CN115537362A - Composite bacterium for regulating human body micro-ecology and preparation method and application thereof - Google Patents

Composite bacterium for regulating human body micro-ecology and preparation method and application thereof Download PDF

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CN115537362A
CN115537362A CN202211473425.5A CN202211473425A CN115537362A CN 115537362 A CN115537362 A CN 115537362A CN 202211473425 A CN202211473425 A CN 202211473425A CN 115537362 A CN115537362 A CN 115537362A
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culture medium
fermentation
bifidobacterium
preparation
bacteria
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冯魏
郑雅丹
杨鹏
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Hangzhou Hengmei Food Technology Co ltd
Nanjing Jienuo Biological Products Co ltd
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Hangzhou Hengmei Food Technology Co ltd
Nanjing Jienuo Biological Products Co ltd
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Abstract

The invention provides a composite bacterium for regulating human microecology and a preparation method and application thereof, relating to the field of probiotics, wherein the preparation method of the composite bacterium comprises the following steps: (1) Inoculating lactobacillus plantarum into a culture medium, and performing aerobic fermentation to obtain a fermentation liquid A; (2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium into a culture medium, and performing anaerobic fermentation to obtain a fermentation liquid B; (3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria; wherein, the culture medium comprises: peptone, cane molasses, yeast extract, dipotassium hydrogen phosphate, sodium citrate, corn oligopeptide powder, cottonseed oligosaccharide, magnesium sulfate pentahydrate, tween-80, nicotinamide, D-sodium erythorbate, arachidonic acid grease and vitamin B 1 . The prepared composite bacteria can regulate intestinal microecology, thereby reducing the disease risk of metabolic diseases such as infectious diseases, obesity, diabetes and the like.

Description

Composite bacterium for regulating human body micro-ecology and preparation method and application thereof
Technical Field
The invention relates to the field of probiotics, in particular to a composite bacterium for regulating human microecology and a preparation method and application thereof.
Background
The human microecosystem comprises 6 microecosystems of oral cavity, skin, urinary tract, respiratory tract, vagina and intestinal tract, wherein the intestinal microecosystem is most important and complex, and according to the literature: jiangjin, lianjuan, the current research situation of human microecology and disease and the information of Wang [ J ] infectious disease, 2016 (5): 7, it is known that intestinal microecology is closely related to infectious diseases, metabolic diseases such as obesity and diabetes, liver diseases, intestinal diseases, etc.
The dysbiosis of human body refers to the balance of microecology between normal microbiota and host, and under the influence of external environment, the state is changed from physiological combination to pathological combination. Dysbiosis includes: (1) dysbacteriosis, namely, the quantity and the density of beneficial bacteria are reduced, and the pellicle barrier effect is weakened; (2) the ease of flora, i.e., the struggle between microbial populations, and (3) the colonization of foreign bacteria. When the human body micro-ecology is disordered, related diseases can be caused, and the balance of the human body micro-ecology is found to be helpful for preventing and treating the diseases according to the research.
Related studies indicate that there is a close relationship between probiotics and human micro-ecology, as in the literature: the study on the interrelation between the lactobacillus and the human body microenvironment [ J ] family medicine, medicine selection, 2017 (12) finds that the lactobacillus as the probiotic plays a vital role in the intestinal microecological balance, disease control and health care of the human body. The literature is as follows: the results show that the two bacteria can effectively improve the micro-ecological environment of the intestinal tract digestion system of the human body, the viable count of the bifidobacteria and the lactobacilli in the volunteers is obviously increased, and the viable count of the clostridium perfringens is obviously reduced. The literature: the correlation between bifidobacterium in intestinal tract and microecological balance was studied in the proceedings of the university of Hebei agriculture [ J ]. 2000.
Based on the above researches, related microbial agents are used for regulating the micro-ecological balance of human bodies, for example, patent CN107308190A discloses a probiotic composition for regulating the micro-ecological balance of human bodies, and a culture, a preparation and a use thereof, wherein the probiotic composition is prepared from lactobacillus paracasei, lactobacillus bulgaricus, lactobacillus fermentum, lactobacillus gasseri, lactobacillus plantarum subspecies plantarii and lactobacillus helveticus, and further can be used for preventing and treating immune system diseases such as infantile eczema and the like and various types of enteritis.
For another example, patent CN101406489B discloses a method for preparing a microecological complex microbial inoculum, which mainly comprises bacillus natto, kluyveromyces lactis, lactobacillus acidophilus and soybean oligosaccharide. The preparation method comprises the following steps: culturing; carrying out amplification culture; transferring into high-bacteria-concentration culture solution for continuous culture and centrifuging; adding auxiliary materials, pre-freezing, vacuum freeze-drying, and then adding soybean oligosaccharide to obtain the microecological compound microbial inoculum which has more total beneficial viable bacteria and survival rate.
However, at present, the single probiotic bacteria have limited ability to regulate the micro-ecology, and although the complex bacteria can regulate the micro-ecology through competition, the complex bacteria also have the problems of low multi-bacterial activity, poor culture medium safety and the like. Therefore, in order to solve the above problems, it is necessary to find a complex bacterium capable of effectively regulating the intestinal micro-ecology.
Disclosure of Invention
The invention provides a composite bacterium for regulating human microecology, a preparation method and application thereof aiming at the problems in the prior art, and the prepared composite bacterium can regulate intestinal microecology so as to reduce the risk of infectious diseases, obesity, diabetes and other metabolic diseases.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of composite bacteria, which comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium, and performing aerobic fermentation to obtain a fermentation liquid A;
(2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium into a culture medium, and performing anaerobic fermentation to obtain a fermentation liquid B;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the compound bacteria;
the culture medium comprises: peptone, cane molasses, yeast extract, dipotassium hydrogen phosphate, sodium citrate, corn oligopeptide powder, cottonseed oligosaccharide, magnesium sulfate pentahydrate, tween-80, nicotinamide, D-sodium erythorbate, arachidonic acid grease and vitamin B 1 。
Further, the culture medium comprises: 4-6g/L peptone, 2-4g/L cane molasses, 3-6g/L yeast extract, 3-6g/L dipotassium hydrogen phosphate, 0.5-3g/L sodium citrate, 8-16g/L corn oligopeptide powder, 0.5-4.5g/L cottonseed oligosaccharide, 0.5-2g/L magnesium sulfate pentahydrate, 0.5-5mL/L Tween-80, 1-6g/L nicotinamide, 35-40mg/L D-sodium erythorbate, 20-40mg/L arachidonic acid oil and fat, vitamin B 1 2-10mg/L。In actual practice, 1L of deionized water was used to make up.
Preferably, the culture medium comprises: 5g/L peptone, 3g/L cane molasses, 5g/L yeast extract, 5g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, 10g/L corn oligopeptide powder, 1g/L cottonseed oligosaccharide, 1g/L magnesium sulfate pentahydrate, 1.5mL/L Tween-80, 2g/L nicotinamide, 36mg/L D-sodium erythorbate, 28mg/L arachidonic acid oil and fat, vitamin B 1 3mg/L. In actual practice, deionized water was used to make up 1L.
Further, the bifidobacteria comprise one or more of bifidobacterium infantis, bifidobacterium animalis, bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum. Preferably bifidobacterium infantis, bifidobacterium animalis, bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum; further preferred are bifidobacterium infantis, bifidobacterium animalis, bifidobacterium adolescentis, bifidobacterium bifidum and bifidobacterium longum in a volume ratio of 1.
Further, the inoculation amount of the lactobacillus rhamnosus, the lactobacillus casei and the bifidobacterium in the culture medium is 2 to 5 percent by volume percentage; the inoculation amount of the lactobacillus plantarum in a culture medium is 1-2%.
Furthermore, the volume ratio of the inoculation amounts of lactobacillus rhamnosus, lactobacillus casei and bifidobacterium is 1.
Further, the temperature of the aerobic fermentation is 35-37 ℃, the time is 24h, and the rotating speed is 150-200r/min; the anaerobic fermentation temperature is 35-37 ℃, the time is 48h, and the rotating speed is 110-150r/min.
Further, the invention also provides the composite bacteria obtained by the preparation method.
Furthermore, the composite bacteria obtained by the preparation method provided by the invention can be used for preparing products for regulating human microecology.
Further, the product for regulating the human body micro-ecology comprises bacterial powder, buccal tablets, fermented milk, soft sweets or beverage.
Furthermore, the raw materials of the product for regulating the human body microecology also comprise auxiliary materials;
further, the auxiliary materials comprise resistant dextrin, isomaltooligosaccharide, galactooligosaccharide, whole milk powder and seaweed powder.
The technical effects obtained by the invention are as follows:
1. according to the lactobacillus rhamnosus, lactobacillus casei, bifidobacterium and lactobacillus plantarum are selected for matched culture, and in actual operation, aerobic fermentation and anaerobic fermentation are respectively carried out on different strains according to actual conditions, so that the power consumption is reduced. Meanwhile, the culture medium is optimized, specifically, the components of the culture medium are reasonably added, the added cottonseed oligosaccharide (also called as cotton seed oligosaccharide) has an excellent effect on the aspect of assisting proliferation of beneficial bacteria, the effect is better when the cottonseed oligosaccharide is used together with D-sodium erythorbate and arachidonic acid grease, and the obtained culture medium can effectively promote the improvement and growth of the activity of the probiotic bacteria, increase the utilization rate of the components such as a carbon source and the like and ensure the stability of bacteria.
2. The compound bacteria finally prepared by the invention can be directly or indirectly matched with auxiliary materials and the like to prepare a preparation which can directly supplement normal physiological bacteria, and meanwhile, lactic acid, acetic acid and other organic acids are generated in the metabolic process of probiotics, so that the pH value of intestinal tracts can be reduced, the intestinal tract acid environment can be formed, pathogenic bacteria in the intestinal tracts can be cleared, the micro-ecology of human bodies can be efficiently regulated, and the intestinal tract micro-ecology can be especially regulated, so that the disease risk of metabolic diseases such as infectious diseases, obesity, diabetes and the like can be reduced.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is to be noted that the cells used in the present invention are: bifidobacterium infantis CCFM687, lactobacillus rhamnosus CCFM0528, lactobacillus plantarum CN2018, lactobacillus casei CN1566, bifidobacterium adolescentis CCFM1173, bifidobacterium animalis CCFM1155, bifidobacterium bifidum CCFM1167, lactobacillus rhamnosus CCFM1219, bifidobacterium longum CCFM760, and other raw materials are common commercial products, so the source of the raw materials is not particularly limited.
Example 1
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 2% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 35 ℃ for 24 hours at a rotation speed of 200r/min to obtain a fermentation liquid A;
(2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium (1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria;
wherein the culture medium comprises: 4g/L peptone, 2g/L cane molasses, 3g/L yeast extract, 3g/L dipotassium hydrogen phosphate, 0.5g/L sodium citrate, 8g/L corn oligopeptide powder, 0.5g/L cottonseed oligosaccharide, 0.5g/L magnesium sulfate pentahydrate, 0.5mL/L tween-80, 1g/L nicotinamide, and D-sodium erythorbate 35mg/L, 20mg/L arachidonic acid oil and fat, vitamin B 1 2mg/L. In actual practice, deionized water was used to make up 1L.
Example 2
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 5% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 37 ℃ for 24 hours at a rotation speed of 150r/min to obtain a fermentation liquid A;
(2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium into a culture medium (the inoculation amount accounts for 2% v/v of a culture solution in the culture medium) in a volume ratio of 1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria;
wherein, the culture medium comprises: 6g/L peptone, 4g/L cane molasses, 6g/L yeast extract, 6g/L dipotassium hydrogen phosphate, 3g/L sodium citrate, 16g/L corn oligopeptide powder, 4.5g/L cottonseed oligosaccharide, 2g/L magnesium sulfate pentahydrate, 5mL/L Tween-80, 6g/L nicotinamide, 40mg/L D-sodium erythorbate, 40mg/L arachidonic acid oil, vitamin B 1 10mg/L. In actual practice, deionized water was used to make up 1L.
Example 3
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 2-5% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 35-37 ℃ for 24h at a rotation speed of 150-200r/min to obtain a fermentation liquid A;
(2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium (1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria;
wherein the culture medium comprises: 5g/L peptone, 3g/L cane molasses, 5g/L yeast extract, 5g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, 10g/L corn oligopeptide powder, 1g/L cottonseed oligosaccharide, 1g/L magnesium sulfate pentahydrate, 1.5mL/L Tween-80, 2g/L nicotinamide, 36mg/L D-sodium erythorbate, 28mg/L arachidonic acid oil and fat, vitamin B 1 3mg/L. In actual practice, deionized water was used to make up 1L.
Example 4
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 2% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 35 ℃ for 24 hours at a rotation speed of 200r/min to obtain a fermentation liquid A;
(2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium infantis into a culture medium (the inoculation amount accounts for 1% v/v of a culture solution in the culture medium) in a volume ratio of 1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the compound bacteria;
wherein, the culture medium comprises: 4g/L peptone, 2g/L cane molasses, 3g/L yeast extract, 3g/L dipotassium hydrogen phosphate, 0.5g/L sodium citrate, 8g/L corn oligopeptide powder, 0.5g/L cottonseed oligosaccharide, 0.5g/L magnesium sulfate pentahydrate, 0.5mL/L Tween-80, 1g/L nicotinamide, 35mg/L sodium D-isoascorbate, 20mg/L arachidonic acid oil and fat, vitamin B 1 2mg/L. In actual practice, 1L of deionized water was used to make up.
Example 5
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 5% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 37 ℃ for 24 hours at a rotation speed of 150r/min to obtain a fermentation liquid A;
(2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium (bifidobacterium animalis and bifidobacterium adolescentis with the volume ratio of 1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria;
wherein the culture medium comprises: 6g/L peptone, 4g/L cane molasses, 6g/L yeast extract, 6g/L dipotassium hydrogen phosphate, 3g/L sodium citrate, 16g/L corn oligopeptide powder, 4.5g/L cottonseed oligosaccharide, 2g/L magnesium sulfate pentahydrate, 5mL/L Tween-80, 6g/L nicotinamide, 40mg/L D-sodium erythorbate, 40mg/L arachidonic acid oil and fat, and vitamin B 1 10mg/L. In actual practice, deionized water was used to make up 1L.
Comparative example 1
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 2% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 35 ℃ for 24 hours at a rotation speed of 200r/min to obtain a fermentation liquid A;
(2) Inoculating bacillus coagulans, lactobacillus acidophilus and bifidobacterium longum (the volume ratio is 1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the compound bacteria;
wherein, the culture medium comprises: 4g/L peptone, 2g/L cane molasses, 3g/L yeast extract, 3g/L dipotassium hydrogen phosphate, 0.5g/L sodium citrate, 8g/L corn oligopeptide powder, 0.5g/L cottonseed oligosaccharide, and,0.5g/L magnesium sulfate pentahydrate, 0.5mL/L tween-80, 1g/L nicotinamide, 35mg/L D-sodium erythorbate, 20mg/L arachidonic acid oil and fat, vitamin B 1 2mg/L. In actual practice, deionized water was used to make up 1L.
That is, the only difference from example 1 was that lactobacillus rhamnosus and lactobacillus casei in the medium were replaced with an equal amount of bacillus coagulans ATCC7050 and an equal amount of lactobacillus acidophilus ATCC4356, respectively.
Comparative example 2
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 2% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 35 ℃ for 24 hours at a rotation speed of 200r/min to obtain a fermentation liquid A;
(2) Inoculating bacillus coagulans, lactobacillus acidophilus and bifidobacterium longum (the volume ratio is 1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria;
wherein, the culture medium comprises: 4g/L peptone, 2g/L cane molasses, 3g/L yeast extract, 3g/L dipotassium hydrogen phosphate, 0.5g/L sodium citrate, 8g/L corn oligopeptide powder, 0.5g/L cottonseed oligosaccharide, 0.5g/L magnesium sulfate pentahydrate, 0.5mL/L Tween-80, 1g/L nicotinamide, 55mg/L arachidonic acid oil and fat, vitamin B 1 2mg/L. In actual practice, deionized water was used to make up 1L.
That is, the only difference from example 1 was that D-erythorbic acid was replaced with an equal amount of arachidonic acid oil and fat.
Comparative example 3
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 2% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 35 ℃ for 24h at a rotation speed of 200r/min to obtain a fermentation liquid A;
(2) Inoculating 1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria;
wherein the culture medium comprises: 4g/L peptone, 2g/L cane molasses, 3g/L yeast extract, 3g/L dipotassium hydrogen phosphate, 0.5g/L sodium citrate, 8g/L corn oligopeptide powder, 0.5g/L cottonseed oligosaccharide, 0.5g/L magnesium sulfate pentahydrate, 0.5mL/L tween-80, 1g/L nicotinamide, 55mg/L D-sodium erythorbate, vitamin B 1 2mg/L. In actual practice, deionized water was used to make up 1L.
That is, the only difference from example 1 was that arachidonic acid oil was replaced with an equal amount of sodium D-isoascorbate.
Comparative example 4
A preparation method of composite bacteria comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium (the inoculation amount accounts for 2% v/v of a culture solution in the culture medium), and performing aerobic fermentation at 35 ℃ for 24h at a rotation speed of 200r/min to obtain a fermentation liquid A;
(2) Inoculating bacillus coagulans, lactobacillus acidophilus and bifidobacterium longum (the volume ratio is 1;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the composite bacteria;
wherein the culture medium comprises: 4g/L peptone, 2g/L cane molasses, 3g/L yeast extract, 3g/L dipotassium hydrogen phosphate, 0.5g/L sodium citrate, 8g/L corn oligopeptide powder, 0.5g/L magnesium sulfate pentahydrate, 0.5mL/L tween-80, 1g/L nicotinamide, vitamin B 1 2mg/L. In actual practice, 1L of deionized water was used to make up.
That is, the difference from example 1 is that arachidonic acid oil and fat, sodium D-isoascorbate, and cottonseed oligosaccharide were not added to the medium.
Application example 1
A product for regulating human microecology: the complex bacteria of example 1 was mixed with resistant dextrin, isomaltooligosaccharide, galactooligosaccharide, whole milk powder, and seaweed powder.
Application example 2
A product for regulating human microecology: the complex bacteria of example 2 were mixed with resistant dextrin, isomaltooligosaccharide, galacto-oligosaccharide, whole milk powder, and seaweed powder.
Application example 3
A product for regulating human microecology: the complex bacteria of example 3 was mixed with resistant dextrin, isomaltooligosaccharide, galactooligosaccharide, whole milk powder, and seaweed powder.
1. Intestinal flora regulation test
Test animals: c57BL/6J mice, male;
the test method comprises the following steps: randomly dividing the mice into a control group and an example group, wherein each group comprises 6 mice, the compound bacteria in each example are gavaged in the example group, the gavage amount is 0.4g/kg d, the time is 2 weeks, and the same amount of normal saline is gavaged in the control group; collecting 0.1g of fresh feces of each mouse before and after gavage for detecting intestinal flora, specifically, diluting the sample with normal saline, oscillating for homogenizing, diluting, adding dropwise into culture medium of 50 μ L, after the culture medium is dried, anaerobically culturing TSC culture medium at 37 deg.C for 48h, and detecting Clostridium perfringens after bacterial colony grows outClostridium perfringensThe number of bacteria (2) is given as a logarithmic value lgCFU g -1 The results are shown in the following table:
TABLE 1 number of intestinal flora in mice (
Figure 10000259507589
±s）
Figure 350225DEST_PATH_IMAGE001
(Note: p < 0.05, p < 0.01 compared to model control group.
2. Defecation test
Test animals: 8 week old KM mice, male;
the test method comprises the following steps:
(1) preparing ink: mixing Arabic gum with water, boiling, adding activated carbon powder, boiling, and cooling;
(2) molding: after the mice are fasted and fed for 1 day without water, 0.025 percent compound diphenoxylate is used for molding;
(2) administering to the subject: after 30min of modeling, the mice successfully modeled are randomly divided into a model control group and an example group, each group comprises 6 mice, the example group is used for respectively intragastrically irrigating the compound bacteria in different examples, the intragastrically irrigating amount is 0.4 g/kg. D, the model control group is used for intragastrically irrigating the equivalent physiological saline, in addition, 6 mice without modeling are selected as a blank group to be administered with the equivalent physiological saline, the defecation condition of the mice is observed after each group of prepared ink mice are administered for 6h, and the average time of first defecation is counted into a table 2.
TABLE 2 defecation in mice
Figure 214276DEST_PATH_IMAGE002
From the test results, the composite bacteria can effectively adjust the intestinal microecological balance and shorten the first particle black discharge time, and the method corresponding to the embodiment 3 is selected as the preparation method of the optimal composite bacteria, and the composite bacteria is matched with human edible auxiliary materials, such as resistant dextrin, isomaltooligosaccharide, galacto-oligosaccharide, whole milk powder, seaweed powder and the like, and the product obtained by matching production can effectively adjust the intestinal microecological of the human body.
In addition to the above cases, the method corresponding to examples 4-5 mainly explores the influence of replacing bifidobacterium species on the product, and the finally obtained product also has similar performance to examples 1-3, which shows that the effect of the bifidobacterium species on the product itself is not greatly influenced, but the effect of the final product is not satisfactory due to the inadaptability of fermentation conditions after the replacement of other strains is attempted.
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. A preparation method of composite bacteria is characterized by comprising the following steps: the method comprises the following steps:
(1) Inoculating lactobacillus plantarum into a culture medium, and performing aerobic fermentation to obtain a fermentation liquid A;
(2) Inoculating lactobacillus rhamnosus, lactobacillus casei and bifidobacterium into a culture medium, and performing anaerobic fermentation to obtain a fermentation liquid B;
(3) Mixing the fermentation liquor A and the fermentation liquor B, centrifuging and concentrating to obtain active bacterial sludge, and drying to obtain the compound bacteria;
the culture medium comprises: peptone, cane molasses, yeast extract, dipotassium hydrogen phosphate, sodium citrate, corn oligopeptide powder, cottonseed oligosaccharide, magnesium sulfate pentahydrate, tween-80, nicotinamide, D-sodium erythorbate, arachidonic acid oil and fat, vitamin B 1 。
2. The method of claim 1, wherein: the culture medium comprises: 4-6g/L peptone, 2-4g/L cane molasses, 3-6g/L yeast extract, 3-6g/L dipotassium hydrogen phosphate, 0.5-3g/L sodium citrate, 8-16g/L corn oligopeptide powder, 0.5-4.5g/L cottonseed oligosaccharide, 0.5-2g/L magnesium sulfate pentahydrate, 0.5-5mL/L tween-80, 1-6g/L nicotinamide, 35-40mg/L sodium D-isoascorbate, 20-40mg/L arachidonic acid oil and fat, vitamin B 1 2-10mg/L。
3. The method of claim 1, wherein: the culture medium comprises: 5g/L peptone, 3g/L cane molasses, 5g/L yeast extract, 5g/L dipotassium hydrogen phosphate, 2g/L sodium citrate, 10g/L corn oligopeptide powder, 1g/L cottonseed oligosaccharide, 1g/L magnesium sulfate pentahydrate, 1.5mL/L Tween-80, 2g/L nicotinamide, 36mg/L sodium D-isoascorbate, 28mg/L arachidonic acid oil and fat, vitamin B 1 3mg/L。
4. The method of claim 1, wherein: the Bifidobacterium includes one or more of Bifidobacterium infantis, bifidobacterium animalis, bifidobacterium adolescentis, bifidobacterium bifidum, and Bifidobacterium longum.
5. The method of claim 1, wherein: according to the volume percentage, the inoculation amount of the lactobacillus rhamnosus, the lactobacillus casei and the bifidobacteria in the culture medium is 2-5 percent; the inoculation amount of the lactobacillus plantarum in the culture medium is 1-2%.
6. The production method according to claim 1, characterized in that: the temperature of the aerobic fermentation is 35-37 ℃, the time is 24h, and the rotating speed is 150-200r/min; the anaerobic fermentation temperature is 35-37 deg.C, the time is 48h, and the rotation speed is 110-150r/min.
7. A complex bacterium obtained by the production method according to any one of claims 1 to 6.
8. Use of the complex bacteria obtained by the preparation method according to any one of claims 1 to 6 in the preparation of a product for regulating human microecology.
9. Use according to claim 8, characterized in that: the dosage form of the product for regulating the human body microecology comprises bacterial powder, buccal tablets, fermented milk, soft sweets or beverage.
10. Use according to claim 8, characterized in that: the raw materials of the product for regulating the human body microecology also comprise resistant dextrin, isomaltooligosaccharide, galacto-oligosaccharide, whole milk powder and seaweed powder.
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