CN104531549B - A kind of lactobacillus fermenti Lactobacillus fermentum strain Zhao and application thereof of adjustable intestinal movement, Constipation - Google Patents

A kind of lactobacillus fermenti Lactobacillus fermentum strain Zhao and application thereof of adjustable intestinal movement, Constipation Download PDF

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CN104531549B
CN104531549B CN201410578613.3A CN201410578613A CN104531549B CN 104531549 B CN104531549 B CN 104531549B CN 201410578613 A CN201410578613 A CN 201410578613A CN 104531549 B CN104531549 B CN 104531549B
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lactobacillus
zhao
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索化夷
李键
骞宇
赵欣
陈炼红
兰道亮
谢婕
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Southwest University
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Abstract

It is CCTCC NO the invention discloses lactobacillus fermenti (Lactobacillus fermentum strain Zhao) deposit number of a kind of adjustable intestinal movement, Constipation:M 2013513 and application thereof, the acid resistance of the bacterial strain is stronger, survival rate has reached 85.12 ± 5.18% after 3h under the simulated gastric fluid of pH3.0, it can slowly be grown under 1.0% concentration cholate, the hydrophobicity that growth efficiency reaches 18.33 ± 0.52, the Lactobacillus fermentum strain Zhao cells of no cholate culture has also reached 70.80 ± 0.61%.Can in the enteron aisle of people normal growth.In-vivo test in mice also shows, lactobacillus fermenti (Lactobacillus fermentum strain Zhao) 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, reduce the SS factors, increase food ration and amount of drinking water, reduce feces volume, defecation granule number and excrement water content downward trend, play the role of with constipation drug similar, shows certain constipation preventive effect.

Description

A kind of lactobacillus fermenti Lactobacillus of adjustable intestinal movement, Constipation Fermentum strain Zhao and application thereof
Technical field
The present invention relates to microorganisms technical field more particularly to the acidified milks of a kind of adjustable intestinal movement, Constipation Bacillus Lactobacillus fermentum strain Zhao 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 it is specific excellent Benign matter and inhereditary feature show extremely strong probiotic properties.Lactobacillus can not only improve food value, improve food Product flavor extends the holding time, can also improve the functional character of food.It can be by decomposing protein, sugar in food Class, synthesis vitamin, improve the digestibility and biological value of food, promote to digest and assimilate.Can also reduce human cholesterol content, Enhance immune function, anti-allergenicity, antitumor, anti-hypertension etc..In recent years, lactobacillus is with its special physiological activity and battalion Support function and as probiotics main source, just have attracted increasing attention.
Constipation is a kind of very universal disease, and the illness rate in crowd is up to 27%, it can influence each age group People.Habitual constipation is to induce middle-aging male hyperlipidemia, cardiovascular and cerebrovascular disease because firmly increasing abdominal pressure, defecation with strength of holding one's breath The arch-criminal of the diseases such as disease, artery sclerosis, tumour and its complication, drastically so as to cause old man's cardio cerebrovascular affection death rate Increase, the problems such as hemorrhoid, anal fissure occurs, it is painful to can't bear.In addition prolonged constipation, largely just slag can be trapped in hair in enteron aisle Ferment rots, and a large amount of toxin and harmful bacteria are absorbed repeatedly by enteron aisle, reaches partes corporis humani position by blood circulation, it is strong to become women 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 improve not in time, irreversible disease may be caused Become.Because constipation incidence is high, the cause of disease is complicated, patient often has manifold vexations, and quality of life can be influenced when constipation is serious.Therefore, it seeks Look for it is a kind of can alternatives to medication carry out the biological products of Constipation to have prodigious realistic meaning, Tiny ecosystem therapy be just fine with one New means solve various problems existing for traditional remedies.
There is a large amount of research report to the physiological function of lactic acid bacteria both at home and abroad, but the functionality of lactic acid bacteria Constipation is ground It is seldom to study carefully report.Research about 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 development functionality dairy products, the added value enriched existing dairy products type, improve dairy products It is significant.Therefore, the lactic acid bacteria with Constipation has prodigious research and application value.
In the document and patent that have disclosed at present or patent application, such as CN101144064A discloses one kind and has The Lactobacillus brevis and application thereof of antimutagenicity, extracellular polysaccharide, for, to improve food quality, increasing it in fermented food Nutrition function.CN1796540A discloses the Bifidobacterium and application thereof with anti-enterovirus and anti-oxidation characteristics, uses In preparing food compositions or pharmaceutical composition, helicobacter pylori or Escherichia coli are killed or inhibited, treats and be induced by it Various diseases.CN102174450A discloses a kind of lactobacillus plantarum of Helicobacter pylori Infection and application thereof, is used to prepare Prevent or mitigate the infection of helicobacter pylori in pharmaceutical composition, leavening, animal feed additive and health products. The oxidation resistant Lactobacillus casei and application thereof that CN101144065A discloses a kind of resisting hydrogen peroxide, removes free radical, for sending out With development functionality dairy products, the added value enriched existing dairy products type, improve dairy products in ferment food and dairy products capsule product. But these patents or patent application are not related to completely with the microorganism for adjusting the performances such as intestinal movement, Constipation.Cause This, also needs to a kind of food with the microorganism for adjusting intestinal movement, Constipation and its containing them at present.
Invention content
To solve the above-mentioned problems, the present invention provides a kind of adjustable intestinal movement, the lactobacillus fermentis of Constipation Lactobacillus fermentum strain Zhao and application thereof mainly provide a kind of adjustable intestinal movement, prevent just Purposes of the secret L fermentum strain and a kind of lactobacillus fermenti of offer in fermented food.
Lactobacillus fermenti (the Lactobacillus of a kind of adjustable intestinal movement provided by the invention, Constipation Fermentum strain Zhao) on October 30th, 2013 it is preserved in China typical culture collection center (CCTCC), it should Collection is located at:The Chinese Wuhan Wuhan Universitys, deposit number are CCTCC NO:M2013513.
The present inventor filters out one plant of lactobacillus fermenti (Lactobacillus from spontaneous fermentation yak yogurt Fermentum strain Zhao), using Microbiological Characteristics such as morphological feature, cultural colony and physiological and biochemical properties to this Bacillus acidi lactici Lactobacillus fermentum strain Zhao are accredited as lactobacillus fermenti, and the bacterial strain is in 2013 It was preserved in China typical culture collection center (CCTCC) on October 30, deposit number is CCTCC NO:M2013513.
The morphological feature of the lactobacillus fermenti is specially:
Thalline feature:In Gram-positive, cell is rod-shaped, and thalline is about 0.5-1.0 μm wide, 1.5-4 μm long, Cheng Dan, Pairs of or chaining, does not form gemma, and both ends are round.
Colony characteristics:Apparent bacterium colony is formed on MRS culture mediums, diameter is round 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 the simulated gastric fluid of pH3.0 after 3h survival rate reached 85.12 ± 5.18%;It can slowly be grown under 1.0% concentration cholate, growth efficiency reaches the 18.33 ± 0.52% of no cholate culture;Hair The hydrophobicity of kefir milk bacilli-cell has reached 70.80 ± 0.61%.
The lactobacillus fermenti derives from traditional fermented food, belongs to generally recognized as safe (Generally Recognized As Safe, GRAS) strain, in fermented food.
The fermented food is in lactic acid bacteria milk beverage, milk powder, capsule product or acidified milk containing lactobacillus fermenti 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 makes its concentration reach 106Cfu/mL or more, it is stored refrigerated at 4 DEG C;
The raw milk is one or more raw milks selected from defatted milk, fresh milk, recovery milk.
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 bacterium amount inoculation lactobacillus fermenti working stock culture 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 is matched according to 1: 3 (V: V) to be added, and is carried out equal Matter 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, vacuum concentration, 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 Lactobacillus fermenti working stock culture is added according to 3-5% (volume), add 3-5% (volume) can symbiosis be used to prepare fermentation The leavening of dairy products, mixed fungus fermentation is subsequently cooled to 4 to titratable acidity 0.6-0.7% in terms of lactic acid 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, in continuous culture two generations of activation, is used as mother culture;The mother culture is pressed into 3- 5% (volume) is inoculated in sterile milk, and 14-16h is to curdled milk for culture, until the viable count about 10 in the curdled milk9Cfu/mL obtains work Make leavening, directly this working stock culture can be added in food, or with can symbiosis the business for preparing acidified milk 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 object 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.
The heating sterilization is the TW10D1000 for example sold using Shanghai Triowin Automation Machinery Limited What type tubular sterilization machine carried out.
The high temperature thermal sterilization is the YC-104 types for example sold using Beijing Yong Chuanjiaye mechanical equipments Co., Ltd What board-like superhigh temperature sterilization machine carried out.
The homogeneous be the small-sized homogenizers of GJB500-40 for example sold using Changzhou homogeneous Machinery Co., Ltd. into Capable.
The concentration is that the vacuum concentration pan for example sold using Shanghai Wei Zhou light industry and machinery Co., Ltd is carried out.
The spray drying be the experiment type spray drier for example sold using Shanghai Triowin Tech. Co., Ltd. into Capable.
The commodity leavening is preferably lactobacillus bulgaricus or streptococcus thermophilus.
In the sense of the present invention, the raw milk is one or more raw materials selected from defatted milk, fresh milk, recovery milk Breast.
What the technical solution 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 The acid resistance of application in product, lactobacillus fermenti (Lactobacillus fermentum strain Zhao) bacterial strain is stronger, Survival rate has reached 85.12 ± 5.18% after 3h under the simulated gastric fluid of pH3.0, can slowly be given birth under 1.0% concentration cholate Long, growth efficiency reaches 18.33 ± 0.52%, the Lactobacillus fermentum strain Zhao of no cholate culture The hydrophobicity of cell has also reached 70.80 ± 0.61%.This shows lactobacillus fermenti (Lactobacillus fermentum Strain Zhao) can in the enteron aisle of people normal growth.In-vivo test in mice is also shown that lactobacillus fermenti (Lactobacilius fermentum strain Zhao) can slow down mouse weight caused by constipation and mitigate trend, accelerate The melena time is arranged, Intestinal propulsive rate, MTL, Gas, ET, AchE, SP and VIP factor water of different degrees of increase serum are improved It is flat, the SS factors are reduced, food ration and amount of drinking water are increased, reduce feces volume, defecation granule number and excrement water content downward trend, Play the role of with constipation drug similar, shows certain constipation preventive effect.
Description of the drawings
For the clearer technical solution for illustrating the embodiment of the present invention, required use in being described below to embodiment Attached drawing be briefly described, it is clear that, drawings in the following description are only some embodiments of the invention, for this For the those of ordinary skill of field, without creative efforts, others are can also be obtained according to these attached drawings Attached drawing.
Fig. 1 lactobacillus fermentis (Lactobacillus fermentum strain Zhao) colonial morphology
The thalli morphology (1000x) of Fig. 2 lactobacillus fermentis (Lactobacillus fermentum strain Zhao)
Fig. 3 mouse weights with experimental period variation
The time required to Fig. 4 separate groups of mice arranges melena for the first time
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 Zhao) of the present invention was October 30 in 2013 Day is preserved in China typical culture collection center, which is located at:The Chinese Wuhan Wuhan Universitys, number CCTCC NO:M2013513.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Whole description, it is clear that the described embodiments are merely a part of the embodiments of the present invention, the embodiment being not all of, and is 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 not making the creative labor Example, shall fall within the protection scope of the present invention.
Embodiment 1:The separation of lactobacillus fermenti (Lactobacillus fermentum strain Zhao), purifying and Preliminary Identification.
The embodiment carries out as steps described below:
(1) separation, purifying of Lactobacillus fermentum strain Zhao.
It is added to 5mL sterile salines with 500 μ L of sterile working pipette samples and mixes the uniform dilution for being made 1: 10, And continue to make a certain proportion of dilution.The dilution for selecting suitable gradient is respectively drawn 100 μ L with sterile pipette tips and is respectively coated In MRS solid plate culture mediums, 30 DEG C of culture 48-72h observe and record colonial morphology, as shown in Figure 1.With oese (or sterilizing Toothpick) from planar surface and internal picking difference colony inoculation in MRS fluid nutrient mediums, set 30 DEG C, the training of 300r/min shaking tables Support 24-48h;It repeats to carry out gram stain smear microscopy after above step continuously activated for 2 generations, as shown in Fig. 2, being determined as G+ bacterium Continue activate, until obtain pure bacterium colony (microscopy is without miscellaneous bacteria), and carry out catalase experiment.G+ and catalase is cloudy Sporeless bacterium, coccus and the streptococcus of property are fixed tentatively as lactic acid bacteria, and are preserved, concrete operations reference《Microbiology Experiment skill Art》.
Experimental result is shown, 56 plants of lactic acid bacteria is isolated from ten samples of Ruoergai country, and viable count of lactobacillus is flat 108The cfu/mL orders of magnitude.
(2) Preliminary Identification of Lactobacillus fermentum strain Zhao.
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, takes pictures.
Catalase is tested:One oese of bacterium colony, is placed in clean tube on picking solid medium, and 3% mistake is added dropwise Hydrogen peroxide solution 2mL, observation result (being the positive in gassing person in half a minute, gassing person is not feminine gender).
The result shows that the bacterial strain isolated and purified out from sample can be containing 5 ‰ CaCO3MRS plating mediums on it is raw It is long, and form molten calcium circle, Gram-positive, catalase test feminine gender in periphery of bacterial colonies.
Embodiment 2:The in-vitro screening of lactobacillus fermenti Lactobacillus fermentum strain Zhao.
(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 the HCl of 1M adjustment pH value, With spare after vacuum pump suction filtration degerming in aseptic operating platform.
Measurement of the probiotics to artificial gastric juice resistance's property:5mL activated strain cultured solutions are taken, in aseptic operating platform In pour into and sterilized 10mL centrifuge tubes, collect thalline through 3000r/min centrifugations 10min, 5mL sterile saline mixings be added Bacteria suspension is made, takes 1mL bacteria suspensions and the simulated gastric fluid of 9mL pH3.0 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.It is surveyed with colony counting method Determine viable count, calculates its survival rate (%):Viable count × 100% of viable count/0h of survival rate (%)=3h.
(2) measurement of probiotics tolerance various concentration cholate.
The survival rate in pH3.0 simulated gastric fluids is selected to do the survey of the growth under different gallbladder salinities in 10% or more strain Examination.Activated strain 5mL is inoculated in liquid-transfering gun by 2% inoculum concentration (100 μ L) containing 0.0% bovine bile (i.e. sky respectively In vain), 0.3% bovine bile, 0.5% bovine bile, 1.0% bovine bile (W/V) MRS-THIO culture mediums (in MRS culture mediums plus 0.2% sodium thioglycolate).It is that control is (nonvaccinated with blank cultures in constant temperature oscillator after 37 DEG C of cultures for 24 hours MRS-THIO culture mediums), the OD values of above-mentioned various concentration culture medium are measured 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.
Selection survival rate strain stronger in 10% or more, bile tolerance in pH3.0 simulated gastric fluids observes bacterium through microscopy It is tested after body growthform is good, by activated bacterial cultures 5mL, 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 buffer solution 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.
It takes the bacterium solution that 4mL adjusts turbidity in the 10mL centrifuge tubes that sterilized, 0.8mL dimethylbenzene is added, control group is not added with Dimethylbenzene, vibrate 30s, pause 10s, after vibrate 30s again, on rack for test tube stand 5~10min layering, remove a layer water phase, with PBS buffer solution is blank control, and A values are measured at 560nm and are recorded.Hydrophobic rate H%=[(A0-A)/A0] × 100 its Middle A0 and A is the A values measured at 560nm with the forward and backward bacterium solution of dimethylbenzene mixing respectively.
Lactic acid bacteria is a part for normal bowel bacterium, one of the most important condition as probiotics, and being can be before stomach and small intestine Section survival, the bacterial strain being screened should be with the characteristics of growth and development under enough acid resistance and acidic environment.
The selection result of Lactobacillus fermentum strain Zhao is as shown in table 1 below:
Table 1
The selection result shows that Lactobacillus fermentum strain Zhao bacterial strains are resistant to the ring of pH3.0 Border, and survival rate is 80% or more.
It will be contacted with the cholate in small intestine by the thalline survived after stomach, this experiment is lactic acid bacteria to the resistivity of cholate For a selection criteria as potential probiotics.The preferable Lactobacillus fermentum strain of acid resistance Zhao bacterium be resistant to various concentration cholate the result is that the cholate to 0.3% has certain tolerance, when gallbladder salinity is increased to When 0.5% and 1.0%, Lactobacillus fermentum strain Zhao thalline have revealed good cholate tolerance.
While except having certain resistivity to cholate in small intestine, lactic acid bacteria need to also have good adherency on mucous membrane of small intestine Property, therefore by the hydrophobic ability of lactic acid bacteria alternately standard, from table 1 it follows that Lactobacillus The hydrophobicity of fermentum strain Zhao bacterium has reached 70.80 ± 0.61%.
Acid resistance, bile tolerance and the preferable Lactobacillus of hydrophobicity have been filtered out by testing us above Fermentum strain Zhao bacterium, the bacterial strain are preserved in China typical culture collection center on October 30th, 2013 (CCTCC), deposit number CCTCC NO:M2013513.
Embodiment 3:Activated carbon inducing constipation is tested.
Feeding of healthy kunming mice, 6 week old, 23 ± 2g of weight, female, 50.It, will be small after feeding basal feed adapts to 5d Mouse is randomly divided into 5 groups according to weight, respectively:Normal group, constipation control group, Bisacody group, lactobacillus bulgaricus group and The Lactobacillus fermentum strain Zhao group groups of high and low given low, every group 10.Raising is in stainless steel In cage, 24 ± 2 DEG C of room temperature holding, relative humidity 50 ± 10%, 12h light and shades rotation (8:00~20:00 illumination).Entirely testing Period, normal group and the equal feeding basal feed of constipation control group, experimental mice press 1.0 × 10 daily respectively9CFU/kg gavages Probiotics.Start every morning 9 after 2 weeks:00 gives 2 DEG C of activated carbons of other 5 groups of intragastric administration on mice a concentration of 10% in addition to normal group Ice water, only, daily gavage is primary, continuous gavage 3 days by 0.2mL/.Until experiment last day, all mouse fasting for 24 hours, then Gavage activated carbon ice water again, 0.2mL/ is only.5 mouse are for measuring changes of weight, food ration, amount of drinking water, first row in every group Go out melena required time, defecation just quantity and moisture in feces.Other 5 mouse of mouse are after gavage activated carbon 30min Dead dissection.
With reference to as shown in Figure 3 and Figure 4 the time required to the mouse weight variation of experiment periods Nei Ge groups, first discharge melena.
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 slows down weight loss, Lactobacillus fermentum strain Zhao 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 for the first time pole significant difference (P < 0.05).It is high and low The average time that the Lactobacillus fermentum strain Zhao groups mouse of given low arranges melena for the first time is low In constipation control group certain constipation preventive effect is shown higher than normal group and constipation therapy drug Bisacody group.
Variation such as the following table 2 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-Zhao:Lactobacillus fermentum strain Zhao.
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 in Lactobacillus fermentum strain Zhao groups, and high concentration group is ingested Amount reduces less.
Variation such as the following table 3 of experiment periods Nei Ge groups mouse amount of drinking water:
Table 3
Every group of mouse n=10ICR, Bisacody:100mg/kgb.w., lactobacillus bulgaricus:1.0×109CFU/kg B.w., LF-Zhao:Lactobacillus fermentum strain Zhao.
Normal group mouse weight is slightly increased with experimental period amount of drinking water, and control group is after 6 days since 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 higher than control group, Lactobacillus Also there is the situation similar with Bisacody in fermentum strain Zhao groups, and high concentration group amount of drinking water reduces less.
The feces volume of experimental period Nei Ge group mouse, variation such as the following table 4 of 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-Zhao:Lactobacillus fermentum strain Zhao.
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 more normal group of moisture in feces, 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 Zhao high concentrations are close to medicine group and normal group.
Embodiment 4:Small intestine activated carbon Promoting Experiment.
Mouse cervical dislocation is put to death, splits the mesenterium of abdominal cavity and separating mouse, from pylorus, down toward ileocecus on clip Whole intestinal tube, is put on blotting paper, and whole small intestine is pulled into straight line, to measure Length of intestine, as " 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.
Total small intestinal length, the Intestinal propulsive rate of experimental period Nei Ge group mouse 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 Zhao group mouse small intestine propulsion rates of dosage are higher than constipation control group, are less than Normal group and constipation therapy drug Bisacody group, show certain constipation preventive effect.
Embodiment 5:The measurement of each factor level in mice serum.
0.2mL rat aorta blood is taken, at 4 DEG C, 3000r/min centrifuges 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 The method of vasoactive intestinal peptide (VIP) test agent box specification measures above-mentioned factor level in serum.
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 Zhao height is organized can different journeys MTL, Gas, ET, AchE, SP and VIP factor level of the increase serum of degree, reduce SS factor levels.So Lactobacillus fermentum strain Zhao have prevention effect to constipation.
Application Example 1:Breast is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain Zhao) Sour bacterium milk drink.
First, lactobacillus fermenti (the Lactobacillus fermentum strain Zhao) original strain is in temperature It is preserved, or is 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 prepare lactobacillus fermenti (the Lactobacillus fermentum of the present invention using two methods Strain Zhao) working stock culture:
First method is that above-mentioned lactobacillus fermenti (Lactobacillus fermentum strain Zhao) is original Strain be inoculated in 12 weight % sterilize at 110 DEG C 10min skimmed milk in, 14-16h is cultivated under the conditions of 37 DEG C to curdled milk, even In continuous culture two generations of activation, it is used as mother culture;The mother culture is inoculated in by 3-5% (volume) in sterile milk, 14- is cultivated 16h is to curdled milk, the viable count about 10 in the curdled milk at this time9Cfu/mL obtains the working stock culture, 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 Zhao) 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 object is inoculated in by 2-4% (volume) in MRS culture mediums, cultivates 16-18h, and 4000r/min is centrifuged under the conditions of 4 DEG C 15min removes supernatant, obtains cell precipitation, and suspension is made with a certain amount of sterile absorbent breast in precipitation, obtains work hair Ferment agent is spare.
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 Zhao) working stock culture is added, Its concentration is set to reach 106Cfu/ml or more, 4 DEG C it is stored refrigerated i.e. obtain the (Lactobacillus containing lactobacillus fermenti Fermentum strain Zhao) viable bacteria lactic acid bacteria milk beverage.
In the present invention, the 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.
The heating sterilization is the TW10D1000 for example sold using Shanghai Triowin Automation Machinery Limited What type tubular sterilization machine carried out.
The high temperature thermal sterilization is the YC-104 types for example sold using Beijing Yong Chuanjiaye mechanical equipments Co., Ltd What board-like superhigh temperature sterilization machine carried out.
Application Example 2:Breast is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain Zhao) Powder.
According to above-mentioned lactic acid bacteria milk beverage preparation method prepare, only 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 Zhao) working stock culture, then the 16h that ferments at 37 DEG C, Obtain lactobacillus fermenti (Lactobacillus fermentum strain Zhao) acidified milk;Then the acidified milk bar Bacterium (Lactobacillus fermentum strain Zhao) acidified milk is added to above-mentioned sterilizing raw milk according to 1: 3 (V: V) In, homogeneous is carried out, is concentrated in vacuo, spray drying obtains (the Lactobacillus fermentum strain containing lactobacillus fermenti Zhao milk powder).
The homogeneous be the small-sized homogenizers of GJB500-40 for example sold using Changzhou homogeneous Machinery Co., Ltd. into Capable.
The concentration is that the vacuum concentration pan for example sold using Shanghai Wei Zhou light industry and machinery Co., Ltd is carried out.
The spray drying be the experiment type spray drier for example sold using Shanghai Triowin Tech. Co., Ltd. into Capable.
Application Example 3:Glue is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain Zhao) 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 Zhao) working stock culture of the kind present invention, at 37 DEG C Ferment 16h, obtains lactobacillus fermenti (Lactobacillus fermentum strain Zhao) acidified milk.By acidified milk bar Bacterium (Lactobacillus fermentum strain Zhao) acidified milk is added in the raw milk after sterilizing with 1: 3 (V: V) Matter obtains milk powder through being concentrated in vacuo, after spray drying treatment, obtained milk powder is attached in capsule, capsule product is made.
Application Example 4:It is prepared and is sent out using lactobacillus fermenti (Lactobacillus fermentum strain Zhao) Kefir milk.
According to above-mentioned lactic acid bacteria milk beverage preparation method prepare, only 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 Zhao) working stock culture, add 3-5% (volume) can symbiosis system Preparation kefir milk commodity leavening, at 37 DEG C then mixed fungus fermentation is cooled down to titratable acidity 0.6-0.7% in terms of lactic acid after mixing To 4 DEG C, then carry out the acidified milk described in stored refrigerated obtain.
The commodity leavening is preferably lactobacillus bulgaricus or streptococcus thermophilus.
In the sense of the present invention, the raw milk is one or more raw materials selected from defatted milk, fresh milk, recovery milk Breast.
The above, only specific embodiments of the present invention, but the feature of the present invention is not limited thereto, it is any to be familiar with being somebody's turn to do In the field of the present invention, the changes or modifications that can be readily occurred in should all cover special in application of the invention below the people of item technology In sharp range.

Claims (2)

1. the lactobacillus fermenti of a kind of adjustable intestinal movement, Constipation, the lactobacillus fermenti is Lactobacillus Fermentum strain zhao were preserved in China typical culture collection center (CCTCC) on October 30th, 2013, protected It is CCTCC NO to hide number:M2013513.
2. purposes of the lactobacillus fermenti as described in claim 1 in preparing fermented food, which is characterized in that the fermentation food Product are lactic acid bacteria milk beverage, milk powder, capsule containing lactobacillus fermenti Lactobacillus fermentum strain zhao It is one or more in product or acidified milk.
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