CN101580809B - Method for preparing direct vat set Lactobacillus casei subsp. rhamnosus freeze-dried powder - Google Patents
Method for preparing direct vat set Lactobacillus casei subsp. rhamnosus freeze-dried powder Download PDFInfo
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Abstract
The invention discloses direct vat set Lactobacillus casei subsp. rhamnosus freeze-dried powder and a preparation method thereof. The method comprises the following steps: propagating Lactobacillus casei subsp. rhamnosus by a two-step method to make cells in a synchronous growth period as possible; and in a production culture medium, controlling pH to between 6.0 and 6.8 with NaOH solution on line, culturing for 16 to 24h at a temperature of between 35 and 37 DEG C, centrifugating, collecting bacterial sludge, adding protective agent solution, pre-freezing for 0.5 to 2.0h at a temperature of between -40 and -65 DEG C, making the thickness of bacteria on the wall of a container between 5 and 15 mm, and drying to prepare the direct vat set freeze-dried powder. The viable count of the freeze-dried powder reaches between 10<11> and 10<12>cfu/g; and the freeze-dried powder has the characteristics of good rehydration, good luster, good fluidity, easy storage and transportation, convenient production and the like, and can be directly used for fermentation production of pickles, sausages, acidophilus milk, L-lactic acid, corn flour, bean meal and peanut meal, and used as a probiotic preparation and the like.
Description
Technical field
The present invention relates to the preparation of lactic acid bacteria freeze drying powder, specifically be meant a kind of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder and preparation method thereof.
Background technology
The contained viable bacteria amount of traditional commercial starter is few, is about 10
7-10
8Individual/g, can not be directly used in production, need just can be used for dairy production through overactivation, amplification, this starter consumption is big, making processes is numerous and diverse, and quality is wayward, easily pollution microbes and phage, bacterial classification increases and changes along with passage number, causes leavened prod quality instability, and is repeatable poor.The direct-throwing lactobacillus starter do not need to refer to through overactivation, amplification and directly applies to a class novel fermentation agent of production, the key of this kind starter is to want viable count to want high, few with the assurance inoculum size, but fast rehydrating after the inoculation can be finished fermenting process in the relatively shorter time.
The key for preparing this starter is to increase the selection of bacterium culture condition, protective material and vacuum-drying condition because lactobacterium casei rhamnosyl subspecies can produce lactic acid during the fermentation, lactic acid run up to a certain degree can lactic acid bacteria inhibiting self growth; The harvest time of this bacterium is also very crucial, microorganism growth is divided into lag period, logarithmic phase, stationary phase and paracme, results adopt centrifugal and lyophilize, and are centrifugal different to the cell injury of different cell ages with lyophilize, and therefore the cell age of results is extremely important; Somatic cells after the results need add protective material and suspend, and the survival rate of somatic cells suspension density after to freeze-drying is influential; Bacterium is very easy to sustain damage in freezing dry process, therefore need to add suitable protective material, and protective material is divided into perviousness and impermeability, have only the perviousness protective material not only to have cell surface, but also can be penetrated into cell interior, thereby effectively stop the inside and outside freezing injury of cell, the freezing injury of protectant in addition retentiveness pair cell also has very important influence; Pre-freeze temperature, pre-freeze time and pre-freeze speed are also very big to the survival rate and the rehydration influence of bacterial cell, therefore generate little ice crystal in the pre-freeze process as far as possible, help the rehydration of lyophilize secondary fermentation agent.
Reported that Lactobacillus casei subsp.rhamnosus 6013 has good bacteriostatic action in the culture supernatant of MRS basic medium to S.aureus in " stability of potential probiotic bacterium lactobacillus rhamnosus in soybean cheese " that people such as Liu Dongmei delivered in " South China Science ﹠ Engineering University's journal (the natural science edition) " fifth phase in 2006.Application number be 200610123898.7 Chinese disclosure of the Invention " a kind of instant pickles that contain live probiotics and preparation method thereof ", its lactobacterium casei rhamnosyl subspecies 6013 can be used for the fermentation of pickles and finish fermentation at 24h.Reported that this bacterium can suppress the generation of nitrite in the pickle fermentation process in " Lactobacillus casei subsp.rhamnosus LCR 719 suppresses the nitrite in the pickles " that people such as Liu Dongmei delivered in " South China Science ﹠ Engineering University's journal (natural science edition) " the 7th phase in 2008.Reported in " lactobacillus rhamnosus fermentation production of L-lactic acid Optimum of culture medium " that people such as Wu Hui delivered in " foodstuffs industry science and technology " fourth phase in 2008 this bacterium can ferment produce L-lactic acid output up to 143.6g/L.For this bacterium can be increased bacterium to it and cultivate in industrial mass production and application, to obtain to be directly used in the high density fermentation agent of production.
The formal name used at school of lactic acid (Lactic acid) is a 2 hydroxy propanoic acid, and structural formula is CH
3CHOHCOOH, relative molecular mass are 90.08, because intramolecularly contains a unsymmetrical carbon, therefore have rotational isomerism, can divide into L-lactic acid and D-lactic acid, when both mix with equal proportion, promptly become mesomeric DL-lactic acid.Because human body has only the L-serum lactic dehydrogenase of metabolism L-lactic acid, the World Health Organization advocates and uses L-lactic acid to replace the DL-lactic acid that generally uses at present in food and pharmaceutical industries, WHO clearly stipulates, the amount that human body absorbs D-lactic acid every day is limited in below the 100mg/kg body weight, and L-lactic acid is not added restriction.It is significant to substitute the food that contains D-lactic acid and DL-lactic acid to study the food that contains L-lactic acid.
Application number be 200710048203.8 Chinese disclosure of the Invention a kind of " technology of preparing of high reactivity pickles direct-throwing microbial inoculum ", be with plant lactobacillus, short lactobacillus, Leuconostoc mesenteroides, yeast blended composite fungus agent according to a certain percentage.But the number of viable of each bacterium in the bacterium powder is not disclosed, do not use lactobacterium casei rhamnosyl subspecies, consuming time when being used for pickle fermentation is 48h, does not also relate to the situation of nitrite in the pickles of fermentation back, does not also have the packaging means and the terms of packing of clear and definite lyophilized powder simultaneously.
The patent No. be 200610010517.4 Chinese disclosure of the Invention " production method of zymophyte agent in direct input type for yoghourt ", be that bacterium such as thermophilus streptococcus, lactobacillus delbruockii subspecies bulgaricus are cultivated respectively, do not use lactobacterium casei rhamnosyl subspecies, this microbial inoculum also is composite fungus agent and only is used for Yoghourt fermentation that number of viable wherein is 10
4-10
10Individual/g, the while does not have the packaging means and the terms of packing of clear and definite lyophilized powder yet.
The patent No. is that 02144963.5 Chinese invention patent discloses " making method of direct putting type ferment agent for sour milk ", is that thermophilus streptococcus and bacillus bulgaricus are carried out mixed culture, the composite fungus agent of making through series of processes.But do not disclose the number of viable of each bacterium in the microbial inoculum, do not use lactobacterium casei rhamnosyl subspecies, this microbial inoculum only is used for Yoghourt fermentation.With nitrogen-filled packaging, but there are not airflow humidity and ambient moisture in the clear and definite wrapping process.
The patent No. is that the Chinese invention patent of 200410088476.1 (Granted publication CN 1285292C) discloses " a kind of direct-throwing composite bacterium powder product and production method thereof that is used to produce pickles ", is composite fungus agent, and the number of viable of bacterium powder is 10
8～10
9Individual/g, do not carry out on-line Control pH in the enlarged culturing process, so can't realize high-density culture, this microbial inoculum is used for pickle fermentation 36～50h consuming time, do not relate to the situation of nitrite in the pickles of fermentation back, the while does not have the packaging means of clear and definite lyophilized powder yet yet.
Above-mentioned prior art is not owing to adopt online regulation and control fermentation pH value; and the condition of selected enrichment medium, results cell age, protective material, pre-freeze and terms of packing etc. are improper; the ubiquity number of viable is not high, shortcomings such as fermentation time length, rehydration speed difference when being used to produce; and do not consider to contain carbohydrates such as lactose, glucose in the lyophilized powder problem of the meeting moisture absorption in storage process.
Summary of the invention
The objective of the invention is in order to solve the deficiency that above-mentioned prior art exists, it is 10 that a kind of viable count content is provided
11～10
12Cfu/g, fermentation time is short when being used to produce, rehydration is good direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder and preparation method thereof.This direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder can be used for fermentative production catsup and pickled vegetables (containing pickles), sausage, sour milk, L-lactic acid, Semen Maydis powder, dregs of beans, peanut meal and is used as probiotics preparation etc.
Purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder comprises the steps and processing condition:
The first step is with lactobacterium casei rhamnosyl subspecies (Lactobacillus casei subsp.rhamnosus 6013, abbreviation LCR6013) lyophilized powder places MRS (by de Man, Rogosa and Sharp invented in nineteen sixty, abbreviation MRS) in the substratum, in 28～30 ℃ of static cultivation 24h～36h, make mother starter; In parts by weight, described MRS culture medium prescription is: 0.8～1.0 part of peptone, 0.5～0.7 part of yeast extract, 1.5～2.0 parts of glucose, 0.1～0.2 part of dibasic ammonium citrate, 0.048～0.058 part in sal epsom, 0.5～1.0 part of extractum carnis, 0.1～0.2 part of dipotassium hydrogen phosphate, 0.015～0.025 part of manganous sulfate, 0.4～0.5 part of sodium acetate, 0.1～0.3 part of tween 80,94.0～96.0 parts of distilled water; Stirring and dissolving evenly the back with 0.08～0.10MPa, the 15～20min that sterilizes, be cooled to 28～30 ℃ standby;
Second step was 2～5: 100 ratio with volume ratio, in mother starter cut into operation starter substratum, in 28～30 ℃ of static cultivation 24h～36h, made working stock culture; In parts by weight, described working stock culture culture medium prescription is: 0.8～1.0 part of peptone, 0.5～0.7 part of yeast extract, 1.5～2.0 parts of glucose, 0.1～0.2 part of dibasic ammonium citrate, 0.048～0.058 part in sal epsom, 0.5～1.0 part of extractum carnis, 0.1～0.2 part of dipotassium hydrogen phosphate, 0.015～0.025 part of manganous sulfate, 0.4～0.5 part of sodium acetate, 0.1～0.3 part of tween 80,6.0～8.0 parts of tomato juices, 4.5～6.0 parts of Radix Dauci Sativae juices, 79.8～85.4 parts of 0.02～0.04 part in xitix and distilled water; Stirring and dissolving evenly the back with 0.08～0.10MPa, the 15～20min that sterilizes, be cooled to 28～30 ℃ standby;
The 3rd step was 2～5: 100 ratio with volume ratio, working stock culture is inserted in the production substratum, cultivate in the fermentor tank under 35～37 ℃, adjusting pH in culturing process with NaOH solution is 6.0～6.8, mixing speed is 100～150rpm, collect after cultivating 16～24h, get the zymophyte suspension; In parts by weight, described production culture medium prescription consists of: 2.5～3.0 parts of peptones, 0.5～0.7 part of yeast extract, 1.5～2.0 parts of glucose, 0.1～0.2 part of dibasic ammonium citrate, 0.048～0.058 part in sal epsom, 0.5～1.0 part of extractum carnis, 0.1～0.2 part of dipotassium hydrogen phosphate, 0.015～0.025 part of manganous sulfate, 0.4～0.5 part of sodium acetate, 0.1～0.3 part of tween 80,6.0～8.0 parts of tomato juices, 4.5～6.0 parts of Radix Dauci Sativae juices, 0.02～0.04 part in xitix, 78.0～83.7 parts of distilled water; Stirring and dissolving evenly the back with 0.08～0.10MPa, the 15～20min that sterilizes, be cooled to 35～37 ℃ standby;
The 4th step, in 5000～8000rpm, centrifugal 20～25min gave up supernatant liquor with the zymophyte suspension, collected bacterium mud;
The 5th step was 3～5: 1 adding complex protection agent solution by the normal saline solution of complex protection agent solution and the volume ratio of bacterium mud in bacterium mud, mixed to be protective material suspension; In parts by weight, described composite protectant solution formula consists of: 5～10 parts of trehaloses, 5～10 parts of skim-milks, 0.9 part in sodium-chlor, 79.1～89.1 parts of distilled water stir after the dissolving, with 0.08～0.10MPa sterilization, 15～20min, it is standby to be cooled to normal temperature;
The 6th step placed tray with protective material suspension, was-40 ℃～-65 ℃ following pre-freeze 0.5h～2.0h, and making protective material suspension thickness is 5～15mm;
The protective material suspension of the 7th step with the 6th step gained places vacuum freeze drier, is-40 ℃～-65 ℃ in condenser temperature, and vacuum tightness is 1.3～13.0Pa, and dry 18～20h gets lyophilized powder, and lyophilized powder moisture weight content is lower than 3%, and viable count reaches 10
11～10
12Cfu/g;
The 8th step, to be 50～60% constant humidity air with humidity returned to normal temperature with the temperature of lyophilized powder, in humidity is 50～60% environment, carries out aluminum-plastic packagedly with the air-flow wrapping machine, and the humidity of the aseptic dry air that screw filling machine is used should not be higher than 20%.
For further realizing the object of the invention, the 3rd step working stock culture is preferably 3: 100 with the volume ratio of producing substratum, and the concentration of NaOH solution is preferably 1～2mol/L, and pH is preferably 6.8, and leavening temperature is preferably 37 ℃.
Trehalose is preferably 10 parts in described the 5th step, and skim-milk is preferably 10 parts, and sodium-chlor is preferably 0.9 part.
The leavening temperature in the first step or second step is preferably 30 ℃, and incubation time is preferably 24h.
The tray thickness in the 6th step is 40～50mm.
The present invention compared with prior art has following advantage and beneficial effect:
(1) working stock culture of the present invention is cultivated successively with two kinds of substratum, makes the activation as far as possible of lactobacterium casei rhamnosyl subspecies, and locates the synchronous growth state.
(2) the used production substratum of the present invention adopts tomato juice, Radix Dauci Sativae juice and xitix can guarantee needed somatomedin in the thalli growth process, promotes the thalline raised growth.
(3) the used lyophilized vaccine of the present invention is the normal saline solution of 5～10% trehaloses and 5～10% skim-milks, has guaranteed that the viable count of lyophilized powder reaches 10
11～10
12Cfu/g.Used substratum all is edible, and protective material skim-milk and trehalose also are edible food ingredients, and acute toxinology experiment shows that this bacterial classification is nontoxic spendable, and this bacterial strain also is a Chinese industrial microbial strains preservation center preservation bacterial classification.
When (4) lyophilized powder of the present invention was used for the production of L-lactic acid, in 37 ℃ of fermentation 96～120h, control pH wherein was 6.5, can make the output of total lactic acid reach 150g/L, and wherein the content of L-lactic acid is more than 91%; When being directly used in the fermentative production of probiotic bacterium pickles (catsup and pickled vegetables), fermentation time is 24～30h, and this bacterium can suppress the generation of nitrite in the fermenting process, and it is 92.3% that fermentation stops back L-lactic acid content; When being used for fermented sausages, at 15～20 ℃ of bottom fermentation 120～240h, the pH that can make fermented sausages is 5.2～5.8, and L-lactic acid relative content is 91.2%, can not use nitrite in the fermenting process; When being used for fermented bean dregs (peanut meal), the water that adds the raw material isodose, at 25～37 ℃ of bottom fermentation 24～72h, can carry the pH that makes fermented bean dregs (peanut meal) is 5.0～6.0, wherein L-lactic acid relative content is 91.5%, protein content can improve 5.5～10.5%, and to make macromolecular protein degradation be below the 24kDa; When being used for cassiri, add the water of raw material isodose, at 25～37 ℃ of bottom fermentation 24～72h, the pH that can make fermented maize is 4.0～5.0, and wherein the relative content of L-lactic acid is 93.0%; When being used for the production of fermented yogurt, in 41 ℃ of fermentation 4.5～5.5h, fermented yogurt tart flavour is pure, and is dense, wherein contains the L-lactic acid more than 91% after fermentation stops; Therefore can be used for fermentation production of L-lactic acid, probiotic bacterium pickles, sausage, dregs of beans, peanut meal, corn and sour milk etc., and to make L-lactic acid relative content be more than 91%.
(5) the lyophilized powder viable count height of the present invention preparation, prevented from caking, rehydration are fine.
(6) preserve conveniently, be easy to be used for explained hereafter, reduce inoculation, spread cultivation link and reduced pollution, guaranteed the stability of quality product, shortened fermentation period, solved the food-safety problem of producing, meet needs of scale production.
Description of drawings
Fig. 1 is the growth curves of lactobacterium casei rhamnosyl subspecies in basic MRS and embodiment 1 modified MRS culture medium;
The change curve of LCR 6013 fermentation production of L-lactic acid that Fig. 2 spreads cultivation for the LCR 6013 lyophilized powder fermentation production of L-lactic acid of embodiment 4 usefulness with traditional method;
Fig. 3 is for detecting the spirogram that contains of L-lactic acid with the high-efficient liquid phase technique of chiral column Chirex 3126 among the embodiment 5;
Fig. 4 a is the L-lactic acid content high-efficient liquid phase chromatogram of the Semen Maydis powder of 8 couples of lactic acid coccus fermentation 48h in the same old way of embodiment;
Fig. 4 b is the L-lactic acid high-efficient liquid phase chromatogram of the Semen Maydis powder of embodiment 8 usefulness lyophilized powder LCR 6013 fermentation 48h.
Embodiment
For better understanding the present invention, below in conjunction with embodiment the present invention is done detailed description further, but the scope of protection of present invention is not limited to the scope that embodiment represents.
The preparation of lyophilized powder:
Embodiment 1
The first step is with lactobacterium casei rhamnosyl subspecies (numbering CICC 6013, Lactobacillus casei subsp.rhamnosus 6013, the preservation of Chinese industrial microbial strains preservation administrative center, address: No. 32, Xiaoyun Road, Chaoyang District, Beijing City, postcode: lyophilized powder 100027) places the MRS substratum, in 30 ℃ of static cultivation 24h, make mother starter; MRS culture medium preparation: peptone 10g, yeast extract 5.0g, glucose 20.0g, dibasic ammonium citrate 2.0g, sal epsom 0.58g, extractum carnis 10.0g, dipotassium hydrogen phosphate 2.0g, manganous sulfate 0.25g, sodium acetate 5.0g, tween 80 1mL, distilled water 944.0g, in 0.08MPa sterilization 20min, be cooled to 30 ℃ standby.
Second step was 3: 100 a ratio with volume ratio, in mother starter cut into operation starter substratum, in 30 ℃ of static cultivation 24h, made working stock culture.Working stock culture culture medium preparation: peptone 10g, yeast extract 5.0g, glucose 20.0g, dibasic ammonium citrate 2.0g, sal epsom 0.58g, extractum carnis 10.0g, dipotassium hydrogen phosphate 2.0g, manganous sulfate 0.25g, sodium acetate 5.0g, tween 80 1mL, tomato juice 80mL, Radix Dauci Sativae juice 60mL, xitix 0.4g, use distilled water 804.0g,, be cooled to 30 ℃ and promptly obtain the working stock culture substratum in 0.08MPa sterilization 20min.
The making of tomato juice or Radix Dauci Sativae juice: tomato or Radix Dauci Sativae are squeezed the juice with hollander, and in 8000rpm, centrifugal 20min collects supernatant liquor, promptly obtains tomato juice or Radix Dauci Sativae juice with expressed juice.
The 3rd step was 2: 100 ratio with volume ratio, working stock culture insert being produced in the substratum, cultivated in the fermentor tank in 37 ℃ under, is that the NaOH solution maintenance pH of 2mol/L is 6.8 in culturing process concentration, mixing speed is 150rpm, collects the zymophyte suspension behind the cultivation 20h; Production culture medium preparation: peptone 30g, yeast extract 5.0g, glucose 20.0g, dibasic ammonium citrate 2.0g, sal epsom 0.58g, extractum carnis 10.0g, dipotassium hydrogen phosphate 2.0g, manganous sulfate 0.25g, sodium acetate 5.0g, tween 80 1mL, tomato juice 80mL, Radix Dauci Sativae juice 60mL, xitix 0.4g, distilled water 790.0g in 0.08MPa sterilization 20min, is cooled to 37 ℃ and promptly obtains producing substratum.
The 4th step, in 8000rpm, centrifugal 20min gave up supernatant liquor with the zymophyte suspension, collected bacterium mud;
The 5th step was 3: 1 adding complex protection agent solutions by complex protection agent solution and bacterium mud volume ratio in bacterium mud, mixed to be protective material suspension; In parts by weight, described composite protectant solution formula consists of: trehalose 10g, and skim-milk 10g, sodium-chlor 0.9g, distilled water 79.1g stirs after the dissolving, and with the 0.08MPa 20min that sterilizes, it is standby to be cooled to normal temperature;
It is the 40mm tray that the 6th step placed thickness with protective material suspension, in-65 ℃ of following pre-freeze 0.5h, makes its thickness on wall of container be about 10mm;
The tray that the 7th step will be spread thalline places vacuum freeze drier, is-65 ℃ in condenser temperature, and vacuum tightness is 1.3Pa, dry 18h, and the moisture content of lyophilized powder is 2.0%, viable count reaches 10
11Cfu/g.
The 8th step, to be 50% constant humidity air with humidity returned to normal temperature with the temperature of lyophilized powder, in humidity is 50% environment, carry out aluminum-plastic packagedly with the air-flow wrapping machine, the humidity of the aseptic dry air that screw filling machine is used is 15%, has guaranteed that lyophilized powder is nonhygroscopic.
By further investigation to lactobacterium casei rhamnosyl subspecies (Lactobacillus casei subsp.rhamnosus 6013) culture condition; fermentation culture conditions, fermentation time, lyophilized vaccine and the vacuum-drying condition of strict control starter; select the expansion fermentation culture twice; make all cells be in synchronous growth period as far as possible; in modified MRS culture medium, carry out enlarged culturing again; on-line Control fermentation pH and strict control harvest time, viable count reaches 10
11Cfu/g, and guaranteed that this cell age cell avoids the centrifugal and freeze dried damage of lyophilize.With fermentation time the logarithmic value of lactobacterium casei rhamnosyl subspecies (Lactobacillus casei subsp.rhamnosus 6013) viable count is made curve such as Fig. 1, as can be seen from Figure 1, in the MRS basic medium, when 20h bacterium number reached maximum, viable count only was 4.50 * 10
8Cfu/mL, and in the enrichment medium of optimizing through promptly entering logarithmic phase after the short lag phase, about 12h, enter logarithmic phase latter stage, 16h bacterium number can reach 1.67 * 10
9Cfu/mL, than the raising of MRS basic medium nearly 4 times.With comparing of MRS basic medium, when growing, pH descends comparatively slow in the MRS basic medium of improvement, the increase of this explanation nitrogenous source organic acid of growth later stage bacterial metabolism that can neutralize, thus help the thalline breeding.LCR 6013 about 16h left and right sides bacterium number in improved culture medium reaches the highest, is about 1.67 * 10
9Cfu/mL, 16～28h is in stationary phase, and viable count remains unchanged substantially.Therefore, best thalline harvest time is between 16～24h.The trehalose of 10% (quality) can make cell survival rate reach 85.8%, and the trehalose that adds 15% (quality) can make its surviving rate reach 91.9%, trehalose than other any sugar to protecting dried biomaterial more effective.And select for use macromole protective material skim-milk as drying medium, and because: (1) skim-milk can prevent cell injury by the composition of stabilizing cell membrane; (2) in freezing dry process, produce vesicular structure, make dehydration be more prone to; (3) skim-milk itself contains protein and can be the effect that cell provides a protective layer.Different MnSO
4Concentration has bigger influence to the thalline survival rate, and suitable concentration can improve the survival rate of thalline.And it is improper in the middle of the existing technology owing to the fermention medium of selecting; not through carrying out fermentative production after twice the enlarged culturing again, there is not online strict control fermentation pH, there is not strict control results cell age; the selection of lyophilized vaccine is more single, and the viable count that obtains at last is all generally lower.
Embodiment 2
The first step is with lactobacterium casei rhamnosyl subspecies (numbering CICC 6013, Lactobacillus casei subsp.rhamnosus 6013, the preservation of Chinese industrial microbial strains preservation administrative center, address: No. 32, Xiaoyun Road, Chaoyang District, Beijing City, postcode: lyophilized powder 100027) places the MRS substratum, in 28 ℃ of static cultivation 36h, make mother starter; MRS culture medium preparation: peptone 8.0g, yeast extract 7.0g, glucose 15.0g, dibasic ammonium citrate 1.0g, sal epsom 0.48g, extractum carnis 5.0g, dipotassium hydrogen phosphate 1.0g, manganous sulfate 0.15g, sodium acetate 4.0g, tween 80 3mL, distilled water 960g, in 0.10MPa sterilization 15min, be cooled to 28 ℃ standby.
Second step was 5: 100 a ratio with volume ratio, in mother starter cut into operation starter substratum, in 28 ℃ of static cultivation 36h, made working stock culture.Working stock culture culture medium preparation: peptone 8.0g, yeast extract 7.0g, glucose 15.0g, dibasic ammonium citrate 1.0g, sal epsom 0.48g, extractum carnis 5.0g, dipotassium hydrogen phosphate 1.0g, manganous sulfate 0.15g, sodium acetate 4.0g, tween 80 3mL, tomato juice 60mL, Radix Dauci Sativae juice 45mL, xitix 0.2g, distilled water 850.0g in 0.1MPa sterilization 15min, is cooled to 28 ℃ and promptly obtains the working stock culture substratum.
The making of tomato juice or Radix Dauci Sativae juice: tomato and Radix Dauci Sativae are squeezed the juice with hollander, and in 8000rpm, centrifugal 20min collects supernatant liquor, promptly obtains tomato juice or Radix Dauci Sativae juice with expressed juice.
The 3rd step was 5: 100 a ratio with volume ratio, working stock culture is inserted produce in the substratum, cultivated in the fermentor tank under 35 ℃, keeping pH in culturing process with the NaOH solution of 1mol/L is 6.5, mixing speed is 100rpm, collects behind the cultivation 24h, is the zymophyte suspension; Production culture medium preparation: peptone 25g, yeast extract 7.0g, glucose 15.0g, dibasic ammonium citrate 1.0g, sal epsom 0.48g, extractum carnis 5.0g, dipotassium hydrogen phosphate 1.0g, manganous sulfate 0.15g, sodium acetate 4.0g, tween 80 3mL, tomato juice 60mL, Radix Dauci Sativae juice 45mL, xitix 0.2g, distilled water 835.0g in 0.10MPa sterilization 15min, is cooled to 35 ℃ and promptly obtains producing substratum.
The 4th step, in 6500rpm, centrifugal 22min gave up supernatant liquor with the zymophyte suspension, collected bacterium mud;
The 5th step was 5: 1 adding complex protection agent solutions by the solution of composite protectant and bacterium mud volume ratio in bacterium mud, mixed to be protective material suspension; The preparation of composite protectant: trehalose 5.0g, skim-milk 5.0g, sodium-chlor 0.9g, distilled water 89.1g stirs after the dissolving, and with the 0.1MPa 15min that sterilizes, it is standby to be cooled to normal temperature;
It is the 50mm tray that the 6th step placed thickness with protective material suspension, is-40 ℃ of following pre-freeze 2.0h, and making its thickness on wall of container is 15mm;
The tray that the 7th step will be spread thalline places vacuum freeze drier, is-40 ℃ in condenser temperature, and vacuum tightness is 13Pa, dry 20h, and the moisture content of lyophilized powder is 3.0%, viable count reaches 10
12Cfu/g.
The 8th step, to be 60% constant humidity air with humidity returned to normal temperature with the temperature of lyophilized powder, in humidity is 60% environment, carry out aluminum-plastic packagedly with the air-flow wrapping machine, the humidity of the aseptic dry air that screw filling machine is used is 20%, has guaranteed that lyophilized powder is nonhygroscopic.
Embodiment 3
The first step is with lactobacterium casei rhamnosyl subspecies (numbering CICC 6013, Lactobacillus casei subsp.rhamnosus 6013, the preservation of Chinese industrial microbial strains preservation administrative center, address: No. 32, Xiaoyun Road, Chaoyang District, Beijing City, postcode: lyophilized powder 100027) places the MRS substratum, in 29 ℃ of static cultivation 30h, make mother starter; MRS culture medium preparation: peptone 9.0g, yeast extract 6.0g, glucose 17.5g, dibasic ammonium citrate 1.5g, sal epsom 0.53g, extractum carnis 7.5g, dipotassium hydrogen phosphate 1.5g, manganous sulfate 0.20g, sodium acetate 4.5g, tween 80 2mL, distilled water 950.0g, in 0.09MPa sterilization 18min, be cooled to 29 ℃ standby.
Second step was 2: 100 a ratio with volume ratio, in mother starter cut into operation starter substratum, in 29 ℃ of static cultivation 30h, made working stock culture.Working stock culture culture medium preparation: peptone 9.0g, yeast extract 6.0g, glucose 17.5g, dibasic ammonium citrate 1.5g, sal epsom 0.53g, extractum carnis 7.5g, dipotassium hydrogen phosphate 1.5g, manganous sulfate 0.20g, sodium acetate 4.5g, tween 80 2mL, tomato juice 70mL, Radix Dauci Sativae juice 53mL, xitix 0.3g, distilled water 828.0g in 0.09MPa sterilization 18min, is cooled to 29 ℃ and promptly obtains the working stock culture substratum.
The making of tomato juice or Radix Dauci Sativae juice: tomato and Radix Dauci Sativae are squeezed the juice with hollander, and in 8000rpm, centrifugal 20min collects supernatant liquor, promptly obtains tomato juice or Radix Dauci Sativae juice with expressed juice.
The 3rd step was 3.5: 100 ratio with volume ratio, working stock culture insert being produced in the substratum, cultivated in the fermentor tank in 36 ℃ under, is 6.0 in culturing process with the NaOH solution maintenance pH of 1mol/L, mixing speed is 120rpm, collects the zymophyte suspension behind the cultivation 16h; Production culture medium preparation: peptone 27.5g, yeast extract 6.0g, glucose 17.5g, dibasic ammonium citrate 1.5g, sal epsom 0.53g, extractum carnis 7.5g, dipotassium hydrogen phosphate 1.5g, manganous sulfate 0.20g, sodium acetate 4.5g, tween 80 2mL, tomato juice 70mL, Radix Dauci Sativae juice 55mL, xitix 0.3g, distilled water 807.0g in 0.09MPa sterilization 18min, is cooled to 36 ℃ and promptly obtains producing substratum.
The 4th step, in 5000rpm, centrifugal 25min gave up supernatant liquor with the zymophyte suspension, collected bacterium mud;
The 5th step was 4: 1 adding complex protection agent solutions by complex protection agent solution and bacterium mud volume ratio in bacterium mud, mixed to be protective material suspension; The preparation of complex protection agent solution: trehalose 7.5g, skim-milk 7.5g, sodium-chlor 0.9g, distilled water 84.1g stirs after the dissolving, and with the 0.09MPa 18min that sterilizes, it is standby to be cooled to normal temperature;
It is the 45mm tray that the 6th step placed thickness with protective material suspension, is-50 ℃ of following pre-freeze 1h, and making its thickness on wall of container is 5mm;
The tray that the 7th step will be spread thalline places vacuum freeze drier, is-50 ℃ in condenser temperature, and vacuum tightness is 7.5Pa, dry 19h, and the moisture content of lyophilized powder is 2.5%, viable count reaches 10
12Cfu/g.
The 8th step, to be 55% constant humidity air with humidity returned to normal temperature with the temperature of lyophilized powder, in humidity is 55% environment, carry out aluminum-plastic packagedly with the air-flow wrapping machine, the humidity of the aseptic dry air that screw filling machine is used is 15%, has guaranteed that lyophilized powder is nonhygroscopic.
The application of lyophilized powder:
The fermentative production of embodiment 4L-lactic acid
With the lyophilized powder of embodiment 1 preparation in seed culture medium, cultivate 24h for 37 ℃, obtain seed liquor, inoculum size with 5% (volume) is inoculated into seed liquor in the fermention medium, cultivate 110h (can be 108h-120h) for 37 ℃, NaOH solution control pH with 2-4mol/L is 6.5, mixing speed is 120rpm (can be 100-150rpm), the output that obtains L-lactic acid reaches 150g/L, as shown in Figure 2, with the content of the L-lactic acid in the high-efficient liquid phase technique detection fermenting process of chiral column Chirex 3126, analytical instrument: the high performance liquid chromatograph system is equipped with Waters 600 pump hyperchannel delivery systems; Waters 717 automatic samplers; Waters 2996 PDA diode-array detectors; Chromatographic column: chiral column Chirex 3126 (D)-penicillami, 4.6mm ID * 250mm L.Analysis condition: moving phase is that the copper-bath solvent of 0.002moL/L is the aqueous isopropanol of 5% (quality), uses preceding through 0.45 μ m membrane filtration, ultrasonic degas; Flow rate of mobile phase is 0.7mL/min; 30 ℃ of column temperatures; Diode array Waters 2996, the detection wavelength is 254nm; Standard and sample solution with preceding all through ultrasonic degas behind the 0.45 μ m membrane filtration; Sample size is 2 μ L; With Empower integration software integration, carry out the peak area method outer marking quantitative.As can be seen from Figure 2, carry out the L-lactic fermentation with highdensity LCR6013 lyophilized powder, L-lactic acid reaches 150g/L after fermenting 108 hours, and the LCR 6013 that traditional method spreads cultivation just reaches 86g/L behind fermentation 130h.
Lactic acid molecules contains a unsymmetrical carbon, therefore has rotational isomerism, divides into L (+)-lactic acid (being called for short L-lactic acid) and D (-)-lactic acid (being called for short D-lactic acid).Because but human body has only the L-serum lactic dehydrogenase of metabolism L-lactic acid, the World Health Organization advocates and uses L-lactic acid to replace the DL-lactic acid that generally uses at present in food and pharmaceutical industries.Therefore the technology of the many fermentation production of L-lactic acid of research energy seems very necessary.Wherein seed culture medium and fermention medium are formulated as follows:
Seed culture medium preparation: glucose 20g/L, peptone 10g/L, extractum carnis 10g/L, yeast powder 5g/L, sodium acetate 2g/L, ammonium citrate 2g/L, tween 80 1mL/L, MgSO
4.7H
2O 0.2g/L, K
2HPO
42g/L, MnSO
4H
2O0.05g/L is settled to 1000mL with distilled water, and pH 6.5, in 0.10MPa sterilization 20min, promptly obtains L-lactic acid-producing seed culture medium.
Fermention medium preparation: glucose 200g/L (or enzymolysis solution of a considerable amount of Semen Maydis powder), yeast powder 5g/L, peptone 10g/L (or enzymolysis solution of a considerable amount of dregs of beans), extractum carnis 15g/L, tween 80 1ml/L, tomato juice 50ml/L, cabbage juice 50ml/L is settled to 1000mL with producing with tap water, and pH 6.5, in 0.10MPa sterilization 20min, promptly obtain the preparation of L-lactic acid-producing fermention medium.
The preparation of tomato juice or cabbage juice: tomato or Chinese cabbage are squeezed the juice with juice extractor, and in 5000-8000rpm, centrifugal 20min collects supernatant liquor, promptly obtains tomato juice or cabbage juice with expressed juice.
Embodiment 5: the probiotic bacterium pickles
Select the not leaf mustard or the Chinese cabbage of mashed leaf, after cleaning up, drain surface-moisture, be cut into certain size and use the boiling water blanching, under gnotobasis, be cooled to 37 ℃, press the dish mass percent and insert 5% lyophilized powder embodiment 1 preparation, stir gently, with the aqua sterilisa sealing,, drain away the water and carry out vacuum packaging in 37 ℃ of fermentation 120h.Competent bubble stain liquid submergence vegetables to be arranged in the pickled vegetable making process, make the dish must not the emersion liquid level,, under clean environment, carry out as far as possible in the entire making process to satisfy milk-acid bacteria anaerobic growth and fermentation.After testing, the nitrite changing conditions of the probiotic bacterium pickles that present embodiment is produced is as shown in table 1, does not contain nitrite.Nitrite is a kind of potential carcinogens, and nitrite can react with proteinic degradation production amine in the food under suitable condition, generates the N-nitroso compound.The acute poisoning effect of nitrite is to cause methemoglobinemia, promptly the low Ferri-hemoglobin of oxygen carrying capacity in the blood of human body is oxidized to methemoglobin, and lose oxygen carrying capacity, thereby cause that histanoxia poisons, and live body and isolated test show that mutagenesis and carcinogenic effect are arranged.Measure with the content (hydrochloric acid-naphthalene-ethylenediamine method of GB/T5009.33-2003 is measured the nitrite method) of nitrite in fermentation of LCR 6013 lyophilized powders and the pickles expressed juice of the different fermentations time of the traditional zymotic that does not add bacterium as shown in table 1, can find out from table 1, the content of the nitrite in the fresh leaf mustard is 2.87mg/L, the pickles of inoculation LCR 6013 lyophilized powders are reduced to 1.35mg/L behind fermentation 24h, detect less than nitrite behind 48h.And nonvaccinated content to the nitrite in the same old way presents first rising, and the peak value 45.300mg/ of nitrite appears in the trend that the back reduces at 48h.As seen, 6013 pairs of abnormal accumulation that suppress nitrite in the pickles of interpolation lyophilized powder LCR are very effective.Measure the content that LCR 6013 lyophilized powders are used for the L-lactic acid of pickle fermentation process with high-efficient liquid phase technique, as shown in Figure 3, among the figure retention time be 9.214min be L-lactic acid, the content that accounts for total lactic acid is 92.3% (detection method is with the detection of Fig. 1).In addition, the pickled vegetable making time weak point that present embodiment utilizes the lyophilized powder of embodiment 1 preparation to make is 24 hours, and traditional method needs 168 hours, add in the pickles of LCR 6013 lyophilized powders, LCR 6013 is dominant microfloras, can grow fast and product acid fast, makes that fermentation has promptly reached fermentation termination to pickles through 24h, pH is 3.50, the good and raciness of color.
The Changing Pattern of nitrite in the table 1 pickle fermentation process
Embodiment 6: fermented sausages
After raw meat picked a bone, shred and cut and mix, by weight, add 3.0% sucrose, 2.0% salt and 1.5% cooking wine, mix the lyophilized powder of back inoculation embodiment 2 preparations, making by weight, viable count reaches 10
7Individual/g, mix the back bowel lavage, tie up under 20 ℃, relative humidity are 95% environment and ferment, when fermenting, stop fermentation to pH 5.0, put into 15 ℃, relative humidity is to carry out afterripening fermentation 7 days under 80% the environment, goes into altar and pickles into various local flavors.Because the viable bacteria content height carries out the dominant microflora fermentation, need not to add nitrite, it is very pure that the fermented sausages of producing is grown smell, and the color nature never contains nitrite.
The production of embodiment 7 fermented bean dregs and peanut meal
By weight, 1: 1 dregs of beans (going back peanut meal) is mixed with water, at 0.10MPa sterilization 20min, be cooled to 37 ℃ of lyophilized powders that insert embodiment 2, making by weight, viable count reaches 10
7Individual/g, in 30 ℃ of closed environment bottom fermentation 36h, pH is 5.0-6.0 after the fermentation ends, and tunning is carried out the hot-air flow drying, 250 ℃ of dry inlet temperature controls, 150 ℃ of mixing temperature controls, 75 ℃ of drop temperature controls.The relative content of L-lactic acid is 91.5%, and Protein content can improve 5.5%, and the dried material moisture controlled is below 12%, and making macromolecular protein degradation is below the 24kDa.Owing to used the lyophilized powder of high viable count,, improved fermentation efficiency and improved protein content, reduced proteinic molecular weight and help digesting and assimilating so shortened fermentation time greatly.
The production of embodiment 8 fermented maizes
By weight, 1: 1 Semen Maydis powder is mixed with water, be cooled to the lyophilized powder that inserts embodiment 3 about 37 ℃, making by weight, viable count reaches 10
7Individual/g, in 30 ℃ of closed environment bottom fermentation 48h, pH is 4.0～5.0 after the fermentation ends, and tunning is carried out the hot-air flow drying, 280 ℃ of dry inlet temperature controls, 180 ℃ of mixing temperature controls, 80 ℃ of drop temperature controls.Shown in Fig. 4 b (detection method is with the detection of Fig. 2), L-lactic acid high-efficient liquid phase chromatogram with the Semen Maydis powder of lyophilized powder LCR 6013 fermentation 48h, can find out that from Fig. 4 b the content that L-lactic acid accounts for total lactic acid is 93.0%, Fig. 4 a is the L-lactic acid content to the corn fermentation of in the same old way lactic acid coccus, can find out (detection method is with the detection of Fig. 2) from Fig. 4 a, the content that L-lactic acid accounts for total lactic acid only is 18.80%.The dried material moisture controlled is below 12%.
The production of embodiment 9 fermented yogurts
By weight, whole milk powder 12.5%, white sugar 8.5%, yoghourt stabilizer 0.3%, be settled to scale with producing with sweet water, behind the uniform mixing, be preheated to 60 ℃ and carry out homogeneous, be cooled to 41 ℃, by the lyophilized powder that inserts 30g in the milk per ton, in 41 ℃ of fermentation 5.0h, the acidity of sour milk reaches 65 ° of T after the fermentation ends, and the content that L-lactic acid accounts for total lactic acid reaches 91.5%.
Lyophilized powder carries out aluminum-plastic packaged with screw filling machine immediately.Being 50% constant humidity air with humidity returns to normal temperature with the temperature of lyophilized powder, in humidity is 50～60% environment, carry out aluminum-plastic packagedly with the air-flow wrapping machine, the humidity of the aseptic dry air that screw filling machine is used should not be higher than 20%, has guaranteed that lyophilized powder is nonhygroscopic.Lyophilized powder viable count height prepared in accordance with the present invention, prevented from caking, rehydration are fine.
Claims (8)
1. the preparation method of a direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder is characterized in that comprising the steps and processing condition:
The first step places the MRS substratum with the lyophilized powder of lactobacterium casei rhamnosyl subspecies, in 28～30 ℃ of static cultivation 24h～36h, makes mother starter; In parts by weight, described MRS culture medium prescription is: 0.8～1.0 part of peptone, 0.5～0.7 part of yeast extract, 1.5～2.0 parts of glucose, 0.1～0.2 part of dibasic ammonium citrate, 0.048～0.058 part in sal epsom, 0.5～1.0 part of extractum carnis, 0.1～0.2 part of dipotassium hydrogen phosphate, 0.015～0.025 part of manganous sulfate, 0.4～0.5 part of sodium acetate, 0.1～0.3 part of tween 80,94.0～96.0 parts of distilled water; Stirring and dissolving evenly the back with 0.08～0.10MPa, the 15～20min that sterilizes, be cooled to 28～30 ℃ standby; Described lactobacterium casei rhamnosyl subspecies are preserved in Chinese industrial microbial strains preservation administrative center, are numbered CICC 6013, called after Lactobacillus casei subsp.rhamnosus 6013;
Second step was 2～5: 100 ratio with volume ratio, in mother starter cut into operation starter substratum, in 28～30 ℃ of static cultivation 24h～36h, made working stock culture; In parts by weight, described working stock culture culture medium prescription is: 0.8～1.0 part of peptone, 0.5～0.7 part of yeast extract, 1.5～2.0 parts of glucose, 0.1～0.2 part of dibasic ammonium citrate, 0.048～0.058 part in sal epsom, 0.5～1.0 part of extractum carnis, 0.1～0.2 part of dipotassium hydrogen phosphate, 0.015～0.025 part of manganous sulfate, 0.4～0.5 part of sodium acetate, 0.1～0.3 part of tween 80,6.0～8.0 parts of tomato juices, 4.5～6.0 parts of Radix Dauci Sativae juices, 79.8～85.4 parts of 0.02～0.04 part in xitix and distilled water; Stirring and dissolving evenly the back with 0.08～0.10MPa, the 15～20min that sterilizes, be cooled to 28～30 ℃ standby;
The 3rd step was 2～5: 100 ratio with volume ratio, working stock culture is inserted in the production substratum, cultivate in the fermentor tank under 35～37 ℃, adjusting pH in culturing process with NaOH solution is 6.0～6.8, mixing speed is 100～150rpm, collect after cultivating 16～24h, get the zymophyte suspension; In parts by weight, described production culture medium prescription consists of: 2.5～3.0 parts of peptones, 0.5～0.7 part of yeast extract, 1.5～2.0 parts of glucose, 0.1～0.2 part of dibasic ammonium citrate, 0.048～0.058 part in sal epsom, 0.5～1.0 part of extractum carnis, 0.1～0.2 part of dipotassium hydrogen phosphate, 0.015～0.025 part of manganous sulfate, 0.4～0.5 part of sodium acetate, 0.1～0.3 part of tween 80,6.0～8.0 parts of tomato juices, 4.5～6.0 parts of Radix Dauci Sativae juices, 0.02～0.04 part in xitix, 78.0～83.7 parts of distilled water; Stirring and dissolving evenly the back with 0.08～0.10MPa, the 15～20min that sterilizes, be cooled to 35～37 ℃ standby;
The 4th step, in 5000～8000rpm, centrifugal 20～25min gave up supernatant liquor with the zymophyte suspension, collected bacterium mud;
The 5th step was 3～5: 1 adding complex protection agent solution by the normal saline solution of complex protection agent solution and the volume ratio of bacterium mud in bacterium mud, mixed to be protective material suspension; In parts by weight, described composite protectant solution formula consists of: 5～10 parts of trehaloses, 5～10 parts of skim-milks, 0.9 part in sodium-chlor, 79.1～89.1 parts of distilled water stir after the dissolving, with 0.08～0.10MPa sterilization, 15～20min, it is standby to be cooled to normal temperature;
The 6th step placed tray with protective material suspension, was-40 ℃～-65 ℃ following pre-freeze 0.5h～2.0h, and making protective material suspension thickness is 5～15mm;
The protective material suspension of the 7th step with the 6th step gained places vacuum freeze drier, is-40 ℃～-65 ℃ in condenser temperature, and vacuum tightness is 1.3～13.0Pa, and dry 18～20h gets lyophilized powder, and lyophilized powder moisture weight content is lower than 3%, and viable count reaches 10
11～10
12Cfu/g;
The 8th step, to be 50～60% constant humidity air with humidity returned to normal temperature with the temperature of lyophilized powder, in humidity is 50～60% environment, carries out aluminum-plastic packagedly with the air-flow wrapping machine, and the humidity of the aseptic dry air that screw filling machine is used should not be higher than 20%.
2. the preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder according to claim 1 is characterized in that: the 3rd step working stock culture is 3: 100 with the volume ratio of producing substratum.
3. the preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder according to claim 1 is characterized in that: the concentration of NaOH solution is 1～2mol/L in described the 3rd step.
4. the preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder according to claim 1 is characterized in that: pH is 6.8 in described the 3rd step.
5. the preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder according to claim 1 is characterized in that: trehalose is 10 parts in described the 5th step, and skim-milk is 10 parts, and sodium-chlor is 0.9 part.
6. the preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder according to claim 1 is characterized in that: the first step or the leavening temperature in second step are 30 ℃, and incubation time is 24h.
7. the preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder according to claim 1 is characterized in that: described the 3rd step leavening temperature is 37 ℃.
8. the preparation method of direct-throwing lactobacterium casei rhamnosyl subspecies lyophilized powder according to claim 1 is characterized in that: the tray thickness in the 6th step is 40～50mm.
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