CN111587893A - Aureobasidium pullulans fermentation product and preparation method thereof, microbial preparation for reducing fungal diseases and method for reducing fungal diseases of agricultural products - Google Patents
Aureobasidium pullulans fermentation product and preparation method thereof, microbial preparation for reducing fungal diseases and method for reducing fungal diseases of agricultural products Download PDFInfo
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Abstract
The present disclosure provides a fermentation product of Aureobasidium pullulans (Aureobasidium pullulans), wherein a method for forming the fermentation product of Aureobasidium pullulans comprises: subjecting at least one pullulan to fermentation culture in a culture medium to form a fermentation broth, wherein the culture medium comprises: a yeast extract; a malt extract; digestive proteins (Peptone); and dextrose (dextrose). The present disclosure also provides a method of preparing a fermentation product of aureobasidium pullulans, a microbial preparation and a method of reducing fungal diseases of agricultural products.
Description
Technical Field
The present disclosure provides a fermentation product of aureobasidium pullulans and a preparation method thereof. The present disclosure further provides a microbial composition. The present disclosure also provides a method of reducing fungal disease in agricultural products.
Background
Taiwan is located at the junction of tropical zone and subtropical zone, the climate is warm and humid, and the agricultural products are easily infected by pathogenic fungi (postharvest fungalpathogen) of infection in the process from harvest to distribution to sale market and purchase by consumers, so that the agricultural products are spoiled. In order to avoid damage to agricultural products in the distribution process after harvesting, disease control and prolonged preservation treatment after harvesting are important issues.
At present, the main post-harvest disease control and prolonged preservation technologies can be mainly divided into physical, chemical and microbiological methods. The physical bacteriostatic fresh-keeping mode is to keep low temperature during the transportation process so as to avoid the breeding of microorganisms. The method does not need to use chemical substances, so the method does not cause harm to human bodies, but has high investment cost of instruments and facilities, consumes a large amount of electric power and is difficult to popularize and apply.
The chemical method is to use chemical substances to achieve the bacteriostatic effect, although instruments and equipment are not needed, the price is low, the effect of the chemical substances is obvious, the chemical substances are remained on agricultural products, and after the chemical substances are eaten by human bodies, the potential risk of toxicity is caused, so that the environmental and health problems can be caused.
In contrast to the above methods, microbiological methods use substances derived from microorganisms such as Nisin, -Polylysine (-Polylysine), Natamycin (Natamycin) and the like (Xing et al, 2015, Liu et al, 2015) derived from microorganisms for bacteriostatic purposes. The microbiological method mainly requires nature and safety, and at present, physical or chemical modes are gradually replaced, but most microorganisms have a small antibacterial range and are limited in control effect on pathogenic bacteria.
It has been pointed out that the mechanism of controlling postharvest diseases of agricultural products by microorganisms can be mainly classified into 1) nutrient competition of pathogenic bacteria, 2) induction of host to produce disease resistance, or 3) secretion of hydrolytic enzymes by microorganisms themselves to decompose the cell walls of pathogenic bacteria, thereby inhibiting the growth of pathogenic bacteria (Davide and Samir, 2016). In addition, studies indicate that 1% pullulan extracellular polysaccharide can reduce the weight loss rate and the rotting rate of nectarines, keep fruits glossy and achieve a good fresh-keeping effect (euyang et al, 2010). However, at present, no microbial bacteriostatic preservative is available for infectious fungal diseases such as penicilliosis, colletotrichum anthracnose acanthosporium, gray mold and the like which are common in taiwan.
Although the existing microorganisms approximately meet the requirements, the single formulation of the microorganisms has a narrow application range to pathogenic bacteria, so that the microorganisms capable of inhibiting bacteria widely are needed.
Disclosure of Invention
The present disclosure provides a fermentation product of Aureobasidium pullulans (Aureobasidium pullulans), wherein a method for forming the fermentation product of Aureobasidium pullulans comprises: subjecting at least one pullulan to fermentation culture in a culture medium to form a fermentation broth, wherein the culture medium comprises, by weight: a yeast extract; a malt extract; digestive protein (peptone); and dextrose (dextrose).
The present disclosure also provides a microbial formulation for reducing fungal disease, comprising: a fermentation product of pullulan as described previously, wherein the microbial composition for reducing fungal diseases has an effect of reducing fungal diseases caused by fungi.
The present disclosure also provides a method for preparing a fermentation product, which uses the aforementioned pullulan fermentation product.
The present disclosure further provides a method of reducing fungal diseases in agricultural products by applying the microbial preparation described above to an agricultural product.
The present disclosure further provides a novel pullulan, which is a pullulan deposited in the biological resource preservation and research center of the food industry development research institute of taiwan treasury farrowing china in 2018, 10 and 26 months, and the deposit number of the pullulan is BCRC 930198.
The present disclosure further provides a novel pullulan, which is a pullulan deposited in the biological resource preservation and research center of the food industry development research institute of taiwan treasury farrowing china in 2018, 10 and 26 months, and the deposit number of the pullulan is BCRC 930199.
Drawings
FIG. 1 is a graph of one example of a pullulan fermentation product against the formation of black specks in the mango peel.
FIGS. 2A-2B show the front faces of colonies of Aureobasidium pullulans strain BCRC930198 cultured with potato dextrose agar at 25 ℃ for 7 days.
FIG. 2C is the front side of a colony of Aureobasidium pullulans strain BCRC930198 cultured with malt extract agar at 25 ℃ for 7 days.
FIGS. 2D-2H show the sporulation structure and the conidium microstructure of Aureobasidium pullulans strain BCRC 930198. The thick black lines in the figure are to scale and represent 10 μm.
FIG. 2I is a microstructure of crystals of Aureobasidium pullulans strain BCRC 930198. The thick black lines in the figure are to scale and represent 10 μm.
FIGS. 2J to 2L are the microscopic structures of conidia of the pullulan Aureobasidium strain BCRC 930198. The thick black lines in the figure are to scale and represent 10 μm.
FIGS. 3A-3B show the front faces of colonies of Aureobasidium pullulans strain BCRC930199 cultured with potato dextrose agar at 25 ℃ for 7 days.
FIG. 3C is the colony front of Aureobasidium pullulans strain BCRC930199 cultured with malt extract agar at 25 ℃ for 7 days.
FIGS. 3D to 3F show the sporulation structure and the conidium microstructure of Aureobasidium pullulans strain BCRC 930199. The thick black line is a scale bar, representing 10 μm.
FIG. 3G is a microstructure of crystals of Aureobasidium pullulans strain BCRC 930199. The thick black line is a scale bar, representing 10 μm.
FIGS. 3H to 3J are the microstructures of conidia of the pullulan Aureobasidium strain BCRC 930199. The thick black line is a scale bar, representing 10 μm.
Detailed Description
In view of the above problems, in the present disclosure, a pullulan fermentation substance bacteriostatic screening method is established from native samples and collected and stored pullulan seed resources in taiwan of china, to screen out pullulan with exopolysaccharide production potential, and two strains of pullulan with fungi inhibition are screened out from the pullulan fermentation substance bacteriostatic screening method to serve as broad-spectrum natural antibacterial microorganisms, so as to overcome the problem that the single formulation type faced by the existing microbial fresh-keeping bacteriostatic method is narrow in application range to pathogenic bacteria, so that the disease control effect is limited.
In a first embodiment of the present disclosure, the present disclosure provides a fermentation product of aureobasidium pullulans (Aureobasidium pullulans). The fermentation product of pullulan of the present disclosure has an effect of inhibiting the growth of fungi, but is not limited thereto.
The method for forming the fermentation product of the pullulan can include, but is not limited to, fermenting and culturing the pullulan by using a culture medium to form a fermentation liquor so as to obtain the fermentation product of the pullulan.
The components of the culture medium may include, but are not limited to, yeast extract, malt extract, digestive protein (peptone), and dextrose (dextrose).
The concentration of the yeast extract can be, but is not limited to, about 1-15g/L, such as about 1-5g/L, 1-8g/L, 1.5-10g/L, 2-6 g/L. In one embodiment, it may be 3 g/L.
The concentration of the malt extract may be about 1-15g/L, such as 1-5g/L, 1-8g/L, 1.5-10g/L, 2-6g/L, but is not limited thereto. In one embodiment, it may be 3 g/L.
The concentration of digestive proteins may be about 1-20g/L, such as 1-15g/L, 1-10g/L, 2-18g/L, 3-12g/L, but is not limited thereto. In one embodiment, it may be 5 g/L.
The concentration of dextrose can be, but is not limited to, about 1-50g/L, e.g., 5-30g/L, 5-20g/L, 3-20g/L, 5-18 g/L. In one embodiment, it may be 10 g/L.
The temperature of the fermentation culture is not particularly limited as long as it is suitable for the growth of pullulan, and may be, for example, between 15 and 30 ℃, for example, between 22 and 28 ℃, but is not limited thereto, and may be, in one embodiment, 25 ℃. The mode of the fermentation culture is not particularly limited, and may be a batch culture or a continuous culture. In one embodiment, the fermentation culture may be performed in a culture tank.
The rotation speed of the culture tank for fermentation culture can be 100-200rpm, but is not limited thereto, and in one embodiment can be 150 rpm.
The culture time of the fermentation culture is 24 to 72 hours, but is not limited thereto, and may be 48 hours in one embodiment.
In another embodiment, the method for producing a fermentation product of pullulan may further comprise a step of inactivating the bacterial cells in the fermentation broth and/or removing the bacterial cells from the fermentation broth after the fermentation broth is produced. The step of inactivating the bacterial cells in the fermentation broth includes, but is not limited to, for example, treating the fermentation product with an acid or an alkali; the step of removing the bacteria from the fermentation liquid includes, but is not limited to, filtering the bacteria to obtain a filtrate, or extracting the fermentation product.
Examples of at least one pullulan employed in embodiments for forming a fermentation product of the disclosed pullulan may include, aureobasidium pullulans, which was deposited in the biological resource preservation and Research Center (Bioresource Collection and Research Center; BCRC) of the institute of food industry development and Research of the financial institute of Taiwan, China at 26.10.2018, the collection is BCRC930198 (which is also aureobasidium pullulans deposited at the german collection for microbial and cell preservation at 13.11.2018 (DSM 32955), aureobasidium pullulans deposited at the german collection for microbial and cell preservation (DSMZ) at 13.11.2018 (DSM 32955), aureobasidium pullulans deposited at BCRC930199 (which is also aureobasidium pullulans deposited at 13.11.2018 at 13.12 at german collection for microbial and cell preservation (DSM 32956), and the like, or any combination thereof, but is not limited thereto.
In one embodiment, the Aureobasidium pullulans is Aureobasidium pullulans deposited in the center for preservation and research of biological resources of institute of food industry development and research of the financial group of people, Taiwan, China (Bioresource Collection and research center; BCRC) in 2018, 10 and 26, and the preservation number is BCRC 930198. In another embodiment, the Aureobasidium pullulans is the Aureobasidium pullulans deposited in the institute for food industry development and Research institute of the Taiwan treasury institute of China (Bioresource Collection and Research Center; BCRC) in 2018, 10.26.under the accession number BCRC 930199. In yet another embodiment, the aforementioned pullulan is a combination of BCRC930198 and BCRC 930199.
The fermentation product of pullulan of the present disclosure described above, since the fermentation product of pullulan of the present disclosure has an effect of inhibiting fungal growth, can be applied to the use of reducing fungal diseases, for example, can inhibit fungal diseases of agricultural products.
The fungal diseases include, but are not limited to, penicilliosis, colletotrichum nodosum, gray mold, blight, black root mold and tomato early blight.
Fungi of the above fungal diseases include, but are not limited to, Penicillium (Penicillium) fungi, Colletotrichum (Colletotrichum) fungi, Botrytis (Botrytis) fungi, humicola (Fusarium) fungi, Rhizopus (Rhizopus) fungi, or Alternaria (Alternaria) fungi, and the like.
The above-mentioned fungi of the genus Penicillium may include, but are not limited to, Penicillium digitatum, Penicillium expansum, Penicillium notatum (Penicillium crutum) and Penicillium italicum.
The colletotrichum species may include, but are not limited to, colletotrichum gloeosporioides (colletotrichum gloeosporioides).
The genus Botrytis may include, but is not limited to, Botrytis cinerea.
The above-mentioned genus Fusarium can include, but is not limited to, Fusarium incarnatum-equiseti, and Momordica charantia (Fusarium oxysporum f.sp. Morcordica).
The genus Rhizopus may include, but is not limited to, Rhizopus stolonifer (Rhizopus stolonifer).
The above genus Alternaria may include, but is not limited to, Alternaria alternata (Alternaria alternata).
In a second embodiment of the present disclosure, the present disclosure also provides a microbial preparation comprising a fermentation product of any of the foregoing pullulan of the present disclosure, which can have an efficacy in reducing fungal diseases caused by fungi.
In one embodiment, the microbial preparation may further comprise a diluent in addition to the fermentation product of Aureobasidium pullulans. In the microbial preparation of the present disclosure, the content of the diluent is not particularly limited and may be determined according to the need. Examples of the diluent may include water, physiological saline, organic solvents, and the like, but are not limited thereto.
In one embodiment, the microbial preparation does not contain a preservative. Since it does not contain any preservative harmful to the human body, the microbial preparation can be used as a fungal bacteriostatic agent which does not cause harm to the human body.
In one embodiment, the content of the fermentation product of pullulan in the microbial preparation may be 1 to 100% by weight, for example, about 5 to 99% by weight, about 10 to 95% by weight, but is not limited thereto.
The microbial preparation can be liquid or powder, and when the microbial preparation is used in liquid form, the fermentation product of pullulan can be directly used, or diluted by a diluent for use. When the powder is used, the fermentation product of the pullulan aureobasidium pullulans can be dried to be made into powder, and when the powder is used, the powder is diluted to the required concentration by a diluent.
In a third embodiment of the present disclosure, the present disclosure also provides a method of reducing fungal spoilage of an agricultural product. The method can include applying any of the foregoing microbial fermentation products of the present disclosure to produce. The mode of application is not limited, and the microbial fermentation product of the present disclosure is only required to be sufficiently contacted with agricultural products.
In one embodiment, the microbial preparation prepared by soaking the agricultural product in the fermentation product and the diluent containing pullulan can be applied for 1 to 60 minutes, such as 20 to 40 minutes, but is not limited thereto. In another embodiment, after the agricultural product is taken out after soaking, the agricultural product can be further kept still, so that the microorganism composition forms a protective film on the surface of the agricultural product. The time for standing may be determined as required, for example, may be adjusted according to the temperature and humidity at that time, and may be, for example, about 2 hours, 1 hour, 30 minutes, 10 minutes, but is not limited thereto. In one embodiment, the resting time may be 30 minutes.
The aforementioned agricultural products include vegetables and/or fruits. The vegetables include rhizomes and leaf vegetables. Root vegetables may include, but are not limited to, red radish, white radish, potato, taro, sweet potato, yam, and the like. Leaf vegetables may include, but are not limited to, Korean, lettuce and the like. The vegetables may also include pumpkin. The fruit may include, but is not limited to, mango, wax apple, banana, strawberry, lychee, date, plum, citrus, apple, pear, juicy peach, and the like.
In a fourth embodiment of the present disclosure, the present disclosure further provides a novel Aureobasidium pullulans, which is Aureobasidium pullulans deposited at the national institute for the preservation and Research of biological resources (BCRC) of the food industry development and Research institute of the financial institute of Taiwan, Taiwan in 2018 and 26 months, and the deposit number of the Aureobasidium pullulans is BCRC 930198. The ITS1-5.8S-ITS2 sequence of the pullulan Brevibacterium BCRC930198 is the sequence identification number of 1.
In a fifth embodiment of the present disclosure, the present disclosure further provides a novel pullulan, which is the pullulan deposited in the biological resources preservation and Research Center of the food industry development Research institute of the financial institute of taiwan, 10 months and 26 days of 2018, and the preservation number of which is BCRC 930199. The ITS1-5.8S-ITS2 sequence of the pullulan Brevibacterium BCRC930199 is the sequence identification number of 2.
The invention provides a microbial composition for preventing and treating diseases of picked fruits, which is prepared by fermenting pullulan and metabolites thereof, has antibacterial effect on common fungal diseases of picked fruits, and comprises penicilliosis, colletotrichum, gray mold, withering soft rot, black root mold and the like. Compared with the common chemical antistaling agent at present, the strain of the invention has short fermentation period, simple preparation procedure, natural and broad-spectrum effect, reduces the fungal diseases of agricultural products, and is suitable for the fresh-keeping of agricultural products.
In order to make the aforementioned and other objects, features, and advantages of the present disclosure more comprehensible, several examples are described below to illustrate fermentation products and microbial compositions of pullulan of the present disclosure, methods of reducing fungal diseases of agricultural products, and pullulan.
[ examples ]
[ screening of potential strains of Aureobasidium pullulans ]
Potential strains with bacteriostatic ability are screened from a pullulan strain library sampled and stored in Taiwan indigenous.
First, pullulan glycerin tube was inoculated with 1% inoculum size to a liquid medium consisting of deionized water containing 3g/L yeast extract, 3g/L malt extract, 5g/L digestive protein (Peptone), and 10g/L dextrose (dextrose). Then culturing for 48 hours at the temperature of 25 ℃ and the rotating speed of 150rpm to obtain the pullulan fermentation product. The pullulan fermentation product is used for carrying out bacteriostatic activity tests.
[ test for antibacterial Activity ]
The bacteriostatic test methods are commonly used in two methods, i.e., a Kirby-Bauer disk-Dilution method and a Dilution method (Dilution method), which can be divided into a Brothdilution method and an Agar Dilution method. From the results of the previous test examples of the paper diffusion method, it was found that the pullulan fermentation product contains a higher extracellular polysaccharide and has a higher viscosity than a common water-soluble sample, and thus the antibacterial component contained in the fermentation product is less likely to diffuse into the medium. Therefore, the agar dilution method was used as the detection method for the bacteriostatic activity test in the subsequent experiments.
Firstly, culturing according to the optimal growth condition of pathogenic fungi, respectively and uniformly mixing different proportions of pullulan fermentation product samples with Malt Extract Agar (MEA) or Potato Dextrose Agar (PDA) culture media, pouring the mixture into a culture dish for standing, and preparing the agar containing the pullulan fermentation product after the culture media are solidified. Next, the mold grown on the plate was cut into about 0.5X0.5cm with a blade2Placing the mould hypha blocks on the prepared agar central surface containing the pullulan aureobasidium fermentation product, culturing at 24-26 ℃ for 7 days, and observing the hypha growth conditionIn the same time, the results of the blank control group were compared, and the Minimum Inhibitory Concentration (MIC) was determined.
Through the bacteriostatic activity test, 6 beta-glucan producing strains with potential are screened, and two strains of pullulan with the best efficacy are selected, wherein the codes of the two strains are 08F0543 and 08F0544 respectively and are used as strains of the subsequent bacteriostatic test.
Sequencing analysis was performed on ITS1-5.8S-ITS2 sequence fragments of rDNA-internal transcription Intervals (ITS) of Aureobasidium pullulans 08F0543 and 08F 0544.
An ITS1-5.8S-ITS2 sequence fragment of 08F0543 is a sequence identification number of 1 (the complete sequence please refer to the sequence identification number of the sequence table of 1); the ITS1-5.8S-ITS2 sequence fragment of 08F0544 has a sequence identification number of 2 (the complete sequence refers to the sequence identification number of the sequence table: 2), and both alignment results show that the fragment is the most similar to Aureobasidium pullulans melanogenesis variant (Aureobasidium pullulans var. melanogenesis) BCRC 34543T (ATCC 12536T CBS105.22T) and Aureobasidium pullulans NRRL Y-12996 (ATCC 42023). Sequence identification numbers 1 and 2 were identical to the sequence of aureobasidium pullulans NRRL Y-12996 (ATCC 42023) (GenBank accession No. hq702508) in nucleotides 59 to 542, and it was found that 08F0543 and 08F0544 belong to aureobasidium pullulans; however, 58 more nucleotides were added to the 3 'end and 80 more nucleotides were added to the 5' end (78% similarity (484/622)). From the above, 08F0543 and 08F0544 are novel pullulan strains.
Aureobasidium pullulans strains 08F0543 and 08F0544 were identified as belonging to Aureobasidium pullulans, whose name is Aureobasidium pullulans melanogenesis variant (Melanogenum), according to the reference Zalar et al (2008).
And this 2 strain was deposited at 26.10.2018 at the biological resource preservation and Research Center of the institute of food industry development, national treasury institute of financial group, taiwan, china (Bioresource Collection and Research Center; BCRC), 08F0543 was deposited at BCRC930198 (which was also deposited at 13.11.2018 at the german Collection of microorganisms and cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ, deposit No. DSM 32955), 08F0544 was deposited at BCRC930199 at aurora prallenii (which was also deposited at 13.11.2018 at the german Collection of microorganisms and cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen gmmh, DSMZ, deposit No. DSM 32956).
The two pullulan strains (accession numbers BCRC930198, BCRC 930199) were analyzed against a known pullulan strain BCRC 930184. The results are shown in Table 1.
[ Table 1]
The melanin production capacity of the pullulan strains BCRC930198 and BCRC930199 is not much different from that of other pullulan strains, and the pullulan strain BCRC930184 is a strain with low melanin production. In addition, the fermentation products of the pullulan strains BCRC930198 and BCRC930199 have obvious bacteriostatic ability, while the pullulan strain BCRC930184 is a strain with high beta-glucan yield. It is also understood from the above physiological and biochemical properties that the pullulan strains BCRC930198, BCRC930199 and BCRC930184 screened by the above antibacterial activity test are different pullulan.
Aureobasidium pullulans strain BCRC930198 was inoculated to malt extract agar and potato dextrose agar for morphological observation. Malt extract agar, cultured at 25 deg.C for 7 days, the colony is smooth and viscous, the center of the colony is in the form of mucus, the color is dark red to red brown (9B3-E5), and the newly grown part around the colony is white (FIG. 2C); after culturing potato dextrose agar at 25 ℃ for 7 days, colonies are dark red to reddish brown (9B3-D4), the colonies are grayish yellow due to the generation of crystals (4B4-C7), and newly grown parts around the colonies are white (FIG. 2A and FIG. 2B). Observed under an optical microscope, the culture medium has transparent and brown to dark brown hyphae, the transparent hyphae are smooth and thin-walled, and have transverse septa, and the culture time can be gradually thickened; brown hypha with transverse septa, which can be generated by dark brown chlamydospores (chlamydospores), and the shape of the hypha is nearly spherical, cylindrical or irregular; spore-forming cells (conidiogenocytes) are undifferentiated, located in the middle or at the end of the clear hyphae, and have small teeth (denticles); there are transparent and black brown conidia (conidia), 1-2 cells, slightly retracted in the septum, large change in shape and size, common lateral budding, and single cell conidia size of 4.7-12.9X 2.3-5.2 μm.
Aureobasidium pullulans strain BCRC930199 was inoculated to malt extract agar and potato dextrose agar for morphological observation. Malt extract agar, cultured at 25 deg.C for 7 days, the colony is smooth and viscous, the center of the colony is in the form of mucus, the color is dark red to red brown (9B3-E5), and the newly grown part around the colony is white (FIG. 3C); after culturing potato dextrose agar at 25 ℃ for 7 days, colonies are dark red to reddish brown (9B3-D4), the colonies are grayish yellow due to the generation of crystals (4B4-C7), and newly grown parts around the colonies are white (FIGS. 3A and 3B). Observed under an optical microscope, the culture medium has transparent and brown to dark brown hyphae, the transparent hyphae are smooth and thin-walled, and have transverse septa, and the culture time can be gradually thickened; brown hypha with transverse septa, which can be generated by dark brown chlamydospores (chlamydospores), and the shape of the hypha is nearly spherical, cylindrical or irregular; spore-forming cells (conidiogenocytes) are undifferentiated, located in the middle or at the end of the clear hyphae, and have small teeth (denticles); there are transparent and black brown conidia (conidia), 1-2 cells, slightly retracted in the septum, large change in shape and size, common lateral budding, and single cell conidia of 5.2-9.3X 2.0-4.8 μm in size.
[ example 1]
The results of the bacteriostatic activity test using the fermentation products of aureobasidium pullulans of the two selected strains of aureobasidium pullulans (accession numbers BCRC930198, BCRC 930199) are shown in table 3.
The fermentation product is prepared by inoculating Aureobasidium pullulans glycerin tube with 1% inoculation amount to liquid medium, wherein the liquid medium comprises deionized water containing 3g/L yeast extract, 3g/L malt extract, 5g/L digestive protein (Peptone) and 10g/L dextrose (dextrose). Then culturing for 48 hours at 25 ℃ and 150rpm to obtain a pullulan fermentation product, and preparing fermentation liquor-agar culture medium of 5%, 12.5% and 25% (volume percentage, v/v%) with the pullulan fermentation product.
When inoculating, separate culture of pullulan aureobasidium pullulans BCRC930198 and BCRC930199 and co-culture of BCRC930198 and BCRC930199 in a volume ratio of 1:1 are respectively used, and the obtained fermentation product is used for subsequent experiments.
The molds grown on the plates were then cut with a razor blade to about 0.5X0.5cm2And (3) placing the mould hypha blocks on the central surface of the prepared culture medium containing the pullulan aureobasidium pullulans fermentation product, observing the growth condition of the hypha after culturing for 7 days at 24-26 ℃, comparing the results of a blank control group, judging the Minimum Inhibitory Concentration (MIC), displaying the results in a table 3, wherein X represents no bacteriostasis, △ represents the color change of the culture medium, and ○ represents complete bacteriostasis.
The fungal pathogens tested included penicillium (penicillium digitatum BCRC30820, penicillium digitatum BCRC32577, penicillium expansum BCRC33503, penicillium epidermidis BCRC32045, penicillium italicum BCRC32575), colletotrichum anthracnose (colletotrichum mucilaginosum BCRC35178), botrytis cinerea (botrytis cinerea BCRC35340), fusarium wilt (fusarium BCRC35290, wilfordii BCRC35046), rhizopus nigricans (rhizopus stolonifer BCRC35134), and alternaria tenuis (alternaria alternata BCRC 35015). The pathogenic bacteria are obtained from biological resource preservation and research center of the development and research institute of food industry, and the strain information and the growth conditions thereof are shown in table 2.
[ Table 2]
| BCRC numbering | Bacterial strains | Oxygen demand | Growth temperature | Culture medium |
| 30820 | Penicillium digitatum | Aerobic treatment | 25℃ | Malt Extract Agar (MEA) |
| 32577 | Penicillium digitatum | Aerobic treatment | 25℃ | Malt Extract Agar (MEA) |
| 33503 | Extended penicillium | Aerobic treatment | 26℃ | Malt Extract Agar (MEA) |
| 32045 | Peel-sticking penicillium | Aerobic treatment | 24℃ | Malt Extract Agar (MEA) |
| 32575 | Penicillium italicum | Aerobic treatment | 25℃ | Malt Extract Agar (MEA) |
| 35178 | Colletotrichum gloeosporioides | Aerobic treatment | 24℃ | Potato Dextrose Agar (PDA) |
| 35340 | Botrytis cinerea | Aerobic treatment | 24℃ | Potato Dextrose Agar (PDA) |
| 35290 | Fusarium | Aerobic treatment | 24℃ | Potato Dextrose Agar (PDA) |
| 35046 | Bitter gourd withering bacteria | Aerobic treatment | 24℃ | Potato Dextrose Agar (PDA) |
| 35134 | Rhizopus stolonifer (pers.: Fr.) karst | Aerobic treatment | 24℃ | Potato Dextrose Agar (PDA) |
| 35015 | Alternaria alternata | Aerobic treatment | 24℃ | Potato Dextrose Agar (PDA) |
[ Table 3]
The results in Table 3 show that 12.5% of culture broth-agar medium having inhibitory effects on Penicillium pathogenic bacteria (Penicillium digitatum BCRC30820, Penicillium digitatum BCRC32577, Penicillium expansum BCRC33503, Penicillium dermatum BCRC32045, and Penicillium italicum BCRC32575) and on Brevibacterium pullulan BCRC930198 and BCRC930199 and co-cultured BCRC930198 and BCRC930199, respectively, had a minimum inhibitory concentration of 12.5%.
Aiming at colletotrichum anthracnose pathogenic bacteria (colletotrichum gloeosporioides BCRC35178), 12.5 percent of pullulan aureobasidium BCRC930198 and BCRC930199 and fermentation liquor-agar culture medium for co-culturing the two have the bacteriostatic effect on the colletotrichum anthracnose pathogenic bacteria, and the minimum inhibitory concentration is 12.5 percent.
Aiming at botrytis cinerea (botrytis cinerea BCRC35340), the minimum inhibition concentration of a fermentation broth-agar culture medium of 25% of pullulan BCRC930198 on the original bacteriostatic effect of the botrytis cinerea is 25%; 12.5 percent and 25 percent of pullulan aureobasidium BCRC930199 and a fermentation broth-agar culture medium for co-culturing BCRC930198 and BCRC930199 have the lowest inhibitory concentration of 12.5 percent on the original bacteriostatic effect of gray mold; 12.5% fermentation liquor-agar culture medium of pullulan aureobasidium BCRC930199 has the lowest inhibitory concentration of 12.5% on the original bacteriostatic effect of gray mold.
Aiming at withering soft rot pathogenic bacteria (fusarium BCRC35290), 12.5 percent of pullulan Brevibacterium BCRC930198 and BCRC930199 and fermentation liquor-agar culture medium for co-culturing the two have bacteriostasis effect on colletotrichum anthracnose pathogenic bacteria, and the minimum inhibition concentration is 12.5 percent. However, against the withered Momordica charantia BCRC35046, the Aureobasidium pullulans BCRC930198, BCRC930199 and the fermentation broth-agar medium for co-culture have no bacteriostatic effect.
The medium has an antibacterial effect on colletotrichum anthracnose pathogenic bacteria, and has a minimum inhibitory concentration of 12.5% against rhizopus nigricans pathogenic bacteria (rhizopus stolonifer BCRC35134), 12.5% of pullulan Brevibacterium BCRC930198 and BCRC930199, and a fermentation broth-agar for co-culturing the two.
The agaricus (alternaria alternata BCRC35015), 12.5% of pullulan aureobasidium BCRC930198 and BCRC930199 and a fermentation broth-agar culture medium for co-culturing the two have an antibacterial effect on the alternaria alternata, and the minimum inhibition concentration is 12.5%.
From the above results, it is known that the fermentation broth-agar culture media of aureobasidium pullulans BCRC930198 and BCRC930199 respectively have bacteriostatic effects on fungi including penicillium, colletotrichum anthracnose, botrytis cinerea, withered soft rot pathogen, rhizopus nigricans pathogen, and streptosporium, and are wide-ranging fungal bacteriostatic agents.
[ example 2]
The fermentation product of aureobasidium pullulans was prepared in the same manner as in example 1 using the fermentation products of aureobasidium pullulans of the two selected strains of aureobasidium pullulans (accession numbers BCRC930198, BCRC 930199), and a 0.01 to 25% serial dilution broth-agar medium was prepared from the fermentation product of aureobasidium pullulans. Bacteriostatic tests were carried out in the same manner as in example 1 using colletotrichum anthracnose pathogen (colletotrichum gloeosporioides BCRC35178) as a test strain, and the results are shown in table 4, X represents no bacteriostatic; and o indicates complete bacteriostasis.
[ Table 4]
From the results in table 4, it was found that the minimum inhibitory concentration of the fermentation product of aureobasidium pullulans BCRC930198 was 7.5%, and the minimum inhibitory concentration of the fermentation product of aureobasidium pullulans BCRC930199 was 2.5%. From the above results, it was found that the fermentation products of aureobasidium pullulans BCRC930198 and BCRC930199 both had the effect of inhibiting the growth of fungi at a low concentration.
[ example 3]
After preparing a mixture of the fermentation products of aureobasidium pullulans BCRC930198 and BCRC930199 prepared in example 1 at a volume ratio of 1:1, cells were inactivated by alkali treatment to obtain a fermentation metabolite. Specifically, sodium hydroxide (NaOH) was added to the fermentation product so that the final concentration of sodium hydroxide was 500 mM.
Using the fermentation metabolites inactivated by the cells, 25%, 50% and 75% of a fermentation metabolite, agar medium, was prepared and tested for bacteriostasis. The results are shown in table 5, X indicates no bacteriostasis; and o indicates complete bacteriostasis.
[ Table 5]
Aiming at the bacteriostasis results of the finger-shaped penicillium BCRC30820, the extended penicillium BCRC33503 and the pellicular penicillium BCRC32045 of the penicillium, the growth diameter range of the central pathogenic bacteria hypha of the plate is obviously smaller along with the increase of the concentration of fermentation metabolites of the pullulan aureobasidium pullulans.
The pullulan Aureobasidium pullulans fermentation metabolite has 50 percent of inhibition concentration on penicillium digitatum BCRC 32577; the inhibition concentration of Penicillium italicum BCRC32575 was 75%.
The bacteriostatic results of botrytis cinerea BCRC35340 aiming at botrytis cinerea pathogenic bacteria, Momordica charantia withering bacteria BCRC35046 and fusarium BCRC35290 aiming at withering and soft-rot pathogenic bacteria and alternaria alternata BCRC35015 aiming at tomato early blight disease show that the growth diameter range of the central pathogenic bacteria hypha of the plate is obviously smaller along with the increase of the concentration of fermentation metabolites of the pullulan aureobasidium pullulans.
Aiming at the inhibition result of rhizopus nigricans pathogenic bacteria rhizopus stolonifer BCRC35134, the inhibition concentration of the fermentation metabolite of the aureobasidium pullulans on the rhizopus stolonifer is 50 percent.
Aiming at the inhibition result of colletotrichum anthracnose colletotrichum BCRC35178 of colletotrichum anthracnose pathogenic bacteria, the inhibition concentration of the pullulan aureobasidium fermentation metabolite to the bacteria is 75%.
From the above results, it is presumed that even when the cells in the pullulan fermentation product are inactivated and the fermentation metabolites are retained, the bacteriostatic component in the fermentation product has an inhibitory effect on the fungal pathogenic bacteria, and thus the bacteriostatic mechanism is derived from the secondary metabolites of pullulan fermentation.
[ example 4]
Colletotrichum gloeosporioides is one of the fungi responsible for the main infective diseases of taiwan fruits. The growth inhibition effect of the pullulan fermentation product on colletotrichum gloeosporioides is tested.
The pullulan fermentation product of example 3 was diluted with distilled water to 25%, 50%, 75% and 100%, respectively. Using Toxomin (23% aqueous suspension) (manufacturer: Waals chemical) as a positive control, mangoes were soaked in the solutions of each group for 1 minute, and then left to stand for 60 minutes. The negative control group was not soaked with any solution.
Then, a 1.6-1.7mm wound was cut on the mango skin using a 3ml syringe, followed by inoculation of colletotrichum gloeosporioides (5. mu.L of the bacterial solution/granule, the concentration of the bacterial solution being 7.1X106 CFU/ml), standing at 13 ℃ for 15 days, and the diameters of black spots formed on the mango skin on days 4, 6, 8, 11, and 15 were measured, respectively. The results are shown in FIG. 1. In addition, the mango peel was observed on day 13, with the incidence of < 30% of fermentation products with soaked pullulan.
From the results shown in fig. 1, it is understood that the lesion ratio of the fruit on day 15 is reduced by 50 to 80% compared with the control group, and the same effect as that of the treatment with the agricultural chemical can be achieved. However, the pullulan fermentation products of the present disclosure are preservative-free and are natural fungal inhibitors.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention.
Preservation of biological materials
1. Pullulan aureobasidium 08F0543
Biological resource preservation and research center of food industry development research institute of Taiwan financial group legal people
26/10/2018
Accession number BCRC930198
2. Aureobasidium pullulans 08F0544
Biological resource preservation and research center of food industry development research institute of Taiwan financial group legal people
26/10/2018
Accession number BCRC930199
SEQUENCE LISTING (SEQUENCE LISTING)
<110> institute for development of food industry for financial group legal people
<120> fermentation product of pullulan aureobasidium pullulans, method for producing the same, microbial preparation for reducing fungal diseases and method for reducing fungal diseases of agricultural products
<160>2
<170>PatentIn version 3.5
<210>1
<211>622
<212>DNA
<213> Aureobasidium pullulans (Aureobasidium pullulans)
<400>1
tcttggtcat ttagaggaag taaaagtcgt aacaaggttt ccgtaggtga acctgcggaa 60
ggatcattaa agagtaaggg tgctcagcgc ccgacctcca accctttgtt gttaaaacta 120
ccttgttgct ttggcgggac cgctcggtct cgagccgctg gggattcgtc ccaggcgagc 180
gcccgccaga gttaaaccaa actcttgtta tttaaccggt cgtctgagtt aaaattttga 240
ataaatcaaa actttcaaca acggatctct tggttctcgc atcgatgaag aacgcagcga 300
aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca tcgaatcttt gaacgcacat 360
tgcgcccctt ggtattccga ggggcatgcc tgttcgagcg tcattacacc actcaagcta 420
tgcttggtat tgggtgccgt ccttagttgg gcgcgcctta aagacctcgg cgaggcctca 480
ccggctttag gcgtagtaga atttattcga acgtctgtca aaggagagga cttctgccga 540
ctgaaacctt ttatttttct aggttgacct cggatcaggt agggataccc gctgaactta 600
agcatatcaa taagcggagg aa 622
<210>2
<211>622
<212>DNA
<213> Aureobasidium pullulans (Aureobasidium pullulans)
<400>2
tcttggtcat ttagaggaag taaaagtcgt aacaaggttt ccgtaggtga acctgcggaa 60
ggatcattaa agagtaaggg tgctcagcgc ccgacctcca accctttgtt gttaaaacta 120
ccttgttgct ttggcgggac cgctcggtct cgagccgctg gggattcgtc ccaggcgagc 180
gcccgccaga gttaaaccaa actcttgtta tttaaccggt cgtctgagtt aaaattttga 240
ataaatcaaa actttcaaca acggatctct tggttctcgc atcgatgaag aacgcagcga 300
aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca tcgaatcttt gaacgcacat 360
tgcgcccctt ggtattccga ggggcatgcc tgttcgagcg tcattacacc actcaagcta 420
tgcttggtat tgggtgccgt ccttagttgg gcgcgcctta aagacctcgg cgaggcctca 480
ccggctttag gcgtagtaga atttattcga acgtctgtca aaggagagga cttctgccga 540
ctgaaacctt ttatttttct aggttgacct cggatcaggt agggataccc gctgaactta 600
agcatatcaa taagcggagg aa 622
Claims (23)
1. A fermentation product of Aureobasidium pullulans, wherein the fermentation product of Aureobasidium pullulans is formed by a process comprising:
fermenting and culturing at least one pullulan aureobasidium pullulans by using a culture medium to form a fermentation liquid,
wherein the medium comprises the following components:
a yeast extract;
a malt extract;
digesting the protein; and
dextrose.
2. The fermentation product of Aureobasidium pullulans according to claim 1, wherein the content of the yeast extract is 1g/L to 15 g/L.
3. The fermentation product of Aureobasidium pullulans according to claim 1, wherein the content of the malt extract is 1g/L to 15 g/L.
4. The fermentation product of Aureobasidium pullulans of claim 1, wherein the content of the digestive protein is 1g/L to 20 g/L.
5. The fermentation product of Aureobasidium pullulans of claim 1, wherein the dextrose content is 1g/L to 50 g/L.
6. The fermentation product of Aureobasidium pullulans of claim 1, wherein the temperature of the fermentation culture is 15-30 ℃.
7. The fermentation product of Aureobasidium pullulans of claim 1, wherein the culture time of the fermentation culture is 24 to 72 hours.
8. The pullulan fermentation product according to claim 1 wherein the pullulan fermentation product is formed by a method further comprising inactivating bacterial cells in a fermentation broth.
9. The fermentation product of aureobasidium pullulans according to claim 1, wherein the aureobasidium pullulans is the aureobasidium pullulans deposited at the institute for food industry development and research institute of taiwan treasury farrow in 2018 at 26.10.78 with the deposit number BCRC930198 and/or the aureobasidium pullulans deposited at the institute for food industry development and research institute of taiwan treasury at 2018 at 26.10.26 with the deposit number BCRC 930199.
10. A microbial formulation for reducing fungal disease comprising:
a fermentation product of Aureobasidium pullulans according to claim 1,
wherein the microbial composition that reduces fungal disease has the effect of reducing fungal disease caused by fungi.
11. The microbial preparation for reducing a fungal disease according to claim 10, wherein the aureobasidium pullulans is the aureobasidium pullulans deposited at the institute for food industry development and research institute of taiwan treasury institute of china at 26.10.2018 with the deposit number BCRC930198 and/or the aureobasidium pullulans deposited at the institute for food industry development and research institute of taiwan treasury at 26.10.2018 with the deposit number BCRC 019939.
12. The microbial formulation of claim 10, wherein the fungus comprises penicillium, colletotrichum, botrytis cinerea, wilting rot pathogen, rhizopus nigricans pathogen, and streptococci.
13. The microbial preparation of claim 10, wherein the fungal disease comprises penicilliosis, colletotrichum anthracnose, gray mold, wilting soft rot, black root mold, and tomato early blight.
14. The microbial formulation for reducing fungal disease of claim 10, further comprising a diluent comprising water, physiological saline and an organic solvent.
15. The microbial formulation for reducing fungal disease of claim 10, wherein the microbial composition is preservative-free.
16. The microbial preparation for reducing a fungal disease according to claim 10, wherein the proportion of the fermentation product of aureobasidium pullulans is 1 to 100% by weight.
17. A method of producing a fermentation product comprising subjecting at least one pullulan to a fermentation culture in a medium to form a fermentation broth, wherein the medium comprises: a yeast extract; a malt extract; digesting the protein; and dextrose.
18. A method of reducing fungal disease of an agricultural product comprising:
applying the microbial preparation of claim 10 to an agricultural product, wherein the agricultural product comprises vegetables and/or fruits.
19. The method of reducing fungal disease of an agricultural product of claim 18, wherein the applying is as a soak, the soak time being 1 to 60 minutes.
20. The method of reducing fungal disease of an agricultural product of claim 18, further comprising the step of removing and allowing the agricultural product to stand after soaking the pullulan fermentation product.
21. The method of reducing fungal disease of an agricultural commodity of claim 18, wherein the fungal disease comprises penicilliosis, colletotrichum anthracnose, gray mold, wilting soft rot, and black root mold.
22. A novel pullulan aureobasidium pullulans is deposited in 26.10.2018 in the biological resource preservation and research center of the food industry development and research institute of the financial group of Taiwan jurisprudence Farmland, and the preservation number is BCRC 930198.
23. A novel pullulan aureobasidium pullulans is deposited in 26.10.2018 in the biological resource preservation and research center of the food industry development and research institute of the financial group of Taiwan jurisprudence Farmland, and the preservation number is BCRC 930199.
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