WO2023070513A1 - 一种益生元和益生菌复合物的组合物及其应用 - Google Patents

一种益生元和益生菌复合物的组合物及其应用 Download PDF

Info

Publication number
WO2023070513A1
WO2023070513A1 PCT/CN2021/127363 CN2021127363W WO2023070513A1 WO 2023070513 A1 WO2023070513 A1 WO 2023070513A1 CN 2021127363 W CN2021127363 W CN 2021127363W WO 2023070513 A1 WO2023070513 A1 WO 2023070513A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
composition
mice
probiotic
compound
Prior art date
Application number
PCT/CN2021/127363
Other languages
English (en)
French (fr)
Inventor
赵玥明
吴桐
赵怡晴
侯艳梅
Original Assignee
海普诺凯营养品有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 海普诺凯营养品有限公司 filed Critical 海普诺凯营养品有限公司
Priority to CN202180004590.3A priority Critical patent/CN114401728B/zh
Priority to PCT/CN2021/127363 priority patent/WO2023070513A1/zh
Publication of WO2023070513A1 publication Critical patent/WO2023070513A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/718—Starch or degraded starch, e.g. amylose, amylopectin
    • 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
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/175—Rhamnosus
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis

Definitions

  • the invention belongs to the technical field of food nutrition, and in particular relates to a composition of a prebiotic and a probiotic compound and an application thereof.
  • Lactobacillus rhamnosus MP108 is the first edible strain for infants and young children with China's independent intellectual property rights approved by the Chinese Health Commission. At present, there is no report on the formation of a probiotic complex with Lactobacillus rhamnosus MP108 and Bifidobacterium lactis HN019. At the same time, there is no report on the combination of the two probiotics and prebiotics, and there is no such combination Patent application activities based on objects.
  • probiotic products usually have 1 to 2 effects, the common ones are improving immunity and relieving constipation, and the formula constructed by this patent has the functions of relieving atopic dermatitis, relieving allergic asthma, relieving bacterial diarrhea, relieving Food Allergies, Digestion Improvement, and Constipation Relief.
  • the invention aims to overcome the deficiencies of the prior art, and provides a composition of prebiotic and probiotic compound and its application.
  • the prebiotics described in the composition of the prebiotic and the probiotic compound are fructooligosaccharide FOS, galactooligosaccharide GOS and resistant dextrin, and the probiotic compound is Lactobacillus rhamnosus and lactobacillus
  • the compound of fidobacterium; the weight ratio of described Lactobacillus rhamnosus, Bifidobacterium lactis, fructo-oligosaccharide FOS, galacto-oligosaccharide GOS and resistant dextrin is (1 ⁇ 9):(0.5 ⁇ 3): (4 ⁇ 30):(55 ⁇ 100):(30 ⁇ 90).
  • the weight ratio of galacto-oligosaccharide GOS, fructo-oligosaccharide FOS and resistant dextrin in the prebiotic is 9:1:7.
  • the probiotic compound is a compound of Lactobacillus rhamnosus MP108 and Bifidobacterium lactis HN019, and the weight ratio of Lactobacillus rhamnosus MP108 and Bifidobacterium lactis HN019 in the probiotic compound is 5 :1.
  • the viable count of Lactobacillus rhamnosus MP108 is 1*10 7 -10 11 CFU/g
  • the viable count of Bifidobacterium lactis HN019 is 3*10 6 -10 10 CFU/g.
  • the preparation method of the composition of the prebiotic and the probiotic compound is as follows: the first step, pour fructo-oligosaccharide, malto-oligosaccharide, and resistant dextrin into a single-column hopper mixer according to the mass ratio for a first-stage pre-preparation. Mix to form a first-level premix, wherein: the speed of the first-level premix is 3-20 rpm, forward for 10-20 minutes, and reverse for 10-20 minutes.
  • the second step is to add Lactobacillus rhamnosus MP108 bacteria powder and Bifidobacterium lactis HN019 bacteria powder to the above-mentioned primary premix and mix them in a single-column hopper mixer. After mixing evenly, a compound probiotic prebiotic combination is formed. materials, wherein: the rotational speed of the secondary mixing is 5-15 rpm, the forward direction is 8-15 minutes, and the reverse direction is 8-15 minutes. Mixing uniformity ⁇ 99%.
  • the present invention takes BALB/c mice as the research object, and the experimental results show that the composition of the prebiotic and the probiotic compound described in the present invention has the effect of alleviating bacterial diarrhea, alleviating constipation, and promoting digestion , has the effect of relieving food allergy, has the effect of relieving allergic asthma, has the effect of relieving specific dermatitis, can inhibit the growth of other bacteria, and the probiotics in this combination have the characteristics of acid and choline resistance.
  • composition of the prebiotic and the probiotic compound can be used to prepare a bacterial diarrhea relieving agent, a constipation relieving agent, a digestion promoting preparation, a food allergy efficacy evaluation preparation, an allergic asthma relieving agent, and a dermatitis relieving agent.
  • the prebiotic composition mentioned in the present invention can promote the growth of the probiotics in the composition, which will make the probiotics in the composition better activated and colonized in the intestinal tract.
  • prebiotics there are many kinds of prebiotics to choose from on the market. Different probiotics have different growth characteristics.
  • Figure 1 Pathological sections of the jejunum of mice in each group (100X);
  • FIG. 1 Pathological sections of the jejunum of mice in each group (X200);
  • FIG. 1 Lung tissue slices (X200) of each group
  • Figure 4 Histopathological sections (100X) of mouse back skin lesions stained with HE.
  • Example 1 Application of the composition in alleviating bacterial diarrhea
  • mice Taking BALB/c mice as the research object, the effects of these samples on the body weight, feces water content, serum immune inflammatory factors, and jejunal histopathology of mice with diarrhea were detected by gavage of different sample solutions to the mice. Efficacy and variability in alleviating bacterial diarrhea.
  • the rotating speed of the secondary mixing is 10 rpm, forward 8 minutes, reverse 8 minutes.
  • the samples were all dissolved in 0.2mL sterile normal saline, and processed by gavage according to the requirements of Table 1, and the blank control group and the model group were gavaged with 0.2ml Sterile saline.
  • streptomycin (5 g/liter) was added to the drinking water for 3 days to disturb the intestinal flora of the mice. Then replace the water containing streptomycin with sterile water, and stop eating 18 hours before modeling.
  • the blank control group was intragastrically administered normal saline 3 times a day, and the model group was intragastrically administered normal saline once, and then 1.2 ⁇ 10 11 CFU/mL ETEC O78:K80 suspension was administered twice a day , each mouse was gavaged with 0.2mL each time, for four consecutive days, with an interval of 2 hours each time, after gavage with ETEC, the mouse cage was replaced, no litter was added, filter paper was spread in the mouse cage, and the mice were observed every 2 hours Diarrhea and mortality rate of 1.2 ⁇ 10 11 CFU/mL ETEC O78:K80 suspension were administered twice a day for each sample group. 0.2 mL was administered orally once, and the rest of the treatment was the same as that of the model group mice.
  • mice had free access to food and water, and the mice in each group were fed with standard feed.
  • the feeding barrier was in a constant temperature and humidity environment, with a temperature of 25 ⁇ 2°C, a humidity of 50% ⁇ 5%, and a light condition of 12 hours. , 12 hours of darkness.
  • double-bacteria sample has the effect of alleviating and reducing the water content of feces, and helps to form feces. Higher doses are more effective.
  • ST is an enterotoxin of Escherichia coli, and the increase of TLR4 will promote the expression of various inflammatory cells.
  • each sample group has a significant decrease compared with the model group (P ⁇ 0.05). It shows that each sample group has the effect of relieving bacterial diarrhea, and restores the expression of ST and TLR4 in mice. And the double-bacteria group is more conducive to the restoration of expression of ST and TLR4 in mice than the single-bacteria group, and the difference is significant (P ⁇ 0.05).
  • mice Taking BALB/c mice as the research object, by administering probiotics to the mice, the effects of these samples on the wet weight of feces, water content of feces, number of feces particles, small intestinal propulsion rate, and time to first black stool discharge of constipated mice were detected. Impact, to determine the efficacy and variability of samples in relieving constipation.
  • the rotating speed of the secondary mixing is 9 rpm, forward 13 minutes, reverse 13 minutes.
  • the samples were all dissolved in 0.2mL sterile normal saline, and processed by intragastric administration according to the requirements of Table 4, and the blank control group and the model group were intragastrically administered 0.2ml Sterile saline.
  • the blank control group was intragastrically administered sterile normal saline twice a day for a total of 8 days; the model group and the sample group were intragastrically administered loperamide hydrochloride every day, and the sample group was intragastrically administered the corresponding sample after 1 hour.
  • mice had free access to food and water, and the mice in each group were fed with standard feed.
  • the feeding barrier was in a constant temperature and humidity environment, with a temperature of 25 ⁇ 2°C, a humidity of 50% ⁇ 5%, and a light condition of 12 hours. , 12 hours of darkness.
  • Feces water content (%) (wet weight of feces - dry weight of feces) / wet weight of feces ⁇ 100%
  • the detection of the first black stool time of mice was carried out, and the detection method was as follows: except the blank control group, all groups of mice were first gavaged with loperamide hydrochloride, 1h Afterwards, the blank control group and the model control group were gavaged with ink, and the remaining groups were gavaged with ink containing samples. From the gavage of ink, the time of the first black stool of each animal was recorded, and the first time of the last mouse in the model group was recorded. The black stool time is the end time, and the treatment group that exceeds the first black stool time of the model group is invalid. The difference between each treatment group and the model group is compared to illustrate the difference in constipation relief in each group.
  • mice After the mice were gavaged for 8 days, they were fasted overnight on the night of the 8th day. On the 9th day, all groups of mice except the blank control group were given loperamide hydrochloride. After 30 minutes, the blank control group and the model control group The first group was gavaged with ink, and the other groups were gavaged with ink containing samples.
  • mice were killed, the abdominal cavity was opened, the mesentery was separated, and the intestinal tube from the pylorus at the upper end to the ileocecum at the lower end was cut, and the small intestine was gently pulled into a straight line, and the intestinal tube was measured
  • the length is the "total length of the small intestine”
  • the length from the pylorus to the front of the ink is the "length of ink advancement”
  • the small intestine advancement rate is calculated according to the following formula.
  • Small intestine propulsion rate (%) ink propulsion length (cm) / total length of small intestine (cm) ⁇ 100%
  • the sample group had a significant improvement (P ⁇ 0.05), and the effect of the double-bacteria sample group was better than that of the single-bacteria sample group (P ⁇ 0.05).
  • the sample group had a significant increase (P ⁇ 0.05), and the effect of the double-bacteria sample group was better than that of the single-bacteria sample group (P ⁇ 0.05).
  • the patented composition has the ability to improve small intestine peristalsis.
  • the bacteria powder sample group has a significant increase (P ⁇ 0.05), and there is no significant difference compared with the blank group (P>0.05), which can increase the level of motilin in mouse serum.
  • MTL motilin
  • Gas gastrin
  • VIP vasoactive intestinal peptide
  • SS somatostatin
  • the patent composition can improve the constipation symptoms of mice by promoting the secretion of MTL and Gas, and at the same time inhibiting the secretion of VIP and SS. same.
  • mice by gavage different samples to the mice, detect the effects of these samples on the mouse body weight growth rate, food utilization rate, and pepsin activity, and determine the efficacy of these samples in aiding digestion and differences.
  • the rotating speed of secondary mixing is 13 rpm, forward 13 minutes, reverse 13 minutes.
  • 6 mice in each group the composition samples were all dissolved in 0.2mL sterile saline, and were gavaged according to the requirements of Table 7, the blank control group and the model The rats in the group were given 0.2ml sterile normal saline.
  • the adaptation period is 1 week. Feed raw materials are crushed with a hammer mill, passed through a 80-mesh sieve, and mixed evenly. Then, 0.01% Y2O3 indicator is added according to the principle of step-by-step amplification. , air-dried at room temperature to constant weight, and mice in each group had free access to water and food during the experiment. The blank control group was fed with normal saline, and the mice in the other groups were fed with the corresponding sample solution (0.2 mL). During the experiment, the body weight and food intake were measured twice a week using the metabolic cage system. At the end of the experiment, body weight, body weight gain, food intake, food utilization rate, total oxygen consumption, and apparent digestibility were calculated.
  • mice Taking BALB/c female mice as the research object, by gavage different samples to the mice, the effects of these samples on weight changes, diarrhea and other allergic symptom scores, intestinal histopathological observations, mast cells and eosinophils were detected. Cell counts, serum OVA-specific immunoglobulin levels, and Th2 inflammatory factor levels were used to determine the efficacy and differences of these products in alleviating food allergy.
  • the rotational speed of secondary mixing is 14 rpm, Forward 10 minutes, reverse 10 minutes.
  • 8 mice in each group the composition samples were all dissolved in 0.2mL sterile normal saline, and treated by gavage according to the requirements in Table 10, and the blank control group and the model group were gavaged 0.2ml sterile saline.
  • Sensitization solution Dissolve 3mg of OVA in 6mL of normal saline, then add 6mL of aluminum hydroxide adjuvant, and mix well.
  • Excitation solution Dissolve 2.8g of OVA in 11.2mL of normal saline and mix well.
  • Administration method In the sensitization stage, intraperitoneal injection, and in the challenge stage, gavage.
  • Sensitization stage On the 3rd and 17th days, 0.1 mL of normal saline containing 50 ⁇ g OVA + 0.1 mL of aluminum hydroxide adjuvant was used for sensitization by intraperitoneal injection. The blank group was replaced by the same amount of normal saline.
  • Provocation phase On the 31st to 43rd days, 0.2 mL of normal saline containing 50 mg OVA was used to stimulate allergy by gavage every other day, and a total of 7 times were stimulated. The blank group was replaced by the same amount of normal saline.
  • mice were fasted 3 hours before gavage with the sample solution, and sensitized or challenged with OVA 1 h after gavage.
  • mice were weighed every week. After intragastric administration on the last day, they were anesthetized after fasting for 12 hours, and 1% pentobarbital sodium was injected intraperitoneally at 0.5 mL/10 g body weight. After anesthesia, blood was collected from the eye sockets. After the blood was collected, the animals were sacrificed by cervical dislocation, dissected, and the jejunum tissues were taken for histopathological analysis. After the samples were collected, they were quickly fixed in formaldehyde fixative solution for 12 hours, rinsed with PBS after taking out, and then dehydrated, packed Buried, sliced.
  • the serum levels of OVA-specific IgE and intestinal tissue inflammatory factors were detected. After the blood sample was taken, let it stand at 4°C for 2h, centrifuge at 3000 ⁇ g for 15min, separate the serum into an enzyme-free tube, and store it in a -80°C refrigerator for later use.
  • composition group had a significant increase (P ⁇ 0.05), and the effect of the double bacteria group was significantly better than that of the single bacteria group (P>0.05).
  • mice Taking BALB/c female mice as the research object, different samples were administered to the mice to detect the serum specific IgG1 level of these samples, IL-5, IL-13 and IL-17A in the alveolar lavage fluid. content, lung pathology, and the impact of fecal short-chain fatty acid content to determine the efficacy and differences of these samples in alleviating allergic asthma (rhinitis).
  • the samples of each sample group were dissolved in 0.2mL sterile normal saline, and were intragastrically administered according to the requirements in Table 14.
  • the blank control group and the model group were intragastrically administered 0.2ml sterile normal saline.
  • mice had free access to food and water, and the mice in each group were fed with standard feed.
  • the feeding barrier was in a constant temperature and humidity environment, with a temperature of 25 ⁇ 2°C, a humidity of 50% ⁇ 5%, and a light condition of 12 hours. , 12 hours of darkness.
  • mice were anesthetized, blood was collected from the eyeball, and after resting for 2 hours, centrifuged at 3000 rpm for 10 minutes, the serum was collected in a clean centrifuge tube, and the HDM-specific IgG1 content in the serum was detected by the HDM-specific IgG1 ELISA kit; Chest cavity and neck, expose mouse trachea and both lungs, ligate right lung, intubate trachea with indwelling needle, draw 0.3mL pre-cooled PBS with 1mL syringe, lavage mouse left lung, carry out 3 times, recover volume ⁇ 80%, collect the lavage fluid in a clean Eppendorf tube.
  • each sample group has a significant decrease (P ⁇ 0.05), and the effect of the dual-bacteria sample group is more significant than that of the single-bacteria sample group.
  • the patent composition can reduce the exudation of eosinophils in the lung tissue of asthmatic mice, reduce the secretion of IL-4, IL-5, IL-13 and IL-17A, and can also secrete the immune negative regulator IL -10 and IFN- ⁇ inhibit the inflammatory response, thereby playing a role in alleviating asthma.
  • the degree of pathological damage in each sample group was significantly reduced compared with the allergic asthma model group, the infiltration of inflammatory cells in the lung tissue and surroundings was significantly reduced, the mild hyperplasia of the mucosal epithelium, the damage of the airway epithelium and the thickening of the alveolar septum were alleviated.
  • the dose group had no significant difference in pathological damage compared with the low dose group.
  • Example 6 Application of the composition in alleviating atopic dermatitis
  • mice Taking C57BL/6 female mice as the research object, by gavage different samples to the mice, the changes of ear swelling degree, skin pathological changes, serum IgE level, skin tissue IL-4, IL- 5. The levels of IL-13, IL-10 and IFN- ⁇ , to determine the efficacy and differences of these samples in alleviating atopic dermatitis (eczema).
  • the modeling method is as follows: the model group and the composition group removed the back body hair of the mice with a hair remover on the 14th day, with an area of about 2.5cm ⁇ 2.5cm.
  • 20 ⁇ L of 0.2% DNFB solution was applied to the back of the mice, and 20 ⁇ L of 0.2% DNFB solution was applied to the right ear. Only the acetone/olive oil matrix solution was applied to the depilated area of the back of the mice in the blank control group. All groups had free access to water and food .
  • each sample group has a significant decrease (P ⁇ 0.05), and the ear swelling rate of the double-bacteria sample group is lower than that of the single-bacteria sample group (P ⁇ 0.05) .
  • each sample group tended to decrease (P ⁇ 0.05).
  • each sample group had a significant increase (P ⁇ 0.05), and the double-bacteria group had a more significant effect than the single-bacteria group (P>0.05).
  • each sample group had a significant decrease (P ⁇ 0.05), and the double-bacteria group had a more significant effect than the single-bacteria group (P>0.05).
  • the composition sample group was improved, and the double-bacteria group was significantly better than the single-bacteria group.
  • the skin sections of mice in the normal group were normal, the tissue structure was basically normal, the cells were arranged in an orderly manner, and the cortex was normal. Intracellular edema; In each sample group, the intracellular edema was not obvious, and there were a few inflammatory cells in the dermis. The whole layer of skin was intact, and the keratinization of the epidermis was weakened.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Pulmonology (AREA)
  • Nutrition Science (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Immunology (AREA)
  • Dermatology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种益生元和益生菌复合物的组合物及其应用，所述组合物中所述的益生元为低聚果糖FOS、低聚半乳糖GOS和抗性糊精，所述益生菌复合物为鼠李糖乳杆菌和乳双歧杆菌的复合物；所述鼠李糖乳杆菌、乳双歧杆菌、低聚果糖FOS、低聚半乳糖GOS和抗性糊精的重量比为(1～9):(0.5～3):(4～30):(55～100):(30～90)。所述的鼠李糖乳杆菌MP108活菌数为1*10 7-10 11CFU/g，乳双歧杆菌HN019的活菌数为3*10 6-10 10CFU/g。所述益生元和益生菌复合物的组合物可以用于制备细菌性腹泻缓解剂、便秘缓解剂、消化促进制剂、食物过敏功效评价制剂、过敏性哮喘缓解剂、皮炎缓解剂。

Description

一种益生元和益生菌复合物的组合物及其应用 技术领域
本发明属于食品营养学技术领域，具体涉及一种益生元和益生菌复合物的组合物及其应用。
背景技术
目前，在实际生产组合物过程中，只加入益生菌菌粉，会导致菌粉粘附在条包壁上无法倒出而产生较大的益生菌损耗。鼠李糖乳杆菌MP108是中国卫健委批准的第一株中国自主知识产权的婴幼儿可食用菌株。目前，并没有以鼠李糖乳杆菌MP108与乳双歧杆菌HN019形成益生菌复合物的报道，同时，也没有将两种益生菌与益生元制成组合物的相关报道，更没有以此组合物为基础进行的专利申请活动。
现有益生菌产品的功效都是直接使用原辅料本身高含量时所具有的功能，而并没有针对所生产的产品进行功能验证。在产品生产工程中，不同的原辅料组成后，不能保证产品的功效。目前益生菌产品所具有的功效通常为1～2个，常见的为提高免疫力和缓解便秘，而本专利所构建的配方具有缓解特应性皮炎、缓解过敏性哮喘、缓解细菌性腹泻、缓解食物过敏、促进消化以及缓解便秘的六大功效。
发明内容
本发明旨在克服现有技术的不足，提供一种益生元和益生菌复合物的组合物及其应用。
为了达到上述目的，本发明提供的技术方案为：
所述益生元和益生菌复合物的组合物中所述的益生元为低聚果糖FOS、低聚半乳糖GOS和抗性糊精，所述益生菌复合物为鼠李糖乳杆菌和乳双歧杆菌的复合物；所述鼠李糖乳杆菌、乳双歧杆菌、低聚果糖FOS、低聚半乳糖GOS和抗性糊精的重量比为(1～9):(0.5～3):(4～30):(55～100):(30～90)。
优选地，所述益生元中低聚半乳糖GOS、低聚果糖FOS、抗性糊精的重量比为9:1:7。
优选地，所述益生菌复合物为鼠李糖乳杆菌MP108和乳双歧杆菌HN019的复合物,所述益生菌复合物中鼠李糖乳杆菌MP108和乳双歧杆菌HN019的重量比为5:1。
优选地，所述的鼠李糖乳杆菌MP108活菌数为1*10 7-10 11CFU/g，乳双歧杆菌HN019的活菌数为3*10 6-10 10CFU/g。
所述益生元和益生菌复合物的组合物的制备方法如下：第一步、将低聚果糖、低聚麦芽糖、抗性糊精按质量比倒入单柱式料斗混合机中进行一级预混，形成一级预混料，其中：一级预混的转速为3-20转/分钟，正向10～20分钟，反向10～20分钟。第二步、将鼠李糖乳杆菌MP108菌粉和乳双歧杆菌HN019菌粉加入上述一级预混料在单柱式料斗混合机中混匀，混合均匀后形成复配益生菌益生元组合物，其中：二级混合的转速为5～15转/分钟，正向8～15分钟，反向8～15分钟。混合均匀度≥99％。
本发明以BALB/c小鼠为研究对象，实验结果表明：本发明所述的益生元和益生菌复合物的组合物具有缓解细菌性腹泻的作用，具有缓解便秘的作用，具有促进消化的作用，具有缓解食物过敏功效的作用，具有缓解过敏性哮喘的作用，具有缓解特定性皮炎的作用，能够抑制其他细菌生长，同时该组合中的益生菌具有耐酸耐胆碱的特征。
所述益生元和益生菌复合物的组合物可以用于制备细菌性腹泻缓解剂、便秘缓解剂、消化促进制剂、食物过敏功效评价制剂、过敏性哮喘缓解剂、皮炎缓解剂。
本发明将鼠李糖乳杆菌MP108和乳双歧杆菌HN019两种菌株以一定比例形成组合物后，能够互相补充单一菌株功效上的不足，使其发挥更好的保护肠道健康的作用。不仅如此，除双菌组合外，本发明中提到的益生元组合物可以促进组合物中的益生菌生长，会 使得组合物中的益生菌更好的在肠道中活化以及定植。目前，市面上有很多种益生元可以选择，根据不同益生菌有不同的生长特性，只有经过科学的实验验证，才能找到最适合双菌组合生长的益生元，并与其形成益生菌益生元组合物，使其在最大限度的活化组合物中益生菌的活性的同时，将组合物中益生菌的功效发挥至最大，进而提高益生菌在宿主中的存活和定植。而本发明也通过相关试验证实，所应用的不同比例GOS和FOS可以同时促进两种益生菌的生长。
附图说明
图1：各组小鼠空肠病理切片(100X)；
图2：各组小鼠空肠病理切片(X200)；
图3：各组肺组织切片(X200)；
图4：小鼠背部皮损HE染色病理切片(100X)。
具体实施方式
实施例1 组合物在缓解细菌性腹泻中的应用
1.内容
以BALB/c小鼠为研究对象，通过对小鼠灌胃不同的样品溶液，检测这些样品对腹泻小鼠的体重、粪便含水量、血清免疫炎症因子、空肠组织病理的影响，确定这些样品在缓解细菌性腹泻方面的功效及差异性。
2.方案
雄性SPF级BALB/c小鼠(6周龄，体重18-20g)适应性喂养一周后，小鼠随机分成包括空白对照组、模型组、单菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：GOS：FOS：抗性糊精＝6:68:25:74，一级预混的转速为15转/分钟，正向12分钟，反向12分钟。二级混合的转速为10转/分钟，正向8分钟，反向8分钟。)，双菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：乳双歧杆菌HN019：FOS：GOS：抗性糊精＝5:1:25：68:74，工艺同单菌组)，样品均溶于0.2mL无菌生理盐水中，按照表1的要求进行灌胃处理，空白对照组和模型组灌胃0.2ml无菌生理盐水。
表1
Figure PCTCN2021127363-appb-000001
第三周开始，在饮用水中加入链霉素(5g/升)处理3天，将小鼠肠道菌群变的紊乱。然后用无菌水代替含有链霉素的水，同时造模前18小时前停止进食。
从第三周第4天开始，空白对照组每天灌胃3次生理盐水，模型组先灌胃1次生理盐水，然后每天灌胃2次1.2×10 11CFU/mL的ETEC O78：K80悬液，每只小鼠每次灌胃0.2mL，连续灌胃四天，每次间隔2小时，灌胃ETEC后更换鼠笼，不添加垫料，在鼠笼中铺滤纸，每隔2h观察小鼠的腹泻情况和死亡率，持续四天；各样品组分别先灌胃1次相应样品溶液，然后每天灌胃2次1.2×10 11CFU/mL的ETEC O78：K80悬液，每只小鼠每次灌胃0.2mL，其余处理同模型组小鼠。
实验期间，小鼠自由进食进水，各组小鼠均饲喂标准饲料，饲养屏障内为恒温恒湿环境，温度为25±2℃，湿度为50％±5％，光照条件为12小时光照，12小时黑夜。
3.具体指标：
①粪便含水量：小鼠处死前一天，尽可能多的收集小鼠粪便，冻干之后测量前后的含水量，粪便含水量(％)＝(粪便湿重-粪便干重)/粪便湿重×100％；
②血清中炎症因子TNF-α、IL-6和IL-10的测定：在最后一次灌胃的24h后，从眶静脉 丛血样采集，血液静置，4000g离心，10min，离心取血清，置于-80℃冰箱保存，酶联免疫吸附方法测定血清中炎症因子的表达水平
③腹泻相关蛋白的测定：采用酶联免疫吸附测定方法检测小鼠血清中AQP3、ST、TLR4和IgA的水平
④空肠病理观察
4.实验结果
4.1粪便含水量
长时间的腹泻会导致小鼠体重下降，粪便不成形。因此通过记录小鼠的粪便含水量能够知晓腹泻症状变化(表2)。
表2 各组小鼠的粪便含水量
Figure PCTCN2021127363-appb-000002
注:*表示与模型组差异显著性(P<0.05)，△表示与单菌样品组差异显著性(P<0.05)下同。
从表2可知，与空白组相比较，模型组因为腹泻造成粪便不成型，含水量过多成稀状，体重下降严重，说明腹泻模型造模成功。
与模型组相比较，双菌样品组的含水量有显著性降低(P<0.05)。且双菌样品组比单菌样品组效果显著提升(P<0.05)。
说明双菌样品有缓解降低粪便含水量的作用，有助于粪便成型。高剂量效果更明显。
4.2对腹泻相关蛋白的影响(表3)
表3 各组小鼠与腹泻相关蛋白水平
Figure PCTCN2021127363-appb-000003
那个表可知，在AQP3和IgA水平上，各样品组与模型组相比较含量有显著性上升(P<0.05)。通过AQP3的上调，控制肠道内水分子的吸收，降低粪便含水量。IgA增加能增强机体的免疫能力。说明各样品组有缓解细菌性腹泻的作用，使小鼠在AQP3和IgA水平上表达恢复。且双菌组更有利于AQP3上调，差异显著(P<0.05)。
ST是大肠杆菌肠毒素，TLR4的增加会促进各种炎性细胞的表达，在ST和TLR4水平上，各样品组与模型组相比较有显著性下降(P<0.05)。说明各样品组有缓解细菌性腹泻的作用，使小鼠在ST和TLR4水平上表达恢复。且双菌组较单菌组更有利于小鼠在ST和TLR4水平上表达恢复，差异显著(P<0.05)。
4.3各组空肠的病理分析
从图1可知，模型组空肠肠壁变薄，肠壁有空隙，肠绒毛变短，肠通道变宽，形态不完整，有少许炎症细胞。其他各样品组相较与模型组，有一定的恢复作用，肠绒毛长度正常，肠壁空隙恢复，细胞浸润不明显。
实施例2 组合物在缓解便秘方面的应用
1.内容：
以BALB/c小鼠为研究对象，通过对小鼠灌胃益生菌，检测这些样品对便秘小鼠的粪便湿重、粪便含水量、粪便颗粒数、小肠推进率、首粒排黑便时间的影响，确定样品在缓解便秘方面的功效及差异性。
2.方案：
6周龄SPF级BALB/c雄性小鼠经过一周的适应期后，按照每组6只小鼠随机分组，包括空白对照组、模型组、单菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：GOS：FOS：抗性糊精＝9.5:55:4:30，一级预混的转速为20转/分钟，正向10分钟，反向10分钟。二级混合的转速为9转/分钟，正向13分钟，反向13分钟。)，双菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：乳双歧杆菌HN019：GOS：FOS：抗性糊精＝9:0.5:55:4:30，工艺同单菌组)，样品均溶于0.2mL无菌生理盐水中，按照表4的要求进行灌胃处理，空白对照组和模型组灌胃0.2ml无菌生理盐水。
表4
Figure PCTCN2021127363-appb-000004
第三周开始，空白对照组每天灌胃无菌生理盐水两次，共灌胃8天；模型组、样品组每天均先灌胃盐酸洛哌丁胺，1h后样品组灌胃相对应的样品溶液0.2mL，对照组和模型组等量灌胃无菌生理盐水，连续灌胃8天。
实验期间，小鼠自由进食进水，各组小鼠均饲喂标准饲料，饲养屏障内为恒温恒湿环境，温度为25±2℃，湿度为50％±5％，光照条件为12小时光照，12小时黑夜。
3.具体指标：
①粪便湿重、粪便颗粒数、粪便含水量的测定；
粪便含水量(％)＝(粪便湿重-粪便干重)/粪便湿重×100％
②首粒排黑便时间的测定；
在小鼠灌胃的最后一天(第8天)进行小鼠首粒排黑便时间的检测，检测方法如下：除空白对照组之外，所有组小鼠先灌胃盐酸洛哌丁胺，1h之后，空白对照组和模型对照组用墨汁灌胃，其余组灌胃含有样品的墨汁，从灌胃墨汁开始，记录每只动物首粒排黑便时间，以模型组最后一只小鼠的首粒黑便时间为终止时间，超过模型组首粒黑便时间的处理组则说明无效，比较各个处理组与模型组之间的差异，用于说明各组在缓解便秘方面的差异。
③小肠推进率的测定；
小鼠经8天灌胃后，于第8天晚上禁食过夜，第9天给除了空白对照组之外的所有组小鼠灌胃盐酸洛哌丁胺，30min后，空白对照组和模型对照组用墨汁灌胃，其余组灌胃含有样品的墨汁，30min后处死小鼠，打开腹腔，分离肠系膜，剪取上端自幽门，下端至回盲肠的肠管，轻轻将小肠拉成直线，测量肠管长度为“小肠总长度”，从幽门到墨汁前沿为“墨汁推进长度”，按照下式计算小肠推进率。
小肠推进率(％)＝墨汁推进长度(cm)/小肠总长度(cm)×100％
④血清IFN-α、TGF-β1、TNF-α、IL-1β、IL-6、IL-10、IL-17A水平；
⑤胃泌素等信号分子检测
4.实验结果
4.1对墨汁推进率、首粒黑便时间和粪便含水量的影响(表5)
表5 墨汁推进率、首粒黑便时间和粪便含水量的影响
Figure PCTCN2021127363-appb-000005
注:*表示与模型组差异显著性(P<0.05)，△表示与单菌样品组差异显著性(P<0.05)下同。
如表5所示，小肠的墨汁推进率方面，与模型组相比，样品组都有显著性提高(P<0.05)，且双菌样品组效果优于单菌样品组(P<0.05)。
在首粒黑便的排便时间方面，与模型组相比，样品组都有显著性提高(P<0.05)，且双菌样品组效果优于单菌样品组(P<0.05)。
在粪便含水量方面，与模型组相比，样品组都有显著性提高(P<0.05)，且双菌样品组效果优于单菌样品组(P<0.05)。
综上所述，该专利组合物具有提高小肠蠕动的能力。
4.2血清中信号分子的影响(表6)
表6 血清中信号分子水平的影响
Figure PCTCN2021127363-appb-000006
由上表可知，与模型组相比，菌粉样品组都有显著性提高(P<0.05)，与空白组相比无显著性差异(P>0.05)，能够提高小鼠血清中胃动素(MTL)、胃泌素(Gas)的含量，降低血管活性肠肽(VIP)、生长抑素(SS)。
综上所述，该专利组合物能通过对MTL、Gas的分泌的促进作用，同时对VIP，SS的分泌有抑制作用，改善小鼠的便秘症状，结果与上文小肠推进率、黑便时间相同。
实施例3 组合物在助消化中的应用
1.内容：
以BALB/c雄性小鼠为研究对象，通过对小鼠灌胃不同的样品，检测这些样品对小鼠体重增长率、食物利用率、胃蛋白酶活性的影响，确定这些样品在助消化方面的功效及差异性。
2.方案
选取6-8周龄，18-22g的SPF级BALB/c雄性小鼠，包括空白对照组、单菌样品组(36.9mg/只/d,鼠李糖乳杆菌MP108：GOS：FOS：抗性糊精＝9.5:99:30:90，一级预混的转速为20转/分钟，正向11分钟，反向11分钟。二级混合的转速为13转/分钟，正向13分钟，反向13分钟。)、双菌样品组(36.9mg/只/d，36.9mg/只/d，鼠李糖乳杆菌MP108：乳双歧杆菌HN019：GOS：FOS：抗性糊精＝7:2.5:99:30:90，工艺同单菌组)，每组6只小鼠，组合物样品均溶于0.2mL无菌生理盐水中,按照表7的要求进行灌胃处理，空白对照组和模型组灌胃0.2ml无菌生理盐水。
表7
Figure PCTCN2021127363-appb-000007
适应期为1周，饲料原料用锤片式粉碎机粉碎,过80目筛,均匀混合,然后按逐级放大的原则加入0.01％的Y2O3指示剂，充分混合均匀后用制粒机制成颗粒饲料，室温风干至恒重，实验期间各组小鼠自由饮水与摄食。空白对照组灌胃生理盐水，其余各组小鼠分别灌胃相应的样品溶液(0.2mL)，实验期间每周利用代谢笼系统测定2次体重和食物摄入量。实验结束时计算体重，体重增重，摄食量，食物利用率，总耗氧量，表观消化率。
实验结束前16h禁食不禁水，利用胃瘘法制备小胃提取小鼠的胃液，用于测定胃蛋白酶的活性。
3.具体指标
①体重增长率；②食物利用率；③酶活性
4.实验结果
4.1小鼠体重增长率和食物利用率(表8)
Figure PCTCN2021127363-appb-000008
Figure PCTCN2021127363-appb-000009
表8 各组小鼠体重增长率和食物利用率
Figure PCTCN2021127363-appb-000010
注:*表示与模型组差异显著性(P<0.05)，△表示与单菌样品组差异显著性(P<0.05)下同。
由表8可知，各样品组与空白组相比较小鼠的食物利用率和体重增长率有显著性提高(P<0.05)，且双菌组比单菌组效果更显著(P<0.05)，说明该组合物能够提升小鼠的食物利用率，增加小鼠的体重。
4.2小鼠胃蛋白酶活力(表9)
表9 各组小鼠胃蛋白酶酶活力
Figure PCTCN2021127363-appb-000011
由表9可知，与空白组相比较，样品组的胃蛋白酶酶活力有显著性提高(P<0.05)，可以提升小鼠的消化能力，双菌组的效果显著优于单菌组(P<0.05)。
实施例4 组合物在缓解食物过敏中的应用
1.内容
以BALB/c雌性小鼠为研究对象，通过对小鼠灌胃不同的样品，检测这些样品对小鼠体重变化，腹泻及其他过敏症状评分，肠组织组织病理学观察，肥大细胞及嗜酸性粒细胞计数，血清OVA特异性免疫球蛋白水平，Th2炎症因子水平的影响，确定这些产品在缓解食物过敏方面的功效及差异性。
2.方案
选取5周龄，体重13～16g的SPF级BALB/c雌鼠，将小鼠随机分为空白对照组、模型组、单菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：GOS：FOS：抗性糊精＝11:70:23：80，一级预混的转速为16转/分钟，正向15分钟，反向15分钟。二级混合的转速为14转/分钟，正向10分钟，反向10分钟。)，双菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：乳双歧杆菌HN019：GOS：FOS：抗性糊精＝9:2:70:23：80，工艺同单菌组)，每组8只，组合物样品均溶于0.2mL无菌生理盐水中,按照表10的要求进行灌胃处理，空白对照组和模型组灌胃0.2ml无菌生理盐水。
表10
Figure PCTCN2021127363-appb-000012
致敏液：取3mg OVA溶解于6mL生理盐水中，再加入6mL氢氧化铝佐剂，混合均匀。
激发液：取2.8g OVA溶解于11.2mL生理盐水中，混合均匀。
给药方式：致敏阶段为腹腔注射，激发阶段为灌胃。
致敏阶段：第3天和第17天使用0.1mL含50μg OVA的生理盐水+0.1mL氢氧化铝佐剂腹腔注射致敏。空白组使用等量的生理盐水代替。
激发阶段：第31～43天每隔一天使用0.2mL的含50mg OVA生理盐水进行灌胃激发过敏，共激发7次。空白组使用等量的生理盐水代替。
小鼠在灌胃样品溶液前3h禁食，灌胃1h后进行OVA的致敏或激发。
实验期间每天观察小鼠的腹泻、过敏症状并进行评分，每周进行称重。最后一天灌胃之后，禁食12h后对其进行麻醉处理，采用1％戊巴比妥钠按照0.5mL/10g体重腹腔注射。麻醉后进行眼眶取血，取血后颈椎脱臼法处死，解剖，取空肠组织进行组织病理分析，取材后迅速将其置于甲醛固定液中固定12h，取出后用PBS冲洗，然后进行脱水、包埋、切片。同时检测小鼠血清OVA特异性IgE及肠组织炎症因子(IL-4、IFN-γ、IL-17、TGF-β等)水平。将取血后的血样4℃下静置2h后，3000×g离心15min，分离血清于无酶管中，置于-80℃冰箱保存备用。
3.具体指标：
①小鼠体重变化
②腹泻及其他过敏症状评分(表11)
表11
Figure PCTCN2021127363-appb-000013
③肠组织组织病理学观察
④血清OVA特异性免疫球蛋白水平
4.实验结果
4.1小鼠体重变化(表12)
表12 各组小鼠体重变化率和血清OVA-IgE水平
Figure PCTCN2021127363-appb-000014
注:*表示与模型组差异显著性(P<0.05)，△表示与单菌样品组差异显著性(P<0.05)下同。
如表12所示，体重变化率方面，与模型组相比，组合物组均有所提高，双菌组效果显著高于单菌组。
在血清OVA-IgE水平方面，与模型组相比，组合物组都有显著性提高(P<0.05)，双菌组效果显著优于单菌组(P>0.05)。
4.2腹泻及其他过敏症状评分(表13)
表13 各组小鼠腹泻及其他过敏症状评分
Figure PCTCN2021127363-appb-000015
如表13所示，在腹泻评分上，与模型组相比较，各组腹泻评分都有显著性降低(P<0.05)。
在过敏评分上，与模型组相比较，样品组的评分显著性降低。
4.3各组空肠的病理分析
从图2可知，模型组空肠肠壁变薄，形态不完整，质壁有大量炎症细胞，肠绒毛变短。其他各样品组相较与模型组，有一定的恢复作用，肠绒毛长度正常，肠壁形态恢复正常，细胞浸润不明显。
实施例5 组合物在缓解过敏性哮喘中的应用
1.内容
以BALB/c雌性小鼠为研究对象，通过对小鼠灌胃不同的样品，检测这些样品对小鼠血清特异性IgG1水平，肺泡灌洗液中IL-5，IL-13和IL-17A的含量，肺部病理，粪便短链脂肪酸含量的影响，确定这些样品在缓解过敏性哮喘(鼻炎)方面的功效及差异性。
2.方案
将SPF级BALB/c雌性小鼠(6周龄)分组，经过一周的适应期后，按照每组8只小鼠随机分组，共分成4组，包括空白对照组、模型组、单菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：GOS：FOS：抗性糊精＝8.8:59:18：32，一级预混的转速为20转/分钟，正向16分钟，反向16分钟。二级混合的转速为13转/分钟，正向8分钟，反向8分钟。)，双菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：乳双歧杆菌HN019：GOS：FOS：抗性糊精＝7.8：1:59:18：32，工艺同单菌组)。各样品组样品均溶于0.2mL无菌生理盐水中，按照表14的要求进行灌胃处理，空白对照组和模型组灌胃0.2ml无菌生理盐水。
表14
Figure PCTCN2021127363-appb-000016
实验期间，小鼠自由进食进水，各组小鼠均饲喂标准饲料，饲养屏障内为恒温恒湿环境，温度为25±2℃，湿度为50％±5％，光照条件为12小时光照，12小时黑夜。
实验结束后，麻醉小鼠，眼球采血，静止2h后，于离心机3000rpm离心10分钟，收集血清于干净的离心管中，通过HDM特异性IgG1ELISA试剂盒检测血清中HDM特异性IgG1的含量；解剖胸腔和颈部，使小鼠气管和双肺暴露，结扎右肺，用留置针进行气管插管，以1mL注射器吸取0.3mL预冷的PBS，灌洗小鼠左肺，进行3次，回收量≥80％，收集灌洗液于干净的Eppendorf管中。于离心机上，4℃，1000rpm，5min，收集上清液，分装于Eppendorf管中，冻存-20℃，通过ELISA试剂盒检测肺泡灌洗液中的IL-5，IL-13和IL-17A的含量；然后将琼脂糖溶液注入小鼠左肺后，取下左肺置于4％的多聚甲醛溶液中固定，进行石蜡包埋，HE染色，送病理科进行肺部炎症程度评估；通过小鼠血清特异性IgG1，肺泡灌洗液中IL-5、IL-13和IL-17A，以及肺部病理炎症程度的变化，全面地评价益生菌对小鼠哮喘症状的影响。
3.具体指标
①血清特异性IgG1水平；
②肺泡灌洗液中IL-4、IL-5、IL-13、IL-17A、IFN-γ、IL-10的含量；
③肺部病理
4.实验结果
4.1对血清特异性IgG1水平的影响(表15)
表15 各组血清中特异性IgG1水平
Figure PCTCN2021127363-appb-000017
注:*表示与模型组差异显著性(P<0.05)，△表示与单菌样品组差异显著性(P<0.05)下同。
由表15可知，在IgG1水平上，与模型组相比，各样品组有都显著性下降(P<0.05)，双菌样品组较单菌样品组效果更显著。
说明该专利组合物能通过降低IgG1水平，缓解小鼠过敏性哮喘的症状。
4.2对肺泡灌洗液中细胞因子水平的影响(表16)
表16 各组肺泡灌洗液中细胞因子水平
Figure PCTCN2021127363-appb-000018
由表16可知，在IL-4、IL-5、IL-13、IL-17A水平上，与模型组相比，各样品组都有显著性下降(P<0.05)。在IL-10和IFN-γ水平上，与模型组相比，各样品组都有显著性提高(P<0.05)。与单菌样品组相比，双菌样品组在IL-4、IL-5、IL-13、IL-17A水平上有显著下降，在IL-10和IFN-γ水平上，有显著提升。
综上，本专利组合物可使哮喘小鼠肺组织嗜酸性粒细胞渗出减少，IL-4、IL-5、IL-13和IL-17A的分泌减少，还可以通过分泌免疫负调因子IL-10和IFN-γ抑制炎症反应，从而发挥缓解哮喘的作用。
4.3肺部病理切片
如图3所示，与空白组相比，模型组小鼠的气管、支气管、肺泡及血管周围可见大量的嗜酸性细胞和淋巴细胞等浸润、黏膜上皮增生明显、气道上皮损伤严重及肺泡间隔增厚等病理表现。
各样品组的病理损伤程度较过敏性哮喘模型组明显减轻，肺组织及周围的炎症细胞浸润明显减少、黏膜上皮轻度增生、气道上皮损伤减轻及肺泡间隔增厚程度减轻，各对应的高剂量组在病理损伤相对低剂量组无明显的差异。
实施例6 组合物在缓解特应性皮炎的应用
1、内容
以C57BL/6雌性小鼠为研究对象，通过对小鼠灌胃不同的样品，检测这些样品对小鼠耳朵肿胀程度的变化，皮肤病理的变化，血清IgE水平，皮肤组织IL-4、IL-5、IL-13、IL-10、IFN-γ的水平，确定这些样品在缓解特应性皮炎(湿疹)方面的功效及差异性。
2、方案
将6周龄雄性C57BL/6J小鼠随机分为4组，每组8只，包括空白对照组、模型组、单菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：GOS：FOS：抗性糊精＝8.9:77:5：89，一级预混的转速为13转/分钟，正向19分钟，反向19分钟。二级混合的转速为8转/分钟，正向14分钟，反向14分钟。)，双菌样品组(36.9mg/只/d，鼠李糖乳杆菌MP108：乳双歧杆菌HN019：GOS：FOS：抗性糊精＝8：0.9：77:5：89，工艺同单菌组)，各样品组样品均溶于0.2mL无菌生理盐水中，按照表17的要求进行灌胃处理，空白对照组和模型组灌胃0.2ml无菌生理盐水。
表17
Figure PCTCN2021127363-appb-000019
Figure PCTCN2021127363-appb-000020
造模方法具体如下：模型组和组合物组第14天用脱毛器脱去小鼠背部体毛，面积约2.5cm×2.5cm。实验第15天各样品组小鼠皮损致敏与激发用0.5％DNFB溶液50μL一次小鼠背部脱毛区及右耳处，然后在第19天，第22天，第25天，第28天在小鼠背部用0.2％DNFB溶液20μL涂搽，右耳处用0.2％DNFB溶液20μL涂搽，空白对照组小鼠背部脱毛区仅涂搽丙酮/橄榄油基质溶液，所有分组均为自由饮水和摄食。
3.具体指标
①小鼠耳朵的肿胀程度
②血清IgE水平
③皮肤组织IFN-γ、CCL11、CCL22的水平
4.实验结果
4.1小鼠耳肿胀率和血清IgE水平(表18)
表18 小鼠耳肿胀率和血清IgE水平
Figure PCTCN2021127363-appb-000021
注:*表示与模型组差异显著性(P<0.05)，△表示与单菌样品组差异显著性(P<0.05)下同。
如表18所示，耳肿胀率方面，与模型组相比，各样品组都有显著性降低(P<0.05)，双菌样品组较单菌样品组耳肿胀率更低(P<0.05)。
在血清IgE水平方面，与模型组相比，各样品组有趋势下降(P<0.05)。
4.2各组小鼠皮肤组织中细胞因子水平(表19)
表19 皮肤组织中INF-γ、CCL11和CCL22水平的影响
Figure PCTCN2021127363-appb-000022
如表19所示，在INF-γ水平，与模型组相比，各样品组都有显著性提高(P<0.05)，双菌组比单菌组效果更显著(P>0.05)。
在CCL11水平，与模型组相比，各样品组都有显著性降低(P<0.05)，双菌组比单菌组效果更显著(P>0.05)。
在CCL22水平，与模型组相比，组合物样品组有提高，双菌组显著优于单菌组。
4.3皮肤组织的病理切片分析
如图4，正常组小鼠皮肤切片正常，组织结构基本正常，细胞排列有序形态正常，皮层正常；模型组小鼠皮肤切片可见大量炎症细胞浸润，表皮层角化严重，大量皮质缺失，细胞间，细胞内水肿；各样品组小鼠皮肤可见细胞内水肿不明显，真皮内有少数炎症细胞，皮肤全层完整，表皮层角化减弱。
以上结果表明，该专利组合物具有缓解特应性皮炎的作用。

Claims (10)

  1. 一种益生元和益生菌复合物的组合物，其特征在于，所述组合物中所述的益生元为低聚果糖FOS、低聚半乳糖GOS和抗性糊精，所述益生菌复合物为鼠李糖乳杆菌和乳双歧杆菌的复合物；所述鼠李糖乳杆菌、乳双歧杆菌、低聚果糖FOS、低聚半乳糖GOS和抗性糊精的重量比为(1～9):(0.5～3):(4～30):(55～100):(30～90)。
  2. 如权利要求1所述的益生元和益生菌复合物的组合物，其特征在于，所述益生元中低聚半乳糖GOS、低聚果糖FOS、抗性糊精的重量比为9:1:7。
  3. 如权利要求1所述的益生元和益生菌复合物的组合物，其特征在于，所述益生菌复合物为鼠李糖乳杆菌MP108和乳双歧杆菌HN019的复合物,所述益生菌复合物中鼠李糖乳杆菌MP108和乳双歧杆菌HN019的重量比为5:1。
  4. 如权利要求1所述的益生元和益生菌复合物的组合物，其特征在于，所述的鼠李糖乳杆菌MP108活菌数为1*10 7-10 11CFU/g，乳双歧杆菌HN019的活菌数为3*10 6-10 10CFU/g。
  5. 如权利要求1所述益生元和益生菌复合物的组合物在制备细菌性腹泻缓解剂中的应用。
  6. 如权利要求1所述益生元和益生菌复合物的组合物在制备便秘缓解剂中的应用。
  7. 如权利要求1所述益生元和益生菌复合物的组合物在制备消化促进制剂中的应用。
  8. 如权利要求1所述益生元和益生菌复合物的组合物在制备食物过敏功效评价制剂中的应用。
  9. 如权利要求1所述益生元和益生菌复合物的组合物在制备过敏性哮喘缓解剂中的应用。
  10. 如权利要求1所述益生元和益生菌复合物的组合物在制备皮炎缓解剂中的应用。
PCT/CN2021/127363 2021-10-29 2021-10-29 一种益生元和益生菌复合物的组合物及其应用 WO2023070513A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202180004590.3A CN114401728B (zh) 2021-10-29 2021-10-29 一种益生元和益生菌复合物的组合物及其应用
PCT/CN2021/127363 WO2023070513A1 (zh) 2021-10-29 2021-10-29 一种益生元和益生菌复合物的组合物及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/127363 WO2023070513A1 (zh) 2021-10-29 2021-10-29 一种益生元和益生菌复合物的组合物及其应用

Publications (1)

Publication Number Publication Date
WO2023070513A1 true WO2023070513A1 (zh) 2023-05-04

Family

ID=81228046

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/127363 WO2023070513A1 (zh) 2021-10-29 2021-10-29 一种益生元和益生菌复合物的组合物及其应用

Country Status (2)

Country Link
CN (1) CN114401728B (zh)
WO (1) WO2023070513A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115161348B (zh) * 2022-04-28 2024-09-13 海普诺凯营养品有限公司 一种具有多功效的后生元组合物
CN115011516B (zh) * 2022-06-13 2023-11-14 东北农业大学 一种具有缓解便秘功效的合生元酸奶及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107468716A (zh) * 2017-08-24 2017-12-15 北京亦贝安生物医药科技有限公司 一种益生菌和益生元复方制剂及其加工方法
CN109170909A (zh) * 2018-10-10 2019-01-11 河北畅然生物科技有限公司 一种改善便秘的复合益生菌和益生元组合物及其制备方法
CN109198646A (zh) * 2018-10-29 2019-01-15 广州普维君健药业有限公司 具有防治肠道炎症性疾病功效的益生菌组合物及其应用
CN110432494A (zh) * 2018-05-03 2019-11-12 广州普维君健药业有限公司 防治婴幼儿过敏性湿疹和/或腹泻的益生菌组合物和应用
CN111345453A (zh) * 2018-12-24 2020-06-30 广西小咕噜生物科技有限公司 一种用于肠道的复合益生菌粉

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111528479A (zh) * 2020-05-15 2020-08-14 深圳爱米基因科技有限责任公司 一种缓解特应性皮炎功能的益生菌和益生元组合物及应用
CN112516265B (zh) * 2020-12-16 2022-06-21 青岛琛蓝生物营养技术有限公司 一种润肠通便的益生菌中草药复方组合物及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107468716A (zh) * 2017-08-24 2017-12-15 北京亦贝安生物医药科技有限公司 一种益生菌和益生元复方制剂及其加工方法
CN110432494A (zh) * 2018-05-03 2019-11-12 广州普维君健药业有限公司 防治婴幼儿过敏性湿疹和/或腹泻的益生菌组合物和应用
CN109170909A (zh) * 2018-10-10 2019-01-11 河北畅然生物科技有限公司 一种改善便秘的复合益生菌和益生元组合物及其制备方法
CN109198646A (zh) * 2018-10-29 2019-01-15 广州普维君健药业有限公司 具有防治肠道炎症性疾病功效的益生菌组合物及其应用
CN111345453A (zh) * 2018-12-24 2020-06-30 广西小咕噜生物科技有限公司 一种用于肠道的复合益生菌粉

Also Published As

Publication number Publication date
CN114401728A (zh) 2022-04-26
CN114401728B (zh) 2024-05-31

Similar Documents

Publication Publication Date Title
JP7497433B2 (ja) ラクトバチルス・パラカゼイｋ５６の腸管炎症の緩和における新規な用途
WO2023070513A1 (zh) 一种益生元和益生菌复合物的组合物及其应用
WO2023070512A1 (zh) 一种益生元和益生菌的组合物及其应用
CN114146101B (zh) 动物双歧杆菌乳亚种BLa80在制备调节肠道动力的药物或食物中的应用
CN111972670A (zh) 一种润肠通便的组合物、固体饮料及应用
CN111004733B (zh) 一种具有缓解便秘功能的凝结芽孢杆菌复合微生态制剂
CN110643524B (zh) 一种具有胃肠道粘膜保护作用的复合益生菌制剂及其应用
WO2021135798A1 (zh) 桑皮苷a及其衍生物在制备保护肠道屏障的药物中的应用
CN114848685A (zh) 抗过敏益生菌组合物及其应用、以及益生菌中药发酵物
WO2018192501A1 (zh) 一种预防肿瘤化疗肠道毒性的微生物菌剂
EP2510932A1 (en) Lactobacillus paracasei NCC2461 (ST11) for use by perinatal maternal administration in the reduction and prevention of allergies in progeny
CN118497046A (zh) 一种提升肠道免疫力的益生菌剂及其应用
Khursheed et al. Multifaceted role of synbiotics as nutraceuticals, therapeutics and carrier for drug delivery
CN114317367A (zh) 一种益生菌组合物、组合物的应用及辅助解酒制剂
Kawase et al. Inhibitory effect of Lactobacillus gasseri TMC0356 and Lactobacillus GG on enhanced vascular permeability of nasal mucosa in experimental allergic rhinitis of rats
TWI480045B (zh) 使用包含有４株乳酸菌菌株的混合物來預防和／或緩解酒精性肝病變
JP6442219B2 (ja) 経口免疫機能調整組成物
US20090110663A1 (en) Method for treatment of bowel disorders
WO2014134830A1 (zh) 可食用组合物以及包含其的食品、该食品的制备方法
CN117568244B (zh) 一种提高消化及营养吸收能力的益生菌剂及其应用
CN111560335A (zh) 一株用于缓解哮喘的乳双歧杆菌及其应用
KR20060119045A (ko) 아토피성 피부염 치료 및/또는 예방용 유산균 및 이를함유하는 조성물
US20220175855A1 (en) Mixture of probiotic bacteria strains for use in the treatment of allergic asthma and with recurrent wheezing, preferably in paediatrict subjects
CN118272277B (zh) 一种能够缓解腹泻的动物双歧杆菌及其应用
US20140037686A1 (en) Lactobacillus paracasei ncc2461 (st11) for use by perinatal maternal administration in the reduction and prevention of allergies in progeny

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21961872

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE