CN112057479A - Probiotic composition capable of relieving diarrhea caused by antibiotics - Google Patents
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Abstract
The invention provides a probiotic composition capable of relieving diarrhea caused by antibiotics, belonging to the technical field of microorganisms, comprising lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus paracasei, bifidobacterium bifidum, resistant dextrin, maltodextrin, inulin and stachyose, wherein the preparation process of lactobacillus rhamnosus freeze-dried powder comprises the steps of activation of thalli, enlarged culture of the thalli, high-density culture, collection of the thalli and freeze-drying, wherein a culture medium used for the high-density culture contains a lotus leaf extract with the mass volume fraction of 0.06-0.1g/L, and the mass fraction of nuciferine in the lotus leaf extract is 4-6%. The probiotic composition capable of relieving diarrhea caused by antibiotics provided by the invention has the advantages of safety, high quality, good effect and lower preparation cost.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a probiotic composition capable of relieving diarrhea caused by antibiotics.
Background
Antibiotic-associated diarrheal (AAD) refers to diarrhea that occurs with the use of antibiotics and cannot be explained for other reasons. Antibiotics cause diarrhea by several mechanisms: (1) abnormalities of carbohydrate metabolism in the large intestine: broad spectrum antibiotics reduce the normal flora in the intestine, reduce the carbohydrate metabolism of the bacteria, and cause osmotic diarrhea due to the deposition of organic acids, cations and carbohydrates in the intestine. (2) Abnormalities in bile acid metabolism: antibiotic administration significantly affects the synthesis of primary bile salts that are not absorbed into the small intestine, which are dehydroxylated by bacteria in the colon to secondary bile acids, which are potent colonic secretor agents and are present in the colon resulting in secretory diarrhea. (3) Direct action of antibiotics: the clavulanate can stimulate the peristalsis of small intestine, the erythromycin can cause abnormal peristalsis, and meanwhile, the antibiotic destroys the protection effect of symbiotic bacteria, so that the diversity of microorganisms in the intestinal tract is reduced, the resistance of colon mucous membrane to pathogenic bacteria is destroyed, and the excessive growth of pathogenic microorganisms is caused.
Intestinal probiotics are a class of living microorganisms that can benefit the host by regulating the microbial balance in the intestine, and are currently widely used in clinical applications. Probiotics can alter the intestinal flora through nutritional competition, reduce osmotic diarrhea by combining immunomodulators or trophic factors as barriers to pathogen receptor binding, and simultaneously, probiotics are involved in immune regulation to counteract the pro-inflammatory effects of pathogenic bacteria.
The prior art discloses a probiotic composition and application thereof as a Chinese invention patent with the publication number of CN 109666615A. The probiotic composition disclosed by the invention consists of bifidobacterium adolescentis CCFM8630, lactobacillus reuteri CCFM8631 and lactobacillus rhamnosus CCFM1044, the effect of relieving metabolic syndrome is obviously better than that of the single use and combination of the CCFM8630 or the CCFM8631, especially in the aspects of reducing epididymis fat content, lowering fasting blood glucose level, reducing the area under a sugar tolerance curve, reducing the content of serum low-density lipoprotein and total cholesterol, improving the oxidation resistance of liver, reducing the content of serum IL-1 beta and the like, the reduction or increase of the content is improved by 9.62% -769.62% compared with that of a single bacterium or double bacterium combination formula, and the three bacterium combination formula can realize obvious synergistic effect.
Disclosure of Invention
The invention aims to provide a probiotic composition which is safe, high in quality, good in effect and low in preparation cost and can relieve diarrhea caused by antibiotics.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the preparation method of the freeze-dried lactobacillus rhamnosus powder comprises the following steps:
s1, activation of the bacterial cells: inoculating the strain in improved MRS liquid culture medium, and anaerobically culturing at 36.5-37.5 deg.C for 12-18h to obtain seed solution;
s2, expanding culture of thalli: transferring the obtained seed liquid to an improved MRS liquid culture medium according to the inoculation amount of 8-10% (v/v), carrying out anaerobic culture at 36.5-37.5 ℃ for 12-18h, then transferring the obtained seed liquid to the improved MRS liquid culture medium according to the inoculation amount of 8-10% (v/v), and carrying out anaerobic culture at 36.5-37.5 ℃ for 12-18h to obtain a secondary seed liquid;
s3, high-density culture: transferring the obtained secondary seed liquid to an improved MRS liquid culture medium according to the inoculation amount of 3-6% (v/v), and carrying out anaerobic culture at 36.5-37.5 ℃ for 18-22h to obtain a lactobacillus rhamnosus bacterial liquid;
s4, thallus collection: centrifuging and filtering the obtained lactobacillus rhamnosus bacterial liquid, collecting the bacteria, resuspending and cleaning the bacteria, and centrifuging to remove supernatant to obtain lactobacillus rhamnosus bacteria;
s5, freeze-drying thalli: adding a freeze-drying protective agent into the lactobacillus rhamnosus thallus and then carrying out freeze-drying;
the modified MRS liquid culture medium in the step S3 also contains a lotus leaf extract with the mass volume fraction of 0.06-0.1g/L, and the mass fraction of nuciferine in the lotus leaf extract is 4-6%. The addition of a certain amount of lotus leaf extract into the culture medium can accelerate the growth of lactobacillus rhamnosus, improve the yield of thalli, improve the yield of freeze-dried bacterial powder, simultaneously improve the content of viable bacteria in the freeze-dried bacterial powder, improve the number of the viable bacteria of lactobacillus rhamnosus entering gastrointestinal tracts, adjust the imbalance of intestinal flora, reduce the level of intestinal inflammation and further relieve the symptoms of the gastrointestinal tracts.
Further, the components of the freeze-drying protective agent comprise: 5-9% of glucose, 2-4% of D-galactose, 2-4% of ascorbic acid and the balance of water.
Further, the addition amount of the freeze-drying protective agent in the step S5 is 8-12% of the volume of the lactobacillus rhamnosus bacterial liquid obtained in the step S3.
Further, each liter of the improved MRS liquid culture medium comprises the following components: 4-5g of yeast powder, 18-22g of glucose, 8-12g of soybean peptone, 4-6g of beef powder, 1.8-2.5g of dipotassium phosphate, 4.5-5g of sodium acetate, 1.8-2g of ammonium citrate tribasic, 0.18-0.22g of magnesium sulfate, 0.04-0.06g of manganese sulfate, 800.8-1 mL of Tween and 999-999.2mL of distilled water.
Providing a probiotic composition comprising a complex probiotic, a resistant dextrin, a maltodextrin, inulin and stachyose;
the composite probiotics comprise lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus paracasei and bifidobacterium bifidum, and the preparation method of the composite probiotics comprises the following steps: compounding and mixing the freeze-dried powder of lactobacillus rhamnosus, the freeze-dried powder of lactobacillus plantarum, the freeze-dried powder of lactobacillus acidophilus, the freeze-dried powder of lactobacillus paracasei and the freeze-dried powder of bifidobacterium bifidum to obtain composite probiotics;
the freeze-dried powder of the lactobacillus rhamnosus is prepared by the preparation method of the freeze-dried powder of the lactobacillus rhamnosus. When the probiotic composition is prepared from the freeze-dried lactobacillus rhamnosus powder prepared by the method, the number of the live lactobacillus rhamnosus entering gastrointestinal tracts can be increased, the imbalance of intestinal flora is adjusted, the level of intestinal inflammation is reduced, and the gastrointestinal tract symptoms are further relieved.
Further, the freeze-dried powder of lactobacillus rhamnosus, the freeze-dried powder of lactobacillus plantarum, the freeze-dried powder of lactobacillus acidophilus, the freeze-dried powder of lactobacillus paracasei and the freeze-dried powder of bifidobacterium bifidum are compounded and mixed in a mass ratio of 4-6:2-3:2-3:1-5: 1-5.
Further, the probiotic composition comprises lactobacillus rhamnosus LR519, lactobacillus plantarum LP45, lactobacillus acidophilus La28, lactobacillus paracasei YMC1069, bifidobacterium bifidum TMC3115, resistant dextrin, maltodextrin, inulin, and stachyose.
Further, the inulin content in the probiotic composition is less than or equal to 20 wt%.
Furthermore, the probiotic composition also comprises trichosanthes root and bitter gourd seed extract (the mass ratio of the bitter gourd seed extract to the bitter gourd seed polysaccharide is 20: 1).
Furthermore, the mass ratio of the trichosanthes root to the balsam pear seed extract is 5.5-7: 1.
Further, the probiotic composition comprises the following components in parts by weight: 2-5 parts of composite probiotics, 30-40 parts of resistant dextrin, 25-35 parts of maltodextrin, 3-6 parts of trichosanthes root, 0.45-0.8 part of balsam pear seed extract, 15-20 parts of inulin and 15-20 parts of stachyose. When the extracts of the radix trichosanthis and the bitter gourd seeds are combined according to the mass ratio of 5.5-7:1, the generation of a metabolite bacteriocin of lactobacillus rhamnosus can be synergistically promoted, the growth of intestinal pathogens is slowed down, the intestinal environment is purified, the imbalance of intestinal flora is well adjusted, the level of intestinal inflammation is reduced, the recovery of gastrointestinal function is promoted, and the gastrointestinal symptoms are relieved.
It should be noted that, in a specific application scenario, the extracts of trichosanthes root and bitter gourd seed may be replaced by fructo-oligosaccharide and xylo-oligosaccharide, and such changes may be adjusted according to actual needs without affecting the protection scope of the present invention.
The invention has the following beneficial effects that a certain amount of lotus leaf extract is added into the high-density culture medium of the lactobacillus rhamnosus: the growth of lactobacillus rhamnosus can be accelerated, the thallus yield is improved, the yield of freeze-dried bacterium powder is improved, the viable bacteria content in the freeze-dried bacterium powder is high, the number of the lactobacillus rhamnosus viable bacteria entering gastrointestinal tracts can be increased, the imbalance of intestinal flora is adjusted, the intestinal inflammation level is reduced, and the gastrointestinal tract symptoms are relieved.
The invention has the following beneficial effects that as the trichosanthes root and the balsam pear seed extract with the mass ratio of 5.5-7:1 are added into the probiotic composition: the lactobacillus rhamnosus metabolite bacteriocin can be synergistically promoted to generate, the growth of intestinal pathogens is slowed down, the intestinal environment is purified, the intestinal flora imbalance is well adjusted, the intestinal inflammation level is reduced, the gastrointestinal function recovery is promoted, and the gastrointestinal symptoms are relieved.
Therefore, the probiotic composition is safe, high in quality, good in effect and low in preparation cost, and can relieve diarrhea caused by antibiotics.
Drawings
FIG. 1 shows the results of measurement of the dry weight of cells in test example 1 of the present invention;
FIG. 2 is a graph showing the results of the measurement of viable cell count before and after lyophilization in test example 1 of the present invention;
FIG. 3 is the measurement result of the diameter of the zone of inhibition in test example 2 of the present invention;
FIG. 4 shows the measurement results of the IL-6 and TNF- α contents in the serum of each group of mice in test example 3 of the present invention;
FIG. 5 shows the measurement results of stool dilution rate in test example 3 of the present invention;
FIG. 6 is a graph showing the measurement results of loose stool grade and diarrhea index in test example 3 of the present invention.
Detailed Description
The present invention is further described in detail with reference to the following examples:
example 1:
1. a preparation method of lactobacillus rhamnosus freeze-dried powder comprises the following steps:
1.1 test materials: lactobacillus rhamnosus LR 519; folium Nelumbinis extract obtained from Xian Dongfeng Biotech limited (specification: nuciferine mass fraction 4%);
preparing an improved MRS liquid culture medium: 4g of yeast powder, 20g of glucose, 10g of soybean peptone, 5g of beef powder, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of ammonium citrate tribasic, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801 mL of tween and 999mL of distilled water.
Preparing a high-density culture medium: 4g of yeast powder, 20g of glucose, 10g of soybean peptone, 5g of beef powder, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801 mL of tween, 0.08g of lotus leaf extract and 999mL of distilled water.
Preparing a freeze-drying protective agent: 7g of glucose, 3g of D-galactose, 3g of ascorbic acid and 87mL of distilled water.
1.2 activation of the cells: inoculating the strain preserved at the temperature of minus 20 ℃ to an improved MRS liquid culture medium, and carrying out anaerobic culture at the temperature of 37 ℃ for 14h to obtain a seed solution;
1.3 amplification culture of the cells: transferring the obtained seed liquid to an improved MRS liquid culture medium according to the inoculation amount of 8% (v/v), carrying out anaerobic culture at 37 ℃ for 14h, then transferring the obtained seed liquid to the improved MRS liquid culture medium according to the inoculation amount of 8% (v/v), and carrying out anaerobic culture at 37 ℃ for 14h to obtain a secondary seed liquid;
1.4 high-density culture: transferring the obtained secondary seed liquid to a high-density culture medium according to the inoculation amount of 4% (v/v), and carrying out anaerobic culture at 37 ℃ for 22h to obtain a lactobacillus rhamnosus bacterial liquid;
1.5 collecting the thallus: centrifuging the obtained lactobacillus rhamnosus bacterial liquid at 4 ℃ at 6000rpm for 10min, removing supernatant, collecting thallus, resuspending and cleaning the thallus to remove residual culture medium, centrifuging at 4 ℃ at 6000rpm for 10min, and removing supernatant to obtain lactobacillus rhamnosus thallus;
1.6 freeze-drying of thalli: adding a freeze-drying protective agent accounting for 8% of the volume of the fermentation liquid before centrifugation into the lactobacillus rhamnosus thallus to prepare a bacterial suspension, pouring the bacterial suspension into a freeze-drying tray, sealing, pre-freezing for 12 hours at minus 80 ℃, drying on a vacuum freeze-drying machine, collecting bacterial powder after freeze-drying is finished, and storing at minus 20 ℃ for later use.
2. A method of preparing a probiotic composition, comprising:
test materials: lactobacillus acidophilus La28, lactobacillus plantarum LP45, lactobacillus paracasei YMC1069, bifidobacterium bifidum TMC 3115; skim milk powder, purchased from the Wandashan Enterprise group Dairy Co., Ltd; trehalose, purchased from Baiyou Biotech limited, Hebei; resistant dextrin, purchased from Baiying biotechnology, Inc., Jiangxi; maltodextrin, available from hebei Qianju Biotech limited; inulin available from Zhengzhou Longhua chemical products, Inc.; stachyose, purchased from jalapia biotechnology limited, guangzhou.
The preparation of the modified MRS liquid culture medium is the same as the above.
Preparing a freeze-drying protective agent: 5g of trehalose, 15g of skim milk powder, 3g of sucrose, 1g of ascorbic acid and 100mL of distilled water.
2.1 preparation of lyophilized powder of Complex Probiotics
Respectively carrying out activation and enlarged culture on the lactobacillus acidophilus La28, lactobacillus plantarum LP45, lactobacillus paracasei YMC1069 and bifidobacterium bifidum TMC3115 according to the methods of 1.2 and 1.3, respectively transferring the obtained secondary seed liquid high-density culture to an improved MRS liquid culture medium according to the inoculation amount of 4% (v/v), and carrying out anaerobic culture at 37 ℃ for 22h to respectively obtain lactobacillus acidophilus liquid, lactobacillus plantarum liquid, lactobacillus paracasei liquid and bifidobacterium bifidum liquid; centrifuging the obtained bacterial liquid at 4 deg.C and 6000rpm for 10min, removing supernatant, collecting thallus, resuspending and cleaning thallus to remove residual culture medium, centrifuging at 4 deg.C and 6000rpm for 10min, and removing supernatant to obtain thallus; respectively adding 8% of freeze-drying protective agent of the volume of the fermentation liquid before centrifugation into lactobacillus acidophilus, lactobacillus plantarum, lactobacillus paracasei and bifidobacterium bifidum to prepare bacterial suspension, pouring the bacterial suspension into a freeze-drying tray, sealing, pre-freezing for 12h at-80 ℃, drying on a vacuum freeze-drying machine, collecting bacterial powder after freeze-drying is finished, and storing at-20 ℃ for later use; and (2) compounding and mixing the freeze-dried powder of the lactobacillus plantarum, the freeze-dried powder of the lactobacillus acidophilus, the freeze-dried powder of the lactobacillus paracasei, the freeze-dried powder of the bifidobacterium bifidum and the freeze-dried powder of the lactobacillus rhamnosus prepared in the step 1 according to the mass ratio of 3:3:2:2:5 to obtain the composite probiotics.
2.2 preparation of probiotic compositions
4g of composite probiotics, 35g of resistant dextrin, 30g of maltodextrin, 15g of inulin and 15g of stachyose are put into a mixer and fully and uniformly stirred to obtain the probiotic composition.
Example 2:
when the freeze-dried lactobacillus rhamnosus powder is prepared, a culture medium used for high-density culture is an improved MRS liquid culture medium. The rest of the process was identical to example 1.
Example 3:
the test material also included: trichosanthis radix, available from Biotech, Inc., Kugua seed extract, available from Saian rui Biotech, Inc. (specification: Kugua seed extract, Kugua seed polysaccharide mass ratio 20: 1).
3.5g of trichosanthes root and 0.7g of balsam pear seed extract are also added when the probiotic composition is prepared, and the rest parts are completely the same as the example 1.
Example 4:
the probiotic composition was prepared with 5.6g of trichosanthes root, the remainder being identical to example 3.
Example 5:
the probiotic composition was prepared with the addition of 4.2g of trichosanthes root, the remainder being identical to example 3.
Example 6:
the test material also included: trichosanthis radix, available from Biotech, Inc., Kugua seed extract, available from Saian rui Biotech, Inc. (specification: Kugua seed extract, Kugua seed polysaccharide mass ratio 20: 1).
4.2g of trichosanthes root and 0.7g of balsam pear seed extract are also added when the probiotic composition is prepared, and the rest parts are completely the same as the example 2.
Test example 1:
1.1 measurement of the Total amount of cells: the Lactobacillus rhamnosus strains collected in the above examples were weighed. Taking the bacterial liquid obtained by high-density culture of lactobacillus rhamnosus in the embodiment for 22h, centrifuging, collecting the thallus, drying and weighing. The results of the measurement of the dry weight of the cells are shown in FIG. 1.
1.2 determination of viable count:
1.2.1 determination of viable count before lyophilization: 1mL of the Lactobacillus rhamnosus cells collected in the above examples was put into 9mL of 0.85% (m/v) physiological saline and shaken up, and the viable cell count was measured by a gradient dilution method using an agar medium. Fully shaking the cultured bacteria liquid on a clean bench, and continuously diluting all samples by 10 times with the dilution degree of 10-1、10-2、10-3、10-4、10-5、10-6And 10-7. Selecting proper dilution, uniformly inoculating 1mL of the selected dilution into a modified MRS agar culture medium which is sterilized, cooled to about 45 ℃, shaking uniformly, culturing at 37 ℃ for 48h, selecting the dilution with about 80 colony counts, selecting 2 dilutions for each sample, performing 3 repeated averaging on each dilution, and calculating the unit viable count (cfu/mL).
And (3) measuring the viable count after freeze-drying: 1mL of freeze-dried powder of lactobacillus rhamnosus thallus is taken, the freeze-dried powder is restored by normal saline with the same volume of 0.85% (m/v) in a centrifuge tube, the freeze-dried powder is fully oscillated until the freeze-dried powder is completely dissolved, and the viable count is determined by adopting an agar culture medium through a gradient dilution method. The results of viable cell count before and after lyophilization are shown in FIG. 2.
As can be seen from figure 1, the dry weight of the thallus in example 1 is obviously higher than that in example 2, and as can be seen from figure 2, the number of viable bacteria before and after freeze-drying in example 1 is obviously higher than that in example 2, which shows that the addition of a certain amount of lotus leaf extract in the culture medium can accelerate the growth of lactobacillus rhamnosus, improve the thallus yield, improve the yield of freeze-dried powder and improve the content of viable bacteria in the freeze-dried powder.
Test example 2:
2.1 determination of bacteriocin content:
the group A fermentation medium is an improved MRS liquid medium and comprises: 4g of yeast powder, 20g of glucose, 10g of soybean peptone, 5g of beef powder, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801 mL of tween and 999mL of distilled water;
the group B fermentation culture medium is prepared by adding 0.5g Trichosanthis radix and 0.1g semen Momordicae Charantiae extract (semen Momordicae Charantiae extract, semen Momordicae Charantiae polysaccharide mass ratio is 20: 1) into group A fermentation culture medium;
the group C fermentation culture medium is prepared by adding 0.8g Trichosanthis radix and 0.1g semen Momordicae Charantiae extract (semen Momordicae Charantiae extract, semen Momordicae Charantiae polysaccharide mass ratio is 20: 1) into group A fermentation culture medium;
the group D fermentation medium is prepared by adding 0.6g Trichosanthis radix and 0.1g semen Momordicae Charantiae extract (semen Momordicae Charantiae extract, semen Momordicae Charantiae polysaccharide mass ratio is 20: 1) into group A fermentation medium;
2.1.1 fermentation culture of the cells: inoculating lactobacillus rhamnosus LR519 stored at-20 ℃ to an improved MRS liquid culture medium, and performing anaerobic culture at 37 ℃ for 14h to obtain a seed solution; and (2) transferring the obtained seed liquid to an improved MRS liquid culture medium according to the inoculation amount of 8% (v/v), carrying out anaerobic culture at 37 ℃ for 14h to obtain a secondary seed liquid, dividing the secondary seed liquid into A, B, C, D groups according to different fermentation culture media, and transferring the obtained secondary seed liquid to different fermentation culture media according to the inoculation amount of 4% (v/v) for anaerobic culture at 37 ℃ for 18h to obtain lactobacillus rhamnosus fermentation liquid.
2.1.2 crude extraction of bacteriocins: taking 400mL of lactobacillus rhamnosus fermentation liquor, centrifuging at 12000rpm for 15min, filtering supernate with a sterile filter membrane of 0.22 mu m, extracting the filtrate and ethyl acetate for 1h according to the volume of 1:1, and performing rotary evaporation and concentration on the upper layer solution obtained by extraction at 47 ℃ to 5% of the volume of the fermentation liquor, namely obtaining the crude bacteriocin extract.
2.1.3 taking Escherichia coli as an indicator bacterium, transferring the activated indicator bacterium into a 1mL centrifuge tube according to the volume ratio of the bacterium liquid to the LB culture medium of 1:100, and culturing at 37 ℃ overnight. Coating 100 mu L of activated escherichia coli liquid on a 20mL LB culture medium plate, punching a hole by using an Oxford cup with the diameter of 6mm, taking 100 mu L/hole bacteriocin crude extract, culturing for 8h at 37 ℃, performing a bacteriostatic test by using ethyl acetate as a control, observing the diameter of a bacteriostatic circle including the diameter of the Oxford cup, and determining the yield of the bacteriocin. The results of the measurement of the zone of inhibition diameter are shown in FIG. 3.
As can be seen from fig. 3, the diameter of the zone of inhibition of group D is significantly higher than that of groups A, B and C, which indicates that the generation of bacteriocin, a metabolite of lactobacillus rhamnosus, can be synergistically promoted when the extracts of trichosanthes root and balsam pear seed are combined in a mass ratio of 5.5-7: 1.
Test example 3:
establishing a model: healthy SPF-grade BALB/c mice, three weeks old, male, 20g, were acclimatized for 6 days, and a blank group and a diarrhea model group were set. Diarrhea model groups a diarrhea model control group and a probiotic composition treatment group prepared in each example above were separated. Continuously gavage 22g/kg (weight of mouse) ampicillin into the mouse for 5d twice a day, wherein each dose is 11.8 g/kg; the blank group of 30 mice is subjected to intragastric administration at the same time and the same volume of physiological saline as reference every day, the mice with non-ideal modeling are removed, the diarrhea model after removal is 30 control groups, each probiotic composition treatment group prepared in the embodiment is subjected to intragastric administration once every 10 am according to the dose of 5.6mg/d, and the continuous intragastric administration is carried out for 12 days. The diarrhea model control group and the blank group were perfused with the same amount of normal saline.
3.1 determination of the intestinal flora: counting the fecal viable bacteria: analyzing the distribution of intestinal flora of diarrhea model mice treated by the probiotic composition, aseptically taking 12d fecal samples of a normal group and a probiotic preparation treatment group, and continuously diluting all the samples by 10 times with the respective dilution degree of 10-1、10-2、10-3、10-4、10-5、10-6And 10-7. Sampling 0.2mL of each dilution, coating on LBS, TPY, EMB, enterococcus agar plate, TSC, anaerobic culturing at 37 deg.C for 48 hr, and respectively culturing lactobacillus, Bacillus bifidus, and intestineBacilli, enterococci, and clostridium perfringens were plate counted.
TABLE 1 mean intestinal flora (lg CFU/g) of diarrhea model mice
3.2 measurement of proinflammatory factor levels:
1.0mL of blood is taken from mouse eyeballs, the mouse eyeballs are placed in a 1.5mL centrifuge tube to stand for 1h, centrifuged for 15min at 3000 Xg, and serum is transferred to another centrifuge tube to be frozen and stored at minus 20 ℃ for standby. An ELISA method is adopted, a standard curve is established according to the kit step operation, and the content of IL-6 and TNF-alpha in the serum of the mouse is measured. The measurement results of IL-6 and TNF-alpha contents in the serum of each group of mice are shown in FIG. 4.
3.3 antidiarrheal test: the test of the number of loose feces before administration is carried out on each group of mice before treatment after modeling: after fasting for 12h, 25mL/kg body weight of ampicillin liquid (0.3 g/mL) was administered to each mouse in each group, and the same amount of physiological saline was administered to the mice in the blank control group. Then, the mice are placed into an observation cage to be observed in a single cage (special filter paper is paved at the bottom of the cage), the number of loose stools, the number of dry stools and the total number of stools in 5 hours of each mouse after the gastric ampicillin liquid medicine is infused (the filter paper at the bottom of the cage is replaced timely according to the situation) are recorded, and the level of the loose stools and the diarrhea index of each mouse are measured. The dry (formed) feces and the wet (wet) feces were judged based on the presence or absence of stains on the filter paper. The rare excrement rate calculation formula: stool dilution rate = number of stools discharged/total number of stools in a certain time/diarrhea index calculation formula: diarrhea index = loose stool rate × loose stool grade, and the judgment criteria for loose stool grade are shown in table 2. After 12 days of the gavage treatment, a loose stool point number experiment is carried out at 13 days, and the level of loose stool, the rate of loose stool and the diarrhea index are calculated. The results of the measurement of the stool dilution rate are shown in FIG. 5. The results of the measurements of loose stool grade and diarrhea index are shown in FIG. 6.
TABLE 2 determination of stool dilution level
As can be seen from table 1, the bifidobacteria and lactobacilli of examples 1 and 2 were all proliferated to different extents, with reduced numbers of enterobacteria, enterococci and clostridium perfringens, and the bifidobacteria and lactobacilli of example 1 were all proliferated to a greater extent and reduced numbers of enterobacteria, enterococci and clostridium perfringens than the model control group; as can be seen from FIG. 4, compared with the model control group, the IL-6 and TNF-alpha contents of the examples 1 and 2 are obviously reduced, and the IL-6 and TNF-alpha contents of the example 1 are obviously lower than those of the example 2, as can be seen from FIG. 5 and FIG. 6, compared with the model control group, the loose stool rate, the loose stool grade and the diarrhea index of the examples 1 and 2 are obviously reduced, and the loose stool rate, the loose stool grade and the diarrhea index of the example 1 are obviously lower than those of the example 2, which shows that the addition of a certain amount of the lotus leaf extract in the culture medium can improve the viable bacteria content in the Lactobacillus rhamnosus freeze-dried powder, improve the number of viable bacteria of Lactobacillus rhamnosus entering the gastrointestinal tract, adjust the intestinal dysbacteriosis, reduce the intestinal inflammation level, and further reduce the gastrointestinal symptoms.
As can be seen from table 1, the bifidobacterium and lactobacillus of examples 1, 2, 3, 4, 5 and 6 are proliferated to different degrees, the numbers of the enterobacteria, the enterococci and the clostridium perfringens are reduced, the proliferation degree of the bifidobacterium and the lactobacillus of example 2 and the reduction amounts of the enterobacteria, the enterococci and the clostridium perfringens are all lower than those of example 6, the proliferation degree of the bifidobacterium and the lactobacillus of examples 1, 3 and 4 and the reduction amounts of the enterobacteria, the enterococci and the clostridium perfringens are all similar and are all lower than those of example 5, and the number of each index flora of example 5 is close to that of the mouse flora of the blank group; as can be seen from FIG. 4, the IL-6 and TNF-alpha contents of examples 1, 2, 3, 4, 5 and 6 are all significantly reduced, and the IL-6 and TNF-alpha contents of example 6 are significantly lower than those of example 2, and the IL-6 and TNF-alpha contents of examples 1, 3 and 4 are not significantly different and are all significantly higher than those of example 5, compared with the model control group; as can be seen from FIGS. 5 and 6, the stool dilution rate, stool dilution grade and diarrhea index of example 1, example 2, example 3, example 4, example 5 and example 6 are all significantly reduced compared with the model control group, and the stool dilution rate, stool dilution grade and diarrhea index of example 6 are all significantly lower compared with example 2, the stool dilution rate, stool dilution grade and diarrhea index of examples 1, 3 and 4 are not significantly different and are all significantly higher compared with example 5, this shows that when the extracts of radix trichosanthis and bitter melon seed are combined according to the mass ratio of 5.5-7:1, can synergistically promote the generation of a metabolite bacteriocin of lactobacillus rhamnosus, slow down the growth of intestinal pathogens, purify the intestinal environment, better adjust the imbalance of intestinal flora, effectively reduce the level of intestinal inflammation, promote the recovery of gastrointestinal function and relieve gastrointestinal symptoms.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (6)
1. A preparation method of freeze-dried lactobacillus rhamnosus powder is characterized by comprising the following steps:
s1, activation of the bacterial cells: inoculating the strain in an improved MRS liquid culture medium, and performing anaerobic culture at 36.5-37.5 ℃ for 12-18h to obtain a seed solution;
s2, expanding culture of thalli: transferring the obtained seed liquid to an improved MRS liquid culture medium according to the inoculation amount of 8-10% of volume fraction, carrying out anaerobic culture at 36.5-37.5 ℃ for 12-18h, then transferring the obtained seed liquid to the improved MRS liquid culture medium according to the inoculation amount of 8-10% of volume fraction, and carrying out anaerobic culture at 36.5-37.5 ℃ for 12-18h to obtain a secondary seed liquid;
s3, high-density culture: transferring the obtained secondary seed liquid to an improved MRS liquid culture medium according to the inoculation amount of 3-6% volume fraction, and carrying out anaerobic culture at 36.5-37.5 ℃ for 18-22h to obtain a lactobacillus rhamnosus bacterial liquid;
s4, thallus collection: centrifuging and filtering the obtained lactobacillus rhamnosus bacterial liquid, collecting the thallus, carrying out heavy suspension cleaning on the thallus by using a PBS buffer solution, and centrifuging to remove a supernatant to obtain the lactobacillus rhamnosus thallus;
s5, freeze-drying thalli: adding a freeze-drying protective agent into the lactobacillus rhamnosus thallus and then carrying out freeze-drying;
the improved MRS liquid culture medium in the step S3 also contains 0.06-0.1g/L of lotus leaf extract, and the mass fraction of nuciferine in the lotus leaf extract is 4-6%.
2. The method of claim 1, wherein the components of the lyoprotectant include: 5-9% of glucose, 2-4% of D-galactose and 2-4% of ascorbic acid.
3. The method of claim 1, wherein: the addition amount of the freeze-drying protective agent in the step S5 is 8-12% of the volume of the lactobacillus rhamnosus bacterial liquid obtained in the step S3.
4. The method of claim 1, wherein: the improved MRS liquid culture medium per liter comprises the following components: 4-5g of yeast powder, 18-22g of glucose, 8-12g of soybean peptone, 4-6g of beef powder, 1.8-2.5g of dipotassium phosphate, 4.5-5g of sodium acetate, 1.8-2g of ammonium citrate tribasic, 0.18-0.22g of magnesium sulfate, 0.04-0.06g of manganese sulfate, 800.8-1 mL of Tween and 999-999.2mL of distilled water.
5. A probiotic composition for alleviating antibiotic induced diarrhea, comprising a complex probiotic, a resistant dextrin, a maltodextrin, an inulin and a stachyose;
the compound probiotics comprise lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus paracasei and bifidobacterium bifidum, and the preparation method of the compound probiotics comprises the following steps: compounding and mixing the freeze-dried powder of lactobacillus rhamnosus, the freeze-dried powder of lactobacillus plantarum, the freeze-dried powder of lactobacillus acidophilus, the freeze-dried powder of lactobacillus paracasei and the freeze-dried powder of bifidobacterium bifidum to obtain composite probiotics;
the freeze-dried powder of the lactobacillus rhamnosus is prepared by the preparation method of any one of claims 1 to 4.
6. The probiotic composition capable of alleviating antibiotic induced diarrhea according to claim 5, wherein: the freeze-dried powder of lactobacillus rhamnosus, the freeze-dried powder of lactobacillus plantarum, the freeze-dried powder of lactobacillus acidophilus, the freeze-dried powder of lactobacillus paracasei and the freeze-dried powder of bifidobacterium bifidum are compounded and mixed in a mass ratio of 4-6:2-3:2-3:1-5: 1-5.
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