CN102283281A - Method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases - Google Patents
Method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases Download PDFInfo
- Publication number
- CN102283281A CN102283281A CN 201110166736 CN201110166736A CN102283281A CN 102283281 A CN102283281 A CN 102283281A CN 201110166736 CN201110166736 CN 201110166736 CN 201110166736 A CN201110166736 A CN 201110166736A CN 102283281 A CN102283281 A CN 102283281A
- Authority
- CN
- China
- Prior art keywords
- fruit
- trehalose
- pichia pastoris
- pichia
- caribbica
- 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.)
- Pending
Links
Images
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases, belonging to the field of fruit postharvest biological control. The method comprises the steps of: activating pichia caribbica, inoculating in an NYTB culture medium added with 0.2-1% of trehalose for inducing culture, and centrifuging to obtain thalli; diluting the thalli with sterile water to prepare 1*10<8>/ml of bacterial suspension; placing fruits in the bacterial suspension, soaking for 1 minute and then immediately taking out, and naturally airing; and placing in a plastic crate, sealing with a preservative film, and storing in an environment at room temperature. The efficacy of controlling the fruit postharvest diseases by the pichia caribbica is increased by using the inducing culture of the trehalose, thus the method has the advantages of simplicity in use, convenience for operation, good effect and low cost. The pichia caribbica subjected to the inducing culture can be used for replacing a chemical bactericide to control the fruit postharvest diseases, can avoid the damage to people due to the use of the chemical bactericide, and has remarkable economic benefit and social benefit.
Description
Technical field
The present invention relates to a kind of utilize trehalose (trehalose) to induce cultivate to improve the Pichia pastoris bacterial classification (
Pichia caribbica) to the method for fruit postharvest diseases preventing efficiency, belong to fruit and adopt the artifact prevention and control field.
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, have the fresh products of 10%-30% to cause rotting in developed country, and in the developing country that lacks the storing refrigerating equipment, fruit is adopted the back rotting rate then up to 40%-50% owing to postharvest disease.Exotic fruit preservation and freshness amount accounts for the fruit total output 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 of freezer and air-conditioned cold store is 700,000 tons, only accounts for 7% of total output.China's fruit is adopted back loss generally at 20%-30%, high person even reach 50%.In March, 2007, State Statistics Bureau's issue " People's Republic of China's national economy and social development statistical communique in 2006 " showed that China's fruit total output reached 17,050 ten thousand tons in 2006.Estimation in view of the above, the loss after China's fruit is adopted will reach tens billion of yuans level every year.
The main cause that fruit rots is the pathogenic effects of pathogenic microorganism, these pathogenic microorganisms mainly contain Botrytis cinerea (
Botrytis cinerea), Rhizopus stolonifer (
Rhizopus stoloniger), mould (
Penicillium spp.), Mucor (
Mucor spp.), chain lattice spore (
Alternaria spp.), aspergillus (
Aspergillus spp.) and soft rotten bacterium (
Erwinia sp.) etc.Pathogenic microorganism to adopt the back fruit harm not only be the heavy losses that it causes fruit quantitatively to cause, thereby and since many disease funguses can secrete the generation many secondary metabolites cause serious food-safety problem, as penicillium expansum (
P. expansum) produce clavacin (patulin), Botrytis cinerea (
B. cinerea) botrydial (botrydial) that produces etc.
The method of rotting after the most frequently used control fruit is adopted at present is that cryopreservation and chemical bactericide are handled.The cryopreservation technology has obtained developing widely and using in China in recent years, but existing refrigerating equipment often can not satisfy the needs of most fruits.Simultaneously, under cryogenic conditions, still have a considerable amount of pathogenic bacteria on fruit, to grow, and cause fruit to rot.For example, can cause multiple fruit gray mold Botrytis cinerea (
Botryris cinerea), also claim botrytis cinerea, also can growth and breeding in the time of-0.5 ℃, cause that many fruit rot, and cause serious economic 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 more can not be suppressed effectively, and it is quite serious to rot.For a long time, the major measure of control fruit postharvest diseases is to use bactericide.Chemical bactericide can play the effect of getting instant result to preventing and treating disease, and still, the use that chemical bactericide is long-term and a large amount of not only causes pathogenic microorganism to develop immunity to drugs, and serious environment pollution impairs human health simultaneously.The portion report of NAS (National Academy of Scinces) shows that at the whole agricultural chemicals that are used for handling food, bactericide constitutes 60% carcinogenic danger.
Raising along with the national economic development and the level of consumption; people are to the enhancing day by day of food security and environmental protection consciousness; number of chemical bactericide (as Captan, Benomyl etc.) has been prohibited and has been used or restriction use on the part fruit product at present, and its direct consequence is that present many fruit postharvest diseases have not had bactericide to be controlled.Therefore, the various countries scientist all explore energetically can replace chemical bactericide safe, the novel fruits postharvest disease is prevented and treated method efficiently.Currently in the world the research of the biological control of carrying out fruit postharvest diseases with antagonistic microbe has been obtained interim achievement, and thought that this is one of method that is hopeful most by the substituted chemistry bactericide.
Along with yeast classification, ecology, physiology, biochemistry and molecular biological research make progress, people improve day by day to the interest of utilizing yeast in the fruit vegetables storing.Domestic and international research shows that many saccharomycete are applied to fruit surface, can prevent and treat the disease that fruit is caused by pathogenic moulds, and these yeast are called as antagonism yeast.The saccharomycete inheritance stability, antimicrobial spectrum is wide, the height of tiring, not general the generation to people and the harmful metabolite of host plant, safe, and low to nutritional requirement, growth is fast, and to multiplely coerce, adverse circumstance has stronger tolerance, insensitive to most of bactericide, can be compatible to other chemistry and physical treatment.Therefore antagonism yeast is subjected to international extensive concern as the fruit postharvest diseases biocontrol microorganisms in recent years.External aspect, Sansone etc. separate from apple surface the rhodotorula glutinis that obtains to by
Botrytis cinereaThe gray mold that causes has remarkable prevention effect, and under laboratory condition, its rotting rate has reduced by 54% compared with the control.The research of Calvente etc. also finds to adopt afterwards to fruit by rhodotorula glutinis, and the mould disease has significant inhibition effect.
China's biologically controlling postharvest disease of fruit research is started late, Zheng Xiaodong etc. have studied the inhibition effect that rhodotorula glutinis rots to the citrus mould that is caused by Penicillium digitatum in the bacteriostatic test in vitro and in vivo, find that rhodotorula glutinis has demonstrated very high bacteriostatic activity in the citrus that control is caused by Penicillium digitatum rots, under 25 ℃ of conditions, the incidence of disease of contrast is 100%, and uses 1 * 10
8The incidence of disease of the bacteria suspension of individual/mL only is 45 %, and along with the raising of yeast concentration, the incidence of disease also has downward trend gradually.Zhang Hongyin etc. have studied rhodotorula glutinis strawberry have been adopted the inhibition effect of back gray mold, strawberry has been carried out the preliminary treatment in early stage after, inoculation 1 * 10
9The bacteria suspension of individual/mL and 1 * 10
5The pathogen pityrosporion ovale suspension of individual/mL after 4 ℃ storage was preserved 2 days down in 7 days and 20 ℃ down, is found the strawberry of inoculation rhodotorula glutinis respectively, and its rotting rate has reduced by 95% and 94.7 % compared with the control respectively.Model green grass or young crops (2001) etc. are screened fruit disease biological control antagonism bacterium and separate, and the result shows: monilia guilliermondii (
Candida guiliermondii), lemon shape Ke Leke yeast (
Kloeckera apiculata), the inferior Dbaly yeast of the Chinese (
Debaryomyces hansenii) the sweet cherry brown rot is all shown significant inhibition effect, monilia guilliermondii and lemon shape Ke Leke yeast can be prevented and treated drupe class fruit soft rot effectively; Tian researchs such as (2004) sprays 1 * 10 before sweet cherry is adopted
8The trichosporon cutaneum of CFU/mL (
Trichosporon pullulans), Cryptococcus laurentii (
Cryptococcus laurentii) and rhodotorula (
Rhodotorula glutinis), respectively at 25 ℃, under 0 ℃ and the air conditioned storage, trichosporon cutaneum is bred quantity on fruit the highest, and fungistatic effect is best; Zhang Hongyin etc. (2005) have studied under the different temperatures, and Cryptococcus laurentii is to the prevention effect of gray mold, and the result shows, under 25 ℃, the incidence of disease of pears gray mold is 16.7%, in conjunction with CaCl
2Result of use is more remarkable.
The current as can be known control that is used to adopt back fruit disease with effective microbe substituted chemistry bactericide has demonstrated great application prospect from domestic and foreign literature, and the biological control of fruit postharvest diseases becomes a new research focus.It is few to be applied to the antagonism bacterial classification class of producing, 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.Main cause is under the commodity production condition that the prevention 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 being applied in the fresh-keeping actual production of fruit storage, also need the further wide microorganism of screening good, the disease-resistant spectrum of antagonistic effect, and further improve the antagonism effectiveness of antagonism yeast, thereby it to be fresh-keeping to be applied to fruit storage.
Chinese patent CN201010240638 discloses a kind of method that rhodotorula glutinis is renderd a service the biological control of fruit postharvest diseases that improves, this method utilizes shitosan to induce the cultivation rhodotorula glutinis, improve the biological control effectiveness of rhodotorula glutinis, thereby reach the purpose of fresh-keeping fruit.The present invention utilizes trehalose to induce and cultivates Cali's Bick Pichia pastoris, improves the biological control of Cali's Bick Pichia pastoris and renders a service, and compares with shitosan, and trehalose is cheap, and processing method is simple, and has biological control effect preferably.
Trehalose is that Wiggers extracted from the ergot of rye first in 1832, find that subsequently it extensively exists in the animals and plants of occurring in nature and microorganism, comprise rudimentary plant, algae, bacterium, fungi and vertebrate, particularly in the fungies such as yeast, mould, mushroom, content is unexpectedly up to more than 15% of dry weight sometimes.Trehalose is the irreducibility disaccharide that is connected with α-1,1 glycosidic bond by the hemiacetal hydroxyl by two glucose molecules, Stability Analysis of Structures, and chemical inertness, avirulence, all highly stable to heat, acid, be the most stable saccharic in the natural disaccharide.It extensively is present in occurring in nature, and its chemical property is stable, and the characteristic of stabilizing cell membrane and protein structure is arranged, and can be used as the protective agent of biomolecule.The biological function of trehalose makes it to have wide and tempting utilization prospect at medical science, agronomy, Food Science, molecular biology etc.
The present invention utilize trehalose improve Cali's Bick Pichia pastoris (
Pichia caribbica) antagonism render a service, thereby reduce the rotting rate of fruit, and then reach the fresh-keeping purpose of fruit storage.
Summary of the invention
The present invention adds the trehalose of variable concentrations in culture medium, Pichia pastoris is induced cultivation, and a kind of method that the Pichia pastoris biological control is renderd a service that improves is provided.Induce the Pichia pastoris of cultivation through trehalose, can suppress the fruit pathogen significantly, prevent that fruit from adopting the back and rotting.The present invention has safety, efficient, low cost and other advantages, can be widely used in the biological control process of fruit postharvest diseases, reduces the loss that fruit postharvest diseases causes.
The present invention provides a kind of method that Pichia pastoris is renderd a service biologically controlling postharvest disease of fruit that improves simultaneously, carries out according to following step: with Cali's Bick Pichia pastoris (
Pichia caribbica) activation, usefulness NYTB medium culture Cali Bick Pichia pastoris (
Pichia caribbica), the centrifugal thalline that obtains; Thalline is prepared into 1 * 10 with the sterilized water dilution
8The bacteria suspension of individual/mL; Fruit is put into bacteria suspension, take out natural air drying behind the immersion 1min at once; Be placed in the plastic crate, after the preservative film sealing, be placed under the room temperature condition and deposit.
Carbon source is the trehalose of 0.2%-1% and the glucose of 0%-0.8% in the wherein said NYTB culture medium, i.e. yeast extract 5g, beef extract 8g, trehalose 2-10g, glucose 0-8g, purified water 1000ml, pH nature.
Wherein said Cali Bick Pichia pastoris (
Pichia caribbica), preservation strain is numbered CGMCC No.3616.
Wherein said fruit preferably apple, peach or strawberry.
Advantage of the present invention:
(1) Cali used in the present invention Bick Pichia pastoris (
Pichia caribbica) be this laboratory screening, its antagonism is renderd a service strong, and is harmless.
(2) trehalose wide material sources used in the present invention are cheap, nontoxic.
(3) the present invention uses trehalose to induce the cultivation Pichia pastoris, can increase the accumulation of its endogenous content of trehalose, thereby improves the antagonistic activity of Pichia pastoris.
(4) the present invention uses trehalose to induce the cultivation Pichia pastoris, compares with traditional dextrose culture-medium, can significantly improve Cali's Bick Pichia pastoris (
Pichia caribbica) antagonistic activity, thereby reduce the rotting rate of fruit, and then reach the fresh-keeping purpose of fruit storage.
(5) the present invention uses trehalose to induce the Pichia pastoris after the cultivation can replace chemical bactericide control fruit postharvest diseases, avoids using the harm of chemical bactericide to the people, has remarkable economic efficiency and social benefit.
Description of drawings
Wherein Fig. 1 is the inhibition effect of the Pichia pastoris of variable concentrations trehalose after inducing to green mold of apple; Annotate: CK: contrast; 0.2: carbon source is with 0.2% trehalose and 0.8% glucose; 0.5: carbon source is with 0.5% trehalose and 0.5% glucose; 0.8: carbon source is with 0.8% trehalose and 0.2% glucose; NYTB: carbon source is with 1% trehalose (representing with NYTB).The incidence of disease is 20 ℃ and places the result who measures behind the 7d.The significance of difference (p=0.05) represented in different letters.
Fig. 2 is the inhibition effect of the Pichia pastoris of variable concentrations trehalose after inducing to the apple gray mold; Annotate: CK: contrast; 0.2: carbon source is with 0.2% trehalose and 0.8% glucose; 0.5: carbon source is with 0.5% trehalose and 0.5% glucose; 0.8: carbon source is with 0.8% trehalose and 0.2% glucose; NYTB: carbon source is with 1% trehalose (representing with NYTB).The incidence of disease is 20 ℃ and places the result who measures behind the 7d.The significance of difference (p=0.05) represented in different letters.
Fig. 3 is the inhibition effect of the Pichia pastoris of variable concentrations trehalose after inducing to peach fruit head mold disease; Annotate: CK: contrast; 0.2: carbon source is with 0.2% trehalose and 0.8% glucose; 0.5: carbon source is with 0.5% trehalose and 0.5% glucose; 0.8: carbon source is with 0.8% trehalose and 0.2% glucose; NYTB: carbon source is with 1% trehalose (representing with NYTB).The incidence of disease is 25 ℃ and places the result who measures behind the 5d.The significance of difference (p=0.05) represented in different letters.
Fig. 4 is the inhibition effect of the Pichia pastoris of variable concentrations trehalose after inducing to peach fruit penicilliosis; Annotate: CK: contrast; 0.2: carbon source is with 0.2% trehalose and 0.8% glucose; 0.5: carbon source is with 0.5% trehalose and 0.5% glucose; 0.8: carbon source is with 0.8% trehalose and 0.2% glucose; NYTB: carbon source is with 1% trehalose (representing with NYTB).The incidence of disease is 25 ℃ and places the result who measures behind the 5d.The significance of difference (p=0.05) represented in different letters.
Fig. 5 utilizes trehalose and shitosan to induce the inhibition effect of the Pichia pastoris of cultivation to strawberry head mold disease; Annotate: CK: contrast; NYDB: with the Pichia pastoris of glucose as the carbon source cultivation; Trehalose: carbon source is with 0.5% trehalose and 0.5% glucose; Shitosan: carbon source is with 0.5% shitosan and 0.5% glucose.The incidence of disease is 20 ℃ and places the result who measures behind the 3d.The significance of difference (p=0.05) represented in different letters.
The specific embodiment
By by following embodiment with more detailed explanation the present invention.Following examples only are illustrative, and the present invention is not subjected to the restriction of these embodiments.
Cali's Bick Pichia pastoris (
Pichia caribbica) be that this laboratory screening obtains, now be stored in the Institute of Microorganism, Academia Sinica of No. 3, the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City that is positioned at the BeiJing, China, China Committee for Culture Collection of Microorganisms's common micro-organisms revenue centre (CGMCC), preservation strain is numbered CGMCC No.3616, adds 2% agar (quality meter) on the basis of NYDA(NYDB culture medium) 4 ℃ of low temperature preservations of culture medium.Yeast extract 5g in the NYDB culture medium, beef extract 8g, glucose 10g, purified water 1000ml, natural pH.
The cultivation program is: the activation of (1) solid: Pichia pastoris is inoculated in the NYDA culture medium, cultivates 48h at 28 ℃; (2) Liquid Culture: the NYDB seed culture medium of 50 ml that in the triangular flask of 250m1, pack into, insert the good Pichia pastoris of a ring activation with oese, at 200rpm, cultivate 20h under 28 ℃ of conditions; (3) induce cultivation: with centrifugal 10min under above-mentioned culture mix 7000 * g condition, the sterile distilled water washed twice, removing culture medium, and with the sterile distilled water yeast cells that suspends again, it is 5 * 10 that blood cell counting plate is regulated cell concentration
8Individual/mL.(carbon source is with 0.2% trehalose and 0.8% glucose to be respectively charged into 50ml NYDB culture medium or trehalose supplementing culture medium in the triangular flask of 250m1; Carbon source is with 0.5% trehalose and 0.5% glucose; Carbon source is with 0.8% trehalose and 0.2% glucose; Carbon source is with 1% trehalose (representing with NYTB)), and add the yeast cells nutrient solution 1ml of above-mentioned concentration, then at 200 rpm, cultivate 24h under 28 ℃ of conditions; (4) centrifugation suspends again: above-mentioned different disposal Yeast Cultivation mixture is under 7000 * g condition, centrifugal 10-15min, and with sterile distilled water washing 2 times, to remove culture medium, again suspend again with sterile distilled water, and to regulate barm cell concentration with blood counting chamber be 1 * 10
9Individual/mL, the physiological saline with the bacterium of going out is diluted to desired concn again.
Use therein trehalose is available from Nanning Jie Woli biological products Co., Ltd.
Embodiment 1: trehalose is induced and is cultivated the inhibition effect of Pichia pastoris to the fruit disease
One, testing program
After fruit is handled, form the wound of the unified size and the degree of depth at each position, fruit surface equator with sterile card punch.Wherein the superficial cut of strawberry is the 3mm(diameter) * 3mm(is dark), the superficial cut of peach fruit is the 5mm(diameter) * 3mm(is dark), the superficial cut of apple is the 5mm(diameter) * 5mm(is dark).Each wound equivalent adds 30 μ l(1) through the yeast cream of NYDB medium culture; (2) yeast cream of cultivating through trehalose supplementing culture medium (carbon source trehalose and glucose); (3) sterilized water (in contrast).Behind the 3h, add in each wound equivalent 30 μ l the pathogen spore (
B. cinereaConcentration is 1 * 10
5Spores ml
-1 P. expansumConcentration is 5 * 10
4Spores/ mL,
R. stoloniferConcentration is 1 * 10
4Spores/ mL) (spores/ mL represents the number of mycotic spore among the 1ml).Naturally after drying, fruit is put into plastic crate and with preservative film sealing, cultivate in the constant incubator (humidity 95%), peach really places 25 ℃, and strawberry and apple place 20 ℃.
Every processing repeats 3 times, repeats 18 fruits at every turn.Whole test repeats 2 times.Through some days cultivation, the record fruit incidence of disease, Libiee restrains Pichia pastoris with this evaluation card
P.caribbicaFungistatic effect.The computing formula of the incidence of disease is as follows:
Fruit sum * 100% of the fruit/inoculation pathogen of the incidence of disease (%)=morbidity
Two, result of the test
According to the above-mentioned steps test, the rotting rate result of statistics fruit is as follows:
1.1 inhibition effect to green mold of apple and gray mold
From Fig. 1 and Fig. 2 as can be seen, Pichia pastoris is after trehalose is induced cultivation, its incidence of disease to mould and grey mold significantly reduces, particularly when the glucose that uses 0.5% trehalose and 0.5% during as the carbon source culture yeasts, the incidence of disease of its mould and grey mold is 11.11% and 8.34% only, is markedly inferior to the contrast apple and induces the mould of the apple that the Pichia pastoris of cultivation handles and the incidence of disease of gray mold without trehalose.
1.2 inhibition effect to peach fruit head mold disease and penicilliosis
Result of the test by Fig. 3 and Fig. 4 shows, renders a service by adding the control that trehalose induces cultivation can significantly improve the disease that Pichia pastoris causes by Botrytis cinerea, Rhizopus stolonifer and penicillium expansum apple and peach fruit, and the best amount of inducing is 0.5%.
Embodiment 2: trehalose is induced and is cultivated the influence of Pichia pastoris to the fruit storage quality
One, testing program
Behind the test fruit picking directly with following processing immersion fruit 1min:(1) the Pichia pastoris suspension of NYDB medium culture; (2) trehalose supplementing culture medium (trehalose with 0.5% and 0.5% glucose) cultivate yeast cream; (3) sterilized water (in contrast).Then fruit is placed in the plastic crate, cultivate in the constant incubator (humidity 95%) with the preservative film sealing air-dry back, strawberry, and apple places 20 ℃, carries out the storage quality analysis after a period of time.Every processing repeats 3 times, repeats 10 fruits at every turn, and whole test repeats 2 times.
The storage quality analytical method is as follows:
(1) weight-loss ratio: weight method.Weight-loss ratio=(quality before handling-storage back quality)/quality * 100% before handling
(2) hardness: adopt the test of TA-XT2i texture analyser, probe diameter is 5mm, and the speed of service before the probe test, in the test, after the test is respectively 4.0mm s
1, 1.0mm s
1, 5.0mm s
1, the strawberry MTD is 6 mm, and the apple MTD is 10mm, and suffered maximum resistance (unit is newton) just was defined as the hardness of fruit when probe inserted fruit.Each fruit is located per 120 ° and is measured 3 times around the equator.
(3) total dissoluble solid: adopt WYT(0-80%) hand-held saccharometer is measured, and measurement result is expressed as the g/100g sample.
(4) titrable acidity (%): adopt titration, take by weighing 20 g pulp from 6 fruits, adding distil water 20ml homogenate washes the 100ml volumetric flask with 40ml distilled water.Place 80 ℃ of water-bath lixiviate 30min, stir once, take out cooling, be settled to scale every 5min.Filter, get 20 ml filtrates and place the 100ml small beaker, drop into a slice litmus red test paper when measuring strawberry, add three phenolphthalein indicators when measuring apple, with 0.1 mol l
1NaOH carries out titration.When becoming indigo plant or solution, litmus paper is titration end-point when red to little.Strawberry represents that to contain the citric acid percentage composition apple is represented with the percentage composition that contains malic acid.Every processing repeats for 3 times.
(5) mensuration of Vit C contents: ultraviolet rapid test method.Get 20g pulp sample from 6 fruit and add 20ml 1% hydrochloric acid homogenate, the 50ml volumetric flask is settled to scale.10000 * g, centrifugal 10 mim under 4 ℃ of conditions, supernatant are used for measuring.Get 0.4 ml extract, put into the 10 ml scale test tubes that fill 0.8 ml, 10% HCl, to scale, shake up with distilled water diluting.With distilled water is blank, measures absorbance at the 243nm place.Other draws the 0.4ml extract, adds 4ml distilled water and 1.6 ml 1moll respectively
1NaOH solution to 10ml to the scale test tube, shake up, leave standstill 15min after.Add 1.6 ml 10%HCl, mixing, adding distil water is settled to scale.With distilled water is blank, measures absorbance at the 243nm place.A by liquid to be measured and alkali treatment liquid to be measured
243What be worth is poor, looks into calibration curve, ascorbic content in the calculation sample.Every processing repeats for 3 times.
(6) brown stain degree: 6 fruit take by weighing 20 g pulp organizations, add the homogenate of 40ml distilled water, 10000 * g, centrifugal 10mim under 4 ℃ of conditions, get supernatant 10ml and add 95% ethanol 15ml, 10000 * g, centrifugal 10mim under 4 ℃ of conditions gets supernatant and measures absorbance in 420nm, represents browning degree with this.Every processing repeats for 3 times.
Two, result of the test
According to the above-mentioned steps test, the result of statistics apple storage quality is as follows:
Pichia pastoris after table 1 trehalose is induced is to the influence of strawberry storage quality
Remarks: the Pichia pastoris of 0:NYDB medium culture; 0.5: trehalose with 0.5% and 0.5% glucose are induced the Pichia pastoris of cultivation as carbon source.Quality determination in the above-mentioned table is 20 ℃ and places the result who measures behind the 5d.The significance of difference (p=0.05) represented in different letters.
As shown in table 1, Pichia pastoris is after 0.5% trehalose is induced cultivation, and the inhibitory action that strawberry is rotted naturally is remarkable, and the rotting rate of strawberry only is 30% of contrast, and is lower than the rotting rate of the strawberry of handling without the Pichia pastoris of inducing cultivation.Compare with the Pichia pastoris of not inducing cultivation, induce the Pichia pastoris of cultivation that weight-loss ratio, hardness, soluble solid, titratable acid, Vc content, the brown stain degree of strawberry are not all produced adverse effect through 0.5% trehalose.
Pichia pastoris after table 2 trehalose is induced is to the influence of apple storing quality
Remarks: the Pichia pastoris of 0:NYDB medium culture; 0.5: trehalose with 0.5% and 0.5% glucose are induced the Pichia pastoris of cultivation as carbon source.Quality determination in the above-mentioned table is 20 ℃ and places the result who measures behind the 40d.The significance of difference (p=0.05) represented in different letters.
As shown in table 2, induce the apple of cultivating the Pichia pastoris processing through 0.5% trehalose, its brown stain degree and titrable acidity significantly reduce, and other index is compared with the contrast apple, and no significant difference does not all produce adverse effect to every index of quality.
Above result of the test when the amount of inducing is 0.5%, can reduce the natural rotting rate of strawberry significantly, and strawberry and apple storage quality not had a negative impact after showing that trehalose is induced the cultivation Pichia pastoris.
Comparative Examples: utilize trehalose and shitosan to induce of the inhibition effect contrast of the Pichia pastoris of cultivation to strawberry head mold disease
One, testing program
Concrete testing program is with the testing program among the embodiment 1, and institute does not exist together for selecting the best parameter 0.5% of inducing, and promptly induces the cultivation Pichia pastoris to do contrast test with 0.5% trehalose and 0.5% shitosan.
Wherein selected fruit is strawberry, pathogenic bacteria be the strawberry head mold (
R. stolonifer).
Two, result of the test
According to the above-mentioned steps test, the rotting rate result of statistics fruit is as follows:
As shown in Figure 5, when using trehalose to induce the cultivation Pichia pastoris, can significantly improve the inhibition effect of Pichia pastoris to strawberry head mold disease, its rotting rate only is 12.5%, compare with disclosed patent CN201010240638, its rotting rate significantly is lower than the Pichia pastoris that the use shitosan is induced cultivation, and its rotting rate is 27.5%, and there were significant differences between the two.And the Pichia pastoris that uses shitosan to induce cultivation Pichia pastoris and common dextrose culture-medium to cultivate is compared, and it does not have significant difference between the two.
Claims (4)
1. one kind is improved the method that Pichia pastoris is renderd a service biologically controlling postharvest disease of fruit, it is characterized in that carrying out according to following step: with Cali's Bick Pichia pastoris (
Pichia caribbica) activation, usefulness NYTB medium culture Cali Bick Pichia pastoris (
Pichia caribbica), the centrifugal thalline that obtains; Thalline is prepared into 1 * 10 with the sterilized water dilution
8The bacteria suspension of individual/mL; Fruit is put into bacteria suspension, take out natural air drying behind the immersion 1min at once; Be placed in the plastic crate, after the preservative film sealing, be placed under the room temperature condition and deposit.
2. improve the method that Pichia pastoris is renderd a service biologically controlling postharvest disease of fruit according to one kind of claim 1, it is characterized in that carbon source is the trehalose of 0.2%-1% and the glucose of 0%-0.8% in the wherein said NYTB culture medium, be yeast extract 5g, beef extract 8g, trehalose 2-10g, glucose 0-8g, purified water 1000ml, pH nature.
3. improve the method that Pichia pastoris is renderd a service biologically controlling postharvest disease of fruit according to one kind of claim 1, it is characterized in that wherein said Cali Bick Pichia pastoris (
Pichia caribbica), preservation strain is numbered CGMCC No.3616.
4. improve the method that Pichia pastoris is renderd a service biologically controlling postharvest disease of fruit according to one kind of claim 1, it is characterized in that wherein said fruit is apple, peach or strawberry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110166736 CN102283281A (en) | 2011-06-21 | 2011-06-21 | Method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110166736 CN102283281A (en) | 2011-06-21 | 2011-06-21 | Method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102283281A true CN102283281A (en) | 2011-12-21 |
Family
ID=45330261
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110166736 Pending CN102283281A (en) | 2011-06-21 | 2011-06-21 | Method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102283281A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820343A (en) * | 2013-12-04 | 2014-05-28 | 江苏大学 | Application of saccharomyces cerevisiae in disease prevention and treatment on postharvest fruits and use method thereof |
| CN103988893A (en) * | 2014-01-21 | 2014-08-20 | 江苏大学 | Method for preventing and treating fruit postharvest diseases by using compound of ascorbic acid and Pichia caribbica |
| CN103988896A (en) * | 2014-05-29 | 2014-08-20 | 江苏大学 | Prevention and treatment on postharvest diseases of fruits by using glycine betaine-induced culture of pichia caribbica |
| CN104988082A (en) * | 2015-04-23 | 2015-10-21 | 新疆鲜宝莱生物科技有限公司 | Debaryomyces hansenii strain and applications thereof |
| CN107460134A (en) * | 2017-06-29 | 2017-12-12 | 江苏大学 | The Pichia guilliermondii of one plant of control the operatic circle postharvest disease |
| CN108118004A (en) * | 2017-12-15 | 2018-06-05 | 北京工商大学 | One plant of happiness cactus Pichia pastoris that can effectively prevent fruit postharvest diseases and its preparation and application method |
| CN110050831A (en) * | 2019-04-28 | 2019-07-26 | 浙江大学 | For controlling the Pichia guilliermondii bacterium suspension of Cherry Tomato Fruit postharvest disease |
| CN111887293A (en) * | 2020-07-14 | 2020-11-06 | 江苏大学 | Application of saccharomyces cerevisiae in disease control, storage and fresh keeping of picked baby cabbage |
| CN115918720A (en) * | 2023-01-06 | 2023-04-07 | 福州大学 | Application of brown algae oligosaccharide in improving prevention and treatment effect of yeast Lg 3 on postharvest diseases of fruits and vegetables |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899400A (en) * | 2010-06-10 | 2010-12-01 | 江苏大学 | Pichiacaribbica, application thereof in fruit storage and refreshment and use method thereof |
-
2011
- 2011-06-21 CN CN 201110166736 patent/CN102283281A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899400A (en) * | 2010-06-10 | 2010-12-01 | 江苏大学 | Pichiacaribbica, application thereof in fruit storage and refreshment and use method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《Proceedings of 2010 First International Conference on Cellular, Molecular Biology, Biophysics and Bioengineering (CMBB)》 20101225 卢黄娉等 《提高拮抗酵母生防效力的研究进展》 第278-281页 2 第7卷, * |
| 《植物学通报》 20011231 刘海波等 《水果采后生物防治拮抗机理的研究进展》 第657-664 1-4 第18卷, 第6期 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820343A (en) * | 2013-12-04 | 2014-05-28 | 江苏大学 | Application of saccharomyces cerevisiae in disease prevention and treatment on postharvest fruits and use method thereof |
| CN103988893A (en) * | 2014-01-21 | 2014-08-20 | 江苏大学 | Method for preventing and treating fruit postharvest diseases by using compound of ascorbic acid and Pichia caribbica |
| CN103988896A (en) * | 2014-05-29 | 2014-08-20 | 江苏大学 | Prevention and treatment on postharvest diseases of fruits by using glycine betaine-induced culture of pichia caribbica |
| CN104988082A (en) * | 2015-04-23 | 2015-10-21 | 新疆鲜宝莱生物科技有限公司 | Debaryomyces hansenii strain and applications thereof |
| CN104988082B (en) * | 2015-04-23 | 2018-07-24 | 新疆鲜宝莱生物科技有限公司 | The inferior Dbaly yeast bacterial strain of one plant of Chinese and its application |
| CN107460134A (en) * | 2017-06-29 | 2017-12-12 | 江苏大学 | The Pichia guilliermondii of one plant of control the operatic circle postharvest disease |
| CN108118004A (en) * | 2017-12-15 | 2018-06-05 | 北京工商大学 | One plant of happiness cactus Pichia pastoris that can effectively prevent fruit postharvest diseases and its preparation and application method |
| CN110050831A (en) * | 2019-04-28 | 2019-07-26 | 浙江大学 | For controlling the Pichia guilliermondii bacterium suspension of Cherry Tomato Fruit postharvest disease |
| CN111887293A (en) * | 2020-07-14 | 2020-11-06 | 江苏大学 | Application of saccharomyces cerevisiae in disease control, storage and fresh keeping of picked baby cabbage |
| CN111887293B (en) * | 2020-07-14 | 2022-10-28 | 江苏大学 | Application of saccharomyces cerevisiae in disease control, storage and fresh keeping of picked baby cabbage |
| CN115918720A (en) * | 2023-01-06 | 2023-04-07 | 福州大学 | Application of brown algae oligosaccharide in improving prevention and treatment effect of yeast Lg 3 on postharvest diseases of fruits and vegetables |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103820343B (en) | Application that saccharomyces cerevisiae is prevented and treated at fruit postharvest diseases and using method | |
| CN102283281A (en) | Method for improving biological control efficacy of pichia caribbica to fruit postharvest diseases | |
| CN107460134B (en) | Pichia guilliermondii strain for controlling pear postharvest diseases | |
| CN101914475B (en) | Lactobacillus used for biological preservation and application thereof | |
| CN101899400B (en) | Pichiacaribbica, application thereof in fruit storage and refreshment and use method thereof | |
| CN102827790B (en) | Bacillus subtilis strain A97, fungicide, and preparation method and application thereof | |
| Cao et al. | Combination of Pichia membranifaciens and ammonium molybdate for controlling blue mould caused by Penicillium expansum in peach fruit | |
| CN108094519B (en) | Grape antagonistic yeast composite biological preservative and preparation method and application thereof | |
| CN101946805A (en) | Biological prevention and control bacterial agent | |
| CN101697750A (en) | Method for preventing fruit postharvest diseases | |
| CN101911968B (en) | Method for enhancing effect of rhodotorula glutinis on biologically controlling postharvest disease of fruit | |
| CN101892167B (en) | Rhodotorula mucilaginosa, application thereof in preventing and curing postharvest disease of fruit and use method thereof | |
| CN101914459B (en) | Peach fruit disease biological antiseptic preservative and application and used cryptococcus laurentii | |
| Zhang et al. | Burdock fructooligosaccharide enhances biocontrol of Rhodotorula mucilaginosa to postharvest decay of peaches | |
| CN104161116B (en) | Induction fruit resistance controls the preparation of disease and corresponding revulsion | |
| CN105861340B (en) | Pichia anomala for controlling postharvest diseases of citrus and grapes | |
| CN103315058A (en) | Application of leuconostoc mesenteroides strain | |
| CN102388955A (en) | Method for processing disease control after strawberry is picked by combining ozone and Cryptococcus laurentii | |
| CN106615071B (en) | Application of pterostilbene in prevention and treatment of grape gray mold and downy mildew of litchi | |
| CN102696755A (en) | Method for improving efficacy of cryptococcus laurentii with control on penicilliosis and patulin of post-picked apples | |
| CN103125590A (en) | Grape biological fresh-keeping agent and preparation method thereof | |
| CN108795782B (en) | Method for preventing and treating pear postharvest diseases and storing and refreshing pear fruits by using abnormal Weikeham bacteria | |
| CN103988896A (en) | Prevention and treatment on postharvest diseases of fruits by using glycine betaine-induced culture of pichia caribbica | |
| Farahani et al. | Effect of two strains of antagonistic yeasts in combination with silicon against two isolates of Penicillium expansum on apple fruit | |
| CN103988893A (en) | Method for preventing and treating fruit postharvest diseases by using compound of ascorbic acid and Pichia caribbica |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20111221 |