CN101693882B - Lactobacillus acidophilus freeze-drying composite protectant and application method thereof - Google Patents
Lactobacillus acidophilus freeze-drying composite protectant and application method thereof Download PDFInfo
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- CN101693882B CN101693882B CN2009102361728A CN200910236172A CN101693882B CN 101693882 B CN101693882 B CN 101693882B CN 2009102361728 A CN2009102361728 A CN 2009102361728A CN 200910236172 A CN200910236172 A CN 200910236172A CN 101693882 B CN101693882 B CN 101693882B
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Abstract
The invention provides lactobacillus acidophilus freeze-drying composite protectant, and ingredients thereof includes: 9-11 parts by weight of dry buttermilk, 10-12 parts by weight of lactose, 0.3-0.6 part by weight of glycerol, 1.5-2.5 parts by weight of monosodium glutamate and 0.15-0.25 part by weight of Vitamin C. A freeze-drying protection method utilizing the lactobacillus acidophilus freeze-drying composite protectant includes steps of adding any type of lactobacillus acidophilus freeze-drying composite protectant into lactobacillus acidophilus liquor to generate protective liquor with the total weight of 130-180 parts by weight, and then freezing and drying. By utilizing the lactobacillus acidophilus freeze-drying composite protectant, higher freeze-driving survival rate of lactobacillus acidophilus is achieved with longer preservation, and the survival rate thereof is not easy to be reduced.
Description
Technical field
The present invention relates to a kind of lactobacillus acidophilus freeze-drying composite protectant and utilize this protectant lactobacillus acidophilus freeze-drying guard method.
Background technology
Because the research of Lactobacterium acidophilum deepens continuously, Lactobacterium acidophilum and meta-bolites thereof are proved healthy and helpful.It can improve or regulate intestinal microflora balance, enhancing body immunizing power, reducing cholesterol level, alleviation lactose intolerance, prevention and treatment diarrhoea, reduces anaphylaxis, prevent fungal infection etc.Therefore, develop the Lactobacterium acidophilum goods, replenish the quantity that Lactobacterium acidophilum grows surely at human intestinal,, improve immunizing power, promote healthy, significant for adjusting the human intestinal microecological balance by the external source mode.Be applied in the industries such as food, healthcare products, medicine now.Because microorganism is difficult to prolonged preservation, cause product viable bacteria preservation term short.Be unfavorable for the transportation and the sale of product.Therefore, the viable bacteria content of raising Lactobacterium acidophilum goods, prolongation viable bacteria preservation term are the key problem in technology problems that needs to be resolved hurrily in the present live lactobacillus acidophilus goods R﹠D process.
Adopting maximum methods now is freezing and lyophil preservation.The freezing goods all need freezing depositing in preservation, transportation and sales process.Expense is higher, transportation inconvenience.Lyophil preservation, not only viable bacteria content height, preservation term are long, and do not need freezing, convenient transportation during preservation.Therefore, adopting the lyophil preservation method to prepare microbial product more and more is subject to people's attention.
Lyophilize is that microorganism cells is freezing at low temperature environment, keeps the certain vacuum tightness of environment then.Make the ice distillation, reach dry at last.At low temperature, microorganism will be in dormant state under the exsiccant condition, and metabolism weakens relatively, be beneficial to the prolonged preservation activity.The cryogenic freezing of freezing dry process and process of vacuum drying can make microorganism cells change.Unsuitable lyophilize is handled and can be reduced microbic activity.Therefore in order to protect the microbial product activity; usually before freeze-drying, add some the material of defencive function is arranged; physics when these materials can change the biological sample lyophilize, chemical environment; alleviate or the infringement of cell when preventing lyophilize or rehydration, keep original various physio-biochemical characteristics and biological activity as far as possible.
At present; disclose some lactobacillus acidophilus freeze-drying protective materials, wherein the reasonable prescription of effect is: skimming milk (16.0%), maltodextrin (10.0%), trehalose (10.0%), L-L-glutamic acid (1.0%), vitamins C (2.0%), glycerine (2.0%), MnSO
4(0.8%) (" research that the freeze-dried Lactobacillus acidophilus powder protective material is selected ", Xu Liping, foodstuffs industry science and technology, 2007 05 phase).Numeric representation in the above bracket is mixed with the weight percent of solution.Though utilize this lactobacillus acidophilus freeze-drying protective material can obtain higher freeze-drying survival rate, survival rate has obvious reduction in the lower time, can not satisfy the long-time requirement that stores of Lactobacterium acidophilum.
Summary of the invention
In order to address the above problem; the invention provides a kind of lactobacillus acidophilus freeze-drying composite protectant, its prescription is as follows: 9～11 weight part skim-milks, 10～12 weight part lactose, 0.3～0.6 weight part glycerine, 1.5～2.5 weight part Sodium Glutamates and 0.15～0.25 weight part vitamins C.
Better formula is as follows: 9.5～10.5 weight part skim-milks, 10～11 weight part lactose, 0.4～0.5 weight part glycerine, 2～2.5 weight part Sodium Glutamates and 0.2～0.25 weight part vitamins C.
Optimum formula is as follows: 10 weight part skim-milks, 10 weight part lactose, 0.5 weight part glycerine, 2 weight part Sodium Glutamates and 0.2 weight part vitamins C.
Described skim-milk can be common commercially available skim-milk, and lipid content is that 0.5～1% (weight) is proper, and predominant quantity content can be protein 30～40%, lactose 50～60%, moisture content 2.5～5%.
Utilize lactobacillus acidophilus freeze-drying composite protectant of the present invention the frozen-dried protective method can for: make and contain described any lactobacillus acidophilus freeze-drying composite protectant in the Lactobacterium acidophilum bacterium liquid; must protect bacterium liquid; gross weight is 130～180 weight parts, lyophilize then.
Utilize lactobacillus acidophilus freeze-drying composite protectant of the present invention the frozen-dried protective method can for: above-mentioned any lactobacillus acidophilus freeze-drying composite protectant is mixed with the protective material aqueous solution; the total amount of water is 70～80 weight parts; then with the described protective material aqueous solution and Lactobacterium acidophilum bacteria suspension with volume ratio 1.5～2.5: 1 mixes, then lyophilize.
Preferably; the method that described lactobacillus acidophilus freeze-drying composite protectant is mixed with the protective material aqueous solution is: skim-milk, lactose and glycerine are added water and heating; pH value to 6.9～7.1 are regulated in the dissolving back; add the Sodium Glutamate and the ascorbic filtration sterilization aqueous solution under the room temperature of sterilization back, mixing promptly gets the protective material aqueous solution.
Utilize lactobacillus acidophilus freeze-drying composite protectant of the present invention, particularly utilize frozen-dried protective method of the present invention, can obtain higher lactobacillus acidophilus freeze-drying survival rate, and preservation term is longer, survival rate is difficult for reducing.
Embodiment
Below by example and in conjunction with Comparative Examples the present invention is further specified.
Example 1
Composite protectant solution formula: skim-milk 10%, lactose 10%, glycerine 0.5%, Sodium Glutamate 2%, vitamins C 0.2%.More than be the massfraction that is made into behind the aqueous solution, down with.
Wherein, skim-milk is a commercially available New Zealand skim-milk, and weight percent content is: fat 0.8%, protein 33.4%, lactose 54.1%, moisture content 3.5%.The skim-milk of below using is the same.
The composite protectant formulations prepared from solutions
1. take by weighing Sodium Glutamate and vitamins C according to quantity and add part dissolved in distilled water, filtration sterilization;
2. take by weighing skim-milk, lactose and glycerine according to quantity and add part distilled water, the heating for dissolving mixing is cooled to room temperature;
3. regulate about pH value to 7.0 with NaOH solution;
4. 115 ℃ of sterilization 10min are cooled to room temperature;
5. add above-mentioned Sodium Glutamate and vitamins C through filtration sterilization, add sterilized water to 100%, mixing gets final product.(mass content of each component in solution is: skim-milk 10%, lactose 10%, glycerine 0.5%, Sodium Glutamate 2%, vitamins C 0.2%)
Example 2:
Composite protectant solution formula: skim-milk 10%, lactose 10%, glycerine 0.3%, Sodium Glutamate 2%, vitamins C 0.2%, distilled water 77.5%.
Method according to example 1 prepares the complex protection agent solution.
Example 3:
Composite protectant solution formula: skim-milk 10%, lactose 12%, glycerine 0.6%, Sodium Glutamate 2.5%, vitamins C 0.2%, distilled water 74.7%.
Method according to example 1 prepares the complex protection agent solution.
Example 4:
Composite protectant solution formula: skim-milk 10%, lactose 10%, glycerine 0.6%, Sodium Glutamate 2.5%, vitamins C 0.25%, distilled water 76.65%.
Method according to example 1 prepares the complex protection agent solution.
Example 5:
Composite protectant solution formula: skim-milk 10%, lactose 10%, glycerine 0.6%, Sodium Glutamate 2.5%, vitamins C 0.15%, distilled water 76.75%.
Method according to example 1 prepares the complex protection agent solution.
Comparative Examples
Prescription (according to: " research that the freeze-dried Lactobacillus acidophilus powder protective material is selected "; Xu Liping; foodstuffs industry science and technology, 2007 05 phases): skim-milk (16.0%), maltodextrin (10.0%), trehalose (10.0%), L-L-glutamic acid (1.0%), vitamins C (2.0%), glycerine (2.0%), MnSO
4(0.8%).
Wherein, maltodextrin is the Hebei China occasion Dian Fentang MD150 of company limited (11＜=DE＜16).
Method according to example 1 prepares the complex protection agent solution.
The following describes above-mentioned each protectant result of use.
At first, preparation Lactobacterium acidophilum bacteria suspension: Lactobacterium acidophilum is inserted in the liquid nutrient medium by the solid slant culture base, cultivated 24h down, activate at 37 ℃.After activating three times, the inoculum size with 2% is seeded to Erlenmeyer flask and carries out enlarged culturing.Take out a small amount of cultured bacterium liquid and carry out microscopy, behind definite no living contaminants, more cultured bacterium liquid is placed whizzer, centrifugal 15min under the rotating speed of 4800r/min takes out centrifuge tube, discharges supernatant liquor, collects bacteria suspension in the pipe bottom with liquid-transfering gun.
Then, add each complex protection agent solution (volume ratio of complex protection agent solution and Lactobacterium acidophilum bacteria suspension is 2: 1) respectively, carry out lyophilization behind the mixing.Viable bacteria level before the freeze-drying is 4.41 * 10
12Cfu/mL.Sampling adds water redissolution (making that protectant concentration and freeze-drying are preceding identical) after the freeze-drying, surveys the viable bacteria level, and 4 ℃ of preservations are surveyed the viable bacteria level respectively after 1 month, 3 months, 6 months, 12 months more respectively then.
Wherein, the method for lyophilization is: the bacteria suspension that will be added with different cryoprotectants is sub-packed in the little ampoule, and every bottle of 0.5mL freezes 1h and bled 3 minutes afterwards in-35 ℃, dry under vacuum tightness 10.67～12.00Pa condition.Elevated temperature to 15 ℃ gradually behind freeze-drying 5～6h, to discharge remaining moisture, the moisture content that makes freeze-dried products is 1.50%～2.50%, is used to measure total number of bacterial colony then.
The method of surveying the viable bacteria level is: respectively the bacterium sample after the freeze-drying is added to 0.5mL with 10% sterilization nonreactive skimmed milk.Carry out 10 gradient dilutions with diluent, select the suitable dilution gradient, draw 10 μ L bacteria suspensions and place on the MRS agar plate, each the dilution gradient do 3 parallel, bacteria suspension evenly is coated with out, then flat board is moved into 37 ℃ of constant temperature culture 48h in the incubator, count after waiting to grow bacterium colony.
The data that record are as follows:
As can be seen from the above table, utilize lyophilized vaccine of the present invention can obtain higher lactobacillus acidophilus freeze-drying survival rate, and preservation term is longer, survival rate is difficult for reducing.
Claims (2)
1. lactobacillus acidophilus freeze-drying composite protectant is characterized in that, it is as follows to fill a prescription: 10 weight part skim-milks, 10 weight part lactose, 0.5 weight part glycerine, 2 weight part Sodium Glutamates and 0.2 weight part vitamins C.
2. lactobacillus acidophilus freeze-drying guard method; it is characterized in that; the described lactobacillus acidophilus freeze-drying composite protectant of claim 1 is mixed with the protective material aqueous solution; the total amount of water is 70～80 weight parts; then with the described protective material aqueous solution and Lactobacterium acidophilum bacteria suspension with volume ratio 1.5～2.5: 1 mixes; lyophilize then
Wherein, the method that the described lactobacillus acidophilus freeze-drying composite protectant of claim 1 is mixed with the protective material aqueous solution is: skim-milk, lactose and glycerine are added water and heating; pH value to 6.9～7.1 are regulated in the dissolving back; add the Sodium Glutamate and the ascorbic filtration sterilization aqueous solution under the room temperature of sterilization back, mixing promptly gets the protective material aqueous solution.
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| CN101971920B (en) * | 2010-09-28 | 2013-02-27 | 北京市农林科学院 | Porcine lactobacillus reuteri lyophilized preparation and preparation method thereof |
| CN105543141B (en) * | 2016-01-14 | 2019-01-01 | 西华大学 | A kind of norcholesterol bacteria preparation and its preparation method and application |
| CN106361778B (en) * | 2016-09-08 | 2019-10-22 | 济南康多宝生物技术有限公司 | A kind of lactobacillus preparation and its application in baby diarrhea treatment |
| CN106754525B (en) * | 2016-12-28 | 2020-11-03 | 石家庄君乐宝乳业有限公司 | Lactobacillus paracasei N1115 freeze-dried powder leavening agent and preparation method thereof |
| CN108570421B (en) * | 2017-03-09 | 2020-12-22 | 北京大北农科技集团股份有限公司 | Lactobacillus plantarum strain and silage starter thereof |
| CN109880776A (en) * | 2019-04-17 | 2019-06-14 | 合肥工业大学 | A kind of direct putting type composite ferment and its preparation method and application |
| CN112646906B (en) * | 2020-12-30 | 2022-06-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Diarrhea-causing escherichia coli standard reference strain containing specific molecular target and detection and application thereof |
| CN113150996A (en) * | 2021-05-07 | 2021-07-23 | 威凯海思(山东)生物工程有限公司 | Lactic acid bacteria composite freeze-drying protective agent and application method thereof |
| CN114836337B (en) * | 2022-03-04 | 2024-04-02 | 广东省实验动物监测所 | Freeze-drying protective agent and freeze-drying powder for pasteurella pneumophila and preparation method thereof |
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