JP5256214B2 - Method for converting lactose containing whey or lactose into arabitol - Google Patents

Description

  The present invention belongs to the technical field of effective use of whey. The present invention also belongs to the technical field of sugar alcohol production. More specifically, it belongs to the technical field of a method for producing a sugar alcohol using whey.

  With the expansion of cheese production in recent years, cheese whey has become surplus. Whey is composed of lactose, soluble proteins, lipids, salts, etc. Whey protein concentrate (WPC) obtained by ultrafiltration (UF) membrane separation and lactose separated from whey UF permeate, It is used as a product on a commercial scale. However, in lactated whey UF permeate, lactose accounts for about 70% of the solid content, but the lactose is still unused, and there is a problem of creating an effective usage method.

  A method of producing wine (alcohol concentration: 9.5%) by adding dextrose, a fermentation raw material, to whey UF permeate and then allowing wine yeast to act, centrifugation, filtration, and desalination has been devised (Palmer, 1979, Non-Patent Document 1). Further, a method has been devised in which Lactobacillus sp. RKY2 is allowed to act on a whey preparation solution containing lactose at 100 g / L to produce lactic acid at 91 g / L (Kim et al., 2005, Non-Patent Document 2). Furthermore, there are also studies that propose the development of uses for cheese whey as a natural polymer raw material (Fialho et al., 1999, Non-Patent Document 3).

  Lactose contained in whey UF permeate and delactose whey UF permeate has begun to attract attention as a new resource, but has not yet been fully examined from the viewpoint of commercial scale and industrialization.

  On the other hand, in sugar alcohols, for example, xylitol, erythritol, lactitol, sorbitol are used as sweeteners, arabitol is attracting attention as a raw material for pharmaceutical production, food additive, lactitol is a food extender, food additive, It is used as a heat-stable protective additive for proteins.

To date, a method for industrially (commercially) producing polysaccharide alcohol using whey has not been sufficiently studied, and a known route for producing xylitol from lactose has not been developed. However, a technique for obtaining glucose and galactose by acting b-galactosidase on lactose has already been established, and a known route for producing xylitol using glucose as a starting material (Ohnishi et al., 1969, Non-patent document 4; Mayer et al., 2002, Non-patent document 5; Suzuki et al., 2002, Non-patent document 6; Sonoko et al., 2000, Non-patent document 7), Recombinant DNA technology (Harkki et al. , 1997, Non-Patent Document 8; Povelainea et al., 2006, Non-Patent Document 9). That is, Ohnishi et al. Convert glucose to arabitol by Debaryomyces hansenii, then convert arabitol to xylulose by Acetobacter suboxydans, and further convert xylulose to xylitol by Candida guilliermondii. Mayer et al. Obtained xylitol by converting xylulose into glucose and then acting on xylitol dehydrogenase using a similar technique. Povelainea et al. Recombined Bacillus subtilis and converted glucose to xylitol in 20% yield. In addition to these documents, there are known bioconversion and catalytic conversion techniques for the production of arabitol, which is an intermediate substance on the pathway. For example, from experiments in which yeast Saccharomyces rouxii was allowed to act on labeled glucose, it is known that arabitol is derived from ribulose produced by dephosphorylating ribulose-5-phosphate as a metabolic intermediate. (Ingram et al., 1965, Non-Patent Document 10). It has also been reported that the yeast Candida famata R28 produces arabitol (5%) from glucose (10%) (Ahmed et al., 1999, Non-Patent Document 11). There is also an example where arabitol is obtained by reacting a ruthenium catalyst with arabinonic acid or lactone (Fabre et al., 2002, Non-Patent Document 12). Except for research examples based on the premise of using recombinant bacteria in consideration of the production of xylitol for foods, the route for obtaining xylitol from lactose can be summarized as shown in FIG. In any case, it is essential to pass through galactose or glucose.
Palmer, GM: “Wine Production from Cheese Whey”, EPA-600 / 2-79-189 (1979) Jin-Nam Kim, Hyang-Ok Kim, Young-Jung Wee, Doman Kim, Hwa-Won Ryu and Jong-Sun Yun: “Lactic Acid Production from Cheese Whey and Corn Steep Liquor by Lactobacillus sp. RKY2,” 27th Symposium on Biotechnology for Fuels and Chemicals, P4-21 (2005) Fialho, AM, Ligia O. Martins, ML. Donval, JH Leitao, MJ Ridout, AJ Jay, VJ Morris and I.Sa-Correia: “Structures and Properties of Gellan Polymers Produced by Sphingomonas paucimobilis ATCC 31461 from Lactose Compared with Those Produced from Glucose and from Cheese Whey ”, Appl. Environmnt'l Microbiol., 65, 2485-2491 (1999) Ohnishi, H. and T. Suzuki: “Microbial Production of Xylitol from Glucose,” Appl. Microbiol., 18, 1031-1035 (1969) Mayer, G., KDKulbe and B. Nidetzky: “Utilization of Xylitol Dehydrogenase in a Combined Microbial / Enzymatic Process for Production of Xylitol from D-Glucose,” Appl. Biochem. Biotechnol., 98-100, 577-589 (2002) Suzuki, S., M. Sugiyama, Y. Mihara, K. Hashiguchi and K. Yokozeki: “Novel Enzymatic Method for the Production of Xylitol from D-Arabitol by Gluconobacter oxydans,” Biosci. Biotechnol. Biochem., 66, 2614- 2620 (2002) Sonoko, TYJojima, N. Tonouchi, R. Fudou and K. Yokozeki: “Microorganism Capable of Producing Xylitol or D-Xylulose,” EP Patent, 0974646 (2000) Harkki, SZ and J. London: “Manufacture of Xylitol using Recombinant hosts, US Patent, 5631150 (1997) Povelainen, M. and ANMiasnikov: “Production of Xylitol by Metabolically Engineered Strain of Bacillus subtilis,” J. Biotechnol., 128, 24-31 (2006) Ingram, JM and WAWood: “Enzymatic Basis for D-Arabitol Prouction by Saccharomyces rouxii,” J. Bacteriol., 89, 1186-1194 (1965) Ahmed, Z., H. Sasahara, SHBhuiyan, T. Saiki, T. Shimonishi, G. Takada, K. Izumori: “Production of D-Lyxose from D-Glucose by Microbial and Enzymatic Reactions,“ J. Biosci. Bioeng. , 88, 676-678 (1999) Fabre, L., P. Gallezot and A. Perrard: “Catalytic Hydrogenation of Arabinonic Acid and Lactones to Arabitol,” J. Catal., 208, 247-254 (2002)

  In the prior art, when converting lactose contained in whey to xylitol, there was only a conversion pathway of “lactose → glucose (+ galactose) → arabitol → xylulose → xylitol”. That is, a step of converting lactose into glucose and galactose using β-galactosidase is essential, and this glucose was converted into arabitol. At this time, unreacted glucose and unnecessary galactose existed in the reaction solution as impurities, and an operation for separating them was necessary.

  Moreover, although arabitol has been attracting attention as a raw material for pharmaceutical production and a food additive, an efficient production method has not been established.

  Therefore, an object of the present invention is to develop a method in which lactose is directly converted into arabitol, thereby preventing generation of glucose and galactose in the reaction solution and eliminating an operation for separating impurities.

  The present invention also focuses on the genus Kluyveromyces, which is a osmotic yeast having lactose consumption activity, and converts high-concentration lactose into ethanol and sugar alcohol (polyhydric alcohol in which the carbonyl group of sugar is reduced). Method development is also an issue.

  The present inventors have found a yeast that enables the conversion of “lactose → arabitol” among the conversion pathways of “lactose → arabitol → xylulose → xylitol” and completed the present invention.

  In the present invention, lactose can be directly converted into arabitol, and glucose and galactose are not present outside the cells (in the reaction solution), so that an operation for separating impurities is unnecessary.

  This makes it possible to provide a large amount of arabitol, which is a raw material for pharmaceutical production and a food additive, from lactose obtained in a large amount at a low price, and further, by converting it into xylitol via arabitol, whey can be effectively used.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Applications Nos. 2008-001592 and 2008-157574, which are the basis of the priority of the present application.

The possibility of the conversion route from lactose to xylitol is shown. The possibility of the metabolic pathway of lactose by microorganisms is shown. This is the result of aerobic culture of the NBRC1903 strain in the basic medium and examining the influence of medium components on sugar metabolism. A solution containing ammonium sulfate: 41.5 g / L, potassium dihydrogen phosphate: 3 g / L, dipotassium hydrogen phosphate: 1 g / L, magnesium sulfate heptahydrate: 1 g / L in basic medium: 50 mL The results of culturing NBRC1903 strain and adding 10 mL each of lactose monohydrate: 250 g / L 12 h and 24 h after the start of culture are shown. The solution which changed the density | concentration of the yeast extract in the basic medium: The result of having culture | cultivated NBRC1903 strain | stump | stock by 2 mL is shown. Medium (aerobic) shaking culture was performed at 200 rpm (reciprocal) and medium aerobic conditions were set. The solution which changed the density | concentration of the yeast extract in the basic medium: The result of having culture | cultivated NBRC1903 strain | stump | stock with 50 mL is shown. Cultivation was performed in a bubble column reactor at an air flow rate of 100 mL / min, and high aerobic conditions were set. A shows the changes over time in the cell concentration (♦), lactose concentration (■) and ethanol concentration (△) of K.lactis NBRC1903 in yeast in the mixture of whey UF permeate and yeast extract, The time-dependent change of the arabitol concentration (●) of the metabolite is shown. The results of examining the effect of complex salt concentration on arabitol production by adding complex salt (sodium chloride: potassium chloride prepared at a weight ratio of 3: 7) to the basic medium. A and B show the results of examining the influence of each salt concentration on arabitol production by adding sodium chloride and potassium chloride to the basic medium, respectively. The result of having investigated the influence of the time-dependent change of the conversion from arabitol to xylulose after aerobic culture (reaction) of G. oxydans NBRC 3172 is shown. The result of having examined C.shehatae IAM12953 culture | cultivation (reaction) and examined the influence of time-dependent change of the conversion from xylulose to xylitol is shown. A detailed microbial metabolism map is shown. By prioritizing the progression to the Pentose phosphate pathway (PPP) over the Embden-Myerhof Parnas pathway (EMPP), arabitol can be produced preferentially instead of ethanol. Production of D-arabitol from lactose by Kluyveromyces lactis and related metabolic pathways Underline, measurement items; production or consumption of H, NAD (P) H2 The results of examining the effect of initial lactose concentration on D-arabitol production by changing the lactose concentration to 20 g / L, 50 g / L, 100 g / L and 200 g / L are shown. ■ indicates residual lactose, ◆ indicates bacterial cells, ● indicates D-arabitol, ▼ indicates glycerol, and Δ indicates the concentration of ethanol. The results of examining the effect of initial salt concentration (osmotic pressure) on D-arabitol production are shown. The result of having examined the influence which initial glutamine concentration and culture temperature have on D-arabitol production is shown. A indicates the concentration of arabitol, and B indicates the amount of arabitol produced per lactose. ◆ indicates culture at 30 ° C, ■ indicates 35 ° C, and ▲ indicates culture at 37 ° C. The result of having examined the influence which microbial cell concentration operation and oxygen supply operation have on D-arabitol production is shown. A shows lactose concentration, B shows arabitol concentration, and C shows changes in ethanol concentration. Batch culture is shown as a control, and the culture volume is 2 mL. In “2X2mL” in (2), the bacterial cell concentration is twice that of the control, and the culture volume is 2 mL. In “2X1mL” of ◆, the bacterial cell concentration is twice that of the control, and the culture volume is 1mL.

1. Conversion of Lactose to Sugar Alcohol The present inventors examined conversion to sugar alcohols having various uses as effective utilization of lactose contained in a large amount in whey. Sugar alcohols include, for example, sorbitol, maltitol, erythritol, xylitol, lactitol, erythritol, which are used for prevention of dental caries, and used for diet. FIG. 2 shows the possibility of metabolic pathways from lactose to ethanol and sugar alcohol. The 6 carbon sugars produced from lactose are glucose and galactose, but when galactose is isomerized to glucose, the glucose passes through the Embden-Myerhof-Parnas (EMP) pathway, and if conditions are met, ethanol, glycerol Can be converted to erythritol. In addition, glucose is converted into pentose via the Pentose phosphate pathway (PPP), and can be converted into pentose sugar alcohols such as arabitol and xylitol if conditions are met. In the conversion of glucose to sugar alcohol, the case of glycerol has been reported in the conversion of lactose to sugar alcohol, but there are not many cases of other sugar alcohols. Therefore, the present inventors have decided to search for microorganisms that can convert lactose into sugar alcohol.

2. Search for microorganisms capable of converting lactose to sugar alcohol from osmophilic microorganisms It is said that osmotic microorganisms generally have a tendency to produce sugar alcohols, and the utilization of the genus Kluyveromyces is a valuable substance from lactose We think that there is a possibility of deriving. Therefore, the present inventors made the cells of the genus Kluyveromyces act on lactose at a high concentration, evaluated the possibility of conversion to ethanol and sugar alcohol, and examined the culture operating variables to derive valuable substances. did. As a result, Kluyveromyces lactis NBRC 0433 and Kluyveromyces lactis NBRC 1903 were found as strains excellent in the conversion of sugar alcohols into arabitol.

3. Whey, whey UF permeate, delactose whey UF permeate, whey hydrolyzate-containing lactose content or conversion from lactose to arabitol Whey, whey UF permeate, delactose whey UF permeate, whey hydrolyzate components An aqueous solution containing lactose or a hyperosmotic medium containing lactose (for example, a fluid exhibiting an osmotic pressure of lactose concentration of 4% by weight or more, preferably lactose of 100 g / L or more and 190 g / L or less), for example, 20 Treatment with Kluyveromyces lactis NBRC 0433 and / or Kluyveromyces lactis NBRC 1903 at ˜39 ° C., preferably 30-37 ° C., more preferably 35-37 ° C. for 24 h or longer, specifically aerobic Lactose can be converted to arabitol by culturing in an intensive manner.

  Specifically, as a culture condition of Kluyveromyces lactis NBRC 0433 and / or Kluyveromyces lactis NBRC 1903, whey, whey UF permeate, dehydration prepared so that the initial concentration (content) of lactose is an aqueous solution of 100 g / L or more. Prepare a medium containing a lactose whey UF permeate, whey hydrolyzate or other lactose-containing material or a high osmotic pressure solution containing lactose and a nitrogen source, 20-39 ° C, preferably 30-37 ° C, more Preferably, aerobic conditions are set at 35 to 37 ° C., and the bacterium is cultured for 24 hours or longer. Here, as the lactose-containing material, one or a mixture of two or more of whey, whey UF permeate, delactose whey UF permeate, whey hydrolyzate, concentrated solution, diluted solution, powder Any one or a mixture of two or more of aqueous solutions, or a mixture of any two or more of these stock solutions, concentrated solutions, diluents, and powders may be used. In addition, in whey, whey UF permeate, delactose whey UF permeate, and whey hydrolyzate, the solid content concentration is usually 6 to 6.5, 5 to 5.5, 5 to 5.5, and 6 to 6.5% by weight, respectively. In any case, the lactose concentration is 4 to 5% by weight. Nitrogen sources include ammonia or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium acetate, and ammonium phosphate, other inorganic and organic nitrogen-containing compounds, or yeast extract (yeast extract), whey, whey hydrolysate, and casein hydrolysis. Things can be used. As the salts, sodium chloride salt and / or potassium chloride salt, magnesium chloride, calcium chloride and the like can be used.

  At this time, in order to promote the production of arabitol, it is important to increase the osmotic pressure, and the initial concentration of lactose is 190 g / L, which corresponds to the limit of solubility of lactose (or lactose monohydrate) in aqueous solution. It is desirable to make it about 200g / L. Moreover, in order to raise osmotic pressure, it is desirable to prepare so that a sodium chloride salt and / or a potassium chloride salt may be included at 0-30 g / L, or 0.3-1.0 Mol / L. Preferably, the composite salt containing sodium chloride and potassium chloride at a weight ratio of 3: 7 has a concentration of 15 g / L or more, preferably 0.3 to 1.0 mol / L, more preferably 0.5 to 1.0 mol / L. Prepare to be L.

  Furthermore, it is desirable that the temperature be kept at a temperature close to the upper limit temperature for growth under important conditions.

  In order to increase the production of D-arabitol, it is desirable not to lower the aerobic conditions. For example, the culture solution can be treated by aeration of air, the culture solution can be treated by gently shaking, or the microorganism can be treated by gently shaking after increasing the concentration of the microorganism in the solution. Specifically, whey, whey UF permeate, delactose whey UF permeate, aqueous solution containing components of whey hydrolyzate or hyperosmotic medium containing lactose (for example, lactose is 4% by weight or more, preferably Fluid having a lactose concentration of 100 g / L or more and 190 g / L or less), for example, preferably at 35 to 37 ° C. for a certain period of time, for example, 24 h, Kluyveromyces lactis NBRC 0433 and / or Kluyveromyces lactis After culturing NBRC 1903 aerobically and increasing the bacterial cell concentration by applying a centrifugal separation treatment or a membrane separation treatment, it can be treated by gently shaking.

  The concentration of the yeast extract is about 40 g / L under the aerobic condition at the shake flask level (also called the medium aerobic condition or the microaerobic condition), and the aerobic condition at the bubble column reactor level (high level). (Aerobic condition) is preferably about 5 to 10 g / L. Instead of the yeast extract, glutamic acid can be added and used. For example, 2 to 10 g / L, preferably about 5 g / L of glutamic acid can be added. According to the present invention, arabitol can be produced from lactose without producing glucose or galactose as by-products outside the cells (in the reaction solution).

4). Production of xylitol from whey, whey UF permeate, delactose whey UF permeate, whey hydrolyzate and other lactose-containing substances or lactose “3. Whey, whey UF permeate, delactose whey UF permeate, whey hydrolysate The arabitol produced by the lactose-containing product such as a degradation product or the conversion of lactose to arabitol can be further converted to xylulose and subsequently xylylose can be converted to xylitol.

4-1. Conversion from arabitol to xylulose By treating arabitol with a microorganism having an ability to convert arabitol to xylulose, it can be converted to xylulose.

  As a strain capable of converting arabitol into xylulose, various microorganisms are already known, and examples include microorganisms belonging to the genus Acetobacter, microorganisms belonging to the genus Gluconobacter, microorganisms belonging to the genus Klebsiella, and the like.

  For the treatment, a method (means) for culturing the above-mentioned microorganism in a medium containing arabitol can be adopted, but an immobilized microbial cell in which the above-mentioned microorganism is immobilized on a carrier can also be used. For example, arabitol can be converted to xylulose by culturing Gluconobacter oxydans NBRC 3172 in a medium containing arabitol. Specifically, for pre-culture, 50 mL of Gluconobacter pre-culture medium (mannitol: 50 g / L, peptone: 10 g / L, yeast extract: 10 g / L) is placed in an Erlenmeyer flask with baffle (500 mL). After inoculating Gluconobacter oxydans NBRC 3172, incubating at 30 ° C, 200rpm, 48h, and removing the supernatant by centrifugation, collecting the cells, and washing with distilled water Thus, the method (means) used for the main culture can be employed. In the main culture, the bacterium is inoculated into an aqueous solution containing arabitol at 30 g / L so that the OD600 (absorbance at 600 nm) is 20, and the reaction is carried out by culturing at 30 ° C. Thereby, arabitol can be converted into xylulose. This conversion efficiency is close to 100%. Ohmomo et al. Performed conversion of arabitol to xylulose using a biocatalyst in which the Gluconobacter suboxydans Kawai strain was immobilized on κ-carrageenan (Ohmomo, S., Y. Tozawa and K. Ueda: “Biotransformation of D-Arabitol to D-Xylulose by Gluconobacter suboxydans Immobilized within k-Carrageenan, ”J. Ferment. Technol., 61, 373-378 (1983)).

4-2. Conversion of xylulose to xylitol It can be converted to xylitol by treating xylulose with a microorganism having the ability to convert xylulose to xylitol.

  Examples of known microorganisms that can convert xylulose to xylitol include microorganisms belonging to the genus Kluyveromyces, microorganisms belonging to the genus Candida, microorganisms belonging to the genus Zygosaccharomyces, microorganisms belonging to the genus Debaryomyces, microorganisms belonging to the genus Pichia, and the like. Specifically, Kluyveromyces lactis NBRC 1903, Candida shehatae IAM 12953, Kluyveromyces marxianus ATCC 26548, Zygosaccharomyces rouxii IAM 12879, Candida melibiosica NBRC 10238, Debaryomyces hansenii IAM 12837, Candida moP 223 Is included.

  For the treatment, a method (means) for culturing the microorganism in a medium containing xylulose can be adopted, but an immobilized microbial cell in which the microorganism is immobilized on a carrier can also be used. For example, xylulose can be converted to xylitol by culturing Kluyveromyces lactis NBRC 1903 and / or Candida shehatae IAM 12953 in a medium containing xylulose. Specifically, first, G. oxydans NBRC 3172 was inoculated and acted on an aqueous solution containing arabitol at 30 g / L to prepare xylulose. Next, in the pre-culture, in a test tube (20 mL), xylitol production medium (xylulose: 30 g / L, yeast extract: 3 g / L, ammonium sulfate: 5 g / L, dipotassium hydrogen phosphate: 2 g / L 2 mg of magnesium sulfate heptahydrate: 1 g / L), inoculated with Kluyveromyces lactis NBRC 1903 and / or Candida shehatae IAM 12953, and after shaking culture at 30 ° C., 200 rpm, 24 h Collect 0.9 mL of the culture solution after the pre-culture, collect the supernatant by centrifugation, add 0.9 mL of the aforementioned xylitol production medium, and culture at 30 ° C. To advance the reaction. This performed bioconversion from xylulose to xylitol. In actual experiments, stationary culture or medium aerobic culture was adopted as the culture condition. In static culture, the mixture was temporarily stirred every 3 h, and a sample was collected after 9 h. For aerobic culture, shaking culture was performed at 200 rpm. Suihko et al. Carried out the conversion of xylulose to xylitol using a biocatalyst immobilized on alginate gel, such as yeast Saccharomyces cerevisiae VTT-C-76029, Candida tropicalie ATCC 1369 or Pachysolen tannophilus (Suihko, ML., And K. Poutanen: “D-Xylulose Fermentation by Free and Immobilized SACCHAROMYCES CEREVISIAE Cells,” Biotechnol. Lett., 6, 189-194 (1984)).

In this example, the yeasts Kluyveromyces lactis var. Lactis NBRC 0433 and NBRC 1903, Kluyveromyces marxianus NBRC 1735 and NBRC 10005 (independent administrative agency, National Institute of Biotechnology, (Purchased from the Genetic Resource Department (NBRC)), and Kluyveromyces marxianus ATCC 26548 and ATCC 52486 (purchased from the American Type Culture Collection (ATCC)). Various strains were stored on agar plates (glucose: 2%, yeast extract: 1%, peptone: 2%, agar: 2%) at 277K (Kelvin, hereinafter the same).

  For preparation of cells to be used in the experiment, in the preculture, in a test tube (20 mL), medium (lactose monohydrate: 10 g / L, yeast extract: 3 g / L, ammonium sulfate: 5 g / L, phosphoric acid After adding 5 mL of dipotassium hydrogen: 2 g / L, magnesium sulfate heptahydrate: 1 g / L), various strains were inoculated and cultured at 200 rpm, 303 K, 12 h by shaking. In the main culture, put 5 mL of basic medium (lactose monohydrate: 200 g / L, yeast extract: 10 g / L) containing high-concentration lactose into a test tube (20 mL), The strain was inoculated and cultured with shaking at 200 rpm, 303K, 72h.

  In the measurement of the bacterial cell concentration, a sample was collected from the culture (bacterial cell culture) and obtained from the turbidity (OD600) at a wavelength of 600 nm using a spectrophotometer (Shimadzu Corporation, UV-120-02). In the culture of the NBRC1903 strain, OD600 corresponds to 0.225 g / L in terms of the dry cell concentration.

  In the evaluation of the characteristics of the strain, 2 mL of the basic medium (lactose monohydrate: 200 g / L, yeast extract: 10 g / L) was put into a test tube (20 mL), then K. lactis NBRC 0433 strain, K Lactis NBRC1903 strain, K. marxianus NBRC1735 strain, K. marxianus NBRC 10005 strain, K. marxianus ATCC 26548 strain, K. marxianus ATCC 52486 strain were inoculated so that the OD600 would be 0.05 at the initial cell concentration. And shaking culture at 200 rpm, 303 K, 72 h.

In the analysis of sugar and sugar alcohol, a sample was centrifuged to settle the cells, and then the supernatant was microfiltered (pore size: 0.2 μm). High-performance liquid chromatography (Shimadzu Corporation, LC-10AD) and a differential refractometer detector (RID-6A) were applied to the solution to measure the concentration of sugar and sugar alcohol. For high performance liquid chromatography, an SZ5532 column (6.0 × 150 mm) (Showa Denko) that separates in normal phase mode and ligand exchange mode was used, and the column temperature was set to 333K. The mobile phase was acetonitrile / water (75/25) and the flow rate was 1.0 mL / min. In the identification and concentration measurement of sugars and sugar alcohols, an effluent peak was prepared in advance using a standard solution, and the peak was used as a comparative control. The results are shown in Table 1.

  Lactose consumption activity was particularly high in cells of K. lactis NBRC0433, K. lactis NBRC1903, and K. marxianus NBRC1735, and ethanol and glycerol were detected as metabolites. The reaction rate of lactose was 91.3% for K. lactis NBRC0433, 89.1% for K. lactis NBRC1903, and 73.9% for K. marxianus NBRC1735. In these three strains, the amount of ethanol produced was similar, but the highest concentration was found in K. lactis NBRC0433, and the concentration reached 59.3 g / L.

  In addition, arabitol was also detected as a metabolite in K. lactis NBRC 0433 strain and K. lactis NBRC 1903 strain cells (2 species). The production of arabitol has not been reported in previous studies and is a completely new finding.

  On the other hand, in the cells of K. marxianus NBRC 10005 strain, K. marxianus ATCC 26548 strain, and K. marxianus ATCC 52486 strain (3 species), lactose consumption activity was low and no metabolite was detected in the reaction solution . These three strains may be kluyver negative (property that can consume carbon source but cannot be fermented) against lactose.

Effect of medium components on sugar metabolism In this example for examining the effect of medium components on sugar metabolism, K. lactis NBRC1903 strain was used.

  Place 50 mL of the basic medium (lactose monohydrate: 200 g / L, yeast extract: 10 g / L) used in Example 1 into a baffled Erlenmeyer flask (500 mL), and then add K. lactis NBRC1903 strain. Then, the cells were inoculated so that the OD600 was 0.0104 at the initial concentration of the bacterial cells, and shaking culture was performed at 200 rpm, 303K for a predetermined time. A well-ventilated silicon stopper was used for the lid of the flask. The result is shown in FIG.

  In Example 2 (FIG. 3), ethanol reached 31.5 g / L, glycerol reached 3.11 g / L, and arabitol reached 3.67 g / L. Compared with Example 1 (Table 1), in Example 2 (FIG. 3), the aerobic conditions are improved, so that the amount of ethanol produced is reduced and the amount of glycerol produced is improved. The amount of arabitol produced was the same in Example 1 and Example 2.

Effect of lactose addition In a conical flask (500 mL) with baffle, complex salt (ammonium sulfate: 41.5 g) in the basic medium (lactose monohydrate: 200 g / L, yeast extract: 10 g / L) used in Example 1 / L, potassium dihydrogen phosphate: 3 g / L, dipotassium hydrogen phosphate: 1 g / L, magnesium sulfate heptahydrate: 1 g / L) The lactis NBRC1903 strain was cultured under the same conditions as in Example 2. Lactose monohydrate: 250 g / L was added at 10 mL each, 12 h and 24 h after the start of culture. The result is shown in FIG.

  Compared to Example 2 (FIG. 3), in Example 3 (FIG. 4), ethanol was low at 24.5 g / L, but glycerol was significantly improved to 7.21 g / L and arabitol to 8.43 g / L. . It is thought that the addition of lactose suppressed the metabolic reaction following the EMP pathway (Embden-Myerhof Parnas pathway) and promoted the metabolic reaction to PPP (Pentose phosphate pathway).

Effect of yeast extract concentration (culture under moderate aerobic conditions)
In a test tube (20 mL), add 2 mL of the solution prepared by changing the concentration of the yeast extract in the basic medium used in Example 1 (lactose monohydrate: 200 g / L, yeast extract: 10 g / L). Then, the K. lactis NBRC1903 strain was inoculated so that the OD600 was 0.010 at the initial cell concentration, and shake culture (reciprocating) was performed at 200 rpm, 303 K, 72 h. At this time, the cells were cultured under moderate aerobic conditions. The result is shown in FIG.

  When the yeast extract was 40 g / L, arabitol reached a maximum value of 12.7 g / L.

Effect of yeast extract concentration (culture under high aerobic conditions)
In a bubble column reactor (30 mmfx200 mm), a solution prepared by changing the concentration of the yeast extract in the basic medium (lactose monohydrate: 200 g / L, yeast extract: 10 g / L) used in Example 1 After injecting 20 mL, inoculate K. lactis NBRC1903 strain so that the OD600 is 0.02 at the initial cell concentration, and continuously supplying air at 100 mL / min from the bottom of the reactor, For 72 hours. At this time, the cells were cultured under high aerobic conditions. The result is shown in FIG.

  When the yeast extract was 5 to 10 g / L, arabitol reached a maximum value of 9 g / L.

Production of arabitol from whey Production of arabitol from whey was examined. A medium containing powder of whey UF permeate was prepared, inoculated with K. lactis NBRC1903 strain, and cultured with shaking. The result is shown in FIG.

  In a medium containing only whey UF permeate powder, the initial concentration of whey UF permeate powder was adjusted to 250 g / L. In this medium, growth of K. lactis NBRC 1903 strain was confirmed. The maximum amount of arabitol reached 4.15 g / L.

Effect of complex salt concentration Prepared by changing the concentration of complex salt (sodium chloride and potassium chloride adjusted to 3: 7 by weight ratio) in the basic medium used in Example 1 in a baffled Erlenmeyer flask (500 mL). After adding 50 mL of the prepared medium, K. lactis NBRC1903 strain was inoculated and cultured by shaking at 50 rpm, 303 K, 90 h, and the influence of the complex salt concentration on arabitol production was examined. The result is shown in FIG.

  As the concentration of the complex salt increased, the production of arabitol increased, and when the concentration of the complex salt was 30 g / L, the maximum value of arabitol reached 2.1 g / L.

Effect of salt concentration Medium prepared by changing the concentration of complex salt (adjusted by changing the weight ratio of sodium chloride and potassium chloride) with the basic medium used in Example 1 in an Erlenmeyer flask with baffle (500 mL) After adding 50 mL of the solution, K. lactis NBRC1903 strain was inoculated and cultured with shaking at 50 rpm, 303 K, 90 h, and the influence of the concentration of each salt on arabitol production was examined. The result is shown in FIG.

  When sodium chloride was 10 g / L, arabitol production reached a maximum of 2.1 g / L. On the other hand, when potassium chloride was 30 g / L, the arabitol production reached a maximum of 2.2 g / L.

Effect of initial lactose concentration on D-arabitol production 2 mL of medium added with initial lactose (monohydrate) concentration of 20-200 g / L and yeast extract added to a concentration of 40 g / L, K. lactis NBRC1903 was inoculated and cultured in batches at 303K under shaking conditions of 200 rpm. The result is shown in FIG.

  ■ indicates residual lactose, ◆ indicates bacterial cells, ● indicates D-arabitol, ▼ indicates glycerol, and Δ indicates the concentration of ethanol. Regarding the production of D-arabitol, the initial lactose concentration is extremely important, and D-arabitol production was confirmed only when the initial lactose concentration was 100 g / L or more. When the initial lactose concentrations were 100 and 200 g / L, the production amounts of D-arabitol after 168 hours were 0.62 g / L and 18.0 g / L, respectively. As a supplementary experiment, K. lactis NBRC1903 was cultured at an initial lactose concentration of 200 g / L, and after 24 hours when the lactose concentration reached 125 g / L, lactose was added to 150 g / L, and the osmotic pressure was increased and cultured. It was. D-arabitol production decreased to 13.1 g / L. Next, K. lactis NBRC1903 was cultured at an initial lactose concentration of 200 g / L, and after 72 hours when the lactose concentration decreased to about 1/10 of the initial concentration, lactose was added to 100 g / L to increase the osmotic pressure. The culture was continued. D-arabitol production was 17.1 g / L. Furthermore, K. lactis NBRC1903 was cultured at an initial lactose concentration of 100 g / L, and after 24 hours when the lactose concentration dropped to about 1/10 of the initial concentration, lactose was added to 100 g / L to increase the osmotic pressure. The culture was continued. D-arabitol production increased to 8.01 g / L (more than 10 times the above 0.62 g / L). Early D-arabitol production appears to be the cellular response of K. lactis NBRC1903 to the high osmotic pressure that high lactose concentrations imply. Glycerol accumulation has been reported in the cellular response of the yeast Saccharomyces cereviae to hyperosmolarity. However, in FIG. 13, it can be seen that the extracellular glycerol concentration of K. lactis NBRC1903 is not significantly improved.

Effect of initial salt concentration on D-arabitol production A 50 mL medium was prepared in a baffled flask (500 mL). In addition to 200 g / L lactose (monohydrate) and 10 g / L yeast extract, salt was added to the medium. This salt is a complex salt containing NaCl and KCl at a mass ratio of 3: 7, and was prepared by adding NaCl and KCl so that the concentration was 0 to 50 g / L. Further, an experiment was conducted in which a single salt of NaCl or KCl was added in a concentration range of 0 to 30 g / L. The result is shown in FIG. In FIG. 14D, the D-arabitol production was plotted against the total ion concentration ci = [Na +] + [K +] + [Cl−] (1) set for each condition. The experimental results for both the complex salt and the single salt appear to be plotted in a single curve. From this, it is inferred that the cell responds according to the osmotic pressure proportional to the ion concentration, and produces D-arabitol.

Effect of initial glutamine concentration and culture temperature on D-arabitol production Using a test tube (20 mL), 2 mL of medium (lactose (monohydrate), 200 g / L; (NH ₄ ) ₂ SO ₄ 3.4 g / L; KH ₂ PO ₀ , 2 g / L; MgSO ₀ · 7H ₀ O, 1 g / L; Glutamine, 2 to 10 g / L; Yeast Nitrogen Base without Amino Acid) Shaking culture was performed at 308 and 310K at 200 rpm, and the effects of glutamine concentration and culture temperature were examined. The result is shown in FIG. Compared to experiments using yeast extract, the overall production of D-arabitol is low, but increasing the culture temperature increases the production of D-arabitol. At 310K (37 ° C), the glutamine concentration is 5 g / L. It can be seen that the production of D-arabitol has reached 17.1 g / L.

Effects of cell concentration and oxygen supply on D-arabitol production
The effects of concentration and oxygen supply operations of K. lactis NBRC1903 on D-arabitol production were examined. The medium contains lactose at 100 g / L or 200 g / L; (NH ₀ ) ₀ SO ₀ , 3.4 g / L; KH ₀ PO ₀ , 2 g / L; MgSO ₀ · 7H ₀ O, 1 g / L; Glutamine, 5 g / L; Using 2 mL of a liquid consisting of Yeast Nitrogen Base without Amino Acid, shaking culture was performed at 200 rpm at a temperature of 310K. After 24 hours from the start of the culture, the cell concentration was doubled by centrifugation and the culture was continued. The results are shown in FIG. When the bacterial cell concentration operation was performed and shaking culture was continued at 200 rpm using a 2 mL medium, the D-arabitol production amount was slightly reduced as compared to the case where the bacterial cell concentration operation was not performed. As a result of the bacterial cell concentration operation, it was considered that the ethanol concentration was improved, the anaerobic property was improved, and the production amount of D-arabitol was reduced. When the bacterial cell concentration operation was performed and shaking culture was continued at 200 rpm using 1 mL of the medium in the liquid volume, the D-arabitol production increased compared to the case where the bacterial cell concentration operation was not performed, and after 120 h. Reached 26.7 g / L. Compared to Example 1 above, a concentration as high as 6.8 times could be obtained.

[Reference Example 1]
Conversion from arabitol to xylulose In this reference example, conversion from arabitol to xylulose was examined.

  Gluconobacter oxydans NBRC 3172 strain on the preservation medium after putting 50mL of medium (mannitol: 50g / L, yeast extract: 10g / L, peptone: 10g / L) into an Erlenmeyer flask with baffle (500mL) Was inoculated by the amount of the tip of the platinum ear, and cultured with shaking at 200 rpm, 303 K, 48 h. After centrifuging and culturing this culture solution, the cells were sedimented and concentrated, and 1 mL of an aqueous solution containing arabitol at 30 g / L was added to a test tube (20 mL), and then the G. oxydans NBRC prepared above was used. The 3172 strain was inoculated so that the OD600 was 20 at the initial concentration of the cells, and shaking culture was performed at 200 rpm, 303K for a predetermined time. The result is shown in FIG.

  Six hours after the start of culture, most of the arabitol reacted and xylulose was produced. The xylulose yield was about 100%. At this time, a trace amount of xylitol was produced as a by-product.

[Reference Example 2]
Conversion of xylulose to xylitol In this reference example, conversion of xylulose to xylitol was examined.

In a test tube (20 mL), medium (xylulose: 32.9 g / L, xylitol: 0.4 g / L, yeast extract: 3 g / L, ammonium sulfate: 5 g / L, dipotassium hydrogen phosphate: 2 g / L, magnesium sulfate After adding 2 mL of heptahydrate: 1 g / L), Kluyveromyces lactis NBRC 1903 strain, Kluyveromyces marxianus ATCC 26548 strain, Candida melibiosica NBRC 10238 strain, Candida mogii NBRC 0436 strain, Candida shehatae IAM 12953 IAM 87953 strain, Zygosaccharom Strain, Debaryomyces hansenii IAM 12837, Pichia farinose IAM 12223, Pichia stipitis IAM 12952 (purchased from the Foundation for Applied Microbiology Research (IAM)) (Reciprocating) shaking culture was performed at 303K for 24 hours. At the end of these cultures, OD600 was 18.7 for Kluyveromyces lactis NBRC 1903, 27.3 for Kluyveromyces marxianus ATCC 26548, 32 for Candida melibiosica NBRC 10238, 16.3 for Candida mogii NBRC 0436, 30.8 for Candida shehatae IAM 12953go It was 3.6 for rouxii IAM 12879 strain, 16.6 for Debaryomyces hansenii IAM 12837 strain, 17.1 for Pichia farinose IAM 12223 strain, and 20.7 for Pichia stipitis IAM 12952 strain. Place 0.9 mL of these cultures in a micro test tube (1.5 mL), centrifuge and wash, and concentrate the cells by sedimentation, then add 0.9 mL of the above medium and add 303K. And static culture was performed. After 3 hours and 6 hours from the start of culture, the culture solution in the microtube was temporarily stirred. The results are shown in Table 2.

  As a comparative control experiment, the above-mentioned cells were inoculated into a test tube (20 mL) and aerobically shaken and cultured at 200 rpm, 30 ° C. for a predetermined time.

  Xylitol production was 0.42 g / L for Kluyveromyces marxianus ATCC 26548, 0.88 g / L for Candida melibiosica NBRC 102388, 1.79 g / L for Candida shehatae IAM 129538, 0.15 g / L for Zygosaccharomyces rouxii IAM 128798, The Pichia farinose IAM 122238 strain was 0.13 g / L, and the Pichia stipitis IAM 129528 strain was 0.10 g / L.

  On the other hand, xylitol was not detected in Kluyveromyces lactis NBRC 19038 strain, Candida mogii NBRC 04368 strain and Debaryomyces hansenii IAM 128378 strain.

  Candida shehatae IAM 129538 strain was collected so that OD600 would be 30.8, put into a micro test tube (1.5 mL), 0.9 mL of the above medium was newly added, and then static culture was performed. The result is shown in FIG.

  Basically, it was stationary culture, but the culture solution was temporarily stirred 3, 6, 9, 12 hours after the start of the culture. In the initial stage, 85.0% of 32.9 g / L of xylulose was consumed, and in the final stage, xylitol reached 6.32 g / L (initial xylitol: 0.54 g / L). At this time, the yield of xylitol was 0.22. The by-product arabitol was in a low concentration.

  The present invention can be used in the technical field of sugar alcohol production.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (6)

A method for converting lactose into arabitol by treating a lactose-containing solution with lactose assimilating / osmotic pressure Kluyveromyces lactis having the ability to convert lactose into arabitol. By treating lactose-containing solution with lactose assimilation and osmotic pressure Kluyveromyces lactis that has the ability to convert lactose into arabitol, lactose can be converted to arabitol without producing glucose and galactose as by-products How to convert to The method according to claim 1 or 2, wherein the lactose assimilating and osmotic Kluyveromyces lactis is Kluyveromyces lactis NBRC 0433 and / or Kluyveromyces lactis NBRC 1903. A method for producing xylitol from a solution containing lactose, comprising the following steps.
(1) A step of converting lactose into arabitol by treating a lactose-containing solution with lactose assimilating / osmotic Kluyveromyces lactis having the ability to convert lactose into arabitol;
(2) A step of converting arabitol into xylulose by treating a solution containing arabitol with a microorganism having the ability to convert arabitol into xylulose,
(3) A step of converting xylulose into xylitol by treating a solution containing xylulose with a microorganism having the ability to convert xylulose into xylitol. A method for producing xylitol from a solution containing lactose, comprising the following steps.
(1) Lactose- utilizing and osmotic Kluyveromyces lactis , which has the ability to convert lactose into arabitol, is converted into arabitol without producing glucose and galactose as by-products by treating the solution containing lactose The process of
(2) A step of converting arabitol into xylulose by treating a solution containing arabitol with a microorganism having the ability to convert arabitol into xylulose,
(3) A step of converting xylulose into xylitol by treating a solution containing xylulose with a microorganism having the ability to convert xylulose into xylitol. The method according to claim 4 or 5 , wherein the lactose assimilating and osmotic Kluyveromyces lactis having the ability to convert lactose into arabitol is Kluyveromyces lactis NBRC 0433 and / or Kluyveromyces lactis NBRC 1903.

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