NOVEL SUCROSE UNASSIMILATING FLOCCULENT YEAST
20180327787 ยท 2018-11-15
Assignee
Inventors
Cpc classification
Y02E50/10
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12P19/14
CHEMISTRY; METALLURGY
International classification
Abstract
An object of the present invention is to provide a sucrose unassimilating yeast which has a flocculation ability and has much of a proven performance of food production. The present invention pertains to a yeast strain expressed by accession number: NITE BP-1587.
Claims
1. Saccharomyces cerevisiae strain NITE BP-1588.
2. The Saccharomyces cerevisiae strain according to claim 1, wherein the strain is employed for a method of producing sugar and ethanol comprising a step of fermenting sugar liquid derived from a plant with yeast.
3. The Saccharomyces cerevisiae strain according to claim 1, wherein the strain is employed for a method of producing sugar and ethanol comprising steps of: removing non-sugar components contained in sugar liquid derived from a plant to clarify the sugar liquid; and adding yeast to the clarified sugar liquid to carry out ethanol fermentation at a given temperature under an anaerobic condition for an appropriate length of time.
4. A method of producing sugar and ethanol comprising a step of fermenting sugar liquid derived from a plant with the Saccharomyces cerevisiae strain according to claim 1.
5. A method of producing sugar and ethanol comprising steps of: removing non-sugar components contained in sugar liquid derived from a plant to clarify the sugar liquid; and adding the Saccharomyces cerevisiae strain according to claim 1 to the clarified sugar liquid to carry out ethanol fermentation at a given temperature under an anaerobic condition for an appropriate length of time.
6. Saccharomyces cerevisiae strain NITE BP-1587 obtained by a breeding method comprising: subjecting a yeast strain deposited under accession number NBRC 10055 and a yeast strain deposited accession number NITE BP-1589 to spore isolation to acquire an isolated spore showing type a or type a as a mating type; mating the isolated spore of the yeast strain deposited under accession number NBRC 10055 and the isolated spore of yeast strain deposited under accession number NITE BP-1589 which have different mating types to create a mated strain; carrying out, as for the created mated strain, tetrad dissection to acquire an isolated spore; and screening the obtained isolated spore to select the Saccharomyces cerevisiae yeast strain and having a character of a sucrose unassimilating property and flocculation.
7. Saccharomyces cerevisiae strain obtained by diploidizing the Saccharomyces cerevisiae strain according to claim 6.
8. A method of breeding a Saccharomyces cerevisiae yeast strain comprising: subjecting a yeast strain deposited under accession number NBRC 10055 and a yeast strain deposited under accession number NITE BP-1589 to spore isolation to acquire an isolated spore showing type a or type a as a mating type; mating the isolated spore of the yeast strain deposited under accession number NBRC 10055 and the isolated spore of the yeast strain deposited under accession number NITE BP-1589 which have different mating types to create a mated strain; carrying out, as for the created mated strain, tetrad dissection to acquire an isolated spore; and screening the obtained isolated spore to select the Saccharomyces cerevisiae yeast strain and having characters of a sucrose unassimilating property and flocculation.
9. A method of breeding a yeast strain according to claim 8, further comprising: diploidizing the obtained Saccharomyces cerevisiae yeast strain.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0015]
[0016]
[0017]
[0018]
[0019]
[0020]
[0021]
[0022]
[0023]
MODE FOR CARRYING OUT THE INVENTION
[0024] As a sucrose unassimilating yeast, Saccharomyces cerevisiae NBRC 10055 strain was prepared. NBRC 10055 strain was purchased from NBRC. NBRC 10055 strain is a Saccharomyces yeast isolated from red wine from Garcia (Spain). When this yeast was isolated, it was found that this yeast has a property of forming membrane after fermentation, a property of mainly fermenting glucose, a property of producing acetic acid under an aerobic condition, and the like.
[0025] In addition, as a flocculent yeast, Saccharomyces cerevisiae SDT strain was prepared. SDT strain is a strain found by the applicant, and confirmed to belong to Saccharomyces cerevisiae. SDT strain is deposited at NITE. Accession number of SDT strain is NITE BP-1589.
[0026] NBRC 10055 strain and SDT strain were independently subjected to spore isolation, to acquire an isolated spore showing type a or type a as a mating type. The isolated spore of NBRC 10055 and the isolated spore of NITE BP-1589 which have different mating types were mated using a manipulator, to create a mated strain. As for the created mated strain, tetrad dissection was carried out using a manipulator after induction of spore formation, to acquire an isolated spore. Next, MAT locus of the mated strain and the isolated spore was amplified by PCR method to carry out analysis, and the mating type was determined.
[0027] Screening was carried out on the obtained isolated spore, and a strain having an objective character of a sucrose unassimilating property and flocculation was selected, to acquire a sucrose unassimilating flocculent yeast of the present invention. This yeast strain is a haploid. The yeast was named GYK-10. The obtained strain was obtained from parent strains, both of which are Saccharomyces cerevisiae, and belongs to Saccharomyces cerevisiae. GYK-10 is deposited at NITE. Accession number of GYK-10 is NITE BP-1587.
[0028] GYK-10 was cultured on a YPD agar medium, and a diploidized yeast on the basis of a property of homothallism was selected. The yeast was named GYK-11. GYK-11 is deposited at NITE. Accession number of GYK-11 is NITE BP-1588.
[0029] A sucrose unassimilating property, a flocculent ability, heat tolerance and acid tolerance of GYK-10 and GYK-11 were tested as follows.
Test of Sucrose Unassimilating Property
[0030] As a test object yeast, GYK-10 and GYK-11, and NBRC 10055 and SDT, which are the parent strains thereof, were prepared.
[0031] A yeast cultured at 30 C. using 100 ml of a YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) was recovered by centrifugation (5,000g, 5 minutes). The recovered yeast (wet weight: 1 g) was added to 100 ml of a sugar liquid prepared simulating sugar cane squeezed juice (1% yeast extract, 2% peptone, 12% sucrose, 1.5% glucose, 1.5% fructose), and the mixture was fermented at 30 C. or 37 C. As for concentrations of saccharides and ethanol contained in the sugar liquid, change with time during fermentation time was recorded. The results are shown in
[0032] According to the test results, it was confirmed that GYK-10 and GYK-11 are sucrose unassimilating, as with NBRC 10055, which is a parent strain.
Test of Flocculation Ability
[0033] As a test object yeast, GYK-10 and GYK-11, NBRC 10055 and SDT, which are the parent strains thereof, and Saccharomyces cerevisiae STX 347-1D strain, which is recognized to have a high flocculation ability, were prepared.
[0034] After the yeast cell was precultured in 5 mL of a YPD liquid medium (30 C., 48 hours, 120 rpm), the yeast cell was washed twice with twenty times the amount of distilled water to the volume of the cell. The washed cell was suspended in 4 mL of distilled water so that the concentration of wet cells was 2%. After 250 L of distilled water was added to 1 mL of the suspension liquid of cells, absorbance at 600 nm was determined (a control). Next, after 250 L of 100 mM calcium chloride was added to 1 mL of the suspension liquid of cells, the mixture was settled for 5 minutes. Thereafter, absorbance at 600 nm was determined (a sample). Absorbance was determined using spectrophotometer UV-1700 (manufactured by SHIMADZU CORPORATION).
[0035] The flocculation ability was determined according to the formula:
C=(1B/A)100
wherein C is a flocculation ability (%); A is absorbance of the control; and B is absorbance of the sample. The results are shown in Table 1 and
TABLE-US-00001 TABLE 1 ABSORBANCE STRAIN (600 nm) NBRC10055 SDT GYK-10 GYK-11 STX347-1D CONTROL (1) 1.707 1.931 1.691 1.856 1.579 CONTROL (2) 1.694 1.913 1.704 1.845 1.608 CONTROL (3) 1.699 1.904 1.699 1.810 1.614 SAMPLE (1) 1.377 1.050 0.162 0.813 0.600 SAMPLE (2) 1.390 1.045 0.167 0.786 0.648 SAMPLE (3) 1.380 1.007 0.154 0.807 0.599 FLOCCULATION 19.3% 45.6% 90.4% 56.2% 62.0% ABILITY (1) (%) FLOCCULATION 17.9% 45.4% 90.2% 57.4% 59.7% ABILITY (2) (%) FLOCCULATION 18.8% 47.1% 90.9% 55.4% 62.9% ABILITY (3) (%) FLOCCULATION 18.7% 46.0% 90.5% 56.3% 61.5% ABILITY (AVERAGE) FLOCCULATION 0.7% 0.9% 0.4% 1.0% 1.6% ABILITY (STANDARD DEVIATION)
[0036] According to the test results, it was confirmed that the flocculent ability of GYK-10 is about twice higher as compared with that of SDT, which is a parent strain thereof, and about 1.5 times higher as compared with that of a general flocculent yeast STX 347-1D.
Test of Heat Tolerance and Acid Tolerance
[0037] As a test object yeast, GYK-10 and GYK-11, and NBRC 10055 and SDT, which are the parent strains thereof, were prepared.
[0038] After the yeast cell was precultured in 1 mL of a YPD liquid medium (30 C., one night, 120 rpm), the yeast cell was washed twice with 1 mL of sterilized water. The washed cell was added to a YPD liquid medium so that the concentration was 110.sup.6 cells/mL. One hundred microliters of the suspension liquid of cells in YPD was added to well of a microtiter plate and underwent main culture, and absorbance at 600 nm was determined every 10 minutes. Absorbance at 600 nm was determined using PLATE READER HiTS-S2 (manufactured by SCINICS CORPORATION).
[0039] At that time, as a test of heat tolerance of the yeast, the suspension liquid of cells in YPD was cultured with adjusting the temperature to 30 C., 37 C. or 40 C. The results are shown in
[0040] Heat tolerance of GYK-10 and GYK-11 is weaker than that of SDT, which is a parent strain, but stronger than that of NBRC 10055. GYK-10 and GYK-11 do not have clear difference in heat tolerance.
[0041] In addition, as a test of acid tolerance of the yeast, the yeast was cultured at 30 C. using a YPD (pH 6.5), YPD (pH 2.0), or YPD (pH 3.0) liquid medium. The results are shown in
[0042] GYK-10 and GYK-11 are capable of growing in YPD of which pH is 2.0, and have acid tolerance stronger than that of SDT and NBRC 10055, which are the parent strains.
Method for Producing Sucrose and Ethanol
[0043] The sucrose unassimilating flocculent yeast of the present invention can be used for, for example, a method for producing sucrose and ethanol described in Patent Document 1. That is to say, first, sugar liquid derived from a plant is fermented using the sucrose unassimilating flocculent yeast of the present invention. The plant to be used at a raw material of the sugar liquid may be a plant which can accumulate sugar components, and examples thereof generally include sugar cane, sugar beet and the like. The sugar liquid may be a squeezed juice, a broth and the like of these plants. Next, the sucrose unassimilating flocculent yeast of the present invention is added to the sugar liquid, and ethanol fermentation is carried out at a given temperature under an anaerobic condition for an appropriate length of time.
[0044] As the sucrose unassimilating flocculent yeast of the present invention to be used for carrying out ethanol fermentation of the sugar liquid, either of GYK-10 or GYK-11 may be used. In addition, both can be concomitantly used. In a preferable embodiment, GYK-10 is used. GYK-10 is excellent in a flocculation property, and the yeast can be easily removed from sugar liquid after ethanol fermentation.
[0045] The sucrose unassimilating flocculent yeast of the present invention is excellent in heat tolerance, and fermentation can progress so long as the temperature is up to about 37 C. Preferable temperature for carrying out ethanol fermentation is 35 C. or higher at which cost of a cooling installation can be reduced, and the sucrose unassimilating yeast of the present invention can carry out fermentation in a low-cost installation.
[0046] The sugar liquid may be clarified by removing non-sugar components contained in the sugar liquid before carrying out ethanol fermentation. By such an operation, impurities contained in the sugar liquid are reduced, and it becomes easy to repeatedly utilize the yeast. The sucrose unassimilating flocculent yeast of the present invention has a flocculation ability, and an efficient method for fermentation wherein a flocculent yeast is always present in a fermenter and fermentation is continuously carried out without separation of yeasts becomes possible.
[0047] The fermented liquid obtained as a result of fermentation contains yeasts, ethanol, water, sucrose, minerals, amino acids and the like. Next, the fermented liquid is concentrated, and ethanol is recovered from the fermented liquid.
[0048] The recovery of ethanol from the fermented liquid can be carried out by a method known to one skilled in the art, and the method is, for example, separation of ethanol by distillation. When ethanol separation by distillation is carried out, the sugar liquid is concentrated at the same time. Thus, it is unnecessary to carry out heat concentration once again in production of sugar, and both time and energy can be saved.
[0049] The production of sugar from the fermented liquid can be carried out by a method known to one skilled in the art, and the method is, for example, crystallization of sugar, or the like. Specifically, the fermented sugar liquid is repeatedly subjected to heat concentration under vacuum suction in small portions (0.5 to 1 kl) to extract sugar crystal having a predetermined size or larger, and the concentrate is then separated into sugar crystal and sugar liquid with a centrifugal machine.
[0050] The sugar liquid separated from the sugar crystal is generally referred to as molasses. The molasses may be mixed with the cleaned liquid in an appropriate amount, to be used again as a fermentation raw material. Thus, the utilization efficiency of the sugar components contained in the sugar liquid is further improved.
EXAMPLES
[0051] The present invention is explained more specifically by means of the examples described below, but the present invention is not limited thereto.
1) Fermentation Test
[0052] After the yeast cell (GYK-10) was precultured in 5 L of a YPD liquid medium (1 L5, 30 C., one night, 100 rpm), main culture was carried out in 100 L of a medium of 4% glucose and 3% yeast extract (100 L, 30 C., one night, stirring with aeration). After completion of culture, stirring with aeration was stopped, and 5.5 kg of flocculated and sedimented yeast was recovered.
[0053] After a medium simulating squeezed juice of sugar cane (Brix 15%, sucrose 9.9%, glucose 2.7%, fructose 2.8%), of which raw materials are brown sugar, glucose and fructose, was prepared, 5.5 kg of the yeast was added thereto, and fermentation was carried out (30 C., 4 hours). After completion of fermentation, stirring was stopped, and the yeast was flocculated and sedimented. Thereafter, the upper fermented liquid was recovered, and a small amount of left yeast was removed by a centrifugal machine, to give a clear liquid.
[0054] Concentrations of sucrose, glucose and fructose in the clarified fermented liquid were determined by liquid chromatography. SCR-101N (manufactured by SHIMADZU CORPORATION) was used as the column, and water was used as the mobile phase. Temperature of the column oven was set to 60 C. The results are shown in Table 2.
TABLE-US-00002 TABLE 2 SUCROSE GLUCOSE FRUCTOSE BEFORE 98.6 g/L 27.4 g/L 28.1 g/L FERMENTATION AFTER 98.6 g/L 0.2 g/L 0.0 g/L FERMENTATION
[0055] As shown in the fermentation test, GYK-10 is flocculated in the course of fermentation of the sugar liquid, and sedimented. In addition, GYK-10 did not assimilate sucrose, but selectively assimilated reducing sugar.
2) Test of Sugar Production
[0056] The sugar liquid obtained by the fermentation test was concentrated under reduced pressure, to obtain a concentrated liquid having Brix of 60%. After the concentrated liquid (Brix 60%, 10 kg) was heated under reduced pressure, 1 kg of sucrose having a particle size of 250 m was added thereto as seed crystal. The mixture was further heated under reduced pressure, to grow the crystal. A mixture of the crystallized sugar and the sugar liquid was centrifugal machined by a 0.354 mm-perforated centrifugal machine at 1,500g for 5 minutes, to recover sugar. Recovery rate of sucrose was 59.4%. The material balance of this method is shown in
[0057] As a control, a sugar liquid on which selective fermentation was not carried out (Brix 15%, sucrose 9.9%, glucose 2.7%, fructose 2.8%) was used instead of the clarified fermented liquid, and sucrose was recovered in the same manner. Recovery rate of sucrose was 43.6%. The material balance of this method is shown in
[0058] As shown in the test of sugar production, the selective fermentation of sugar liquid improved recovery efficiency of sucrose.
[Accession Number]
[0059] Saccharomyces cerevisiae GYK-10 strain was deposited at Incorporated Administrative Agency National Institute of Technology and Evaluation Patent Microorganisms Depositary, located in #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan, on Apr. 11, 2013, and given accession number NITE BP-1587.
[0060] Saccharomyces cerevisiae GYK-11 strain was deposited at Incorporated Administrative Agency National Institute of Technology and Evaluation Patent Microorganisms Depositary, located in #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan, on Apr. 11, 2013, and given accession number NITE BP-1588.
[0061] Saccharomyces cerevisiae SDT strain was deposited at Incorporated Administrative Agency National Institute of Technology and Evaluation Patent Microorganisms Depositary, located in #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan, on Apr. 11, 2013, and given accession number NITE BP-1589.