Meyerozyma guilliermondii PJ15 and its applications

Abstract

The present invention relates to the field of microbial technology, and particularly to Meyerozyma guilliermondii PJ15 and its application. During the research process of the present invention, it was found that Meyerozyma guilliermondii has an inhibitory effect on Penicillium crustosum and is a biocontrol agent that can be effectively applied to the preservation of wogan oranges. Through preservation experiments, it was found that compared with the control group without spraying the preservative, the preservation duration of wogan oranges can be extended by at least about 20 days when sprayed with the preservative prepared from the metabolites of PJ15. If it is sprayed within 1 hour after picking and wrapped with plastic wrap, the preservation duration of wogan oranges can be extended by about 45 days, which can greatly improve the preservation period of wogan oranges and provide good guarantee for the long-distance transportation of wogan oranges. It is a good biological preservative. At the same time, during the research, it was also found that when the extract of wogan orange leaves and wogan orange peels is added and activated during the production of secondary metabolites, the preservation effect on wogan oranges is further enhanced, and it has a better antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to Meyerozyma guilliermondii PJ15 and its applications.

Background Art

[0002] As a common fruit widely favored in the southern region, Wogan oranges are not only easy to plant, but also have the advantages of delicious taste, juicy, and moderate sugar content, and have good sales in the market. As a hybrid of oranges and tangerines, the preservation effect of Wogan oranges themselves after picking is better than that of oranges and tangerines. However, in the humid and warm weather in the south, Wogan oranges are also prone to rot. During the research on the preservation of Wogan oranges, it was found that Penicillium digitatum is the main pathogenic bacterium causing the rot of Wogan oranges. Therefore, how to prepare a preservative for Penicillium digitatum strains is the focus of current research on the preservation of Wogan oranges, and biocontrol agents are the focus of research in the field of fruit preservation. In the prior art, through continuous screening and comparison by technicians, many strains with fruit preservation effects have been obtained, such as Bacillus, endophytic fungi, AM fungi and other different strains. However, the screening of biocontrol bacteria is a huge experimental process, and during the specific screening, technicians also found that even strains of the same genus or even the same species have completely different antagonistic effects on different pathogenic bacteria, which leads to different types of fruit preservation applications for the screened biocontrol bacteria. For example, a certain biocontrol bacterium may have a control effect on diseases caused by Penicillium, but has no control effect on diseases caused by other strains. Therefore, in order to improve the utilization of biocontrol strains, a large number of screenings to obtain various strains with preservation effects on different diseases and different fruits are the key to the research of biocontrol agents.

[0003] Rich germplasm resources can provide more selectable strains for the field of biocontrol agent processing. The Meyerozyma guilliermondii of the present application belongs to yeasts. Although yeasts have been reported to be used in the protection and preservation fields of certain fruits and vegetables, it was also found during the actual screening of bacteria that some yeasts do not have obvious effects on the protection and preservation of certain fruits and vegetables. Therefore, starting from the research on the preservation of Wogan oranges, it is necessary to screen out a biocontrol strain that can inhibit the growth of Penicillium digitatum and can be effectively applied to the preservation of Wogan oranges.

Summary of the Invention

[0004] In view of the above, it is necessary to provide a biocontrol strain that can inhibit the growth of Penicillium digitatum and has a preservation effect on Wogan oranges. After research and identification, this strain is Meyerozyma guilliermondii.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The strain Meyerozyma guilliermondii PJ15, with the deposit number GDMCC NO.62572, is deposited at: Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit date is June 28, 2022.

[0007] The present invention also includes an antibacterial agent comprising the Meyerozyma guilliermondii PJ15.

[0008] The present invention also includes a preservative comprising the Meyerozyma guilliermondii PJ15.

[0009] The present invention also includes the use of the Meyerozyma guilliermondii PJ15 or the antibacterial agent in inhibiting the growth of Penicillium crustosum.

[0010] The present invention also includes the use of the Meyerozyma guilliermondii PJ15, the antibacterial agent or the preservative in the preservation of mandarin oranges.

[0011] Furthermore, the method comprises: obtaining the YPD liquid culture medium fermentation product of the Meyerozyma guilliermondii PJ15, filtering, and taking the filtrate to obtain the preservative.

[0012] Furthermore, the YPD liquid culture medium is also added with Wogan peel leaves and / or Wogan leaf liquid.

[0013] Furthermore, the volume ratio of the Wogan leaf liquid to the Wogan peel liquid is 1:3.

[0014] The present invention also includes a method for preserving Wogan oranges by using the preservative, which comprises: preparing the preservative and water to obtain a preservative liquid, spraying the liquid on the surface of the Wogan oranges, and wrapping the liquid with a layer of preservative film.

[0015] Furthermore, the time for spraying the preservative is within 1 hour after the oranges are picked.

[0016] The present invention has the following beneficial effects:

[0017] In the process of research, it was found that Meyerozyma guilliermondii has an inhibitory effect on Penicillium crustosum, and it is a biocontrol agent that can be effectively applied to the preservation of Wogan oranges. Through preservation experiments, it was found that compared with the control group without spraying the preservative, the preservation period of Wogan oranges sprayed with the preservative prepared from the metabolites of PJ15 can be extended by at least about 20 days. If it is sprayed within 1 hour after picking and wrapped with plastic wrap, the preservation period of Wogan oranges can be extended by about 45 days, which can greatly improve the preservation period of Wogan oranges and provide good guarantee for the long-distance transportation of Wogan oranges. It is a good biological preservative. At the same time, in the research, it was also found that when producing secondary metabolites, after adding the extracts of Wogan orange leaves and peels for activation, the preservation effect on Wogan oranges is further enhanced, with better antibacterial effect.

Description of the Drawings

[0018] Figure 1 It is the morphological diagram of strain PJ15 of the present invention on a petri dish;

[0019] Figure 2 It is the morphological diagram of strain PJ15 of the present invention under a microscope;

[0020] Figure 3 It is the plate confrontation experiment diagram of the screened strain against Penicillium crustosum; the left side is the control group and the right side is the experimental group;

[0021] Figure 4 It is the biological anti-corrosion effect diagram of strain PJ15 on Wogan oranges; among them, Figure ① is the state of Wogan oranges on the 0th day, Figure ② is the state of Wogan oranges on the 7th day, Figure ③ is the state of Wogan oranges on the 14th day, and Figure ④ is the state of Wogan oranges on the 21st day; in each figure, the upper row is the control group and the lower row is the experimental group.

Detailed Embodiments

[0022] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0023] Any feature disclosed in this specification (including any additional claims, abstract) is only an example of a series of equivalent or similar features unless specifically stated otherwise.

[0024] Example 1:

[0025] Screening of strain Meyerozyma guilliermondii PJ15:

[0026] Meyerozyma guilliermondii PJ15, with the preservation number of GDMCC NO. 62572, is preserved in: Guangdong Provincial Culture Collection of Microorganisms, Address: 5th Floor, Building 59, No. 100 Compound, Middle Xianlie Road, Guangzhou, and the preservation date is June 28, 2022.

[0027] The above strain was isolated from the grape fruit epidermis in 2021.

[0028] The isolation method is as follows: Cut the grape skin sample into pieces, add 10 times the mass of sterile water and vortex for 20 min, and perform serial dilution of the sterile water by 10 -1 , 10 -2 , 10 -3 , 10 -4 . Take 100 μL of each gradient and spread it on Rose Bengal medium. Incubate in a constant temperature incubator at 30 °C for 1 - 3 days, and observe the colony growth. Use a sterile toothpick to pick the single colonies suspected to be yeast on the Rose Bengal medium with a colony morphology and inoculate them onto a PDA plate. Use Penicillium crustosum, the citrus fruit rot pathogen, as an indicator bacterium on the PDA plate for a plate confrontation experiment to preliminarily screen for yeasts that can antagonize molds. Incubate in a constant temperature incubator at 30 °C for about 1 month, observe the colony growth, and select the strains with mold inhibition to continue the plate confrontation experiment for re-screening.

[0029] The isolated strains were subjected to morphological classification and molecular biological identification, as follows:

[0030] 1. Morphological classification of the strain

[0031] Morphological classification of the strain:

[0032] As Figure 1 shown, the colony morphological characteristics of strain PJ15 on YPD medium are: round colonies with a white and smooth surface, with a bulge, and a regular edge. As Figure 2 shown, the strain is oval under the microscope. The width of the oval cells is about 1 - 5 μm, and the length is about 5 - 20 μm. Asexual reproduction is all budding.

[0033] 2. Molecular biological identification

[0034] The above strain was sequenced and verified: Universal primers were used to amplify the gene sequence of the strain, and the purified PCR product was sequenced. The ITS sequence of strain PJ15 was obtained through sequencing, specifically as shown in SEQ ID NO. 1. After comparison in the NCBI database, this sequence has 100% similarity with the strains of the species Meyerozyma guilliermondii. Therefore, it was identified as Meyerozyma guilliermondii.

[0035] Example 2:

[0036] This example mainly studies the confrontation effect of strain PJ15 against Penicillium crustosum:

[0037] 1. Confrontation effect against Botrytis cinerea:

[0038] ① Preparation of pathogen suspension: Inoculate the pathogen onto a PDA solid plate and incubate it statically at 30 °C for 2 - 3 d until fungal spores grow. Use an inoculation loop to scrape an appropriate amount of spores and transfer them to sterile water. Count the spores with a hemocytometer, and then adjust the final concentration to 10 6 spores / ml spore suspension for standby.

[0039] ② Inoculate the above pathogen suspension onto the PDA medium. In the control group, only the pathogen Penicillium crustosum was inoculated in the center; while in the experimental group, the pathogen was inoculated in the center of the PDA medium, and the corresponding screened strains: PJ14, PJ15, PJ16, and PJ18 were inoculated at its 4 ends.

[0040] The results are as Figure 3 shown: In the figure, the left side is the control group plate, and the right side is the experimental group plate. It can be seen that the size of the Penicillium crustosum colonies on the experimental group plate is significantly smaller than that of the control group. Thus, it shows that strains PJ14, PJ15, PJ16, and PJ18 may all have an inhibitory effect on Botrytis cinerea.

[0041] Example 3:

[0042] For the further verification of PJ14, PJ15, PJ16, and PJ18 screened in Example 2, whether the strains can prevent the decay of Wogan oranges.

[0043] Use an inoculation needle to pick up the hyphae of Penicillium crustosum and inoculate them on the epidermis of Wogan oranges. A total of 6 Wogan oranges are inoculated, with 3 being the experimental group and 3 being the control group. The control group and the experimental group are treated as follows:

[0044] Experimental group: Spray the bacterial suspension containing strain PJ15 on the epidermis of Wogan oranges, repeat the spraying every 24 h, the spraying amount is 78 μl, and the viable count of the bacterial suspension is 5×10 7 cfu / ml.

[0045] Control group: Spray distilled water on the epidermis of Wogan oranges, repeat the dripping every 24 h, and the dripping amount is 78 μl.

[0046] Cultivate the above Wogan oranges at room temperature, and continuously observe the decay situation of Wogan oranges from day 0 to day 21.

[0047] The experimental results are as follows Figure 4 shown in the figure. In the figure, ① is the decay situation of ponkan at 0 d, ② is the decay situation of ponkan at 7 d, ③ is the decay situation of ponkan at 14 d, and ④ is the decay situation of ponkan at 21 d. In each figure, the ponkans above are the control group, and the ponkans below are the experimental group. It can be clearly seen that the ponkans began to decay at 7 d, and the size of the bacterial plaque in the control group was significantly larger than that in the experimental group. Thus, it shows that the strain PJ15 of the present application has an inhibitory effect on the blue mold of ponkan.

[0048] Using the same experimental method, the strains PJ14, PJ16 and PJ18 were experimentally verified. Finally, it was found that: the strains PJ14 and PJ16 had no obvious anti-corrosion effect; while for the strain PJ18, a small amount of decay phenomenon began to appear at 21 d.

[0049] Thus, it shows that the strain PJ15 has a good effect of inhibiting the growth of Penicillium crustosum compared with other strains screened in the same batch, and can well preserve ponkan.

[0050] Example 4:

[0051] Prepare a preservative using the strain PJ15 for preserving ponkan.

[0052] Inoculate the strain PJ15 into YPD culture medium with an inoculation amount of 1%, culture for 24 h, after the culture is completed, perform centrifugal separation, take the liquid for sterile filtration, and then prepare a preservative solution by mixing it with water at a volume ratio of 1:1.

[0053] Spray the surface of the ponkan picked within 1 h (including 1 h) with the preservative solution, and then wrap it with a layer of plastic wrap to complete the preservation process.

[0054] It was found in actual preservation operations that spraying the surface of the ponkan with the preservative solution 1 h later had a much lower preservation effect than spraying within 1 h. Spraying the preservative solution within 1 h could extend the preservation period by about 15 d.

[0055] Spraying the preservative solution within 1 h extended the preservation days of the ponkan by about 45 d compared with not spraying the preservative solution, and the preservation effect was good.

[0056] Example 5:

[0057] This example studies the influence of different plant components on the preservation effect of the metabolites of the strain PJ15 on ponkan, specifically as follows:

[0058] Plant components: cactus, ponkan leaves, ponkan peel, ginger.

[0059] Step 1: Remove the thorns from the cactus, wash the ginger without peeling it, wash the leaves and peel of ponkan oranges, then mash them separately, add water according to a solid-liquid ratio of 1:4, boil, keep boiling for 30 min, cool to room temperature and filter, and take the filtrate to obtain the corresponding plant liquid.

[0060] Step 2: Based on the YPD liquid medium, add the corresponding plant liquid prepared in Step 1 to the YPD liquid medium at a volume percentage of 5%; use the group without adding the plant liquid as the control group, then inoculate the strain PJ15 into each group of raw materials at an inoculation amount of 1%, culture at room temperature for 48 h, after the culture is completed, centrifuge and separate, take the liquid for sterile filtration, and then prepare a fresh-keeping liquid by mixing it with water at a volume ratio of 1:2.

[0061] Step 3: Spray the surface of ponkan oranges picked within 1 h (including 1 h) with the fresh-keeping liquid prepared in Step 2, and use the spraying of an equal amount of distilled water as the blank group. Spray 3 ponkan oranges in each group. After spraying, do not wrap them with plastic wrap and expose them to the air. Observe the decay situation of the ponkan oranges, and record the average proportion of the surface decay of the ponkan oranges on the 20th day and 30th day, as shown in the following table:

[0062] Table 1 Effects of plant components on the preservation of ponkan oranges

[0063] Group Cactus group Wogan leaf group Wogan peel group Ginger group Control group Blank group Days with black spots The 15th day The 20th day The 17th day The 8th day The 15th day The 7th day Rotting ratio on the 20th day 13.0％ 1％ 3.0％ 98.2％ 24.1％ 100％ Rotting ratio on the 30th day 100％ 26.0％ 75.2％ 100％ 100％ 100％

[0064] As can be seen from the table, compared with the control group, the cactus + ginger group has no obvious advantage in the preservation of ponkan oranges. After adding ginger, the preservation effect of the fresh-keeping liquid is not much different from that of the blank group, and even worse than that of the control group without adding the plant liquid. The possible reason is that the preservation components of the biological preservative are generally polysaccharide components of microbial metabolism. After adding the plant liquid, due to the different components of the plant liquid, some plant liquids will promote the growth of microorganisms, and some will inhibit the growth of microorganisms. Different growth abilities of microorganisms will lead to different amounts of polysaccharide secretion, and ultimately affect the preservation effect of their metabolites. It can be seen that after the leaves and peel of ponkan oranges in the plant liquid stimulate the fermentation of the strain PJ15, the preservation effect of ponkan oranges is better, indicating that these two components have a certain promoting effect on the secretion of extracellular products of the strain PJ15. The cactus group is not much different from the control group, indicating that the cactus liquid has no effect on the secretion of extracellular products of the strain PJ15. The ginger group is worse than the control group and not much different from the blank group, indicating that the ginger group has a certain inhibitory effect on the secretion of extracellular products of the strain PJ15. This may be because ginger has an inhibitory effect on the growth of the strain PJ15.

[0065] Therefore, the applicant selects the leaves and peel of ponkan oranges as additives, adds them to the YPD liquid medium to prepare a fresh-keeping liquid for the preservation of ponkan oranges, specifically as follows:

[0066] Step 1: Wash the leaves and peels of Wogan oranges, then mash them separately, add water according to a solid-liquid ratio of 1:4, boil, keep boiling for 30 min, cool to room temperature and filter. Take the filtrate to obtain the corresponding plant liquid; then mix according to the ratio in the following table to get the plant liquid:

[0067] Table 2 Volume ratio of the plant liquid of Wogan orange leaves and the plant liquid of Wogan orange peels

[0068] Grouping Volume ratio Group 1 Wogan leaf:Wogan peel = 1:1 Group 2 Wogan leaf:Wogan peel = 1:2 Group 3 Wogan leaf:Wogan peel = 1:3 Group 4 Wogan leaf:Wogan peel = 2:1 Group 5 Wogan leaf:Wogan peel = 3:1

[0069] Step 2: Based on the YPD liquid medium, add the corresponding plant liquid prepared in Step 1 to the YPD liquid medium at a volume percentage of 5%; use the group without adding the plant liquid as the control group. Then inoculate the strain PJ15 into each group of raw materials at an inoculation amount of 1%, culture at room temperature for 48 h, after the culture ends, centrifuge and separate, take the liquid for sterile filtration, and then prepare a fresh-keeping liquid by mixing it with water at a volume ratio of 1:2.

[0070] Step 3: Spray the surface of the Wogan oranges picked within 1 h (including 1 h) with the fresh-keeping liquid prepared in Step 2, and use the spraying of an equal amount of distilled water as the blank group. Spray 3 Wogan oranges in each group. After spraying, do not wrap with plastic wrap and expose them to the air. Record the time when black spots start to appear on the epidermis of each group of Wogan oranges, as shown in the following table:

[0071] Table 3 Influence of different ratios of plant liquid on the preservation of Wogan oranges

[0072]

[0073] The above results show that the fresh-keeping liquid prepared with the additive of the ratio of Wogan orange leaves and Wogan orange peels can improve the fresh-keeping effect on Wogan oranges. Without wrapping with plastic wrap, the fresh-keeping time can be extended to about 20 d. The best fresh-keeping effect is in Group 3, that is, the volume ratio of Wogan orange leaf liquid to Wogan orange peel liquid is 1:3.

[0074] In summary, the Meyerozyma guilliermondii PJ15 strain of the present invention and its metabolites have an obvious inhibitory effect on Penicillium crustosum, and at the same time have a protective effect on the rot of Wogan oranges. The fresh-keeping agent prepared with this strain can significantly improve the fresh-keeping effect on Wogan oranges, and the fresh-keeping time can be extended by about 45 d, with good effects. It is an excellent strain widely applicable to plant protection and fruit fresh-keeping.

[0075] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Meyerozyma guilliermondii ( Meyerozyma guilliermondii ) PJ15, characterized in that The Meyerozyma guilliermondii ( Meyerozyma guilliermondii ) PJ15 has a deposit number of GDMCC NO.62572.

2. An antibacterial agent comprising the Meyerozyma guilliermondii ( Meyerozyma guilliermondii ) PJ15 as claimed in claim 1.

3. A preservative comprising the Meyerozyma guilliermondii ( Meyerozyma guilliermondii ) PJ15 as claimed in claim 1.

4. Use of the Meyerozyma guilliermondii ( Meyerozyma guilliermondii ) PJ15 as claimed in claim 1 or the antibacterial agent as claimed in claim 2 for inhibiting the growth of Penicillium crustosum ( Penicillium crustosum ).

5. Use of the Meyerozyma guilliermondii ( Meyerozyma guilliermondii ) PJ15 as claimed in claim 1, the antibacterial agent as claimed in claim 2 or the preservative as claimed in claim 3 for preserving Wogan oranges.

6. A method for preparing the preservative as claimed in claim 3, characterized in that The method is as follows: Obtain the fermentation product of the YPD liquid medium of Meyerozyma guilliermondii ( Meyerozyma guilliermondii ), filter it, and take the filtrate to obtain the preservative.

7. The method according to claim 6, characterized in that The YPD liquid medium is also added with ponkan peel liquid and / or ponkan leaf liquid.

8. The method according to claim 7, characterized in that The volume ratio of the ponkan leaf liquid to the ponkan peel liquid is 1:

3.

9. A method for preserving Wogan oranges by using the preservative as claimed in claim 3, characterized in that The method is as follows: Prepare a preservative solution by mixing a preservative and water, spray it on the surface of ponkans, and wrap them with a layer of plastic wrap.

10. The method according to claim 9, characterized in that The time for spraying the preservative is within 1 h after the ponkans are picked.

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