CN113082145B - Method for increasing AKK (alkyl ketene dimer) number - Google Patents
Method for increasing AKK (alkyl ketene dimer) number Download PDFInfo
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- CN113082145B CN113082145B CN202110426353.8A CN202110426353A CN113082145B CN 113082145 B CN113082145 B CN 113082145B CN 202110426353 A CN202110426353 A CN 202110426353A CN 113082145 B CN113082145 B CN 113082145B
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Abstract
The invention discloses a method for increasing the number of AKK bacteria, belonging to the technical field of microorganisms. Pericarpium Citri Tangerinae, radix Codonopsis, longan, Glycyrrhrizae radix, fructus Lycii, rhizoma Dioscoreae and/or flos Chrysanthemi can be used as nutritional supplement, or its effective component extract can be used as nutritional supplement. The dietary supplements with homology of medicine and food provided by the invention can improve the number of AKK bacteria in vivo or in vitro, can be added into a culture medium as an auxiliary material to improve the yield of AKK bacteria, and is used for large-scale production of industrial culture; the dietary supplement safe to human bodies can improve the quantity of AKK bacteria in intestinal tracts of patients by oral administration, and achieves the purposes of preventing and treating diseases with reduced quantity of AKK bacteria, such as diabetes, hypertension, hyperlipidemia, diseases of liver systems and cardiovascular and cerebrovascular systems, tumor adjuvant therapy and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms.
Background
AKK is a gram-negative anaerobic bacterium, and is a next generation probiotic. During early life, AKK bacteria colonize the gut and levels within a year approach those observed in healthy adults, accounting for approximately 1-3% of the overall microbiota, one of the most numerous single species in the human gut flora. While AKK bacteria use mucin as an energy source, AKK bacteria maintain intestinal mucus layer thickness and intestinal barrier integrity. The AKK bacteria can improve the symptoms of diabetes, obesity, liver, intestinal inflammation and other diseases and is beneficial to treatment. The AKK bacteria with high abundance in intestinal tract can improve the immune checkpoint inhibitor immunotherapy effect of tumors. In addition, AKK bacteria have been reported to have a negative correlation in diseases such as autism and epilepsy. Therefore, finding a dietary supplement capable of promoting the growth of AKK bacteria is of great importance for the prevention and treatment of diseases.
Meanwhile, the term of homology of medicine and food is gradually known, and related research and discussion are increasing. The homology of medicine and food is the summary of the relation between medicine and food and the application of modern people, which means that the food and the medicine have the same source, and have component homology and theoretical homology, and many foods have edibility and medicinal property, so the health care food can be used for health preservation and health care and can prevent and treat diseases.
AKK (AKK-K) is an anaerobic bacterium, and has attracted attention as a good star bacterium in recent years, but the culture conditions for the industrial production of AKK are immature at present. Difficult culture, slow growth, low yield and the like.
Disclosure of Invention
The invention aims to provide a dietary supplement from homology of medicine and food, which can promote the growth of AKK bacteria. In vitro, the method can be used for large-scale production of AKK bacteria suitable for human use, and is convenient and economical; in vivo, the product can be used as a dietary supplement, is safe to take, and plays a certain role in preventing and treating certain diseases by improving the abundance of AKK bacteria in intestinal tracts.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for increasing the amount of AKK bacteria comprises using pericarpium Citri Tangerinae, radix Codonopsis, longan, Glycyrrhrizae radix, fructus Lycii, rhizoma Dioscoreae and/or flos Chrysanthemi as nutritional supplement, or using its effective component extract in combination with AKK bacteria as nutritional supplement.
Dried orange peel, codonopsis pilosula, longan, liquorice and/or medlar are used as a nutritional supplement for increasing the number of AKK bacteria in anaerobic culture conditions, or an extract of the effective components of the dried orange peel, the codonopsis pilosula, the longan, the liquorice and/or the medlar are used as a nutritional supplement.
Pericarpium Citri Tangerinae, radix Codonopsis, longan, Glycyrrhrizae radix or fructus Lycii is used as single nutritional supplement for increasing AKK bacteria amount in anaerobic culture condition, or its effective component extract is used as single nutritional supplement.
When the water decoction is used:
the concentration of each traditional Chinese medicine in the medicine-containing culture medium is respectively as follows: 0.002g/ml to 0.04g/ml of dried orange peel; 0.002g/ml to 0.03g/ml of medlar; longan, 0.006g/ml to 0.03 g/ml; 0.006g/ml to 0.06g/ml of codonopsis pilosula; licorice root, 0.002g/ml to 0.02 g/ml.
One of three combinations of medlar, longan, radix codonopsitis, liquorice and radix codonopsitis and longan is used as a nutritional supplement for improving the number of AKK bacteria in anaerobic culture conditions, or an effective component extract of the nutritional supplement is used as the nutritional supplement;
the mass ratio of the medlar to the longan in the composition is 0.2-3: 0.6-3; the mass ratio of the codonopsis pilosula to the liquorice in the composition is 0.6-6: 0.2-2; the mass ratio of the codonopsis pilosula to the longan in the composition is 0.6-6: 0.6-3;
when the water decoction is used:
the culture medium comprises medlar and longan: 0.002-0.03g/ml of medlar; longan, 0.006-0.03 g/ml;
in a combined culture medium containing codonopsis pilosula and liquorice: radix Codonopsis, 0.006-0.06 g/ml; licorice root, 0.002-0.02 g/ml;
in a combined culture medium containing radix codonopsitis and longan: radix Codonopsis, 0.006-0.06 g/ml; longan, 0.006-0.03 g/ml.
Use of radix Codonopsis + longan, longan or fructus Lycii in combination with AKK bacteria as nutritional supplement for improving intestinal AKK and reducing the number of maleThe effective component extract is taken in combination with AKK bacteria as a nutritional supplement; the mass ratio of the codonopsis pilosula to the longan is 1-3.9: 0.1-1.95. When the decoction of Chinese medicinal liquid is used as nutritional supplement, the dosage of fructus Lycii is 6-12 g/Kg/day, and the dosage of arillus longan is 9-15 g/Kg/day. The preparation method comprises decocting radix Codonopsis and arillus longan in water at a ratio of 9-30 g/Kg/day and 9-15 g/Kg/day. The combined administration method comprises combining the above Chinese medicinal water decoction with AKK bacteria, wherein the dosage of AKK bacteria is 1 time per day, 10 times per time9-1010CFU。
The radix codonopsitis or the medlar and AKK bacteria are taken together to be used as a nutritional supplement for women with the function of improving the intestinal AKK quantity and reducing the intestinal AKK quantity, or the effective component extract of the radix codonopsitis or the medlar and AKK bacteria are taken together to be used as a nutritional supplement. When the traditional Chinese medicine decoction is used as a nutritional supplement, the dosage of radix Codonopsis is 9-30 g/Kg/day, and the dosage of fructus Lycii is 6-12 g/Kg/day. The combined administration method comprises combining the above Chinese medicinal water decoction with AKK bacteria, wherein the dosage of AKK bacteria is 1 time per day, 10 times per time9-1010CFU。
The Chinese yam, the medlar, the chrysanthemum and the AKK bacteria are taken in combination to be used as a nutritional supplement for improving the number of the AKK bacteria in intestinal tracts of normal people, or the effective component extract of the Chinese yam, the medlar, the chrysanthemum and the AKK bacteria are taken in combination to be used as a nutritional supplement; the mass ratio of the Chinese yam to the medlar to the chrysanthemum is 0.1-3.9:0.1-1.6: 0.1-1.3. When the water decoction containing Chinese yam, medlar and chrysanthemum is used as a nutritional supplement, the dosage of Chinese yam is 15-30 g/Kg/day, the dosage of medlar is 6-12 g/Kg/day, and the dosage of chrysanthemum is 5-10 g/Kg/day. The combined administration method comprises combining the above Chinese medicinal water decoction with AKK bacteria, wherein the dosage of AKK bacteria is 1 time per day, 10 times per time9-1010CFU。
The application of dried orange peel, codonopsis pilosula, longan, liquorice, medlar, Chinese yam and/or chrysanthemum as a nutritional supplement or the application of an effective component extract of the dried orange peel, the codonopsis pilosula, the longan, the liquorice, the medlar, the Chinese yam and/or the chrysanthemum as a nutritional supplement in increasing the number of AKK bacteria.
First, experimental verification
(1) Selecting 19 kinds of Chinese medicinal materials from the homology of medicine and food, including radix Codonopsis, Glycyrrhrizae radix, fructus Lycii, longan, rhizoma Dioscoreae, pericarpium Citri Tangerinae, semen lablab album, rhizoma Phragmitis, rhizoma Polygonati Odorati, Atractylodis rhizoma, radix Angelicae sinensis, semen Nelumbinis, Coicis semen, semen Sojae Preparatum, fructus Jujubae, semen Ziziphi Spinosae, Poria, radix rehmanniae and flos Chrysanthemi. All the alternative dietary supplements are in the list of traditional Chinese medicines published by Weijian Wei of China, which are both foods and medicines.
(2) Screening the concentration range of Chinese medicinal materials
Preparation of a culture medium: 0.02g/ml of radix codonopsitis; licorice root, 0.02 g/ml; 0.02g/ml of medlar; longan, 0.02 g/ml; 0.06g/ml of yam; 0.04g/ml of dried orange peel; white hyacinth bean, 0.02 g/ml; reed rhizome, 0.06 g/ml; 0.02g/ml of polygonatum; 0.02g/ml of largehead atractylodes rhizome; 0.02g/ml of angelica; lotus seed 0.02 g/ml; 0.06g/ml of coix seed; fermented soya beans, 0.02 g/ml; 0.02g/ml of Chinese date; 0.02g/ml of fried jujube kernels; 0.03g/ml of tuckahoe; radix rehmanniae, 0.03 g/ml.
The culture medium comprises medlar and longan: 0.02g/ml of medlar; longan, 0.02 g/ml;
in a combined culture medium containing codonopsis pilosula and liquorice: 0.02g/ml of radix codonopsitis; licorice root, 0.02 g/ml;
in a combined culture medium containing radix codonopsitis and longan: 0.02g/ml of radix codonopsitis; longan, 0.02 g/ml.
② the gavage dosage of the mice: 1.3 g/Kg/day of radix codonopsis pilosulae; 1.3 g/Kg/day of medlar; longan: 1.3 g/Kg/day; 3.9 g/Kg/day of Chinese yam; 1.3 g/Kg/day of chrysanthemum.
When the decoction containing the codonopsis pilosula and the longans simultaneously is prepared, the dosage of the codonopsis pilosula is 1.3 g/Kg/day, and the dosage of the longans is 1.3 g/Kg/day.
(3) Experiment for culturing AKK bacteria by using various traditional Chinese medicines
Step 1, preparing a liquid-free culture medium: the preparation method comprises the following steps of (1) sterilizing 2.45g of BHI, 0.3-0.5g of mucin and 100ml of distilled water at 121 ℃ for 20min under high pressure, adding 1ml of 0.3% L-cysteine into each 100ml of culture medium, uniformly mixing, and standing for at least 1 hour for later use;
step 5, preparing a drug-free solid culture medium: the ratio of BHI to mucin is 2.45g, mucin 0.3-0.5g, agar 1g and distilled water 100 ml. Sterilizing at 121 deg.C under high pressure for 20min, adding 0.3% L-cysteine 1ml into each 100ml culture medium, mixing, pouring into flat plate (about 15ml per dish) by aseptic technique, and standing for coagulation for at least 1 hr;
after 772 hours, the plates were removed, dishes with colonies ranging from 30 to 300 colonies/dish were selected, the number of colonies per dish was counted, and in the case of high density colony plates, the number of colonies was roughly counted after counting the representative 1/8 to 1/4 areas.
(4) Experiment for improving abundance of AKK bacteria in intestinal tract of mouse by dietary supplement
Healthy female and male Kunming mouse, female mouse weight 40-45g, male mouse weight 45-50g, 4 weeks old. The experimental animals are randomly divided into 5 groups, namely an AKK bacteria group, an AKK + radix codonopsis group, an AKK + medlar group, an AKK + longan group and an AKK + radix codonopsitis + longan group, wherein each group comprises 10 animals, and the animals are respectively 5 animals. The experimental animals were drunk with water combined with antibiotics for 2 weeks (antibiotic mixed solution of 1mg/ml ampicillin, 5mg/ml streptomycin and 0.25mg/ml vancomycin). Gavage is carried out 2 weeks later, once a day, and 0.1ml (10 ml) of AKK bacteria is gavage for each mouse9CFU/time), the decoction of each kind of traditional Chinese medicine is prepared according to the dosage, and each mouse is gavaged with 0.1ml of liquid medicine for 14 days. Collecting feces after 14 days of gastric lavage, extracting DNA of the feces, and detecting the content of AKK bacteria in different groups by fluorescent quantitative PCR.
Experimental results A
The 19 candidate medicinal and edible traditional Chinese medicines are added into AKK extracellular anaerobic culture medium at the concentration, and colony counting is carried out after 72h of culture (the result is shown in figure 1 and table 1, the colony counting result of each group of plate culture medium, and the data are in the form of mean value plus or minus standard deviation 'x plus or minus SEM', compared with a control group). The traditional Chinese medicine for improving the AKK bacteria quantity comprises dried orange peel (4.8 times), radix codonopsitis (4.04 times), liquorice (3.35 times), longan (3.06 times), medlar (2.68 times), yam (2.27 times), white hyacinth bean (1.69 times), reed rhizome (1.42 times) and polygonatum (1.22 times); the traditional Chinese medicines for reducing the number of AKK bacteria comprise radix rehmanniae (0.9 times), parched semen Ziziphi Spinosae (0.86 times), Poria (0.8 times), semen Sojae Preparatum (0.56 times), semen Nelumbinis (0.5 times), Coicis semen (0.38 times), and fructus Jujubae (0.23 times); the angelica and the white atractylodes rhizome have no influence on the quantity of AKK bacteria. Therefore, the dried orange peel, the codonopsis pilosula, the longan, the liquorice and the medlar are used as single dietary supplements capable of increasing the number of AKK bacteria in anaerobic culture conditions.
Further verification shows that the colony numbers of 3 dietary supplement combinations comprising medlar, longan (3.67 times), codonopsis pilosula, longan (3.54 times) and codonopsis pilosula, liquorice (2.14 times) are higher than those of a drug-free control group, and the differences are obvious, which indicates that the dietary supplement combinations can improve the number of AKK bacteria. Therefore, medlar, longan, radix codonopsitis, liquorice, radix codonopsitis and longan are used as dietary supplement combinations capable of increasing the number of AKK bacteria in anaerobic culture conditions.
TABLE 1 results of colony counts of AKK bacteria cultured by dietary supplements of each group
Note: the fold relationship is the specific value of fold change of each traditional Chinese medicine culture group compared with the control.
Experimental results II
Clinically, chronic oral administration of antibiotics results in dysbacteriosis. Meanwhile, "rich and precious diseases" such as diabetes, hypertension, hyperlipidemia and the like, "metabolic diseases" and "chronic diseases" of a liver system and a cardiovascular system, and "serious diseases" such as tumors and the like have the conditions of intestinal flora imbalance and reduced AKK bacteria number. Therefore, sterile enteric-coated mice prepared by subjecting Kunming mice to water intervention with antibiotics for 14 days[11]The dietary supplement was used as a subject to evaluate whether or not the amount of AKK in the cells could be increased.
The results showed that after 14 days, there was no significant difference in the total number of bacteria in the intestinal tract of the male mice groups (a in fig. 2); compared with the single AKK group, the AKK + medlar (1.3 times), the AKK + longan (2 times), the AKK + radix codonopsitis and longan (8.5 times) groups all improve the abundance of AKK bacteria in the male rat intestinal tract (B in figure 2), which indicates that the medlar, the longan, the radix codonopsitis and longan are taken as dietary supplements and the AKK bacteria simultaneously under the condition of flora imbalance, so that the number of the AKK bacteria in the male rat intestinal tract can be increased.
There was no significant difference in the total number of bacteria in the intestinal tracts of the groups of female mice (C in fig. 2); compared with the single AKK group, the AKK + radix codonopsitis (1.5 times) and the AKK + medlar (2 times) groups improve the abundance of AKK bacteria in the intestinal tract of the female mouse (D in figure 2), which shows that under the condition of dysbacteriosis, the radix codonopsitis and medlar are taken as dietary supplements and the AKK bacteria simultaneously, so that the quantity of the AKK bacteria in the intestinal tract of the female mouse can be improved.
Therefore, for the population with reduced intestinal AKK quantity, the codonopsis pilosula, the longan and the medlar are used as dietary supplements suitable for increasing the quantity of intestinal AKK bacteria in men; the medlar and the codonopsis pilosula are used as dietary supplements suitable for increasing the number of intestinal AKK bacteria of women.
Experimental results III
Healthy female C57BL/6 mice, weighing 20-23g, 12-13 weeks old. The experimental animals were randomly divided into 3 groups, namely, AKK group, AKK + dietary supplement (yam + lycium barbarum + chrysanthemum) group, and dietary supplement group, with 4 animals per group. The experimental animals were gavaged once a day, and each mouse was gavaged with 0.1ml of bacterial solution (10)9CFU/time), the decoction is 0.1ml per mouse, and lasts for 2 weeks; collecting feces after 14 days of gastric lavage, extracting DNA of the feces, and detecting the content of AKK bacteria in different groups by fluorescent quantitative PCR.
The results showed that there was no significant difference in the total bacterial count in the feces of mice in the AKK group, AKK + dietary supplement group, dietary supplement group 3 groups after 14 days of gavage (fig. 3A); compared with the AKK group, the AKK bacteria abundance of the AKK + dietary supplement group is obviously increased (3.5 times), and the AKK bacteria abundance of the dietary supplement group is reduced (0.4 times) (fig. 3B). The results show that the combination of the Chinese yam, the medlar and the chrysanthemum can be used as a dietary supplement to be taken together with AKK bacteria, so that the number of the AKK bacteria in the intestinal tract of a normal mouse can be increased; however, the combination of the three components taken alone as a dietary supplement can reduce the number of AKK bacteria in the intestinal tract of normal mice.
Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:
(1) the invention aims to provide several medicinal and edible dietary supplements which can improve the quantity of AKK bacteria in vivo or in vitro.
(2) The economic medicinal and edible food can be used as an auxiliary material to be added into a culture medium to improve the yield of AKK bacteria, and is used for large-scale production of industrial culture.
(3) The dietary supplement safe to human bodies can improve the quantity of AKK bacteria in intestinal tracts of patients by oral administration, and achieves the purposes of preventing and treating diseases with reduced quantity of AKK bacteria, such as diabetes, hypertension, hyperlipidemia, diseases of liver systems and cardiovascular and cerebrovascular systems, tumor adjuvant therapy and the like.
Drawings
FIG. 1 shows the results of the growth of AKK plate medium cultured by dietary supplements of each group;
FIG. 2 shows the result of DNA fluorescent quantitative PCR detection of AKK bacteria content in different groups on mouse feces after 14 days of antibiotic water-dryness after gastric lavage;
fig. 2 a. total number of bacteria in the male mouse;
in fig. 2, b. in the male mice, the AKK bacteria growth fold for each AKK + dietary supplement group compared to the AKK group alone;
total number of bacteria in the female mouse c in fig. 2;
in fig. 2, in vivo, the AKK bacteria of each AKK + dietary supplement group increased by a multiple compared to the AKK group alone;
FIG. 3 shows the result of DNA fluorescent quantitative PCR detection of AKK bacteria content in different groups on mouse feces 14 days after gastric lavage of normal mice;
total number of bacteria in fig. 3;
in fig. 3b, the AKK bacteria of the AKK + dietary supplement group and the dietary supplement group varied in fold compared to the AKK group alone.
Detailed Description
Liquid-free liquid medium configuration used in the examples: the preparation method comprises the following steps of sterilizing 2.45g of BHI, 0.3-0.5g of mucin and 100ml of distilled water at 121 ℃ for 20min under high pressure, adding 1ml of 0.3% L-cysteine into each 100ml of culture medium, uniformly mixing, and standing for at least 1 hour for later use.
Preparation of drug-free solid medium in examples: the ratio of BHI to mucin is 2.45g, mucin 0.3-0.5g, agar 1g and distilled water 100 ml. Autoclaving at 121 deg.C for 20min, adding 0.3% L-cysteine 1ml per 100ml culture medium, mixing, pouring into flat plate (about 15ml per dish) by aseptic technique, and standing for coagulation for at least 1 hr.
Example 1
One of three combinations of medlar, longan, radix codonopsitis, liquorice and radix codonopsitis and longan is used as a nutritional supplement to improve the number of AKK bacteria in anaerobic culture conditions.
The test steps are as follows:
step 1: preparing a liquid-free culture medium for later use; preparing the traditional Chinese medicine decoction.
Step 2: adding various Chinese medicinal water decoctions into the prepared liquid culture medium to obtain medicinal culture medium; the concentration of each traditional Chinese medicine in the medicine-containing culture medium is as follows:
the culture medium comprises medlar and longan: 0.022g/ml of medlar; longan, 0.022 g/ml;
in a combined culture medium containing codonopsis pilosula and liquorice: 0.022g/ml of radix codonopsitis; licorice, 0.022 g/ml;
in a combined culture medium containing radix codonopsitis and longan: 0.022g/ml of radix codonopsitis; longan, 0.022 g/ml.
And step 3: 3ml of the prepared liquid drug-containing culture medium is taken and added into corresponding test tubes respectively, and 1ml of AKK bacteria (ATCC BAA-835) liquid with the same concentration is added into each test tube respectively.
And 4, step 4: placing a test tube containing the bacterial liquid and a drug-containing culture medium into an anaerobic jar, and culturing for 48 hours in a constant-temperature incubator at 37 ℃;
and 5: and (4) preparing a drug-free solid culture medium for later use.
Step 6: performing gradient dilution on each group of bacterial fluid cultured for 48 hours, and sucking each group of dilution with the dilution degree of 10-5And 10-6200ul of the bacterial liquid is added on the prepared solid plate culture medium, and the obtained product is put into an anaerobic jar and cultured in a constant temperature incubator at 37 ℃ for 72 hours.
And 7: plates were removed after 72 hours, dishes with colonies ranging from 30-300/dish were selected, the number of colonies per dish was counted, and in high density colony plates, the number of colonies was roughly counted after counting representative 1/8-1/4 regions.
And (3) test results:
the medlar and longan combination can improve the AKK bacteria quantity by 3.8 times, the codonopsis pilosula and longan combination can improve the AKK bacteria quantity by 3.54 times, the codonopsis pilosula and liquorice combination can improve the AKK bacteria quantity by 2.3 times, the colony number of 3 dietary supplement combinations is higher than that of a drug-free control group, and the difference is obvious, so that the dietary supplement combinations can improve the AKK bacteria quantity.
Example 2
Pericarpium Citri Tangerinae, radix Codonopsis, longan, Glycyrrhrizae radix or fructus Lycii is used as single nutritional supplement to increase AKK bacteria amount in anaerobic culture condition.
The test steps are as follows:
step 1: preparing a liquid-free culture medium for later use; preparing the traditional Chinese medicine decoction.
Step 2: adding various Chinese medicinal water decoctions into the prepared liquid culture medium to obtain medicinal culture medium;
the concentration of each traditional Chinese medicine in the medicine-containing culture medium is respectively as follows: 0.038g/ml of dried orange peel; 0.022g/ml of medlar; longan, 0.022 g/ml; 0.022g/ml of radix codonopsitis; licorice, 0.022 g/ml.
And step 3: 3ml of the prepared liquid drug-containing culture medium is taken and added into corresponding test tubes respectively, and 1ml of AKK bacteria (ATCC BAA-835) liquid with the same concentration is added into each test tube respectively.
And 4, step 4: placing a test tube containing the bacterial liquid and a drug-containing culture medium into an anaerobic jar, and culturing for 48 hours in a constant-temperature incubator at 37 ℃;
and 5: and (4) preparing a drug-free solid culture medium for later use.
Step 6: performing gradient dilution on each group of bacterial fluid cultured for 48 hours, and sucking each group of dilution with the dilution degree of 10-5And 10-6200ul of the bacterial liquid is added on the prepared solid plate culture medium, and the obtained product is put into an anaerobic jar and cultured in a constant temperature incubator at 37 ℃ for 72 hours.
And 7: plates were removed after 72 hours, dishes with colonies ranging from 30-300/dish were selected, the number of colonies per dish was counted, and in high density colony plates, the number of colonies was roughly counted after counting representative 1/8-1/4 regions.
And (3) test results:
pericarpium Citri Tangerinae can increase AKK bacteria amount by 4.5 times, radix Codonopsis can increase AKK bacteria amount by 4.2 times, Glycyrrhrizae radix can increase AKK bacteria amount by 3.5 times, arillus longan can increase AKK bacteria amount by 3.2 times, and fructus Lycii can increase AKK bacteria amount by 2.8 times
Example 3
Radix codonopsitis, longan and AKK bacteria are taken simultaneously to improve the intestinal AKK quantity and reduce the AKK bacteria quantity in a male body.
The test steps are as follows:
step 1: preparing a traditional Chinese medicine water decoction containing the combination of the codonopsis pilosula and the longan, wherein the dosage of the codonopsis pilosula is 1.56 g/Kg/day, and the dosage of the longan is 1.56 g/Kg/day.
Step 2: selecting healthy male Kunming mouse with the weight of 45-50g and the age of 4 weeks; the experimental animals were randomly divided into 2 groups, namely, AKK group, AKK + radix Codonopsis + longan group, and each group had 5 animals. The experimental animals were drunk with water combined with antibiotics for 2 weeks (antibiotic mixed solution of 1mg/ml ampicillin, 5mg/ml streptomycin and 0.25mg/ml vancomycin). Gavage is carried out 2 weeks later, once a day, and 0.1ml (10 ml) of AKK bacteria is gavage for each mouse9CFU/time), preparing the Chinese medicinal water decoction according to the dosage, and feeding 0.1ml of the medicinal liquid into each mouse for 14 days. Collecting feces after 14 days of gastric lavage, extracting DNA of the feces, and detecting the content of AKK bacteria in different groups by fluorescent quantitative PCR.
And (3) test results:
after 14 days, the total number of bacteria in intestinal tracts of all groups of the male mice has no obvious difference; compared with a single AKK group, the AKK + radix codonopsis and longan combination improves the abundance of AKK bacteria to 8.7 times, so that the radix codonopsis and longan are taken as dietary supplements and the AKK bacteria are taken simultaneously under the condition of dysbacteriosis, and the number of the AKK bacteria in intestinal tracts of male mice can be improved.
Example 4
The medlar or longan and AKK bacteria are taken simultaneously to improve the intestinal AKK quantity and reduce the AKK bacteria quantity in men.
The test steps are as follows:
step 1: preparing decoction of Chinese medicinal materials, wherein the dosage of fructus Lycii is 1.56 g/Kg/day, and the dosage of arillus longan is 1.56 g/Kg/day.
Step 2: selecting healthy male Kunming mouse with the weight of 45-50g and the age of 4 weeks; the experimental animals were randomly divided into 3 groups, namely, AKK bacteria group, AKK + medlar group, and AKK + longan group, and each group had 5 animals. The experimental animals were drunk with water combined with antibiotics for 2 weeks (antibiotic mixed solution of 1mg/ml ampicillin, 5mg/ml streptomycin and 0.25mg/ml vancomycin). Gavage is carried out 2 weeks later, once a day, and 0.1ml (10 ml) of AKK bacteria is gavage for each mouse9CFU/time), preparing the Chinese medicinal water decoction according to the dosage, and feeding 0.1ml of the medicinal liquid into each mouse for 14 days. Collecting feces after 14 days of gastric lavage, extracting DNA of the feces, and detecting the content of AKK bacteria in different groups by fluorescent quantitative PCR.
And (3) test results:
after 14 days, the total number of bacteria in intestinal tracts of all groups of the male mice has no obvious difference; compared with a single AKK group, the AKK + medlar combination improves the abundance of AKK bacteria to 1.43 times and the AKK + longan combination improves the abundance of AKK bacteria to 2.13 times, so that the medlar or longan is taken as a dietary supplement and the AKK bacteria simultaneously under the condition of dysbacteriosis, and the number of the AKK bacteria in intestinal tracts of male mice can be improved.
Example 5
Radix Codonopsis or fructus Lycii is administered with AKK bacteria to increase intestinal AKK amount and reduce the amount of AKK bacteria in female body.
The test steps are as follows:
step 1: preparing a traditional Chinese medicine decoction, wherein the dosage of the medlar is 1.56 g/Kg/day, and the dosage of the radix codonopsitis is 1.56 g/Kg/day.
Step 2: selecting healthy female Kunming mouse, wherein the female mouse has the weight of 40-45g and the age of 4 weeks; the experimental animals were randomly divided into 3 groups, namely, AKK group, AKK + radix Codonopsis group, and AKK + fructus Lycii group, and each group had 5 animals. The experimental animals were drunk with water combined with antibiotics for 2 weeks (antibiotic mixed solution of 1mg/ml ampicillin, 5mg/ml streptomycin and 0.25mg/ml vancomycin). Gavage is carried out 2 weeks later, once a day, and 0.1ml (10 ml) of AKK bacteria is gavage for each mouse9CFU/time), preparing the Chinese medicinal water decoction according to the dosage, and feeding 0.1ml of the medicinal liquid into each mouse for 14 days. Collecting feces 14 days after gastric lavage, extracting feces DNA, and performing fluorescent quantitative PCR detection on different groupsThe content of AKK bacteria.
And (3) test results:
after 14 days, the total number of bacteria in intestinal tracts of each group of female mice has no obvious difference; compared with a single AKK group, the AKK + radix codonopsitis group improves the abundance of AKK bacteria to 1.63 times that of the AKK + medlar group and improves the abundance of AKK bacteria to 2.2 times that of the AKK group, so that the radix codonopsitis and medlar are taken as dietary supplements and are taken together with the AKK bacteria under the condition of dysbacteriosis, and the quantity of the AKK bacteria in intestinal tracts of female mice can be improved.
Example 6
The Chinese yam, the medlar, the chrysanthemum and the AKK bacteria are taken simultaneously to improve the quantity of the AKK bacteria in intestinal tracts of normal people.
The test steps are as follows:
step 1: preparing a Chinese medicinal water decoction containing Chinese yam, medlar and chrysanthemum, wherein the dosage of the Chinese yam is 3.64 g/Kg/day, the dosage of the medlar is 1.56 g/Kg/day, and the dosage of the chrysanthemum is 1.17 g/Kg/day.
Step 2:
healthy female C57BL/6 mice were selected, weighing 20-23g, 12-13 weeks old. The experimental animals were randomly divided into 3 groups, namely, AKK group, AKK + dietary supplement (yam + lycium barbarum + chrysanthemum) group, and dietary supplement group, with 4 animals per group. The experimental animals were gavaged once a day, and each mouse was gavaged with 0.1ml of bacterial solution (10)9CFU/time), the decoction is 0.1ml per mouse, and lasts for 2 weeks; collecting feces after 14 days of gastric lavage, extracting DNA of the feces, and detecting the content of AKK bacteria in different groups by fluorescent quantitative PCR.
And (3) test results:
the results show that after 14 days of gastric lavage, the total bacterial quantity in the mouse feces of the AKK group, the AKK + dietary supplement group and the dietary supplement group 3 has no obvious difference; compared with the AKK group, the AKK bacteria abundance of the AKK + Chinese yam + Chinese wolfberry + chrysanthemum group is obviously increased to 3.3 times, and the AKK bacteria abundance of the Chinese yam + Chinese wolfberry + chrysanthemum group is reduced to more than 0.38 times, so that the combination of the Chinese yam + Chinese wolfberry + chrysanthemum is taken as a dietary supplement to be combined with the AKK bacteria, and the AKK bacteria amount in the intestinal tract of a normal mouse can be increased; however, the combination of the three components taken alone as a dietary supplement can reduce the number of AKK bacteria in the intestinal tract of normal mice.
Claims (3)
1. Application of one of pericarpium citri reticulatae water extract, longan water extract or liquorice water extract and AKK bacteria in preparation of nutritional supplement for increasing the number of AKK bacteria in vitro under anaerobic conditions.
2. Application of a combination of radix codonopsis water extract, longan water extract and AKK bacteria in preparation of a nutritional supplement for improving the number of intestinal AKK bacteria of men with decreased number of intestinal AKK bacteria.
3. Application of a combination of a Chinese yam water extract, a Chinese wolfberry water extract, a chrysanthemum water extract and AKK bacteria in preparation of a nutritional supplement for improving the number of intestinal AKK bacteria of a female with normal number of intestinal flora.
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