CN101822288A - Preparation method of vacuum freeze-dried product of marineyeast Rhodosporidium paludigenum Fell and Tallman - Google Patents
Preparation method of vacuum freeze-dried product of marineyeast Rhodosporidium paludigenum Fell and Tallman Download PDFInfo
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- 241000007102 Rhodotorula paludigena Species 0.000 title abstract description 22
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 58
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000003223 protective agent Substances 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 9
- 239000002054 inoculum Substances 0.000 claims abstract description 9
- 239000000047 product Substances 0.000 claims description 32
- 210000004027 cell Anatomy 0.000 claims description 25
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 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 claims description 8
- 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 claims description 8
- 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 claims description 8
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- 239000006228 supernatant Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 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 description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 235000020183 skimmed milk Nutrition 0.000 claims description 5
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- 230000002068 genetic effect Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 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 description 2
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Abstract
The invention discloses a preparation method of a vacuum freeze-dried product of marineyeast Rhodosporidium paludigenum Fell and Tallman. The method comprises the following steps of: inoculating the marineyeast Rhodosporidium paludigenum Fell and Tallman into a yeast media according to the inoculum size of 1-5 percent; culturing for 20-50 h at the speed of 100-300 r/min and the temperature of 20-30 DEG C; collecting the thallus of the marineyeast Rhodosporidium paludigenum Fell and Tallman through centrifuging; removing a supernate; cleaning with sterile water 2-3 times; counting by a blood counting method; adjusting by using a protective agent to form a cell suspension with the density of 1*109 to 1*1011 cells/ml; incubating the cell suspension for 0.5-3h; and then carrying out vacuum freeze-drying to obtain the vacuum freeze-dried product, wherein the vacuum degree is 0.005-0.01 mbar, the drying temperature is -55 to -65 DEG C, and the drying time is 12-36h. The vacuum freeze-dried product prepared by the method can ensure that the survival rate of R.paludigenum is not less than 80 percent; the invention has simple production process, high safety and no pollution, can obviously reduce diseases of fruits and vegetables, and can play important roles in storage and fresh-keeping of various picked fruits and vegetables and development of products related to R.paludigenum.
Description
Technical field
The present invention relates to a kind of marine yeast Rhodospridium paludigenum Fell ﹠amp; The preparation method of the vacuum freeze-dried product of Tallman.
Background technology
China is not only world fruits and vegetables big producing country, also is pesticide producing and use big country.Frequent, excessive for a long time use chemistry fungicide, microbial environment, contaminated air and water quality, the enhancing disease and pest resistance of meeting heavy damage soil, thereby the foreign exchange earning of influence agricultural has endangered the healthy of the common people, seriously restricts the sustainable development of China's agricultural.Along with being rooted in the hearts of the people day by day of food security idea, increasing chemical bactericide is limited to use.The bio-preservative that develops more high performance cheaps, safety non-toxic becomes the task of top priority.
Developed country's the most frequently used non-chemically fresh-keeping means at present is cold chain technology.The CHINESE REGION disparate development, the cold chain technology all has higher requirement to equipment, cost, and universal in the whole nation in the short time have than big difficulty.Antagonism yeast does not produce antibiotic, does not need to purchase special installation, easy and simple to handle, and be beneficial to and apply, be one of storage fruit and vegetable method of the most potential instead of chemical bactericide
[1]
Existing result of study shows, the main mechanism of action of antagonism bacterium is the competition in nutrition and space between cell and the pathogen, and the prevention effect of antagonism bacterium depends on the working concentration and the growth activity of cell
[2]Biological preservation has accumulated a lot of successful experience in the laboratory, but business-like biological prevention and control agent has comprised production, packing, transportation, sale and all too many levels such as finally use on different fruits.Therefore, biocontrol microorganisms will really be realized industrialization, and necessary form is fit to the product form of merchandise sales.
Vacuum freeze drying has lot of advantages, shrinks for a short time such as the microbial cell in the dry run, and activity is strong etc. after the high and rehydration of product dissolubility, comparatively is suitable for preparing active antagonism yeast dry powder
[3]The viability of microorganism in freeze-drying process is subjected to influence of various factors, comprises initial concentration
[4], protectant kind and concentration
[5]In addition, the initial concentration of freeze-drying also has very big influence to microbial survival power and production cost
[6]
Studies show that marine yeast Rhodospridium paludigenum adopts the back fungal disease to multiple fruit and has significant inhibition effect
[7,8]Consider the advantage of marine yeast aspect adverse circumstance adaptability, R.paludigenum has the potentiality that the market development is used.At present, the domestic and international report that does not also have about the active dry powder of marine yeast R.paludigenum vacuum freeze drying.
List of references:
[1]Droby，S.，Wisniewski，M.E.，Macarisin，D.，Wilson，C.，2009.Twenty?years?ofpostharvest?biocontrol?research：Is?it?time?for?a?new?paradigm？PostharvestBiology?and?Technolpgy?52：137-145.
[2]Janisiewicz，W.J.，Korsten?L.，2002.Biological?control?of?postharvest?diseases?offruits.Annual?Review?of?Phytopathology?52：4406-4413.
[3]Powell，K.A.，1992.Biocontrol?product?fermentation，formulation?and?marketing.In?Biological?Control?of?Plant?Diseases?ed.Tjamos，E.S.pp.381-387.New?York：Plenum?Press.
[4]Bozoglu，T.F.，Ozilgen，M.，Bakir，U.，1987.Survival?kinetics?of?lactic?acidstarter?cultures?during?and?after?freeze?drying.Enzyme?Microbial?Technology?9，531-537.
[5]Sinha，R.N.，Shukla，A.K.，Madan?Lal?and?Ranganathan，B.，1982.Rehydrationof?freeze-dried?cultures?of?lactic?streptococci.Jounal?of?Food?Science?47，668-669.
[6]Palmfeldt，J.，
P.，
B.，2003.Optimisation?of?initial?cellconcentration?enhances?freeze-drying?tolerance?of?Pseudomonas?chlororaphis.Cryobiology?47，21-29.
[7]Wang，Y.F.，Bao，Y.H.，Shen，D.H.，Feng，W.，Yu，T.，Zhang，J.，Zheng?X.D.，2008.Biocontrol?of?Alternaria?alternata?on?Cherry?Tomato?Fruit?by?Use?ofmarine?yeast?Rhodosporidium?paludigenum?Fell?&?Tallman，International?Journalof?Food?Microbiology?123，234-239.
[8]Wang，Y.F.，Yu，T.，Li，Y.C.，Cai，D.G，Liu，X.，Lu，H.P.，Zheng，X.D.，2009.Postharvest?biocontrol?of?Alternaria?alternata?in?Chinese?winter?jujube?byRhodosporidium?paludigenum.Journal?of?Applied?Microbiology?107，1492-1498.
Summary of the invention
The purpose of this invention is to provide a kind of marine yeast Rhodospridium paludigenum Fell ﹠amp; The preparation method of the vacuum freeze-dried product of Tallman.
Marine yeast Rhodospridium paludigenum Fell ﹠amp; The preparation method of the vacuum freeze-dried product of Tallman is: the inoculum concentration by 1%～5% is with marine yeast Rhodospridium paludigenumFell ﹠amp; Tallman is inoculated in the Yeast Cultivation base, at 20～30 ℃, cultivates 20～50 hours for 100～300 rev/mins, by centrifugal collection marine yeast Rhodospridium paludigenum Fell ﹠amp; The Tallman thalline, abandoning supernatant with sterilized water washing 2～3 times, is counted with counting method of blood cell, and is adjusted to 1 * 10 with protective agent
9～1 * 10
11The cells/ml cell suspending liquid is hatched cell suspending liquid 0.5-3 hour, carries out vacuum freeze drying again, obtains vacuum freeze-dried product; Vacuum is 0.005～0.01mbar, and baking temperature is-55～-65 ℃, 12～36 hours drying times.
Described marine yeast Rhodospridium paludigenum Fell ﹠amp; Tallman is in Britain international standard fungal studies institute (International Mycological Institute) international agricultural and genetic resources preservation center, biological center (CABI Genetic Resource Collection) preservation, and preserving number is IMI 394084.
Consisting of of described Yeast Cultivation base: beef extract 5～10g/L, dusty yeast 3～6g/L, glucose 8～12g/L, sodium chloride 10～40g/L.
Described protective agent is: the quality percentage composition be 1～5% sucrose, quality percentage composition be 1～5% trehalose, quality percentage composition be 1～5% skimmed milk power or quality percentage composition be in 0.5～1% the sodium chloride one or more.
The vacuum freeze-dried product of the present invention preparation can keep survival rate 〉=80% of R.paludigenum.Production technology of the present invention is simple, and is pollution-free, safe, can obviously reduce diseases of garden stuff, will be at the postharvest storage of multiple fruits and vegetables fresh-keeping and relate in the product development of R.paludigenum and playing a significant role.
The specific embodiment
Embodiment 1
Protective agent of the present invention is to the influence of marine yeast R.paludigenum vacuum freeze-dried product viable bacteria rate
Inoculum concentration by 1% is with marine yeast Rhodospridium paludigenum Fell ﹠amp; Tallman is inoculated in the Yeast Cultivation base, at 20 ℃, cultivates 50 hours for 100 rev/mins, by centrifugal collection marine yeast Rhodospridium paludigenum Fell ﹠amp; The Tallman thalline, abandoning supernatant with sterilized water washing 2 times, is counted with counting method of blood cell, and is adjusted to 1 * 10 with 1% trehalose
9The cells/ml cell suspending liquid is hatched cell suspending liquid 0.5 hour, carries out vacuum freeze drying again, obtains vacuum freeze-dried product; Vacuum is 0.005mbar, and baking temperature is-55 ℃, 36 hours drying times.
Take by weighing above-mentioned vacuum freeze-dried product 0.1g and add in the physiological saline of 100ml sterilization, hatch 30min under the normal temperature after, dilute certain gradient, get 0.1ml suspension and evenly coat Yeast Cultivation base flat board, 28 ℃ are calculated clump count after cultivating 48h.Viable bacteria rate computing formula: viable bacteria rate=viable count/total bacteria count * 100%.
As can be seen from Table 1, added protectant marine yeast Rhodospridium paludigenum Fell﹠amp; The freeze drying example of Tallman is the pale red powder, has the special odor of yeast, free from extraneous odour, and viable bacteria rate 〉=80% is higher than protectant control sample viable bacteria rate of not adding significantly.
Table 1 trehalose is to the influence of R.paludigenum vacuum freeze-dried product viable bacteria rate
Embodiment 2
Protective agent of the present invention is to the influence of marine yeast R.paludigenum vacuum freeze-dried product viable bacteria rate
Inoculum concentration by 5% is with marine yeast Rhodospridium paludigenum Fell ﹠amp; Tallman is inoculated in the Yeast Cultivation base, at 30 ℃, cultivates 20 hours for 300 rev/mins, by centrifugal collection marine yeast Rhodospridium paludigenum Fell ﹠amp; The Tallman thalline, abandoning supernatant, with sterilized water washing 3 times, with the counting method of blood cell counting, and the protective agent that mixes with 2.5% trehalose, 2.5% skimmed milk, 2.5% sucrose and 0.85% sodium chloride is adjusted to 1 * 10
11The cells/ml cell suspending liquid is hatched cell suspending liquid 3 hours, carries out vacuum freeze drying again, obtains vacuum freeze-dried product; Vacuum is 0.01mbar, and baking temperature is-65 ℃, 12 hours drying times.Add up the viable bacteria rate according to the method described above.
As can be seen from Table 2, added protectant marine yeast Rhodospridium paludigenum Fell﹠amp; The freeze drying example of Tallman is the pale red powder, has the special odor of yeast, free from extraneous odour, and viable bacteria rate 〉=90% is higher than protectant control sample viable bacteria rate of not adding significantly.
Table 2 protective agent is to the influence of R.paludigenum vacuum freeze-dried product viable bacteria rate
Embodiment 3
Protective agent of the present invention is to the influence of marine yeast R.paludigenum vacuum freeze-dried product viable bacteria rate
Inoculum concentration by 2% is with marine yeast Rhodospridium paludigenum Fell ﹠amp; Tallman is inoculated in the Yeast Cultivation base, at 28 ℃, cultivates 36 hours for 200 rev/mins, by centrifugal collection marine yeast Rhodospridium paludigenum Fell ﹠amp; The Tallman thalline, abandoning supernatant, with sterilized water washing 3 times, with the counting method of blood cell counting, and the protective agent that mixes with 2.5% trehalose and 2.5% sucrose is adjusted to 1 * 10
10The cells/ml cell suspending liquid is hatched cell suspending liquid 1 hour, carries out vacuum freeze drying again, obtains vacuum freeze-dried product; Vacuum is 0.01mbar, and baking temperature is-65 ℃, 24 hours drying times.Add up the viable bacteria rate according to the method described above.
As can be seen from Table 3, added protectant marine yeast Rhodospridium paludigenum Fell﹠amp; The freeze drying example of Tallman is the pale red powder, has the special odor of yeast, free from extraneous odour, and viable bacteria rate 〉=83% is higher than protectant control sample viable bacteria rate of not adding significantly.
Table 3 protective agent is to the influence of R.paludigenum vacuum freeze-dried product viable bacteria rate
Embodiment 4
The shelf life of marine yeast R.paludigenum vacuum freeze-dried product of the present invention
Inoculum concentration by 3% is with marine yeast Rhodospridium paludigenum Fell ﹠amp; Tallman is inoculated in the Yeast Cultivation base, at 26 ℃, cultivates 48 hours for 200 rev/mins, by centrifugal collection marine yeast Rhodospridium paludigenum Fell ﹠amp; The Tallman thalline, abandoning supernatant, with sterilized water washing 3 times, with the counting method of blood cell counting, and the protective agent that mixes with 2.5% trehalose, 2.5% skimmed milk and 2.5% sucrose is adjusted to 5 * 10
10The cells/ml cell suspending liquid is hatched cell suspending liquid 2 hours, carries out vacuum freeze drying again, obtains vacuum freeze-dried product; Vacuum is 0.01mbar, and baking temperature is-60 ℃, 24 hours drying times.Above-mentioned cold sample is preserved in 4 ℃ of refrigerators, every sampling in 30 days, the viable bacteria rate of working sample.Viable bacteria rate assay method is the same.
As can be seen from Table 4, added protectant marine yeast Rhodospridium paludigenum Fell﹠amp; The vacuum freeze-dried product of Tallman is in the preceding April of storage, and the viable bacteria rate does not significantly descend.
Table 4 marine yeast R.paludigenum vacuum freeze-dried product shelf life
Embodiment 5
Marine yeast R.paludigenum vacuum freeze-dried product of the present invention is adopted the inhibition effect of back penicilliosis to fruit
Inoculum concentration by 4% is with marine yeast Rhodospridium paludigenum Fell ﹠amp; Tallman is inoculated in the Yeast Cultivation base, at 26 ℃, cultivates 30 hours for 250 rev/mins, by centrifugal collection marine yeast Rhodospridium paludigenum Fell ﹠amp; The Tallman thalline, abandoning supernatant with sterilized water washing 2 times, with the counting method of blood cell counting, is adjusted to 1 * 10 with 5% trehalose, 5% skimmed milk, 5% sucrose as protective agent respectively
10The cells/ml cell suspending liquid is hatched cell suspending liquid 1 hour, carries out vacuum freeze drying again, obtains vacuum freeze-dried product; Vacuum is 0.01mbar, and baking temperature is-65 ℃, 24 hours drying times.Add up the viable bacteria rate according to the method described above.
Viable bacteria rate per sample, making concentration with the just above-mentioned sample ligand of certain sterilized water is 1 * 10
8The yeast cells suspension of cells/ml.Present embodiment is a test material with pears and apple.Fruit derives from orchard, suburb, Hangzhou.
Pathogen: penicillium expansum (Penicillium expansum)
Fruit is stabbed back inoculation sample cell suspension and sterilized water in contrast.Inoculum concentration 30 μ l are.After 2 hours, inoculating 15 μ l concentration is 5 * 10
4The pathogen spore suspension of individual/mL.Fruit is handled back 20 ℃ and is put storage 5 days down in storage, observes the incidence of disease of fruit.
The prevention effect of penicilliosis was shown in table 5 and 6 after marine yeast R.paludigenum vacuum freeze-dried product was adopted fruit, and these goods can effectively reduce the incidence of disease of fruit penicilliosis.
Table 5 marine yeast R.paludigenum vacuum freeze-dried product is to the inhibition effect of pears penicilliosis
Table 6 marine yeast R.paludigenum vacuum freeze-dried product is to the inhibition effect of green mold of apple
Claims (4)
1. marine yeast Rhodospridium paludigenum Fell﹠amp; The preparation method of the vacuum freeze-dried product of Tallman is characterized in that by 1%～5% inoculum concentration marine yeast Rhodospridium paludigenum Fell﹠amp; Tallman is inoculated in the Yeast Cultivation base, at 20～30 ℃, cultivates 20～50 hours for 100～300 rev/mins, by centrifugal collection marine yeast Rhodospridium paludigenum Fell﹠amp; The Tallman thalline, abandoning supernatant with sterilized water washing 2～3 times, is counted with counting method of blood cell, and is adjusted to 1 * 10 with protective agent
9～1 * 10
11The cells/ml cell suspending liquid is hatched cell suspending liquid 0.5-3 hour, carries out vacuum freeze drying again, obtains vacuum freeze-dried product; Vacuum is 0.005～0.01mbar, and baking temperature is-55～-65 ℃, 12～36 hours drying times.
2. a kind of marine yeast Rhodospridium paludigenum Fell﹠amp according to claim 1; The preparation method of the vacuum freeze-dried product of Tallman is characterized in that described marine yeast Rhodospridium paludigenum Fell﹠amp; Tallman is in Britain international standard fungal studies institute (InternationalMycological Institute) international agricultural and genetic resources preservation center, biological center (CABI GeneticResource Collection) preservation, and preserving number is IMI 394084.
3. a kind of marine yeast Rhodospridium paludigenum Fell﹠amp according to claim 1; The preparation method of the vacuum freeze-dried product of Tallman is characterized in that consisting of of described Yeast Cultivation base: beef extract 5～10g/L, dusty yeast 3～6g/L, glucose 8～12g/L, sodium chloride 10～40g/L.
4. a kind of marine yeast Rhodospridium paludigenum Fell﹠amp according to claim 1; The preparation method of the vacuum freeze-dried product of Tallman is characterized in that described protective agent is: the quality percentage composition be 1～5% sucrose, quality percentage composition be 1～5% trehalose, quality percentage composition be 1～5% skimmed milk power or quality percentage composition be in 0.5～1% the sodium chloride one or more.
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| CN102228078A (en) * | 2011-05-02 | 2011-11-02 | 浙江大学 | Biological preservative for citrus based on antagonistic activity of rhodosporidium paludigenum fell&tallman |
| CN102246850A (en) * | 2011-05-04 | 2011-11-23 | 浙江大学 | Biological preservative for oranges |
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| CN105104500A (en) * | 2015-10-10 | 2015-12-02 | 太仓市荣德生物技术研究所 | Meat preservative |
| CN106070583A (en) * | 2016-06-28 | 2016-11-09 | 浙江大学 | Induction fruit resistance controls the method for disease and preparation used |
| WO2021035503A1 (en) * | 2019-08-27 | 2021-03-04 | 湖南农业大学 | Freeze-drying protective agent for luminescent bacteria, freeze-dried luminescent bacteria powder, and application thereof in online monitoring of comprehensive water toxicity |
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| CN102224842A (en) * | 2011-05-02 | 2011-10-26 | 浙江大学 | Orange biological preservative based on antagonistic activities of three antagonistic yeasts |
| CN102228078A (en) * | 2011-05-02 | 2011-11-02 | 浙江大学 | Biological preservative for citrus based on antagonistic activity of rhodosporidium paludigenum fell&tallman |
| CN102224843A (en) * | 2011-05-04 | 2011-10-26 | 浙江大学 | Orange biological preserving agent based on yeast, sodium bicarbonate and elicitor activity |
| CN102246850A (en) * | 2011-05-04 | 2011-11-23 | 浙江大学 | Biological preservative for oranges |
| CN102246850B (en) * | 2011-05-04 | 2012-11-07 | 浙江大学 | Biological preservative for oranges |
| CN102224843B (en) * | 2011-05-04 | 2012-11-07 | 浙江大学 | Orange biological preserving agent based on yeast, sodium bicarbonate and elicitor activity |
| CN103614307A (en) * | 2013-11-13 | 2014-03-05 | 中国水产科学研究院南海水产研究所 | Solid ocean red yeast preparation as well as preparation method and application thereof |
| CN105104500A (en) * | 2015-10-10 | 2015-12-02 | 太仓市荣德生物技术研究所 | Meat preservative |
| CN106070583A (en) * | 2016-06-28 | 2016-11-09 | 浙江大学 | Induction fruit resistance controls the method for disease and preparation used |
| CN106070583B (en) * | 2016-06-28 | 2019-12-24 | 浙江大学 | Method for controlling diseases by inducing fruit resistance and preparation used by method |
| WO2021035503A1 (en) * | 2019-08-27 | 2021-03-04 | 湖南农业大学 | Freeze-drying protective agent for luminescent bacteria, freeze-dried luminescent bacteria powder, and application thereof in online monitoring of comprehensive water toxicity |
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