WO2021219001A1 - 用于提高免疫力的组合物 - Google Patents

用于提高免疫力的组合物 Download PDF

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WO2021219001A1
WO2021219001A1 PCT/CN2021/090531 CN2021090531W WO2021219001A1 WO 2021219001 A1 WO2021219001 A1 WO 2021219001A1 CN 2021090531 W CN2021090531 W CN 2021090531W WO 2021219001 A1 WO2021219001 A1 WO 2021219001A1
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bifidobacterium
composition
amount
individual
probiotic
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PCT/CN2021/090531
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English (en)
French (fr)
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黄秀娟
陈家亮
徐之璐
邓咏欣
梁巧仪
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香港中文大学
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Priority to KR1020227041617A priority Critical patent/KR20230004809A/ko
Priority to US17/921,788 priority patent/US20230165912A1/en
Priority to JP2022566193A priority patent/JP2023523470A/ja
Publication of WO2021219001A1 publication Critical patent/WO2021219001A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
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    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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    • A—HUMAN NECESSITIES
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    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Definitions

  • This application generally relates to the fields of medicine, food, and health products. Specifically, the present application provides a composition and its use based on the regulation of the intestinal flora to improve individual immunity or assist in the treatment and prevention of diseases or improve the efficacy of diseases.
  • the intestinal flora is not only related to digestive function, but also related to the body's ability to resist pathogen infections, autoimmune diseases and other diseases, and even to the response to drug treatments.
  • probiotics In the intestinal flora, there is a type of bacteria that are beneficial to the body, called probiotics.
  • probiotics are designated to colonize the human body and change the composition of a certain part of the host's flora, a type of active microorganism that is beneficial to the host.
  • Probiotics can regulate the host mucosa and immune function of the system or regulate the balance of the intestinal flora to promote nutrient absorption and maintain intestinal health, thereby producing beneficial effects on health.
  • Common probiotics include bifidobacteria, lactobacilli, yeasts and so on.
  • prebiotics Some substances are closely related to probiotics and are called prebiotics.
  • prebiotics refer to organic substances that are not digested and absorbed by the host but can selectively promote the metabolism and proliferation of probiotics in the body, thereby improving the health of the host.
  • prebiotics should pass through the upper digestive tract, most of which are not digested but can be fermented by the intestinal flora. The most important thing is that prebiotics can stimulate the growth of beneficial bacteria without stimulating harmful bacteria with potential pathogenicity or spoilage activity.
  • Common prebiotics are oligosaccharides, also known as dietary fiber.
  • the present application provides a probiotics composition, including Bifidobacterium bifidum and Bifidobacterium longum.
  • the ratio of the amounts of Bifidobacterium bifidum and Bifidobacterium longum is 1: (0.21-2.36) in terms of colony forming units.
  • the probiotic composition further comprises Bifidobacterium adolescentis.
  • the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum is (0.57-3.56):1:(0.21-2.36) in terms of colony forming units. In some specific solutions, the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum is (0.75-1):1:(0.75-1) in terms of colony forming units.
  • the probiotic composition further comprises Lactobacillus rhamnosus.
  • the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus in terms of colony forming units is (0.57-3.36):1:(0.21-2.36) ):1.
  • the probiotic composition is in a unit dosage form, and the amount of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium, Bifidobacterium longum, and Lactobacillus rhamnosus is independently 10 4 in colony forming units. To the order of 10 12 CFU. In some embodiments, the total amount of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is on the order of 10 6 to 10 12 CFU.
  • the probiotic composition is for administration to an adult, and the amount of Bifidobacterium adolescentis is 2.59 ⁇ 10 5 -4.49 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium bifidum is 1.26 ⁇ 10 5 –7.35 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium longum is 2.23 ⁇ 10 5 –7.02 ⁇ 10 11 CFU; and/or the amount of Lactobacillus rhamnosus is 1.26 ⁇ 10 5 –2.59 ⁇ 10 11 CFU.
  • the probiotic composition is for administration to children, and the amount of Bifidobacterium adolescentis is 2.05 ⁇ 10 5 -4.55 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium bifidum is 1.47 ⁇ 10 5- 3.6 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium longum is 7.55 ⁇ 10 4 -2.5 ⁇ 10 11 CFU; and/or the amount of Lactobacillus rhamnosus is 1.47 ⁇ 10 5 -3.6 ⁇ 10 11 CFU.
  • the probiotic composition does not contain other probiotics other than the probiotics described in the various embodiments of the present application. In some embodiments, the probiotic composition does not contain probiotics other than Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus. In some embodiments, the probiotic composition does not contain bifidobacteria other than Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum.
  • the present application provides a prebiotics composition comprising xylo-oligosaccharides, galacto-oligosaccharides and corn dietary fiber.
  • the ratio of the amount of xylo-oligosaccharide, galactooligosaccharide, and corn dietary fiber by weight is (0.25-5): (0.75-4): (0.5-1). In some specific embodiments, the ratio of the amount of xylo-oligosaccharide, galactooligosaccharide, and corn dietary fiber by weight is (0.25-0.5): (2-4): (0.5-0.75).
  • the prebiotic composition is in a unit dosage form, and the total amount of xylo-oligosaccharides, galacto-oligosaccharides, and corn fiber is 0.1-12 g by weight. In some embodiments, the prebiotic composition is in a unit dosage form, and the total amount of xylo-oligosaccharides, galacto-oligosaccharides, and corn fiber is 0.1-5 g by weight.
  • the amount of xylo-oligosaccharides is 0.01g-6g; and/or the amount of galacto-oligosaccharides is 0.04g-9.6g; and/or the amount of corn dietary fiber is 0.01g-6g.
  • the prebiotic composition does not contain prebiotic components other than xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber.
  • the present application provides a dietary composition (also referred to as a "synbiotics" composition in some cases), comprising the probiotic composition of the first aspect and the prebiotic composition of the second aspect.
  • the dietary composition comprises Bifidobacterium adolescentis, Bifidobacterium bifidum and Bifidobacterium longum as probiotics, and xylooligosaccharides, galacto-oligosaccharides and corn dietary fiber as prebiotics, wherein In terms of colony forming units (CFU), the ratio of the amount of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium and Bifidobacterium longum is (0.75-1):1:(0.75-1), and the three bacteria total about 2 ⁇ 10 11 CFU, and by weight, the ratio of xylo-oligosaccharide, galacto-oligosaccharide and corn dietary fiber is (0.25-0.5): (2-4): (0.5-0.75) and the ratio of xylo-oligosaccharide, low
  • CFU colony forming units
  • the probiotic composition or prebiotic composition or dietary composition is formulated for oral administration.
  • oral administration includes oral administration, mixing with oral products, tube feeding, and the like.
  • the probiotic composition or prebiotic composition or dietary composition is a food supplement, food additive or food.
  • the probiotic composition or prebiotic composition or dietary composition is formulated as a powder, granule, tablet or capsule.
  • the probiotic composition or prebiotic composition or dietary composition is administered to an individual to assist in the prevention and/or treatment of pathogen infection, or to enhance the therapeutic effect of pathogen infection , Or improve the individual's immunity, or balance the individual's intestinal microecology (including increasing the abundance of microorganisms, increasing the ideal bacterial species and/or reducing the undesirable bacteria).
  • the pathogen is a virus, bacteria or fungus.
  • the pathogen is a respiratory disease virus, such as the new coronavirus (COVID-19), influenza, and respiratory syncytial virus.
  • this application provides the probiotic composition of the first aspect, or the prebiotic composition of the second aspect, or the dietary composition of the third aspect in preparation for assisting in the prevention and/or treatment of pathogen infection or Use in dietary products or medicines that enhance the therapeutic effect of an individual's pathogen infection or improve the individual's immunity or balance the individual's intestinal microecology (including increasing microbial abundance, increasing ideal bacterial species and/or reducing undesirable bacteria).
  • the pathogen is a virus, bacteria or fungus.
  • the pathogen is a respiratory disease virus, such as COVID-19, influenza, and respiratory syncytial virus.
  • this application provides methods for assisting in the prevention and/or treatment of individual pathogen infections or enhancing the therapeutic effect of individual pathogen infections or enhancing the individual’s immunity or balancing the individual’s intestinal microbiota (including increasing microbial abundance,
  • the method of increasing ideal bacterial species and/or reducing undesirable bacteria) comprises administering the probiotic composition of the first aspect, or the prebiotic composition of the second aspect, or the dietary composition of the third aspect to the individual.
  • the pathogen is a virus, bacteria or fungus.
  • the pathogen is a respiratory disease virus, such as COVID-19, influenza, and respiratory syncytial virus.
  • Figure 1 shows the relationship between ideal bacterial species identified in previous studies and various probiotics.
  • the ideal bacterial species has the potential to enhance immunity, and its abundance is negatively correlated with the severity of the COVID-19 disease or the SARS-CoV-2 viral load.
  • the circle represents the positive correlation between the ideal bacterial species and the abundance of probiotics, the size indicates the strength of the positive correlation, and the box encircles the target probiotics identified in this application.
  • Figure 2 shows part of the results of the cohort study of Example 2, where Figure A shows the positive rates and overall positive rates of Bifidobacterium adolescentis, Bifidobacterium bifidum and Bifidobacterium longum in each cohort study; Figure B shows The stool sample of the subject contains three types of bifidobacteria, any two of the three types of bifidobacteria, or only one or none of the three types of bifidobacteria.
  • Figure 3 shows part of the results of the synbiotic composition and standard treatment of COVID-19 patients in the clinical study of Example 3.
  • Figure A shows the clinical symptom relief scores in the first and second weeks (the score is defined as 20) And antibody formation;
  • Figure B shows the quantification of immune response markers in plasma (converted to log10 display). For all box plots, the center is drawn by the measured median value, and the upper and lower boundaries of the box plot correspond to In the first and third percentiles. The p value is determined from both sides. A p value of ⁇ 0.05 is considered statistically significant (Wilcoxon rank sum test);
  • Figure C shows the percentage decrease in the inflammatory immune response markers at week 5 compared with baseline.
  • the shape represents the decrease in the median percentage, and the p value is determined by the two-sided Wilcoxon rank sum test;
  • Figure D shows the probiotic concentration at baseline, 2 weeks and 5 weeks after taking the synbiotic composition for the first time, and the concentration is determined by qPCR. And after log10 transformation (ng/ ⁇ l), p value ⁇ 0.05 is considered to be statistically significant (Wilcoxon rank sum test).
  • Figure 4 shows the study design scheme of Example 4.
  • Figure 5 shows the 3 types of bifidobacteria in the synbiotic composition of the healthy people in Example 4 and the COVID-9 patients in the synbiotic composition group and the standard treatment group at baseline, 2, 4, and 5 weeks
  • the total relative abundance of probiotics (Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum).
  • the upper graph is a summary graph, and the lower graph is a graph at each time point.
  • the relative abundance (percentage) is displayed in the form of conversion to log10.
  • the p-values between the relative abundances at the second, fourth, and fifth week and the baseline were determined by the Wilcoxon rank sum test.
  • Figure 6 shows the Shannon diversity index of healthy people in Example 4 and COVID-9 patients in the synbiotic composition group and standard treatment group at baseline, 2, 4, and 5 weeks.
  • the p value between the second, fourth, and fifth week and the baseline Shannon Diversity Index was determined by the Wilcoxon rank sum test.
  • Figure 7 shows the ideal bacterial species (A) and non-ideal bacteria of healthy people in Example 4 and COVID-9 patients in the synbiotic composition group and standard treatment group at baseline, 2, 4, and 5 weeks
  • the total relative abundance of species (B) where Figure A shows the total relative abundance of ideal bacterial species (the species with higher abundance in non-COVID-19 people), and Figure B shows the total relative abundance of non-ideal bacterial species (in COVID-19). -19 patients with higher abundance) total relative abundance.
  • the relative abundance (percentage) is displayed in the form of conversion to log10.
  • the p-values between the relative abundances at the second, fourth, and fifth week and the baseline were determined by the Wilcoxon rank sum test.
  • Figure 8 shows the species with differences in abundance at baseline, at 2, 4, and 5 weeks between the synbiotic composition group and the standard treatment group in Example 4 (LDA>2, p ⁇ 0.05).
  • the levels of a variety of ideal bacterial species (box marks) in the synbiotic composition group were significantly higher, while the undesirable bacterial species in the synbiotic composition group (Klebsiella pneumoniae, Xiaowei Rongococcus and Escherichia coli) are significantly lower than the standard treatment group.
  • the corresponding relationship between square chromaticity and LDA value is shown in the figure. If the LDA value is positive, it means that the level of the species in the synbiotic composition treatment group is significantly higher, and a darker color represents a greater difference. If the LDA value is negative, it means that the species is significantly higher in the standard treatment group, and lighter colors represent greater differences.
  • probiotic/prebiotic/synbiotic compositions are provided. It is expected that these compositions can effectively enhance the body’s immunity and help prevent and/or treat pathogen infections, such as respiratory pathogen infections, such as Novel coronavirus (COVID-19), influenza, respiratory syncytial virus, etc.
  • pathogen infections such as respiratory pathogen infections, such as Novel coronavirus (COVID-19), influenza, respiratory syncytial virus, etc.
  • this application provides a probiotic composition comprising Bifidobacterium bifidum and Bifidobacterium longum.
  • probiotic composition refers to a composition with probiotics as the active ingredient, and does not exclude the presence of auxiliary components required for the cultivation, isolation and purification of probiotics and/or auxiliary ingredients for formulating the composition according to the desired purpose .
  • the ratio of the amounts of Bifidobacterium bifidum and Bifidobacterium longum is 1: (0.21-2.36) in terms of colony forming units.
  • Colony forming unit is a form of characterizing the amount of microorganisms that is common in the art. Unless otherwise specified, the amount of microorganisms described in this application is calculated in colony forming units.
  • the ratio of the amount of Bifidobacterium bifidobacterium to Bifidobacterium longum can be 1: (0.21, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.36).
  • the composition is for administration to an adult, and the ratio of the amounts of Bifidobacterium bifidum and Bifidobacterium longum is 1: (0.36-2.36).
  • the composition is for administration to children, and the ratio of the amounts of Bifidobacterium bifidum and Bifidobacterium longum is 1: (0.21-1.7).
  • the probiotic composition further comprises Bifidobacterium adolescentis.
  • the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum is (0.57-3.56):1:(0.21-2.36) in terms of colony forming units.
  • the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium and Bifidobacterium longum is (0.57, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 , 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.56): 1: (0.21, 0.3, 0.4 , 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.36).
  • the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum is (0.75-1):1:(0.75-1) in terms of colony forming units.
  • the composition is for administration to an adult, and the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium and Bifidobacterium longum is (1-3.56):1:(0.86-2.36).
  • the composition is for administration to children, and the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium and Bifidobacterium longum is (0.57-3.09):1:(0.21-1.7).
  • the probiotic composition further comprises Lactobacillus rhamnosus.
  • Short-chain fatty acids SCFA, such as butyric acid and propionic acid
  • SCFA Short-chain fatty acids
  • Lactobacillus rhamnosus can increase the production of SCFA in the intestine
  • the combination of Lactobacillus rhamnosus with Bifidobacterium bifidum and Bifidobacterium longum is expected to increase the total SCFA production.
  • oral Lactobacillus rhamnosus can increase the content of Bacteroides and Fischeri in the intestine.
  • the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is (0.57-3.56):1:(0.21-2.36):1.
  • the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus in terms of colony forming units is (0.57, 0.6, 0.7, 0.8, 0.9, 1.0 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 , 3.56): 1: (0.21, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3 , 2.36): 1.
  • the composition is used to administer to an adult, the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is (1-3.56):1:(0.86 -2.36): 1.
  • the composition is used for administration to children, and the ratio of the amounts of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is (0.57-3.09):1:(0.21- 1.7): 1.
  • the probiotic composition is in a unit dosage form, and the amount of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium, Bifidobacterium longum, and Lactobacillus rhamnosus is independently 10 4 in colony forming units. To the order of 10 12 CFU. In some embodiments, the total amount of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is on the order of 10 6 to 10 12 CFU.
  • the total amount of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is about 2 ⁇ 10 11 CFU. It should be understood that the probiotic composition of the present application may not all contain Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus. Therefore, the “total amount” here refers to these four types of probiotics. The total amount of bacteria present in the probiotic composition.
  • unit dosage form refers to a composition for a single administration dose that is packaged separately or separately, and can usually be present in a single tablet, capsule, or powder/granule bag, etc. In some embodiments, for ease of administration, the unit dosage form is prepared as a composition containing a daily dose.
  • the probiotic composition is for administration to an adult, and the amount of Bifidobacterium adolescentis is 2.59 ⁇ 10 5 -4.49 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium bifidum is 1.26 ⁇ 10 5 –7.35 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium longum is 2.23 ⁇ 10 5 –7.02 ⁇ 10 11 CFU; and/or the amount of Lactobacillus rhamnosus is 1.26 ⁇ 10 5 –2.59 ⁇ 10 11 CFU.
  • the probiotic composition is for administration to children, and the amount of Bifidobacterium adolescentis is 2.05 ⁇ 10 5 -4.55 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium bifidum is 1.47 ⁇ 10 5- 3.6 ⁇ 10 11 CFU; and/or the amount of Bifidobacterium longum is 7.55 ⁇ 10 4 -2.5 ⁇ 10 11 CFU; and/or the amount of Lactobacillus rhamnosus is 1.47 ⁇ 10 5 -3.6 ⁇ 10 11 CFU.
  • the probiotic composition does not contain other probiotics other than the probiotics described in the various embodiments of the present application.
  • free should be understood as “essentially free”, and it does not exclude the presence of traces or traces of other probiotics due to factors such as strain cultivation, isolation, and purification.
  • the amount of other probiotics does not exceed 5% of the total probiotics of the composition, and preferably does not exceed 1%.
  • the probiotic composition does not contain probiotics other than Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus. In some embodiments, the probiotic composition does not contain bifidobacteria other than Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium longum.
  • the present application provides a prebiotic composition comprising xylo-oligosaccharides, galacto-oligosaccharides and corn dietary fiber.
  • prebiotic composition refers to a composition that uses prebiotics as the active ingredient, and does not exclude the presence of auxiliary ingredients introduced due to the synthesis, separation, and purification of prebiotics and/or auxiliary materials for formulating the composition according to the desired purpose Element.
  • the ratio of the amounts of xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber is (0.25-5): (0.75-4): (0.5-1) by weight. In some embodiments, the ratio of the amounts of xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber is (0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1) by weight.
  • the prebiotic composition is in a unit dosage form, and by weight, the total amount of xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber is 0.1-12 g, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12g.
  • the prebiotic composition is in a unit dosage form, and the amount of xylo-oligosaccharides is 0.01g-6g; and/or the amount of galacto-oligosaccharides is 0.04g-9.6g; and/or corn dietary fiber The amount is 0.01g-6g.
  • Such unit dosage forms can be administered to adults or children.
  • the total amount of xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber can be controlled to be 0.1-5 g, which is the total amount after being combined with food It is expected that the desired value can be achieved, for example, about 12 g.
  • the prebiotic composition does not contain prebiotic components other than xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber.
  • free should be understood as “essentially free”, which does not exclude the presence of trace or trace amounts of other prebiotics due to factors such as the synthesis, extraction, separation, and purification of prebiotics.
  • the amount of other prebiotics does not exceed 5% of the total prebiotics of the composition, and preferably does not exceed 1%.
  • the present application provides a dietary composition (sometimes also referred to as a "synbiotics" composition), comprising the probiotic composition of the first aspect and the prebiotic composition of the second aspect.
  • a dietary composition sometimes also referred to as a "synbiotics" composition
  • a dietary composition does not need to be separately formulated with the probiotic composition of the first aspect and the prebiotic composition of the second aspect, and then the two are mixed or combined.
  • a dietary composition covers all the features of one embodiment of the probiotic composition of the first aspect and all the features of one embodiment of the prebiotic composition of the second aspect, it belongs to the dietary composition of the third aspect of the present application .
  • the dietary composition of the present application may have the following formula (daily dosage, which may be provided in unit dosage form):
  • the dietary composition comprises Bifidobacterium adolescentis, Bifidobacterium bifidum and Bifidobacterium longum as probiotics, and xylooligosaccharides, galacto-oligosaccharides and corn dietary fiber as prebiotics, wherein In terms of colony forming units (CFU), the ratio of the amount of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium and Bifidobacterium longum is (0.75-1):1:(0.75-1), and the three bacteria total about 2 ⁇ 10 11 CFU, and by weight, the ratio of xylo-oligosaccharide, galacto-oligosaccharide and corn dietary fiber is (0.25-0.5): (2-4): (0.5-0.75) and the ratio of xylo-oligosaccharide, low
  • CFU colony forming units
  • the probiotic composition or prebiotic composition or dietary composition is formulated for oral administration.
  • oral administration includes oral administration, mixing with oral products, tube feeding, and the like.
  • the probiotic composition or prebiotic composition or dietary composition is a food supplement, food additive or food.
  • the probiotic composition or prebiotic composition or dietary composition is formulated as a powder, granule, tablet or capsule.
  • the main application mode of the probiotic composition or prebiotic composition or dietary composition of the present application is to administer the gastrointestinal tract of an individual.
  • Direct oral administration is a more convenient way, but for some special individuals (such as bedridden patients), the administration can also be assisted by means such as tube feeding.
  • the product form of the probiotic composition or prebiotic composition or dietary composition of the present application can be various, for example, can be prepared as a separate dietary supplement (such as capsules, tablets, powders, granules), with meals or It is not taken with meals; it can also be prepared as various solid/semi-solid foods, prepared powder/granular foods, beverages and other additive products that are added or formulated before being ingested by the individual; it can also be used as various solid/semi-solid foods, Reconstitute the direct components of powder/granule foods and beverages.
  • a separate dietary supplement such as capsules, tablets, powders, granules
  • the probiotic composition or prebiotic composition or dietary composition is administered to an individual to assist in the prevention and/or treatment of pathogen infection, or to enhance the therapeutic effect of pathogen infection , Or improve the individual's immunity, or balance the individual's intestinal microecology (including increasing the abundance of microorganisms, increasing the ideal bacterial species and/or reducing the undesirable bacteria).
  • the pathogen is a virus, bacteria or fungus.
  • the pathogen is a respiratory disease virus, such as COVID-19, influenza, and respiratory syncytial virus.
  • this application provides the probiotic composition of the first aspect, or the prebiotic composition of the second aspect, or the dietary composition of the third aspect in preparation for assisting in the prevention and/or treatment of pathogen infection or Use in dietary products or medicines that enhance the therapeutic effect of an individual's pathogen infection or improve the individual's immunity or balance the individual's intestinal microecology (including increasing microbial abundance, increasing ideal bacterial species and/or reducing undesirable bacteria).
  • the pathogen is a virus, bacteria or fungus.
  • the pathogen is a respiratory disease virus, such as COVID-19, influenza, and respiratory syncytial virus.
  • the preparation of the probiotic composition or prebiotic composition or dietary composition of the present application can refer to the conventional processing methods of probiotics or prebiotic products in the art.
  • various probiotics or prebiotic ingredients can be mixed into the product sequentially or simultaneously or as a frozen intervention mixture by conventional processing techniques.
  • this application provides methods for assisting in the prevention and/or treatment of individual pathogen infections or enhancing the therapeutic effect of individual pathogen infections or enhancing the individual’s immunity or balancing the individual’s intestinal microbiota (including increasing microbial abundance,
  • the method of increasing ideal bacterial species and/or reducing undesirable bacteria) comprises administering the probiotic composition of the first aspect, or the prebiotic composition of the second aspect, or the dietary composition of the third aspect to the individual.
  • the pathogen is a virus, bacteria or fungus.
  • the pathogen is a respiratory disease virus, such as COVID-19, influenza, and respiratory syncytial virus.
  • This example describes the first phase cohort study conducted by the inventor.
  • the inventor publicly recruited 546 healthy Hong Kong adults. The study has been approved by the Joint Committee of Clinical Research Ethics of the Chinese University of Hong Kong-New Territories East Hospital Network (The Joint CUHK-NTEC CREC, CREC Number: 2016.707). All subjects signed written informed consent, donated stool samples, and provided demographic information through questionnaire surveys. The stool samples of the subjects were stored at -80°C for bacterial group analysis.
  • RSC PureFood GMO and Authentication Kit extract fecal DNA. Approximately 100 mg of each stool sample was washed with 1 ml ddH 2 O in advance and centrifuged at 13000 g for 1 minute. The pellet was resuspended in 800 ⁇ LTE buffer (pH 7.5), 1.6 ⁇ l 2-mercaptoethanol and 500U lyase (Sigma) were added, and incubated at 37°C for 60 minutes. The sample was then centrifuged at 13000g for 2 minutes, and the supernatant was discarded. After pretreatment, then use RSC PureFood GMO and Authentication Kit (Promega), extract DNA according to product instructions.
  • DNeasy PowerSoil kit (QIAGEN) was used to extract fecal DNA. Extract DNA from 0.1g stool sample, and then use Qubit dsDNA BR kit (Thermo Fisher Scientific) to determine the concentration of extracted DNA. The DNA samples are sent to a sequencing service provider (Novogene HK Company Limited, Wanchai, Hong Kong) for library preparation and paired shotgun metagenomic sequencing (Illumina NovaSeq 6000). Each sample returns an average of 7.5GB of original data.
  • QIAGEN DNeasy PowerSoil kit
  • Ideal bacterial species used for correlation analysis include: Akkermansia muciniphila, Alipis onderdonkii, Anaerostipes hadrus, Bacteroides dorei, Bacteroides massiliensis, Bacteroides ovatus, Bacteroides ovatus, Bacteronides thetaiotao Bifidobacterium pseudocatenulatum, Eubacterium limosum, Eubacterium rectal, Eubacterium ventriosum, Faecalibacterium prausnitzii, Roseburia hominis, intestinal tract Roseburia intestinalis, Eubacterium hallii (Eubacterium hallii).
  • Use Trimmomatic (v0.38) default parameters for quality filtering and trimming of metagenomics readings. Then, the host DNA (reference genome: hg38) was removed by Kneaddata (v0.7.2, https://bitbucket.org/biobakery/kneaddata/wiki/Home). Use MetaPhlAn26 (v2.6.0) for species-level metagenomic annotation. Then, the relative abundance generated by MetaPhlAn2 is transformed by the centered log ratio (clr). Use the R package corrplot v0.78 to calculate and plot the Pearson correlation coefficient.
  • the inventors calculated the average relative abundance of Bifidobacterium adolescentis, Bifidobacterium bifidum and Bifidobacterium longum in adults and children in study cohorts 1-3.
  • the proportions of these probiotics are based on the natural proportions of various probiotics in healthy people, and their proportions are relatively fixed among about 1500 healthy Chinese people in the study cohort 1-3. Therefore, simulating the proportion of bacterial species in healthy people may increase the chance of bacterial species colonizing in the intestine.
  • the prebiotics selected by the inventors include xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber.
  • Xylooligosaccharides also known as xylo-oligosaccharides, refer to functional oligosaccharides composed of 2-10 xylose molecules connected by ⁇ -1,4 glycosidic bonds. Xylooligosaccharides are excellent proliferation factors for bifidobacteria. Xylooligosaccharides have very obvious effects on the proliferation of Bifidobacterium bifidum and Bifidobacterium adolescentis.
  • Bifidobacterium adolescentis, Bifidobacterium infantis, and Bifidobacterium bifidum all utilize xylo-oligosaccharides by producing xylosidase and arabinosidase, and their ability to hydrolyze xylo-oligosaccharides depends on their xylanase.
  • the efficiency of the carbohydrase digestion system The effect of xylo-oligosaccharides on the proliferation of bifidobacteria and the yield of short-chain fatty acids after fermentation decreased with the increase of the molecular weight of xylo-oligosaccharide components.
  • Xylooligosaccharides have the characteristics of acid resistance, high temperature resistance, strong stability, and good compatibility, and can be well used in food.
  • Galacto-oligosaccharide and corn dietary fiber can promote the growth of a variety of bifidobacteria.
  • Galactooligosaccharide is a new type of functional substance, and its molecular structure is generally connected with 1-7 galactosyl groups on the galactose or glucose molecule. It is a kind of functional oligosaccharide with natural properties. Its palatability, water solubility and stability are good, and after entering the human body, it can proliferate the probiotic bacteria in the human intestine, especially bifidobacteria, and can also inhibit the growth of spoilage bacteria.
  • the probiotics in the intestines use galacto-oligosaccharides to proliferate while also producing large amounts of extracellular polysaccharides.
  • Exopolysaccharide not only has anti-tumor activity and immune activity, but also promotes the long-term colonization of probiotics in the intestine.
  • the corn dietary fiber can absorb part of the water and promote the intestinal peristalsis to accelerate the excretion of feces, thereby reducing the pressure of the rectum and preventing and reducing intestinal diseases.
  • bifidobacteria have a fermentative effect on corn dietary fiber.
  • Corn dietary fiber can be rapidly fermented by microorganisms in the cecum to produce short-chain fatty acids.
  • the inventors further gave the appropriate proportions of three prebiotics, among which the proportions of xylo-oligosaccharides, galacto-oligosaccharides, and corn dietary fiber can be (0.25-5): (0.75-4): (0.5-1).
  • This embodiment describes an expanded second-stage cohort study conducted by the inventor based on the research cohort 2 of embodiment 1.
  • Study cohort HC (ie, study cohort 2 of Example 1)
  • the QIAamp DNeasy PowerSoil kit was used for fecal DNA extraction.
  • the inventor publicly recruited 219 healthy Hong Kong adults. The study has been approved by the Joint Committee of Clinical Research Ethics of the Chinese University of Hong Kong-New Territories East Hospital Network (The Joint CUHK-NTEC CREC, CREC Number: 2017.369). All subjects signed written informed consent, donated stool samples, and provided demographic information through questionnaire surveys. The stool samples of the subjects were stored at -80°C for bacterial group analysis. According to the manufacturer's instructions, use the QIAamp DNA Stool Mini kit for fecal DNA extraction.
  • the inventors included 78 stool samples of healthy Hong Kong adults from a healthy control group in a COVID-19 study. According to the manufacturer's instructions, use Maxwell RSC PureFood GMO and Authentication kits for fecal DNA extraction.
  • the stool samples of these subjects were stored at -80°C for microbiome analysis. According to the manufacturer's instructions, use Maxwell RSC PureFood GMO and Authentication kits for fecal DNA extraction.
  • This example includes stool samples from healthy subjects collected from five independent cohort studies. According to the manufacturer's instructions, use QIAamp DNeasy PowerSoil kit separation kit, Maxwell RSC PureFood GMO and Authentication kit or QIAamp DNA Stool Mini kit for fecal DNA extraction. Use NanoDrop spectrophotometer and gel electrophoresis to determine the quality and quantity of DNA.
  • the DNA library is constructed through the process of end repair, purification and PCR amplification. After constructing the DNA library, the NextSeq platform in the inventor's laboratory used a 150bp paired-end sequencing strategy to sequence the DNA library. On average, each sample can obtain 12Gb data for further analysis. All experimental procedures are in accordance with the unified standards of the inventor's laboratory.
  • the inventors used Fastp to perform quality filtering on the sequencing fragments of the metagenome, PolyG tail modification and adaptor modification, and deleted sequencing fragments of 50 bases or less. Then use KneadData to remove the human genes in the sequencing fragments of the quality trimmed metagenomics, and then use MetaPhlAn2 to analyze the metagenomics at the species level. All non-zero levels will be regarded as positive levels.
  • the inventor calculated the incidence of each species and its combination.
  • the inventors used Pearson correlation analysis to explore the correlation between species level and age and gender. The correlation between species level and age was analyzed by Pearson correlation, and the correlation between species level and gender was evaluated by Spearman correlation.
  • the inventor further analyzed the correlation between the levels of the three bifidobacteria species and age and sex. The results show that age growth is negatively correlated with these three types of bifidobacteria. There is a significant correlation between male sex and low levels of Bifidobacterium longum (Table 2). These results suggest that most people are suitable for supplementing the probiotic/prebiotic/synbiotic composition of the present application, especially the older people and the male population.
  • This example describes the use of the synbiotic composition of the present application to improve the symptoms of hospitalized COVID-19 patients and regulate immune response markers.
  • the coronavirus disease-2019 (COVID-19) caused by the SARS-CoV-2 virus not only targets the lungs, but also targets multiple other organs, including the intestines. Intestinal microbes can regulate the host's immune response, so it may affect the severity and prognosis of COVID-19 patients.
  • the gut microbiome of COVID-19 patients becomes imbalanced, such as the reduction of symbiota and the increase of opportunistic pathogens, which are all related to the severity of COVID-19 and the shedding of fecal SARS-CoV-2 virus.
  • SARS-CoV-2 is less infectious when the content of probiotics produced by short-chain fatty acids in stool samples is higher, which highlights the potential beneficial effects of beneficial bacteria in combating SARS-CoV-2 infection.
  • a treatment plan that rebalances the gut microbiome of COVID-19 patients may improve clinical outcomes.
  • the inventor expects that the synbiotic composition of the present application can improve the clinical symptoms of COVID-19 patients, and therefore designed and conducted a preliminary study to evaluate the effect of this synbiotic composition on COVID-19 in hospitalized COVID-19 patients. 19 clinical symptoms, blood immune markers and the influence of fecal microbiome. The inventors compared these results with COVID-19 hospitalized patients (control group) who received standard treatment during the same period.
  • the formula of the synbiotic composition used in this study is as follows:
  • the synbiotic composition contains Bifidobacterium adolescentis, Bifidobacterium bifidum and Bifidobacterium longum as probiotics, and xylo-oligosaccharides, galacto-oligosaccharides and corn dietary fiber as prebiotics, in which colony forming units (CFU ), the ratio of the amount of Bifidobacterium adolescentis, Bifidobacterium bifidobacterium and Bifidobacterium longum is controlled as (0.75-1):1:(0.75-1), and the total control of the three bacteria is about 2 ⁇ 10 11 CFU, and by weight, the ratio of xylo-oligosaccharides, galacto-oligosaccharides and corn dietary fiber is controlled to (0.25-0.5): (2-4): (0.5-0.75) The total amount of lactose and corn dietary fiber is controlled at 1.2-1.5g.
  • CFU colony
  • the enrolled COVID-19 patients will receive standard treatment or the synbiotic composition of this application within 48 hours of admission.
  • Subjects will receive standard treatment or take synbiotic composition capsules for 28 days.
  • the main result is a comprehensive evaluation of the following three indicators: the overall symptom questionnaire assesses whether the symptoms are relieved, whether respiratory support is required, and the antibody production from the start of treatment to the fifth week.
  • Questions 1 to 19 and 26 of the General Symptom Questionnaire evaluate COVID-19-related symptoms including fever, respiratory symptoms and general symptoms. The lowest score is 20 (normal) and the highest score is 80 (most symptoms). Severe), the score for complete symptom relief is 20 points. Questions 20 to 25 of Table 3 evaluate gastrointestinal (GI) symptoms, and the score for complete remission of GI symptoms is 6 points.
  • GI gastrointestinal
  • the SARS-CoV-2 immunoglobulin G (IgG) antibody was tested within 2 weeks after admission.
  • the inventor will evaluate the patient's clinical symptoms every two days until the symptoms disappear or the patient is discharged from the hospital.
  • the inventor also included another group of patients who received standard treatment at the same time into the study as a comparison.
  • the inventor collected blood samples at baseline and 5 weeks after taking the synbiotic composition, and used
  • the immunomultiplex analysis method detected the immune response markers in the baseline and 5th week plasma of the standard treatment group and the synbiotic group.
  • the inventor also collected stool and quality of life questionnaires at baseline, 2, 4, and 5 weeks after taking the synbiotic composition.
  • Subjects receiving intensive care or using ventilators are allergic or intolerant to intervention products or their components, have a known history of endocarditis or active endocarditis, have recently received CAPD or hemodialysis, or are recruited All pregnant subjects were excluded.
  • Subjects suffering from any diseases that prevent oral probiotics or increase the risks associated with probiotics are also excluded. These risks include, but are not limited to, inability to swallow or risk of aspiration and no other method of administration (e.g., no G/ J tube), known to increase the risk of infection due to immunosuppression, such as organ or hematopoietic stem cell transplant history, neutropenia (ANC ⁇ 500 cells/ ⁇ l) or HIV and CD4 ⁇ 200 cells/ ⁇ l.
  • Subjects who were hospitalized between August 2020 and October 2020 were assigned to the synbiotic composition group or standard treatment group. Subjects discharged from the hospital in July 2020 will be assigned to the standard treatment group.
  • Stool samples and quality of life questionnaires were collected at baseline and the first synbiotic composition or at 2, 4, and 5 weeks after enrollment. This research was conducted in accordance with the "Declaration of Helsinki".
  • IL-6 interleukin
  • TNF- ⁇ tumor necrosis factor
  • M -CSF macrophage colony stimulating factor
  • CXCL-10 CXC chemokine ligand 10
  • MCP-1 monocyte chemoattractant protein 1
  • MIG interferon- ⁇ -induced mononuclear factor
  • the inventors discovered for the first time that the intestinal tract of COVID-19 patients lacked a series of beneficial bacteria and after the SARS-CoV-2 virus was cleared from the respiratory tract, the activity of viral infection and replication continued in the intestinal tract.
  • the inventor Using big data analysis and machine learning, the inventor has developed a probiotic formula that targets the imbalance of the intestinal microecology.
  • the abundance of probiotics increased significantly at the second week, which confirmed that the probiotics had been successfully delivered to the intestinal tract.
  • Studies have shown that the levels of immune response markers in severely ill patients with COVID-19 are elevated. These markers include IL-6, IL-1RA, IL-18, TNF- ⁇ , M-CSF, CXCL10, MCP-1, and MIG.
  • the synbiotic composition can reduce the levels of the above eight immune response markers in the 5th week plasma sample.
  • the patient's COVID-19 symptoms were also relieved in the 2nd and 5th weeks after the treatment of the synbiotic composition.
  • the treatment of the synbiotics composition may enhance the host's immune response to SARS-CoV2, which is mainly manifested in suppressing the cytokines that increase in the early stage of COVID-19 infection.
  • This result indicates that the synbiotics targets the intestinal flora.
  • the treatment provided the basis.
  • the inventor's research suggests that the synbiotics have the effect of early immune intervention, which provides reference and hope for the application of the synbiotics composition to enhance the body's immunity against COVID-19 and other emerging virus infections.
  • Example 3 This example is an extended study conducted on the basis of Example 3.
  • the inventor included 25 COVID-19 patients taking the synbiotic composition and 10 patients receiving standard treatment from Example 3.
  • the inventor also included 69 patients receiving standard treatment and 78 healthy people as a control group.
  • metagenomics analysis we found that the synbiotic formulation of the present application allows the gut microflora imbalance back into balance, and at or near normal levels.
  • RT-qPCR quantitative reverse transcription polymerase chain reaction
  • the inventors collected stool samples of the synbiotic composition and subjects in the standard treatment group at different time points (baseline, 2, 4, and 5 weeks). In addition, the inventor collected a stool sample at a time point from a healthy control group.
  • the DNA library was constructed through the process of end repair, purification and PCR amplification. After constructing the DNA library, the NextSeq platform in the inventor's laboratory used a 150bp paired-end sequencing strategy to sequence the DNA library. On average, each sample can obtain 12Gb data for further analysis. All experimental procedures are in accordance with the unified standards of the inventor's laboratory.
  • the species with negatively correlated SARS-CoV-2 viral load is defined as "ideal bacterial species" (relevant studies are recorded in U.S. Provisional Patent Applications 63/016,759 and 63/025,310).
  • the species with relatively high abundance in the intestines of COVID-19 patients is defined as "undesirable bacterial species.”
  • Use LEfSe software linear discriminant analysis [LDA] effect size) to analyze differences between microbial groups. Species with LDA greater than 2 and p ⁇ 0.05 were considered to be significantly different between groups.
  • COVID-19 patients receiving the synbiotic composition include Bifidobacterium adolescentis, Eubacterium rectum, Rumenococcus and Bifidobacterium longum and other ideal bacterial species are significantly more abundant ( Figure 8).

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Abstract

一种包含两岐双岐杆菌（Bifidobacterium bifidum）和长双岐杆菌（Bifidobacterium longum）的益生菌组合物，可以进一步包含青春双歧杆菌（Bifidobacterium adolescentis）和鼠李糖乳杆菌（Lactobacillus rhamnosus）。一种包含低聚木糖、低聚半乳糖和玉米膳食纤维的益生元组合物，以及一种包含上述益生菌组合物和益生元组合物的膳食组合物。一种上述组合物在制备用于辅助预防和/或治疗个体的病原体感染或增强个体的病原体感染的治疗效果或提高个体的免疫力或平衡个体的肠道微生态的膳食产品或药品中的用途。所述组合物可以用于COVID-19患者。

Description

用于提高免疫力的组合物
相关申请
本申请要求中国专利申请202010657312.5、202011259564.9和202110223880.9号的优先权，通过援引加入的方式将三篇专利文献的整体内容并入本文。本申请还要求2020年4月28日提交的美国临时申请63/016,759号、2020年5月15日提交的美国临时申请63/025,310号和2020年8月12日提交的美国临时申请63/064,821号的优先权，通过援引加入的方式将三篇美国临时专利申请的整体内容并入本文。
技术领域
本申请大体涉及药品、食品、保健品领域。具体而言，本申请提供了基于对于肠道菌群的调节而提高个体免疫力或辅助治疗和预防疾病或提高疾病疗效的组合物及其用途。
背景技术
人体或动物体的肠道菌群中存在种类多样、组成复杂的细菌，并且对于肠道菌群的研究报道也很多。肠道菌群不仅仅与消化功能相关，而且对于机体抵抗病原体感染、自体免疫疾病等疾病的能力，甚至对于药物治疗的反应均有关联性。
肠道菌群中有一类对于机体有益的细菌，被称为益生菌。通常，益生菌指定殖在人体内，改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。益生菌能通过调节宿主粘膜与系统免疫功能或通过调节肠道内菌群平衡，促进营养吸收保持肠道健康的作用，从而产生有利于健康作用。常见的益生菌包括双歧杆菌、乳杆菌、酵母菌等。
有一些物质与益生菌关系密切，被称为益生元。通常，益生元指一些不被宿主消化吸收却能够选择性地促进体内益生菌的代谢和增殖，从而改善宿主健康的有机物质。一般情况下，益生元应在通过上消化道时，大部分不被消化而能被肠道菌群所发酵的。最重要的是益生元能刺激有益菌群的生长，而不刺激有潜在致病性或腐败活性的有害细菌。常见的益生元有低聚糖类，也称为膳食纤维。
基于益生菌/益生元调节肠道菌群，进而促进机体健康水平(例如提高免疫力)是本领域一直以来的课题。
发明概述
第一方面，本申请提供了一种益生菌(probiotics)组合物，包含两岐双岐杆菌(Bifidobacterium bifidum)和长双岐杆菌(Bifidobacterium longum)。
在一些实施方案中，以菌落形成单位计，两岐双岐杆菌和长双岐杆菌的量的比值为1：(0.21-2.36)。
在一些实施方案中，益生菌组合物还包含青春双歧杆菌(Bifidobacterium adolescentis)。
在一些实施方案中，以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.57-3.56)：1：(0.21-2.36)。在一些具体方案中，以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.75-1)：1：(0.75-1)。
在一些实施方案中，益生菌组合物还包含鼠李糖乳杆菌(Lactobacillus rhamnosus)。
在一些实施方案中，以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量的比值为(0.57-3.36)：1：(0.21-2.36)：1。
在一些实施方案中，益生菌组合物为单位剂量形式，并且以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量独立地为10 4至10 12CFU的量级。在一些实施方案中，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的总量为10 6至10 12CFU的量级。
在一些实施方案中，所述益生菌组合物用于给予成年人，并且青春双歧杆菌的量为2.59×10 5-4.49×10 11CFU；和/或两岐双岐杆菌的量为1.26×10 5–7.35×10 11CFU；和/或长双岐杆菌的量为2.23×10 5–7.02×10 11CFU；和/或鼠李糖乳杆菌的量为1.26×10 5–2.59×10 11CFU。
在一些实施方案中，益生菌组合物用于给予儿童，并且青春双歧杆菌的量为2.05×10 5–4.55×10 11CFU；和/或两岐双岐杆菌的量为1.47×10 5–3.6×10 11CFU；和/或长双岐杆菌的量为7.55×10 4–2.5×10 11CFU；和/或鼠李糖乳杆菌的量为1.47×10 5–3.6×10 11CFU。
在一些实施方案中，益生菌组合物不含本申请的各个实施方案所写明的益生菌之外的其他益生菌。在一些实施方案中，益生菌组合物不含青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌之外的益生菌。在一些实施方案中，益生菌组合物不含青春双歧杆菌、两岐双岐杆菌、长双岐杆菌之外的双歧杆菌。
第二方面，本申请提供了一种益生元(prebiotics)组合物，包含低聚木糖、低聚半乳糖和玉米膳食纤维。
在一些实施方案中，以重量计，低聚木糖、低聚半乳糖，玉米膳食纤维的量的比值为(0.25-5)：(0.75-4)：(0.5-1)。在一些具体实施方案中，以重量计，低聚木糖、低聚半乳糖，玉米膳食纤维的量的比值为(0.25-0.5)：(2-4)：(0.5-0.75)。
在一些实施方案中，益生元组合物为单位剂量形式，并且以重量计，所述低聚木糖、低聚半乳糖，玉米纤维的总量为0.1-12g。在一些实施方案中，益生元组合物为单位剂量形式，并且以重量计，所述低聚木糖、低聚半乳糖，玉米纤维的总量为0.1-5g。
在一些实施方案中，低聚木糖的量为0.01g-6g；和/或低聚半乳糖的量为0.04g-9.6g；和/或玉米膳食纤维的量为0.01g-6g。
在一些实施方案中，益生元组合物不包含低聚木糖、低聚半乳糖，玉米膳食纤维之外的益生元组分。
第三方面，本申请提供了一种膳食组合物(一些情况下也称为“合生元(synbiotics)”组合物)，包含第一方面的益生菌组合物和第二方面的益生元组合物。
在一些具体实施方案中，膳食组合物包含作为益生菌的青春双歧杆菌、两岐双岐杆菌和长双岐杆菌以及作为益生元的低聚木糖、低聚半乳糖和玉米膳食纤维，其中以菌落形成单位(CFU)计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.75-1)：1：(0.75-1)，并且三种菌总计约2×10 11CFU，并且以重量计，低聚木糖、低聚半乳糖和玉米膳食纤维的比为(0.25-0.5)：(2-4)：(0.5-0.75)并且低聚木糖、低聚半乳糖和玉米膳食纤维的总量为1.2-1.5g。
在第一至第三方面的一些实施方案中，所述益生菌组合物或益生元组合物或膳食组合物被配制为用于经口施用。在一些实施方案中，经口施用包括口服、与口服类产品混合、管饲等方式。
在第一至第三方面的一些实施方案中，所述益生菌组合物或益生元组合物或膳食组合物为食品补充剂、食品添加剂或食品。
在第一至第三方面的一些实施方案中，所述益生菌组合物或益生元组合物或膳食组合物被配制为粉剂、颗粒剂、片剂或胶囊剂。
在第一至第三方面的一些实施方案中，所述益生菌组合物或益生元组合物或膳食组合物施用于个体，用于辅助预防和/或治疗病原体感染、或增强病原体感染的治疗效果、或提高个体的免疫力、或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)。在一些实施方案中，病原体为病毒、细菌或真菌。在一些实施方案中，病原体为呼吸道疾病病毒，例如新冠病毒(COVID-19)、流行性感冒、呼吸道合胞病毒。
第四方面，本申请提供了第一方面的益生菌组合物、或第二方面的益生元组合物、或第三方面的膳食组合物在制备用于辅助预防和/或治疗个体的病原体感染或增强个体的病原体感染的治疗效果或提高个体的免疫力或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)的膳食产品或药品中的用途。在一些实施方案中，病原体为病毒、细菌或真菌。在 一些实施方案中，病原体为呼吸道疾病病毒，例如COVID-19、流行性感冒、呼吸道合胞病毒。
第五方面，本申请提供了用于辅助预防和/或治疗个体的病原体感染或增强个体的病原体感染的治疗效果或提高个体的免疫力或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)的方法，包括向所述个体给予第一方面的益生菌组合物、或第二方面的益生元组合物、或第三方面的膳食组合物。在一些实施方案中，病原体为病毒、细菌或真菌。在一些实施方案中，病原体为呼吸道疾病病毒，例如COVID-19、流行性感冒、呼吸道合胞病毒。
附图简要说明
图1显示了前期研究鉴定的理想细菌物种和各种益生菌之间的关系。理想细菌物种有增强免疫力的潜在能力，并且其丰度与COVID-19疾病严重程度或SARS-CoV-2病毒载量呈负相关。圆圈代表理想细菌物种和益生菌的丰度呈正相关，大小指示正相关的强度，方框圈出本申请鉴定得出的目标益生菌。
图2显示了实施例2的队列研究的部分结果，其中A图显示了青春双歧杆菌、两歧双歧杆菌和长双歧杆菌在各个队列研究中的阳性率及总体阳性率；B图显示了受试者的粪便样品包含三种双歧杆菌，三种双歧杆菌中的任何两种、或三种双歧杆菌中仅一种或不包含任何一种的发生率。
图3显示了实施例3的临床研究中合生元组合物和标准治疗的COVID-19患者的部分结果，其中A图显示了第1周和第2周的临床症状缓解评分(评分定义为20)以及抗体形成情况；B图显示了血浆中免疫反应标志物的定量(转换为log10显示)，对于所有箱形图，中心都是通过测量的中值绘制的，而箱形图的上下边界分别对应于第一和第三百分位数。p值由两面确定，p值<0.05被认为具有统计学显著性(Wilcoxon秩和检验)；C图显示了与基线相比，第5周的炎性免疫反应标志物的百分比下降量，各个条形表示中位数百分比下降量，p值由双面Wilcoxon秩和检验确定；D图显示了在基线、首次服用合生元组合物的2周和5周后的益生菌浓度，浓度通过qPCR确定，并在log10转化后显示(ng/μl)，p值<0.05被认为具有统计学显著性(Wilcoxon秩和检验)。
图4显示了实施例4的研究设计方案。
图5显示了实施例4中健康人以及在合生元组合物组和标准治疗组的COVID-9患者在基线、第2、第4和第5周的合生元组合物中的3种双歧杆菌属益生菌(青春双歧杆菌、两岐双岐杆菌和长双 岐杆菌)的总相对丰度，其中上方的图为汇总图，下方的图为各个时间点的图。相对丰度(百分比)以转换成log10的方式显示。第2、第4和第5周与基线相对丰度间的p值由Wilcoxon秩和检验确定。
图6显示了实施例4中健康人以及在合生元组合物组和标准治疗组的COVID-9患者在基线、第2、第4和第5周的香农多样性指数。第2、第4和第5周与基线香农多样性指数间的p值由Wilcoxon秩和检验确定。
图7显示了实施例4中的健康人以及在合生元组合物组和标准治疗组的COVID-9患者在基线、第2、第4和第5周的理想细菌物种(A)和不理想细菌物种(B)的总相对丰度，其中A图显示了理想细菌物种(在非COVID-19人中丰度较高的物种)的总相对丰度，B图显示了不理想细菌物种(在COVID-19患者中丰度较高)的总相对丰度。相对丰度(百分比)以转换成log10的方式显示。第2、第4和第5周与基线相对丰度间的p值由Wilcoxon秩和检验确定。
图8显示了实施例4中合生元组合物组和标准治疗组之间在基线、第2、第4和第5周时丰度有差异的物种(LDA>2,p<0.05)。与标准治疗组相比，合生元组合物组中多种理想细菌物种(方框标记)水平显著更高，而在合生元组合物组中的不理想细菌物种(肺炎克雷伯菌、小韦荣球菌和大肠杆菌)，则明显低于标准治疗组。方块色度与LDA数值对应关系如图解。若LDA值为正，则表示该物种在合生元组合物治疗组水平显著更高，更深的颜色代表更大差异。若LDA值为负，则表示该物种在标准治疗组水平显著更高，更浅的颜色代表更大差异。
发明详细描述
本申请的发明人对于肠道菌群、特别是益生菌进行了大量深入的研究，并发现益生菌与可增强免疫力并可促进预防和治疗病原体感染(例如呼吸道感染)的理想细菌物种呈显著正相关，从而基于上述发现，提供了益生菌/益生元/合生元组合物，预期这些组合物能有效增强机体免疫力，并且有助于预防和/或治疗病原体感染，例如呼吸道病原体感染，如新型冠状病毒(COVID-19)，流行性感冒，呼吸道合胞病毒等。
在下文中，根据一些具体实施方案来进一步阐述本申请的内容。然而，所列举的具体实施方案仅出于例示目的，而不旨在限制本申请的范围。本领域技术人员会认识到，以下某一实施方案中的具体特征可以用于任何其他实施方案，只要其不背离本申请的主旨即可。
除非另外说明，本申请中的术语的含义与本领域技术人员通 常理解的含义相同。本文所引用的所有专利文献、学术论文及其他公开出版物，其中的全部内容整体并入本文作为参考。
应当理解，本文中给出的具体数值不仅可作为单独的数值理解，还应当认为提供了某一范围的端点值，并且可以相互组合提供其他范围。例如，当公开了组合物的某组分的含量为1、2或3g时，相当于还公开了该组分的含量可以为1-2g、1-3g或2-3g。
第一方面，本申请提供了一种益生菌组合物，包含两岐双岐杆菌(Bifidobacterium bifidum)和长双岐杆菌(Bifidobacterium longum)。
本文所述的“益生菌组合物”指以益生菌作为活性成分的组合物，不排除存在由于益生菌的培养、分离和纯化所需要的辅助成分和/或根据希望目的配制组合物的辅料成分。
在一些实施方案中，以菌落形成单位计，两岐双岐杆菌和长双岐杆菌的量的比值为1：(0.21-2.36)。
菌落形成单位(CFU)是本领域常见的表征微生物的量的形式。除非另作规定，本申请中描述微生物的量时以菌落形成单位计算。
例如，两岐双岐杆菌和长双岐杆菌的量的比值为可以为1：(0.21、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.36)。在一些实施方案中，组合物用于给予成年人，两岐双岐杆菌和长双岐杆菌的量的比值为1：(0.36-2.36)。在一些实施方案中，组合物用于给予儿童，两岐双岐杆菌和长双岐杆菌的量的比值为1：(0.21-1.7)。
在一些实施方案中，益生菌组合物还包含青春双歧杆菌(Bifidobacterium adolescentis)。
在一些实施方案中，以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.57-3.56)：1：(0.21-2.36)。在一些实施方案中，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.57、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.56)：1：(0.21、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.36)。在一些具体方案中，以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.75-1)：1：(0.75-1)。在一些实施方案中，组合物用于给予成年人，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(1-3.56)：1：(0.86-2.36)。在一些实施方案中，组合物用于给予儿童，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.57-3.09)：1：(0.21-1.7)。
在一些实施方案中，益生菌组合物还包含鼠李糖乳杆菌(Lactobacillus rhamnosus)。短链脂肪酸(SCFA，例如丁酸和丙酸)会影响T细胞、巨噬细胞和树突状细胞的分化或功能，这对于维持免疫稳态具有意义。鼠李糖乳杆菌能增加肠道中SCFA的产量，鼠李糖乳杆菌与两歧双歧杆菌和长双歧杆菌中的组合预期能增加总的SCFA产生量。此外据报道，口服鼠李糖乳杆菌可以增加肠道中拟杆菌属和费氏杆菌属的含量。
在一些实施方案中，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量的比值为(0.57-3.56)：1：(0.21-2.36)：1。在一些实施方案中，以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量的比值为(0.57、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.56)：1：(0.21、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.36)：1。在一些实施方案中，组合物用于给予成年人，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量的比值为(1-3.56)：1：(0.86-2.36)：1。在一些实施方案中，组合物用于给予儿童，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量的比值为(0.57-3.09)：1：(0.21-1.7)：1。
在一些实施方案中，益生菌组合物为单位剂量形式，并且以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量独立地为10 4至10 12CFU的量级。在一些实施方案中，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的总量为10 6至10 12CFU的量级。在一些具体实施方案中，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的总量为约2×10 11CFU。应当理解，本申请的益生菌组合物未必全部包含青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌，因此，此处的“总量”是指这四种益生菌中存在于益生菌组合物中的总量。
本文所述的“单位剂量形式”是指独立或分离包装的单次施用剂量的组合物，通常可以存在于单个片剂、胶囊或粉末/颗粒袋等。在一些实施方案中，为了便于施用，单位剂量形式被制备为含有每日剂量的组合物。
在一些实施方案中，所述益生菌组合物用于给予成年人，并且青春双歧杆菌的量为2.59×10 5-4.49×10 11CFU；和/或两岐双岐杆菌的量为1.26×10 5–7.35×10 11CFU；和/或长双岐杆菌的量为2.23×10 5–7.02×10 11CFU；和/或鼠李糖乳杆菌的量为1.26×10 5–2.59×10 11CFU。
在一些实施方案中，益生菌组合物用于给予儿童，并且青春双 歧杆菌的量为2.05×10 5–4.55×10 11CFU；和/或两岐双岐杆菌的量为1.47×10 5–3.6×10 11CFU；和/或长双岐杆菌的量为7.55×10 4–2.5×10 11CFU；和/或鼠李糖乳杆菌的量为1.47×10 5–3.6×10 11CFU。
在一些实施方案中，益生菌组合物不含本申请的各个实施方案所写明的益生菌之外的其他益生菌。
在此类实施方案的技术背景下，“不含”应当理解为“基本不含”，不排除由于菌株的培养、分离和纯化等因素导致存在微量或痕量的其他益生菌。在一些具体实施方案中，其他益生菌的量占组合物全部益生菌总量不超过5％，优选不超过1％。
在一些实施方案中，益生菌组合物不含青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌之外的益生菌。在一些实施方案中，益生菌组合物不含青春双歧杆菌、两岐双岐杆菌、长双岐杆菌之外的双歧杆菌。
第二方面，本申请提供了一种益生元组合物，包含低聚木糖、低聚半乳糖和玉米膳食纤维。
本文所述的“益生元组合物”指以益生元作为活性成分的组合物，不排除存在由于益生元的合成、分离、纯化等所引入的辅助成分和/或根据希望目的配制组合物的辅料成分。
在一些实施方案中，以重量计，所述低聚木糖、低聚半乳糖、玉米膳食纤维的量的比值为(0.25-5)：(0.75-4)：(0.5-1)。在一些实施方案中，以重量计，所述低聚木糖、低聚半乳糖、玉米膳食纤维的量的比值为(0.25、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0)：(0.75、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0)：(0.5、0.6、0.7、0.8、0.9、1.0)。在一些具体实施方案中，以重量计，低聚木糖、低聚半乳糖，玉米膳食纤维的量的比值为(0.25-0.5)：(2-4)：(0.5-0.75)。
在一些实施方案中，益生元组合物为单位剂量形式，并且以重量计，低聚木糖、低聚半乳糖、玉米膳食纤维的总量为0.1-12g，例如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12g。
在一些实施方案中，益生元组合物为单位剂量形式，低聚木糖的量为0.01g-6g；和/或低聚半乳糖的量为0.04g-9.6g；和/或玉米膳食纤维的量为0.01g-6g。这样的单位剂量形式可以给予成年人或儿童。
在一些实施方案中，当益生元组合物作为食物的补充剂或添加剂时，低聚木糖、低聚半乳糖、玉米膳食纤维的总量可以控制为0.1-5g，与食物搭配之后的总量预期可以达到期望值，例如约12g。
在一些实施方案中，益生元组合物不包含低聚木糖、低聚半乳糖、玉米膳食纤维之外的益生元组分。
在此类实施方案的技术背景下，“不含”应当理解为“基本不含”，不排除由于益生元的合成、提取、分离和纯化等因素导致存在微量或痕量的其他益生元。在一些具体实施方案中，其他益生元的量占组合物全部益生元总量不超过5％，优选不超过1％。
第三方面，本申请提供了一种膳食组合物(有时也称为“合生元(synbiotics)”组合物)，包含第一方面的益生菌组合物和第二方面的益生元组合物。
本领域技术人员应当理解，上述膳食组合物不需要是单独配制了第一方面的益生菌组合物和第二方面的益生元组合物，然后将两者混合或配合。只要一种膳食组合物涵盖第一方面的益生菌组合物的一个实施方案的全部特征和第二方面的益生元组合物的一个实施方案的全部特征，即属于本申请第三方面的膳食组合物。
作为非限制性的一个实例，本申请的膳食组合物可以具有以下配方(每日用量，可以提供于单位剂量形式)：
Figure PCTCN2021090531-appb-000001
在一些具体实施方案中，膳食组合物包含作为益生菌的青春双歧杆菌、两岐双岐杆菌和长双岐杆菌以及作为益生元的低聚木糖、低聚半乳糖和玉米膳食纤维，其中以菌落形成单位(CFU)计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.75-1)：1：(0.75-1)，并且三种菌总计约2×10 11CFU，并且以重量计，低聚木糖、低聚半乳糖和玉米膳食纤维的比为(0.25-0.5)：(2-4)：(0.5-0.75)并且低聚木糖、低聚半乳糖和玉米膳食纤维的总量为1.2-1.5g。
在第一至第三方面的一些实施方案中，所述益生菌组合物或 益生元组合物或膳食组合物被配制为用于经口施用。在一些实施方案中，经口施用包括口服、与口服类产品混合、管饲等方式。
在第一至第三方面的一些实施方案中，所述益生菌组合物或益生元组合物或膳食组合物为食品补充剂、食品添加剂或食品。
在第一至第三方面的一些实施方案中，所述益生菌组合物或益生元组合物或膳食组合物被配制为粉剂、颗粒剂、片剂或胶囊剂。
本申请的益生菌组合物或益生元组合物或膳食组合物的主要应用方式是给予个体的胃肠道。直接经口施用是较为方便的方式，但是对于某些特殊个体(例如卧床患者)而言，也可以通过管饲等方式辅助施用。
本申请的益生菌组合物或益生元组合物或膳食组合物的产品形式可以多种多样，例如，可以制备为单独的膳食补充剂(例如胶囊、片剂、粉剂、颗粒剂)，随餐或不随餐服用；也可以制备为各种固体/半固体食品、冲调粉末/颗粒食品、饮料等在被个体摄入之前加入或调配的添加型产品；也可以作为各种固体/半固体食品、冲调粉末/颗粒食品、饮料直接的组成成分。
在第一至第三方面的一些实施方案中，所述益生菌组合物或益生元组合物或膳食组合物施用于个体，用于辅助预防和/或治疗病原体感染、或增强病原体感染的治疗效果、或提高个体的免疫力、或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)。在一些实施方案中，病原体为病毒、细菌或真菌。在一些实施方案中，病原体为呼吸道疾病病毒，例如COVID-19、流行性感冒、呼吸道合胞病毒。
第四方面，本申请提供了第一方面的益生菌组合物、或第二方面的益生元组合物、或第三方面的膳食组合物在制备用于辅助预防和/或治疗个体的病原体感染或增强个体的病原体感染的治疗效果或提高个体的免疫力或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)的膳食产品或药品中的用途。在一些实施方案中，病原体为病毒、细菌或真菌。在一些实施方案中，病原体为呼吸道疾病病毒，例如COVID-19、流行性感冒、呼吸道合胞病毒。
在不存在冲突的情况下，本申请的益生菌组合物或益生元组合物或膳食组合物的制备可以参照本领域中益生菌或益生元类产品的常规加工方式。例如，可以通过常规加工技术将各种益生菌或益生元成分依次或同时或作为冻干预混物混合到产品中。
第五方面，本申请提供了用于辅助预防和/或治疗个体的病原体感染或增强个体的病原体感染的治疗效果或提高个体的免疫力或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种 和/或减少不理想细菌)的方法，包括向所述个体给予第一方面的益生菌组合物、或第二方面的益生元组合物、或第三方面的膳食组合物。在一些实施方案中，病原体为病毒、细菌或真菌。在一些实施方案中，病原体为呼吸道疾病病毒，例如COVID-19、流行性感冒、呼吸道合胞病毒。
实施例
以下实施例仅用于说明而非限制本申请范围的目的。
实施例1
本实施例描述了发明人进行的第一阶段队列研究。
方法
研究队列1
发明人从香港(n＝61)和云南省(n＝881)招募了942名健康中国人。该研究获得香港中文大学-新界东医院联网临床研究伦理联席委员会(The Joint CUHK-NTEC CREC,CREC编号:2016.407)和昆明医学院附属第一医院研究伦理委员会的批准(编号：2017.L.14)。所有受试者均签署书面知情同意书。研究对象的粪便样品保存在-80℃进行细菌组分析。
研究队列2
发明人公开招募了546名健康香港成年人。该研究已获得香港中文大学-新界东医院联网临床研究伦理联席委员会的批准(The Joint CUHK-NTEC CREC,CREC编号:2016.707)。所有受试者均签署书面知情同意书，捐赠粪便样本，并通过问卷调查提供人口统计学信息。研究对象的粪便样品保存在-80℃进行细菌组分析。
研究队列3
发明人招募了64名健康儿童。该研究已获得香港中文大学-新界东医院联网临床研究伦理联席委员会的批准(The Joint CUHK-NTEC CREC,CREC编号:2016.607)。所有受试者均签署书面知情同意书,捐赠粪便样本，并通过问卷调查提供人口统计学信息。研究对象的粪便样品保存在-80℃进行细菌组分析。
队列1和队列3的粪便DNA提取和DNA测序
使用
Figure PCTCN2021090531-appb-000002
RSC PureFood GMO和Authentication试剂盒(Promega)提取粪便DNA。将约100mg的每个粪便样品预先用1ml ddH 2O洗涤，并通过以13000g离心1分钟。将沉淀物重悬于800μLTE缓冲液(pH 7.5)中，加入1.6μl 2-巯基乙醇和500U裂解酶(Sigma)，并在37℃下孵育60分钟。然后将样品以13000g离心2分钟，弃去上清液。预处理后，随后使用
Figure PCTCN2021090531-appb-000003
RSC PureFood GMO和 Authentication试剂盒(Promega)，根据产品说明提取DNA。将1ml CTAB缓冲液添加到沉淀中并振荡30s，然后将溶液在95℃加热5分钟。此后，将样品用珠子(Biospec，真菌为0.5mm，细菌为0.1mm，1：1)以高速振荡研磨15分钟。此后，加入40μl蛋白酶K和20μl RNA酶，并在70℃孵育10分钟。然后通过以13000g离心5分钟获得上清液，并将其置于用于DNA提取的
Figure PCTCN2021090531-appb-000004
仪器中。提取的粪便DNA通过Ilumina Novoseq 6000(Novogen，北京，中国)用于超深度宏基因组学测序。每个样本平均获得12G数据。
队列2的粪便DNA提取和DNA测序
根据制造商的说明，使用DNeasy PowerSoil试剂盒(QIAGEN)提取粪便DNA。取0.1g粪便样本中提取DNA，然后使用Qubit dsDNA BR试剂盒(Thermo Fisher Scientific)测定提取的DNA浓度。将DNA样本发送到测序服务提供商(香港湾仔，Novogene HK Company Limited)进行文库制备和配对猎枪宏基因组测序(Illumina NovaSeq 6000)，每个样本平均返回7.5GB的原始数据。
细菌物种间的相关性分析
根据本申请的发明人团队前期的研究成果，肠道中包括多种拟杆菌及假链状双歧杆菌(Bifidobacterium pseudocatenulatum)在内的多种细菌与COVID-19病人的疾病严重程度或SARS-CoV-2病毒载量呈负相关，提示这些菌种(本文中也称为“理想细菌物种”)对COVID-19有保护作用(相关研究记载于美国临时专利申请63/016,759和63/025,310中，通过引用的方式将它们的内容整体并入本文，用于所有目的)。然而，理想细菌物种中的大部分目前尚未被批准应用于食品中。因此，发明人将目前被批准应用食品的菌种与理想细菌物种在人群中的含量做相关性分析。用于相关性分析的理想细菌物种包括：Akkermansia muciniphila,Alistipes onderdonkii,Anaerostipes hadrus,Bacteroides dorei,马赛拟杆菌(Bacteroides massiliensis),椭圆拟杆菌(Bacteroides ovatus),多形拟杆菌(Bacteroides thetaiotaomicron),假小链双歧杆菌(Bifidobacterium pseudocatenulatum),粘液真杆菌(Eubacterium limosum),直肠真杆菌(Eubacterium rectale),凸腹真杆菌(Eubacterium ventriosum),Faecalibacterium prausnitzii,人罗斯拜瑞氏菌(Roseburia hominis),肠道罗斯拜瑞氏菌(Roseburia intestinalis),霍氏真杆菌(Eubacterium hallii)。
相关性分析在研究队列1和2中进行，具体方式如下：
使用Trimmomatic(v0.38)默认参数对宏基因组学读数进行质量过滤和修剪。然后，通过Kneaddata(v0.7.2，https： //bitbucket.org/biobakery/kneaddata/wiki/Home)去除宿主DNA(参考基因组：hg38)。使用MetaPhlAn26(v2.6.0)进行物种级宏基因组注释。随后对MetaPhlAn2生成的相对丰度进行中心化对数比变换(Centered log ratio，clr)。使用R包corrplot v0.78计算并绘制皮尔逊相关系数。
结果
益生菌种类的鉴定
在研究队列1和2的相关性分析中，一些益生菌的相对丰度与理想细菌物种具有显著相关性，其中两岐双岐杆菌、长双岐杆菌青春双歧杆菌与多种理想细菌物种呈正相关(图1和表1)，提示补充此三种益生菌能增加至少一部分理想细菌物种在肠道中的含量，改善肠道健康，从而降低感染风险或疾病严重程度，例如增强免疫力、预防和治疗呼吸道感染。
表1
细菌物种 NCBI:txid
青春双歧杆菌 1680
两歧双歧杆菌 1681
长双歧杆菌 216816
益生菌的比例
发明人计算了研究队列1-3的成年人和儿童中的青春双歧杆菌，两歧双歧杆菌和长双歧杆菌的平均相对丰度。这些益生菌的比例是基于各种益生菌在健康人群中的自然比例，它们在研究队列1-3的约1500名健康中国人群中比例相对固定。因此，模拟健康人群中菌种的比例可能增加菌种在肠道中定植的机会。在此基础上，将青春双歧杆菌，双歧双歧杆菌和长双歧杆菌在合生元中的比例分别设计为成人(1-3.56)：1：(0.86-2.36)，儿童(0.57-3.09)：1：(0.21-1.7)：1。
益生元的选择和比例
益生元与益生菌通常是相辅相成的，因此在鉴定和选择益生菌之后，添加益生元而配合为合生元是有利的。针对上述研究中鉴定的益生菌种类，发明人针对性选择的益生元包括低聚木糖、低聚半乳糖，玉米膳食纤维。
低聚木糖又称木寡糖，是指2-10个木糖分子以β-1,4糖苷键连接而成的功能性低聚糖。低聚木糖是优良的双歧杆菌增殖因子。低聚木糖对两歧双歧杆菌和青春双歧杆菌的增殖效果非常明显。有报道称，青春双歧杆菌、婴儿双歧杆菌和两歧双歧杆菌均通过产生木糖苷酶和 阿拉伯糖苷酶利用低聚木糖，且其对低聚木糖的水解能力取决于其木聚糖酶酶解系统的效率。低聚木糖对双歧杆菌的增殖作用和发酵后短链脂肪酸的产量随低聚木糖组分分子质量的增加而下降。体外消化试验证明，低聚木糖经过唾液至小肠黏膜酶液后的残留率达到了99.6％，可充分被大肠内双歧杆菌发酵，且低聚木糖对双歧杆菌的增殖效果是其他功能性低聚糖的10～20倍。低聚木糖有耐酸、耐高温、稳定性强、配伍性好等特点，能很好地应用在食品方面。
低聚半乳糖和玉米膳食纤维可以促进多种双歧杆菌的生长。低聚半乳糖是一种新型的功能性物质，其分子结构一般是在半乳糖或葡萄糖分子上连接1-7个半乳糖基。它是具有天然属性的功能性低聚糖的一种。其适口性、水溶性和稳定性都较好，而且其进入人体后可以使人体肠道中的益生菌尤其是双歧杆菌增殖，同时也能抑制腐败菌的生长。肠道中的益生菌利用低聚半乳糖增殖的同时还能产生大量胞外多糖。胞外多糖不仅有抗肿瘤活性、免疫活性，还能促进益生菌在肠道内长期定植。玉米膳食纤维进入胃后可吸收部分水分并促进肠道加快蠕动，加速排出粪便，从而降低直肠的压力，并预防和减少肠道疾病。同时，双歧杆菌对于玉米膳食纤维具有的发酵作用，玉米膳食纤维可以在盲肠中迅速地被微生物发酵，产生短链脂肪酸。
根据益生菌的比例，发明人进一步给出了三种益生元的合适比例，其中低聚木糖、低聚半乳糖、玉米膳食纤维的比例可以为(0.25-5)：(0.75-4)：(0.5-1)。
实施例2
本实施例描述了发明人基于实施例1的研究队列2进行的扩展式第二阶段队列研究。
方法
研究队列HC(即，实施例1的研究队列2)
对于研究对象的粪便样品，根据制造商的说明，使用QIAamp DNeasy PowerSoil试剂盒进行粪便DNA提取。
研究队列CR1
发明人公开招募了219名健康香港成年人。该研究已获得香港中文大学-新界东医院联网临床研究伦理联席委员会的批准(The Joint CUHK-NTEC CREC,CREC编号:2017.369)。所有受试者均签署书面知情同意书，捐赠粪便样本，并通过问卷调查提供人口统计学信息。研究对象的粪便样品保存在-80℃进行细菌组分析。根据制造商的说明，使用QIAamp DNA Stool Mini试剂盒进行粪便DNA提取。
研究队列CR2
发明人从一组无症状受试者中随机抽取了30名健康香港成年人的粪便样本，这些受试者均接受了结肠镜检查且结果正常。该研究已获得香港中文大学-新界东医院联网临床研究伦理联席委员会的批准(The Joint CUHK-NTEC CREC,CREC编号:2017.198)。所有受试者均签署书面知情同意书，同意捐赠粪便样本。在微生物组分析之前，将来自研究对象的粪便样品储存在-80C。根据制造商的说明，使用Maxwell RSC PureFood GMO和Authentication试剂盒进行粪便DNA提取。
研究队列cov
发明人从一项COVID-19研究的健康对照组纳入了78个健康香港成年人的粪便样本。根据制造商的说明，使用Maxwell RSC PureFood GMO和Authentication试剂盒进行粪便DNA提取。
研究队列LeanHC
发明人从研究队列HC中随机选择了67名BMI<23的健康香港成年人。将这些研究对象的粪便样品储存在-80℃以用做微生物组分析。根据制造商的说明，使用Maxwell RSC PureFood GMO和Authentication试剂盒进行粪便DNA提取。
粪便样本和DNA提取
本实施例包括了从五个独立的队列研究收集的健康受试者的粪便样本。根据制造商的说明，使用QIAamp DNeasy PowerSoil试剂盒分离试剂盒，Maxwell RSC PureFood GMO和Authentication试剂盒或QIAamp DNA Stool Mini试剂盒进行粪便DNA提取。使用NanoDrop分光光度计和凝胶电泳来确定DNA的质量和数量。
元基因组测序
通过末端修复，纯化和PCR扩增的过程构建DNA文库。构建完DNA文库后，在发明人实验室中的NextSeq平台使用150bp配对末端测序策略对DNA文库进行测序。平均每个样本可获得12Gb数据以进行进一步分析。所有实验程序均按照发明人实验室的统一标准。
宏基因组学数据的数据处理和统计分析
发明人使用Fastp对元基因组的测序片段进行质量过滤，PolyG尾部修饰和适配器修饰，并删除了50个碱基或以下的测序片段。然后用KneadData清除品质修剪后的宏基因组的测序片段中的人类基因，再使用MetaPhlAn2分析物种水平的宏基因组，所有非零的水平 均会被是为正水平。发明人计算了每个物种及其组合的发生率。发明人使用Pearson相关分析探讨物种水平与年龄和性别之间的相关性。物种水平与年龄之间的相关性通过Pearson相关性进行分析，物种水平与性别之间的相关性通过Spearman相关性进行评估。
结果
在上述健康人队列的总体分析结果中，青春双歧杆菌、两歧双歧杆菌和长双歧杆菌的阳性率分别为68.2％，20.3％和81.7％(参见图2中A图)。少于20％的健康受试者的肠道中同时有这三种双歧杆菌物种，46.3％的健康受试者的肠道中有这三种双歧杆菌中的其中两种，高达36.3％的健康受试者的肠道中只有三种双歧杆菌物种中的其中一种或全无(参见图2中B图)。发明人进一步分析了三种双歧杆菌物种水平与年龄和性别的相关性。结果表明年龄增长与这三种双歧杆菌都呈负相关。男性性别与长双歧杆菌的低水平有显著的相关性(表2)。这些结果提示，大部分人群都适合补充本申请的益生菌/益生元/合生元组合物，尤其是年龄较大的人群和男性人群。
表2总队列中三种双歧杆菌与年龄和性别的相关性分析结果
Figure PCTCN2021090531-appb-000005
实施例3
本实施例描述了施用本申请的合生元组合物对住院COVID-19患者的症状改善和免疫反应标志物调节。据报道，由SARS-CoV-2病毒引起的冠状病毒疾病-2019(COVID-19)不仅针对肺部，也针对其他多个器官，包括肠道。肠道微生物可以调节宿主的免疫反应，因此有可能影响COVID-19患者的严重程度及预后。COVID-19患者的肠道微生物组变得不平衡，例如共生体减少、机会病原体增加，这都与COVID-19的严重程度和粪便SARS-CoV-2病毒脱落有关。此外据报道，粪便样本中短链脂肪酸产生的益生菌的含量较高时，SARS-CoV-2感染性较低，这突出了有益细菌在对抗SARS-CoV-2感染中的潜在有益作用。重新平衡COVID-19患者的肠道微生物组的治疗方案有可能改善临床效果。
发明人预期本申请的合生元组合物能改善COVID-19患者的临床症状，并因此设计和进行了一项初步研究，以评估在住院的COVID-19患者中这种合生元组合物对COVID-19的临床症状、血液免疫标志物和粪便微生物组的影响。发明人将这些结果与在同一时期接受标准治疗的COVID-19住院患者(对照组)进行了比较。此研究中使用的合生元组合物的配方如下:
合生元组合物包含作为益生菌的青春双歧杆菌、两岐双岐杆菌和长双岐杆菌以及作为益生元的低聚木糖、低聚半乳糖和玉米膳食纤维，其中以菌落形成单位(CFU)计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值控制为(0.75-1)：1：(0.75-1)，并且三种菌总计控制为约2×10 11CFU，并且以重量计，低聚木糖、低聚半乳糖和玉米膳食纤维的比控制为(0.25-0.5)：(2-4)：(0.5-0.75)并且低聚木糖、低聚半乳糖和玉米膳食纤维的总量控制为1.2-1.5g。
研究概述
发明人将在2020年8月13日至2020年10月9日期间威尔士亲王医院收治的SARS-CoV-2阳性的成年患者纳入研究。入组的COVID-19患者会在入院48小时内接受标准治疗或本申请的合生元组合物。受试者会接受标准治疗或服用合生元组合物胶囊28天。
主要结果是以下3项指标的综合评估：总体症状调查表评估症状是否缓解，是否需要呼吸支持，及从治疗开始到第5周的抗体产生情况。
总体症状调查表(表3)中第1到第19和第26题评估COVID-19相关症状包括发热，呼吸道症状和一般症状，最低分为20分(正常)，最高分为80分(症状最重)，症状完全缓解的评分为20分。表3的第20到25题评估胃肠道(GI)症状，GI症状完全缓解的评分为6分。
患者入院后2周内进行SARS-CoV-2免疫球蛋白G(IgG)抗体检测。发明人每两天会对患者进行一次临床症状评估，直到症状消失或患者出院为止。发明人也将同一时间接受标准治疗的另一组患者纳入研究作为比较。在所有受试者中，发明人在基线和服用合生元组合物5周后收集血液样本，并使用
Figure PCTCN2021090531-appb-000006
免疫多重分析法检测了标准治疗组和合生元组基线和第5周血浆中的免疫反应标志物。发明人也分别在基线，服用合生元组合物后的2、4和5周收集了粪便和生活质量调查表。
表3总体症状调查表
今天以下症状对您的影响程度
请圈出最合适的答案(1-4)。
Figure PCTCN2021090531-appb-000007
研究方法
受试者招募
本研究已获得香港中文大学-新界东医院联网临床研究伦理联席委员会(The Joint CUHK-NTEC CREC,CREC Ref.No:2020.407)的批准，并在临床试验注册中心注册(NCT04581018)。所有受试者均 签署书面知情同意书。入组的受试者包括合生元组合物组中的25名受试者和标准治疗组(SC)中的30名受试者。发明人招募了在2020年8月13日至10月9日期间，经逆转录酶聚合酶链反应(PCR)试验确诊为SARS-CoV-2感染的18岁或以上并在香港威尔士医院住院的患者。接受重症监护或使用呼吸机的受试者，对干预产品或其成分过敏或不耐受，有已知心内膜炎病史或活动性心内膜炎，近期接受CAPD或血液透析，或在招募时怀孕的受试者均被排除。受试者如患有任何会阻止口服益生菌或增加与益生菌有关的风险的疾病也被排除，这些风险包括但不限于无法吞咽或有误吸风险且没有其他给予方法(例如，没有G/J管)，已知的由于免疫抑制而增加的感染风险，例如器官或造血干细胞移植史，中性粒细胞减少症(ANC<500细胞/μl)或HIV和CD4<200细胞/μl。
在2020年8月至2020年10月期间住院的受试者被分配到合生元组合物组或标准治疗组。2020年7月起出院的受试者被分配到标准治疗组。在本研究中，受试者按指示将合生元组合物胶囊与食物一起服用28天，并每两天评估一次临床症状，直到症状消失或出院。血液样本在基线和首次服用合生元组合物或入组后5周收集。粪便样本和生活质量调查表在基线和首次合生元组合物或入组后第2、4和5周收集。本研究是根据《赫尔辛基宣言》进行的。
血液免疫标志物
将全血样品静置60分钟后，在4℃下无制动1500x g离心10分钟，收集血浆。未稀释的血浆被转移到15毫升聚丙烯锥形管中，然后分装并保存在-80C下用于后续研究。使用Custom Premix Human Cyto Panel A 47 Plex(Millipore，#HCYTA-60K-47C)检测血浆细胞因子和趋化因子的水平。所有样品均在首次解冻时进行检测。
结果
本研究纳入25例服用合生元组合物的COVID-19患者和30例接受标准治疗的患者。患者临床特征见表3，两组之间的年龄，合并症，基线症状评分和疾病严重程度均无显著差异。在诊断时，两组之间的生命体征，低氧血症和炎症标志物相似(数据未显示)。
与标准治疗组相比，合生元组合物组中达到完全症状缓解的比例明显更高(图3中A图，第一周为64％vs 10％；p＝<0.001；第二周为100％vs 52％；p<0.001)，抗体阳性率也明显更高(第16天为88％vs 63％)；p＝0.037)。标准治疗组的8名受试者(26.7％)和合生元组合物组的1名受试者(4％)出院前IgG抗体检测从未呈阳性。 所有接受合生元组合物的患者的胃肠道(GI)症状到第2周都得到缓解(图3中A图)。通过EuroQol视觉模拟量表(EQ-VAS)和EuroQol指数评分(EQ指数评分)评估(具体数据未显示)，合生元组合物组的受试者在第四周的生活质量与基线相比有显著改善(EQ-VAS第四周和基线分数分别为81.5和69.75，p＝0.034；EQ指数评分第四周和基线分别为0.839和0.805，p<0.0005)，但标准治疗组则没有观察到这种改善。两组均无严重不良反应。在合生元组合物组中，观察到一些不良事件，包括轻度头晕症状(4％)，癣感染(4％)，高血压(4％)。通过常规血液检查发现1名受试者(4％)患有慢性淋巴细胞性白血病。在标准治疗组中，3名受试者(10％)患有便秘，1名受试者(3.3％)患有轻度手部炎症。在合生元组合物组中，八种主要免疫反应标志物：白血球介素(IL-6，IL-1RA，IL-18)，肿瘤坏死因子(TNF-α)，巨噬细胞集落刺激因子(M-CSF)，CXC趋化因子配体10(CXCL-10，也称为IP10)，单核细胞趋化蛋白1(MCP-1)和干扰素-γ诱导的单核因子(MIG)，在第5周时的水平与基线时的相应水平相比显著降低。相反，在标准治疗组中，这些免疫反应标志物中的多种没有显著变化(图3中B图)。与基线水平比，合生元组合物组在第5周时，IL-6、CXCL-10、IL1RA、MIG、TNF-α和M-CSF的百分比下降量显著高于对照组(图3中C图)。在合生元组合物组中，第2周和第5周的青春双歧杆菌和长双歧杆菌的丰度与基线相比显著增加(图3中D图)。
表4合生元组合物组和标准治疗组的患者特征表
Figure PCTCN2021090531-appb-000008
Figure PCTCN2021090531-appb-000009
IQR:症状基线中位数
讨论
这项初步研究发现，在住院COVID-19的患者中，使用本申请的合生元配方可以减轻胃肠道症状并抑制炎症细胞因子水平。本研究的理论基础是，在COVID-19患者中使用合生元组合物可以重新平衡肠道微生物组，从而减轻疾病的严重程度，并改善生活质量。由于在这之前尚未有对COVID-19患者进行过有关合生元的抗SARS-CoV-2效应的研究，因此相关事前数据有限。
发明人首次发现COVID-19患者的肠道缺乏了一系列有益细菌并且在呼吸道清除了SARS-CoV-2病毒后，病毒感染和复制的活动在肠道中仍然持续。通过使用大数据分析和机器学习，发明人开发了一种针对肠道微生态失衡的益生菌配方。在合生元组合物组中，益生菌的丰度在第2周时显著增加，这证实了益生菌已成功递送至肠道。研究显示COVID-19重症患者的免疫反应标志物水平升高，这些标志物包括IL-6，IL-1RA，IL-18，TNF-α，M-CSF，CXCL10，MCP-1，MIG。发明人发现合生元组合物可以降低上述8种免疫反应标志物在第5周的血浆样品中的水平。同时，在合生元组合物治疗后第2周和第5周， 患者的COVID-19症状也得到缓解。这些结果提示合生元组合物治疗可能会增强宿主对SARS-CoV2的免疫反应，主要表现在能压抑COVID-19感染早期升高的细胞因子，这一结果为合生元针对肠道菌群进行靶向治疗提供了依据。发明人的研究提出了合生元有早期免疫干预的作用，为应用合生元组合物提升机体免疫力抗击COVID-19和其他新兴病毒感染带来了借鉴和希望。
实施例4
本实施例为实施例3的基础上所进行的延伸研究。发明人从实施例3中纳入25例服用合生元组合物的COVID-19患者和10例接受标准治疗的患者，此外发明人另外纳入69例接受标准治疗的患者以及78例健康人作为对照组。透过 元基因组学分析发现，本申请的合生元配方可以让失衡的肠道微生态回复平衡，并达到或接近正常人的水平。
研究方法
受试者招募
发明人从实施例3纳入25例服用合生元组合物的COVID-19患者和10例接受标准治疗的患者，此外发明人另外纳入69例接受标准治疗的患者。这研究已获得香港中文大学-新界东医院联网临床研究伦理联席委员会(The Joint CUHK-NTEC CREC,CREC Ref.No:2020.076)的批准，并根据赫尔辛基宣言进行。所有受试者均签署书面知情同意书。这些COVID-9患者是在2020年2月至2020年5月间，从香港威尔斯亲王医院和联合基督教医院招募。医院工作人员收集了这些患者的鼻咽拭子，并由实验室通过定量逆转录聚合酶链反应(RT-qPCR)确认为SARS-CoV-2阳性。发明人还从一项香港人群肠道微生物组调查研究中纳入78例健康人作为对照组，这些健康人是通过广告或结肠镜检查试验招募的。
粪便样本收集
按图4所描述，发明人在不同时间点(基线、第2、第4和第5周)收集了合生元组合物及标准治疗组的受试者的粪便样本。另外，发明人从健康对照组收集了一个时间点的粪便样本。
粪便样本和DNA提取
根据制造商的说明，使用QIAamp DNeasy PowerSoil试剂盒，Maxwell RSC PureFood GMO和Authentication试剂盒或QIAamp DNA Stool Mini试剂盒进行粪便DNA提取。使用NanoDrop分光光度计和 凝胶电泳来确定DNA的质量和数量。
元基因组测序
通过末端修复、纯化和PCR扩增的过程构建DNA文库。构建完DNA文库后，在发明人实验室中的NextSeq平台使用150bp配对末端测序策略对DNA文库进行测序。平均每个样本可获得12Gb数据以进行进一步分析。所有实验程序均按照发明人实验室的统一标准。
宏基因组学数据的数据处理和统计分析
使用Fastp对元基因组的测序片段进行质量过滤，PolyG尾部修饰和适配器修饰，并删除了50个碱基或以下的测序片段。然后用KneadData清除品质修剪后的宏基因组的测序片段中的人类基因，再使用MetaPhlAn2分析物种水平的宏基因组，所有非零的水平均会被是为正水平。发明人计算了每个物种及其组合的发生率。如同实施例1的描述，根据发明人前期的研究成果，将肠道中包括多种拟杆菌及假链状双歧杆菌(Bifidobacterium pseudocatenulatum)在内的多种细菌与COVID-19病人的疾病严重程度或SARS-CoV-2病毒载量呈负相关的物种定义为“理想细菌物种”(相关研究记载于美国临时专利申请63/016,759和63/025,310)。另外，将COVID-19病人肠道中相对丰度较高的物种定义为“不理想细菌物种”。使用R软件内的Vegan包计算α生物多样性指数(香农多样性指数，Shannon diversity index)。使用LEfSe软件(linear discriminant analysis[LDA]effect size)分析微生物组间差异。LDA大于2且p<0.05的物种被认为组间有显著差异。
结果
与基线比，接受标准治疗的COVID-19患者在第2周和第4周时，本申请的合生元配方中的3种双歧杆菌属益生菌的总丰度略有下降；相比之下，在接受合生元组合物的患者中，第2周和第4周时的3种双歧杆菌属益生菌的总丰度则显著增加(图5)。与基线相比，接受标准治疗的COVID-19患者的香农多样性指数在第2周、第4周和第5周显著下降；相比之下，在接受合生元组合物的COVID-19患者中，其香农多样性指数则维持在与基线相似的高水平(图6)。
在接受标准治疗的COVID-19患者中，与基线比，第2周和第4周时的理想细菌物种的总丰富度显著降低；相比之下，在接受合生元组合物的COVID-19患者中，与基线比，第2周和第4周时的理想细菌物种的总丰富度显著增加(图7中A图)。同时，与基线比，在第2、第4和第5周时,接受合生元组合物的COVID-19患者中的不理 想细菌物种的总丰富度显著降低，而接受标准治疗的患者则没有降低(图7中B图)。
此外，与接受标准治疗的COVID-19患者相比，接受合生元组合物的COVID-19患者中包括青春双歧杆菌、直肠真杆菌、瘤胃球菌和长双歧杆菌等理想细菌物种显著较为丰富(图8)。
上文对本申请的各项发明的示例性实施方案进行了描述，但是，在不脱离本申请的实质和范围的情况下，本领域技术人员能够对本申请描述的示例性实施方案进行修改或改进，由此得到的变形方案或等同

Claims (23)

  1. 一种益生菌组合物，包含两岐双岐杆菌(Bifidobacterium bifidum)和长双岐杆菌(Bifidobacterium longum)。
  2. 如权利要求1所述的益生菌组合物，其中以菌落形成单位计，两岐双岐杆菌和长双岐杆菌的量的比值为1：(0.21-2.36)。
  3. 如权利要求1或2所述的益生菌组合物，还包含青春双歧杆菌(Bifidobacterium adolescentis)。
  4. 如权利要求3所述的益生菌组合物，其中以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.57-3.56)：1：(0.21-2.36)，优选(0.75-1)：1：(0.75-1)。
  5. 如权利要求1-4中任一项所述的益生菌组合物，还包含鼠李糖乳杆菌(Lactobacillus rhamnosus)。
  6. 如权利要求5所述的益生菌组合物，其中以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量的比值为(0.57-3.56)：1：(0.21-2.36)：1。
  7. 如权利要求1-6中任一项所述的益生菌组合物，其中所述益生菌组合物为单位剂量形式，并且以菌落形成单位计，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的量独立地为10 4至10 12CFU的量级；任选地，青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌的总量为10 6至10 12CFU的量级。
  8. 如权利要求7所述的益生菌组合物，其中所述益生菌组合物用于给予成年人，并且
    青春双歧杆菌的量为2.59×10 5-4.49×10 11CFU；和/或
    两岐双岐杆菌的量为1.26×10 5-7.35×10 11CFU；和/或
    长双岐杆菌的量为2.23×10 5-7.02×10 11CFU；和/或
    鼠李糖乳杆菌的量为1.26×10 5-2.59×10 11CFU。
  9. 如权利要求7所述的益生菌组合物，其中所述益生菌组合物用于给予儿童，并且
    青春双歧杆菌的量为2.05×10 5-4.55×10 11CFU；和/或
    两岐双岐杆菌的量为1.47×10 5-3.6×10 11CFU；和/或
    长双岐杆菌的量为7.55×10 4-2.5×10 11CFU；和/或
    鼠李糖乳杆菌的量为1.47×10 5-3.6×10 11CFU。
  10. 如权利要求1-9中任一项所述的益生菌组合物，其中所述益生菌组合物不含青春双歧杆菌、两岐双岐杆菌、长双岐杆菌、鼠李糖乳杆菌之外的益生菌，例如不含青春双歧杆菌、两岐双岐杆菌、长双岐杆菌之外的双歧杆菌。
  11. 一种益生元组合物，包含低聚木糖、低聚半乳糖和玉米膳食纤维。
  12. 如权利要求11所述的益生元组合物，其中以重量计，所述低聚木糖、低聚半乳糖和玉米膳食纤维的量的比值为(0.25-5)：(0.75-4)：(0.5-1)，优选(0.25-0.5)：(2-4)：(0.5-0.75)。
  13. 如权利要求11或12所述的益生元组合物，其中所述益生元组合物为单位剂量形式，并且以重量计，所述低聚木糖、低聚半乳糖和玉米膳食纤维的总量为0.1-12g，例如0.1-5g。
  14. 如权利要求13所述的益生元组合物，其中
    低聚木糖的量为0.01g-6g；和/或
    低聚半乳糖的量为0.04g-9.6g；和/或
    玉米膳食纤维的量为0.01g-6g。
  15. 如权利要求11-14中任一项所述的益生元组合物，其不包含低聚木糖、低聚半乳糖，玉米膳食纤维之外的益生元组分。
  16. 一种膳食组合物，包含权利要求1-10中任一项所述的益生菌组合物和权利要求11-15中任一项所述的益生元组合物；优选地，所述膳食组合物包含作为益生菌的青春双歧杆菌、两岐双岐杆菌和长双岐杆菌以及作为益生元的低聚木糖、低聚半乳糖和玉米膳食纤维，其中以菌落形成单位(CFU)计，青春双歧杆菌、两岐双岐杆菌和长双岐杆菌的量的比值为(0.75-1)：1：(0.75-1)，并且三种菌总计约2×10 11CFU，并且以重量计，低聚木糖、低聚半乳糖和玉米膳食纤维的比为(0.25-0.5)：(2-4)：(0.5-0.75)并且低聚木糖、低聚半乳糖和玉米膳食纤维的总量为1.2-1.5g。
  17. 如权利要求1-10中任一项所述的益生菌组合物、或权利要求11-15中任一项所述的益生元组合物、或权利要求16所述的膳食组合物，其被配制为用于经口施用，例如口服、与口服类产品混合、管饲。
  18. 如权利要求1-10、17中任一项所述的益生菌组合物、或权利要求11-15、17中任一项所述的益生元组合物、或权利要求16或17所述的膳食组合物，其为食品补充剂、食品添加剂或食品。
  19. 如权利要求1-10、17-18中任一项所述的益生菌组合物、或权利要求11-15、17-18中任一项所述的益生元组合物、或权利要求16-18中任一项所述的膳食组合物，其被配制为粉剂、颗粒剂、片剂或胶囊剂。
  20. 如权利要求1-10、17-19中任一项所述的益生菌组合物、或权利要求11-15、17-19中任一项所述的益生元组合物、或权利要求16-19中任一项所述的膳食组合物，其施用于个体用于辅助预防和/或治疗病原体感染、或增强病原体感染的治疗效果、提高个体的免疫力、或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)。
  21. 如权利要求20所述的益生菌组合物、或益生元组合物、或膳食组合物，其中所述病原体为病毒、细菌或真菌，例如呼吸道疾病病毒，例如新冠病毒(COVID-19)、流行性感冒、呼吸道合胞病毒。
  22. 权利要求1-10、17-19中任一项所述的益生菌组合物、或权利要求11-15、17-19中任一项所述的益生元组合物、或权利要求16-19中任一项所述的膳食组合物在制备用于辅助预防和/或治疗个体的病原体感染或增强个体的病原体感染的治疗效果或提高个体的免疫力或平衡个体的肠道微生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)的膳食产品或药品中的用途。
  23. 用于辅助预防和/或治疗个体的病原体感染或增强个体的病原体感染的治疗效果或提高个体的免疫力或平衡个体的肠道微 生态(包括增加微生物丰度、增加理想细菌物种和/或减少不理想细菌)的方法，包括向所述个体给予权利要求1-10、17-19中任一项所述的益生菌组合物、或权利要求11-15、17-19中任一项所述的益生元组合物、或权利要求16-19中任一项所述的膳食组合物。
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