WO2016088589A1 - ラクターゼ溶液及びそれを用いた乳製品 - Google Patents
ラクターゼ溶液及びそれを用いた乳製品 Download PDFInfo
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- WO2016088589A1 WO2016088589A1 PCT/JP2015/082784 JP2015082784W WO2016088589A1 WO 2016088589 A1 WO2016088589 A1 WO 2016088589A1 JP 2015082784 W JP2015082784 W JP 2015082784W WO 2016088589 A1 WO2016088589 A1 WO 2016088589A1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C7/00—Other dairy technology
- A23C7/04—Removing unwanted substances other than lactose or milk proteins from milk
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- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
- A23C9/1206—Lactose hydrolysing enzymes, e.g. lactase, beta-galactosidase
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- 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
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01108—Lactase (3.2.1.108)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44747—Composition of gel or of carrier mixture
Definitions
- the present invention relates to a lactase solution and milk and dairy products using the same.
- Lactose intolerance is a condition in which lactose cannot be successfully decomposed innately and thus exhibits various symptoms such as abdominal pain and diarrhea due to lactose in foods such as dairy products.
- Lactose is a disaccharide composed of galactose and glucose.
- lactose contained in milk or the like is previously decomposed into galactose and glucose by the enzyme lactase.
- a lactase solution used for degrading lactose such as milk is conventionally cultured by cultivating lactase-producing microorganisms, extracting lactase from the cells, removing contaminants derived from the culture, and then purifying the stabilizer. Manufactured by adding and sterilizing by filtration.
- Patent Document 1 Japanese Patent Publication No. 60-183964 discloses an invention relating to a method for producing lactase from a culture of a strain having Kluyveromyces lactis. According to this method, after the yeast cells are self-digested, the obtained crude enzyme solution is passed through a DEAE cellulose column and eluted with a salt concentration gradient, so that two active categories (lactase A and lactase B) are obtained. can get. It is disclosed that these two active categories have little difference in various properties including temperature stability, except that the pH stability is slightly different, and that the enzyme preparation may consist of a mixture of these. Yes.
- this lactase is a polypeptide which consists of 1025 amino acids, and the molecular weight is estimated to be 117,618 (nonpatent literature 1).
- the lactase described in Patent Document 1 has an optimum temperature of 40 to 50 ° C., is inactivated at 50 ° C. for 10 minutes, 45% at pH 7.0, and 100% at 55 ° C. for 10 minutes. Has been.
- the fact that lactose contained in milk was actually degraded using this enzyme is not described. Accordingly, there is no description of the problem of the activity decrease when lactase is added to the raw material milk, particularly the problem of enzyme deactivation when a heat load of 40 ° C. or higher is applied in the milk.
- An object of the present invention is to provide a lactase solution excellent in thermal stability.
- lactase has high thermal stability by increasing the fraction of lactase that forms a band of about 120 kDa in SDS polyacrylamide gel electrophoresis (SDS-PAGE).
- SDS-PAGE SDS polyacrylamide gel electrophoresis
- a lactase solution wherein the fraction of lactase having a molecular weight of about 120 kDa as determined by SDS polyacrylamide gel electrophoresis is 20% or more;
- the sum of the fraction of the 120 kDa lactase and the fraction of the lactase fraction with a molecular weight of about 80 kDa as determined by SDS polyacrylamide gel electrophoresis is 30% or more [1.
- the lactase solution according to [3] The lactase solution according to [1], wherein the fraction of lactase having a molecular weight of about 50 kDa as determined by SDS polyacrylamide gel electrophoresis is 70% or less; [4] The sum of the fraction of the lactase fraction of about 120 kDa and the fraction of the fraction of about 80 kDa lactase is the ratio of the fraction of lactase having a molecular weight of about 50 kDa as determined by SDS polyacrylamide gel electrophoresis.
- the salting-out treatment includes A saturation step of saturating the recovered lactase with a salting-out agent of 10 to 90%;
- a lactase solution in which lactose decomposition activity is hardly lowered even when it is added to raw milk and subjected to a heat load of 40 ° C. or higher.
- FIG. 1 is a diagram showing the results of SDS-PAGE of various lactase solutions.
- Lane 1 Example 1-1
- Lane 2 Example 1-2
- Lane 3 Comparative Example 1-1
- Lane 4 Comparative Example 1-2
- Lane 5 Comparative Example 2
- Lane 6 Comparative Example 3
- Lane M molecular weight standard.
- Lanes 1 and 2 and lanes 3 and 4 show the results of different lots.
- FIG. 2 is a diagram showing the results of SDS-PAGE (panel A) and Western blotting (panel B) after lactose decomposition reaction at 43 ° C. for 2 hours after adding a lactase solution to raw milk.
- FIG. 3A is a diagram showing the change over time in the amount of lactose when a lactase solution is added to raw milk and reacted at 37, 40, 43, 46 and 49 ° C.
- FIG. The plots in the figure indicate the results in Example 1, ⁇ in Comparative Example 1, ⁇ in Comparative Example 2, and ⁇ in Comparative Example 3.
- FIG. 3A is a diagram showing the change over time in the amount of lactose when a lactase solution is added to raw milk and reacted at 37, 40, 43, 46 and 49 ° C.
- FIG. The plots in the figure indicate the results in Example 1, ⁇ in Comparative Example 1, ⁇ in Comparative Example 2, and ⁇ in Comparative Example 3.
- FIG. 3A is a diagram showing the change over time in the amount of lactose when a lactase solution is added to raw milk and reacted at 37, 40, 43, 46 and 49 ° C.
- FIG. The plots in the figure indicate the results in Example
- FIG. 3B is a diagram showing the change over time in the lactose decomposition rate when a lactase solution is added to raw milk and reacted at 37, 40, 43, 46 and 49 ° C.
- FIG. The plots in the figure indicate the results in Example 1, ⁇ in Comparative Example 1, ⁇ in Comparative Example 2, and ⁇ in Comparative Example 3.
- Example 5 is a graph showing changes over time in the amount of lactose (Panel A) and the rate of lactose degradation (Panel B) when lactase solutions having different proportions of 120 kDa fraction are added to raw milk and reacted at 49 ° C. is there.
- ⁇ indicates the results in Example 1
- ⁇ indicates the results in Example 2
- ⁇ indicates the results in Example 3
- * indicates the results in Example 4
- * indicates the results in Example 5
- ⁇ indicates the results in Comparative Example 1.
- the lactase solution of the present invention desirably has a lactase activity of 10 to 100,000 NLU / g.
- “NLU” is Neutral Lactase Unit.
- the method for measuring the activity is as follows. Measured by hydrolysis of the substrate o-nitrophenyl- ⁇ -galactopyranoside (ONPG) to o-nitrophenol and galactose. The reaction is terminated by the addition of sodium carbonate. The o-nitrophenol formed turns yellow in alkaline medium and the change in absorbance is used to measure enzyme activity (expressed in NLU / g). This procedure was published in the US Food Chemicals Standards Collection (FCC), 4th Edition, July 1, 1996, pages 801-802 / Lactase (neutral) ( ⁇ -galactosidase) activity. Yes.
- the lactase solution used in the present invention can be recovered from a microorganism and purified by the following method.
- the manufacturing method of the lactase solution of this invention passes through the following four processes. That is, (1) Microbe culture process, (2) Lactase recovery process from microorganisms, (3) a lactase purification step, and (4) a lactase activity adjustment step.
- a known culture medium may be used for the microorganism culturing step, and a known strain may be used.
- the culture conditions are also known and can be appropriately selected as necessary.
- This extraction step is not particularly limited as long as lactase can be transferred to the outside of the cell, and a known extraction method can be used.
- lactase is an enzyme secreted to the outside of cells by gene transfer, mutation, etc., this culture step contains lactase, so this extraction step is unnecessary.
- Patent Document 1 Patent Document 2 and Patent Document 3 all have the same purification of the lactase solution by chromatography. By performing this chromatography, lactase can be purified and lactase activity can be improved. However, as will be described later, when lactase is purified using chromatography (partition chromatography or molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, etc.), lactase, which is originally 120 kDa, is converted into 80 kDa and 50 kDa lactase. Each was found to be decomposed.
- chromatography partition chromatography or molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, etc.
- lactase Although any molecular weight lactase has lactase activity, lactase undergoes degradation, especially when the fraction of 50 kDa increases, resulting in a decrease in lactase thermal stability, resulting in degradation of lactose at relatively high temperatures. There was a problem that it was difficult to proceed.
- the lactase of the present invention can be obtained by using salting-out and desalting treatment in the purification process. That is, after lactase is precipitated by salting out, the precipitate is recovered and redissolved, and the salt contained in the precipitate is desalted to obtain the lactase of the present invention. Salting-out, precipitation recovery / re-dissolution and desalting may be performed continuously. Further, as long as the lactase of the present invention is obtained, other purification means (including chromatography and activated carbon treatment) can be used in combination.
- Examples of the salting-out agent for salting out include ammonium sulfate, sodium sulfate, potassium phosphate, magnesium sulfate, sodium citrate, sodium chloride, and potassium chloride, and one or more of these can be used.
- ammonium sulfate as a salting-out agent to a solution containing lactase, it is preferably 10 to 90% saturated, more preferably 30 to 70% saturated.
- an amount corresponding to the amount of ammonium sulfate added may be used.
- Lactase can be precipitated from a solution containing lactase by adding a salting-out agent such as ammonium sulfate.
- the conditions from the addition of the salting-out agent to the precipitation of lactase are preferably left at 1 to 40 ° C. for 1 to 80 hours.
- the pH condition at this time is preferably 4-9. More preferably, it is 4 to 25 ° C. (room temperature), 1 to 48 hours, and pH 5 to 8. In addition, about the lower limit of temperature conditions, what is necessary is just the temperature which the solution containing lactase does not coagulate.
- the solid lactase is redissolved in water or a buffer solution, and desalted by dialysis or ultraconcentration.
- the step of adjusting lactase activity is not limited as long as the activity of lactase can be adjusted.
- water an aqueous solution containing salts, addition of a stabilizer, and the like.
- the lactase solution of the present invention can also be obtained by mixing a commercially available lactase solution and the lactase solution obtained by the above-mentioned method in a range where the fraction of molecular weight by SDS polyacrylamide gel electrophoresis satisfies a predetermined ratio. Is possible.
- the molecular weight of lactase in the lactase solution can be estimated by SDS-PAGE using 10% polyacrylamide gel.
- a lactase solution is diluted with purified water as necessary, mixed 1: 1 with Sample-Buffer for SDS-PAGE, and a sample for electrophoresis is prepared by heat treatment at 95 ° C. for 5 minutes. Apply standard and electrophoresis sample to 10% acrylamide gel and run. Standard uses BIO-RAD # 161-0313 (Prestained). The gel after electrophoresis is subjected to protein staining with a CBB staining solution (APRO SP-4010).
- the lactase solution of the present invention was dried using a TEFCO polyacrylamide gel drying kit (trade name Clear® Solution) after staining with SDS-PAGE and CBB.
- the dried gel is taken in as a gray scale image with a scanner GT-X820 manufactured by EPSON, and the concentration (protein amount) of each band is measured with ImageJ software (NIH, Bethesda, MD).
- the lactase solution of the present invention is characterized in that the fraction having a molecular weight of about 120 kDa measured by the above method is high.
- the fraction of lactase having a molecular weight of about 120 kDa as determined by SDS polyacrylamide gel electrophoresis refers to a position corresponding to about 120 kDa compared to the mobility of the molecular weight standard after electrophoresis by the above method ( Refers to the fraction of molecules that form a band (between about 100 kDa and about 150 kDa).
- the lactase solution of the present invention has a fraction of about 120 kDa as measured by ImageJ software (NIH, Bethesda, MD) after SDS-PAGE and CBB staining, which is 20% or more, preferably 50% or more, more preferably 80%. Above, most preferably 90% or more. Although an upper limit is not specifically limited, For example, it is 100%. This ratio is calculated by the following method.
- the concentration of each band was quantified by using ImageJ software (NIH, Bethesda, MD), about 120 kDa (100-150 kDa), about 80 kDa (80-100 kDa), about 50 kDa (49-54 kDa) ) And about 30 kDa (28 to 32 kDa), and the ratio of the band of about 120 kDa is calculated, assuming that the entire main band including the band corresponding to about 30 kDa (28 to 32 kDa) is 100%.
- ImageJ software NASH, Bethesda, MD
- the values when the total of the four bands is 100% are described.
- the sum of the fraction of about 80 kDa calculated by the same method as described above and the ratio of the fraction of 120 kDa is preferably 30% or more, more preferably 60%. Or more, more preferably 90% or more. Although an upper limit is not specifically limited, For example, it is 100%.
- lactase I When about 120 kDa lactase (hereinafter sometimes referred to as lactase I) is degraded, it becomes about 80 kDa lactase (hereinafter sometimes referred to as lactase II), and when lactase I or lactase II is degraded, about 50 kDa lactase (hereinafter referred to as lactase). III). Both lactase I and lactase II in the lactase solution have lactase activity and heat resistance. Lactase III has lactase activity but has insufficient heat resistance. In any fraction, lactase activity is equivalent.
- lactase II is a degradation product of lactase I
- the ratio of lactase I in the lactase solution is larger than that of lactase II from the viewpoint of heat resistance.
- the ratio of lactase III in the lactase solution is preferably 70% or less, more preferably 40% or less, and even more preferably 10% or less. Although a lower limit is not specifically limited, For example, it is 0%.
- the value obtained by dividing the sum of the ratio of lactase I and the ratio of lactase II by the ratio of lactase III is preferably 0.5 or more, more preferably 1.0 or more, and further preferably 5.0 or more. preferable. If it is less than 0.5, the heat resistance tends to be insufficient. Since it is most preferable that the lactase solution does not contain lactase III at all, that is, it is preferably zero, the upper limit value is not limited.
- Raw material milk is a target to which a lactase solution is added.
- known raw material milk may be used.
- the raw milk includes those before sterilization and those after sterilization.
- the raw material milk should just use milk.
- the raw milk is composed of water, raw milk, pasteurized milk, skim milk, whole milk powder, skim milk powder, butter milk, butter, cream, whey protein concentrate (WPC), whey protein isolate (WPI). ), ⁇ (alpha) -La, ⁇ (beta) -Lg, and the like.
- the lactose contained in the raw material milk can be decomposed by adding the lactase solution of the present invention to the raw material milk.
- the decomposition temperature is 1 to 60 ° C., and the decomposition time is 10 minutes to 24 hours.
- the method for producing lactose-decomposed fermented milk is as follows. 1. Method of fermenting milk after lactase is decomposed by adding lactase to milk before sterilization, and lactase is deactivated simultaneously with heat sterilization of milk (JP-A-5-501197). 2. a method of fermenting milk after lactase is decomposed by adding lactase to pasteurized milk, and lactase is deactivated by heat treatment; 3. Method of fermenting milk after degrading lactose in milk with immobilized lactase (Japanese Patent Laid-Open Nos. 46-105593 and 59-162833). There is a method of fermenting raw materials that have been previously lactose-decomposed or lactose-removed using pasteurized milk.
- the lactase solution according to the present invention is particularly suitable for dairy production.
- dairy products refer to milk such as ice and long life milk, yogurt, fresh cream, sour cream, cheese and the like.
- the lactase solution according to the present invention can be preferably used when a heat load of 40 ° C. or higher is applied.
- An example of such an application is yogurt.
- Lactase solution (Example 1) After sterilizing a liquid medium containing 7% corn steep liquor and 2% lactose (pH 5.5 after sterilization), Kluyveromyces lactisNo. 013-2 (ATCC 8585 strain) was inoculated and cultured at 30 ° C. for 24 hours with aeration of 12000 L / min. After culturing, the mixture was allowed to stand for 4 hours while cooling, and then the supernatant was removed from the upper part of the tank to obtain 1500 kg of bacterial cells that aggregated and settled at the bottom of the tank.
- Example 2 The lactase solution of Example 1 and the following Comparative Example 1 were mixed at a weight ratio of 80:20 to obtain the lactase solution of Example 2 (5,000 NLU / g).
- Example 3 The lactase solution of Example 1 and the following Comparative Example 1 were mixed at a weight ratio of 60:40 to obtain the lactase solution of Example 3 (5,000 NLU / g).
- Example 4 The lactase solution of Example 1 and the following Comparative Example 1 were mixed so that the weight ratio was 40:60 to obtain the lactase solution (5,000 NLU / g) of Example 4.
- Example 5 The lactase solution of Example 1 and the following Comparative Example 1 were mixed so that the weight ratio was 20:80 to obtain the lactase solution of Example 5 (5,000 NLU / g).
- Comparative Example 1 The commercial name “GODO-YNL2SS” (5000 NLU / g, manufactured by Godo Shusei Co., Ltd.), which is a commercially available lactase preparation, was used as the lactase solution of Comparative Example 1.
- Comparative Example 2 The trade name “MAXILACT LG5000” (manufactured by DSM, 5000 NLU / g), which is a commercially available lactase preparation, was used as the lactase solution of Comparative Example 2.
- a 10% acrylamide gel (4% stacking gel, gel thickness 1 mm, migration distance 50 mm) is subjected to a molecular weight standard and a sample for electrophoresis, and at the Marisol Industrial Migration Co., the electrophoresis front is 10 mA constant current and separating 20 mA. Electrophoresis was performed until the vicinity was reached. BIO-RAD # 161-0313 (Prestained) was used as the molecular weight standard (lane M). The gel after electrophoresis was subjected to protein staining with a CBB staining solution (APRO SP-4010) for 1 hour.
- the solution after the reaction is diluted 20 times with purified water (W / V), mixed with Sample Buffer for SDS-PAGE in the same manner as above, and sample for electrophoresis by heat treatment at 95 ° C for 5 minutes.
- W / V purified water
- Sample Buffer for SDS-PAGE sample for electrophoresis by heat treatment at 95 ° C for 5 minutes.
- a molecular weight standard and a sample for electrophoresis were provided on two 10% acrylamide gels and migrated simultaneously. BIO-RAD # 161-0313 (Prestained) was used as the molecular weight standard (lane M).
- a nitrocellulose membrane (BIO-RAD # 162-0114) was used as a transfer membrane for Western blotting, and transfer was performed by a wet method. After the transfer, the membrane was subjected to a blocking operation with a block ace solution (4 g of Snow Brand Dairy Block Ace powder / 100 mL of purified water) and then washed with Tween-PBS.
- a block ace solution (4 g of Snow Brand Dairy Block Ace powder / 100 mL of purified water
- the lactase solution obtained in Example 1 was subjected to SDS-PAGE, and a 120 kDa band was cut out from the gel and finely crushed, and then mixed with Difco's adjuvant complete Freund to make an emulsion. This was injected subcutaneously into the base of the tail of Balb-C mice a total of 3 times. After confirming an increase in antibody titer in the serum, the centrifuged supernatant of the collected whole blood was used as an anti-lactase polyclonal antibody. With this antibody, bands of 120, 80 and 50 kDa of lactase were detectable.
- the above-mentioned anti-lactase antibody was reacted at room temperature for 2 hours in a solution diluted 1,000 times with Block Ace diluent (Block Ace solution diluted 10 times with purified water). After washing 4 times with Tween-PBS, the secondary antibody (Gort-mouse IgG (H + L) -HRP; Southern Biotech 1034-05) was diluted 5,000 times with Block Ace dilution solution at room temperature. Reacted for hours. After washing with Tween-PBS, staining with 3,3′-diaminobenzidine (DAB) was performed. As the DAB substrate, DAB buffer tables (MERCK 1.029224.0001) were used.
- Lactose degradation test 1 The lactase solution of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were each final concentration 0.05% (w / v) (2.5 NLU / 100 mL milk) on UHT pasteurized milk (130 ° C., 2 seconds) Lactose decomposition reaction was performed at 49 ° C, 46 ° C, 43 ° C, 40 ° C and 37 ° C.
- FIG. 3A shows the result of the change over time in the amount of lactose
- FIG. 3B shows the rate of lactose degradation.
- Lactose degradation test 2 Using the lactase solutions of Examples 1 to 5 and Comparative Example 1, a lactose decomposition reaction was carried out at 49 ° C. according to the method of the lactose decomposition test described above. The obtained results are shown in FIG. Panel A shows the result of the change over time in the lactose amount, and panel B shows the lactose decomposition rate. It was confirmed that lactose decomposition progressed as the band of about 120 kDa increased.
- a lactase solution having a main band of about 120 kDa in SDS-PAGE is 20% or more is more heat-resistant than a lactase solution having a main band of molecular weight of about 80 kDa, about 50 kDa and about 30 kDa in SDS-PAGE. Can be said to be expensive.
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Abstract
Description
また、Kluyveromyces lactisのラクターゼの遺伝子解析によれば、このラクターゼは1025アミノ酸からなるポリペプチドであり、分子量は117,618と推定されている(非特許文献1)。
〔1〕 SDSポリアクリルアミドゲル電気泳動による分子量が約120kDaのラクターゼの画分の割合が、20%以上であることを特徴とするラクターゼ溶液;
[2] 前記120kDaのラクターゼの画分の割合と、SDSポリアクリルアミドゲル電気泳動による分子量が約80kDaのラクターゼの画分の割合と、の和が、30%以上であることを特徴とする[1]に記載のラクターゼ溶液;
[3] SDSポリアクリルアミドゲル電気泳動による分子量が約50kDaのラクターゼの画分の割合が、70%以下であることを特徴とする[1]に記載のラクターゼ溶液;
[4] 前記約120kDaのラクターゼの画分の割合と、前記約80kDaのラクターゼの画分の割合と、の和を、SDSポリアクリルアミドゲル電気泳動による分子量が約50kDaのラクターゼの画分の割合で除した値が、0.5以上であることを特徴とする[1]~[3]のいずれか一項に記載のラクターゼ溶液;
[5] 乳製品製造用であることを特徴とする[1]~[4]のいずれか一項に記載のラクターゼ溶液;
[6] [1]~[5]のいずれか一項に記載のラクターゼ溶液を含有する乳製品;
[7] [1]~[5]のいずれか一項に記載のラクターゼ溶液を原料乳に添加し、当該原料乳に含まれる乳糖を1~60℃で分解させることを特徴とする原料乳の処理方法;
[8] 微生物を培養する培養工程と、
前記培養工程により得られた培養物からラクターゼを回収する回収工程と、
前記回収工程により回収したラクターゼを精製する精製工程と
を含むことを特徴とするラクターゼ溶液の製造方法であって、
前記精製工程が、
塩析処理及び脱塩処理を行う工程
を1又は複数回含むことを特徴とするラクターゼ溶液の製造方法;
[9] 前記塩析処理が、
前記回収したラクターゼに、塩析剤を10~90%飽和させる飽和工程と、
前記飽和工程後に、ラクターゼを4~40℃で1~80時間放置する放置工程と
を含むことを特徴とする[8]に記載のラクターゼ溶液の製造方法;
[10] 前記塩析処理が、pH4~9において行われることを特徴とする[8]又は[9]に記載のラクターゼ溶液の製造方法;
が提供される。
本発明のラクターゼ溶液の製造方法は以下の4つの工程を経る。すなわち、
(1)微生物の培養工程、
(2)微生物からのラクターゼ回収工程、
(3)ラクターゼの精製工程、及び
(4)ラクターゼ活性の調整工程
である。
(1)微生物の培養工程については公知の培地を使用し、公知の菌株を使用すればよい。培養条件も公知であり、必要に応じて適宜選択することができる。
(2)微生物からのラクターゼ回収工程については、細胞内酵素である場合、ラクターゼを抽出する工程を含む必要がある。この抽出工程は、ラクターゼを細胞外に移行させることができる方法であれば特に限定されず、公知の抽出方法を使用することができる。他方、遺伝子導入、変異等によりラクターゼを細胞外に分泌される酵素とすれば、その培養液にラクターゼが含まれることになるため、本抽出工程は不要である。
(3)ラクターゼの精製工程は本発明のラクターゼ溶液を得るために重要である。特許文献1、特許文献2及び特許文献3は、いずれもクロマトグラフィーによってラクターゼ溶液を精製することが共通する。このクロマトグラフィーを行うことによって、ラクターゼの精製が進みラクターゼ活性を向上させることができる。しかし、後述するように、クロマトグラフィー(分配クロマトグラフィー又は分子篩クロマトグラフィー、吸着クロマトグラフィー又はイオン交換クロマトグラフィーなど)を使用してラクターゼを精製すると、本来120kDaであるラクターゼが、80kDa及び50kDaのラクターゼにそれぞれ分解されることが判明した。いずれの分子量のラクターゼもラクターゼ活性を有するが、ラクターゼが分解を受けることによって、特に50kDaの画分の割合が増すと、ラクターゼの熱安定性が低下する結果、比較的高い温度で乳糖の分解が進みにくくなる問題が生じるものであった。
ラクターゼを含む溶液に、塩析剤としての硫酸アンモニウムを添加する場合、10~90%飽和とすることが好ましく、30~70%飽和とすることがさらに好ましい。他の塩析剤を使用する場合、当該硫酸アンモニウムの添加量に相当する量を使用すればよい。
ラクターゼ溶液中のラクターゼI及びラクターゼIIは、いずれもラクターゼ活性及び耐熱性を有する。ラクターゼIIIはラクターゼ活性を有するが耐熱性が不十分である。いずれの画分であってもラクターゼ活性は同等である。
(実施例1)
コーン・スティープ・リカー7%、ラクトース2%を含有する液体培地を加圧殺菌後(殺菌後のpH5.5)、Kluyveromyces lactisNo.013-2(ATCC 8585株)を植菌し、30℃にて24時間、12000L/minの通気で培養した。培養終了後冷却しながら4時間放置後、タンク上部から上澄液を除き、タンク底部に凝集沈降した菌体1500kgを得た。次いでここに得られた菌体のうち1500gを水道水で洗浄後、トルエン80mlを加え混和後、1500mlの0.05Mリン酸緩衝液(pH7.0)を加え撹拌均一化し、密栓を施して30℃、15時間放置し自己消化せしめた。
この消化液を遠心分離して得た上清2500mlに等容の冷アセトンを加え一夜放置した。生じた沈殿を遠心分離にて集め600mlの水道水に溶かし、濃縮前酵素溶液を得た。
この濃縮前酵素溶液600mlを4℃に冷却しながら、硫酸アンモニウム粉末を60分間かけて少しずつ添加し、50%飽和水溶液を得た。当該水溶液を80時間4℃で放置(静置)しラクターゼを沈殿させた後、濾過によって固液分離し、固体状のラクターゼを回収した。当該ラクターゼを600mlの水道水に再溶解させた後、限外濃縮を行った。脱塩されたラクターゼに水道水を添加し、終濃度が50%となるようにグリセリンを添加し実施例1のラクターゼ溶液(5,000NLU/g)を得た。
(実施例2)
実施例1のラクターゼ溶液と下記比較例1とを、重量比が80:20となるように混合して、実施例2のラクターゼ溶液(5,000NLU/g)を得た。
(実施例3)
実施例1のラクターゼ溶液と下記比較例1とを、重量比が60:40となるように混合して、実施例3のラクターゼ溶液(5,000NLU/g)を得た。
(実施例4)
実施例1のラクターゼ溶液と下記比較例1とを、重量比が40:60となるように混合して、実施例4のラクターゼ溶液(5,000NLU/g)を得た。
(実施例5)
実施例1のラクターゼ溶液と下記比較例1とを、重量比が20:80となるように混合して、実施例5のラクターゼ溶液(5,000NLU/g)を得た。
市販のラクターゼ製剤である商品名「GODO-YNL2SS」(合同酒精社製、5000NLU/g)を比較例1のラクターゼ溶液とした。
(比較例2)
市販のラクターゼ製剤である商品名「MAXILACT LG5000」(DSM社製、5000NLU/g)を比較例2のラクターゼ溶液とした。
(比較例3)
市販のラクターゼ製剤である商品名「MAXILACT LGX5000」(DSM社製、5000NLU/g)を比較例3のラクターゼ溶液とした。
ラクターゼ溶液をミリQ水で10NLU/gに希釈し、SDS-PAGE用Sample Buffer(0.125M Tris-HCl pH6.8、0.0125% ブロモチモールブルー、20% グリセリン、2.5% SDS、2.5% 2-メルカプトエタノール)と1:1に混合し、95℃、5分間の加熱処理により泳動用サンプルを調製した。10%アクリルアミドゲル(4%スタッキングゲル、ゲル厚1mm、泳動距離50mm)に分子量スタンダード及び泳動用サンプルを供し、マリソル産業泳動漕にて、スタッキング10mA定電流、セパレーティング20mAにて泳動前線がゲル下端付近に達するまで泳動した。分子量スタンダード(レーンM)にはBIO-RAD#161-0313(プレステインド)を使用した。泳動後のゲルは、CBB染色液(APRO SP-4010)によるタンパク染色を1時間行った。
ラクターゼ活性を有するのは、約120kDa、約80kDa及び約50kDaのバンドであり、約30kDaのバンドはラクターゼ活性を有するものではなかった。
上述する方法により、泳動後のゲルについて、バンドの定量により割合を算出した。結果を表1に示す。表1は全てのバンドを含む値を記載した。さらに、表2には、主要な4つのバンドのみを全体としたそれぞれのバンドの割合を算出した結果を示した。表1~表4において、各実施例、比較例等の値を合計しても100にならないのは、得られた測定結果の小数点第2位を四捨五入したためである。
表1、2には示していないが、上記実施例1等と同様にして、特許文献1の実施例1を追試することによって得られたA区分及びB区分について、電気泳動を行ったところ、比較例2、3と同様の傾向を示した。
表1
表2
UHT殺菌(130℃、2秒)牛乳(商品名「明治おいしい牛乳」、株式会社明治製)に実施例1のラクターゼ溶液(レーン1及びレーン2)、比較例1(レーン3及びレーン4)、比較例2(レーン5)又は比較例3(レーン6)を、それぞれ終濃度0.05%(W/V)となるように添加し、43℃で2時間乳糖分解反応を行った。反応後の溶液を精製水で20倍(W/V)に希釈して、上記と同様にSDS-PAGE用Sample Bufferと1:1に混合し、95℃、5分間の加熱処理により泳動用サンプルを調製した。10%アクリルアミドゲル2枚に分子量スタンダード及び泳動用サンプルを供し、同時に泳動した。分子量スタンダード(レーンM)はBIO-RAD#161-0313(プレステインド)を使用した。
一次抗体としては以下の様に自家調製した抗ラクターゼポリクローナル抗体を使用した。実施例1で得られたラクターゼ溶液のSDS-PAGEを行い、ゲルより120kDaのバンドを切り出して細かく砕いた後にDifco社製アジュバント コンプリート フロイントと混合してエマルジョン化した。これをBalb-Cマウスの尾部根元に計3回皮下注射し、血清中抗体価の上昇を確認後、採取した全血の遠心分離上清を抗ラクターゼポリクローナル抗体とした。この抗体でラクターゼの120、80、50kDaのバンドが検出可能であった。
上記の抗ラクターゼ抗体をブロックエース希釈液(ブロックエース溶液を精製水で10倍希釈)で1,000倍に希釈した液中で、室温で2時間反応させた。Tween-PBSで4回洗浄した後、2次抗体(Gort a-mouse IgG(H+L)-HRP;SouthernBiotech 1034-05)をブロックエース希釈液で5,000倍に希釈した液中で、室温で2時間反応させた。Tween-PBSで洗浄後、3,3’-ジアミノベンジジン(DAB)による染色を行った。DAB基質はDAB buffer tablets(MERCK 1.02924.0001)を使用した。
UHT殺菌牛乳(130℃、2秒)に実施例1のラクターゼ溶液、比較例1、比較例2及び比較例3をそれぞれ終濃度0.05%(w/v)(2.5NLU/100mL牛乳)となるように添加し、49℃、46℃、43℃、40℃及び37℃で乳糖分解反応を行った。乳糖分解反応前の乳糖の含量と、反応中の乳糖の含量を経時的にHPLC(Transgenomic CARBOSep CHO620カラム)で測定した(Waters Alliance HPLCシステム、カラム温度:85℃、溶媒:H2O、流速:0.5mL/min、検出器:Waters2414RIディテクター)。乳糖分解率は、以下のように算出した。乳糖分解率(%)=100-(各実施例又は各比較例のラクターゼ溶液を用いた乳糖分解反応後の牛乳に含まれる乳糖含量/各実施例又は各比較例のラクターゼ溶液を用いた乳糖分解反応前の牛乳に含まれる乳糖含量)×100)
実施例1のラクターゼ溶液と比較例1のラクターゼ溶液を上述する割合で混合して得たラクターゼ溶液、実施例2~5について以下検討を行った。
実施例2~5について、上述した方法により、電気泳動を行った後、各バンドの定量を行った。電気泳動の結果を図4、定量の結果を表3に示す。表3は全てのバンドを含む値を記載した。さらに表4には、主要な4つのバンドのみを全体としたそれぞれのバンドの割合を算出した結果を示した。尚、比較のため、実施例1及び比較例1についても同時に泳動及び定量を行った結果を示した。
表3
表4
実施例1~5及び比較例1のラクターゼ溶液を用いて、上述した乳糖分解試験の方法により、49℃で乳糖分解反応を行った。得られた結果を図5に示した。
パネルAは乳糖量、パネルBは乳糖分解率の経時変化の結果を示している。約120kDaのバンドが増えるにつれ、乳糖分解が進むことを確認した。
Claims (10)
- SDSポリアクリルアミドゲル電気泳動による分子量が約120kDaのラクターゼの画分の割合が、20%以上であることを特徴とするラクターゼ溶液。
- 前記120kDaのラクターゼの画分の割合と、SDSポリアクリルアミドゲル電気泳動による分子量が約80kDaのラクターゼの画分の割合と、の和が、30%以上であることを特徴とする請求項1に記載のラクターゼ溶液。
- SDSポリアクリルアミドゲル電気泳動による分子量が約50kDaのラクターゼの画分の割合が、70%以下であることを特徴とする請求項1に記載のラクターゼ溶液。
- 前記約120kDaのラクターゼの画分の割合と、前記約80kDaのラクターゼの画分の割合と、の和を、SDSポリアクリルアミドゲル電気泳動による分子量が約50kDaのラクターゼの画分の割合で除した値が、0.5以上であることを特徴とする請求項1~3のいずれか一項に記載のラクターゼ溶液。
- 乳製品製造用であることを特徴とする請求項1~4のいずれか一項に記載のラクターゼ溶液。
- 請求項1~5のいずれか一項に記載のラクターゼ溶液を含有する乳製品。
- 請求項1~5のいずれか一項に記載のラクターゼ溶液を原料乳に添加し、当該原料乳に含まれる乳糖を1~60℃で分解させることを特徴とする原料乳の処理方法。
- 微生物を培養する培養工程と、
前記培養工程により得られた培養物からラクターゼを回収する回収工程と、
前記回収工程により回収したラクターゼを精製する精製工程と
を含むことを特徴とするラクターゼ溶液の製造方法であって、
前記精製工程が、
塩析処理及び脱塩処理を行う工程
を1又は複数回含むことを特徴とするラクターゼ溶液の製造方法。 - 前記塩析処理が、
前記回収したラクターゼに、塩析剤を10~90%飽和させる飽和工程と、
前記飽和工程後に、ラクターゼを4~40℃で1~80時間放置する放置工程と
を含むことを特徴とする請求項8に記載のラクターゼ溶液の製造方法。 - 前記塩析処理が、pH4~9において行われることを特徴とする請求項8又は9に記載のラクターゼ溶液の製造方法。
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EP3228704A1 (en) | 2017-10-11 |
EP3228704A4 (en) | 2018-05-16 |
US20170367357A1 (en) | 2017-12-28 |
BR112017011607A2 (pt) | 2018-01-09 |
AU2015356272A1 (en) | 2017-06-29 |
JP6441382B2 (ja) | 2018-12-19 |
US10368558B2 (en) | 2019-08-06 |
MX2017007197A (es) | 2017-08-28 |
CN107109384B (zh) | 2021-06-08 |
AU2015356272B2 (en) | 2019-08-08 |
JPWO2016088589A1 (ja) | 2017-11-30 |
CN107109384A (zh) | 2017-08-29 |
CO2017006191A2 (es) | 2017-09-20 |
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