CN101418270A - The Lactobacillus casei Zhang high-density cultivation method, use them to prepare the method for freeze-dried vaccine powder and resulting freeze-dried vaccine powder and uses thereof - Google Patents
The Lactobacillus casei Zhang high-density cultivation method, use them to prepare the method for freeze-dried vaccine powder and resulting freeze-dried vaccine powder and uses thereof Download PDFInfo
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Abstract
The present invention relates to a strain and have the high density fermentation technology that benefit is given birth to the lactobacterium casei (Lactobacillus casei Zhang) of effect, comprise the technology of preparing of the high vigor freeze-dried vaccine powder of the control of proliferated culture medium and optimization of fermentation condition and this bacterial strain.Can obtain 10 by the present invention
11The freeze-dried vaccine powder of the lactobacterium casei of the above viable count of cfu/mL can directly apply to the preparation and the application of this bacterial strain probiotics leaven and probiotics preparation.
Description
[technical field]
The present invention relates to microorganism culturing and applied technical field.More specifically, the present invention relates to the Lactobacillus casei Zhang high-density cultivation method, also relate to the method for using described Lactobacillus casei Zhang fermented liquid to prepare the freeze-dried vaccine powder, also relate to and adopt resulting lyophilized powder of described freeze-dried vaccine powder, preparation method thereof and their purposes.
[background technology]
As everyone knows, as a kind of probiotic bacterium, Bacterium lacticum is with the good probiotic properties that it was possessed, such as: acid resistance, tolerance bile acide, in digestive tube, decide reproductive growth and alleviate that lactose is not restrained oneself disease, the different sudden change of resistance, reduced serum cholesterol, antitumor, inhibition enteric pathogenic bacteria and anti-Helicobacter pylori, improve intestinal microflora and enhance immunity function etc. and be widely used in the fields such as food fermentation, animal-feed and health care.
High-density culture technology (High Cell Density Culture) is the high density fermentation technology known of people just.Because the highest cell concentration that each bacterial classification can reach differs bigger, so existing document is not made definite definition to high-density culture.Generally speaking, high-density culture is meant the fermentation density of using certain culture technology or device raising thalline, the more traditional training method of cell density is increased significantly, thereby reach the minimizing volume of culture, production cycle can also be shortened, the final specific production rate that improves specific product reduces production costs thereby reduce facility investment, improves the competitive power on market.People (J Ferment Bioeng such as Hayakawa K, 1990,70 (6): 404-408) custom-designed biological fermentation device is adopted in the research of milk-acid bacteria high-density culture, adopt membrane technique and fermentor tank coupling connection, constantly remove by filter lactic acid, hold back thalline and return fermentor tank realization somatic cells circulation cultivation, the highest cell density can be up to 10
11CFU/mL, this method need expend a large amount of substratum, and only limits to laboratory level, is difficult to expand to the production application scale, therefore is unsuitable for promoting the use of.For example CN 200510094737.5 relates to a kind of cell culture processes and biological reaction apparatus thereof, relates in particular to a kind of cell high-density circulating or continous way cultural method and biological reaction apparatus thereof.It is characterized in that on bio-reactor, installing additional inorganic membrane filtration assembly and feed supplement jar, and the three is connected to a bioreactor system that can be used for cell cycle formula high-density culture or the cultivation of high-density continous way by certain control loop.CN 200710004941.2 relates to a kind of high-density cultivation method of milk-acid bacteria, with plant lactobacillus, lactobacillus bulgaricus and pediococcus acidilactici is bacterial classification, use special milk-acid bacteria substratum 3% soy peptone, 2% yeast lixiviate powder, 0.5% sodium acetate, composite trace element liquid, vitamin complex liquid etc., regulate initial pH value and be 7.0-7.2; And stream adds Na in culturing process
2CO
3, KOH, NaOH or ammoniacal liquor makes the pH value stabilization of nutrient solution at 6.0-6.8.Obtain the lactic acid bacteria culture solution of high reactivity, high density, the viable lactic acid bacteria number reaches 9.0 * 10 in the nutrient solution
9-1.0 * 10
10Cfu/ml.People such as Huang Liangchang (Chinese dairy industry, 2002,30 (1): 12-15), people's (modern food science and technology such as Jin Zhiqiang, 2006,22 (2): 4-8) still mainly adopt batch culture mode on the optimization basis of medium component, this method is simple, and realization scale is easily amplified, but resulting cell density is not high, common 10
9CFU/mL.
Along with starter commercially produce and use increase sharply, the elementary object of industrial fermentation has turned to the cost with minimum to obtain the maximum output value and profit naturally, and the engineering means of realizing this goal are to set up corresponding efficient fermentation pattern at each particular procedure.Therefore, carry out suitability for industrialized production, the technology and the technology of non-genetic modification bacterium of large scale culturing and engineering bacteria just seem increasingly important.The key that milk-acid bacteria concentrates the starter preparation is will realize it is carried out high reactivity, highdensity cultivation.
The present invention is directed to a strain from Inner Mongol traditional function fermented-milk---separate the koumiss; and through external acid resisting test; anti-cholate test; the lactobacterium casei with potential prebiotic effect (Lactobacillus casei Zhang) that a series of experiment sievings such as decreasing cholesterol test obtain; by optimizing liquid nutrient medium and the methods such as control of culture condition being realized high density fermentation; this high density fermentation liquid is through centrifugal collection thalline; add the protective material postlyophilization and obtain the freeze-dried vaccine powder; these can be applied in probiotics leaven and the probiotics preparation by the freeze-dried vaccine powder; improve probiotics leaven and probiotics preparation quality, the broadened application scope.
[summary of the invention]
[technical problem that will solve]
The object of the present invention is to provide the fermentation process in high density of lactobacterium casei (Lactobacillus casei Zhang).
Another object of the present invention is to provide the preparation method of Lactobacillus casei Zhang freeze-dried vaccine powder.
Another object of the present invention is to provide Lactobacillus casei Zhang freeze-dried vaccine powder.
Another object of the present invention is to provide the purposes of Lactobacillus casei Zhang freeze-dried vaccine powder in probiotics leaven and probiotics preparation.
[technical scheme]
Described lactobacterium casei (Lactobacillus casei Zhang) is the probiotic bacterium that separates the acidproof and anti-bile acide that obtains from koumiss; This bacteria strain on April 21st, 2006 in the preservation of common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is CGMCC 1697.Lactobacillus casei Zhang has good probiotic properties as a kind of probiotic bacterium, through studies show that in a large number, this bacterial strain has good acid resistance, artificial gastrointestinal fluid tolerance and cholate tolerance and (opens and equality, China's dairy industry, 2006,34 (4): 6-11), immunity system is had significant regulatory function (opens and equality, China's dairy industry, 2006,34 (10): 4-8; Tuo Ya opens and equality, Food science, 2006,27 (11): 488-491), feed and raise this thalline and can significantly reduce high lipid food rat blood serum cholesterol and low-density lipoprotein content (Yun Yueying opens and equality, microbiology circular, 2006,33 (3): 60-64).
The objective of the invention is to be achieved through the following technical solutions:
The present invention relates to a kind of high-density cultivation method of Lactobacillus casei Zhang, it is characterized in that this method steps is as follows:
A. actication of culture:
Lactobacterium casei (the Lactobacillus casei Zhang) CGMCC 1697 of freezing preservation is inoculated in the MRS liquid nutrient medium, cultivated 18-24 hour down for 37 ℃ in temperature, so going down to posterity to cultivate obtains described activation Lactobacillus casei Zhang bacterial classification 1-2 time;
B. culture medium preparation:
Take by weighing following raw material at first, respectively: 60-90 weight part one DEXTROSE MONOHYDRATE, 8-12 weight part yeast powder, 8-12 weight part soy peptone, 3.0-3.5 weight part dipotassium hydrogen phosphate, 13-15 weight part sodium acetate, 2.0-2.5 weight part Trisodium Citrate, 0.8-1.0 weight part MgSO
4.7H
2O, 0.04-0.08 weight part MnSO
4.5H
2O and 0.8-1.0 weight part tween 80;
Then, above-mentioned raw materials is dissolved in 1000 weight parts waters, stirs, re-use neutralizing agent its pH value is adjusted to 6.4-6.6, sterilized 15 minutes down at temperature 120-122 ℃ again, obtain described substratum like this;
C. the preparation of strain fermentating liquid:
To use described substratum weight, the activation Lactobacillus casei Zhang bacterial classification inoculation that step a) is obtained according to 1-3 weight % is cultivated down for 37 ℃ in temperature and to be obtained the Lactobacillus casei Zhang strain fermentating liquid in 8-10 hour in the described substratum that obtains in step b);
D. fermentation:
To use described substratum weight, the described Lactobacillus casei Zhang strain fermentating liquid that step c) is obtained according to 4-10 weight % is inoculated in the described substratum that step b) obtains, under temperature 35-37 ℃, fermented 8-16 hour, in described fermenting process, add neutralizing agent the pH of this fermentation media is remained on 5.8-6.5, keep little logical oxygen or anaerobic condition simultaneously by stream;
E. stop fermentation:
The pH of described fermentation media no longer reduces with neutralizing agent stream and adds when stopping, and just stops fermentation, reaches 10 so obtain described Lactobacillus casei Zhang viable count
10The lactobacterium casei high density fermentation liquid that cfu/ml is above.
A preferred embodiment of the invention, described neutralizing agent are one or more neutralizing agents that are selected from NaOH, KOH or ammoniacal liquor, and the concentration of described neutralizing agent is 15-40 weight %.
In the present invention, preferably in described fermenting process, add neutralizing agent the pH of this fermentation media is remained on 5.8-6.2 by stream.
In the present invention, preferably described Lactobacillus casei Zhang strain fermentating liquid is inoculated in the described substratum that step b) obtains, and ferments 10 hours under 37 ℃ of temperature.
According to another kind of preferred implementation of the present invention, described little logical oxygen is blowing air 0.02-0.05vvm or every two hours measures blowing air once with 0.03-0.1vvm.
According to another kind of preferred implementation of the present invention, described anaerobically fermenting is logical nitrogen 0.02-0.05vvm or every two hours leads to nitrogen once with the 0.03-0.1vvm amount.
The invention still further relates to a kind of preparation method of Lactobacillus casei Zhang freeze-dried vaccine powder, it is characterized in that the step of this method is as follows:
A. concentrating of described Lactobacillus casei Zhang high density fermentation liquid:
Allow the Lactobacillus casei Zhang high density fermentation liquid at room temperature under the 5000-12000g condition centrifugal 5-30 minute of method noted earlier preparation, obtain Lactobacillus casei Zhang thalline concentrated solution;
B. add protective material:
The Lactobacillus casei Zhang thalline concentrated solution that obtains toward step a) adds the protective material aqueous solution of 5-10 concentration 18-25 weight % doubly by weight, obtains Lactobacillus casei Zhang thalline suspension;
C. dry: with above-mentioned steps b) the described Lactobacillus casei Zhang thalline suspension that obtains carries out lyophilize under temperature-10 ℃～-50 ℃, obtain described Lactobacillus casei Zhang freeze-dried vaccine powder.
According to another kind of preferred implementation of the present invention, described protective material is selected from skimmed milk powder, glycerine, vitamins C, L-glutamic acid, sorbyl alcohol, trehalose or lactose.
Preferably, described protective material is selected from skimming milk, vitamins C, L-glutamic acid, trehalose or lactose.
More preferably, described protective material is selected from skimming milk, vitamins C, L-glutamic acid or lactose.
The invention still further relates to the Lactobacillus casei Zhang freeze-dried vaccine powder that adopts aforesaid method to obtain, it is characterized in that it contains 10
11The total viable count of described Lactobacillus casei Zhang that cfu/ml is above.
The invention still further relates to the purposes of described buttermilk bacillus Zhang freeze-dried vaccine powder in probiotics leaven or probiotics preparation.
To illustrate in greater detail the present invention below.
The present invention relates to a kind of high-density cultivation method of Lactobacillus casei Zhang.
The high-density cultivation method step of described Lactobacillus casei Zhang is as follows:
A. actication of culture:
Lactobacterium casei (the Lactobacillus casei Zhang) CGMCC 1697 of freezing preservation is inoculated in the MRS liquid nutrient medium, cultivated 18-24 hour down for 37 ℃ in temperature, so going down to posterity to cultivate obtains described activation Lactobacillus casei Zhang bacterial classification 1-2 time.
Wherein, described MRS liquid nutrient medium is composed as follows: 10g peptone, 10g extractum carnis, 5g yeast soak powder, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g trisodium citrate, 1g tween 80,200mg sal epsom, 54mg manganous sulfate and 1000mL distilled water.Above-mentioned these raw materials with the 1000mL dissolved in distilled water after, re-use mineral alkali with its pH regulator to 6.5, the 15min that sterilizes under 121 ℃ of temperature then obtains the MRS liquid nutrient medium like this.
Described peptone by protein through enzyme, acid, basic hydrolysis and a kind of water soluble mixt of forming by peptone, peptone peptide ammino acid that obtains.It is the main basic ingredient of microbiological culture media, and main effect is to provide nitrogenous source and VITAMIN for microorganism growth.The peptone that the present invention uses for example be Xiamen prosperous Long Da chemical reagents corporation with trade(brand)name peptone product sold, or Beijing extensive and profound in meaning star biotechnology responsibility company limited is with trade(brand)name peptone product sold.
Described extractum carnis be with fresh beef through a kind of pale brown look of rejecting fat, digestion, filtering, concentrating and obtaining to tan paste.Extractum carnis contains creatine, creatinine, polypeptide class, amino acids, ucleotides, organic acid, mineral substance class and vitamins water-soluble substances.Its main effect is to provide carbon source, the energy, phosphoric acid salt and VITAMIN for microorganism.The extractum carnis that the present invention uses for example be Beijing bispin biological medium products factory with trade(brand)name beef extract product sold, or Beijing extensive and profound in meaning star biotechnology responsibility company limited is with trade(brand)name extractum carnis powder product sold.
It is to be raw material with the yeast that described yeast soaks powder, adopts autolysis method or adds enzyme hydrolysis method, the product that contains amino acid, peptide, polypeptide and yeast cell water soluble component that obtains through separation, decolouring, spraying drying.It for example is that a part product sold soaks with the trade(brand)name yeast in Beijing extensive and profound in meaning star biotechnology responsibility company limited that the yeast that the present invention uses soaks powder, or the prosperous Long Da in Xiamen chemical reagents corporation is with trade(brand)name yeast powder product sold.
The technology that above-mentioned other raw material all is present technique field product that know, that can obtain from the market.
Here the device that uses of strain activation and culture is the culture apparatus that has temperature control and timing device that those skilled in the art know, and for example the culture apparatus sold with the biochemical incubator of trade(brand)name of Shanghai Yiheng Scientific Instruments Co., Ltd or Shanghai laboratory apparatus Co., Ltd., Factory are with the culture apparatus of trade(brand)name constant incubator sale.
B. culture medium preparation:
Take by weighing following raw material at first, respectively: 60-90 weight part one DEXTROSE MONOHYDRATE, 8-12 weight part yeast powder, 8-12 weight part soy peptone, 3.0-3.5 weight part dipotassium hydrogen phosphate, 13-15 weight part sodium acetate, 2.0-2.5 weight part Trisodium Citrate, 0.8-1.0 weight part MgSO
4.7H
2O, 0.04-0.08 weight part MnSO
4.5H
2O and 0.8-1.0 weight part tween-80;
Wherein said yeast powder is that yeast soaks powder as previously described.
Described soy peptone is the peptone that obtains with soy-protein as previously described.
Then, above-mentioned raw materials is dissolved in 1000 weight parts waters, stirs, re-use neutralizing agent its pH value is adjusted to 6.4-6.6, sterilized 15-17 minute down at temperature 120-122 ℃ again, obtain described substratum like this.
Described neutralizing agent is one or more neutralizing agents that are selected from NaOH, KOH or ammoniacal liquor.Preferably, described neutralizing agent is NaOH or ammoniacal liquor, and more preferably described neutralizing agent is NaOH.
The concentration of described neutralizing agent is 15-40 weight %.Their concentration can be 30-40 weight % when for example using NaOH or KOH.Its concentration is 15-25 weight % when using ammoniacal liquor.
C. the preparation of strain fermentating liquid:
To use described substratum weight, the activation Lactobacillus casei Zhang bacterial classification inoculation that step a) is obtained according to 1-3 weight % is cultivated down for 37 ℃ in temperature and to be obtained the Lactobacillus casei Zhang strain fermentating liquid in 8-10 hour in the described substratum that obtains in step b).The viable count that adopts survey 600nm light absorption value method to measure this Lactobacillus casei Zhang strain fermentating liquid reaches 10
8More than the CFU/mL.
Preferably, described inoculum size is to use described substratum weight 2 weight %.
The culture apparatus that this step is used is the culture apparatus that has temperature control and timing device that those skilled in the art know, the culture apparatus that for example culture apparatus sold with the biochemical incubator of trade(brand)name of Shanghai Yiheng Scientific Instruments Co., Ltd, or Shanghai laboratory apparatus Co., Ltd., Factory is sold with the trade(brand)name constant incubator.
D. fermentation:
On the zymotechnique basis of Miao Shuing, under different neutralizing agents, different buffer salinity, different pH, different substrate (glucose) concentration and different aeration conditions, carry out fermentation test successively, in front according to the maximum specific growth rate (μ under the different fermentations condition
Max), cell density (in dry cell weight, DCW) and the viable count situation, is determined the Lactobacillus casei Zhang fermentation condition that suits.
According to logistic equation growth kinetics model (Gu Ruixia, Liu Aiping, Luo Chengxiang, " microbiology circular ", 2001,28 (4): 35-39) calculate μ
MaxAs follows:
In the formula: C
x---cell density, with dry weight basis, the g/L of unit; μ
Max---maximum specific growth rate, the h of unit
-1C
X, max---maximum cell density; T---fermentation time, the h of unit.To formula (1) integration and be converted into linear equation and get:
By experimental data with ln[C
x/ (C
M-C
x)] to time t mapping, obtaining slope is μ
Max
1, the selection of neutralizing agent:
Four kinds of different alkali lye (6mol/L NaOH, 6mol/L KOH, 25% ammoniacal liquor and 30% Na have been studied
2CO
3) do the fermentation test that neutralizing agent carries out, it the results are shown in accompanying drawing 1.Different neutralizing agents use the effect of ammoniacal liquor best for the growth effect difference of L.casei Zhang, the μ of thalline fermentation
Max, cell density and viable count all be higher than the ferment effect with other three kinds of neutralizing agents; Na
2CO
3Effect be slightly poorer than ammoniacal liquor, but both significant differences not significantly (P＜0.05), the effect of NaOH solution is the poorest.Subsequent experimental adopts ammoniacal liquor as neutralizing agent.
2, substratum buffering salt content is optimized:
In substratum, contain 20g/L one DEXTROSE MONOHYDRATE, 10.4g/L yeast powder, 10.4g/L soy peptone, 1.0g/L MgSO
4.7H
2O, 54mg/L MnSO4.5H
2O and tween 80 1.0g/L and buffering salt, it contains 3.6g/L K
2HPO
4, 15g/L sodium acetate and 2.4g/L Trisodium Citrate, this buffering salt consumption is designated as " 1buffer ", reducing in the substratum buffering salt consumption reaches to half (0.5buffer) and 1/4 (0.25 buffer) and does not add buffering salt (0 buffer) and ferment, the results are shown in accompanying drawing 2, reduce or not add the substratum ferment effect of buffering salt good not as good as the ferment effect of former substratum.Be more or less the same μ though add the final cell density of fermented liquid (dry cells) of different concns buffering salt
MaxBut there is significant difference (P＜0.05) with viable count.Along with the minimizing of buffering salt addition, μ
MaxAll decrease with viable count.
3, the influence of condition of different pH to fermenting:
Studied the growing state of L.casei Zhang in pH 5.3-6.8 scope, shown in 3, viable count changes in the scope of pH 5.6-6.5 not quite with reference to the accompanying drawings, but viable count significantly reduces (P＜0.05), μ after being higher or lower than this pH scope
MaxReach the highest when pH 5.9, be higher or lower than this pH value, growth velocity is remarkable reduction (P＜0.05).
4, the growth of L.casei Zhang under the different carbon source concentrations:
Under pH 5.9 conditions, further study for the concentration of glucose in the substratum, from accompanying drawing 4 as can be seen, when glucose concn was increased to 100g/L by 20g/L, cell density and viable count improved with glucose concn, are up to 3.5 * 10
10CFU/mL improves glucose concn when higher again, and cell density no longer increases, the dead increase of thalline when the 150g/L glucose concn is cultivated, and viable count significantly descends.μ
MaxAlong with glucose concn increases and significantly reduces (p＜0.05), high concentration glucose has certain restraining effect to the growth of thalline, but in the scope of glucose concn 50-100g/L μ
MaxChange little.
5, the influence of different aeration conditions:
From accompanying drawing 5 as seen, there is certain influence in different aeration conditions for the growth of L.casei Zhang.The anaerobic condition culture effect slightly is better than aerated culture generally.But the anaerobically fermenting that adopts different ventilating modes to carry out, the effect difference, cell density, viable count and the specific growth rate that intermittently logical condition of nitrogen gas bottom fermentation obtains is higher than continuously logical nitrogen fermentation.Intermittently blowing air (little logical oxygen condition) bottom fermentation effect slightly is inferior to intermittently nitrogen, is better than the effect of logical nitrogen continuously, and other aeration condition ferment effects are not good.
6, training method
Begin in fermentation that stream adds fresh culture in fermented liquid during 6h, realize fed batch cultivation, from accompanying drawing 6 as can be seen, with the feed supplement of 100mL/h speed, viable count and cell density reach maximum in succession when fermenting to 12h and 14h, and continuing to cultivate then, viable count and cell density begin to descend.The ferment effect of 100mL/h fed-batch fermentation and batch culture (substratum glucose concn 80g/L) does not have significant difference (P＜0.05).When feed supplement speed increased to 200mL/h, fermentation just reached peak value at 8h, continued feed supplement and dilution effect occurred, and cell density and viable count descend.
This shows, glucose concn is adjusted into 80-100g/L, the lactic acid that stream adds in the ammoniacal liquor and fermentation produces in the fermenting process, make pH remain on slant acidity (pH5.9), method by intermittently logical nitrogen (every 2h ventilation 2min) keeps the environment anaerobism, the following 37 ℃ of heat-preservation fermentation 10-12h of batch culture mode, cell density is than optimizing preceding (4.8 * 10
9CFU/mL) improve more than 7 times, dry cell weight reaches 7g/L, viable count 3.5 * 10
10CFU/mL.Can satisfy probiotic composition and produce desired high cell density.
E. stop fermentation:
The pH of described fermentation media no longer reduces with neutralizing agent stream and adds when stopping, and just stops fermentation, reaches 10 so obtain described Lactobacillus casei Zhang viable count
10The lactobacterium casei high density fermentation liquid that cfu/ml is above.
The measuring method of described Lactobacillus casei Zhang viable count is the measuring method that the flat board that adopts those skilled in the art to know pours into, for example with MRS substratum pour plate counting.
The invention still further relates to a kind of preparation method of Lactobacillus casei Zhang freeze-dried vaccine powder, it is characterized in that the step of this method is as follows:
A. concentrating of described Lactobacillus casei Zhang high density fermentation liquid:
Use the Lactobacillus casei Zhang high density fermentation liquid at room temperature under the 5000-12000g condition centrifugal 5-30 minute of the high-density cultivation method preparation of the Lactobacillus casei Zhang of describing in front, obtain Lactobacillus casei Zhang thalline concentrated solution.
Described Lactobacillus casei Zhang thalline concentrated solution is a fermented liquid through the centrifugal product that obtains after removing supernatant liquor.
B. add protective material:
The Lactobacillus casei Zhang thalline concentrated solution that obtains toward step a) adds the protective material aqueous solution of 5-10 concentration 18-40 doubly by weight, obtains Lactobacillus casei Zhang thalline suspension.
On meaning of the present invention, described protective material should be appreciated that it is that the protection thalline is avoided or alleviated the destruction of frozen drying to thalline in freezing dry process.
Described protective material is selected from skimmed milk powder, glycerine, vitamins C, L-glutamic acid, sorbyl alcohol, trehalose or lactose.
Preferably, described protective material is selected from skimmed milk powder, vitamins C, L-glutamic acid, trehalose or lactose.
More preferably, described protective material is selected from skimmed milk powder, vitamins C, L-glutamic acid or lactose.
The concentration of the described protective material aqueous solution is 18-40 weight %.Preferably, 18-35%.20-35% more preferably.
C. dry: with above-mentioned steps b) the described Lactobacillus casei Zhang thalline suspension that obtains carries out lyophilize under temperature-10--50 ℃, obtain described Lactobacillus casei Zhang freeze-dried vaccine powder.
Described lyophilize is known to those skilled in the art.Generally speaking, described lyophilize temperature-10--50 ℃ with vacuum 1.3-13 handkerchief under carry out, the moisture content that should reach the freeze-dried vaccine powder after described lyophilize is not higher than 8%.
Described Lactobacillus casei Zhang thalline suspension lyophilize use equipment is that Tianli Deep Freezing Equipment Co., Ltd., Beijing is with the large-scale medicinal vacuum freeze drier of trade(brand)name equipment that sell or that Hangzhou intention vacuum freeze factory sells with the trade(brand)name vacuum freeze drier.This equipment is characterised in that and is controlled under the cryogenic vacuum condition, is that moisture content realizes dry with distillation, and the vigor of the fine low protection thalline of energy is damaged less.
The invention still further relates to the Lactobacillus casei Zhang freeze-dried vaccine powder that adopts aforesaid method to obtain, it is characterized in that it contains 10
11The total viable count of described Lactobacillus casei Zhang that cfu/ml is above.The total viable count of described Lactobacillus casei Zhang is the measuring method that the flat board that adopts those skilled in the art to know pours into, for example with MRS substratum pour plate counting.
The invention still further relates to the purposes of described buttermilk bacillus Zhang freeze-dried vaccine powder in probiotics leaven or probiotics preparation.
Described probiotics leaven for example can be one or more probiotics leavens that are selected from Lactobacterium acidophilum, lactobacillus bulgaricus, thermophilus streptococcus or bifidus bacillus.Usually, can directly be added to buttermilk bacillus Zhang freeze-dried vaccine powder of the present invention equably in the described probiotics leaven, perhaps adopt alternate manner to be added in the described probiotics leaven according to the 1-30% amount.
Described probiotics preparation for example can be one or more probiotics preparations that are selected from Lactobacterium acidophilum, lactobacterium casei or bifidus bacillus.Usually, can directly be added to buttermilk bacillus Zhang freeze-dried vaccine powder of the present invention equably in the described probiotics preparation, perhaps adopt alternate manner to be added in the described probiotics preparation according to the 1-30% amount.
Lactobacillus casei Zhang of the present invention is the probiotic bacterium that separates the acidproof and bile tolerance that obtains from koumiss.Before without the overpopulation culture studies in common MRS substratum conventional its viable count only 10 of cultivating
8Cfu/mL far can not satisfy the requirement to high viable count of probiotics leaven and probiotics preparation.And the Lactobacillus casei Zhang freeze-dried vaccine powder that adopts method of the present invention to obtain after high-density culture can directly be added to some probiotics preparations or probiotics leaven, this Lactobacillus casei Zhang not only keeps it active fully, and can bring into play it because of Lactobacillus casei Zhang content height many benefits such as cholesterol, enhancing immunity are given birth to effect.
[beneficial effect]
Lactobacillus casei Zhang of the present invention is strong to acid and cholate tolerance, and has the living functions of multiple benefit such as decreasing cholesterol, inhibition tumour, raising immunizing power, adopts fermentation process in high density of the present invention can obtain 10
10The total viable count fermented liquid of described Lactobacillus casei Zhang that cfu/mL is above is cultivated in the more conventional MRS substratum, and incubation time can shorten 5～10 hours, viable count (4.8 * 10
8Cfu/mL) improve more than 50 times, high density fermentation liquid can obtain 10 through lyophilize
11The freeze-dried vaccine powder of the high vigor of cfu/mL viable count can satisfy the requirement of giving birth to the required high vigor of effect for probiotic bacterium performance benefit, directly applies to probiotics preparation or probiotics leaven.
[description of drawings]
The different neutralizing agents of Fig. 1 are to the influence of growth
The different buffer salinities of Fig. 2 are for the influence of L.casei Zhang growth
The growing state of L.casei Zhang under Fig. 3 condition of different pH
The growing state of L.casei Zhang under the different glucose concn of Fig. 4
The growing state of L.casei Zhang under the different aeration conditions of Fig. 5
Fig. 6 fed batch cultivation and batch culture cell density and viable count change, and wherein ■ and represent the dry cell weight and the viable count of 100mL/h fed batch cultivation respectively; ● and zero dry cell weight and the viable count of representing the 200mL/h fed batch cultivation respectively; ▲ represent the dry cell weight of batch culture (substratum glucose concn 80g/L) and viable count to change respectively with △
[embodiment]
The invention will be further described and do not limit protection scope of the present invention for following embodiment.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Embodiment 1: the high density fermentation of Lactobacillus casei Zhang
This embodiment carries out according to following step:
A. the activation of bacterial classification:
The lactobacterium casei (Lactobacillus casei Zhang) of-40 ℃ of freezing preservations is inoculated in the 5ml MRS liquid nutrient medium.The culture device that use is sold with the biochemical incubator of trade(brand)name by Shanghai Yiheng Scientific Instruments Co., Ltd was cultivated 18 hours down for 37 ℃ in temperature, and so going down to posterity to cultivate obtains the activatory bacterial classification 2 times.
Described MRS liquid nutrient medium is composed as follows: the peptone that 10g is sold with the trade(brand)name peptone by Beijing extensive and profound in meaning star biotechnology responsibility company limited, the extractum carnis that 10g is sold with the trade(brand)name beef extract powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited, 5g soaks powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited with the yeast that the trade(brand)name yeast soaks the powder sale, 20g glucose, the 2g dipotassium hydrogen phosphate, the 5g sodium acetate, the 2g trisodium citrate, the 1g tween 80,200mg sal epsom, 54mg manganous sulfate and 1000mL distilled water.Above-mentioned these raw materials with the 1000mL dissolved in distilled water after, re-use mineral acid with its pH regulator to 6.5, the 15min that sterilizes under 121 ℃ of temperature then obtains described MRS liquid nutrient medium like this.
B. culture medium preparation:
Take by weighing following raw material: 70g one DEXTROSE MONOHYDRATE, 8g respectively and soak the yeast powder that powder sells, soy peptone, 3.0g dipotassium hydrogen phosphate, 13g sodium acetate, 2.0g Trisodium Citrate, the 1.0g MgSO that 12g is sold with the trade(brand)name soy peptone by the prosperous Long Da in Xiamen chemical reagents corporation with the trade(brand)name yeast by the prosperous Long Da in Xiamen chemical reagents corporation
4.7H
2O, 0.05g MnSO
4.5H
2O and 0.88g tween-80;
Above-mentioned raw materials is dissolved in 1000 weight parts waters, stirs, re-use concentration 1mol/L aqueous hydrochloric acid its pH value is adjusted to 6.5, sterilized 15 minutes down for 121 ℃ in temperature again, obtain described substratum like this.
C. the preparation of strain fermentating liquid:
To use described substratum weight, the activation Lactobacillus casei Zhang bacterial classification inoculation that step a) is obtained according to 2 weight % is in the described substratum that obtains in step b), the culture device of using Shanghai Yiheng Scientific Instruments Co., Ltd to sell with the biochemical incubator of trade(brand)name was cultivated 10 hours down for 37 ℃ in temperature, obtain the Lactobacillus casei Zhang strain fermentating liquid, the viable count that adopts survey 600nm light absorption value method to measure this Lactobacillus casei Zhang strain fermentating liquid reaches 10
8More than the CFU/mL.
D. fermentation:
To use described substratum weight, the described Lactobacillus casei Zhang strain fermentating liquid that step c) is obtained according to 5 weight % is inoculated in the described substratum that step b) obtains, use the have temperature control of Shanghai high machine biotechnology company limited with the sale of trade(brand)name BIFO series of biologic fermentor tank, ventilation and stream add the fermentation unit of assembly, under 37 ℃ of temperature, fermented 10 hours, in described fermenting process, add concentration 15% ammoniacal liquor by stream simultaneously the pH of this fermentation media is remained on 5.9, with every 2 hours flow velocitys with 0.04vvm blowing air 2 minutes in fermentation media, keep the condition of little logical oxygen.These air are the dry airs by 0.22 μ m membrane filtration purifying treatment, pass through described fermentation media with micro bubbles form homodisperse ground.
E. stop fermentation:
The pH of described fermentation media no longer reduces with neutralizing agent stream and adds when stopping, and just stops fermentation, and the viable count that adopts MRS culture medium flat plate dump method to measure this Lactobacillus casei Zhang strain fermentating liquid reaches 2.8 * 10
10CFU/mL.Reach 2.8 * 10 so obtain described Lactobacillus casei Zhang viable count
10The lactobacterium casei high density fermentation liquid of cfu/ml.
Embodiment 2: the high density fermentation of Lactobacillus casei Zhang
This embodiment carries out according to following step:
A. the activation of bacterial classification:
The lactobacterium casei (Lactobacillus casei Zhang) of-40 ℃ of freezing preservations is inoculated in the 5ml MRS liquid nutrient medium.The culture device that use is sold with the biochemical incubator of trade(brand)name by Shanghai Yiheng Scientific Instruments Co., Ltd was cultivated 18 hours down for 37 ℃ in temperature, and so going down to posterity to cultivate obtains the activatory bacterial classification 2 times.
Described MRS liquid nutrient medium is composed as follows: the peptone that 10g is sold with the trade(brand)name peptone by Beijing extensive and profound in meaning star biotechnology responsibility company limited, the extractum carnis that 10g is sold with the trade(brand)name beef extract powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited, 5g soaks powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited with the yeast that the trade(brand)name yeast soaks the powder sale, 20g glucose, the 2g dipotassium hydrogen phosphate, the 5g sodium acetate, the 2g trisodium citrate, the 1g tween 80,200mg sal epsom, 54mg manganous sulfate and 1000mL distilled water.Above-mentioned these raw materials with the 1000mL dissolved in distilled water after, re-use mineral acid with its pH regulator to 6.5, the 15min that sterilizes under 121 ℃ of temperature then obtains described MRS liquid nutrient medium like this.
B. culture medium preparation:
Take by weighing following raw material: 80g one DEXTROSE MONOHYDRATE, 10g respectively and soak the yeast powder that powder sells, soy peptone, 3.0g dipotassium hydrogen phosphate, 13g sodium acetate, 2.0g Trisodium Citrate, the 1.0g MgSO that 10g is sold with the trade(brand)name soy peptone by the prosperous Long Da in Xiamen chemical reagents corporation with the trade(brand)name yeast by the prosperous Long Da in Xiamen chemical reagents corporation
4.7H
2O, 0.054g MnSO
4.5H
2O and 1.00g tween-80;
Above-mentioned raw materials is dissolved in 1000 weight parts waters, stirs, re-use concentration 1mol/L aqueous hydrochloric acid its pH value is adjusted to 6.5, sterilized 15 minutes down for 121 ℃ in temperature again, obtain described substratum like this.
C. the preparation of strain fermentating liquid:
To use described substratum weight, the activation Lactobacillus casei Zhang bacterial classification inoculation that step a) is obtained according to 2 weight % is in the described substratum that obtains in step b), the culture device of using Shanghai Yiheng Scientific Instruments Co., Ltd to sell with the biochemical incubator of trade(brand)name was cultivated 10 hours down for 37 ℃ in temperature, obtain the Lactobacillus casei Zhang strain fermentating liquid, the viable count that adopts survey 600nm light absorption value method to measure this Lactobacillus casei Zhang strain fermentating liquid reaches 10
8More than the CFU/mL.
D. fermentation:
To use described substratum weight, the described Lactobacillus casei Zhang strain fermentating liquid that step c) is obtained according to 5 weight % is inoculated in the described substratum that step b) obtains, use the have temperature control of Shanghai high machine biotechnology company limited with the sale of trade(brand)name BIFO series of biologic fermentor tank, ventilation and stream add the fermentation unit of assembly, under 37 ℃ of temperature, fermented 10 hours, in described fermenting process, add concentration 20% ammoniacal liquor by stream simultaneously the pH of this fermentation media is remained on 6.0, in fermentation media, to lead to nitrogen 2 minutes, keep the condition of little logical oxygen every 2 hours flow velocitys with 0.04vvm.The high pressure nitrogen of these nitrogen gas purities 99.9% passes through described fermentation media with micro bubbles form homodisperse ground.
E. stop fermentation:
The pH of described fermentation media no longer reduces with neutralizing agent stream and adds when stopping, and just stops fermentation, and the viable count that adopts MRS culture medium flat plate dump method to measure this Lactobacillus casei Zhang strain fermentating liquid reaches 3.5 * 10
10CFU/mL.Reach 3.5 * 10 so obtain described Lactobacillus casei Zhang viable count
10The lactobacterium casei high density fermentation liquid of cfu/ml.
Embodiment 3: the Lactobacillus casei Zhang common fermentation
This embodiment carries out according to following step:
A. the activation of bacterial classification:
The lactobacterium casei (Lactobacillus casei Zhang) of-40 ℃ of freezing preservations is inoculated in the 5ml MRS liquid nutrient medium.The culture device that use is sold with the biochemical incubator of trade(brand)name by Shanghai Yiheng Scientific Instruments Co., Ltd was cultivated 18 hours down for 37 ℃ in temperature, and so going down to posterity to cultivate obtains the activatory bacterial classification 2 times.
Described MRS liquid nutrient medium is composed as follows: the peptone that 10g is sold with the trade(brand)name peptone by Beijing extensive and profound in meaning star biotechnology responsibility company limited, the extractum carnis that 10g is sold with the trade(brand)name beef extract powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited, 5g soaks powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited with the yeast that the trade(brand)name yeast soaks the powder sale, 20g glucose, the 2g dipotassium hydrogen phosphate, the 5g sodium acetate, the 2g trisodium citrate, the 1g tween 80,200mg sal epsom, 54mg manganous sulfate and 1000mL distilled water.Above-mentioned these raw materials with the 1000mL dissolved in distilled water after, re-use mineral acid with its pH regulator to 6.5, the 15min that sterilizes under 121 ℃ of temperature then obtains described MRS liquid nutrient medium like this.
B. preparation of fermentation liquid:
To use described MRS substratum weight, the activation Lactobacillus casei Zhang bacterial classification inoculation that step a) is obtained according to 2 weight % is in described MRS substratum, the culture device of using Shanghai Yiheng Scientific Instruments Co., Ltd to sell with the biochemical incubator of trade(brand)name was cultivated 18 hours down for 37 ℃ in temperature, and the viable count that adopts MRS culture medium flat plate dump method to measure this Lactobacillus casei Zhang strain fermentating liquid reaches 4.5 * 10
8CFU/mL.Reach 4.5 * 10 so obtain described Lactobacillus casei Zhang viable count
8The lactobacterium casei fermented liquid of cfu/ml
Embodiment 4: the Lactobacillus casei Zhang common fermentation
This embodiment carries out according to following step:
A. the activation of bacterial classification:
The lactobacterium casei (Lactobacillus casei Zhang) of-40 ℃ of freezing preservations is inoculated in the 5ml MRS liquid nutrient medium.The culture device that use is sold with the biochemical incubator of trade(brand)name by Shanghai Yiheng Scientific Instruments Co., Ltd was cultivated 18 hours down for 37 ℃ in temperature, and so going down to posterity to cultivate obtains the activatory bacterial classification 2 times.
Described MRS liquid nutrient medium is composed as follows: the peptone that 10g is sold with the trade(brand)name peptone by Beijing extensive and profound in meaning star biotechnology responsibility company limited, the extractum carnis that 10g is sold with the trade(brand)name beef extract powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited, 5g soaks powder by Beijing extensive and profound in meaning star biotechnology responsibility company limited with the yeast that the trade(brand)name yeast soaks the powder sale, 20g glucose, the 2g dipotassium hydrogen phosphate, the 5g sodium acetate, the 2g trisodium citrate, the 1g tween 80,200mg sal epsom, 54mg manganous sulfate and 1000mL distilled water.Above-mentioned these raw materials with the 1000mL dissolved in distilled water after, re-use mineral acid with its pH regulator to 6.5, the 15min that sterilizes under 121 ℃ of temperature then obtains described MRS liquid nutrient medium like this.
B. preparation of fermentation liquid:
To use described MRS substratum weight, the activation Lactobacillus casei Zhang bacterial classification inoculation that step a) is obtained according to 1 weight % is in described MRS substratum, the culture device of using Shanghai Yiheng Scientific Instruments Co., Ltd to sell with the biochemical incubator of trade(brand)name was cultivated 20 hours down for 37 ℃ in temperature, and the viable count that adopts MRS culture medium flat plate dump method to measure this Lactobacillus casei Zhang strain fermentating liquid reaches 4.8 * 10
8CFU/mL.Reach 4.8 * 10 so obtain described Lactobacillus casei Zhang viable count
8The lactobacterium casei fermented liquid of cfu/ml
Embodiment 5:
The preparation of lactobacterium casei (Lactobacillus casei Zhang) freeze-dried vaccine powder:
Lactobacterium casei high density fermentation liquid is obtained concentrating thalline through 10000g is centrifugal; add protective material solution in concentrated thalline and protective material skimmed milk powder solution ratio 1:10; go in the freeze drier lyophilize after mixing 36 hours, and obtained lactobacterium casei freeze-dried vaccine powder.The viable count of bacterium powder can reach 10
11More than the cfu/g, can directly apply to probiotics leaven or probiotics preparation.
Embodiment 6:
Lactobacterium casei (Lactobacillus casei Zhang) freeze-dried vaccine powder is applied to probiotics leaven:
Lactobacterium casei freeze-dried vaccine powder joined to be mixed by lactobacillus bulgaricus and thermophilus streptococcus by 10 weight % deliver directly in the type starter, mix the probiotics leaven that can obtain containing lactobacterium casei (Lactobacillus casei Zhang).This starter can be used for the process for processing of fermented yogurt or leben.
Embodiment 7:
Lactobacterium casei freeze-dried vaccine powder is applied to probiotics preparation:
Lactobacterium casei freeze-dried vaccine powder is joined in the medical probiotics preparation matrix of bifidus bacillus by 10 weight %, make capsule or compressing tablet after mixing, can obtain containing lactobacterium casei 10
10Probiotic bacterium capsule or the probiotic tablet of cfu/g.
Claims (10)
1, a kind of high-density cultivation method of Lactobacillus casei Zhang is characterized in that this method steps is as follows:
A. actication of culture:
Lactobacterium casei (the Lactobacillus casei Zhang) CGMCC 1697 of freezing preservation is inoculated in the MRS liquid nutrient medium, cultivated 18-24 hour down for 37 ℃ in temperature, so going down to posterity to cultivate obtains described activation Lactobacillus casei Zhang bacterial classification 1-2 time;
B. culture medium preparation:
Take by weighing following raw material at first, respectively: 60-90 weight part one DEXTROSE MONOHYDRATE, 8-12 weight part yeast powder, 8-12 weight part soy peptone, 3.0-3.5 weight part dipotassium hydrogen phosphate, 13-15 weight part sodium acetate, 2.0-2.5 weight part Trisodium Citrate, 0.8-1.0 weight part MgSO
4.7H
2O, 0.04-0.08 weight part MnSO
4.5H
2O and 0.8-1.0 weight part tween-80;
Then, above-mentioned raw materials is dissolved in 1000 weight parts waters, stirs, re-use neutralizing agent its pH value is adjusted to 6.4-6.6, sterilized 15 minutes down at temperature 120-122 ℃ again, obtain described substratum like this;
C. the preparation of strain fermentating liquid:
To use described substratum weight, the activation Lactobacillus casei Zhang bacterial classification inoculation that step a) is obtained according to 1-3 weight % is cultivated down for 37 ℃ in temperature and to be obtained the Lactobacillus casei Zhang strain fermentating liquid in 8-10 hour in the described substratum that obtains in step b);
D. fermentation:
To use described substratum weight, the described Lactobacillus casei Zhang strain fermentating liquid that step c) is obtained according to 4-10 weight % is inoculated in the described substratum that step b) obtains, under temperature 35-37 ℃, fermented 8-16 hour, in described fermenting process, add neutralizing agent the pH of this fermentation media is remained on 5.8-6.5, keep little logical oxygen or anaerobic condition simultaneously by stream;
E. stop fermentation:
The pH of described fermentation media no longer reduces with neutralizing agent stream and adds when stopping, and just stops fermentation, reaches 10 so obtain described Lactobacillus casei Zhang viable count
10The lactobacterium casei high density fermentation liquid that cfu/ml is above.
2. method according to claim 1 is characterized in that described neutralizing agent is one or more neutralizing agents that are selected from NaOH, KOH or ammoniacal liquor, and the concentration of described neutralizing agent is 15-40 weight %.
3. method according to claim 1 is characterized in that in described fermenting process adding neutralizing agent by stream remains on 5.8-6.2 with the pH of this fermentation media.
4. according to the described method of claim 1, it is characterized in that described Lactobacillus casei Zhang strain fermentating liquid is inoculated in the described substratum that step b) obtains, under 37 ℃ of temperature, fermented 10 hours.
5. method according to claim 1 is characterized in that described little logical oxygen is blowing air 0.02-0.05vvm or every two hours measures blowing air once with 0.03-0.1vvm.
6. method according to claim 1 is characterized in that described anaerobically fermenting is logical nitrogen 0.02-0.05vvm or every two hours leads to nitrogen once with the 0.03-0.1vvm amount.
7. the preparation method of a Lactobacillus casei Zhang freeze-dried vaccine powder is characterized in that the step of this method is as follows:
A. concentrating of described Lactobacillus casei Zhang high density fermentation liquid:
Allow according to the Lactobacillus casei Zhang high density fermentation liquid of the described method of each claim among claim 1-6 preparation at room temperature under the 5000-12000g condition centrifugal 5-30 minute, obtain Lactobacillus casei Zhang thalline concentrated solution;
B. add protective material:
The Lactobacillus casei Zhang thalline concentrated solution that obtains toward step a) adds the protective material aqueous solution of 5-10 concentration 18-25 weight % doubly by weight, obtains Lactobacillus casei Zhang thalline suspension;
C. dry: with above-mentioned steps b) the described Lactobacillus casei Zhang thalline suspension that obtains carries out lyophilize under temperature-10 ℃～-50 ℃, obtain described Lactobacillus casei Zhang freeze-dried vaccine powder.
8. method according to claim 7 is characterized in that described protective material is selected from skimmed milk powder, glycerine, vitamins C, L-glutamic acid, sorbyl alcohol, trehalose or lactose.
9. the Lactobacillus casei Zhang freeze-dried vaccine powder that obtains according to claim 7 or 8 described methods is characterized in that it contains 10
11The total viable count of described Lactobacillus casei Zhang that cfu/ml is above.
10. the purposes of buttermilk bacillus Zhang freeze-dried vaccine powder according to claim 9 in probiotics leaven or probiotics preparation.
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| CNA2008101758567A CN101418270A (en) | 2008-11-06 | 2008-11-06 | The Lactobacillus casei Zhang high-density cultivation method, use them to prepare the method for freeze-dried vaccine powder and resulting freeze-dried vaccine powder and uses thereof |
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