. According to the processing method of example 4, when the soybean lecithin and the concentrated whey protein are added at the concentrations of 0.74% and 2.54%, respectively, the log value of viable count of lactobacillus paracasei L9 is improved to 9.5 from less than 1.0 before optimization after 2.5 hours of action under the condition of pH 2.0.
Drawings
FIG. 1: effect of different pH stress for 2.5h on growth of lactobacillus paracasei L9 strain.
FIG. 2: effect of bile salt stress for 2.5h at various concentrations on growth of lactobacillus paracasei L9 strain.
FIG. 3: response surface analysis influences of simultaneous addition of soybean lecithin and concentrated whey protein with different concentrations on growth of lactobacillus paracasei L9 strain stressed by 0.3% of bile salt for 2.5 h.
Detailed Description
The present invention will be further illustrated by, but is not limited to, the following examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
A method for improving the bile salt tolerance of lactobacillus paracasei L9, comprising the steps of:
the lactobacillus paracasei L9 strain is from China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 9800. The preservation specification is as follows: and (3) classification and naming: lactobacillus paracasei; latin name: lactobacillus paracasei; the biological material of the reference: l9; the preservation organization: china general microbiological culture Collection center; the preservation organization is abbreviated as: CGMCC; address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is as follows: 10 month 22 days 2014; registration number of the preservation center: CGMCC No. 9800.
(1) MRS liquid medium: the specific method comprises the steps of adding 20g of glucose, 10g of casein peptone, 10g of beef extract, 5g of yeast extract powder, 5g of anhydrous sodium acetate, 2g of ammonium citrate tribasic, 2g of dipotassium hydrogen phosphate, 1mL of tween-80, 0.25g of manganese sulfate and 0.58g of magnesium sulfate into 1000mL of water, mixing the magnesium sulfate and the manganese sulfate with the rest components after separately dissolving, and sterilizing at 121 ℃ for 20min for later use.
(2) MRS solid medium: the specific method comprises adding agar with mass volume ratio of 1.5% into MRS liquid culture medium, and sterilizing at 121 deg.C for 20 min.
(3) Preparing a soybean lecithin-MRS liquid culture medium: the specific method comprises adding soybean lecithin with final concentration of 0.2%, 0.4%, 0.6%, 0.8%, 1.0% (mass/volume) into MRS liquid culture medium, and autoclaving at 121 deg.C for 20 min.
(4) Preparing a bile salt-MRS liquid culture medium: the specific method comprises adding ox bile salt with final concentration of 0.1%, 0.2%, and 0.3% (mass volume ratio) into MRS liquid culture medium, and autoclaving at 121 deg.C for 20 min.
(5) Inoculation: the activated lactobacillus paracasei L9 is respectively inoculated in MRS liquid culture medium and soybean lecithin-MRS liquid culture medium by the inoculation amount of 2 percent, and is statically cultured for 18 hours at the temperature of 38 ℃.
(6) Bile salt-MRS liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing for 18h in an MRS liquid culture medium and a soybean lecithin-MRS liquid culture medium, centrifuging at 12000rpm for 5min, removing supernatant, suspending cell sediment in 1mL of cholate-MRS liquid culture medium, and culturing at 38 ℃ for 2.5 h.
(7) Measuring the number of viable bacteria after treatment: after each treated strain is centrifuged at 12000rpm for 5min, the supernatant is removed, 1mL of physiological saline is added, isocratic gradient dilution is carried out, the mixture is poured into a flat plate containing 20mL of MRS solid medium by a pouring method, and the colony number is measured after the mixture is cultured for 36h at 38 ℃. The number of viable lactobacillus paracasei L9 bacteria obtained after the treatment of the soybean lecithin-MRS liquid culture medium is the number of viable bacteria after the treatment, and the number of viable bacteria of lactobacillus paracasei L9 bacteria obtained after the treatment of the MRS liquid culture medium is the number of viable bacteria of a control group.
The calculation method of the bile salt tolerance rate comprises the following steps: bile salt tolerance (%). viable cell count after treatment/viable cell count of control group × 100%.
The effect of different concentrations of soybean lecithin on the growth of lactobacillus paracasei L9 strain at different concentrations of bile salt stressed for 2.5h is shown in table 1.
Table 1:
the inventor determines the influence of soybean lecithin with different concentrations on the viable count of lactobacillus paracasei under different cholate conditions, and the experimental result according to table 1 shows that when the concentration of the soybean lecithin is added for a certain time, the viable count of the lactobacillus paracasei is gradually reduced along with the rise of the cholate concentration. The addition amount of the soybean lecithin has no obvious influence on the survival rate of the strain under the condition of 0.1 percent of bile salt, but under the condition of 0.2 percent of bile salt, when the addition amount of the soybean lecithin is 0.2 to 0.6 percent, the number of viable bacteria is increased along with the increase of the addition amount, and the change of the number of viable bacteria is not obvious when the addition amount is continuously increased; under the condition of 0.3% of bile salt, when the addition amount of the soybean lecithin is 0.2% -0.8%, the number of viable bacteria is increased along with the increase of the addition amount, and then when the addition amount of the soybean lecithin is continuously increased, the number of viable bacteria is not obviously changed. That is, compared with the method without adding soybean lecithin, the tolerance rate of lactobacillus paracasei under the conditions of 0.2% and 0.3% of bile salt can be increased to 24% and 0.28% from 0.04% and 0% respectively by adding the soybean lecithin. Therefore, the soybean lecithin can obviously improve the bile salt tolerance of lactobacillus paracasei L9. However, the viable count of the strain under the condition of 0.3 percent of bile salt is two orders of magnitude different from that of a control group, and the tolerance rate is still far lower than 1 percent.
Example 2
A method for improving the bile salt tolerance of lactobacillus paracasei L9, comprising the steps of:
the lactobacillus paracasei L9 strain is from China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 9800.
(1) Preparing an MRS liquid culture medium and an MRS solid culture medium: same as in step (1) and step (2) of example 1.
(2) Preparing a concentrated whey protein-MRS liquid culture medium: to MRS liquid medium (1000mL of water added with 2 times the amount of each normal substance) sterilized at 121 ℃ for 20min and concentrated 2 times was mixed a final volume of concentrated whey protein filtered with 0.22 μm Polyethersulfone (PES) at 2 times the desired final concentration. The final concentration of the concentrated whey protein in the MRS liquid culture medium is 1.5%, 2.0%, 2.5%, 3.0% and 3.5% (mass to volume ratio), respectively.
(3) Preparing a bile salt-MRS liquid culture medium: same as in step (4) of example 1.
(4) Inoculation: the activated lactobacillus paracasei L9 is respectively inoculated in MRS liquid culture medium and concentrated lactalbumin-MRS liquid culture medium by the inoculation amount of 2 percent, and the mixture is kept stand and cultured for 18 hours at the temperature of 38 ℃.
(5) Bile salt-MRS liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing for 18h in an MRS liquid culture medium and a concentrated lactalbumin-MRS liquid culture medium, centrifuging for 5min at 12000rpm, removing supernatant, suspending cell sediment in 1mL of cholate-MRS liquid culture medium, and culturing for 2.5h at 38 ℃.
(6) Measuring the number of viable bacteria after treatment: after each treated strain is centrifuged at 12000rpm for 5min, the supernatant is removed, 1mL of physiological saline is added, isocratic gradient dilution is carried out, the mixture is poured into a flat plate containing 20mL of MRS solid medium by a pouring method, and the colony number is measured after the mixture is cultured for 36h at 38 ℃. The number of viable bacteria of lactobacillus paracasei L9 after treatment was obtained in the concentrated whey protein-MRS liquid medium, and the number of viable bacteria of a control group of lactobacillus paracasei L9 was obtained in the MRS liquid medium.
The calculation method of the bile salt tolerance rate comprises the following steps: bile salt tolerance (%). viable cell count after treatment/viable cell count of control group × 100%.
The effect of different concentrations of concentrated whey protein on the growth of different concentrations of the L9 strain of Lactobacillus paracasei stressed by bile salts for 2.5h is shown in Table 2.
Table 2:
the inventor simultaneously determines the influence of the concentrated whey protein with different concentrations on the viable count of lactobacillus paracasei under different cholate conditions, and the experimental result according to the table 2 shows that when the concentrated whey protein is added for a certain concentration, the viable count of lactobacillus paracasei gradually decreases along with the rise of the cholate concentration. When the concentration of the bile salt is 0.1%, the addition of concentrated whey protein has no obvious influence on the viable count of the strain; when the concentration of the bile salt is 0.2 percent and 0.3 percent, the addition of 2.5 percent of concentrated whey protein can effectively improve the viable count of the strain, and the action effect after the addition amount is more than 2.5 percent has no obvious difference with that when the addition amount is 2.5 percent. The results generally indicate that concentrated whey protein can significantly improve the tolerance rate of the lactobacillus strains at the lethal concentration of bile salts. That is, compared with the condition without adding concentrated whey protein, the tolerance rate of lactobacillus paracasei can be increased from 0.04% to 0% and from 0.3% to 0.22% to 0.02% respectively by adding soybean lecithin. But the result also shows that the single addition of the concentrated whey protein can obviously improve the strain tolerance rate of the lactic acid bacteria under the condition of lethal bile salt, but the viable count still differs from a control group by 3-4 orders of magnitude, and the tolerance rate is far lower than 1%.
According to the two single-factor experimental results, the effect of improving the survival rate of the strains sensitive to the bile salts by adding the single-factor strain is not obvious. However, it is known that the cell wall of lactic acid bacteria is mainly composed of teichoic acid/polysaccharides and peptidoglycan consisting of proteins, and the cell membrane is composed of different types of protein and phospholipid bilayers. The direct damage of bile salt to cells results from the amphiphilic character of the bile salt, especially the strong fat solubility which destroys the cell structure and function and causes cell death. Therefore, in terms of a lethal mechanism of bile salts on cells, the damage of the bile salts on the cell structures of the lactic acid bacteria cannot be effectively relieved by adding the lipid or protein substances alone, and the two substances are combined and added at the same time, so that the effect of relieving the damage of the bile salts on the cell structures is better. Therefore, the influence on the tolerance of the lactobacillus bile salt when the soybean lecithin and the concentrated whey protein are simultaneously added is considered and the optimal adding proportion is optimized by designing a response surface method of central combination design.
Example 3
A method for improving the bile salt tolerance of lactobacillus paracasei L9, comprising the steps of:
the lactobacillus paracasei L9 strain is from China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 9800.
(1) The preparation methods of the MRS liquid culture medium, the MRS solid culture medium and the bile salt-MRS liquid culture medium are the same as the steps (1), (2) and (4) in the example 1.
(2) 0.3% bile salt-MRS liquid medium: the specific method comprises adding 0.3% (mass volume ratio) of ox bile salt into MRS liquid culture medium, and autoclaving at 121 deg.C for 20 min.
(3) Preparing a soybean lecithin-concentrated lactalbumin-MRS liquid culture medium: soybean lecithin-MRS liquid medium (1000mL of water added with 2 times of the amount of each normal substance) sterilized at 121 ℃ for 20min and concentrated 2 times was mixed with a final volume of concentrated whey protein filtered with 0.22 μm Polyethersulfone (PES) at 2 times the desired final concentration.
(4) Inoculation: the activated lactobacillus paracasei L9 is respectively inoculated in MRS liquid culture medium and soybean lecithin-concentrated lactalbumin-MRS liquid culture medium by the inoculation amount of 2 percent, and is statically cultured for 18 hours at the temperature of 38 ℃.
(5) Bile salt-MRS liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing for 18h in an MRS liquid culture medium and a soybean lecithin-concentrated lactalbumin-MRS liquid culture medium, centrifuging at 12000rpm for 5min, removing supernatant, suspending cell sediment in 1mL of bile salt-MRS liquid culture medium, and culturing for 2.5h at 38 ℃.
(6) Measuring the number of viable bacteria after treatment: after each treated strain is centrifuged at 12000rpm for 5min, the supernatant is removed, 1mL of physiological saline is added, isocratic gradient dilution is carried out, the mixture is poured into a flat plate containing 20mL of MRS solid medium by a pouring method, and the colony number is measured after the mixture is cultured for 36h at 38 ℃. The number of the treated viable bacteria of lactobacillus paracasei L9 was obtained in soybean lecithin-concentrated whey protein-MRS liquid medium, and the number of the viable bacteria of a control group of lactobacillus paracasei L9 was obtained in MRS liquid medium.
The calculation method of the bile salt tolerance rate comprises the following steps: bile salt tolerance (%). viable cell count after treatment/viable cell count of control group × 100%.
The center combination Design response surface experimental software is Design expert 8.0.6, and the software used for statistical analysis is GraphPad Prism.
Based on the result obtained by the central combined Design experiment, a second-order regression model of the influence of different concentrations of two substances on the viable count of lactobacillus paracasei L9 after the lactobacillus paracasei L9 acts for 2.5 hours under the condition of 0.3% bile salt is obtained through simulation by Design Export 8.0.6 software, and the corresponding regression equation is as follows:
R＝-3.94+13.75A+6.56B+1.99AB-12.74A2-1.58B2
wherein R, A and B represent the log of viable count, the concentration of soybean lecithin and the concentration of concentrated whey protein, respectively.
After a second order regression model was obtained, we evaluated the effectiveness of the model based on statistical significance by analysis of variance. The model has a F value of 375.12 and "Prob >An F "value of less than 0.0001 indicates that the model is extremely significant, i.e., the probability of noise resulting in an F value of 375.12 for the model is only 0.01%. All parameters A, B, AB, A in the model2And B2The effects on the response value R were all significant (pA ═ 0.0001, pB)<0.0001,pAB＝0.0006,pA2<0.0001,pB2<0.0001). In addition, regression model mismatching value (p ═ 0.0929)>0.05) not significant. Further, fitting analysis is carried out on the regression equation, and the result shows that R20.9963, the predicted equation of the model is shown to explain 99.63% variation of L9 viable count of Lactobacillus paracasei. The "PredR-squared" value was 0.9783, which is similar to the "Adj R-squared" value (0.9936), and the "Adeq precision" value was 54.859 > 4, indicating that the model signal-to-noise ratio is reliable. Therefore, the second order regression model is suitable for describing the relationship between the addition concentration of the soybean lecithin and the concentrated whey protein and the log value of the viable count of lactobacillus paracasei L9 after the lactobacillus paracasei L9 acts for 2.5 hours under the condition of 0.3% bile salt.
Then, we constructed a three-dimensional graph of the effect of soy lecithin and concentrated whey protein on the response values, as shown in fig. 3, and the results showed that the response values changed with the changes in the added concentrations of soy lecithin and concentrated whey protein, and that the model had and only had an optimal value. Meanwhile, the contour plot also shows that the interaction between soy lecithin and WPC 80 (whey protein concentrate) also has a significant effect on the response values (pAB ═ 0.0006 < 0.05).
According to the second order regression equation, when the added concentrations of the soybean lecithin and the concentrated whey protein are 0.74 percent and 2.54 percent respectively, the equation has the optimal solution, and after the lactobacillus paracasei L9 acts for 2.5 hours under the condition of 0.3 percent of the concentration of the bile salt, the viable count can reach 2.89 multiplied by 109CFU/mL。
The effect of the addition of soy lecithin and concentrated whey protein on the growth of the L9 strain of Lactobacillus paracasei under 0.3% bile salt stress for 2.5h is shown in Table 3.
Table 3:
the results of the experiments in Table 3 show that the log viable count of Lactobacillus paracasei L9 is 9.47 and the corresponding viable count is 2.97X 10 under the same conditions9CFU/mL, and predicted viable count of 2.89X 109There was no significant error between CFU/mL. The result also shows that the model can effectively reflect the correlation between the number logarithm value of the viable bacteria of the strain under the condition of 0.3% bile salt by the soybean lecithin and the concentrated whey protein.
Through the experiment, after the lactobacillus paracasei L9 acts for 2.5 hours under the condition of 0.3% of bile salt, the log value of the viable count is improved to 9.47 from less than 1.0 before optimization, namely the survival rate is improved to 52.5% from nearly 0, and compared with the situation that one substance is singly added, the tolerance of the lactobacillus paracasei L9 bile salt is effectively improved by simultaneously adding the two substances.
Example 4
A method for improving acid resistance of lactobacillus paracasei L9, comprising the following steps:
the lactobacillus paracasei L9 strain is from China general microbiological culture Collection center (CGMCC), CGMCC No. 9800.
(1) The preparation methods of the MRS liquid medium, the MRS solid medium, and the soybean lecithin-concentrated whey protein-MRS liquid medium are the same as those in step (1) and step (2) of example 1, and the preparation method of the soybean lecithin-concentrated whey protein-MRS liquid medium is the same as that in step (3) of example 3.
(2) pH 2.0 MRS liquid medium: the specific method comprises adjusting pH of MRS liquid culture medium to 2.0 with 1.0mol/L HCl solution, and autoclaving at 121 deg.C for 20 min.
(3) Inoculation: the activated lactobacillus paracasei L9 is respectively inoculated in MRS liquid culture medium and soybean lecithin-concentrated lactalbumin-MRS liquid culture medium by the inoculation amount of 2 percent, and is statically cultured for 18 hours at the temperature of 38 ℃.
(4) pH 2.0 MRS liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing for 18h in an MRS liquid culture medium and a soybean lecithin-concentrated lactalbumin-MRS liquid culture medium, centrifuging at 12000rpm for 5min, removing supernatant, suspending cell sediment in 1mL of MRS liquid culture medium with the pH value of 2.0, and culturing for 2.5h at the temperature of 38 ℃.
(5) Measuring the number of viable bacteria after treatment: after each treated strain is centrifuged at 12000rpm for 5min, the supernatant is removed, 1mL of physiological saline is added, isocratic gradient dilution is carried out, the mixture is poured into a flat plate containing 20mL of MRS solid medium by a pouring method, and the colony number is measured after the mixture is cultured for 36h at 38 ℃. The number of the treated viable bacteria of lactobacillus paracasei L9 was obtained in soybean lecithin-concentrated whey protein-MRS liquid medium, and the number of the viable bacteria of a control group of lactobacillus paracasei L9 was obtained in MRS liquid medium.
The calculation method of the pH 2.0 tolerance rate comprises the following steps: tolerance (%). the number of viable bacteria after treatment/the number of viable bacteria in the control group × 100%.
The inventors further investigated the tolerance of the optimized culture medium to lactic acid bacteria under the condition of low pH on the basis of optimizing the lactic acid bacteria bile salt tolerance culture medium, and the results are shown in Table 4, wherein the log value of the viable count of the lactic acid bacteria is 9.6 after the lactic acid bacteria is cultured in the normal MRS liquid culture medium for 18 hours, and the log value of the viable count of the lactic acid bacteria is reduced to 4.1 after the lactic acid bacteria is acted for 2.5 hours under the condition of pH 2.0. After the same strain is cultured in an MRS liquid culture medium simultaneously added with 0.74 percent of soybean lecithin and 2.54 percent of concentrated whey protein for 18 hours, the logarithmic value of the viable count is 9.5 after the strain acts for 2.5 hours under the condition of pH 2.0. That is, the method is also effective in improving the tolerance of Lactobacillus paracasei under low pH conditions.
The effect of the addition of soy lecithin and concentrated whey protein on the growth of the L9 strain of Lactobacillus paracasei under pH2.0 stress for 2.5h is shown in Table 4.
Table 4:
the results of the experiments in Table 4 show that, when 0.74% of soybean lecithin and 2.54% of whey protein were not added, the log of viable count of Lactobacillus paracasei after 2.5 hours at pH2.0 was 4.14, and the tolerance was close to 0, and when 0.74% of soybean lecithin and 2.54% of whey protein were added, the log of viable count of Lactobacillus paracasei after 2.5 hours at pH2.0 was 9.51, and the tolerance was increased to 70.8%. Namely, the tolerance rate of the lactobacillus paracasei under the condition of low pH can be effectively improved by simultaneously adding the soybean lecithin and the concentrated whey protein in a certain proportion.
Example 5
A method for treating streptococcus thermophilus to improve the acid resistance and the bile salt resistance of the streptococcus thermophilus by adding soybean lecithin and concentrated whey protein, which comprises the following steps:
wherein the streptococcus thermophilus is from China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 14756. The preservation specification is as follows: and (3) classification and naming: streptococcus thermophilus; latin name: streptococcus thermophilus; the biological material of the reference: LSR-Q-G1; the preservation organization: china general microbiological culture Collection center; the preservation organization is abbreviated as: CGMCC; address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is as follows: 26 months 9 in 2017; registration number of the preservation center: CGMCC No. 14756.
(1) Preparing a TJA liquid culture medium: adding 5g of yeast extract, 10g of beef extract, 20g of lactose, 2g of glucose, 2g of dipotassium hydrogen phosphate, 1g of tween-80, 5g of anhydrous sodium acetate and 50mL of tomato juice into 1000mL of water, and sterilizing at 121 ℃ for 15min for later use.
(2) Preparing a TJA solid culture medium: adding 1.5% agar into TJA liquid culture medium, and sterilizing at 121 deg.C for 15 min.
(3) The preparation method of the soybean lecithin-concentrated whey protein-TJA liquid culture medium comprises the following steps: a soybean lecithin-TJA broth (1000mL of water with 2 times the amount of each of the substances added to the medium as the normal substance) sterilized at 121 ℃ for 20min and concentrated 2 times was mixed with a final volume of concentrated whey protein filtered with 0.22 μm Polyethersulfone (PES) at 2 times the desired final concentration.
(4) Preparing a 0.3% bile salt-TJA liquid culture medium: the specific method comprises adding 0.3% (mass volume ratio) of ox bile salt into TJA liquid culture medium, and autoclaving at 121 deg.C for 20 min.
(5) preparing a pH 2.0 TJA liquid culture medium: the specific method comprises adjusting pH of TJA liquid culture medium to 2.0 with 1.0mol/L HCl solution, and autoclaving at 121 deg.C for 20 min.
(6) Inoculation: the activated streptococcus thermophilus is respectively inoculated in a TJA liquid culture medium and a soybean lecithin-concentrated whey protein-TJA liquid culture medium according to the inoculation amount of 2 percent, and is statically cultured for 18 hours at the temperature of 38 ℃.
(7) pH 2.0 TJA liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing for 18h in a TJA liquid culture medium and a soybean lecithin-concentrated whey protein-TJA liquid culture medium, centrifuging for 5min at 12000rpm, removing supernatant, and suspending cell precipitates in 1mL of TJA liquid culture medium with the pH value of 2.0, and culturing for 2.5h at the temperature of 38 ℃.
(8) 0.3% bile salt-TJA liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing in TJA and soybean lecithin-concentrated whey protein-TJA culture medium for 18h, centrifuging at 12000rpm for 5min, removing supernatant, suspending cell precipitate in 1 mL0.3% bile salt-TJA liquid culture medium, and culturing at 38 ℃ for 2.5 h.
(9) Measuring the number of viable bacteria after treatment: centrifuging each treated strain at 12000rpm for 5min, removing supernatant, adding 1mL of physiological saline, diluting with isocratic gradient, pouring into a plate containing 20mL of TJA solid culture medium by pouring, culturing at 38 deg.C for 36h, and measuring colony count. And obtaining the number of the treated viable bacteria of the streptococcus thermophilus in a soybean lecithin-concentrated whey protein-TJA liquid culture medium, and obtaining the number of the viable bacteria of a control group of the streptococcus thermophilus in the TJA liquid culture medium.
The tolerance rate calculation method comprises the following steps: tolerance (%). the number of viable bacteria after treatment/the number of viable bacteria in the control group × 100%.
The effect of the simultaneous addition of soy lecithin and concentrated whey protein on the growth of Streptococcus thermophilus under 0.3% bile salt stress for 2.5h and pH2.0 stress for 2.5h is shown in Table 5.
Table 5:
according to the experimental results shown in Table 5, when 0.74% of soybean lecithin and 2.54% of whey protein are not added, the number of viable bacteria of the streptococcus thermophilus after 2.5 hours of action under the conditions of 0.3% of bile salt and pH2.0 is respectively 0 and 2.45, and the tolerance rate is close to 0. After 0.74% of soybean lecithin and 2.54% of whey protein are added, the log values of viable count of the streptococcus thermophilus are respectively increased to 8.47 and 8.57 after the streptococcus thermophilus acts for 2.5 hours under the conditions of 0.3% of bile salt and pH2.0, and the tolerance rate is more than 100%. This shows that the simultaneous addition of a certain proportion of soybean lecithin and concentrated whey protein can also significantly improve the tolerance rate of streptococcus thermophilus under the conditions of high bile salt content and low pH.
Example 6
A method for treating Lactobacillus bulgaricus to improve acid and bile salt resistance by adding soybean lecithin and concentrated whey protein comprises the following steps:
the lactobacillus bulgaricus is from China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 14750. The preservation specification is as follows: and (3) classification and naming: lactobacillus delbrueckii subsp bulgaricus; latin name: butganius, bacillus delbrueckii subsp; the biological material of the reference: LSR-L-L1; the preservation organization: china general microbiological culture Collection center; the preservation organization is abbreviated as: CGMCC; address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is as follows: 26 months 9 in 2017; registration number of the preservation center: CGMCCNo.14750.
(1) The MRS liquid medium and the MRS solid medium were prepared in the same manner as in steps (1) and (2) of example 1, the 0.3% bile salt-MRS liquid medium was prepared in the same manner as in step (2) of example 3, and the soybean lecithin-whey protein concentrate-MRS liquid medium was prepared in the same manner as in step (3) of example 3, and the pH 2.0 MRS liquid medium was prepared in the same manner as in step (2) of example 4.
(2) Inoculation: the activated lactobacillus bulgaricus is respectively inoculated in an MRS liquid culture medium and a soybean lecithin-concentrated whey protein-MRS liquid culture medium by the inoculation amount of 2 percent, and is statically cultured for 18 hours at the temperature of 38 ℃.
(3) pH 2.0 MRS liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing for 18h in MRS and soybean lecithin-concentrated lactalbumin-MRS liquid culture medium, centrifuging at 12000rpm for 5min, removing supernatant, suspending cell precipitate in 1mL of MRS liquid culture medium with pH of 2.0, and culturing at 38 ℃ for 2.5 h.
(4) 0.3% bile salt-MRS liquid medium treatment: respectively sucking 1mL of fermentation liquor after culturing for 18h in MRS and soybean lecithin-concentrated whey protein-MRS culture media, centrifuging for 5min at 12000rpm, removing supernatant, suspending cell precipitate in 1mL of 0.3% bile salt-MRS liquid culture medium, and culturing for 2.5h at 38 ℃.
(5) Measuring the number of viable bacteria after treatment: after each treated strain is centrifuged at 12000rpm for 5min, the supernatant is removed, 1mL of physiological saline is added, isocratic gradient dilution is carried out, the mixture is poured into a flat plate containing 20mL of MRS solid medium by a pouring method, and the colony number is measured after the mixture is cultured for 36h at 38 ℃. And obtaining the treated viable count of the lactobacillus bulgaricus in a soybean lecithin-concentrated whey protein-MRS liquid culture medium, and obtaining the control group viable count of the lactobacillus bulgaricus in an MRS liquid culture medium.
The tolerance rate calculation method comprises the following steps: tolerance (%). the number of viable bacteria after treatment/the number of viable bacteria in the control group × 100%.
The effect of simultaneous addition of soy lecithin and concentrated whey protein on the growth of Lactobacillus bulgaricus under 0.3% bile salt stress for 2.5h and pH2.0 stress for 2.5h is shown in Table 6.
Table 6:
according to the experimental results in Table 6, when 0.74% of soybean lecithin and 2.54% of whey protein are not added, the logarithmic viable count values of the Lactobacillus bulgaricus after 2.5 hours of action under the conditions of 0.3% of bile salt and pH2.0 are respectively 0 and 3.20, and the tolerance rate is close to 0. After 0.74% of soybean lecithin and 2.54% of whey protein are added, the logarithm value of the viable count of the lactobacillus bulgaricus is respectively increased to 8.62 and 8.64 after the lactobacillus bulgaricus acts for 2.5 hours under the conditions of 0.3% of bile salt and pH2.0, and the tolerance rate is more than 100%. This shows that the tolerance rate of lactobacillus bulgaricus under the conditions of high bile salt and low pH can be obviously improved by adding a certain proportion of soybean lecithin and concentrated whey protein.
The inventors have found, through experiments with examples 1 to 6, that the method of the present invention is applicable to various lactic acid bacteria including lactobacillus bulgaricus, streptococcus thermophilus, and lactobacillus paracasei. That is to say, 0.74 percent of soybean lecithin and 2.54 percent of concentrated whey protein are simultaneously added into the basic culture medium, so that the tolerance of the lactic acid bacteria under the conditions of high bile salt content and low pH value can be remarkably improved.
Claims (5)
1. A method for improving the tolerance of bile salt of lactic acid bacteria is characterized by comprising the following steps: the method comprises the step of culturing the lactic acid bacteria by using a culture medium containing soybean lecithin and concentrated whey protein, wherein the mass volume ratio of the final concentration of the soybean lecithin in the culture medium is 0.6-1.0%, and the mass volume ratio of the final concentration of the concentrated whey protein in the culture medium is 2.0-3.0%.
2. The method of claim 1, wherein the method comprises: the mass volume ratio of the final concentration of the soybean lecithin in the culture medium is 0.74 percent, and the mass volume ratio of the final concentration of the concentrated whey protein in the culture medium is 2.54 percent.
3. The method of claim 1, wherein the method comprises: the lactic acid bacteria include Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus paracasei and Lactobacillus plantarum.
4. The method of claim 1, wherein the method comprises: the culture medium is a liquid culture medium or a solid culture medium.
5. The method of claim 4, wherein the method comprises: the culture medium comprises an MRS liquid culture medium, a TJA liquid culture medium, a soybean lecithin-MRS liquid culture medium, a concentrated lactalbumin-MRS liquid culture medium, a soybean lecithin-concentrated lactalbumin-MRS liquid culture medium, an MRS solid culture medium and a TJA solid culture medium.
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| CN113278554A (en) * | 2021-05-24 | 2021-08-20 | 四川大学 | Method for improving acid resistance of lactic acid bacteria by using mixed bacteria biological membrane |
| CN113699073A (en) * | 2021-08-31 | 2021-11-26 | 中国农业大学 | Method for improving tolerance of lactobacillus bile salt and application |
| CN114468052A (en) * | 2020-11-13 | 2022-05-13 | 内蒙古伊利实业集团股份有限公司 | Children yoghurt with high probiotic survival rate after digestion and application |
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| CN103614332A (en) * | 2013-11-14 | 2014-03-05 | 江南大学 | Method for improving tolerance of lactic acid bacteria cholate |
| CN108697138A (en) * | 2015-12-17 | 2018-10-23 | Cj第制糖株式会社 | The coating method for the lactic acid bacteria that enteron aisle survival rate improves |
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| CN103614332A (en) * | 2013-11-14 | 2014-03-05 | 江南大学 | Method for improving tolerance of lactic acid bacteria cholate |
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| CN114468052A (en) * | 2020-11-13 | 2022-05-13 | 内蒙古伊利实业集团股份有限公司 | Children yoghurt with high probiotic survival rate after digestion and application |
| CN113278554A (en) * | 2021-05-24 | 2021-08-20 | 四川大学 | Method for improving acid resistance of lactic acid bacteria by using mixed bacteria biological membrane |
| CN113278554B (en) * | 2021-05-24 | 2023-05-09 | 四川大学 | Method for improving acid resistance of lactic acid bacteria by using mixed bacteria biological film |
| CN113699073A (en) * | 2021-08-31 | 2021-11-26 | 中国农业大学 | Method for improving tolerance of lactobacillus bile salt and application |
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