CN101948776A - High-density cultivation of functional probiotic lactobacillus and preparation method of deep-frozen product thereof - Google Patents
High-density cultivation of functional probiotic lactobacillus and preparation method of deep-frozen product thereof Download PDFInfo
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Abstract
The invention discloses high-density cultivation of functional probiotic lactobacillus and a preparation method of a deep-frozen product thereof, and relates to the cultivation of the lactobacillus and the preparation method of the deep-frozen product thereof. The invention solves the problems that the traditional lactobacillus has small viable bacteria content in the product, large dosage and bad fermentation effect in the production process and the freeze-dried preparation thereof has higher manufacturing cost and higher using cost. The cultivation comprises the steps of: (1) inoculating the aerotolerant naturalized functional probiotic lactobacillus into an enrichment medium to obtain the fermentation liquor; (2) eluting and flocculating; and (3) carrying out membrane concentration to finish the cultivation. The preparation method comprises the steps of: (1) mixing and pre-cooling a membrane concentration product and a cryoprotectant to obtain the mixture; and (2) pre-freezing the mixture, then crushing, packing and storing to obtain the finished product. The viable count of the lactobacillus in the invention is 5.4*1010 CFU/g, therefore, the dosage is decreased, the fermentation effect is good; and the deep-frozen product has low production cost, simple process and high activity and achieves the purpose of directly using.
Description
Technical field
The present invention relates to Bacterium lacticum and cultivate and freeze deeply the preparation method of product.
Background technology
Lactobacillus is in milk-acid bacteria, Gram-positive, sporeless bacterium.The main end product that decomposes sugar is a lactic acid, and the nonfermented lactic acid salt is seldom pathogenic, is distributed widely in the animals and plants leavened prod that contains carbohydrate, also sees in oral cavity, vagina and the enteron aisle of warm-blooded animal; The ability of decomposing sugar is strong, and the ability of decomposing protein class is extremely low.
At present, mostly Bacterium lacticum production is to adopt centrifugal spissated mode, has that treatment capacity is less, whizzer is got the shortcoming that the material program is numerous and diverse, the thalline mortality ratio is higher and yield rate is lower, causes that viable bacteria content is little in the product, consumption big, ferment effect is poor; And its product is mainly freeze-dried preparation, and manufacturing cost and use cost is all higher.
Summary of the invention
The objective of the invention is to have in order to solve existing Bacterium lacticum production that viable bacteria content is little in the product, consumption big, ferment effect is poor, and the equal problem of higher of the manufacturing cost and use cost of its freeze-dried preparation, and functional beneficial natural disposition Bacterium lacticum high-density culture is provided and freezes the preparation method of product deeply.
Functional beneficial natural disposition Bacterium lacticum high-density culture is carried out according to the following steps: one, will be inoculated in the enrichment medium of fermentor tank through the functional beneficial natural disposition Bacterium lacticum of oxytolerant domestication, in temperature is that 35～37 ℃, pH value are to cultivate 15～17h under 6.5～6.8 the condition, fermented liquid; Two, fermented liquid is in the time of 0～20 ℃, add mass concentration with weight such as fermented liquid and be 10% sodium citrate solution or mass concentration and be 10% sodium radio-phosphate,P-32 solution fermented liquid is carried out proteic wash-out, add mass concentration again and be 0.2～2% chitosan and carry out the flocculation of thalline; Three, to use the aperture be that 100～200nm, area are 0.5m to the fermented liquid after the flocculation
2Ceramic membrane, be that 110KPa carries out membrane concentration and handles the cycles of concentration of the fermented liquid to the flocculation and reach 6 at transmembrane pressure, obtain containing the membrane concentration product of functional beneficial natural disposition Bacterium lacticum, promptly finish functional beneficial natural disposition Bacterium lacticum high-density culture; Wherein inoculum size is 2% in the step 1; Enrichment medium is by the skimmed milk powder of 80～120g in the step 1, the trypsin hydrolyzing whey powder of 40～60g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 30～60g, the wort of 30～60g, 10～30g lactose, 0.50～0.70g or yeast powder, 6.8～7.2mg and the L-cysteine hydrochloride of 50～54mg add distilled water and are settled to 1000mL and form; The mode that membrane concentration is handled in the step 3 is two ceramic-film tube series connection.
The preparation method that functional beneficial natural disposition Bacterium lacticum freezes product deeply carries out according to the following steps: one, mix with cryoprotectant by weight 1: 1～2 membrane concentration products that will contain functional beneficial natural disposition Bacterium lacticum, under the condition of 0～10 ℃ of temperature, carry out precooling treatment before 10～12h freezing, mixture; Two, mixture is placed the refrigerator tray of-70 ℃ of refrigerators to carry out pre-freeze, temperature until mixture reaches-50 ℃, keep taking out behind the 30min, pulverize with granulator then, with aseptic aluminium foil carton package and place-49.5～50.5 ℃ environment to preserve, promptly finish the preparation that functional beneficial natural disposition Bacterium lacticum freezes product deeply again; Wherein the trehalose of the sucrose of the L-glutamic acid of the vitamins C of the glycerine of the skimmed milk powder of cryoprotectant 10～20g, 1～3g, 1～2g, 0.5～1g or glycine, 5g and 3～15g adds distilled water and is settled to the 1000mL composition in the step 1; Mixture places the size of the refrigerator tray of-70 ℃ of refrigerators to be long 30cm * wide 30cm * high 20mm in the step 2.
The viable count that functional beneficial natural disposition Bacterium lacticum high-density culture among the present invention, gained contain Bacterium lacticum in the membrane concentration product of functional beneficial natural disposition Bacterium lacticum is 5.4 * 10
10CFU/g, improve more than 10 times than existing viable count, the Bacterium lacticum consumption reduces, only getting 0.005%～0.010% is used with ferment agent for sour milk and can satisfies the requirement that probiotics fermention breast is produced, can be used as fine probiotic bacterium throw type leaven, and ferment effect is good, and used VITAMIN B4 in the enrichment medium simultaneously is for having increased the survival rate of functional beneficial natural disposition Bacterium lacticum in the follow-up preparation process of freezing product deeply.The present invention can be suitable for Lactobacterium acidophilum, lactobacillus rhamnosus or the lactobacillus paraceasi that benefit is given birth to function.
Functional beneficial natural disposition Bacterium lacticum freezes the preparation method of product deeply among the present invention; the membrane concentration product of functional beneficial natural disposition Bacterium lacticum high-density culture gained is processed; reduce the load of freezing treatment like this; simultaneously; cryoprotectant after the optimization also can improve the survival rate of functional beneficial natural disposition Bacterium lacticum, compares with freeze-dried products to have the low and active high advantage of production cost; reach direct application target, and technology is simple.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the functional beneficial natural disposition Bacterium lacticum high-density culture of present embodiment is carried out according to the following steps: one, will be inoculated in the enrichment medium of fermentor tank through the functional beneficial natural disposition Bacterium lacticum of oxytolerant domestication, in temperature is that 35～37 ℃, pH value are to cultivate 15～17h under 6.5～6.8 the condition, fermented liquid; Two, fermented liquid is in the time of 0～20 ℃, add mass concentration with weight such as fermented liquid and be 10% sodium citrate solution or mass concentration and be 10% sodium radio-phosphate,P-32 solution fermented liquid is carried out proteic wash-out, add mass concentration again and be 0.2～2% chitosan and carry out the flocculation of thalline; Three, to use the aperture be that 100～200nm, area are 0.5m to the fermented liquid after the flocculation
2Ceramic membrane, be that 110KPa carries out membrane concentration and handles the cycles of concentration of the fermented liquid to the flocculation and reach 6 at transmembrane pressure, obtain containing the membrane concentration product of functional beneficial natural disposition Bacterium lacticum, promptly finish functional beneficial natural disposition Bacterium lacticum high-density culture; Wherein inoculum size is 2% in the step 1; Enrichment medium is by the skimmed milk powder of 80～120g in the step 1, the trypsin hydrolyzing whey powder of 40～60g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 30～60g, the wort of 30～60g, 10～30g lactose, 0.50～0.70g or yeast powder, 6.8～7.2mg and the L-cysteine hydrochloride of 50～54mg add distilled water and are settled to 1000mL and form; The mode that membrane concentration is handled in the step 3 is two ceramic-film tube series connection.
The functional beneficial natural disposition Bacterium lacticum of oxytolerant domestication in the present embodiment step 1, be from spontaneous fermentation food or human intestine, to isolate functional beneficial natural disposition Bacterium lacticum, be Lactobacterium acidophilum, lactobacillus rhamnosus or lactobacillus paraceasi, then by acclimation and screening gained under little oxygen condition continuously, concrete operations are with reference to Zhang Xu, Wei Zhongshan, Li Huali. isolation identification of bifidus bacillus and oxytolerant domestication. modern food science and technology, 2009, Vol.25, No.1:31-37; Domestication: select Gram-positive, catalase test feminine gender, gelatin liquification test feminine gender, negative, fructose-6-phosphate salt phosphoketolase (F6PPK) the test male bacterium colony of glucose aerogenesis test, be inoculated in the sterilized PTY liquid nutrient medium, postvaccinal triangular flask is positioned in the anaerobism incubator, wash 2 times with nitrogen, the employing air-permeable envelope seals, and cultivates 72h then in 37 ℃ of anaerobism incubators.The back bacterium liquid that will increase continues to cultivate 6～8h at aerobic environment, aerobic conditions progressively increases the oxygen content in the incubator when cultivating, judge what of oxygen level with the colour-change of indicator, behind passage and attenuation repeatedly, be chosen in that the curdled milk time is short in the degreasing milk medium, viable count is higher and storage in still can keep the bacterial strain of higher viable count.
Used ceramic membrane can be regenerated in the present embodiment step 3, reclaiming process is: 50 ℃ hot water cleans 5min, mass concentration is 0.5% sodium hydroxide solution cleaning 10min, 50 ℃ hot water cleans 5min, mass concentration is 0.5% nitric acid cleaning 10min, 50 ℃ hot water cleans 5min, promptly finishes ceramic membrane regeneration.
Present embodiment adopts the stream addition to replenish enrichment medium.
Embodiment two: what present embodiment and embodiment one were different is to adopt neutralizing agent to regulate the pH value of enrichment medium in the step 1, and neutralizing agent is that mass concentration is that 10～15% sodium hydroxide, mass concentration are that 10～15% ammoniacal liquor or mass concentration are 20% Na
2CO
3Cultivate in the step 1 in the process of 15～17h and need add lactose, the lactose additional amount is 3～5 times of the neutralizing agent neutral and alkali material molar weight that consumed, adopts the sodium salt that produces in the D301 type resin absorption culturing process simultaneously.Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment is different with embodiment one or two is to be that 35 ℃, pH value are to cultivate 17h under 6.5 the condition in temperature in the step 1, fermented liquid.Other step and parameter are identical with embodiment one or two.
Embodiment four: present embodiment is different with embodiment one or two is to be that 37 ℃, pH value are to cultivate 15h under 6.8 the condition in temperature in the step 1, fermented liquid.Other step and parameter are identical with embodiment one or two.
Embodiment five: present embodiment is different with embodiment one or two is to be that 36 ℃, pH value are to cultivate 16h under 6.6 the condition in temperature in the step 1, fermented liquid.Other step and parameter are identical with embodiment one or two.
Embodiment six: present embodiment is different with one of embodiment one to five be in the step 1 enrichment medium by the skimmed milk powder of 80g, the trypsin hydrolyzing whey powder of 40g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 30g, the wort of 30g, 10g lactose, 0.50g or yeast powder, 6.8mg and the L-cysteine hydrochloride of 50mg add distilled water and are settled to 1000mL and form.Other step and parameter are identical with one of embodiment one to five.
Embodiment seven: present embodiment is different with one of embodiment one to five be in the step 1 enrichment medium by the skimmed milk powder of 120g, the trypsin hydrolyzing whey powder of 60g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 60g, the wort of 60g, 30g lactose, 0.70g or yeast powder, 7.2mg and the L-cysteine hydrochloride of 54mg add distilled water and are settled to 1000mL and form.Other step and parameter are identical with one of embodiment one to five.
Embodiment eight: present embodiment is different with one of embodiment one to five be in the step 1 enrichment medium by the skimmed milk powder of 90～110g, the trypsin hydrolyzing whey powder of 45～55g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 40～50g, the wort of 40～50g, 15～25g lactose, 0.55～0.65g or yeast powder, 6.9～7.1mg and the L-cysteine hydrochloride of 51～53mg add distilled water and are settled to 1000mL and form.Other step and parameter are identical with one of embodiment one to five.
Embodiment nine: present embodiment is different with one of embodiment one to five be in the step 1 enrichment medium by the skimmed milk powder of 100g, the trypsin hydrolyzing whey powder of 50g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 45g, the wort of 45g, 20g lactose, 0.60g or yeast powder, 7mg and the L-cysteine hydrochloride of 52mg add distilled water and are settled to 1000mL and form.Other step and parameter are identical with one of embodiment one to five.
Embodiment ten: present embodiment is different with one of embodiment one to nine is that to add mass concentration in the step 2 be that 0.2% chitosan carries out the flocculation of thalline.Other step and parameter are identical with one of embodiment one to nine.
Embodiment 11: present embodiment is different with one of embodiment one to nine is that to add mass concentration in the step 2 be that 1% chitosan carries out the flocculation of thalline.Other step and parameter are identical with one of embodiment one to nine.
Embodiment 12: present embodiment is different with one of embodiment one to nine is that to add mass concentration in the step 2 be that 0.5～1.5% chitosan carries out the flocculation of thalline.Other step and parameter are identical with one of embodiment one to nine.
Embodiment 13: present embodiment is different with one of embodiment one to nine is that to add mass concentration in the step 2 be that 2% chitosan carries out the flocculation of thalline.Other step and parameter are identical with one of embodiment one to nine.
Embodiment 14: the preparation method that the functional beneficial natural disposition Bacterium lacticum of present embodiment freezes product deeply carries out according to the following steps: one, mix with cryoprotectant by weight 1: 1～2 membrane concentration products that will contain functional beneficial natural disposition Bacterium lacticum, under the condition of 0～10 ℃ of temperature, carry out precooling treatment before 10～12h freezing, mixture; Two, mixture is placed the refrigerator tray of-70 ℃ of refrigerators to carry out pre-freeze, temperature until mixture reaches-50 ℃, keep taking out behind the 30min, pulverize with granulator then, with aseptic aluminium foil carton package and place-49.5～50.5 ℃ environment to preserve, promptly finish the preparation that functional beneficial natural disposition Bacterium lacticum freezes product deeply again; Wherein the trehalose of the sucrose of the L-glutamic acid of the vitamins C of the glycerine of the skimmed milk powder of cryoprotectant 10～20g, 1～3g, 1～2g, 0.5～1g or glycine, 5g and 3～15g adds distilled water and is settled to the 1000mL composition in the step 1; Mixture places the size of the refrigerator tray of-70 ℃ of refrigerators to be long 30cm * wide 30cm * high 20mm in the step 2.
Embodiment 15: what present embodiment and embodiment 14 were different is to mix with cryoprotectant by weight the membrane concentration product that will contain functional beneficial natural disposition Bacterium lacticum at 1: 1 in the step 1; under the condition of 0 ℃ of temperature, carry out precooling treatment before 12h freezing, mixture.Other step and parameter are identical with embodiment 14.
Embodiment 16: what present embodiment and embodiment 14 were different is to mix with cryoprotectant by weight the membrane concentration product that will contain functional beneficial natural disposition Bacterium lacticum at 1: 2 in the step 1; under the condition of 10 ℃ of temperature, carry out precooling treatment before 10h freezing, mixture.Other step and parameter are identical with embodiment 14.
Embodiment 17: what present embodiment and embodiment 14 were different is to mix with cryoprotectant by weight the membrane concentration product that will contain functional beneficial natural disposition Bacterium lacticum at 1: 1.5 in the step 1; under the condition of 5 ℃ of temperature, carry out precooling treatment before 11h freezing, mixture.Other step and parameter are identical with embodiment 14.
Embodiment 18: present embodiment is different with one of embodiment 14 to 17 is that the trehalose of the sucrose of the L-glutamic acid of vitamins C, 0.5g of glycerine, the 1g of skimmed milk powder, the 1g of cryoprotectant 10g in the step 1 or glycine, 5g and 3g adds distilled water and is settled to 1000mL and forms.Other step and parameter are identical with one of embodiment 14 to 17.
Embodiment 19: present embodiment is different with one of embodiment 14 to 17 is that the trehalose of the sucrose of the L-glutamic acid of vitamins C, 1g of glycerine, the 2g of skimmed milk powder, the 3g of cryoprotectant 20g in the step 1 or glycine, 5g and 15g adds distilled water and is settled to 1000mL and forms.Other step and parameter are identical with one of embodiment 14 to 17.
Embodiment 20: present embodiment is different with one of embodiment 14 to 17 is that the trehalose of the sucrose of the L-glutamic acid of vitamins C, 0.6～0.9g of glycerine, the 1.2～1.8g of skimmed milk powder, the 1.5～2.5g of cryoprotectant 12～18g in the step 1 or glycine, 5g and 5～10g adds distilled water and is settled to 1000mL and forms.Other step and parameter are identical with one of embodiment 14 to 17.
Embodiment 21: present embodiment is different with one of embodiment 14 to 17 is that the trehalose of the sucrose of the L-glutamic acid of vitamins C, 0.8g of glycerine, the 1.5g of skimmed milk powder, the 2g of cryoprotectant 15g in the step 1 or glycine, 5g and 8g adds distilled water and is settled to 1000mL and forms.Other step and parameter are identical with one of embodiment 14 to 17.
Embodiment 22: the functional beneficial natural disposition Bacterium lacticum high-density culture of present embodiment is carried out according to the following steps: one, will be inoculated in the enrichment medium of fermentor tank through the functional beneficial natural disposition Bacterium lacticum of oxytolerant domestication, in temperature is that 36 ℃, pH value are to cultivate 16h under 6.6 the condition, fermented liquid; Two, fermented liquid is in the time of 10 ℃, add mass concentration with weight such as fermented liquid and be 10% sodium citrate solution or mass concentration and be 10% sodium radio-phosphate,P-32 solution fermented liquid is carried out proteic wash-out, add mass concentration again and be 1% chitosan and carry out the flocculation of thalline; Three, the fermented liquid after the flocculation uses the aperture to be 0.5m as 150nm, area
2Ceramic membrane, be that 110KPa carries out membrane concentration and handles the cycles of concentration of the fermented liquid to the flocculation and reach 6 at transmembrane pressure, obtain containing the membrane concentration product of functional beneficial natural disposition Bacterium lacticum, promptly finish functional beneficial natural disposition Bacterium lacticum high-density culture; Wherein inoculum size is 2% in the step 1; Enrichment medium is by the skimmed milk powder of 100g in the step 1, the trypsin hydrolyzing whey powder of 50g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 45g, the wort of 45g, 20g lactose, 0.60g or yeast powder, 6.8mg and the L-cysteine hydrochloride of 52mg add distilled water and are settled to 1000mL and form; The mode that membrane concentration is handled in the step 3 is two ceramic-film tube series connection.
The preparation method that functional beneficial natural disposition Bacterium lacticum freezes product deeply carries out according to the following steps: one, mix with cryoprotectant by weight the membrane concentration product that will contain functional beneficial natural disposition Bacterium lacticum at 1: 1.5, under the condition of 5 ℃ of temperature, carry out precooling treatment before 11h freezing, mixture; Two, mixture is placed the refrigerator tray of-70 ℃ of refrigerators to carry out pre-freeze, temperature until mixture reaches-50 ℃, keep taking out behind the 30min, pulverize with granulator then, with aseptic aluminium foil carton package and place-50 ℃ environment to preserve, promptly finish the preparation that functional beneficial natural disposition Bacterium lacticum freezes product deeply again; Wherein the trehalose of the sucrose of the L-glutamic acid of the vitamins C of the glycerine of the skimmed milk powder of cryoprotectant 15g, 2g, 1.5g, 0.8g or glycine, 5g and 10g adds distilled water and is settled to the 1000mL composition in the step 1; Mixture places the size of the refrigerator tray of-70 ℃ of refrigerators to be long 30cm * wide 30cm * high 20mm in the step 2.
Gained freezes product deeply in the present embodiment, and wherein viable count is 5.4 * 10 after tested
10CFU/g, the amount that is used with 0.005%-0.010% with ferment agent for sour milk is inoculated in sterilization skimming milk and the fresh milk, condition bottom fermentation 3.5h at 41～43 ℃, solid and the 70 ° of T of product acid of curdling, structural state, excellent flavor, the viable bacteria number average of probiotic bacterium is greater than 1.0 * 10 in the quality guaranteed period of 21 days sour milks
6CFU/g can be used as fine probiotic bacterium throw type leaven.
Claims (10)
1. functional beneficial natural disposition Bacterium lacticum high-density culture, it is characterized in that functional beneficial natural disposition Bacterium lacticum high-density culture carries out according to the following steps: one, will be inoculated in the enrichment medium of fermentor tank through the functional beneficial natural disposition Bacterium lacticum of oxytolerant domestication, in temperature is that 35～37 ℃, pH value are to cultivate 15～17h under 6.5～6.8 the condition, fermented liquid; Two, fermented liquid is in the time of 0～20 ℃, add mass concentration with weight such as fermented liquid and be 10% sodium citrate solution or mass concentration and be 10% sodium radio-phosphate,P-32 solution fermented liquid is carried out proteic wash-out, add mass concentration again and be 0.2～2% chitosan and carry out the flocculation of thalline; Three, to use the aperture be that 100～200nm, area are 0.5m to the fermented liquid after the flocculation
2Ceramic membrane, be that 110KPa carries out membrane concentration and handles the cycles of concentration of the fermented liquid to the flocculation and reach 6 at transmembrane pressure, obtain containing the membrane concentration product of functional beneficial natural disposition Bacterium lacticum, promptly finish functional beneficial natural disposition Bacterium lacticum high-density culture; Wherein inoculum size is 2% in the step 1; Enrichment medium is by the skimmed milk powder of 80～120g in the step 1, the trypsin hydrolyzing whey powder of 40～60g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 30～60g, the wort of 30～60g, 10～30g lactose, 0.50～0.70g or yeast powder, 6.8～7.2mg and the L-cysteine hydrochloride of 50～54mg add distilled water and are settled to 1000mL and form; The mode that membrane concentration is handled in the step 3 is two ceramic-film tube series connection.
2. functional beneficial natural disposition Bacterium lacticum high-density culture according to claim 1, it is characterized in that in the step 1 adopting neutralizing agent to regulate the pH value of enrichment medium, neutralizing agent is that mass concentration is that 10～15% sodium hydroxide, mass concentration are that 10～15% ammoniacal liquor or mass concentration are 20% Na
2CO
3Cultivate in the step 1 in the process of 15～17h and need add lactose, the lactose additional amount is 3～5 times of the neutralizing agent neutral and alkali material molar weight that consumed, adopts the sodium salt that produces in the D301 type resin absorption culturing process simultaneously.
3. functional beneficial natural disposition Bacterium lacticum high-density culture according to claim 1 and 2 is characterized in that in the step 1 in temperature being that 36 ℃, pH value are to cultivate 16h under 6.6 the condition, fermented liquid.
4. functional beneficial natural disposition Bacterium lacticum high-density culture according to claim 3, it is characterized in that enrichment medium is by the skimmed milk powder of 90～110g in the step 1, the trypsin hydrolyzing whey powder of 45～55g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 40～50g, the wort of 40～50g, 15～25g lactose, 0.55～0.65g or yeast powder, 6.9～7.1mg and the L-cysteine hydrochloride of 51～53mg add distilled water and are settled to 1000mL and form.
5. functional beneficial natural disposition Bacterium lacticum high-density culture according to claim 3, it is characterized in that enrichment medium is by the skimmed milk powder of 100g in the step 1, the trypsin hydrolyzing whey powder of 50g, the VITAMIN B4 of the yeast extract paste of the tomato juice of 45g, the wort of 45g, 20g lactose, 0.60g or yeast powder, 7mg and the L-cysteine hydrochloride of 52mg add distilled water and are settled to 1000mL and form.
6. according to claim 4 or 5 described functional beneficial natural disposition Bacterium lacticum high-density culture, it is characterized in that adding in the step 2 mass concentration and be 0.5～1.5% chitosan and carry out the flocculation of thalline.
7. according to claim 4 or 5 described functional beneficial natural disposition Bacterium lacticum high-density culture, it is characterized in that adding in the step 2 mass concentration and be 2% chitosan and carry out the flocculation of thalline.
8. prepare the method that the functional beneficial natural disposition Bacterium lacticum of gained of functional beneficial natural disposition Bacterium lacticum high-density culture as claimed in claim 1 freezes product deeply, it is characterized in that the preparation method that functional beneficial natural disposition Bacterium lacticum freezes product deeply carries out according to the following steps: one, mix with cryoprotectant by weight 1: 1～2 membrane concentration products that will contain functional beneficial natural disposition Bacterium lacticum, under the condition of 0～10 ℃ of temperature, carry out precooling treatment before 10～12h freezing, mixture; Two, mixture is placed the refrigerator tray of-70 ℃ of refrigerators to carry out pre-freeze, temperature until mixture reaches-50 ℃, keep taking out behind the 30min, pulverize with granulator then, with aseptic aluminium foil carton package and place-49.5～50.5 ℃ environment to preserve, promptly finish the preparation that functional beneficial natural disposition Bacterium lacticum freezes product deeply again; Wherein the trehalose of the sucrose of the L-glutamic acid of the vitamins C of the glycerine of the skimmed milk powder of cryoprotectant 10～20g, 1～3g, 1～2g, 0.5～1g or glycine, 5g and 3～15g adds distilled water and is settled to the 1000mL composition in the step 1; Mixture places the size of the refrigerator tray of-70 ℃ of refrigerators to be long 30cm * wide 30cm * high 20mm in the step 2.
9. functional beneficial natural disposition Bacterium lacticum according to claim 8 freezes the preparation method of product deeply; it is characterized in that mixing with cryoprotectant by weight the membrane concentration product that will contain functional beneficial natural disposition Bacterium lacticum at 1: 1.5 in the step 1; under the condition of 5 ℃ of temperature, carry out precooling treatment before 11h freezing, mixture.
According to Claim 8 or 9 described functional beneficial natural disposition Bacterium lacticum freeze the preparation method of product deeply, the trehalose that it is characterized in that the sucrose of the L-glutamic acid of vitamins C, 0.8g of glycerine, the 1.5g of skimmed milk powder, the 2g of cryoprotectant 15g in the step 1 or glycine, 5g and 8g adds distilled water and is settled to 1000mL to be formed.
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| CN102978143A (en) * | 2012-12-21 | 2013-03-20 | 哈尔滨工业大学 | Lactobacillus freeze-dried product and preparation method of same |
| CN103013832A (en) * | 2012-12-21 | 2013-04-03 | 哈尔滨工业大学 | Deep-frozen lactobacillus product and production method thereof |
| CN105154368A (en) * | 2015-09-28 | 2015-12-16 | 内蒙古蒙牛乳业（集团）股份有限公司 | Lactobacillus plantarum multiplying culture medium and lactobacillus plantarum multiplying method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978143A (en) * | 2012-12-21 | 2013-03-20 | 哈尔滨工业大学 | Lactobacillus freeze-dried product and preparation method of same |
| CN103013832A (en) * | 2012-12-21 | 2013-04-03 | 哈尔滨工业大学 | Deep-frozen lactobacillus product and production method thereof |
| CN105154368A (en) * | 2015-09-28 | 2015-12-16 | 内蒙古蒙牛乳业（集团）股份有限公司 | Lactobacillus plantarum multiplying culture medium and lactobacillus plantarum multiplying method |
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