CN102453681B - Protective agent for vacuum freeze drying of lactobacillus johnsonii, and application thereof - Google Patents
Protective agent for vacuum freeze drying of lactobacillus johnsonii, and application thereof Download PDFInfo
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- CN102453681B CN102453681B CN201110419979.2A CN201110419979A CN102453681B CN 102453681 B CN102453681 B CN 102453681B CN 201110419979 A CN201110419979 A CN 201110419979A CN 102453681 B CN102453681 B CN 102453681B
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Abstract
The invention belongs to the technical field of biology, and in particular relates to a protective agent for vacuum freeze drying of lactobacillus johnsonii, and application thereof. The protective agent provided by the invention contains no sorbitol substances, has lower cost, can provide effective protection for lactobacillus johnsonii in a vacuum freeze drying process, and is favorable to improvement of the utilization efficiency of a lactobacillus microbial feed additive, the protective rate of the protective agent is high, and the yield of produced live bacteria is above 109.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of protective material for Lactobacillus johnsonii vacuum lyophilization and application thereof.
Background technology
Lactobacillus johnsonii (Lactobacillus johnsonii) is a kind of important Bacterium lacticum, belongs to probiotic bacterium, and it is mainly reflected in following three aspects: to the prebiotic function of animal: 1. regulation and control natural animal system of defense and immunity function effect; 2. prevention animal atopic dermatitis effect; 3. exclusiveness Competition.But it is harsh that Lactobacillus johnsonii requires growth conditions, at very easily inactivation of manufacture, duration of storage.
Vacuum Freezing & Drying Technology is to preserve one of effective means of fermentation probiotic bacterium, but in vacuum-freeze-dry process, owing to also having a large amount of moisture in cell, when cryopreservation cell, moisture in born of the same parents solidifies in two ways: one is to form ice crystal, and another kind is to form unformed vitrifying state.In the time that the form with ice crystal is solidified, the volume of water increases and the mechanical force of ice crystal increases cell interior electrolyte concentration, and pH changes, protein and enzyme denaturation, thus cause cell interior structural damage, finally make necrocytosis; And in vitrified solidification process, water molecules is not reset, can not produce the variation of structure and volume, thereby can not cause tissue and cell damage due to machinery or solution effect, the cell after recovery still has vigor.In born of the same parents, water molecules solidifies in ice crystal and vitreous state running balance.Therefore, to make exactly the moisture form with vitreous state as much as possible solidify in the key of cell freeze drying technology, reduce intracellular moisture simultaneously, reduce the formation of ice crystal.
In probiotics bacterial powder process of vacuum drying, add suitable protective material, be conducive to microorganism and resist freezing mechanical wounding and long-term storage, become the inevitable requirement of preserving probiotic bacterium.Can be divided into two kinds according to the difference of protective material role, perviousness protective material and impermeability protective material.Perviousness protective material refers to the organism that number molecular weight is less, is easy to water molecules in permeates cell membranes and born of the same parents, thereby reduces cell freezing point, improves the permeability of cytolemma to water molecules simultaneously.When frozen, can impel moisture in born of the same parents to ooze out, thereby reduce the formation of intracellular ice crystal.Impermeability protective material is some macromolecular substance soluble in water, can not be penetrated in born of the same parents, but can reduce the outer water content of born of the same parents, reduces the formation of the outer ice crystal of born of the same parents.Two kinds of protectant mode of action differences are used in conjunction with in refrigerating process, can effectively improve the survival rate of freeze-dried vaccine powder.In freeze-drying process, also usually add antioxidant, in case the injury of oxidation effect to cell, keep cytoactive and improve the stability of cell.
At present, about the research of lyophilized vaccine mainly concentrates on medicine field, be applied to that the research of the lyophilized vaccine of producing microorganism feed addictive active bacteria formulation is few and result difference is large, have the shortcoming that viable count is not high.In some protective material, have glucitols material, protection effect improves, but the cost of glucitols material is higher, has limited its application and current needleless protective material to Lactobacillus johnsonii vacuum lyophilization in agriculture production.
Summary of the invention
The invention provides a kind of protective material for Lactobacillus johnsonii (Lactobacillus johnsonii) vacuum lyophilization; reduce the damage of lyophilize to thalline; improve freeze-drying after product viable count and utilising efficiency, can prepare highly active Lactobacillus johnsonii freeze-dried vaccine powder.
Another object of the present invention is to provide this protective material for Lactobacillus johnsonii vacuum lyophilization in the application of preparing Lactobacillus johnsonii freeze-dried vaccine powder.
The object of the invention is to be achieved through the following technical solutions:
The invention provides a kind of protective material for Lactobacillus johnsonii (Lactobacillus johnsonii) vacuum lyophilization, every gram of wet thallus or wet bacterium mud use the protective material of following weight proportion: skimming milk 0.25-0.5g, trehalose 0.25-0.5g, Zulkovsky starch 0.25-1g, Sodium Glutamate 0.25-1g, glycerine 0.25-0.5g.
For better reaching invention effect of the present invention: every gram of wet thallus or wet bacterium mud use the protective material of following weight proportion: skimming milk: trehalose: Zulkovsky starch: Sodium Glutamate: glycerine=1: 1: 1: 1: 1.
For better reaching invention effect of the present invention: every gram of wet thallus or wet bacterium mud also can use the protective material of following weight proportion: skimming milk: trehalose: Zulkovsky starch: Sodium Glutamate: glycerine=2: 1: 2: 4: 1.
For better reaching invention effect of the present invention: every gram of wet thallus or wet bacterium mud also can use the protective material of following weight proportion: skimming milk: trehalose: Zulkovsky starch: Sodium Glutamate: glycerine=2: 2: 4: 1: 2.
Protective material for Lactobacillus johnsonii vacuum lyophilization of the present invention can be applicable to prepare Lactobacillus johnsonii freeze-dried vaccine powder.
Further, prepare the process of Lactobacillus johnsonii freeze-dried vaccine powder as follows: (1) Lactobacillus johnsonii kind subbase is cultivated; (2) high density fermentation; (3) collection of zymophyte mud; (4) preparation of freeze-dried vaccine powder.
Further, the process of preparing Lactobacillus johnsonii freeze-dried vaccine powder is as follows: (1) Lactobacillus johnsonii kind subbase is cultivated: from flat board, single bacterium colony of picking Lactobacillus johnsonii is to the MRS substratum of 50mL, 37 degree constant temperature culture 18h, then be forwarded in the MRS substratum of 500mL with 5% inoculum size, continue the lower static cultivation 18h of 37 degree, obtain kind of a subbase; (2) high density fermentation: the parameter of high density fermentation arranges as follows: ammoniacal liquor regulates and maintains pH value 5.0; 50 revs/min of rotating speeds; Temperature 37 is spent; Tank pressure keeps 0.05MPa; Fermentation time 12 hours; (3) collection of zymophyte mud: centrifugal 10 minutes of fermented liquid 4000g, supernatant discarded; With physiological saline, thalline is resuspended; Centrifugal 10 minutes of 4000g, supernatant discarded; (4) preparation of freeze-dried vaccine powder: add the protective material of 5 times of volumes in the bacterium mud of preparation, mix balance 30 minutes; Spend pre-freeze 3 hours in-80; Freeze-drying 30-36 hour in vacuum freeze drier, freeze-drying parameter arranges as follows: vacuum tightness 0.1Mba-0.2Mba; Condenser temperature is extremely-55 degree of-50 degree, to obtain final product.
Beneficial effect
The present invention is the preservation of the preservation of milk-acid bacteria, especially Lactobacillus johnsonii, and a new protective material prescription is provided; In this protective material, without glucitols material, cost is lower; This protective material can play effective provide protection in vacuum lyophilization process to Lactobacillus johnsonii, protection ratio is high, and the viable bacteria yield of producing is 10
9above, be conducive to the raising of lactobacillus microorganism fodder additives utilising efficiency.
, it should be understood that described embodiment is only for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by the following example.
Embodiment
Embodiment 1-embodiment 18
(1) preparation of kind subbase
Single bacterium colony of picking Lactobacillus johnsonii (Lactobacillus johnsonii) is to MRS substratum (10 grams of the peptones of 50mL, 10 grams of meat extracts, 5 grams of yeast extracts, 2 grams of dipotassium hydrogen phosphates, 2 grams of dibasic ammonium citrates, 5 grams of sodium acetates, 20 grams of glucose, soil temperature 1mL, 0.58 gram of magnesium sulfate heptahydrate, four 0.25 gram of water manganous sulfate, distilled water 1L, pH6.2-6.4) in, 37 degree constant temperature culture 18 hours, then 5% inoculum size is forwarded in the MRS substratum of 500mL, continues the lower static cultivation of 37 degree 18 hours, value OD
600nmbe 5.0 to obtain kind of a subbase.
(2) high density fermentation
1% inoculum size, by kind of the fermentor tank of subbase access 5L, keeps tank pressure as 0.05MPa take nitrogen in fermenting process, and rotating speed is 50 revs/min, and temperature is 37 degree, and it is 5.0 that Feeding ammonia water keeps pH; Cultivate and after 4 hours, start stream and add glucose and continue to cultivate 8 hours.
(3) preparation of the collection of zymophyte mud and (4) freeze-dried vaccine powder
Fermented liquid is in 4 degree, and 4000g collects thalline for centrifugal 10 minutes; Supernatant discarded; precipitation is resuspended by stroke-physiological saline solution; by above-mentioned condition recentrifuge; supernatant discarded; spend respectively the resuspended thalline of protective material of the present invention (in table 1) of the various proportionings of preheating with 5 times of volumes 37; stir balance 30 minutes, be placed in-80 degree refrigerator pre-freeze 3 hours, be placed in vacuum freeze drier.Lyophilisation condition is vacuum tightness 0.2mba, and condenser temperature is-55 degree, and freeze-drying 30 hours, obtains Lactobacillus johnsonii lyophilized powder.
(5) survival rate is measured
Take 1 gram of dry bacterial powder and be dissolved in 9mL physiological saline, mix; 10 times are diluted to 10 successively
-10; Each extent of dilution is got 100 microlitre coating MRS nutrient agar (10 grams of peptones, 10 grams of meat extracts, 5 grams of yeast extracts, 2 grams of dipotassium hydrogen phosphates, 2 grams of dibasic ammonium citrates, 5 grams of sodium acetates, 20 grams of glucose, soil temperature 1mL, 0.58 gram of magnesium sulfate heptahydrate, four 0.25 gram of water manganous sulfate, 15 grams, agar, distilled water 1L, pH6.2-6.4), each gradient do three parallel, 37 degree are cultivated 18 hours, adopt the method for plate culture count, draw the viable count in fermented liquid and freeze-drying sample, and calculate the survival rate (the results are shown in Table 1) of milk-acid bacteria.
Table 1 protective material formulating of recipe and result
Interpretation of result: protectant formula of embodiments of the invention 1, embodiment 13 and embodiment 14, the Lactobacillus johnsonii viable bacteria rate drawing is the highest.Protective material of the present invention can play effective provide protection in vacuum lyophilization process to Lactobacillus johnsonii, viable count is up to 3.73 × 10
9.
The protection effect comparison of the different protective materials of embodiment 19 to Lactobacillus johnsonii
The present invention has studied except protective material under equal experiment condition, and other experiment material and consumption be all with embodiment 1, and experimental technique is also in the situation identical with embodiment 1, the protectant protection effect of protective material of the prior art and the present invention etc.The results are shown in Table 2.
The different protective material protection effects of table 2 Lactobacillus johnsonii and cost compare
Claims (1)
1. a Lactobacillus johnsonii freeze-dried vaccine powder, preparation method thereof, is characterized in that, preparation process is as follows:
(1) Lactobacillus johnsonii kind subbase is cultivated: from flat board, single bacterium colony of picking Lactobacillus johnsonii is to the MRS substratum of 50mL, 37 degree constant temperature culture 18h, then be forwarded in the MRS substratum of 500mL with 5% inoculum size, continue the lower static cultivation 18h of 37 degree, obtain kind of a subbase;
(2) high density fermentation: the parameter of high density fermentation arranges as follows: ammoniacal liquor regulates and maintains pH value 5.0; 50 revs/min of rotating speeds; Temperature 37 is spent; Tank pressure keeps 0.05MPa; Fermentation time 12 hours;
(3) collection of zymophyte mud: centrifugal 10 minutes of fermented liquid 4000g, supernatant discarded; With physiological saline, thalline is resuspended; Centrifugal 10 minutes of 4000g, supernatant discarded;
(4) preparation of freeze-dried vaccine powder: add the protective material of 5 times of volumes in the bacterium mud of preparation, mix balance 30 minutes; Spend pre-freeze 3 hours in-80; Freeze-drying 30-36 hour in vacuum freeze drier, freeze-drying parameter arranges as follows: vacuum tightness 0.1Mba-0.2Mba; Condenser temperature is extremely-55 degree of-50 degree, to obtain final product,
Wherein, every gram of wet thallus or wet bacterium mud use the protective material of following weight proportion: skimming milk 0.25-0.5g, trehalose 0.25-0.5g, Zulkovsky starch 0.25-1g, Sodium Glutamate 0.25-1g, glycerine 0.25-0.5g,
Wherein, every gram of wet thallus or wet bacterium mud use the protective material of following weight proportion: skimming milk: trehalose: Zulkovsky starch: Sodium Glutamate: glycerine=1: 1: 1: 1: 1, or
Every gram of wet thallus or wet bacterium mud use the protective material of following weight proportion: skimming milk: trehalose: Zulkovsky starch: Sodium Glutamate: glycerine=2: 1: 2: 4: 1, or
Every gram of wet thallus or wet bacterium mud use the protective material of following weight proportion: skimming milk: trehalose: Zulkovsky starch: Sodium Glutamate: glycerine=2: 2: 4: 1: 2.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102696885A (en) * | 2012-06-08 | 2012-10-03 | 北京大北农科技集团股份有限公司 | Application of Lactobacillus johnsonii to improvement of weaned pig production performance |
| CN103667063A (en) * | 2013-12-12 | 2014-03-26 | 福建省农业科学院农业工程技术研究所 | Composite protective agent of MLF (Malolactic Fermentation) lactic acid bacteria freeze-dried starter culture and preparation method of composite protective agent |
| CN103689595A (en) * | 2013-12-12 | 2014-04-02 | 天津科技大学 | Soft lactic acid bacteria capsule and preparation method thereof |
| CN109757730B (en) * | 2019-01-31 | 2021-06-04 | 山东环亿生物科技有限公司 | Composition with functions of reducing blood fat, blood pressure and blood sugar and preparation method thereof |
| CN111500512A (en) * | 2020-04-17 | 2020-08-07 | 张大生 | Three recombinant lactic acid bacteria for expressing avian influenza virus protein and construction method and application thereof |
| CN111534467B (en) * | 2020-05-15 | 2024-07-02 | 郑州和合生物工程技术有限公司 | Lactobacillus johnsonii with auxiliary effect of inhibiting tumor cell proliferation, and preparation method and application of lactobacillus johnsonii |
| CN112457990A (en) * | 2020-12-18 | 2021-03-09 | 中国科学院合肥物质科学研究院 | Freeze-drying protective agent and application thereof |
| CN114292788A (en) * | 2021-12-30 | 2022-04-08 | 江苏德禧生物科技有限公司 | Preparation method of active probiotics by freeze drying |
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