CN1793330A - Process for preparing high actived alctic acid bacteria product - Google Patents
Process for preparing high actived alctic acid bacteria product Download PDFInfo
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Abstract
The invention relates to new type high activity lactic acid bacteria produce manufacturing method. It uses Lactobacillus plantarum HSC235 CCTCC No.M204051 to culture the new strain, and gain the high activity lactic aid bacteria by fermentation medium culturing, enzyme inducting matter offering type, fermentation technology condition selecting. Active bacteria number in lyophilized product can reach 10<SUP>10</SUP> per gram. The transformation ratio of linoleic acid can reach 42.7%. The new type lactic acid bacteria micro ecology active bacteria preparation can be compounded with other component to form composite high effective micro ecology preparation. And as probiotics, it will have very extensive application prospect in foods, medicine, and micro ecology feed industries.
Description
(1) technical field the present invention relates to a kind of plant lactobacillus (Lactobacillusplantarum HSC 235) of the lactobacillus (Lactobacillus) that is used for microbial transformation linolic acid generation conjugated linolic acid newly, and utilizes this lactobacillus-fermented to produce the method for high-activity lactic acid bacteria goods.
(2) background technology is present, and what the probiotics application that utilizes little ecological theory to develop was wider is the lactic acid bacteria class probiotics, and product forms has oral pasty state agent, water solube powder, liquor etc.Oneself is generally acknowledged the effect of probiotics, and it promotes the propagation of profitable strain by suppressing pernicious bacteria in the enteron aisle, eliminates enterogenous endotoxin and enhancing immunity system and prevents intestinal tract disease, thereby improve the balance of little ecology in the enteron aisle.It had been both nontoxic, have no side effect, noresidue, no resistance, did not also pollute the environment simultaneously, had broad application prospects in fields such as food, medicine, fodder additivess.
Zhou Deqing (present situation of China's probiotics and development imagination. industrial microorganism, 1999,1:34～43) be the probiotic composition of representative with the enteral microecological formulation now, mainly select following 3 bacterioids for use.
(1) genus bifidobacterium of strictly anaerobic: according to the latest news, this belongs to existing 32 kinds, the person mainly contains 5 kinds wherein to be applied to making the enteral microecological formulation, i.e. bifidus longum bb, bifidobacteria infantis, bifidobacterium adolescentis, two fork bifidus bacillus and bifidobacterium breve.(2) general anaerobism is aerotolerant lactobacillus: that has reported so far has 56 kinds.Be usually used in there are 10 kinds approximately in the enteral microecological formulation, as Lactobacterium acidophilum (L.acidophilus), plant lactobacillus, short lactobacillus (L.brevis), lactobacterium casei (L.casei) and lactobacillus delbruockii subspecies bulgaricus (L.delbrueckii subsp.bulgaricus is once called as " lactobacillus bulgaricus ") etc.(3) some amphimicrobian coccuses: as belong to enterococcus faecalis in the enterococcus spp (Enterococcus) and faecium (E.faecium is once called as " streptococcus faecalis ") etc.; Belong to the Lactococcus lactis breast subspecies (L.lactis subsp.lactis is once called as " streptococcus uberis ") of lactococcus (Lactococcus) and lactococcus lactis subsp (L.lactis subsp.cremoris is once called as " junket suis ") etc.; And belong to the saliva chain coccus thermophilous subspecies (S.salivarius subsp.thermophilus is once called as " thermophilus streptococcus ") of streptococcus (S.treptococcus) and intermediate chain coccus (S.intermedius) etc.
Japan and the American-European lactobacillus inoculation that is used for sour milk and probiotic agent mainly are some from the lactobacillus inoculation of human body and bifidus bacillus as Lactobacterium acidophilum, lactobacillus rhamnosus, Lactobacillus rogosae, plant lactobacillus, buttermilk bacillus, Zhan Shi lactobacillus, bifidobacterium breve, bifidus longum bb and bifidobacterium and lactoenterococcus etc.Also having yeast also is one quite to like the probiotic bacterium of gazing at, yeast is because of can being used simultaneously with microbiotic by the general antibiotic group that influences, other advantage be acidproof, thermotolerance is stronger, also learn and kill by bile, all can survive at the anaerobic-aerobic environment, for making and storing favourable.Day body also uses clostridium butyricum (Cl.butyricum) unusual with treatment intestinal tract infections and intestinal function as the probiotic bacterium medicament.The heat-resisting acid resistance of its gemma is strong, takes in the back in enteron aisle germination propagation, can detect this bacterium in the ight soil after 1-2 days, completely dissolve after 3-5 days.Having 150 kinds in the existing 224 kinds of specific protective foodss of Japan is whole intestines class healthcare products.Mainly be the compound of bifidus bacillus in the milk-acid bacteria and correlation factor (as oligose, mushroom class extract, milk ketose etc.).(Hu Xuezhi, the research and the market profile of external probiotics.Industrial microorganism, 2002,3:56-61)
The application clinically of medicinal viable lactic acid bacteria preparation is mainly used at present and prevents and treats diarrhoea, enteritis, dysentery, hepatitis, liver cirrhosis, vaginitis, constipation, tract function disorder, reducing blood-fat, tetter and urinary system etc.Can mainly contain as medicinal bifidus bacillus: bifidobacterium breve, bifidus longum bb, bifidumbacterium bifidum, bifidobacteria infantis and bifidobacterium adolescentis etc.The lactobacillus that can be used as medicine mainly contains: Lactobacterium acidophilum, lactobacterium casei, short lactobacillus, plant lactobacillus etc.Can do medicinal faecalis mainly contains: enterococcus faecalis, faecium.
Report such as Peng Kaisong (research of aquatic products beneficial microorganism and application.World agriculture, 2004, what 11:51-53) think domestic stand-alone development mainly is some single bacterial strains: as photosynthetic bacterium, genus bacillus, Bdellovibrio etc.; Compound formulation mainly is imitated or introduces external commodity.
Yang Rude etc. (develop new and effective little ecological active bacteria formulation with microencapsulation; ACAD J GCP 2003; 2:87-90) adopt fluidization and nozzle bonded microencapsulation; the short factor of giving birth to of 3 kinds of bifidus bacilluss, efficient viable bacteria protective material and bifid together as core substance, is encapsulated in and makes acid resistant enteric coated capsule (or microballoon) in the nontoxic capsule material.Micro-capsule is handled 4h in manual simulation's gastric juice (pH 1.5～2.0), bifidobacteria viable bacteria number wherein still remains on more than 44%; Handle 12min in simulated intestinal fluid, the whole disintegrations of micro-capsule discharge active bacterium, preserve down 3 months (be equivalent under the normal temperature more than 1 year) at 37 ℃, and viable count wherein is still greater than 10
9Individual/g.Solved deactivation and the short problem of product normal temperature preservation period to living bifidobacteria such as hydrochloric acid in gastric juice, digestive ferment and microbiotic effectively.
(the research of Lactobacterium acidophilum and bifidus bacillus enteric active bacteria formulation such as Liu Jingsheng, China's dairy industry, 2003,1:14-16) bifidus bacillus and Lactobacterium acidophilum being carried out optimum pH stream adds neutralization and cultivates, after vacuum lyophilization is handled, utilize corresponding mechanical means and special art breading, develop the serial active bacteria formulations such as enteric soft capsules, enteric hard capsule and common hard capsule of bifidus bacillus and Lactobacterium acidophilum.And these preparations have been carried out physical behavior, stomach and intestine disintegration, simulation stomach and intestine comprehensive test, and normal temperature preserves test and wait and analyze detection, the characteristic and the performance index of above-mentioned various preparations have been made objective evaluation.
Chen Tietao etc. [the industrialized producing technology research of bifidumbacterium bifidum and Lactobacterium acidophilum probiotics, Chinese dairy industry, 2002,30 (5)] have studied the industrialized producing technology of bifidumbacterium bifidum and Lactobacterium acidophilum probiotics.Fermention medium and fermentation condition are studied and optimized, and determined fermented milk's freeze drying process condition.Bifidumbacterium bifidum reaches 10 with the viable count of having a liking in the product
9-10
10/ g.
Probiotics not only has wide use in the health care of food product, have the industry of breed that using value is quite widely also arranged as a kind of fodder additives.Discovering in recent years, the life-time service microbiotic is made fodder additives, all brings many serious problems to livestock industry production, human health and environment etc.: the microecological balance that 1. destroys animal and bird intestines; 2. cause some bacterium to develop immunity to drugs, the harm people and animals; 3. antibiotic chemical residual is threatening human health.Utilize the probiotics of little ecological theory development, promptly livestock and poultry are used probiotics, are a kind of fodder additivess that contains living microorganism.It had been both nontoxic, have no side effect, noresidue, no resistance, did not also pollute the environment simultaneously, had broad application prospects.At present, study morely, using wider is the lactic acid bacteria class probiotics, and product forms has oral pasty state agent, water solube powder, liquor etc.Livestock and poultry are generally acknowledged with oneself quilt of effect of probiotics, it promotes the propagation of profitable strain by suppressing pernicious bacteria in the animal and bird intestines, eliminates enterogenous endotoxin and enhancing immunity system and prevents intestinal tract disease, thereby improve the balance of little ecology in the enteron aisle, reach the purpose that improves livestock and poultry production performance.
Conjugated linolic acid has the fat and the protein that can promote the body fat mass in human body cellular accumulation, and carbohydrate etc. are transported in the motor tissue cells such as muscle, association tissue, internal organ, keep energy, adjust metabolic function etc.Have anti-oxidantly, suppress to produce prostaglandin E2, and functions such as impede protein kinase c activation (grain and grease, 1999,1:53-54).Wu Jihua, Qiu Aiyong (the physiological function of conjugated linolic acid, Chinese oil, 2001,6:45-47), experimentation on animals shows that conjugated linolic acid is a kind of very strong cancer-resisting substance, has atheroma formation, anti-diabetic, antianaphylaxis, adjusting immunity, promotes growth, reduces body fat and increase cutability and influences physiological function such as bone formation.
Lin Shuying etc. (Chinese oil, 2003, the 11:55-57 conjugated linolic acid is in the Application in Food Industry prospect) think that as anti-cancer health-care food CLA is a kind of potential anticarcinogen.The people of a 70kg may need to take in the sickness rate that about 115～310g CLA could reduce tumour every day; As the diet food additive, human body can not synthesize CLA, must obtain from food, and ruminating animal product such as milk, cheese and meat are the main sources of CLA.In American-European countries, particularly the U.S. with CLA as the auxiliary healthy food material of motion food and nutrition, towards the sales staff who takes exercises with oxygen uptake body building.If add about 0.1%～0.5% CLA in the food, because of it can enter the phosphatide mucous membrane of cell, nutrition is transported in the motor tissue cell repeatedly from adipocyte, make the people that wish to reduce body fat like this when carrying out motion such as walking, can not only reduce body fat, promote metabolism, improve muscle tone simultaneously, make muscle flourishing more; CLA is novel food preservatives.The growth that has research to think that the sodium salt, sylvite of CLA can mould fungus inhibitions, and have no side effect, character is relatively stable, does not have the restriction on using, and can be used for industries such as food, makeup, can be used as benzoic substitute.Both can reach the effect of antiseptic and inhibiting bacteria function, and can play the good health care effect again, this just can cater to the demand of human consumer for nontoxic natural antiseptic agent.
Shen Jihong etc. (research of conjugated linoleic acid microcapsule, the hi-tech communication, 2002,4:97-99) utilize spray drying technology, make the conjugated linolic acid microencapsulation.For the influence to the microencapsulation quality product of different embedding mediums and conjugated linolic acid proportioning, and the thermostability of product and dissolution rate are studied.The result shows, suitable conjugated linolic acid and embedding medium proportioning can reduce the surface oil of product; At the sour side's conjugated linolic acid of the best: embedding medium I: embedding medium II=6: 7: 4 o'clock, the oxidation-resistance of product was better than the raw material conjugated linolic acid, and dissolution rate also meets the pharmacopeia requirement.
Lv Guishan etc. studied the applied research of conjugated linolic acid in liquid milk product (Guangzhou foodstuffs industry science and technology, Vol.20No.3:15-17).In liquid milk product, add conjugated linolic acid (the Conjugated linoleic Acid of different content, CLA) and different CLA product form (CLA, CLA ethyl ester, CLA triglyceride level) back is to the influence of product sensory quality (but acceptance), and the storage stability of CLA is studied.Result of study shows that when the addition of CLA surpassed 1 ‰, product promptly has the distinctive astringent taste of a kind of CLA, and was difficult to allow the people accept; When using the CLA triglyceride level, the not influence of organoleptic quality to product makes that on the contrary product is more smooth, and fat aroma is denseer; The CLA triglyceride level has satisfactory stability in storage.
By above-mentioned documents and materials as seen, have the active lactobacillus powder of higher conversion linolic acid generation conjugated linolic acid and do not see as yet that with preparation report is arranged as a kind of research and development of novel little ecological active bacteria formulation.With the little ecological active bacteria formulation of the novel lactic acid bacteria express of this high activity of conversion again with other composition composite after, can make the probiotics of composite highly effective, in food, medicine and micro-ecological feed industry very wide application prospect will be arranged.
(3) summary of the invention task of the present invention provides and a kind ofly can effectively transform the microorganism strains that linolic acid is a conjugated linolic acid, and a kind of method of utilizing this new microbial fermentation to produce the high-activity lactic acid bacteria goods is provided.
Of the present inventionly can effectively transform the new microorganism strains that linolic acid is a conjugated linolic acid, be kind: HSC235 of plant lactobacillus (Lactobacillusplantarum HSC 235) of lactobacillus (Lactobacillus), this bacterial strain is to adopt the screening of MRS milk-acid bacteria culture of isolated to obtain from natural fermented vegetables.
Oneself is preserved in Chinese typical culture collection center this bacterial strain on July 16th, 2004, be called for short CCTCC, preservation CCTCC No.M204051.
The bacterial characteristics of this bacterial strain is as follows:
(1) form:
1) thin rod shape, 0.5 micron～0.8 micron of diameter, long 1 micron～5 microns;
2) cultivating the starting stage, cell is elongated rod shape;
3) mobility: do not move;
4) sporulation: do not form spore;
5) gramstaining: the positive;
6) acid resistance: the positive.
(2) growth conditions (30 ℃) on various substratum:
1) MRS Agar Plating: poor growth, bacterium colony is rounded, and is irregular,
Smooth surface, oyster white;
2) MRS agar slant culture-medium: poor growth, tarnish, oyster white;
3) MRS liquid nutrient medium: well-grown, the liquid muddiness has precipitation.
(3) physiological characteristic:
1) wood sugar :+
2) trehalose :+
3) sucrose :+
4) sorbyl alcohol :+
5) saligenin :+
6) ribose :+
7) rhamnosyl :-
8) raffinose :+
9) close disaccharides :+
10) seminose :+
11) N.F,USP MANNITOL :+
12) maltose :+
13) lactose :+
14) gluconate :+
15) semi-lactosi :+
16) fructose :+
17) polychrom :+
18) cellobiose :+
19) pectinose :+
20) amygdaloside :+
21) lactic acid opticity: DL
22) catalase :-
23) produce the indoles ability :-
24) decompose casein :-
According to above bacterial characteristics, and this bacterial strain HSC235 culture can be defined as plant lactobacillus Lactobacillus plantarum based on its situation of utilizing to substrate.
The preservation on the MRS substratum of this bacterial classification, used substratum contains usually: carbon source (for example glucose, lactose, whey), nitrogenous source (as), organic nutrient substance is (as yeast extract, peptone, the white peptone of pancreas, phytone, extractum carnis, corn steep liquor), inorganic nutrients composition (as phosphoric acid salt, magnesium, potassium, zinc, iron, cobalt and manganese) reaches the linolic acid as inductive substance, perhaps contain linoleic other material, perhaps linolic acid analog etc.All can grow under 20 ℃～40 ℃ culture temperature, the suitableeest growth temperature is 28 ℃～37 ℃.Culture condition: pH value 4.0～7.0, the suitableeest growth pH value is 6.0～6.6.This bacterial classification was cultivated 1 day～3 days containing on the bright linoleic MRS substratum under the anaerobic condition, and the gained cell has the linolic acid of conversion and linoleic acid material for gripping linoleic ability altogether.
The present invention prepares the method for high-activity lactic acid bacteria goods, comprising:
(1) bacterial strain CCTCC No.M204051 is carried out regular bevel and cultivate activation and seed culture;
(2) preparation fermention medium, and after sterilization, connect bacterial classification, fermentative medium formula, each components contents is percent weight in volume, i.e. g/L:
Yeast extract: 5.0-15.0
Peptone: 10.0-30.0
Sodium-chlor: 8.5
Glucose: 20.0-30.0
Linolic acid: 8-30
Trace element: an amount of
Tween 80: 1ml/L～5ml/L
All the other are water;
(3) zymotechnique
A. connect seed liquor, inoculum size is 5%～15% (v/v is etc. the ratio of unit volume) of fermentation culture based sols, and the optical density(OD) of seed liquor (OD value) is 1～3;
B. in the fermenting process, long bacterium temperature is 28 ℃～30 ℃, and the time is 60h～66h;
C. take sugaring mode at a slow speed, the sugaring amount is 100g-350g (sends out total 10 liters of meters of fermentating liquid volume), and 6h begins after inoculation, adds in 48h;
D. linolic acid generally can not directly add in the above-mentioned fermention medium, and through with other dispersant after could add, and linoleic concentration in the control fermented liquid adopts the mode that slowly adds;
E. adopt in the fermenting process and add sodium hydroxide and hydrochloric acid Automatic Balance Regulation pH value, the pH value is maintained between 6.0～6.6;
(4) bacterium mud is collected and is dry: fermentation ends, and fermented liquid washs three times (7000 rev/mins, 20min, 4 ℃) by high speed freezing centrifuge with the deionization sterilized water, centrifugal collecting cell, with protective material, vacuum lyophilization promptly gets the viable bacteria powder.
Trace element is sodium acetate, anhydrous 5g in the fermention medium of the present invention, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, CoCl
20.002g.
The present invention has designed the presentation mode of enzyme induction thing, certain density linolic acid is inhibited for the growth of bacterium, and linolic acid is less in the solvability of fermention medium, therefore with the linolic acid dissolving or be embedded in one of cyclodextrin, starch, bovine serum albumin-Yelkin TTS, bovine serum albumin, Yelkin TTS, glycerine, Tegin 55G material, row adds again.Its addition manner is: linolic acid starting point concentration induced concentration is controlled in the 0.4g/L in the fermented liquid, and intermittent injecting again behind inoculation culture 12h adds in the 48h.
Fermentation ends, through separation, collection, drying, the freeze-dried products that makes is that viable count can reach 10 in the viable bacteria powder
10Individual/g.The transformation efficiency that thalline transforms linolic acid generation conjugated linolic acid can reach 42.7%.The viable lactic acid bacteria counting is pressed GB/T 4789.35-2003 regulation and is measured, and conjugated linolic acid adopts vapor-phase chromatography (GC) and determined by ultraviolet spectrophotometry (Wu Jihua, Qiu Aiyong, the gc analysis conjugated linoleic acid isomers, Chinese oil, 2002,27:65-67; Shigenobu Kishino, Jun Ogawa.Conjugated Linoleic Acid Production from Linoleic Acid by Lactic AcidBacteria.Journal of American Oil Chemistry Society.2002,79:159-163; JohnAG, Roach M, Mossoba M.Chromatographic separation and identification ofconjugated linoleic acid isomers.Analytica Chimica Acia, 2002,465:207-226; Zhang Yagang, Fan Li etc., the application of ultraviolet-visible spectrophotometry in the conjugated linolic acid quantitative analysis, Xinjiang petroleum College's journal, 2002,14:59-64).
The present invention has following characteristics:
Adopt bacterial strain HSC235 of the present invention, thalline yield height, thalline OD value is 2～6.3, and gained bacterium powder viable count can reach 10
10Individual/g; The gained thalline has the ability that linolic acid generates conjugated linolic acid that transforms; Linolic acid adopts chaotropic agent to protect dissolving, reduces the restraining effect of its cell growth, has strengthened its inducing action on the other hand, has improved transformation efficiency, and it can reach 17%～42.7% (substrate quality transformation efficiency) to linoleic transformation efficiency; And leavening temperature is low, has reduced energy consumption, saves cost.Adopt gained lactobacillus powder of the present invention to can be used as probiotic bacterium and use, to improve the content of conjugated linolic acid in the prominent host at aspects such as food, biological fodders.
(4) embodiment is intended to illustrate rather than limit the scope of the invention below the specific embodiments.
Embodiment 1:
The culture presevation substratum:
MRS medium component (1L is metering with preparation): Tryptones 10g, extractum carnis 10g, yeast extract 5g, glucose 20g, tween 80 1ml, K
2HPO
42g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g.(pH=6.0～6.2), with distilled water preparation, 121 ℃ of moist heat sterilizations 20 minutes;
Seed culture medium: with the culture presevation substratum;
The preparation of seed liquor:, insert in the seed culture medium and cultivate 37 ℃ of culture temperature through above-mentioned slant medium activatory bacterial classification CCTCC No.M204051, shaking speed 120rpm, incubation time 1 day obtains seed culture fluid, and cell density OD value is controlled between 1～3 in the seed culture fluid.
Fermention medium (1L is metering with preparation, if preparation 10L fermention medium, then corresponding group component also increases by 10 times, in following examples also by that analogy): yeast extract 5g, peptone 10g, sodium-chlor 8.5g, glucose 20g, linolic acid 8g, sodium acetate, anhydrous 5g, MgSO
47H
2O0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, CoCl
20.002g, tween-80 1ml, all the other are water, pH6.5, it is standby to sterilize.
The presentation mode of enzyme induction thing (linolic acid):
With the linolic acid in fermention medium dissolving or be embedded in and be added in the cyclodextrin in the fermented liquid, linolic acid starting point concentration induced concentration is controlled at 0.2g/L in the fermented liquid; All the other are added in the fermented liquid during the fermentation, promptly after meeting seed liquor cultivation and fermentation 12h, remaining dissolving or be embedded in linolic acid amount in the cyclodextrin divides three intermittent injectings to go in the fermented liquid, add in the 48h, linoleic total add-on is 8g/L in the fermented liquid.
Zymotechnique:
In fermentor tank, inoculate sub-nutrient solution by above-mentioned formulated 10L fermented liquid through sterilizing after, the inoculum size of bacterial classification is 5% of a culture medium solution, i.e. 500mL, and optical density(OD) (OD value) is 1;
The linolic acid that the 10L fermention medium is required dissolves or is embedded in the cyclodextrin, is added in the fermented liquid, and linolic acid starting point concentration induced concentration is 0.2g/L in the control fermented liquid; Remaining linolic acid is added in the fermented liquid during the fermentation again;
Regulate pH6.0 with sodium hydroxide, the beginning cultivation and fermentation;
Behind beginning cultivation and fermentation 6h, glucose 100g (at first be mixed with 30% solution disinfection) is added fermentor tank at a slow speed, in 48h, add;
After meeting seed liquor cultivation and fermentation 12h, with remaining dissolving or be embedded in linolic acid amount in the cyclodextrin, divide three intermittent injectings to go in the fermented liquid, add in the 48h, linoleic total add-on is 8g/L in the fermented liquid;
Continue fermentation, total time is 60h, and long bacterium cultivation and inducing temperature are controlled at about 30 ℃ in the whole fermentation process; Adopt sodium hydroxide and hydrochloric acid to regulate pH in the fermenting process, the pH value in the fermenting process is maintained between 6.0～6.6.
Bacterium mud is collected and is dry: fermentation ends; fermented liquid by high speed freezing centrifuge with (7000 rev/mins of deionization sterilized water washings three times; 20min, 4 ℃), centrifugal collecting cell; (select glycerine with protective material; skim-milk, trehalose, any one material in the N.F,USP MANNITOL is as protective material); vacuum lyophilization promptly gets freeze-dried products---the viable bacteria powder.Viable count gained bacterium powder viable count can reach 2.1*10 in the freeze-dried products
10Individual/g, the transformation efficiency that thalline transforms linolic acid generation conjugated linolic acid can reach 17%.
Embodiment 2:
Slant medium: with embodiment 1;
The preparation of seed culture medium and seed liquor: with embodiment 1;
Fermention medium: yeast extract 10g, peptone 20g, sodium-chlor 8.5g, glucose 25g, linolic acid 12g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, CoCl
20.002g, tween-80 3ml, all the other are water, pH value 6.0, it is standby to sterilize.
The presentation mode of inductor:
Linolic acid in the fermention medium is dissolved earlier or be embedded in the mono-glycerides, be added in the fermented liquid again, linolic acid starting point concentration induced concentration is controlled at 0.3g/L in the fermented liquid; After connecing seed liquor and cultivating 12h, with remaining dissolving or be embedded in linolic acid in the mono-glycerides, divide three intermittent injectings to go in the fermented liquid, add in the 48h, linoleic total add-on is 12g/L in the fermented liquid.
Zymotechnique (no longer repeating linoleic adding mode in the zymotechnique below):
In fermentor tank, inoculate sub-nutrient solution by above-mentioned formulated 10L fermented liquid through sterilizing after, the inoculum size of bacterial classification is 10% of a culture medium solution, i.e. 1L, and optical density(OD) (OD value) is 2; Regulate pH6.0 with sodium hydroxide, the beginning cultivation and fermentation; Beginning adds fermentor tank to glucose 200g (at first be mixed with 30% solution disinfection) at a slow speed after cultivating 6h, adds in 48h; Fermentation time 72h, long bacterium and inducing temperature are 28 ℃ in the fermenting process; Adopt sodium hydroxide and hydrochloric acid to regulate pH in the fermenting process, the pH value in the fermenting process is maintained between 6.0～6.6 all the time.
Bacterium mud is collected and is dry: process is with embodiment 1, and viable count gained bacterium powder viable count can reach 5.7*10 in the freeze-dried products
10Individual/g.The transformation efficiency that thalline transforms linolic acid generation conjugated linolic acid can reach 24.1%.
Embodiment 3:
Slant medium: with embodiment 1;
The preparation of seed culture medium and seed liquor: with embodiment 1;
Fermention medium: yeast extract 15g, peptone 30g, sodium-chlor 8.5g, glucose 30g, linolic acid 20g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, CoCl
20.002g, tween-80 5ml, all the other are water, pH value 6.6, it is standby to sterilize.
The presentation mode of enzyme induction thing:
With the linolic acid in fermention medium dissolving or be embedded in bovine serum albumin-Yelkin TTS, be added into again in the fermented liquid, linolic acid starting point concentration induced concentration is controlled at 0.4g/L (being to contain the 4g linolic acid in every liter of fermented liquid) in the fermented liquid; All the other are added in the fermented liquid during the fermentation, promptly after meeting seed liquor fermentation culture 12h, divide three intermittent injectings to go in the fermented liquid more remaining dissolving or the linolic acid that is embedded in bovine serum albumin-Yelkin TTS, add in the 48h, the linoleic total add-on of fermented liquid is 20g/L.
Zymotechnique (no longer repeating linoleic adding mode in the zymotechnique below):
In fermentor tank, inoculate sub-nutrient solution by above-mentioned formulated 10L fermented liquid through sterilizing after, the inoculum size of bacterial classification is 15% of a culture medium solution, i.e. 1.5L, and optical density(OD) (OD value) is 2.5; Regulate pH6.0 with sodium hydroxide, the beginning cultivation and fermentation; After beginning to cultivate 6h, glucose 350g (at first be mixed with 30% solution disinfection) is added fermentor tank at a slow speed, in 48h, add; Long bacterium and inducing temperature are 30 ℃ in the fermenting process, fermentation total time 66h; Adopt sodium hydroxide and hydrochloric acid to regulate pH in the fermenting process, the pH value in the fermenting process is maintained between 6.0～6.6 all the time.
Bacterium mud is collected and is dry: process is with embodiment 1, and viable count gained bacterium powder viable count can reach 9.7*10 in the freeze-dried products
10Individual/g.The transformation efficiency that thalline transforms linolic acid generation conjugated linolic acid can reach 42.7%.
Embodiment 4:
Slant medium: with embodiment 1;
The preparation of seed culture medium and seed liquor: with embodiment 1;
Fermention medium: yeast extract 15g, peptone 30g, sodium-chlor 8.5g, glucose 30g, linolic acid 30g, sodium acetate, anhydrous 5g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.05g, dibasic ammonium citrate 2g, CoCl
2: 0.002g, tween-80 4ml, all the other are water, pH value 6.0, it is standby to sterilize.
The presentation mode of enzyme induction thing:
With the linolic acid in fermention medium dissolving or be embedded in starch, or Yelkin TTS, or glycerine, or in a kind of material of bovine serum albumin, be added into again in the fermented liquid, linolic acid starting point concentration induced concentration is controlled at 0.4g/L in the fermented liquid; After connecing seed liquor and cultivating 12h, with remaining dissolving or be embedded in that linolic acid divides three intermittent injectings to go in the fermented liquid in starch, the Yelkin TTS, add in the 48h, the linoleic total add-on of fermented liquid is 30g/L.
Zymotechnique (no longer repeating linoleic adding mode in the zymotechnique below):
In fermentor tank, inoculate sub-nutrient solution by above-mentioned formulated 10L fermented liquid through sterilizing after, the inoculum size of bacterial classification is 15% of a culture medium solution, i.e. 1.5L, and optical density(OD) (OD value) is 3; Regulate pH6.0 with sodium hydroxide, begin to cultivate; After beginning to cultivate 6h, glucose 350g (at first be mixed with 30% solution disinfection) is added fermentor tank, time spent 20h at a slow speed; Long bacterium and inducing temperature are 30 ℃ in the fermenting process, and fermenting is time 66h; Adopt sodium hydroxide and hydrochloric acid to regulate pH in the fermenting process, the pH value in the fermenting process is maintained between 6.0～6.6.
Bacterium mud is collected and is dry: process is with embodiment 1, and viable count gained bacterium powder viable count can reach 9.3*10 in the freeze-dried products
10Individual/g.The transformation efficiency that thalline transforms linolic acid generation conjugated linolic acid can reach 32.7%.
Claims (5)
1. one kind can effectively transform linolic acid for gripping linoleic microorganism strains altogether, is plant lactobacillus (Lactobacillus plantarum HSC 235) CCTCC No.M204051, utilizes this bacterial strain to prepare the method for high-activity lactic acid bacteria goods, comprising:
(1) preparation fermention medium, each components contents is percent weight in volume, i.e. g/L: yeast extract: 5.0-15.0, peptone: 10.0-30.0, sodium-chlor: 8.5, glucose: 20.0-30.0, linolic acid: 8-30, sodium acetate, anhydrous: 5, MgSO
47H
2O:0.2, MnSO
4H
2O:0.05, dibasic ammonium citrate: 2, CoCl
20.002, tween 80: 1ml/L-5ml/L, all the other are water, the pH value is 6.0～6.6;
(2) with bacterial strain CCTCC No.M204051, HSC235 carries out regular bevel activation, seed culture, and seed liquor inserts in the above-mentioned fermention medium and cultivates, ferments, zymotechnique:
A. the seed liquor inoculum size is 5%～15% of a fermentation culture based sols, and the optical density(OD) OD value of seed liquor is 1～3;
B. culture condition in the fermenting process: long bacterium temperature is 28 ℃～30 ℃, and fermentation time is 60h～72h;
C. take to add at a slow speed sugared mode, the sugaring amount is per 10 liters of fermented liquid 100g-350g, and 6h begins after inoculation, adds in the 48h;
D. adopt the linoleic mode that slowly adds;
E. adopt in the fermenting process and add sodium hydroxide and hydrochloric acid Automatic Balance Regulation pH value, the pH value is maintained between 6.0～6.6;
(3) fermentation ends, fermented liquid be through centrifuge dehydration, collecting cell, with protective material, vacuum lyophilization, the viable bacteria powder.
2. preparation method according to claim 1, it is characterized in that the linolic acid dissolving in the fermention medium or be embedded among a kind of in cyclodextrin, Tegin 55G, bovine serum albumin-Yelkin TTS, starch, bovine serum albumin, Yelkin TTS, the glycerine material that row adds again.
3. preparation method according to claim 1 and 2, it is characterized in that dissolving or embedding after the linolic acid addition manner be: linolic acid starting point concentration induced concentration is controlled in the 0.4g/L in the fermented liquid, and intermittent injecting again behind the inoculation culture 12h adds in the 48h.
4. preparation method according to claim 1 is characterized in that fermentation ends, fermented liquid by high speed freezing centrifuge with (7000 rev/mins of deionization sterilized water washings three times; 20min, 4 ℃), centrifugal collecting cell; with protective material, vacuum lyophilization promptly gets the viable bacteria powder.
5. preparation method according to claim 4 is characterized in that described protective material is a kind of in glycerine, skim-milk, trehalose, the N.F,USP MANNITOL.
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