Aureobasidium pullulans for efficiently producing pullulan and melanin by utilizing yellow wine lees and application of aureobasidium pullulans

Abstract

The invention discloses aureobasidium pullulans for efficiently producing pullulan and melanin by utilizing yellow wine lees and application of the aureobasidium pullulans, and belongs to the technical field of microbial fermentation. The aureobasidium pullulans capable of efficiently utilizing the yellow wine lees component to synthesize the pullulan and the melanin is obtained through screening, the strain can well grow in a culture medium with the yellow wine lees component, and the yield of the pullulan and the yield of the melanin are remarkably superior to those of other microorganisms of the same genus and species. The invention further provides a fermentation method suitable for the strain, different target products can be produced by the strain by controlling different fermentation pH values, and the strain has higher flexibility in the industrial application process.

Description

Technical Field

[0001] The invention relates to a budding Aureobasidium pullulans strain for efficiently producing pullulan and melanin by using yellow wine lees and application thereof, belonging to the technical field of microbial fermentation. Background Art

[0002] Pullulan is an extracellular water-soluble mucoid polysaccharide similar to glucan. Its chemical structure is composed of maltosyl units connected by α-1,4 and α-1,6 glycosidic bonds. This unique structure gives pullulan unique physical and chemical properties, making it an ideal candidate for biodegradable materials. In addition, pullulan is non-immunogenic, non-mutagenic, non-carcinogenic and non-toxic. It has broad application potential in many fields such as food, medicine, light chemical industry and agriculture. It is a high-tech biological product with great research value and application prospects.

[0003] Melanin is a brownish black to dark black pigment commonly found in the metabolites of various animals, plants and microorganisms. It is a heterogeneous polyaromatic compound with many unique organic chemical properties, so it is widely used in light industry, fermentation industry production, pharmaceutical industry, agriculture and animal husbandry, cotton textile and food industry. In recent years, melanin produced by the metabolism of fungi, bacteria, yeast, etc. after fermentation has become a research hotspot and practical direction for people to study and apply melanin in depth.

[0004] Aureobasidium pullulans is a polymorphic fungus with a very complex life history. It generally exists in five forms: yeast, spores, swollen spores, thick-walled spores and mycelium. Aureobasidium pullulans can use various sugars, glycerol, pectin, tannic acid, beet pulp and sugarcane bagasse as carbon sources to produce pullulan, and can also produce melanin with high economic value.

[0005] At present, there are still some problems in the production of pullulan and melanin using Aureobasidium pullulans. For example, Aureobasidium pullulans will also produce melanin while producing pullulan, which will lead to insufficient utilization of raw materials in the production process, and different fermentation products of Aureobasidium pullulans need to be separated and purified, increasing production costs. Studies have shown that Aureobasidium pullulans can survive in an environment with a pH of 2-11, but due to changes in environmental pH, there are significant differences in its biomass and the amount of secondary metabolites synthesized. Under acidic pH conditions, the product metabolic pathway of Aureobasidium pullulans will flow more toward the production of melanin, and the acidic environment will also hydrolyze pullulan, ultimately leading to a decrease in pullulan production; under alkaline pH conditions, Aureobasidium pullulans will reduce the production of melanin, and the product metabolic pathway will flow more toward the production of pullulan. If it is possible to reduce the production of by-products by controlling the fermentation conditions without inhibiting the growth of Aureobasidium pullulans, and achieve a single high yield of pullulan or melanin, it will not only greatly reduce the cost of separation and purification of the fermentation products, but also provide support and inspiration for the industrial production of pullulan and melanin using Aureobasidium pullulans. Summary of the invention

[0006] The invention provides a budding Aureobasidium pullulans strain, which can effectively utilize yellow wine lees to efficiently produce pullulan and melanin.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention provides Aureobasidium pullulans LL1, which was deposited in the China Center for Type Culture Collection (CCTCC) on December 22, 2023, with a deposit number of CCTCC NO: M 20232646, and a deposit address of Wuhan University, Wuhan, China.

[0009] The present invention also provides a microbial preparation containing the Aureobasidium pullulans LL1.

[0010] The present invention also provides a method for directionally regulating the fermentation products of Aureobasidium pullulans, wherein the method controls the pH of the fermentation environment so that the Aureobasidium pullulans LL1 described in claim 1 produces pullulan or melanin; the pH control of the fermentation environment comprises: controlling the pH to 8.0-9.0 to prepare pullulan, or controlling the pH to 6.0-7.0 to prepare melanin.

[0011] In one embodiment, the method is to culture the Aureobasidium pullulans LL1 in a culture medium containing rice wine grains or rice wine grains hydrolyzate.

[0012] In one embodiment, the amount of the rice wine lees or the rice wine lees hydrolyzate added to the culture medium is ≥50 g / L.

[0013] In one embodiment, the amount of the rice wine lees or the rice wine lees hydrolyzate added to the culture medium is 50 g / L to 200 g / L.

[0014] In one embodiment, the pH of the culture environment is controlled to be 8.0 to 9.0, or 8.4 to 8.6.

[0015] In one embodiment, the rice wine lees is dried, crushed and then added to the culture medium.

[0016] In one embodiment, the rice wine lees hydrolyzate is prepared as follows: the dried and crushed rice wine lees powder is treated at 95° C. for 3 hours in an environment containing sulfuric acid and phosphoric acid, then subjected to ultrasonic treatment for 30 minutes, neutralized with sodium hydroxide solution, and the solid residue is separated to obtain a filtrate.

[0017] The present invention also provides a method for producing pullulan by using budding Aureobasidium pullulans, wherein the budding Aureobasidium pullulans is fermented in a culture medium containing yellow wine lees or a lees extract, and the pH of the fermentation environment is controlled to be alkaline.

[0018] In one embodiment, the pH of the fermentation environment is controlled to be 8.0-9.0.

[0019] The present invention also provides a method for producing melanin by using budding Aureobasidium pullulans, wherein the budding Aureobasidium pullulans is fermented in a culture medium containing yellow wine lees or a lees extract, and the pH of the fermentation environment is controlled to be acidic.

[0020] In one embodiment, the pH of the fermentation environment is controlled to be 6.0-7.0.

[0021] In one embodiment, the method comprises the following steps:

[0022] (1) Activation culture: The Aureobasidium strain is cultured aerobically, and then the cultured inoculum is transferred to an agar plate for activation culture;

[0023] (2) Seed culture: The strain obtained by activation culture was inoculated into the seed culture solution and cultured at 28°C and 160 rpm for 72 h;

[0024] (3) Fermentation culture: The seed liquid is inoculated into the fermentation medium for fermentation culture; the fermentation culture is carried out in a shake flask, and the shake flask fermentation conditions are: inoculation amount 3%, shaker speed 160r / min, and culture temperature 28°C.

[0025] In one embodiment, the method further comprises extracting pullulan from the fermentation broth: centrifuging the fermentation broth at 4°C, 10,000 r / min in a centrifuge for 10 min, taking the supernatant and precipitating it with 2 volumes of anhydrous ethanol, placing it in a 4°C refrigerator for 24 h, then centrifuging at 4°C at 10,000 r / min to collect the precipitate, washing it twice with anhydrous ethanol, and drying it in an oven at 65°C to constant weight.

[0026] In one embodiment, the culture medium for seed culture contains: sucrose 20 g / L, yeast extract 2.5 g / L, (NH4)2SO4 0.6 g / L, K2HPO4 5 g / L, NaCl 1 g / L, MgSO4 0.2 g / L.

[0027] In one embodiment, the culture medium for fermentation culture contains: 50 g / L sucrose, 2.5 g / L yeast extract powder, 0.6 g / L (NH4)2SO4, 5 g / L K2HPO4, 1 g / L NaCl, 0.2 g / L MgSO4, and rice wine lees powder or rice wine lees hydrolyzate is added at 20% of the total volume of the culture medium.

[0028] Beneficial effects:

[0029] (1) The present invention screened and obtained a budding Aureobasidium pullulans strain that can efficiently utilize rice wine lees components to synthesize pullulan and melanin. The strain can grow well in a culture medium containing rice wine lees components, providing basic conditions for the production of pullulan and melanin with the addition of rice wine lees components, and the yields of pullulan and melanin are significantly better than those of other microorganisms of the same genus and species.

[0030] (2) The present invention also provides a fermentation method suitable for the strain, by controlling different fermentation pH, the strain can produce different target products; under shake flask culture conditions with a pH of 8.5, the strain can produce 45.46±0.58 g / L pullulan in a fermentation medium containing rice wine lees hydrolyzate; under shake flask culture conditions with a pH of 6.5, the strain can produce 17.59±0.14 g / L melanin in a fermentation medium containing rice wine lees hydrolyzate.

[0031] (3) The strain and fermentation method of the present invention can realize the comprehensive utilization of the waste raw material yellow rice wine lees in the brewing field, and promote industrial green production and increase output value.

[0032] Biomaterial Deposit

[0033] Aureobasidium pullulans LL1, classified and named: Aureobasidium pullulans LL1, was deposited in the China Center for Type Culture Collection on December 22, 2023, with the deposit number CCTCCNO: M20232646, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The colony morphology (left) and microscope morphology (right) of Aureobasidium bracteata.

[0035] Figure 2 These are microscopic images of budding Aureobasidium pullulans in fermentation medium, medium with added distiller's grains powder, and medium with added distiller's grains hydrolyzate.

[0036] Figure 3 These are pictures of budding Aureobasidium being cultured in a fermenter (left) and the colony morphology of budding Aureobasidium being cultured in a fermenter for 7 days (right).

[0037] Figure 4 This is a photo of the finished product of pullulan produced by Aureobasidium pullulans of the present invention.

[0038] Figure 5 This is a photo of the finished product of the melanin produced by Aureobasidium pullulans of the present invention. DETAILED DESCRIPTION

[0039] The present invention is described below by specific embodiments. Unless otherwise stated, the technical means used in the present invention are methods known to those skilled in the art. In addition, this embodiment should be understood as illustrative, rather than limiting the scope of the present invention, the essence and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications of the material composition and dosage in these schemes also fall within the scope of protection of the present invention.

[0040] Seed culture medium: sucrose 20g / L, yeast extract powder 2.5g / L, (NH4)2SO4 0.6g / L, K2HPO4 5.0g / L, NaCl1.0g / L, MgSO4 0.2g / L.

[0041] Fermentation medium: sucrose 50 g / L, yeast extract powder 2.5 g / L, (NH4)2SO4 0.6 g / L, K2HPO4 5.0 g / L, NaCl 1.0 g / L, MgSO4 0.2 g / L.

[0042] Culture medium with added rice wine lees powder: 50 g / L rice wine lees powder, 2.5 g / L yeast extract powder, 0.6 g / L (NH4)2SO4, 5.0 g / L K2HPO4, 1.0 g / L NaCl, and 0.2 g / L MgSO4.

[0043] Culture medium with added rice wine lees hydrolyzate: 50 mL / L rice wine lees hydrolyzate, 2.5 g / L yeast extract powder, 0.6 g / L (NH4)2SO4, 5.0 g / L K2HPO4, 1.0 g / L NaCl, and 0.2 g / L MgSO4.

[0044] Preparation of yellow wine lees powder: fresh yellow wine lees are taken, placed in an oven at 55°C for drying, and crushed with a grinder to obtain yellow wine lees powder.

[0045] Preparation of rice wine lees hydrolyzate: take rice wine lees powder, mix it with deionized water in a ratio of 1:10, add 2% mixed acid (1% sulfuric acid and 1% phosphoric acid), treat at 95°C for 3h, then perform ultrasonic treatment for 30min, neutralize with sodium hydroxide solution, separate the solid residue, and obtain a filtrate.

[0046] Pullulan detection method: centrifuge the fermentation broth at 4°C, 10,000 r / min in a centrifuge for 10 minutes, take the supernatant and precipitate it with 2 volumes of anhydrous ethanol, place it in a 4°C refrigerator for 24 hours, then collect the precipitate by centrifugation at 4°C at 10,000 r / min, wash twice with anhydrous ethanol, and dry it in a 65°C oven to constant weight.

[0047] Melanin detection method: centrifuge the fermentation broth at 4°C, 4000r / min in a centrifuge for 10 minutes, take the supernatant and measure the pH value. If the pH value is greater than 3, add 1mol / L hydrochloric acid solution to adjust the pH value to 2-3, then place the supernatant in a 60°C water bath for 30 minutes, then add an equal volume of anhydrous ethanol and stir to fully precipitate the melanin, filter through filter paper, wash the resulting precipitate repeatedly with distilled water to remove residual sugar, and dry in an oven at 65°C to constant weight.

[0048] Example 1 Screening of Aureobasidium pullulans and study of its fermentation characteristics

[0049] 1. Screening of strains:

[0050] (1) Bacteria sample collection

[0051] Samples were collected from soil beneath cherry trees in Yuantouzhu, Wuxi City, Jiangsu Province, China (31°N, 120°E).

[0052] (2) Enrichment culture

[0053] Use seed culture medium for enrichment culture to promote the growth and reproduction of budding Aureobasidium.

[0054] (3) Pure strain separation

[0055] The bacterial liquid after enrichment culture is diluted in a gradient manner and spread on a solid culture medium. After culturing for a period of time, colonies with characteristics such as morphology and color are picked out.

[0056] (4) Strain identification

[0057] The target strain was identified by comparing its 16S rRNA sequence with a database of known strains.

[0058] (II) Study on the fermentation characteristics of the strain:

[0059] In order to study the fermentation characteristics of Aureobasidium pullulans, three different culture media were selected, including the original fermentation medium, the medium with added rice wine lees powder, and the medium with added rice wine lees hydrolyzate, and fermentation culture was carried out under two different pH environments of pH 6.5 and pH 8.5. The specific steps are as follows:

[0060] (1) Seed liquid culture: A loop of Aureobasidium pullulans LL1 was selected from the glycerol tube and spread on a solid plate for activation. The plate was cultured at 28°C for 96 h. A single colony was then selected and transferred to a liquid culture medium. The plate was cultured at 28°C, 180 r / min, and shaken for 72 h as the primary seed liquid.

[0061] (2) Fermentation culture: The first-level seed liquid was inoculated into the fermentation medium, the medium with added rice wine lees powder, and the medium with added rice wine lees hydrolyzate, respectively. The pH value was controlled at 6.5, the inoculation amount was 3%, and the medium was placed in a shaker and cultured at 28°C and 160 rpm for 72 h.

[0062] The composition of the culture medium to which the yellow wine lees powder is added is:

[0063] Sucrose 50g / L, yeast extract powder 2.5g / L, (NH4)2SO4 0.6g / L, K2HPO4 5.0g / L, NaCl 1.0g / L, MgSO40.2g / L, and rice wine lees powder are added at a mass of 20% of the total volume of the culture medium.

[0064] The composition of the culture medium to which the yellow wine lees hydrolyzate is added is:

[0065] Sucrose 50g / L, yeast extract powder 2.5g / L, (NH4)2SO4 0.6g / L, K2HPO4 5.0g / L, NaCl 1.0g / L, MgSO40.2g / L, the volume of rice wine lees hydrolyzate is added according to 20% of the total volume of the culture medium.

[0066] (3) Determination of fermentation products: The yields of pullulan and melanin were determined, and the yield differences under different conditions were compared. The results are shown in Table 1.

[0067] Table 1 Pullulan and melanin yields under fermentation conditions of different culture media

[0068]

[0069] As shown in Table 1, the original strains can grow and produce pullulan and melanin in the culture medium with added lees powder and lees hydrolyzate. However, there are obvious differences in the yield of pullulan and melanin produced by fermentation of different culture medium types. The fermentation characteristics of budding short-stalked mold under different pH environments also show obvious differences. When cultured in fermentation medium, the pullulan yield and melanin yield of budding short-stalked mold at pH 6.5 were higher than those at pH 8.5, indicating that budding short-stalked mold is not conducive to growth and fermentation in fermentation medium under alkaline conditions. However, after adding yellow wine lees powder or yellow wine lees hydrolyzate, it can be seen that the yield of pullulan at pH 8.5 is higher than that at pH 6.5, while the yield of melanin at pH 6.5 is higher than that at pH 8.5.

[0070] The study on the fermentation characteristics of Aureobasidium pullulans showed that after adding yellow wine lees powder or yellow wine lees hydrolyzate, Aureobasidium pullulans under alkaline conditions can efficiently produce pullulan without obvious melanin production. After adding yellow wine lees powder or yellow wine lees hydrolyzate, Aureobasidium pullulans under acidic conditions can efficiently produce melanin.

[0071] Example 2 Strain fermentation using yellow wine lees powder fermentation medium shake flask

[0072] Fermentation was carried out as follows:

[0073] (1) Bacterial Activation

[0074] The screened strains were inoculated into PDA solid medium and cultured at 28°C for 72 h;

[0075] (2) Seed solution preparation

[0076] The colony on the PDA solid culture medium in step (1) was inoculated into a conical flask containing 100 mL of liquid seed culture medium, and the culture was carried out at a shaking speed of 160 rpm and 28° C. for 72 h until the strain reached the logarithmic growth phase to obtain a seed solution; the composition of the liquid seed culture medium was: 20 g / L sucrose, 2.5 g / L yeast extract powder, 0.6 g / L (NH4)2SO4, 5.0 g / L K2HPO4, 1.0 g / L NaCl, and 0.2 g / L MgSO4.

[0077] (3) Liquid fermentation

[0078] The seed liquid is inoculated into a 500mL Erlenmeyer flask containing 200mL of liquid fermentation medium at an inoculation rate of 3% (v / v), the pH value is set to two values ​​of 6.5 and 8.5, the shaking speed is 160rpm, and the culture is carried out at 28°C for 6 days to obtain a fermentation liquid; the composition of the liquid fermentation medium is: 50g / L rice wine lees powder, 2.5g / L yeast extract powder, 0.6g / L (NH4)2SO4, 5.0g / L K2HPO4, 1.0g / L NaCl, and 0.2g / L MgSO4.

[0079] The pullulan and melanin after fermentation were tested, and the results showed that at a pH of 6.5, the pullulan production was about 4.45±0.51 g / L, and the melanin production was 16.22±0.37 g / L. At a pH of 8.5, the pullulan production was about 33.47±0.29 g / L, and the melanin production was 0.44±0.09 g / L.

[0080] Example 3 Strain fermentation using rice wine lees hydrolyzate fermentation medium shake flask

[0081] Fermentation was carried out as follows:

[0082] (1) Bacterial Activation

[0083] The screened strains were inoculated into PDA solid medium and cultured at 28°C for 72 h.

[0084] (2) Seed solution preparation

[0085] The colonies on the PDA solid culture medium were inoculated into a conical flask containing 100 mL of liquid seed culture medium, and the culture was carried out at a shaking speed of 160 rpm and 28° C. for 72 h until the strain was in the logarithmic growth phase to obtain a seed solution; the composition of the liquid seed culture medium was: 20 g / L sucrose, 2.5 g / L yeast extract powder, 0.6 g / L (NH4)2SO4, 5.0 g / L K2HPO4, 1.0 g / L NaCl, and 0.2 g / L MgSO4.

[0086] (3) Liquid fermentation

[0087] The seed liquid is inoculated into a 500mL Erlenmeyer flask containing 200mL of liquid fermentation medium at an inoculation rate of 3% (v / v), the pH value is set to two values ​​of 6.5 and 8.5, the shaking speed is 160rpm, and the fermentation liquid is obtained at 28°C for 6 days; the composition of the liquid fermentation medium is: 50mL / L of rice wine lees hydrolyzate, 2.5g / L of yeast extract powder, 0.6g / L of (NH4)2SO4, 5.0g / L of K2HPO4, 1.0g / L of NaCl, and 0.2g / L of MgSO4.

[0088] The pullulan and melanin after fermentation were tested, and the results showed that at a pH of 6.5, the pullulan production was about 3.95±0.51 g / L, and the melanin production was 17.59±0.14 g / L. At a pH of 8.5, the pullulan production was about 45.46±0.58 g / L, and the melanin production was 0.21±0.04 g / L.

[0089] Example 4 Pilot fermentation to produce pullulan and melanin

[0090] The pilot fermentation to produce pullulan and melanin was carried out in a 5L fermenter as follows:

[0091] (1) Fermentation tank pilot test using yellow wine lees powder

[0092] Preparation of medium for tank loading: For a 5L fermentation tank, 2.5L of fermentation medium with the same formulation as that of Example 2 needs to be prepared according to 50% of the liquid volume. The pH is adjusted, sterilized at 121°C for 20 min, and cooled.

[0093] Cultivation of seed solution: Take a slant culture of the budding Aureobasidium strain and transfer it all to the seed culture medium. Cultivate it in a shaking incubator at a constant temperature of 28°C and 160 rpm for 72 hours.

[0094] Fermentation of pullulan in a fermenter: The obtained seed solution was transferred to a 5L fermenter at a 3% inoculation rate, pH 8.5, constant temperature 28°C, stirring 160 rpm, and ventilation volume 0.4 m 3 / h and fermented for 7 days, the pullulan production in the fermenter was about 38.71±0.44g / L.

[0095] Fermentation tank production of fermented melanin: The obtained seed liquid was transferred to a 5L fermentation tank at a 3% inoculation rate, pH 6.5, constant temperature 28℃, stirring 160rpm, ventilation volume 0.4m 3 / h fermentation for 7 days, the melanin production in the fermentation tank was 14.66±0.31g / L.

[0096] (2) Fermentation tank pilot test using rice wine lees hydrolyzate

[0097] Preparation of medium for tank loading: For a 5L fermentation tank, 2.5L of medium is required based on 50% of the liquid volume. The formula is the same as that of the fermentation medium in Example 3. The pH is adjusted, sterilized at 121°C for 20 min, and cooled.

[0098] Cultivation of seed solution: Take a slant culture of the budding Aureobasidium strain and transfer it all to the seed culture medium. Cultivate it in a shaking incubator at a constant temperature of 28°C and 160 rpm for 72 hours.

[0099] Fermentation of pullulan in a fermenter: The obtained seed solution was transferred to a 5L fermenter at a 3% inoculation rate, pH 8.5, constant temperature 28°C, stirring 160 rpm, and ventilation volume 0.4 m 3 / h and fermented for 7 days, the pullulan production in the fermenter was about 49.82±0.79g / L.

[0100] Fermentation tank production of fermented melanin: The obtained seed liquid was transferred to a 5L fermentation tank at a 3% inoculation rate, pH 6.5, constant temperature 28℃, stirring 160rpm, ventilation volume 0.4m 3 / h fermentation for 7 days, the melanin production was 16.78±0.45g / L.

[0101] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Aureobasidium pullulans LL1, which was deposited in the China Center for Type Culture Collection on December 22, 2023, with the deposit number of CCTCC NO: M 20232646.

2. A microbial preparation containing the budding Aureobasidium pullulans LL1 according to claim 1.

3. Use of the budding Aureobasidium pullulans LL1 according to claim 1 in the fermentation production of pullulan and / or melanin.

4. The use according to claim 3, characterized in that: Fermentation is carried out in a culture medium containing rice wine lees or rice wine lees hydrolyzate.

5. A method for directional regulation of fermentation products of Aureobasidium pullulans, characterized in that: The budding Aureobasidium pullulans LL1 described in claim 1 is made to produce pullulan or melanin by controlling the pH of the fermentation environment; the pH control of the fermentation environment includes: controlling the pH to 8.0-9.0 to prepare pullulan, or controlling the pH to 6.0-7.0 to prepare melanin.

6. The method according to claim 5, characterized in that The fermentation environment includes but is not limited to a culture medium containing yellow wine lees or yellow wine lees hydrolyzate.

7. The method according to claim 6, characterized in that The amount of the yellow wine lees or yellow wine lees hydrolyzate added to the culture medium is ≥50 g / L.

8. The method according to claim 6 or 7, characterized in that: The yellow wine lees are dried, crushed and then added into the culture medium.

9. The method according to claim 6 or 7, characterized in that: The yellow wine lees hydrolyzate is prepared by the following method: treating the dried and crushed yellow wine lees powder at 90-100° C. for 2-3 hours in an environment containing sulfuric acid and phosphoric acid, then ultrasonically treating it for 20-40 minutes, neutralizing it with a sodium hydroxide solution, and separating the solid residue to obtain the yellow wine lees hydrolyzate.

10. Use of the budding Aureobasidium pullulans LL1 according to claim 1, the microbial preparation according to claim 2, or the method according to any one of claims 3 to 9 in the preparation of pullulan with low pigment content, or a product containing pullulan, or melanin, or a product containing melanin.

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