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
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- 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
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- cryptococcus laurentii
- laurentii
- dried product
- vacuum freeze
- protective material
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- 241000222051 Papiliotrema laurentii Species 0.000 title claims abstract description 80
- 241000894006 Bacteria Species 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 240000004808 Saccharomyces cerevisiae Species 0.000 title abstract description 55
- 239000005557 antagonist Substances 0.000 title 1
- 239000000725 suspension Substances 0.000 claims abstract description 21
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 13
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 8
- 229930006000 Sucrose Natural products 0.000 claims abstract description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 8
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims abstract description 8
- 235000013923 monosodium glutamate Nutrition 0.000 claims abstract description 8
- 239000001632 sodium acetate Substances 0.000 claims abstract description 8
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 8
- 229940073490 sodium glutamate Drugs 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 5
- 239000000463 material Substances 0.000 claims description 40
- 230000001681 protective effect Effects 0.000 claims description 40
- 238000004108 freeze drying Methods 0.000 claims description 32
- 238000011534 incubation Methods 0.000 claims description 24
- 235000020183 skimmed milk Nutrition 0.000 claims description 17
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- 239000008103 glucose Substances 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 7
- 229960004249 sodium acetate Drugs 0.000 claims description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 230000003042 antagnostic effect Effects 0.000 abstract description 38
- 238000004321 preservation Methods 0.000 abstract description 17
- 238000000034 method Methods 0.000 abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 10
- 244000005700 microbiome Species 0.000 abstract description 8
- 229960004793 sucrose Drugs 0.000 abstract description 6
- 239000008121 dextrose Substances 0.000 abstract description 3
- 239000003223 protective agent Substances 0.000 abstract 2
- 230000012447 hatching Effects 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000008267 milk Substances 0.000 abstract 1
- 235000013336 milk Nutrition 0.000 abstract 1
- 210000004080 milk Anatomy 0.000 abstract 1
- 238000009777 vacuum freeze-drying Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 25
- 239000000047 product Substances 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 20
- 230000004083 survival effect Effects 0.000 description 18
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 9
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 9
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 9
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 238000007710 freezing Methods 0.000 description 7
- 230000008014 freezing Effects 0.000 description 7
- 238000003860 storage Methods 0.000 description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- 235000009754 Vitis X bourquina Nutrition 0.000 description 3
- 235000012333 Vitis X labruscana Nutrition 0.000 description 3
- 240000006365 Vitis vinifera Species 0.000 description 3
- 235000014787 Vitis vinifera Nutrition 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000000443 biocontrol Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
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- 238000003306 harvesting Methods 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- VQVUBYASAICPFU-UHFFFAOYSA-N (6'-acetyloxy-2',7'-dichloro-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(OC(C)=O)C=C1OC1=C2C=C(Cl)C(OC(=O)C)=C1 VQVUBYASAICPFU-UHFFFAOYSA-N 0.000 description 2
- 244000144730 Amygdalus persica Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 244000007021 Prunus avium Species 0.000 description 2
- 235000010401 Prunus avium Nutrition 0.000 description 2
- 235000006040 Prunus persica var persica Nutrition 0.000 description 2
- 241000223253 Rhodotorula glutinis Species 0.000 description 2
- 244000126002 Ziziphus vulgaris Species 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 235000013681 dietary sucrose Nutrition 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 235000019263 trisodium citrate Nutrition 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 241000220225 Malus Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- CUBCNYWQJHBXIY-UHFFFAOYSA-N benzoic acid;2-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=CC=C1.OC(=O)C1=CC=CC=C1O CUBCNYWQJHBXIY-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- -1 ctive Species 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 239000000417 fungicide Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 208000018875 hypoxemia Diseases 0.000 description 1
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- 238000011081 inoculation Methods 0.000 description 1
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- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 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.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 * |
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