CN110218681A - One plant of lactobacillus fermenti KP101 and its application - Google Patents
One plant of lactobacillus fermenti KP101 and its application Download PDFInfo
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- CN110218681A CN110218681A CN201910555559.3A CN201910555559A CN110218681A CN 110218681 A CN110218681 A CN 110218681A CN 201910555559 A CN201910555559 A CN 201910555559A CN 110218681 A CN110218681 A CN 110218681A
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- lactobacillus fermenti
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- lactobacillus
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- milk
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- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical compound [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 description 1
- 229940046307 sodium thioglycolate Drugs 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- 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
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
-
- 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
-
- 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
- A23L2/52—Adding ingredients
-
- 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
-
- 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
- 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
- A23V2400/143—Fermentum
-
- 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
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/249—Thermophilus
-
- 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
- C12R2001/225—Lactobacillus
Abstract
The invention discloses one plant of lactobacillus fermenti KP101, its deposit number is CCTCC No.2019085；And its application in terms of preparing fermented yoghourt and probiotics solid beverage；Lactobacillus fermenti KP101 of the present invention is resistant to acid stress, tolerance cholate is coerced and tolerance oxidative stress is good, anti-pathogenic infection, adjusting metabolic disorder, adjusting hyperlipemia etc., while Weight-reducing and lipid-lowering；Animal experiments show that can be effectively reduced increasing for TG, LDL-c in the serum as caused by high fat diet, respectively 50.5%, 67.0%；It is horizontal that HDL-C in liver can be significantly improved, increment rate 78.9%；Reduce the content of TC, TG and LDL-C in liver, respectively 36.2%, 64.6% and 42.8%；Also there is extremely significant effect for reducing fat, reduce its perirenal fat coefficient and abdominal fat coefficient is respectively 97.2% and 95.5%.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to one plant of lactobacillus fermenti KP101 and its application.
Background technique
Hyperlipemia and its complication have become one of global incidence and the highest disease of the death rate, but current treatment
Means are also limited to very much, and there are two types of main Clinical Processing methods, first is that diet intervention, second is that drug therapy.The core of diet intervention
The heart is high-fat and high-cholesterol diet the intake of limitation.But this method is only effective in a short time and is difficult to adhere to for a long time, controls
Therapeutic effect is gradually deteriorated；Although drug therapy is more obvious, often side effect is big and expensive for cholesterol-lowering drug.Probiotics
As cheap, a highly-safe substance, by taking probiotics preparation regulation and control serum cholesterol level pair
There is very big development prospect in the prevention of hyperlipemia and adjuvant treatment.
Currently, having been found that numerous probiotics such as Activities of Some Plants lactobacillus by a large amount of animal experiments and clinical test, doing
Lactobacillus paracasei, Bifidobacterium etc. all have effects that adjust human serum cholesterol levels.Nowadays, have a large number of researchers to benefit
Norcholesterol function of the raw bacterium in experimental animal body is evaluated and has been studied.By the study found that probiotics can mainly show
Writing reduces that experimental animal serum TC and LDL-C are horizontal, do not find also probiotics can the HDL-C level to experimental animal serum produce
Life significantly affects.Since the eating habit of China resident and inherent cause etc. and the presence of external ethnic group are poor, for being introduced from foreign countries
Probiotics strain may not necessarily entirely appropriate China resident take.Temporarily the norcholesterol without independent intellectual property right is prebiotic in China
Bacteria strain or Related product, therefore research and develop the probiotics strain that suitable China resident with independent intellectual property rights takes
And its product is for promoting China's probiotics industry development and national healthy being promoted to be of great significance.
Summary of the invention
The lactobacillus fermenti KP 101 that there is fat-reducing and antihyperglycemic it is an object of the present invention to provide one plant and its application.
One plant of lactobacillus fermentiLactobacillus fermentum KP 101, its deposit number is CCTCC No.
M 2019085。
A kind of fermented yoghourt, it is prepared by the following method:
1) activation of bacterial strain: the lactobacillus fermenti KP101 and streptococcus thermophilus for being CCTCC No. M 2019085 by deposit number
Freeze-dried vaccine powder be inoculated in germ-free milk culture medium, cultivated respectively by optimum temperature, pass on 2 ~ 3 times, each inoculum concentration 1
~ 10%, viable count reaches 106CFU/mL or more；
2) preparation of fermented yoghourt: by the ratio of lactobacillus fermenti KP101 and streptococcus thermophilus 1 ~ 2:1 by volume after activation
Mixing, is seeded in milk according to 1% ~ 10% inoculum concentration, and ferment 3 ~ 5h at 38 ~ 45 DEG C, cooling at 0 ~ 4 DEG C, is sent out
Ferment Yoghourt；
It is seeded in milk described in step 2 according to 3% ~ 5% inoculum concentration, ferment 3 ~ 5h at 40 ~ 43 DEG C；
Milk described in step 2, is prepared by the following method: the sugar of mass fraction 5% ~ 10%, mistake is added in filtering, purification
Filter, stirs evenly, is heated to 55 ~ 65 DEG C, 3 ~ 10min of homogeneous at 18 ~ 22MPa, and sterilize 5 ~ 10min under the conditions of 95 ~ 100 DEG C
Afterwards, cooling.
A kind of probiotics solid beverage, it includes the component of following parts by weight: 10 parts of lactobacillus fermenti freeze-dried powder, oligomeric
5 ~ 10 parts of isomaltose, 5 ~ 10 parts of soyabean oligosaccharides, 5 ~ 15 parts of fruity powder；The lactobacillus fermenti, its deposit number are
CCTCC No. M 2019085；
The fruity powder is blueberry powder, Cranberry powder or hawthorn powder；
The lactobacillus fermenti freeze-dried powder, is prepared by the following method: lactobacillus fermenti KP101 is activated, and expands culture,
It is centrifuged 8 ~ 15min at 3000 ~ 5000r/min, is washed with water, supernatant is abandoned in centrifugation, obtains thallus；By thallus and freeze drying protectant
1 ~ 3:1 is mixed by volume, and freeze-drying obtains lactobacillus fermenti freeze-dried powder；
The freeze drying protectant, by mass percentage include following component: skimmed milk power 10%-12%, cellulose 7%-9%,
Trehalose 3%-5%, Pulan polysaccharide 6%-8%, surplus are water.
The present invention provides one plant of lactobacillus fermenti KP101, its deposit number is CCTCC No. M 2019085；And
Its application in terms of preparing fermented yoghourt and probiotics solid beverage；It is lactobacillus fermenti KP101 of the present invention tolerance acid stress, resistance to
It is coerced by cholate and tolerance oxidative stress is good, anti-pathogenic infection, adjusting metabolic disorder, adjusting hyperlipemia etc. subtract simultaneously
Fertile lipid-loweringing；Animal experiments show that can be effectively reduced increasing for TG, LDL-c in the serum as caused by high fat diet, respectively
50.5%,67.0%；It is horizontal that HDL-C in liver can be significantly improved, increment rate 78.9%；Reduce TC, TG and LDL-C in liver
Content, respectively 36.2%, 64.6% and 42.8%；Also there is extremely significant effect for reducing fat, reduce its perirenal fat coefficient and abdomen
Fat coefficient is respectively 97.2% and 95.5%.
Detailed description of the invention
Fig. 1 o-phthalaldehyde removing cholesterol rate result；
Fig. 2 bile salt hydrolase qualitative and quantitative measurement result；
The 16S rRNA result of gene sequence determination of Fig. 3 bacterial strain；
The acid-resistant property result of Fig. 4 bacterial strain；
The bile tolerance characterization result of Fig. 5 bacterial strain；
The antioxidant activity result of Fig. 6 bacterial strain；
TC, TG, HDL, LDL measurement result of Fig. 7 mice serum；
ALT, AST measurement result of Fig. 8 mice serum；
TC, TG, HDL, LDL measurement result of Fig. 9 mouse liver；
ALT, AST measurement result of Figure 10 mouse liver；
Figure 11 mouse the 8th week weight, abdominal fat coefficient, perirenal fat coefficient.
Specific embodiment
The screening and identification of 1 lactobacillus fermenti KP101 of embodiment
1, the screening of bacterial strain
By purchase in the pickles sample in market, lactic acid bacteria separation is carried out in laboratory；By pickled vegetable fermentation liquor with 10-1Gradient it is dilute
It releases, is coated on MRS agar plate, for 24 hours, picking single colonie separates Anaerobic culturel in flat lining out, and purifying training is repeated
It supports, selects gram positive bacterial strain with Gram's stain observation colonial morphology；The multi-strain bacteria strain that will be selected, then in liquid MRS
37 DEG C of cultures for 24 hours, carry out connecing bacterium according to the inoculum concentration of culture solution 3% in culture medium, and continuous activation is passed on three times, to guarantee thallus
Vigorous growth vigor；
Liquid MRS culture medium contains peptone 10.0g, beef extract 10.0g, 5.0 g of yeast extract, diammonium hydrogen citrate [(NH4)
2HC6H5O7] 2.0 g, glucose (C6H12O6H2O) 20.0g, Tween 80 1.0mL, sodium acetate (CH3COONa
3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO43H2O) 2.0g, magnesium sulfate (MgSO47H2O) 0.58g, manganese sulfate
(MnSO4H2O) 0.25g.
2, the preparation of bacteria suspension
By above-mentioned activation and the bacterium solution 4000r/min that expands culture is centrifuged 10min, with brine 2 times of 0.85%, from
The heart abandons supernatant, obtains bacterial sediment, is suspended in isometric physiological saline, obtains bacteria suspension.
3, the preparation of bacterial strain screening culture medium and reagent
BSH screening and culturing medium: 2% agar, 0.3% bovine bile, 0.2% sulfydryl second are added on the basis of liquid MRS culture medium
Sour sodium, the calcium chloride of 0.37g/L；
MRS-THIO-OX-CHOL culture medium: 0.2% sodium thioglycolate, 0.3% ox are added on the basis of liquid MRS culture medium
Cholate, concentration are the cholesterol solution of 100 μ g/ml；
Cholesterol solution is prepared: being accurately weighed cholesterol powder 0.5g, under heating conditions, is sufficiently dissolved simultaneously with dehydrated alcohol
It is settled to 50mL, cholesterol solution concentration is 10.0mg/mL at this time, in an aseptic environment, is filtered with the micropore that aperture is 0.45 μm
After film degerming, it is added in sterilized liquid MRS culture medium according to 1% amount, at this point, cholesterol is dense in liquid MRS culture medium
Degree is 0.1mg/mL；
O-phthalaldehyde working solution: accurately weighing o-phthalaldehyde 50mg, with dehydrated alcohol constant volume 50mL, so that concentration is 1mg/
ML is refrigerated spare；
Mixed acid is prepared: dense H2SO4It is mixed with glacial acetic acid 1:1.
4, the good bacterial strain of the outer norcholesterol ability of o-phthalaldehyde method screen body
The drafting of standard curve: taking five clean tubes, and number 1-5 is separately added into 0,0.1,0.2,0.3,0.4mL in order
Cholesterol solution, then after being separately added into 0.5,0.4,0.3,0.2,0.1mL glacial acetic acid by the sequence of 1-5, be separately added into
The O-phthalic aldehyde reagent of 0.2mL makes its concussion sufficiently dissolution, stands the mixing for being separately added into 4.0mL after reacting 10min again
Acid is uniformly mixed, and being placed at room temperature for 10min makes its colour developing, and reaction solution is placed in 96 orifice plates with liquid-transfering gun, measures it with microplate reader
Light absorption value at 550nm, using the concentration of cholesterol as abscissa, absorbance OD550 value is that ordinate draws standard curve；It returns
Return equation :+0.0793 R of y=3.8445x2 = 0.9903
It measures the OD value of sample: bacteria suspension is linked into the MRS-THIO-OX-CHOL culture medium equipped with 10mL with 3% inoculum concentration
In, it is cultivated for 24 hours under the conditions of 37 °C；At this point, utilizing the culture solution of o-phthalaldehyde method measurement fermentation 0h rapidly；Bacterium will just have been connect
Culture medium is centrifuged 10min under the conditions of 9000r/min, takes the supernatant of 0.25mL, and the o-phthalaldehyde solution of 0.1mL is added,
It fullys shake, after standing reaction 10min, the mixed acid solution of 2m L is added, stands 10min, reaction solution is placed in 96 orifice plates,
Its OD value is measured at 550nm with microplate reader；During the cultivation process, respectively 6,12 and for 24 hours when sample, utilize above-mentioned adjacent benzene two
Formaldehyde Absorption Method for Determination of Low fermentation liquid 6,12 and OD value for 24 hours；Then fermentation liquid is determined according to the equation of the standard curve fit of cholesterol
The content of middle cholesterol, and the removal efficiency (%) of final cholesterol is measured according to the following formula；
Formula: the removal efficiency of cholesterol: V=(B-A)/B × 100%；Wherein, A is the gallbladder after each test strain ferments in supernatant
Sterol content；B is the cholesterol level before each test strain ferments in supernatant；
As a result: the removal efficiency of the variation of thallus incubation time, bacterial strain cholesterol in vitro also changes therewith, this shows cholesterol
The growth conditions of removal efficiency and thallus have certain relationship；Removing cholesterol rate of the bacterial strain in 6h, 12h and for 24 hours be respectively
44.09%, 77.59% and 84.58%, show that its good Lowering cholesterol effect in vitro, the bacterial strain can be applied in animal body
Test, further measures its reducing blood lipid ability (Fig. 1) in vivo.
5, bile salt hydrolase vitality test
BSH qualitative determination: the BSH screening and culturing medium of sterilizing is poured into aseptic flat board, by aseptic filter paper after culture medium solidification
Piece is uniformly put on culture medium, and activated 10 μ L of bacterium solution to be measured is slowly added in the sterile of about 3~4 mm of diameter with liquid-transfering gun
On filter paper, after bacterium solution fully absorbs, whether after 72 h are cultivated under 37 DEG C of anaerobic conditions, observing around filter paper has white
Sediment generates, if there is white precipitate can the preliminary identification bacterium contain BSH；
BSH quantitative determination: the BSH Elisa kit using purchase in the preferred biology in Shanghai is measured；
As a result: there is obvious and distinct milky white precipitate around bacterial strain, bacterial strain has good bile salt hydrolase activity,
Quantitative determination obtains total enzyme activity and specific enzyme activity is respectively 65.13% and 9.33%；It further demonstrates that, bacterial strain has good drop gallbladder
Sterol ability can should have in experiment in vivo (Fig. 2).
6, strain idenfication
It will be singled out that external norcholesterol ability is higher, and the bacterial strain with stronger bile salt hydrolase vigor is sent to the raw work in Shanghai
Company is sequenced, and is identified by 16SrDNA；It as a result is to show the parent on highest molecular systematics with lactobacillus plantarum
Edge relationship (Fig. 3)；Therefore lactobacillus fermenti KP101, Latin name are named asLactobacillus fermentum KP 101, now it is stored in Wuhan City's China typical culture collection center (Wuhan, China university), the preservation time is January 24 in 2019
Day, number: CCTCC No. M 2019085.
2 probiotics tolerance test of embodiment
1, acid resistance is evaluated
Bacterial strain is inoculated in liquid MRS culture medium according to 3% inoculum concentration respectively, in 37 DEG C of culture 18h.Culture solution is in 4 DEG C 4000
rpm·min-1Be centrifuged 10min, obtain bacterium mud with 0.85% brine twice, then with sterile saline by bacterium mud
It suspends, guarantees that the bacterial strain of activation is about 108CFU·mL-1, then according to 3% inoculum concentration by bacterial suspension inoculation in pH be 2.0,
It in 3.0 MRS culture medium, is sampled respectively in 0h, 2h, 4h, carries out 10 times of dilutions with physiological saline, be coated with after diluting debita spissitudo
In MRS solid medium, 37 DEG C of constant temperature incubation 48h, colony count, each three, concentration work parallel, is averaged, calculates
Viable count and survival rate；
As a result: under conditions of pH2.0, survival rate of the bacterial strain after 4h remains to reach 90% or more, but under conditions of pH3.0,
The survival volume of bacterial strain, which has, significantly to be increased, and illustrates it with good acid-fast ability (Fig. 4).
2, bile tolerance is evaluated
It would be about 10 respectively8 CFU·mL-1It is respectively 0% that the bacterial strain of activation, which is inoculated in gallbladder salinity with 3.0% inoculum concentration,
0.2%, in 0.3% MRS culture medium, 37 DEG C of constant temperature incubations are measured in 0,2,4 h respectively using gradient dilution colony counting method
Its viable count, each concentration are made three and are averaged in parallel, and viable count and survival rate are calculated；
As a result: under the conditions of 0.3% cholate, survival rate of the bacterial strain after 4h can reach 105.07%, in 0.5% cholate condition
Under, survival rate of the bacterial strain after 4h reaches 102.24%, illustrates it with good bile tolerance ability (Fig. 5).
3, metabolite biocidal property is evaluated
The bacteriostatic experiment of Metabolite is carried out using Odontothrips loti.By activated Escherichia coli, staphylococcus aureus
10 are diluted to salmonella bacterium solution5 CFU·mL-1, take 0.1mL to be applied on LB plate, sterilized Oxford cup be put in flat
In plate, two are placed in parallel, takes the fermented supernatant fluid of activated aimed strain, 200 μ L is drawn respectively and is put into Oxford cup,
Then plate is slowly put in 37 DEG C of constant incubators, cultivate 24 h, observe and measure antibacterial circle diameter size.Parallel laboratory test three
It is secondary；
It the results are shown in Table 1, it can be seen from the table suppression of the lactobacillus fermenti KP101 to Escherichia coli, staphylococcus aureus, salmonella
Bacterium diameter is respectively 9.34mm, 15.1mm, 10.39mm, shows its good antagonistic property.
4, drug resistance is evaluated
Aimed strain accesses MRS fluid nutrient medium, 37 DEG C of cultures to logarithmic growth phase, sterile absorption 0.1mL by 1% inoculum concentration
To MRS agar plate, coating uniformly, chooses 6 kinds of common drug sensitive test papers: tetracycline, vancomycin, ampicillin, to block that mould
Element, gentamicin, penicillin, aseptic nipper clamping are put to the MRS plate for having been coated with bacterium solution, and each plate places 3 kinds of differences
Drug sensitive test paper, 37 DEG C of 24 h of culture；Table 2 the result shows that: lactobacillus fermenti KP101 being capable of tetracycline resistance, streptomysin, kanamycins
And rifampin；But it is sensitive to gentamicin, chloramphenicol, antibiotic erythromycin, but still in the range of safe concentration.
5, in-vitro simulated artificial gastro-intestinal Fluid evaluation
By bacteria suspension 1mL, it is transferred to pH3.0 simulated gastric fluid 9mL, 37 DEG C of Anaerobic culturels, from 0h, the separately sampled plate of 2h, 4h is coated with
It counts.It is accessed in the 9mL simulated intestinal fluid of pH8.0 after drawing 1mL bacterium solution in gastric juice culture solution again, obtains 10mL liquid, 37 DEG C
Anaerobic culturel, from 0h, the separately sampled plate coating of 2h, 4h is counted.
3 antioxidant activity tests of embodiment
1、H202Resistance test
The pure culture of lactobacillus fermenti KP101 is inoculated into the hydrogen peroxide (0.5,1.0 mmol/L) for being supplemented with various concentration
MRS meat soup in；Light absorption value from 0h at 2h measurement 600nm, calculates survival rate；
As a result: bacterial strain is in concentration-dependent relation to the tolerance of hydrogen peroxide, bacterial strain to low concentration hydrogen peroxide (0.5mM) have compared with
Strong tolerance, 37 DEG C of culture 4h survival rates increased significantly, and high-strength hydrogen peroxide (1mM) survival rate is increased it is less,
The survival rate of 8h bacterial strain is also not less than 90%, and it is stronger resistance to show that lactobacillus fermenti KP101 all has various concentration hydrogen peroxide
By property (Fig. 6 a).
2, DPPH free radical is removed
Bacterium solution 4000r/min is centrifuged 10min, respectively obtains fermented supernatant fluid and somatic cells, by somatic cells with 0.85%
It brine 2 times, is suspended in isometric physiological saline, obtains somatic cells liquid；Fermented supernatant fluid, somatic cells
Liquid, fermentation liquid are respectively provided with control group, sample sets and blank group, and respectively by fermented supernatant fluid, somatic cells liquid, fermentation liquid is according to such as
Lower sequence adds reagent:
1) control group: 2mL 0.04g/L DPPH ethanol solution+2mL dehydrated alcohol；
2) sample sets: 2mL 0.04g/L DPPH ethanol solution+2mL sample solution (fermented supernatant fluid, somatic cells
Liquid or fermentation liquid)；
3) blank group: 2mL sample solution+2mL dehydrated alcohol；
After sufficiently reaction 20min, each group liquid is added in 96 orifice plates, its OD517 is measurednm；
Scavenging activity formula is as follows:
Scavenging capacity %=[1- (sample-blank)/control] × 100%；
As a result (Fig. 6 b) shows the DPPH clearance rate of the cell-free extract of lactobacillus fermenti KP101, fermented supernatant fluid, bacteria suspension
Respectively 33.9%, 88.7%, 48.6%.
3, superoxide anion merit rating is removed
It is measured using purchase in the ultra-oxygen anion free radical kit that Nanjing is built up；As a result (Fig. 6 c) shows acidified milk bar
The cell-free extract of bacterium KP101, fermented supernatant fluid, bacteria suspension ultra-oxygen anion free radical Scavenging activity be respectively
39.45U/L、136.02 U/L、110.24 U/L。
4, scavenging hydroxyl merit rating
It is measured using purchase in the hydroxy radical kit that Nanjing is built up；As a result (Fig. 6 d) shows lactobacillus fermenti KP101
Cell-free extract, fermented supernatant fluid, bacteria suspension Hydroxyl radical-scavenging ability be respectively 701.7U/L, 588.7U/L,
839.5U/L.To sum up, show that lactobacillus fermenti KP101 has preferable oxidation resistance.
4 animal experiment of embodiment
1, the raising and selection of animal
C57BL/6N male mice 30 of healthy adult are selected, mouse are randomly assigned to be 3 groups, every group 10, feeding is different
Diet is respectively as follows:
A group: Normal group (Control): normal diet；
B group: hyperlipidemia model group (HFD): high lipid food；
C group: intervention group (HFD+KP101): the fermentation liquid of 5% lactobacillus fermenti KP101 is added into high lipid food powder and fills
Divide drying after being uniformly mixed that solid feed is made.
The fermentation liquid of the lactobacillus fermenti KP101 is that lactobacillus fermenti KP101 is cultivated 18h under the conditions of 37 DEG C,
It is inoculated with according to the amount of culture solution 3%, continuously activates three generations；By above-mentioned activation and expand the seed liquor 4000r/min of culture from
Heart 10min after centrifugation, is abandoned supernatant, obtains bacterial sediment, be suspended in isometric freeze drying protectant with sterile water washing 2 times
In, obtain 1010The bacteria suspension of CFU/mL；Bacteria suspension is linked into the training of the MRS-THIO-OX-CHOL equipped with 10mL with 3% inoculum concentration
It supports in base, is cultivated for 24 hours under the conditions of 37 °C.
Intervention group needs daily to be mixed and stirred for high lipid food with corresponding intervention object uniformly, to intervene object according to 50g/kg
The dosage of BW/d is given, and guarantees the enough intakes for intervening object.Mouse freely ingests, drinks water, and feeding environment is to follow for 12 hours
Ring illumination, temperature are 20 ± 2 DEG C, humidity 50 ± 5%.Record food ration daily weighs weekly a mouse weight and replaces pad
Material.Test period is 8 weeks, and fasting 12 hours before putting to death, weight before recording extremely opens mouse peritoneal and thoracic cavity, rapidly after anesthesia
Organs of Mice and fat are taken out, weighs and records.
2, the preparation of mouse sample
Blood sample preparation: using eyeball blood sampling collect blood sample, room temperature for a period of time after, with 3000rpmmin-1
Under the conditions of be centrifuged 10 min, the serum isolated is placed in 1.5 mL EP pipes, is stored in spare in -80 DEG C of refrigerators.
Liver: being added the physiological saline of 9 times of volumes by the preparation of liver homogenate liquid, and 10% liver homogenate is made in sufficiently homogenate, will
It is with low-temperature and high-speed centrifuge with 3000 rpmmin-1Under the conditions of be centrifuged 10min, take supernatant, be placed in -80 DEG C of refrigerators and be used for
Analysis.
3, the detection of biochemical indicator
Total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein in mice serum and liver
(LDL), the indexs such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) are built up according to purchased from Nanjing
Kit specification operated.
As a result it and analyzes: compared with the control group, the horizontal significant raising of TC, LDL and TG in the serum of high fat diet mouse；And
It is horizontal (Fig. 7 abc) that the intervention of lactobacillus fermenti KP101 can significantly reduce serum TC, TG and LDL.HDL can be by excessive gallbladder
Sterol is removed from atherosclerotic plaque prevents vascular wall from depositing.Compared with Normal group, high in fat group of mice serum HDL is horizontal
Significant to increase, this may be since body is the stress reaction for reducing serum cholesterol level and making；Relative to high in fat group
Speech, lactobacillus fermenti KP101 intervention group mice serum HDL level have no significant changes, this illustrates lactobacillus fermenti KP101 to blood
The influence of clear HDL is less (Fig. 7 d).Therefore, the blood induced by high fat diet can be effectively relieved in the intervention of lactobacillus fermenti KP101
Anomalous lipid metablism.
Compared with Normal group, high fat diet causes ALT and AST level in mice serum to increase and all have conspicuousness
Difference.Lactobacillus fermenti KP101 effectively inhibits the raising of serum alt and AST level, reduces 27.09% and 14.17% respectively
(Fig. 8).
Compared with normally group mouse, high fat diet causes mouse liver TC, TG and LDL level different degrees of liter occur
Height, this shows that high fat diet not only causes metabolism disorder of blood lipid, and different degrees of damage is also generated to liver metabolism.And it is fermented
After lactobacillus KP101 intervenes, mouse liver TC, TG and LDL level has decline, reduces 13.4%, 50.5% and 67.0% respectively
(Fig. 9 abc).However, compared with normal diet group mouse, high fat diet cause mouse liver HDL level significantly reduce (p <
0.001)；Compared with high fat diet group mouse, the horizontal significantly raising of lactobacillus fermenti KP101 intervention group mouse liver HDL (p <
0.01) (Fig. 9 d).
Compared with Normal group, high fat diet causes ALT and AST level in mouse blood liver to increase and have conspicuousness
Difference.Lactobacillus fermenti KP101 effectively inhibits the raising of ALT and AST level in liver, reduces 33.34% He respectively
33.02%.The result shows that lactobacillus fermenti KP101 has certain alleviation to hepatic injury caused by high fat diet and inflammatory reaction
With improvement result (Figure 10).
Compared with normally group mouse, after high in fat group of mouse weight dramatically increases, and fermented lactobacillus KP101 intervenes,
Weight but significantly reduces (Figure 11 a).Compared with Normal group mouse, high fat diet improves mouse abdominal adipose coefficient significantly
With perirenal fat coefficient, and fermented lactobacillus KP101 intervene after, stomach fat and perirenal fat coefficient reduce respectively
95.5% and 97.2%, wherein perirenal fat coefficient declines significant (Figure 11 bc).
The bacterial strain of the present invention of embodiment 5 is compared with existing lactobacillus fermenti performance
Based on the indices measurement result of lactobacillus fermenti KP101 of the present invention, compared with existing lactobacillus fermenti,
It can be seen that lactobacillus fermenti KP101 of the present invention according to result listed by table 4 and achieve good effect；
Based on the above embodiments with table comparison result, lactobacillus fermenti KP101 of the present invention has the good tolerance acid side of body simultaneously
Compel, tolerance cholate stress, tolerance oxidative stress, anti-pathogenic infection, adjusts metabolic disorder, adjusts a variety of health such as hyperlipemia
Effect, while the significant effect in terms of Weight-reducing and lipid-lowering.A large number of studies show that lactobacillus fermenti can effectively adjust inflammatory bowel
Disease adjusts intestinal microflora, treats hyperlipemia.Radha Yadav etc. is the study found that afterfermentation lactobacillus MTCC:
The buffalo milk of 5898- fermentation, has the potentiality for reducing cholesterol, can reduce total cholesterol in rat blood serum (TC), glycerol three
The content of ester (TG) and low-density lipoprotein (LDL-c)；But lactobacillus fermenti KP101 of the present invention is to mice serum TG, LDL-
The regulating power of c is more significant, can effectively reduce increasing for TG, LDL-c in the serum as caused by high fat diet, respectively
50.5%,67.0%.Lactobacillus fermenti KP101 can not only reduce the content of TC, TG, LDL-C in serum, and can significantly mention
HDL-C is horizontal in high liver, increment rate 78.9%, the content of TC, TG and LDL-C in reduction liver, respectively 36.2%,
64.6% and 42.8%；Meanwhile lactobacillus fermenti KP101 also has extremely significant effect for reducing fat, can be significantly reduced its kidney week
Fat coefficient and abdominal fat coefficient are respectively 97.2% and 95.5%；Currently, for lactobacillus fermenti in terms of Weight-reducing and lipid-lowering
Research it is less, mostly study lactobacillus fermenti in terms of content.Lactobacillus fermenti KP101 of the present invention, is demonstrated by
Its good ability that can reduce the obesity of the fatty as caused by high fat diet.
The preparation of 6 lactobacillus fermenti KP101 fermented yoghourt of embodiment
(1) preparation of milk: qualified fresh milk filtration, purification will be examined, is proportionally added into the white granulated sugar of 5%-10%, filtered
It is preheated to 60 DEG C after mixing evenly, it is cold after high-pressure homogeneous 5min under conditions of 20MPa, the 5-10min that sterilizes under the conditions of 95 DEG C
Stand-by；
(2) the freeze-dried vaccine powder of lactobacillus fermenti KP101 and streptococcus thermophilus the activation of bacterial strain: is inoculated in germ-free milk culture medium
In, it is cultivated, is passed on 2 ~ 3 times, each inoculum concentration 3%, viable count reaches 10 by optimum temperature respectively6CFU/mL or more；
(3) preparation of fermented yoghourt: respectively by lactobacillus fermenti KP101 and streptococcus thermophilus by volume 1:1 ratio mix,
Leavening is made, is seeded in the milk cooled down according to the inoculum concentration of 3%-5%, cultivation and fermentation 4h under the conditions of 42 DEG C
Afterwards, cooling storage under the conditions of being placed on 4 DEG C.Lactobacillus fermenti KP101 viable count reaches 106CFU/mL or more.
The preparation of 7 probiotics solid beverage of embodiment
(1) prepared by lactobacillus fermenti KP101 thallus: lactobacillus fermenti KP101 being cultivated 18h under the conditions of 37 DEG C, according to culture
The amount of liquid 3% is inoculated with, and three generations is continuously activated.By above-mentioned activation and expand culture seed liquor 4000r/min be centrifuged 10min,
With sterile water washing 2 times, after centrifugation, supernatant is abandoned, bacterial sediment is obtained, is suspended in isometric freeze drying protectant, obtains
1010The bacteria suspension of CFU/mL；
(2) prepared by protective agent: add skimmed milk power 10%-12%, cellulose 7%-9%, trehalose 3%-5% respectively in aqueous solution,
Pulan polysaccharide 6%-8% sterilizes under conditions of 20min at 80 DEG C, and freeze drying protectant is made；
(3) freeze-drying prepares lactobacillus fermenti freeze-dried powder；
(4) serial probiotics solid beverage (3g) is formulated: blueberry flavor solid beverage: lactobacillus fermenti freeze-dried powder 1 g, oligomeric different
Maltose 0.5-1.0g, soyabean oligosaccharides 0.5-1.0g, blueberry powder 0.5-1.5g；Cranberry flavor solids beverage: acidified milk bar
Bacterium freeze-dried powder 1 g, oligoisomaltose 0.5-1.0g, soyabean oligosaccharides 0.5-1.0g, Cranberry powder 0.5-1.5g；Hawthorn flavor
Solid beverage: lactobacillus fermenti freeze-dried powder 1 g, oligoisomaltose 0.5-1.0g, soyabean oligosaccharides 0.5-1.0g, hawthorn powder
The probiotics solid beverage of different taste is made in 0.5-1.5g；
(5) product specification: solid beverage uses 3g for 1 packet, and the viable count of the lactobacillus fermenti KP101 of addition is 1010CFU/
mL。
Claims (8)
1. one plant of lactobacillus fermentiLactobacillus fermentum KP 101, its deposit number is CCTCC No. M
2019085。
2. a kind of fermented yoghourt, it is prepared by the following method:
1) activation of bacterial strain: the lactobacillus fermenti KP101 and streptococcus thermophilus for being CCTCC No. M 2019085 by deposit number
Freeze-dried vaccine powder be inoculated in germ-free milk culture medium, cultivated respectively by optimum temperature, pass on 2 ~ 3 times, each inoculum concentration 1
~ 10%, viable count reaches 106CFU/mL or more；
2) preparation of fermented yoghourt: by the ratio of lactobacillus fermenti KP101 and streptococcus thermophilus 1 ~ 2:1 by volume after activation
Mixing, is seeded in milk according to 1% ~ 10% inoculum concentration, and ferment 3 ~ 5h at 38 ~ 45 DEG C, cooling at 0 ~ 4 DEG C, is sent out
Ferment Yoghourt.
3. a kind of fermented yoghourt according to claim 2, it is characterised in that: described in step 2 according to 3% ~ 5% inoculation
Amount is seeded in milk, and ferment 3 ~ 5h at 40 ~ 43 DEG C.
4. a kind of fermented yoghourt according to claim 3, it is characterised in that: milk described in step 2 is by following sides
Method preparation: the sugar of mass fraction 5% ~ 10% is added in filtering, purification, and filtering stirs evenly, is heated to 55 ~ 65 DEG C, 18 ~
It is cooling after 3 ~ 10min of homogeneous under 22MPa, the 5 ~ 10min that sterilizes under the conditions of 95 ~ 100 DEG C.
5. a kind of probiotics solid beverage, it includes the component of following parts by weight: 10 parts of lactobacillus fermenti freeze-dried powder, oligomeric different
5 ~ 10 parts of maltose, 5 ~ 10 parts of soyabean oligosaccharides, 5 ~ 15 parts of fruity powder；The lactobacillus fermenti, its deposit number are
CCTCC No. M 2019085。
6. a kind of probiotics solid beverage according to claim 5, it is characterised in that: the fruity powder be blueberry powder,
Cranberry powder or hawthorn powder.
7. a kind of probiotics solid beverage according to claim 6, it is characterised in that: the lactobacillus fermenti freeze-drying
Powder is prepared by the following method: lactobacillus fermenti KP101 is activated, expand culture, at 3000 ~ 5000r/min be centrifuged 8 ~
15min, it is washed with water, supernatant is abandoned in centrifugation, obtains thallus；By thallus and freeze drying protectant, 1 ~ 3:1 is mixed by volume, and freezing is dry
It is dry, obtain lactobacillus fermenti freeze-dried powder.
8. a kind of probiotics solid beverage according to claim 7, it is characterised in that: the freeze drying protectant, by matter
Measure percentages include following component: skimmed milk power 10%-12%, cellulose 7%-9%, trehalose 3%-5%, Pulan polysaccharide 6%-8%,
Surplus is water.
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| CN112940967B (en) * | 2021-02-06 | 2022-06-21 | 中南大学 | Lactobacillus fermentum MF423, fermented rice bran extract thereof and application of lactobacillus fermentum MF423 and fermented rice bran extract |
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| CN115989870A (en) * | 2021-10-19 | 2023-04-21 | 百岳特生物技术（上海）有限公司 | Use of lactobacillus fermentum or its metabolites for preparing a composition for increasing muscle and reducing lipid |
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