HK1251253B - AUREOBASIDIUM PULLULANS, CULTURING MEDIUM AND METHOD FOR PRODUCING β-GLUCAN, A CULTURE OF AUREOBASIDIUM PULLULANS CONTAINING β-GLUCAN AND A COMPOSITION - Google Patents

Description

Black yeast, culture medium and method for producing β-glucan, and black yeast culture and composition containing β-glucan

Technical Field

The present invention relates to a method for producing beta-glucan, and in particular to a method for producing beta-glucan in high yield by using black yeast that does not produce melanin and a fermentation medium.

Background Art

Black yeast (Aureobasidium pullulans), also known as dark golden basidiomycete or budding aureobasidium, is a yeast-like fungus that produces polysaccharides, such as β-glucan, around its cell wall.

β-glucan is already approved as a food ingredient by the Taiwan Food and Drug Administration and the Ministry of Health, Labor and Welfare of Japan. It is also a Generally Recognized As Safe (GRAS) food additive by the U.S. FDA, eliminating food safety concerns during its development.

Extracting active ingredients like β-glucan from raw materials such as barley, oats, yeast, and mushrooms requires extensive chemical processing. During these processing steps, many beneficial trace components may be lost, resulting in the product failing to achieve the desired effect. β-glucan produced in plants, on the other hand, can vary in content depending on whether the source is wild or cultivated, as well as the conditions and environment in which the plant is grown. Compared to processes using raw materials such as barley, oats, yeast, and mushrooms, producing β-glucan using black yeast eliminates the extraction process and reduces waste generation because β-glucan is secreted extracellularly. Furthermore, producing β-glucan using black yeast avoids the aforementioned drawbacks of plant-based production.

However, despite the advantages of using black yeast to produce β-glucan, the production of β-glucan by black yeast fermentation is still limited due to the following reasons:

(1) Low yield: Black yeast produces soluble extracellular polysaccharides during fermentation. However, as the polysaccharide production increases, the fermentation liquid tends to form a viscous gel. This high viscosity can easily cause problems such as aeration, dissolved oxygen, and stirring, which can cause mass transfer or heat transfer problems, thereby affecting the production of β-glucan.

(2) The melanin contained in the polysaccharide is difficult to remove: The extracellular polysaccharide produced by the fermentation of black yeast strains generally contains melanin, so the product has a poor appearance. If the melanin is to be further removed, it will increase the inconvenience of the process, thereby affecting the subsequent production process of the product.

Summary of the Invention

In view of the above problems in the prior art, the object of the present invention is to provide a black yeast having a high β-glucan yield, and the β-glucan produced contains almost no melanin.

According to one object of the present invention, a black yeast (Aureobasidium pullulans) is provided, which was deposited in the Patent Microorganisms Depositary, National Institute of Technology and Evaluation, Japan on October 19, 2016, with the deposit number NITE BP-02372.

According to another object of the present invention, a culture medium for producing β-glucan is provided, which may comprise: a carbon source, a nitrogen source, and ascorbic acid; wherein the carbon source may be selected from: lactose, fructose, maltose, glucose, galactose, xylose, xylitol, inulin, sorbitol, trehalose, sucrose, molasses, and combinations thereof; and the nitrogen source may be selected from: peptone, yeast extract, urea, potassium nitrate, sodium nitrate, ammonium sulfate, lecithin, and combinations thereof.

Preferably, the carbon source may be sucrose or glucose.

Preferably, the concentration of sucrose may be 10 to 70 g/L based on the total volume of the fermentation medium. Preferably, the concentration of glucose may be 25 to 150 g/L based on the total volume of the fermentation medium.

Preferably, the nitrogen source may be lecithin. Preferably, the concentration of lecithin may be less than or equal to 6 g/L based on the total volume of the fermentation medium. Preferably, the concentration of ascorbic acid may be less than or equal to 6 g/L based on the total volume of the fermentation medium.

Preferably, the initial pH value of the culture medium may be 4 to 8. More preferably, the initial pH value of the culture medium may be 5 to 6.

According to another object of the present invention, a culture medium for producing β-glucan is provided, comprising: a carbon source and ascorbic acid; wherein the carbon source can be selected from: lactose, fructose, maltose, glucose, galactose, xylose, xylitol, inulin, sorbitol, trehalose, sucrose, molasses and combinations thereof; and wherein the culture medium may not contain a nitrogen source.

According to another object of the present invention, a method for producing β-glucan is provided, which comprises: culturing the black yeast in a fermentation medium for fermentation.

Preferably, the fermentation medium may be the medium described above.

Preferably, the black yeast may be cultured at a temperature of 15 to 30°C.

Preferably, the fermentation is carried out at a rotation speed of 150 to 350 rpm.

Preferably, the fermentation is carried out under a ventilation rate of 1 to 2 vvm.

Preferably, the method may further include growing the black yeast in a seed culture medium to a stationary phase before fermentation.

Preferably, the seed culture medium may comprise granulated yeast extract, malt extract, peptone from soybean (enzymatic digest) and dextrose.

According to another object of the present invention, a black yeast culture containing β-glucan is provided, which can be prepared by the above method.

According to another object of the present invention, a composition is provided, which may comprise the yeast culture and an optional carrier.

In general, the present invention provides at least the following advantages:

(1) The black yeast strains according to the embodiments of the present invention are characterized by not producing melanin, thus eliminating the need for subsequent melanin removal. Furthermore, by improving the culture medium composition and culture method of the present invention, the yield of β-glucan can be effectively increased.

(2) By improving the production process of β-glucan and the composition of the culture medium, the fermentation broth of black yeast can be used entirely for the preparation of β-glucan products, so as to obtain products with high β-glucan content. In addition to being effective, the products can also be easy to store and process, while reducing the discharge of process waste liquid, thus achieving the goals of energy conservation, carbon reduction and environmental friendliness.

The above and other objects, features and advantages of the present invention will become apparent after referring to the following detailed description and preferred embodiments and the accompanying drawings attached hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the morphological characteristics and microscopic structure of black yeast 12F0291. (A) Front view of colony, MEA, 25°C, 5 days; (B) Back view of colony, MEA, 25°C, 5 days; (C) Front view of colony, PDA, 25°C, 5 days; (D) Back view of colony, PDA, 25°C, 5 days; (E) Hyphae and conidial structures (Bar = 10 μm); (F) Conidia (Bar = 10 μm); (G) Chlamydospores (Bar = 5 μm).

FIG2 shows the results of measuring the concentration of β-glucan produced using fermentation media with different initial pH values.

FIG3 shows the results of measuring the concentration of β-glucan produced in fermentation media containing different carbon sources.

FIG4 shows the results of measuring the concentration of β-glucan produced in fermentation media containing different sucrose concentrations.

FIG5 shows the results of measuring the concentration of β-glucan produced in fermentation media containing different glucose concentrations.

FIG6 shows the results of measuring the concentration of β-glucan produced in GCL fermentation medium with different nitrogen sources.

FIG7 shows the results of measuring the concentration of β-glucan produced by culturing black yeast using fermentation media with or without a nitrogen source.

FIG8 is a schematic diagram showing the process of thawing black yeast from a frozen strain glycerol tube and culturing and fermenting it.

FIG9 shows the appearance of black yeast colonies and shake flasks cultured in YM medium or fermentation medium after fermentation.

FIG10 shows the bacterial count (expressed as _OD600 absorbance) of black yeast after culturing in YM medium for 1 or 2 days.

FIG11 shows the appearance of black yeast colonies after being cultured in YM medium for 48 hours.

FIG12 shows the analysis results of the β-glucan content in the fermentation broth after fermentation with black yeast for 2, 5, 6, and 8 days.

In the following detailed description, in order to explain the present invention, numerous specific details are provided to provide a thorough understanding of the disclosed embodiments. However, it will be apparent that one or more embodiments can be practiced without these specific details. In other cases, known structures and processes are shown in schematic form to simplify the drawings.

DETAILED DESCRIPTION

The details of each embodiment of the present invention are as follows. Other features of the present invention will be clearly presented through the detailed description of each embodiment and the scope of the claims below.

Without further elaboration, it is believed that those skilled in the art can utilize the present invention to its fullest extent based on the foregoing description. Therefore, it is understood that the following description is merely for illustrative purposes and is not intended to limit the remaining disclosure in any way.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the articles "a" and "an" refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

Screening of potential black yeast strains

Initial screening:

After activating frozen strains of black yeast (Aureobasidium pullulans) stored in cryovials, they were spread on a screening medium (composition shown in Table 1 below) and incubated at 25°C for 5-7 days. Colony morphology and color were observed, and strains showing a significant color change after incubation were selected. A total of 530 strains were screened in the initial screening, and 30 promising candidate strains were identified for subsequent screening.

Table 1 Primary screening culture medium (YM agar + sucrose + aniline blue)

pH 6.2±0.2

Screening of candidate strains

From the 30 candidate strains selected in the initial screening, potential production strains were further selected for production trials. Briefly, after the candidate strains were activated, they were cultured with shaking in YMS medium containing sucrose (the composition is shown in Table 2 below) at 25°C for four days. Next, samples of the culture medium were taken and the pullulan was broken down using the enzyme pullulanase. The total sugar content was then measured using the phenol-sulfuric acid assay. Strains with the highest production of non-pullulan polysaccharides were selected.

Table 2 YMS medium (YM broth + sucrose)

pH 6.2±0.2

Next, the black yeast strains screened for their potential to produce β-glucan were activated using YM medium (Difco ^™ ) and then initially tested in shake flasks using YMS medium. The culture conditions were 25°C and 150 rpm. β-glucan levels in the shake flask fermentation broth were measured after 72 hours of culture. The measurement method is as follows, and the results are shown in Table 3. LQ, a black yeast fermentation broth purchased from ADEKA, Japan, with a β-glucan content of 1% (10 g/L), served as the positive control group.

Measurement method of black yeast β-glucan

Using the Megazyme Total Dietary Fiber Assay kit, the fermentation broth was first added to an appropriate volume of buffer and mixed thoroughly. Alpha-amylase, protease, and amyloglucosidase were then added sequentially for treatment. The precipitate was then separated from the solution by precipitation with four volumes of alcohol. The precipitate was collected, washed with alcohol, and dried. The dried precipitate was hydrolyzed with strong acid and high temperature. After acid-base neutralization, glucose was measured and the β-glucan content was calculated. The detailed steps are as follows:

When the sample to be tested is a fermentation broth, weigh 5 g of the sample and add 3.5 mL of 0.08 M phosphate buffer (pH 6.0) and mix thoroughly.

After adding 500 μL of α-amylase (60 mg/mL PB buffer) to the sample, react in a 95°C water bath for 30 minutes, shaking every 15 minutes to mix evenly. After cooling the sample to 60°C, adjust the pH to 7.5±0.1 (about 1000 μL) with 0.275M NaOH, then add 50 μL of proteolytic enzyme and react in a 60°C water bath for 30 minutes. After adjusting the pH to 4.3±0.1 (about 800 μL) with 0.325M HCl, add 50 μL of amyloglucosidase and react in a 60°C water bath for 30 minutes to obtain the enzyme-treated sample.

After enzyme treatment, the sample was filled with 95% EtOH to a volume of 40 mL. The sample was allowed to stand at room temperature for one hour to precipitate the β-glucan. The β-glucan was then separated from the solution by sedimentation. The sample was then centrifuged (10,000 g) for 10 minutes to remove the supernatant. The precipitate was collected and rinsed with 4 mL of 80% EtOH. The sample was centrifuged (10,000 g) for another 5 minutes to remove the supernatant, and then vacuum dried at room temperature.

Add 1 mL of 72% (w/v) sulfuric acid to the overnight dried sample and allow to stand at room temperature for 60 minutes before adding 14 mL of deionized water. Heat in a boiling water bath (100°C) for 2 hours. If any large residues remain, autoclave sterilization is performed (121°C, 20 minutes). After cooling, neutralize the hydrolyzate with 5 mL of 5N NaOH and dilute to 25 mL with deionized water. Glucose content is then determined using a glucose analysis kit (Glucose PAP Kit).

During the enzymatic treatment, the α-linked polysaccharides in the fermentation broth are broken down by α-amylase, and the macromolecular proteins present in the fermentation broth are also broken down by proteolytic enzymes. Finally, amyloglucosidase breaks down the short-chain polymerized glucose chains into glucose. The long polymerized glucose chains that remain unbroken by the enzyme (remaining as β-linked glucans) are then separated from the solution by alcohol precipitation and dried. After drying, the glucose content is measured by hydrolysis with sulfuric acid and high temperature, which is the content of β-linked glucans. Finally, the following formula is used to calculate the glucan weight (W _BGP ) based on the glucose concentration (C _glu ) measured by this enzymatic method.

W _BGP = C _glu * 0.9 * 1000 * 0.025, where 0.025 is the dilution factor (25 mL/1 L) and 1000 is the unit factor (mg/g).

Table 3 β-glucan measurement results of black yeast potential strains after preliminary shake flask culture for 3 days

According to the β-glucan concentration in Table 3 above, the best production strain 6 was finally screened out and named black yeast 12F0291.

Strain identification

According to one embodiment of the present invention, a black yeast strain 12F0291 with β-glucan production potential was screened out from the black yeast strain library of the Food Industry Development Research Institute and was deposited in the Japan Patent Microorganism Depository of Japan on October 19, 2016, with the deposit number NITE BP-02372.

Black yeast strain 12F0291 was isolated and sampled from plant leaves in Xinpu Township, Hsinchu County, Taiwan, on June 17, 2008. As shown in Figures 1(A) and (B), when incubated at 25°C on MEA (Malt Extract Agar, Blakeslee's Formula) medium (Table 4), the colonies were smooth, viscous, and covered with a transparent mucus. Their color changed from grayish-yellow to orange to brown, and the surrounding areas were yellowish-white. As shown in Figures 1(C) and (D), when incubated at 25°C on PDA (Potato Dextrose Agar, Difco ^™ ) medium (Table 5), the colonies were smooth, viscous, and covered with a transparent mucus. Their color changed from yellowish-white to orange-red, and the surrounding areas were yellowish-white.

Table 4 MEA medium (MEA, Blakeslee's Formula)

Table 5 PDA culture medium (PDA, Difco ^™ )

Referring to Figures 1(E) to (G), microscopic observation of black yeast strain 12F0291 reveals smooth hyphae with transverse septa that are slightly constricted near the septa. The hyphae are thin to thick-walled, with diameters ranging from 3.4 to 5.8 μm. Over time, some hyphae gradually turn orange-brown. Undifferentiated conidiogenous cells are located in the middle or at the ends of the transparent hyphae, occasionally branching out from them. These cells possess denticle-like structures and bear conidia. These conidia have smooth outer walls and vary in shape, measuring 4.0-11.0 μm by 3.0-6.0 μm. Chlamydospores are transparent or orange-brown, nearly spherical or elliptical in shape, and measuring 4.7-9.0 μm by 2.9-6.5 μm.

Sequence alignment of the rDNA ITS1-5.8S-ITS2 fragment of black yeast 12F0291 showed that black yeast 12F0291 was most similar to Aureobasidium pullulans var. melanogenum BCRC 34543T (=CBS 105.22T) (BCRC database no. BCRC34543_05042009_FD_ITS) and Aureobasidium pullulans NRRLY-12996 (=ATCC 42023) (GenBank accession no. HQ702508).

Based on the morphological characteristics of the colony, spore-forming structure, conidia, and rDNA ITTS-5.8S-ITSS2 sequence analysis, the black yeast 12F0291 was identified as Aureobasidium pulllulans, and was identified as Aureobasidium pullululans var. melanogenum.

Fermentation medium for β-glucan production

To address the aforementioned issues, one embodiment of the present invention relates to a culture medium for producing β-glucan. The culture medium may contain a carbon source, a nitrogen source, and ascorbic acid. The carbon source may be selected from the group consisting of lactose, fructose, maltose, glucose, galactose, xylose, xylitol, inulin, sorbitol, trehalose, sucrose, molasses, and combinations thereof. The nitrogen source may be selected from the group consisting of peptone from soybean enzymatic digest, granulated yeast extract, urea, potassium nitrate, sodium nitrate, ammonium sulfate, lecithin, and combinations thereof.

In a preferred embodiment, the carbon source may be sucrose or glucose. In a more preferred embodiment, the concentration of sucrose may be 10-70 g/L or the concentration of glucose may be 25-150 g/L, the concentrations being calculated based on the total volume of the culture medium. In another preferred embodiment, the nitrogen source may be lecithin, and the concentration of lecithin may be less than or equal to 6 g/L.

According to one embodiment of the present invention, the culture medium for producing beta-glucan can be prepared by the following method. When preparing the culture medium, ascorbic acid is added, and at this time, the pH is about 3.0, and then the pH value is adjusted to 4.0 to 8.0, preferably 5.0 to 6.0, using NaOH _(aq) , and then sterilized by autoclave (Autoclave), and then accessed after cooling. In another embodiment, the pH value of the culture medium can also be adjusted to 12.0 when preparing the culture medium, and after autoclave sterilization, the ascorbic acid solution is filtered with a 0.22 μm filter in a sterile operating table and added to the sterilized culture medium, so that the pH value of the culture medium is between 4.0 and 8.0, preferably between 5.0 and 6.0, and then accessed to the seed. In a preferred embodiment, the concentration of ascorbic acid may be less than or equal to 6 g/L.

According to one embodiment of the present invention, a thawed glycerol tube of black yeast 12F0291 was activated in YM medium (the composition of which is shown in Table 6 below) at a 0.3% (v/v) inoculum. Seed culture was performed under conditions of a temperature of 25° C., a rotation speed of 150 rpm, and a culture temperature of 48 hours. The medium was then inoculated into the following fermentation media (Tables 7 to 13). After culturing for 2 days at a temperature of 25° C., a rotation speed of 150 rpm, and a temperature of 25° C., the β-glucan content in each fermentation medium was measured. The measurement method is the same as described above and will not be repeated here.

Table 6 YM medium (YM broth, Difco ^™ )

pH 6.2±0.2

In one embodiment, black yeast strain 12F0291 was inoculated into SCL fermentation media with initial pH values of 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 for fermentation to produce β-glucan, and the β-glucan concentrations produced during fermentation were measured. The "initial pH" herein refers to the pH of the culture medium after preparation and before any incubation. Please refer to FIG2 , which shows the results of measuring the β-glucan concentrations produced using fermentation media with different initial pH values. As shown, black yeast strain 12F0291 produced significantly higher levels of β-glucan in fermentation media with initial pH values of 4.0 to 8.0. In particular, black yeast strain 12F0291 cultured in fermentation media with initial pH values of 5.0 and 6.0 produced the highest β-glucan concentrations, reaching 7.95 g/L and 8.23 g/L, respectively.

In one embodiment, black yeast strain 12F0291 was inoculated into fermentation media containing different carbon sources for fermentation to produce β-glucan. Specifically, in this embodiment, black yeast strain 12F0291 was inoculated into fermentation media containing sucrose (composition shown in Table 7), glucose (composition shown in Table 8), or molasses (composition shown in Table 9) as the carbon source for fermentation, and the β-glucan concentration produced by the fermentation was measured. Please refer to FIG3 , which shows the results of measuring the β-glucan concentration produced in fermentation media containing different carbon sources. As shown in the figure, black yeast strain 12F0291 was able to produce β-glucan regardless of whether sucrose (SCL), glucose (GCL), or molasses (MCL) was used as the carbon source. However, the black yeast strain 12F0291 cultured in fermentation media containing sucrose (SCL) and glucose (GCL) as the carbon sources produced the highest β-glucan production, approximately 9 g/L.

Table 7 SCL fermentation medium

pH 5.55

Table 8 GCL fermentation medium

pH 5.51

Table 9 MCL fermentation medium

pH 5.54

In a preferred embodiment, black yeast strain 12F0291 was inoculated into fermentation media containing varying sucrose concentrations to produce β-glucan. Specifically, in this embodiment, black yeast strain 12F0291 was inoculated into fermentation media containing 0, 10, 20, 30, 40, and 50 g/L of sucrose (the composition is shown in Table 10), and the β-glucan concentration produced by the fermentation was measured. Please refer to Figure 4 for the results, which shows the concentration of β-glucan produced by black yeast strain 12F0291 in fermentation media containing varying sucrose concentrations. As shown in the figure, the concentration of β-glucan produced by fermentation increases with the sucrose concentration in the fermentation medium. In this embodiment, the highest β-glucan concentration, approximately 8 g/L, was achieved in the fermentation medium containing 50 g/L of sucrose.

Table 10 Fermentation medium with different concentrations of sucrose

pH 5.55～5.60

In a preferred embodiment, black yeast strain 12F0291 was inoculated into fermentation media containing different glucose concentrations for fermentation to produce β-glucan. Specifically, in this embodiment, black yeast strain 12F0291 was inoculated into fermentation media containing 25, 50, 75, 100, 125, and 150 g/L of glucose (the composition is shown in Table 11), and the β-glucan concentration produced by the fermentation was measured. Please refer to Figure 5 for the results, which shows the concentration of β-glucan produced by black yeast strain 12F0291 in fermentation media containing different glucose concentrations. As shown in the figure, the concentration of β-glucan produced by fermentation increases roughly with the glucose concentration in the fermentation medium. In this embodiment, the highest β-glucan concentration, approximately 9.31 g/L, was achieved in a fermentation medium containing 100 g/L of glucose.

Table 11 Fermentation medium with different concentrations of glucose

pH 5.56～5.62

In one embodiment, the GCL fermentation medium shown in Table 8 was used as the basal medium, and various nitrogen sources, including urea, peptone from soybean enzymatic digest, granulated yeast extract, potassium nitrate (KNO ₃ ), sodium nitrate (NaNO ₃ ), and ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), were added. In one embodiment, the nitrogen source concentrations were 2, 4, and 8 g/L, respectively. The initial pH values of the culture medium with different nitrogen source concentrations were adjusted to the values shown in Table 12. After 48 hours of culture in YM medium, the culture fluid was inoculated at a 20% (v/v) inoculum into GCL fermentation medium containing various nitrogen sources. The β-glucan content was analyzed after 48 hours of culture in a shake flask at 25°C and 150 rpm.

Table 12 GCL fermentation medium containing different nitrogen sources

Please refer to Figure 6 for the results, which show the concentration of β-glucan produced by black yeast strain 12F0291 in GCL fermentation media containing different nitrogen sources. As shown in the figure, black yeast strain 12F0291 was able to produce β-glucan in fermentation media containing different nitrogen sources. Notably, in the three groups (Groups 3, 4, and 5) supplemented with peptone (from soybean enzymatic digest), granulated yeast extract, and potassium nitrate ( _KNO₃ ), the β-glucan concentration showed a gradual decrease with increasing nitrogen source concentration. In the two groups (Groups 2 and 6) supplemented with urea and sodium nitrate (NaNO ₃ ), the group supplemented with a nitrogen source concentration of 4 g/L had higher β-glucan production, but the increase in β-glucan production was less pronounced compared to the group supplemented with a nitrogen source concentration of 2 g/L. The lowest β-glucan concentration was achieved in the group supplemented with 8 g/L of nitrogen source. In the group supplemented with lecithin alone (Group 1), black yeast 12F0291 produced approximately 8 g/L of β-glucan after 48 hours of fermentation, a yield approximately four times higher than that of the group supplemented with ammonium sulfate.

Fermentation medium for producing β-glucan without nitrogen source

In this example, black yeast strain 12F0291 was inoculated into a fermentation medium devoid of a nitrogen source for fermentation to produce β-glucan. Specifically, in this example, black yeast strain 12F0291 was cultured in a medium designed for β-glucan production. The medium contained a carbon source and ascorbic acid; the carbon source can be selected from the group consisting of lactose, fructose, maltose, glucose, galactose, xylose, xylitol, inulin, sorbitol, trehalose, sucrose, and combinations thereof. The medium may not contain a nitrogen source.

In one specific embodiment, lecithin was used as the nitrogen source for fermentation by black yeast strain 12F0291. Specifically, black yeast strain 12F0291 was inoculated into fermentation media containing lecithin (e.g., SCL fermentation medium in Table 7) as a nitrogen source or without a nitrogen source (e.g., SC fermentation medium in Table 13), and the resulting β-glucan concentrations were measured. Please refer to Figure 7, which shows the β-glucan concentrations produced by black yeast strain 12F0291 cultured in fermentation media containing and without a nitrogen source. As shown, black yeast strain 12F0291 produced β-glucan in both fermentation media containing and without lecithin. However, black yeast strain 12F0291 cultured in fermentation media containing lecithin produced a higher β-glucan production, approximately 9.37 g/L, while black yeast strain 12F0291 cultured in fermentation media without lecithin produced approximately 2.74 g/L.

Table 13 SC fermentation medium

Process for producing β-glucan

In this embodiment, a method for producing β-glucan is provided, comprising culturing the black yeast in a fermentation medium for fermentation. In one embodiment, the fermentation medium can be any of the aforementioned culture media, and the culture can be performed under the following conditions:

In one embodiment, the black yeast can be cultured at a temperature of 15 to 37°C; preferably, it can be cultured at a temperature of 20 to 35°C; most preferably, it can be cultured at a temperature of 23 to 30°C. In one embodiment, the culture can be carried out at a rotation speed of 100 to 450 rpm; preferably, it can be cultured at a rotation speed of 120 to 400 rpm; most preferably, it can be cultured at a rotation speed of 150 to 350 rpm. In one embodiment, the culture can be carried out at a ventilation rate of 0.5 to 3 vvm; preferably, it can be cultured at a ventilation rate of 0.8 to 2.5 vvm; most preferably, it can be cultured at a ventilation rate of 1 to 2 vvm.

Please refer to Table 14 below, which lists several exemplary culture conditions. Black yeast was cultured and fermented under these culture conditions. Subsequently, the β-glucan content in the fermented culture broth was measured. It was found that black yeast was able to produce β-glucan under these culture conditions (not shown).

Table 14 Different fermentation conditions of black yeast

Specifically, please refer to Figures 8 to 12. Figure 8 shows a schematic flow diagram for thawing, culturing, and fermenting black yeast 12F0291 and potential strains 3, 4, and 5 listed in Table 3 from frozen glycerol tubes. The frozen black yeast strains in the glycerol tubes were inoculated with a 0.3% inoculum into YM medium listed in Table 6. After 48 hours of seed culture in a shake flask at 25°C and 150 rpm, the culture broth was inoculated into high-GCL fermentation medium listed in Table 15 and fermented at 25°C and 150 rpm for 2, 5, 6, and 8 days, respectively, to produce β-glucan. In a preferred embodiment, the black yeast was cultured in YM medium and allowed to grow to the stationary phase before fermentation in the fermentation medium.

Table 15 High GCL fermentation medium

Figures 9 to 11 show the colony appearance, shake flask appearance, and bacterial count results of black yeast cultured in YM medium or fermentation medium after fermentation. Referring to Figure 9 , when cultured in YM medium (in the middle), the bacterial broths of all four strains were beige in color, with strain 4 appearing slightly darker. However, in high-GCL fermentation medium (bottom of the figure), the bacterial broth of strain 4 was slightly brown, that of strain 5 was slightly yellow, and that of black yeast 12F0291 and strain 3 was off-white.

Please refer to Figure 10 and Table 16, which show the _OD600 and viable cell count results of bacterial cultures after 24 and 48 hours of seed culture, respectively. As can be seen, the four strains used in this example exhibited similar growth rates after 24 hours of culture, with no significant differences. However, after 48 hours of culture, strain 4 exhibited a significantly lower _OD600 , indicating a slower growth rate than strain 3. The growth rates of black yeast 12F0291 and strain 5 were similar.

Table 16 Viable bacteria count results after seed culture for 48 hours

Please refer to Figure 12, which shows the analysis results of β-glucan content in the fermentation broth after culturing the strains for 2, 5, 6, and 8 days. As shown in the figure, the β-glucan content in the fermentation broth of black yeast strain 12F0291, strains 4, and 5 increases with fermentation time. After 8 days of culturing, strain 4 produced a β-glucan content of approximately 3 g/L, lower than the β-glucan content produced by the other three strains after 8 days of fermentation. Notably, strain 3 produced approximately 8 g/L of β-glucan after 2 days of culturing. However, after 5 days of culturing, the β-glucan content in the fermentation broth dropped to 4 g/L, then increased significantly to 16 g/L after 8 days of culturing. In summary, although the amount of β-glucan produced by each strain varied, the methods and fermentation medium according to the present invention were all capable of producing β-glucan.

In one embodiment, a black yeast culture containing β-glucan is provided, produced by the aforementioned method. It is noteworthy that the culture produced by the aforementioned method can be further centrifuged or filtered to remove the black yeast cells. In one embodiment, the black yeast culture without cell removal can be used in feed, pet health products, biological preparations, and antagonistic yeasts. In another embodiment, the black yeast culture without cell removal can be used in food, liquid beverages, beauty drinks, and cosmetics.

Therefore, in another embodiment, a composition is provided, comprising the black yeast culture and an optional carrier. The composition can be used in fields including, but not limited to, pharmaceuticals, food, and the like.

The composition according to the present invention can be prepared into a dosage form suitable for parenteral, topical or oral use using techniques well known to those skilled in the art.

Preferably, the composition can be prepared into a dosage form suitable for oral administration, including but not limited to: solution, suspension, emulsion, powder, tablet, pill, syrup, lozenge, troche, chewing gum, capsule, slurry and the like.

As used herein, the term "carrier" may refer to a carrier that does not cause allergic reactions or other undesirable effects in the subject to which it is administered. In the present application, the carrier may include one or more agents selected from the following: a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent, an excipient, a stabilizer, a chelating agent, a diluent, a gelling agent, a preservative, a lubricant, or the like.

Therefore, the black yeast culture or the composition containing the same can be used as a food additive and added during the preparation of raw materials by known methods, or formulated into food products for consumption by humans and non-human animals.

According to an embodiment of the present invention, the types of food products include, but are not limited to, milk powder, beverages, confectionery, candies, fermented foods, animal feeds, health foods, dietary supplements, jellies, infant formulas, dressings, mayonnaise, spreads, creams, sauces, puddings, ice-cream, bakery products, ketchup, mustard, antistaling agent, biocontrol agent (BCA) or antagonist yeast, etc.

In summary, the black yeast according to the embodiments of the present invention has the advantage of not producing melanin (as shown in FIG9 ). Furthermore, by improving the composition of the culture medium and the culture method, the yield of β-glucan can be effectively increased.

Furthermore, by improving the β-glucan production process and culture medium composition formula, the fermentation broth of black yeast can be entirely used to prepare β-glucan products, resulting in products with high β-glucan content. In addition to their efficacy, these products are also easy to store and process, while also reducing process waste liquid emissions, achieving energy conservation, carbon reduction, and environmental friendliness. In addition to preserving β-glucan, they also retain the functional substances contained in black yeast.

The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or variations that do not depart from the spirit and scope of the present invention shall be included in the scope defined by the scope of the patent application.

Claims (18)

1. A black yeast (Aureobasidium pullulans) that was deposited on October 19, 2016 at the Patent Microbial Collection Center of the Technical Base for Product Evaluation of Japan, with accession number NITE BP-02372. 2. A fermentation medium for the production of β-glucan, comprising a carbon source, lecithin, and ascorbic acid; The carbon source is selected from lactose, fructose, maltose, glucose, galactose, xylose, xylitol, inulin, sorbitol, trehalose, sucrose, molasses, and combinations thereof. 3. The fermentation medium according to claim 2, wherein the carbon source is sucrose or glucose. 4. The fermentation medium according to claim 3, wherein the concentration of sucrose is 10-70 g/L based on the total volume of the fermentation medium. 5. The fermentation medium of claim 3, wherein the concentration of glucose is 25-150 g/L based on the total volume of the fermentation medium. 6. The fermentation medium of claim 2, wherein the concentration of lecithin is less than or equal to 6 g/L based on the total volume of the fermentation medium. 7. The fermentation medium of claim 2, wherein the concentration of ascorbic acid is less than or equal to 6 g/L based on the total volume of the fermentation medium. 8. The fermentation medium of claim 2, wherein the initial pH of the medium is 4 to 8. 9. The fermentation medium of claim 8, wherein the initial pH of the medium is 5 to 6. 10. A method for producing β-glucan, comprising: culturing the black yeast of claim 1 in a fermentation medium for fermentation. 11. The method of claim 10, wherein the fermentation medium is the fermentation medium of any one of claims 2 to 9. 12. The method of claim 10, wherein the black yeast is cultured at a temperature of 15 to 30°C. 13. The method of claim 10, wherein the fermentation is carried out at a rotation speed of 150 to 350 rpm. 14. The method of claim 10, wherein the fermentation is carried out under an aeration rate of 1 to 2 vvm. 15. The method of claim 10, further comprising proliferating the black yeast to a stationary phase in a seed culture medium before fermentation. 16. The method of claim 15, wherein the seed culture medium comprises yeast extract, malt extract, peptone, dextrose, and water. 17. A black yeast culture containing β-glucan, which is prepared by the method of any one of claims 10 to 16. 18. A composition comprising a black yeast culture as claimed in claim 17, and optionally a carrier.