CN114426570A - Application of milk fat globule membrane protein in improving stress resistance of lactobacillus acidophilus - Google Patents
Application of milk fat globule membrane protein in improving stress resistance of lactobacillus acidophilus Download PDFInfo
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- CN114426570A CN114426570A CN202111479096.0A CN202111479096A CN114426570A CN 114426570 A CN114426570 A CN 114426570A CN 202111479096 A CN202111479096 A CN 202111479096A CN 114426570 A CN114426570 A CN 114426570A
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- milk fat
- lactobacillus acidophilus
- fat globule
- globule membrane
- membrane protein
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Abstract
The invention discloses the application of Milk Fat Globule Membrane Protein (MFGMP) in improving the acid stress resistance of lactobacillus acidophilus, the invention takes lactobacillus acidophilus CICC6074 as a research object, provides a proper acid stress condition, improves the acid stress resistance of lactobacillus acidophilus by adding milk fat globule membrane protein externally, and further determines various physiological indexes under the acid stress condition; the exogenously added globulins can obviously improve the growth performance of lactobacillus acidophilus and the survival rate under the condition of acid stress, keep the integrity of the thallus structure, effectively regulate the stability of intracellular pH, enhance the acid stress resistance of the thallus and provide development basis for the development and application of milk fat globulins and probiotics in the aspects of functional foods, medicines and the like.
Description
Technical Field
The invention belongs to the field of microbial protective agents, and particularly relates to application of milk fat globule membrane protein in improving the acid stress resistance of lactobacillus acidophilus.
Background
Lactic Acid Bacteria (LAB) are a general term for bacteria that can ferment glucose and lactose and other carbohydrates to produce a large amount of lactic acid, produce no spores, are anaerobic or facultative anaerobic, and most of lactic acid bacteria are gram-positive bacteria, do not move or move very little, and have mostly cocci or bacilli in their bacterial morphology. The lactobacillus is widely present in the digestive system and the respiratory system of human bodies and animals, and can regulate the normal flora of gastrointestinal tracts of organisms and keep the microecological balance; relieving lactose intolerance and promoting nutrient absorption; barrier function, regulating microecological balance of intestinal flora; reducing cholesterol and blood pressure; immunoregulation, anticancer, antitumor, etc. In addition, lactic acid bacteria produce superoxide dismutase (SOD), which can remove superoxide anion generated during metabolism, thereby delaying aging. The metabolite lactic acid can also inhibit the generation of harmful bacteria metabolite ammonia, amine, hydrogen sulfide, indole, etc.; in addition, the lactic acid bacteria can generate various volatile flavor substances in the fermentation process, and also have the effects of improving the flavor of the food, improving the storage stability of the food and the like.
The growth performance and the metabolic capability of the lactobacillus are limited to a certain extent due to the inevitable influence of various environmental stresses on the industrial production and the gastrointestinal system of a human body as the probiotic, and the function of the probiotic is seriously influenced. Various environmental stresses encountered during the survival of lactic acid bacteria mainly include pH (acid) stress, salt stress, osmotic stress, oxygen stress, freezing stress, and the like. Firstly, lactic acid bacteria are used as microorganisms which are anaerobic or facultative anaerobic and take lactic acid as a main metabolite, and oxygen stress and acid stress are mainly environmental stresses faced by the lactic acid bacteria in the fermentation process; secondly, in the downstream processing, storage and transportation processes of the lactic acid bacteria, oxygen stress and acid stress are mainly faced; finally, after the lactobacillus enters the gastrointestinal tract of a human body, the stress of gastric acid and bile salt becomes a main stress condition. While in all stress environments that lactic acid bacteria are exposed to, acid stress is undoubtedly the most damaging to lactic acid bacteria. Although most lactic acid bacteria can adapt to the acid bias environment to make the lactic acid bacteria become dominant strains due to the metabolism of the lactic acid bacteria, the normal growth and metabolism of the lactic acid bacteria can be influenced along with the continuous reduction of the pH value of the environment, particularly after the lactic acid bacteria enter a human body, the pH value of gastric acid can be as low as 2.5-3, and the lactic acid bacteria can perform the self-probiotic effect on the human body only by tolerating the stress of the acid environment of the gastrointestinal tract and living. Therefore, the resistance mechanism of lactic acid bacteria under acid stress and the finding of a method for improving the acid resistance of lactic acid bacteria become the focus of attention in academia and industry.
Milk Fat Globule Membrane (MFGM), which means that fat globules in milk consist of a core of triglycerides surrounded by a thin membrane called milk fat globule membrane. Most milk fat globule membranes are composed of membrane-specific proteins, primarily glycoproteins, and phospholipids and sphingolipids. Milk fat globule membrane proteins mainly include MUC1, xanthine dehydrogenase/chloride (XDH/XO), peptone 3(PP3), and cremophil protein (BTN). Wherein, the Fatty Acid Binding Protein (FABP) has the function of inhibiting breast cancer cells, and BRCA can inhibit tumors. The acidophilic lipoprotein (BTN) can inhibit multiple sclerosis acidophilic milk protein (BTN) and can inhibit autism to a certain extent. Xanthine dehydrogenase/chloride (XDH/XO) is bacteriostatic and can be used as gastrointestinal antibacterial agent. The milk fat globule membrane protein can relieve venous atherosclerosis and prevent Coronary Heart Disease (CHD). The milk fat globule membrane can be used for food processing due to its high nutritive value, and can not only improve the nutritive value of food, but also improve the quality of food. The milk fat globule membrane protein has not been reported to have resistance to lactic acid bacteria acid stress and protection against gastrointestinal tolerance of lactic acid bacteria.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing the application of milk fat globule membrane protein in improving the stress resistance of lactobacillus acidophilus.
The technical scheme is as follows: in order to protect lactic acid bacteria and enable the lactic acid bacteria to exert better probiotic characteristics, the invention provides application of milk fat globule membrane protein in improving the stress resistance of lactobacillus acidophilus.
Wherein the Lactobacillus acidophilus is Lactobacillus acidophilus CICC 6074.
Wherein the acid stress condition is pH 2-4.
Wherein, preferably, the acid stress condition is culture in MRS medium at pH 2.5.
The invention also comprises the application of the milk fat globule membrane protein in the preparation of probiotics or gastrointestinal tract protective agents or cell membrane protective agents. The milk lipocorm protein relieves the cell roughness and rupture of lactobacillus acidophilus when encountering acid stress, shows the protective effect on cell membranes, and further improves the resistance of lactobacillus acidophilus in acid stress.
The invention also includes the use of milk fat globule membrane proteins in the preparation of a medicament for maintaining the dynamic balance of intracellular pH or enhancing intracellular H in Lactobacillus acidophilus+-use of an ATPase active agent. The milk fat globule membrane protein is used for lactobacillus acidophilus, can more effectively adjust the change of intracellular pH, and maintains the dynamic balance of the intracellular pH, thereby ensuring the application of the lactobacillus acidophilus in survival under the condition of acid stress. The milk fat globule membrane protein is used for lactobacillus acidophilus under acid stress condition, and can obviously enhance intracellular H of lactobacillus acidophilus+The activity of ATPases, which may also be responsible for the increased resistance of milk lipocorms to stress by Lactobacillus acidophilus.
The milk fat globule membrane protein of the invention contains abundant protein and amino acid which enter cells and cause the content of the amino acid in the acidophilic lactobacillus cell to be obviously increased.
The invention also comprises the application of the milk fat globule membrane protein in preparing the synthetic medicine for promoting the peptidoglycan. The invention also comprises the application of the milk fat globule membrane protein in preparing the medicine for reducing purine metabolism, ribosome synthesis, ABC transfer related protein and reducing DNA synthesis, translation and transfer of thalli. After the milk fat globule membrane protein is used for lactobacillus acidophilus under the acid stress condition, the integrity of a cell structure is maintained by promoting the synthesis of peptidoglycan through a protein component analysis strain; the protein related to purine metabolism, ribosome synthesis and ABC transfer is reduced, the DNA synthesis, translation and transfer of thalli are reduced, and the growth speed of the thalli is reduced so as to adapt to an acidic environment; up-regulating amino acid metabolism, DNA repair proteins to relieve the acidic environment in the cell and repair damaged DNA to reduce the damage of acid stress to the strain.
The invention also comprises the application of the milk fat globule membrane protein in preparing the medicine for up-regulating the amino acid metabolism and DNA repair protein.
The invention improves the acid stress resistance of lactobacillus acidophilus by adding milk fat globule membrane protein externally, and the milk fat globule membrane protein and probiotics are developed and applied in the aspects of functional foods, medicines and the like.
Has the advantages that: the milk fat globule membrane protein of the invention protects lactobacillus acidophilus from acid hypochondriumCompared with casein, lactalbumin and bovine serum albumin, the exogenous addition of lactoglobulin can obviously improve the growth performance of lactobacillus acidophilus and the survival rate under the condition of acid stress, and the cell survival rate is 1.9 times that of the unadditized lactobacillus acidophilus. Meanwhile, the bacterial strain added with milk fat globule membrane protein keeps higher pHi and H+The ATP enzyme activity and the amino acid content can effectively regulate the stability of intracellular pH and enhance the acid stress resistance of the thalli; the addition of milk fat globule membrane protein can better maintain the integrity of the thallus structure, and reduce the outflow of intracellular substances and the entry of extracellular harmful substances, thereby effectively reducing the damage of acid stress to cells; the addition of milk fat globule membrane protein can change the expression of regulation protein of cell metabolism and energy generation, promote the expression of DNA replication, transcription and translation related protein, promote the generation of a large amount of DNA repair protein, maintain the normal metabolism of thalli in acid stress, and reduce the damage of acid stress to cells.
Drawings
FIG. 1 is a graph of the survival rate of Lactobacillus acidophilus CICC6074 under different pH conditions;
FIG. 2 is a graph showing the effect of different proteins on the survival rate of Lactobacillus acidophilus CICC6074 under acid stress;
FIG. 3 is a graph showing the effect of milk fat globule membrane protein at different concentrations on the growth of Lactobacillus acidophilus;
FIG. 4 is a graph showing the effect of different concentrations of milk fat globule membrane protein on the survival rate of Lactobacillus acidophilus under acid stress;
FIG. 5 is a graph of the effect of milk fat globule membrane protein on intracellular pH of Lactobacillus acidophilus following acid stress;
FIG. 6H of Lactobacillus acidophilus after acid stress of milk lipocorm protein+-map of atpase activity;
FIG. 7 is a scanning electron micrograph of Lactobacillus acidophilus after acid stress of milk lipocorm protein;
FIG. 8 is a graph showing the total amino acid content of Lactobacillus acidophilus after acid stress on milk lipocorm protein;
FIG. 9 the effect of milk fat globule membrane protein on the intracellular content of amino acids of Lactobacillus acidophilus after acid stress;
FIG. 10 is a graph of comparison results of differential proteins GO, COG and KEGG of Lactobacillus acidophilus after acid stress of milk fat globule membrane protein, FIG. 10 is an AGO functional classification graph; FIG. 10BCOG functional classification diagram; figure 10C upregulated differential protein KEGG metabolic pathway classification map; figure 10D downregulation of differential prominent proteins KEGG metabolic pathway classification map. Fig. 10B represents different english letters: (A) RNA processing and modification; (B) chromatin structure and dynamics; (C) energy generation and conversion; (D) cell cycle control, cell division, chromosome division; (E) amino acid transport and metabolism; (F) nucleotide transport and metabolism; (H) coenzyme transport and metabolism; (I) lipid transport and metabolism; (J) translation, ribosomal structure, and biogenesis; (K) transcription; (L) replication, recombination and repair; (M) cell wall/membrane/envelope biogenesis; (N) cell activity; (O) post-translational modifications, protein conversions, chaperones; (P) inorganic ion transport and metabolism; (Q) biosynthesis, transport and catabolism of secondary metabolites; (R) general function prediction; (S) the function is unknown; (T) a signal transduction mechanism; (U) intracellular trafficking, secretion, and vesicle trafficking; (V) defense mechanisms; (W) extracellular structures; (Y) a nuclear structure; (Z) cytoskeleton.
FIG. 11 expression profiles of F0F1-ATPase, P-type-ATPase and peptidoglycan synthesis-related proteins of milk lipocorm protein after acid stress on Lactobacillus acidophilus;
FIG. 12 is a graph showing the expression profiles of amino acid metabolism-related proteins after the stress protection of milk fat globule membrane proteins against Lactobacillus acidophilus acid;
FIG. 13 is a DNA replication and transcription associated protein expression profile of milk fat globule membrane proteins protected against Lactobacillus acidophilus acid stress;
FIG. 14 shows the expression profiles of proteins related to DNA and protein repair after the stress protection of milk fat globule membrane protein against Lactobacillus acidophilus acid;
FIG. 15 effect of different proteins on survival rate of Lactobacillus acidophilus CICC6074 under bile salt stress;
FIG. 16 Effect of different proteins on survival of Lactobacillus acidophilus CICC6074 acid stress.
Detailed Description
The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.
The milk fat globule membrane protein in the embodiment of the invention is Hilmar TM 7500 whey protein powder, which is purchased from Hilmar company, and the Hilmar TM 7500 has the following product components: 70% of protein, 6.5% of phospholipid, 15% of fat, 3-6% of lactose, 4.5% of water and 3.0% of ash.
Example 1 Effect of different proteins on survival under lactic acid stress conditions
1 Effect of different pH on survival Rate of Lactobacillus acidophilus CICC6074
Taking a glycerol stock solution of lactobacillus acidophilus CICC6074 (purchased from China center for culture Collection of industrial microorganisms) at the temperature of-80 ℃, selecting a proper amount of the stock solution by using an aseptic toothpick, streaking the stock solution on an MRS agar culture medium, culturing the stock solution in an incubator at the temperature of 37 ℃ until a single colony is formed, then selecting the single colony in 5mL of the MRS liquid culture medium, statically culturing the single colony at the temperature of 37 ℃ until the single colony is in a stable initial stage (18h), inoculating the single colony into the sterilized MRS liquid culture medium according to the inoculum size of 2 percent (v/v), and statically culturing the single colony at the temperature of 37 ℃ until the single colony is in the stable initial stage (18h) to ensure that the thallus reaches 1.9 multiplied by 109CFU/mL. Centrifuging at 4 deg.C for 10min at 3000g, and collecting thallus. The collected thalli are resuspended in MRS broth culture medium with pH of 2.0, 2.5, 3.0 and 4.0, incubated at 37 ℃ for 3h, bacterial liquid is collected every 1h, and viable bacteria count is carried out according to GB4789.2-2010 national food safety Standard food microbiology inspection colony count determination method. The tolerance is calculated as follows:
N1: viable count under the condition of enduring treatment
N0: pH 6.4 (Normal) MRS Broth Medium
2 influence of different proteins on the survival rate of Lactobacillus acidophilus CICC6074 under stress conditions:
taking Lactobacillus acidophilus CICC6074 (purchased from China Industrial culture Collection of microorganisms) at-80 deg.CCenter), selecting a proper amount of stock solution with a sterile toothpick to perform streaking on an MRS agar culture medium, culturing in an incubator at 37 ℃ until a single colony is formed, then selecting the single colony in 5mL of the MRS liquid culture medium, and performing static culture at 37 ℃ until the thalli reaches 1.9 multiplied by 10 for 18 hours at a stable initial stage9CFU/mL. Inoculating into sterilized MRS liquid culture medium at an inoculum size of 2% (v/v), and standing at 37 deg.C until the initial stage of stabilization (18 h). Centrifuging at 4 deg.C for 10min at 3000g, collecting thallus, washing thallus with pre-cooled sterilized normal saline for three times, collecting, suspending the thallus in MRS culture medium containing 10mg/mL whey protein, casein, bovine serum albumin and milk fat globule membrane protein and having pH of 2.5, standing the thallus suspension, forcing the thallus suspension in 37 deg.C incubator for 3h, and taking out bacterial liquid plate at intervals of 1h, and coating to analyze viable count.
The survival rate of the lactic acid bacteria under each condition is calculated by the following formula.
N1: viable count under the condition of enduring treatment
N0: pH 6.4 (Normal) MRS Broth Medium
3 Experimental data processing and analysis
The experimental data were statistically analyzed using SPSS19.0 statistical software, and the one-way analysis of variance examined the differences in significance between groups, with data expressed as mean + -SD and different letters indicating significant differences at levels P < 0.05.
4 results and analysis
4.1 Effect of different pH on survival rates of Lactobacillus acidophilus CICC6074
As shown in fig. 1, it can be seen from fig. 1 that the survival rate of lactobacillus acidophilus decreases with decreasing pH, and when pH is 4, the survival rate of lactobacillus acidophilus shows a trend of increasing at 1h of stress and a trend of decreasing after 2h, but the decreasing trend is not significant; when the pH value is 3, the survival rate of the lactobacillus acidophilus is wholly in a descending trend, the survival rate of the lactobacillus acidophilus is kept about 90 percent all the time, and the descending trend is not obvious; when the pH value is 2.5, the survival rate of the lactic acid bacteria is remarkably reduced along with the prolonging of the stress time, and after the stress time is 3 hours, the survival rate is reduced to 36.65%; when pH is 2, lactobacillus acidophilus hardly grows in an acid stress environment, and its survival rate drops to 0. From the above results, it can be concluded that the survival rate was significantly decreased when pH2.5 and decreased to 36.65% after 3h of stress, approaching a sublethal level, and thus pH2.5 was selected as the acid stress treatment condition.
4.2 Effect of different proteins on survival Rate of Lactobacillus acidophilus CICC6074 under acid stress
As shown in fig. 2, it can be seen from fig. 2 that the survival rate of the lactic acid bacteria added with casein, whey protein and bovine serum albumin after 1h treatment is not significantly different from that of the blank MRS, and the survival rate is significantly improved only by adding milk fat globule membrane protein; after 2h of treatment, compared with a blank MRS group, the survival rate of the lactobacillus added with different proteins is obviously improved, but the group added with casein and milk fat globule membrane protein has an obvious effect; after 3 hours of treatment, the group added with the whey protein and the bovine serum albumin has no significant difference with the blank MRS group, the group added with the casein and the milk fat globule membrane protein still has better effect, and the protection effect of the added milk fat globule membrane protein is far better than that of the added casein. Therefore, compared with other proteins, the milk fat globule membrane protein can obviously improve the acid stress resistance of lactobacillus acidophilus.
Example 2 Effect of milk fat globule membrane proteins on survival under stress by Lactobacillus acidophilus acid
1 Effect of milk fat globule membrane protein on the growth of Lactobacillus acidophilus CICC6074
Taking a glycerol stock solution of lactobacillus acidophilus CICC6074 (purchased from China center for culture collection of industrial microorganisms) at a temperature of-80 ℃, picking a proper amount of the stock solution by using an aseptic toothpick, streaking the stock solution on an MRS agar culture medium, culturing the stock solution in an incubator at a temperature of 37 ℃ until a single colony is formed, then picking the single colony in a 5mL MRS liquid culture medium, carrying out static culture at the temperature of 37 ℃ until the initial stage is stable (18h), inoculating the single colony into the sterilized MRS liquid culture medium respectively containing 0, 2.5, 5 and 10mg/mL of milk fat globule membrane protein according to the inoculum size of 2 percent (v/v), uniformly mixing and distributing the sterilized MRS liquid culture medium into sterilized centrifuge tubes, carrying out static culture at the temperature of 37 ℃, randomly and respectively taking out bacterial solutions under the protection of different concentrations of protein at intervals of 4h to carry out plate coating analysis (n is 3), and detecting the change condition of the viable count of the bacterial solutions at each time point.
2 Effect of milk fat globule membrane proteins with different concentrations on survival rate of Lactobacillus acidophilus CICC6074 under acid stress
Taking a glycerol stock solution of lactobacillus acidophilus CICC6074 (purchased from China center for culture Collection of industrial microorganisms) at the temperature of-80 ℃, selecting a proper amount of the stock solution by using a sterile toothpick, carrying out lineation on an MRS agar culture medium, culturing in an incubator at the temperature of 37 ℃ until a single colony is formed, then selecting the single colony in 5mL of the MRS liquid culture medium, and carrying out static culture at the temperature of 37 ℃ until the thalli reaches 1.9 multiplied by 10 (18h) at the initial stage of stabilization9CFU/mL. Inoculating 2% of the strain into an MRS liquid culture medium, performing static culture at 37 ℃ for 18h, centrifuging at 4 ℃ for 10min at 3000g, collecting thalli, washing the thalli with precooled sterilized normal saline for three times, collecting the thalli, respectively suspending the obtained thalli in the MRS culture medium containing milk fat globule membrane proteins (0, 1, 2.5, 5 and 10mg/L) with different concentrations and the pH value of 2.5, performing static stress in an incubator at 37 ℃ for 3h, and taking a bacterial liquid plate at intervals of 1h to coat and analyze the number of viable bacteria in the bacterial liquid culture medium. The survival rate of the lactic acid bacteria under each condition is calculated by the following formula.
N1: viable count under the condition of enduring treatment
N0: pH 6.4 (Normal) MRS Broth Medium
3 Experimental data processing and analysis
The experimental data were statistically analyzed using SPSS19.0 statistical software, and the one-way analysis of variance examined the differences in significance between groups, with data expressed as mean + -SD and different letters indicating significant differences at levels P < 0.05.
4 results and analysis
4.1 Effect of milk fat globule membrane protein on the growth of Lactobacillus acidophilus
As shown in FIG. 3, it can be seen from FIG. 3 that when the milk lipocorm protein concentration is 2.5mg/mL, there is no significant difference from the growth curve of lactobacillus in MRS blank, while milk lipocorm protein of 5mg/mL and 10mg/mL has significant promotion effect on the growth of lactobacillus acidophilus and there is a certain concentration dependence. This is probably because the abundant proteins in milk fat globule membrane proteins provide certain nutrients for the growth of lactic acid bacteria and promote their growth.
4.2 Effect of milk fat globule membrane protein on survival Rate of Lactobacillus acidophilus CICC6074 acid stress
As shown in FIG. 4, the cells were separately resuspended in MRS medium containing milk fat globule membrane proteins (0, 1, 2.5, 5, 10mg/L) at different concentrations and pH2.5 for stress culture. Compared with a blank control, the survival capacity of lactobacillus acidophilus is remarkably improved by adding the milk fat globule membrane protein, and after the lactobacillus acidophilus is cultured for 1h under stress, the survival rate is not reduced but is in an increasing trend except for a blank MRS group and a milk fat globule membrane protein group with the addition concentration of 1 mg/mL; after the milk lipocorm protein group with the addition concentration of 1mg/mL and the blank MRS group have no significant difference after the stress culture for 2 hours, the survival rates of other groups are all significantly improved, and when the milk lipocorm protein addition concentration is 10mg/mL, the viable count is 2.43 multiplied by 107The survival rate is 88.65 percent, which is 1.64 times of that of the blank MRS group; after the milk fat globule membrane protein group with the addition concentration of 1mg/mL and 2.5mg/mL and the blank MRS group have no significant difference after the stress culture for 3 hours, the survival rate of the other two groups is significantly improved, and particularly when the addition concentration is 10mg/mL, the viable count is 1.74 multiplied by 106The survival rate was 73.64%, which was 1.9-fold higher than that of the blank group. Therefore, the survival condition of lactobacillus acidophilus CICC6074 under the acid stress condition can be effectively improved by adding the milk fat globule membrane protein, certain concentration dependence exists, and the milk fat globule membrane protein with the concentration of 10mg/mL is selected as the subsequent addition concentration according to the result.
Example 3 Effect of milk fat globule membrane proteins on intracellular and extracellular stress of Lactobacillus acidophilus
1 determination of intracellular pH:
taking out growth to the beginning of stabilizationThe concentration reaches 1.9 multiplied by 10 in the period (18h)9And (3) respectively resuspending the CFU/mL thallus in a common MRS culture medium, an MRS culture medium with the pH of 2.5 and an MRS culture medium containing 10mg/mL milk fat globule membrane protein and the pH of 2.5 for 3 hours, and washing for later use.
Determination of the standard curve for pHi (intracellular pH): the growth is carried out until the concentration of the stabilizer reaches 1.9X 109CFU/mL cells were resuspended in HEPES buffer for standard curve, and 1mol/L of valinomycin and nigericin were added to balance cell pHi (intracellular pH) and pHex (ambient pH). The cells were washed again and collected, resuspended in a buffer for standard curves of pH 4, 5, 6, 7 and 8, respectively, and 1. mu.L of BCECFAM (purchased from Shanghai Binyun Biotech Co., Ltd.: S1006) as a pH fluorescent probe was added, and the mixture was subjected to a water bath at 30 ℃ for 20min in the dark, and centrifuged to collect the cells and washed with the same buffer. Then, the fluorescence intensity (Itotal) of the bacterial suspension and the fluorescence intensity (Ifiltrate) of the supernatant were measured at excitation wavelengths of 490nm and 440nm, emission wavelengths of 525nm, and a slit width of 5nm, respectively. The fluorescence intensity I was calculated according to the following formula, and a standard curve was plotted with pH as abscissa and 1gI as ordinate, the standard curve formula being as follows: y 0.0288x +0.5901, R2＝0.9907。
Determination of the pHi of the sample: the bacterial cells were resuspended in 50 mmol. multidot.L-1 HEPES-K (pH 8.0) buffer, and 1. mu.L of the pH fluorescent probe BCECF AM (2 ', 7' -bis- (2-carboxyethyl) -5- (and-6) -carboxyfluorescein, acetoxymethyl ester) was added, washed with an equal volume of 50mmol/L phosphate buffer in a dark water bath for 20min at 30 ℃ and resuspended. Then, the fluorescence intensity (I) of the bacterial suspension is respectively measured when the excitation wavelength is 490nm, the emission wavelength is 525nm and the slit width is 5nmtotal) And fluorescence intensity (I) of the supernatantfiltrate). pHi was calculated from the standard curve. (fluorescence intensity reflects pH value according to pH fluorescence probe BCECF AM kit)
2 H+Determination of the ATPase Activity
Taking 10mL of the mixture to grow until the concentration of the mixture in the stationary phase reaches 1.9 multiplied by 109And (3) respectively resuspending the CFU/mL thallus in an MRS culture medium containing 0 and 10mg/mL milk fat globule membrane proteins and having the pH of 2.5 and a common MRS culture medium for 3h under stress. The acid-stressed bacterial suspension was shaken, centrifuged (12000g, 10min, 4 ℃), washed with physiological saline and resuspended (cell: physiological saline: 1: 9). And (3) carrying out ultrasonic wall breaking on the thalli under the ice bath condition until no obvious thalli form is observed under a microscope, and centrifuging to take supernatant. Taking the obtained liquid to be measured according to the formula H+Instructions for ATPase activity kit, adding reagents in sequence, finally zeroing with distilled water at 660nm, and measuring the absorbance of each tube with an ultraviolet spectrophotometer. 3 observing the condition of the cell membrane of the lactobacillus by a scanning electron microscope
The concentration is up to 1.9X 109Inoculating lactobacillus acidophilus CICC6074 of CFU/mL into an MRS broth culture medium (10.0g/L peptone, 8.0g/L beef extract powder, 4.0g/L yeast extract powder, 20.0g/L glucose, 2.0g/L dipotassium hydrogen phosphate, 2.0g/L diammonium hydrogen citrate, 5.0g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04g/L manganese sulfate and 1.0g/L Tween 80) in an inoculation amount of 2%, standing and culturing at 37 ℃ for 18h, centrifuging at 4 ℃ for 10min at 3000g, collecting thalli, respectively suspending the obtained thalli in an MRS culture medium containing 0 and 10mg/mL milk fat globule membrane protein and having a pH value of 2.5 and a normal MRS culture medium, and standing and stressing the thalli suspension in a 37 ℃ culture box for 3 h. Three groups of lactic acid bacteria were subsequently washed 3 times with PBS. Glutaraldehyde is added respectively and fixed overnight at normal temperature, 3000g is centrifuged for 15min, and the supernatant is removed. Washing with precooled PBS for 3 times, performing gradient dehydration for 8-10 min with prepared 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol, treating with isoamyl acetate for 20min for replacement, and freeze-drying the thallus. And adhering a carbon conductive adhesive tape on the sample table, uniformly coating the freeze-dried samples on the adhesive tape respectively, and putting the adhesive tape into the sample chamber for gold spraying treatment. The three groups of samples were placed in a scanning electron microscope for observation.
4 determination of intracellular amino acid content of milk fat globule membrane protein under stress of lactobacillus acidophilus acid
50mL of the solution is taken to grow until the stationary phase concentration reaches 1.9 multiplied by 109CFU/mL of the cells were resuspended in a medium containing 0 and 10mg/mL milk fat globule membrane protein, MRS culture medium with pH of 2.5 and common MRS culture medium for 3h, and centrifuging and washing for later use. And taking the thallus precipitate, suspending the thallus precipitate in 20mL of 6mol/L HCl solution, hydrolyzing for 22-24 h at 110 ℃, taking out, cooling, and transferring to a 25mL colorimetric tube for constant volume. Taking 1mL of clear liquid, drying in a water bath at 85 ℃, adding 1mL of water, and drying. 10mL of 0.02mol/L HCl was added and shaken well. mu.L of the mixture was aliquoted and added with 250. mu.L of 0.1mol/L phenylacetonitrile and 250. mu.L of 1mol/L triethylamine acetonitrile for derivatization for 1 hour. Adding 2mL of n-hexane, shaking and standing. Separating the layers, separating the lower layer, passing through 0.45 μm organic film, and analyzing with high performance liquid chromatograph. The chromatographic conditions are as follows: column C18 SHISEIDO, 4.6mm 250mm 5 μm; the column temperature is 40 ℃; the detection wavelength is 254 nm; the flow rate was 1 mL/min. Mobile phase A: 0.1mol/L anhydrous sodium acetate and 0.1mol/L acetonitrile, and after mixing uniformly, adjusting the pH value to 6.5. Mobile phase B: 80% acetonitrile. Sample introduction amount: 10 μ L. The elution procedure is shown in table 1.
TABLE 1 elution procedure
5 TMT proteomics analysis
5.1 preparation of cells
Inoculating activated Lactobacillus acidophilus CICC6074 into MRS culture medium at an inoculum size of 2%, standing at 37 deg.C for 18h, centrifuging at 4 deg.C for 10min, and collecting thallus. The collected cells were separately resuspended in MRS medium having a pH of 2.5 and MRS medium having a pH of 2.5 containing 10mg/mL milk fat globule membrane protein, cultured at 37 ℃ for 3 hours, then washed with physiological saline and the cell pellet was collected and frozen with liquid nitrogen for use.
5.2 extraction of proteins
Transferring the collected thallus precipitate to an MP shaking tube, adding a proper amount of extraction button (1% SDS, 200mM DTT, 50mM Tris-HCl, pH 8.8) and protease inhibitor, and vortexing and mixing uniformly. Shaking for 3 times with high throughput tissue grinder for 40s each time, then cracking on ice for 30min, vortexing every 5min for 5-10s, incubating at 100 deg.C for 10min, and cooling on ice. After the sample was completely cooled, the supernatant was centrifuged (12000g/min, 20min, 4 ℃) and the pre-cooled acetone was added at 1: 4 and precipitated overnight at-20 ℃. The next day, at 4 deg.C, 12000g was centrifuged for 20min to discard the supernatant, 90% pre-cooled acetone was added, the mixture was mixed well and centrifuged to discard the supernatant, and the process was repeated twice. Adding protein lysis solution (8M urea, 1% SDS, containing protease inhibitor) into the precipitate, vortexing and shaking to dissolve the precipitate, and centrifuging at 12000g for 20min at 4 deg.C to collect protein supernatant.
5.3 reductive alkylation and enzymatic hydrolysis of samples
A protein sample (100. mu.g, mycoprotein extracted at 5.2 above) was taken, 100mM TEAB (triethylammonium bicarbonate solution) was added to give a final concentration of 100mM, and then 0.5M TCEP (tris (2-carboxyethyl) phosphine) was added to give a final concentration of 10mM, and the reaction was carried out at 37 ℃ for 60 min. IAM (iodoacetamide) was added to a final concentration of 40mM and the reaction was carried out for 40min at room temperature with exclusion of light. Precooled acetone (acetone: sample 6: 1(v/v)) was added to each tube and precipitated at-20 ℃ for 4h, after the reaction was finished 10000.g was centrifuged for 20min and the precipitate was taken. The precipitate was dissolved well with 100. mu.L of TEAB at a concentration of 100mM, and trypsin was added at a ratio of 1: 50(m/m) enzyme to protein for enzymatic hydrolysis overnight.
5.4 TMT labelling and peptide fragment isolation
TMT marking: the labeling of the polypeptide samples was performed using the TMT 10Plex kit. TMT alone was labeled as follows for six polypeptide samples (MRS medium at pH2.5 and MRS medium at pH2.5 containing milk lipocorm protein, 3h samples were cultured under stress, three samples in parallel each): pH-1(128C label), pH-2(129N label), pH-3 (129V label), pH-MFGM1(130N), pH-MFGM2(130C), pH-MFGM3 (131). Taking out the TMT label reagent according to the quantity of the sample, centrifugally adding acetonitrile after the temperature is restored to room temperature, and carrying out vortex centrifugation. One tube of TMT reagent was added per 100. mu.g of polypeptide sample, and the reaction was allowed to stand at room temperature for 2h, followed by addition of hydroxylamine, and left at room temperature for 30min, and finally dried by vacuum concentration.
Peptide fragment separation: the labeled polypeptide sample was reconstituted with UPLC loading buffer (2% acetonitrile, pH 10) and subjected to high pH liquid phase separation using reverse phase C18 column. A total of 20 fractions were collected according to peak shape and time, combined into 10 fractions, and concentrated by centrifugation in vacuo. The chromatographic conditions are as follows: a chromatographic column: ACQUITYUPLC BEH C18, 2.1mm × 150mm × 1.7 μm; the detection wavelength is 214 nm; the flow rate was 200. mu.L/min. Mobile phase A: 2% acetonitrile, ammonia to adjust pH to 10. Mobile phase B: 80% acetonitrile, ammonia to pH 10. Sample introduction amount: 10 μ L. The elution procedure is shown in table 1.
5.5 proteomics data analysis
All LC-MS/MS raw data files were analyzed using a Proteome scanner TM Software 2.4. And during library search, submitting the file to a protocol discover server, selecting the established database, and then searching the database. Subsequently, a spectrum and a peptide fragment list of the significance identification are obtained. According to the protein abundance level, if the expression amount of a certain protein in the experimental group is different from that of the Control group by more than 1.2 or less than 0.83 and the P-value is statistically tested to be less than 0.05, the protein is considered as a differentially expressed protein between the samples of the Control normal group and the pH stressed group. One or more groups of proteins are analyzed in NCBI databases, and functional metabolic pathways of differential proteins are analyzed in GO, COG and KEGG databases.
6 Experimental data processing and analysis
The experimental data were statistically analyzed using SPSS19.0 statistical software, and the one-way analysis of variance examined the differences in significance between groups, with data expressed as mean + -SD and different letters indicating significant differences at levels P < 0.05.
7 results and analysis
7.1 Effect of milk fat globule membrane protein on intracellular pH
As shown in fig. 5, after 3h of stress under acid stress, the pHi of the blank MRS group and the group added with milk fat globule membrane protein decreased, but the intracellular pHi of lactobacillus acidophilus added with milk fat globule membrane protein was maintained at 5.79, which is significantly higher than the cell pHi of blank MRS not added with milk fat globule membrane protein: only 5.50; furthermore, there was no significant difference in the decrease of pHi of Lactobacillus acidophilus to which milk fat globule membrane protein was added, compared to the intracellular pHi of normal cells. The result shows that the lactobacillus acidophilus added with the milk fat globule membrane protein can more effectively adjust the change of the intracellular pH value and maintain the dynamic balance of the intracellular pH value, thereby ensuring the survival of the lactobacillus acidophilus under the condition of acid stress.
7.2 addition of milk fat globule membrane protein to intracellular H+Influence of ATPase Activity
The results are shown in FIG. 6, in which H is present in Lactobacillus acidophilus cells under normal conditions+Intracellular H of Lactobacillus acidophilus with an ATPase activity of 32.16U/mg protein, without addition of milk lipocorm protein+ATP enzyme activity of only 3.67U/mg protein, intracellular H of Lactobacillus acidophilus supplemented with milk lipocorm protein+The ATP enzyme activity was 21.64U/mg protein, and although it was significantly different from that of the cells under normal conditions, intracellular H was observed in comparison with cells to which lactofat globule membrane protein had not been added+The activity of ATPase was increased 5.9-fold. Therefore, the addition of milk fat globule membrane protein can obviously enhance intracellular H in lactobacillus acidophilus under the condition of acid stress+-ATPase activity, which may also be responsible for the increased stress resistance of Lactobacillus acidophilus by milk lipococcal membrane proteins.
7.3 morphological Effect of milk fat globule membrane proteins on the stress of Lactobacillus acidophilus acid
As a result, as shown in FIG. 7, FIG. 7A shows the cell morphology in the normal state, the structure was well maintained, and the cell membrane was completely smooth; the figure B shows the shape of the thallus after acid stress for 3 hours at pH2.5, and the surface of the thallus of the cell becomes rough, similar to the surface of balsam pear, and partial thallus even has the phenomenon of overflowing of intracellular substances caused by the breakage of cell membranes; and the figure C shows the form of the thallus added with milk fat globule membrane protein and stressed for 3h by pH2.5, and shows that the thallus structure is kept more complete, only a small part of thallus has rough surface, and the phenomenon of overflowing of intracellular substances caused by cell membrane disruption does not occur. It can be seen that when lactobacillus acidophilus is subjected to acid stress, milk lipocorm protein relieves cell roughness and rupture, shows protective effect on cell membrane, and thus improves resistance of lactobacillus acidophilus in acid stress.
7.4 Effect of milk fat globule membrane proteins on intracellular amino acid content under stress of Lactobacillus acidophilus acid
The results are shown in fig. 8, the intracellular amino acid content of lactobacillus acidophilus not added with milk lipoglobulin slightly decreased after 3h of stress, but there was no significant difference compared to the strain under normal conditions; the content of the amino acid in the lactobacillus acidophilus added with the milk fat globule membrane protein is obviously increased, is 2.19 times of that of the bacterial strain under normal conditions, and is 2.3 times of that of the lactobacillus acidophilus which is not added with the milk fat globule membrane protein and is subjected to acid stress, which is probably the total content change of the amino acid caused by the fact that the milk fat globule membrane protein contains abundant protein and amino acid which enter cells.
In lactic acid bacteria, arginine can be used to produce NH via the ADI pathway under the action of arginine deiminase3ATP and CO2Wherein NH3Can be used to neutralize intracellular H+ATP may be used as H+ATPase of H+The arginine is discharged to provide energy in vitro, so that the accumulation of intracellular arginine can effectively enhance the survival rate of the thalli under the condition of acid stress. As can be seen from fig. 9A, the intracellular arginine content of lactobacillus acidophilus, to which milk lipocorm protein was not added after 3h of stress, was slightly decreased, but there was no significant difference compared to the strain under normal conditions; the content of the amino acid in the lactobacillus acidophilus added with the milk fat globule membrane protein is obviously increased, is 2.31 times of the content of the amino acid in the lactobacillus acidophilus added with the milk fat globule membrane protein under the normal condition, and is 2.42 times of the content of the amino acid in the lactobacillus acidophilus added with the milk fat globule membrane protein and subjected to acid stress.
Glutamate can consume one molecule of H through the glutamate decarboxylase pathway (GAD)+And gamma-aminobutyric acid with stronger alkalinity is generated to reduce the intracellular pH value, thereby improving the survival rate of the thalli. In the aspartate decarboxylase pathway (AspD), aspartate consumes intracellular H under the action of aspartate decarboxylase+Producing alanine, and simultaneously producing glutamic acid and NH by aspartic acid under the action of asparaginase3On the one hand NH3Can be used to neutralize intracellular H+On the other hand, the produced glutamate can re-enter the GAD pathway, consuming intracellular H+. Thus, the assay measures the intracellular glutamate, aspartate and alanine content of Lactobacillus acidophilus. It can be seen from the figure thatCompared with the strain under the normal condition, the content of three glutamic acids in the lactobacillus acidophilus cell without adding milk fat globule membrane protein is slightly reduced, but no significant difference exists; the content of amino acids in the lactobacillus acidophilus cells added with milk lipocorm protein is obviously increased, wherein the content of glutamic acid is more obviously increased, probably because aspartic acid generates more glutamic acid under the action of asparaginase.
7.5 differential protein analysis
Through TMT analysis, 538 different proteins are identified, wherein 439 proteins are up-regulated, 99 proteins are down-regulated, and the up-regulated proteins are more than the down-regulated proteins, which indicates that milk fat globule membrane proteins can cope with acid stress environment by up-regulating the expression amount of certain proteins of lactic acid bacteria. The results are shown in fig. 10, the differential proteins were compared with GO, COG, KEGG databases, respectively, and the functions of the differential proteins were enriched and annotated. The up-regulated significantly different proteins are mainly distributed in L replication, recombination and repair, J translation, ribosome structure and biosynthesis, transport and metabolism of F nucleotide, biosynthesis of M cell wall and cell membrane, transport and metabolism of E amino acid, transport and metabolism of G carbohydrate and the like; downregulation of significantly different proteins is mainly distributed in J translation, ribosomal structure and biosynthesis, transport and metabolism of G carbohydrates, K transcription, etc. The differential proteins between the two groups are brought into a KEGG metabolic pathway diagram, the statistical result of the up-regulated significant proteins is shown in FIG. 10C, and the statistical result is mainly distributed in a global and schematic diagram of a metabolic lower level, sugar metabolism, nucleotide metabolism and amino acid metabolism, membrane transport of an environmental information processing lower level, replication and repair of a genetic information processing lower level and the like; the statistical results of the down-regulated significantly different proteins are shown in fig. 10D, and are mainly distributed in translation at the lower level of genetic information processing, global and profile and carbohydrate metabolism at the lower level of metabolism, membrane transport at the lower level of environmental information processing, and the like.
7.6 Effect of differential proteins on Strain acid stress
7.6.1 Effect of milk fat globule membrane proteins on cellular metabolism
FIG. 11 is the expression pattern of protein related to the synthesis of Lactobacillus acidophilus F0F1-ATPase, P-ATPase and peptidoglycan under the acid stress environment after the treatment of milk fat globule membrane protein. F0F1-ATPase (H + -ATPase) is membrane-bound protease, and after milk fat globule membrane protein is added, subunits related to F0F1-ATPase, such as AtpB, are up-regulated, which indicates that milk fat globule membrane protein may promote the expression of F0F1-ATPase to consume ATP in cells, thereby removing H in cells+Is discharged out of the body to maintain the intracellular pH near neutral. In addition, the expression level of the P-ATPase is obviously increased in the strain added with the milk fat globule membrane protein, which indicates that the milk fat globule membrane protein can promote the expression of the P-ATPase so as to maintain the intracellular pH. Penicillin Binding Proteins (PBPs) are the major proteins involved in peptidoglycan assembly at this stage. From the proteome analysis, it was found that the expression levels of the penicillin binding proteins PBP1B and PBP2A were significantly increased by adding milk fat globule membrane protein. Therefore, the addition of milk fat globule membrane protein may promote the third stage of peptidoglycan synthesis, thereby promoting peptidoglycan synthesis, and thus being beneficial to maintaining the integrity of cell structure, and the scanning electron microscope results also show that the addition of milk fat globule membrane protein can better maintain the integrity of cells, thereby better resisting acid stress and reducing the damage of acid stress to cells.
7.6.2 cell metabolism and energy production related protein
The results are shown in fig. 12, and after adding milk fat globule membrane protein, glutamate decarboxylase pathway-associated protein such as glutamate/gamma-aminobutyric acid antiporter (GadC) is significantly up-regulated, which indicates that milk fat globule membrane protein can promote glutamate decarboxylase (GAD) pathway of lactobacillus acidophilus and consume intracellular H+The pH value in the cells is improved, and the acid resistance of the cells is improved. Aspartic acid can form asparagine (reversibly) under the action of asparaginase or further form alanine under the action of aspartate decarboxylase, and the expression level of the asparaginase (AnsA) is remarkably increased and is not greatly changed through analysis. In addition, aspartate eventually produces oxaloacetate under the action of aspartate racemase (RacD), whereas oxaloacetate undergoes glutamate formation after the TCA cycle and glutamine formation under the action of glutamine synthetase, NH being produced during this process3Can neutralize intracellular H+And the intracellular pH balance is maintained. At the same time, glutamine can be added in glutamine-fructose-6-phosphorusThe acid-salt transaminase (GlmS) generates D-glutamine-6 phosphate as a precursor substance of amino sugar and nucleotide sugar metabolic pathways, then UDP-MurNAc necessary for peptidoglycan synthesis is generated through amino sugar and nucleotide sugar metabolic pathways, 5-phosphoribosyl-imine is generated under the action of phosphoribosyltransferase (PurL) to participate in purine metabolism, carbamoyl phosphate is generated under the action of carbamoyl phosphate synthase large subunit (CarB) to participate in pyrimidine metabolism and arginine biosynthesis, and the expression levels of the three proteins are obviously up-regulated after milk fat globule membrane protein is added. In conclusion, the addition of milk lipocalin promotes the amino acid metabolism of Lactobacillus acidophilus, which on the one hand consumes or neutralizes intracellular H+The relative balance of intracellular pH is maintained, and on the other hand, the generation of precursor substances of a basal metabolic pathway is promoted, so that the normal metabolism of the bacteria is maintained, and the acid resistance of the bacteria is improved.
7.6.2 DNA replication, RNA synthesis and translation of related proteins
The results are shown in fig. 13, after adding milk fat globule membrane proteins, the expression levels of the subunits related to DNA-activating enzyme (DnaG), DNA polymerase i (pol i), ribonuclease (RNaseHI, RNaseH II) and DNA polymerase iii (pol iii) are all significantly up-regulated, indicating that milk fat globule membrane proteins can promote DNA replication and maintain normal growth and metabolism of the bacteria. Meanwhile, 3 proteins related to RNA synthesis are respectively RNA polymerase B subunits alpha, epsilon and delta guided by DNA, and the expression is slightly reduced after the milk fat globule membrane protein is added, which indicates that the transcription process of thalli is reduced after the milk fat globule membrane protein is added. Analysis of the protein with the significant difference shows that 21 proteins are related to a translation process, 3 of the proteins are glutamic acid aminotransferase subunit (Fmt), methionyl tRNA transformylase (GatC) participate in the process of synthesizing aminoacyl-tRNA synthetase, the expression level of uracil phosphoribosyl transferase (transcription inhibitor) is significantly reduced, and the rest 18 proteins are related to ribosome metabolism. The results show that the addition of milk fat globule membrane protein plays a certain role in promoting the function of ribosome synthetic protein, not only maintains the normal physiological function of cells, but also possibly generates repair protein to repair the forced damage of thalli in acid stress.
7.6.3 DNA and protein repair proteins
As shown in FIG. 14, after adding milk fat globule membrane protein, 24 different proteins were found to be involved in DNA repair, which respectively involved in DNA mismatch repair, homologous recombination, base excision repair, nucleotide excision repair, and the like. After the milk fat globule membrane protein is added, the main proteins of the DNA repair process, MutS, MutL, helicase (UvrD) and related subunits of DNA polymerase III, are all significantly up-regulated. After adding milk fat globule membrane protein, the important component proteins participating in DNA homologous recombination repair are as follows: meanwhile, the expression levels of major enzymes for base excision repair, such as glycosylase (Mpg), MutM (simultaneously having glycosylase and endonuclease activities), Nth (simultaneously having glycosylase and endonuclease activities), Ung (capable of recognizing and removing aminated bases and repairing damaged structures) and DNA polymerase I, in the thallus are all obviously up-regulated. From proteome analysis, it was found that the expression level of the transcription coupled repair factor translocase (Mfd) was significantly up-regulated, and in addition, the expression levels of UvrB, UvrD and DNA polymerase I were also significantly up-regulated after the addition of milk fat globule membrane protein, indicating that milk fat globule membrane protein might promote transcription coupled repair. In conclusion, the improvement of the survival rate of lactobacillus acidophilus CICC6074 in the environment with the pH value of 2.5 by the milk fat globule membrane protein is probably realized by promoting the expression of DNA repair protein so as to repair DNA and protein damage caused by acid stress and improve the resistance of the bacteria to the acid stress environment.
Comparative example 1 Effect of Casein, whey protein, bovine serum albumin, milk fat globule membrane protein on the bile salt stress and acid stress survival of Lactobacillus acidophilus CICC6074, respectively
We examined the influence of casein, whey protein, bovine serum albumin, milk fat globule membrane protein on the bile salt stress and acid stress survival ability of Lactobacillus acidophilus CICC6074, respectively, the experimental steps are as follows:
1.1 Lactobacillus acidophilus activation and culture
Taking a lactobacillus acidophilus CICC6074 glycerol stock solution at the temperature of minus 80 ℃, picking a proper amount of the stock solution with a sterile toothpick, streaking on an MRS agar culture medium, culturing in an incubator at the temperature of 37 ℃ until a single colony grows out, then picking the single colony in 5mL of the MRS liquid culture medium, carrying out static culture at the temperature of 37 ℃, inoculating a second generation bacterium which is cultured and activated to a stable period (18h) into the sterilized MRS liquid culture medium according to the inoculation amount of 2% (v/v), and carrying out static culture at the temperature of 37 ℃ until the stable period (18 h).
1.2 survival Rate of Lactobacillus acidophilus CICC6074 at different concentrations of bile salts
Inoculating Lactobacillus acidophilus CICC6074 second-generation bacterium which is cultured and activated to a stable phase (18h) into a sterilized MRS liquid culture medium according to the inoculation amount of 2% (v/v), standing and culturing at 37 ℃ for 18h, centrifuging at 4 ℃ for 10min at 3000g, and collecting the bacterium. The collected thalli are suspended in MRS broth with 0.1%, 0.2% and 0.3% of bile salt concentration, incubated for 3h at 37 ℃, bacterial liquid is collected every 1h, and viable bacteria count is carried out according to GB 4789.2-2010. The calculation formula is as follows:
1.3 Effect of different proteins on survival Rate of Lactobacillus acidophilus CICC6074 bile salt under stress conditions
Inoculating lactobacillus acidophilus CICC6074 second-generation bacteria which are cultured and activated to a stable period (18h) into an MRS broth culture medium in an inoculation amount of 2%, standing and culturing at 37 ℃ for 18h, centrifuging at 4 ℃ for 10min at 3000g, collecting the bacteria, washing the bacteria with precooled sterilized normal saline for three times, collecting the bacteria, respectively suspending the obtained bacteria in the MRS culture medium which contains 10mg/mL of lactalbumin, casein, bovine serum albumin and milk fat globule membrane protein and has the concentration of bile salt of 0.2%, standing and stressing the bacteria suspension in an incubator at 37 ℃ for 3h, taking bacteria liquid every 1h, and counting live bacteria according to GB 4789.2-2010. The calculation formula is as follows:
referring to fig. 15, it can be seen from fig. 15 that after 3 hours of treatment, the survival rates of the lactic acid bacteria added with different proteins are significantly different, the group effect of the milk lipocorm protein is the best, and the survival rate of the lactobacillus acidophilus is the highest. Other whey protein, bovine serum albumin and the like also have certain capability of ensuring the survival of the lactobacillus acidophilus in the bile salt environment.
The results of the effect of different proteins on the survival rate of lactobacillus acidophilus under acid stress are shown in fig. 16, and it can be seen in fig. 16 that the group effect of adding milk fat globule membrane protein is the best after 3h treatment, the acid stress resistance of lactobacillus acidophilus is the strongest, and the survival rate of lactobacillus acidophilus is the highest. Other whey protein, bovine serum albumin and casein have poor capacity of improving the bile salt resistance of lactobacillus acidophilus.
Claims (9)
1. Use of milk fat globule membrane protein for improving stress resistance of lactobacillus acidophilus.
2. The use according to claim 1, wherein said lactobacillus acidophilus is lactobacillus acidophilus CICC 6074.
3. The use according to claim 1, wherein the acid stress condition is pH 2-4.
4. The use according to claim 1, wherein the acid stress condition is culture in MRS medium at pH 2.5.
5. Use according to claim 1, characterized in that milk fat globule membrane protein is used for the preparation of probiotics or gastrointestinal tract protective agents or cell membrane protective agents.
6. Use according to claim 1, characterized in that milk fat globule membrane protein is used in the preparation of a medicament for maintaining the homeostasis of intracellular pH or for enhancing the intracellular H of Lactobacillus acidophilus+-use of an ATPase active agent.
7. Use according to claim 1, characterized in that the milk fat globule membrane protein is used for the preparation of a medicament promoting peptidoglycan synthesis.
8. The use according to claim 1, wherein the milk fat globule membrane protein is used in the manufacture of a medicament for down-regulating purine metabolism, ribosome synthesis, ABC transport associated proteins, and reducing DNA synthesis, translation and transport in bacterial cells.
9. Use according to claim 1, characterized in that milk fat globule membrane protein is used for the manufacture of a medicament for upregulating amino acid metabolism, DNA repair proteins.
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