WO2009088037A1 - Procédé de conversion d'une matière contenant du lactose telle que le lactosérum ou du lactose en arabitol - Google Patents

Procédé de conversion d'une matière contenant du lactose telle que le lactosérum ou du lactose en arabitol Download PDF

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WO2009088037A1
WO2009088037A1 PCT/JP2009/050133 JP2009050133W WO2009088037A1 WO 2009088037 A1 WO2009088037 A1 WO 2009088037A1 JP 2009050133 W JP2009050133 W JP 2009050133W WO 2009088037 A1 WO2009088037 A1 WO 2009088037A1
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whey
lactose
arabitol
solution
permeate
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PCT/JP2009/050133
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Japanese (ja)
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Kazuhisa Ohtaguchi
Hiroyuki Itou
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Tokyo Institute Of Technology
The Food Science Institute Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

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  • 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.
  • Whey protein concentrate obtained by ultrafiltration (UF) membrane separation and lactose separated from whey UF permeate, It is used as a product on a commercial scale.
  • WPC Whey protein concentrate
  • 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 applications 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.
  • 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.
  • 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, T. Y. Jojima, N. Tonouchi, R.
  • 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 conversion of “lactose ⁇ arabitol” among the conversion pathways of “lactose ⁇ arabitol ⁇ xylulose ⁇ xylitol” and completed 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 not necessary.
  • the results of culturing NBRC1903 strain and adding 10 mL each of lactose monohydrate: 250 10g / L 12 h and 24 h after the start of culture are shown.
  • ⁇ indicates residual lactose
  • ⁇ indicates bacterial cells
  • ⁇ indicates D-arabitol
  • ⁇ indicates glycerol
  • ⁇ 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
  • B indicates the amount of arabitol produced per lactose.
  • ⁇ indicates culture at 30 ° C
  • ⁇ indicates 35 ° C
  • ⁇ 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
  • C shows changes in ethanol concentration.
  • Batch culture is shown as a control, and the culture volume is 2 mL.
  • the culture volume is 2 mL.
  • the bacterial cell concentration is twice that of the control, and the culture volume is 2 mL.
  • the culture volume is 1mL.
  • 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.
  • EMP Embden-Myerhof-Parnas
  • 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.
  • PPP Pentose phosphate pathway
  • the present inventors have decided to search for microorganisms that can convert lactose into sugar alcohol.
  • whey, whey UF permeate dehydrated, prepared so that the initial concentration (content) of lactose is 100 g / L or more.
  • a medium containing lactose whey UF permeate, whey hydrolyzate or other lactose-containing substance or a high osmotic pressure aqueous solution containing lactose and add nitrogen source to 20-39 ° C, preferably 30-37 ° C.
  • 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.
  • the solid content is usually 6 to 6.5, 5 to 5.5, 5 to 5.5, and 6 to 6.5% by weight, respectively.
  • 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.
  • 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.
  • the concentration of which is 15 g / L or more, preferably 0.3 to 1.0 mol / L, more preferably 0.5 to 1.0 mol / L.
  • the temperature is preferably maintained at a temperature close to the upper limit of growth temperature under important conditions, and can be set to, for example, 35 to 37 ° C, more preferably 37 ° C.
  • 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.
  • 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.
  • 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.
  • glutamic acid can be added and used.
  • 2 to 10 g / L, preferably about 5 g / L of glutamic acid can be added.
  • arabitol can be produced from lactose without producing glucose or galactose as by-products outside the cells (in the reaction solution).
  • microorganisms As strains 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.
  • a method for culturing the above-mentioned microorganisms in a medium containing arabitol can be employed, but an immobilized microbial cell in which the above-mentioned microorganisms are immobilized on a carrier can also be used.
  • an immobilized microbial cell in which the above-mentioned microorganisms are immobilized on a carrier can also be used.
  • arabitol can be converted to xylulose.
  • Gluconobacter pre-culture medium mannitol: 50 g / L, peptone: 10 g / L, yeast extract: 10 g / L
  • Gluconobacter pre-culture medium mannitol: 50 g / L, peptone: 10 g / L, yeast extract: 10 g / L
  • baffle 500 mL
  • Gluconobacter oxydans NBRC 3172 incubate at 30 °C, 200rpm, 48h, remove the supernatant by centrifugation, collect the bacteria, and wash with distilled water
  • the method (means) used for the main culture can be employed.
  • 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.
  • 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 ⁇ -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)).
  • a method for culturing the microorganism in a medium containing xylulose can be adopted, or an immobilized microbial cell in which the microorganism is immobilized on a carrier can also be used.
  • xylulose can be converted to xylitol by culturing Kluyveromyces lactis NBRC 1903 and / or Candida shehatae IAM 12953 in a medium containing xylulose.
  • xylulose was prepared by inoculating G.Goxydans NBRC 3172 in an aqueous solution containing arabitol at 30 g / L.
  • 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.
  • NBRC Genetic Resource Department
  • ATCC American Type Culture Collection
  • Various strains were stored on agar plates (glucose: 2%, yeast extract: 1%, peptone: 2%, agar: 2%) at 277K (Kelvin, hereinafter the same).
  • 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).
  • OD600 corresponds to 0.225 g / L in terms of the dry cell concentration.
  • K. lactis NBRC 0433 strain K Lactis NBRC1903, K. marxianus NBRC1735, K. marxianus NBRC 10005, K. marxianus ATCC 26548, K. marxianus ATCC 52486 were inoculated at an initial cell concentration of 0.05.
  • shaking culture at 200 rpm, 303 K, 72 h.
  • Lactose consumption activity was particularly high in K. lactis NBRC0433 strain, K. lactis NBRC1903 strain and K. marxianus NBRC1735 strain, and ethanol and glycerol were detected as metabolites.
  • the reaction rate of lactose was 91.3% in K. lactis NBRC0433, 89.1% in K. lactis NBRC1903, and 73.9% in K. marxianus NBRC1735.
  • the amount of ethanol produced was similar, but K. lactis NBRC0433 strain had the highest concentration, reaching 59.3 g / L.
  • arabitol was also detected as a metabolite in the cells of K.2lactis NBRC 0433 strain and K. lactis NBRC 1903 strain (2 species). The production of arabitol has not been reported in previous studies and is a completely new finding.
  • Example 2 Put 50 mL of the basic medium (lactose monohydrate: 200 g / L, yeast extract: 10 g / L) used in Example 1 into an Erlenmeyer flask with baffle (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.
  • the basic medium lactose monohydrate: 200 g / L
  • yeast extract 10 g / L
  • Example 2 In Example 2 (FIG. 3), ethanol reached 31.5 g / L, glycerol reached 3.11 g / L, and arabitol reached 3.67 g / L.
  • Example 1 Table 1
  • Example 2 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.
  • 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)
  • 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.
  • Example 3 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 that traces EMP pathway (Embden-Myerhof Parnas pathway) and promoted the metabolic reaction to PPP (Pentose phosphate pathway).
  • EMP pathway Embden-Myerhof Parnas pathway
  • PPP Pentose phosphate pathway
  • the initial concentration of the whey UF permeate powder was adjusted to 250 ⁇ g / L.
  • the growth of K. lactis NBRC 1903 strain could be confirmed.
  • the maximum amount of arabitol reached 4.15 g / L.
  • ⁇ indicates residual lactose
  • ⁇ indicates bacterial cells
  • ⁇ indicates D-arabitol
  • ⁇ indicates glycerol
  • ⁇ indicates ethanol concentration.
  • 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.
  • the production amounts of D-arabitol after 168 hours when the initial lactose concentrations were 100 and 200 g / L were 0.62 g / L and 18.0 g / L, respectively.
  • K 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.
  • K. lactis NBRC1903 was cultured at an initial lactose concentration of 200 g / L, and after 72 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 was 17.1 g / L.
  • 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 impair. 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 has not improved much.
  • 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.
  • D-arabitol 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.
  • 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.
  • the cell concentration was doubled by centrifugation and the culture was continued.
  • the results are shown in FIG.
  • 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.
  • the ethanol concentration was improved, the anaerobic property was improved, and the production amount of D-arabitol was reduced.
  • Gluconobacter oxydans NBRC 3172 strain on the storage medium 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 settled and concentrated, and then 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.
  • 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.
  • 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, and 1.79 g / L for Zygosaccharomycesacrouxii 0.15 It was 0.13 g / L for Pichia farinose IAM 122238 and 0.10 g / L for Pichia stipitis IAM 129528.
  • xylitol could not be 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 in 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.
  • the present invention can be used in the technical field of sugar alcohol production.

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Abstract

Dans les techniques classiques de conversion du lactose contenu dans le lactosérum en xylitol, un procédé de conversion du lactose en glucose et en galactose avec la β-galactosidase est absolument nécessaire, et le glucose est ensuite converti en arabitol. Pendant le procédé, le produit de réaction peut être contaminé par du glucose n'ayant pas réagi ou du galactose indésirable en tant qu'impureté et, par conséquent, une opération de séparation du glucose n'ayant par réagi ou du galactose indésirable est également requise. Ainsi, l'objectif est de développer un procédé de conversion du lactose en arabitol directement, qui peut empêcher la production de glucose ou de galactose et pour lequel une quelconque opération de séparation des impuretés n'est pas nécessaire. Une nouvelle souche de levure a été trouvée, qui permet la conversion du lactose en arabitol.
PCT/JP2009/050133 2008-01-08 2009-01-08 Procédé de conversion d'une matière contenant du lactose telle que le lactosérum ou du lactose en arabitol WO2009088037A1 (fr)

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