CN106754619A - It is a kind of that the method that traditional Chinese medicinal components promote growth of probiotics is added in grain culture medium - Google Patents
It is a kind of that the method that traditional Chinese medicinal components promote growth of probiotics is added in grain culture medium Download PDFInfo
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Abstract
The invention provides a kind of method for promoting growth of probiotics and improving number of live bacteria of probiotics.Methods described is fermented during probiotics to be inoculated into the MRS lactic acid bacteria culturing mediums of the improvement for being added with different traditional Chinese medicinal components or blended grain swelling powder culture medium.5g mulberry-leaf extracts or Cordyceps sinensis polysaccharide or plurality of Chinese mixture are added in every 1 liter of culture medium.Probiotics is Lactobacillus rhamnosus or various mixing probiotics.The method of the invention can significantly improve the fermenting speed of probiotics in zymotic fluid and the viable count of probiotics；Addition mulberry-leaf extract or Cordyceps sinensis polysaccharide ferment 12 hours after viable count up to 109 CFU/mL；Viable count is up to 4.1 × 10 after the grain culture medium fermentation of the traditional Chinese medicinal components of addition8CFU/mL.The experimental technique using number of live bacteria of probiotics in flow cytometer detection muddiness fermentation system is established simultaneously.
Description
Technical field
The present invention relates to field of microbial fermentation, specifically, it is related to one kind that traditional Chinese medicinal components are added in grain culture medium
The method for promoting growth of probiotics.
Background technology
Probiotics is that a class is colonized in human body intestinal canal, reproductive system, can produce definite health efficacy so as to improve host
Microecological balance, the general name of the microorganism of the work played an important role to health is widely used in food, health products and medicine
In production.LGG（ATCC 53103）It is a kind of Lactobacillus rhamnosus of patent, early eighties are by two American professors from health
It is isolated in human body, is also one of probiotics of current whole world most study.Substantial amounts of experiment in vitro, zoopery, human body
Experiment and epidemiological investigation fully demonstrate the security of LGG.Feature aspect, LGG have significantly balance and change
Kind gut flora, prevention and treatment diarrhoea, improve immunity of organisms, it is pre- it is hypo-allergenic, with reference to and exclude the work(such as toxin, pre- anti-caries
Energy.Additionally, used as a kind of probiotics, LGG also has some advantages of itself：Such as stomach juice-resistant, resistance to bile, can colonize in human body
Enteron aisle, final products（Yoghourt, milk etc.）Viable count keeps constant grade (Wang Shibin, Liu Jinjie, Qiao Lan, Cheng Lin, benefit in shelf-life
The research of raw bacterium LGG, Guangzhou Food Industry science and technology [J], 2003, s1,55-58).Research finds that probiotics is in human body intestinal canal
Quantity health care and therapeutic efficiency played to probiotics play decisive role.Except probiotics breeds in vivo, processed
Ensure outside the factor such as its survival and holding bioactivity with storage period in journey, enough viable counts are Probiotic supplement products
Play the prerequisite of its validity.
Vegetalitas cereal materialses be culture medium, as fermentation substrate, cereal not only can for the growth of probiotics is provided must
Carbon source and nitrogen source, the reduction fermentation costs wanted；Cereal after by fermentation can significantly improve its nutritive value and functional characteristic,
The fragrance ingredients such as lactic acid, ethyl acetate, the acetaldehyde produced after fermentation can improve the local flavor and fragrance of cereal products.Zhao Lina with
10% oat malt-oatmeal（Oat malt powder：Oatmeal=2:1）Skimmed milk powder with 1% is culture medium, and 9 h of fermentation can make plant
The viable count of thing lactobacillus and lactobacillus acidophilus reaches 8.75 Log CFU/mL（Zhao Lina, the development of oat malt probiotic beverage
[D], Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology, 2015）.Zhou little Li have studied seven plants of growth feelings for examination lactic acid bacteria in Oats paste (a kind of food) matrix
Condition, as a result shows that seven plants of bacterium can grow in oat.And after malt is added, Lactobacillus helveticus grows 10 in cereal matrix
Viable count after hour is 2.29 × 108 cfu/ml（Zhou little Li, lactic acid bacteria growth characteristics research [D], the south of the River in oat matrix
University, 2012）.Be inoculated into corn juice for saccharomyces cerevisiae, lactobacillus acidophilus, lactobacillus bulgaricus by white horse with a black mane extra extra, finds this
Three plants of bacterium can carry out breeding fermentation by fermentation substrate of corn（Luo Chaochao, Lu Zhi bravely wait several probiotics fermention cereal beverage systems of
Make technical study Northeast Agricultural Universities journals 2011.42 (5): 127~129）.There is a kind of cereal culture of patent literature
The method of base fermentation propagation probiotics, is that bifidobacterium bifidum, short double is cultivated after the treatment such as enzymolysis with corn, oat, soybean
Discrimination bacillus, lactobacillus acidophilus, Lactobacillus casei and lactobacillus bulgaricus, 38 DEG C ferment 22 hours, and zymotic fluid pH can drop to
4, and the Bifidobacterium of propagation can be obtained（Chinese Patent Application No.：200810015537.X）.Have patent literature a kind of with
The method of waxy corn culture medium fermentation probiotics, such as fermentation tank after going kind of skin to be beaten waxy corn, by 1:1 inoculating lactic acid strain
28 DEG C of L041 and L042 high-efficiency ferments slow fermentation 70-80 h, fermentation terminal pH are 2.9-3.3（Chinese Patent Application No.：
200510047702.6）.
Cereal can provide growth necessary nutriment as probiotics fermention matrix for it, Effective multiplication probiotics, but
These grain culture mediums were both needed to carry out the pretreatment of complexity before fermented and cultured is carried out, including went kind of a skin, boiling, enzymolysis etc. multiple
Miscellaneous handling process.Corn expanding powder is obtained through high temperature, high pressure swelling, and the damage of nutrition can be to greatest extent reduced in maturing process
Consumption.Can make the starch granules in cereal expanded through the HTHP of expanded generation, high shear force and elite, open starch molecule
Connect, increase the surface area of chyme particle, improving the digestibility of starch；Simultaneously it is expanded make protein peptide chain be cracked into peptide and
Amino acid, greatly improves the absorptivity of protein.Can effectively simplify zymotechnique using puffed cereal, shorten fermentation period,
So as to reduce fermentation costs.
The current research direction for improving probiotics viable bacteria sum be concentrated mainly on zymotechnique optimization and compound bacteria it is highly dense
Degree fermentation.The viable count of probiotics can be not only improved to benefit materials are added in fermentation medium, acidified milk can also have concurrently beneficial
The function of material.Be added to the probiotics such as inulin, soyabean oligosaccharides and FOS in Yoghourt fermented by Lin Ying etc., as a result
Show, probiotic has a significant impact to the propagation of Bifidobacterium, wherein most have to the propagation of Bifidobacterium with soyabean oligosaccharides again
Effect, number of viable is from 0.49 × 108Cfu/ml Effective multiplications are to 1.2 × 108cfu/ml（Lin Ying, probiotics health care in beam generation
Research [J] the Guangzhou Food Industries science and technology that ferment agent for sour milk is prepared and probiotic influences on thalline quantity, 2003:41-43）.Have
A kind of method for improving number of live bacteria of probiotics of patent literature, is that Cordceps militaris extract is added into conventional lactic acid bacteria culture
In base, viable count of lactobacillus from 1.5 × 10 can be made8Cfu/ml brings up to 1.3 × 109 Cfu/ml (Chinese Patent Application No.：
200910038553.5).Liu Chengguo etc. probiotics strain is inoculated into be added with purple potato slurry fermentation cow's milk matrix in sent out
Ferment, can significantly improve probiotics fermention breast in probiotics viable count, obtain probiotics fermention breast maximum viable count from
109-1010 Cfu/ml brings up to 1010-1012 cfu/ml.Soybean protein hydrolyate and yeast extract complexing agent pair are added in skimmed milk
Streptococcus thermophilus and lactobacillus bulgaricus growth have significant facilitation, and addition propagation complexing agent can make lactic acid bacterium number
The 1-2 order of magnitude is improved than degreasing milk medium.(the soybean protein hydrolyates such as white phoenix tail feather, Qu Lingtong, Zhang Bolin promote lactic acid bacteria
Breed research [J] the food industry science and technology of fermentation, 2012,33 (16):209-212).Li Hui wait quietly have studied the addition Radix Astragali it
The change of the upgrowth situation of lactobacillus acidophilus afterwards, they are added to the Radix Astragali extractive solution for preparing in the MRS culture mediums of improvement, grind
Study carefully the influence that various concentrations Radix Astragali extractive solution grows to lactobacillus acidophilus, as a result show：When addition Radix Astragali extractive solution concentration is
During 0.032g/mL, there is obvious facilitation to lactobacillus acidophilus growth, 48h OD are increased to 0.8 from 0.6（Li Huijing, acidophilus
Lactobacillus and Radix Astragali correlation research [D], Anhui Polytechnic University, 2013）.Zhao Yuping etc. research various concentrations licorice polysaccharide and
Influence of the Codonopsis pilosula polysaccharide to Lactobacillus rhamnosus, finds the proliferative effect of the Codonopsis pilosula polysaccharide to Lactobacillus rhamnosus of 1.5% (m/v)
Maximum, its viable count maximum can reach 3.29 × 109Cfu/mL, improves 3 times or so, and it can be made to shift to an earlier date 2~3 h and reach
Acid amount is produced to maximum, shortens fermentation time, preferably improve its acidproof, bile tolerance and resistant to elevated temperatures ability (Zhao Yuping, Jia Jian
Ripple, influence [J] of the herbal polysaccharide to Lactobacillus rhamnosus property, food science and technology, 200,8, P13-15).The research such as Xu Haiyan is low
The influence that xylan grows to lactobacillus, Bifidobacterium and gut flora.The xylo-oligosaccharide of result 1% is to Lactobacillus plantarum, sandlwood
Sugared lactobacillus, bifidobacterium longum and bifidobacterium adolescentis have obvious proliferation function.（Xu Haiyan, Xin Guoqin, Cao Yinsheng, Wu Xiang
Jade, Wang Mengjuan, Gu Wei, influence of the xylo-oligosaccharide to probiotics and people's gut flora, the phase P500- of study of pharmacy 2013 the 9th
503）
Current proliferation of probiotics technology achieves certain achievement, is a kind of efficient, letter to benefit materials are added in fermentation medium
The method of the viable count of raising probiotics just.Fermenting and producing cost can be not only reduced, even hair can be shortened in some situations
The ferment cycle.The research of the additive for possessing specific function but more is not also a lot.
Many foods are medicine, and absolute line of demarcation is had no between them.Chinese yam, property is sweet flat, reinforced the kidney with strengthening the spleen and tonifying the lung,
Effect of supplementing qi and nourishing yin controlling nocturnal emission with astringent drugs.The root of kudzu vine, property is pungent sweet and cool, has both evacuated the perverse trend of flesh natural fibre line of meat channels and collaterals, inspires again on the gas of the clear sun of taste
OK, promote the production of body fluid to quench thirst, Shengyang Zhixie.Radix polygonati officinalis, it is in sweet flavor and mend, it is flat and few inclined, enter lung channel lung-yin nourishing and can moisten the lung and relieve the cough；Enter stomach
Nourishing stomach-YIN and can promote the production of body fluid to quench thirst.Navy bean, sweet tepor, can invigorating the spleen for eliminating dampness, and can dampness elimination and in and relieve summer heat, moreover it is possible to relieve the effect of alcohol
Poison.The Radix Astragali, sweet temperature rise is mended, main with righting gas, and can remove water heresy, has both been apt to bowl spares gas, has been risen and lift clear sun, tonifying lung gas, benefit is apt to again and defends solid
Table.The root of large-flowered skullcap, bitter cold is let out clearly and dry, the fire of kind clearing lung-heat and large intestine, except upper dampness-heat in middle jiao；It is simultaneous enter blood system and cooling blood and hemostasis.
Grape seed extract（GSE）, belong to polyphenols, also known as OPC.Since the eighties in 20th century, people are to it
The research such as a large amount of biochemistry, pharmacological activity is carried out.Result shows that GSE is the most strong antioxidant of Scavenging ability, is so far
The most strong effective oxygen free radical scavenger and lipid peroxidation inhibitor found untill the present, can prevent and treat more than 80 kinds because of freedom
The disease that base causes, including heart disease, cancer and illness etc. arthritis（Zhao Chaoying, the small graceful grape seed extracts original cyanine of Yao
Alimentary health-care function (summary) [J] Chinese food health magazines of element, 2000,12 (6):38-41.）.Because its resource is rich
The features such as richness, determined curative effect, Small side effects, favor is enjoyed in Europe, Japan, in recent years fashionable U.S. again.Mulberry leaf contain various
Active chemical componentses, with hypoglycemic, reducing blood lipid, anti-inflammatory, anti-aging, it is antiviral the effects such as（Ou Yangzhen, Chen Jun mulberry leaf
Chemical composition and its Advance on Pharmacological Activities [J] Journal of Jiangsu University natural science editions, 2003,24 (6):39-44.）.
Cordyceps sinensis has had history very long as the traditional rare medicinal herbs of China, Cordyceps sinensis polysaccharide as Chinese caterpillar fungus main active
One of, with various pharmacological activity.Artifical fermentation cordyceps polysaccharide has antitumor, hypoglycemic, anti-oxidant and raising immunity energy
Various pharmacological activity such as power.But the research by the Cordyceps sinensis polysaccharide of probiotics fermention is still more rare.
But, currently there is the research of proliferation function to probiotics still without document report on traditional Chinese medicinal components, in addition
The probiotics of medicine component also has no report.
The content of the invention
In order to solve the problems, such as prior art, it is an object of the invention to provide a kind of side for improving number of live bacteria of probiotics
Method.
In order to realize the object of the invention, promote growth of probiotics the invention provides a kind of in vitro culture and improve probiotics
The method of viable count, methods described is：Probiotics strain is inoculated into and is added with the culture medium of traditional Chinese medicinal components solution, carry out benefit
The propagation fermentation of raw bacterium.
Preceding method concrete technical scheme is comprised the following steps：
（1）The preparation of traditional Chinese medicinal components solution：The various traditional Chinese medicinal components of precise are configured to the concentrated solution of traditional Chinese medicinal components, and stirring is to molten
Solution, 115 DEG C of 30 min of sterilizing；
（2）The configuration of enriched medium：By step（1）The traditional Chinese medicinal components concentrated solution for preparing adds to the modified MRS after sterilizing
In culture medium or grain culture medium；Prepare a control group for being not added with traditional Chinese medicinal components simultaneously
（3）The preparation of probiotics seed liquor：The probiotics strain that will be saved in, seed liquor is made after recovery, expansion；
（4）The Zengjing Granule of probiotics：By step（3）The probiotics seed liquor for obtaining, is inoculated into step（2）The increasing bacterium training for obtaining
Carry out increasing bacterium fermented and cultured in foster base.
（5）The measure of viable count：To preliminary fermentation culture medium zymotic fluid of the clarification without turbid phenomenons such as precipitations using conventional
OD600Optical density counting method.The process of fermentation determines a viable count since 0 h at interval of a period of time, is finally somebody's turn to do
The maximum viable count of probiotics.Meanwhile, flow cytometry is used, the muddy fermentation body to optical density detection can not be carried out
System carries out viable count detection.
More preferably, the step（1）In, the root of large-flowered skullcap, Chinese yam, the root of kudzu vine, radix polygonati officinalis, navy bean, grape pip, green tea, the Radix Astragali are with 100
The concentration of mg/mL is configured to the DMSO concentrated solutions of traditional Chinese medicinal components.DMSO solution can improve the dissolubility of traditional Chinese medicinal components, and by it
Ratio afterwards is added to Zengjing Granule system, and DMSO can't suppress the growing multiplication of probiotics.Mulberry-leaf extract, Cordyceps sinensis polysaccharide
（50%）The water concentrated solution of traditional Chinese medicinal components is configured to the concentration of 100 mg/mL.
More preferably, the step（2）In, step（1）Middle gained traditional Chinese medicinal components solution adds to increasing bacterium and ferments by certain volume
In culture medium, make the final concentration of mg/mL of 0.1 mg/mL to 5 of Chinese medicine.Described modified lactic acid bacteria culture medium（Modified MRS）Configuration
Well after 30 min that sterilized under 115 DEG C, 0.1 Mpa.Modified MRS formula is as follows：
| Composition | Consumption（g） |
| Peptone | 20.0 |
| Beef extract | 10.0 |
| Yeast extract | 5.0 |
| Diammonium hydrogen citrate [（NH4）2HC6H5O7] | 4.0 |
| Glucose (C6H12O6·H2O) | 20.0 |
| Tween-80 | 3.0 mL |
| Sodium acetate（CH3COONa·3H2O） | 5.0 |
| Dipotassium hydrogen phosphate（K2HPO4·3H2O） | 2.0 |
| Magnesium sulfate（MgSO4·7H2O） | 0.25 |
| Manganese sulfate（MnSO4·H2O） | 0.05 |
| Agar | 18.0 |
| Distilled water | 1 000 mL |
| pH | 7.0 |
Described grain culture medium formula is as follows：8 kind of 80~100 corn expanding powder of mesh（Oat, buckwheat, sorghum rice, maize,
The seed of Job's tears, mung bean, black soya bean, soya bean）Fermentation medium is configured to according to 8% total solid content, various grain component contents in culture medium
It is equal.Sterilize 25-30 min under 115 DEG C of the grain culture medium for having configured, 0.1 Mpa.
More preferably, the step（3）In, by probiotics（Lactobacillus plantarum, lactobacillus bulgaricus, Lactobacillus casei, excrement
Enterococcus, Lactobacillus salivarius, Lactobacillus rhamnosus, lactobacillus fermenti）Strain frozen stock solution, is added with the ratio of 1% (v/v) respectively
To in modified MRS culture medium, 37 DEG C of static gas wave refrigerators carry out the recovery of strain for 12 hours；Will be multiple in the ratio of 10%~20% (v/v)
Soviet Union's bacterium solution is inoculated into modified MRS culture medium, and 37 DEG C of static gas wave refrigerators carry out the preparation of probiotics seed liquor for 12 hours.
More preferably, by step 3) the probiotics seed liquor that obtains is inoculated into step 2 according to the ratio of 3% (v/v)) obtain
Zengjing Granule is carried out in enriched medium.When mixed fungus fermentation, the total addition level of various bacterium seed liquors is 3% (v/ of fermentation system
v).Zengjing Granule temperature is 37oC, the Zengjing Granule time is 16-24 h.
The present invention with probiotics as fermented bacterium, by adding in enriched medium the traditional Chinese medicinal components solution through processing,
Under preference temperature, the growth of probiotics and viable count during Zengjing Granule are significantly improved, so as to be prepared for after prebiotic
Bacterium probiotics provides a large amount of live probiotics thalline.In addition, appropriate traditional Chinese medicinal components are such as added in probiotics, not only
The growth of part probiotics in human gastrointestinal tract can be promoted, other health-care effects are may also operate as.The present invention is also comprehensively utilized
The traditional Chinese medicinal components resource of low cost, and effect of the traditional Chinese medicinal components in number of live bacteria of probiotics is improved is found that, and to part
The probiotics of kind has obvious cultivation effect.Work during fermented grain system can also be significantly improved by adding traditional Chinese medicinal components
Bacterium number, while increasing the nutrition composition of fermented grain product, increased the many-sided adjustment effect to health.
Brief description of the drawings
Fig. 1 is effect of the various concentrations mulberry-leaf extract to Lactobacillus rhamnosus in-vitro multiplication.
Fig. 2 is various concentrations Cordyceps sinensis polysaccharide（50%）Effect to Lactobacillus rhamnosus in-vitro multiplication.
Fig. 3 is the viable bacteria of probiotics in flow cytomery fermented grain system and dead bacterium number amount.
Fig. 4 is the proliferation function for adding traditional Chinese medicinal components to probiotics in fermented grain culture medium.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Embodiment 1
1) preparation of traditional Chinese medicinal components solution：By mulberry-leaf extract or Cordyceps sinensis polysaccharide（50%）Added with the concentration of 100 mg/mL respectively
Enter to be configured in distilled water the water concentrated solution of traditional Chinese medicinal components, stirring to dissolving, 115 DEG C of sterilizing 30min.
2) configuration of enriched medium：The traditional Chinese medicinal components solution that step (1) is prepared is added into improvement by finite concentration
In MRS culture mediums, make traditional Chinese medicinal components final concentration of respectively 0.5 mg/mL, 1 mg/mL, 5 mg/mL.Described improvement lactic acid
Bacterium culture medium（Modified MRS）Configuration is good after natural cooling after 30 min that sterilized under 115 DEG C, 0.1 Mpa, sterilizing.Formula is as follows：
| Composition | Consumption（g） |
| Peptone | 20.0 |
| Beef extract | 10.0 |
| Yeast extract | 5.0 |
| Diammonium hydrogen citrate [（NH4）2HC6H5O7] | 4.0 |
| Glucose (C6H12O6·H2O) | 20.0 |
| Tween-80 | 3.0 mL |
| Sodium acetate（CH3COONa·3H2O） | 5.0 |
| Dipotassium hydrogen phosphate（K2HPO4·3H2O） | 2.0 |
| Magnesium sulfate（MgSO4·7H2O） | 0.25 |
| Manganese sulfate（MnSO4·H2O） | 0.05 |
| Agar | 18.0 |
| Distilled water | 1 000 mL |
| pH | 7.0 |
3) preparation of probiotics seed liquor：The Lactobacillus rhamnosus strain that will be preserved, seed liquor is made after expanding through recovery.Will
Lactobacillus rhamnosus kind is added in modified MRS culture medium with the ratio of 1% (v/v), and 37 DEG C of static gas wave refrigerators carry out bacterium in 12 hours
The recovery planted；Recovery bacterium solution is inoculated into modified MRS culture medium in the ratio of 10% (v/v), 37 DEG C of static gas wave refrigerators 12 hours
Carry out the preparation of Lactobacillus rhamnosus seed liquor.
4) Zengjing Granule of probiotics：The probiotics seed liquor that step (3) is obtained is inoculated into the increasing that step (2) is obtained
Increasing bacterium is carried out in bacterium culture medium.Seed liquor adds to enriched medium according to the ratio of 3% (v/v).Zengjing Granule temperature is 37 DEG C,
The Zengjing Granule time is 16 h.
5）The measure of viable count：The process of fermentation since 0 h, using conventional 10 times of dilution plate Anaerobic culturel counting methods
A viable count is determined at interval of a period of time, the maximum viable count of the probiotics is finally obtained.
Embodiment 2
1) preparation of traditional Chinese medicinal components solution：The root of large-flowered skullcap, Chinese yam, the root of kudzu vine, radix polygonati officinalis, navy bean, grape pip, green tea, the Radix Astragali are added
In DMSO, stirring to dissolving, 115 DEG C of sterilizing 30min.The traditional Chinese medicinal components are configured to traditional Chinese medicinal components with the concentration of 100 mg/mL
DMSO concentrated solutions.
2) configuration of enriched medium：The traditional Chinese medicinal components solution that step (1) is prepared is added into cereal by finite concentration
In culture medium, make final concentration of 0.1 mg/mL of traditional Chinese medicinal components.Described grain culture medium collocation method is：By 8 kind 80~100
Purpose corn expanding powder（Oat, buckwheat, sorghum rice, maize, the seed of Job's tears, mung bean, black soya bean, soya bean）Configured according to 8% total solid content
Into fermentation medium, various grain component contents are equal wherein in grain culture medium.115 DEG C of the grain culture medium for having configured, 0.1
Sterilize 25-30 min under Mpa.
3) preparation of probiotics seed liquor：The probiotics strain that will be preserved（It is Lactobacillus plantarum, lactobacillus bulgaricus, dry
Lactobacillus paracasei, enterococcus faecalis, Lactobacillus salivarius, Lactobacillus rhamnosus, lactobacillus fermenti）, seed liquor is made after expanding through recovery.
Lactobacillus rhamnosus kind is added in modified MRS culture medium with the ratio of 1% (v/v), 37 DEG C of static gas wave refrigerators are carried out for 12 hours
The recovery of strain；Recovery bacterium solution is inoculated into modified MRS culture medium in the ratio of 10% (v/v), 37 DEG C of static gas wave refrigerators 12 are small
The preparation of Shi Jinhang probiotics bacterial seed liquors.Described modified lactic acid bacteria culture medium（Modified MRS）Configuration it is good after 115 DEG C, 0.1
Sterilize 30 min under Mpa, natural cooling after sterilizing.Formula is as follows：
| Composition | Consumption（g） |
| Peptone | 20.0 |
| Beef extract | 10.0 |
| Yeast extract | 5.0 |
| Diammonium hydrogen citrate [（NH4）2HC6H5O7] | 4.0 |
| Glucose (C6H12O6·H2O) | 20.0 |
| Tween-80 | 3.0 mL |
| Sodium acetate（CH3COONa·3H2O） | 5.0 |
| Dipotassium hydrogen phosphate（K2HPO4·3H2O） | 2.0 |
| Magnesium sulfate（MgSO4·7H2O） | 0.25 |
| Manganese sulfate（MnSO4·H2O） | 0.05 |
| Agar | 18.0 |
| Distilled water | 1 000 mL |
| pH | 7.0 |
4) Zengjing Granule of probiotics：The probiotics seed liquor that step (3) is obtained is inoculated into the increasing bacterium training that step (2) is obtained
Increasing bacterium is carried out in foster base.Seed liquor adds to enriched medium according to the ratio of 3% (v/v).Zengjing Granule temperature is 37 DEG C, increases bacterium
Incubation time is 20 h.
5）The measure of viable count：Using probiotics bacterium number in Flow cytometry method measure zymotic fluid, cereal hair is obtained
The viable count of probiotics and dead bacterium number in zymotic fluid.The concrete operations of Flow cytometry method are as described below：
A. preparation of samples, appropriate dilution is carried out by the zymotic fluid of taking-up, makes the quantity model of viable count in bacteria liquid sample to be detected
It is trapped among 5.5 × 105~6.5 × 105 CFU/mL.Fermentation broth sample after dilution is filtered through 300 mesh gauzes, is taken to be checked after dilution
The μ L of sample 200 were added in the EP pipes of 1.5 mL, in 37 DEG C of constant incubator warm bath 3 minutes.
B. sample dyeing, adds 2 μ L 100 × Sybr Green I and 2 μ L 100 × PI, 37 in the μ L of measuring samples 200
DEG C lucifuge is dyeed 10 minutes.Sybr Green I and PI (Propidium Iodide) are two kinds of common DNA fluorescent dyes.
Sybr Green I can penetrate the cell membrane of living cells and dead cell, and PI can not well penetrate the cell membrane of living cells.Profit
With Sybr Green I and PI stained bacterias, bacterium can be divided into three groups：The bacterium living of Sybr Green I (+) PI (-),
The dead bacterium of Sybr Green I (+) PI (+), and the damage bacterium between two groups.
C. C6 Accuri absolute countings, C6 Accuri flow cytometry analysis samples, threshold value is set to FSC-H and is more than
10000, flow velocity is selected at a slow speed, and sampling quantity is set to 60 μ L, carries out absolute counting detection.
D. with FL1-H as abscissa, FL2-A is ordinate, draws cell density figure.By analysis water-like density map, bacterium solution
And the bacterium solution after heating, the cell point group according to these samples, circle gating counting.
The maximum viable count of the probiotics cereal fermentation liquor obtained according to the present invention can reach 4.1 × 108 CFU/mL, and not
Add the maximum viable count 1.4 × 10 that the zymotic fluid of traditional Chinese medicinal components composition is obtained8 CFU/mL is compared, and is increased significantly.
Although above oneself is through the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. a kind of method for promoting growth of probiotics and improving number of live bacteria of probiotics, it is characterised in that methods described is：Will be prebiotic
Bacterium strain is inoculated into the culture medium for being added with traditional Chinese medicinal components, carries out fermented and cultured, using the method for cereal culture probiotics, and
The method for using traditional Chinese medicinal components to improve number of live bacteria of probiotics, it is characterised in that methods described is：This kind of use corn expanding powder enters
Row probiotic's culture, and integration of drinking and medicinal herbs traditional Chinese medicinal components are added, carry out fermented and cultured.
2. method according to claim 1, it is characterised in that the described method comprises the following steps:
1) preparation of traditional Chinese medicinal components solution：Using the distillation various traditional Chinese medicinal components of water dissolves, traditional Chinese medicinal components concentrated solution is made；
2) activation of prebiotic strain and the preparation of seed liquor；Recovery activation and expansion are carried out to the strain for preserving successively, is planted
Sub- liquid；
3) preparation of the culture medium of addition traditional Chinese medicinal components：It is combined as fermentation medium by the use of corn expanding powder；And to changing
Step 1 is added in good MRS culture mediums or corn expanding powder culture medium) the traditional Chinese medicinal components solution for preparing, is gone out successively
Bacterium, cooling；
4) fermented and cultured of probiotics：By step 2) probiotics that obtains is inoculated into step 3) training of addition traditional Chinese medicinal components that obtains
Fermented and cultured is carried out in foster base；
5) the number of live bacteria of probiotics detection of muddy fermentation system：Dyeed using Sybr Green I and PI, be with FL1-H
Abscissa, FL2-A is ordinate, draws cell density figure, and carry out number of bacteria statistics.
3. method according to claim 2, it is characterised in that step 2) described in probiotics be Lactobacillus rhamnosus or plant
Thing lactobacillus, lactobacillus bulgaricus, Lactobacillus casei, enterococcus faecalis, Lactobacillus salivarius, Lactobacillus rhamnosus, acidified milk bar
Bacterium etc. compare mixed bacteria liquid.
4. method according to claim 2, it is characterised in that step 3) described in the traditional Chinese medicinal components addition of culture medium be
Mass fraction 0.1%-0.5%（m/v）, 115oC sterilizing 30min, refrigerate standby.
5. method according to claim 4, it is characterised in that the step 3) in the MRS culture mediums configuration of improvement complete
After adjust pH, 115 in high-pressure sterilizing potoC sterilizing 30min, taking-up is cooled to 37oC or so.
6. method according to claim 2, it is characterised in that step 4) described in the inoculum concentration of probiotics recovery inoculation be
1%, the inoculum concentration of expansion is 10%-20%, and the inoculum concentration of fermentation is 3%.
7. method according to claim 6, it is characterised in that the step 4) in the fermentation temperature of fermentation be 37oC, fermentation
Time is 6h-14h.
8. the method that the method described in any one of claim 1-7 improves growth of probiotics and viable count.
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| CN109576203A (en) * | 2018-12-20 | 2019-04-05 | 吉林省浦生泰生物技术有限责任公司 | A kind of compound probiotic culture medium and preparation method thereof based on Chinese medicine additive |
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