CN117814304A - Preparation method and application of intestinal microecological active liquid - Google Patents
Preparation method and application of intestinal microecological active liquid Download PDFInfo
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- CN117814304A CN117814304A CN202311834400.8A CN202311834400A CN117814304A CN 117814304 A CN117814304 A CN 117814304A CN 202311834400 A CN202311834400 A CN 202311834400A CN 117814304 A CN117814304 A CN 117814304A
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- active liquid
- culture medium
- mixed solution
- lactobacillus
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to the technical field of intestinal tract active liquid, in particular to a preparation method of intestinal tract microecological active liquid and a product thereof, comprising the following preparation steps: s1, culturing lactobacillus plantarum in a culture medium to obtain lactobacillus plantarum mixed solution; cheese milkCulturing bacillus in a culture medium to obtain a lactobacillus mixed solution; culturing lactobacillus acidophilus and escherichia coli in a seed culture medium C to obtain a lactobacillus acidophilus and escherichia coli mixed solution; s2, placing the lactobacillus plantarum mixed solution into a production tank for culture, and maintaining the ventilation rate to be 5-6m 3 Stirring, adding lactobacillus casei mixed solution, co-culturing, adding lactobacillus acidophilus and escherichia coli mixed solution, and continuously culturing to obtain symbiotic fermentation liquor; s3, uniformly stirring the symbiotic fermentation liquid and the sterilized raw milk, fermenting, demulsification and stirring after the fermentation is finished, standing, filtering, taking whey, continuing fermenting, and concentrating to obtain a concentrated solution; s4, mixing the concentrated solution, glucose and sodium benzoate to obtain the intestinal microecological active liquid.
Description
Technical Field
The application relates to the technical field of intestinal tract active liquid, in particular to a preparation method and application of intestinal tract microecological active liquid.
Background
The microorganisms in human bodies are various in variety and huge in quantity and are distributed at different positions of the bodies, wherein the content of the microorganisms in the gastrointestinal tract accounts for about 78% of the total microorganisms. The microorganisms in the gastrointestinal tract are divided into three types, namely beneficial bacteria, neutral bacteria and harmful bacteria, which are required to keep the human body interdependence and mutual restriction to form a dynamic balance state, namely microecological balance. The microecological balance is to have enough strain diversity, enough strain quantity and absolute advantage of beneficial bacteria. Once one of them is destroyed, the microecological balance is unbalanced, which can lead to illness of human body.
When the human body is affected by age, environment, diet, medication and other factors, the intestinal microorganisms are unbalanced, such as reduced flora, reduced number of beneficial bacteria, increased number of harmful bacteria and the like, so that the synthesis of nutrients, malabsorption, abnormal blood sugar and blood lipid metabolism, increased inflammatory factors, abnormal toxin removal, reduced immune function, unbalanced immunity and unbalanced neuroendocrine are caused, thereby causing various diseases. Therefore, maintaining the micro-ecological balance in the intestinal tract is extremely important.
Disclosure of Invention
In order to maintain the balance of microecology in the intestinal tract, the application provides a preparation method and application of an intestinal tract microecology active liquid.
In a first aspect, the present application provides a method for preparing an intestinal microecological active solution, which adopts the following technical scheme:
the preparation method of the intestinal microecological active liquid comprises the following preparation steps:
s1, culturing lactobacillus plantarum in a seed culture medium at a temperature of 35-40 ℃ for 20-24 hours to obtain lactobacillus plantarum mixed solution;
culturing lactobacillus casei in a seed culture medium at 30-35 ℃ for 20-24 hours to obtain lactobacillus casei mixed solution;
culturing lactobacillus acidophilus and escherichia coli in a seed culture medium C at 30-35 ℃ for 16-20 hours to obtain lactobacillus acidophilus and escherichia coli mixed solution;
wherein, the preparation process of the seed culture medium C is as follows:
2-5 parts of glucose, 4-9 parts of peptone, 2-5 parts of beef extract, 2-6 parts of yeast extract, 0.1-0.5 part of monopotassium phosphate, 0.1-0.4 part of citric acid diamine, 0.1-1 sodium acetate, 4-6 parts of tween, 0.1-0.4 part of magnesium sulfate, 0.1-0.3 part of manganese sulfate, 0.1-0.3 part of calcium carbonate and 95-100 parts of distilled water in parts by weight, and sterilizing for 30-50min at the temperature of 100-120 ℃ and the pressure of 0.09-0.12MPa to prepare a C seed culture medium;
s2, placing the lactobacillus plantarum mixed solution into a production tank for culture, and maintaining the ventilation rate to be 5-6m 3 Stirring at 150-200rpm, maintaining the culture temperature at 35-40deg.C and pH at 7-8, adding lactobacillus casei mixed solution, co-culturing for 5-6 hr, adding lactobacillus acidophilus and escherichia coli mixed solution, and continuously culturing for 36-40 hr to obtain symbiotic fermentation liquor;
s3, uniformly stirring the symbiotic fermentation liquid and the sterilized raw milk, fermenting for 20-24 hours at the fermentation temperature of 35-40 ℃, after the fermentation is finished, demulsifying and stirring, stirring for 30-60 minutes, standing for 40-48 hours, filtering, taking whey, continuously fermenting, and concentrating to obtain a concentrated solution;
s4, mixing the concentrated solution, glucose and sodium benzoate to obtain the intestinal microecological active liquid.
By adopting the technical scheme, the prepared intestinal microecological active liquid can be directly taken orally, has no side effect, utilizes the interaction of lactobacillus plantarum, lactobacillus casei, lactobacillus acidophilus and escherichia coli, can ferment substances such as sucrose, disaccharide and the like to produce acid, lactobacillus casei can utilize various monosaccharides, fructose and the like, lactobacillus acidophilus can utilize substances such as lactose, sucrose, fructose and the like to ferment, and the produced substances such as lactic acid, bacteriocin, hydrogen peroxide and the like have natural antibacterial effect, and have obvious inhibition effect on staphylococcus aureus, salmonella, listeria monocytogenes and the like, thereby regulating intestinal flora, ensuring that escherichia coli has a mutually-beneficial symbiotic relationship in intestinal tracts, can competitively resist pathogenic bacteria attack, and promote the synthesis of vitamins. The above strains cooperate to regulate microecological balance, promote digestion and nutrient absorption, prevent and treat nutrient deficiency (vitamins, amino acids, calcium and lactose), prevent and treat canker sore and oral odor, promote excretion of toxic substances, clean intestinal tract, moisten intestine and relieve constipation, prevent and treat diarrhea, constipation, flatulence, dyspepsia, irritable bowel syndrome and other intestinal diseases, and can help human body to comprehensively repair and reestablish intestinal microecological balance after administration.
The symbiotic fermentation liquid and the sterilized raw milk are fermented together, and concentrated solution is prepared through the steps of demulsification, standing, filtration and the like, so that the concentrated solution is convenient for intestinal absorption and can be directly taken. Cow milk can directly enter human body, and the concentration of lactobacillus plantarum, lactobacillus casei, lactobacillus acidophilus and escherichia coli can be diluted by fermenting symbiotic fermentation liquid and sterilized raw cow milk together so as to be orally taken.
And S4, the taste of the intestinal microecological active liquid can be regulated, glucose is used for regulating the taste, a small amount of sodium benzoate can reduce strain deterioration, and the intestinal microecological active liquid is convenient to take, and simultaneously provides nutrients for the intestinal microecological active liquid, so that the intestinal microecological active liquid is beneficial to keep living bacteria.
Preferably, the seed medium A in step 1) is prepared as follows:
according to parts by weight, 1-6 parts of beef extract, 4-8 parts of peptone, 0.1-0.5 part of sodium chloride, 0.1-0.3 part of ammonium sulfate, 1-2 parts of monopotassium phosphate and 5-10 parts of lactose are mixed, added into 95-100 parts of distilled water, uniformly mixed, pH value is regulated to 6.5-7.5, and then sterilized for 30-40min under the conditions that the temperature is 120-130 ℃ and the pressure is 0.09-0.12MPa, so that the seed culture medium A is prepared.
By adopting the technical scheme, enough nutrient components are provided for the lactobacillus plantarum, and the A seed culture medium is sterilized under the dual actions of high temperature and pressure, so that the influence of other microorganisms or harmful bacteria on the cultivation of the lactobacillus plantarum is further prevented, and the cultivated lactobacillus plantarum is high in cleanliness. During the cultivation period, the growth curve 0D600 needs to be detected every 2 hours, and the growth curve is drawn to reach the middle or later period of logarithmic growth, so that the culture medium can be inoculated into a production tank.
Preferably, the seed medium B in step 2) is prepared as follows:
according to parts by weight, 4-10 parts of peptone, 2-6 parts of glucose, 1-5 parts of yeast extract, 1-4 parts of beef extract, 0.1-0.3 part of sodium chloride, 0.1-0.3 part of potassium dihydrogen phosphate and 95-100 parts of distilled water are uniformly mixed, pH is regulated to 5.5-6.5, and then sterilization is carried out for 30-50min under the conditions that the temperature is 100-120 ℃ and the pressure is 0.09-0.12MPa, so that the seed culture medium B is prepared.
By adopting the technical scheme, enough nutrient components are provided for the lactobacillus, and the B seed culture medium is sterilized under the dual actions of high temperature and pressure, so that the influence of other microorganisms or harmful bacteria on the cultivation of the lactobacillus is further prevented, and the cultivated lactobacillus has high cleanliness. During the cultivation period, the growth curve 0D600 needs to be detected every 2 hours, and the growth curve is drawn to reach the middle or later period of logarithmic growth, so that the culture medium can be inoculated into a production tank.
Preferably, the preparation process of the C seed medium is as follows:
according to weight portions, 2 to 5 portions of glucose, 4 to 9 portions of peptone, 2 to 5 portions of beef extract, 2 to 6 portions of yeast extract, 0.1 to 0.5 portion of monopotassium phosphate, 0.1 to 0.4 portion of citric acid diamine, 0.1 to 1 portion of sodium acetate, 4 to 6 portions of tween, 0.1 to 0.4 portion of magnesium sulfate, 0.1 to 0.3 portion of manganese sulfate, 0.1 to 0.3 portion of calcium carbonate and 95 to 100 portions of distilled water are sterilized for 30 to 50 minutes under the conditions that the temperature is 100 to 120 ℃ and the pressure is 0.09 to 0.12MPa, and the C seed culture medium is prepared.
By adopting the technical scheme, enough nutrient components are provided for the lactobacillus casei, and the C seed culture medium is sterilized under the dual actions of high temperature and pressure, so that the influence of other microorganisms or harmful bacteria on the cultivation of the lactobacillus casei is further prevented, and the cultivated lactobacillus casei is high in cleanliness. During the cultivation period, the growth curve 0D600 needs to be detected every 2 hours, and the growth curve is drawn to reach the middle or later period of logarithmic growth, so that the culture medium can be inoculated into a production tank.
Preferably, the preparation process of the culture medium in the production tank is as follows:
3 to 6 parts of corn steep liquor, 0.1 to 0.4 part of ammonium sulfate, 2 to 5 parts of beef extract, 0.1 to 0.3 part of magnesium sulfate, 0.2 to 0.4 part of dipotassium hydrogen phosphate, 0.2 to 0.6 part of citric acid diamine, 0.2 to 0.7 part of zinc chloride, 0.3 to 0.5 part of calcium chloride and 95 to 100 parts of distilled water are fully stirred and dissolved, then the mixture is sealed and heated to 80 to 100 ℃, the pressure is regulated to 0.09 to 0.12MPa, and the mixture is sterilized for 1 to 2 hours, so that the culture medium in the production tank is prepared.
By adopting the technical scheme, a nutrient-rich environment is provided for the common fermentation of lactobacillus plantarum, lactobacillus casei, lactobacillus acidophilus and escherichia coli, so that the lactobacillus casei, lactobacillus casei and escherichia coli can be fully fermented. And the culture medium in the production tank is sterilized under the dual actions of high temperature and pressure, so that the influence of other microorganisms or harmful bacteria on the cultivation of mixed bacteria is further prevented, and the normal fermentation is ensured.
Preferably, the content of lactobacillus in the concentrated solution is more than or equal to 10 6 CFU/g(ml)。
Preferably, the content of lactobacillus casei is 1.2×10 or more 6 CFU/g (ml), content of Lactobacillus acidophilus greater than or equal to 1.2X10 6 CFU/g (ml) and E.coli content of (0.6-0.7) x 10 6 CFU/g(ml)。
By adopting the technical scheme, the intestinal microecological active liquid can selectively stimulate the growth and metabolites of bacteria (including bacillus, lactobacillus, saccharomycetes and escherichia coli) beneficial to the health of hosts in the intestinal tract, optimize the flora in the intestinal tract and be beneficial to balancing the microecology in the intestinal tract.
Preferably, the fermentation is continued in step S3 at a temperature of 35-40deg.C for 20-24h, and prebiotics are added during fermentation, wherein the amount of prebiotics is 1-2% of whey weight.
Preferably, the prebiotics are fructo-oligosaccharides, inulin, galacto-oligosaccharides, xylo-oligosaccharides, mannooligosaccharides, xylitol, mannitol, sorbitol and the like. The prebiotics can be used for fermenting strains to improve nutrition.
Preferably, the milk protein rate of the raw cow milk is more than or equal to 5.0%, the milk fat rate is more than or equal to 5.1%, the total colony count is less than or equal to 20 ten thousand CFU/mL, and the somatic cell count is less than or equal to 40 ten thousand/mL.
By adopting the technical scheme, the whey separation rate is high, and the content of various fungi in the obtained concentrated solution is high, so that the subsequent fermentation is facilitated.
Preferably, the weight ratio of the concentrated solution, the glucose and the sodium benzoate is (10-15): (1-3): 0.01.
by adopting the technical scheme, the dosage of the concentrated solution, glucose and sodium benzoate is optimized, the taste of the intestinal microecological active solution is adjusted, the direct drinking is facilitated, meanwhile, the sodium benzoate can prevent the deterioration of the intestinal microecological active solution in the storage process, and the glucose can provide nutrients for the intestinal microecological active solution, so that the survival bacteria can be preserved.
Preferably, the lactobacillus plantarum mixed solution, the lactobacillus casei mixed solution and the lactobacillus acidophilus and escherichia coli mixed solution are prepared according to the weight ratio of (3-6): (8-12): 7.
by adopting the technical scheme, the dosages of the lactobacillus plantarum mixed solution, the lactobacillus casei mixed solution and the lactobacillus acidophilus and escherichia coli mixed solution are optimized, so that intestinal microecological active liquid is thought to be absorbed more easily, the strain can play the maximum role, and the balance of flora in the intestinal tract is adjusted rapidly.
In a second aspect, the application provides an application of an intestinal microecological active liquid, which adopts the following technical scheme:
the intestinal microecological active liquid is prepared by the preparation method of the intestinal microecological active liquid in the first aspect, and the intestinal microecological active liquid is directly taken orally by adopting the technical scheme, so that the intestinal microecological active liquid is convenient and quick to use. The intestinal microecological active liquid can improve diarrhea caused by antibiotic treatment, improve mood abnormality, improve sleep, balance immunity, resist bacteria and diminish inflammation, and prevent and inhibit chronic inflammation.
Preferably, the intestinal microecological active liquid can be orally taken after being mixed with warm water with the volume of 6-8 times, and the temperature of the warm water is lower than 40 ℃.
And meanwhile, proper amount of white sugar, honey, xylitol, milk, fruit juice and the like can be added according to personal preference for blending and then drinking.
In summary, the present application has the following beneficial effects:
1. the method comprises the steps of respectively culturing a lactobacillus plantarum mixed solution, a lactobacillus casei mixed solution, lactobacillus acidophilus and escherichia coli mixed solution, then co-culturing the lactobacillus plantarum mixed solution, the lactobacillus casei mixed solution, the lactobacillus acidophilus and the escherichia coli mixed solution according to specific conditions and sequences to obtain a symbiotic fermentation liquid, fermenting symbiotic fermentation and sterilized raw cow milk, demulsification, standing, filtering, taking whey, continuing fermentation, and concentrating to obtain a concentrated solution; mixing the concentrated solution with glucose and sodium benzoate to obtain intestinal microecological active solution; the intestinal active liquid prepared by the application can promote digestion and nutrient absorption by regulating microecological balance, and can help human body to comprehensively repair and reconstruct the microecological balance of the intestinal tract after being taken. By combining lactobacillus plantarum, lactobacillus casei, lactobacillus acidophilus and escherichia coli, living bacteria can smoothly reach the intestinal tract through a gastric acid barrier, and the balance of flora in the intestinal tract is regulated, namely the microecology of the intestinal tract reaches an equilibrium state through three steps of flora change, flora optimization and flora stabilization.
Detailed Description
Preparation example
Preparation example 1
The preparation process of the seed culture medium A is as follows:
10.00g of beef extract, 40.00g of peptone, 1.00g of sodium chloride, 1.00g of ammonium sulfate, 10.00g of monopotassium phosphate and 50.00g of lactose are mixed, added into 950.00g of distilled water, uniformly mixed, pH value is regulated to 6.5, and then sterilized for 30min under the conditions of 120 ℃ and 0.09MPa of pressure to prepare the seed culture medium A.
Preparation examples 2 to 3 differ from preparation example 1 in the amount of raw materials used for preparing the seed medium A, and the specific differences of preparation examples 1 to 3 are shown in Table 1:
TABLE 1 preparation examples 1-3 amounts of preparation raw materials
Preparation example 4
The preparation process of the seed culture medium B is as follows:
uniformly mixing 40.00g of peptone, 20.00g of glucose, 10.00g of yeast extract, 10.00g of beef extract, 1.00g of sodium chloride, 1.00g of monopotassium phosphate and 950g of distilled water, adjusting the pH to 5.5, and sterilizing at the temperature of 100 ℃ under the pressure of 0.09MPa for 30min to prepare the seed culture medium B.
Preparation examples 5 to 6 differ from preparation example 4 in the amount of raw materials used for preparing the seed medium A, and the specific differences of preparation examples 4 to 6 are shown in Table 2:
TABLE 2 preparation examples 4-6 amounts of preparation raw materials
| Experimental materials and procedure | Preparation example 4 | Preparation example 5 | Preparation example 6 |
| Peptone (g) | 40.00 | 80.00 | 100.00 |
| Glucose (g) | 20.00 | 40.00 | 60.00 |
| Yeast paste (g) | 10.00 | 30.00 | 50.00 |
| Beef extract (g) | 10.00 | 25.00 | 40.00 |
| Sodium chloride (g) | 1.00 | 2.00 | 3.00 |
| Potassium dihydrogen phosphate (g) | 1.00 | 2.00 | 3.00 |
| Distilled water (g) | 950.00 | 960.00 | 1000.00 |
| pH | 5.5 | 6 | 6.5 |
| Temperature (. Degree. C.) | 100 | 120 | 110 |
| Pressure intensity (MPa) | 0.09 | 0.11 | 0.12 |
| Sterilizing time (min) | 30 | 40 | 50 |
Preparation example 7
The preparation process of the seed culture medium C is as follows:
2 to 5 parts of glucose, 4 to 9 parts of peptone, 2 to 5 parts of beef extract, 2 to 6 parts of yeast extract, 0.1 to 0.5 part of monopotassium phosphate, 0.1 to 0.4 part of diamine citrate, 0.1 to 1 sodium acetate, 4 to 6 parts of tween, 0.1 to 0.4 part of magnesium sulfate, 0.1 to 0.3 part of manganese sulfate, 0.1 to 0.3 part of calcium carbonate and 95 to 100 parts of distilled water, wherein the pH value is 6.5 to 7, and then sterilizing for 30 to 50 minutes under the conditions of the temperature of 100 to 120 ℃ and the pressure of 0.09 to 0.12MPa to prepare the C seed culture medium.
Preparation examples 8 to 9 differ from preparation example 7 in the amount of raw materials used for preparing the seed medium A, and the specific differences of preparation examples 7 to 9 are shown in Table 3:
TABLE 3 preparation examples 7-9 amounts of preparation raw materials
| Experimental materials and procedure | Preparation example 7 | Preparation example 8 | Preparation example 9 |
| Glucose (g) | 20.00 | 35.00 | 50.00 |
| Peptone (g) | 40.00 | 70.00 | 90.00 |
| Yeast paste (g) | 20.00 | 40.00 | 60.00 |
| Potassium dihydrogen phosphate (g) | 1.00 | 3.00 | 5.00 |
| Citric acid diamine (g) | 1.00 | 3.00 | 4.00 |
| Tween (g) | 40.00 | 50.00 | 60.00 |
| Magnesium sulfate (g) | 1.00 | 3.00 | 4.00 |
| Manganese sulfate (g) | 1.00 | 2.00 | 3.00 |
| Calcium carbonate (g) | 1.00 | 2.00 | 3.00 |
| Distilled water (g) | 950.00 | 960.00 | 1000.00 |
| pH | 6.5 | 7 | 7 |
| Temperature (. Degree. C.) | 100 | 120 | 110 |
| Pressure intensity (MPa) | 0.09 | 0.11 | 0.12 |
| Sterilizing time (min) | 30 | 40 | 50 |
Preparation example 10
The preparation process of the culture medium in the production tank is as follows:
30.00g of corn steep liquor, 1.00g of ammonium sulfate 20.00g of beef extract, 1.00g of magnesium sulfate, 2.00g of dipotassium hydrogen phosphate, 2.00g of citric acid diamine, 2.00g of zinc chloride, 3.00g of calcium chloride and 950g of distilled water are fully stirred and dissolved, then the mixture is sealed and heated to 80 ℃, the pressure is regulated to 0.09MPa, and the mixture is sterilized for 1 hour to prepare the culture medium in the production tank.
Preparation examples 11 to 12 differ from preparation example 10 in the amounts of raw materials used for the culture medium in the production tank, and the specific differences of preparation examples 10 to 12 are shown in Table 4:
TABLE 4 preparation examples 10-12 amounts of raw materials for preparation
Examples
Example 1
A preparation method of an intestinal microecological active liquid comprises the following preparation steps:
s1, culturing lactobacillus plantarum in a seed culture medium A at a temperature of 35 ℃ for 24 hours to obtain lactobacillus plantarum mixed solution;
culturing lactobacillus casei in a seed culture medium B at 33 ℃ for 20 hours to obtain lactobacillus casei mixed solution; culturing lactobacillus acidophilus and escherichia coli in a seed culture medium C at a temperature of 33 ℃ for 16 hours to obtain a lactobacillus acidophilus and escherichia coli mixed solution;
s2, placing the lactobacillus plantarum mixed solution into a production tank for culture, and maintaining the ventilation rate at 5m 3 Stirring at 150r/min, maintaining the culture temperature at 35 ℃ and the pH at 7, adding lactobacillus casei mixed solution, co-culturing for 5h, adding lactobacillus acidophilus and escherichia coli mixed solution, and continuing culturing for 36h to obtain symbiotic fermentation liquor;
s3, uniformly stirring the symbiotic fermentation liquid and sterilized raw milk, fermenting for 20 hours at the fermentation temperature of 35 ℃, after the fermentation is finished, demulsifying and stirring, stirring for 30 minutes, standing for 40 hours, filtering, taking whey, weighing, continuously fermenting at the fermentation temperature of 35 ℃ for 20 hours, adding prebiotics in the fermentation process, wherein the dosage of the prebiotics is 1% of the weight of the whey, and concentrating to obtain concentrated solution;
s4, mixing the concentrated solution, glucose and sodium benzoate to obtain the intestinal microecological active liquid.
The milk protein rate of raw cow milk is 5.0%, the milk fat rate is 5.1%, the total colony count is 20 ten thousand CFU/mL, and the somatic cell count is 40 ten thousand/mL.
The weight ratio of the concentrated solution to glucose to sodium benzoate is 10:1:0.01.
the weight ratio of the lactobacillus plantarum mixed solution to the lactobacillus casei mixed solution to the lactobacillus acidophilus to the escherichia coli mixed solution is 3:8:7.
the content of lactobacillus plantarum in the concentrated solution is 1.1×10 6 CFU/g (ml), lactobacillus casei content of 1.2X10 6 CFU/g (ml), lactobacillus acidophilus content of 1.2X10 6 CFU/g (ml) and E.coli content of 0.6X10 6 CFU/g(ml)。
The prebiotics are fructo-oligosaccharide, inulin and galacto-oligosaccharide, and the weight ratio of the fructo-oligosaccharide, the inulin and the galacto-oligosaccharide is 1:2:3.
examples 2-3 differ from example 1 in the parameters of the preparation of the intestinal microecological active liquid, the specific differences of examples 1-3 are shown in Table 5:
TABLE 5 specific parameters of intestinal microecological active liquid in examples 1-3
Example 4
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: a seed medium was from preparation 2 and the rest of the experimental procedure was identical to example 1.
Example 5
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: b seed medium was from preparation 5, and the rest of the experimental procedure was identical to example 1.
Example 6
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: the seed medium C was from preparation 8 and the rest of the experimental procedure was identical to that of example 1.
Example 7
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: the culture medium in the production tank was obtained in preparation 11, and the rest of the experimental procedures were the same as in example 1.
Example 8
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: the weight ratio of the lactobacillus plantarum mixed solution to the lactobacillus casei mixed solution to the lactobacillus acidophilus and the escherichia coli mixed solution is 3:8:3, the remaining experimental steps were identical to example 1.
Example 9
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: the weight ratio of the lactobacillus plantarum mixed solution to the lactobacillus casei mixed solution to the lactobacillus acidophilus and the escherichia coli mixed solution is 3:15:3, the remaining experimental steps were identical to example 1.
Example 10
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: the weight ratio of the lactobacillus plantarum mixed solution to the lactobacillus casei mixed solution to the lactobacillus acidophilus and the escherichia coli mixed solution is 1:8:3, the remaining experimental steps were identical to example 1.
Example 11
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: the milk protein rate of raw cow milk is 3.0%, the milk fat rate is 2.4%, the total colony count is 18 ten thousand CFU/mL, the somatic cell count is 41 ten thousand/mL, the rest experimental steps are the same as those of example 1,
comparative example
Comparative example 1
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: in step S2, the mixture of Lactobacillus plantarum, lactobacillus casei and Lactobacillus acidophilus and Escherichia coli is placed in a production tank for culturing, and aeration rate is maintained at 5m 3 Stirring at 150 r/min/h/50L, culturing at 35deg.C, maintaining pH at 7 until the propagule is Bacillus empty, and culturing for 41 hr, wherein the rest experimental steps are the same as those of example 1.
Comparative example 2
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: in step S2, when Lactobacillus plantarum was cultured until the propagule was bacillus empty, lactobacillus casei mixed solution and Lactobacillus acidophilus and E.coli mixed solution were added, and the culture was continued for 41 hours, and the rest of the experimental procedures were the same as in example 1.
Comparative example 3
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: in step S1, the culture of Lactobacillus plantarum is omitted, in step S2, the culture is performed in a production tank, and the aeration rate is maintained at 5m 3 Stirring at 150r/min, culturing at 35deg.C, maintaining pH at 7, ventilating for the same time as in example 1, adding lactobacillus casei mixed solution, co-culturing for 5 hr, adding lactobacillus acidophilus and escherichia coli mixed solution, and culturing for 36 hr to obtain symbiotic fermentation broth, wherein the rest experimental steps are the same as in example 1.
Comparative example 4
The preparation method of the intestinal microecological active liquid is different from the embodiment 1 in that: in step S3, distilled water was used instead of raw milk, and the rest of the experimental procedures were the same as in example 1.
Performance test
The intestinal microecological active solutions prepared in examples 1 to 11 and comparative examples 1 to 4 were tested for their effects on treating diarrhea, insomnia and ulcerative enteritis.
Detection method/test method
The procedure for the diarrhea test in mice was as follows:
construction of an experimental mouse diarrhea model: 18-22g of mice 170 were selected. Model animals were randomly divided into 17 groups (normal, drug-applied (15 total, 10 per group), model control group), where the drug-applied group and model control group model diarrhea models. After the mice are fasted for 4 hours and each mouse is filled with 0.2-0.3mL of castor oil, the mice are placed in a single-culture cage, and filter paper or general white water absorbing paper is filled under the cage net. The number of points of the mice for eliminating rotten feces and thin feces in a certain time is observed.
After obvious diarrhea symptoms appear, respectively dissolving the intestinal microecological active liquid obtained in the examples 1-11 or the comparative examples 1-4 in sterile distilled water, and filling the intestinal microecological active liquid into mice in a group of administration mode by stomach irrigation; normal and model control groups were perfused with an equal amount of physiological saline, and the defecation conditions of the mice were observed within 4 hours after the gastric lavage, including the number of times of defecation, the diarrhea rate (the number of animals in defecation was a percentage of the total number of animals in the group) and the defecation conditions of the mice were recorded (the number of times of defecation per mouse was counted, the average was taken, and the whole number was taken according to the rounding method). The effect of the intestinal microecological active liquid on diarrhea is shown by the number of times of defecation and the diarrhea rate of mice. Experimental data are shown in table 6:
table 6 diarrhea test results in mice
| Examples or comparative examples | Number of times of defecation | Diarrhea Rate (%) |
| Example 1 | 10 | 40 |
| Example 2 | 11 | 40 |
| Example 3 | 10 | 40 |
| Example 4 | 10 | 30 |
| Example 5 | 10 | 40 |
| Example 6 | 11 | 40 |
| Example 7 | 10 | 40 |
| Example 8 | 14 | 80 |
| Example 9 | 13 | 60 |
| Example 10 | 14 | 70 |
| Example 11 | 14 | 60 |
| Comparative example 1 | 18 | 100 |
| Comparative example 2 | 16 | 90 |
| Comparative example 3 | 15 | 90 |
| Comparative example 4 | 14 | 80 |
| Normal group | 6 | 0 |
| Model control group | 20 | 100 |
Compared with a model control group, the number of times of defecation of the mice after taking the intestinal microecological active liquid is reduced, the diarrhea rate is reduced, and the antidiarrheal effect is obvious.
Example 1 and comparative examples 1 to 3 illustrate that the antidiarrheal effect of the intestinal microecological active liquid can be improved by the method for preparing the intestinal microecological active liquid according to the present application.
Example 1 and comparative example 4 demonstrate that antidiarrheal effect of the intestinal microecological active liquid can be improved by preparing a concentrate from raw cow's milk and a symbiotic fermentation broth.
The procedure for the ulcerative enteritis test in rats was as follows:
building an experimental rat ulcerative enteritis model: 170 Wistar rats weighing 200-220g were selected and were male and female halves. The dorsal part of the rat was dehaired, and 2% DNCB-acetone solution (2, 4-dinitrochlorobenzene 2 g/acetone 100 ml) was dropped on the dorsal part of the rat 1 time per day, each time 5 drops, and the sensitization was continued for 14 days. On day 15, a catheter 3mm in diameter was inserted into the intestinal canal through the anus by 8cm, and 0.25ml of 0.1% DNCB alcohol (DNCB 1g/50% alcohol 1000 ml) was injected, resulting in a model of acute ulcerative colitis, which was chronic after 2-5 weeks.
Randomly dividing model animals into three groups (normal group, administration group (15 groups each, 10 groups each) and model control group), respectively dissolving the intestinal microecological active liquid obtained in the above examples 1-11 or comparative examples 1-4 in sterile distilled water, and injecting the intestinal microecological active liquid into mice of administration group by adopting a stomach-filling administration mode; the control group was filled with an equal amount of physiological saline. After 4 weeks, the animals were sacrificed immediately after the animals were opened to access 9-12cm sections of intestine on the anus.
As a result of the experiment, the intestinal mucosa crypt of the model control group is disordered or disappeared, goblet cells are reduced, the proliferation of the lamina propria and submucosa fibers is serious, and chronic inflammatory cell infiltration occurs.
The intestinal mucosa of all mice in examples 1-7 had recovered to normal performance, i.e. was consistent with the normal group; essentially, but not completely, the intestinal mucosa of the mice in examples 8-11 was restored; the intestinal mucosa of all mice in comparative examples 1-3 was not restored; the intestinal mucosa of only 2 mice in comparative example 4 was not restored. The results show that the preparation of the intestinal microecological active liquid helps the mice to recover intestinal health.
The mice sleep test steps are as follows:
170 mice weighing 18-25g were selected, and the model animals were randomly divided into three groups (normal group, drug group (15 total groups, 10 each) and model control group). The small dose of caffeine (2 mg/kg) was injected intraperitoneally into the drug administration group and model control group at 7 hours and 30 minutes each day; the blank group was injected with physiological saline (0.1 ml/10 g) intraperitoneally for 7 days. The intestinal microecological active liquid dissolved in sterile water is irrigated with a group stomach according to a program from the 8 th day; model control and placebo control were only programmed to grasp untreated.
Observing whether the mice are lying or not during sleeping, and whether the mice have sniffs, lift limbs, tidy hair and walk; during the period of activities, the conditions of eating and playing, including irritation, biting, easy frightening, slow response, etc. Sleep is indicated by the disappearance of the eversion and the reflection. If the number exceeds 3060s, the person cannot turn over, namely, the turning over reflection is considered to disappear, and the person goes to sleep. The recovery of the eversion and the reflection is the arousal of the animal. The number of animals falling asleep in the model control group and the subject group was recorded. The influence of the intestinal microecological active liquid on the sleeping condition is represented by the number of the animals entering the sleep. Experimental data are shown in table 7:
TABLE 7 mouse sleep test results
| Examples or comparative examples | Number of animals sleeping |
| Example 1 | 7 |
| Example 2 | 7 |
| Example 3 | 8 |
| Example 4 | 7 |
| Example 5 | 7 |
| Example 6 | 7 |
| Example 7 | 8 |
| Example 8 | 6 |
| Example 9 | 5 |
| Example 10 | 6 |
| Example 11 | 5 |
| Comparative example 1 | 3 |
| Comparative example 2 | 4 |
| Comparative example 3 | 1 |
| Comparative example 4 | 4 |
| Normal group | 9 |
| Model control group | 3 |
Compared with a model control group, the intestinal microecological active liquid prepared by the application can obviously improve the sleeping condition of mice.
Example 1 and comparative examples 1 to 3 illustrate that the method for preparing an intestinal microecological active liquid according to the present application can improve the sleep-aiding effect of the intestinal microecological active liquid.
Example 1 and comparative example 4 demonstrate that the sleep-aiding effect of the intestinal microecological active liquid can be improved by preparing a concentrate from raw cow's milk and a symbiotic fermentation liquid.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. A preparation method of an intestinal microecological active liquid is characterized by comprising the following steps: the preparation method comprises the following preparation steps:
s1, culturing lactobacillus plantarum in a seed culture medium at a temperature of 35-40 ℃ for 20-24 hours to obtain lactobacillus plantarum mixed solution;
culturing lactobacillus casei in a seed culture medium at 30-35 ℃ for 20-24 hours to obtain lactobacillus casei mixed solution;
culturing lactobacillus acidophilus and escherichia coli in a seed culture medium C at 30-35 ℃ for 16-20 hours to obtain lactobacillus acidophilus and escherichia coli mixed solution;
wherein, the preparation process of the seed culture medium C is as follows:
2-5 parts of glucose, 4-9 parts of peptone, 2-5 parts of beef extract, 2-6 parts of yeast extract, 0.1-0.5 part of monopotassium phosphate, 0.1-0.4 part of citric acid diamine, 0.1-1 sodium acetate, 4-6 parts of tween, 0.1-0.4 part of magnesium sulfate, 0.1-0.3 part of manganese sulfate, 0.1-0.3 part of calcium carbonate and 95-100 parts of distilled water in parts by weight, and sterilizing for 30-50min at the temperature of 100-120 ℃ and the pressure of 0.09-0.12MPa to prepare a C seed culture medium;
s2, placing the lactobacillus plantarum mixed solution into a production tank for culture, and maintaining the ventilation rate to be 5-6m 3 Stirring at 150-200rpm, culturing at 35-40deg.C and pH of 7-8, adding Lactobacillus casei, and mixingMixing the solutions, co-culturing for 5-6h, adding mixed solution of lactobacillus acidophilus and escherichia coli, and continuously culturing for 36-40h to obtain symbiotic fermentation liquor;
s3, uniformly stirring the symbiotic fermentation liquid and the sterilized raw milk, fermenting for 20-24 hours at the fermentation temperature of 35-40 ℃, after the fermentation is finished, demulsifying and stirring, stirring for 30-60 minutes, standing for 40-48 hours, filtering, taking whey, continuously fermenting, and concentrating to obtain a concentrated solution;
s4, mixing the concentrated solution, glucose and sodium benzoate to obtain the intestinal microecological active liquid.
2. The method for preparing the intestinal microecological active liquid according to claim 1, wherein in the step 1), the seed culture medium A is prepared by the following steps:
according to parts by weight, 1-6 parts of beef extract, 4-8 parts of peptone, 0.1-0.5 part of sodium chloride, 0.1-0.3 part of ammonium sulfate, 1-2 parts of monopotassium phosphate and 5-10 parts of lactose are mixed, added into 95-100 parts of distilled water, uniformly mixed, pH value is regulated to 6.5-7.5, and then sterilized for 30-40min under the conditions that the temperature is 120-130 ℃ and the pressure is 0.09-0.12MPa, so that the seed culture medium A is prepared.
3. The method for preparing the intestinal microecological active liquid according to claim 2, wherein in the step 2), the seed culture medium is prepared as a seed culture medium B as follows:
according to parts by weight, 4-10 parts of peptone, 2-6 parts of glucose, 1-5 parts of yeast extract, 1-4 parts of beef extract, 0.1-0.3 part of sodium chloride, 0.1-0.3 part of potassium dihydrogen phosphate and 95-100 parts of distilled water are uniformly mixed, pH is regulated to 5.5-6.5, and then sterilization is carried out for 30-50min under the conditions that the temperature is 100-120 ℃ and the pressure is 0.09-0.12MPa, so that the seed culture medium B is prepared.
4. A method for preparing an intestinal microecological active liquid according to claim 3, wherein the preparation process of the culture medium in the production tank is as follows:
3 to 6 parts of corn steep liquor, 0.1 to 0.4 part of ammonium sulfate, 2 to 5 parts of beef extract, 0.1 to 0.3 part of magnesium sulfate, 0.2 to 0.4 part of dipotassium hydrogen phosphate, 0.2 to 0.6 part of citric acid diamine, 0.2 to 0.7 part of zinc chloride, 0.3 to 0.5 part of calcium chloride and 950 to 1000 parts of distilled water are fully stirred and dissolved, then the mixture is sealed and heated to 80 to 100 ℃, the pressure is regulated to 0.09 to 0.12MPa, and the mixture is sterilized for 1 to 2 hours, thus obtaining the culture medium in the production tank.
5. The method for preparing an intestinal microecological active liquid according to claim 3, wherein the content of lactobacillus in the concentrated liquid is 10 or more 6 CFU/g（ml）。
6. The method for preparing the intestinal microecological active liquid according to claim 1, wherein the method comprises the following steps: and (3) continuing fermentation in the step (S3) at 35-40 ℃ for 20-24 hours, wherein prebiotics are added in the fermentation process, and the dosage of the prebiotics is 1-2% of the weight of whey.
7. The method for preparing the intestinal microecological active liquid according to claim 1, wherein the method comprises the following steps: the milk protein rate of raw cow milk is more than or equal to 5.0%, the milk fat rate is more than or equal to 5.1%, the total colony count is less than or equal to 20 ten thousand CFU/mL, and the somatic cell count is less than or equal to 40 ten thousand/mL.
8. The method for preparing the intestinal microecological active liquid according to claim 7, wherein the method comprises the following steps: the weight ratio of the concentrated solution to the glucose to the sodium benzoate is (10-15): (1-3): 0.01.
9. the method for preparing the intestinal microecological active liquid according to claim 7, wherein the method comprises the following steps: the lactobacillus plantarum mixed solution, the lactobacillus casei mixed solution and the lactobacillus acidophilus and escherichia coli mixed solution are prepared according to the weight ratio of (3-6): (8-12): 7.
10. an application of an intestinal microecological active liquid is characterized in that: the intestinal microecological active liquid is used as an oral liquid, and is prepared by the preparation method of the intestinal microecological active liquid according to any one of claims 1 to 9.
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