JP2003000197A - Composition containing polysaccharide derived from yeast and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition containing a polysaccharide derived from a yeast, containing a large amount of β-glucan in an intact state having high bioactivities, and further to provide a method for producing the composition. SOLUTION: This composition containing the polysaccharide derived from the yeast is obtained by subjecting the yeast to bacteriolysis by allowing an exogenous enzyme containing a proteolytic enzyme to act on the yeast. The total activity of the glucanase of the exogenous enzyme is preferably <=30 unit/g-dried weight of the raw material yeast. The polysaccharide preferably contains >=6 wt.% β-glucan.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a composition containing a yeast-derived polysaccharide having a high β-glucan content in an intact state and a method for producing the same.

[0002]

2. Description of the Related Art The cell wall of yeast has a complicated structure and is composed of polysaccharides, proteins, lipids, etc., which are mainly composed of glucan and sonnan.

Polysaccharides such as glucan have attracted attention for their physiological effects as dietary fiber, and in particular, water-insoluble β-1,3.
Glucans and β-1,6 glucans are known to have antibacterial properties and immune enhancing functions. It is said that such a physiological activity of β-glucan is stronger when the β-glucan molecule is not bound to other components, that is, when it is free and the molecular weight is larger (Yakugaku Zasshi. 120
(5) 413-431 (2000)).

Polysaccharides contained in the yeast cell wall are lysed by autolyzing with salt, sugar, ethyl acetate, etc., or by lysing yeast with cell wall lytic enzymes such as cellulase, hemicellulase, pectin degrading enzyme and glucanase. It was contained in the residue (insoluble fraction other than yeast extract) after recovering the soluble fraction (yeast extract), and has not been used so far.

[0005]

As a method for increasing the purity of polysaccharides such as glucan contained in the residue during the production of yeast extract as described above, there is a method of treating with an alkali or the like. Production of the above yeast extract In the method, that the focus is on extracting yeast extract from the raw material yeast, and commonly used cell wall lytic enzyme has glucanase activity, the amount of β-glucan contained in the residue itself. It was low (usually less than 5%), and the molecular weight of β-glucan decreased due to the decomposition, and a sufficient physiologically active effect could not be expected.

[0006] Therefore, an object of the present invention is to provide a yeast-derived polysaccharide-containing composition containing a large amount of β-glucan in an intact state with high physiological activity and a method for producing the same.

[0007]

In order to achieve the above object, one of the present invention comprises a polysaccharide obtained by lysing yeast by causing an enzyme agent containing a proteolytic enzyme to act on yeast. It is a yeast-derived polysaccharide-containing composition characterized in that.

According to the above invention, it is possible to provide a composition containing a polysaccharide derived from yeast containing a large amount of β-glucan in an intact state with high physiological activity.

In the above invention, the total amount of glucanase activity of the enzyme preparation is 30 uni per dry yeast raw material.
It is preferably t / g or less. According to this aspect,
A composition containing a yeast-derived polysaccharide containing a large amount of β-glucan having a higher molecular weight can be provided.

Further, it is preferable that the polysaccharide contains 6% by mass or more of β-glucan. According to this aspect, the physiological activity possessed by β-glucan can be expected by incorporating the composition into foods and drinks, pharmaceuticals, quasi-drugs and the like, and ingesting and administering the composition.

Another aspect of the present invention is a method for producing a yeast-derived polysaccharide-containing composition, which comprises lysing yeast with an enzyme agent containing a proteolytic enzyme.

According to the above invention, a composition containing a yeast-derived polysaccharide containing a large amount of β-glucan in an intact state with high physiological activity can be obtained in good yield.

In the above invention, the total amount of glucanase activity of the enzyme preparation is 30 uni per dry mass of raw material yeast.
It is preferably t / g or less. According to this aspect,
A composition containing a yeast-derived polysaccharide containing a large amount of β-glucan having a higher molecular weight can be obtained in good yield.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The yeast used in the present invention is not particularly limited as long as it is a yeast that can be generally eaten, and examples thereof include Saccharomyces cerevisiae used as baker's yeast, brewer's yeast, wine yeast, etc., Yeasts belonging to Saccharomyces genus, Torulaspora genus, Candida genus and the like represented by Saccharomyces ubaum and Saccharomyces exiguses can be mentioned. There are no particular restrictions on the conditions for culturing the above-mentioned yeast, and it may be carried out under the usual culturing conditions suitable for each yeast. That is, for example, in the case of baker's yeast, it may be cultivated by using general molasses as a main raw material and by using auxiliary raw materials such as a nitrogen source and a phosphorus source. In addition, commercially available raw yeast, dry yeast and the like can also be used.

The enzyme agent used in the present invention contains a proteolytic enzyme, and other enzymes such as glucanase,
Mannase, cellulase and the like can be included.

Examples of the above-mentioned proteolytic enzyme include endoprotease and exoprotease. Specifically, for example, as commercially available products, "Samoase PC" (trade name, manufactured by Daiwa Kasei Co., Ltd.), "Protin A" (trade name, manufactured by Daiwa Kasei Co., Ltd.), "YL-NL" (trade name) , Manufactured by Amano Pharmaceutical Co., Ltd., and the like, but are not limited thereto.

The term "bacteriolysis" in the present invention means that a part or all of the cell wall of yeast is decomposed by using an enzyme agent containing a proteolytic enzyme as described above. By using an enzyme agent containing a proteolytic enzyme as described above, a yeast-derived polysaccharide containing a large amount of β-glucan in an intact state with high physiological activity can be obtained.

The amount of the enzyme agent to be used can be appropriately selected depending on the reaction conditions and the like, but it is preferable that the total amount of glucanase activity of the proteolytic enzyme and other enzymes used in combination is 30 unit / g or less per dry mass of the raw material yeast. Total amount of glucanase activity per dry mass of raw material yeast is 30 uni
If it exceeds t / g, the obtained yeast cell wall-derived β-glucan is unnecessarily decomposed to impair the intact state, and the yield of the yeast-derived polysaccharide is reduced, which is not preferable.

The glucanase activity referred to in the present invention means the turbidity of the reaction solution (specifically, a wavelength of 660 nm when the enzyme is allowed to act on 1 mg of freeze-dried cells of Saccharomyces cerevisiae (IFO 0309) at 35 ° C. The amount of enzyme that reduces the absorbance at 1) by 1% per minute is defined as 1 unit.

The yeast-derived polysaccharide-containing composition of the present invention can be prepared, for example, as follows.

The culture solution in which the yeast has been cultivated, or the yeast recovered from the culture solution, is suspended in water, and is 0.01% by mass or more, preferably 0.05% by mass, based on the dry mass of the yeast.
After adding the above enzyme agents and reacting at 20 to 70 ° C. for 1 to 36 hours, the enzyme is inactivated by heat treatment or the like. This reaction solution as it is, or after performing a separation operation such as centrifugation, the insoluble fraction is collected, washed with water as necessary, and then appropriately concentrated and dried to obtain the yeast-derived polysaccharide-containing composition of the present invention. be able to.

The yeast-containing polysaccharide-containing composition of the present invention comprises β
-The glucan content is preferably 6% by mass or more, and more preferably 15% by mass or more. If the β-glucan content is less than the above range, a sufficient bioactive effect may not be obtained. Note that β-glucan refers to a polysaccharide composed of glucose in which glucose is bound in a β-type structure, and specifically, β-1,4 glucan (cellulose), β-1,3. Examples include glucan and β-1,6 glucan. However, since the cellulose content in yeast is low, in the present invention, β-1,3 is mainly used.
And β-1,6 glucan.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.
In addition, the glucanase activity (unit / g) and the total amount of glucanase activity (un of the enzymes used in each Example and Comparative Example were measured.
It / g raw material dry yeast) is shown in Table 1.

[0024]

[Table 1]

Example 1 Baker's yeast belonging to the genus Saccharomyces as a raw material yeast (trade name "Kaneka Red Yeast", Kanegafuchi Chemical Industry Co., Ltd.)
30 g (10 g as a dry matter) was suspended in 70 ml of water and heat-treated at 90 ° C. for 60 minutes to inactivate the enzyme in the bacterial cells.

The suspension was cooled to 50 ° C., and proteolytic enzyme (0.2% by mass of “YL-N” was added to dry cells.
L "(trade name, manufactured by Amano Pharmaceutical Co., Ltd.) and 0.5% by mass of" Protin AY-10 "(trade name, manufactured by Daiwa Kasei Co., Ltd.)
) Was added and the enzyme reaction was carried out at 50 ° C. for 24 hours with stirring, and then the enzyme was inactivated by heating at 95 ° C. for 30 minutes.

The reaction solution was centrifuged to collect the insoluble fraction, which was dispersed in water and spray-dried to obtain 4.2 g of a yeast-derived polysaccharide-containing composition (hereinafter referred to as sample A).

After carrying out the enzyme reaction in the same manner as above, the reaction solution in which the enzyme has been deactivated is concentrated without being centrifuged and then spray-dried to give a yeast-derived polysaccharide-containing composition (hereinafter , Sample B) was obtained.

Example 2 In Example 1, 0.5% by mass of glucanase (trade name "Tunicase Fn", manufactured by Amano Pharmaceutical Co., Ltd.) was used in place of the proteolytic enzyme "YL-NL". The enzyme reaction was performed in the same manner as in Example 1 to deactivate the enzyme.
Then, after concentrating the reaction solution without centrifugation,
It was spray-dried to obtain 9.1 g of a yeast-containing polysaccharide-containing composition (hereinafter referred to as sample C).

Example 3 In Example 1, a proteolytic enzyme "YL-15" (trade name, manufactured by Daiwa Kasei Co., Ltd.) having a higher glucanase activity was used in place of the proteolytic enzyme "YL-NL". 5
The enzyme reaction was carried out in the same manner as in Example 1 except that the amount was used to deactivate the enzyme. Then, the reaction solution was concentrated without being centrifuged, and then spray-dried to obtain 9.0 g of a yeast-derived polysaccharide-containing composition (hereinafter referred to as sample D).
Got

Glucanase (trade name “Tunicase Fn”,
The enzyme reaction was carried out in the same manner as in Example 1 except that only 0.5% by mass of Daiwa Kasei Co., Ltd. was used to inactivate the enzyme. Then, this reaction liquid was concentrated without being centrifuged and then spray-dried to obtain 9.2 g of a yeast-derived polysaccharide-containing composition (hereinafter referred to as sample E).

The amount of β-glucan, nitrogen content, and glucose content in the samples obtained in each of the above examples (indicates the degree to which β-glucan in the starting yeast was decomposed by the glucanase activity of the enzyme used). Was measured by the following method. The results are shown in Table 2.

Amount of β-glucan: Obtained after solubilizing carbohydrate in a sample with amylase and precipitating a polysaccharide with 80 (v / v)% ethanol to separate and remove water-soluble monosaccharides and oligosaccharides. The resulting precipitate was hydrolyzed with hydrochloric acid, and glucose was quantified by a colorimetric method, and 90% of the total glucose amount was defined as the β-glucan amount. Nitrogen content: Measured by the Kjeldahl method.

Glucose content: CarboPak PA1 (4 mmφ
× 250 mm) (trade name, manufactured by Dionex) was used to measure by ion chromatography.

[0035]

[Table 2]

From Table 2, it can be seen that each sample of the examples using the enzyme preparation containing the proteolytic enzyme has a higher β-glucan amount and β-glucan yield than the samples of the comparative examples using only glucanase. I understand. In addition, since each sample of the examples has a lower glucose content than the sample of the comparative example, it can be seen that β-glucan is hardly decomposed.

Test Example In the above samples A and C and Example 1, after the enzymatic reaction was completed, heat treatment was performed to inactivate the enzyme, and centrifugation was performed to dry the recovered soluble fraction ( Less than,
1 g of the soluble fraction) was suspended in purified water
After 0 ml, it was sterilized by autoclave, and E. coli powder was suspended in water and then autoclaved (Lyop
hilized cells of St. K-12, manufactured by Sigma),
The effect of improving physiological activity was examined by the following method. In addition,
The effect of improving physiological activity is that human β
It was judged whether to induce and / or enhance the expression of defensin 2 (hereinafter referred to as hBD-2).

Human epidermal keratinocytes (manufactured by Nissui Pharmaceutical Co., Ltd.), which had been cultured to confluence in a 6-well multiplate (manufactured by Falcon), had the above-mentioned samples A, C and soluble fractions at a final concentration of 0 respectively. .5 and 5 mg / m
1 and E. coli powder were added to a final concentration of 0.75 mg / ml, and the mixture was cultured at 37 ° C. in a 5% CO ₂ atmosphere.
The one to which PBS was added served as a control.

After about 16 hours, after removing the medium and washing with PBS, total RNA was extracted from the recovered cultured cells using RNeasy (trade name, manufactured by Qiagen), and each RNA was extracted.
Quantitative RT-PCR was performed using
The mRNA of 2) was amplified.

The quantitative RT-PCR method was carried out using the hBD-2 cDNA.
Sequence No. 1 based on the sequence (Accession No. Z71389)
(QRThBD2f) shown in SEQ ID NO: 2, the primer (qRThBD2r) shown in SEQ ID NO: 2, and the probe (hBD2probe) shown in SEQ ID NO: 3 as an ABIPRISM TaqMan ^™ probe (manufactured by PerkinElmer Japan Co., Ltd.) was prepared and used. The ABI PRISM TaqMan ^TM probe is 5 '
It is a probe with a reporter fluorescent dye attached to the end and a quencher fluorescent dye attached to the 3'end. It does not emit fluorescence because the reporter fluorescent dye and quencher fluorescent dye are in close proximity before the reaction, but during the extension cycle of the PCR reaction To DN
The 5'-3 'exonuclease activity of A polymerase liberates the reporter fluorescent dye and fluoresces. Therefore, it is possible to confirm the accumulation of PCR products by monitoring the fluorescence intensity.

The conditions for the quantitative RT-PCR method are TaqMan EZ
Reverse transcription reaction (60 ° C: 30 minutes, 95 ° C: 5 minutes) using RT-PCR Kit (manufactured by Perkin Elmer Japan Co., Ltd.)
40 cycles, PCR reaction (94 ° C: 20 seconds, 60
ABIPRISM 7700 sequ for 45 cycles of 1 ℃)
The PCR product was detected with an ence detector (manufactured by PerkinElmer Japan Co., Ltd.). Then, the expression level of hBD-2 mRNA was determined using analysis software (Sequence Detector v1.6.3, manufactured by Perkin Elmer Japan Co., Ltd.). As an internal standard for the amount of template mRNA,
GAPDH (gleceraldehyde-3-phosphate dehydrogena
se) mRNA of TaqMan ^TM GAPDH Contorol Rege
It was measured at the same time using nts (manufactured by Perkin Elmer Japan Co., Ltd.). Then, the expression level of the control is 10
The relative expression level was determined as 0%. The result is shown in FIG.

From FIG. 1, the effect of inducing and / or enhancing hBD-2 mRNA was confirmed by adding samples A and C.

On the other hand, no effect of inducing and / or enhancing hBD-2 mRNA was observed in the soluble fraction sample.

[0044]

As described above, according to the present invention,
By lysing yeast with an enzymatic agent containing a proteolytic enzyme, a yeast-derived polysaccharide-containing composition containing a large amount of β-glucan in an intact state with high physiological activity can be provided.

[0045]

[Sequence list]                                SEQUENCE LISTING        <110> Morinaga Seika Kabusiki kaisya       Kanegabuti kagaku kougyou Kabusiki kaisya <120> Composition for inducing and / or enhancing expression of mammalian antimicrobial peptide, containing the composition Food and drink, pharmaceuticals and quasi-drugs <130> MP-1322 <140> <141> <160> 3 <170> PatentIn Ver. 2.1 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> A nucleotide synthesized based on the nucleotide sequence of hBD-2 cDNA. <400> 1 cttccaggtg tttttggtgg ta 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> A nucleotide synthesized based on the nucleotide sequence of hBD-2 cDNA. <400> 2 ggagccatat gtcatccagt c 21 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A nucleotide synthesized based on the nucleotide sequence of hBD-2 cDNA. <400> 3 aggcgatcct gttacctgcc ttaagag 27

[Brief description of drawings]

FIG. 1 hBD-2 mRN of yeast-derived polysaccharide-containing composition
It is a chart showing the result of the expression induction and / or enhancement test of A.

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Claims (5)

[Claims] 1. A yeast-containing polysaccharide-containing composition comprising a polysaccharide obtained by lysing yeast by allowing yeast to act with an enzyme agent containing a proteolytic enzyme. 2. The total amount of glucanase activity of the enzyme preparation is
30 unit / g or less per dry mass of raw material yeast,
The yeast-containing polysaccharide-containing composition according to claim 1. 3. The yeast-derived polysaccharide-containing composition according to claim 1, wherein the polysaccharide contains 6% by mass or more of β-glucan. 4. A method for producing a yeast-derived polysaccharide-containing composition, which comprises lysing yeast with an enzyme agent containing a proteolytic enzyme. 5. The total amount of glucanase activity of the enzyme preparation is
30 unit / g or less per dry mass of raw material yeast,
The method for producing the yeast-derived polysaccharide-containing composition according to claim 4.