CN101892167B - Rhodotorula mucilaginosa, application thereof in preventing and curing postharvest disease of fruit and use method thereof - Google Patents
Rhodotorula mucilaginosa, application thereof in preventing and curing postharvest disease of fruit and use method thereof Download PDFInfo
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- CN101892167B CN101892167B CN2010101981317A CN201010198131A CN101892167B CN 101892167 B CN101892167 B CN 101892167B CN 2010101981317 A CN2010101981317 A CN 2010101981317A CN 201010198131 A CN201010198131 A CN 201010198131A CN 101892167 B CN101892167 B CN 101892167B
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Abstract
The invention discloses Rhodotorula mucilaginosa which has efficacies of prevention and cure for postharvest disease of fruit, and application and use method thereof in storage and refreshment of fruits. The strain of the invention is CGMCC No.3617. When in use, Rhodotorula mucilaginosa is activated, cultured through fermentation and centrifuged to obtain thalluses, the thalluses are diluted by sterile water to prepare into bacterial suspension of 1.0*107-numbered thalluses/ml to 1.0*109-numbered thalluses/ml. Fruits are put into the bacterial suspension to soak for about 30s to 2min, then taken out immediately to perform air drying, put in a plastic crate, sealed by a preservative film, and then stored under room temperature or in cold storage. In the invention, antagonistic yeast is used to prevent and cure postharvest disease of fruit, the invention has simple use, convenient operation, good effect and low cost, thereby avoiding dangers to people or pollution to environment from using chemical bactericide, thus the invention has significant economic benefits and social benefits.
Description
Technical field
The present invention relates to a kind of rhodotorula mucilaginosa bacterial classification (Rhodotorula mucilaginosa) and this bacterial classification application and method of use thereof to the fruit postharvest diseases control.
Background technology
Fruit because of the loss of rotting to cause is huge, also is the problem that the whole world is paid close attention in harvesting, transportation and sales, storage.It is reported, 10% one 30% fresh prod is arranged because post-harvest diseases causes rotting in developed country, and in the developing country that lacks the storing refrigerating plant, fruit is adopted the back rotting rate then up to 40%-50%.Exotic fruit preservation and freshness amount accounts for the fruit ultimate production more than 60%, and China's fruit storage amount is more than 800 ten thousand tons at present, and less than 15% of total amount, wherein the storage capacity of refrigerator chamber and air adjusting storeroom is 700,000 tons, only accounts for 7% of ultimate production.China's fruit is adopted the back rotting rate generally at 20%-30%, high person even reach 50%.Fruit is rotten mainly owing to the pathogenic effects of pathogenic micro-organism, and these pathogenic micro-organisms mainly contain Botrytis cinerea (Botrytiscinerea), Rhizopus stolonifer (Rhizopus stoloniger), penicillium (Penicillium spp.), Mucor (Mucor spp.), chain lattice spore (Alternaria spp.), aspergillus (Aspergillus spp.) and soft rotten bacterium (Erwinia sp.) etc.
Septic method was that low temperature and chemical bactericide are handled after the most frequently used control fruit was adopted at present.Though technique of refrigeration has obtained developing widely and using in China in recent years, thereby under coldcondition, still have a considerable amount of pathogenic bacterium to grow to cause rotten.For example, can cause the Botrytis cinerea (Botrytis cinerea) of multiple fruit gray mold, also claim botrytis cinerea, can also growth and breeding in the time of-0.5 ℃, cause that many fruit rot, and cause serious financial loss.And those are to the fruit of low-temperature sensitive, and because of preserving being not less than under the temperature that product is damaged to plants caused by sudden drop in temperature, the growth of pathogen, breeding can not be suppressed effectively, and it is quite serious to rot.The use of chemical bactericide can be played the effect of getting instant result though prevent and treat disease, in kill pathogenic bacteria, has also killed useful microbe, and the chemical bactericide on the fruit is residual that HUMAN HEALTH is impacted, environment is polluted.The portion report of AAS (National Academy of Scinces) shows that at the sterilant that is used for handling food, 60% has carcinogenic danger.Various sterilization agent (like Captan, Benomyl etc.) has at present been prohibited by EPA and has been used or use in portion water fruit product limit.For health and the safety that guarantees agricultural-food, the various countries scientist can replace the fruit postharvest diseases of chemical bactericide to prevent and treat new technology in positive exploration.In recent years, developed countries such as the U.S., Italy have obtained interim achievement to the research of biologically controlling postharvest disease of fruit, and the application potential of biological control is huge, make it become a kind of effective ways of substituted chemistry sterilant.
China's biologically controlling postharvest disease of fruit research is started late.Fan Qing etc. (2001) screen fruit disease biological control antagonism bacterium and separate; The result shows: monilia guilliermondii (Candida guiliermondii), lemon shape Ke Leke yeast (Kloeckera apiculata), the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii) all show significant inhibition effect to the sweet cherry brown heart, and monilia guilliermondii and lemon shape Ke Leke yeast can be prevented and treated drupe class fruit soft rot effectively; Field generation equality (2004) research sprays 1 * 10 before sweet cherry is adopted
8The trichosporon of CFU/mL (Trichosporon pullulans); Cryptococcus laurentii (Cryptococcus laurentii) and rhodotorula glutinis (Rhodotorula glutinis); Respectively at 25 ℃; Under 0 ℃ and the storage in controlled atmosphere, trichosporon is bred quantity on fruit the highest, and fungistatic effect is best; Zhang Hongyin etc. (2005) have studied under the differing temps, and Cryptococcus laurentii is to the control effect of gray mold, and the result shows, under 25 ℃, the sickness rate of pears gray mold is 16.7%, in conjunction with CaCl
2Result of use is more remarkable.
Can know that from domestic and foreign literature the current control that is used for fruit postharvest diseases with effective microbe substituted chemistry sterilant has demonstrated great application prospect, the biological control of fruit postharvest diseases becomes a new research focus.The antagonism bacterial classification class that has been applied to produce is few, have only a few launch such as Biosave and Aspire, and China does not have still at present the antagonism bacterium to be applied to actual production but up to the present.Major cause is under the commercial prodn condition that the control effect of the antagonistic microbe of having reported at present often is not so good as the effect of chemical bactericide, thereby does not reach fresh-keeping requirement.These factor affecting the antagonism bacterium as fruit and vegetable fresh-keeping agent use aborning.In order as soon as possible antagonistic microbe to be applied in the fresh-keeping actual production of fruit storage; Also need the further wide mikrobe of screening good, the disease-resistant spectrum of antagonistic effect; Need deeply inquire into the mechanism of antagonistic microbe control fruit postharvest diseases simultaneously; Further improve the bacteriostasis of antagonistic microbe on this basis, carry out the research of antagonistic microbe enlarged culturing, preservation and commercialization utilisation technology.
Summary of the invention
The present invention is in order to overcome above-mentioned deficiency of the prior art, and the contriver provides a strain rhodotorula mucilaginosa (Rhodotorula mucilaginosa) bacterial strain on the basis of broad research in early stage, and it has the thing preventing efficiency than Johnson & Johnson.
Middle bar Agro-ecology orchard screening and separating has the antagonism yeast Y20 bacterial strain of remarkable preventive and therapeutic effect to a strain to fruit postharvest diseases from the Zhengjiang City for this bacterial strain system, identifies through phylogenetic systematics, is accredited as rhodotorula mucilaginosa (Rhodotorula mucilaginosa).
Rhodotorula mucilaginosa Y20 bacterial strain of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on January 29th, 2010, and preservation strain is numbered CGMCC No.3617.
The present invention also provides the purposes of this rhodotorula mucilaginosa bacterial strain, can prevent and treat and preservation and freshness with its post-harvest diseases that carries out to fruit; Be particularly suitable for the post-harvest diseases control and the preservation and freshness of fruit such as the operatic circle, peach fruit, strawberry, apple.
The present invention provides a kind of rhodotorula mucilaginosa that utilizes to carry out fruit postharvest diseases control and storage preservation method simultaneously; Step is following: with rhodotorula mucilaginosa Y20 activation, fermentation culture, and the centrifugal thalline that obtains; Thalline is prepared into 1.0 * 10 with the sterilized water dilution
7Individual/ml-1.0 * 10
9The bacteria suspension of individual/ml; Fruit is put into bacteria suspension, soak after the time about 30 seconds-2 minutes and take out at once, air-dry again; Be placed in the plastic crate, after the preservative film sealing, be placed under room temperature or the refrigerated condition and deposit.
Rhodotorula mucilaginosa Y20 bacterial strain of the present invention adopts conventional strain inclined plane method for preserving, and adopting the substratum first-selection is NYDA substratum (Carnis Bovis seu Bubali cream 0.8%, YE 0.5%; Glucose 1%; Agar 2%), also can use potato agar substratum (yam 200 grams, glucose 20 grams; Agar 15～20 grams, 1000 milliliters in tap water).In pH7.0～9.0,26 ℃～28 ℃, 36h cultivated.When fermentation culture, can be to fermention medium NYDB or malt extract medium with the slant strains direct inoculation, pH 7.0～9.0, and 26 ℃～28 ℃ of temperature under the culture condition of shaking speed 180r/min, are cultivated 40～48h.Generally about 40h, can reach maximum biomass.
Rhodotorula mucilaginosa of the present invention (Rhodotorula mucilaginosa) Y20; Can produce the putrid pathogenic bacteria of rotting for obvious suppression fruit; Prevent to take place to rot after fruit from adopting; And have do not produce toxin, can with advantage such as common uses of chemical bactericide, can be widely used in the biological control process of fruit postharvest diseases the loss that the minimizing post-harvest diseases causes.
Through by following embodiment with more detailed explanation the present invention.Following examples only are illustrative, and the present invention does not receive the restriction of these embodiments.
Description of drawings wherein Fig. 1 is rhodotorula mucilaginosa Y205.8S rDNA-ITS region sequence evolutionary relationship figure.
Embodiment
Bacterial strain of the present invention obtains through following method, and repeatedly the sampling of middle bar Agro-ecology garden orangery and peach garden comprises from the Zhengjiang City: orange blade, flower, orange fruit and orangery soil; Peach blade, flower, peach fruit and peach garden soil.Through separation, screening, cultivation, isolated test and live test, obtain having the anti-microorganism strains Y20 that renders a service than Johnson & Johnson.Through morphology cultivation, physiological and biochemical property test and small subunit 5.8S rDNA and internal transcribed spacer district ITS1 and the base sequence analysis of ITS2 district, be accredited as rhodotorula mucilaginosa (Rhodotorula mucilaginosa).
Bacterial strain rhodotorula mucilaginosa Y20 bacterial strain of the present invention (strain number: CGMCC No.3617) have following Microbiological Characteristics:
1. morphological feature
(1) at YEPD solid medium dull and stereotyped (yeast powder 10g, peptone 20g, glucose 20g; Zero(ppm) water 1000mL, agar 20g, pH6.0; 115 ℃ of moist heat sterilization 20min) cultivate 72h for last 28 ℃, bacterium colony be oval, neat in edge, orange red, smooth surface, quality evenly, be prone to provoke.
(2) in malt juice liquid medium, behind the cultivation 48h, do not form mould, bacterium liquid is muddy, and nonfermented has deposition, microscopy yeast cell ovalize, cell size (3.5-5.3) * (2.7-4.5) μ m, budding, monolateral sprouting.
(3) having on the Corn Meal Agar substratum of deckglass about 120-144h, microscopy can not form pseudohypha.
2. physiological property
Bacterial strain carbon source of the present invention can be assimilated glucose, SANMALT-S, semi-lactosi, sucrose fiber, wood sugar, can not assimilation starch.Nitrogenous source can assimilate nitrate salt, can grow for 37 ℃.Its physiological property result sees the following form 1.
3. molecular genetics is identified
Screening bacterial strain rhodotorula mucilaginosa Y20 small subunit 5.8S rDNA-ITS region sequence is analyzed, on GenBank, retrieved, confirm that Y20 is rhodotorula mucilaginosa (Rhodotorula mucilaginosa).According to the homologous strain that retrieves, the Mege4 program of Application of DNA Star software, it is shown in Figure 1 to make up organic evolution relational tree such as Figure of description.
The physiological and biochemical property of table 1 rhodotorula mucilaginosa
Rhodotorula mucilaginosa Y20 of the present invention adopts conventional strain inclined plane method for preserving, and adopting the substratum first-selection is NYDA substratum (Carnis Bovis seu Bubali cream 0.8%, YE 0.5%, glucose 1%, agar 2%), also can use the potato agar substratum.In pH7.0～9.0,26 ℃～28 ℃, 36h cultivated.When fermentation culture, can be to fermention medium NYDB or malt extract medium with the slant strains direct inoculation, pH7.0～9.0,26 ℃～28 ℃ of temperature under the culture condition of shaking speed 180r/min, are cultivated 40～48h.Generally about 40h, can reach maximum biomass.
Embodiment 1: utilize rhodotorula mucilaginosa to carry out post-harvest diseases control and the storage preservation method of fruit (the operatic circle and peach fruit)
With yeast-inoculated after the activation be equipped with 50mlNYDB (yeast extract paste 5g, beef extract 8g, glucose 10g, purified water 1000ml, in triangular flask pH7.0-9.0), 28 ℃, 180r/min shaking table are cultivated 48h.Get the centrifugal 10min of bacterium liquid 12000r/min, abandon supernatant, sterilized water cleans 3 times repeatedly, and it is 1 * 10 that the blood counting chamber counting is made into concentration
8The bacteria suspension of individual/ml.Use punch tool to make a call to the hole of 3 diameters as 3m (wide) * 3mm (deeply) at the fruit waist, the antagonism bacteria concentration that 30 μ l are annotated in every hole is 1 * 10
8Individual/the ml cell suspension, contrast fruit injects 30 μ l sterilized waters.Inoculate pathogenic bacteria Botrytis cinerea (Botrytis cinerea) and Penicilllum expansum (Penicillium expansum) spore suspension 20 μ l behind the 2h respectively.After drying fruit is packed in the plastic crate, preservative film seals and keeps 95% humidity, storage at room temperature, the morbidity rotting rate of statistics fruit behind the 10d.Each handles 8 fruits, test repetition 3 times.According to the above-mentioned steps test, the rotting rate result of statistics fruit sees the following form 2 behind the 10d.
Table 2 rhodotorula mucilaginosa Y20 is to the inhibition effect of ash arrhizus bacteria and mould germ
Estimate the inhibition effect of each strain antagonism bacterium to peach and two kinds of pathogenic bacterias of pear fruit, the result sees table 2.Can find out that from table control group is all morbidities, can find out that Y20 has the obvious suppression effect to the back disease of rotting of adopting of peach and pears.
Embodiment 2: utilize rhodotorula mucilaginosa to carry out control of fruit (the operatic circle) post-harvest diseases and storage preservation method
One, testing program
With yeast-inoculated after the activation be equipped with 50ml NYDB (yeast extract paste 5g, beef extract 8g, glucose 10g, purified water 1000ml, in triangular flask pH7.0-9.0), 28 ℃, 180r/min shaking table are cultivated 48h.Get the centrifugal 10min of bacterium liquid 12000r/min, abandon supernatant, sterilized water cleans 3 times repeatedly, and it is 1 * 10 that the blood counting chamber counting is made into concentration
8The bacteria suspension of individual/ml.The operatic circle is dipped to 1 * 10
8In the bacteria suspension of individual/ml, the contrast the operatic circle is dipped in the sterilized water, and the time is 30 seconds-2 minutes.After drying fruit is packed in the plastic crate, preservative film seals and keeps 95% humidity, storage at room temperature.The rotting rate of statistics fruit and the quality of measuring fruit behind the 45d.Each handles 8 fruits, test repetition 3 times.After the operatic circle storage finished, every group was taken out some the operatic circles respectively, carried out organoleptic quality and measured.Sense organ is measured and is at room temperature carried out (20 ℃), handles for every group and when measuring each index, gets 5 fruit, the test triplicate.
The quality determination method is following:
(1) rate of weight loss: adopt weighting method.
(2) hardness: measure with TA-XT2i texture analysis appearance, use the probe of diameter as 5mm, probe is 5.0 mm/second to the movement velocity of fruit, and the bounce (N) that probe punctures the maximum that fruit runs into is defined as the hardness of fruit.
(3) total dissoluble solid content: measure with the hand-held saccharometer, adopt hand-held saccharometer to measure, measure the result and be expressed as the g/100g sample.
(4) titratable acidity: adopt volumetry, from 6 fruit samplings, take by weighing the 10g sample, adding distil water grinds rapidly; Be settled to 100ml, filter, get 10ml filtrating; Carry out titration to pH8.1 with 0.1mol/l NaOH, calculate after the titration, the result representes with the percentage ratio that contains Hydrocerol A.
(5) Vc content
Vc content: 2,6-Dichlorophenol indophenol method.Get the 5g sample from 6 fruit and add 5ml 2% oxalic acid grinding homogenate, pour the 100ml volumetric flask into,, add lml 30% zinc sulfate, shake up with 1% oxalic acid washing residue.Add 1ml15% yellow prussiate of potash (depigmentation) again,, shake up filtration with 1% oxalic acid constant volume.Get filtered liq 4ml, add 2 to each pipe then, 6-Dichlorophenol indophenol dye solution adds YLENE 5ml immediately, shakes about 0.5min rapidly, leaves standstill back YLENE and separates with water layer, and upper strata xylene extraction liquid is measured absorbancy under the 500nm wavelength.OD value according to measuring liquid is looked into typical curve, obtains the content of corresponding vitamins C (Vc), and the result representes with milligram Vc/100g fruit.
Two, test-results
According to the above-mentioned steps test, the natural rotting rate of statistics the operatic circle and the result that influences of storage quality see the following form 3 behind the 45d.
Table 3 rhodotorula mucilaginosa rots to suppress the influence of effect and storage quality thereof naturally to the operatic circle
Remarks: the result who behind room temperature (20 ℃) held 45d, measures when sickness rate in the above-mentioned table and quality determination.The significance of difference represented in different letters.
Can find out that from table 3 through the operatic circle that rhodotorula mucilaginosa was managed, after (20 ℃) were placed 45d at normal temperatures, the sickness rate of pear fruit significantly was lower than control group (p＜0.05).Do not produce adverse influence for rate of weight loss, hardness.The operatic circle soluble solid content that rhodotorula mucilaginosa was handled, titratable acid content all obviously are superior to control group (p＜0.05), and other index does not then all have a negative impact.
Above test-results shows through rhodotorula mucilaginosa to be handled, and can obviously reduce the natural rotting rate of the operatic circle, and storage quality is not exerted an adverse impact.
Claims (4)
1. rhodotorula mucilaginosa (Rhodotorula mucilaginosa) Y20 CGMCC No.3617.
2. the purposes of the described rhodotorula mucilaginosa bacterial strain of claim 1 is characterized in that with it fruit being carried out post-harvest diseases control and preservation and freshness.
3. the purposes of rhodotorula mucilaginosa bacterial strain according to claim 2 is characterized in that being suitable for the post-harvest diseases control and the preservation and freshness of the operatic circle, peach fruit, strawberry, apple.
4. use the described rhodotorula mucilaginosa of claim 1 to carry out the method for fruit postharvest diseases control and preservation and freshness; Step is following: with rhodotorula mucilaginosa Y20 activation, fermentation culture, and the centrifugal thalline that obtains; Thalline is prepared into 1.0 * 10 with the sterilized water dilution
7Individual/ml-1.0 * 10
9The bacteria suspension of individual/ml; Fruit is put into bacteria suspension, take out after soaking time of 30 seconds-2 minutes at once, air-dry again; Be placed in the plastic crate, after the preservative film sealing, be placed under room temperature or the refrigerated condition and deposit.
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102283280A (en) * | 2011-06-21 | 2011-12-21 | 江苏大学 | Method for improving biological prevention and control effect of rhodotorula mucilaginosa on fruit postharvest diseases |
| CN102283282B (en) * | 2011-06-21 | 2013-05-08 | 江苏大学 | Method for improving biological control efficacy of rhodotorula mucilaginosa to fruit postharvest diseases by burdock fructooligosaccharide |
| CN102550653A (en) * | 2011-11-15 | 2012-07-11 | 江苏大学 | Method for biologically controlling postharvest diseases of fruits by combining salicylic acid with Rhodotorula mucilaginosa |
| CN104109638B (en) * | 2014-03-25 | 2016-08-17 | 河南恒瑞源实业有限公司 | The method that one Rhodotorula mucilaginose strain strain ZZR-1# and this bacterial strain produce at |
| CN105112312B (en) * | 2015-09-14 | 2018-08-17 | 浙江大学 | Tibet rhodotorula mucilaginosa and its application in Shuijing Pear cryopreservation |
| CN107937290B (en) * | 2017-12-15 | 2021-01-19 | 北京工商大学 | Zygosaccharomyces bailii for preventing and treating postharvest diseases of fruits as well as preparation method and application thereof |
| CN110591932B (en) * | 2019-10-21 | 2021-03-30 | 福州大学 | Yeast MA for controlling postharvest diseases of fruits and vegetables and use method thereof |
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|---|---|---|---|---|
| US5525132A (en) * | 1991-04-11 | 1996-06-11 | Daratech Proprietary Limited | Yeasts as a biocontrol for microbial diseases of fruit |
| CN101319194A (en) * | 2007-10-24 | 2008-12-10 | 大连理工大学 | Rhodotorula mucilaginosa with novel metabolic characteristic and application of the same in biodegradation |
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2010
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5525132A (en) * | 1991-04-11 | 1996-06-11 | Daratech Proprietary Limited | Yeasts as a biocontrol for microbial diseases of fruit |
| CN101319194A (en) * | 2007-10-24 | 2008-12-10 | 大连理工大学 | Rhodotorula mucilaginosa with novel metabolic characteristic and application of the same in biodegradation |
Non-Patent Citations (1)
| Title |
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| 郑春莉.R.mucilaginosa，S.albidoflavus和M.luteus对硝基苯的好氧生物降解.《博士学位论文》.2007,1－150. * |
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