CN104651272B - Lactobacillus casei Lactobacillus casei strain Qian working stock cultures products and its enteron aisle adjust dietetic use - Google Patents
Lactobacillus casei Lactobacillus casei strain Qian working stock cultures products and its enteron aisle adjust dietetic use Download PDFInfo
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- CN104651272B CN104651272B CN201510027686.8A CN201510027686A CN104651272B CN 104651272 B CN104651272 B CN 104651272B CN 201510027686 A CN201510027686 A CN 201510027686A CN 104651272 B CN104651272 B CN 104651272B
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- 210000000130 stem cell Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- ADNPLDHMAVUMIW-CUZNLEPHSA-N substance P Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CCCN=C(N)N)C1=CC=CC=C1 ADNPLDHMAVUMIW-CUZNLEPHSA-N 0.000 description 1
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/00—Lactic or propionic acid bacteria
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Abstract
The invention discloses a kind of Lactobacillus casei Lactobacillus casei strain Qian (CCTCC No.M2013514) working stock culture products and its enteron aisle to adjust dietetic use, the Lactobacillus casei working stock culture product and its downstream product acid resistance are stronger, survival rate has reached 62.51 ± 7.35% after handling 3h under the simulated gastric fluid of pH3.0, it can slowly be grown under 1.0% concentration cholate, growth efficiency reaches the 7.86 ± 0.45% of no cholate culture, the hydrophobicity of Lactobacillus casei strain Qian cells has also reached 51.30 ± 4.28%, the normal growth in the enteron aisle of people；Mouse weight caused by constipation can be slowed down and mitigate trend, accelerate row's melena time, improve Intestinal propulsive rate, turn down the TRPV1 of mouse, the gene expression dose of NOS, feces volume, defecation granule number and excrement water content downward trend are reduced, plays the role of with constipation drug similar, shows certain constipation preventive effect.
Description
Technical field
The present invention relates to microorganisms technical field more particularly to the cheese breasts of a kind of adjustable intestinal movement, Constipation
Bacillus Lactobacillus casei strain Qian and application thereof.
Background technology
Lactic acid bacteria (lactic acid bacteria, LAB), which is one kind, to utilize fermentable carbohydrate to generate largely
The common name of the bacterium of lactic acid.This kind of bacterium is extremely wide in distributed in nature, has abundant species diversity.Lactic acid bacteria is not only
Nutritive value of food can be improved, flavour of food products is improved, improves food preservation and added value.In addition to only a few, wherein absolutely
Most of is all flora essential in human body and with important physiological function, is widely present in the enteron aisle of human body.
Numerous studies show that lactic acid bacteria can adjust body gastrointestinal tract normal flora, keep microecological balance, improve food digestion rate.
Lactic acid bacteria is also the main source of probiotic strain, and such as Lactobacillus casei, lactobacillus plantarum, Bifidobacterium benefit has now been found
Raw bacteria strain has plurality of health care functions.Serum cholesterol is such as reduced, endotoxin is controlled, inhibits spoilage organisms growth and breeding in enteron aisle
With the generation of spoilage product, nutriment, stimulation tissue development etc. are manufactured.
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.Because constipation incidence is high, the cause of disease is complicated, often there are many bitter by patient
It is angry, quality of life can be influenced when constipation is serious.Therefore, find it is a kind of can the alternatives to medication biological products that carry out Constipation have very
Big realistic meaning, Tiny ecosystem therapy are just to solve various problems existing for traditional remedies with a brand-new means.
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 CN1796540A is disclosed with anti-enteron aisle
The Bifidobacterium and application thereof of pathogenic bacteria and anti-oxidation characteristics is used to prepare food compositions or pharmaceutical composition, kills or presses down
Helicobacter pylori processed or Escherichia coli treat the various diseases being induced by it.CN101144064A discloses a kind of with anti-
The Lactobacillus brevis and application thereof of Mutagenicity, extracellular polysaccharide, for, to improve food quality, increasing its battalion in fermented food
It supports and learns function.The oxidation resistant Lactobacillus casei and its use that CN101144065A discloses a kind of resisting hydrogen peroxide, removes free radical
On the way, it is used in fermented food and dairy products capsule product with development functionality dairy products, enriches existing dairy products type, raising dairy products
Added value.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.But
These patents or patent application are not related to completely with the microorganism for adjusting the performances such as intestinal movement, Constipation.Therefore, mesh
Before also need to it is a kind of have adjust intestinal movement, the microorganism of Constipation and its food containing them.
Invention content
To solve the above-mentioned problems, the present invention provides a kind of adjustable intestinal movement, the Lactobacillus caseis of Constipation
And application thereof, mainly provide a kind of adjustable intestinal movement, Constipation lactobacillus casei bacterial strain and provide a kind of described dry
Purposes of the Lactobacillus paracasei in fermented food.
One plant of Lactobacillus casei (Lactobacillus casei strain are filtered out from spontaneous fermentation yak yogurt
Qian), using Microbiological Characteristics such as morphological feature, cultural colony and physiological and biochemical properties to the Bacillus acidi lactici
(Lactobacillus casei strain Qian) is accredited as Lactobacillus casei, which protects on October 25th, 2013
It is hidden in China typical culture collection center (the Chinese Wuhan Wuhan Universitys), deposit number CCTCC NO:M2013514.
The morphological feature of the Lactobacillus casei is specially：
Thalline feature：In Gram-positive, cell is rod-shaped, and thalline is about 0.5-1.0 μm wide, 2.5-4 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 casei body：
The bacterial strain acid resistance of Lactobacillus casei is stronger, and survival rate reaches after handling 3h under the simulated gastric fluid of pH3.0
62.51 ± 7.35%, can slowly be grown under 1.0% concentration cholate, growth efficiency reach no cholate culture 7.86 ±
The hydrophobicity of 0.45%, Lactobacillus casei strain Qian cells has also reached 51.30 ± 4.28%, in people
Enteron aisle in normal growth.
The Lactobacillus casei derives from traditional fermented food, belongs to generally recognized as safe (Generally Recognized As
Safe, GRAS) strain.
The realization rate of the object of the invention is：
A kind of Lactobacillus casei Lactobacillus casei strain Qian working stock culture products, Chinese Typical Representative
Culture collection deposit number CCTCC NO:Object is made using following step in M2013514：1) by above-mentioned cheese
Lactobacillus (Lactobacillus casei strain Qian) original strain is inoculated in MRS fluid nutrient mediums, in 37 DEG C of items
12-16h is cultivated under part to be activated, it is continuous to activate for two generations, activation culture object is then inoculated in MRS trainings by 2-4% (volume)
It supports in base, cultivates 16-18h, 4000r/min centrifuges 15min under the conditions of 4 DEG C, removes supernatant, obtains cell precipitation, will sink
It forms sediment and suspension is made with a certain amount of sterile absorbent breast, obtain working stock culture；2) direct by 1) gained target operation leavening
Be added in food or with can the commercial fermentation agent for preparing acidified milk of symbiosis be used together and prepare acidified milk.
The commercial fermentation agent is lactobacillus bulgaricus or streptococcus thermophilus.
It is a further object of the present invention to provide a kind of Lactobacillus casei working stock culture product enteron aisles to adjust Constipation food
Treat purposes.The specific means used for：
A kind of Lactobacillus casei working stock culture product enteron aisle adjusting Constipation dietetic use, is added to as leavening
In food addition or with can the commercial fermentation agent for preparing acidified milk of symbiosis be used together and prepare acidified milk, to obtain adjusting intestines
Road moves Constipation health-care effect, optimal use dosage 1.0 × 109CFU pers kilogram of body weight.
The Lactobacillus casei working stock culture product enteron aisle adjusts Constipation dietetic use, and the food includes the mankind
Food, animal feed or health products.
The edible object includes the mankind and cultivated animals, and method addition is adjusted edible object in daily bread
The health-care effect of intestinal movement Constipation.
The fermented food is in lactic acid bacteria milk beverage, milk powder, capsule product or acidified milk containing Lactobacillus casei
It is one or more.
The lactic acid bacteria milk beverage containing Lactobacillus casei is prepared as steps described below：
The original strain of the Lactobacillus casei 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 casei working stock culture is prepared using the original strain of the Lactobacillus casei；
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 casei working stock culture makes its concentration reach 106Cfu/mL or more, it is stored refrigerated at 4 DEG C.
The milk powder containing Lactobacillus casei is prepared as steps described below：
The original strain of the Lactobacillus casei 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 casei working stock culture is prepared using the original strain of the Lactobacillus casei, raw milk adds at 95 DEG C
Then the thermal sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C is cooled to 37 DEG C, then connects bacterium amount with the 4% of raw milk volume and connect
The Lactobacillus casei working stock culture is planted, ferment 16h at 37 DEG C, obtains lactobacillus casei fermented milk；The cheese breast bar
Raw milk after bacterium acidified milk and sterilizing is according to 1:3(V:V) proportioning is added, and carries out homogeneous, is concentrated in vacuo, spray drying is contained
The milk powder of Lactobacillus casei.
The capsule product containing Lactobacillus casei is prepared as steps described below：
The original strain of the Lactobacillus casei 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 casei working stock culture is prepared using the original strain of the Lactobacillus casei；
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 casei working stock culture, ferment 16h at 37 DEG C, obtains lactobacillus casei fermented milk；By cheese breast
Raw milk after bacillus fermentation breast and sterilizing is according to 1:3(V:V)) proportioning is added, homogeneous, is concentrated in vacuo, spray drying treatment
After obtain milk powder, the capsule product containing Lactobacillus casei will be obtained.
The acidified milk containing Lactobacillus casei is prepared as steps described below：
The original strain of the Lactobacillus casei 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 casei working stock culture is prepared using the original strain of the Lactobacillus casei；Raw milk adds at 95 DEG C
The thermal sterilization 20min or high temperature thermal sterilization 2s at 140 DEG C, is then cooled to 37 DEG C, and cheese breast is added according still further to 3-5% (volume)
Bacillus working stock culture, add 3-5% (volume) can symbiosis the leavening for being used to prepare fermented dairy product, 37 after mixing
Mixed fungus fermentation is subsequently cooled to 4 DEG C, then carry out stored refrigerated contained to titratable acidity 0.6-0.7% in terms of lactic acid at DEG C
The acidified milk of Lactobacillus casei.
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 be it is a kind of be isolated from it is adjustable in Maizhokunggar County herdsman man of Tibet Autonomous Region spontaneous fermentation yak yogurt
Save intestinal movement, Constipation Lactobacillus casei and using the bacterial strain as leavening or the acidified milk of addition, healthy food and
Application in animal health product, the acid resistance of Lactobacillus casei (Lactobacillus casei strain Qian) bacterial strain
Relatively strong, survival rate has reached 62.51 ± 7.35% after handling 3h under the simulated gastric fluid of pH3.0, can under 1.0% concentration cholate
Slowly to grow, growth efficiency reaches 7.86 ± 0.45%, the Lactobacillus casei strain of no cholate culture
The hydrophobicity of Qian cells has also reached 51.30 ± 4.28%, the normal growth in the enteron aisle of people.In-vivo test in mice also table
Bright, Lactobacillus casei (Lactobacillus casei strain Qian) working stock culture product and its downstream product can
Slow down mouse weight caused by constipation and mitigate trend, accelerate row's melena time, improves Intestinal propulsive rate, different degrees of increase blood
Clear MTL, Gas, ET, AchE, SP and VIP factor level, the reduction SS factors, increase food ration and amount of drinking water, reduction feces volume,
Defecation granule number and excrement water content downward trend, play the role of similar with constipation drug, and it is pre- to show certain constipation
Anti- 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 caseis (Lactobacillus casei strain Qian) colonial morphology；
The thalli morphology (l000x) of Fig. 2 Lactobacillus caseis (Lactobacillus casei strain Qian)；
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 defecation form of Fig. 5 separate groups of mice；
The total small intestinal length of Fig. 6 separate groups of mice；
C-Kit, SCF, GAPDH gene expression amount of Fig. 7 separate groups of mice
TRPV1, GDNF, NOS, GAPDH gene expression amount of Fig. 8 separate groups of mice
The Lactobacillus casei (Lactobacillus casei strain Qian) of the present invention was protected on October 25th, 2013
It is hidden in China typical culture collection center, number is CCTCC NO:M2013514.
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 casei (Lactobacillus casei strain Qian), purifying and preliminary
It is fixed.
The embodiment carries out as steps described below：
(1) separation, purifying of Lactobacillus casei strain Qian.
It is added to the mixing of 5mL sterile salines and is made with 500 μ L of sterile working absorption traditional zymotic yak yoghourt sample
1：10 uniform dilution, and continue to make a certain proportion of dilution.The dilution for selecting suitable gradient, is respectively drawn with sterile pipette tips
100 μ L are respectively coated in MRS solid plate culture mediums, and it is as shown in Figure 1 to observe and record colonial morphology by 30 DEG C of culture 48-72h.
With oese (or sterilizing toothpick) from planar surface and internal picking difference colony inoculation in MRS fluid nutrient mediums
In, 30 DEG C are set, 300r/min shaking table cultures 24-48h；It repeats to carry out smear Gram's staining after above step continuously activated for 2 generations
Microscopy is as shown in Figure 2.
It is determined 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, more than 58 plants of lactic acid bacteria, sample lactic acid bacteria are isolated from ten samples of Maizhokunggar County
Viable count is average 108The cfu/mL orders of magnitude.Wherein Lactobacillus casei strain Qian are one of them bacterium
Strain.
(2) Preliminary Identification of Lactobacillus casei strain Qian.
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：Picking solid
One oese of bacterium colony, 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).
58 strains of lactic acid bacteria are purified into the result shows that being isolated from sample, these bacterial strains can be containing 5 ‰ CaCO3MRS
It is grown on plating medium, and molten calcium circle, Gram-positive, catalase test feminine gender is formed in periphery of bacterial colonies.
Embodiment 2：The in-vitro screening of Lactobacillus casei (Lactobacillus casei strain Qian).
(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, l.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) hydrophobicity measures.
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 casei strain Qian is as shown in table 1 below：
Note：LC-Qian：Lactobacillus casei strain Qian LB：Lactobacillus
bulgaricus
Table 1
The selection result shows that Lactobacillus casei strain Qian survival rates under pH3.0 gastric juice reach
62.51 ± 7.35%.
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 casei strain Qian of acid resistance
Be resistant to various concentration cholate the result is that the cholate to 0.3% has certain tolerance, when gallbladder salinity be increased to 0.5% and
When 1.0%, Lactobacillus casei strain Qian have embodied 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, as can be seen from the table, Lactobacillus casei
The hydrophobicity of strain Qian has reached 51.30 ± 4.28%.
Acid resistance, bile tolerance and the preferable Lactobacillus casei of hydrophobicity have been filtered out by testing us above
Lactobacillus casei strain Qian。
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 casei strain Qian groups of high and low given low, every group 10.Raising is in Rotating Stainless Steel Cage, room
24 ± 2 DEG C of temperature holding, relative humidity 50 ± 10%, 12h light and shades rotation (8:00~20:00 illumination).In entire experimental period, just
Often group and the equal feeding basal feed of constipation control group, experimental mice press 1.0 × 10 daily respectively9CFU/kg gavage probiotics.2
Start every morning 9 after week:00 gives 2 DEG C of activated carbon ice water of other 5 groups of intragastric administration on mice a concentration of 10% in addition to normal group,
Only, daily gavage is primary, continuous gavage 3 days by 0.2mL/.Until experiment last day, all mouse fasting for 24 hours, then gavage again
Activated carbon ice water, 0.2mL/ is only.5 mouse are for measuring changes of weight, defecation just quantity and moisture in feces in every group.It is small
Other 5 mouse of mouse put to death dissection after gavage activated carbon 30min.
The mouse weight variation of experiment periods Nei Ge groups is as shown in Figure 3 and Figure 4.
Normal group (Normal) mouse weight is put on weight increase with experimental period, and constipation control group (Control) was at 6 days
Afterwards since constipation weight loss occurs in gavage activated carbon, Bisacody (bisacodyl) constipation medicine group slows down weight loss,
Lactobacillus casei strain Qian 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 casei strain Qian groups mouse of given low arranges melena for the first time is below just
Secret control group shows certain constipation preventive effect higher than normal group and constipation therapy drug Bisacody group.
The feces volume of experimental period Nei Ge group mouse, the variation such as table 2, defecation shape of defecation granule number and moisture in feces
State such as Fig. 5.
Note：LC-Qian：Lactobacillus casei strain Qian
Table 2
Every group of mouse n=10ICR, Bisacody:100mg/kg b.w., lactobacillus bulgaricus:1.0×109CFU/kg
B.w., LC-Qian:Lactobacillus casei strain Qian.
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 casei
The defecation granule number and moisture in feces of strain Qian 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 6.
It is 89.2 ± 6.0% in constipation therapy drug Bisacody group mouse small intestine averagely propulsion rate, constipation control group is small
Mouse Intestinal propulsive rate has pole significant difference (P ＜ 0.05), the Lactobacillus of high and low given low for 21.3 ± 2.2%
Casei strain Qian groups and lactobacillus bulgaricus group mouse small intestine propulsion rate be respectively 65.8 ± 4.8%, 52.4 ±
5.1% and 40.3 ± 4.3% is higher than constipation control group, is less than constipation therapy drug Bisacody group, shows 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 table 3 in experiment periods Nei Ge groups mice serum.
Table 3
Relative to constipation control group, what Lactobacillus casei strain Qian height group can be different degrees of
These constipation negative correlation factor levels of MTL, Gas, ET, AchE, SP and VIP of increase serum, reduce the SS constipation positive correlation factors
It is horizontal.So Lactobacillus casei strain Qian have prevention effect to constipation.
Embodiment 6：The measurement of constipation related gene expression amount
It is grasped according to specification using the total RNA extraction reagent box (Invitrogen, the U.S.) of kit reagent chorista
Make.Purifying is operated using RNeasy kits (Qiagen, Germany) according to specification.CDNA is incubated 1h at 37 DEG C by 2 μ g total serum IgEs
It is synthesized with avian myeloblastosis virus reverse transcriptase (GE, Britain) reverse transcription.Gene primer sequence such as table 4 for nucleic acid amplification
Shown, polymerase chain reaction (PCR) is in thermal cycle (Eppendorf, Germany).PCR product is electric in 1.0% Ago-Gel
Swimming, is imaged with ethidium bromide staining.
The gene primer sequence of 4 nucleic acid amplification of table
Interstitial cells of Cajal (ICC) is the mesenchymal cell between a kind of enteric nervous system and smooth muscle, it is being adjusted
The nerve signal of section intestinal smooth cell plays an important role.Cytoplasmic tyrosine kinase (C-Kit) is the special marks of ICC
Note, it is the key that ICC proliferation.Stem cell factor (SCF) is the key protein of ICC growths.There is no SCF, ICC that can not give birth to
It is long.Transient receptor potential channel vanillic aldehyde hypotype -1 (TRPV1) is closely related with defecation and absorption, its high expression can cause intestines
Road damages.Constipation patient can express excessively high TRPV1.The neurotrophic factor (GDNF) of Deiter's cells source property is a kind of work
Property rho factor, can control the growth and development of nerve cell, protection and repair impaired nerve fibre.It has simultaneously
Adjust the function of nerve cell.NOS is the key enzyme for generating endogenous NO, it is widely present in stomach intestinal tissue from esophagus to anus
Door sphincter plays an important role adjusting gastrointestinal movement.
The c- of mouse can be turned up in Lactobacillus casei strain Qian high low dose groups compared to the control group
The mRNA and protein expression level of Kit, SCF, GDNF turn down the TRPV1 of mouse, gene expression dose such as Fig. 7, Fig. 8 of NOS.
From interpretation of result, it may be seen that with obvious effects be higher than lactobacillus bulgaricus.
Application Example 1：Lactic acid bacteria is manufactured using Lactobacillus casei (Lactobacillus casei strain Qian)
Milk drink.
First, Lactobacillus casei (the Lactobacillus casei strain Qian) original strain is in temperature -75
It is preserved with 30 weight % glycerine suspensions at DEG C, or is saved backup in the form of being freeze-dried bacterium powder at 4 DEG C of temperature.
Then, Lactobacillus casei (the Lactobacillus casei strain of the present invention are prepared with the following method
Qian) working stock culture：
Above-mentioned Lactobacillus casei (Lactobacillus casei strain Qian) original strain is inoculated in MRS liquid
In body culture medium, culture 12-16h is activated under the conditions of 37 DEG C, continuous to activate for two generations, and activation culture object is then pressed 2-
4% (volume) is inoculated in MRS culture mediums, cultivates 16-18h, and 4000r/min centrifuges 15min under the conditions of 4 DEG C, removes supernatant
Night obtains cell precipitation, and suspension is made with a certain amount of sterile absorbent breast in precipitation, obtains working stock culture, can be direct
This working stock culture is added in food, or with can symbiosis the commercial fermentation agent such as bulgarian milk for preparing acidified milk
Bacillus is used together with streptococcus thermophilus and prepares acidified milk.
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 casei (Lactobacillus casei strain Qian) working stock culture is added, it is made
Concentration reaches 106Cfu/ml or more, 4 DEG C it is stored refrigerated i.e. obtain the casei of Lactobacillus containing Lactobacillus casei
The lactic acid bacteria milk beverage of strain Qian viable bacterias.
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：Milk powder is manufactured using Lactobacillus casei (Lactobacillus casei strain Qian).
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 casei (Lactobacillus casei strain Qian) working stock culture, then the 16h that ferments at 37 DEG C, obtain
Lactobacillus casei (Lactobacillus casei strain Qian) acidified milk；Then the Lactobacillus casei
(Lactobacillus casei strain Qian) acidified milk is according to 1:3(V:V it) is added in above-mentioned sterilizing raw milk, carries out
Homogeneous is concentrated in vacuo, spray drying obtains the breast containing Lactobacillus casei (Lactobacillus casei strain Qian)
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:Capsule system is manufactured using Lactobacillus casei (Lactobacillus casei strain Qian)
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 casei (Lactobacillus casei strain Qian) working stock culture of the kind present invention, ferments at 37 DEG C
16h obtains Lactobacillus casei (Lactobacillus casei strain Qian) acidified milk.By Lactobacillus casei
(Lactobacillus casei strain Qian) acidified milk is with 1:3(V:V homogeneous in the raw milk after sterilizing, warp) is added
It is concentrated in vacuo, obtains milk powder after spray drying treatment, obtained milk powder is attached in capsule, capsule product is made.
Application Example 4：It is prepared and is fermented using Lactobacillus casei (Lactobacillus casei strain Qian)
Breast.
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 cheese breast bar is added according still further to 3-5% (volume)
Bacterium (Lactobacillus casei strain Qian) working stock culture, add 3-5% (volume) can symbiosis preparation hair
Kefir milk commodity leavening, 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 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. a kind of Lactobacillus casei Lactobacillus casei strain Qian working stock culture products, Chinese Typical Representative training
Support object collection deposit number CCTCC NO:Object is made using following step in M2013514：1) by above-mentioned cheese breast
Bacillus Lactobacillus casei strain Qian original strains are inoculated in MRS fluid nutrient mediums, under the conditions of 37 DEG C
Culture 12-16h is activated, continuous to activate for two generations, and activation culture object is then inoculated in MRS trainings by 2-4% percents by volume
It supports in base, cultivates 16-18h, 4000r/min centrifuges 15min under the conditions of 4 DEG C, removes supernatant, obtains cell precipitation, will sink
It forms sediment and suspension is made with a certain amount of sterile absorbent breast, obtain working stock culture product；2) by 1) gained target operation leavening
Product be directly appended in food or with can the commercial fermentation agent for preparing acidified milk of symbiosis be used together and prepare fermented milk products
Or food.
2. Lactobacillus casei Lactobacillus casei strain Qian working stock cultures according to claim 1
Product, which is characterized in that the commercial fermentation agent is lactobacillus bulgaricus or streptococcus thermophilus；The fermented food be containing
It is one or more in the lactic acid bacteria milk beverage of Lactobacillus casei, milk powder, capsule product or acidified milk.
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| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
| CN103184183A (en) * | 2013-04-19 | 2013-07-03 | 甘肃农业大学 | Preparation method of lactobacillus casei dry powder |
| CN104031856A (en) * | 2013-03-08 | 2014-09-10 | 中国农业大学 | Lactobacillus casei, its use, functional food composition and preparation method of functional food composition |
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| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
| CN104031856A (en) * | 2013-03-08 | 2014-09-10 | 中国农业大学 | Lactobacillus casei, its use, functional food composition and preparation method of functional food composition |
| CN103184183A (en) * | 2013-04-19 | 2013-07-03 | 甘肃农业大学 | Preparation method of lactobacillus casei dry powder |
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| Title |
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| 牦牛酸乳中乳酸菌的研究进展及前景展望;吴春生等;《食品工业》;20121231;第33卷(第9期);129-133 * |
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