CN107227275A - A kind of lactobacillus fermenti HY01 and application thereof - Google Patents
A kind of lactobacillus fermenti HY01 and application thereof Download PDFInfo
- Publication number
- CN107227275A CN107227275A CN201710545674.3A CN201710545674A CN107227275A CN 107227275 A CN107227275 A CN 107227275A CN 201710545674 A CN201710545674 A CN 201710545674A CN 107227275 A CN107227275 A CN 107227275A
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- milk
- lactobacillus fermenti
- lactobacillus
- culture
- fermenti
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses a kind of lactobacillus fermenti (Lactobacillus fermentum）HY01, deposit number is CCTCCM2015792) and its purposes in the fermented food for preparing adjustable function of intestinal canal or/and Constipation, it is 104.60 ± 6.50% that the bacterial strain handles survival rate after 3 h in pH 3.0 simulated gastric fluid, and be able to can be grown under 0.3% gallbladder salinity, growth efficiency is 21.10 ± 0.24% without cholate culture, with good external tolerance.In-vivo test in mice shows, lactobacillus fermenti (Lactobacillus fermentum) HY01 can dramatically increase the dejecta moisture of mice with constipation；Improve small intestine activated carbon propulsion rate and shorten the time of the first discharge melena of mice with constipation；Mitigate the degree of injury of intestinal villi, so as to promote intestines peristalsis.In addition, on serum levels,Lactobacillus fermentumHY01 processing can significantly improve the level of SST in MTL, GT, ET, AchE, SP, VIP level in serum and reduction serum, therefore, lactobacillus fermenti (Lactobacillus fermentum) HY01 has the effect of Constipation.
Description
Technical field
The present invention relates to microbial technology field, more particularly to a kind of lactobacillus fermenti (Lactobacillus
Fermentum) HY01 and its purposes in the fermented food of adjustable function of intestinal canal or/and Constipation is prepared.
Background technology
Constipation is a kind of common and complicated symptom, and clinical manifestation is mainly that feces volume is reduced, excrement is dry and hard, difficult defecation
With defecation frequency reduce etc..Although the research of the constipation therapy enriched, epidemiology and pathogenesis, it is fixed to will be clear that
Adopted constipation is still very difficult.Definition of the traditional Chinese and western medicine to constipation depends on defecation situation, but is due to that dietary structure and defecation are practised
Used difference, people are to the cognitive diversity of constipation and the difference of evaluation in addition, and this is according to still not enough fully, so as to face
The diagnosis and treatment of bed bring difficulty.At present, it is RomeⅢdiagnostic criteria for the diagnostic criteria that functional consitipation is more approved,
It referred to past 12 middle of the month, at least 12 weeks（Continue, accumulate）There is the symptom of following 2 or more：（1）More than 1/4
It is nervous that defecation occurs in defecation；（2）There is lumpy or scleroma in defecation more than 1/4；（3）Defecation more than 1/4 has defecation not to the utmost
Sensation；（4）There is the sensation of anorectal obstruction in the defecation more than 1/4；（5）Defecation more than 1/4 need with hand help with
Beneficial to defecation；（6）Defecation frequency is less than 3 times weekly.Epidemiological study shows that constipation has very high generation in crowd
Rate, up to 27%, the crowd of different regions, different sexes and all ages and classes is possible to be affected by it.In terms of the angle of region, west
Fang Guojia constipation patients are higher than oriental countries constipation patient；In terms of sex angle, women is people at highest risk, data statistics result table
Bright, Beijing area adult men and women's illness rate ratio is 1:4.59, In Guangzhou Area is 1:1.77, Efficiency in Buildings in Tianjin Area is 1:2.75；From the age
Angle sees that gerontal patient is more than adolescent patients, and overall condition is：With the increase at age, patient is more and more.Prolonged constipation meeting
A large amount of just slags is trapped in enteron aisle, after fermenting and rotting, cause a large amount of toxin and harmful bacteria to be absorbed repeatedly by enteron aisle, menses
Spread after liquid circulation, finally endanger human health.In addition, constipation can also make child's gastrointestinal bacterial flora unbalance, thought slowness, mood
It is irritable, malnutritive, become thin, immunity degradation and growth retardation etc..Given birth to many worries of patient's increase with patient is had a strong impact on
Bioplasm amount.
In clinical studies, it is still unclear at present because constipation pathogenesis is complex.So that treatment method is more,
The treatment of current constipation is still based on drug therapy, and the medicine of primary treatment is broadly divided into DA receptor antagonists, 5- hydroxyl colors
Amine receptor activator, motilin agonists and several classes of cathartic, conventional DA acceptor inhibitors have domperidone（Also known as fourth
Quinoline）And Metoclopramide（Also known as metoclopramide）, the two therapeutic effect to constipation is poor；In 5-hydroxytryptamine receptor activator, west
Husky Billy is clinically to apply earliest 5-hydroxytryptamine receptor activator, but is currently based on Cisapride and has developed high selectivity
The receptor stimulating agent Mosapride of serotonin 4；Erythromycin is most widely used in motilin agonists, but it has
Many side reactions, such as nausea, stomachache and diarrhoea；And cathartic because its can rapid recovery constipation symptom, therefore by constipation
The general choice of patient.Even so, clinically still without generally acknowledged and effective treatment method, so find a kind of safe,
Good effect and the small treatment concept of toxic side effect are favored by domestic and foreign scholars.In addition drug therapy has many drawbacks, example
Irritant laxative is such as used for a long time（Folium sennae, rheum officinale, aloe）Melanosis coli, emollient cathartic can be caused in-convenience in use
（Anus is used）, bulk cathartic is easily caused flatulence, and probiotics agents treatment have side effect it is relatively low, effectively and body can be made many-sided by
Beneficial the characteristics of, therefore as treatment concept new in constipation therapy.Zhou Xiaodan researchs show lactobacillus paracasei LC-01 to constipation
It has also been found that the bacterial strain has the work of Constipation in the effect that mouse tool relaxes bowel, fourth sage's research bifidobacterium longum BBMN68
With.It is reported that the probiotics for being usually used in constipation therapy has the newborn bar of bifidobacterium longum, Lactobacillus casei, Lactobacillus rhamnosus, acidophilus
Bacterium and streptococcus thermophilus；Therefore, lactic acid bacteria of the screening with Constipation function has important to the research for improving function of intestinal canal
Meaning.
The content of the invention
In order to solve the above problems, first purpose of the invention is to provide a kind of lactobacillus fermenti（Lactobacillus
fermentum）HY01, the lactobacillus fermenti can adjust function of intestinal canal, Constipation.
Lactobacillus fermenti that the present invention is provided (Lactobacillus fermentum) HY01 is on December 29th, 2015
It is preserved in China typical culture collection center (CCTCC), depositary institution address：Chinese Wuhan Wuhan Universitys, deposit number
For CCTCCM2015792.
The bacterial strain be filtered out from spontaneous fermentation yak yogurt one plant of lactobacillus fermenti (Lactobacillus fermentum) HY01, using Microbiological Characteristics such as morphological feature, cultural colony and physiological and biochemical properties to the Bacillus acidi lacticiLactobacillus fermentumHY01 is accredited as lactobacillus fermenti.
The biological characteristics of the lactobacillus fermenti is as follows：
1）Morphological feature：Thalline feature, in Gram-positive, cell is shaft-like, no gemma, and two ends are circular；Colony characteristicses,
Obvious bacterium colony is formed on MRS solid mediums, circular, neat in edge, milky, bacterium colony projection, surface wettability is smooth, no
Chromogenesis；
2）Feature is resistant in vivo:It is 104.60 ± 6.50% that the survival rate after 3 h is handled in pH 3.0 simulated gastric fluid；
Growth efficiency under 0.3% gallbladder salinity is 21.10 ± 0.24 without cholate culture.
Second object of the present invention be to provide the lactobacillus fermenti HY01 for prepare regulation function of intestinal canal or/and
Purposes in the fermented food of Constipation.
The fermented food is in lactic acid bacteria milk beverage, milk powder, capsule product and acidified milk containing lactobacillus fermenti
It is one or more.
Further, the lactic acid bacteria milk beverage is prepared as steps described below：
Raw milk heats sterilization 15 ~ 30 min or s of high temperature thermal sterilization 2 ~ 5 at 125 ~ 140 DEG C at 95 ~ 100 DEG C, is cooled to
4 DEG C, the working stock culture containing the lactobacillus fermenti is added, its concentration is reached 106 More than cfu/mL, is protected in 2 ~ 8 DEG C of refrigerations
Deposit.
Further, the milk powder is prepared as steps described below：
Raw milk heats sterilization 15 ~ 30 min or high temperature thermal sterilization 2 ~ 5 s, Ran Houleng at 125 ~ 140 DEG C at 95 ~ 100 DEG C
But to 37 DEG C, then with the 4% of raw milk volume working stock culture of the bacterium amount inoculation containing the lactobacillus fermenti is connect, at 35 ~ 40 DEG C
Ferment 16 h, obtains lactobacillus fermenti acidified milk；Raw milk after the lactobacillus fermenti acidified milk and sterilizing is according to 1:3（V:
V）Proportioning is added, and carries out homogeneous, is concentrated in vacuo, spray drying obtains the milk powder containing lactobacillus fermenti..
Further, the capsule product is prepared as steps described below：
By raw milk in 125 ~ 140 DEG C of s of high temperature thermal sterilization 2 ~ 5,35 ~ 40 DEG C are subsequently cooled to, is connect with the 4% of raw milk volume
Working stock culture of the bacterium amount inoculation containing the lactobacillus fermenti, ferment 15 ~ 20 h at 35 ~ 40 DEG C, obtains lactobacillus fermenti hair
Kefir milk；By the raw milk after the lactobacillus fermenti acidified milk and sterilizing according to 1:3~1:5（V:V））Proportioning is added, homogeneous,
Milk powder is obtained after vacuum concentration, spray drying treatment, the capsule product containing lactobacillus fermenti will be obtained.
Further, the acidified milk is prepared as steps described below：
Raw milk heats sterilization 15 ~ 30 min or high temperature thermal sterilization 2 ~ 5 s, Ran Houleng at 125 ~ 140 DEG C at 95 ~ 100 DEG C
But to 35 ~ 40 DEG C, according still further to 3 ~ 5%（Volume）The working stock culture containing lactobacillus fermenti is added, 3 ~ 5% are added（Volume）Can
Being used for of symbiosis prepares the leavening of fermented dairy product, after mixing at 35 ~ 40 DEG C mixed fungus fermentation to titratable acidity in terms of lactic acid
0.6 ~ 0.7%, it is subsequently cooled to 2 ~ 8 DEG C, then carry out stored refrigerated obtaining the acidified milk containing lactobacillus fermenti.
Further, one or more of the raw milk in defatted milk, fresh milk and recovery milk.
Further, the preparation method of the working stock culture containing lactobacillus fermenti comprises the following steps：
The original strain of lactobacillus fermenti is preserved at -80 DEG C of temperature in magnetic bead form, or to freeze at 2 ~ 8 DEG C of temperature
The form for drying bacterium powder is saved backup;
The original strain of the lactobacillus fermenti is inoculated in 12%（Weight）Skimmed milk in, 110 DEG C sterilizing 10 min, 37
14 ~ 16 h are cultivated under the conditions of DEG C to curdled milk, continuous culture two generations of activation, as mother culture；The mother culture is pressed 3 ~ 5%
（Volume）It is inoculated in sterile milk, 14 ~ 16 h are to curdled milk for culture, the viable count about 10 into the curdled milk9 Cfu/mL, obtains work
Leavening；
Or the original strain of the lactobacillus fermenti is inoculated in MRS fluid nutrient mediums, 12 ~ 16h is cultivated under the conditions of 37 DEG C
Activated, two generations of continuous activation, activation culture thing is then pressed 2 ~ 4%（Volume）It is inoculated in MRS culture mediums, culture 16 ~ 18
H, 3000 r/min centrifuge 15 min under the conditions of 4 DEG C, remove supernatant, obtain cell precipitation, by precipitation sterile absorbent breast
Suspension is made, working stock culture is obtained.
The beneficial effects of the present invention are：The present invention is isolated from Sichuan Province Hongyuan County herdsman family spontaneous fermentation yak for one kind
Adjustable function of intestinal canal, the lactobacillus fermenti of Constipation in yogurt, lactobacillus fermenti (Lactobacillus fermentum) HY01 bacterial strain tolerances are stronger, it is 104.60 that the survival rate after 3 h is handled in pH 3.0 simulated gastric fluid
±6.50%；Growth efficiency under 0.3% gallbladder salinity is 21.10 ± 0.24 without cholate culture.This shows lactobacillus fermenti
(Lactobacillus fermentum) HY01 can well grow in the enteron aisle of people.In-vivo test in mice shows, ferments
Lactobacillus (Lactobacillus fermentum) HY01 can increase the dejecta moisture of mice with constipation；Improve small intestine activity
The time of the first discharge melena of charcoal propulsion rate and shortening mice with constipation；Mitigate the degree of injury of intestinal villi, so as to promote intestines
Wriggle in road.In addition, on serum levels,Lactobacillus fermentumHY01 processing can be significantly improved in serum
SST level, the effect with Constipation in MTL, GT, ET, AchE, SP, VIP level and reduction serum.Due to this hair
Bright lactobacillus derives from traditional fermented food, belongs to generally recognized as safe (Generally Recognized As Safe, GRAS) bacterium
Kind, available in fermented food.
Brief description of the drawings
Fig. 1 be lactobacillus fermenti (Lactobacillus fermentum）HY01 colonial morphologies.
Fig. 2 be lactobacillus fermenti (Lactobacillus fermentum) HY01 thalli morphology（1000×）.
Fig. 3 is the change of separate groups of mice dejecta moisture.
Fig. 4 is the time that separate groups of mice discharges melena for the first time.
Fig. 5 is the change of separate groups of mice activated carbon propulsion rate.
Fig. 6 is separate groups of mice intestinal villi pathological observation.
Fig. 7 is inflammation-related factor level in separate groups of mice serum.
The present invention lactobacillus fermenti (Lactobacillus fermentum) HY01 is preserved on December 29th, 2015
China typical culture collection center, depositary institution address：Chinese Wuhan Wuhan Universitys, deposit number is
CCTCCM2015792。
Embodiment
The invention will be further described with specific embodiment below in conjunction with the accompanying drawings, so that those skilled in the art can be with
It is better understood from the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Embodiment 1：Lactobacillus fermenti (Lactobacillus fermentum) HY01 separation, purifying and Preliminary Identification
（1）Lactobacillus fermentumHY01 separation, purifying
Added with the mL of sterile working pipette samples 1 to 9 mL sterile salines and mix the uniform dilution for being made 1: 10, and after
It is continuous to make a certain proportion of dilution.The dilution of suitable gradient is selected, being respectively coated MRS with 100 μ L of each absorption of sterile pipette tips consolidates
In body plating medium, 37 DEG C of 48 h of culture, observed and recorded colonial morphology, as shown in Figure 1.
With oese from the different colony inoculation of planar surface picking in MRS fluid nutrient mediums, put 37 DEG C, 100 r/min
The h of shaking table culture 18；Repeat to carry out gram stain smear microscopy after above step continuously activated for 2 generations, as shown in Fig. 2 being defined as
The continuation activation of G+ bacterium, until obtaining pure bacterium colony（Microscopy is without miscellaneous bacteria）And preserve, concrete operations reference《Microbiology Experiment skill
Art》.Experimental result is shown, 43 plants of lactic acid bacteria is isolated from 12 parts of samples of Sichuan Hongyuan.
（2）Lactobacillus fermentum HY01 Preliminary Identification
Gram's staining：Colonies typical smear is taken, Gram's staining is carried out, in the oily Microscopic observation thalli morphology of microbioscope
And its arrangement mode.Picking gets a clear view, the typical bacterial strain of form, is taken pictures.As a result show, isolated and purified from sample
The bacterial strain gone out is Gram-positive.
Embodiment 2：Lactobacillus fermentiLactobacillus fermentum HY01 in-vitro screening
（1）Probiotics is resistant to the screening of the simulated gastric fluids of pH 3.0
The preparation of simulated gastric fluid：NaCl 0.2%, pepsin 0.35%, it is after 3.0, in sterile behaviour with 1M HCl adjustment pH value
Make degerming rear standby with vavuum pump suction filtration in platform；
Measure of the probiotics to artificial gastric juice resistance's property：The activated good strain cultured solutions of 5 mL are taken, in aseptic operating platform
Pour into the 10 mL centrifuge tubes that sterilized, centrifuging 10 min through 3000 r/min collects thalline, adds 5 mL sterile salines and mixes
It is even that bacteria suspension is made, take 1mL bacteria suspensions and 9 mL pH 3.0 simulated gastric fluid to mix, shake up, be placed in constant temperature oscillator and cultivate
(37 DEG C, 100r/min), and sampled respectively in 0 h and 3 h, it is coated with 37 DEG C of 48 h of culture with MRS agar mediums.Use flat board
Counting method determines viable count, calculates its survival rate (%)：Survival rate (%)=3h viable count/0h viable count × 100%.
（2）Probiotics is resistant to the measure of various concentrations cholate
The mL of strain 5 activated is inoculated in containing 0.0% bovine bile respectively by 2% inoculum concentration (100 μ L) with liquid-transfering gun（It is i.e. empty
The MRS-THIO culture mediums of 0.05 % in vain), 0.1 %, 0. 2 % and 0. 3 % bovine biles (W/V) (Jia 0.2% in MRS culture mediums
Sodium thioglycolate).In constant temperature oscillator after 37 DEG C of 24 h of culture, using blank cultures as control (nonvaccinated MRS-
THIO culture mediums), the OD values of above-mentioned various concentrations culture medium are determined respectively, calculate tolerance of the bacterial strain to cholate.Cholate is resistant to
OD value × 100% of OD values/blank cultures of power=culture medium containing cholate.
Lactic acid bacteria is a part for normal bowel bacterium, as one of most important condition of probiotics, and being can be before stomach and small intestine
Section survival, the bacterial strain being screened should have the characteristic grown under enough acid resistance and sour environment.
Lactobacillus fermentum HY01The selection result it is as shown in table 1 below：
Table 1
The selection result shows,Lactobacillus fermentumHY01 bacterial strains are resistant to pH3.0 environment, and survival rate is
104.60±6.50%。
It will be contacted by the thalline survived after stomach with the cholate in small intestine, this experiment is resistivity of the lactic acid bacteria to cholate
For a selection standard as potential probiotics.Acid resistance is preferableLactobacillus fermentumHY01 with
The increase of gallbladder salinity, growth efficiency is gradually reduced, and the growth efficiency under 0.3% gallbladder salinity remains at more than 20%.
Us are tested more than, and to have filtered out acid resistance, bile tolerance ability preferableLactobacillus fermentumHY01, the bacterial strain is preserved in China typical culture collection center (CCTCC) on December 29th, 2015, protects
Hide numbering CCTCCM2015792.
Embodiment 3：Activated carbon inducing constipation is tested
Forage feed based on 40 female KM mouse, the g of body weight 20 ± 2, rearing conditions, 12 h light and shades alternating（9：00~
21：00 illumination）, temperature is 25 ± 2 DEG C.After raising 5 days, be randomly divided into 4 groups, i.e. normal group, it is constipation control group, low dense
Degree group（108CFU/kg·BW）With high concentration group（109CFU/kg·BW）, every group 10.It is totally 10 days experimental period, during which each
Free water is with ingesting all the time for group mouse, and experiment starts first 7 days, in addition to normal group and constipation control group, various concentrations treatment group
Gavage respective concentration bacterium solution, gavage amount is determined according to mouse weight.Afterwards, in addition to normal group, Constipation Model group, low concentration group and
1 10 g/100 mL of the daily gavage of all mouse of high concentration group 0 DEG C of activated carbon frozen water, every mL of mouse stomach 0.2,
Continuous gavage 3 days, until last 1 day of experiment, all h of mouse fasting 24.After end, every mL of mouse stomach 0.2 work
Property charcoal frozen water, every group of mouse take 5 be served only for determining first discharge melena the time required to, remaining mouse stomach activated carbon frozen water 30
Dissection is put to death after min, for small intestine activated carbon Promoting Experiment.During experiment, a small amount of mouse fresh excreta is collected daily, for commenting
The change of valency stool in mice water content, specific method is：The fresh excreta being collected into is placed in 105 DEG C of air dry ovens and dried
To constant weight, the moisture content in stool in mice is drawn according to the weight of the change of quality before and after constant weight and fresh excreta.
The time of the dejecta moisture of experimental period Nei Ge group mouse and first discharge melena is as shown in Figure 3 and Figure 4.
Each group dejecta moisture no significant difference is tested before modeling.After each group gavage activated carbon frozen water is tested（Normal group
Except）, the dejecta moisture of model group mouse is decreased obviously, and difference also occurs in the dejecta moisture of mouse in high and low concentration group
The reduction of degree, but the excrement water content of high and low concentration group is above model group, wherein, the mice with constipation of high concentration treatment group
Dejecta moisture highest, illustrates that high and low concentration group can improve the moisture content in mice with constipation excrement, and high concentration in various degree
The effect that group improves mice with constipation dejecta moisture is more preferable.
The time that normal group mouse discharges melena for the first time is most short, and the model group of activated carbon frozen water induction discharges melena for the first time
Time it is most long, there is significant difference in the two.The time that high and low concentration group discharges melena for the first time is all remarkably higher than normal group, but
Substantially less than model group, and high concentration group discharges melena time substantially less than low concentration group for the first time.ExplanationLactobacillus fermentumThe high and low concentration group processing of HY01 can shorten the time that mice with constipation discharges melena for the first time in various degree, have
Promote the effect of mice with constipation defecation.
Embodiment 4：Small intestine activated carbon propulsion rate is tested
Mouse is taken off after cervical vertebra puts to death and dissect, on clip from pylorus, under to caecum whole small intestine, straight line is pulled into measure
Its length, represents total small intestinal length（cm）；In addition continue to measure the distance from pylorus activated carbon distalmost end into small intestine, represent to live
Property charcoal promote length（cm）.Propulsion rate is calculated as follows.Propulsion rate (%)=(activated carbon advance distance)/(total small intestinal length)
×100.Total small intestinal length, the Intestinal propulsive rate of experimental period Nei Ge group mouse are as shown in Figure 5.
Normal group small intestine activated carbon propulsion rate is maximum, and model group small intestine activated carbon propulsion rate is minimum, and the two exists notable
Difference, illustrates the success of activated carbon frozen water inducing constipation model.The small intestine activated carbon propulsion rate of high and low concentration group is significantly higher than model
Group, and high concentration activated carbon propulsion rate is higher than low concentration group.Its middle and high concentration group is with normal group without significant difference, explanationLactobacillus fermentumHY01 processing can promote intestines peristalsis, improve the effect of small intestine activated carbon propulsion rate.
Embodiment 5：Mouse small intestine fine hair pathological observation
Mouse is taken off after cervical vertebra execution, is taken small intestine to be soaked with 10% formalin solution, is fixed, and does conventional H E sections.Experiment periods
Interior each group mouse Colon pathological observation is as shown in Figure 6.
The uniform neat, not damaged of the intestinal villi of normal group mouse, and the fracture of model group intestinal villi, atrophy equivalent damage ten
Divide serious, and pass throughLactobacillus fermentumThe mice with constipation of HY01 processing, mice with constipation intestinal villi damage journey
Degree is eased, explanationLactobacillus fermentumHY01 processing can improve activated carbon inducing constipation mouse small intestine
The damage of fine hair, has facilitation to Constipation.
Embodiment 6：The measure of each factor level in mice serum
Rat aorta blood is taken, at 4 DEG C, 3000 r/min centrifugation 10min take upper serum.According to motilin（MTL）, gastrin
（GT）, Endothelin（ET）, growth hormone release inhibiting hormone（SST）, serum acetylcholinesterase（AchE）, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2（SP）And vasoactive intestinal peptide
（VIP）The method of test agent box specification determines above-mentioned factor level in serum.
Each factor level is as shown in Figure 7 in experiment periods Nei Ge groups mice serum.
Relative to constipation control group,Lactobacillus fermentumHY01 can significantly improve MTL in serum,
SST level in GT, ET, AchE, SP, VIP level and reduction serum, soLactobacillus fermentum HY01
There is facilitation to prevention colitis.
Application Example 1：Using lactobacillus fermenti (Lactobacillus fermentum) HY01 manufacture lactic acid bacteria milks
Beverage
First, the lactobacillus fermenti (Lactobacillus fermentum) HY01 original strains at -80 DEG C of temperature with
Magnetic bead form is preserved, or is saved backup at 4 DEG C of temperature in the form of being freeze-dried bacterium powder；
It is then possible to using two methods prepare the present invention lactobacillus fermenti (Lactobacillus fermentum) HY01
Working stock culture：
First method be by above-mentioned lactobacillus fermenti (Lactobacillus fermentum) HY01 original strains are inoculated in
12%（Weight）In 110 DEG C of skimmed milks for sterilizing 10 min, 14 ~ 16 h are cultivated under the conditions of 37 DEG C to curdled milk, continuous culture
Activated for two generations, as mother culture；The mother culture is pressed 3 ~ 5%（Volume）It is inoculated in sterile milk, 14 ~ 16 h of culture are extremely
Curdled milk, the now viable count about 10 in the curdled milk9 Cfu/mL, obtains described working stock culture, can be directly by this work
Leavening is added in food, or with can the commercial fermentation agent such as lactobacillus bulgaricus for preparing acidified milk of symbiosis and thermophilic
Streptococcus, which is used together, prepares acidified milk;
Second method be by above-mentioned lactobacillus fermenti (Lactobacillus fermentum) HY01 original strains are inoculated in
In MRS fluid nutrient mediums, 12 ~ 16 h of culture are activated under the conditions of 37 DEG C, two generations of continuous activation, then by activation culture
Thing presses 2 ~ 4%（Volume）It is inoculated in MRS culture mediums, cultivates 16 ~ 18 h, 3000 r/min centrifuges 15 min under the conditions of 4 DEG C,
Supernatant is removed, cell precipitation is obtained, suspension is made with a certain amount of sterile absorbent breast in precipitation, working stock culture is obtained standby
With.
Then, raw milk heats the sterilization 20 min or s of high temperature thermal sterilization 2 at 140 DEG C at 95 DEG C, then cools down
To 4 DEG C, add foregoing lactobacillus fermenti (Lactobacillus fermentum) HY01 working stock cultures, make
Its concentration reaches 106 More than cfu/ml, 4 DEG C it is stored refrigerated i.e. obtain containing lactobacillus fermenti (Lactobacillus fermentum) HY01 viable bacterias lactic acid bacteria milk beverage.
Raw milk described in the present embodiment is defatted milk；
Described MRS fluid nutrient mediums are known to those skilled in the art, be Suo Laibao companies sale be used for breast
The culture medium of bacillus culture；
Described heating sterilization is the TW10D1000 type pipes for example sold using Shanghai Triowin Automation Machinery Limited
What formula sterilization machine was carried out；
Described high temperature thermal sterilization is the YC-104 plate-types for example sold using Beijing Yong Chuanjiaye plant equipment Co., Ltd
What superhigh temperature sterilization machine was carried out.
Application Example 2：Using lactobacillus fermenti (Lactobacillus fermentum) HY01 manufacture milk powder
Raw milk is heated to the sterilization 20 min or s of high temperature thermal sterilization 2 at 140 DEG C at 95 DEG C, 37 DEG C are then cooled to,
Again with the 4% of raw milk volume connect bacterium amount inoculation Application Example 1 described in lactobacillus fermenti (Lactobacillus fermentum) HY01 working stock cultures, then 16 h that ferment at 37 DEG C, obtain lactobacillus fermenti (Lactobacillus fermentum) HY01 acidified milks；Then described lactobacillus fermenti (Lactobacillus fermentum) HY01 fermentations
Breast 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 containing lactobacillus fermenti
(Lactobacillus fermentum) HY01 milk powder.
Raw milk described in the present embodiment is fresh milk；
Described homogeneous is that the small-sized homogenizers of GJB500-40 for example sold using Changzhou homogeneous Machinery Co., Ltd. are carried out
's；
Described concentration is that the vacuum concentration pan for example sold using Shanghai Wei Zhou light industry and machinery Co., Ltd is carried out；
Described spray drying is that the experiment type spray drier for example sold using Shanghai Triowin Tech. Co., Ltd. is carried out.
Application Example 3:Using lactobacillus fermenti (Lactobacillus fermentum) HY01 manufacture capsule products
By raw milk in 140 DEG C of s of high temperature thermal sterilization 2,37 DEG C are subsequently cooled to, bacterium amount inoculation is connect with the 4% of raw milk volume
Described in Application Example 1 lactobacillus fermenti (Lactobacillus fermentum) HY01 working stock cultures, at 37 DEG C
16 h of lower fermentation, obtain lactobacillus fermenti (Lactobacillus fermentum) HY01 acidified milks.By lactobacillus fermenti
(Lactobacillus fermentum) HY01 acidified milks are with 1: 3（V∶V）Homogeneous in the raw milk after sterilizing is added, through vacuum
Milk powder is obtained after concentration, spray drying treatment, obtained milk powder is attached in capsule capsule product is made.
Raw milk described in the present embodiment is recovery milk.
Application Example 4：Using lactobacillus fermenti (Lactobacillus fermentum) HY01 prepares acidified milk
According to above-mentioned lactic acid bacteria milk beverage preparation method prepare, simply raw milk heat at 95 DEG C sterilize 20 min or
The s of high temperature thermal sterilization 2 at 140 DEG C, is then cooled to 37 DEG C, according still further to 3 ~ 5%（Volume）Add the hair described in Application Example 1
Kefir milk bacillus (Lactobacillus fermentum) HY01 working stock cultures, add 3 ~ 5%（Volume）Can symbiosis preparation
Acidified milk commodity leavening, mixed fungus fermentation, to titratable acidity in terms of lactic acid 0.6 ~ 0.7%, is subsequently cooled at 37 DEG C after mixing
4 DEG C, then carry out the acidified milk described in stored refrigerated obtain.
Raw milk described in the present embodiment is the mixture of defatted milk and recovery milk；
Described commodity leavening is preferably lactobacillus bulgaricus or streptococcus thermophilus.
Embodiment described above is only the preferred embodiment to absolutely prove the present invention and being lifted, protection model of the invention
Enclose not limited to this.Equivalent substitute or conversion that those skilled in the art are made on the basis of the present invention, in the present invention
Protection domain within.Protection scope of the present invention is defined by claims.
Claims (10)
1. a kind of lactobacillus fermenti (Lactobacillus fermentum) HY01, it is characterised in that the lactobacillus fermenti in
On December 29th, 2015 is preserved in China typical culture collection center (CCTCC), and deposit number is CCTCCM2015792.
2. lactobacillus fermenti HY01 as claimed in claim 1, it is characterised in that the biological characteristics of the lactobacillus fermenti is such as
Under：
1）Morphological feature：Thalline feature, in Gram-positive, cell is shaft-like, no gemma, and two ends are circular；Colony characteristicses,
Obvious bacterium colony is formed on MRS solid mediums, circular, neat in edge, milky, bacterium colony projection, surface wettability is smooth, no
Chromogenesis；
2）Feature is resistant in vivo:It is 104.60 ± 6.50% that the survival rate after 3 h is handled in pH 3.0 simulated gastric fluid；
Growth efficiency under 0.3% gallbladder salinity is 21.10 ± 0.24 without cholate culture.
3. lactobacillus fermenti HY01 as claimed in claim 1 or 2 is in the hair for preparing regulation function of intestinal canal or/and Constipation
Purposes in ferment food.
4. lactobacillus fermenti HY01 as claimed in claim 3 purposes, it is characterised in that the fermented food is to contain fermentation
One or more in lactic acid bacteria milk beverage, milk powder, capsule product and the acidified milk of lactobacillus.
5. lactobacillus fermenti HY01 as claimed in claim 4 purposes, it is characterised in that the lactic acid bacteria milk beverage be according to
What following step was prepared：
Raw milk heats sterilization 15 ~ 30 min or s of high temperature thermal sterilization 2 ~ 5 at 125 ~ 140 DEG C at 95 ~ 100 DEG C, is cooled to
4 DEG C, the working stock culture containing the lactobacillus fermenti is added, its concentration is reached 106 More than cfu/mL, is protected in 2 ~ 8 DEG C of refrigerations
Deposit.
6. lactobacillus fermenti HY01 as claimed in claim 4 purposes, it is characterised in that the milk powder is as steps described below
Prepare：
Raw milk heats sterilization 15 ~ 30 min or high temperature thermal sterilization 2 ~ 5 s, Ran Houleng at 125 ~ 140 DEG C at 95 ~ 100 DEG C
But to 37 DEG C, then with the 4% of raw milk volume working stock culture of the bacterium amount inoculation containing the lactobacillus fermenti is connect, at 35 ~ 40 DEG C
Ferment 16 h, obtains lactobacillus fermenti acidified milk；Raw milk after the lactobacillus fermenti acidified milk and sterilizing is according to 1:3（V:
V）Proportioning is added, and carries out homogeneous, is concentrated in vacuo, spray drying obtains the milk powder containing lactobacillus fermenti.
7. lactobacillus fermenti HY01 as claimed in claim 4 purposes, it is characterised in that the capsule product is according to following
What step was prepared：
By raw milk in 125 ~ 140 DEG C of s of high temperature thermal sterilization 2 ~ 5,35 ~ 40 DEG C are subsequently cooled to, is connect with the 4% of raw milk volume
Working stock culture of the bacterium amount inoculation containing the lactobacillus fermenti, ferment 15 ~ 20 h at 35 ~ 40 DEG C, obtains lactobacillus fermenti hair
Kefir milk；By the raw milk after the lactobacillus fermenti acidified milk and sterilizing according to 1:3~1:5（V:V））Proportioning is added, homogeneous,
Milk powder is obtained after vacuum concentration, spray drying treatment, the capsule product containing lactobacillus fermenti will be obtained.
8. lactobacillus fermenti HY01 as claimed in claim 4 purposes, it is characterised in that the acidified milk is according to following steps
Suddenly prepare：
Raw milk heats sterilization 15 ~ 30 min or high temperature thermal sterilization 2 ~ 5 s, Ran Houleng at 125 ~ 140 DEG C at 95 ~ 100 DEG C
But to 35 ~ 40 DEG C, according still further to 3 ~ 5%（Volume）The working stock culture containing lactobacillus fermenti is added, 3 ~ 5% are added（Volume）Can
Being used for of symbiosis prepares the leavening of fermented dairy product, after mixing at 35 ~ 40 DEG C mixed fungus fermentation to titratable acidity in terms of lactic acid
0.6 ~ 0.7%, it is subsequently cooled to 2 ~ 8 DEG C, then carry out stored refrigerated obtaining the acidified milk containing lactobacillus fermenti.
9. the purposes of the lactobacillus fermenti HY01 as any one of claim 5~8, it is characterised in that described raw material
One or more of the breast in defatted milk, fresh milk and recovery milk.
10. the purposes of the lactobacillus fermenti HY01 as any one of claim 5~8, it is characterised in that described containing fermentation
The preparation method of the working stock culture of lactobacillus comprises the following steps：
The original strain of lactobacillus fermenti is preserved at -80 DEG C of temperature in magnetic bead form, or to freeze at 2 ~ 8 DEG C of temperature
The form for drying bacterium powder is saved backup;
The original strain of the lactobacillus fermenti is inoculated in 12%（Weight）Skimmed milk in, 110 DEG C sterilizing 10 min, 37
14 ~ 16 h are cultivated under the conditions of DEG C to curdled milk, continuous culture two generations of activation, as mother culture；The mother culture is pressed 3 ~ 5%
（Volume）It is inoculated in sterile milk, 14 ~ 16 h are to curdled milk for culture, the viable count about 10 into the curdled milk9 Cfu/mL, obtains work
Leavening；
Or the original strain of the lactobacillus fermenti is inoculated in MRS fluid nutrient mediums, 12 ~ 16h is cultivated under the conditions of 37 DEG C
Activated, two generations of continuous activation, activation culture thing is then pressed 2 ~ 4%（Volume）It is inoculated in MRS culture mediums, culture 16 ~ 18
H, 3000 r/min centrifuge 15 min under the conditions of 4 DEG C, remove supernatant, obtain cell precipitation, by precipitation sterile absorbent breast
Suspension is made, working stock culture is obtained.
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| CN109628346A (en) * | 2018-12-29 | 2019-04-16 | 重庆第二师范学院 | Lactobacillus fermenti CQPC04 and its application in the food that preparation improves constipation |
| WO2020259564A1 (en) * | 2019-06-28 | 2020-12-30 | 江南大学 | Multifunctional lactobacillus capable of relieving pfoa toxic effects and application thereof |
| CN113234612A (en) * | 2021-02-05 | 2021-08-10 | 重庆第二师范学院 | Lactobacillus fermentum ZS40 having preventive effect on colitis |
| CN113508907A (en) * | 2021-04-28 | 2021-10-19 | 汉臣氏（沈阳）儿童制品有限公司 | Application of heat-resistant lactobacillus fermentum in preparation of defecation promoting food or medicine |
| CN114015630A (en) * | 2021-12-21 | 2022-02-08 | 重庆市天友乳业股份有限公司 | Lactobacillus fermentum TY-G04 with effect of relaxing bowel and application thereof |
| CN116478256A (en) * | 2021-01-19 | 2023-07-25 | 西南大学 | Bacteriocin produced by lactobacillus fermentum and application thereof |
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| CN113234612A (en) * | 2021-02-05 | 2021-08-10 | 重庆第二师范学院 | Lactobacillus fermentum ZS40 having preventive effect on colitis |
| CN113508907A (en) * | 2021-04-28 | 2021-10-19 | 汉臣氏（沈阳）儿童制品有限公司 | Application of heat-resistant lactobacillus fermentum in preparation of defecation promoting food or medicine |
| CN114015630A (en) * | 2021-12-21 | 2022-02-08 | 重庆市天友乳业股份有限公司 | Lactobacillus fermentum TY-G04 with effect of relaxing bowel and application thereof |
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Application publication date: 20171003 |