CN104498383B - A kind of lactobacillus fermenti Lactobacillus fermentum strain suo and application thereof of adjustable intestinal movement, Constipation - Google Patents
A kind of lactobacillus fermenti Lactobacillus fermentum strain suo and application thereof of adjustable intestinal movement, Constipation Download PDFInfo
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- CN104498383B CN104498383B CN201410578559.2A CN201410578559A CN104498383B CN 104498383 B CN104498383 B CN 104498383B CN 201410578559 A CN201410578559 A CN 201410578559A CN 104498383 B CN104498383 B CN 104498383B
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- 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
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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
- A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
-
- 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
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- 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
It is CCTCC NO the invention discloses lactobacillus fermenti (Lactobacillus fermentum strain suo) deposit number of a kind of adjustable intestinal movement, Constipation：M 2013511 and application thereof, the acid resistance of the bacterial strain is stronger, survival rate has reached 92.46 ± 4.06% after 3h under pH3.0 simulated gastric fluid, can slowly it be grown under 1.0% concentration cholate, growth efficiency reaches the 17.36 ± 1.19% of no cholate culture, the hydrophobicity of Lactobacillus fermentum strain suo cells has also reached 68.44 ± 2.48%, the normal growth in the enteron aisle of people.Lactobacillus fermentum strain suo can slow down mouse weight caused by constipation and mitigate trend, accelerate row's melena time, improve Intestinal propulsive rate, MTL, Gas, ET, AchE, SP and VIP factor level of different degrees of increase serum, the SS factors are reduced, increase food ration and amount of drinking water, reduce feces volume, defecation granule number and excrement water content downward trend, similar effect is played to constipation medicine, shows certain constipation preventive effect.
Description
Technical field
The present invention relates to microbial technology field, more particularly to a kind of adjustable intestinal movement, the acidified milk of Constipation
Bacillus Lactobacillus fermentum strain suo and application thereof.
Background technology
Lactobacillus is in close relations with human lives, is to be widely used in food fermentation, industrial lactic fermentation and health care
One of beneficial microbe in field.Many kinds in the lactobacillus and Bifidobacterium that have recognized all have specific excellent
Benign matter and inhereditary feature, show extremely strong probiotic properties.Lactobacillus can not only improve food value, improve food
Product flavor, extend the holding time, the functional character of food can also be improved.It can be by decomposing the protein in food, sugar
Class, synthesis vitamin, improve the digestibility and biological value of food, promote to digest and assimilate.Can also reduce human cholesterol content,
Strengthen immunologic function, anti-allergenicity, antitumor, anti-hypertension etc..In recent years, lactobacillus is with its special physiologically active and battalion
Function is supported as the main source of probiotics, is just had attracted increasing attention.
Constipation is a kind of very universal disease, and the illness rate in crowd is up to 27%, and it can influence each age group
People.Habitual constipation defecation with strength of holding one's breath, is to induce middle-aging male high fat of blood, cardiovascular and cerebrovascular disease because firmly increasing abdominal pressure
The arch-criminal of the diseases such as disease, artery sclerosis, tumour and its complication, so as to cause old man's cardio cerebrovascular affection death rate drastically
Increase, there is the problems such as hemorrhoid, anal fissure, it is painful to can't bear.Other prolonged constipation, largely just slag can be trapped in hair in enteron aisle
Ferment, rot, a large amount of toxin and harmful bacteria are absorbed repeatedly by enteron aisle, reach partes corporis humani position by blood circulation, turn into women and be good for
Health and beautiful arch-criminal.Constipation can also make child's gastrointestinal bacterial flora unbalance, cause energy not concentrated, thought slowness, nutrition
It is bad, mood is irritable, becomes thin, immunity degradation, easy catching a cold, growth retardation, such as nurse one's health not in time, irreversible disease may be caused
Become.Because the constipation incidence of disease is high, the cause of disease is complicated, patient is often had manifold vexations, and quality of life can be influenceed when constipation is serious.Therefore, seek
Look for it is a kind of can the alternatives to medication biological products that carry out Constipation have very big realistic meaning, Tiny ecosystem therapy is just fine with one
New means solve various problems existing for traditional remedies.
There is substantial amounts of research report to the physiological function of lactic acid bacteria both at home and abroad, but the feature of lactic acid bacteria Constipation is ground
It is seldom to study carefully report.Research on lactic acid bacteria Constipation to many subjects such as Food Science, preventive medicine and microecology all
There is very big value.Fermented dairy product is the important channel that people take in lactic acid bacteria, screen it is excellent have Constipation and
The lactic acid bacteria strains of safety, will have for development functionality dairy products, the added value enriched existing dairy products species, improve dairy products
It is significant.Therefore, the lactic acid bacteria with Constipation has very big research and application value.
In disclosed document and patent or patent application at present, such as CN101144064A discloses one kind and had
The Lactobacillus brevis and application thereof of antimutagenicity, extracellular polysaccharide, for improving food quality in fermented food, increases it
Nutrition function.CN1796540A discloses Bifidobacterium with anti-enterovirus and anti-oxidation characteristics and application thereof, uses
In preparing food compositions or pharmaceutical composition, kill or suppress helicobacter pylori or Escherichia coli, treat what is be induced by it
Various diseases.CN102174450A discloses a kind of Lactobacillus plantarum of Helicobacter pylori Infection and application thereof, for preparing
Prevent or mitigate the infection of helicobacter pylori in pharmaceutical composition, leavening, animal feed additive and health products.
CN101144065A discloses a kind of resisting hydrogen peroxide, the oxidation resistant Lactobacillus casei for removing free radical and application thereof, for sending out
With development functionality dairy products, the added value enriched existing dairy products species, improve dairy products in ferment food and dairy products capsule product.
But these patents or patent application are not related to the microorganism with performances such as regulation intestinal movement, Constipations completely.Cause
This, also needs to a kind of with regulation intestinal movement, the microorganism of Constipation and its food containing them at present.
The content of the invention
In order to solve the above problems, the invention provides a kind of adjustable intestinal movement, the lactobacillus fermenti of Constipation
And application thereof, a kind of adjustable intestinal movement, the L fermentum strain of Constipation and a kind of hair of offer are mainly provided
Purposes of the kefir milk bacillus in fermented food.
A kind of adjustable intestinal movement provided by the invention, the lactobacillus fermenti (Lactobacillus of Constipation
Fermentum strain suo) on October 25th, 2013 it is preserved in China typical culture collection center (CCTCC), the guarantor
The address at Tibetan center is located at：Chinese Wuhan Wuhan Universitys, deposit number are CCTCC NO：M 2013511.
One plant of lactobacillus fermenti (Lactobacillus fermentum is filtered out from spontaneous fermentation yak yogurt
Strain suo), using Microbiological Characteristics such as morphological feature, cultural colony and physiological and biochemical properties to the Bacillus acidi lactici
(Lactobacillus fermentum strain suo) is accredited as lactobacillus fermenti, and the bacterial strain is on October 25th, 2013
It is preserved in China typical culture collection center (CCTCC), deposit number CCTCCNO：M 2013511.
The morphological feature of the lactobacillus fermenti is specially：
Thalline feature：In Gram-positive, cell is shaft-like, and thalline is about 0.5-1.0 μm wide, 1.5-4 μm long, Cheng Dan,
Paired or chaining, does not form gemma, and both ends are circular.
Colony characteristicses：Obvious bacterium colony is formed on MRS culture mediums, diameter is circular between 0.5-2.0mm, and edge is whole
Together, milky, transparent, surface wettability is smooth, not chromogenesis.
Tolerance is characterized as in the lactobacillus fermenti body：
The bacterial strain acid resistance of lactobacillus fermenti is stronger, under pH3.0 simulated gastric fluid after 3h survival rate be 92.46 ±
4.06%；Can slowly it be grown under 1.0% concentration cholate, growth efficiency reaches the 17.36 ± 1.19% of no cholate culture；Hair
The hydrophobicity of kefir milk bacilli-cell is 68.44 ± 2.48%.
The lactobacillus fermenti derives from traditional fermented food, category generally recognized as safe (Generally Recognized As
Safe, GRAS) strain, in fermented food.
The fermented food is in the lactic acid bacteria milk beverage containing lactobacillus fermenti, milk powder, capsule product or acidified milk
It is one or more.
The lactic acid bacteria milk beverage containing lactobacillus fermenti is prepared as steps described below：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with 30% (weight) glycerine suspensions, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
Raw milk heats the sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is cooled to 4 DEG C, described in addition
Lactobacillus fermenti working stock culture, its concentration is set to reach 106More than cfu/mL, it is stored refrigerated at 4 DEG C.
The milk powder containing lactobacillus fermenti is prepared as steps described below：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with 30% (weight) glycerine suspensions, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
Raw milk heats the sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is then cooled to 37 DEG C, then
The described lactobacillus fermenti working stock culture of bacterium amount inoculation is connect with the 4% of raw milk volume, ferment 16h at 37 DEG C, is fermented
Lactobacillus ferment breast；Raw milk after the lactobacillus fermenti acidified milk and sterilizing matches according to 1: 3 (V: V) to be added, and is carried out equal
Matter, it is concentrated in vacuo, spray drying obtains the milk powder containing lactobacillus fermenti.
The capsule product containing lactobacillus fermenti is prepared as steps described below：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with 30% (weight) glycerine suspensions, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
By raw milk in 140 DEG C of high temperature thermal sterilization 2s, 37 DEG C are subsequently cooled to, connecing bacterium amount with the 4% of raw milk volume connects
The kind lactobacillus fermenti working stock culture, ferment 16h at 37 DEG C, obtains lactobacillus fermenti acidified milk；By the acidified milk
Raw milk after bacillus fermentation breast and sterilizing is according to 1: 3 (V: V)) proportioning addition, homogeneous, it is concentrated in vacuo, spray drying treatment
After obtain milk powder, the capsule product containing lactobacillus fermenti will be obtained.
The acidified milk containing lactobacillus fermenti is prepared as steps described below：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with 30% (weight) glycerine suspensions, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
Raw milk heats the sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is then cooled to 37 DEG C, then
According to 3-5% (volume) add lactobacillus fermenti working stock culture, add 3-5% (volume) can symbiosis be used for prepare fermentation
The leavening of dairy products, mixed fungus fermentation to titratable acidity 0.6-0.7% in terms of lactic acid, is subsequently cooled to 4 at 37 DEG C after mixing
DEG C, then carry out stored refrigerated obtaining the acidified milk containing lactobacillus fermenti.
The preparation method of the lactobacillus fermenti working stock culture includes：
The original strain of the lactobacillus fermenti is inoculated in the skimmed milk of 12% (weight), 110 DEG C of sterilizing 10min,
14-16h is cultivated under the conditions of 37 DEG C to curdled milk, continuous culture two generations of activation, as mother culture；The mother culture is pressed into 3-
5% (volume) is inoculated in sterile milk, culture 14-16h to curdled milk, now the viable count about 10 in the curdled milk9Cfu/mL, obtain
Working stock culture, directly this working stock culture can be added in food, or with can symbiosis the business for preparing acidified milk
Industry leavening such as lactobacillus bulgaricus is used together with streptococcus thermophilus prepares acidified milk；Or
The original strain of the lactobacillus fermenti is inoculated in MRS fluid nutrient mediums, 12- is cultivated under the conditions of 37 DEG C
16h is activated, continuous to activate for two generations, is then inoculated in activation culture thing in MRS culture mediums by 2-4% (volume), is cultivated
16-18h, 4000r/min centrifuges 15min under the conditions of 4 DEG C, removes supernatant, cell precipitation is obtained, by precipitation sterile absorbent
Suspension is made in breast, obtains working stock culture.
Described heating sterilization is the TW10D1000 for example sold using Shanghai Triowin Automation Machinery Limited
What type tubular sterilization machine was carried out.
Described high temperature thermal sterilization is the YC-104 types for example sold using Beijing Yong Chuanjiaye plant equipment Co., Ltd
What board-like superhigh temperature sterilization machine was carried out.
Described homogeneous is that the small-sized homogenizers of GJB500-40 for example sold using Changzhou homogeneous Machinery Co., Ltd. are entered
Capable.
Described concentration is that the vacuum concentration pan for example sold using Shanghai Wei Zhou light industry and machinery Co., Ltd is carried out.
Described spray drying is that the experiment type spray drier for example sold using Shanghai Triowin Tech. Co., Ltd. is entered
Capable.
Described commodity leavening is preferably lactobacillus bulgaricus or streptococcus thermophilus.
In the sense of the present invention, described raw milk is one or more selected from defatted milk, fresh milk, the raw material of recovery milk
Breast.
What the technical scheme of the embodiment of the present invention was brought has the beneficial effect that：
The present invention is a kind of adjustable enteron aisle fortune being isolated from Sichuan Province Hongyuan County herdsman man spontaneous fermentation yak yogurt
The dynamic, lactobacillus fermenti of Constipation and using the bacterial strain as the leavening or acidified milk of addition, healthy food and animal health
Application in product, the acid resistance of lactobacillus fermenti (Lactobacillus fermentum strain suo) bacterial strain is stronger,
Survival rate has reached 92.46 ± 4.06% after 3h under pH3.0 simulated gastric fluid, can slowly be given birth under 1.0% concentration cholate
Long, 17.36 ± 1.19%, the Lactobacillus fermentum strain suo that growth efficiency reaches no cholate culture are thin
The hydrophobicity of born of the same parents has also reached 68.44 ± 2.48%.This shows lactobacillus fermenti (Lactobacillus fermentum
Strain suo) can in the enteron aisle of people normal growth.In-vivo test in mice is also shown that lactobacillus fermenti (Lactobacillus
Fermentum strain suo) mouse weight mitigation trend caused by constipation can be slowed down, accelerate row's melena time, improve small
Alvine pushing rate, MTL, Gas, ET, AchE, SP and VIP factor level of different degrees of increase serum, reduces the SS factors, increase
Food ration and amount of drinking water, feces volume, defecation granule number and excrement water content downward trend are reduced, is played to constipation medicine similar
Effect, show certain constipation preventive effect.
Brief description of the drawings
For the technical scheme of the clearer explanation embodiment of the present invention, required use in being described below to embodiment
Accompanying drawing be briefly described, it is clear that, drawings in the following description are only some embodiments of the present invention, for this
For the those of ordinary skill of field, on the premise of not paying creative work, it can also be obtained according to these accompanying drawings other
Accompanying drawing.
Fig. 1 lactobacillus fermentis (Lactobacillus fermentum strain suo) colonial morphology；
The thalli morphology (1000x) of Fig. 2 lactobacillus fermentis (Lactobacillus fermentum strain suo)；
Fig. 3 mouse weights with experimental period change；
The time required to Fig. 4 separate groups of mice arranges melena first；
The total small intestinal length of Fig. 5 separate groups of mice；
The Intestinal propulsive rate of Fig. 6 separate groups of mice；
Each factor level in Fig. 7 separate groups of mice serum.
The lactobacillus fermenti (Lactobacillus fermentum strain suo) of the present invention was October 25 in 2013
Day is preserved in China typical culture collection center, and the address of the collection is located at：Chinese Wuhan Wuhan Universitys, numbering are
CCTCC NO：M 2013511.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Whole description, it is clear that described embodiment be only the present invention part of the embodiment, the embodiment being not all of, based on this hair
Embodiment in bright, the every other implementation that those of ordinary skill in the art are obtained under the premise of creative work is not paid
Example, belongs to the scope of protection of the invention.
Embodiment 1：The separation of lactobacillus fermenti (Lactobacillus fermentum strain suo), purifying and just
Step is fixed.
The embodiment is carried out as steps described below：
(1) Lactobacillus fermentum strain suo separation, purifying.
Added with the μ L of sterile working absorption traditional zymotic yak yoghourt sample 500 to the mixing of 5mL sterile salines and be made 1
: 10 uniform dilution, and continue to make a certain proportion of dilution.The dilution of suitable gradient is selected, is respectively drawn with sterile pipette tips
100 μ L are respectively coated in MRS solid plate culture mediums, 30 DEG C of culture 48-72h, it is as shown in Figure 1 to observe and record colonial morphology.
With oese (or sterilizing toothpick) from planar surface and internal picking difference colony inoculation in MRS fluid nutrient mediums
In, 30 DEG C are put, 300r/min shaking table cultures 24-48h；Repeat to carry out smear Gram's staining after above step continuously activated for 2 generations
Microscopy is as shown in Figure 2.
It is defined as G+ bacterium to continue to activate, until obtaining pure bacterium colony (microscopy is without miscellaneous bacteria), and carries out catalase experiment.
The sporeless bacterium of G+ and negative catalase, coccus and streptococcus are fixed tentatively as lactic acid bacteria, and preserved, concrete operations ginseng
According to《Microbiology Experiment technology》.
Experimental result is shown, is pacified from Hongyuan in ten samples in bent area and is isolated more than 58 plants of lactic acid bacteria, sample lactic acid
Bacterium viable count is average 108The cfu/mL orders of magnitude.Wherein Lactobacillus fermentum strain suo are that numbering is
54 bacterial strain.
(2) Lactobacillus fermentum strain suo Preliminary Identification.
Gram's staining：Colonies typical smear is taken, Gram's staining is carried out, in microbioscope oil Microscopic observation thalline
Form and its arrangement mode.Picking gets a clear view, the typical bacterial strain of form, is taken pictures.Catalase is tested：Picking solid
The oese of bacterium colony one, is placed in clean tube on culture medium, 3% hydrogenperoxide steam generator 2mL is added dropwise, observation result is (in half a minute
Interior gassing person is the positive, and gassing person is not feminine gender).
As a result show, isolated from sample and be purified into 58 strains of lactic acid bacteria, these bacterial strains can be containing 5 ‰ CaCO3MRS
Grown on plating medium, and molten calcium circle, Gram-positive, catalase test feminine gender are formed in periphery of bacterial colonies.
Embodiment 2：The in-vitro screening of lactobacillus fermenti (Lactobacillus fermentum strain suo).
(1) screening of probiotics tolerance pH3.0 simulated gastric fluids.
The preparation of simulated gastric fluid：NaCl 0.2%, pepsin 0.35%, after being 3.0 with 1M HCl adjustment pH value,
It is degerming rear standby with vavuum pump suction filtration in aseptic operating platform.
Measure of the probiotics to artificial gastric juice resistance's property：The activated good strain cultured solutions of 5mL are taken, in aseptic operating platform
In pour into the 10mL centrifuge tubes that sterilized, collect thalline through 3000r/min centrifugations 10min, add 5mL sterile salines and mix
Bacteria suspension is made, takes 1mL bacteria suspensions and 9mL pH3.0 simulated gastric fluid to mix, shakes up, is placed in constant temperature oscillator and cultivates (37
DEG C, 300r/min), and sampled respectively in 0h and 3h, pour into 37 DEG C of culture 48h with MRS agar mediums.Surveyed with colony counting method
Determine viable count, calculate its survival rate (%)：Survival rate (%)=3h viable count/0h viable count × 100%.
(2) measure of probiotics tolerance various concentrations cholate.
The growth that strain of the survival rate more than 10% is done under different gallbladder salinities in pH3.0 simulated gastric fluids is selected to survey
Examination.The strain 5mL activated is inoculated in containing 0.0% bovine bile (i.e. sky respectively by 2% inoculum concentration (100 μ L) with liquid-transfering gun
In vain), 0.3% bovine bile, 0.5% bovine bile, the MRS-THIO culture mediums of 1.0% bovine bile (W/V) (add in MRS culture mediums
0.2% sodium thioglycolate).It is (nonvaccinated to compare using blank cultures in constant temperature oscillator after 37 DEG C of culture 24h
MRS-THIO culture mediums), the OD values of above-mentioned various concentrations culture medium are determined respectively, calculate tolerance of the bacterial strain to cholate.Cholate
OD value × 100% of OD values/blank cultures of tolerance=culture medium containing cholate.
(3) bacterial concentration adjusts.
More than 10%, the strain that bile tolerance is stronger, bacterium is observed through microscopy for selection survival rate in pH3.0 simulated gastric fluids
Tested after body growthform is good, the bacterial cultures 5mL that will have been activated, the 10mL that sterilized is poured into aseptic operating platform
In centrifuge tube, thalline is collected with 3000r/min centrifugations 10min.With 5mL PBS (50mM, pH6.5) buffer solution washing thalline,
3000r/min centrifuges 10min, and repeated washing is twice.Using PBS as blank control, strain subject bacterium is adjusted with buffer solution
Bulk concentration, it is about 1.00 to make its A0 value under 560nm wavelength.
The bacterium solution that 4mL adjusts turbidity is taken to add 0.8mL dimethylbenzene, control group is not added with the 10mL centrifuge tubes that sterilized
Dimethylbenzene, vibrate 30s, pause 10s, after vibrate 30s again, on rack for test tube stand 5~10min layering, remove a layer aqueous phase, with
PBS is blank control, and A values are measured under 560nm and are recorded.Hydrophobic rate H%=[(A0-A)/A0] × 100 its
Middle A0 and A are to mix the A values that forward and backward bacterium solution measures to obtain under 560nm with dimethylbenzene respectively.
Lactic acid bacteria is one of a part for normal bowel bacterium, the most important condition as probiotics, and being can be before stomach and small intestine
Section survival, the bacterial strain being screened should have the characteristic grown under enough acid resistance and sour environment.
Lactobacillus fermentum strain suo the selection result is as shown in table 1 below：
Table 1
The selection result shows, there is 4 plants of environment for being resistant to pH3.0 in lactic bacteria strain, and survival rate is all more than 50%, its
In No. 54 bacterium Lactobacillus fermentum strain suo acid resistances it is most strong, survival rate reaches under pH3.0 gastric juice
92.46 ± 4.06%.
It will be contacted by the thalline survived after stomach with the cholate in small intestine, resistivity of this experiment lactic acid bacteria to cholate
For a selection standard as potential probiotics.The preferable No. 54 bacterium Lactobacillus fermentum of acid resistance
The result of strain suo tolerance various concentrations cholate is that have certain tolerance to 0.3% cholate, when gallbladder salinity liter
When up to 0.5% and 1.0%, No. 54 bacterium Lactobacillus fermentum strain suo have embodied good cholate
Tolerance.
While except having certain resistivity to cholate in small intestine, lactic acid bacteria need to also have good adhesion on mucous membrane of small intestine
Property, therefore by the hydrophobic ability of lactic acid bacteria alternately standard, as can be seen from the table, No. 54 bacterium Lactobacillus
Fermentum strain suo hydrophobicity has reached 68.44 ± 2.48%.
Us are tested more than acid resistance, bile tolerance and preferable No. 54 bacterium of hydrophobicity, this bacterial strain are filtered out and ordered
Entitled Lactobacillus fermentum strain suo lactobacillus fermentis, the bacterial strain is in preservation on October 25 in 2013
In China typical culture collection center (abbreviation CCTCC), deposit number CCTCC NO：M 2013511.
Embodiment 3：Activated carbon inducing constipation is tested.
Feeding of healthy kunming mice, 6 week old, 23 ± 2g of body weight, female, 50., will be small after feeding basal feed adapts to 5d
Mouse is randomly divided into 5 groups according to body weight, is respectively：Normal group, constipation control group, Bisacody group, lactobacillus bulgaricus group and
The Lactobacillus fermentum strain suo groups of high and low given low, every group 10.Raising is in Rotating Stainless Steel Cage
In, 24 ± 2 DEG C of room temperature holding, relative humidity 50 ± 10%, 12h light and shades rotation (8：00~20：00 illumination).In whole experiment week
Phase, normal group and the equal feeding basal feed of constipation control group, experimental mice press 1.0 × 10 daily respectively9CFU/kg gavages benefit
Raw bacterium.Start every morning 9 after 2 weeks：00 to 2 DEG C of activated carbon ice that other 5 groups of mouse stomach concentration are 10% in addition to normal group
Water, 0.2mL/ only, daily gavage once, continuous gavage 3 days.Until experiment last day, all mouse fasting 24h, Ran Houzai
Gavage activated carbon frozen water, 0.2mL/ is only.5 mouse are used to determine changes of weight, food ration, amount of drinking water, first discharge in every group
Melena required time, discharge feces just quantity and moisture in feces.Other 5 mouse of mouse are put to death after gavage activated carbon 30min
Dissection.
The time required to the mouse weight change of experiment periods Nei Ge groups, first discharge melena as shown in Figure 3 and Figure 4.
Normal group (Normal) mouse weight is put on weight increase with experimental period, control group (Control) after 6 days by
There is constipation weight loss in gavage activated carbon, Bisacody (bisacodyl) constipation medicine group slow down weight loss,
Lactobacillus fermentum strain suo groups also slow down weight loss, and high concentration group weight loss is less.
Compared with normal group, constipation control group mice arranges the melena time first pole significant difference (P ＜ 0.05).It is high and low
The average time that the Lactobacillus fermentum strain suo groups mouse of given low arranges melena for the first time is low
In constipation control group, higher than normal group and constipation therapy medicine Bisacody group, certain constipation preventive effect is shown.
The change such as table 2 below of experiment periods Nei Ge groups mouse diet intake：
Table 2
Every group of mouse n=10ICR, Bisacody：100mg/kg b.w., lactobacillus bulgaricus：1.0×109CFU/kg
B.w., LF-Suo：Lactobacillus fermentum strain suo.
Normal group (Normal) mouse weight is slightly increased with experimental period food ration, and control group is after 6 days due to gavage
There is the reduction of constipation food ration in activated carbon, and Bisacody constipation medicine group food ration is less than normal group higher than control group,
Also there is the situation similar with Bisacody, high concentration group food ration in Lactobacillus fermentum strain suo groups
Reduce less.
The change such as table 3 below of experiment periods Nei Ge groups mouse amount of drinking water：
Table 3
Every group of mouse n=10ICR, Bisacody：100mg/kg b.w., lactobacillus bulgaricus：1.0×109CFU/kg
B.w., LF-Suo：Lactobacillus fermentum strain suo.
Normal group mouse weight is slightly increased with experimental period amount of drinking water, and control group is after 6 days because gavage activated carbon goes out
Existing constipation amount of drinking water is reduced, and Bisacody constipation medicine group amount of drinking water is less than normal group, Lactobacillus higher than control group
Also there is the situation similar with Bisacody in fermentum strain suo groups, and high concentration group amount of drinking water reduces less.
The change such as table 4 below of the feces volume of experimental period Nei Ge group mouse, defecation granule number and moisture in feces：
Table 4
Every group of mouse n=10ICR, Bisacody：100mg/kg b.w., lactobacillus bulgaricus：1.0×109CFU/kg
B.w., LF-Suoi Lactobacillus fermentum strain suo.
The feces volume of preceding 6 days each groups, defecation granule number and moisture in feces difference are little.After 6 days after constipation induction, just
Normal pack variable is decreased obviously without significant change, control group feces volume, defecation granule number and moisture in feces compared with normal group, than
Sand can pyridine and each probiotic group feces volume, defecation granule number and fecal water are higher than control group, Lactobacillus
The defecation granule number and moisture in feces of fermentum strain suo high concentrations are close to medicine group and normal group.
Embodiment 4：Small intestine activated carbon Promoting Experiment.
Mouse is taken off into cervical vertebra to put to death, splits the mesenterium of abdominal cavity and separating mouse, on clip from pylorus, under to ileocecus
Whole intestinal tube, is put on blotting paper, and whole piece small intestine is pulled into straight line, to measure Length of intestine, is " total small intestinal length ", from deep and remote
Door to activated carbon juice forward position is " activated carbon propulsion length ".By measuring the propulsion length of activated carbon in small intestine, it is calculated as follows
Propulsion rate.Propulsion rate (%)=(activated carbon advance distance)/(total small intestinal length) × 100.
The total small intestinal length of experimental period Nei Ge group mouse, Intestinal propulsive rate are as shown in Figure 5 and Figure 6.
Compared with normal group, constipation control group mice Intestinal propulsive rate has pole significant difference (P ＜ 0.05), high and low gavage
The Lactobacillus fermentum strain suo group mouse small intestine propulsions rates of dosage are higher than constipation control group, less than just
Often group and constipation therapy medicine Bisacody group, show certain constipation preventive effect.
Embodiment 5：The measure of each factor level in mice serum.
0.2mL rat aorta blood is taken, at 4 DEG C, 3000r/min centrifugation 10min, takes upper serum.According to motilin
(MTL), gastrin (Gas), Endothelin (ET), growth hormone release inhibiting hormone (SS), serum acetylcholinesterase (AchE), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP) and
Above-mentioned factor level in the method measure serum of vasoactive intestinal peptide (VIP) test agent box specification.
Each factor level is as shown in Figure 7 in experiment periods Nei Ge groups mice serum.
Relative to constipation control group, Lactobacillus fermentum strain suo height is organized can be in various degree
Increase serum MTL, Gas, ET, AchE, SP and VIP factor level, reduce SS factor levels.So Lactobacillus
Fermentum strain suo have prevention effect to constipation.
Application Example 1：Breast is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain suo)
Sour bacterium milk drink.
First, lactobacillus fermenti (the Lactobacillus fermentum strain suo) original strain is in temperature
Preserved, or preserved in the form of being freeze-dried bacterium powder at 4 DEG C of temperature standby with 30 weight % glycerine suspensions at -75 DEG C of degree
With.
It is then possible to lactobacillus fermenti (the Lactobacillus fermentum of the present invention are prepared using two methods
Strain suo) working stock culture：
First method is that above-mentioned lactobacillus fermenti (Lactobacillus fermentum strain suo) is original
Strain is inoculated in 12 weight % in 110 DEG C of skimmed milks for sterilizing 10min, and 14-16h is cultivated under the conditions of 37 DEG C to curdled milk, is connected
Continuous culture two generations of activation, as mother culture；The mother culture is inoculated in sterile milk by 3-5% (volume), cultivates 14-
16h is to curdled milk, the now viable count about 10 in the curdled milk9Cfu/mL, described working stock culture is obtained, can directly will be this
Working stock culture is added in food, or with can symbiosis the commercial fermentation agent such as lactobacillus bulgaricus for preparing acidified milk and
Streptococcus thermophilus, which is used together, prepares acidified milk.
Second method is that above-mentioned lactobacillus fermenti (Lactobacillus fermentum strain suo) is original
Strain is inoculated in MRS fluid nutrient mediums, and 12-16h is cultivated under the conditions of 37 DEG C and is activated, continuous to activate for two generations, then will
Activation culture thing is inoculated in MRS culture mediums by 2-4% (volume), cultivates 16-18h, and 4000r/min is centrifuged under the conditions of 4 DEG C
15min, supernatant is removed, obtains cell precipitation, suspension is made with a certain amount of sterile absorbent breast in precipitation, obtains work hair
Ferment agent is standby.
Then, raw milk heats the sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is then cooled to 4
DEG C, foregoing lactobacillus fermenti (Lactobacillus fermentum strain suo) working stock culture is added,
Its concentration is set to reach 106More than cfu/ml, 4 DEG C it is stored refrigerated i.e. obtain Lactobacillus containing lactobacillus fermenti
The lactic acid bacteria milk beverage of fermentum strain suo viable bacterias.
In the present invention, described MRS fluid nutrient mediums are known to those skilled in the art, are Suo Laibao public affairs
Take charge of the culture medium for lactobacillus culture of sale.
Described heating sterilization is the TW10D1000 for example sold using Shanghai Triowin Automation Machinery Limited
What type tubular sterilization machine was carried out.
Described high temperature thermal sterilization is the YC-104 types for example sold using Beijing Yong Chuanjiaye plant equipment Co., Ltd
What board-like superhigh temperature sterilization machine was carried out.
Application Example 2：Breast is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain suo)
Powder.
According to above-mentioned lactic acid bacteria milk beverage preparation method prepare, simply raw milk heat at 95 DEG C sterilize 20min or
The high temperature thermal sterilization 2s at 140 DEG C, is then cooled to 37 DEG C, then with the 4% of raw milk volume to connect bacterium amount inoculation foregoing
Lactobacillus fermenti (Lactobacillus fermentum strain suo) working stock culture, then the 16h that fermented at 37 DEG C, are obtained
To lactobacillus fermenti (Lactobacillus fermentum strain suo) acidified milk；Then described lactobacillus fermenti
(Lactobacillus fermentum strain suo) acidified milk is added in above-mentioned sterilizing raw milk according to 1: 3 (V: V), entered
Row homogeneous, it is concentrated in vacuo, spray drying obtains (Lactobacillus fermentum strain suo) containing lactobacillus fermenti
Milk powder.
Described homogeneous is that the small-sized homogenizers of GJB500-40 for example sold using Changzhou homogeneous Machinery Co., Ltd. are entered
Capable.
Described concentration is that the vacuum concentration pan for example sold using Shanghai Wei Zhou light industry and machinery Co., Ltd is carried out.
Described spray drying is that the experiment type spray drier for example sold using Shanghai Triowin Tech. Co., Ltd. is entered
Capable.
Application Example 3：Glue is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain suo)
Capsule product.
By raw milk in 140 DEG C of high temperature thermal sterilization 2s, 37 DEG C are subsequently cooled to, connecing bacterium amount with the 4% of raw milk volume connects
Lactobacillus fermenti (Lactobacillus fermentum strain suo) working stock culture of the kind present invention, is issued at 37 DEG C
Ferment 16h, obtain lactobacillus fermenti (Lactobacillus fermentum strain suo) acidified milk.By lactobacillus fermenti
(Lactobacillusfermentum strain suo) acidified milk adds homogeneous in the raw milk after sterilizing with 1: 3 (V: V),
Milk powder is obtained through being concentrated in vacuo, after spray drying treatment, obtained milk powder is attached in capsule capsule product is made.
Application Example 4：Prepared and sent out using lactobacillus fermenti (Lactobacillus fermentum strain suo)
Kefir milk.
According to above-mentioned lactic acid bacteria milk beverage preparation method prepare, simply raw milk heat at 95 DEG C sterilize 20min or
The high temperature thermal sterilization 2s at 140 DEG C, is then cooled to 37 DEG C, and foregoing acidified milk bar is added according still further to 3-5% (volume)
Bacterium (Lactobacillus fermentum strain suo) working stock culture, add 3-5% (volume) can symbiosis system
Preparation kefir milk commodity leavening, mixed fungus fermentation to titratable acidity 0.6-0.7% in terms of lactic acid, then cools down at 37 DEG C after mixing
To 4 DEG C, then carry out the acidified milk described in stored refrigerated obtain.
Described commodity leavening is preferably lactobacillus bulgaricus or streptococcus thermophilus.
In the sense of the present invention, described raw milk is one or more selected from defatted milk, fresh milk, the raw material of recovery milk
Breast.
It is described above, it is only the specific embodiment of the present invention, but the feature of the present invention is not limited thereto, and it is any to be familiar with being somebody's turn to do
The people of item technology is in field of the present invention, the change or modification that can readily occur in, should all cover special in application of the invention below
In sharp scope.
Claims (6)
1. the lactobacillus fermenti (Lactobacillus fermentum suo) of a kind of adjustable intestinal movement, Constipation, in
It is preserved in China typical culture collection center (CCTCC) on October 25th, 2013, deposit number is CCTCC NO：
M2013511；
The lactobacillus fermenti derives from traditional fermented food, category generally recognized as safe (Generally Recognized As Safe,
GRAS) strain, in fermented food；
The fermented food is one kind in the lactic acid bacteria milk beverage containing lactobacillus fermenti, milk powder, capsule product or acidified milk
It is or a variety of；
The lactic acid bacteria milk beverage containing lactobacillus fermenti is prepared as steps described below：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with the glycerine suspensions of percentage by weight 30%, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
Raw milk heats the sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is cooled to 4 DEG C, adds the fermentation
Lactobacillus working stock culture, its concentration is set to reach 106More than cfu/mL, it is stored refrigerated at 4 DEG C；
Described raw milk is one or more selected from defatted milk, fresh milk, the raw milk of recovery milk；
The preparation method of the lactobacillus fermenti working stock culture includes：
The original strain of the lactobacillus fermenti is inoculated in the skimmed milk that percentage by weight is 12%, 110 DEG C of sterilizings
10min, 14-16h is cultivated under the conditions of 37 DEG C to curdled milk, continuous culture two generations of activation, as mother culture；By female fermentation
Agent is inoculated in sterile milk by percent by volume 3-5%, and 14-16h is to curdled milk for culture, the viable count 10 into the curdled milk9cfu/
ML, obtain working stock culture；Or
The original strain of the lactobacillus fermenti is inoculated in MRS fluid nutrient mediums, 12-16h is cultivated under the conditions of 37 DEG C and is entered
Row activation, it is continuous to activate for two generations, then activation culture thing is inoculated in MRS culture mediums by percent by volume 2-4%, cultivated
16-18h, 4000r/min centrifuges 15min under the conditions of 4 DEG C, removes supernatant, cell precipitation is obtained, by precipitation sterile absorbent
Suspension is made in breast, obtains working stock culture.
2. lactobacillus fermenti as claimed in claim 1, it is characterised in that the morphological feature of the lactobacillus fermenti is specific
For：
Thalline feature：In Gram-positive, cell is shaft-like, and thalline is 0.5-1.0 μm wide, 1.5-4 μm long, Cheng Dan, in pairs or
Person's chaining, does not form gemma, and both ends are circular；
Colony characteristicses：Obvious bacterium colony is formed on MRS culture mediums, diameter is circular between 0.5-2.0mm, neat in edge, breast
White, transparent, surface wettability is smooth, not chromogenesis.
3. lactobacillus fermenti as claimed in claim 1, it is characterised in that tolerance is characterized as in the lactobacillus fermenti body：
The bacterial strain acid resistance of lactobacillus fermenti is stronger, and survival rate is 92.46 ± 4.06% after 3h under pH3.0 simulated gastric fluid；
Can slowly it be grown under 1.0% concentration cholate, growth efficiency reaches the 17.36 ± 1.19% of no cholate culture；Acidified milk bar
The hydrophobicity of bacterium cell is 68.44 ± 2.48%.
4. lactobacillus fermenti as claimed in claim 1, it is characterised in that the milk powder containing lactobacillus fermenti is under
State what step was prepared：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with the glycerine suspensions of percentage by weight 30%, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
Raw milk heats the sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is then cooled to 37 DEG C, then with original
Expect that the 4% of newborn volume connects the described lactobacillus fermenti working stock culture of bacterium amount inoculation, ferment 16h at 37 DEG C, obtains acidified milk bar
Bacterium acidified milk；Raw milk after the lactobacillus fermenti acidified milk and sterilizing matches according to volume ratio 1: 3 to be added, and carries out homogeneous,
It is concentrated in vacuo, spray drying obtains the milk powder containing lactobacillus fermenti.
5. lactobacillus fermenti as claimed in claim 1, it is characterised in that the capsule product containing lactobacillus fermenti be by
It is prepared according to following step：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with the glycerine suspensions of percentage by weight 30%, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
By raw milk in 140 DEG C of high temperature thermal sterilization 2s, 37 DEG C are subsequently cooled to, bacterium amount inoculation institute is met with the 4% of raw milk volume
Lactobacillus fermenti working stock culture is stated, ferment 16h at 37 DEG C, obtains lactobacillus fermenti acidified milk；By the lactobacillus fermenti
Raw milk after acidified milk and sterilizing adds according to the proportioning of volume ratio 1: 3, homogeneous, after vacuum concentration, spray drying treatment
To milk powder, the capsule product containing lactobacillus fermenti will be obtained.
6. lactobacillus fermenti as claimed in claim 1, it is characterised in that the acidified milk containing lactobacillus fermenti be according to
What following step was prepared：
The original strain of the lactobacillus fermenti is preserved at -75 DEG C of temperature with the glycerine suspensions of percentage by weight 30%, or
Person is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
Lactobacillus fermenti working stock culture is prepared using the original strain of the lactobacillus fermenti；
Raw milk heats the sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is then cooled to 37 DEG C, according still further to
Volume ratio 3-5% add lactobacillus fermenti working stock culture, add volume ratio 3-5% can symbiosis be used for prepare acidified milk system
The leavening of product, mixed fungus fermentation to titratable acidity 0.6-0.7% in terms of lactic acid, is subsequently cooled to 4 DEG C at 37 DEG C after mixing, then
Carry out stored refrigerated obtaining the acidified milk containing lactobacillus fermenti.
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| CN105132330A (en) * | 2015-09-14 | 2015-12-09 | 西南大学 | Lactobacillus fermentum strain Lee capable of preventing gastric ulcers and application of lactobacillus fermentum strain Lee |
| CN105154362A (en) * | 2015-09-14 | 2015-12-16 | 西南民族大学 | Lactobacillus fermentum suo strain capable of preventing gastrohelcoma and health-care application thereof |
| CN106906165B (en) * | 2017-04-25 | 2020-07-24 | 重庆第二师范学院 | Lactobacillus plantarum and application thereof in preparation of food for preventing constipation |
| CN107164263A (en) * | 2017-05-27 | 2017-09-15 | 西南大学 | A kind of adjustable function of intestinal canal, the lactobacillus fermenti HY01 for preventing colitis and application thereof |
| CN107227275A (en) * | 2017-07-06 | 2017-10-03 | 西南大学 | A kind of lactobacillus fermenti HY01 and application thereof |
| CN110699271B (en) * | 2018-07-10 | 2022-11-01 | 善恩康生物科技（苏州）有限公司 | Lactobacillus plantarum CQPC02 and application thereof in preparation of food for improving constipation |
| CN109628346B (en) * | 2018-12-29 | 2022-07-12 | 善恩康生物科技（苏州）有限公司 | Lactobacillus fermentum CQPC04 and application thereof in preparing fermented food |
| CN110938563B (en) * | 2019-12-05 | 2020-09-29 | 北京伯恩世纪科技发展有限公司 | Lactobacillus BJ-REBORN001 and application thereof in preparation of helicobacter pylori inhibiting fermentation broth |
| CN113508907A (en) * | 2021-04-28 | 2021-10-19 | 汉臣氏（沈阳）儿童制品有限公司 | Application of heat-resistant lactobacillus fermentum in preparation of defecation promoting food or medicine |
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