JPS6366199B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel yeast with unique sugar utilization. In general, yeast has hexokinase, an enzyme that phosphorylates glucose, fructose, and mannose in order to introduce them into the metabolic circuit, or glucokinase, an enzyme that phosphorylates glucose and mannose, as constituent enzymes in its sugar metabolism. It is known that there are However, conventionally known yeast does not have galactokinase, which is an enzyme that phosphorylates galactose, as a constituent enzyme, and galactokinase is induced and produced only when the yeast is precultured in a medium containing galactose. . Furthermore, looking at the sugar utilization of conventional yeast, for example, in a mixed sugar solution of glucose and galactose, even when yeast is pre-cultured as described above to induce production of galactokinase, it is not possible to preferentially utilize glucose. It is customary. Additionally, yeast that can utilize glucose typically also utilize fructose and mannose to the same extent. In other words, it can be said that the sugar utilization of conventional yeast is poor in the utilization of galactose, and that it utilizes glucose, fructose, and mannose to the same extent. Therefore, a method is known in which galactose is produced from whey, which is a byproduct during cheese production, by applying the sugar utilization of yeast. That is, in this method, for example, Saccharomyces cerevisiae is added to a mixture containing glucose and galactose obtained by decomposing lactose in whey with acid or enzyme (lactase).
Glucose is selectively broken down by the action of galactose, and only galactose is collected. However, on the other hand, it is possible to decompose and remove only galactose from a liquid containing glucose and galactose, such as that obtained by treating milk or whey with the enzyme lactase, or to act on a sugar mixture containing fructose, such as glucose, with the enzyme glucose isomerase. When producing isomerized sugar solution, only glucose is decomposed from the isomerization reaction product solution in which glucose and fructose have reached a 1:1 equilibrium.
It would also be useful to remove it to produce fructose. Conventionally, microorganisms that have the above-mentioned effects, namely galactokinase as a constituent enzyme, have superior utilization of galactose than that of glucose, and also lack hexokinase. No microorganism has been found in nature that can utilize glucose but cannot utilize fructose. In view of the usefulness of microorganisms that have the above-mentioned unique sugar utilization ability, the present inventor conducted a wide search for microorganisms with such characteristics in the natural world, and as a result, discovered yeast having the above-mentioned characteristics from commercially available kefir grains. , arrived at the present invention. Incidentally, kefir grains are the seed mother of fermented milk kefir, which is widely consumed mainly in Eastern Europe, and has been used as a drink since ancient times, so the yeast isolated from it is not suitable for hygiene and safety reasons. There is no problem in using it for food. The present invention will be explained in detail below. First, the isolation method and mycological properties of the yeast of the present invention will be described. (1) Separation from kefir grains and pure culture Kefir grains soaked in water are washed with water.
Skimmed milk sterilized at 121℃ for 10 minutes after draining
Inoculate 10g per 100ml and statically culture at 20-25°C for 2-3 days. The above culture is repeated five times or more by subculturing, and when the vitality of the bacteria is restored, the kefir grains are separated from the skim milk and washed with water. The obtained kefir grains are ground in a mortar and then suspended in physiological saline. Next, this suspension was diluted appropriately using a yeast isolation medium, such as a commercially available potodextrose agar medium (PH3.5).
Pour culture at 25°C for 3 days. Colonies produced by this culture are picked and purified three times or more on a plate medium having the same composition as the above medium. Next, a lactose fermentability test is performed on the isolated yeast, and strains that do not ferment lactose are selected. This selected yeast is further cultured using a medium containing both glucose and galactose to confirm that it is a strain that preferentially uses galactose over glucose. The yeast obtained in this way is KY−
Name it 5. (2) Mycological properties (a) Growth status in each medium Culture observed in YM liquid medium at 26°C Gas generation No pellicle formation Shape of precipitate Compact precipitate turbid state Moderate Size 3-6 x 7-12μ Growth form Multipolar budding Cultured on YM agar medium at 26℃ Findings Medium growth Shape of the colony periphery Entire 〃 Surface 〃 Smooth 〃 Properties Buttery ( (b) Formation of ascospores Gypsum medium, modified Gorodkova medium, glucose peptone medium were used, but ascospore formation did not occur. (C) Formation of extruded spores Not formed. (d) Physiological test Optimal growth conditions Temperature 25℃, PH5.5-6.5 Range of growth conditions Temperature 12-30℃, PH3.0-7.5 Assimilation of nitrates No assimilation Decomposition of urea None Formation of carotenoids None Production of starch-like substances None Degradability of glucosides None (e) Presence or absence of sugar assimilation [Table] The above mycological properties of the yeast KY-5 according to the present invention are similar to that of Torulopsis except that it lacks the ability to ferment fructose and raffinose.・Most similar to Torulopsis holmii , while the standard strain of Torulopsis holmii
holmii IFO0660 ferments fructose and raffinose (1/3), the yeast KY
-5 is considered to be a unique strain of Torulopsis formii, and therefore Torulopsis formii KY-5
It was named. Yeast KY-5 has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession number 5766. Next, the characteristics of sugar utilization of yeast KY-5 according to the present invention will be explained. The sugar utilization behavior was investigated by culturing yeast KY-5 in a medium containing various sugars, and the results are shown below. (a) Yeast KY-5 was inoculated into a medium containing 5% by weight of glucose and 5% by weight of galactose as a sugar source, static culture was performed at 25°C, and the amount of residual sugar in the medium was measured over time. The results are shown in Figures 1a and b. Furthermore, Fig. 1a shows the sugar utilization over time when yeast KY-5, which has been previously subcultured in a medium containing glucose, is inoculated, and Fig. 1b shows the utilization of sugar over time when yeast KY-5 was subcultured in a medium containing glucose. This figure shows the utilization of sugar over time when inoculating a subculture of -5 in advance in a medium containing galactose. As seen in Figures 1a and b, galactose is always preferentially utilized in a mixture of glucose and galactose, regardless of the presence or absence of pre-incubation. This pattern of sugar utilization is also the same when shaking culture is used instead of static culture. In addition, in conventionally known yeast, when the yeast is pre-cultured with galactose, the utilization of galactose increases, but does not exceed the utilization of glucose. Moreover, when known yeasts are precultured with sugars other than galactose, the utilization of galactose is lost. That is, yeast KY-5 according to the present invention is essentially different from known yeasts in terms of galactose utilization. (b) Yeast KY-5 with 5% by weight each of glucose, galactose, fructose and mannose
inoculated into each medium containing each as a sugar source, and
The amount of residual sugar in each medium was measured over time when cultured statically at °C. The results are shown in Figure 2 a and b.
Shown below. Furthermore, Figure 2a shows the sugar utilization over time when yeast KY-5 was previously subcultured in a medium containing available sugars other than fructose, such as glucose. FIG. 2b shows the sugar utilization over time when yeast KY-5, which had been sufficiently acclimatized and subcultured in a medium containing fructose, was inoculated. As seen in Figure 2 a and b, yeast KY
-5 has the highest galactose utilization in all cases, and does not utilize fructose unless it is pre-cultured with fructose. Furthermore, this pattern of sugar utilization remained the same even when shaking culture was used instead of static culture. Incidentally, considering that known yeasts that can utilize glucose also utilize fructose, yeast KY-5 also exhibits sugar utilization that is essentially different from conventional yeast in this respect. It can be said that it is a thing. (c) Yeast KY-5 was inoculated into a medium containing 5% by weight of sucrose and cultured stationary at 25°C, and the amount of residual sugar in the medium was measured over time. The results are shown in Figure 3. FIG. 3 shows the sucrose utilization behavior over time when yeast KY-5 was previously subcultured in a medium containing an available sugar other than fructose, such as glucose, and was inoculated. As shown in Figure 3, yeast KY-5 uses only the glucose produced after decomposing sucrose with its sucrose-degrading enzyme and does not use fructose, so fructose accumulates in the medium over time. . That is, it can be seen that yeast KY-5 selectively utilizes only glucose and does not utilize fructose in a sugar solution containing a mixture of glucose and fructose. Next, Table 1 below shows the results of a comparison of the sugar phosphorylating enzyme of yeast KY-5 according to the present invention with that of Saccharomyces cerevisiae . [Table] As shown in Table 1 above, galactokinase in yeast KY-5 is present as a constituent enzyme at the same time as glucokinase, but hexokinase is missing; on the other hand, galactokinase is missing in Saccharomyces cerevisiae. It can be seen that it is produced in an inducible manner only when precultured in a medium containing galactose. As mentioned above, the yeast KY-5 according to the present invention was taxonomically identified as Torulopsis formii, and therefore, the yeast KY-5 of the present invention was identified as Torulopsis formii, the standard strain of Torulopsis formii, Torulopsis formii IFO0660, and
IFO1629 was tested for sugar utilization in a medium containing glucose and galactose, but did not show preferential utilization of galactose. or,
A medium containing fructose and glucose was also used in the same way. That is, in view of the above-mentioned essential difference in sugar utilization, yeast KY-5 according to the present invention can be said to be a new and unique strain of the known Torulopsis kirmii. Since the yeast KY-5 according to the present invention has the above-mentioned specific sugar utilization, it can be applied to various fields. For example, yeast KY-5 can remove galactose from a galactose-containing mixed sugar solution and selectively extract fructose from a fructose-containing solution mixed with other sugars, which was considered almost impossible or at least very difficult in the past. This can be carried out advantageously by applying . Incidentally, to remove galactose from a sugar mixture containing galactose, yeast without lactose utilization (lactose non-fermenting yeast) was used to remove lactylose (β-1.3 between galactose and fructose).
A method for removing galactose from a mixture of lactose and galactose has been reported (Japanese Patent Publication No. 51-23573), but when the present invention is applied, galactose can be removed from a mixture of lactose and galactose, and It is also possible to remove glucose from a mixture of lactose and glucose or a mixture of fructose and glucose. The present invention and its effects will be specifically explained below by way of examples. Note that % indicating the amount in each example indicates weight unless otherwise specified. Example 1 A solution of 10% reduced whey powder was heated to remove protein and then treated with mold lactase powder to decompose almost 100% of the lactose in the whey. After adjusting the decomposition solution containing glucose (approximately 3%) and galactose (approximately 3%) to pH 5.6, it was heated and sterilized again at 90°C for 30 minutes, and then cultured as shown below.
Torulopsis holmii KY-5 (hereinafter referred to as yeast KY-5)
) and perform aeration and agitation culture. The yeast added here is a 1N-10% whey powder solution.
After adjusting the pH to 4.5 with HCl and heating, add 3% glucose or 3% galactose to the deproteinized solution.
300ml of medium adjusted to pH 5.6 and sterilized at 121℃ for 15 minutes
Seed yeast KY-5 from an agar slant was contacted with 27
The cells were cultured with shaking at ℃ for 2 days and added as bacterial cells collected by centrifugation. It is advantageous to add a larger amount of these microbial cells because the culture time will be shortened. After culturing with aeration at 26°C for 1 to 3 days, remove the yeast cells when the galactose is completely consumed, and if necessary, incubate at 90°C for 30 days.
After degassing, dealcoholization, and activated carbon treatment by heating for minutes, neutralization with 1N NaOH and concentration, glucose syrup (glucose content 30-33%) is produced that is galactose-free or has a significantly reduced galactose content , galactose content 0-2
%) can be obtained. This syrup can be used as a raw material for refining glucose, but
Dairy products such as condensed milk, ice cream, yogurt, snacks, etc.
It can be used as an additive to confectionery. Example 2 Whey treated with lactase as in Example 1
450ml of yeast KY- was placed in a 900ml continuous culture fermenter and precultured in whey medium supplemented with 1% galactose.
Inoculate 5% of the culture solution from No. 5. Next, the airflow rate is 2.5
vvm, stirring speed 300rpm, feed 10ml/hr temperature
Cultivate continuously for 72 hours at 25°C, and immediately separate and remove bacterial cells from the effluent. The resulting whey liquid is concentrated 10 times to obtain syrup with a glucose content of 32% and a galactose content of 1.5%. Example 3 Whey or whey permeate obtained by deproteinizing whey by UF membrane treatment was prepared at pH 6.5 in advance.
Using lactase obtained from Saccharomyces lactis , approximately 50% of the lactose in whey is degraded. The sugar composition in the liquid is 3% lactose, 1.5% glucose, and 1.5% galactose. 5% yeast KY-5 was added to the whey or whey permeate thus obtained.
Inoculate and incubate at 28°C for 3 hours, then immediately refrigerate. When the product temperature has completely reached 5-7℃, the bacterial cells are removed by centrifugation, and the sugar composition is 3% lactose and glucose.
A whey beverage containing 1.5% galactose and 0% galactose with a slight alcohol odor is obtained. If the culture time is unnecessarily long, glucose will also be consumed for fermentation, resulting in a product with a high alcohol content but inferior sweetness and flavor, so it is important to stop the culture when the galactose disappears. It is. Example 4 100% of the lactose contained in whey or whey permeate is decomposed in advance using lactase (glucose: 3.1%, galactose: 3.1%). Concentrate this liquid 2 to 3 times, sterilize it, and add 5% yeast KY-5.
and as an example known as wine yeast
When 3% of Saccharomyces cerevisiae OC-2 was simultaneously inoculated and cultured statically at 25°C, galactose was preferentially fermented by KY-5 and glucose was preferentially fermented by OC-2, and the sugar in the substrate was completely fermented within 24 hours. By eliminating the seeds, we were able to shorten the fermentation time using either single yeast to less than 2/3.
Furthermore, the flavor was also superior to that fermented with a single bacteria. Example 5 Milk 1 was HTST pasteurized, cooled, and then yogurt bacteria Lactobacillus bulgaricus and Streptococcus thermophilus
thermophilus) mixed starter at 2%,
Incubate at 37°C. When culturing only yogurt bacteria in this way, the lactose in the milk is naturally consumed, but when about 30% of the lactose is consumed, galactose begins to be released (VsO′leary and JH
Woychick: J.Food Soi., 41 , 791 (1976)),
Yeast KY-5 is added at 0.3 to 1.0% at the same time as or after inoculation of yogurt bacteria and before the milk coagulates.
After culturing at 37° C. for 4 to 5 hours, the mixture is transferred to a refrigerator and kept for at least 5 hours to ferment with yeast to obtain yogurt with improved flavor. Example 6 Milk 1 to which a cell-free extract of lactic acid bacterium Lactobacillus bulgaricus (L. bulgaricus) SBT 2110 was added in advance (0.05% as crude enzyme dry matter) was kept at 5°C and lactose was extracted with lactic acid bacterium lactase. 70%
At the same time as the crude enzyme is decomposed, the protein or lipolytic enzyme coexisting in the crude enzyme is also activated. After holding for 16 hours, yogurt was produced using the yogurt mixture and yeast KY-5 in the same manner as in Example 5, resulting in a product with a lower lactose content and free galactose compared to the product obtained in Example 5. Because of the presence of a large amount of yeast, it is possible to obtain yogurt with greater flavor improvement with yeast KY-5. Example 7 Defatting agent or whey is treated with lactase in advance to decompose 50% or more of the lactose contained therein into glucose and galactose, which is then made into powder using, for example, a spray dryer. The lactose decomposed skimmed milk powder or whey powder obtained in this way is mixed with wheat flour used for bread dough preparation in a ratio of 1:1 to 2:1 to form a new bread dough material, and 5% commercially available baker's yeast and yeast KY -5 1 to 5% is added and mixed, and after fermentation at 25°C or lower, bread is manufactured according to a conventional method. Example 8 Skim milk powder or whey powder is mixed with wheat for bread dough in a ratio of 1:1 to 2:1 to prepare a new bread dough material, and the lactic acid bacterium Lactobacillus bulgaricus SBT is added thereto.
After adding and mixing 2% of dried bacterial cells of 2110 and pre-fermenting at 35℃ for 4 hours, 5% of commercially available baker's yeast and yeast KY-
5. Add and mix 1 to 5%, ferment at below 25°C, and then manufacture bread according to a conventional method. Example 9 A whey solution with a solid content concentration of 30% is heated at PH4.4 to remove protein, and then 10 clear wheys are obtained. 1% mold lactase is added to this whey and reacted at 50°C for 2 hours to completely decompose lactose. After adjusting the pH to 6.0, 2% Streptomyces glucose isomerase was added and the reaction was continued at 50°C for 40 hours until 40-45% of glucose was added.
Convert % to fructose. After cooling the whey solution to 25°C, 10% yeast KY-5, which had been precultured using glucose or galactose as a sugar source, was added and cultured for 24 hours to decompose the galactose and then glucose in the whey, and then re-incubate the whey solution. PH
By adjusting the concentration to 7.0 and desalting and concentrating, a fructose syrup containing neither galactose nor fructose can be obtained. Example 10 When fruit juice such as grape or orange is adjusted to pH 5.6 and yeast KY-5 pre-cultured using glucose or galactose as a sugar source is added and cultured,
Only free glucose or glucose constituting sucrose in the fruit juice is utilized to obtain a fruit juice product with increased fructose purity. It can be used as a sugar source in snacks and desserts for children.

[Brief explanation of drawings]

Figures 1a and 1b show the sugar utilization of the yeast of the present invention over time in a medium containing equal amounts of glucose and galactose, and Figures 2a and 2b show the sugar utilization of the yeast of the present invention in a medium containing equal amounts of glucose and galactose. Figure 3 shows the utilization of sucrose by the yeast of the present invention over time.

Claims (1)

[Scope of Claims] 1. Has glucokinase and galactokinase as constituent enzymes, is deficient in hexokinase, and can be cultured in a mixed sugar solution containing galactose even when precultured in a medium containing available sugars other than galactose. Torulopsis formii has the property of preferentially utilizing galactose in
holmii) KY-5. 2. Torulopsis formii KY-5 according to claim 1, which has the characteristic of not utilizing fructose unless precultured in a culture medium containing fructose.