CN103141722B - Preparation method of lactobacillus rhamnosus soybean milk direct vat set starter - Google Patents
Preparation method of lactobacillus rhamnosus soybean milk direct vat set starter Download PDFInfo
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Abstract
The invention discloses a preparation method of lactobacillus rhamnosus soybean milk direct vat set starter. Lactobacillus rhamnosus is subjected to high-density culture by a low-price enrichment medium, centrifugal concentration and separation, mixed dissolution with high-efficiency composite protective agent and lyophilization to prepare the lactobacillus casei soybean milk direct vat set starter. According to the preparation method, the viable organism of the prepared starter is over 1*10<11>cfu/g; after the starter is sealed under normal pressure and preserved at 4 DEG C for 1 year, the content of the viable organism is still over 10<10>cfu/g, and a higher fermentation activity is stilled maintained; and soybean milk is fermented at 37 DEG C with the inoculum size being lower than one ten-thousandth, and the milk can be cured after 4 to 5 hours, wherein the soybean yoghurt product fermented by the starter has the acidity being between 70oT and 80oT, the pH value being about 4.6, and good sense and flavor. The starter can be used in the fermented dairy products industry and vegetable protein processing industry.
Description
Technical field
The preparation method who the present invention relates to a kind of lactobacillus rhamnosus soybean yogurt throw type leaven, belongs to cultured milk prod and vegetable-protein processing technique field.
Background technology
Probiotic bacterium means that with people and animal and plant body keep symbiotic relationship host is had to the general name of a large quasi-microorganism of health-promoting effect.At present, internationally recognized and typical probiotic lactobacillus human body symbiosis mainly comprise: take first-generation probiotic lactobacillus that bifidus bacillus and Lactobacterium acidophilum be representative and the novel probiotic lactobacillus of the s-generation that lactobacillus rhamnosus and lactobacterium casei be representative of take.Than first-generation probiotic lactobacillus, the novel probiotic lactobacillus of the s-generation is oxytolerant, stomach juice-resistant, bile tolerance more; Leavening property is better, viability is higher.
At present, lactobacillus rhamnosus leavened prod is mainly cow's milk product, and its fermented vegetable protein yogurt product at home and abroad still in the research starting stage, and lacks the good special bacteria of production performance.
China has natural soybean resource advantage, and soybean not only can compare favourably with cow's milk aspect the various functional ingredient of nutritive health-care and the somatomedin of promotion probiotic bacterium breeding, and not containing cholesterol and lactose.Therefore, the beneficial characteristics of developing comprehensive probiotic bacterium and soybean, integrate the novel probiotic bacterium of the s-generation (as lactobacillus rhamnosus) the fermented soybean milk product of sense organ local flavor and multiple nutrients nourishing function, be developing direction and the new growth point of current cultured milk prod industry and soyabean processing industry, will lead market and the human consumer new trend to heath food demand.
The preparation of starter is key link and the top priority of developing novel fermentation milk-product.Tradition cultivation type starter exists viable bacteria content low by (10
7～10
8cfu/mL), inoculum size large (2%～5%), preservation term are short (4 ℃, 1～2d), production process is many, labour intensity is large, culture cycle is long, bacterial classification is very easily degenerated and the drawback such as pollution, this will directly cause, and cultured milk prod quality product is unstable and production efficiency is low.Thereby novel fermentation agent---efficient concentration type throw type leaven arises at the historic moment.Efficient concentration type throw type leaven be milk-acid bacteria through deep liquid high-density culture, concentrating and separating, miscible with protective material medium, again through freezing or lyophilize, sterile packed, make direct-throwing cold storage starter or direct-throwing freeze dried fermenting preparation.There is viable bacteria content high by (10
10～10
12cfu/g), long (4 ℃ of the freeze dried fermenting preparations of preservation term, >1), the more traditional cultivation type starter of inoculum size reduces by 1000～10000 times, in cultured milk prod is produced, omitted the preparation section of starter, investment and the space of having reduced bacterial classification workshop, the degeneration that has prevented bacterial classification and pollution, can make the labour productivity of cultured milk prod industry and quality product greatly improve.Direct-throwing cold storage starter, in application, maintains freezing expense higher, and transportation is inconvenient, excessively strong to the dependency at starter center.And direct-throwing freeze dried fermenting preparation, preservation and convenient transportation, mechanization degree is high, is convenient to industrialized mass production, is the main development direction of throw type leaven from now on.
The principal element that affects efficient concentration type direct-throwing freeze dried fermenting preparation cell survival rate and storage stability has: strain properties, substratum, culture condition and harvesting time, initiator cell concentration, lyophilized vaccine and pH value thereof, chilling rate, drying process, residual moisture, packaged form, preservation temperature etc.Wherein, optimizing the cheap enrichment medium of screening and efficient lyophilized vaccine is the gordian technique of development direct-throwing freeze dried fermenting preparation.Research shows: milk-acid bacteria is very harsh to nutritional requirement, the milk-acid bacteria substratum (as MRS, M17 etc.) that laboratory is conventional, and complicated component, makes loaded down with trivial details, expensive.At present, although conventional degreasing milk medium and whey medium economy are easy to get, cheap, degreasing milk medium viscosity is large, is difficult for concentrating and separating thalline; And easily there is the thermally denature of whey-protein in whey medium and produce precipitation in heat sterilization process.Therefore, according to china natural resources advantage, actively seek wide material sources, the cheap raw material that economy is easy to get, optimizes the enrichment medium that screening is applicable to milk-acid bacteria raised growth, is one of gordian technique realizing China's large-scale industrial production direct-throwing freeze dried fermenting preparation.Lyophilized vaccine is the most complicated, the key factor of difficult selection, and it not only affects the cell survival rate of milk-acid bacteria in freeze-drying process, also affects the cell stability between preservation term, and its formula and preparation method thereof belongs to trade secret.Therefore, optimize the efficient lyophilized vaccine of screening be develop direct-throwing freeze dried fermenting preparation gordian technique two.
As fully visible, actively develop the efficient concentration type direct-throwing probiotic bacterium soy acid milk fermentation agent with autonomous innovation of alternative imported product, for improving the quality of fermentation soybean milk product and functional, promote the technical progress of China's efficient concentration type direct-throwing probiotics leaven industrialization, promote the development of China's fermented soybean milk-product and vegetable-protein secondary industry, significant.
Application number be 200710156323.X Chinese disclosure of the invention " a kind of Lactobacterium acidophilum, with method and the fermented soy milk beverage of this strain fermentation soya-bean milk ", be that a strain Lactobacterium acidophilum is carried out to the fermentation of soya-bean milk and soyabean milk beverage, but its starter form used is the conventional liquid starter that skimming milk is cultivated.Application number be 200910040265.3 Chinese disclosure of the invention " preparation method of direct vat set Lactobacillus casei subsp. rhamnosus freeze-dried powder ", but its MRS medium component used is complicated, makes loaded down with trivial detailsly, cost is higher; The vacuum-drying time needs 18 ~ 20h; And clearly whether this bacterium is not applicable to fermented soybean yogurt yet.
Summary of the invention
The preparation method who the object of this invention is to provide a kind of lactobacillus rhamnosus soybean yogurt throw type leaven, to realize the commercial scale production of lactobacillus rhamnosus throw type leaven.
The applicant has carried out collection, the separation screening work of a large amount of novel probiotic bacteriums, growth to novel probiotic bacterium in Soybean Milk, fermentation character etc. study in detail, discovery comes from the lactobacillus rhamnosus AS1.2466 of Institute of Microorganism, Academia Sinica, in Soybean Milk substratum, there is good growth and fermentation character, cultivate 12 hours viable counts and can reach 10
9cfu/mL, and it has been carried out to follow-up soybean yogurt throw type leaven preparation method's research.
The applicant is the feature to nutritional requirement harshness for milk-acid bacteria, by lot of experiments, selects colloidal state cheap and easy to get---and jerusalem artichoke is raw material, has studied the preparation technology of jerusalem artichoke juice basic medium; And take lactobacillus rhamnosus AS1.2466 as test strain, studied at jerusalem artichoke juice basic medium and added the impact of single nutritional factor on lactobacillus rhamnosus cell yield, further utilized optimization of orthogonal test to filter out the compound enrichment medium of jerusalem artichoke juice of lactobacillus rhamnosus; By the impact of factor on lactobacillus rhamnosus cell yield such as research inoculum size, culture temperature, matrix Initial pH, training methods, further utilize optimization of orthogonal test to determine the processing condition of lactobacillus rhamnosus multiplication culture; Study centrifugal force, the impact of centrifugation time on cell centrifugation rate of loss and survival rate, determined the suitableeest centrifuging process condition of the highest viable bacteria recovery rate; Meanwhile, the applicant, on lot of experiments basis, has studied the impact of different composite protectants on lactobacillus rhamnosus freeze-drying survival rate, optimizes and has filtered out anti-cryodesiccated complex protection agent prescription; The lactobacillus rhamnosus lyophilisate that the anti-cryodesiccated complex protection agent prescription that optimization is filtered out is made, by cell accelerated test, further filters out not only anti-lyophilize but also long keeping good complex protection agent prescription.By research, add lactobacillus rhamnosus bacteria suspension chilling rate impact on cell survival rate in refrigerating process of composite protectant, determined the freeze drying process of reasonable; By research lactobacillus rhamnosus freeze dried fermenting preparation in difference, seal the lower packing of mode (normal pressure is sealed up for safekeeping, vacuum preservation) up for safekeeping, in the viable count of the lower storage of differing temps (18 ℃, 4 ℃) and the variation of fermentative activity, determined storage conditions and the preservation term of freeze dried fermenting preparation.
Concrete, the preparation method of lactobacillus rhamnosus soybean yogurt throw type leaven of the present invention, comprises the following steps:
(1) multiplication culture of lactobacillus rhamnosus
1. the preparation of the cheap enrichment medium of lactobacillus rhamnosus
A. the preparation of lactobacillus rhamnosus jerusalem artichoke juice basic medium
Raw material: jerusalem artichoke, distilled water;
Preparation technology: (a) jerusalem artichoke is cleaned, weighs; (b) mass volume ratio of raw material jerusalem artichoke and water is 1:1; (c) heat the enzyme that goes out: be heated to 100 ℃, boil 5min; (d) adding water smashs to pieces: add appropriate 80 ℃ of hot water, in stamp mill, smash jerusalem artichoke and water to pieces pulp; (e) filter: with 100 order filter cloth press filtrations; (f) filter residue adds hydraulic pressure squeezing: filter residue is added appropriate 80 ℃ of hot water press filtrations; (g) filtered juice is mixed constant volume: the filtered juice of twice filtration is mixed, be settled to 1kg jerusalem artichoke produce 1L juice (being defined as 100% jerusalem artichoke juice) with distilled water; (h) allotment: it is 10% that heating is concentrated into sugar degree; With NaOH solution, regulate pH to 7.0, standby;
B. the preparation of the compound enrichment medium of lactobacillus rhamnosus
Proportioning raw materials: lactobacillus rhamnosus jerusalem artichoke juice basic medium 100mL, extractum carnis 0.3～0.6g, peptone 0.3～0.5g, soy peptone 0.3～0.6g, corn steep liquor 0.2～0.4g, glycerine 0.1mL, pH6.5～7.0;
Preparation technology: add extractum carnis, peptone, soy peptone, corn steep liquor, glycerine in jerusalem artichoke juice basic medium, regulate pH value to 6.5～7.0 with NaOH solution, 121 ℃, 15min sterilizing are cooling standby;
2. lactobacillus rhamnosus multiplication culture
Lactobacillus rhamnosus AS1.2466 is through MRS(Man Rogosa Sharpe) after liquid nutrient medium activation, with 0.1%～0.5%(approximately 1 * 10
6cfu/mL～5 * 10
6cfu/mL) in the compound enrichment medium of jerusalem artichoke juice that inoculum size access pH value is 6.5～7.0, under aerobic conditions, cultivate 16h～18h to logarithm latter stage or stable initial stage for 35～37 ℃, viable count can reach 4～5 * 10
9cfu/mL, the high-concentration culturing thing of acquisition lactobacillus rhamnosus;
(2) concentrating and separating of lactobacillus rhamnosus cell
By lactobacillus rhamnosus high-concentration culturing thing 5000～7500r/min(2550g～5738g at normal temperatures) centrifugal 10～20min carries out cell concentration separation, centrifugal rear abandoning supernatant, obtaining viable bacteria recovery rate is the lactobacillus rhamnosus concentrating cells higher than 95%;
(3) in lactobacillus rhamnosus concentrating cells, add composite protectant
Complex protection agent prescription: distilled water 100mL, skimmed milk powder 10g, lactose 3～4g, sucrose 3～4g, tween 80 1～1.2g, glycerine 0.1～0.2g, yeast powder 1～1.2g, serum 0.8～1.1g, vitamin-E 0.3～0.6g, pH value 6.8～7.0, filters or heating degerming;
Composite protectant preparation technology: 1. add skimmed milk powder, lactose, sucrose, tween 80, glycerine, yeast powder in distilled water, heating for dissolving mixes, and is cooled to room temperature; 2. with NaOH solution, regulate pH value to 6.8～7.0; 3. 112～115 ℃ of sterilizing 10min, are cooled to room temperature; 4. under aseptic condition, add serum and vitamin-E after degerming after filtration, mix;
Composite protectant adding technology: add the composite protectant of bacterial strain high-concentration culturing object long-pending 20～30% in lactobacillus rhamnosus concentrating cells, make lactobacillus rhamnosus bacteria suspension, mix;
(4) freeze-dry process of lactobacillus rhamnosus throw type leaven
By having added protectant lactobacillus rhamnosus bacteria suspension, be sub-packed in freeze-drying dish or bottle thickness 0.75cm ,-20 ℃ of freezing 12h; In freeze drier at vacuum tightness 3～10Pa, condenser temperature-55～-45 ℃ vacuum lyophilization 11～13h, make lactobacillus rhamnosus freeze dried fermenting preparation finished product, the viable count of freeze dried fermenting preparation can reach 1 * 10
11cfu/g is above, water content is 2～3%.
The beneficial effect that the present invention obtains is as follows:
1. the lactobacillus rhamnosus soybean yogurt throw type leaven that prepared by the present invention, its viable bacteria content can reach 1 * 10
11more than cfu/g; Normal pressure seals up for safekeeping, preservation 1 year at 4 ℃, and its viable bacteria content is still 10
10more than cfu/g, still keep higher fermentative activity.
2. the lactobacillus rhamnosus soybean yogurt throw type leaven that prepared by the present invention, can be used as the throw type leaven use of producing soybean yogurt, the more traditional cultivation type starter of inoculum size reduces by 10000～100000 times, in industrial production, omitted the preparation section of starter, investment and the space in bacterial classification workshop have been reduced, prevent degeneration and the pollution of bacterial classification, can improve labour productivity and the quality product of cultured milk prod industry and vegetable-protein secondary industry.
3. the lactobacillus rhamnosus soybean yogurt throw type leaven that prepared by the present invention, carries out 37 ℃ of soybean-contg yoghurt fermentations with ten thousand/following inoculum size, and 4～5h gets final product curdled milk, the soybean-contg yoghurt goods of its fermentation, and acidity reaches 70～80
oT, pH value is 4.6 left and right, sense organ raciness.Can be used for cultured milk prod industry and vegetable-protein secondary industry.
Accompanying drawing explanation
Fig. 1 is that the viable count of preserving 1 year under 4 ℃ and-18 ℃ of conditions after the lactobacillus rhamnosus soybean yogurt throw type leaven normal pressure of embodiment 1,2,3 preparation is sealed up for safekeeping changes.
Embodiment
Following examples are used for illustrating the present invention.
The preparation method of 1 one kinds of lactobacillus rhamnosus soybean yogurt throw type leavens of embodiment, the method realizes by following steps:
(1) multiplication culture of lactobacillus rhamnosus
1. the preparation of the cheap enrichment medium of lactobacillus rhamnosus
A. the preparation of lactobacillus rhamnosus jerusalem artichoke juice basic medium
Proportioning raw materials: jerusalem artichoke, distilled water;
Preparation technology: (a) jerusalem artichoke is cleaned, weighs; (b) mass volume ratio of material water is 1:1; (c) heat the enzyme that goes out: be heated to 100 ℃, boil 5min; (d) adding water smashs to pieces: add appropriate 80 ℃ of hot water, in stamp mill, smash jerusalem artichoke and water to pieces pulp; (e) filter: with 100 order filter cloth press filtrations; (f) filter residue adds hydraulic pressure squeezing: filter residue is added appropriate 80 ℃ of hot water press filtrations; (g) mix filtered juice constant volume: the filtered juice of twice filtration is mixed, with distilled water, be settled to 1kg jerusalem artichoke and produce 1L juice (being defined as 100% jerusalem artichoke juice); (h) allotment: it is 10% left and right that heating is concentrated into sugar degree; With NaOH solution, regulate pH to 7.0 left and right;
B. the preparation of the compound enrichment medium of lactobacillus rhamnosus
Proportioning raw materials: lactobacillus rhamnosus jerusalem artichoke juice basic medium 100mL, extractum carnis 0.6g, peptone 0.3g, soy peptone 0.3g, corn steep liquor 0.4g, glycerine 0.1mL, pH6.5～7.0;
Preparation technology: add extractum carnis 0.6g, peptone 0.3g, soy peptone 0.3g, corn steep liquor 0.4g, glycerine 0.1mL in 100mL jerusalem artichoke juice basic medium, regulate pH value to 6.5～7.0 with NaOH solution, 121 ℃ of 15min sterilizings are cooling standby;
2. the processing condition of lactobacillus rhamnosus multiplication culture
Lactobacillus rhamnosus is after the activation of MRS liquid nutrient medium, with 0.5%(approximately 5 * 10
6cfu/mL) in the compound enrichment medium of jerusalem artichoke juice that inoculum size access pH value is 6.5～7.0, under aerobic conditions, cultivate 16h to logarithm latter stage or stable initial stage for 37 ℃, viable count can reach 4.1 * 10
9cfu/mL, the high-concentration culturing thing of acquisition lactobacillus rhamnosus;
(2) concentrating and separating of lactobacillus rhamnosus cell
Lactobacillus rhamnosus high-concentration culturing thing adopts 7500r/min(5738g at normal temperatures) centrifugal 10～20min carries out cell concentration separation, and centrifugal rear abandoning supernatant, obtains the lactobacillus rhamnosus concentrating cells that viable bacteria recovery rate is 95.08%;
(3) in lactobacillus rhamnosus concentrating cells, add composite protectant
Complex protection agent prescription: distilled water 100mL, skimmed milk powder 10g, lactose 3g, sucrose 4g, tween 80 1.2g, glycerine 0.2g, yeast powder 1.0g, serum 0.8g, vitamin-E 0.6g, pH value 6.8～7.0, filters or heating degerming;
The preparation technology of complex protection agent prescription: 1. in 100mL distilled water, add skimmed milk powder 10g, lactose 3g, sucrose 4g, tween 80 1.2g, glycerine 0.2g, yeast powder 1.0g, heating for dissolving is mixed, and is cooled to room temperature; 2. with NaOH solution, regulate pH value to 6.8～7.0; 3. 112～115 ℃ of sterilizing 10min, are cooled to room temperature; 4. adopt aseptic technique to add serum 0.8g and the vitamin-E 0.6g after filtration sterilization, mix;
In lactobacillus rhamnosus concentrating cells, add composite protectant technique: in lactobacillus rhamnosus concentrating cells, add the composite protectant of bacterial strain high-concentration culturing object long-pending 30%, make lactobacillus rhamnosus bacteria suspension, mix;
(4) freeze-dry process of lactobacillus rhamnosus throw type leaven
By the protectant lactobacillus rhamnosus bacteria suspension of interpolation packing freeze-drying dish/bottle, thickness 0.75cm ,-20 ℃ of freezing 12h; In freeze drier at vacuum tightness 3～10Pa, condenser temperature-55～-45 ℃ vacuum lyophilization 13h, make lactobacillus rhamnosus freeze dried fermenting preparation.The viable count of freeze dried fermenting preparation can reach 1.11 * 10
11cfu/g is above, water content is 2.0%.
The preparation method of 2 one kinds of lactobacillus rhamnosus soybean yogurt throw type leavens of embodiment, the method realizes by following steps:
(1) multiplication culture of lactobacillus rhamnosus
1. the preparation of the cheap enrichment medium of lactobacillus rhamnosus
A. the proportioning raw materials of lactobacillus rhamnosus jerusalem artichoke juice basic medium and preparation technology are with embodiment 1.
B. the preparation of the compound enrichment medium of lactobacillus rhamnosus
Proportioning raw materials: lactobacillus rhamnosus jerusalem artichoke juice basic medium 100mL, extractum carnis 0.5g, peptone 0.5g, soy peptone 0.5g, corn steep liquor 0.3g, glycerine 0.1mL, pH6.5～7.0;
Preparation technology: add extractum carnis 0.5g, peptone 0.5g, soy peptone 0.5g, corn steep liquor 0.3g, glycerine 0.1mL in 100mL jerusalem artichoke juice basic medium, regulate pH value to 6.5～7.0 with NaOH solution, 121 ℃ of 15min sterilizings are cooling standby;
2. the processing condition of lactobacillus rhamnosus multiplication culture
Lactobacillus rhamnosus is after the activation of MRS liquid nutrient medium, with 0.1%(approximately 5 * 10
6cfu/mL) in the compound enrichment medium of jerusalem artichoke juice that inoculum size access pH value is 6.5～7.0, under aerobic conditions, cultivate 18h to logarithm latter stage or stable initial stage for 35 ℃, viable count can reach 4.5 * 10
9cfu/mL, the high-concentration culturing thing of acquisition lactobacillus rhamnosus;
(2) concentrating and separating of lactobacillus rhamnosus cell
Lactobacillus rhamnosus high-concentration culturing thing adopts 5000r/min(2550g at normal temperatures) centrifugal 20min carries out cell concentration separation, and centrifugal rear abandoning supernatant, obtains the lactobacillus rhamnosus concentrating cells that viable bacteria recovery rate is 97.58%;
(3) in lactobacillus rhamnosus concentrating cells, add composite protectant
Complex protection agent prescription: distilled water 100mL, skimmed milk powder 10g, lactose 3g, sucrose 3g, tween 80 1.0g, glycerine 0.1g, yeast powder 1.0g, serum 1.0g, vitamin-E 0.6g, pH value 6.8～7.0, filters or heating degerming;
The preparation technology of complex protection agent prescription: 1. in 100mL distilled water, add skimmed milk powder 10g, lactose 3g, sucrose 3g, tween 80 1.0g, glycerine 0.1g, yeast powder 1.0g, heating for dissolving is mixed, and is cooled to room temperature; 2. with NaOH solution, regulate pH value to 6.8～7.0; 3. 112～115 ℃ of sterilizing 10min, are cooled to room temperature; 4. adopt aseptic technique to add serum 1.0g and the vitamin-E 0.5g after filtration sterilization, mix;
In lactobacillus rhamnosus concentrating cells, add composite protectant technique: in lactobacillus rhamnosus concentrating cells, add the composite protectant of bacterial strain high-concentration culturing object long-pending 20%, make lactobacillus rhamnosus bacteria suspension, mix;
(4) freeze-dry process of lactobacillus rhamnosus throw type leaven
By the protectant lactobacillus rhamnosus bacteria suspension of interpolation packing freeze-drying dish/bottle, thickness 0.75cm ,-20 ℃ of freezing 12h; In freeze drier at vacuum tightness 3～10Pa, condenser temperature-55～-45 ℃ vacuum lyophilization 12h, make lactobacillus rhamnosus freeze dried fermenting preparation.The viable count of freeze dried fermenting preparation can reach 1.42 * 10
11cfu/g is above, water content is 2.3%.
The preparation method of 3 one kinds of lactobacillus rhamnosus soybean yogurt throw type leavens of embodiment, the method realizes by following steps:
(1) multiplication culture of lactobacillus rhamnosus
1. the preparation of the cheap enrichment medium of lactobacillus rhamnosus
A. the proportioning raw materials of lactobacillus rhamnosus jerusalem artichoke juice basic medium and preparation technology are with embodiment 1.
B. the preparation of the compound enrichment medium of lactobacillus rhamnosus
Proportioning raw materials: lactobacillus rhamnosus jerusalem artichoke juice basic medium 100mL, extractum carnis 0.3g, peptone 0.5g, soy peptone 0.3g, corn steep liquor 0.4g, glycerine 0.1mL, pH6.5～7.0;
Preparation technology: add extractum carnis 0.3g, peptone 0.5g, soy peptone 0.3g, corn steep liquor 0.4g, glycerine 0.1mL in 100mL jerusalem artichoke juice basic medium, regulate pH value to 6.5～7.0 with NaOH solution, 121 ℃ of 15min sterilizings are cooling standby;
2. the processing condition of lactobacillus rhamnosus multiplication culture
Lactobacillus rhamnosus is after the activation of MRS liquid nutrient medium, with 0.3%(approximately 5 * 10
6cfu/mL) in the compound enrichment medium of jerusalem artichoke juice that inoculum size access pH value is 6.5～7.0, under aerobic conditions, cultivate 17h to logarithm latter stage or stable initial stage for 37 ℃, viable count can reach 4.2 * 10
9cfu/mL, the high-concentration culturing thing of acquisition lactobacillus rhamnosus;
(2) concentrating and separating of lactobacillus rhamnosus cell
Lactobacillus rhamnosus high-concentration culturing thing adopts 6000r/min(3672g at normal temperatures) centrifugal 15min carries out cell concentration separation, and centrifugal rear abandoning supernatant, obtains the lactobacillus rhamnosus concentrating cells that viable bacteria recovery rate is 96.14%;
(3) in lactobacillus rhamnosus concentrating cells, add composite protectant
Complex protection agent prescription: distilled water 100mL, skimmed milk powder 10g, lactose 4g, sucrose 3g, tween 80 1.1g, glycerine 0.1g, yeast powder 1.0g, serum 1.1g, vitamin-E 0.3g, pH value 6.8～7.0, filters or heating degerming;
The preparation technology of complex protection agent prescription: 1. in 100mL distilled water, add skimmed milk powder 10g, lactose 4g, sucrose 3g, tween 80 1.1g, glycerine 0.1g, yeast powder 1.0g, heating for dissolving is mixed, and is cooled to room temperature; 2. with NaOH solution, regulate pH value to 6.8～7.0; 3. 115 ℃ of sterilizing 10min, are cooled to room temperature; 4. adopt aseptic technique to add serum 1.1g and the vitamin-E 0.3g after filtration sterilization, mix;
In lactobacillus rhamnosus concentrating cells, add composite protectant technique: in lactobacillus rhamnosus concentrating cells, add the composite protectant of bacterial strain high-concentration culturing object long-pending 25%, make lactobacillus rhamnosus bacteria suspension, mix;
(4) freeze-dry process of lactobacillus rhamnosus throw type leaven
By the protectant lactobacillus rhamnosus bacteria suspension of interpolation packing freeze-drying dish/bottle, thickness 0.75cm ,-20 ℃ of freezing 12h; In freeze drier at vacuum tightness 3～10Pa, condenser temperature-55～-45 ℃ vacuum lyophilization 11h, make lactobacillus rhamnosus freeze dried fermenting preparation.The viable count of freeze dried fermenting preparation can reach 1.17 * 10
11cfu/g is above, water content is 2.7%.
Table 1
Table 1 for sealing up for safekeeping at normal pressure, the impact on soy acid milk fermentation and quality of the lactobacillus rhamnosus soybean yogurt throw type leaven of-18 ℃ of storages.
Claims (1)
1. a preparation method for lactobacillus rhamnosus soybean yogurt throw type leaven, is characterized in that comprising the following steps:
(1) multiplication culture of lactobacillus rhamnosus
1. the preparation of the cheap enrichment medium of lactobacillus rhamnosus
A. the preparation of lactobacillus rhamnosus jerusalem artichoke juice basic medium
Raw material: jerusalem artichoke, distilled water;
Preparation technology: (a) jerusalem artichoke is cleaned, weighs; (b) mass volume ratio of raw material jerusalem artichoke and water is 1:1; (c) heat the enzyme that goes out: be heated to 100 ℃, boil 5min; (d) adding water smashs to pieces: add appropriate 80 ℃ of hot water, in stamp mill, smash jerusalem artichoke and water to pieces pulp; (e) filter: with 100 order filter cloth press filtrations; (f) filter residue adds hydraulic pressure squeezing: filter residue is added appropriate 80 ℃ of hot water press filtrations; (g) filtered juice is mixed constant volume: the filtered juice of twice filtration is mixed, be settled to 1kg jerusalem artichoke produce 1L juice with distilled water; (h) allotment: it is 10% that heating is concentrated into sugar degree, regulates pH to 7.0 with NaOH solution, standby;
B. the preparation of the compound enrichment medium of lactobacillus rhamnosus
Proportioning raw materials: lactobacillus rhamnosus jerusalem artichoke juice basic medium 100mL, extractum carnis 0.3～0.6g, peptone 0.3～0.5g, soy peptone 0.3～0.6g, corn steep liquor 0.2～0.4g, glycerine 0.1mL, pH value to 6.5～7.0;
Preparation technology: in jerusalem artichoke juice basic medium, add extractum carnis, peptone, soy peptone, corn steep liquor, glycerine, with NaOH solution, regulate pH value to 6.5～7.0,121 ℃, 15min sterilizing, cooling, standby;
2. lactobacillus rhamnosus multiplication culture
Lactobacillus rhamnosus AS1.2466 is after the activation of MRS liquid nutrient medium, in the compound enrichment medium of lactobacillus rhamnosus that 0.1%～0.5% inoculum size access pH value of take is 6.5～7.0, under aerobic conditions, cultivate 16h～18h to logarithm latter stage or stable initial stage for 35～37 ℃, obtain the high-concentration culturing thing of lactobacillus rhamnosus;
(2) concentrating and separating of lactobacillus rhamnosus cell
By lactobacillus rhamnosus high-concentration culturing thing at normal temperatures the centrifugal 10～20min of 5000～7500r/min carry out cell concentration separation, centrifugal rear abandoning supernatant, obtaining viable bacteria recovery rate is the lactobacillus rhamnosus concentrating cells higher than 95%;
(3) in lactobacillus rhamnosus concentrating cells, add composite protectant
Complex protection agent prescription: distilled water 100mL, skimmed milk powder 10g, lactose 3～4g, sucrose 3～4g, tween 80 1～1.2g, glycerine 0.1～0.2g, yeast powder 1～1.2g, serum 0.8～1.1g, vitamin-E 0.3～0.6g, pH value 6.8～7.0, filters or heating degerming;
Composite protectant preparation technology: 1. add skimmed milk powder, lactose, sucrose, tween 80, glycerine, yeast powder in distilled water, heating for dissolving mixes, and is cooled to room temperature; 2. with NaOH solution, regulate pH value to 6.8～7.0; 3. 112～115 ℃ of sterilizing 10min, are cooled to room temperature; 4. under aseptic condition, add serum and vitamin-E after degerming after filtration, mix;
Composite protectant adding technology: add the composite protectant of lactobacillus rhamnosus high-concentration culturing thing 20%～30% in lactobacillus rhamnosus concentrating cells, make lactobacillus rhamnosus bacteria suspension, mix;
(4) freeze-dry process of lactobacillus rhamnosus throw type leaven
By having added protectant lactobacillus rhamnosus bacteria suspension, be sub-packed in freeze-drying dish or bottle thickness 0.75cm ,-20 ℃ of freezing 12h; In freeze drier at vacuum tightness 3～10Pa, condenser temperature-55～-45 ℃ vacuum lyophilization 11～13h, make lactobacillus rhamnosus soybean yogurt throw type leaven finished product.
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| CN106665837A (en) * | 2016-12-16 | 2017-05-17 | 常州市鼎日环保科技有限公司 | Preparation method of high-activity ready-to-use yoghurt starter |
| CN111820349A (en) * | 2019-04-17 | 2020-10-27 | 吉林省绿色食品工程研究院 | Direct-throwing fruit and vegetable juice lactobacillus accelerant |
| CN110106119B (en) * | 2019-05-28 | 2021-07-06 | 北京科拓恒通生物技术股份有限公司 | Lactobacillus rhamnosus M9 separated from breast milk and application thereof |
| CN114451485B (en) * | 2022-01-26 | 2022-12-30 | 江苏省淡水水产研究所 | Traditional Chinese medicine polysaccharide extract freeze-drying protective agent, direct-vat-set starter and preparation method thereof |
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