JPH01117778A - Yeast having antibacterial activity - Google Patents

Abstract

PURPOSE:To provide specific five yeasts having antibacterial activity to bacteria, capable of preventing contamination by wild bacteria or wild yeasts and useful in production of liquors, alcohol, etc. CONSTITUTION:Metschnikowia pulcherrima (e.g., G-94-3), Candida pintolopesii (e.g., B-48-2), Candida fennica (e.g., N-6-1) and Candida sp. A-31-2 (FERM P-1441) are separated from fruit skin of grape as yeast having antibacterial activities against bacteria. Further Pichia membranaefaciens (e.g., S-105-1) is separated from tree liquid of Fagus crenata. Contamination by wild bacteria, etc., can be prevented by producing liquors, etc., using the above-mentioned five yeast strains and further these yeast cells can be directly utilized for agricultural chemical, animal medicine, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to yeast having antibacterial activity. Yeast is used in the production of foods and alcoholic beverages such as wine, and the present invention can be used in the field of producing foods and alcoholic beverages. It also has potential applications in fields such as medicine and agrochemicals.

Conventional yeasts with antibacterial activity include Aureobasidium, which produces naphthaquinone antibiotics.
A new species of yeast related to the genus sidium [Journal of Antibiotics (J, Antibiotic
s) 37, Nα4.325 (1984)], yeasts belonging to the genera Kluyveromyces and Ioeckera [Applied Empirical Microbiology (
^pp, 8nviron, hat crohiol, )50
, 5.1330 (1985)] and yeast belonging to the genus Satucharomyces [SBC Journal 42, NcL4.
164 (1984)) are known.

Problems to be Solved by the Invention In general, the production of alcoholic beverages and alcohol requires a fermentation process using yeast. In this fermentation process, contamination by wild yeasts and wild bacteria such as lactic acid bacteria and acetic acid bacteria is often a problem. Among these, excellent killer wine yeast has already been put into practical use to solve the problem of contamination by wild yeast [Journal of Fermentation].
John Technology (J, Ferm, Techn)
ol, ) 63, No. 5.421 (1985), WeinWissenschaft
chaft) k4.25B (1985), International Congress of Wine and Wine
However, although the killer substances produced and secreted by killer yeast are effective against yeast, they do not exhibit antibacterial activity against bacteria. Therefore, the problem of bacterial contamination cannot be solved by known killer yeasts.

Therefore, the breeding of brewer's yeast that has antibacterial activity against both yeast and bacteria is very important for the production of alcoholic beverages and alcohol.

Means for Solving the Problems The present inventor conducted a search for yeast that has antibacterial activity against bacteria, and as a result, obtained four strains of yeast that had antibacterial activity from grape skin and one strain from beech sap. and completed the present invention.

The present invention will be explained in detail below.

The present invention is directed to the use of Matschnikovia pulcherima (Matschnikowiap
ulcherrima), candy goobi7)
o /<'7-(Candida pintolop
esii), Candida Fueniriki (Candida
fennica) and Pichia membranaefaciens
ns), and Candida species (Can
dida sp,) A-31-2 (FBRM BP-1
No. 441).

Specifically, the yeast strains of the present invention are Metunikovia pulcherima G-94-3 and Candida pulcherima G-94-3, respectively.
Species 8-31-2, Pichia membranefaciens S-105-1, + Yandida pintropesii 8-48-2, and Candida fuenica N-6-1, with the following mycological properties: shows.

(2) Metunicovia pulcherima G-94-3 This strain was isolated from grape skins in Yamagata Prefecture, and its mycological properties are as follows.

When cultured on a malt extract agar medium at 25°C, the colonies exhibit a cream color. Two weeks after culturing, formation of a reddish-brown pigment is observed. The reddish brown pigment is not a carotenoid pigment. In optical microscopy observation,
Spherical, subglobular, or oval vegetative cells are observed, and true hyphae are not observed. Vegetative cells proliferate by multipolar budding. Aerobic.

Diluted v8 agar (Pitt and Miller,
(1968), when colonies cultured at 20°C were observed under an optical microscope, asci were observed. The baby is
sphaeropedu uncurate
nculate), that is, it consists of a spherical structure with a cylindrical tube that is thinner than its diameter;
7 μm, the tube is 9-25 μm long, 1-1.5 μm in diameter
It is. Inside the ascus, there are 1 to 2 needle-shaped ascospores. This strain cannot assimilate nitrate, and strong insect acidity from glucose is not observed.

This strain was tested in The Yeasts, a taxonomic study, 3rd edition.
omicstudy, third revised
and enlarged edition.

Elsevier 5science Publicat
ion B, V,, N, J, W.

, Kreger-van Rij, 1984)
As a result of searching according to the above mycological properties, Metschnikowia purcherima
a pulcherrima) and Metunikovia pulcherrima G-94-3 (hereinafter G-94-
F [! R.M.B.
It was deposited as No. P-1438.

■The Candida sp. A-31-2 strain was isolated from grape skins in the Yamanashi region.
Its mycological properties are as follows.

When cultured on a malt extract agar medium at 25°C, the colonies exhibit a glossy cream-white color, and no carotenoid pigment production is observed.

Under optical microscopy, it is subspherical or elliptical;
A vegetative cell size of 2.5 to 4.5 μm in length and 1 to 3 μm in width can be accommodated. Vegetative cells proliferate by multipolar budding. True hyphae and pseudohyphae cannot be tolerated. Aerobic.

Teleomorphs are not observed. This strain cannot assimilate nitrate and inositol.

As a result of searching for this strain using "The East's Taxonomic Study - Third Edition", it was concluded from the above mycological properties that it belongs to the genus Candida.

This strain was isolated from Candida sp.
sp, ) ^-31-2 (hereinafter referred to as ^-31-2), and submitted the FORM to the Microtech Institute on August 7, 1988.
It was deposited as BP-1441.

(2) Candida Pinto Strain - 8-48-2 This strain was isolated from the skin of a grape in Shirane Town, Yamanashi Prefecture, and its mycological properties are as follows.

When cultured on a malt extract agar medium at 25°C, the colonies exhibit a cream color. In light microscopic observation, only subglobular or ovoid vegetative cells are observed, and true hyphae and pseudohyphae are not observed. Vegetative cells proliferate by multipolar budding. Aerobic. No teleomorphs were observed, and no carotenoid pigments were produced. This strain is negative in the diazonium blue B (08B) test and cannot assimilate nitrate. It cannot assimilate various sugars such as inosyl), erythritol, maltose, mannitol, galactose, trehalose, cellobiose, and xylose.

It has the ability to ferment glucose, but lacks the ability to ferment sucrose. The growth temperature range is 10-37℃. It exhibits vitamin auxotrophy and cannot grow in vitamin-free media.

When this strain was searched according to "The Yeasts a Taxonomic Study - 3rd Edition," it was found that Candida pintropesii was found based on the mycological properties mentioned above.
a pintolopesii var.

pintolopesi i), named Candida medium pintolopesi 8-48-2 (hereinafter referred to as B-48-2), and submitted it to the Microtech Institute on August 7, 1988.
It was deposited as BRM BP-1440.

(2) Pichia membranefaciens S-105-1 This strain was isolated from the sap of a beech tree in Miyamachi, Yamanashi Prefecture, and its mycological properties are as follows.

When cultured on malt extract agar medium at 25°C, the colonies exhibit a creamy yellow color with a dull shine.

Light microscopy reveals subglobular or oblong vegetative cells as well as oval or oblong asci containing usually four ascospores. Vegetative cells proliferate by multipolar budding. Ascospores are colorless and spherical or hemispherical. True hyphae are not observed. Pseudohyphae grow well, and ascospores are frequently observed within these cells. Aerobic. No formation of carotenoid pigments was observed. This strain cannot assimilate nitrate, maltose,
Unable to assimilate galactose, trehalose, mannitol, salicin, and potassium hycluconate. There is no gluconis fermentation ability, and strong insect acidity is not observed.

As a result of searching for this strain according to "The East's Taxonomy Study - Third Edition," it was determined that it was Pichia membranefaciens based on the mycological properties mentioned above.
a membranaefaciens), and Pichia membranefaciens S-105-1 (hereinafter referred to as S
-105-1) and was sent to the Microtech Institute in 1988.
It was deposited as FBRM No. 0P-1439 on August 7, 2013.

■, Candida fuenica N-6 (This strain was isolated from grape fruits in the Yamanashi region, and its mycological properties are as follows.

When cultured on a malt extract agar medium at 25°C, the colonies appear white. In optical microscopic observation, true hyphae grow well, and pseudohyphae are also occasionally observed. The vegetative cells are formed in a budding type rather than a true hyphae or a pseudohyphae. At the same time, vegetative cells exhibit a multipolar budding mode of growth.

The vegetative cells are spherical, oval, or oval.

Aerobic. No teleomorphs were observed, and no carotenoid pigments were produced.

This strain shows negative in the DBB test and cannot assimilate nitrate. Furthermore, various sugars such as inositol, rhamnose, lactose, and galactitol cannot be assimilated, but erythritol and raffinose can be assimilated.

As a result of searching for this strain according to "The East's Taxonomic Study - Third Edition," it was determined that it was Candida fuenica from the above mycological properties.
F[! RM BP-144
Deposited as No. 2.

The G-94-3 strain obtained here has a broad antibacterial spectrum, and the N-6-1 strain has an antibacterial effect against bacteria and yeast.
The strain exhibits antibacterial activity against bacteria, yeast, and mold. Also,
Two strains, B-48-2 and S-105-1, can only be used in the presence of methylene blue.
S 5ubtilis) exhibits antibacterial activity. These yeast strains, particularly G-94-3 and Hachi-31-2, can be used as materials for introducing the above-mentioned antibacterial activity into brewing yeast, for example, by cell fusion, genetic manipulation, etc. Furthermore, the antibacterial substances produced by these or the yeast cells themselves can be used as agricultural chemicals, veterinary drugs, medicines, food preservatives, and the like.

The yeast having antibacterial activity of the present invention can be cultured according to a conventional yeast culture method.

For example, carbon sources include glucose, fructose,
Among sucrose, glycerin, maltose, galactose, sorbitol, etc., those that can be assimilated by each yeast strain are used. Nitrogen sources include NIl, C/2, (NH
,)2So, ,casamino acid, yeast extract, peptone,
Meat extract, malt extract, batatotributone, corn stable liquor, soybean meal, Pharmamedia, etc. Other nutritional sources include K2HPO, KLP.
O,, NaC1, MgSO4, vitamin B5, MgCj
'2, biotin, etc. can be used. Furthermore, fruit juice obtained from fruits such as grapes or concentrated juice thereof can also be used as the medium. Cultivation is carried out at p+ 2-8, temperature 10-35°C (preferably pII 5.0-6.5, temperature 20-30°C) for 24-90 hours. When any strain is cultured statically, the antibacterial activity is weak or undetectable, so aerated culture is preferable to static culture.

In addition, for any strain, when stored on a solid medium such as a slant, the antibacterial activity decreases (especially B-48-2,
In S-105-1, the decline is quite rapid), so store it at low temperature in glycerin (final glycerin concentration 10-40%).
(-30 to -80°C) is desirable.

Examples of the present invention are shown below.

Example 1 Two grape skins picked from a field and 0.5 g of beech sap collected from a beech forest were each treated with 6100 units/ml of penicillin and 100 units of streptomycin.
The mixture was added to 3 ml of YP media (glucose 2%, peptone 2%, yeast extract 1%, pH 6.0) containing g/m+, shaken well, and then statically cultured at 25° C. for 7 days. After that, transfer the culture solution to YPD agar medium (YPO medium with 2% agar).
(pH 6.0) was diluted and applied so that the number of yeast cells per petri dish was 100 to 150, and incubated at 25°C. After 5 days, the resulting colonies were transferred to Nutriendo broth agar medium containing 1% glucose (2% Far Eastern powder broth, 2% agar, pH 6).
, 0; hereinafter referred to as NB agar medium) at 105 cells/m
M1070 to Bacillus subtilis l to become l
was replicated onto a plate inoculated with and cultured at 25°C for 2 to 4 days. Colonies exhibiting antibacterial activity (A circle of inhibition of Bacillus subtilis growth was observed around the colony. Only those with a clear inhibition circle were selected to exclude cases of poor growth of Bacillus subtilis due to competition for nutrients. After single cell isolation was performed, the antibacterial activity was assayed using the following three methods.

The antibacterial activity of various yeasts isolated from single cells was assayed using the following three methods, and G-94-
3, A-31-2, S-105-1, B-48-2 and N-6-1 were selected.

(1) Method■ Yeast strains cultured on YPD agar at 5°C above ground for 3 days were injected into one platinum loop at 105 cells/m of the test bacteria shown in Table 1.
The cells were coated on an agar medium containing + and cultured at 30°C for 2 days, and when Leuconostoc oenos was used as the test bacterium, at 25°C for 3 to 4 days. Test bacteria 10' here
An agar medium containing cells/m+ means that bacteria other than lactic acid bacteria are 1
NB agar containing % glucose and 50mM citrate-phosphate buffer, lactic acid bacteria on BL agar (pH 7).
, 2, but for Leuconostoc oenos, adjust the pH to 4.8 with hydrochloric acid. Eiken! For mold, malt extract agar medium (malt extract 2%, peptone 0.
1%, glucose 2%, agar 2%; pH 5,0), and YPD agar medium (pf 16.0) containing 100 mM citric acid-phosphate buffer for yeast, respectively, with the test bacteria at 105 cells/m+. This is what I did.

After culturing, the growth-inhibited area of the test bacteria that appeared around the smeared yeast strain was observed. Antibacterial activity was expressed as follows according to the degree of inhibition of growth of test bacteria.

+++: Very strong activity, ++: Strong activity, +: Moderate activity, ±: Slight activity, -2 No activity 2) Method ■ (Well test-1) 28°C in YPD medium for 2 days Each yeast strain cultured with shaking was placed in an L-shaped test tube containing 5 ml of YPD medium.
Inoculate to a bacterial concentration of Xl0' cells/ml and incubate at 28°C.
Shaking culture was performed.

G-94-3 was cultured for 24 hours, B-48-2, S-1
05-1 and N-6-1 were carried out for 36 hours, and A-31-2 was carried out for 60 hours.

After culturing, each culture solution was filtered through a 0° 45 μ cellulose acetate membrane filter, concentrated approximately 5 times by freeze-drying, and each concentrated solution was mixed with 105 cells/ml of test bacteria as in Method I. A hole was made on an agar plate inoculated with (
(diameter 10+nm), inject 2004 each into 3
Cultured at 0°C. Two days later, the diameter of the growth inhibition circle of the test bacteria that had formed around the hole was measured and determined as the strength of the antibacterial activity (
Those with no activity were indicated as -).

(3) Method ■ (Well Test-2) Contain methylene blue in the agar medium inoculated with the test bacteria (
Antibacterial activity was measured in the same manner as method ① except that the concentration was 0.003% for yeast and mold plates and 0.0015% for bacteria plates. However, in this method, the strength of antibacterial activity was determined by measuring the diameter of a circle formed around the hole due to methylene blue incorporated into the dead cells (those with no activity were indicated as -).

Table 1 shows the results of testing the antibacterial activity using Methods ■, ■, and ■. Strain A-31-2 exhibits antibacterial activity against bacteria, yeast, and mold. The N-6-1 strain exhibits antibacterial activity against bacteria and yeast, and the G-94-3 strain exhibits antibacterial activity against bacteria. In addition, strains S-105-1 and B-48-2 exhibit antibacterial activity against Bacillus subtilis only in the presence of methylene blue.

Example 2 Each yeast strain cultured with shaking at 28°C for 2 days in YPD medium was inoculated into two L-shaped test tubes each containing 5 ml of YPD medium at a bacterial concentration of 1X10' cells/ml. One was cultured with shaking and the other was cultured with aeration at 28°C. At 24.36.48.60 hours after the start of culture, 0.5 ml of culture solution was collected from each L-shaped test tube, filtered through a 0.45μ cellulose acetate membrane filter, and each p solution was on N8 agar medium (containing 1% glucose and 50mM citrate-phosphate buffer, pH 6,0) inoculated with 105 cells/ml of Bacillus subtilis IAM1070 and YPD agar medium inoculated with Candida glabrata IFo 0622 ( 1
Contains 00mM citric acid-phosphate buffer, pH 6.0
) Inject 2004 each into the holes drilled above, and add 30
Cultured at ℃. For determination of antibacterial activity, ^-31-2, G-
For 94-3 and N-6-1, Example 1 Method ■
8-48-2 and S-105-1 were carried out according to Method ①. The results are shown in Table 2.

For all yeast strains in Table 2, shaking culture has higher antibacterial activity. In the ^-31-2 strain, antibacterial activity continued to increase up to 60' hours. Moreover, the anti-yeast activity remained constant after 36 hours. The G-94-3 strain had its peak activity at 24 hours, and the other three strains had their activity peak at 36 hours.

Example 3 It was confirmed that the antibacterial activity exhibited by each yeast strain was not due to organic acids or higher alcohols present in the culture solution.
In order to confirm the i
31-2 at 60 hours, G-94-3 at 24 hours, B
-48-2, S-105-1 and N-6-1 were analyzed for organic acids, higher alcohols, etc. in the culture fluid collected at 36 hours, and anti- Bacterial activity was assayed. As a control, each alcohol and organic acid were added to YP in an amount equal to the analytical value of the culture solution.
The antibacterial activity of the sample added to medium D was similarly tested.

The antibacterial activity assay method is Method 2 for B-48-2 and S-105-1, and Method H for other strains.
It was done by Acetic acid, ethanol, and β-7 ethyl alcohol were assayed using methods ① and ②.

Organic acid analysis was performed using liquid chromatography (column: 5
hodex C-811, detector: visible, mouth V detector U
VIDBC100-VT, detection method: BT8 color method), ethanol analysis using gas chromatography method [column:
20% to 8P-201/FIusionT (60~8
0mesh), Detector: PI [L detector], Higher alcohol analysis was performed using gas chromatography method [Column: P
EG IIT 5%-Uniport HP 60/8
0, Detector: FI port detector (220°C), Column temperature:
65-190℃ (5℃/min temperature increase system), Inje
controller temperature: 220°C].

The analysis results of the culture solution are shown in Table 3, and the results of the antibacterial activity assay are shown in Table 4.

E9/-Luha 2.0% (V/V) + 73 concentration, acetic acid 1000mg/β, β-phenethyl alcohol 3
00 mg/j! Bacillus subtilis IAM 1070 and Candida glabrata even at concentrations of
IFo 0622 showed no antibacterial activity.

Furthermore, other alcohols and organic acids were not found to contribute at all to the antibacterial activity exhibited by these five strains.

From these facts, the antibacterial activity exhibited by these yeast strains is
It is clear that this is not caused by organic acids such as acetic acid, higher alcohols, ethanol, etc.

Effects of the Invention By using the yeast strain of the present invention, contamination with wild bacteria, wild yeast, etc. can be prevented, which is a great advantage in the production of alcoholic beverages and alcohol. Furthermore, the present invention provides the possibility of using the yeast cells themselves as agricultural chemicals and veterinary medicines, and the use of the produced antibacterial substances as agricultural chemicals, veterinary medicines, pharmaceuticals, food additives, and the like.

Claims (1)

[Claims] Metschnikowia pulchria has antibacterial activity against bacteria.
errima), Candida pintropesii (Ca.
ndida pintolopesii), Candida fennica and PiChiame
mbranaefaciens), and Candida sp. A-31-
2 Microtechnical Research Institute No. 1441.