TWI718203B - The use of the combination package in the preparation of food, health products or nutraceuticals - Google Patents

Abstract

A combination package is used for preparing food, medicine, health care product or nutraceutical, said food, medicine, health care product or nutraceutical is used to reduce the drug resistance genome of intestinal flora or reduce antibiotic resistance in antibiotic treatment, said The combination package includes the following compositions, each of which is in the form of a dosage unit for ease of dosage administration and uniformity, and the dosage unit form is a single-dose physically dispersed unit: the first composition includes: whole grains; the second composition Including: bitter gourd, soluble dietary fiber and oligosaccharide; the third composition includes; soluble dietary fiber and oligosaccharide. Through the reasonable adjustment of the patient's diet and nutrition, the goal of reducing or improving the drug resistance genome of the intestinal flora is achieved.

Description

The use of the combination package in the preparation of food, health products or nutraceuticals

The invention relates to the fields of microorganisms and food, medicines and health products. More specifically, the present invention relates to the application of a combination package including bitter melon powder in the preparation of foods, medicines, health products, and nutraceuticals for reducing antibiotic resistance genes or gut microflora resistome in the intestinal flora.

Since the beginning of the use of antibiotics in the 1940s, the use of antibiotics has greatly reduced deaths caused by infections and prolonged people's life expectancy. However, due to the abuse and improper use of antibiotics, antibiotic resistance has developed rapidly in microorganisms, and antibiotic resistance has become one of the important factors threatening human health in the world. The problem of antibiotic resistance has greatly increased the expenditure and investment in public health, prolonged the hospitalization time of patients, and caused an increase in treatment failure and an increase in mortality (Cited Reference 1).

In China, the abuse of antibiotics is serious, and antibiotic resistance has become a problem that cannot be ignored.

Many previous studies on antibiotic resistance focused on the analysis of clinical pathogens. However, recently, more and more researches have turned to "drug-resistant genomes." The concept of drug-resistant genome was proposed by Vanessa M.D'Costa in an article on soil antibiotic resistance genes in 2006, which represents the sum of all antibiotic resistance genes in a specific environment (reference 2). A large number of antibiotic resistance genes are stored in the human intestines (Citation 3). These resistance genes are not only It can be exchanged between commensal intestinal bacteria, and can also be transferred to opportunistic pathogens (Citation 4). In a large-scale population study, researchers used total genome sequencing technology to detect the human intestinal drug resistance genome, and identified thousands of antibiotic resistance genes in it (Cited Reference 5).

For obese individuals, dietary intervention is a commonly used method to reduce weight. At the same time, diet has also been reported to be the main factor regulating the intestinal flora (Citation 6, Citation 7). In a study published in our laboratory, we found that patients with genetic obesity and simple obesity, after undergoing dietary intervention consisting of whole grains, traditional Chinese medicine ingredients, and prebiotics, their weight was significantly reduced and their physiology The indicators and inflammation have been significantly improved. At the same time, in this dietary intervention experiment, the structure and function of the intestinal flora have undergone significant changes, including the reduction of toxin-producing bacteria and the increase of beneficial bacteria (reference 8).

So far, there has not been a method that can effectively reduce the resistant genome of the intestinal flora.

By using the overall genomics analysis method centered on the genome, the present inventors unexpectedly discovered that by taking a combination package (dietary intervention), patients can be used to support beneficial intestinal bacteria, inhibit conditional pathogenic bacteria, and reduce the number of pathogenic bacteria in the intestine. The number and abundance of antibiotic resistance genes in the flora.

Therefore, the present invention provides an application of a combination package in the preparation of foods, medicines, health products, or nutraceuticals. The foods, medicines, health products, or nutraceuticals are used to reduce the drug-resistant genome of the intestinal flora or in the treatment of antibiotics. To reduce antibiotic resistance, the combination package includes the following compositions, each of which is in the form of a dosage unit for convenient dosage administration and uniformity, and the dosage unit form is a single-dose physically dispersed unit: The first composition includes: whole grains; the second composition includes: bitter gourd, soluble dietary fiber and oligosaccharide; the third composition includes: soluble dietary fiber and oligosaccharide.

The present invention also provides a method for reducing the drug resistance genome of the intestinal flora or reducing antibiotic resistance in antibiotic treatment, comprising the following steps: administering a combination package to an object in need, the combination package including the following compositions, each combination The substance is a dosage unit form for convenient dosage administration and uniformity. The dosage unit form is a single-dose physically dispersed unit: the first composition includes: whole grains; the second composition includes: bitter gourd, soluble dietary fiber, and Oligosaccharides; The third composition includes: soluble dietary fiber and oligosaccharides.

According to another aspect of the present invention, the present invention also provides a combination package for reducing the drug resistance genome of the intestinal flora or reducing antibiotic resistance in antibiotic therapy. The combination package includes the following compositions, each of which is convenient The dosage unit form of dosage administration and uniformity, the dosage unit form is a single-dose physically dispersed unit: the first composition includes: whole grains; the second composition includes: bitter gourd, soluble dietary fiber and oligosaccharides ; The third composition includes: soluble dietary fiber and oligosaccharides.

In some embodiments, the whole grains include coix seed, oats, buckwheat, lentils, corn, adzuki beans, soybeans, yams, dates, peanuts, lotus seeds, and goji berries.

In some embodiments, the combination package is for human patients.

In some embodiments, the first composition is taken as a staple food in the form Selected from rice, noodles, porridge or rice. Before preparing rice, noodles, porridge or meals with the first composition, the seeds, fruits or other plant parts of the first composition are crushed into particles, of which 1-70%, 15-70%, 25-70% , 30-70%, 50-70% of the particle diameter is 0.65mm or more.

In some embodiments, the weight of the coix seed accounts for 5-35%, 10-30%, or 15-25% of the weight of the first composition, and the weight of oats accounts for 5- 35% of the weight of the first composition. 30% or 10-20%, the weight of the buckwheat accounts for 5-50%, 8-40% or 10-30% of the weight of the first composition, and the weight of the lentils accounts for the weight of the first composition The weight of the yam accounts for 5-30%, 8-20% or 10-15% of the weight of the first composition.

In some embodiments, the weight of protein accounts for 5-40% or 10-20% of the weight of the first composition, and the weight of carbohydrate accounts for 30-80% or 50-70% of the weight of the first composition. , The weight of fat accounts for 0.5-30% or 2-15% of the weight of the first composition, the weight of dietary fiber accounts for 0.5-30% or 2-15% of the weight of the first composition, and the weight of vitamins The weight of the first composition is 0.1-5% or 0.5-1%, and the weight of the minerals is 0.1-2% or 0.8-1.2% of the weight of the first composition.

In some embodiments, each 100 grams of the first composition provides 320-400 kcal total calories.

In some embodiments, every 100 grams of the first composition contains: VA 3-857 ugRE, VD 0.01-5ugRE, VE 2-79.09mg, VB1 0.01-1.89mg, VB2 0.01-1.4mg, VB60.01 -1.2mg, VB12 0.1-2.4mg, VC 1-1170mg, niacin 0.5-28.4mg, Ca 60-2458mg, P 200-1893mg, K 350-1796mg, Na 8-2200mg, Mg 100-350mg, Fe 2- 20mg.

In some embodiments, the buckwheat in the first composition includes: common buckwheat or tartary buckwheat.

In some embodiments, the buckwheat in the first composition includes: Fagopyrum seeds.

In some embodiments, the oats in the first composition include: Oats seeds.

In some embodiments, the yam in the first composition includes: dried yam.

In some embodiments, the second composition is prepared as a powder and is taken 0.25 to 1 hour before a meal.

In some embodiments, the daily dose of the second composition is 5-100 grams, 40-60 grams, or 30-80 grams, which is prepared with water.

In some embodiments, the bitter melon in the second composition includes: whole fruit powder of a plant of the genus Momordica.

In some embodiments, the whole plant fruit powder is produced by freeze drying or spray drying.

In some embodiments, the bitter melon in the second composition includes: bitter melon extract.

In some embodiments, the soluble dietary fiber in the second composition includes: Fibersol-2, resistant starch, polydextrose, cellulose, hemicellulose, pectin or gum.

In some embodiments, the oligosaccharides in the second composition include: fructooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalt-oligosaccharides, soybean oligosaccharides, glucose oligosaccharides, water Threose or lactulose oligosaccharide.

In some embodiments, the weight ratio of the bitter gourd to the dietary fiber and the oligosaccharide is 10:1 to 1:1, such as 9:1, 8:1 or 7:1.

In some embodiments, the weight of the bitter melon powder accounts for 15-99.8%, 30-95%, or 45-90% of the weight of the second composition, and the weight of the soluble dietary fiber accounts for the weight of the second composition 0.1-51%, 1-40%, 3-30% or 5-20% of the weight, the weight of the oligosaccharide accounts for the second 0.1-34%, 1-25%, 2-20% or 3-15% of the weight of the composition.

In some embodiments, the third composition is prepared as a powder, which is taken 2-5 hours before a meal or taken at the same time as breakfast.

In some embodiments, the daily dose of the third composition is 5-200 grams, 30-100 grams, or 50-150 grams, prepared with 300-1500 milliliters of water.

In some embodiments, the soluble dietary fiber in the third composition includes: Fibersol-2, resistant starch, polydextrose, cellulose, hemicellulose, pectin or gum.

In some embodiments, the weight ratio of dietary fiber and oligosaccharide in the third composition is 6:1 to 1:6, such as 6:4.

In some embodiments, the oligosaccharides in the third composition include: fructo-oligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalt-oligosaccharides, soybean oligosaccharides, glucose oligosaccharides, water Threose, or lactulose oligosaccharide. In some embodiments, the soluble dietary fiber in the third composition includes: guar gum, pectin, konjac flour, and other fermentable dietary fibers (Fibersol-2, resistant starch, hemicellulose), etc. Prebiotics.

Compared with the prior art, the present invention has the following beneficial effects: the composition of the present invention can be used to balance the structure of the intestinal flora in patients, improve or reduce antibiotic resistance genes in the intestinal flora; it can be administered to patients in need in multiple ways , To achieve the purpose of restoring health.

Figure 1 shows the changes in the overall structure of the intestinal drug resistance genome after dietary intervention. (a) The PCA score chart derived from the data of 399 ARGs shows significant separation in the samples before and after the diet intervention (log-transformed, PERMONOVA P=0.0187, replacement=9999). (b) The total number and abundance of ARGs. The box represents the interquartile range (IQR) between the first and third quartiles (25% and 75%, respectively), and the line in the box represents the median. Representatives of whiskers The lowest and highest value within 1.5 times the IQR of the first and third quartiles, respectively. The sample is shown as the point on the left. Two-tailed Wilcoxon paired signed-rank test (n=35) was used for statistical analysis. (c) The incidence of ARGs and ARGs corresponding to the type of resistance gene.

Figure 2 is a heat map showing 96 significantly altered ARGs and their responses to dietary interventions. The color of the dot corresponds to the log-converted abundance of ARGs. These ARGs are organized according to the type of antibiotic resistance gene.

Figure 3 shows antibiotic resistance genes that are significantly changed after dietary intervention. (a) The number of antibiotic resistance gene types in our diet experiment (before and after) and in three additional groups (China, Denmark and Spain, from literature [5]). The frame represents the interquartile range (IQR) between the first and third quartiles (25% and 75%, respectively), and the line in the frame represents the median. Must represent the lowest and highest values within 1.5 times the IQR of the first and third quartiles, respectively. (b) 36 significantly changed gene types. The line/triangle markers represent the median and 25%/75% quartiles of gene type abundance. (c) Significantly altered abundance of ARGs assigned to antibiotic compounds (corrected P <0.05). Assign resistance genes to antibiotics based on ARDB. The genes that confer resistance to the same antibiotic compound are added in abundance. (d) The abundance of ARGs assigned to each antibiotic category. According to ARDB, antibiotic genes are assigned to antibiotic categories. Add the abundance of genes that confer resistance to the same antibiotic class. For the statistical analysis of (a) paired comparison, two-tailed Mann-Whitney U test and two-tailed Weiss paired signed rank test were used (before n=35, after n=35, China n=38, Denmark n= 85, Spain n=39); for (b), (c) and (d), the Weiss paired signed-rank test (n=35) was performed. *Corrected P<0.05, **corrected P<0.01, ***corrected P<0.001 (Benjamini & Hochberg 1995).

Figure 4 shows the changes in the abundance of antibiotic resistance genes that exert resistance through three different mechanisms 化. Through the ARDB database, antibiotic resistance genes are assigned to corresponding resistance mechanisms. Add the abundance of antibiotic resistance genes that exert resistance through the same mechanism. The box represents the interquartile range (IQR) between the first and third quartiles (25% and 75%, respectively), and the line in the box represents the median. Must represent the lowest and highest values within 1.5 times the IQR of the first and third quartiles, respectively. The sample is shown as the point on the left. Two-tailed Weiss paired signed-rank test (n=35) was used for statistical analysis. P value correction (Benjamini & Hochberg 1995).

Figure 5 shows the changes of antibiotic resistance gene carriers after dietary intervention. Principal component analysis based on 120 antibiotic resistance gene carriers showed that the samples before and after intervention had a certain trend of differentiation (log transformation, PERMONOVA P=0.0584, permutations=9999).

700基因)。各ARG攜帶者豐度變化的方向被分到四類中，由校正的P<0.05確定顯著性(雙尾威氏配對符號秩檢驗法，n=120，Benjamini & Hochberg 1995)。 Figure 6 shows the distribution network of ARGs among ARG carriers. The circle in the figure represents the antibiotic resistance category, and the other shapes represent different phyla (square: Proteobacteria, triangle: Bacteroides, diamond: Firmicutes). The size of the shape represents two types of ARG carriers (large: >700 genes and small:

700 genes). The directions of changes in the abundance of each ARG carrier were divided into four categories, and the significance was determined by the adjusted P<0.05 (two-tailed Weiss paired signed-rank test, n=120, Benjamini & Hochberg 1995).

The following describes the embodiments of the present invention in detail: this embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation manners and specific operating procedures, but the scope of protection of the present invention is not limited to the following implementations example.

In a population diet intervention experiment targeting the intestinal flora. We performed in-depth overall genome sequencing on 70 stool samples from 35 children, obtaining an average of 84 million high-quality sequenced fragments, and comparing drug-resistant genomes at multiple levels analysis. A total of 399 antibiotic resistance genes were identified from the set of established gut microbial genes (cited reference 8) containing more than two million. After dietary intervention, the overall structure of the intestinal drug resistance genome has undergone significant changes (Figure 1 part a), and the overall number and abundance are significantly reduced (Figure 1 part b, Figure 2). After the intervention 48 antibiotic resistance genomes disappeared completely, and 77% of antibiotic resistance gene abundances were significantly reduced or tended to decrease.

We grouped 399 antibiotic resistance genes into the resistance gene category, obtained 131 resistance gene categories, and compared them with data from adults in China, Denmark, and Spain. According to existing reports, among the three populations, Chinese adults have the most types and abundance of drug-resistant genomes (reference 5). We found that the samples before the intervention contained significantly more resistance gene types than Chinese adults (part a in Figure 3), that is, the intestinal resistance group of these individuals had a very high diversity before the intervention. And consistent with the results observed in the existing literature, we have also seen the wide range of resistance categories of TetW, TetQ, TetO, TetM, Tet40, Tet32, ErmB and BacA in the intestinal drug resistance genome of the studied objects in individuals. Existence (references 5, 9, 10), and tetracycline resistance genes are also the most abundant in the drug-resistant genome (reference 5). After the intervention, the abundance of 36 resistance gene categories changed significantly, of which 33 were significantly reduced (Figure 3, part b). According to the corresponding relationship between the resistance genes in the database and the antibiotics that produced resistance, we found that after the intervention, the resistance potential to 10/47 antibiotic compounds was significantly reduced (part c in Figure 3), and 8/ The resistance potential of 15 major antibiotics has decreased significantly (Figure 3, part d).

Bacterial antibiotic resistance genes mainly generate resistance through three mechanisms: 1) reduce the concentration of antibiotics in bacterial cells by weakening permeability or efflux pump; 2) modify antibiotic targets to protect them; and 3 ) Hydrolyze or modify the antibiotic molecule to make the antibiotic inactive (Citation 11). After the intervention, the sum of the abundance of resistance genes that produced resistance through the two mechanisms of target protection and efflux pump was significantly reduced (Figure 4). Through the Canopy algorithm (reference 12) to merge and classify the gut microbial genes, the "common abundance gene set" can be obtained. (CAG)", then de novo assembly and classification status identification of CAG. Through the above steps, we were able to retrospectively obtain the carriers of 201 antibiotic resistance genes (their abundance accounts for more than 86% of the total drug resistance genome), a total of There are 120 CAGs, of which 24 CAGs have high-quality genome drafts, and 30 CAGs can determine their taxonomic status at the species/genus level. The overall structure of these CAGs has a clear trend of change after intervention (Figure 5). The distribution network of antibiotic resistance genes in its carriers (Figure 6), it can be seen that antibiotic resistance genes exist in Firmicutes, Bacteroides and Proteobacteria, which indicates this type of Genes are widely distributed in the bacterial kingdom. Among them, bacteria of the genus Klebsiella, Enterobacter and Escherichia are connected to a variety of antibiotic resistance groups and exist in the form of hot spots. These bacteria are potential opportunities for multiple drug resistance. Pathogenic bacteria (cited documents 13 and 14) have the gene potential to develop resistance to a variety of antibiotics including β-lactams, tetracyclines, sulfonamides and macrolides. According to existing reports Infections caused by multi-drug resistant pathogens not only cause higher mortality and more treatment failures, but also cause more economic losses to the health system (cited reference 15). Through dietary intervention, 69% Carriers of antibiotic resistance genes are significantly reduced or there is a tendency to decrease, especially those multi-drug resistant bacteria, including CAG00001a (Escherichia/Shigella sp.), CAG00002 (Escherichia/Shigella sp.), CAG00146 (Klebsiella sp.), CAG00008 (Klebsiella pneumoniae) and CAG00356 (Enterobacter sp.).

In order to deal with the global threat of antibiotic resistance, people have made a lot of efforts from different angles. Our research results prove that dietary intervention targeting the intestinal flora can not only regulate the intestinal flora and improve the health of individuals, but also significantly reduce the types of drug resistance genes contained in their intestinal drug resistance genomes. And abundance.

Accordingly, the present invention provides a method as a response to the threat of antibiotic resistance. One of the objectives of the present invention is to apply a combination package to the preparation of food, medicine, health care products or Nutraceuticals, the foods, medicines, health care products or nutraceuticals used to reduce the drug resistance genome of the intestinal flora or to reduce antibiotic resistance in antibiotic therapy, the combination package includes the following compositions, each composition is convenient for dosage Dosage unit form for administration and uniformity, the dosage unit form is a single-dose physically dispersed unit: the first composition includes: whole grains; the second composition includes: bitter gourd, soluble dietary fiber and oligosaccharides; The third composition includes: soluble dietary fiber and oligosaccharides.

In some embodiments, the whole grain is selected from the group consisting of coix seed, oats, buckwheat, lentils, corn, adzuki beans, soybeans, yam, jujube, peanuts, lotus seeds, wolfberry, or a combination of the above. In a preferred embodiment, the lentils are white lentils. In another preferred embodiment, the corn is yellow corn.

The "antibiotic" in the context of the present invention can refer to any antibiotic known in the art, for example selected from the following antibiotic classes: β-lactam, aminoglycoside, glycylcycline, macrolide, chloramphenicol, Tetracyclines, quinolones, polypeptides, aminonucleosides, glycopeptides, lincosamide, streptocin, sulfonamides, fosfomycin or a combination of the above.

Whole grains or whole grains (whole grains) refer to whole grains including bran, endosperm, and germ, or plant foods that preserve all the nutrients of natural plant foods during processing. The whole grains used in the present invention may include, but are not limited to, barley, oats, buckwheat, lentils, corn, adzuki beans, soybeans, yams, jujubes, peanuts, lotus seeds and wolfberry, and other whole grains that can be used such as millet, sorghum, Sweet potatoes and so on. The various components of the three compositions used in the present invention can all be purchased through conventional commercial channels.

For example, as a nutritional product, it may be in the form of a dietary supplement. As a medicine, It can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition. "Pharmaceutically acceptable carriers" include solvents, dispersants, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents, etc., suitable for drug administration.

The combination package can be formulated into a dosage form suitable for its planned route of administration. See, for example, U.S. Patent No. 6,756,196. Examples of the route of administration include oral administration. The combination package may be in the form of tablets, capsules, rice, porridge or rice for oral administration, and it would be advantageous to formulate an oral composition in the form of a dosage unit that facilitates dosage administration and uniformity. The term "dosage unit form" refers to a physically discrete unit suitable as a single dose for the treatment of a subject, each unit containing a predetermined amount of active ingredient in combination with the required pharmaceutical carrier and intended to produce the desired therapeutic effect.

Through the use of systems biology strategies, combined analysis of the overall intestinal genome characteristics, the characteristics of the co-metabolic profile of the host and the intestinal flora, and the transplantation of the patient’s intestinal flora to sterile mice, we have observed that the composition and composition of the intestinal flora after intervention Significant changes in function, significant improvement of host health, reduction of toxic and harmful substances in co-metabolites, and the patient's flora before intervention will cause inflammatory reactions in sterile mice and cause more fat accumulation. These results indicate that our dietary intervention can increase the beneficial bacteria in the intestines, reduce harmful bacteria, improve the imbalanced flora, reduce harmful substances in host co-metabolites, and thereby improve human health.

Example 1: Dietary intervention for genetic obesity and simple obesity

In order to study the contribution of intestinal microflora disorders in human obesity and related metabolic disorders, we recruited a group of obese children and children who were obese due to dietary factors, and carried out WTP dietary intervention on them in the hospital (Cited Reference 8).

Nutritional intervention was carried out at Guangzhou Women and Children's Medical Center in Guangdong Province. Except for being overweight, being given antibiotics for more than 3 days, or participating in weight loss projects, the subjects participating in the nutritional intervention have not suffered from gastrointestinal disease, gastrointestinal surgery or chronic disease in the previous 3 months. Said camp The subject of nutrition intervention can be determined by the judgment of the subject or health care professional, and can be subjective (e.g. opinion) or objective (e.g. measurable by testing or diagnostic methods). For example, the subject of the intervention may be a subject with a low level of beneficial intestinal bacteria and a high level of pathogenic bacteria, or a subject diagnosed with metabolic syndrome.

"Nutrition intervention" is defined as the purpose of reducing, relieving, remedying, preventing or improving the symptoms of metabolic syndrome and restoring health through reasonable adjustments to the subject's diet and nutrition.

Approved by the Ethics Committee of the School of Life and Biotechnology, Shanghai Jiaotong University, we conducted open labeling research and self-control research. The registration number of the clinical trial in the Chinese Clinical Trial Registry is ChiCTR-ONC-12002646, and the written consent of the child’s guardian has been obtained. We also conducted a questionnaire survey to collect information on demographic characteristics, health status, disease history, gastrointestinal conditions, eating habits, and physical activity. Based on the "Chinese Food Composition Table (2002 Edition)", questionnaires with meal frequency and 24-hour diet records were used to calculate basic nutritional intake.

We recruited 37 obese children (age range 2-16 years). The subjects underwent a 30-day dietary intervention in the Guangdong Provincial Maternity and Child Health Hospital.

The WTP diet refers to giving whole grains, traditional Chinese medicated diet and prebiotics to children. Specifically, in this embodiment, the WTP diet refers to providing a combination package, including a first composition, a second composition, and a third composition. Each composition is in dosage unit form for ease of dosage administration and uniformity, and the dosage unit form is a single dose of physically discrete units. The first composition, the second composition, and the third composition are prepared by a food manufacturer: Perfect (China) Co., Ltd.

The first composition is a pre-cooked mixture of 12 kinds of whole grains selected from the group consisting of whole grains rich in dietary fiber, including: coix seed (coix), oats, buckwheat, white lentils, yellow corn, red beans, soybeans, yam, and glutinous rice Dates, peanuts, lotus seeds and wolfberry are prepared by food manufacturers in the form of canned porridge (each can has a net weight of 370g). Each can of the first composition contains 100g of ingredients (including 59g Carbohydrate, 15g protein, 5g fat, 6g fiber; Vitamin and mineral content: VE 2.8mg/kg; VB2 0.082mg/100g; Vc 0.3mg/100g; Niacin (niacinamide) 220ug/100g; Folic acid 11.1 ug/100g; Sodium 67mg/kg; Potassium 1800mg/kg; Copper 1mg/kg; Magnesium 337mg/kg; Iron 9mg/kg; Zinc 5mg/kg; Manganese 5mg/kg; Calcium 158mg/kg; Phosphorus 74.2mg/100g; Iodine 0.12mg/kg; selenium 0.016mg/kg; inositol 90mg/kg; linoleic acid 0.28g/100g; a-linolenic acid 0.01g/100g) and 336kcal calories (including 70% carbohydrate, 17% protein, 13% fat), see Table 1 below for specific ingredient content. Among them, the total calories can be determined by the oxygen bomb determination ability correction method, the protein content can be determined by the Kjeldahl micro method, the carbohydrate content can be determined by high pressure liquid chromatography, the fat content can be determined by the Soxhlet extraction method, and the diet The content of fiber can be measured by the neutral detergent method, the content of vitamins can be measured by high pressure liquid chromatography, and the content of minerals can be measured by spectrophotometry.

The first composition is used as a staple food to be prepared as rice, noodles, porridge or rice for administration. After cooking methods such as steaming and boiling, the starch in it is not easy to gelatinize, and it is not easy to raise blood sugar. Children are given enough first composition to satisfy hunger and meet the standard nutritional requirements for their age group. The standard nutritional requirements are stipulated in the Chinese Dietary Nutrient Reference Intakes recommended by the Chinese Nutrition Society (CNS, 2012) "(DRI). The diet record of each patient was used to calculate the nutritional intake based on the "Chinese Food Composition Table" (2002).

The second composition was given in the form of a reconstituted powder (20 g per bag), and the specific ingredients and contents are shown in Table 1 below. Among them, oligosaccharides are sugar molecules formed by two or more (generally 2-10) monosaccharide units connected by glycosidic bonds, such as fructooligosaccharides or isomaltose oligosaccharides. After the second composition is made into a powdered preparation, it is reconstituted and eaten with warm water, and 60 grams of the second composition is taken every day.

The third composition is administered in the form of a reconstituted powder (20 g per bag), and the specific ingredients and contents are shown in Table 1 below. Prepare and consume with warm water, and take 60 grams of the third composition every day.

Table 1. Ingredients and contents of the three compositions

The representative administration period of the combination package of the present invention is one week to several months, such as one week, two weeks, one month, two months, four months, and eight months. After the level of intestinal beneficial bacteria in the subject begins to rise and the level of conditional pathogenic bacteria begins to decrease, the dosage of the composition can be gradually reduced. The administration can be terminated when the structure of the intestinal flora returns to normal.

We found that: (1) Diet intervention can relieve genetic obesity and simple obesity and improve the patient's biochemical indicators; after 30 days of intervention, all relevant clinical indicators have been significantly improved in obesity and simple obesity patients caused by genetic factors; (2) The intestinal flora produced little inflammation and fat accumulation in mice after the intervention; (3) Diet intervention changed the structure and function of the intestinal flora.

Example 2

Data set used in the embodiment

In a study we have published (Citation 8), 17 children with genetic obesity and 21 children with simple obesity received dietary intervention targeting the intestinal flora. We used the Illumina platform to perform overall genome sequencing on their stool samples (simple obese children: 0 days and 30 days; genetically obese children: 0 days, 30 days, 60 days, and 90 days). The original sequencing data can be downloaded from NCBI's SRA database (acceptance number: SRP045211). In our pairing study like this, we Due to the incompleteness of the data or the identification of outliers, the data of three individuals were removed (GD10 individuals lacked 30-day data, and GD20 and GD26 individuals were outliers). In the remaining 35 obese children's 0-day and 30-day samples, there were an average of 84 million high-quality sequencing fragments.

Identification of antibiotic resistance genes

We downloaded the core 7828 antibiotic resistance gene protein sequences in the ARDB database. The protein sequences in the non-redundant gut microbial gene collection we constructed before are compared with the protein sequences of these antibiotic resistance genes. The result of the comparison requires E-value to be less than 1e-10, and 70% of the aligned sequence is required. It is covered, and the identity of the comparison is at least 80% (Cited Document 5).

Genome assembly of a large co-abundance gene collection (CAG)

All genes satisfying 90% of the total abundance distribution in more than three samples, based on their abundance, we use the canopy algorithm to classify them. We assemble all the large CAGs carrying antibiotic resistance genes by the same means as published papers (Cited Reference 8). Brief description of the process: By comparing with CAG-specific genes, we collect sample-specific and CAG-specific sequencing short sequences, and then assemble them de novo using the Velvet tool. We introduce 6 indicators for the Human Health Group Project (HMP) to judge high-quality genome drafts.

Classification status recognition of CAG

For large CAGs that meet at least five HMP high-quality genome draft standards, we use CVtree3.0 web server and SpecI to determine the classification status (cited reference 8). For small CAGs and large CAGs without high-quality assembly products, we compare the genes in each CAG with 7,991 reference genome sequences at the nucleic acid and protein levels. The comparison results are filtered by E-value (nucleic acid level: less than 1e10, protein level: less than 1e-5) and sequence coverage (>70%). According to the reported taxonomic status recognition threshold (reference 17), CAG is recognized at the species or genus level (species level: at the DNA level, 90% of the genes can be compared with the same species with 95% identity) Genome; Genus level: At the DNA and protein level, 80% of the genes are met at the same time Can be compared to the same genus).

result

Altered antibiotic resistance gene

In this dietary intervention project for hereditary and simple obesity, we have published an article in which we constructed a non-redundant gene set containing ~2 million human intestinal flora ( Reference 8). By using the BLAST comparison tool and using the ARDB database as the reference sequence library (cited reference 18), we identified a total of 399 antibiotic resistance genes in this gene set.

Principal component analysis based on the overall structure of antibiotic resistance genes shows that there are significant differences before and after intervention, and such differences can be tested by substitutional MANOVA (part a in Figure 1) (9999 substitutions, P value = 0.0187). The total number of resistance genes was significantly reduced from 185.54±10.56 before intervention (if there is no other statement, the data are expressed in the form of mean±standard deviation) to 151.91±7.37 (part b in Figure 1). Compared with the samples before the intervention, 48 antibiotic resistance genes disappeared in the samples after the intervention, but 15 new antibiotic resistance genes appeared (Table 1A), which indicates the dynamic gains and losses of antibiotic resistance genes during the intervention process. Although new antibiotic resistance genes appeared, the number of samples carrying these genes (1.6±1.1, mean±standard deviation) was significantly less than the number of samples carrying antibiotic resistance genes that disappeared after intervention (2.9±2.1, mean±standard) difference).

Moreover, after the intervention, the total abundance of antibiotic resistance genes also decreased significantly, from 4,075.51±313.91 to 3410.52±306.90 (part b in Figure 1). Among all 399 antibiotic resistance genes, the abundance of 86 decreased significantly after the intervention, and they accounted for 33.39%±3.39% and 24.48%±2.79% of the total abundance of drug-resistant genomes before and after the intervention. There are 222 anti- The abundance of antibiotic resistance genes decreased after intervention, and they accounted for 47.90%±4.48% (before intervention) and 41.22%±5.68% (after intervention) of the total drug resistance genome. Although the abundance of 10 antibiotic resistance genes increased significantly after intervention, their abundance only accounted for 1.96%±0.94% (before intervention) and 8.94%±3.33% (after intervention) of the total resistance gene group. The antibiotic resistance genes that were significantly reduced after the intervention mainly belonged to the tet, mdt and erm families, and the increased antibiotic resistance genes were mainly members of the pbp and erm families (Figure 2 and Table 2 below).

The type of resistance gene changed and the corresponding antibiotic

The 399 antibiotic resistance genes identified can be further classified into 131 different resistance classes. Fifteen of these 131 resistance categories are present in all 70 samples. They are TetW, TetQ, TetO, TetM, Tet40, Tet32, MdtF, MdtE, ErmF, ErmB, B12e_cfxa, BacA, AcrB, AcrA And Aac6Ie. There are 11 categories that only appeared in the samples before the intervention, and 4 were only in the samples after the intervention (Part c of Figure 1). In the pre-intervention samples of obese children, the number of resistant categories was significantly higher than that of adults in China, Denmark, and Spain (Figure 3, part a). After the intervention, the number of resistance categories decreased significantly. Compared with Chinese adults, there is no significant difference, but it is still significantly higher than the number of resistance categories contained in Danish and Spanish adults (part a in Figure 3).

Among the 131 resistance categories, 36 abundances changed significantly after intervention (Figure 3, part b). On average, 33 significantly reduced categories, their abundances totaled from 1364.2 ± 188.85 Reduced to 761.6±78.81. The abundance of only three categories increased significantly after the intervention. They are ErmX, which is resistant to lincosamides, macrolides and streptocin b, and TetL and TetL, which are resistant to tetracyclines. PBP2b that is resistant to penicillin. From the perspective of widespread distribution in the sample, of the 36 categories with significant changes, 8 did not change, and 16 decreased after the intervention (Figure 3, part b).

When we matched all resistance categories to the antibiotics they participated in the resistance, we got the potential resistance of 47 antibiotics. 10 of these 47 were significantly reduced after intervention. They are chloramphenicol, norfloxacin, puromycin, erythromycin, fosfomycin, vancomycin, polymyxin, enoxacin, Fosmidomycin and kasugamycin (panel 3 part c). At the level of antibiotics, 8 of the 15 categories showed significant abundance in the samples after the intervention, including streptomycins, macrolides, peptides, sulfonamides, quinolones, and amides. Alcohols and glycopeptides (Figure 3, part d).

Bacterial antibiotic resistance genes mainly generate resistance through three mechanisms: 1) reduce the concentration of antibiotics in bacterial cells by weakening permeability or efflux pump; 2) modify antibiotic targets to protect them; and 3) Hydrolyze or modify the antibiotic molecule to make the antibiotic inactive (reference 11). Based on the information in the ARDB database, 399 antibiotic genes were assigned to these three mechanisms according to the mechanism of resistance. After the intervention, the sum of the abundance of resistance genes that produced resistance through the two mechanisms of target protection and efflux pump was significantly reduced (Figure 4). The erythromycin ribosomal methylase (erm) family develops resistance to antibiotics such as macrolides, lincosamides, and streptomycin by methylating 16S ribosomal RNA genes (reference 19). Dietary intervention significantly reduced the abundance of ErmB, ErmG and ErmQ in the erm family. In addition, the abundance of TetO and TetPB, which protect ribosomes from translational restriction of tetracycline, was also significantly reduced after the intervention. The abundance of other types of resistance, including sul2, arna, ksga, and vangu, that generate resistance by protecting the target has also been significantly reduced. The bacterial efflux pump system can transfer a lot of antibiotics to the outside of the cell, thereby reducing The concentration of intracellular antibiotics. After the intervention, the abundance of the multi-drug resistance pumps of ErmD and 10 Mdt (E, F, G, H, K, L, M, N, O, and P) families, and the AcrAB-TolC multi-drug pump complex The abundances of are all significantly reduced. This result implies a reduction in potentially multi-drug resistant bacteria. In addition, specific efflux pumps include Bcr for efflux bacitracin, MacB for efflux macrolides, Tete40 and TetC for tetracycline efflux, and RosA-RosB complex for efflux fosfomycin. (Cited Reference 20), significantly reduced after dietary intervention.

Identify carriers of antibiotic resistance genes

In published studies, we rely on the canopy algorithm to classify non-redundant intestinal bacterial gene sets into the "common abundance gene set (CAG)" based on the correlation based on gene abundance (Cited References 8, 12 ). According to the number of genes contained, CAG is further divided into two categories. A large CAG containing more than 700 genes is considered to be a bacterial genome, and a small CAG containing no more than 700 genes may be an incomplete bacterial genome or a phage. In the entire drug resistance genome, 201 antibiotic resistance genes can be found in their carriers. They come from 38 large CAGs and 80 small CAGs. The abundances of these 201 resistance genes account for the total resistance genes before and after intervention. 86.60%±1.11% and 87.45%±1.99% of the drug genome. On average, the abundance of resistance genes in 38 large CAGs was 87.45%±1.99% (before intervention) and 334.54±61.70 (after intervention). The abundance of resistance genes in 80 small CAGs were 2985.58±246.93 (before intervention) and 2665.39±272.79 (after intervention). A total of 198 antibiotic resistance genes have been traced back in CAG. The total abundance of resistance genes in this part was significantly reduced from 532.63±60.82 to 425.13±103.35 after the intervention.

We have performed de novo assembly for each CAG carrying antibiotic resistance genes (see "Genome Assembly of Large Co-abundance Gene Set (CAG)" above for details). The 24 genome assembly products meet at least 5 of the 6 quality standards of the Human Microbiome Project (HMP) for the draft genome. Through the CVtree3.0 web server (cited reference 21) and the specI tool (cited reference 22), the phylogenetic information of the 24 draft genomes was determined. The remaining 14 large CAGs and 80 The taxonomic identification of a small CAG is achieved by comparison with a reference genome (see "CAG Classification Status Identification" above for details), of which 8 large CAGs and 22 small CAGs can be classified at the species/genus level Status information. Among the 118 CAGs carrying antibiotic resistance genes, the first three CAGs with the largest number are CAG00002 (Escherichia/Shigella sp.), CAG00001 and CAG00008 (grams of pneumonia). Klebsiella pneumonia), they carry 41, 37 and 20 antibiotic resistance genes, respectively. At the nucleic acid level, through comparison with the reference genome, we found that 51.5% of the genes in CAG00001 can be compared to Escherichia/Shigella, 16.7% to Weissella (Weissella), and 14.9% to Enterococcus (intestinal). Coccus), this result indicates the heterogeneity of CAG00001. By increasing the parameter "--max_canopy_dist" in the canopy tool from 0.9 to 0.95, we performed the second round of classification of the genes in CAG00001. Four sub-categories are obtained, which are labeled CAG00001a to CAG00001d. CAG00001a and CAG00001c are Escherichia/Shigella sp, but CAG00001b and CAG00001d cannot obtain information on their taxonomic status at the species/genus level. CAG00001a, b, c, and d carry 28, 3, 5, and 0 antibiotic resistance genes, respectively. Because of stricter classification parameter settings, three resistance genes could not be classified into CAG00001~d. Since a gene can be classified into more than one CAG, the total number of antibiotic resistance genes carried in CAG00001a~d will be greater than 34.

Based on gene classification, genome assembly and classification status identification, we finally got 120 (CAG00001a~c+117 CAGs carrying resistance genes) antibiotic resistance gene carriers. The component analysis based on the abundance of resistance gene carriers showed that the samples before and after the intervention had a certain trend of differentiation (Figure 2) (replacement MANOVA test, 9999 replacements, P=0.0584). The procrustes analysis combining the principal component analysis results of 399 resistance genes and the principal component analysis results of the resistance gene carriers showed that the changes in the drug resistance genome were significantly related to the changes in these carriers (Monte-Carlo test, P <0.001, M ² =0.23). Considering that these 120 resistance gene carriers carry a relatively high abundance of resistance genes, this implies that the structural changes of the overall drug resistance genome are mainly contributed by these carriers.

Distribution of antibiotic resistance genes and their carriers

Based on the affiliation of antibiotic resistance genes and their carriers, a network of resistance genes belonging to the specific population we studied was constructed (Figure 6). Antibiotic resistance genes exist in many bacterial phyla, including Firmicutes, Bacteroides and Proteobacteria, which indicates that antibiotic resistance genes are widely distributed in the bacterial kingdom. Bacteria in Firmicutes contain the smallest antibiotic resistance gene load, and basically each bacteria contains only one type of resistance gene. The BacA gene type that is resistant to bacitracin is carried in large numbers by bacteria in Firmicutes and appears as a hot spot in the network diagram. A total of 6 Lactobacillus (Lactobacillus), 3 Eubacterium (Eubacterium) and 3 Clostridia carry this gene category. Bacteria in the Bacteroides phylum mainly (8/11) carry resistance genes that are resistant to β-lactam antibiotics, including php2b, BL3_ccra, BL2e_cbla, BL2e_cepa and BL2e_cfxa. The resistance gene category BL2e-cepa only exists in Bacteroides, which is consistent with the results of the ARDB database and implies the potential taxonomic specificity of this resistance gene category. Bacteria in Enterobacteriaceae, especially Klebsiella, Enterobacter and Escherichia, exist in the form of hot spots in the distribution network. 10 mdt (E, F, G, H, K, L, M, N, O, and P) families of multi-drug resistant efflux pumps and AcrAB-TolC multi-drug resistant pump complexes are specifically controlled by these genera Bacteria carry. And the types of resistance genes carried by these bacteria can develop resistance to a wide range of antibiotics, including β-lactams, tetracyclines, sulfonamides and macrolides. After the intervention, the abundance of these bacteria was significantly reduced or reduced, especially CAG00001a (Escherichia/Shigella sp.), CAG00002 (Escherichia/Shigella sp.), CAG00146 (Klebsiella sp.), CAG00008 (Klebsiella pneumoniae) and CAG00356 ( Enterobacter sp.).

All publications cited in this specification are incorporated by reference in their entirety.

Citation

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Claims (21)

The use of a combination package in the preparation of foods, health products or nutritional products, the food, health products or nutritional products in the treatment of antibiotics to reduce antibiotic resistance, the combination package includes the following composition, wherein each composition is A dosage unit form for convenient dosage administration and uniformity, the dosage unit form is a single-dose physically dispersed unit: the first composition includes: whole grains; the second composition includes: bitter gourd, soluble dietary fiber and oligomer Sugar; a third composition, including: soluble dietary fiber and oligosaccharides, wherein, in the first composition, the weight of the protein accounts for 5-40% or 10-20% of the weight of the first composition, and carbon water The weight of the compound accounts for 30-80% or 50-70% of the weight of the first composition, the weight of fat accounts for 0.5-30% or 2-15% of the weight of the first composition, and the weight of dietary fiber accounts for The weight of the first composition is 0.5-30% or 2-15%, the weight of vitamins is 0.1-5% or 0.5-1% of the weight of the first composition, and the weight of minerals is the weight of the first composition. 0.1-2% or 0.8-1.2% by weight of the substance; wherein, in the first composition, the whole grains include Huiren 20wt%, oats 15wt%, buckwheat 15wt%, white lentils 10wt%, yellow corn 5wt% %, red bean 5wt%, soybean 5wt%, yam 10wt%, jujube 5wt%, peanut 5wt%, lotus seed 5wt% and wolfberry 5wt%; in the second composition, the bitter melon is relative to the dietary fiber and the The weight of oligosaccharides The ratio is 10:1 to 1:1; the weight ratio of dietary fiber and oligosaccharide in the third composition is 6:1 to 1:6; and the antibiotic is selected from β-lactam, aminoglycoside, Glycylcyclines, macrolides, chloramphenicols, tetracyclines, quinolones, peptides, aminonucleosides, glycopeptides, lincoramide, streptomycin, sulfonamides, fosfomycin and A group composed of the above combinations. The use described in item 1 of the scope of patent application is characterized in that the first composition is taken as a staple food in the form of rice, noodles, porridge or rice. The use described in item 2 of the scope of patent application is characterized in that before preparing rice, noodles, porridge or meals with the first composition, the seeds, fruits or other plant parts of the first composition are crushed into particles , Where 1-70%, 15-70%, 25-70%, 30-70%, or 50-70% of the particles have a diameter of 0.65mm or more. The use described in item 1 of the scope of the patent application is characterized in that every 100 grams of the first composition provides 320-400 kcal total calories. The use described in item 1 of the scope of patent application is characterized in that, per 100 grams of the first composition contains: VA 3-857ugRE, VD 0.01-5ugRE, VE 0.2-79.09mg, VB1 0.01-1.89mg, VB2 0.01-1.4mg, VB60.01-1.2mg, VB12 0.1-2.4mg, VC 0.1-1170mg, niacin 0.1-28.4mg, Ca 10-2458mg, P 50-1893mg, K 100-1796mg, Na 5-2200mg , Mg 30-350mg, Fe 0.5-20mg. The use described in item 1 of the scope of the patent application is characterized in that the first composition The buckwheat in includes: common buckwheat or tartary buckwheat. The use described in item 1 of the scope of patent application is characterized in that the buckwheat in the first composition comprises: seeds of the genus Fagopyrum. The use described in item 1 of the scope of the patent application is characterized in that the oats in the first composition comprise oat seeds. The use described in item 1 of the scope of patent application is characterized in that the yam in the first composition includes: dried yam. The use described in item 1 of the scope of the patent application is characterized in that the second composition is made into a reconstituted powder and taken 0.25 to 1 hour before a meal. The use described in item 10 of the scope of the patent application is characterized in that the daily dose of the second composition is 5-100 g, 40-60 g or 30-80 g, prepared with water. The use described in item 1 of the scope of the patent application is characterized in that the bitter gourd in the second composition comprises: whole fruit powder of a plant of the genus Momordica. The use described in item 12 of the scope of the patent application is characterized in that the whole plant fruit powder is produced by freeze drying or spray drying. The use described in item 1 of the scope of the patent application is characterized in that the bitter gourd in the second composition comprises: bitter gourd extract. The use described in item 1 of the scope of the patent application is characterized in that the second composition The soluble dietary fiber in is selected from the group consisting of Fibersol-2, resistant starch, polydextrose, cellulose, hemicellulose, pectin and gum. The use described in item 1 of the scope of the patent application is characterized in that the oligosaccharides in the second composition are selected from the group consisting of fructooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, Soybean oligosaccharides, oligodextrose, stachyose, and lactulose oligosaccharides. The use described in item 1 of the scope of the patent application is characterized in that the weight of the bitter gourd accounts for 15-99.8%, 30-95% or 45-90% of the weight of the second composition, and the soluble dietary fiber The weight of the oligosaccharide accounts for 0.1-51%, 1-40%, 3-30% or 5-20% of the weight of the second composition, and the weight of the oligosaccharide accounts for 0.1-34% of the weight of the second composition %, 1-25%, 2-20% or 3-15%. The use described in item 1 of the scope of the patent application is characterized in that the third composition is made into a reconstituted powder, which is taken 2-5 hours before a meal or taken at the same time as breakfast. The use described in item 18 of the scope of patent application is characterized in that the daily dose of the third composition is 5-200 g, 30-100 g or 50-150 g, prepared with 300-1500 ml of water. The use according to item 1 of the scope of patent application, characterized in that the soluble dietary fiber in the third composition is selected from Fibersol-2, resistant starch, polydextrose, cellulose, hemicellulose, fruit Group of gums and gums. The use described in item 1 of the scope of the patent application is characterized in that the oligosaccharides in the third composition are selected from the group consisting of fructooligosaccharides, galacto-oligosaccharides, xylo-oligosaccharides, isomalto-oligosaccharides, Soybean oligosaccharides, oligodextrose, stachyose, and lactulose oligosaccharides.

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