CN107723257A - A kind of animal bifidobacteria and its food compositions - Google Patents
A kind of animal bifidobacteria and its food compositions Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
-
- 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
-
- 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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The present invention relates to a kind of microorganism mushroom, more particularly to a kind of animal bifidobacteria to hydrochloric acid in gastric juice with well tolerable property.Animal bifidobacteria CG B1 applied for China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation on 09 05th, 2016.Preserving number：CGMCCNo.12943.Rule of origin healthy old men enteron aisle at the age of one hundred years old of the present invention.Proved through animal experiment, mice with constipation intestines peristalsis speed can be promoted, alleviate mice with constipation symptom.Colitis in mice can significantly be alleviated, intestinal inflammatory level is reduced, reduce serum levels of inflammatory cytokines, by improving intestinal tight connexin expression, intestinal barrier integrity is improved, there is prevention and improvement result to type II diabetes.Suitable for food, medicine and/or health products.
Description
Technical field
The invention belongs to technical field of food science, particularly belongs to microorganism field, relates generally to a kind of animal bifid bar
Bacteria strain and food compositions.
Background technology
Glycometabolism is an importance of body metabolism, and it plays an important role in human energy metabolism is maintained,
Nowadays the chronic disease of carbohydrate metabolism disturbance and its correlation turns into a global problem, and I, the occurred frequently of type II diabetes arranged
For No. second killer after cancer.IDDM is due to that the β cells of excreting insulin are directly destroyed and cause insulin
Caused by hyposecretion, type II diabetes be then because in long-term process of insulin resistance, insulin lose sensitiveness and
Induce.Type II diabetes belongs to chronic metabolic disease, in diabetic, account for feeling most, proportion is about
95%.
Animal bifidobacteria (Bifidobacterium) is also referred to as bifidobacterium lactis, is in people and many mammalian guts
One of dominant bacteria.Belong to flora in microecology.The Di Seer (Tissier) of French Pasteur's Institute in 1899 is first
Be isolated to the bacterium in being defecated from breast-fed babies, and point out its nutrition to nursing infant and prevention intestines problem have it is important
Effect.The bacterium is physiological bacterium important in humans and animals enteron aisle.It is a series of to participate in immune, nutrition, digestion and protection etc.
Physiology course, play important function.
Many researchs have shown that animal bifidobacteria has improvement gut flora, improve immunity, the effect of reducing inflammation, also
There are some researchs to show, animal bifidobacteria also has the effect for reducing blood glucose, and research finds to supplement animal bifid bar in the diet
Bacterium can produce active influence to glucose tolerance.Glucose tolerance can also be improved, also reduce rat model blood glucose
Effect.
The content of the invention
The purpose of the present invention is to obtain a kind of source healthy population enteron aisle, there is prevention and improvement result to type II diabetes
Animal bifidobacteria CG-B1.
A kind of the first aspect of the present invention, there is provided Bifidobacterium.In a preferential example, it is animal bifidobacteria CG-
B1, preserving number：CGMCCNo.12943.Preservation date：On 09 05th, 2016.Depositary institution：Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center.Preservation place：China, Beijing, Institute of Microorganism, Academia Sinica.
In another preference, the animal bifidobacteria has good tolerance to hydrochloric acid in gastric juice.
The second aspect of the present invention, there is provided the purposes of animal bifidobacteria of the present invention, it is used for preparation group
Compound, described composition have prevention and improvement result to type II diabetes animal.
Above-mentioned composition includes milk powder 100-112.5kg or fresh milk 800-900kg, PURE WHEY 0-8kg, stabilizer 0-
5kg, leavening 100-200DUC, CG-B1 strain 100-200DUC, white granulated sugar 70-80kg, 1 ton of water.
The animal bifidobacteria bacterial strain of the present invention can promote mice with constipation intestines peristalsis speed, alleviate mice with constipation disease
Shape.
The animal bifidobacteria bacterial strain of the present invention can significantly alleviate colitis in mice, reduce intestinal inflammatory water
It is flat, serum levels of inflammatory cytokines is reduced, by improving intestinal tight connexin expression, improves intestinal barrier integrity.
Brief description of the drawings
Fig. 1 is tolerance test block diagrams of the animal bifidobacteria CG-B1 of the embodiment of the present invention 2 to hydrochloric acid in gastric juice；
Fig. 2 is influence linear graphs of the animal bifidobacteria CG-B1 of the embodiment of the present invention 3 to each group rat fasting blood-glucose；
Fig. 3 is influence linear graphs of the animal bifidobacteria CG-B1 of the embodiment of the present invention 3 to each group rat 2h-plasma glucose；
Each group Oral Administration in Rats glucose glucose tolerance curve figure when Fig. 4 is the modeling of the embodiment of the present invention 4 success；
Area-graph under each group Oral Administration in Rats glucose glucose tolerance curve when Fig. 5 is the modeling of the embodiment of the present invention 4 success；
Fig. 6 is that the animal bifidobacteria CG-B1 of the embodiment of the present invention 5 improves glucose tolerance curve to each group Oral Administration in Rats glucose
Figure；
Fig. 7 is that the animal bifidobacteria CG-B1 of the embodiment of the present invention 5 improves glucose tolerance curve to each group Oral Administration in Rats glucose
Lower area-graph；
Fig. 8 is influence columns of the animal bifidobacteria CG-B1 of the embodiment of the present invention 6 to each group rat glycosylated hemoglobin
Figure.
Wherein：+<0.05 normal group, *<0.05 control group
Embodiment
The present inventor is through repeatedly screening and cultivates, isolated one plant of animal bifidobacteria bacterial strain from centenarian's excrement
CG-B1, the bacterial strain have good tolerance to hydrochloric acid in gastric juice.Proved through animal experiment, mice with constipation intestines peristalsis can be promoted fast
Degree, alleviate mice with constipation symptom.Colitis in mice can significantly be alleviated, intestinal inflammatory level is reduced, it is scorching to reduce serum
Inflammation factor, by improving intestinal tight connexin expression, improve intestinal barrier integrity.
For animal bifidobacteria CG-B1 on 09 05th, 2016, application China Committee for Culture Collection of Microorganisms was general
Logical microorganism center preservation.Preserving number：CGMCCNo.12943.
" Bifidobacterium of the present invention " used herein refers to animal bifidobacteria CG-B1, preserving number：CGMCCNo.12943.Ying Li
Solving these terms also includes being derived from animal bifidobacteria CG-B1 bacterial strains, especially has well tolerable property characteristic to hydrochloric acid in gastric juice
Derivative strain.
Animal bifidobacteria CG-B1 Basic Biological Character and the representative microbial of the known kind are essentially identical,
It belongs to bifidobacterium animalis acid subspecies, and Gram-positive is shaft-like, and form is irregular, anodontia hail, strict anaerobes, different
Point is that microbial strains of the present invention have well tolerable property characteristic to hydrochloric acid in gastric juice, can promote mice with constipation intestines peristalsis speed,
Alleviate mice with constipation symptom.Can significantly alleviate colitis in mice, it is horizontal to reduce intestinal inflammatory, reduce inflammation because
Son, by improving intestinal tight connexin expression, improve intestinal barrier integrity.
Specifically, acid resistance experiment proves that animal bifidobacteria CG-B1 of the invention handles 3 under the conditions of PH2.5
Hour, significant changes do not occur for viable count.
Animal experiment proves that after 4 weeks CG-B1 of diabetes rat are given, fasting blood-glucose and 2h-plasma glucose level are without bright
It is aobvious to reduce, but oral glucose glucose tolerance curve and TG-AUC show that CGB1 can significantly reduce diabetes rat
90min and 120min blood glucose value and sugar tolerance can be significantly improved.CG-B1 can be obviously improved type II diabetes simultaneously
Dose-dependent effect is presented between symptom and each dosage group that rat body weight mitigates.
Below in conjunction with specific embodiment, the present invention is expanded on further, it should be appreciated that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention.
Embodiment 1
Animal bifidobacteria CG-B1 is obtained from the separation of healthy centenarian's enteron aisle within 2007.
Separation method：Anaerobism dilution is placed in 4 DEG C of preservations after collecting old man's excrement, and strain isolation is completed in 2 hours.Profit
With 37 DEG C of NPNL agar mediums Anaerobic culturel 48 hours, bifidobacterium strain is separated.
Authentication method：The doubtful bacterium colony of picking carries out Gram's staining, is observed in 1000 × oil mirror, and G+ is presented in bacterial strain, does not produce
Gemma, shape are in quarter butt or irregular shape.Bacterium colony is increased into bacterium using MRS culture mediums anaerobism, is extracted and tried using bacterial genomes
Agent box extracts bacterial strain STb gene, expands bacterial strain 16s rDNA total lengths using bacterium 16s rDNA universal primers, PCR primer is purified
Afterwards, deliver to sequencing company and carry out sequence analysis, sequence results are retrieved using ncbi database Blast programs, are accredited as
Bifidobacterium animalis subsp.lactis, are named as CG-B1.
Animal bifidobacteria CG-B1 physio-biochemical characteristics：
Animal bifidobacteria CG-B1 Basic Biological Character and the representative microbial of the known kind are essentially identical,
It belongs to bifidobacterium animalis acid subspecies, and Gram-positive is shaft-like, and form is irregular, anodontia hail, strict anaerobes.
Embodiment 2
Tolerances of the animal bifidobacteria CG-B1 to hydrochloric acid in gastric juice
It can be seen in FIG. 1 that being handled 3 hours under the conditions of PH2.5, significant changes do not occur for viable count.
Embodiment 3
Influences of the CG-B1 to fasting blood-glucose and 2h-plasma glucose
(a) test material
Strain subject：Bifidobacterium animalis subsp.lactisCG-B1(CGMCC No.12943),
By the preservation of morning twilight dairy industry Co., Ltd of Shenzhen.
Counting alive microbial：CG-B1 strain culturings 12h bacterium solution is subjected to gradient dilution with sterile saline, inclined with flat board
Note method, MRS solid mediums, 37 DEG C of Anaerobic culturel 48h, the viable count for determining bacterium solution are 1.0 × 109CFU/mL。
Bacterial strain processing：By CG-B1 from glycerol tube with 1.0 × 107CFU/mL inoculum concentration is inoculated into MRS Liquid Cultures
Base, activate to the third generation, 37 DEG C of Anaerobic culturel 12h, culture terminates rear 4000g centrifugations 15min, collects thalline.The thalline of harvest
Wash twice and be resuspended in physiological saline with PBS, obtain viable bacteria suspension.In use, bacteria suspension is adjusted to test required concentration.
Experimental animal
SPF levels male SD rat is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., body weight 240 ± 10g, and 6~8
Week old.Rat feeding moves in China Agricultural University's genetically modified organism edible safety supervision and inspection center SPF level Animal Houses
Thing quality certification number：SCXK (capital) 2012-0001.
(b) experimental index and detection method
(1) fasting blood sugar (Fasting blood glucose, FBG) and 2h-plasma glucose value (Postprandial
2h blood glucose, PBG) measure
Once in a week, each group Rat Fast can't help water 12h, determines tail vein blood glucose value with blood glucose meter, is fasting blood sugar；
Corresponding feed is given after measure fasting blood-glucose, after giving feed 2h, tail vein blood glucose value is determined with blood glucose meter again, is postprandial 2h
Blood glucose value.
(2) oral glucose tolerance (OGTT) determines
Carried out with putting to death first three day of rat within first day after modeling judgement.Each group Rat Fast can't help water 12h, by 2g/kg
The glucose solution of body weight gavage 50% (w/v), and 0min, 15min, 30min, 60min, 90min, 120min are determined respectively
Caudal vein blood glucose value.Draw change of blood sugar curve, and calculate area under glucose tolerance curve (Area Under Curve,
AUC)。
(3) blood preparation is left and taken
The 3rd day after second of OGTT experiment, water 12h, etherization are can't help in fasting, and angular vein takes blood, and blood sample is put respectively
In 5mL EDTA anticoagulant tubes and 1.5mL liquaemin anticoagulant tubes, the blood sample in EDTA anticoagulant tubes is used to determine glycosylated hemoglobin,
Blood sample 3000g centrifugation 10min separated plasmas in 1.5mL liquaemin anticoagulant tubes, the interior preservation of -80 DEG C of refrigerators are to be measured.
(4) glycosylated hemoglobin determines
The blood sample being stored in EDTA anticoagulant tubes is delivered in Beijing and uses biochemistry with Co., Ltd of blue rich clinical examination institute
Analysis-e/or determining.
(c) data and statistical analysis
All experimental datas are represented in the form of average ± standard deviation (Mean ± SD).Data conspicuousness is poor between group
It is different that one-way analysis of variance (One-way ANOVA) is carried out using statistic software SPSS 20.0, with p<0.05 or p<0.01 represents
Difference has statistical significance.
Since feeding high lipid food to treatment four weeks after put to death, each dosage group rat serum of normal group, model group, CG-B1
Sugar level situation of change is as shown in Figures 2 and 3.
As seen from Figure 2, during high lipid food is fed, each group rat fasting blood-glucose has no significant change；STZ is induced
Into in mould two weeks, all modeling group rat fasting blood-glucoses significantly increase (p<0.01), because high glucose and high fat diet induced
Although insulin resistance occur in the peripheral tissues of rat during surrounding, now compensatory hyperinsulinaemia is inhibited on blood glucose
Rise；After injecting STZ, because STZ is to the selective killing effect of beta cells of isolated rat islets, the secretion of insulin have impact on, therefore modeling
Group rat shows fasting blood-glucose and substantially risen.Model group and the fasting blood-glucose change after modeling of CGB1 low dose groups are steady, nothing
Obvious fluctuation.The variation tendency of the middle and high dosage groups of CGB1 is identical, begins to decline within the 3rd week after modeling, at the end of experiment,
The middle and high treatment group's rat fasting blood-glucoses of CGB1 are respectively 18.6 ± 1.2mmol/L and 18.7 ± 0.9mmol/L, are below model
Group 20.1 ± 1.0mmol/L of rat fasting blood-glucose, but without significant difference (p>0.05).
As seen from Figure 3, each horizontal variation tendency of dosage group rat 2h-plasma glucose of model group and CG-B1 is identical, nothing
Obvious fluctuation (p>0.05), illustrate probiotics CG-B1 to treatment diabetes rat 2h-plasma glucose without obvious effect.
Embodiment 4
Influences of the CG-B1 to oral Glucose tolerance test
Each group rat sugar tolerance during modeling success
Modeling success after each group rat sugar tolerance situation as shown in Figure 4 and Figure 5.
In 15~30min it can be seen from Fig. 4 and Fig. 5 after gavage glucose, each group rat blood sugar becomes in rising
Gesture, normal rats show normal resistance to sugared phenomenon, and the blood glucose of the normal rats ascensional range in 0~30min is gentle,
Gradually reduced after 30min, fasting level is returned to after 2h；The blood glucose of remaining each group rat significantly rises (p in 30min<
0.01) top, is reached in 30min, blood glucose gradually reduces after 30min, and blood sugar level is still higher after 2h.In addition,
TG-AUC statistical result showed, model group and each dosage treatment group AUC of CGB1 are significantly higher than normal group (p<0.01), say
There is the situation (p of serious impaired glucose tolerance in bright modeling group rat<0.01) spy of type II diabetes sugar tolerance reduction, is met
Sign.Meanwhile model group is identical with each dosage group rat glucose tolerance curve variation tendencies of CGB1, there was no significant difference between group (p>
0.05).When showing modeling success, all rat occurring degrees are suitable.
Embodiment 5
Improvement results of the CG-B1 to rat sugar tolerance
Before Fig. 6 and Fig. 7 is puts to death rat, the comparison of each group rat sugared tolerance.
As shown in Figure 6 and Figure 7, model group and each dosage group oral glucose tolerance peak of curves (30min) of CG-B1 are significantly high
In normal group, show to still suffer from obvious impaired glucose tolerance.CG-B1 is low, middle dose group and model group oral glucose tolerance curve peak
It is not notable to be worth (30min) difference.CG-B1 high dose group oral glucose tolerance peak of curves (30min) are 31.5 ± 1.0mmol/L,
Less than 32.7 ± 1.1mmol/L of model group, but there was no significant difference.CGB1 high dose group oral glucose tolerance curve 90min and
120min blood glucose value is substantially less than model group (p<0.05).TG-AUC statistical result showed, model group AUC are significantly big
In normal group AUC (p<0.01), CGB1 is low, middle dose group AUC and model group AUC differences not significantly (p>0.05), the high agent of CGB1
Amount group AUC is substantially less than model group AUC (p<0.01).
Embodiment 6
Influences of the CG-B1 to glycosylated hemoglobin
Glycosylated hemoglobin (HbA1c) is one of diagnosis most important index of diabetes, in glycosylated hemoglobin red blood cell
The product that is combined with blood glucose of hemoglobin, the reaction is irreversible, and directly proportional to blood sugar concentration, and can stablize and be kept for 120 days
Left and right, therefore the blood glucose situation that glycosylated hemoglobin can reflect within 3 months.Each group rat glycated hemoglobin levels change
Situation is as shown in Figure 8.
As shown in figure 8, model group and each dosage group HbA1c levels of CG-B1 are all remarkably higher than normal group (p<0.01).With mould
Type group compares, and each dosage group blood plasma HbA1c levels of CG-B1 decrease and dosage effect between group be present, but difference is not notable.
Embodiment 7
Food compositions containing animal bifidobacteria CG-B1
1st, it is formulated：
Fresh milk 110kg, white granulated sugar 75kg, stabilizer 3kg, PURE WHEY 5kg, leavening 100DCU, CG-B1 strain
1 ton of 150DUC, water.
2nd, production craft step explanation：
(1) pipeline, is connected, fresh milk is taken out and heats up 60-80 DEG C；
(2), white sugar, stabilizer etc. are added in milk, after stirring, notify laboratory personnel to detect；
(3), sterilized after sterilizing equipment decontaminating apparatus cleaning with hot water circuit, ensure that equipment is in germ-free condition, then cooled down with frozen water；
(4), by raw material preheating to 70 DEG C or so rear homogeneous, homogenization pressure 200bar；
(5), the material after homogeneous is after 95 DEG C keep 300s to sterilize, supercooling row, and milk is crossed after being cooled to 40-45 DEG C to buffering
Cylinder；
(6) after, dissolving strain with sterilized water, it is inoculated with, after stirring 10-15min, overheat row makes milk temperature rise to 40~45
DEG C progress is filling；
(7) fermented after, canned through 42~44 DEG C of insulations, about 5 hours or so, through chemically examine it is qualified after, be pushed into Cold storage in the refrigerator.
Claims (3)
- A kind of 1. animal bifidobacteria, it is characterised in that：It is bifidobacterium animalis subspecies CG-B1, preserving number： CGMCCNo.12943.Preservation date：On 09 05th, 2016.Depositary institution：China Committee for Culture Collection of Microorganisms is general Logical microorganism center.
- A kind of 2. animal bifidobacteria according to claim 1, it is characterised in that：The animal bifidobacteria has to hydrochloric acid in gastric juice There is good tolerance.
- A kind of 3. food compositions containing animal bifidobacteria described in claim 1, it is characterised in that：Including milk powder 100- 112.5kg or fresh milk 800-900kg, white granulated sugar 70-80kg, PURE WHEY 0-8kg, stabilizer 0-5kg, leavening 100- 1 ton of 200DUC, CG-B1 strain 100-200DUC, water.
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Cited By (11)
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| CN108570429A (en) * | 2018-05-08 | 2018-09-25 | 西藏高原之宝牦牛乳业股份有限公司 | A kind of animal bifidobacteria and preparation method thereof |
| CN109527089A (en) * | 2018-12-19 | 2019-03-29 | 扬州市扬大康源乳业有限公司 | A kind of probiotics fresh milk and preparation method thereof |
| CN110893194A (en) * | 2019-11-20 | 2020-03-20 | 内蒙古伊利实业集团股份有限公司 | Novel application of bifidobacterium lactis BL-99 in inhibiting intestinal inflammation |
| CN111135197A (en) * | 2018-11-02 | 2020-05-12 | 中国农业大学 | Application of animal bifidobacterium A6 in preparing health-care products |
| CN112618577A (en) * | 2020-12-15 | 2021-04-09 | 深圳君拓生物科技有限公司 | Effect of animal bifidobacterium on improving tumor immunotherapy response |
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| CN111135197A (en) * | 2018-11-02 | 2020-05-12 | 中国农业大学 | Application of animal bifidobacterium A6 in preparing health-care products |
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| CN113736697A (en) * | 2021-09-01 | 2021-12-03 | 哈尔滨葵花药业有限公司 | Bifidobacterium lactis for preventing or treating colitis and application thereof |
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