CN114568701B - Composition containing fructo-oligosaccharide and probiotics and application thereof - Google Patents
Composition containing fructo-oligosaccharide and probiotics and application thereof Download PDFInfo
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- CN114568701B CN114568701B CN202011381265.2A CN202011381265A CN114568701B CN 114568701 B CN114568701 B CN 114568701B CN 202011381265 A CN202011381265 A CN 202011381265A CN 114568701 B CN114568701 B CN 114568701B
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- bifidobacterium
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- bifidobacterium longum
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Classifications
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
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- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/385—Concentrates of non-alcoholic beverages
- A23L2/39—Dry compositions
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention provides a composition containing fructo-oligosaccharide and probiotics and application thereof. The probiotics in the composition containing fructo-oligosaccharide and probiotics are bifidobacterium animalis BB12 and/or bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496. The invention also provides application of the composition in promoting proliferation of bifidobacterium longum subspecies infantis GB-1496. The research of the invention discovers that the multiplication of the bifidobacterium infantis can be obviously promoted by adopting the FOS as a carbon source and adopting the bifidobacterium animalis BB12 and/or the bifidobacterium lactis HN019 to compound with the bifidobacterium longum subspecies infantis GB-1496, and the multiplication quantity is obviously higher than that of the single combination of the FOS and the bifidobacterium animalis BB12, the bifidobacterium lactis HN019 or the bifidobacterium longum subspecies infantis GB-1496.
Description
Technical Field
The invention relates to a composition containing fructo-oligosaccharide and probiotics and application thereof, belonging to the technical field of health-care food.
Background
BB12 belongs to bifidobacterium animalis and is a probiotic with the most extensive research history and product application. BB-12 is resistant to gastric acid and bile and can survive in an environment of pH2, mainly because BB12 has a gene encoding bile salt hydrolase that hydrolyzes bound bile salts in the intestinal tract to form amino acids and free bile acids [4]. There have been several studies demonstrating that BB-12 can survive the gastrointestinal tract and that BB-12 can be detected in the feces of term infants fed with the partially hydrolyzed whey formula supplemented with BB-12 at 6 weeks. BB-12 is detected in the feces of a subject after a healthy adult consumes BB-12 or BB-12+galacto-oligosaccharides for one week. The BB-12 level in the feces of the yogurt containing BB-12 and inulin was significantly higher than that of the control group, and 90% of BB-12 was viable bacteria [7] for adults for 4 weeks. Clinical trials in different populations have shown that BB12 has good safety and tolerability, including children 1-5 years [8], healthy adults with recent antibiotics [9], women from early gestation to lactation [10].
There are a number of clinical studies reporting various benefits that it brings to the human body. First, BB12 may bring about intestinal comfort. The infants with intestinal colic are supplemented with BB12 (10≡9 cfu/day), and crying time is reduced by more than 50% and more than that of the control group [11]. BB-12 was added to low lactose and whey protein partially hydrolyzed formula to feed infants with hernia for 1 month, crying of the infants in the intervening group continued significantly lower than the control group, and all parameters of the quality of life (QOL) questionnaire improved significantly [12]. For elderly and healthy adults in nursing homes, supplementation with BB-12 increases intestinal peristalsis [37] and frequency of bowel movement, especially the uncomfortable gastrointestinal symptoms that are insufficient to go to hospital consultation [38]. Second, BB12 is beneficial for immune health. The milk powder added with BB-12 has a preventive effect on acute diarrhea when fed to infants [39], and domestic clinical experiments show that compared with the treatment effects of BB-12, bacillus subtilis bigeminal viable bacteria (mammy love) and a blank control group in patients with children diarrhea, the results show that BB-12 is effective in treatment, and the effect is not significantly different from mammy love [40]. Partially hydrolyzed whey protein-supplemented milk powder supplemented with BB12 (10≡6cfu) was studied in healthy term infants, resulting in an increase in fecal IgA total in vaginally delivered formula fed infants. In formula fed infants produced by caesarean section, anti-rotavirus specific IgA was also elevated, and anti-poliovirus specific IgA was elevated in the feces of all probiotic-supplemented infants [41]. The premature infants were fed milk powder containing BB12 (1.6X10A 9 on the first three days, 4.8X10A 9cfu on the fourth day), and the pH of the faeces was significantly lower than that of the placebo group. Short chain fatty acids in the infant's faeces were elevated, with lower levels of calprotectin, whereas the faecal IgA levels were higher in this group compared to placebo [43]. BB-12 maintains the health of the gastrointestinal flora of premature infants, and premature infants were intervened with BB-12 for 3 weeks, both by fluorescence in situ hybridization and bacterial culture, and were found to significantly increase the number of intestinal bifidobacteria, while reducing the numbers of enterobacteria and clostridia [44]. Following long-term follow-up of neonates who were post-natally supplemented with BB-12, the respiratory tract infection was found to be significantly lower in infants at 8 months than in the control group [45], while the respiratory tract infection was still significantly lower in infants at 2 years [46]. The yogurt ice cream added with the milk BB12 before the fermentation process can improve IL-2 secretion and NK cytotoxicity and has less upper respiratory tract infection fever days [47] when the yogurt ice cream is eaten by healthy adults. Thirdly, BB requests beneficial oral health. BB12 tablets (10≡10 cfu/day) were administered to infants from 1-2 months to 2 years old, and the oral mutans streptococcus number was evaluated at 8 months, with BB12 group being the lowest [48].
HN019 is a probiotic separated from dairy products and has strong intestinal function regulating capability. In adult population with constipation problems, continuous 28-day supplementation with HN019 had significant intestinal vibrating effect on no more than 3 times per week of bowel movement [55]. The total intestinal transit time decreases with increasing dose and the number of gastrointestinal discomfort symptoms decreases with increasing dose for adults with gastrointestinal discomfort symptoms given HN019 daily [56]. Second, HN019 may stimulate the activated immune system. Children aged 1-3 years were enrolled in the community to compare milk with the same milk supplemented with oligosaccharides and HN019. The results suggest that probiotics have no effect on diarrhea following 1 year. The incidence of diarrhea is reduced by 21%, the incidence of pneumonia is reduced by 24%, and the incidence of severe acute lower respiratory tract infection (ALRI) is reduced by 35% [56]. The supplementation of HN019 (1.72×10≡10-1.8×10≡9-billion) to pregnant women can affect the change of immune related substances in umbilical cord blood and breast milk [57]. After the old people eat the milk added with HN019 (1.5X10-11 cfu), the phagocytic capacity of polymorphonuclear cells of the test group subjects is obviously increased, and the phagocyte-mediated bactericidal activity is enhanced [58]. Another study found that the administration of HN019 (2.5X10-10 cfu) increased the number of two white blood cells (polymorphonuclear and natural killer cells) [59]. The elderly take HN019 (two doses of 5X 10≡10 and 5X 10≡9 cfu), and the total, helper (CD4+), activated (CD25+) T lymphocytes and natural killer cells in the blood of the subjects are increased. After eating bifidobacterium lactis HN019, the in vitro phagocytic capacity of mononuclear and polymorphonuclear phagocytes and the tumor killing activity of natural killer cells are also improved. The greatest change in immune function occurs in subjects with poor pre-treatment immune responses. The two dose results were similar [60]. HN019 (5 x 10≡9, 1 x 10≡9, 6.5 x 10≡7 cfu) can significantly increase the number of bifidobacteria, lactobacilli and enterococci, and is beneficial to the immunity of the elderly. And the three dose groups are similar, the lowest dose can change the number of beneficial intestinal bacteria [61]. The supplementation of HN019 can increase CD56 positive lymphocytes in peripheral blood, and the killing activity of PBMC on K562 cells is also improved in vitro test. And subjects over 70 years old improved significantly over subjects under 70 years old. These results indicate that the consumption of probiotic laboratories in milk-based diets may be beneficial for elderly consumers against some of the detrimental effects on cellular immunity during immune aging [62]. Again, a series of studies evaluate that HN019 gives negative results for what should be allergic [63-66], and more elaborate studies indicate that HN019 is useful for allergic symptoms in a small number of people, and that some people with specific SNP may have an effect of preventing and treating allergy [67-68]. Finally, the supplementation of HN019 may also improve inflammation and periodontal pocket development in chronic periodontitis [69], improve anaemia [70] and reduce cardiovascular disease risk in metabolic syndrome patients [71].
Bi-07 reduces bacterial translocation and improves inflammation by restoring intestinal mucosal integrity [52], a specific mechanism possibly through inhibition of TNF expression [53] and binding to plasmin [54].
Breast milk contains not only abundant nutrients but also viable microbial flora. For breast-fed infants, breast milk micro-ecology is a key influencing factor for the intestinal micro-ecology development of infants. The most abundant species in the intestinal microecology of breast-fed infants is bifidobacteria in early life. Further microbiological analysis revealed that the intestinal flora of breast-fed infants is different from that of adults and is enriched with bifidobacterium infantis. Previous studies have shown that the efficacy of bifidobacterium infantis mainly includes the following aspects:
first, bifidobacterium infantis has a strong ability to utilize HMO, has advantages in intestinal colonisation, and unlike other bifidobacteria infantis contains a broader range of genes related to HMO transport and metabolism in the genome [13-19], and in vitro studies bifidobacterium infantis grows better in the presence of HMO than other strains [19,25], so bifidobacterium infantis strains have unique advantages over other conventional probiotics. The bifidobacterium infantis can also metabolize the produced short chain fatty acids to reduce intestinal pathogens and promote the growth and development of intestinal epidermal cells [36]. Short chain fatty acids are commonly acetic, propionic, butyric and lactic acids, which, besides being absorbed as energy substances by intestinal cells, contribute to the integrity of the intestinal epidermal cells [42]. Second, bifidobacterium infantis have anti-inflammatory effects, and intestinal inflammation in premature infants is an important cause of reduced Necrotizing Enterocolitis (NEC) in premature infants. The study using intestinal cells as a model shows that the bifidobacterium infantis can induce the intestinal cells to express anti-inflammatory cytokines [20-23], and the rat animal experiment shows that the addition of the bifidobacterium infantis can reduce the inflammation of NEC [24]. Bifidobacteria are often absent in the gut of premature infants [26], and the incidence of NEC in breast-fed premature infants is significantly lower than in formula-fed infants [27]. Unlike term breast milk, preterm breast milk contains a higher concentration of fucosylated HMO [28]. Comparing the feeding effect of bifidobacterium infantis and bifidobacterium lactis in the premature infant population, it was found that the ability of bifidobacterium infantis to colonize was stronger than that of bifidobacterium lactis, both in formula feeding and breast feeding, whereas the highest number of bifidobacterium faecalis was that of the group of bifidobacterium infantis fed breast milk [29]. A series of clinical studies have found that supplementation of preterm infants with bifidobacteria infancy can reduce the risk of NEC occurrence [31-35]. Finally, the advantage of adding bifidobacterium infantis is also reflected in immunity, and in a full term clinical study of Bengala, it is found that the number of bifidobacterium infantis in the intestinal tract of the infant is positively correlated with the vaccine response in the early four months of life, that is, the higher the number of bifidobacterium infantis is, the higher the corresponding index of the vaccine response is [30-31], and the trend still exists in the 2 years after birth [31], and it is speculated that bifidobacterium infantis helps to activate immune function and immune memory function.
Prebiotics are a class of carbohydrates that are indigestible by the human body and which interact with the intestinal flora in the intestinal tract of the host to produce substances or effects beneficial to the host [3]. More prebiotics currently studied include: galacto-oligosaccharides, fructo-oligosaccharides, inulin, isomerized lactose, polydextrose, xylo-oligosaccharides, and the like.
Fructo-oligosaccharide (FOS) is an excellent water-soluble dietary fiber, and is widely used in fruits, vegetables and the like. Fructooligosaccharides can now be generally divided into two types, mainly based on the raw materials and processes used for their manufacture. An inulin fructo-oligosaccharide is prepared from inulin extracted from herba Cichorii or Jerusalem artichoke by enzymolysis or acidolysis to obtain functional oligosaccharide with beta (2-1) glycosidic bond linkage and polymerization degree of 2-9, and has main structure form of Fn type (F is fructose, n is number of fructose), and also contains a certain amount of GFn type sucrose fructo-oligosaccharide; the other is sucrose fructo-oligosaccharide, the raw material is sucrose, and the structural form of the sucrose fructo-oligosaccharide, the sucrose pentasaccharide, the sucrose hexasaccharide and the mixture thereof are GFn type. The two fructooligosaccharides differ slightly in structure but have substantially identical physiological functions. The chemical structures of GFn type and Fn type are shown below.
The supplementation of FOS to premature infants is safe, and it can be observed that bifidobacteria in feces are increased, feces are softer, the number of times of defecation is more, and the number of pathogenic bacteria is reduced [72]. Supplementing FOS to children with chronic constipation in the age of 2-5 years can significantly improve stool consistency [73].
FOS is helpful for the promotion of immunity. For non-breast-fed children of 4-24 months of age, 0.55g FOS is supplemented daily, diarrhea-related fever, fever onset event, antibiotic use, and infection duration decrease [74 ]]. Administration of food containing FOS (dose not specifically defined) to elderly people has improved antibody response to viral vaccine, reduced influenza hospitalization and influenza vaccine side effects [75 ]]Administering 8 g/day to middle aged 45-53 yearsSynergy1 (FOS-rich inulin) improves immune response to influenza vaccine [76 ]]. FOS can improve emotion and cognition. The healthy adult (19-30 years old) is happier in emotion, less in dyspepsia, less in hunger, and improvement of episodic memory (recall and recognition) 5 g/day with inulin rich in fructo-oligosaccharides [77 ]]. Finally, 10g inulin rich in fructo-oligosaccharides is administered, and healthy adults have a tendency to lower total cholesterol in blood and rise HDL, which is beneficial for cardiovascular health [78 ]]。
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Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a composition capable of promoting proliferation of bifidobacterium longum subspecies infancy GB-1496 and application thereof.
To achieve the above object, the present invention provides a composition comprising Fructooligosaccharides (FOS) and probiotics, wherein the probiotics consist of bifidobacterium longum subspecies infantis GB-1496, and bifidobacterium animalis BB12 and/or bifidobacterium lactis HN019.
According to a specific embodiment of the present invention, in the composition of the present invention, the bifidobacterium longum subspecies infancy GB-1496 is bifidobacterium subspecies infancy (Bifidobacterium longum subsp. The strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation unit address is: 430072, university of martial arts, chinese; preservation date: 04 th 2011; preservation number: cctccc NO: m2011122, classification nomenclature: bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp.infantis).
The bifidobacterium longum subspecies infancy CCTCC No. M201122 has better free radical scavenging capability and reducing capability, and can induce Caco-2 cells to increase the expression of antioxidant enzyme, so that the bifidobacterium longum subspecies infancy GB-1496 strain has an antioxidant activity effect, and can reduce the concentration of free radicals so as to inhibit organ aging.
According to a particular embodiment of the invention, preferably, the probiotic is a combination of bifidobacterium longum subspecies infancy GB-1496 and bifidobacterium animalis BB 12. More preferably, the ratio of viable count of bifidobacterium animalis BB12 to bifidobacterium longum subspecies infancy GB-1496 is 10:1-1:10; preferably 1:1; alternatively, the ratio of colony numbers of bifidobacterium animalis BB12 and bifidobacterium longum subspecies infantis GB-1496 is 10:1-1:10; preferably 1:1.
According to a particular embodiment of the invention, preferably the probiotic is a combination of bifidobacterium longum subspecies infancy GB-1496 and bifidobacterium lactis HN019. More preferably, the ratio of viable count of bifidobacterium lactis HN019 to bifidobacterium longum subspecies infancy GB-1496 is 10:1-1:10; preferably 1:1, or alternatively, the ratio of colony numbers of bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496 is 10:1-1:10; preferably 1:1.
According to a particular embodiment of the invention, preferably, in the above composition, the fructooligosaccharides: bifidobacterium animalis BB12 and/or bifidobacterium lactis HN019: the proportion of bifidobacterium longum subspecies infantis GB-1496 is 1g: (10 6 -10 13 )cfu：(10 6 -10 13 ) cfu; wherein the amounts of the bifidobacterium animalis BB12, the bifidobacterium lactis HN019 and the bifidobacterium longum subspecies infantis GB-1496 are based on colony numbers or viable bacteria numbers.
According to a specific embodiment of the present invention, preferably, the fructooligosaccharide is contained in an amount of 0.1 to 20g in the above composition.
The composition of the present invention may be prepared by directly mixing fructooligosaccharides with probiotics.
The invention also provides a nutritional composition comprising the above-described fructooligosaccharide and probiotic-containing composition. The nutritional composition may also contain other food or medicine allowable raw materials, adjuvants, and additives. In preparing the nutritional composition, the fructo-oligosaccharide and probiotic-containing composition may be directly mixed with other raw materials, adjuvants, and additives.
The invention also provides a food product comprising the above fructooligosaccharide and probiotic containing composition and/or the above nutritional composition. The food can be infant food or senile food. According to the specific embodiment of the invention, the food can comprise one or more than two of milk powder, complementary food, beverage and the like, and can also contain other raw materials, auxiliary materials and additives which are allowed to be added.
The invention also provides a medicament comprising the composition containing fructooligosaccharides and probiotics and/or the nutritional composition. The medicine can also contain other allowable raw materials, auxiliary materials and additives.
The invention also provides application of the combination of fructo-oligosaccharide, bifidobacterium animalis BB12 and/or bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496 in promoting proliferation of the bifidobacterium longum subspecies infancy GB-1496.
According to a specific embodiment of the invention, in the above application, the fructooligosaccharides: bifidobacterium animalis BB12 and/or bifidobacterium lactis HN019: the proportion of bifidobacterium longum subspecies infantis GB-1496 is 1g: (10 6 -10 13 )cfu：(10 6 -10 13 ) cfu; wherein the amounts of the bifidobacterium animalis BB12, the bifidobacterium lactis HN019 and the bifidobacterium longum subspecies infantis GB-1496 are based on colony numbers or viable bacteria numbers.
The invention also provides application of the composition containing fructo-oligosaccharide and probiotics in promoting proliferation of bifidobacterium longum subspecies infancy GB-1496.
According to a particular embodiment of the invention, the above promotion is preferably intended to promote the proliferation of bifidobacterium longum subspecies infantis GB-1496 in the gut.
Bifidobacterium infantis is one of the earliest established bifidobacteria species in the infant's intestinal tract. Finding an effective method for promoting proliferation and dominant proliferation of bifidobacterium infantis, and is very important for intestinal colonization and further exerting other effects.
The research of the invention discovers that the multiplication of the bifidobacterium infantis can be obviously promoted by adopting the FOS as a carbon source and adopting the bifidobacterium animalis BB12 and/or the bifidobacterium lactis HN019 to compound with the bifidobacterium longum subspecies infantis GB-1496, and the multiplication quantity is obviously higher than that of the single combination of the FOS and the bifidobacterium animalis BB12, the bifidobacterium lactis HN019 or the bifidobacterium longum subspecies infantis GB-1496. Moreover, in case of using FOS, bifidobacterium animalis BB12 and/or bifidobacterium lactis HN019 in combination with bifidobacterium longum subspecies infancy GB-1496, the bifidobacterium longum subspecies infancy GB-1496 exhibits advantageous proliferation.
Drawings
FIG. 1 shows the results of analysis of the significance of differences in promotion of bifidobacterium proliferation (OD 600 values) when cultured with FOS as the carbon source using HN019, GB-1496 alone and a combination of both.
FIG. 2 shows the results of PCR detection when FOS was used as a carbon source and HN019, GB-1496 and a combination of both were used.
FIG. 3 shows the results of analysis of the significance of differences (OD 600 values) in promoting bifidobacterium proliferation when cultured with FOS as the carbon source using BB12, GB-1496 alone, and a combination of both.
FIG. 4 shows the results of PCR detection when FOS was used as a carbon source and B12 alone, GB-1496 and a combination of both were used.
Microbial preservation for patent procedures:
GB-1496 Strain of the present invention
Preservation date: 10/04/2011
Preservation unit: china Center for Type Culture Collection (CCTCC)
Deposit unit address: 430072 of university of Wuhan, china
Preservation number: cctccc NO: m2011122
Classification naming: bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp. Infantis)
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.
The bifidobacterium longum subspecies of babies GB-1496 is human breast milk, and is preserved in China Center for Type Culture Collection (CCTCC), and the preservation unit address is: 430072, university of martial arts, chinese; preservation date: 04 th 2011; preservation number: cctccc NO: m2011122, classification nomenclature: bifidobacterium longum subspecies infantis (Bifidobacterium longum subsp.infantis).
The taxonomic characteristics of the strains were confirmed based on the 16S rDNA sequence analysis and the API bacteria identification system analysis results. The morphological and general characteristics of Bifidobacterium longum subspecies infant GB-1496 are detailed in Table 1.
TABLE 1
The bifidobacterium longum subspecies infantis GB-1496 strain was maintained at-80℃in MRS medium containing 20% glycerol. Before use, the mixture was activated twice at 37℃with MRS broth (DIFCO) containing 0.05% L-cysteine (24 hours).
Experimental example
(1) Preparing MRS basal medium: (1) weighing: weighing beef powder 5.0g, tryptone 10.0g, yeast powder 4.0g, tween 80 1.0mL, K 2 HPO 4 2.0g、CH 3 COONa 5.0g、C 6 H 5 O 7 (NH 4 ) 3 2.0g、MgSO 4 0.2g、MnSO 4 0.05g, 15.0g of agar powder and 1000mL of distilled water. (2) Mixing evenly: the weighed substances are uniformly mixed, heated and uniformly stirred. (3) Adjusting pH: with 1 mol/mL -1 NaOH adjusts the pH of the culture medium to 7.0-7.2. (4) Dissolving: pouring the dissolved culture medium into a triangular flask, adding a certain volume of water, and heating for dissolution. (5) And (5) subpackaging: pouring the prepared culture solution into a 250mL triangular flask, and wrapping the flask mouth with newspaper. (6) And (3) sterilization:the culture medium is sterilized by high pressure steam for 15min and then cooled for standby.
(2) On the premise of the same initial culture amount, the growth curve of GB-1496, HN019 and compound bacteria (GB-1496:HN019=1:1) in the carbon source of FOS is compared with the growth curve of the bacteria in the culture medium containing 1wt% of FOS and 0.05% of cysteine.
The results show that: when FOS is used as a single carbon source, the combination of GB-1496 and HN019 can synergistically promote the total amount of bifidobacteria to increase, and the total amount of the bifidobacteria is obviously different in 24 hours, and the total amount of the bifidobacteria is 80% higher than that obtained by culturing an equivalent single strain. The specific results are shown in fig. 1 and table 2, wherein in fig. 1, # represents the difference from HN019 alone, and x represents the difference from GB-1496 alone, and the combination of GB-1496 and HN019, vs GB-1496 x: p <0.01; combination of GB-1496 and HN019 vs HN019# #: p <0.01.
As can be seen from fig. 1: in the case of FOS as a carbon source, the combination of GB-1496 and HN019 is adopted, and the proliferation amount of the total bifidobacterium is significantly higher than that of the single GB-1496 and HN019.
TABLE 2
As can be seen from the data in table 2: the proliferation amount obtained by using the mixed strain is 1.5 times of the sum of the proliferation amounts of HN019 and GB-1496 which are cultured independently under the premise of the same culture initiation amount, and the unexpected improvement is obtained.
(3) PCR detection
In 24h, the proportion of two bacteria in the compound bacteria is detected by a PCR method, and the primer design is based on a 16SrRNA sequence, so that the bifidobacterium lactis and the bifidobacterium longum subspecies infantis can be distinguished specifically.
Wherein, the primers for specific differentiation of bifidobacterium lactis are as follows:
Bani-tF：TCACGACAAGTGGGTTGCCA(SEQ ID NO：1)
Bani-tR：GTTGATCGGCAGCTTGCCG(SEQ ID NO：2)。
primers specific for differentiating bifidobacterium longum subspecies infantis are as follows:
Binf-tF：ATCCGTCCGACCCAGACC(SEQ ID NO：3)
Binf-tR：CTCGACATCCTCACGGCC(SEQ ID NO：4)。
the results are shown in FIG. 2. As can be seen from fig. 2: although the proliferation rate of HN019 is faster than that of GB-1496 in single strain culture, the proportion of two bacteria in the compound bacteria is detected in 24 hours, and as a result, the bifidobacterium infantis GB-1496 is dominant in mixed bacteria co-cultured by taking FOS as a carbon source and the mixed bacteria co-cultured by GB-1496 and HN019, and the bifidobacterium infantis is dominant in growth.
(4) Growth in MRS+0.05% cysteine medium containing 1wt% glucose (Glu), isomerized Lactose (LOS), GOS, FOS, 2' FL and lactose, respectively, was compared to the growth curves of GB-1496, BB12 and the complex bacteria (GB-1496: BB12=1:1) in the FOS carbon source under the same initial amount of culture.
When FOS is used as a single carbon source, the combination of GB-1496 and BB12 can synergistically promote the total amount of bifidobacteria to increase, and the total amount of the bifidobacteria is obviously different in 24 hours, and is 110% higher than that obtained by culturing the same amount of single strain. The specific results are shown in fig. 3 and table 3, wherein in fig. 3, # represents the difference from BB12 alone, # represents the difference from GB-1496 alone, and the combination of GB-1496 and BB12, vs GB-1496: p <0.01; combination of GB-1496 and BB12 vs HN019# #: p <0.01.
TABLE 3 Table 3
As can be seen from the data in table 3: on the premise of the same initial culture amount, the proliferation amount obtained by adopting the mixed strain is 2.1 times of the sum of the proliferation amounts of BB12 and GB-1496 which are cultured independently, and the unexpected improvement is obtained.
(5) PCR detection
In 24h, the proportion of two bacteria in the compound bacteria is detected by a PCR method, and the primer design is based on a 16SrRNA sequence, so that bifidobacterium animalis and bifidobacterium longum subspecies infantis can be distinguished specifically.
Wherein, the primers for specific differentiation of bifidobacterium lactis are as follows:
Bani-tF：TCACGACAAGTGGGTTGCCA(SEQ ID NO：1)
Bani-tR：GTTGATCGGCAGCTTGCCG(SEQ ID NO：2)。
primers specific for differentiating bifidobacterium longum subspecies infantis are as follows:
Binf-tF：ATCCGTCCGACCCAGACC(SEQ ID NO：3)
Binf-tR：CTCGACATCCTCACGGCC(SEQ ID NO：4)。
the results are shown in FIG. 4. As can be seen from fig. 4: although the proliferation rate of BB12 is faster than that of GB-1496 in single strain culture, the proportion of two bacteria in the compound bacteria is detected in 24 hours, and the result shows that the bifidobacterium infantis GB-1496 is dominant in mixed bacteria co-cultured by using FOS as a carbon source and the mixed bacteria are dominant growing bacteria.
Example 1
The present example provides a composition comprising FOS, bifidobacterium animalis BB12 and bifidobacterium longum subspecies infancy GB-1496 comprising FOS, bifidobacterium animalis BB12, bifidobacterium longum subspecies infancy GB-1496, wherein GB-1496: BB12 = 1:1.
example 2
The present example provides a composition comprising FOS, bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496 comprising FOS, bifidobacterium lactis HN019, bifidobacterium longum subspecies infancy GB-1496, wherein GB-1496: BB12 = 1:1.
example 3
This example provides a set of nutritional compositions comprising the composition provided in example 1 comprising FOS, bifidobacterium animalis BB12 and bifidobacterium longum subspecies infancy GB-1496. The nutritional composition has remarkable promoting effect on proliferation of bifidobacterium longum subspecies of infants GB-1496 in intestinal tracts, and can enable the bifidobacterium longum subspecies of infants GB-1496 to exhibit dominant growth.
Example 4
This example provides a set of nutritional compositions comprising the composition provided in example 2 comprising FOS, bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496. The nutritional composition has remarkable promoting effect on proliferation of bifidobacterium longum subspecies of infants GB-1496 in intestinal tracts, and can enable the bifidobacterium longum subspecies of infants GB-1496 to exhibit dominant growth.
Example 5
This example provides a group of pediatric foods comprising the composition comprising FOS, bifidobacterium animalis BB12 and bifidobacterium longum subspecies infancy GB-1496 provided in example 1, the composition comprising FOS, bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496 provided in example 2, and the nutritional composition provided in example 3 or 4, respectively, in combination with one or both of the above-mentioned composition comprising FOS, bifidobacterium animalis BB12 and bifidobacterium subspecies infancy GB-1496, the composition comprising FOS, bifidobacterium lactis HN019 and bifidobacterium subspecies infancy GB-1496, and the nutritional composition may be added alone or in any combination and ratio. The food can be infant food or senile food, such as milk powder, beverage, infant complementary food, etc.
The food has remarkable promoting effect on proliferation of Bifidobacterium longum subspecies baby GB-1496 in intestinal tract, and can promote growth of Bifidobacterium longum subspecies baby GB-1496.
Example 6
This example provides a group of drugs comprising the composition comprising FOS, bifidobacterium animalis BB12 and bifidobacterium longum subspecies infancy GB-1496 provided in example 1, the composition comprising FOS, bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496 provided in example 2, and one or a combination of two of the nutritional compositions provided in example 3 or 4, wherein the composition comprising FOS, bifidobacterium animalis BB12 and bifidobacterium subspecies infancy GB-1496, the composition comprising FOS, bifidobacterium lactis HN019 and bifidobacterium subspecies infancy GB-1496, and the nutritional composition may be added singly or in any combination and any ratio.
The medicine has remarkable promoting effect on proliferation of Bifidobacterium longum subspecies baby GB-1496 in intestinal tract, and can promote growth of Bifidobacterium longum subspecies baby GB-1496.
Sequence listing
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Claims (19)
1. A composition comprising fructooligosaccharides and probiotics, wherein the probiotics consist of bifidobacterium longum subspecies infantis GB-1496 and bifidobacterium animalis BB12 or bifidobacterium lactis HN 019;
wherein the fructooligosaccharides: bifidobacterium animalis BB12 or bifidobacterium lactis HN019: the proportion of bifidobacterium longum subspecies infantis GB-1496 is 1g: (10 6 -10 13 ）cfu：（10 6 -10 13 ）cfu。
2. The composition of claim 1, wherein the probiotic is a combination of bifidobacterium longum subspecies infantis GB-1496 and bifidobacterium animalis BB 12.
3. The composition of claim 2, wherein the ratio of viable count of bifidobacterium animalis BB12 to bifidobacterium longum subspecies infancy GB-1496 is 10:1-1:10.
4. the composition of claim 2, wherein the ratio of viable count of bifidobacterium animalis BB12 to bifidobacterium longum subspecies infancy GB-1496 is 1:1.
5. The composition of claim 1, wherein the probiotic is a combination of bifidobacterium longum subspecies infancy GB-1496 and bifidobacterium lactis HN019.
6. The composition according to claim 5, wherein the ratio of viable count of bifidobacterium lactis HN019 to bifidobacterium longum subspecies infancy GB-1496 is 10:1-1:10.
7. the composition according to claim 5, wherein the ratio of viable count of bifidobacterium lactis HN019 to bifidobacterium longum subspecies infancy GB-1496 is 1:1.
8. the composition according to any one of claims 1 to 7, wherein the fructooligosaccharide is present in an amount of 0.1 to 20g.
9. A nutritional composition comprising the composition according to any one of claims 1-8.
10. A food product comprising the composition of any one of claims 1-8 or the nutritional composition of claim 9.
11. The food product of claim 10, wherein the food product is a pediatric food product or an geriatric food product.
12. The food product of claim 10, wherein the food product comprises one or a combination of two or more of milk powder, a complementary food, a beverage.
13. A medicament comprising the composition of any one of claims 1-8 or the nutritional composition of claim 9.
14. Use of a combination of fructooligosaccharides, bifidobacterium animalis BB12 and bifidobacterium longum subspecies infancy GB-1496 for promoting proliferation of bifidobacterium subspecies infancy GB-1496;
wherein the fructooligosaccharides: bifidobacterium animalis BB12: the proportion of bifidobacterium longum subspecies infantis GB-1496 is 1g: (10 6 -10 13 ）cfu：（10 6 -10 13 ）cfu。
15. The use according to claim 14, wherein the promotion is in promoting proliferation of bifidobacterium longum subspecies infantis GB-1496 in the gut.
16. Use of a combination of fructo-oligosaccharides, bifidobacterium lactis HN019 and bifidobacterium longum subspecies infancy GB-1496 to promote proliferation of bifidobacterium subspecies infancy GB-1496;
wherein the fructooligosaccharides: bifidobacterium lactis HN019: the proportion of bifidobacterium longum subspecies infantis GB-1496 is 1g: (10 6 -10 13 ）cfu：（10 6 -10 13 ）cfu。
17. The use according to claim 16, wherein the promotion is in promoting proliferation of bifidobacterium longum subspecies infantis GB-1496 in the gut.
18. Use of a composition comprising fructooligosaccharides and probiotics according to any of claims 1-8 for promoting proliferation of bifidobacterium longum subspecies infancy GB-1496.
19. The use according to claim 18, wherein the promotion is in promoting proliferation of bifidobacterium longum subspecies infantis GB-1496 in the gut.
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