TWI712689B - CULTURING MEDIUM AND METHOD FOR PRODUCING β-GLUCAN, A CULTURE OF AUREOBASIDIUM PULLULANS AND A COMPOSITION COMPRISING THE SAME - Google Patents

Abstract

The present application provides a culturing medium for producing β-glucan. The culturing medium comprises: a carbon source, a lecithin and an ascorbic acid. The present application further provides a method for producing β-glucan, comprising culturing Aureobasidium pullulans in the culturing medium so as to ferment. The yield of the β-glucan may be increased and the waste may be reduced by the improvements of the culturing medium and the method of producing the β-glucan.

Description

Medium and method for producing β-glucan, black yeast culture and containing it Composition

The present invention relates to a fermentation medium, in particular to a method for producing β-glucan by black yeast through the fermentation medium, so as to achieve a method for producing β-glucan with high yield.

Aureobasidium pullulans , also known as Aureobasidium pullulans or Aureobasidium pullulans , is a yeast-like fungus. The periphery of the black yeast cell wall produces polysaccharides, such as β-glucan.

Beta-glucan has been one of the raw materials for food use announced by the Taiwan Food and Drug Administration and the Japanese Ministry of Health, Labour and Welfare. It is also a generally recognized as safe (GRAS, Generally Recognized As Safe) food additive announced by the US FDA. There is no doubt about food safety in development.

When extracting effective ingredients such as β-glucan from sources such as barley, oats, yeast, and mushrooms, it must go through a treatment process that uses a variety of chemicals. In these processing procedures, many beneficial trace components may be lost, and the product fails to obtain the expected effect. The β-glucan produced by plants will affect the content of β-glucan depending on whether it is wild or cultivated, or depending on the conditions and environment of plant growth. Compared to the processes of sources such as barley, oats, yeast and mushrooms, the use of black yeast When β-glucan is produced, since β-glucan is secreted to the outside of the cell, the extraction process can be omitted and the production of waste liquid can be reduced. In addition, the production of β-glucan by black yeast does not have the disadvantage of uncovering β-glucan produced by plants.

However, although the use of black yeast to produce β-glucan has the above advantages, the production of β-glucan through the fermentation of black yeast is still limited due to the following reasons:

(1) Low yield: soluble extracellular polysaccharides are produced during the fermentation of black yeast, but as the production of polysaccharides increases, the overall fermentation broth is likely to form a viscous gel. This high-viscosity characteristic is likely to cause aeration , Dissolved oxygen, stirring and other mass or heat transfer issues, which in turn affect the production of β-glucan.

(2) Polysaccharides containing melanin are difficult to remove: the extracellular polysaccharides produced by the fermentation of black yeast strains generally contain melanin, so the product sales are poor. If the melanin is to be further removed, it will increase the inconvenience of the manufacturing process, and further affect the subsequent production process of its commercialization.

In view of the above-mentioned problems of the prior art, the purpose of the present invention is to provide a black yeast fermentation medium, which can enable the black yeast to have higher β-glucan production.

According to one objective of the present invention, a black yeast fermentation medium is provided, which includes: carbon source, lecithin and ascorbic acid; wherein the carbon source is selected from the following groups: lactose, fructose, maltose, glucose, galactose , Xylose, xylitol, inulin, sorbitol, trehalose, sucrose, molasses and combinations thereof.

Preferably, the carbon source may be sucrose or glucose.

Preferably, the concentration of sucrose can be 10-70 g/L; it is based on the total volume of the aforementioned fermentation medium. Preferably, the concentration of glucose can be 25~150g/L; it is based on the aforementioned fermentation medium Based on the total volume.

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

The initial pH of the medium can be 4-8. More preferably, the initial pH value of the medium may be 5-6.

Preferably, the black yeast ( Aureobasidium pullulans ) can be deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on September 22, 2015, with the deposit number BCRC 930184.

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

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

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

Preferably, the fermentation is carried out under the condition of aeration of 1 to 2 vvm.

Preferably, it may further include propagating the black yeast in the seed culture medium to the stationary phase before the fermentation.

Preferably, the inoculum culture medium may include yeast extract granulated, malt extract, peptide from soybean (enzymatic digest), dextrose and water.

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

According to another object of the present invention, a composition is provided, which may include the aforementioned yeast Bacteria culture, and optionally carrier.

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

(1) Through the improvement of the medium composition and culture method of the present invention, the yield of β-glucan can be effectively increased.

(2) Through the production process of β-glucan and the improvement of the medium composition formula, the fermentation broth of black yeast can be used for the preparation of β-glucan products to obtain products with high β-glucan content In addition to its efficacy, the product can also be easily stored, processed and used, and at the same time, it can reduce the discharge of waste liquid in the process to achieve the goals of energy saving, carbon reduction and environmental friendliness.

The above and other objects, features, and advantages of the present invention will become apparent with reference to the following detailed description, preferred embodiments, and drawings attached to the text.

Figure 1 shows the morphological characteristics and microstructure of the black yeast 12F0291. (A) Colony front, MEA, 25°C, 5 days; (B) Colony back, MEA, 25°C, 5 days; (C) Colony front, PDA, 25°C, 5 days; (D) Colony back, PDA, 25℃, 5 days; (E) hyphae and sporulation structure (Bar=10μm); (F) conidia (Bar=10μm); (G) thick-walled spores (Bar=5μm).

Figure 2 shows the results of determination of the concentration of β-glucan produced by fermentation media with different initial pH values.

Figure 3 shows the results of determination of the concentration of β-glucan produced in fermentation media with different carbon sources.

Figure 4 shows the results of determination of the concentration of β-glucan produced in fermentation media with different concentrations of sucrose.

Figure 5 shows the results of determination of the concentration of β-glucan produced in fermentation media with different concentrations of glucose.

Figure 6 shows the results of determination of the concentration of β-glucan produced in GCL fermentation media with different nitrogen sources.

Fig. 7 Measured results of the concentration of β-glucan produced by culturing black yeast with or without lecithin.

Fig. 8 is a schematic diagram showing the process of thawing, culturing and fermentation of the glycerol tube of the self-frozen black yeast strain.

Figure 9 shows the appearance of colonies and shake flasks after cultivation and fermentation of black yeast in YM medium or fermentation medium.

Figure 10 shows the cell mass (expressed in OD ₆₀₀ absorbance) of the black yeast after culturing in YM medium for 1 or 2 days.

Figure 11 shows the colony appearance of the black yeast after being cultured in YM medium for 48 hours.

Figure 12 shows the analysis results of β-glucan content in the fermentation broth after fermenting the black yeast for 2, 5, 6, and 8 days.

In the following detailed description, in order to explain the present invention, many specific details are provided in order to thoroughly understand the disclosed embodiments. However, it is obvious that one or more embodiments may be implemented without the specific details. In other cases, in order to simplify the drawings, the conventional structure and process will be shown in a schematic manner.

The details of each specific example of the present invention are described below. Other features of the invention will It is clearly presented through the detailed description and the scope of patent application in the following specific examples.

Without further elaboration, it is believed that those with ordinary knowledge in the technical field of the present invention can use the present invention to the widest extent based on the foregoing description. Therefore, it can be understood that the following description is only for illustrative purposes, and is not intended to limit the remaining disclosure content in any way.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs.

The article "a" used herein refers to one or more (ie, at least one) grammatical acceptors of the article.

Screening of Potential Black Yeast Strains

Initial screening:

After activating the frozen strain of Aureobasidium pullulans stored in the frozen vial, apply it to the primary screening medium (the ingredients are shown in Table 1 below) and incubate at 25°C for 5-7 days. Observe the colony shape and color, and select Strains with obvious color changes after cultivation. In the preliminary screening, a total of 530 strains were screened, and 30 potential candidate strains were obtained for subsequent screening.

pH 6.2±0.2

Screening of candidate strains

From the 30 candidate strains selected in the preliminary screening, the potential production strains were further screened for production tests. In brief, after the candidate strain was activated, it was cultured with shaking in YMS medium containing sucrose (the composition is shown in Table 2 below), and cultured at 25°C for 4 days. Next, after sampling the culture medium, pullulanase is used to decompose pullulan, and then the total sugar content is determined by Phenol-sulfuric acid assay to select strains with higher non-pullulan polysaccharide production.

pH 6.2±0.2

Next, the black yeast strains with the potential to produce β-glucan, which were screened out above, were activated with YM medium (Difco ^TM ), and then the YMS medium was used for preliminary shake flask test. The culture conditions were 25° C., 150 rpm, and 72 Measure the β-glucan in the fermentation broth of the shake flask after hours. The measurement method is as follows, and the measurement results are shown in Table 3 below. Among them, LQ is a black yeast fermentation broth product purchased from ADEKA, Japan, and the product specification is β- The dextran content is 1% (10g/L), as a positive control group.

Black yeast beta-glucan measurement method

Use the Total Dietary Fiber Assay kit (Megazyme), first take the fermentation broth and add an appropriate volume of buffer to mix well, and then add α-amylase and protease in sequence After treatment with three enzymes and Amyloglucosidase (Amyloglucosidase), precipitation with 4 times the volume of alcohol, the β-glucan is separated from the solution, the precipitate is collected and washed with alcohol and dried. The dried precipitate is Strong acid and high temperature hydrolysis treatment, after acid-base neutralization, determine glucose, and calculate β-glucan content. The detailed steps are as follows: when the sample to be tested is fermentation broth, weigh 5 g of the sample, add 3.5 mL, 0.08 M phosphate buffer (pH 6.0), and mix it thoroughly.

The sample was added to 500μL of α-amylase (60mg/mL PB buffer) and reacted in a water bath at 95°C for 30 minutes, shaking every 15 minutes to make the mixture uniform. 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 (approximately 800 μL) with 0.325M HCl, 50 μL of amyloglucosidase was added and reacted in a 60°C water bath for 30 minutes to obtain an enzyme-treated sample.

After the enzyme treatment sample was added to 40mL with 95% EtOH, let it stand at room temperature for one hour to precipitate β-glucan, and make β-glucan separate from the solution, and then centrifuge (10,000g) for 10 minutes Remove the supernatant. After collecting the precipitate, add 4mL 80% EtOH to rinse, and then centrifuge (10,000g) for 5 minutes to remove the supernatant, then vacuum and dry it at room temperature.

Add 1 mL of 72% (w/v) sulfuric acid to the sample dried overnight, and then add 14 mL of deionized water after standing at room temperature for 60 minutes. Treat it in a boiling water bath (100°C) for 2 hours. If there are large pieces of residue, perform Autoclave sterilization (121°C, 20min). After the hydrolyzate was cooled, it was neutralized with 5 mL of 5N NaOH, and the volume was adjusted to 25 mL with deionized water, and then the glucose content was measured with the glucose PAP kit.

In the process of enzyme treatment, the α-bonded polysaccharide in the fermentation broth is decomposed by α-amylase, and the large-molecule proteins in the fermentation broth are also decomposed by proteolytic enzymes, and finally the short-chain polymerized glucose chain is used for starch After the glucosidase is decomposed into glucose, the polymerized glucose long chain that has not been decomposed by the above enzymes (the remaining β-bonded glucan at this time) is then separated from the solution by alcohol precipitation and separated for a drying step. After drying, the glucose content is measured by sulfuric acid and high temperature hydrolysis, which is the content of β-bonded glucan. Finally, the following formula is used to calculate the glucan weight (W _BGP ) based on the glucose concentration (C _glu ) measured by the enzyme method.

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

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

Strain identification

According to an embodiment of the present invention, a black yeast strain 12F0291 with β-glucan production potential was screened from the black yeast strain bank of the Food Industry Development Institute and deposited in China on September 22, 2015 The Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China, the deposit number is BCRC 930184.

Black yeast 12F0291 was isolated and sampled from plant leaves in Xinpu Town, Hsinchu County, Taiwan on June 17, 2008. As shown in part (A) and (B) of Figure 1, the black yeast 12F0291 is placed in the MEA (Malt Extract Agar, Blakeslee's Formula) medium shown in Table 4 below. When cultured at 25°C, the colony is smooth and sticky. And the surface has transparent mucus, the color changes from gray-yellow to orange to brown, and the surrounding colonies are yellow-white. Please refer to Figure 1(C) and (D). Place the black yeast 12F0291 in the PDA (Potato Dextrose Agar, Difco ^TM ) medium shown in Table 5 below. When cultured at 25°C, the colony is smooth and viscous. , And the surface has transparent mucus, the color changes from yellow-white to orange-red, and the surrounding colonies are yellow-white.

Please refer to Figure 1(E) to (G). Observing the black yeast 12F0291 through a microscope, its hyphae are smooth and septa, with slight constriction near the septum, thin to thick, with a diameter of 3.4-5.8μm. As the culture time increases, part of the hypha gradually turns orange-brown. Conidiogenous cells are undifferentiated, located in the middle or the end of the transparent hyphae, and occasionally grow from branches, with denticles, conidia, smooth outer walls and variable shapes. The size is 4.0-11.0×3.0-6.0μm. Chlamydospores (chlamydospores) are transparent or orange-brown, approximately spherical or elliptical in shape, and 4.7-9.0×2.9-6.5 μm in size.

Sequence alignment of rDNA ITS1-5.8S-ITS2 fragments of black yeast 12F0291 showed that black yeast 12F0291 and Aureobasidium pullulans var. melanogenum BCRC 34543T (=CBS 105.22T) (BCRC database no.BCRC34543_05042009_FD_ITS) and Aureobasidium pullulans NRRL Y-12996 (=ATCC 42023) (GenBank accession no.HQ702508) is the closest.

Based on the aforementioned morphological characteristics such as colony, sporulation structure, conidia, and rDNA ITTS-5.8S-ITSS2 sequence analysis results, it was identified that the black yeast 12F0291 belongs to Aureobasidium pulllulans and is Aureobasidium pulllulans var. melanogenum .

Fermentation medium for producing β-glucan

In order to solve the aforementioned problems, an embodiment of the present invention relates to a medium for producing β-glucan. The culture medium may include: carbon source, nitrogen source and ascorbic acid; wherein the carbon source may be selected from the following groups: lactose, fructose, maltose, glucose, galactose, xylose, xylitol, inulin, sorbitol, Trehalose, sucrose, molasses and combinations thereof; and the nitrogen source can be selected from the following groups: peptide from soybean, enzymatic digest, yeast extract granulated, urea, potassium nitrate, sodium nitrate, Ammonium sulfate, lecithin (lecithin) and combinations thereof.

In a preferred embodiment, the carbon source can be sucrose or glucose. In a more preferred embodiment, the concentration of sucrose may be 10 to 70 g/L or the concentration of glucose may be 25 to 150 g/L, and the concentration is 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 an embodiment of the present invention, the medium for producing β-glucan can be prepared by the following method. Ascorbic acid is added when preparing the medium, and the pH is about 3.0 at this time. Then use NaOH _(aq) to adjust the pH to 4.0 to 8.0, preferably 5.0 to 6.0, then autoclave (Autoclave) sterilization, and then connect after cooling. Into the seed bacteria. In another embodiment, the pH value of the medium can also be adjusted to 12.0 when preparing the medium. After autoclave sterilization, the ascorbic acid solution is filtered in a sterile operating table with a 0.22μm filter and then added to the sterilized medium , Make the pH value of the culture medium between 4.0 and 8.0, preferably between 5.0 and 6.0, and then insert the inoculum. In a preferred embodiment, the concentration of ascorbic acid may be less than or equal to 6 g/L.

According to an embodiment of the present invention, the thawed black yeast 12F0291 glycerol tube was activated with 0.3% (v/v) inoculation amount into YM medium (the ingredients are shown in Table 6 below), and the temperature was 25°C and the rotation speed Incubate at 150 rpm and 48 hours for inoculation, and then inoculate the following (Table 7 to Table 13) different fermentation media. Test the β-glucan in each fermentation media at a temperature of 25°C, a rotation speed of 150 rpm, and culture for 2 days. The measurement method is the same as that described above, so I won’t repeat it here.

pH 6.2±0.2

In one example, the black yeast 12F0291 was inoculated into SCL fermentation medium 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 fermentation production was determined. The concentration of β-glucan. The "initial pH value" as used herein refers to the pH value of a culture medium when it has been configured and has not undergone any cultivation. Please refer to Figure 2, which is a measurement result of the concentration of β-glucan produced by fermentation media with different initial pH values. As shown in the figure, in the fermentation medium with the initial pH value of 4.0 to 8.0, the black yeast 12F0291 can obviously produce more β-glucan, and the fermentation medium with the initial pH value of 5.0 and 6.0 The black yeast 12F0291 produced the most β-glucans, with concentrations of 7.95g/L and 8.23g/L, respectively.

In one embodiment, the black yeast 12F0291 is inoculated into fermentation media with different carbon sources for fermentation to produce β-glucan. Specifically, in this example, black yeast 12F0291 was inoculated with sucrose (composition shown in Table 7), glucose (composition shown in Table 8) or molasses (Molasses, composition shown in Table 9) as Fermentation is carried out in the fermentation medium of the carbon source, and the concentration of β-glucan produced by the fermentation is measured. Please refer to Figure 3, which is the measurement results of the concentration of β-glucan produced in fermentation media with different carbon sources. As shown in the figure, whether it is a fermentation medium that uses sucrose (SCL), glucose (GCL) or molasses (MCL) as a carbon source, the black yeast 12F0291 can produce β-glucan, which is cultivated in the use of sucrose (SCL) and glucose (GCL) as a carbon source in the fermentation medium, the black yeast 12F0291 can produce more β-glucan, about 9g/L.

Table 7 SCL fermentation medium

pH 5.5-5.6

pH 5.5-5.6

pH 5.5-5.6

In a preferred embodiment, the black yeast 12F0291 is inoculated in a fermentation medium with different concentrations of sucrose for fermentation to produce β-glucan. Specifically, in this example, the black yeast 12F0291 was respectively inoculated into a fermentation medium containing 0, 10, 20, 30, 40, and 50 g/L (the ingredients are shown in Table 10) for fermentation, and the measurement was performed. The concentration of β-glucan produced by fermentation. Please refer to Figure 4 for the results, which is the result of measuring the concentration of β-glucan produced by the fermentation medium of different concentrations of sucrose using black yeast 12F0291. As shown in the figure, the concentration of β-glucan produced by fermentation increases with the concentration of sucrose in the fermentation medium. In this example, the highest concentration of β-glucan can be obtained in the fermentation medium with 50 g/L sucrose, which is about 8 g/L.

pH 5.5-5.6

In a preferred embodiment, the black yeast 12F0291 is inoculated into a fermentation medium with different concentrations of glucose for fermentation to produce β-glucan. Specifically, in this example, the black yeast 12F0291 was respectively inoculated into a fermentation medium containing 25, 50, 75, 100, 125, and 150 g/L glucose (the ingredients are shown in Table 11) for fermentation, and Determine the concentration of β-glucan produced by fermentation. Please refer to Figure 5 for the results, which is the measurement result of the concentration of β-glucan produced by the fermentation medium of different concentrations of glucose using the black yeast 12F0291. As shown in the figure, the concentration of β-glucan produced by fermentation roughly increases with the concentration of glucose in the fermentation medium. In this example, the highest concentration of β-glucan can be obtained in the fermentation medium with 100g/L glucose. Dextran, about 9.31g/L.

pH 5.5-5.6

In one embodiment, the GCL fermentation medium shown in Table 8 is used as the basic medium, and urea, protein decomposition product (Peptone from soybean, enzymatic digest), yeast extract granulated, and nitric acid are added separately. Different nitrogen sources such as potassium (KNO ₃ ), sodium nitrate (NaNO ₃ ), and ammonium sulfate ((NH ₄ ) ₂ SO ₄ ). In one embodiment, the concentration of the nitrogen source may be 2, 4, and 8 g/L, respectively, and the initial pH value of the culture medium with different concentration of the nitrogen source may be adjusted sequentially as shown in Table 12. The bacteria solution after 48 hours of culture in YM medium will be inoculated into GCL fermentation medium containing different nitrogen sources at 20% (v/v) inoculation amount, and cultured in shake flask at 25°C, 150rpm The β-glucan content was analyzed after 48 hours.

Please refer to Figure 6 for the results, which is the measurement result of the concentration of β-glucan produced by the black yeast 12F0291 in the GCL fermentation medium with different nitrogen sources. As shown in the figure, in the fermentation medium containing different nitrogen sources, the black yeast 12F0291 can be fermented to produce β-glucan. It is worth noting that the three groups (groups 3, 4 and 5) of protein decomposition (Peptone from soybean, enzymatic digest), yeast extract (Yeast extract granulated) and potassium nitrate (KNO ₃ ) (groups 3, 4 and 5) will be added As the concentration of nitrogen source increases, the concentration of β-glucan tends to gradually decrease. In the two groups of urea (Urea) and sodium nitrate (NaNO ₃ ) (groups 2, 6), the group with a nitrogen source concentration of 4g/L has higher β-glucan production, but it is relatively In the group with a nitrogen source concentration of 2g/L, the yield of β-glucan was not greatly improved, and in the group with a nitrogen source concentration of 8g/L, the concentration of β-glucan was the lowest. In the group that only added lecithin (group 1), the black yeast 12F0291 was fermented for 48 hours to produce about 8g/L of β-glucan, and its yield was higher than that of the group that added ammonium sulfate About 4 times.

Fermentation medium for producing β-glucan without nitrogen source

In this example, the black yeast 12F0291 was inoculated in a fermentation medium containing no nitrogen source for fermentation to produce β-glucan. Specifically, in this embodiment, the black yeast 12F0291 is cultured in a medium for producing β-glucan for fermentation. The medium includes a carbon source and ascorbic acid; the carbon source can be selected from the following group: lactose, fructose, maltose, glucose, galactose, xylose, xylitol, inulin, sorbitol, trehalose, sucrose and combinations thereof. Among them, the medium may not contain a nitrogen source.

In a specific embodiment, lecithin is used as the nitrogen source used in the fermentation of black yeast 12F0291. That is to say, the black yeast 12F0291 is respectively inoculated into a fermentation medium containing lecithin (such as the SCL fermentation medium in Table 7) as a nitrogen source or without a nitrogen source (components such as the SC fermentation medium in Table 13) for fermentation, and Determine the concentration of β-glucan produced by fermentation. Please refer to FIG. 7, which is the measurement result of the concentration of β-glucan produced by culturing the black yeast 12F0291 in a fermentation medium with or without a nitrogen source. As shown in the figure, whether it is a fermentation medium containing or without lecithin, the black yeast Both 12F0291 can produce β-glucan. However, the black yeast 12F0291 cultivated in the fermentation medium containing lecithin can produce more β-glucan, about 9.37g/L, and the black yeast cultivated in the fermentation medium without lecithin 12F0291 can produce about 2.74g/L glucan.

pH 5.5-5.6

Production process of β-glucan

In this embodiment, a method for producing β-glucan is proposed, which includes culturing the aforementioned black yeast in a fermentation medium for fermentation. In an embodiment, the fermentation medium can be various media as mentioned above, and can be cultured 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 To cultivate. In one embodiment, the culture can be carried out at a rotating speed of 100-450 rpm; preferably, the culture can be carried out at a rotating speed of 120-400 rpm; most preferably, the culture can be carried out at a rotating speed of 150-350 rpm. In one embodiment, the culture can be carried out under the condition of ventilation volume of 0.5~3vvm; preferably, the culture can be carried out under the condition of ventilation volume of 0.8~2.5vvm; best, it can be cultured under the condition of ventilation volume of 1~2vvm. Cultivate under the conditions.

Please refer to Table 14 below, which lists several exemplary specific culture conditions under which the black yeast was cultured and fermented. Then, by measuring the content of β-glucan in the fermentation broth, it can be found that the black yeast can produce β-glucan under these culture conditions (not shown).

In detail, please refer to Figure 8 to Figure 12. Figure 8 is a schematic diagram showing the process of thawing, culturing, and fermenting the glycerol tube of the black yeast strain 12F0291 and the potential strains 3, 4, and 5 shown in Table 3. Inoculate the frozen black yeast strain in the strain glycerol tube into the YM medium in Table 6 with 0.3% inoculation amount. After incubating in a shake flask for 48 hours at 25°C and 150 rpm, the bacterial solution is changed to inoculation In the high GCL fermentation medium shown in Table 15, fermentation was carried out at 25° C. and 150 rpm for 2, 5, 6, and 8 days to produce β-glucan. In a preferred embodiment, the black yeast is cultured in YM medium to allow the bacteria to proliferate to a stationary phase, and then ferment in the fermentation medium.

pH 5.5-5.6

As shown in Figures 9 to 11, the black yeast is cultured in YM medium or fermentation medium, the colony appearance after fermentation, the appearance of the shake flask and the count result of the bacterial count. Please refer to FIG. 9, when cultured in YM medium (in the figure), the color of the bacterial liquid of the four strains is beige, and the color of strain No. 4 is slightly darker. However, in the high GCL fermentation medium (bottom side of the figure), the bacterial liquid of strain number 4 is slightly brown, the bacterial liquid of strain number 5 is slightly yellow, and the bacterial liquid of black yeast 12F0291 and strain number 3 is off-white.

Please refer to Figure 10 and Table 16, which are the results of the determination of the OD ₆₀₀ and the count of the number of viable bacteria of the inoculum culture for 24 and 48 hours, respectively. It can be seen that the growth rates of the four strains used in this example after 24 hours of cultivation are similar, and there is no significant difference. However, after 48 hours of culture, the OD ₆₀₀ of the strain with strain number 4 was significantly lower, indicating that its growth rate was lower than that of the strain with strain number 3. The growth rate of the black yeast 12F0291 and the strain No. 5 are similar.

Please refer to Figure 12, which is the analysis result of β-glucan content in the fermentation broth after the aforementioned strains were cultured for 2, 5, 6 and 8 days. As can be seen from the figure, black yeast 12F0291 and strain number 4, strain number 5 The content of β-glucan in the fermentation broth of the strain will increase as the fermentation time increases. The β-glucan content of the strain No. 4 after being cultured for 8 days was about 3g/L, which was lower than the β-glucan content produced by the remaining 3 strains after 8 days of fermentation. It is worth noting that the content of β-glucan produced by the strain No. 3 was about 8g/L after 2 days of culture, but after 5 days of culture, the content of β-glucan in the fermentation broth decreased 4g/L, and then increased to 16g/L after 8 days of culture. To sum up, although the amount of β-glucan produced by each strain is different, it is true that the aforementioned black yeast strain can produce β-glucan by using the method and fermentation medium according to the embodiment of the present invention.

In one embodiment, a black yeast culture containing β-glucan is provided, which is prepared by the aforementioned method. It is worth noting that the culture prepared according to the aforementioned method can be further centrifuged or filtered to remove the black yeast cells. In one aspect, the black yeast culture without removing the bacteria can be applied to feed, pet health products, biological preparations, and antagonistic yeasts. In another embodiment, the black yeast culture with the bacteria removed can be applied to foods, liquid beverages, beauty drinks, cosmetics and skin care products.

Therefore, in another embodiment, a composition is provided, which includes the aforementioned black yeast culture and a selective carrier. The composition can be applied to, including but not limited to, the fields of medicine or food.

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

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

As used herein, the term "carrier" may refer to a carrier that, when administered, does not cause an allergic reaction or other undesirable effects in the administered individual. In the present application, the carrier may include one or more reagents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer, binding agent , Excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant or the like Things.

Therefore, the black yeast culture or the composition containing it can be used as a food additive, and can be added during the preparation of raw materials by a conventional method, or formulated for human and non-human animals. Ingested food products.

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, and animal feeds. , Health foods, dietary supplements, jellys, infant formulas, dressings, mayonnaise, spreads, creams ), sauces, puddings, ice-cream, bakery products, ketchup, mustard, antistaling agent, biocontrol Agnet, BCA) or antagonisitic yeast, etc.

In summary, the black yeast according to an embodiment of the present invention has the advantage of not producing melanin (as shown in FIG. 9). And, through the improvement of the aforementioned medium composition and culture method, It can effectively increase the yield of β-glucan.

Furthermore, through the production process of β-glucan and the improvement of the medium composition formula, the fermentation broth of the black yeast can be used for the preparation of β-glucan products to obtain products with high β-glucan content. In addition to its efficacy, the product can also be easily stored, processed and used, while reducing the discharge of process waste, achieving the goals of energy saving, carbon reduction and environmental friendliness. In addition to retaining β-glucan, it can also retain the content of black yeast Functional substance.

The above description is only exemplary and not restrictive. Any equivalent modifications or changes made to the present invention without departing from the spirit and scope of the present invention shall be included in the scope defined by the scope of the patent application.

【Biological Material Deposit】

Biological Resources Conservation and Research Center, Food Industry Development Institute of the Republic of China, September 22, 2015, the deposit number is BCRC 930184.

Claims (13)

A black yeast fermentation medium, comprising: a carbon source, a lecithin (lecithin) and an ascorbic acid; wherein the carbon source is sucrose or glucose, wherein the concentration of the sucrose is 10~70g/L, and the concentration of the glucose is 25~150g/L; it is based on the total volume of the aforementioned fermentation medium. The fermentation medium described in item 1 of the scope of patent application, wherein the concentration of the lecithin is less than or equal to 6 g/L; it is based on the total volume of the aforementioned fermentation medium. The fermentation medium as described in item 1 of the scope of patent application, wherein the concentration of the ascorbic acid is less than or equal to 6 g/L; it is based on the total volume of the aforementioned fermentation medium. The fermentation medium described in item 1 of the scope of the patent application, wherein one of the mediums has an initial pH value of 4-8. The fermentation medium described in item 4 of the scope of patent application, wherein one of the mediums has an initial pH value of 5-6. As for the fermentation medium described in item 1 of the scope of patent application, the black yeast was deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on September 22, 2015, and the deposit number is BCRC 930184. A method for producing β-glucan, which comprises: cultivating a black yeast in the fermentation medium described in any one of items 1 to 6 in the scope of the patent application for fermentation. The method described in item 7 of the scope of patent application, wherein the black yeast is cultured at a temperature of 15 to 30°C. The method described in item 7 of the scope of patent application, wherein the fermentation is carried out at a rotation speed of 150~350rpm. As the method described in item 7 of the scope of patent application, the fermentation is carried out under the condition of aeration of 1 to 2 vvm. As the method described in item 7 of the scope of patent application, before fermentation, the black yeast is propagated in a bacterial culture medium to a stable period. The method described in item 11 of the scope of patent application, wherein the culture medium includes yeast extract granulate, malt extract, peptide from soybean, enzymatic digest, dextrose ( dextrose) and water. According to the method described in item 7 of the scope of patent application, the black yeast was deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on September 22, 2015, and the deposit number is BCRC 930184.

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