CN100352914C - Vacuum freeze-dried product of antagonist bacteria C.laurentii of yeast and its preparation method - Google Patents
Vacuum freeze-dried product of antagonist bacteria C.laurentii of yeast and its preparation method Download PDFInfo
- Publication number
- CN100352914C CN100352914C CNB2005100900650A CN200510090065A CN100352914C CN 100352914 C CN100352914 C CN 100352914C CN B2005100900650 A CNB2005100900650 A CN B2005100900650A CN 200510090065 A CN200510090065 A CN 200510090065A CN 100352914 C CN100352914 C CN 100352914C
- Authority
- CN
- China
- Prior art keywords
- cryptococcus laurentii
- laurentii
- dried product
- vacuum freeze
- protective material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a vacuum freeze-dried product of yeast antagonistic bacteria C. laurentii and a preparation method thereof. The vacuum freeze-dried product is obtained by collecting thallus of the yeast antagonistic bacteria C. laurentii, which is cultivated for 24 to 48h, making the thallus into bacterium suspension of which the concentration is from 5*10<8> to 5*10<9> cells/mL by protective agents, hatching the bacterium suspension for 1 to 2h, and carrying out a vacuum freeze-drying step. The protective agents are dextrose of which the mass percentage is from 5 to 10%, cane sugar, sodium acetate, defatted milk powder or a water solution of sodium glutamate. The vacuum freeze-dried product of the yeast antagonistic bacteria C. laurentii, which is prepared by the method of the present invention, has high bioactivity and has the viable bacterium rate of 80%. The present invention has the advantages of simple preparation process and low cost, and has feasibility of industrial production; the present invention can perform important functions in the preservation of the yeast antagonistic bacteria C. laurentii and other microorganisms and the preparation of associated products.
Description
Technical field
The present invention relates to vacuum freeze-dried product of microorganism and preparation method thereof, (Cryptococcus laurentii is called for short: a kind of vacuum freeze-dried product C.laurentii) and preparation method thereof particularly to relate to the antagonistic yeast Cryptococcus laurentii.
Background technology
Keep the blodynamic mode of yeast to mainly contain at present: the cultivation preservation of going down to posterity, the whiteruss cryopreservation, the inclined-plane cryopreservation, semi-solid puncture preservation, (Ashwood-Smith MJ.Preservation of microorganisms by freezing such as liquid nitrogen freezing preservation and lyophil preservation, freeze-drying and desiccation.In:Ashwood-Smith M J (Eds) .Low Temperature Preservation in Medicine andBiology.Tunbridge Wells:Pitman Medical, 1980, pp219-252.; Li Zhongqing compiles the microbial strains preservation technology. Beijing: and Science Press, 1989, pp19-28).Because having preservation term, the lyophil preservation method grows, can avoid other living contaminants, be convenient to transport and be easy to realize advantages such as commercialization production, thereby become present preservation bacterium, yeast fungal spore and mould is the most effective, one of successful method (Li Hua, Luo Yane, Liu Yanlin. the progress of vacuum lyophilization microorganism. the microbiology circular, 2002,29:78-82).Yet, be not that all microorganisms all are suitable for the vacuum lyophilization preservation, both enabled to adopt the microorganism of vacuum lyophilization preservation, its survival rate also may be lower than 0.1% (Benny J F, Hennebert G L.Viability andstability of yeast cells and filamentous fungus spores during freeze drying:effects of protectants and cooling rates.Mycologia, 1991,83:805-815.).It is reported, the factor that influences microorganism vacuum lyophilization effect mainly comprises: suspension concentration, pre-freeze speed, protectant kind and (Bozoglu T F such as concentration and rehydration mode, Ozilgen M, Bakir U.Survivalkinetics of lactic acid starter cultures during and after freez drying.EnzmymeMicrobial Technology, 1987,9:531-537.; Champage C P, Gardner N, Brochu E, Beaulier Y.The freeze-drying of lactic acid bacteria:a review.Can.Inst.Sci.Technol.J., 1991,24:118-126).In order to obtain ideal vacuum lyophilization effect, need screen preparation method and protective material thereof effectively.But do not see that as yet pair report of antagonistic yeast Cryptococcus laurentii vacuum lyophilization mode effect is arranged both at home and abroad at present.
Studies show that: the antagonistic yeast Cryptococcus laurentii all has obvious inhibition effect (Haibo Liu to the main post-harvest diseases of fruits such as grape, peach, apple, winter jujube, sweet cherry, Tian Shiping, state of Qin's political affairs, Xu Yong. Cryptococcus laurentii is to the antagonistic effect of grape post-harvest diseases. Scientia Agricultura Sinica, 2002,35:831-835.; Lin Li, Tian Shiping, state of Qin's political affairs, Xu Yong. two kinds of antagonistic yeasts to the peach fruit storage during the prevention effect of main disease. Scientia Agricultura Sinica, 2003,36:1535-1539.); Can tolerate low temperature, main holding conditions after fruit such as hypoxemia and high carbon dioxide is adopted (Qin G Z, Tian S P.Biocontrol of postharvest diseases of jujube fruitby Cryptococcus laurentii combined with a low dosage of fungicides underdifferent storage conditions.Plant Dis., 2004a, 88:497-501.), can Yu Molybdenum acid ammonium, sodium bicarbonate, the sterilant of Whitfield's ointment and low dosage (Qin et al., 2004a) be used (WanY K, Tian S P, Qin G Z.Enhancement of biocontrol activity of yeasts by addingsodium bicarbonate or ammonium molybdate to control postharvest disease ofjujube fruits.Lett.Appl.Microbiol., 2003,37:1-5.), and can spray (Tian S P adopting preceding field, Qin G Z, Xu Y.Survival of antagonistic yeasts under field conditionsand their biocontrol ability against postharvest diseases of sweet cherry.Postharyest Biol.Technol., 2004a, In press.), have a good application prospect.
Summary of the invention
The vacuum freeze-dried product that the purpose of this invention is to provide a kind of antagonistic yeast Cryptococcus laurentii.
The vacuum freeze-dried product of antagonistic yeast Cryptococcus laurentii provided by the present invention is a thalline of collecting the Cryptococcus laurentii through cultivating 24-48h, and it is made concentration with protective material is 5 * 10
8-5 * 10
9The bacteria suspension of individual cell/mL is hatched 1-2h, carries out the product that obtains after the vacuum lyophilization again; Described protective material is glucose, sucrose, sodium-acetate, skim-milk or the Sodium Glutamate aqueous solution of 5-10% for the quality percentage composition.
The culture condition of described Cryptococcus laurentii is; Inoculum density by 2% is inoculated in the antagonistic yeast Cryptococcus laurentii in the special culture media, at 25 ℃, and shaking culture under the 200rpm; The prescription of described special culture media is: citric acid 40, soy peptone 15, sal epsom 1.23, calcium chloride 0.56, unit: gL
-1
The concentration of bacteria suspension is preferably 5 * 10
8Individual cell/mL, incubation time is preferably 2h, and it is 5% skim milk powder aqueous solution that protective material is preferably the quality percentage composition.
Second purpose of the present invention provides a kind of preparation method of vacuum freeze-dried product of above-mentioned antagonistic yeast Cryptococcus laurentii.
The preparation method of the vacuum freeze-dried product of Cryptococcus laurentii provided by the present invention is a thalline of collecting the Cryptococcus laurentii through cultivating 24-48h, and it is made concentration with protective material is 5 * 10
8-5 * 10
9The bacteria suspension of individual cell/mL is hatched 1-2h, carries out obtaining after the vacuum lyophilization vacuum freeze-dried product of Cryptococcus laurentii again; Described protective material is glucose, sucrose, sodium-acetate, skim-milk or the Sodium Glutamate aqueous solution of 5-10% for the quality percentage composition.
In above-mentioned preparation method, the culture condition of described Cryptococcus laurentii is; Inoculum density by 2% is inoculated in Cryptococcus laurentii in the special culture media, at 25 ℃, and shaking culture under the 200rpm; The prescription of described special culture media is: citric acid 40, soy peptone 15, sal epsom 1.23, calcium chloride 0.56, unit: gL
-1
The concentration of bacteria suspension is preferably 5 * 10
8Individual cell/mL, incubation time is preferably 2h, and it is 5% skim milk powder aqueous solution that protective material is preferably the quality percentage composition.
Described vacuum lyophilization condition can adopt the vacuum lyophilization condition of general microbial product, as drying temperature-45 ℃, vacuum tightness 0.54mPa, time of drying 24h.
The present invention at first determines to influence the main factor of antagonistic yeast Cryptococcus laurentii vacuum lyophilization effect by orthogonal experiment; and deeply inquire into the blodynamic influence of different growth phases and protective material on this basis to freeze-dried product, obtain the effective ways of Cryptococcus laurentii vacuum lyophilization.Antagonistic yeast Cryptococcus laurentii vacuum freeze-dried product with method preparation of the present invention has higher biological activity, and the viable bacteria rate can reach 80%.Preparation technology of the present invention is simple, with low cost, has the feasibility of suitability for industrialized production, will play a great role in the preparation of the preservation of antagonistic yeast Cryptococcus laurentii and other microorganism and related products thereof.
Description of drawings
Fig. 1 is for identifying the experimental result of different protective materials to the influence of antagonistic yeast Cryptococcus laurentii vacuum lyophilization effect
Fig. 2 for identify different protective materials to antagonistic yeast Cryptococcus laurentii vacuum freeze-dried product shelf-lives (0 ℃, March) influence experimental result
The experimental result of Fig. 3 for identifying that different protective materials exert an influence to cell membrane integrity after 0 ℃ of storage of antagonistic yeast Cryptococcus laurentii vacuum freeze-dried product March and active oxygen
Embodiment
Be ordinary method among the following embodiment if no special instructions.
Embodiment 1,
One, the preservation of antagonistic yeast Cryptococcus laurentii kind, activation and fermentation culture thereof
1, the separation of bacterial classification and preservation
Separate acquisition antagonistic yeast Cryptococcus laurentii kind from the apple fruit surface, place glycerine-20 ℃ preservation (Haibo Liu, Tian Shiping, state of Qin's political affairs, Xu Yong. Cryptococcus laurentii is to the antagonistic effect of grape post-harvest diseases. Scientia Agricultura Sinica, 2002,35:831-835).
2, the activation of bacterial classification
Draw a small amount of bacterium liquid in the glycerine pipe of from-20 ℃, preserving, in NYDA substratum (glucose 10, soy peptone 5, extractum carnis 1, yeast extract 7.5, agar 18, unit: gL
-1) bed board and cultivation under 28 ℃, well-grown antagonistic yeast that takes a morsel behind the 72h is seeded in NYDB substratum (glucose 10, soy peptone 5, extractum carnis 1, yeast extract 7.5, unit: gL
-1) in further activation, bed board, and on the NYDA substratum, separate single bacterium colony.
3, culture of strains
Picking list bacterium colony inserts special culture media (citric acid 40, soy peptone 15, sal epsom 1.23, calcium chloride 0.56, the unit: gL of Cryptococcus laurentii
-1) in (containing the 200mL nutrient solution in the 1000mL triangular flask), shaking culture (25 ℃, 200rpm) 12-18h.
4, fermentation culture
The bacterial classification inoculation of step 3 being cultivated with 2% inoculum density carries out enlarged culturing under the same conditions in 120L fermentor tank (containing the 80L nutrient solution).
Two, influence the factor analysis experiment of antagonistic yeast Cryptococcus laurentii vacuum lyophilization effect
Adopt four factors, the orthogonal experiment L of three levels
9(4
3); determine the influence of different incubation times, suspension concentration, protective material kind and incubation time to Cryptococcus laurentii vacuum lyophilization effect; find the main factor of antagonistic yeast vacuum lyophilization effect, it is carried out the mode of vacuum lyophilization so that further determine.Orthogonal experiment L
9(4
3) selected different factors and the design of level be as shown in table 1, is contrast with antagonistic yeast R.glutinis:
Table 1 influences the orthogonal experiment L of antagonistic yeast Cryptococcus laurentii and R.glutinis vacuum lyophilization effect
9(3
4) factor and level
| Factor and level | Incubation time (h) | The concentration of yeast suspension (individual cell/mL) | Incubation time (h) | Protective material (5%) |
| 1 2 3 | 24 48 72 | 1×10 8 5×10 8 5×10 9 | 1 2 3 | The trehalose G/W |
Concrete experimental technique is: the method with step 1 is cultivated Cryptococcus laurentii; every kind of bacterium is established three different fermented incubation times (24,48 and 72h); after cultivating end; centrifugal collection thalline; with thalline aseptic water washing 3 times, again by being made into difference with different protective materials through the thalline that different time is cultivated shown in the table 1
The bacteria suspension of concentration, hatch different time after, place-18 ℃ of refrigerator and cooled to freeze, behind the 24h freezing sample put into drying in the freeze drier (FD-1 type, Beijing rich doctor Kanggong department).Drying temperature is-45 ℃, vacuum tightness 0.54mPa, and be 24h time of drying.Add the sterilized water rehydration immediately behind dry the end, and carry out 1/10,1/100,1/1000,1/10000 gradient dilution, bed board is cultivated 2d for 28 ℃, the statistics colony number.Experimental data adopts the method for general quadrature analysis to carry out (Montagomery, D C.Design and analysis of experiments, 4th ed.NewYork:Wiley, 1996, pp 627-631), and experimental result is as shown in table 2:
The different incubation times of table 2, bacteria suspension concentration, incubation time and protective material are to the Orthogonal experiment results and the intuitive analysis table of antagonistic yeast Cryptococcus laurentii lyophilize effect
| The experiment group number | Incubation time (h) | The concentration of yeast suspension (individual cell/mL) | Incubation time (h) | Protective material (5%) | Survival rate (%) |
| 1 2 3 4 5 6 7 8 9 K 1 a K 2 K 3 k 1 b k 2 k 3 R c | 24 24 24 48 48 48 72 72 72 18.57 16.41 6.67 9.28 8.21 3.34 5.95 | 1×10 8 5×10 8 5×10 9 1×10 8 5×10 8 5×10 9 1×10 8 5×10 8 5×10 9 5.88 16.53 19.24 2.94 8.27 9.62 6.68 | 1 2 3 2 3 1 3 1 2 16.20 16.89 8.56 8.10 8.45 4.28 4.17 | Trehalose G/W water trehalose glucose G/W trehalose 1.55 34.76 5.35 0.77 17.38 2.67 16.61 | 0.27 22.55 5.03 1.44 0.70 22.48 7.11 1.55 1.35 |
aK
i A=∑ survival rate A
i. experimental data is the mean value of three repeated experiments
bk
i A=K
i A/ 2. experimental data is the mean value of three repeated experiments
cR
i A=maximum (k
i A)-minimum (k
i A). experimental data is the mean value of three repeated experiments
Table 2 data show; each factor is followed successively by the size that influences of Cryptococcus laurentii lyophilize effect: protective material kind＞bacteria suspension concentration＞yeast incubation time＞protective material incubation time; in four factors of experiment, the protective material kind has the greatest impact to Cryptococcus laurentii vacuum lyophilization effect.
Average between each factor different levels is compared, and the result shows that vacuum lyophilization effect difference in yeast is cultivated 24-48h of Cryptococcus laurentii is little, and the yeast suspension concentration also has only from 1 * 10
8Individual cell/mL brings up to 5 * 10
8Influence is bigger during individual cell/mL, and the protective material incubation time is little to cryodesiccated influence in 1-2h.Therefore the vacuum lyophilization condition of preliminary definite Cryptococcus laurentii is: yeast incubation time 24-48h, suspension concentration are 5 * 10
8-5 * 10
9Individual cell/mL, incubation time 2h.Because protective material kind having the greatest impact to the lyophilize effect; consider simultaneously between the different factors and also may have interaction; on this basis, further identify the influence of incubation time and protectant kind and concentration in the following embodiments to Cryptococcus laurentii lyophilize effect.
Embodiment 2, different protective material are to the blodynamic influence of antagonistic yeast vacuum freeze-dried product
It is centrifugal to cultivate the antagonistic yeast Cryptococcus laurentii liquid of 24h and 48h respectively with method therefor among the embodiment 1, collecting precipitation, aseptic water washing 3 times.Use the protective material (glucose, lactose, sucrose, trehalose, Trisodium Citrate, sodium-acetate, skim-milk and Sodium Glutamate) of different concns to suspend respectively precipitation; water belongs with yin contrast with the unprotect agent; with the nutrient solution of yeast after 24h and 48h cultivation is contrast, and making final bacteria suspension concentration is 5 * 10
8Individual cell/mL behind the incubated at room 1-2h, puts into-18 ℃ of refrigerator and cooled and freezes 24h, and it is dry then freezing sample to be put into FD-1 type freeze drier, drying temperature-45 ℃, vacuum tightness 0.54mPa, time 24h.
Behind dry the end sample is taken out, a part is used the volume of sterilized water rehydration to the lyophilize immediately, adopts the dilution-plate method counting, investigate different protective materials to the yeast lyophilize after the influence of survival rate.Another part freeze drying example is packed, is sealed with centrifuge tube, preserves after 3 months down for 0 ℃, uses the sterilized water rehydration again, and the dilution-plate method counting is to identify the influence of different lyophilize conditions to antagonistic yeast freeze-dried product shelf-lives.
Partial data to different protective material screening experiment of single factor utilizes the SPSS software analysis; by the ANOVA mode, adopt the multiple variance analysis of Duncan formula (Duncan ' s multiple range tests) method that the otherness between handling is analyzed on P＜0.05 level.Experimental result as shown in Figure 1, show lyophilize after, only the Cryptococcus laurentii survival rate that suspends with sterilized water is between 3.9-5.1%, wherein the yeast survival rate of cultivating behind the 24h is higher.And during with the nutrient solution suspension cell, no matter cultivate 24h or 48h, the viable bacteria rate after the Cryptococcus laurentii lyophilize is all below 0.5%.
Survival rate after the lyophilize of antagonistic yeast Cryptococcus laurentii also has notable difference because of protectant kind is different with concentration.In four kinds of sugar; have only dextrose plus saccharose apparent in view to the provide protection of Cryptococcus laurentii; both are when protective material concentration is 10%, effect during incubation time 24h is best, and nonetheless, the survival rate after the freeze-drying of antagonism bacterium Cryptococcus laurentii also has only 46.8% and 31.8%.And the yeast survival rate of handling with lactose and trehalose all is lower than 15%.In three kinds of sodium salts, the action effect of Trisodium Citrate is relatively poor; Effect when 5% sodium-acetate and Sodium Glutamate, cultivation 48h is better, and the viable bacteria rate reaches 33.9% and 28.2% respectively.Skim-milk has the better protecting effect equally to the antagonistic yeast Cryptococcus laurentii, and under the protection of 5% skim-milk, the survival rate when cultivating 48h after the yeast freeze-drying reaches 80%.
The concentration effect influence that the different growth phases of yeast Cryptococcus laurentii are protected agent is very big.When cultivating 24h, increase the survival rate that dextrose plus saccharose concentration all can significantly improve Cryptococcus laurentii, and trehalose concentration reduces significantly on the contrary from 5% viable count of bringing up to 10% o'clock Cryptococcus laurentii.After yeast is cultivated 48h, except that glucose, increase the lyophilize effect that protective material concentration all significantly reduces Cryptococcus laurentii.
Equally, the different growth phases of antagonistic yeast Cryptococcus laurentii also have notable difference to the influence of its lyophilize effect because of protectant kind is different with concentration.When being protective material, prolong the survival rate that incubation time all can significantly improve Cryptococcus laurentii, and the protective material of different concns all is like this with Sodium Glutamate and skim-milk.And when being protective material with glucose and trehalose, the survival rate of antagonistic yeast Cryptococcus laurentii all prolongs and significantly reduces along with incubation time.
Embodiment 3, different protective material are to the influence of the freeze-dried product storage tolerance of Cryptococcus laurentii
Storage is after 3 months down at 0 ℃ for the freezing dry products of Cryptococcus laurentii of embodiment 2 acquisitions, and the survival rate of the antagonism bacterium Cryptococcus laurentii freeze drying example that each is handled is decline (Fig. 2) significantly all.When the Cryptococcus laurentii incubation time was 24h, the freeze-dried products survival rate of all processing was all reduced to below 3% after 3 months.When yeast is cultivated 48h; except lactose, trehalose and three kinds of protectant different concns freeze-dried products viable bacteria rates of sodium acetate all are lower than 1%, other protective material is 5% o'clock in concentration, and survival rate is between the 8-19%; and protective material concentration is 10% o'clock, and survival rate is then reduced between the 0.8-6.7%.
Embodiment 4, different protective material are to the influence of freezing dry products cell membrane integrity of antagonistic yeast Cryptococcus laurentii and active oxygen
Cell membrane integrity and the active oxygen of measuring the freezing dry products of Cryptococcus laurentii of embodiment 2 acquisitions with the viable cell fluorescent marker method produce.Result Bright Field (light field) as shown in Figure 3 is all cells sum, and the cell of PI mark is red (film system destruction), H
2The cell of DCFDA mark is green (producing ROS), and (Integrated uses PI to two dyestuffs, H
2The common mark of DCFDA) cell of mark is yellow (dead).A: skim-milk (5%), 48h; B: skim-milk (10%), 48h; C: skim-milk (5%), 24h; D: sterilized water.Bar，20μm。); show in storage under 0 ℃ after 3 months; for cultivating the Cryptococcus laurentii of 48h; with 5% skim-milk is that most of saccharomycetic cytolemma are kept perfectly in protectant freeze-drying sample; raising skim-milk concentration (10%) then can promote the destruction of yeast cell film; but all have in two kinds of processing the part cell can produce ROS (Fig. 3 A, B).Shorten the cell proportion that yeast incubation time (24h) can improve PI (+) in the Cryptococcus laurentii equally; freeze-dried products in 0 ℃ the storage 3 months after nearly all cell all dyeed by PI; and can't detect the cell (Fig. 3 C) that produces ROS, this with do not add protectant processing be more or less the same (Fig. 3 D).These differences between the different treatment and the basically identical as a result that utilizes plate dilution method to obtain.
Claims (9)
1, the vacuum freeze-dried product of Cryptococcus laurentii (Cryptococcus laurentii) is to collect through cultivating the Cryptococcus laurentii body of 24-48h, and it is made concentration with protective material is 5 * 10
8-5 * 10
9The bacteria suspension of individual cell/mL is hatched 1-2h, carries out the product that obtains after the vacuum lyophilization again; Described protective material is glucose, sucrose, sodium-acetate, skim-milk or the Sodium Glutamate aqueous solution of 5-10% for the quality percentage composition.
2, the vacuum freeze-dried product of Cryptococcus laurentii according to claim 1, it is characterized in that: the culture condition of described Cryptococcus laurentii is: the inoculum density by 2% is inoculated in Cryptococcus laurentii in the special culture media, at 25 ℃, shaking culture under the 200rpm; The prescription of described special culture media is: citric acid 40, soy peptone 15, sal epsom 1.23, calcium chloride 0.56, unit: gL
-1
3, the vacuum freeze-dried product of Cryptococcus laurentii according to claim 1 and 2 is characterized in that: described protective material is 5% skim milk powder aqueous solution for the quality percentage composition.
4, the vacuum freeze-dried product of Cryptococcus laurentii according to claim 1 and 2 is characterized in that: the concentration of described bacteria suspension is 5 * 10
8Individual cell/mL, incubation time are 2h.
5, the preparation method of the vacuum freeze-dried product of the described Cryptococcus laurentii of a kind of claim 1 is to collect through cultivating the Cryptococcus laurentii body of 24-48h, and it is made concentration with protective material is 5 * 10
8-5 * 10
9The bacteria suspension of individual cell/mL is hatched 1-2h, carries out obtaining after the vacuum lyophilization vacuum freeze-dried product of Cryptococcus laurentii again; Described protective material is glucose, sucrose, sodium-acetate, skim-milk or the Sodium Glutamate aqueous solution of 5-10% for the quality percentage composition.
6, preparation method according to claim 5 is characterized in that: the culture condition of described Cryptococcus laurentii is: the inoculum density by 2% is inoculated in Cryptococcus laurentii in the special culture media, at 25 ℃, and shaking culture under the 200rpm; The prescription of described special culture media is: citric acid 40, soy peptone 15, sal epsom 1.23, calcium chloride 0.56, unit: gL
-1
7, according to claim 5 or 6 described preparation methods, it is characterized in that: described protective material is 5% skim milk powder aqueous solution for the quality percentage composition.
8, according to claim 5 or 6 described preparation methods, it is characterized in that: the concentration of described bacteria suspension is 5 * 10
8Individual cell/mL, incubation time are 2h.
9, according to claim 5 or 6 described preparation methods, it is characterized in that: described vacuum lyophilization condition is: drying temperature-45 ℃, vacuum tightness 0.54mPa, time of drying 24h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100900650A CN100352914C (en) | 2005-08-11 | 2005-08-11 | Vacuum freeze-dried product of antagonist bacteria C.laurentii of yeast and its preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100900650A CN100352914C (en) | 2005-08-11 | 2005-08-11 | Vacuum freeze-dried product of antagonist bacteria C.laurentii of yeast and its preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1733898A CN1733898A (en) | 2006-02-15 |
| CN100352914C true CN100352914C (en) | 2007-12-05 |
Family
ID=36076516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100900650A Expired - Fee Related CN100352914C (en) | 2005-08-11 | 2005-08-11 | Vacuum freeze-dried product of antagonist bacteria C.laurentii of yeast and its preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100352914C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101822288A (en) * | 2010-04-02 | 2010-09-08 | 浙江大学 | Preparation method of vacuum freeze-dried product of marineyeast Rhodosporidium paludigenum Fell and Tallman |
| CN102140424B (en) * | 2010-12-30 | 2012-11-14 | 广东环凯微生物科技有限公司 | Preparation method of quantitative microorganism freeze-dried product |
| CN104161116B (en) * | 2014-07-04 | 2016-06-29 | 浙江大学 | Induction fruit resistance controls the preparation of disease and corresponding revulsion |
| CN107325970B (en) * | 2016-01-15 | 2022-07-05 | 西南大学 | Method for vacuum freeze drying of pichia membranaefaciens and application thereof |
| CN106106721B (en) * | 2016-06-28 | 2020-03-17 | 浙江大学 | Method for controlling diseases by inducing fruit resistance and used preparation |
| CN110205252A (en) * | 2019-06-20 | 2019-09-06 | 江苏大学 | The method of spray drying preparation antagonism yeast solid pharmaceutical preparation |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2148648C1 (en) * | 1999-01-05 | 2000-05-10 | Санкт-Петербургская Государственная Химико-Фармацевтическая Академия | Method of heteropolysaccharide producing |
| CN1507878A (en) * | 2002-12-15 | 2004-06-30 | 东北农业大学 | Method for preparing lactobacillus dry-powder product |
| CN1544614A (en) * | 2003-11-19 | 2004-11-10 | 中国科学院植物研究所 | Bacteria for biocontrol of disease and its application |
| CN1543796A (en) * | 2003-11-19 | 2004-11-10 | 中国科学院植物研究所 | Method for preventing and treating postharvest disease of fruit and vegetable using sodium silicate or potassium silicate and liquid for preventing and treating postharvest disease of fruit and vegeta |
-
2005
- 2005-08-11 CN CNB2005100900650A patent/CN100352914C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2148648C1 (en) * | 1999-01-05 | 2000-05-10 | Санкт-Петербургская Государственная Химико-Фармацевтическая Академия | Method of heteropolysaccharide producing |
| CN1507878A (en) * | 2002-12-15 | 2004-06-30 | 东北农业大学 | Method for preparing lactobacillus dry-powder product |
| CN1544614A (en) * | 2003-11-19 | 2004-11-10 | 中国科学院植物研究所 | Bacteria for biocontrol of disease and its application |
| CN1543796A (en) * | 2003-11-19 | 2004-11-10 | 中国科学院植物研究所 | Method for preventing and treating postharvest disease of fruit and vegetable using sodium silicate or potassium silicate and liquid for preventing and treating postharvest disease of fruit and vegeta |
Non-Patent Citations (1)
| Title |
|---|
| 罗伦隐球酵母对葡萄采后病害的拮抗效果 刘海波等.中国农业科学，第35卷第7期 2002 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1733898A (en) | 2006-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107034149A (en) | A kind of kluyveromyces marxianus bacterium freezes the preparation method and its usage of microbial inoculum | |
| CN100352914C (en) | Vacuum freeze-dried product of antagonist bacteria C.laurentii of yeast and its preparation method | |
| CN103249830A (en) | Starter culture compositions | |
| CN101974450A (en) | Leuconostoc mesenteroides and application thereof | |
| CN107287121A (en) | A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method | |
| CN105524867A (en) | Lactobacillus plantarum and application thereof in native grass silage | |
| CN106434463A (en) | Preparation method of lactobacillus rhamnosus lyophilized powder | |
| CN103992978A (en) | Leuconostoc pseudomesenteroides and method for co-producing dextran and mannitol therefrom | |
| CN113215016B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN102453681B (en) | Protective agent for vacuum freeze drying of lactobacillus johnsonii, and application thereof | |
| CN104877936A (en) | Fibrinolytic-enzyme-producing Bacillus amyloliquefaciens strain separated from traditional fermented black beans | |
| CN106497842B (en) | A kind of preparation method and application of lactic acid bacteria and aspergillus niger mixed solid fermentation preparation | |
| CN108208654A (en) | A kind of preparation method of the fruit ferment powder of high activity | |
| CN103602609A (en) | High-yield strain for producing L-alanine by fermentation and preparation method thereof | |
| CN100344750C (en) | Vacuum freeze-dried product of antagonist bacteria of yeast and its preparation method | |
| CN109234215A (en) | A kind of Lactobacillus rhamnosus less salt culture medium and cultural method | |
| CN113234597A (en) | Culture method for improving freeze-drying stress resistance of bifidobacterium and application thereof | |
| CN102719502A (en) | Method for producing L-alanine by mutating lactate-production bacteria | |
| CN109266553A (en) | A kind of freeze drying protectant, the method and application that Pu'er tea direct putting type freeze-drying microbial inoculum is prepared with it | |
| CN103289931B (en) | Bacillus vallismortis strain SJ and application thereof in preparation of tobacco antiviral preparation and promoter | |
| CN109182231A (en) | A kind of lactic acid bacteria culturers long term storage method | |
| CN108913618A (en) | A kind of bacillus amyloliquefaciens JSPB14 and its application | |
| CN111363695B (en) | Apple tree rot biocontrol microbial inoculum and preparation method and application thereof | |
| CN101240241A (en) | Method for preserving stropharia rugoso-annulata strain | |
| CN107523525B (en) | Bacillus belgii and application thereof in prevention and treatment of walnut canker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |