TWI732462B - Fermentation products from aureobasidium pullulans, methods of manufacturing the same, microbial agents for reducing fungal diseases, and methods of reducing fungal diseases of agricultural products - Google Patents

Abstract

A fermentation product fromAureobasidium pullulans is provided. The method of manufacturing the fermentation product fromAureobasidium pullulans includes fermenting and culturing at least oneAureobasidium pullulans by a medium to form a fermentation liquid. The composition of medium includes a yeast extract, a malt extract, a peptone, and a dextrose. A method of manufacturing the fermentation product fromAureobasidium pullulans , a microbial agent for reducing fungal diseases, and a method of reducing fungal diseases of agricultural products are also provided.

Description

Aureobasidium pullulans fermented product and preparation method thereof, microbial preparation for reducing fungal diseases and method for reducing fungal diseases of agricultural products

The present disclosure provides a fermented product of pullulan pullulan and a preparation method thereof. The present disclosure also provides a microbial composition. The present disclosure also provides a method for reducing fungal diseases of agricultural products.

Taiwan is located at the junction of tropical and subtropical, with a warm and humid climate. Agricultural products are susceptible to infection by postharvest fungal pathogens (postharvest fungal pathogens), which can cause agricultural products to rot during the process from harvest to distribution to sales markets and purchases by consumers. Bad. In order to avoid damage to agricultural products during the distribution process after harvest, disease prevention and prolonged preservation treatment after harvest are important issues.

At present, the main post-harvest disease prevention and prolonged preservation techniques can be divided into physical, chemical and microbiological methods. The physical antibacterial preservation method is to maintain low temperature during transportation to avoid microbial breeding. This method does not require the use of chemical substances, so it will not cause harm to the human body, but the investment cost of equipment and facilities is high, and it consumes a lot of electricity, which is difficult to popularize and apply. The chemical method uses chemical substances to achieve the antibacterial effect. Although there is no need to use equipment and therefore the price is low, and the effect of chemical substances is significant, but the chemical substances remain on agricultural products, there is a potential risk of toxicity after human consumption, which will cause Environmental and health issues. Compared with the above methods, the microbial method uses substances derived from microorganisms to achieve antibacterial purposes, such as nisin (Nisin), ε-polylysine (ε-Polylysine), and Natamycin (Natamycin) derived from microorganisms. ) Et al. (Xing et al., 2015, Liu et al., 2015). The microbiological law mainly appeals to nature and safety, and has gradually replaced physical or chemical methods. However, most microorganisms have a small antibacterial range and their control effects on pathogenic bacteria are limited.

Studies have pointed out that the mechanism of using microorganisms to prevent and control diseases after harvest of agricultural products can be classified into 1) nutritional competition of pathogens, 2) inducing host resistance to disease, or 3) microorganisms themselves secrete hydrolytic enzymes to decompose the cell walls of pathogens, thereby inhibiting the growth of pathogens (Davide and Samir, 2016). In addition, studies have pointed out that 1% Aureobasidium pullulans exopolysaccharide can reduce the weight loss and rot rate of nectarines, maintain the luster of the fruit, and achieve better preservation effects (Ouyang et al., 2010). However, there is currently no microbial antibacterial antistaling agent against common infectious fungal diseases in Taiwan, such as Penicillium, Colletotrichum, Botrytis.

Although the existing microorganisms generally meet the requirements, their single-prescription formulations have a narrow application range to pathogenic bacteria, so microorganisms that can be broadly effective in inhibiting bacteria are required.

The present disclosure provides a fermented product of Aureobasidium pullulans (Aureobasidium pullulans), wherein the method for forming the fermented product of Aureobasidium pullulans includes: performing at least one Aureobasidium pullulans with a culture medium. Fermentation is cultivated to form a fermented broth, wherein the medium components include: yeast extract; malt extract; digestive protein (peptone); and dextrose (dextrose).

The present disclosure also provides a microbial preparation for reducing fungal diseases, including the aforementioned fermented product of Aureobasidium pullulans, wherein the microbial composition for reducing fungal diseases has the effect of reducing fungal diseases caused by fungi.

The present disclosure also provides a preparation method of fermented product, which uses the fermented product of Aureobasidium pullulan as described above.

The present disclosure also provides a method for reducing fungal diseases of agricultural products by applying the aforementioned microbial preparation to an agricultural product.

This disclosure further provides a novel pullulans pullulans ( Aureobasidium pullulans ), which was deposited at the Bioresource Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018. Mold, its deposit number is BCRC 930198.

This disclosure also provides a novel pullulans pullulans ( Aureobasidium pullulans ), which was deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018. Mold, its deposit number is BCRC 930199.

In view of the above-mentioned issues, in this disclosure, from the Aureobasidium pullulans collected and preserved in Taiwan, a screening method for the antibacterial fermentation of Aureobasidium pullulans has been established to screen out the common plants with the potential for production of extracellular polysaccharides. Aureobasidium pullulan, and two strains of Aureobasidium pullulan with inhibitory fungi were screened out. As a broad-acting natural antibacterial microorganism, it overcomes the narrow application scope of single formulations to pathogenic bacteria faced by the current microbial preservation and antibacterial method. And the problem that the effect of disease control is limited.

In a first aspect of the present disclosure, the present disclosure provides a fermented product of Aureobasidium pullulans (Aureobasidium pullulans). The fermented product of Aureobasidium pullulans disclosed in the present disclosure has the effect of inhibiting the growth of fungi, but is not limited thereto.

The method for forming the above-mentioned fermented product of Aureobasidium pullulans may include, but is not limited to, subjecting Aureobasidium pullulans to a fermented culture in a medium to form a fermented broth to obtain the general fermented product. The fermented product of Aureobasidium lulanum.

The components of the above-mentioned medium may include yeast extract, malt extract, peptone and dextrose, but are not limited thereto.

The concentration of the yeast extract may be about 1-15 g/L, such as about 1-5 g/L, 1-8 g/L, 1.5-10 g/L, 2-6 g/L, but is not limited thereto. In one embodiment, it can be 3g/L.

The concentration of the malt extract may be about 1-15 g/L, such as 1-5 g/L, 1-8 g/L, 1.5-10 g/L, 2-6 g/L, but is not limited thereto. In one embodiment, it can be 3g/L.

The concentration of the digested protein may be about 1-20 g/L, for example, 1-15 g/L, 1-10 g/L, 2-18 g/L, 3-12 g/L, but is not limited thereto. In an embodiment, it may be 5 g/L.

The concentration of dextrose may be about 1-50 g/L, such as 5-30 g/L, 5-20 g/L, 3-20 g/L, 5-18 g/L, but is not limited thereto. In an embodiment, it may be 10 g/L.

The above fermentation temperature is not particularly limited as long as it is suitable for the growth of Aureobasidium pullulans, for example, between 15-30°C, such as 22-28°C, but not limited to this. In one embodiment, it can be 25°C.

There is also no particular limitation on the above-mentioned fermentation culture method, and it can be batch culture or continuous culture. In one embodiment, the ferment culture can be carried out by a culture tank.

The rotation speed of the above-mentioned fermentation tank for fermentation culture may be 100-200 rpm, but is not limited to this, and may be 150 rpm in one embodiment.

The culture time of the above fermented culture is 24-72 hours, but is not limited to this, and may be 48 hours in one embodiment.

Furthermore, in another embodiment, the aforementioned method of forming the fermented product of Aureobasidium pullulans may further include the step of inactivating the bacteria in the fermented broth after forming the fermented broth and/ Or remove the bacteria from the fermented liquid. The step of inactivating the bacteria in the fermented liquor includes but not limited to, for example, treating the fermented product with acid or alkali; the step of removing the bacteria from the fermented liquor, including but not limited to filtering the bacteria to obtain the filtrate, or It extracts the fermented product.

At least one example of the at least one pullulan pullulan used in the embodiment of the ferment product of pullulan pullulan used in the present disclosure may include the food deposited at the Republic of China Consortium on October 26, 2018 Aureobasidium pullulans from the Bioresource Collection and Research Center (BCRC) of the Industrial Development Institute, and its deposit number is BCRC 930198 (it was also deposited in Germany on November 13, 2018. Aureobasidium pullulans from the preservation center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), deposit number is DSM 32955), Aureobasidium pullulans with deposit number BCRC 930199 (which is also on November 13, 2018 Aureobasidium pullulans deposited daily at the German Center for Microorganism and Cell Preservation (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), deposit number is DSM 32956), etc., or any combination of the above, but not limited to this. In one embodiment, the aforementioned Aureobasidium pullulan is a Plucol which was deposited with the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute of the Republic of China on October 26, 2018. Aureobasidium pullulans, its deposit number is BCRC 930198. In another embodiment, the aforementioned Aureobasidium pullulan was deposited with the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute of the Republic of China on October 26, 2018. Aureobasidium pullulans, its deposit number is BCRC 930199. In yet another embodiment, the aforementioned Aureobasidium pullulan is a combination of BCRC 930198 and BCRC 930199.

The above-mentioned fermented product of Aureobasidium pullulans in the present disclosure, because the fermented product of Aureobasidium pullulans disclosed in the present disclosure has the effect of inhibiting the growth of fungi, it can be applied to the purpose of reducing fungal diseases, such as inhibiting agricultural products Fungal disease.

The above-mentioned fungal diseases include, but are not limited to, Penicillium, Colletotrichum, Botrytis, Wilt and Soft Rot, Rhizopus nigricans, and Tomato Early Blight.

The above fungal fungal diseases including but not limited to Penicillium (Penicillium) fungi, the genus Colletotrichum anthrax (Colletotrichum) fungi, the genus Botrytis cinerea (Botrytis-) fungi, soft rot withering genus (of Fusarium) fungi, Rhizopus black ( Rhizopus fungi or Alternaria fungi, etc.

The aforementioned Penicillium fungi may include, but are not limited to, Penicillium digitatum , Penicillium expansum , Penicillium crustosum , and Penicillium italicum .

The aforementioned Colletotrichum gloeosporioides may include, but is not limited to, Colletotrichum gloeosporioides.

The aforementioned Botrytis genus may include but is not limited to Botrytis cinerea .

The genus Fusarium incarnatum-equiseti can include but is not limited to Fusarium incarnatum-equiseti and Fusarium oxysporum f. sp. Momordicae.

The aforementioned Rhizopus nigricans may include, but are not limited to, Rhizopus stolonifer.

The aforementioned Alternaria genus may include, but is not limited to, Alternaria alternata .

In a second aspect of the present disclosure, the present disclosure also provides a microbial preparation comprising any of the fermented products of Aureobasidium pullulans of the present disclosure. The microbial preparation has the effect of reducing fungal diseases caused by fungi.

In one embodiment, in addition to the fermented product of Aureobasidium pullulan, the microbial preparation may further include a diluent. In the microbial preparation disclosed in the present disclosure, the content of the diluent is not particularly limited, and can be determined according to requirements. Examples of the diluent may include water, physiological saline, organic solvents, etc., but are not limited thereto.

In one embodiment, the microbial preparation does not contain preservatives. Since it does not contain preservatives harmful to the human body, this microbial preparation can be used as a fungal antibacterial agent that does not cause harm to the human body.

In an embodiment, the content of the fermented product of Aureobasidium pullulan in the microbial preparation may be 1-100% by weight, for example, about 5-99% by weight, or about 10-95% by weight, but is not limited thereto.

The microbial preparation can be in liquid or powder form. When using in liquid form, the fermented product of Aureobasidium pullulan can be used directly or diluted with a diluent. In powder form, the fermented product of pullulan pullulan can be dried into powder form, and then diluted with diluent to the required pH concentration when it is to be used.

In a third embodiment of the present disclosure, the present disclosure also provides a method for reducing fungal damage of agricultural products. This method may include applying any of the microbial fermentation products of the present disclosure to agricultural products. There is no restriction on the method of application, as long as the microbial fermented product disclosed in the present disclosure is fully contacted with the agricultural product.

In one embodiment, the application is to immerse agricultural products in the microbial preparation prepared by the fermented product of pullulan pullulan and the diluent. The immersion time may be 1-60 minutes, such as 20-40 minutes, but not Limited to this. In another embodiment, after soaking, after the agricultural product is taken out, the agricultural product can be further left to stand still, so that the microorganisms are formed on the surface of the agricultural product to form a protective film. The time of standing can be determined according to needs, for example, can be adjusted according to the current temperature and humidity, for example, it can be about 2 hours, 1 hour, 30 minutes, 10 minutes, but is not limited to this. In one embodiment, the standing time may be 30 minutes.

The aforementioned agricultural products include vegetables and/or fruits. Vegetables include roots and stems and leafy vegetables. Root vegetables may include, but are not limited to, carrots, white radishes, potatoes, taro, sweet potatoes, yams, and the like. Leafy vegetables may include, but are not limited to, cabbage, lettuce, lettuce, and the like. Vegetables can also include pumpkin. Fruits may include, but are not limited to, mango, lotus mist, banana, strawberry, lychee, date, plum, citrus, apple, pear, peach, etc.

In the fourth implementation aspect of this disclosure, this disclosure also provides a novel pullulan pullulan, which was deposited at the Bioresource Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018 (Bioresource Collection and Research Center; BCRC) Aureobasidium pullulans, its deposit number is BCRC 930198. The ITS1-5.8S-ITS2 sequence of Aureobasidium pullulan BCRC 930198 is sequence identification number: 1.

In the fifth implementation aspect of this disclosure, this disclosure also provides a novel Aureobasidium pullulan, which was deposited with the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018. Bioresource Collection and Research Center; BCRC) Aureobasidium pullulan, its deposit number is BCRC 930199. The ITS1-5.8S-ITS2 sequence of Aureobasidium pullulan BCRC 930199 is sequence identification number: 2.

The present disclosure provides a microbial composition for preventing and curing diseases after fruit harvest. It is mainly produced by Aureobasidium pullulans and its metabolite fermented raw materials, which has antibacterial effect on common fungal diseases of fruits after harvest. Including Penicillium, Colletotrichum, Botrytis, Blight and Soft Rot and Rhizopus nigricans. Compared with the current common chemical preservatives, the strain of the present invention has a short fermentation cycle, simple preparation procedures, natural and wide-ranging effects, reduces fungal diseases of agricultural products, and is suitable for agricultural products preservation.

In order to make the above and other purposes, features, and advantages of this disclosure more obvious and understandable, the following examples are given to illustrate the pullulan pullulan ferment product and microbial composition described in this disclosure, as well as the reduction of agricultural products. Methods of fungal disease and Aureobasidium pullulan.

[Example]

[Screening of potential strains of Aureobasidium pullulan] From the strain bank of Aureobasidium pullulan sampled and preserved in Taiwan, the potential strains with antibacterial ability were screened. First, a pullulan pullulan glycerol tube was inoculated into a liquid medium with 1% of the inoculation amount. The composition of the liquid medium was deionized water containing 3g/L yeast extract, 3g/L malt extract, and 5g/L Digest protein (Peptone) and 10g/L dextrose (dextrose). Then, it was cultured for 48 hours at 25° C., rotation speed 150 rpm, and Aureobasidium pullulans fermentation product was obtained. Use this pullulan pullulan ferment product for antibacterial activity test.

[Antibacterial activity test] Antibacterial test methods, the more commonly used are the Kirby-Bauer disk-diffusion method and the dilution method. The dilution method can be divided into broth dilution and agar. Two kinds of ester dilution method (Agar dilution). From the results of the previous test example of the paper ingot diffusion method, it is known that the fermented product of pullulan pullulan contains higher extracellular polysaccharides and the viscosity is higher than that of the general water-soluble sample. Therefore, the antibacterial contained in the fermented product The ingredients are less likely to diffuse into the culture medium. Therefore, the subsequent test uses the agar dilution method as the detection method for the antibacterial activity test.

Firstly, culture was carried out according to the best conditions for the growth of pathogenic fungi. Different ratios of pullulan pullulan fermented product samples were combined with malt extract agar (MEA) or potato dextrose agar (potato dextrose agar). dextrose agar (PDA) medium is mixed evenly and then poured into a petri dish and allowed to stand. When the medium is solidified, agar containing pullulan pullulan fermented product is prepared. Then, the mold growing on the plate was cut into a size of about 0.5x0.5cm ² with a blade, and the mold hyphae block was placed on the prepared agar center surface containing the pullulan pullulan fermented product. Observe the mycelial growth after 7 days of culture at 24~26 ℃, and compare the results of the blank control group to determine the minimum inhibitory concentration (MIC).

Based on the above-mentioned antibacterial activity test, a total of 6 potential β-glucan producing strains were screened, among which 2 strains of Aureobasidium pullulan with the best efficacy were selected, and their codes were 08F0543 and 08F0544 as the follow-up antibacterial experiments. Strains.

Aureobasidium pullulan 08F0543 and 08F0544 were subjected to rDNA-internal transcribed spacer (ITS) ITS1-5.8S-ITS2 sequence fragments for sequencing analysis. The ITS1-5.8S-ITS2 sequence fragment of 08F0543 is the sequence identification number: 1 (for the complete sequence, please refer to the sequence identification number of the sequence table: 1); the ITS1-5.8S-ITS2 sequence fragment of 08F0544 is the sequence identification number: 2 (the complete sequence Please refer to the sequence identification number of the sequence table: 2). The comparison results of the two show that they are compatible with Aureobasidium pullulans var. melanogenum BCRC 34543T (=ATCC 12536 ^T =CBS 105.22T) and common Aureobasidium pullulans var. melanogenum. Aureobasidium pullulans NRRL Y-12996 (=ATCC 42023) is the closest. Sequence ID: 1 and Sequence ID: 2 Compared with the sequence of Aureobasidium pullulan NRRL Y-12996 (=ATCC 42023) (GenBank accession no. HQ702508), the 59-542 nucleotides are the same. This shows that 08F0543 and 08F0544 belong to Aureobasidium pullulans; however, there are 58 nucleotides more at the 3'end and 80 nucleotides more at the 5'end (78% similarity (484/622)). From the above, it can be seen that 08F0543 and 08F0544 are novel strains of Aureobasidium pullulan. According to the reference Zalar et al. (2008), Aureobasidium pulllulans strains 08F0543 and 08F0544 were identified as Aureobasidium pulllulans var. melanogenum which belonged to Aureobasidium pulllulans var. melanogenum. And these two strains were deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute of the Republic of China on October 26, 2018. The deposit number of 08F0543 is BCRC 930198 (which is also It was deposited with the German Center for Microbiology and Cell Preservation (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ, deposit number is DSM 32955) on November 13, 2018. The deposit number of 08F0544 is BCRC 930199 of Aureobasidium pullulans (its also It was deposited at the German Center for Microbiology and Cell Preservation (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ, with the deposit number DSM 32956) on November 13, 2018.

The two Aureobasidium pullulans strains (accession numbers BCRC 930198, BCRC 930199) and the known Aureobasidium pullulans strain BCRC 930184 were analyzed. The results are shown in Table 1.

[Table 1] Strains BCRC 930198 BCRC 930199 BCRC 930184 Separate source Bamboo Leaf (Hsinchu/Xinpu Town) Bamboo Leaf (Hsinchu/Xinpu Town) blade Melanin production capacity Not much different from other pullulan Aureobasidium pullulans Not much different from other pullulan Aureobasidium pullulans Low melanin production Strain characteristics Significant antibacterial ability Significant antibacterial ability High yield of β-glucan

Aureobasidium pullulan strains BCRC 930198 and BCRC 930199 have little difference in melanin production ability from other Aureobasidium pullulans, while Aureobasidium pullulan strain BCRC 930184 is a strain with low melanin production. In addition, the fermentation products of Aureobasidium pullulan strains BCRC 930198 and BCRC 930199 have significant bacteriostatic ability, while Aureobasidium pullulan strain BCRC 930184 is a strain with high β-glucan production. It can also be understood from the above physiological and biochemical characteristics that the Aureobasidium pullulan strains BCRC 930198, BCRC 930199 and BCRC 930184 selected by the aforementioned antibacterial activity test are different Aureobasidium pullulans.

Aureobasidium pullulans strain BCRC 930198 was inoculated on malt extract agar and potato dextrose agar for morphological observation. Malt extract agar, cultured at 25°C for 7 days, the colony is smooth and viscous, the center of the colony is slimy, the color is dark red to reddish brown (9B3-E5), the new part around the colony is white (Figure 2C); potato Dextrose agar, cultured at 25°C for 7 days, the colonies are dark red to reddish brown (9B3-D4), the colonies will be gray-yellow due to the production of crystals (4B4-C7), and the new parts around the colonies are white (Figure 2A) , Figure 2B). Observed under an optical microscope, it has transparent and brown to dark-brown hyphae. The transparent hyphae are smooth, thin-walled, and have transverse septa, which will gradually thicken as the culture time increases; brown hyphae, septa, and dark brown thick-walled spores can be seen (chlamydospores), the shape is nearly spherical, cylindrical to irregular; conidiogenous cells (conidiogenous cells) are undifferentiated, located in the middle or end of the transparent hyphae, with small teeth (denticle); with transparent and dark brown conidia ( conidia), 1-2 cells, slightly indented at the diaphragm, with large changes in shape and size. Lateral budding is common. The size of single-celled conidia is 4.7-12.9 × 2.3-5.2 μm.

Aureobasidium pullulans strain BCRC 930199 was inoculated on malt extract agar and potato dextrose agar for morphological observation. Malt extract agar, cultured at 25°C for 7 days, the colony is smooth and viscous, the center of the colony is slimy, the color is dark red to reddish brown (9B3-E5), the new part around the colony is white (Figure 3C); potato Dextrose agar, cultured at 25°C for 7 days, the colonies are dark red to reddish brown (9B3-D4), the colonies will be gray-yellow due to the production of crystals (4B4-C7), and the new parts around the colonies are white (Figure 3A) , Figure 3B). Observed under an optical microscope, it has transparent and brown to dark-brown hyphae. The transparent hyphae are smooth, thin-walled, and have transverse septa, which will gradually thicken as the culture time increases; brown hyphae, septa, and dark brown thick-walled spores can be seen (chlamydospores), the shape is nearly spherical, cylindrical to irregular; conidiogenous cells (conidiogenous cells) are undifferentiated, located in the middle or end of the transparent hyphae, with small teeth (denticle); with transparent and dark brown conidia ( conidia), 1-2 cells, slightly indented at the diaphragm, with large changes in shape and size. Lateral budding is common, and the size of single-celled conidia is 5.2-9.3 × 2.0-4.8 μm.

[Example 1] The fermentation products of Aureobasidium pullulans selected from two selected strains of Aureobasidium pullulan (Accession Nos. BCRC 930198, BCRC 930199) were used to test the antibacterial activity, and the results are shown in the table 3. The preparation method of the fermented product is to inoculate the pullulan pullulan glycerol tube with 1% of the inoculation amount into the liquid medium. The liquid medium is composed of deionized water containing 3g/L yeast extract and 3g/L malt extract Food, 5g/L digested protein (Peptone) and 10g/L dextrose (dextrose). Then cultured for 48 hours at 25°C, 150 rpm, and 48 hours to obtain Aureobasidium pullulans fermented products. From the Aureobasidium pullulans fermented products, 5%, 12.5% and 25% (volume percentage, v/v) %) of fermentation broth-agar medium. During the inoculation, separate cultivation of Aureobasidium pullulan BCRC 930198 and BCRC 930199 and a co-cultivation method of mixing BCRC 930198 and BCRC 930199 in a volume ratio of 1:1 were used to carry out the fermentation product obtained. Follow-up experiment. Next, the mold growing on the plate was cut into a size of about 0.5x0.5cm ² with a blade, and the mold hyphae block was placed on the central surface of the prepared culture medium containing pullulan pullulan fermented product, and the value was 24~26 Observe the results of mycelial growth after 7 days of culture at ℃, and compare the results of the blank control group to determine the minimum inhibitory concentration (MIC). The results are shown in Table 3. X means no bacteriostasis; △ means the culture medium has changed color; ○ means complete bacteriostasis.

The fungal pathogens tested included Penicillium (Penicillium digitatum BCRC30820, Penicillium digitatum BCRC32577, Penicillium expanded BCRC33503, Penicillium spp. BCRC32045, Penicillium italy BCRC32575), Colletotrichum anthracis (Colletotrichum gloeosporioides) Colletotrichum sp. BCRC35178), Botrytis cinerea (Botrytis cinerea BCRC35340), Soft rot fungus (Fusarium BCRC35290, Bitter gourd blight BCRC35046), Rhizopus nigricans (Rhizopus stolonifer BCRC35134), and Alternaria alternata (Alternaria alternata) BCRC35015). The above-mentioned test pathogens were obtained from the Biological Resources Conservation and Research Center of the Food Industry Development Institute. The information of the strains and their growth conditions are shown in Table 2.

[Table 2] BCRC number Strains Oxygen demand Growth temperature Culture medium 30820 Penicillium digitatum Aerobic 25°C Malt Extract Agar (MEA) 32577 Penicillium digitatum Aerobic 25°C Malt Extract Agar (MEA) 33503 Penicillium expanding Aerobic 26°C Malt Extract Agar (MEA) 32045 Penicillium verticillata Aerobic 24°C Malt Extract Agar (MEA) 32575 Penicillium italy Aerobic 25°C Malt Extract Agar (MEA) 35178 Colletotrichum gloeosporioides Aerobic 24°C Potato Dextrose Agar (PDA) 35340 Botrytis cinerea Aerobic 24°C Potato Dextrose Agar (PDA) 35290 Fusarium Aerobic 24°C Potato Dextrose Agar (PDA) 35046 Momordica charantia Aerobic 24°C Potato Dextrose Agar (PDA) 35134 Rhizopus stolonifer Aerobic 24°C Potato Dextrose Agar (PDA) 35015 Alternaria alternata Aerobic 24°C Potato Dextrose Agar (PDA)

[table 3] BCRC number Strains Aureobasidium pullulans BCRC 930198 sample concentration (v/v%) Aureobasidium pullulan BCRC 930199 sample concentration (v/v%) Aureobasidium pullulan BCRC 930198 + BCRC 930199 sample concentration (v/v%) 5% 12.5% 25% 5% 12.5% 25% 5% 12.5% 25% 30820 Penicillium digitatum △ ○ ○ △ ○ ○ △ ○ ○ 32577 Penicillium digitatum △ ○ ○ △ ○ ○ △ ○ ○ 33503 Penicillium expanding △ ○ ○ △ ○ ○ △ ○ ○ 32045 Penicillium verticillata △ ○ ○ △ ○ ○ △ ○ ○ 32575 Penicillium italy △ ○ ○ △ ○ ○ △ ○ ○ 35178 Colletotrichum gloeosporioides △ ○ ○ △ ○ ○ △ ○ ○ 35340 Botrytis cinerea x x ○ x ○ ○ x ○ ○ 35290 Fusarium △ ○ ○ x ○ ○ △ ○ ○ 35046 Momordica charantia x x x x x x x x x 35134 Rhizopus stolonifer x ○ ○ x ○ ○ x ○ ○ 35015 Alternaria alternata x ○ ○ x ○ ○ x ○ ○

From the results in Table 3, it can be seen that for the pathogenic bacteria of Penicillium (Penicillium digitatum BCRC30820, Penicillium digitatum BCRC32577, Penicillium digitatum BCRC33503, Penicillium spp. BCRC32045, Penicillium italy BCRC32575), 12.5% of pullulan short Aureobasidium BCRC 930198, BCRC 930199 and co-cultured fermentation broth-agar medium of BCRC 930198 and BCRC 930199 have antibacterial effects on Penicillium, with the lowest inhibitory concentration of 12.5%.

For the pathogen of Colletotrichum anthracis (Colletotrichum gloeosporioides BCRC35178), 12.5% of Aureobasidium pullulans BCRC 930198, BCRC 930199 and co-cultivation of both broth-agar medium The spore pathogen has a bacteriostatic effect, and the minimum inhibitory concentration is 12.5%.

Against the pathogen of Botrytis cinerea (Cinerea Botrytis BCRC35340), 25% of Aureobasidium pullulans BCRC 930198 fermentation broth-agar medium has the original antibacterial effect of Botrytis cinerea, the minimum inhibitory concentration is 25%; 12.5% and 25% Aureobasidium pullulans BCRC 930199 and co-cultured fermentation broth-agar medium of BCRC 930198 and BCRC 930199 have the original antibacterial effect on Botrytis cinerea, the minimum inhibitory concentration is 12.5%; 12.5% Aureobasidium pullulans BCRC 930199's fermentation broth-agar medium has the original antibacterial effect on Botrytis cinerea, and the minimum inhibitory concentration is 12.5%.

Against the withering soft rot pathogen (Fusarium BCRC35290), 12.5% pullulan Aureobasidium pullulans BCRC 930198, BCRC 930199 and co-cultivation of the two fermentation broth-agar medium has antibacterial effect on the pathogen of Colletotrichum anthracis, with the lowest inhibition The concentration is 12.5%. However, for bitter gourd blight bacteria BCRC35046, Aureobasidium pullulans BCRC 930198, BCRC 930199 and the co-cultivation broth-agar medium of the two have no antibacterial effect.

Against the pathogen of Rhizopus nigricans (Rhizopus stolonifer BCRC35134), 12.5% Aureobasidium pullulans BCRC 930198, BCRC 930199 and co-cultivation of both fermentation broth-agar medium have antibacterial effects on the pathogen of Colletotrichum anthracis , The minimum inhibitory concentration is 12.5%.

Against Alternaria alternata (Alternaria alternata BCRC35015), 12.5% Aureobasidium pullulans BCRC 930198, BCRC 930199 and co-cultivation of the two fermentation broth-agar medium have antibacterial effect on Alternaria alternata , The minimum inhibitory concentration is 12.5%.

From the above results, it can be seen that the fermentation broth-agar medium of Aureobasidium pullulans BCRC 930198 and BCRC 930199 are respectively suitable for penicillium, Colletotrichum anthracis, Botrytis cinerea, withering soft rot pathogen, Rhizopus niger pathogen and streptomyces. Spores and other fungi have antibacterial effects and are a wide range of fungal antibacterial agents.

[Example 2] Using the selected two strains of Aureobasidium pullulans (accession numbers BCRC 930198, BCRC 930199) of Aureobasidium pullulans fermentation products of Aureobasidium pullulans were prepared in the same manner as in Example 1. The fermented product, a 0.01-25% series diluted fermented broth-agar medium is prepared from the pullulan pullulan fermented product. The pathogen of Colletotrichum anthracis (Colletotrichum gloeosporioides BCRC35178) was used as the test strain, and the bacteriostatic test was carried out in the same way as in Example 1. The results are shown in Table 4, X means no bacteriostasis; ○ Indicates complete bacteriostasis.

[Table 4] Concentration of fermented product (v/v%) Aureobasidium pullulans BCRC 930198 antibacterial results Aureobasidium pullulan BCRC 930199 antibacterial results 0.05 x x 0.25 x x 0.5 x x 1.25 x x 2.5 x ○ 5 x ○ 7.5 ○ ○ 10 ○ ○ 12.5 ○ ○ 25 ○ ○

From the results in Table 4, it can be seen that the minimum inhibitory concentration of the fermentation product of Aureobasidium pullulan BCRC 930198 is 7.5%, and the minimum inhibitory concentration of the fermentation product of Aureobasidium pullulan BCRC 930199 is 2.5%. From the above results, it can be seen that the fermentation products of Aureobasidium pullulans BCRC 930198 and BCRC 930199 can inhibit the growth of fungi at low concentrations.

[Example 3] The fermentation products of Aureobasidium pullulans BCRC 930198 and BCRC 930199 prepared in Example 1 were prepared into a mixture at a volume ratio of 1:1, and then alkali treatment was performed to inactivate the bacteria to obtain the fermentation metabolites. Specifically, sodium hydroxide (NaOH) is added to the fermented product so that the final concentration of sodium hydroxide is 500 mM. Using the above-mentioned fermented metabolites to inactivate the bacteria, prepare 25%, 50% and 75% fermented metabolites-agar medium for antibacterial test. The results are shown in Table 5, X means no bacteriostasis; ○ means complete bacteriostasis.

[table 5] BCRC number Strains Pullulan pullulan fermentation metabolite BCRC 930198+BCRC 930199 (v/v%) 25% 50% 75% 30820 Penicillium digitatum x x x 32577 Penicillium digitatum x ○ ○ 33503 Penicillium expanding x x x 32045 Penicillium verticillata x x x 32575 Penicillium italy x x ○ 35178 Colletotrichum gloeosporioides x x ○ 35340 Botrytis cinerea x x x 35290 Fusarium x x x 35046 Momordica charantia x x x 35134 Rhizopus stolonifer x ○ ○ 35015 Alternaria alternata x x x

The antibacterial results of Penicillium digitatum BCRC30820, Penicillium expanded BCRC33503, and Penicillium sticky-shaped BCRC32045. As the concentration of Aureobasidium pullulans fermented metabolites increases, the mycelial growth diameter range of the pathogenic fungus in the center of the plate Significantly smaller. The inhibitory concentration of the fermentation metabolite of Aureobasidium pullulan against Penicillium digitatum BCRC32577 is 50%; for Penicillium spp. BCRC32575, the inhibitory concentration is 75%.

The inhibitory results against Botrytis cinerea BCRC35340, the pathogen of Botrytis cinerea, and Bitter Melon BCRC35046, Fusarium BCRC35290, and Alternaria alternata BCRC35015, which is the pathogen of Botrytis cinerea, along with the fermentation metabolites of Aureobasidium pullulans The increase of the concentration, the smaller the diameter range of the pathogenic fungus growth in the center of the plate.

In response to the inhibition results of Rhizopus nigricans pathogen Rhizopus stolonifer BCRC35134, the inhibitory concentration of Aureobasidium pullulan fermentation metabolites for this bacteria was 50%.

In response to the inhibition results of Colletotrichum gloeosporioides BCRC35178, the pathogen of Colletotrichum gloeosporioides, the inhibitory concentration of the fermentation metabolite of Aureobasidium pullulan against this fungus was 75%.

It can be seen from the above results that even if the bacteria in the fermentation product of Aureobasidium pullulans are inactivated and the fermentation metabolites are retained, the antibacterial components in it also have an inhibitory effect on fungal pathogens. Therefore, it can be inferred that the antibacterial mechanism comes from The secondary metabolite of pullulan pullulan ferment.

[Example 4]

Colletotrichum gloeosporioides Colletotrichum is one of the main infectious diseases of Taiwan fruits. Test the growth inhibitory effect of pullulan pullulan ferment products on Colletotrichum gloeosporioides. The pullulan pullulan ferment product of Example 3 was diluted with distilled water to 25%, 50%, 75%, and 100%, respectively. Using Yatomin (23% water suspension) (manufacturer: Guanghua Chemical) as the positive control group, the mangoes were soaked in the solutions of each group for 1 minute, and then taken out and left to stand for 60 minutes. The negative control group is not soaked in any solution. Then, cut a 1.6-1.7mm wound on the mango epidermis with a 3ml syringe, and inoculate Colletotrichum gloeosporioides with Colletotrichum gloeosporioides (apply 5μL bacterial solution/particle, bacterial solution concentration of 7.1x10 ⁶ CFU/ml). Placed at 13˚C for 15 days, the diameters of black spots formed on the mango epidermis on the 4th, 6th, 8th, 11th and 15th days were measured. The results are shown in Figure 1. In addition, the results of observing the mango skin on the 13th day showed that the incidence of soaked pullulan pullulan ferment products was >30%.

From the results in Figure 1, it can be seen that the diameter of the fruit lesions on the 15th day was reduced by 50-80% compared with the control group, which can achieve the same effect as the pesticide treatment. However, the pullulan pullulan ferment product disclosed in the present disclosure does not contain preservatives and is a natural fungal inhibitor.

Although the present invention has been disclosed in several preferred embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the art can make any changes without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

no.

Figure 1 is a graph showing the effect of the pullulan pullulan ferment product on inhibiting the formation of dark spots on mango epidermis according to an embodiment. Figure 2A-Figure 2B shows the colony front of Aureobasidium pullulans strain BCRC 930198 cultivated on potato dextrose agar at 25°C for 7 days. Figure 2C shows the colony front of Aureobasidium pullulans strain BCRC 930198 cultivated with malt extract agar at 25°C for 7 days. Figure 2D-Figure 2H shows the sporulation structure and conidia microstructure of Aureobasidium pullulans strain BCRC 930198. The thick black line in the figure is a scale bar, representing 10 μm. Figure 2I shows the microstructure of the crystal of Aureobasidium pullulans strain BCRC 930198. The thick black line in the figure is a scale bar, representing 10 μm. Figure 2J-Figure 2L shows the microstructure of the conidia of Aureobasidium pullulans strain BCRC 930198. The thick black line in the figure is a scale bar, representing 10 μm. Figure 3A-Figure 3B shows the colony front of Aureobasidium pullulan strain BCRC 930199 cultivated on potato dextrose agar at 25°C for 7 days. Figure 3C shows the colony front of Aureobasidium pullulans strain BCRC 930199 cultivated with malt extract agar at 25°C for 7 days. Figure 3D-Figure 3F shows the sporulation structure and conidia microstructure of Aureobasidium pullulans strain BCRC 930199. The thick black line is the scale bar, representing 10 μm. Figure 3G shows the microstructure of the crystal of Aureobasidium pullulans strain BCRC 930199. The thick black line is the scale bar, representing 10 μm. Figure 3H-Figure 3J shows the microstructure of the conidia of Aureobasidium pullulans strain BCRC 930199. The thick black line is the scale bar, representing 10 μm.

[Domestic hosting information] 1. Aureobasidium pullulan 08F0543 Biological Resources Conservation and Research Center, Food Industry Development Institute, Republic of China Foundation October 26, 1987 The registration number is BCRC 930198 2. Aureobasidium pullulan 08F0544 Biological Resources Conservation and Research Center, Food Industry Development Institute, Republic of China Foundation October 26, 1987 BCRC 930199

Claims (21)

A fermented product of Aureobasidium pullulans (Aureobasidium pullulans), wherein the method for forming the fermented product of Aureobasidium pullulans comprises: fermenting at least one Aureobasidium pullulans with a medium Cultured to form a fermented broth, wherein the medium components include: yeast extract; malt extract; digestive protein (peptone); and dextrose (dextrose), the pullulan pullulan is in 2018 Aureobasidium pullulans deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Research Institute of the Republic of China on October 26, and its deposit number is BCRC 930198 and/or in 2018 Aureobasidium pullulans deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Research Institute of the Republic of China on October 26, and its deposit number is BCRC 930199. The fermented product of Aureobasidium pullulans as described in item 1 of the scope of the patent application, wherein the content of the yeast extract is about 1 g/L-15 g/L. The fermented product of pullulan pullulan as described in item 1 of the scope of the patent application, wherein the content of the malt extract is about 1 g/L-15 g/L. The fermented product of Aureobasidium pullulans as described in item 1 of the scope of the patent application, wherein the content of the digested protein is about 1 g/L-20 g/L. The fermented product of Aureobasidium pullulans described in item 1 of the scope of the patent application, wherein the dextrose content is about 1 g/L-50 g/L. The fermented product of Aureobasidium pullulans as described in item 1 of the scope of the patent application, wherein the fermented culture temperature is about 15-30°C. The fermented fermentation product of Aureobasidium pullulans described in item 1 of the scope of the patent application, wherein the culture time of the fermented culture is about 24-72 hours. The fermented product of Aureobasidium pullulans described in item 1 of the patent application, wherein the method for forming the fermented product of Aureobasidium pullulans further includes inactivating the bacteria in the fermented liquid. A microbial preparation for reducing fungal diseases, comprising: the fermented product of Aureobasidium pullulan as described in item 1 of the scope of patent application, wherein the Aureobasidium pullulan was deposited in China on October 26, 2018 Aureobasidium pullulans from the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China, its deposit number is BCRC 930198 and/or it was deposited at the Food Industry Development Institute of the Republic of China on October 26, 2018. The Aureobasidium pullulan of the Resource Conservation and Research Center has a deposit number of BCRC 930199, in which the microbial composition for reducing fungal diseases has the effect of reducing fungal diseases caused by fungi. The microbial preparation for reducing fungal diseases as described in item 9 of the scope of patent application, wherein the fungi include Penicillium, Colletotrichum anthracis, Botrytis cinerea, Fusarium wilt pathogen, Rhizopus nigricans, and Alternaria spp. The microbial preparation for reducing fungal diseases as described in item 9 of the scope of the patent application, wherein the fungal diseases include penicillium, anthracnose trichomes, gray mold, wilting soft rot, rhizopus nigricans and tomato early blight. As described in item 9 of the scope of patent application, the microbial preparation for reducing fungal diseases further includes a diluent including water, physiological saline, and an organic solvent. The microbial preparation for reducing fungal diseases as described in item 9 of the scope of patent application, wherein the microbial composition does not contain preservatives. The microbial preparation for reducing fungal diseases as described in item 9 of the scope of patent application, wherein the proportion of the fermented product of Aureobasidium pullulan is 1-100% by weight. A method for preparing a fermented product, comprising subjecting at least one pullulan pullulan to a fermented culture in a medium to form a fermented broth, wherein the medium components include: yeast extract; malt extract; Digestive protein (peptone); and dextrose (dextrose), the pullulan pullulan was deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018 Aureobasidium pullulans, its deposit number is BCRC 930198 and/or Aureobasidium pullulans deposited at the Bioresource Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018, and its deposit number is BCRC 930199. A method for reducing fungal diseases of agricultural products, comprising: applying the microbial preparation described in item 9 of the scope of patent application to an agricultural product, wherein the agricultural product includes vegetables and/or fruits. The method for reducing fungal diseases of agricultural products as described in item 16 of the scope of patent application, wherein the application is as soaking, and the soaking time is 1-60 minutes. The method for reducing fungal diseases of agricultural products as described in item 16 of the scope of the patent application further includes the steps of taking out the agricultural product after soaking the pullulan pullulan fermented product, and leaving it to stand. The method for reducing fungal diseases of agricultural products as described in item 16 of the scope of the patent application, wherein the fungal diseases include penicillium, anthracnose trichomes, gray mold, wilting soft rot and rhizopus nigricans. A novel pullulan pullulan (Aureobasidium pullulan), which is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018, and its deposit The number is BCRC 930198. A novel pullulan pullulan (Aureobasidium pullulan), which is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 26, 2018, and its deposit The number is BCRC 930199.

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