CN111321105B - Method for improving effect of yeast in inhibiting fruit diseases by using acetylglucosamine - Google Patents
Method for improving effect of yeast in inhibiting fruit diseases by using acetylglucosamine Download PDFInfo
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Abstract
The invention discloses a culture medium for improving the effect of yeast on inhibiting fruit diseases by utilizing acetylglucosamine, which comprises a YEPD activation culture medium, an SM primary seed culture medium and a GlcNAc-SM secondary seed culture medium. The invention also provides a method for preparing a preparation for improving the effect of yeast on inhibiting fruit diseases by using the culture medium, which comprises the following steps: activating a strain; carrying out amplification culture; centrifuging, adjusting the concentration of yeast to 1 × 10 with sterilized distilled water7cell/mL to obtain the preparation. The invention also provides the application of the preparation: the bactericidal composition is used for preventing and treating the penicillium disease of pear fruits.
Description
Technical Field
The invention relates to the technical field of disease control after fruit picking, in particular to a biological preservative and fresh-keeping technology for improving the disease control effect of rhodosporidium marinum on pear fruits by N-acetyl-D-glucosamine.
Background
With the improvement of living standard, the dietary structure and consumption concept of people are obviously changed. The requirements on the yield and quality of fruits are constantly increasing. Meanwhile, labels such as "green", "organic", "pollution-free" and the like have also become a point of attention of consumers.
Fungal diseases are one of the main causes of great losses of picked fruits and vegetables. Among them, penicilliosis of fruits is one of the most important pathogenic fungi, and penicillium expansum also produces mycotoxins, resulting in serious food safety problems. Nowadays, the abuse of chemical agents is more and more realized to cause serious environmental and health problems, antagonistic yeast is utilized to inhibit the postharvest fungal diseases of fruits, and the antagonistic yeast is taken as a novel safe and environment-friendly biological disease prevention and preservation technology and is widely concerned at home and abroad in recent years. However, the bacteriostatic efficacy of the bio-control yeast after being harvested is still in a large gap with that of the chemical bactericide, so that further deep research on the mechanism of the bio-control yeast for inhibiting fruit diseases is urgently needed to further improve the bacteriostatic efficacy.
A patent entitled 'method for degrading patulin by using enzymes', patent number ZL201410065286.1, invented by China discloses a method for degrading patulin by using enzymes based on marine rhodosporidium toruloides. Chinese patent No. ZL 201410085393.0 discloses a biological fresh-keeping liquid based on gamma-aminobutyric acid combined biocontrol yeast, and antagonistic yeast comprises Rhodosporidium paludigenum Fell and Tallman. Chinese application patent 'method for inhibiting fruit postharvest diseases by inducing resistance and preparation used therein', application No. 201610522546.2, discloses a preparation for inhibiting fruit postharvest diseases by inducing resistance, which is composed of rhodosporidium marinum cell wall and water. Chinese application patent "chemical fungicide for controlling citrus postharvest diseases containing biocontrol yeast", application No. 201310224105.0, discloses a chemical fungicide for controlling citrus postharvest diseases containing biocontrol yeast, wherein the biocontrol yeast is Rhodosporidium paludigenum Fell and Tallman. Chinese application patent application No. 201110112540.5, Citrus biological antistaling agent based on activity of Rhodosporidium toruloides and fruit elicitor, discloses a Citrus biological antistaling agent composed of Rhodosporidium toruloides suspension, gibberellin/kinetin, salicylic acid, auxin and water. Chinese application patent No. 200610155062.5, Marine Yeast for biological prevention and treatment of fruit and vegetable postharvest diseases, and its preparation method and application, discloses a marine Rhodosporidium toruloides yeast capable of effectively inhibiting main fungal diseases of fruit and vegetable postharvest such as Alternaria alternata, Botrytis cinerea, Penicillium expansum, Penicillium digitatum, and hirsutella spore.
The Rhodosporidium parvum rhodosporium pallidum Fell & Tallman is a yeast deposited in International society of agriculture and biology center Gene Resource Collection (CABI Genetic Resource Collection) of International Institute of fungi in the United kingdom under the accession number IMI 394084. It can be seen from the above patents that the Rhodosporidium toruloides has a great application potential, and researchers have already performed physiological regulation on the antagonistic yeast by adding elicitors, thereby achieving the purpose of expanding the biocontrol effect against pathogenic bacteria.
N-acetyl-D-glucosamine (GlcNAc) is an amide derivative of the monosaccharide glucose. It is a secondary amide between glucosamine and acetic acid. Is of great significance in many biological systems. Is an integral part of the cell wall of microorganisms. In addition, N-acetylglucosamine is also reducing and has many important physiological functions in vivo, such as anti-inflammatory, anti-tumor and anti-oxidant activities. It can reduce the damage of cartilage matrix, and is effective medicine for treating osteoarthritis and rheumatoid arthritis. In addition, N-acetylglucosamine has wide applications in the food, chemical and cosmetic industries, such as food antioxidants, infant and toddler food additives, and diabetic sweeteners. International studies in recent years have shown that GlcNAc metabolism is involved in important physiological functions, particularly as a signal molecule and a nutrient (sensory) molecule, in addition to being a structural molecule in organisms, including microorganisms such as yeast.
A patent entitled "a method for purifying N-acetylglucosamine" of China, patent number ZL 201710038401.X, provides a method for purifying N-acetylglucosamine by desalting and impurity removal by an electrodialysis method. A Chinese patent of invention entitled fermentation method for improving the yield of N-acetylglucosamine, patent No. ZL 201610592555.9, discloses a fermentation method for improving the yield of N-acetylglucosamine by adding sodium cyclamate solution into fermentation liquor during fermentation. A patent No. ZL 201610384090.8 issued in China for a method for preparing N-acetyl-D-glucosamine from chitin discloses a method for preparing N-acetyl-D-glucosamine from chitin, which comprises 7 process steps of dissolving, filtering, degrading, crystallizing, filtering, cleaning and drying. Chinese application patent 'N-acetylglucosamine capsule preparation and preparation method thereof', application No. 201910846210.5, discloses an N-acetylglucosamine capsule preparation and preparation method thereof, belonging to the technical field of pharmaceutical preparation. Chinese application patent application No. 201810302095.0, namely Corynebacterium glutamicum for producing N-acetylglucosamine and application thereof, discloses Corynebacterium glutamicum for producing N-acetylglucosamine and application thereof, and belongs to the field of metabolic engineering. Chinese application patent No. 201910790130.2, a method for improving the content and conversion rate of N-acetylglucosamine in fermentation broth, discloses a method for improving the content and conversion rate of N-acetylglucosamine in fermentation broth. Chinese application patent application No. 201510412636.1, application of N-acetylglucosamine in honey product and honey product containing N-acetylglucosamine, discloses application of N-acetylglucosamine as additive in honey product with remarkable effect in crowd experiment, and novel honey product containing N-acetylglucosamine. The Chinese application patent 'application number 201810193361.0 of N-acetyl-D-glucosamine in the preparation of drugs and health products for preventing and treating uremia' provides an application of N-acetyl-D-glucosamine in the preparation of drugs and health products for preventing and treating uremia and a drug thereof, belonging to the field of medicines. The Chinese application patent 'application number 201810198767.8 of N-acetyl-D-glucosamine in preparing medicines for treating orthopedic diseases and medicines and health products' provides an application number 201810198767.8 of N-acetyl-D-glucosamine in preparing medicines for treating orthopedic diseases and medicines and health products, and relates to the field of medicines. The Chinese application patent 'application number of N-acetyl-D-glucosamine in preparing medicine and health care products for preventing and treating encephalatrophy' and the application number of the medicine '201810193362.5' provides application of N-acetyl-D-glucosamine in preparing medicine and health care products for preventing and treating encephalatrophy and the medicine, and belongs to the field of medicines.
As a food-grade additive, the N-acetylglucosamine is mature from preparation to application in the industrial degree and is a substance which is generally recognized as safe and has a certain health-care effect on human bodies. Therefore, the method has considerable research and application values for the development and the utilization of the method.
However, there is no report of the use of N-acetyl-D-glucosamine in yeast culture media to improve the biological activity of yeast.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for improving the control efficiency of the biocontrol yeast on fruit diseases, and the culture medium provided by the invention can be used for inducing and improving the control efficiency of the biocontrol yeast (the marine rhodosporidium toruloides IMI 394084) on the fruit diseases.
In order to solve the technical problems, the invention provides a culture medium for improving the effect of yeast on inhibiting fruit diseases by using acetylglucosamine, which comprises the following components in parts by weight: comprises a YEPD activation culture medium, an SM primary seed culture medium and a GlcNAc-SM secondary seed culture medium;
YEPD activation medium is: adding water into peptone (20 +/-0.5) g, yeast powder (10 +/-0.5) g, glucose (20 +/-0.5) g and agar (20 +/-0.5) g to a constant volume of 1000mL, and sterilizing to obtain a YEPD activated culture medium;
the SM primary seed culture medium is: glucose (10 +/-0.5 g), ammonium sulfate (5 +/-0.1 g), dipotassium hydrogen phosphate (2 +/-0.1 g), magnesium sulfate (3 +/-0.1) g and zinc sulfate (0.005 g), adding water to a constant volume of 1000mL, and sterilizing to obtain the SM primary seed culture medium.
The GlcNAc-SM secondary seed culture medium is as follows: adding 1g of N-acetylglucosamine into water, dissolving the solution in a 0.22 mu m filter membrane, sterilizing the solution to obtain a solution, adding 10 +/-0.5 g of glucose, 5 +/-0.1 g of ammonium sulfate, 2 +/-0.1 g of dipotassium hydrogen phosphate, 3 +/-0.1 g of magnesium sulfate and 0.005g of zinc sulfate, adding water to a constant volume of 1000mL, and sterilizing the solution to obtain a GlcNAc-SM secondary seed culture medium.
Description of the drawings:
the amount of water added to 1g of N-acetylglucosamine is at least such that the N-acetylglucosamine is dissolved.
The invention also provides a method for preparing a preparation for improving the effect of yeast on inhibiting fruit diseases by using the culture medium, which comprises the following steps:
(1) activation of bacterial strains
Standing and culturing at (28 +/-1) DEG C, and carrying out subculture on the marine cochliobolus ophioides (used as biocontrol yeast) on a YEPD activated culture medium for 30-48 h, and repeating subculture once under the same conditions;
(2) expanding culture
Inoculating the yeast obtained in the step (1) into an SM primary seed culture medium in an inoculation amount of 1% (volume%), and culturing for (24 +/-1) h under the culture conditions of (200 +/-50) rpm and (28 +/-1) ° C; then inoculating 2% (volume%) of the seed culture into a GlcNAc-SM secondary seed culture medium, and culturing (24 +/-1) h under the same culture conditions as above;
(3) centrifugal separation
Centrifuging the bacterial liquid obtained in the step (2) and then washing with water (so as to remove the culture medium); adjusting the concentration of yeast to 1 × 10 with sterilized distilled water7cell/mL to obtain the preparation.
As an improvement of the above method: the marine Rhodosporidium toruloides is Rhodosporidium paludigenum Fell & Tallman deposited under the accession number IMI 394084.
As a further improvement of the method, the step (3) is as follows: centrifuging the bacterial liquid obtained in the step (2) for 5min at the speed of 3000 Xg, and washing with sterilized distilled water for three times so as to remove the culture medium; counting by using a blood cell plate counting method and adjusting the concentration of yeast to 1X 10 with sterilized distilled water7cells/mL to obtain the preparation.
The invention also provides the application of the preparation prepared by the method: the bactericidal composition is used for preventing and treating the penicillium disease of pear fruits.
The invention has the following technical advantages:
1. the selected exciton N-acetylglucosamine is a food-grade additive, so that the exciton N-acetylglucosamine has the technical advantage of environmental protection.
2. The disease inhibiting capability of antagonistic yeast with the original biocontrol effect is further improved by only using 0.1% of GlcNAc, the operation is simple, the cost performance is high, and the negative influence brought by chemical bactericides is avoided.
3. The traditional YEPD culture medium in a laboratory is improved by the primary seed culture medium and the secondary seed culture medium, common and cheap inorganic ammonium sulfate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 3g and zinc sulfate 0.005g are used for replacing the original organic part of yeast powder 10g and peptone 20g, the dosage of glucose is halved, and the culture medium with more economic benefits is provided on the premise of ensuring the biomass and the growth speed of yeast.
Drawings
The following further describes embodiments of the present invention with reference to the drawings.
FIG. 1 shows the inhibitory effect of GlcNAc-induced Rhodosporidium toruloides on Penicillium disease in pear fruit at different concentrations; the results are day 4 storage. Wherein, the picture (a) is the disease rate of the pear fruit, and the picture (b) is the lesion diameter. Different letters represent differential significance (P ═ 0.05).
FIG. 2 shows the inhibitory effect of Rhodosporidium toruloides induced by different low GlcNAc on Penicillium disease in pear; the results are day 4 storage. Wherein, the picture (a) is the disease rate of the pear fruit, and the picture (b) is the lesion diameter. Different letters represent differential significance (P ═ 0.05).
Fig. 3 shows the control effect of different induction culture times R.paludigenum on pear penicilliosis; the results are day 4 storage. Wherein, the picture (a) is the disease rate of the pear fruit, and the picture (b) is the lesion diameter. Different letters represent differential significance (P ═ 0.05).
Fig. 4 shows the control effect of mono-substance GlcNAc treatment and r.paludigenum treatment on penicilliosis in pear fruits; the results are day 3 storage. Wherein, the picture (a) is the disease rate of the pear fruit, and the picture (b) is the lesion diameter. Different letters represent differential significance (P ═ 0.05).
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1, method for preparing a preparation for inducing and improving the fruit disease control efficacy of biocontrol yeast, the media used are three as follows:
YEPD activation medium is: 20g of peptone, 10g of yeast powder, 20g of glucose and 20g of agar, adding water to a constant volume of 1000mL, and pouring the mixture after sterilization to obtain a YEPD activated culture medium;
the SM primary seed culture medium is: 10g of glucose, 5g of ammonium sulfate, 2g of dipotassium hydrogen phosphate, 3g of magnesium sulfate and 0.005g of zinc sulfate, adding water to a constant volume of 1000mL, and sterilizing to obtain an SM primary seed culture medium;
the GlcNAc-SM secondary seed culture medium is as follows: adding 1g of N-acetylglucosamine into 10mL of water, dissolving the solution in the water, sterilizing the solution by using a 0.22 mu m water system filter membrane to obtain a solution, adding 10g of glucose, 5g of ammonium sulfate, 2g of dipotassium hydrogen phosphate, 3g of magnesium sulfate and 0.005g of zinc sulfate, adding water to a constant volume of 1000mL, and sterilizing the solution to obtain a GlcNAc-SM secondary seed culture medium. That is, the content of N-acetylglucosamine in the GlcNAc-SM secondary seed medium was 0.1%.
The sterilization of the culture medium is conventional high temperature sterilization, and is generally performed at 1.1 atmospheric pressure and 121 ℃ for 20 min.
The preparation method of the preparation comprises the following steps:
1) activation of the strain
Rhodosporidium paludigenum IMI 394084 was passaged for 2 passages on YEPD activated medium, i.e.:
taking one loop of Rhodosporidium paludigenum IMI 394084 to perform static culture on YEPD activated culture medium in a culture dish (the volume is about 15ml) for 48h at the temperature of 28 ℃, and repeating subculture once under the same condition; obtaining activated yeast;
2) and the enlargement culture
Inoculating the activated yeast obtained in the step 1) into an SM primary seed culture medium in an inoculation amount of 1% (volume%), and culturing for 24h under the culture conditions of 200rpm and 28 ℃; inoculating the obtained culture into a GlcNAc-SM secondary seed culture medium by an inoculation amount of 2 percent (volume percent), and culturing for 24 hours under the same culture condition to obtain a bacterial liquid;
3) and centrifugal separation
Centrifuging the bacterial liquid obtained in the step 2) for 5min at the speed of 3000 Xg, and washing with sterilized distilled water for three times to remove the culture medium; counting by using a blood cell plate counting method and adjusting the concentration of yeast to 1X 10 with sterilized distilled water7cells/mL to obtain the preparation.
Comparative example 1, zero concentration of N-acetylglucosamine:
the content of N-acetylglucosamine in the GlcNAc-SM secondary seed medium of example 1 was changed from 0.1% to 0 (i.e., same as SM medium), and the rest was the same as in example 1; obtaining the preparation.
Comparative example 2 high concentration of N-acetylglucosamine
The content of N-acetylglucosamine in the GlcNAc-SM secondary seed culture medium of example 1 was changed from 0.1% to 0.5%, 1.0%, 2.0%, respectively, and the rest was the same as example 1; respectively obtaining different preparations.
Comparative example 3 Low concentration of N-acetylglucosamine
The content of N-acetylglucosamine in the GlcNAc-SM secondary seed culture medium of example 1 was changed from 0.1% to 0.001% and 0.01%, respectively, and the rest was the same as example 1; respectively obtaining different preparations.
Comparative example 4 different incubation times
The cultivation time of example 1 in GlcNAc-SM secondary seed medium was changed from 24h to each of: 48h, 72h and 96h, the remainder being equivalent to example 1; respectively obtaining different preparations.
Experiment 1, control effect of different concentrations of N-acetylglucosamine (GlcNAc) on pear penicilliosis by induced culture of R
1. Experimental Material
Pear varieties: crystal pear
Pathogenic bacteria: penicillium expansum (Penicillium expansum), was activated on Potato Dextrose Agar (PDA) for 7 days at 25 ℃ for future use.
2. Treatment of
(1) Pre-treating pear fruits: selecting fruits with intact epidermis, cleaning with tap water, then soaking in 0.1% (volume%) sodium hypochlorite solution for disinfection for 2 minutes, taking out, washing with tap water, washing to remove residual sodium hypochlorite on the surface of pear, and air drying for later use.
(2) The fruit surface was perforated with an autoclave perforator, about 3mm in diameter and about 2mm in depth. Add 30. mu.l of 10 with pipette7Cells/ml r.paludigenum (preparations prepared using 0, 0.1%, 0.5%, 1.0%, 2.0% GlcNAc-SM medium of example 1 and comparative example 1, comparative example 2, respectively) were treated with an equal amount of sterile water as a control CK.
(3) After standing and air-drying at room temperature (25 ℃) for 2 hours, 1X 10 was placed on each wound4Spore/ml Penicillium expansum PMu.l of the sum bacterial suspension is stored at room temperature (25 ℃) after the treatment, is sealed by a PE preservative film for moisturizing treatment, is placed in a room with constant temperature (25 ℃) and constant humidity, is observed and recorded at regular time, and is compared, and the result is expressed by average morbidity (%) and average lesion diameter (mm). The 9 fruits were selected as one treatment group for 3 replicates and the experiment was repeated twice.
The control effect of different concentrations of N-acetylglucosamine (GlcNAc) on the penicilliosis of pear fruits by culturing R.paludigenum under induction is shown in FIG. 1.
The abscissa in fig. 1 represents the following treatments:
CK: sterilized distilled water;
SM: a preparation prepared at a concentration of 0% GlcNAc;
0.1% GlcNAc: a preparation prepared at a concentration of 0.1% GlcNAc;
0.5% GlcNAc: a preparation prepared at a concentration of 0.5% GlcNAc;
1.0% GlcNAc: a preparation prepared at a concentration of 1.0% GlcNAc;
2.0% GlcNAc: the resulting preparation was prepared at a concentration of 2.0% GlcNAc.
3. Results
The results of the fourth day are shown in fig. 1, on the basis that the rhodosporidium toruloides originally has certain biocontrol effect, the effect is remarkably improved after the induction culture of the N-acetylglucosamine with the concentration of 0.1 percent, and the effects are respectively remarkably reduced compared with the control medium and the culture medium without the addition. This trend is also shown in the results of lesion diameter. The mean lesion diameters of the control, SM and 0.1% concentration experimental groups were 16.11mm, 4.70mm and 1.76mm, respectively. While the relatively high induction concentrations, a few, do not show significant advantages. It can be seen that 0.1% of the excitons were rather effective in all concentrations studied.
1. Experimental Material
Pear varieties: crystal pear
Pathogenic bacteria: penicillium expansum (Penicillium expansum), was activated on Potato Dextrose Agar (PDA) for 7 days at 25 ℃ for future use.
2. Treatment of
(1) Pre-treating pear fruits: selecting fruits with intact epidermis, cleaning with tap water, then soaking in 0.1% (volume%) sodium hypochlorite solution for disinfection for 2 minutes, taking out, washing with tap water, washing to remove residual sodium hypochlorite on the surface of pear, and air drying for later use.
(2) The fruit surface was perforated with an autoclave perforator, about 3mm in diameter and about 2mm in depth. Add 30. mu.l of 10 with pipette7Cells/ml r.paludigenum (preparations prepared using 0, 0.001%, 0.01%, 0.1% GlcNAc-SM medium of example 1, comparative example 3, respectively) were treated with an equal amount of sterile water as a control CK.
(3) After standing and air-drying at room temperature (25 ℃) for 2 hours, 5X 10 of the gel was placed on each wound430 μ l of spore/ml Penicillium expansum P.exsusum suspension was stored at room temperature (25 ℃ C.) after completion of the treatment, and was sealed with a PE wrap film for moisturizing treatment, and placed in a room at a constant temperature (25 ℃ C.) and a constant humidity, and the results were observed and recorded at regular time intervals and were compared and expressed as average incidence (%) and average lesion diameter (mm). The 9 fruits were selected as one treatment group for 3 replicates and the experiment was repeated twice.
The control effect of low-concentration N-acetylglucosamine (GlcNAc) induced culture of R.paludigenum on pear penicilliosis is shown in FIG. 2.
The abscissa in fig. 2 represents the following treatments:
CK: sterilized distilled water;
SM: a preparation prepared at a concentration of 0% GlcNAc;
0.001% GlcNAc: a preparation prepared at a concentration of 0.001% GlcNAc;
0.01% GlcNAc: a preparation prepared at a concentration of 0.01% GlcNAc;
0.1% GlcNAc: the resulting preparation was prepared at a concentration of 0.1% GlcNAc.
3. Results
As a result, as shown in FIG. 2, the incidence and lesion diameter recorded on the fourth day were the same, and the control ability of red wintergreen to penicilliosis on pear fruits was improved to various degrees after the induction with low dose of GlcNAc in the culture, and the incidence rates of CK, SM, 0.001% GlcNAc, 0.01% GlcNAc, and 0.1% GlcNAc were 100.00%, 83.98%, 76.15%, 77.00%, and 65.82%, respectively, which were significantly different. Among them, the biocontrol effect was the best when the GlcNAc concentration in the medium was 0.1%.
Experiment 3 control effect of different induction culture time R.paludigenum on pear fruit penicilliosis
1. Experimental Material
Pear varieties: crystal pear
Pathogenic bacteria: penicillium expansum (Penicillium expansum), was activated on Potato Dextrose Agar (PDA) for 7 days at 25 ℃ for future use.
2. Treatment of
(1) Pre-treating pear fruits: selecting fruits with intact epidermis, cleaning with tap water, then soaking in 0.1% (volume) sodium hypochlorite solution for disinfection for 2 minutes, taking out, washing with tap water, washing to remove residual sodium hypochlorite on the surface of pear, and air drying for later use.
(2) The fruit surface was perforated with an autoclave perforator, about 3mm in diameter and about 2mm in depth. Add 30. mu.l of 10 with pipette7Cells/ml r. paludigenum (preparations prepared using 24h, 48h, 72h, 96h incubation times for example 1, comparative example 4, respectively) were treated with an equal amount of sterile water as control CK.
(3) After standing and air-drying at room temperature (25 ℃) for 2 hours, 5X 10 of the gel was placed on each wound430 μ l of spore/ml Penicillium expansum P.exsusum suspension was stored at room temperature (25 ℃ C.) after completion of the treatment, and was sealed with a PE wrap film for moisturizing treatment, and placed in a room at a constant temperature (25 ℃ C.) and a constant humidity, and the results were observed and recorded at regular time intervals and were compared and expressed as average incidence (%) and average lesion diameter (mm). The 9 fruits were selected as one treatment group for 3 replicates and the experiment was repeated twice.
The control effect of different induction culture time R.paludigenum on pear penicilliosis is shown in figure 3.
The abscissa in fig. 3 represents the following processes:
CK: sterilized distilled water;
SM 24: a preparation prepared by culturing GlcNAc at a concentration of 0% for 24 h;
GlcNAc 24: a preparation prepared by culturing GlcNAc at a concentration of 0.1% for 24 h;
GlcNAc 48: a preparation prepared by culturing 0.1% GlcNAc for 48 h;
GlcNAc 72: a preparation prepared by culturing 0.1% GlcNAc for 72 h;
GlcNAc 96: the preparation prepared by culturing the culture medium for 96h at the concentration of 0.1% GlcNAc.
3. Results
The results are shown in FIG. 3, which shows the inhibitory effect of P.expansum on pear fruit by Rhodosporidium toruloides obtained by culturing at different induction times in the medium on day four. As can be seen, there was no significant difference in the effect 24-96h after induction culture. The incidence rate is about 40 percent, and the diameter of the disease spot is between 2 and 3 mm. In consideration of economic efficiency, 24h induction is also the best scheme of the invention.
Experiment 4, comparison of control effects of single-substance GlcNAc treatment and R.paludigenum treatment on pear penicilliosis
1. Experimental Material
Pear varieties: crystal pear
Pathogenic bacteria: penicillium expansum (Penicillium expansum), was activated on Potato Dextrose Agar (PDA) for 7 days at 25 ℃ for future use.
2. Treatment of
(1) Pre-treating pear fruits: selecting fruits with intact epidermis, cleaning with tap water, then soaking in 0.1% (volume%) sodium hypochlorite solution for disinfection for 2 minutes, taking out, washing with tap water, washing to remove residual sodium hypochlorite on the surface of pear, and air drying for later use.
(2) The fruit surface was perforated with an autoclave perforator, about 3mm in diameter and about 2mm in depth. Add 30. mu.l of 10 with pipette7Cells/ml R.paludigenum (example 1, comparative example, respectively)1, prepared using 0, 0.1% GlcNAc-SM medium), with an equivalent amount of sterile water as a control CK and an equivalent amount of 0.1% GlcNAc solution as a control;
(3) after standing and air-drying at room temperature (25 ℃) for 2 hours, 1X 10 was placed on each wound430 μ l of spore/ml Penicillium expansum P.exsusum suspension was stored at room temperature (25 ℃ C.) after completion of the treatment, and was sealed with a PE wrap film for moisturizing treatment, and placed in a room at a constant temperature (25 ℃ C.) and a constant humidity, and the results were observed and recorded at regular time intervals and were compared and expressed as average incidence (%) and average lesion diameter (mm). The 9 fruits were selected as one treatment group for 3 replicates and the experiment was repeated twice.
The control effect of single substance GlcNAc treatment and r.paludigenum treatment on penicilliosis in pear fruit is shown in fig. 4.
The abscissa in fig. 4 represents the following processes:
CK: sterilized distilled water;
0.1% GlcNAc: a 0.1% GlcNAc solution;
a preparation prepared by culturing the Rp-SM with the concentration of 0% GlcNAc for 24 h;
Rp-GlcNAc-SM: a preparation prepared at a concentration of 0.1% GlcNAc by incubation for 24 h.
3. Results
The results are shown in fig. 4, and the recorded results on the third day show that: the single addition of 0.1% N-acetylglucosamine solution does not reduce the incidence of diseases, and can eliminate the possibility that the substance can inhibit penicillium expansum. Therefore, the substance is proved to play a role in inhibiting diseases through physiological regulation and control of biocontrol yeast rhodosporidium toruloides.
In summary, the following steps: based on a GlcNAc-SM secondary seed culture medium, in the concentration range of 0.001-2%, when the concentration of N-acetylglucosamine is 0.1%, after culturing for 24h, the rhodosporidium toruloides is prepared into a microbial inoculum, at the moment, the control effect on penicillium expansum is the best, and meanwhile, the economic benefit is the greatest, so that the method has a larger practical application space.
Description of the drawings: the experiments 1 to 4 adopt crystal pears of different batches.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (4)
1. A culture medium for improving the effect of yeast on inhibiting fruit diseases by utilizing acetylglucosamine is characterized in that: comprises a YEPD activation culture medium, an SM primary seed culture medium and a GlcNAc-SM secondary seed culture medium;
YEPD activation medium is: 20 +/-0.5 g of peptone, 10 +/-0.5 g of yeast powder, 20 +/-0.5 g of glucose and 20 +/-0.5 g of agar, adding water to a constant volume of 1000mL, and sterilizing to obtain a YEPD activated culture medium;
the SM primary seed culture medium is: glucose 10 + -0.5 g, ammonium sulfate 5 + -0.1 g, dipotassium hydrogen phosphate 2 + -0.1 g, magnesium sulfate 3 + -0.1 g, and zinc sulfate 0.005g, adding water to a constant volume of 1000mL, and sterilizing to obtain SM primary seed culture medium;
the GlcNAc-SM secondary seed culture medium is as follows: adding 1g of N-acetylglucosamine into water, dissolving the solution with a 0.22 mu m filter membrane, sterilizing the solution to obtain a solution, adding 10 +/-0.5 g of glucose, 5 +/-0.1 g of ammonium sulfate, 2 +/-0.1 g of dipotassium phosphate, 3 +/-0.1 g of magnesium sulfate and 0.005g of zinc sulfate, adding water to a constant volume of 1000mL, and sterilizing the solution to obtain a GlcNAc-SM secondary seed culture medium.
2. A method for preparing a preparation for improving the fruit disease-inhibiting effect of yeast by using the medium according to claim 1, comprising the steps of:
(1) activation of bacterial strains
Standing and culturing the rhodosporidium marinum on a YEPD activated culture medium for 30-48 h at the temperature of 28 +/-1 ℃, and repeatedly carrying out subculture once under the same conditions;
the marine rhodosporidium toruloides is marine rhodosporidium toruloides with a preservation number of IMI 394084Rhodosporidium paludigenum Fell & Tallman；
(2) Expanding culture
Inoculating the yeast obtained in the step (1) into an SM primary seed culture medium in an inoculation amount of 1%, and culturing for 24 +/-1 h under the culture conditions of 200 +/-50 rpm and 28 +/-1 ℃; then inoculating the strain into a GlcNAc-SM secondary seed culture medium with the inoculation amount of 2%, and culturing for 24 +/-1 h under the same culture conditions;
(3) centrifugal separation
Centrifuging the bacterial liquid obtained in the step (2) and washing with water; adjusting the concentration of yeast to 1 × 10 with sterilized distilled water7cell/mL to obtain the preparation.
3. The method according to claim 2, wherein the step (3) is:
centrifuging the bacterial liquid obtained in the step (2) for 5min at the speed of 3000 Xg, and washing with sterilized distilled water for three times so as to remove the culture medium; counting by using a blood cell plate counting method and adjusting the concentration of yeast to 1X 10 with sterilized distilled water7cells/mL to obtain the preparation.
4. Use of a formulation prepared by the method of claim 2 or 3: the bactericidal composition is used for preventing and treating the penicillium disease of pear fruits.
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