CN109234205A - A kind of the preservation protective agent and freeze-drying store method of lactobacillus acidophilus - Google Patents
A kind of the preservation protective agent and freeze-drying store method of lactobacillus acidophilus Download PDFInfo
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- CN109234205A CN109234205A CN201811321260.3A CN201811321260A CN109234205A CN 109234205 A CN109234205 A CN 109234205A CN 201811321260 A CN201811321260 A CN 201811321260A CN 109234205 A CN109234205 A CN 109234205A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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Abstract
The present invention relates to a kind of preservation protective agent of lactobacillus acidophilus and freeze-drying store methods; method is mainly achieved the goal by adjusting protectant formula and optimization freeze drying process; store method includes: that lactobacillus acidophilus is carried out increasing bacterium in culture dish; protective agent is added into culture dish; spreading rod obtains mixed bacteria liquid after smoothening, mixed bacteria liquid is sub-packed in 10mL penicillin bottle, every bottle of 0.8~1mL of packing; it covers after standing 30 minutes, sequentially pre-freeze, freezing and vacuum drying.The protective agent includes following components: trehalose 4~5%; skimmed milk powder 10~12%; sodium glutamate 0.4~0.5%, potassium tripolyphosphate 2~3%, gelatin 0.1~0.15%; ascorbic acid 3~4%; borate buffer solution (BBS, pH8.4) 1~3%, the lactobacillus acidophilus purity is high after present invention freeze-drying; viable count is high, and retention cycle is long.
Description
Technical field
The present invention relates to cell preservation technique fields, and in particular to a kind of preservation protective agent of lactobacillus acidophilus and freezing are dry
Dry store method.
Background technique
Lactobacillus acidophilus belongs to lactobacillus, Gram-positive bacillus, and it is a kind of anaerobism or facultative that form, which is in elongated rod-shaped,
The microorganism of anaerobism, acid resistance is strong, can in the environment that other lactic acid bacterias cannot grow growth and breeding.It can be carried out using a variety of sugar
Fermentation.Lactobacillus acidophilus is widely present in the enteron aisle of people and some animals, belongs to beneficial microbe colony, when there are a fixed numbers
When amount, it can advantageously adjust the balance between harmful microorganism flora beneficial in enteron aisle, to the physiological function for maintaining gastrointestinal tract
It plays an important role.
With the development of aquaculture in recent years, the problem of antibiotic, is continuously emerged, probiotics constantly start by
The attention of people, lactobacillus acidophilus are exactly that a kind of strain being wherein widely used needs in lactobacillus acidophilus production process
Freeze-drying preservation is carried out to Lactobacillus acidophilus species, that is, the microbial cell for being intended to save makes in vacuum condition and frozen state
Ice distillation, finally reaches drying, however, thallus will receive mechanical damage, carefully after being freeze-dried in the state of unprotect
After birth permeability changes, and dynamic equilibrium is destroyed, and bacterium will be dead, is used for the freezing of lactobacillus acidophilus currently on the market
Drying protectant majority is single substance, cannot be protected comprehensively to lactobacillus acidophilus, and protecting effect is general, while big at present
Partly lyophilized technique also more falls behind, and therefore, screening maximum can improve Survival probability of bacteria and increase answering for pot-life
Technique with protective agent and freeze-drying, it is just particularly important.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of lactobacillus acidophilus
Save protective agent and freeze-drying store method.
The purpose of the present invention can be achieved through the following technical solutions: a kind of preservation protective agent of lactobacillus acidophilus, should
Protective agent includes the composition of following weight percent:
In mentioned component, skimmed milk powder be dissolved in the solution after water can saltout in supercooled state, protein is denaturalized it is few, can shape
At protective film, cell and extraneous contact area are reduced, protects cell wall, while ingredient therein such as lactalbumin, lactose
Deng can play a protective role；Trehalose can form unique protective film in cell surface under conditions of high and cold, effectively protect
Protein molecule is protected, and protein structure can be kept more stable by acting on protein hydrated sheath, not vulnerable to extraneous adverse environment
Influence；Sodium glutamate has antioxidation, inhibits the formation of triacylglycerol and free radical, can prevent caused by cell membrane
Irreversible destruction；The hydroxyl of ascorbic acid can form hydrogen bond between the phosphoric acid group of the phospholipid molecule in cell wall,
To prevent cell membrane to be damaged because of dehydration, in addition ascorbic acid still has oxidation resistant effect；Potassium tripolyphosphate can be
Bound water molecule in intracellular fluid weakens the crystallization process of water in freeze-drying process, reduces the crystallization water to the detrimental effect of cell；
The colloid structure of gelatin can play skeleton function, protected protein matter molecule in freeze-drying process, while can improve to a certain extent
The structural stability of protein and enzyme；Borate buffer solution can also play the role of to a certain extent it is oxidation resistant, while can
Prevent the excessive descent of mixed bacteria liquid ph.The present invention will appeal different types of protective agent and mix to form compounding protection
Agent determines that optimal protective agent matches by orthogonal experiment, and each component can play respective effect in freezing dry process,
There is synergistic effect again each other, such as high score subclass protective agent (skimmed milk powder, trehalose, gelatin) is by being wrapped in outside thallus
Layer reduces it and is exposed to extraneous area, can directly protect thallus, and mass exchange, which can also be formed, inside and outside inhibition thallus makes low point
The environment that the protective agent (sodium glutamate, potassium tripolyphosphate, ascorbic acid) of subclass can preferably play a role；And boric acid salt buffer
The effect that liquid adjusts ph also can make other protective agents preferably play a role at suitable ph, improve to lactobacillus acidophilus
Protecting effect, the survival rate after effectively promoting lactobacillus acidophilus freeze-drying, meanwhile, it improves and is protected under the conditions of 4 DEG C and -20 DEG C
The survival rate of strain when depositing.
A kind of freeze-drying store method carrying out lactobacillus acidophilus using protective agent as described above, comprising the following steps:
(1) lactobacillus acidophilus is placed in culture dish and carries out culture increasing bacterium, then added in the culture solution of the culture dish
Enter protective agent, obtains mixed bacteria liquid after mixing；
(2) mixed bacteria liquid is sub-packed in penicillin bottle, is stood；
(3) penicillin bottle equipped with mixed bacteria liquid is successively subjected to pre-freeze, freezing and vacuum drying, completes to save.
Preferably, in the mixed bacteria liquid, protectant weight percent is 20.5%~27.62%.
Preferably, 0.8~1mL of mixed bacteria liquid is housed in each penicillin bottle, packing is then 20~24 hours too many
It can not be freeze-dried and be finished, then be easy that " boiling " phenomenon occurs under vacuum very little.
Preferably, the time of the standing is 20~30min.Since small-molecule substance penetrating cell film can just play inhibition
The effect of eutectic phenomena intracellular, small-molecule substance penetrating cell film need some times, and the time is too short can not to make protectant function
Effect performs to most preferably, and due to containing skimmed milk power in protective agent, " Yoghourt cure " phenomenon will occur if stood for a long time,
Instead by low protectant effect.
Preferably, the temperature of the pre-freeze is -25~-30 DEG C, and the pre-freeze time is 1~2h.The excessively high time mistake of pre-freezing temperature
When long, the extracellular ice crystal of formation is larger, and in addition overlong time is easy hair " solute damage " phenomenon, so that thallus cytoplasm is occurred dense
Contracting, plasmolysis, thallus shrinkage and disrepair phenomenon influence greatly life entity, and pre-freezing temperature is too low, then easily forms intracellular ice
Crystalline substance is the main reason for generating cellular damage.
Preferably, the temperature of the freezing is -55~-60 DEG C, and the time of freezing is 3~4h.
Preferably, the vacuum drying temperature is -55~-60 DEG C, and vacuum drying vacuum degree is 0~0.1Pa, vacuum
The dry time be 20~for 24 hours.
Strain purity after improving freeze-drying above improves after freeze-drying strain under same preservation condition
Survival rate reduces the preservation cost of strain for production above, while reducing the time of rejuvenation after strain thaws, significantly
Save manpower and material resources.
Compared with prior art, the beneficial effects of the present invention are embodied in following several respects:
(1) protective agent of the invention can intuitively understand the vigor and purity of strain before preservation, promote final be made
Freeze-dried powder in strain vigor and purity；
(2) the strain purity after improving freeze-drying improves after freeze-drying strain under same preservation condition
Survival rate reduces the preservation cost of strain for production above, while reducing the time of rejuvenation after strain thaws, significantly
Save manpower and material resources.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
1, prepared by lactobacillus acidophilus seed liquor
The target lactobacillus acidophilus for being stored in inclined-plane is inoculated into the Shake flask medium of 100mL, it is closed in 36~38 DEG C
Under the conditions of stationary culture 20~24 hours.As seed liquor.
The wherein formula of Medium of shaking flask fermentation are as follows: peptone 10g/L, yeast powder 5g/L, glucose 20g/L, sodium acetate
5g/L, Triammonium citrate 2g/L, dipotassium hydrogen phosphate 2g/L.
2, prepared by lactobacillus acidophilus culture dish
After the seed liquor dilution after fermentation, it is seeded on culture dish with rubbing method, under the conditions of 36~37 DEG C, is placed in and detests
Culture 20~24 hours in oxygen cylinder.
Wherein culture dish formula are as follows: 8~10g/L of peptone, 3~5g/L of powdered beef, 4~5g/L of yeast powder, glucose 15
~20g/L, 0.8~1mL/L of Tween 80,2~3g/L of dipotassium hydrogen phosphate, 3~5g/L of sodium acetate, 2~3g/L of Triammonium citrate,
0.1~0.2g/L of magnesium sulfate, 0.02~0.05g/L of manganese sulfate, 15~18g/L of agar.
3, prepared by lactobacillus acidophilus mixed bacteria liquid
By 1 concentration of table configuration control group and totally 6 groups of protective agents of sample 1-5 after each ingredient sterilizing, 5mL is pipetted with pipettor
On protective agent to culture dish, after spreading rod is smeared uniformly, mixed bacteria liquid is dispensed to 5 bottles of 10mL penicillin bottles with 1mL liquid-transfering gun
In, every bottle of 0.8~1mL mixed bacteria liquid, every group of 10 bottles of mixed bacteria liquids, each group takes 1 sample to measure initial total viable count, remaining
After sample covers packing to freeze dryer pallet, 20~30 minutes are stood, is immediately placed in freeze drier freezes later.
4, it is freeze-dried
Freeze dryer pallet is put into freeze dryer, first pre-freeze 1~2 hour under conditions of temperature is -25~-30 DEG C, it
Adjusting temperature afterwards is -55~-60 DEG C, continues freezing 3~4 hours, lasting to rise later in the case where vacuum degree is the pressure of 0~0.1Pa
Magnificent moisture 20~24 hours.
5, the survival rate of strain after detection each group protective agent is freeze-dried
After freeze-drying, every group of viable count for taking 1 sample to measure same day freeze-dried powder, every later group is divided into 2 lot samples, every batch of
4 bottles of sample, saved respectively in 4 DEG C and -20 DEG C of under the conditions ofs, at 1 month, 6 months, 12 months, 24 months every group take a sample
Total viable count is measured, calculates survival rate in conjunction with initial total viable count.
Table 1 protects agent prescription
The survival rate (%) saved under the conditions of 24 DEG C of table
| Holding time (moon) | Control group | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 |
| 0 | 78.9 | 96.20 | 96.72 | 97.15 | 97.94 | 97.20 |
| 1 | 69.72 | 79.06 | 79.53 | 79.64 | 80.68 | 78.84 |
| 6 | 51.91 | 60.61 | 60.49 | 61.65 | 62.24 | 61.12 |
| 12 | 36.27 | 44.95 | 44.62 | 44.64 | 45.30 | 43.88 |
| 24 | 10.35 | 16.92 | 17.28 | 16.58 | 17.12 | 18.72 |
The survival rate (%) saved under the conditions of 3-20 DEG C of table
| Holding time (moon) | Control group | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 |
| 0 | 78.9 | 96.20 | 96.72 | 97.15 | 97.94 | 97.20 |
| 1 | 70.78 | 84.43 | 84.15 | 84.21 | 85.44 | 84.34 |
| 6 | 59.49 | 71.64 | 71.79 | 71.29 | 72.03 | 71.35 |
| 12 | 47.34 | 58.58 | 58.15 | 59.72 | 58.95 | 59.36 |
| 24 | 35.16 | 46.41 | 47.19 | 46.52 | 47.28 | 46.91 |
Note: the calculating of survival rate: plating dilutions are carried out to bacterium powder in penicillin bottle and are coated with count detection total bacteria count, in conjunction with right
The initial total viable count that should be organized carries out the counting of survival rate, survival rate=(the initial total viable count of measurement total bacteria count/correspondence group) *
100%.
6, it can be obtained in this experimental study, by the way that Lactobacillus acidophilus species can be made using protective agent used in the experiment
The survival rate on the same day reaches 96-97% after freeze-drying, under the conditions of 4 DEG C, remains to reach 16~18%, -20 after saving 24 months
It under the conditions of DEG C, remains to reach 46~47% after saving 24 months, the survival rate after substantially increasing lactobacillus acidophilus freeze-drying
And hold capacity.
Claims (8)
1. a kind of preservation protective agent of lactobacillus acidophilus, which is characterized in that the protective agent includes the composition of following weight percent:
2. a kind of freeze-drying store method for being carried out lactobacillus acidophilus using protective agent as described in claim 1, feature are existed
In, comprising the following steps:
(1) lactobacillus acidophilus is placed in culture dish and carries out culture increasing bacterium, be then added and protect in the culture solution of the culture dish
Agent is protected, obtains mixed bacteria liquid after mixing；
(2) mixed bacteria liquid is sub-packed in penicillin bottle, is stood；
(3) penicillin bottle equipped with mixed bacteria liquid is successively subjected to pre-freeze, freezing and vacuum drying, completes to save.
3. a kind of freeze-drying store method of lactobacillus acidophilus according to claim 2, which is characterized in that described is mixed
In combined bacteria liquid, protectant weight percent is 20.5%~27.62%.
4. a kind of freeze-drying store method of lactobacillus acidophilus according to claim 2, which is characterized in that each described
Penicillin bottle in be equipped with 0.8~1mL of mixed bacteria liquid.
5. a kind of freeze-drying store method of lactobacillus acidophilus according to claim 2, which is characterized in that the standing
Time be 20~30min.
6. a kind of freeze-drying store method of lactobacillus acidophilus according to claim 2, which is characterized in that the pre-freeze
Temperature be -25~-30 DEG C, the pre-freeze time be 1~2h.
7. a kind of freeze-drying store method of lactobacillus acidophilus according to claim 2, which is characterized in that the freezing
Temperature be -55~-60 DEG C, time of freezing is 3~4h.
8. a kind of freeze-drying store method of lactobacillus acidophilus according to claim 2, which is characterized in that the vacuum
Dry temperature be -55~-60 DEG C, vacuum drying vacuum degree be 0~0.1Pa, the vacuum drying time be 20~for 24 hours.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261298A (en) * | 2022-09-06 | 2022-11-01 | 福建益昕葆生物制药有限公司 | Domestication method of high-temperature-resistant lactobacillus plantarum and preparation method of lactobacillus plantarum powder |
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| CN101486986A (en) * | 2009-02-12 | 2009-07-22 | 上海谱莱生物技术有限公司 | Preparation of freeze-dried Lactobacillus acidophilus powder |
| CN101875911A (en) * | 2010-06-23 | 2010-11-03 | 陕西科技大学 | Protective agent in the freeze drying process of Lactobacillus acidophilus |
| CN101914518A (en) * | 2010-08-02 | 2010-12-15 | 天津商业大学 | Method for preparing Lactobacillus casei microcapsule bacterial powder by spray drying |
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2018
- 2018-11-07 CN CN201811321260.3A patent/CN109234205A/en active Pending
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| CN101486986A (en) * | 2009-02-12 | 2009-07-22 | 上海谱莱生物技术有限公司 | Preparation of freeze-dried Lactobacillus acidophilus powder |
| CN101875911A (en) * | 2010-06-23 | 2010-11-03 | 陕西科技大学 | Protective agent in the freeze drying process of Lactobacillus acidophilus |
| CN101914518A (en) * | 2010-08-02 | 2010-12-15 | 天津商业大学 | Method for preparing Lactobacillus casei microcapsule bacterial powder by spray drying |
| CN101962616B (en) * | 2010-09-29 | 2012-06-27 | 陕西科技大学 | Method for preparing lactobacillus acidophilus powder by freeze-drying in vacuum |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261298A (en) * | 2022-09-06 | 2022-11-01 | 福建益昕葆生物制药有限公司 | Domestication method of high-temperature-resistant lactobacillus plantarum and preparation method of lactobacillus plantarum powder |
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Application publication date: 20190118 |