Classifications

• • 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
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters
  • C12P7/6445—Glycerides
  • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/98—Preparation of granular or free-flowing enzyme compositions
• • 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
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Definitions

• Lipases are widely used in the reactions such as esterification of various carboxylic acids such as fatty acids with alcohols such as mono-alcohol and polyalcohol, and trans-esterification between plural carboxylates.
• the trans-esterification method is an important technology not only as reforming animal and plant fatty oils but also as methods for producing various fatty esters, sugar esters and steroids.
• esterification can be conducted under the mild condition such as at room temperature to about 70° C. Therefore, the lipase can better inhibit side reactions and reduce energy costs compared with the existing chemical reactions.
• Patent Literature 1 Japanese Patent Publication No. Sho 60-98984
• Patent Literature 3 Japanese Patent Publication No. Hei 2-138986
• Patent Literature 7 Japanese Patent Publication No. 2000-106873
• An object of the present invention is to provide a lipase powder wherein the lipase activity and stability are improved.
• Another object of the present invention is to provide a lipase powder wherein the 1,3-selectivity of the lipase is improved.
• a still another object of the present invention is to provide a method for producing the lipase powder.
• a further object of the present invention is to provide a trans-esterification method of fatty oil, which comprises using the lipase powder.
• Lipase activity and stability are extremely improved by granulating the lipase with a solid content of animal milk to obtain the powder thereof.
• the lipase is a 1,3-specific lipase
• the 1,3-selectivity is extremely improved.
• the present invention has been completed on the basis of these findings.
• the present invention provides a lipase powder which is a granulated product containing a lipase and a solid content (solid material) of animal milk.
• the present invention also provides a lipase composition wherein the lipase powder is immersed or impregnated in fatty oil.
• the present invention further provides a method for producing a lipase powder which comprises adding animal milk or cream derived from animal milk to an aqueous solution containing a lipase, and spray-drying, freeze-drying or solvent-precipitating the mixture thereof.
• the present invention further provides a lipase for trans-esterification or esterification containing the lipase powder.
• the present invention further provides a trans-esterification method of fatty oil, which comprises using the lipase for the trans-esterification.
• the lipase used in the present invention includes a lipoprotein lipase, a monoacylglycerol lipase, a diacylglycerol lipase, a triacylglycerol lipase, a galactolipase, a phospholipase and the like.
• the triacylglycerol lipase is preferred.
• Microorganism which produces these lipases includes, without limited to bacteria, yeast, filamentous bacterium, actinomyces and the like, Psudomonas sp., Alcaligenes sp., Arthrobacter sp., Staphylococcus sp., Torulopsis sp., Escherichia sp., Micotorula sp., Propionibacterum sp., Chromobacterum sp., Xanthomonas sp., Lactobacillus sp., Clostridium sp., Candida sp., Geotrichum sp., Sacchromycopsis sp., Nocardia sp., Fusarium sp., Aspergillus sp., Penicillium sp., Mucor sp., Rhizopus sp., Phycomycese sp., Puccinia sp., Bacillus
• a 1,3-specific lipase derived from Rhizopus sp. and Thermomyces sp. is preferred; in particular, a 1,3-specific lipase derived from Rhizopus oryzae and Thermomyces lanugenousus is more preferred.
• the animal milk used in the present invention includes cow milk, goat milk and the like.
• the cow milk is preferred, in particular, the solid content of animal milk is preferably a solid content of cow milk or cream derived from cow milk.
• the solid content of animal milk is preferably 0.1 to 20 times, more preferably 1 to 20 times mass of lipase.
• the lipase powder according to the present invention must comprise a lipase and a solid content of animal milk.
• the lipase powder may comprise, in addition to these components, a lipase culture component.
• the lipase powder according to the present invention has a water content of 10% by weight or less, in particular, from 6.5 to 8.5% by weight.
• the particle size of the lipase powder according to the present invention can be optional, it is preferable that 90% by weight or more of the lipase powder has the particle size of 1 to 100 ⁇ m. In this connection, it is preferable that an average particle size thereof be 20 to 80 ⁇ m, more preferably 20 to 50 ⁇ m. In addition, the lipase powder is preferably spherical.
• the particle size of the lipase powder can be determined by, for example, Particle Size Distribution Analyzer (LA-500) of HORIBA, Ltd.
• the lipase powder according to the present invention can be obtained by, for example, adding animal milk or cream derived from animal milk to an aqueous solution containing a lipase, and spray-drying, freeze-drying or solvent-precipitating the mixture thereof.
• solvent-precipitation examples include ethanol, acetone, methanol, isopropyl alcohol and hexane, and a mixture thereof.
• ethanol and acetone are preferable since these solvents can further improve activity of lipase powder.
• the drying after solvent-precipitation can be conducted by, for example, drying under reduced pressure.
• examples of the aqueous solution containing a lipase include a lipase culture solution from which a cell body is removed, a purified culture solution thereof, a solution in which the lipase powder obtained from these culture solutions is dissolved and dispersed again; a solution in which the commercially available lipase powder is dissolved and dispersed again; and a commercially available liquid lipase.
• low-molecular-weight components such as salts are removed from the solution.
• low-molecular-weight components such as sugar are removed from the solution.
• a lipase culture solution includes, for example, aqueous solutions containing soybean flour, peptone, corn steep liquor, K 2 HPO 4 , (NH 4 ) 2 SO 4 , MgSO 4 /7H 2 O and the like.
• concentrations thereof are as follows: the soybean flour is 0.1 to 20% by weight and preferably 1.0 to 10% by weight; peptone is 0.1 to 30% by weight and preferably 0.5 to 10% by weight; the corn steep liquor is 0.1 to 30% by weight and preferably 0.5 to 10% by weight; K 2 HPO 4 is 0.01 to 20% by weight and preferably 0.1 to 5% by weight; (NH 4 ) 2 SO 4 is 0.01 to 20% by weight and preferably 0.05 to 5% by weight; and MgSO 4 /7H 2 O is 0.01 to 20% by weight and preferably 0.05 to 5% by weight.
• the culture conditions thereof should be controlled as follows: the culture temperature is 10 to 40° C. and preferably 20 to 35° C.; the quantity of airflow is 0.1 to 2.0 VVM and preferably 0.1 to 1.5 VVM; the rotation speed for stirring is 100 to 800 rpm and preferably 200 to 400 rpm; pH is 3.0 to 10.0 and preferably 4.0 to 9.5.
• the separation of a cell body is preferably conducted by centrifugation, the membrane filter procedure and the like.
• the removal of the low-molecular-weight components such as salts and sugar can be treated with ultrafiltration membranes. Specifically, after the treatment with ultrafiltration membranes, the aqueous solution containing a lipase is concentrated so as to become 1 ⁇ 2 volume thereof, and then, the same amount of a phosphate buffer as that of the concentrated solution is added thereto. By repeating these procedures once to 5 times, the aqueous solution containing a lipase can be obtained, from which the low-molecular-weight components are removed.
• the centrifugation is preferably controlled to 200 to 20,000 ⁇ g.
• the pressure applied to the membrane filter is preferably controlled by microfiltration membranes, the filter press and the like to become not more than 3.0 kg/m 2 .
• cell breakage thereof is conducted by the homogenizer, Waring blender, the ultrasonic disruption, the French press, the ball mill and the like; then the cell residues are removed by centrifugation, the membrane filter procedure and the like.
• the rotation speed of the homogenizer for stirring is 500 to 30,000 rpm and preferably 1,000 to 15,000 rpm.
• the rotation speed of Waring blender is 500 to 10,000 rpm and preferably 1,000 to 5,000 rpm.
• the time for stirring is 0.5 to 10 minutes and preferably 1 to 5 minutes. It is preferable that the ultrasonic disruption is conducted under the condition of 1 to 50 KHz and more preferably 10 to 20 KHz. It is preferable that the ball mill has glass pellets having the diameter of 0.1 to 0.5 mm.
• the aqueous solution containing a lipase is that containing 5 to 30% by weight of lipase as a solid content.
• the solid content of the added animal milk or cream derived from animal milk is preferably 0.1 to 20 times, more preferably 0.3 to 10 times, most preferably 0.3 to 5 times mass of the solid content of the aqueous solution containing a lipase.
• the concentrations of the solid content in the aqueous solution containing a lipase and the solid content of the animal milk or the cream derived from animal milk can be determined as Brix. % by using, for example, the sugar content analyzer (Refractormeter) (CIS Corporation., Ltd.: BRX-242).
• pH of the aqueous solution containing a lipase is adjusted to the range of from 6 to 7.5 after animal milk or cream derived from animal milk is added.
• pH is preferably adjusted to 7.0 or less, more preferably the range of from 6.5 to 7.0.
• pH adjusting is conducted immediately before the drying step such as spray-drying, pH adjusting can be conducted in any previous steps. It is possible that pH of the aqueous solution containing a lipase is preliminarily adjusted in such a manner that pH immediately before the drying step is in the above-mentioned range.
• an alkali metal hydroxide such as sodium hydroxide.
• the aqueous solution containing a lipase may be concentrated.
• the concentration methods are not particularly limited and they include evaporator, flash evaporator, the concentration by ultrafiltration, the concentration by microfiltration, salting out by inorganic salts, precipitation methods with solvents, absorption methods with ion-exchange cellulose and the like, and water absorption methods with water-absorbing gels.
• the concentration by ultrafiltration and evaporator are preferable.
• the module for the concentration by ultrafiltration is preferably a flat membrane or a hollow fiber membrane having a fractioned molecular weight of 3,000 to 100,000 and more preferably 6,000 to 50,000.
• the materials of the membrane are preferably polyacrylonitrile, polysulfonic and the like.
• spray drying is conducted by spray-dryers such as nozzle countercurrent flow, disk countercurrent flow, nozzle concurrent flow and disk concurrent flow, and the disk concurrent flow is more preferable.
• the spray-drying is preferably controlled as follows: the rotation speed of the atomizer is 4,000 to 20,000 rpm; and heating is 100 to 200° C. for inlet temperature and 40 to 100° C. for outlet temperature.
• Freeze-drying is also preferable, for example, it is preferable that the freeze-drying is conducted by a tray stepwise type freeze-drying with a freeze-drying machine for small amount, which is laboratory size. Furthermore, the lipase powder can be prepared by drying under reduced pressure.
• the lipase powder thus prepared can be used as itself. However, it is preferable, from the point of handling, that it is used as a lipase composition wherein the lipase powder is immersed or impregnated in fatty oil.
• the mass of the fatty oil in the lipase composition is preferably 0.1 to 20 times and more preferably 1 to 20 times mass of the lipase powder.
• the lipase composition can be easily obtained by adding the fatty oil to the lipase powder produced by spray-drying and the like; and then uniformly stirring the mixture by a stirrer, three-one motor, and the like. It can also be easily obtained by preliminarily adding the fatty oil to a powder recovering region of a spray-dryer; uniformly stirring the mixture after the recovering; and then removing the excess fatty oil by filtration.
• the fatty oils for immersing or infiltrating the lipase powder are not particularly limited. They include vegetable oils such as canola oil, soybean oil, higholeic sunflower oil, olive oil, safflower oil, corn oil, palm oil and sesame-seed oil; triacylglycerols such as triolein(glycerol trioleate), tricaprilyn(glycerol trioctanoate), triacetin(glycerol triacetate)and tributyrin(glycerol tributyrate); and the mixture of one or more thereof such as fatty ester and sterol ester.
• vegetable oils such as canola oil, soybean oil, higholeic sunflower oil, olive oil, safflower oil, corn oil, palm oil and sesame-seed oil
• triacylglycerols such as triolein(glycerol trioleate), tricaprilyn(glycerol trioctanoate), triacetin(gly
• the lipase is a 1,3-specific lipase, in particular, a lipase derived from Rhizomucor miehei and Alcaligenes sp.
• 1,3-selectivity of said lipase is extremely improved according to the present invention. Therefore, said lipase powder can be suitably used as a lipase for trans-esterification and for esterification.
• the trans-esterification of the fatty oil and the like, trans-esterification of the fatty oil and fatty acid ester, trans-esterification of alcoholysis and acidolysis, or esterification of glycerin and fatty acid can be effectively conducted by the ordinary method using the lipase powder.
• the present invention provides a lipase powder having improved lipase activity and stability.
• the lipase is a 1,3-specific lipase
• 1,3-selectivity of a 1,3-specific lipase is extremely improved, and the fatty acid residue which is located on the second position of triglyceride as a raw material can be retained in the trans-esterification manufacture at an extremely high percentage.
• the low-molecular-weight components were removed by using the UF module (ASAHI KASEI CHEMICALS CORPORATION: SIP-0013) from a liquid lipase (Trade name: Palatase 20000L) of Novozymes Japan Ltd, in which a lipase derived from Rhizomucor miehei was dissolved and dispersed in an aqueous solution to obtain an aqueous solution 1 containing a lipase (the concentration of the solid content: 20.1% by weight).
• liquid lipase (Palatase 20000L) was treated with ultrafiltration modules under cooling with ice and concentrated so as to become 1 ⁇ 2 volume thereof.
• the solid content of the cow milk is 0.64 times mass of the solid content of the aqueous solution 1 containing a lipase.
• the solution was sprayed by using a spray-dryer (SD-1000: TOKYO RIKAKIKI Co., Ltd.) under the conditions of inlet temperature: 130° C., the air content for drying: 0.7 to 1.1 m 3 /min, and spray pressure: 11 to 12 kpa to obtain lipase powder.
• the shape of the thus-obtained lipase powder was spherical, 90% by weight or more of the lipase powder has a particle size of 1 to 100 ⁇ m and the average particle size thereof was 7.6 ⁇ m.
• the particle size was determined by Particle Size Distribution Analyzer (LA-500) of HORIBA, Ltd.
• the concentration of the solid content of the aqueous solution containing a lipase and the concentration of the solid content of the cow milk were determined by the following method.
• the concentrations were determined as Brix. % by using the sugar content analyzer (Refractormeter) (CIS Corporation.: BRX-242).
• Example 2 To the lipase concentrated solution obtained in Example 1, the same amount of water as that of the concentrated solution was added to obtain the aqueous solution 2 containing a lipase (The volume ratio of the lipase concentrated solution to water was 1:1). To obtain a lipase powder, the same procedure was conducted as that of Example 1 except that the aqueous solution 2 containing a lipase was used instead of the aqueous solution 1 containing a lipase. The volume ratio of the lipase concentrated solution: water: the milk was 0.5:0.5:1. The solid content of the cow milk was 1.05 times mass of the solid content of the aqueous solution (UF) containing a lipase.
• UF aqueous solution
• Example 2 To the lipase concentrated solution obtained in Example 1, the same amount of 0.01M phosphate buffer (pH 7) as that of the concentrated solution was added to obtain the aqueous solution 3 containing a lipase (The volume ratio of the lipase concentrated solution to the buffer was 1:1). To obtain a lipase powder, the same procedure was conducted as that of Example 1 except that the aqueous solution 3 containing a lipase was used instead of the aqueous solution 1 containing a lipase. The volume ratio of the lipase concentrated solution:the phosphate buffer:the cow milk was 0.5:0.5:1. The solid content of the cow milk was 1.03 times mass of the solid content of the aqueous solution containing a lipase.
• Example 2 To the lipase concentrated solution obtained in Example 1, the same amount of 0.01M phosphate buffer (pH 8) as that of the concentrated solution was added to obtain the aqueous solution 4 containing a lipase (The volume ratio of the lipase concentrated solution to the buffer was 1:1). To 20 ml of the aqueous solution 4 containing a lipase, 10 ml of cow milk (available from Koiwai Dairy Products Co. Ltd., “Koiwai Gyu-nyu Oisisa Shitate”: the concentration of the solid content is 12.9% by weight) was added. The pH of the solution thus obtained was adjusted with an aqueous solution of sodium hydroxide to become the pH 6.8 to 6.9. The volume ratio of the lipase concentrated solution:the phosphate buffer:the cow milk was 0.5:0.5:0.5. The solid content of the cow milk was 0.52 times mass of the solid content of the aqueous solution containing a lipase.
• Example 2 To the lipase concentrated solution obtained in Example 1, the same amount of 0.01M phosphate buffer (pH 8) as that of the concentrated solution was added to obtain the aqueous solution 5 containing a lipase (The volume ratio of the lipase concentrated solution to the buffer was 1:1). To 20 ml of the aqueous solution 5 containing a lipase, 2 ml of cream fraiche (Trade name: Hokkaido Junsei Cream 35; available from Takanashi Milk Co. Ltd.; the concentration of the solid content is 43% by weight) was added. The pH of the solution thus obtained was adjusted with an aqueous solution of sodium hydroxide to become the pH 6.8 to 6.9.
• 0.01M phosphate buffer pH 8
• the volume ratio of the lipase concentrated solution:the phosphate buffer:the cream fraiche was 0.5:0.5:0.1.
• the solid content of the cream fraiche was 0.34 times mass of the solid content of the aqueous solution containing a lipase.
• Example 2 To the lipase concentrated solution obtained in Example 1, the same amount of 0.01M phosphate buffer (pH 8) as that of the concentrated solution was added to obtain the aqueous solution 6 containing a lipase (The volume ratio of the lipase concentrated solution to the buffer was 1:1). To 20 ml of the aqueous solution 6 containing a lipase, 20 ml of Jersey cow milk (“Aso Shokoku Jersey 4.5 Milk”; available from Aso Agriculture Corporative Association; the concentration of the solid content was 13.2% by weight) was added. The pH of the solution thus obtained was adjusted with an aqueous solution of sodium hydroxide to become the pH 6.8 to 6.9.
• 0.01M phosphate buffer pH 8
• the volume ratio of the lipase concentrated solution:the phosphate buffer:the cow milk was 0.5:0.5:1.
• the solid content of the milk was 1.06 times mass of the solid content of the aqueous solution containing a lipase. Thereafter, a lipase powder was obtained as Example 1.
• Example 2 The same procedure was conducted as that of Example 1 except that freeze-drying was conducted as powdering means instead of spray-drying to obtain lipase powder.
• the freeze-drying was conducted as follows. An aqueous solution containing a lipase whose pH was adjusted to 6.8 to 6.9 was poured into a recovery flask and frozen by dry ice methanol. Then, the frozen material was freeze-dried by using a freeze-dryer (FDU-830) of TOKYO RIKAKIKAI CO, LTD at 0.15 Torr for 1 to 2 day(s). After drying, the resultant was lightly crushed in a mortar to obtain lipase powder.
• FDU-830 of TOKYO RIKAKIKAI CO, LTD at 0.15 Torr for 1 to 2 day(s).
• Example 3 To obtain a lipase powder, the same procedure was conducted as that of Example 3 except that the cow milk was not added. The volume ratio of the lipase concentrated solution to the buffer was 1:1.
• the activity of the lipase powder thus obtained was determined by the following method. The results were shown in Table 1.
• the reaction rate constant K was determined from the reaction rates of each samples at each time by using the analysis software (orijin ver.6.1). At this time, the value of the final reaction rate is changeable.
• the reactivity of the 1.3 position was calculated when the reactivity of the second position was regarded as 1.
• the vaporizing chamber Temperature of the detector: 370° C.
• the reaction using 5 g of tricaprilyn and 5 g of triolein was conducted at 60° C. for 24 to 72 hours. Initial decreasing levels of activity for each batch were plotted and the half-life period was calculated from total reaction time and the decreasing level of activity.
• the half-life period of the lipase powder obtained by Example 1 was 913 hours and that of Comparative Example 1 was 234 hours. Therefore, the stability of the lipase powder according to the present invention was improved twice or more.
• a lipase powder of Meito Sangyo Co., Ltd. (Trade name: Lipase QL, derived from Alcaligenes sp.) was suspended in water to obtain an aqueous solution containing a lipase (the concentration of the solid content: 2.0% by weight).
• an aqueous solution containing a lipase the concentration of the solid content: 2.0% by weight.
• cow milk available from Koiwai Dairy Products Co. Ltd., “Koiwai Gyu-nyu Oishisa Shitate”, the concentration of the solid content: 12.9% by weight
• the volume ratio of the aqueous solution containing a lipase to the cow milk was 10:1, and the solid content of the cow milk was 0.65 times mass of the solid content of the aqueous solution containing a lipase.
• the pH of the solution thus obtained was adjusted with an aqueous solution of sodium hydroxide to become the pH 6.8 to 6.9.
• This solution was sprayed by using a spray-dryer (SD-1000: TOKYO RIKAKIKAI Co., Ltd.) under the conditions of inlet temperature: 130° C., the air content for drying: 0.7 to 1.1 m 3 /min, and spray pressure: 11 to 12 kpa to obtain a lipase powder.
• the shape of the thus-obtained lipase powder was spherical, 90% by weight or more of the lipase powder has a particle size of 1 to 100 ⁇ m and the average particle size thereof was 351 ⁇ m.
• the particle size was determined by Particle Size Distribution Analyzer (LA-500) of HORIBA, Ltd.
• Example 8 To obtain a lipase powder, the same spray-drying procedure was conducted as that of Example 8 except that the cow milk was not added.
• SD-1000 TOKYO RIKAKIKAI Co., Ltd.
• the resultant was sprayed by using a spray-dryer (SD-1000: TOKYO RIKAKIKAI Co., Ltd.) under the conditions of inlet temperature: 130° C., the air content for drying: 0.7 to 1.1 m 3 /min, and spray pressure: 11 to 12 kpa to obtain a lipase powder.
• SD-1000 TOKYO RIKAKIKAI Co., Ltd.
• the obtained precipitate was collected by a centrifuge machine (Beckman Co., Ltd.: GS-6KR) under the condition of 3000 rpm for 10 minutes and then, the drying step was conducted under a reduced pressure by a dry machine (available from TOKYO RIKAKIKAI Co., Ltd.: FDU-830) for 16 to 20 hours to obtain a lipase powder.
• a spray-dryer SD-1000: TOKYO RIKAKIKAI Co., Ltd.
• the same lipase powder (Lipase F-AP15) as that used in Comparative Example 4 was re-suspended in water in 15% by weight concentration, and to 10 ml of the lipase solution, 10 ml of cow milk (available from Meiji Dairies Co., Ltd., “Meiji Oishii Gyu-nyu”, the concentration of the solid content: 12.9 mass %) was added.
• the resultant was sprayed by using a spray-dryer (SD-1000: TOKYO RIKAKIKAI Co., Ltd.) under the conditions of inlet temperature: 130° C., the air content for drying: 0.7 to 1.1 m 3 /min, and spray pressure: 11 to 12 kpa to obtain a lipase powder.
• SD-1000 TOKYO RIKAKIKAI Co., Ltd.
• a liquid lipase (Trade name: Lipozyme Tl 100L) of Novozymes Japan Ltd, which was derived from Thermomyces lanugenousus was sprayed by using a spray-dryer (SD-1000: TOKYO RIKAKIKAI Co., Ltd.) under the conditions of inlet temperature: 130° C., the air content for drying: 0.7 to 1.1 m 3 /min, and spray pressure: 11 to 12 kpa to obtain a lipase powder.
• SD-1000 TOKYO RIKAKIKAI Co., Ltd.
• the obtained precipitate was collected by a centrifuge machine (Beckman Co., Ltd.: GS-6KR) under the condition of 3000 rpm for 3 minutes and then, the drying step was conducted under a reduced pressure by a dry machine (available from TOKYO RIKAKIKAI Co., Ltd.: FDU-830) for 16 to 20 hours to obtain a lipase powder.
• Example 5 Tl 100 L ⁇ spray dry 100
• Example 6 Tl 100 L ⁇ ethanol-precipitation 0

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