CN114451450A - Hansenula polymorpha capable of antagonizing botrytis cinerea and application thereof - Google Patents
Hansenula polymorpha capable of antagonizing botrytis cinerea and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
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- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The invention discloses Hanseniaspora uvarum (Hanseniaspora uvarum) capable of antagonizing botrytis cinerea and application thereof, and particularly relates to the fields of microbial culture and application. The method comprises the steps of separating, identifying and preserving yeast strains; preparing a sample bacterium suspension; preparing and culturing different gradients of diluent; screening strains with different colony colors and morphologies for preservation; after multiple screening, Hanseniaspora uvarum (Hanseniaspora uvarum) LY3 with good antagonistic effect on Botrytis cinerea is obtained. The in vitro screening experiment shows that the in vitro inhibition rate of LY3 on botrytis cinerea reaches 100%. The minimum concentration measurement for inhibiting the growth of the mould and the exploration of mechanisms for producing hydrogen sulfide and the like further prove that the bacteria can keep good bacteriostatic activity in the in-vivo environment.
Description
Technical Field
The invention relates to the field of microbial culture and application, in particular to hansenula polymorpha capable of antagonizing botrytis cinerea and application thereof.
Background
The mould pollution causes a great amount of fruits and vegetables to rot and deteriorate after being picked every year, the waste is serious, and the mould pollution is concerned globally. Among them, Botrytis cinerea (Botrytis cinerea) has strong survivability in the environment, wide host range, various invasion modes and extremely difficult control, and is one of the most widely-affecting pathogenic bacteria for fruits and vegetables. Gray mold is a fungal disease caused by Botrytis cinerea, and the prevention and treatment of gray mold at present mainly depends on chemical sterilization means, such as benzimidazole bactericides, N-phenyl carbamate bactericides, anilinopyrimidine bactericides and stable chlorine dioxide (ClO)2) And some novel chemical agents. However, the excessive use and abuse of chemical bactericides can easily cause chemical residues in food or toxicity to other organisms, and can cause harm to the environment, food safety and human health, thereby causing various public health problems. In addition, the control effect is weakened along with the increase of the drug resistance of pathogenic bacteria. Therefore, the development of a novel safe, efficient and pollution-free bacteriostatic agent is particularly urgent. The existing research shows that the antagonistic yeast has great potential to become a novel biological bacteriostatic agent due to simple nutritional requirement, stronger growth and reproduction capability than pathogenic fungi under extreme conditions, stable genetic property and more superiority in biological prevention and control.
Biological control is a novel fruit and vegetable disease control technology which has been widely paid attention to in recent 30 years, and most researches are carried out on antagonistic bacteria such as yeast. Researches show that the antagonistic yeast plays a biological prevention and treatment role mainly through nutrition and space competition, resistance induction, heavy parasitic action, metabolites and the like, and has certain requirements on the minimum inhibitory concentration of the antagonistic bacteria.
Most of antagonistic yeast strains obtained by the existing research are from low-altitude areas, the mining degree of natural microbial resources above the altitude of 1900 m is low, the antagonistic yeast screening source is single, the number of original samples is small, and the coverage of the types of the samples related to a specific ecological system is not comprehensive. The research sample is from a vineyard with more than 1900 meters, the grape fruits, leaves and soil are covered, the sample amount of the primarily screened strain reaches 791 strains, and the original sample is rich.
Disclosure of Invention
Therefore, the invention provides the Hansenula polymorpha capable of antagonizing botrytis cinerea from high-altitude areas and application thereof, so as to solve the problem that the grape fresh-keeping and biological control in the existing research are less.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to one aspect of the invention, the invention provides application of Hanseniaspora uvarum (Hanseniaspora uvarum) in preparation of products for antagonizing botrytis cinerea to achieve fruit and vegetable fresh-keeping.
According to another aspect of the present invention, there is provided a yeast screening method comprising the steps of:
selecting sample leaves, cutting the sample leaves into small pieces, putting the small pieces into a triangular flask containing sterile deionized water, and fully and uniformly mixing to obtain four parts of sample bacterial suspension;
step two, diluting the sample bacterial suspension with sterile deionized water to obtain diluents with different gradients;
step three, coating each gradient diluent in a YEPD + chloramphenicol culture medium, and culturing in a constant-temperature incubator; selecting 20-30 single colonies for each species according to the color and the morphology of the colonies on the culture medium, streaking on a YEPD + chloramphenicol culture medium again, culturing under the same conditions, and screening strains with different colony colors and morphologies for preservation;
step four, selecting representative strains, inoculating the representative strains into YEPD liquid culture medium for activation, diluting the strains with sterile deionized water after shaking culture, and separating and purifying yeast strain single strains;
and fifthly, inoculating the separated and purified single bacterial colony of the yeast strain into a YEPD liquid culture medium for activation, streaking and inoculating the single bacterial colony on a WL nutrient agar culture medium, and culturing in a constant temperature incubator.
Further, in the first step, the sample blade is chardonnay fresh blade with an altitude of 1900-3000 m in Yunnan Deckini.
Further, in the first step, fully mixing the mixture for 2-3 hours under the conditions of 25-30 ℃ and 120-180 r/min.
Further, in the second step, the dilution times are 10 times and 100 times respectively.
Further, in the third step, the constant temperature incubator is used for culturing for 2-4 days under the condition of the constant temperature incubator of 25-30 ℃.
Further, in the fourth step, the dilution times are 10 times and 100 times respectively.
Further, in the fourth step, the shaking culture condition is 120-180 r/min shaking culture at 25-30 ℃ for 48-96 h.
The invention has the following advantages:
the research sample is selected from vineyards in Yunnan Deckini wine production areas with the altitude of 1900-3000 m, including grape berries, grape leaves and soil, 791 strains of yeast are obtained through separation and purification, and according to the results of cluster analysis and biological identification, all strains are divided into 9 genera and 14 varieties. After screening, Hanseniaspora uvarum (Hanseniaspora uvarum) LY3 with good antagonistic effect on Botrytis cinerea is obtained. The in vitro screening experiment shows that the in vitro inhibition rate of LY3 on the botrytis cinerea reaches 100%. The minimum concentration measurement for inhibiting the growth of the mould and the exploration of mechanisms for producing hydrogen sulfide and the like further prove that the bacteria can keep good bacteriostatic activity in the in-vivo environment. As shown in the research, LY3 has excellent performance in vivo and in vitro tests and has great potential to become a biological bacteriostatic agent.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a characteristic of LY3 colonies on WL medium provided by the present invention;
FIG. 2 is a graph showing the microscopic cell characteristics of LY3 at WL provided by the present invention;
FIG. 3 is a sequencing peak diagram provided by the present invention, waveforms with different colors represent different bases, the distance between peaks is uniform, and there is no mixed bacteria interference at the bottom, which indicates that the sequencing result is stable and clear.
FIG. 4 shows the blast comparison result provided by the present invention, and a value of E is 0, which indicates a perfect match.
FIG. 5 is a test for inhibition of Botrytis cinerea provided herein, wherein A is the LY35 inhibition test, B is the control inhibition test, C is the LY3 inhibition test, and D is the LY1 inhibition test;
FIG. 6 shows the production of hydrogen sulfide (H) by LY3 provided by the present invention2S) comparing the capacity of the image; wherein A is LY 3; b is LY 2; c is LY 6; d is LY 30;
FIG. 7 is a graph showing the measurement of β -glucosidase activity in a control group according to the present invention;
FIG. 8 is a graph showing the measurement of β -glucosidase activity of LY3 provided by the present invention;
FIG. 9 is a graph comparing the effect of different concentrations of LY3 metabolite on Botrytis cinerea provided by the present invention, wherein A is a control, B is 0.05mL, C is 0.5mL, and D is 5 mL;
FIG. 10 is a graph showing the results of an in vivo test control group of grapes provided by the present invention;
FIG. 11 is a graph showing the results of in vivo test LY3 on grapes provided by the present invention;
FIG. 12 is a comparison graph of LY3 in vivo bacteriostasis test provided by the present invention, wherein A is empty PDA medium, B is LY3 supernatant, and C is LY3 stock solution;
FIG. 13 is a graph comparing the change of grapes on day 12 according to the present invention; wherein A is a control group, and B is LY3 group;
FIG. 14 shows the change of decay rates of CK group and LY3 group within 12 days under the storage condition of 25-30 deg.C;
FIG. 15 shows the decay scores of CK group and LY3 group within 12 days under the storage condition of 25-30 deg.C;
FIG. 16 shows the changes of the sensory evaluation of CK group and LY3 group within 12 days under the storage condition of 25-30 ℃;
FIG. 17 shows the hardness changes of CK group and LY3 group within 12 days under the storage condition of 25-30 deg.C;
FIG. 18 shows the change of weight loss of CK group and LY3 group within 12 days under the storage condition of 25-30 deg.C;
FIG. 19 shows the change of browning rate of fruit stalks between CK group and LY3 group within 12 days under the condition of 25-30 deg.C storage.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 Hanseniaspora uvarum (Hanseniaspora uvarum) antagonistic to Botrytis cinerea and use thereof
The LY3 strain is collected from a wine production area with an altitude of 1900-3000 m in Texas;
taking fresh leaves of Chardonnay as samples, randomly and accurately weighing 5g, cutting into small pieces, filling into a triangular flask containing 50mL of sterile deionized water, fully mixing for 2-3 h at 25-30 ℃ at 120-180 r/min, and performing quadruplicate mixing.
And (3) respectively diluting the bacterial suspensions of the different samples by 10 times and 100 times, sucking 100 mu L of each gradient diluent, coating the gradient diluent on a YEPD + chloramphenicol culture medium, culturing for 2-4 days in a constant-temperature incubator at 25-30 ℃, selecting 20-30 single bacterial colonies for each variety according to the color and the shape of the bacterial colonies on the culture medium, streaking on the YEPD + chloramphenicol culture medium again, culturing under the same conditions, and screening bacterial strains with different bacterial colony colors and shapes for preservation.
Selecting a representative strain, inoculating the representative strain into a YEPD liquid culture medium, activating, carrying out shake culture at the temperature of 25-30 ℃ for 48-96 h at the speed of 120-180 r/min, diluting the strain into 10 times and 100 times by using sterile deionized water, and observing the individual morphological characteristics of the strain under a microscope. Inoculating the separated and purified single bacterial colony of the yeast strain into a YEPD liquid culture medium for activation, then carrying out streak inoculation on a WL nutrient agar culture medium, placing the culture medium in a constant-temperature incubator at 28 ℃ for 5-7 days, and then photographing, wherein LY3 has the characteristic of the bacterial colony on the WL culture medium as shown in figure 1; LY3 is shown in FIG. 2 as characteristic of microscopic cells at WL; the color and morphology of colonies were observed and recorded, and cluster analysis was performed based on the color and morphology of colonies. Inoculating the strain into a YEPD liquid culture medium for activation, performing shake culture at the temperature of 25-30 ℃ at a speed of 120-150 r/min for 48-96 h, diluting the strain into 10 times and 100 times by using sterile deionized water, and observing the individual morphological characteristics of the strain under a microscope.
Example 2 minimal concentration validation of yeasts against mold growth
The minimum concentration of the saccharomycetes for inhibiting the growth of the moulds is explored in the early stage of the experiment, and researches find that the cell concentration of the saccharomycetes is 103、104、105、106The CFU/mL is gradually increased, the hypha growth diameter of the botrytis cinerea on the PDA culture medium is gradually reduced, and the cell concentration is 106At CFU/mL, the inhibitor can completely inhibit botrytis cinereaHyphae grow.
Example 3 Hanseniaspora uvarum (Hanseniaspora uvarum) antagonistic to Botrytis cinerea and use thereof
The LY3 strain is collected from a wine production area with an altitude of 1900-3000 m in Texas;
taking fresh leaves of Chardonnay as samples, accurately weighing 5g at random, cutting into small pieces, placing into a triangular flask containing 50mL of sterile deionized water, mixing at 28 deg.C and 150r/min, and mixing thoroughly for 2.5h in quadruplicate.
And (3) diluting the bacterial suspensions of the different samples by 10 times and 100 times respectively, sucking 100 mu L of each gradient diluent, coating the gradient diluent on a YEPD + chloramphenicol culture medium, culturing for 4d in a constant-temperature incubator at 25 ℃, selecting 20-30 single bacterial colonies for each variety according to the color and the form of the bacterial colonies on the culture medium, streaking on the YEPD + chloramphenicol culture medium again, culturing under the same conditions, and screening bacterial strains with different bacterial colony colors and forms for preservation.
Selecting a representative strain, inoculating the representative strain into a YEPD liquid culture medium, activating, carrying out shake culture at the temperature of 25-30 ℃ for 48-96 h at the speed of 120-180 r/min, diluting the strain into 10 times and 100 times by using sterile deionized water, and observing the individual morphological characteristics of the strain under a microscope. Inoculating the separated and purified single bacterial colony of the yeast strain into a YEPD liquid culture medium for activation, then carrying out streak inoculation on a WL nutrient agar culture medium, placing the culture medium in a constant temperature incubator for 5-7d at 28 ℃, and then photographing; the color and morphology of colonies were observed and recorded, and cluster analysis was performed based on the color and morphology of colonies. Inoculating the strain into YEPD liquid culture medium for activation, performing shaking culture at 28 deg.C for 64h at 130r/min, diluting with sterile deionized water to 10 times and 100 times, and observing individual morphological characteristics under microscope.
Experimental example 1 molecular biological identification
LY3 fungal DNA was sequenced, and a total of 1 primer pair was designed. The PCR product was purified with shrimp alkaline enzyme (SAP) (from Promega) and exonuclease I (EXO I) (from epicentre) and sequenced with the BigDye3.1 kit from ABI.
The method comprises the following operation steps:
DNA samples were subjected to quality inspection and concentration estimation by electrophoresis using 1% agarose 1. mu.L, and then diluted to a working concentration of 5-10 ng/. mu.L based on the estimated concentration, and not diluted for samples without significant DNA bands.
PCR reaction
a) PCR primer
ITS1 CTTGGTCATTTAGAGGAAGTAA (sequence Listing <210>1, NO.1seq, 2Ambystoma laterale x Ambystoma jeffersonia-1)
ITS4 TCCTCCGCTTATTGATATGC (sequence Listing <210>1, NO.1seq, 2Ambystoma laterale x Ambystoma jeffersonia-1)
b) PCR conditions
The PCR reaction system was 20. mu.L, comprising 1 XGC buffer I (TAKARA), 2.5mM Mg2+0.2mM dNTP, 0.2. mu.M each primer, 1U HotStarTaq polymerase (TAKARA) and 1. mu.L of DNA template. The circulation parameter is 95 ℃ and 5 min; 35 cycles (94 ℃, 20 s; 55 ℃, 40 s; 72 ℃, 1 min); 72 ℃ for 2 min; keeping at 4 ℃.
PCR purification
To 8 PCR products, 0.5U SAP and 4U Exo I were added. 37 ℃, 60min, 75 ℃, 15 min.
4. Sequencing reactions
Sequencing primer:
ITS1 CTTGGTCATTTAGAGGAAGTAA (sequence Listing <210>1, NO.1seq, 2Ambystoma laterale x Ambystoma jeffersonia-1)
ITS4 TCCTCCGCTTATTGATATGC (sequence Listing <210>2, NO.2seq, 2Ambystoma laterale x Ambystoma jeffersonia-2)
The reaction mixture included 3. mu.L of LBigDye3.1 mixture, 2. mu.L of sequencing primer (1. mu.M) and 1-2. mu.L of LPCR purified product. The circulation parameter is 96 ℃ and 1 min; 28 cycles (96 ℃, 10 s; 50 ℃, 5 s; 60 ℃, 4 min); keeping at 4 ℃.
5) The sequencing products were loaded onto an ABI3730XL sequencer and the Polyphred software was used for data analysis. Sequencing results of sample LY3 Blast results at NCBI were Hansenula polymorpha (Hanseniaspora uvarum) as shown in FIGS. 3 and 4. The gene sequence is shown in a sequence table <210>3, NO.3seq, 2Ambystoma laterale x Ambystoma jeffersonia-3.
Experimental example 2 inhibitory Effect of LY3 on Botrytis cinerea
10mL of PDA medium was added to the petri dish followed by 5mL of 10 concentration PDA medium6CUF/mL yeast, triplicate, 5 μ L of fresh spore suspension of the mold to be tested was inoculated onto the petri dish. After cultivation, the inhibition rate is calculated by measuring the diameter of a mould colony by adopting a cross method:
DC: diameter of mold growth without added yeast;
DA: diameter of mold growth after yeast addition.
As shown in figure 5, in an in vitro environment, the inhibition rate of LY3 on pathogenic bacteria Botrytis cinerea (Botrytis cinerea) reaches 100%, and is obviously superior to other yeasts.
Experimental example 3 LY3 production of hydrogen sulfide (H)2S) ability
Dibbling 3 mu L of each test strain suspension on a BIGGY solid culture medium, culturing at 30 ℃ for 4-7 d, continuously observing the color change of a bacterial colony, and judging the H yield of each test strain according to the color depth2The ability of S. White, cream color 2, light brown, brown 4, dark brown 5, black 6. Higher number, darker color, indicates H2The stronger the S-producing capacity, 3 in parallel per group, 4 test strains were spotted per medium. As can be seen from FIG. 6, LY3 has a stronger H production2And S capability.
EXAMPLE 4 determination of beta-glucosidase Activity
Agar medium (5.0g arbutin, 20.0g peptone, 10.0g yeast extract, 12.0g agar) was dissolved in 990mL deionized water, adjusted to pH 5.0 on a magnetic stirrer at 100 ℃ and autoclaved at 121 ℃ for 20 min. 2mL of sterilized 1% (v/v) ferric ammonium citrate solution was added to 100mL of the medium. And (3) coating the yeast to be tested on the cooled culture dish, culturing for 2-3 d at 25-30 ℃, paralleling each treatment group, and repeating the experiment twice. This beta-glucosidase converts arbutin in the dish to epidermin and glucose. The released epidermin forms a black iron complex with the iron ammonium, and a dark brown halo is observed in the beta-glucosidase positive yeast.
As shown in FIGS. 7 and 8, the entirety of the test dish coated with LY3 turned brown, and the color change was significant compared to the control, indicating that LY3 can produce β -glucosidase.
Experimental example 5 Effect of LY3 metabolites on the growth of Botrytis cinerea
To examine the effect of other potential metabolites from yeast primary or secondary metabolism antagonizing yeast production, molds were grown in media containing yeast culture supernatants. And (3) carrying out shake culture on the yeast culture in 50mL of yeast extract peptone glucose solid culture medium at 25-30 ℃ at 120-150 r/min for 5-7 d. The supernatant was collected by centrifugation at 3500rpm at 4 ℃ and sterilized by filtration through a 0.45-. mu.m sterile water filtration membrane. Subsequently, 5mL, 0.5mL, 0.05mL of the supernatant and distilled water were mixed with gentle: (A)<45 ℃) and concentrating the 5X PDA culture medium, and cooling for later use. Followed by inoculation with 20. mu.L of 104The diameter of a colony is observed after the botrytis cinerea (B.cinerea) spores/mL are cultured for 4-5 days at 25-30 ℃. Each treatment group was replicated in duplicate for 3 replicates.
As shown in FIG. 9, in the culture medium of the supernatant of LY3 in which 5mL, 0.5mL and 0.05mL were mixed, the colony diameter of Staphylococcus griseus decreased as the concentration of the supernatant increased, compared to the control group. Indicating that the bacteriostatic effect gradually weakens with the decrease of the concentration of the LY3 metabolite.
Experimental example 6 in vivo test
Selecting mature and healthy grape fruit grains with consistent sizes, and shearing off grape stalks by using scissors, wherein the fruit stalks with the length of about 2mm are left at the joint of the tail ends of the grape stalks and the grape fruit grains, so as to prevent the red grape fruit grains from drying, breaking skins and contaminating bacteria. And (3) soaking and sterilizing the mixture for 2-3 min by using a sodium hypochlorite solution with the concentration of 2%, then washing away the residual sodium hypochlorite by using sterile normal saline, and naturally airing the mixture in a sterile operating platform for later use. Puncturing the equatorial part of the fruit with sterilized iron wire, wherein the diameter of the wound is 2-3 mm, the depth is 2-3 mm, and 10 μ L of antagonistic yeast suspension (10 μ L) is added into each wound by a liquid transfer gun8CFU/mL), stand in the aseptic work station 2 toAfter 3h, no flowing bacterial suspension was left in and around the lesion of fruit pieces. Then 10. mu.L of a pathogen spore suspension (10. mu.L) was added to the wound6spores/mL) is placed at room temperature for 2 h-3 h, the fruit wound and the surrounding are sealed by a preservative film after no flowing bacterial suspension exists, the fruit wound and the surrounding are placed in an incubator at 28 ℃/93RH for constant-temperature moisture-preserving culture, the size of the lesion of the wound is measured after 5-6 d, the morbidity and the disease index are counted, and sterile water and corresponding pathogenic bacteria spore suspension are added into a control group. Each group of 12 fruits, each treatment group 3 in parallel, the experiment was repeated twice. The grading criteria for the degree of fruit set incidence are shown in Table 1.
d: the number of rotten grapes in the group;
f: the total number of grapes in the group;
a: number of fruits at each disease stage
b: relative grade value
d: total number of fruits in treatment group
e: highest grade value
TABLE 1 grading Standard of the degree of disease of fruit pieces
FIGS. 10 and 11 show that, after the 5-day in vivo test, no Botrytis cinerea hyphae grow on the wound of the LY 3-treated group, and the overall integrity of the grape is higher than that of the control group; the decay rate and decay score of LY3 in Table 2 were also superior to those of the CK control blank. Shows that LY3 has good effect of inhibiting botrytis cinerea.
TABLE 2 decay Rate and decay score of LY3 antagonism of Botrytis cinerea in vivo experiments
Note: the rotting rate is the number of rotted grapes/total number of grapes; rot is divided into Σ (grade value of the grade, the number of grapes of the grade)/(maximum grade, the total number of grapes of the grade)
Experimental example 7 antagonistic action on fruit
Cleaning fresh grapes with 70% alcohol, puncturing the grapes with a sterile needle at intervals, and soaking the grapes in a prepared solution for 2-3 h: 10mL of yeast suspension; 10mL of culture supernatant is subjected to sterile filtration; YPD culture medium 10mL, take out, air-dry, cut into halves, place and spread inoculated with pathogenic bacteria (10)6spores/mL) at room temperature for 7-8 days, and if an obvious inhibition zone is generated, the bacteria have a good inhibition effect.
As shown in FIG. 12, the grapes soaked in the LY3 supernatant and the raw liquid treated group both inhibited the growth of Botrytis cinerea significantly compared to the control group, while the inhibition zone of the raw liquid containing viable bacteria was more significant compared to the supernatant.
Experimental example 8 preservation test
Taking sweet seedless grapes freshly picked in a vineyard as samples, selecting mature healthy grape fruit grains with consistent sizes, shearing off grape stalks by using scissors, and reserving the fruit stalks with the length of about 2mm at the joint of the tail ends of the fruit stalks and the fruit grains so as to prevent the grape fruit grains from drying up, breaking skins and contaminating bacteria. Spray 10 of the experimental group8(CFU/mL) 50mL of yeast suspension, 50mL of normal saline sprayed on a control group, 30 fruits in each group are placed in a preservation box to be stored at the room temperature of 25 ℃, and the physical and chemical indexes and microbial flora change of the grapes are observed in 0, 4, 8 and 12 days.
As shown in FIG. 13, the grapes of the control group sprayed with clear water on the twelfth day were severely mildewed, while the treatment group sprayed with LY3 was relatively well preserved.
The decay rates of the blank control group and LY3 group were varied within 12 days under simulated room temperature storage conditions at 25 ℃ to 28 ℃ (P <0.05), and the results are shown in FIG. 14. Shows that spraying LY3 bacterial liquid can effectively reduce the rotting rate of the grape fruits during the storage period.
The decay scores of the blank control group and the LY3 group were changed within 12 days under simulated room temperature storage conditions at 25 ℃ to 28 ℃ (P <0.05), and the results are shown in FIG. 15. Spraying LY3 bacterial liquid can effectively reduce the rotting degree of the grape fruits during the storage period.
Experimental example 9 evaluation of acceptability
Fresh grapes were subjected to sensory evaluation by an evaluation panel. The acceptability of fresh grapes, according to their sensory acceptability according to the gloss, colour, odour, appearance and overall acceptability parameters, is evaluated with reference to the 9-part table of Santos et al for fresh grapes: a score of "1" indicates extreme dislike and complete unacceptable; "5" is divided into a critical value, meaning neither liked nor disliked; the "9" score indicates extreme liking and is fully acceptable. The results showed that the acceptability of the sensory panel on day 1 was 100% for both groups of grapes. On day 4, the evaluation 7 points for the LY 3-treated group accounted for 44.4% or more, while the evaluation 5 points for the CK control group accounted for 88.9% or more. On day 8, 77.8% was evaluated by 5 or more points for the LY 3-treated group, and only 11.1% was evaluated by 5 or more points for the CK control group. On day 12, 55.6% above the LY 3-treated 5 fraction, but only 11.1% above the CK control 3 fraction.
As shown in fig. 16, after 12 days of grape storage, the final sensory score of LY 3-treated group was around 50% of the initial score, while the control CK group was less than one third, indicating that LY 3-treated group was more acceptable than the control CK group on subjective visual evaluation.
Experimental example 10 physical and chemical indexes
A5 x 5cm peel is removed from the surface of the grape, and the hardness of the grape fruit is measured by a fruit hardness tester GY-3. The fruit was pierced with a probe of 3mm diameter at a constant force and a depth of 10 mm. A total of 30 fruits were tested per treatment combination and the maximum force was recorded.
As can be seen from fig. 17, both groups showed a decrease in fruit firmness, and the blank control group was lower than LY3 throughout storage, with the difference being statistically significant after the fourth day; the change in fruit weight loss is shown in fig. 18, the fruit weight in the control group and LY3 group decreased consistently during storage, and the difference between the two groups was not statistically significant in weight loss; as can be seen from fig. 19, the control and LY3 groups showed almost complete browning of the fruit stalks over time, and the browning levels of the fruit stalks at 8 th and 10 th days were significantly different from those of LY3 group (P < 0.05).
In summary, spraying LY3 yeast culture solution was effective in maintaining the quality of the grape fruit during storage.
Along with the rise of the altitude, the temperature is reduced, the temperature difference between day and night is large, the ultraviolet ray is strong, and the soil type is also different. Microorganisms living in this special environment also have great development value due to their unique physiological functions, metabolites, and the like. The research sample is selected from vineyards of Yunnan Deckini wine producing areas with the altitude of 1900-3000 m, including grape berries, grape leaves and soil, 791 strains of yeast are obtained through separation and purification, and all strains are divided into 9 genera and 14 varieties according to the results of cluster analysis and biological identification. After screening, Hanseniaspora uvarum (Hanseniaspora uvarum) LY3 with good antagonistic effect on Botrytis cinerea is obtained. In-vitro screening experiments show that the in-vitro inhibition rate of LY3 on botrytis cinerea reaches 100%. The minimum concentration measurement for inhibiting the growth of the mould and the exploration of mechanisms for producing hydrogen sulfide and the like further prove that the bacteria can keep good bacteriostatic activity in the in-vivo environment. As shown in the research, LY3 has excellent performance in vivo and in vitro tests and has great potential to become a biological bacteriostatic agent.
Biological control is used as an important means of green control, powerful technical support can be provided for agricultural production and food safety, and the development of a high-quality, safe, stable and efficient biological preparation is the final aim of the research. Researches are started from a regulation and control mechanism for inhibiting the growth and the propagation of the botrytis cinerea by antagonistic saccharomycetes, and feasibility of the botrytis cinerea as a biological bacteriostatic agent in the field of foods is explored from a brand new view angle. The development and utilization of the novel biological bacteriostatic agent are very challenging subjects, the research is an important direction for reducing mould pollution and improving food safety after fruit and vegetable harvesting, and the research is also an important measure for combined innovation and deep fusion of life science and health research. The construction of a healthy environment is one of the key targets in the healthy Chinese 2030 runaways of China, and the prevention and treatment of harmful organisms are the important civil matters related to the health of people. In conclusion, the research has important significance from both academic value and practical value.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Lanzhou university
<120> Hansenula polymorpha capable of antagonizing botrytis cinerea and application thereof
<141> 2022-02-17
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA/RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-1
<400> 1
cttggtcatt tagaggaagt aa 22
<210> 2
<211> 20
<212> DNA/RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-2
<400> 2
<210> 3
<211> 712
<212> DNA/RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum-3
<400> 3
ccgtaggtga acctgcggaa ggatcattag attgaattat cattgttgct cgagttcttg 60
tttagatctt ttacaataat gtgtatcttt attgaagatg tgcgcttaat tgcgctgctt 120
ctttagagtg tcgcagtgaa agtagtcttg cttgaatctc agtcaacgct acacacattg 180
gagtttttta ctttaattta attctttctg ctttgaatcg aaaggttcaa ggcaaaaaac 240
aaacacaaac aattttattt tattataatt ttttaaacta aaccaaaatt cctaacggaa 300
attttaaaat aatttaaaac tttcaacaac ggatctcttg gttctcgcat cgatgaagaa 360
cgtagcgaat tgcgataagt aatgtgaatt gcagatactc gtgaatcatt gaatttttga 420
acgcacattg cgcccttgag cattctcagg ggcatgcctg tttgagcgtc atttccttct 480
caaaagataa tttattattt tttggttgtg ggcgatactc agggttagct tgaaattgga 540
gactgtttca gtctttttta attcaacact tagcttcttt ggagacgctg ttctcgctgt 600
gatgtattta tggatttatt cgttttactt tacaagggaa atggtaacgt accttaggca 660
aagggttgct tttaatattc atcaagtttg acctcaaatc aggtaggatt ac 712
Claims (8)
1. An application of Hanseniaspora uvarum (Hanseniaspora uvarum) in fruit and vegetable product preservation, which can antagonize botrytis cinerea, is provided.
2. The method for screening yeast as claimed in claim 1, wherein the method comprises the steps of:
selecting sample leaves, cutting the sample leaves into small pieces, putting the small pieces into a triangular flask containing sterile deionized water, and fully and uniformly mixing to obtain four parts of sample bacterial suspension;
step two, diluting the sample bacterial suspension with sterile deionized water to obtain diluents with different gradients;
step three, coating each gradient diluent in a YEPD + chloramphenicol culture medium, and culturing in a constant-temperature incubator; selecting 20-30 single colonies from each variety according to the color and the form of the colonies on the culture medium, streaking on a YEPD + chloramphenicol culture medium again, culturing under the same conditions, and screening representative colonies according to the color and the form for preservation;
selecting representative strains, inoculating the strains into YEPD liquid culture medium for activation, after shaking culture, diluting the strains with sterile deionized water, and observing the strain morphology under a microscope;
and fifthly, inoculating the separated and purified single bacterial colony of the yeast strain into a YEPD liquid culture medium for activation, then streaking and inoculating the single bacterial colony on a WL nutrient agar culture medium, placing the culture medium in a constant temperature incubator for culture, and recording the form and the color.
3. The yeast screening method according to claim 2, wherein in the first step, the sample leaf is obtained from fresh leaf of Chardonnay at an altitude of 1900-3000 m in south Yunnan Textilene.
4. The yeast screening method according to claim 2, wherein in the first step, the mixing is performed under the conditions of 25-30 ℃ and 120-180 r/min for 2-3 h.
5. The method for screening yeast according to claim 2, wherein the dilution ratio in the second step is 10 times and 100 times, respectively.
6. The yeast screening method according to claim 2, wherein in the third step, the constant temperature incubator is used for incubation at 25-30 ℃ for 2-4 days.
7. The method for screening yeast according to claim 2, wherein the dilution ratio in the fourth step is 10 times and 100 times, respectively.
8. The yeast screening method according to claim 2, wherein in the fourth step, the shake culture is performed at a temperature of 25-30 ℃ and at a speed of 120-180 r/min for 48-96 h.
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| 张永玲: "云南德钦优良野生酵母菌的筛选及其生物防治性能研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》, no. 1, pages 4 - 12 * |
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