CN105132329B - It is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain Lee and its health purpose - Google Patents
It is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain Lee and its health purpose Download PDFInfo
- Publication number
- CN105132329B CN105132329B CN201510587031.6A CN201510587031A CN105132329B CN 105132329 B CN105132329 B CN 105132329B CN 201510587031 A CN201510587031 A CN 201510587031A CN 105132329 B CN105132329 B CN 105132329B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus
- gastric ulcer
- lee
- lactobacillus fermentum
- fermentum strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses it is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain Lee and its health purpose, deposit number is that the acid resistance of the bacterial strain of CCTCC NO:M 2013512 is stronger, survival rate has reached 40.48 ± 1.84% after 3h under the simulated gastric fluid of pH3.0, and survival rate reaches 60% under 0.3% concentration cholate；The hydrophobicity of Lactobacillus fermentum strain Lee cell has also reached 15.17 ± 6.20%, the normal growth in the enteron aisle of people.Lactobacillus fermentum strain Lee can reduce gastric ulcer area, reduce gastric ulcer degree, reduce the secretion of gastric acid, the level of different degrees of increase amicine (SS) and vasoactive intestinal peptide (VIP) factor, reduce motilin (MOL) and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP) factor level, it lowers interleukin-6 (IL-6), interleukin 12 (IL-12), the level of tumor necrosis factor-alpha (TNF-α) and interferon-γ (IFN-γ).Frustrating with the drawing of Gastric Ulcer Treatment Aomei plays the role of similar, shows certain gastric ulcer preventive effect.
Description
Technical field
The present invention relates to microorganisms technical field, more particularly to it is a kind of can preventing gastric ulcer lactobacillus fermenti
Lactobacillus fermentum strain Lee and application thereof.
Background technique
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 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 simultaneously
Flavour of food products is assigned, the addition in food such as flavors and fragrances is reduced, extends the holding time, the functionality of food can also be improved
Matter.It can improve the digestibility and biological value of food, promote by decomposing the protein in food, carbohydrate, synthesis vitamin
It digests and assimilates, it is anti-to treat constipation, prevent and treat cell senescence.Human cholesterol content can also be reduced, blood lipid is improved, enhances immune function
Energy, anti-allergenicity, antitumor, anti-hypertension etc..In recent years, lactobacillus is formed with its special physiological activity and trophic function
For the main source of probiotics, just have attracted increasing attention.
Gastric ulcer is one of the most common type in peptic ulcer, and in China, disease incidence is higher, can betide any age, especially
With between twenty and fifty in the majority.Gastric ulcer shows as mucosa tissue damage, can also cause tissue and inflammation occurs.The clinical manifestation of gastric ulcer
It is in periodical attack repeatedly for Upper abdominal pain, show as dull pain, cusalgia or hungry sample pain, most people may occur in which various disappear more
Change undesirable symptom, but some is also without any symptom, until there is complication.Serious complication mainly have bleeding, perforation,
Canceration etc. can lead to 2.5%~8.0% case fatality rate if handling not in time.Under the symptom of appearance gastric ulcer not due to stomach
Suitable, patients diet is difficult, and chronic gastric ulcer patients chronic high is difficult, can also cause malnutrition, immunity degradation the problems such as.
Some patientss may occur in which that ulcer deteriorates, and seriously affect patient body Health and Living quality.In general gastric ulcer itself will not
Cause death, generally after drug therapy, remission or disappearance, but Ulcer cicatrization quality is poor, easily recurs after drug withdrawal,
Great harm is brought to the physical and mental health of patient.Therefore, find it is a kind of can alternatives to medication carry out the biology system of preventing gastric ulcer
Product have very big realistic meaning, and Tiny ecosystem therapy is just to solve a variety of existing for traditional remedies ask with a brand-new means
Topic.
There is a large amount of research report to the physiological function of lactic acid bacteria both at home and abroad, but to the functionality of lactic acid bacteria preventing gastric ulcer
Research report is seldom.Research about lactic acid bacteria preventing gastric ulcer is to many such as Food Science, preventive medicine and microecology
There is very big value in section.Fermented dairy product is the important channel that people take in lactic acid bacteria, screens excellent having and prevents stomach
The lactic acid bacteria strains of ulcer and safety for development functionality dairy products, enrich existing dairy products type, improve the additional of dairy products
Value will be of great significance.Therefore, the lactic acid bacteria with preventing gastric ulcer has very big research and application value.
In the document and patent that have disclosed at present or patent application, such as CN104489687A discloses one plant of pair and does
Lactobacillus paracasei is compounded with Chinese medicinal material extract, can be used for preventing and treating nonalcoholic fatty liver.CN104450586A is disclosed
A kind of Lactobacillus casei and combinations thereof, provided Lactobacillus casei is to glycopeptide class, monobactams and carbapenem
Class antibiotic and sulfanilamide (SN) have tolerance, suitable for food, drug and/or health care product.CN103333847A discloses one plant of hair
Kefir milk bacillus can promote the conversion and decomposition of internal cholesterol, can effective reducing blood lipid.CN103756926A discloses one plant of tool
There is acid resistance, there is the lactobacillus plantarum of preferable tolerance to copper ion in vivo and in vitro, can be used for preparing and alleviate Copper electrodissolution
In pharmaceutical composition and fermented food.CN1796540A discloses the Bifidobacterium with anti-enterovirus and anti-oxidation characteristics
And application thereof, it is used to prepare food compositions or pharmaceutical composition, kills or inhibit helicobacter pylori or Escherichia coli, treatment
The various diseases being induced by it.CN104430847A and CN104381440A uses identical acidified milk prevent constipation health purpose.
But these patents or patent application be not related to completely the microorganism with preventing gastric ulcer performance or its.Therefore, mesh
It is preceding also to need a kind of microorganism with preventing gastric ulcer and its food containing them.
Summary of the invention
To solve the above-mentioned problems, the object of the present invention is to provide it is a kind of can preventing gastric ulcer lactobacillus fermenti and its use
On the way, mainly provide it is a kind of can preventing gastric ulcer L fermentum strain and provide a kind of lactobacillus fermenti in fermented food
In purposes.
One kind provided by the invention can preventing gastric ulcer lactobacillus fermenti (Lactobacillus fermentum
Strain Lee) China typical culture collection center (the Chinese Wuhan Wuhan University) is preserved on October 25th, 2013,
Deposit number is CCTCC NO:M 2013512.
One plant of lactobacillus fermenti (Lactobacillus fermentum is filtered out from spontaneous fermentation yak yogurt
Strain Lee), using Microbiological Characteristics such as morphological feature, cultural colony and physiological and biochemical properties to the Bacillus acidi lactici
(Lactobacillus fermentum strain Lee) is accredited as lactobacillus fermenti.
The morphological feature of the lactobacillus fermenti specifically:
Thallus feature: being in Gram-positive, and cell is rod-shaped, about 0.5-1.0 μm of thallus wide, 1.5-4 μm long, Cheng Dan,
Pairs of or chaining, does not form gemma, and both ends are round.
Colony characteristics: forming apparent bacterium colony on MRS culture medium, and diameter is round between 0.5-2.0mm, and edge is whole
Together, milky, transparent, surface wettability is smooth, not chromogenesis.
Feature is resistant in the lactobacillus fermenti body are as follows: the bacterial strain acid resistance of lactobacillus fermenti is stronger, in the artificial of pH3.0
Survival rate is 40.48 ± 1.84% after 3h under gastric juice；Survival rate reaches 60% under 0.3% concentration cholate；Lactobacillus fermenti is thin
The hydrophobicity of born of the same parents is 15.17 ± 6.20%.
The object of the invention is also to provide can preventing gastric ulcer Lactobacillus fermentum strain Lee
The health purpose of lactobacillus fermenti: lactobacillus fermenti working stock culture made from the lactobacillus fermenti is added to cream system fermentation food
In product, the preparation step of the lactobacillus fermenti working stock culture includes: to be inoculated in the original strain of the lactobacillus fermenti
In MRS fluid nutrient medium, 12-16h is cultivated under the conditions of 37 DEG C and is activated, it is continuous to activate for two generations, then by activation culture object
It is inoculated in MRS culture medium by 2-4% (volume), cultivates 16-18h, 4000r/min is centrifuged 15min, removal under the conditions of 4 DEG C
Supernatant obtains cell precipitation, and suspension is made with sterile absorbent cream in precipitating, obtains working stock culture.
The cream fermented food includes lactic acid bacteria milk beverage, milk powder, capsule product and acidified milk.
The lactic acid bacteria milk beverage containing lactobacillus fermenti is prepared as steps described below:
The original strain of the lactobacillus fermenti is saved at -75 DEG C of temperature with 30% (weight) glycerol suspensions, or
Person is saved backup in the form of being freeze-dried bacterium powder at 4 DEG C of temperature；
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 milk powder containing lactobacillus fermenti is prepared as steps described below:
The original strain of the lactobacillus fermenti is saved at -75 DEG C of temperature with 30% (weight) glycerol suspensions, or
Person is saved backup in the form of being freeze-dried bacterium powder at 4 DEG C of temperature；
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 cream；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, vacuum concentration, spray drying obtain 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 saved at -75 DEG C of temperature with 30% (weight) glycerol suspensions, or
Person is saved backup in the form of being freeze-dried bacterium powder at 4 DEG C of temperature；
By raw milk in 140 DEG C of high temperature thermal sterilization 2s, 37 DEG C are subsequently cooled to, bacterium amount is connect with the 4% of raw milk volume and 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 cream and sterilizing is according to 1:3 (V:V)) proportion 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 saved at -75 DEG C of temperature with 30% (weight) glycerol suspensions, or
Person is saved backup in the form of being freeze-dried bacterium powder at 4 DEG C of temperature；
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, culture 14-16h to curdled milk, at this time the viable count about 10 in the curdled milk9Cfu/mL is obtained
This working stock culture can directly be added in food by working stock culture, or with can symbiosis the quotient 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 medium, cultivates 12- 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 medium by 2-4% (volume), is cultivated
16-18h, 4000r/min is centrifuged 15min under the conditions of 4 DEG C, removes supernatant, cell precipitation is obtained, by precipitating sterile absorbent
Suspension is made in cream, 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 type for example sold using Beijing Yong Chuanjiaye mechanical equipment Co., Ltd
What board-like superhigh temperature sterilization machine carried out.
The homogeneous be for example using Changzhou homogeneous Machinery Co., Ltd. sell the small-sized homogenizer of GJB500-40 into
Capable.
The concentration is for example to be carried out using the vacuum concentration pan that Shanghai Wei Zhou light industry and machinery Co., Ltd sells.
The spray drying be for example using Shanghai Triowin Tech. Co., Ltd. sell experiment type spray drier 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 for being selected from defatted milk, fresh milk, recovery milk
Cream.
The technical solution bring of the embodiment of the present invention has the beneficial effect that:
The present invention bursts for a kind of stomach that prevents being isolated from Sichuan Province Ruoergai country herdsman man spontaneous fermentation yak yogurt
The lactobacillus fermenti of ulcer and using the bacterial strain as answering in leavening or the acidified milk of addition, healthy food and animal health product
With the acid resistance of lactobacillus fermenti (Lactobacillus fermentum strain Lee) bacterial strain is stronger, in the people of pH3.0
Survival rate has reached 40.48 ± 1.84% after 3h under work gastric juice, and survival rate reaches 60% under 0.3% concentration cholate,
The hydrophobicity of Lactobacillus fermentum strain Lee cell has also reached 15.17 ± 6.20%.This shows to send out
Kefir milk bacillus (Lactobacillus fermentum strain Lee) can in the enteron aisle of people normal growth.Examination in Mice Body
It tests and is also shown that lactobacillus fermenti (Lactobacillus fermentum strain Lee) can reduce gastric ulcer area, drop
Low gastric ulcer degree, reduce the secretion of gastric acid, different degrees of increase amicine (SS) and vasoactive intestinal peptide (VIP) because
The level of son reduces motilin (MOL) and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP) factor level, lowers interleukin-6 (IL-6), and leucocyte is situated between
- 12 (IL-12) of element, the level of tumor necrosis factor-alpha (TNF-α) and interferon-γ (IFN-γ).With Gastric Ulcer Treatment Aomei
Drawing, which is frustrated, to be played the role of similar, shows certain gastric ulcer preventive effect.
Since lactobacillus fermenti derives from traditional fermented food, belong to generally recognized as safe (Generally Recognized As
Safe, GRAS) strain, it can be used in fermented food.
Detailed description of the invention
It, below will be to required use in embodiment description for the clearer technical solution for illustrating the embodiment of the present invention
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, it can also be obtained according to these attached drawings others
Attached drawing.
Fig. 1 lactobacillus fermenti (Lactobacillus fermentum strain Lee) colonial morphology；
The thalli morphology (l000x) of Fig. 2 lactobacillus fermenti (Lactobacillus fermentum strain Lee)；
Fig. 3 each group mouse gastric ulcer degree (ulcer area size)；
Fig. 4 each group mouse gastric ulcer degree (ulcer form)；
Fig. 5 Mouse Stomach liquid measure；
Fig. 6 mouse gastric juice pH；
Each factor level in Fig. 7 experiment periods Nei Ge group mice serum；
Each cytokine levels in Fig. 8 experiment periods Nei Ge group mice serum.
Lactobacillus fermenti (Lactobacillus fermentum strain Lee) of the invention was October 30 in 2013
Day is preserved in China typical culture collection center, and number is CCTCC NO:M 2013512.
Specific embodiment
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, those of ordinary skill in the art's every other implementation obtained under that premise of not paying creative labor
Example, shall fall within the protection scope of the present invention.
Embodiment 1: the separation of lactobacillus fermenti (Lactobacillus fermentum strain Lee), purifying and just
Step is fixed.
The embodiment carries out as steps described below:
(1) separation, purifying of Lactobacillus fermentum strain Lee.
It is added with sterile working absorption 500 μ L of traditional zymotic yak yoghourt sample to the mixing of 5mL sterile saline and is made
The uniform dilution of 1:10, 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 medium, 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 medium
In, 30 DEG C are set, 300r/min shaking table culture 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 saved, concrete operations ginseng
According to " Microbiology Experiment technology ".
Experimental result is shown, isolates 56 plants of lactic acid bacteria from ten samples in the county Ruo Gaier, sample viable lactic acid bacteria
Number is average 108The cfu/mL order of magnitude.
(2) Preliminary Identification of Lactobacillus fermentum strain Lee.
Gram's staining: taking colonies typical smear, Gram's staining is carried out, in microbioscope oil microscopic observation thallus
Form and its arrangement mode.Picking gets a clear view, the typical bacterial strain of form, takes pictures.Catalase experiment: picking solid
One oese of bacterium colony, is placed in clean tube on culture medium, and 3% hydrogenperoxide steam generator 2mL is added dropwise, and observes result (in half a minute
Interior gassing person is the positive, and gassing person is not feminine gender).
56 strains of lactic acid bacteria are purified into the result shows that isolating from sample, these bacterial strains can contain 5 ‰ CaCO3MRS
It is grown on plating medium, and forms 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 Lee).
(1) screening of probiotics tolerance pH3.0 simulated gastric fluid.
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 solution is taken, in aseptic operating platform
In pour into the 10mL centrifuge tube that sterilized, through 3000r/min centrifugation 10min collect thallus, be added 5mL sterile saline mix
Bacteria suspension is made, takes 1mL bacteria suspension 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, 37 DEG C of culture 48h are poured into MRS agar medium.It is surveyed with colony counting method
Determine viable count, calculates its survival rate (%): survival rate (%)=3h viable count/0h viable count × 100%.
(2) measurement of probiotics tolerance various concentration cholate.
Select in pH3.0 simulated gastric fluid survival rate 10% or more strain do the growth under different gallbladder salinities survey
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
It is white), 0.3% bovine bile, 0.5% bovine bile, l.0% bovine bile (W/V) MRS-THIO culture medium (in MRS culture medium 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 medium), the OD value of above-mentioned various concentration culture medium is measured respectively, calculates bacterial strain to the tolerance of cholate.Cholate
Tolerance=culture medium containing cholate OD value/blank cultures OD value × 100%.
(3) bacterial concentration adjusts.
Selection survival rate strain stronger in 10% or more, bile tolerance in pH3.0 simulated gastric fluid, 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, thallus is collected with 3000r/min centrifugation 10min.With 5mL PBS (50mM, pH6.5) buffer washing thalline,
3000r/min is centrifuged 10min, and repeated washing is twice.Using PBS buffer solution as blank control, strain subject bacterium is adjusted with buffer
Bulk concentration, making its A0 value under 560nm wavelength is about 1.00.
0.8mL dimethylbenzene is added in the 10mL centrifuge tube that sterilized in the bacterium solution for taking 4mL to adjust turbidity, and control group is not added
Dimethylbenzene, vibrate 30s, pause 10s, after vibrate 30s again, on rack for test tube stand 5~10min layering, take lower layer's water phase, with
PBS buffer solution is blank control, and A value is measured at 560nm and is recorded.Hydrophobic rate H%=[(A0-A)/A0] × 100 its
Middle A0 and A is the A value for mixing forward and backward bacterium solution with dimethylbenzene and measuring at 560nm respectively.
Lactic acid bacteria is a part of 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 have the characteristic of growth and development under enough acid resistances and acidic environment.
The selection result of Lactobacillus fermentum strain Lee is as shown in table 1 below:
Table 1
The selection result shows that Lactobacillus fermentum strain Lee bacterial strain is resistant to the environment of pH3.0,
And survival rate, all 50% or more, survival rate has reached 40.48 ± 1.84% under pH3.0 gastric juice.
It will be contacted with the cholate in small intestine by the thallus 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
Lee bacterial strain 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 Lee has 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
The hydrophobicity of fermentum strain Lee has reached 15.17 ± 6.20%.
Acid resistance, bile tolerance and the preferable Lactobacillus of hydrophobicity have been filtered out by testing us above
Fermentum strain Lee lactobacillus, the bacterial strain are preserved in China typical culture collection on October 30th, 2013
The heart (abbreviation CCTCC), deposit number CCTCC NO:M 2013512.
Embodiment 3: reserpine induction gastric ulcer experiment.
SPF grades of kunming mices of Feeding of healthy, 23 ± 2g of weight, male, 50.It, will be small after feeding basal feed adapts to 7d
Mouse is randomly divided into 5 groups according to weight, be respectively as follows: normal group, gastric ulcer control group, Aomei drawing frustrate group, Lactobacillus
Bulgaricus group and Lactobacillus fermentum strain Lee group, every group 10.It raises in Rotating Stainless Steel Cage,
24 ± 2 DEG C of room temperature holding, relative humidity 50 ± 10%, 12h light and shade rotation (8:00~20:00 illumination).In entire experimental period,
Normal group and the daily stomach-filling 0.2mL physiological saline of gastric ulcer control group, Lactobacillus bulgaricus group with
The experiment mice of Lactobacillus fermentum strain Lee group presses 1.0 × 10 daily respectively9CFU/kg stomach-filling
The corresponding bacterium solution of 0.2mL.Aomei drawing frustrates group and frustrates dilution by the drawing of 30mg/kg stomach-filling 0.2mL Aomei daily, during the experiment,
Do not occur mouse and occurs dead situation to stomach-filling substance discomfort.Each group mouse freely ingests and drinks water during experiment, experiment periods
Surrounding.After four weeks, in addition to normally group mouse, remaining four groups of mouse implements abdominal cavity subcutaneous injection 0.2mL reserpine suspension
(10mg/kg).After injections 24 hours, 5 groups of mouse are all put to death into dissection, the full stomach of mouse is won, is soaked into formalin,
It is taken pictures after 10min with digital camera, and analyzes gastric ulcer area with ImageJ software, calculate ulcer index.
Experiment periods Nei Ge group mouse gastric ulcer degree is as shown in Figure 3 and Figure 4.
Reserpine (10mg/kg) induces gastric ulcer each group mouse gastric ulcer degree such as the following table 2:
A~eDifferent letters indicate there is statistical difference (p < 0.05), lower same
Table 2
To observation mouse stomach ulcer area after mouse reserpine induction gastric ulcer, as shown in Table 2, the stomach of each group mouse
There are significant difference (P ﹤ 0.05) for ulcer area.The ulcer of control group is the most serious, and ulcer area reaches (7.08 ± 1.52)
mm2, fill food Lactobacillus bulgaricus bacterium solution and Lactobacillus fermentum strain Lee bacterium solution
Mouse gastric ulcer area drop to (5.22 ± 0.32) and (2.31 ± 0.41) mm respectively2.It can see by 2 experimental data of table
It is a kind for the treatment of Gastric Ulcer Treatment with good drug effect that Aomei drawing, which is frustrated,.Lactobacillus fermentum strain Lee
It can reduce gastric ulcer area, reduce gastric ulcer degree, effect is greater than common lactobacillus bulgaricus.
Embodiment 4: reserpine (10mg/kg) induces the measurement of the gastric juice amount and Gastric pH of gastric ulcer each group mouse.
Mouse cervical dislocation is put to death, the mouse gastric juice after collecting dissection measures mouse gastric secretion, measures stomach with pH meter
The pH value of liquid.Experiment periods Nei Ge group Mouse Stomach liquid measure and gastric juice pH are as shown in Figure 5 and Figure 6.
The gastric juice amount and Gastric pH such as the following table 3 of reserpine (10mg/kg) induction gastric ulcer each group mouse:
Table 3
Stomach largely secretes gastric acid after gastric ulcer occurs, and gastric acid further stimulates gastric ulcer to aggravate, and the two, which has, to be positively correlated
Property.And pH value is reduced since gastric acid increases, and is negatively correlated with gastric ulcer degree.Lactobacillus fermentum
Strain Lee alleviates gastric ulcer degree, declines gastric juice amount compared with control group, and pH value increases, effect ratio Lactobacillus
Bulgaricus is more significant.
Embodiment 5: the measurement of each factor level in mice serum.
Cervical dislocation puts to death mouse, takes rat aorta blood plasma 0.2mL, is centrifuged 10min in 4 DEG C, 3000r/min, takes upper layer blood
Clearly.By method measurement motilin (MOL), amicine (SS), Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP) and the vasoactive of test agent box specification
Level of peptide (VIP) factor in serum.Each factor level is as shown in Figure 7 in experiment periods Nei Ge group mice serum.
Reserpine (10mg/kg) induces the motilin (MOL) of gastric ulcer each group mouse, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SP), growth hormone release inhibiting hormone (SS)
It is horizontal such as the following table 4 with vasoactive intestinal peptide (VIP):
Table 4
Relative to gastric ulcer control group, the different degrees of reduction of Lactobacillus fermentum strain Lee energy
MOL, SP, the factor level of serum increase SS and VIP factor level.So Lactobacillus fermentum Suo is to stomach
Ulcer has prevention effect.
Embodiment 6: the measurement of cytokine levels in mice serum.
Cervical dislocation puts to death mouse, takes rat aorta blood plasma 0.2mL, is centrifuged 10min in 4 DEG C, 3000r/min, takes upper layer blood
Clearly.Using quantitative elisa kit for detecting, by enzyme-linked immunization ultraviolet specrophotometer, serum is detected at 450nm wavelength
Middle tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin 12 (IL-12) and interferon-γ
(IFN-γ) is horizontal.Each cytokine levels are as shown in Figure 8 in experiment periods Nei Ge group mice serum.
Reserpine (10mg/kg) induces the tumor necrosis factor-alpha (TNF-α) of gastric ulcer each group mouse, interleukin-6
(IL-6), interleukin 12 (IL-12) and interferon-γ (IFN-γ) are horizontal such as the following table 5:
Table 5
IL-6 is the cell factor secreted by T.B cell and mononuclear macrophage, is had in various autoimmune disease
It is abnormal, and a variety of abnormal antibodies in ulcers body related with the pathologic process of these diseases and coincident with severity degree of condition, it is immunized multiple
Closing object and merging cause pathogeny imcrobe infection can generate by different approaches stimulation one macrophage of monokaryon and discharge TNF-α to blood and follow
In ring, causes TNF-α in blood to increase the TNF-α and inflammatory cell interaction that increases, aggravate inflammatory reaction, while can stimulate
In blood and inflammatory cell secretes other cell factors, and promoting damage, the IL-6 of high activity has with the internal abnormal antibodies that generate simultaneously
It closes, by one antibody response of antigen, activating complement makes tissue damage that be further aggravated it is confirmed that ulcers are in the presence of immune
Dysfunction, TNF-α and IL-6 play an important role .IL-12 in the generation of ulcer and progression of the disease and IFN-γ is proinflammatory
Disease cell factor, IL-12 can promote the development and proliferation of T cell and NK cell, and these cells is stimulated to generate IFN-γ, IFN-
γ also will increase the secretion of IL-12.Existing research shows that IFN-γ and IL-12 can pass through the participation ulcer of interaction
Morbidity and treatment.In clinical studies, IL-6, IL-12, the horizontal journey that can mitigate gastric ulcer of TNF-α and IFN-γ are lowered
Degree.Lactobacillus fermentum strain Lee shows to lower IL-6, IL-12, the level of TNF-α and IFN-γ
Ability, effect be significantly better than Lactobacillus bulgaricus, it is believed that Lactobacillus fermentum
Strain Lee has good inhibition gastric ulcer effect, and achievees the effect that draw close to drug Aomei and frustrate.
Application Example 1: cream is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain Lee)
Sour bacterium milk drink.
Firstly, lactobacillus fermenti (the Lactobacillus fermentum strain Lee) original strain is in temperature
It is saved at -75 DEG C of degree with 30 weight % glycerol suspensions, or preservation is standby in the form of being freeze-dried bacterium powder at 4 DEG C of temperature
With.
It is then possible to prepare lactobacillus fermenti of the invention (Lactobacillus fermentum using two methods
Strain Lee) working stock culture:
First method is that above-mentioned lactobacillus fermenti (Lactobacillus fermentum strain Lee) is original
Strain is inoculated in the skimmed milk that 12 weight % sterilize 10min at 110 DEG C, and 14-16h is cultivated under the conditions of 37 DEG C to curdled milk, is connected
In continuous culture two generations of activation, it is used as mother culture；The mother culture is inoculated in sterile milk by 3-5% (volume), cultivates 14-
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 Lee) is original
Strain is inoculated in MRS fluid nutrient medium, 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 MRS culture medium by 2-4% (volume), 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 cream in precipitating, 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, mentioned-above lactobacillus fermenti (Lactobacillus fermentum strain Lee) working stock culture is added,
Its concentration is set to reach 106Cfu/ml or more stored refrigerated obtains Lactobacillus containing lactobacillus fermenti at 4 DEG C
The lactic acid bacteria milk beverage of fermentum strain Lee viable bacteria.
In the present invention, the MRS fluid nutrient medium is known to those skilled in the art, is 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 type for example sold using Beijing Yong Chuanjiaye mechanical equipment Co., Ltd
What board-like superhigh temperature sterilization machine carried out.
Application Example 2: cream is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain Lee)
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 mentioned-above
Lactobacillus fermenti (Lactobacillus fermentum strain Lee) working stock culture, then the 16h that ferments at 37 DEG C, obtain
To lactobacillus fermenti (Lactobacillus fermentum strain Lee) acidified milk；Then the lactobacillus fermenti
(Lactobacillus fermentum strain Lee) acidified milk is added in above-mentioned sterilizing raw milk according to 1:3 (V:V), into
Row homogeneous, vacuum concentration, spray drying obtain (Lactobacillus fermentum strain Lee) containing lactobacillus fermenti
Milk powder.
The homogeneous be for example using Changzhou homogeneous Machinery Co., Ltd. sell the small-sized homogenizer of GJB500-40 into
Capable.
The concentration is for example to be carried out using the vacuum concentration pan that Shanghai Wei Zhou light industry and machinery Co., Ltd sells.
The spray drying be for example using Shanghai Triowin Tech. Co., Ltd. sell experiment type spray drier into
Capable.
Application Example 3: glue is manufactured using lactobacillus fermenti (Lactobacillus fermentum strain Lee)
Capsule product.
By raw milk in 140 DEG C of high temperature thermal sterilization 2s, 37 DEG C are subsequently cooled to, bacterium amount is connect with the 4% of raw milk volume and connects
Kind lactobacillus fermenti (Lactobacillus fermentum strain Lee) working stock culture of the invention, issues at 37 DEG C
Ferment 16h obtains lactobacillus fermenti (Lactobacillus fermentum strain Lee) acidified milk.By lactobacillus fermenti
Homogeneous in the raw milk after sterilizing is added with 1:3 (V:V) in (Lactobacillus fermentum strain Lee) acidified milk,
Milk powder is obtained after vacuum concentration, spray drying treatment, obtained milk powder is attached in capsule, capsule product is made.
Application Example 4: lactobacillus fermenti (Lactobacillus fermentum strain Lee) preparation hair is utilized
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 mentioned-above acidified milk bar is added according still further to 3-5% (volume)
Bacterium (Lactobacillus fermentum strain Lee) 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 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 for being selected from defatted milk, fresh milk, recovery milk
Cream.The above, only specific embodiments of the present invention, but feature of the invention is not limited thereto, it is any to be familiar with this skill
In the field of the present invention, the changes or modifications that can be readily occurred in should all cover and apply for a patent model of the invention below the people of art
In enclosing.
Claims (4)
1. purposes of the lactobacillus fermenti bacterium solution in the health food that preparation improves reserpine induction gastric ulcer, lactobacillus fermenti are
Lactobacillus fermentum strain Lee, deposit number are CCTCC NO:M2013512.
2. purposes according to claim 1, which is characterized in that Lactobacillus fermentum Lee reduces stomach and bursts
Ulcer area, reduces gastric ulcer degree, and effect is greater than common lactobacillus bulgaricus.
3. purposes according to claim 1, which is characterized in that Lactobacillus fermentum Lee can different journeys
The motilin MOL and Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 factor level of the reduction serum of degree, increase amicine SS and the vasoactive intestinal peptide VIP factor
It is horizontal.
4. purposes according to claim 1, which is characterized in that adjusted to white under Lactobacillus fermentum Lee thin
Born of the same parents' interleukin IL-6, interleukins IL-12, tumor necrosis factor TNF-alpha, interferon IFN-γ are horizontal, and effect is significantly better than guarantor
Add Leah lactobacillus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510587031.6A CN105132329B (en) | 2015-09-14 | 2015-09-14 | It is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain Lee and its health purpose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510587031.6A CN105132329B (en) | 2015-09-14 | 2015-09-14 | It is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain Lee and its health purpose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105132329A CN105132329A (en) | 2015-12-09 |
| CN105132329B true CN105132329B (en) | 2019-04-16 |
Family
ID=54717903
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510587031.6A Expired - Fee Related CN105132329B (en) | 2015-09-14 | 2015-09-14 | It is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain Lee and its health purpose |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105132329B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103648511A (en) * | 2011-06-08 | 2014-03-19 | 有机平衡有限公司 | Spray-dried lactobacillus stems / cells and the use of same against helicobacter pylori |
| CN104430847A (en) * | 2014-07-17 | 2015-03-25 | 西南民族大学 | Lactobacillus fermentum Lee working leavening agent product and constipation-prevention and health application thereof |
-
2015
- 2015-09-14 CN CN201510587031.6A patent/CN105132329B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103648511A (en) * | 2011-06-08 | 2014-03-19 | 有机平衡有限公司 | Spray-dried lactobacillus stems / cells and the use of same against helicobacter pylori |
| CN104430847A (en) * | 2014-07-17 | 2015-03-25 | 西南民族大学 | Lactobacillus fermentum Lee working leavening agent product and constipation-prevention and health application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Preventive effect of Lactobacillus fermentum Lee on activated carbon-induced constipation in mice;Yu Qian et al.;《Experimental and Therapeutic Medicine》;20141111;第9卷;摘要，第273页左栏第2段 * |
| 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);第129-133页 * |
| 牦牛酸乳分离发酵乳杆菌发酵豆浆的胃溃疡预防效果研究;赵欣 等;《食品科学》;20141231;第35卷(第17期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105132329A (en) | 2015-12-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111484957B (en) | Bifidobacterium animalis subsp lactis i797, and separation and purification method and application thereof | |
| CN104498383B (en) | A kind of lactobacillus fermenti Lactobacillus fermentum strain suo and application thereof of adjustable intestinal movement, Constipation | |
| CN104531549B (en) | A kind of lactobacillus fermenti Lactobacillus fermentum strain Zhao and application thereof of adjustable intestinal movement, Constipation | |
| CN104430847B (en) | Lactobacillus fermenti Lactobacillus fermentum Lee working stock cultures products and its Constipation health purpose | |
| CN109182207B (en) | Lactobacillus acidophilus La-SJLH001 with probiotic functions of regulating blood sugar level, cholesterol level and the like and application thereof | |
| CN110106122B (en) | Lactobacillus plantarum capable of improving sleep and application thereof | |
| CN104381440B (en) | Lactobacillus fermenti Lactobacillus fermentum strain Lee working stock cultures products and its Constipation health purpose | |
| CN101160134B (en) | Selection and use of lactic acid bacteria for reducing inflammation in mammals | |
| CN105132328B (en) | It is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain suo and application thereof | |
| CN107164263A (en) | A kind of adjustable function of intestinal canal, the lactobacillus fermenti HY01 for preventing colitis and application thereof | |
| CN114085792B (en) | Lactobacillus paracasei for preventing and treating colon cancer and application thereof | |
| CN110157649A (en) | It is a kind of to have effects that improve the lactobacillus paracasei L9 of people's constipation and its application | |
| CN107227275A (en) | A kind of lactobacillus fermenti HY01 and application thereof | |
| CN114381411B (en) | Lactococcus lactis JYLL-60 and application thereof in preparation of product for improving immunity | |
| CN109481476A (en) | Application of the lactobacillus fermenti CQPC04 in the food or drug that preparation improves ulcerative colitis | |
| CN110484459B (en) | Lactobacillus plantarum and application thereof | |
| CN116970539B (en) | Lactobacillus murine complex, composition and application thereof | |
| CN104997813B (en) | The preparation method of prebiotic probiotics | |
| CN116445356B (en) | Bifidobacterium animalis subspecies BA67 for regulating intestinal flora and enhancing immunity and application thereof | |
| CN104651271B (en) | Lactobacillus casei Lactobacillus casei strain Qian working stock cultures products and its enteron aisle adjust health purpose | |
| CN105154362A (en) | Lactobacillus fermentum suo strain capable of preventing gastrohelcoma and health-care application thereof | |
| CN105274032B (en) | A kind of antagonism campylobacter jejuni and the lactobacillus reuteri for inhibiting its flaA gene expression | |
| CN109362882B (en) | Lactobacillus fermentum CQPC07 and application thereof in preparing food or medicine for improving ulcerative colitis | |
| CN105132330A (en) | Lactobacillus fermentum strain Lee capable of preventing gastric ulcers and application of lactobacillus fermentum strain Lee | |
| CN105132329B (en) | It is a kind of can preventing gastric ulcer lactobacillus fermenti Lactobacillus fermentum strain Lee and its health purpose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190416 Termination date: 20190914 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |