Patent Description:
Lactose intolerance indicates a condition exhibiting various symptoms such as abdominal pain and diarrhea caused by lactose present in a food product such as a dairy product when there is congenital insufficiency of decomposing lactose. Lactose is a disaccharide consisting of galactose and glucose. To cope with lactose intolerance, decomposing in advance the lactose contained in milk or the like to galactose and glucose using the lactase enzyme is carried out in the food manufacturing industry.

A lactase solution which is used for decomposing lactose contained in milk or the like is conventionally produced as follows. After culturing lactase-producing microorganisms, lactase is extracted from cells or collected from extracellularly secreted solution , and is purified by removing impurities originating from the culture, and is sterilized by filtration after adding a stabilizer, thereby producing the lactase solution.

The lactase solution produced as above is stored, sold, and transported under a refrigerated condition (<NUM> or lower). After that, the lactase solution is added to milk or a dairy product by a user. As for a method of adding lactase, there are two main methods, that is, a method of adding it before sterilization of milk or the like and a method of adding it after sterilization. In the case of the former, a step for removing microorganisms by filtration is not necessarily required. However, in the case of the latter, a step for sterilizing the lactase by filtration is required. Milk obtained after addition of lactase is filled in a container and sold as a commercial product.

With regard to a production process of adding lactase to milk after sterilization as described above, it has been known that a lactase solution easily causes filter clogging during a filter sterilization step, and this is the reason of having a significant decrease in working efficiency. To cope with such a problem, for example, Patent Literature <NUM> (<CIT>) describes that before deterioration products of proteins and polysaccharides as a cause of clogging are formed, the lactase solution is sterilized by filtration immediately after collecting and purifying the lactase solution. Although this method can be carried out at the time of producing the lactase solution, it cannot be carried out once the deterioration products are formed. That is because, when the lactose solution prepared by this method is filtered after storage or transport, there is a case of having an occurrence of clogging. For example, after the lactase which has been produced by the method is transported to a user, and the lactase is subjected to a filter sterilization step so that a user can add the lactase to milk or the like, an occurrence of filter clogging may be yielded. When clogging occurs, there is a problem in that the production step needs to be stopped and the filter needs to be changed, and thus a significant decrease in working efficiency is caused. When an individual production step for producing milk is continuously present, in particular, it is not possible to stop the filter sterilization step only, and thus it becomes necessary to stop entire steps for producing milk. As a result, the milk production efficiency is significantly lowered, and thus an improvement is required in this regard.

Accordingly, the method described in Patent Literature <NUM> is not a solution to the problem with regard to clogging during filtering of the lactase solution which has been prepared as a commercial product.

Furthermore, Patent Literature <NUM> (<CIT>) describes that maintaining the concentration of polysaccharides and oligosaccharides contained in a lactase solution at a certain value or lower, and in particular, removing these materials by chromatography. However, even when the concentration of polysaccharides and oligosaccharides is kept at a certain value or lower, similarly, there is a case in which the clogging problem associated with filtering after the lactase solution has been prepared as a commercial product still occurs in some cases. Patent Literature <NUM> (<CIT>) describes a method for reducing fouling of ultrafiltration membranes used for removing virus particles from aqueous solutions.

Therefore, the above problems are not fully solved yet.

An object of the present invention is to provide a method for reducing clogging of a filtering device (filter) during a filtering step prior to addition of a previously formulated lactase solution to milk for use, and a lactase solution which is less prone to causing clogging of a filtering device.

The present inventors have found that the clogging phenomenon is caused and also accelerated by physical stimulation such as stirring or shaking and/or temperature load. It has been further found that the clogging phenomenon can be prevented by reducing an interaction between the materials included in the lactase solution, and the present invention is completed accordingly.

Thus, the present invention provides a lactase solution according to claim <NUM>. Advantageous embodiments are subject of the dependent claims.

According to the present invention, it is possible to solve the problem of filter clogging during the filtering step before adding a lactase solution to milk for use. Accordingly, filter exchange caused by clogging and frequency of sterilization or the like of a production line accompanying the filter exchange is reduced, and thus the working efficiency can be greatly enhanced. The present invention is also excellent in that, compared to a method of prior art based on removal of clogging materials or the like, it is more convenient and has no regeneration of clogging materials during storage of the lactase solution.

The lactase solution according to the present invention essentially contains a lactase and an aggregation inhibitor for preventing aggregation of the lactase and other proteins contained in the lactase solution, and optionally contains additives such as a stabilizer. Hereinbelow, descriptions are given in the following order - (<NUM>) constitutional components of a lactase solution, (<NUM>) composition of a lactase solution (in particular, concentration of an aggregation inhibitor), (<NUM>) properties of a lactase solution, (<NUM>) method for preparing a lactase solution, and (<NUM>) method of using a lactase solution and use of a lactase solution.

Lactase has been isolated from a broad range of organisms including microorganisms. In many cases, the lactase is an intracellular or extracellular component of a microorganism such as Kluyveromyces or Bacillus. Kluyveromyces, in particular, K. fragilis, K. lactis, and the yeast of genus Candida, genus Torula, genus of Torulopsis or the like are a common source of yeast enzyme lactase, and B. coagulans or B. circulans is a well known source of microbial lactase. Several kinds of a lactase preparation derived from those organisms are commercially obtainable. As all of those lactases have optimum pH of pH = <NUM> to pH = <NUM>, they are so-called a neutral lactase. Furthermore, an extracellular lactase is produced by Aspergillus niger, Aspergillus oryzae, or Penicillium multicolor, and in the specification of <CIT>, an example of such lactase produced by Aspergillus oryzae is described. The enzyme characteristics of lactase including optimum pH and optimum temperature vary depending on species. In general, an extracellular lactase is a so-called acidic lactase which has low optimum pH, that is, pH = <NUM> to pH = <NUM>.

In the present invention, it is preferable to use a neutral lactase and an acidic lactase. It is particularly preferable to use a neutral lactase derived from genus Kluyveromyces and an acidic lactase derived from genus Aspergillus.

The lactase to be used in the present invention can be a lactase which has been recovered and purified from a microorganism by a common method of a prior art. The lactase to be used in the present invention may be any one of an intracellular lactase and an extracellular lactase. In the case of the former, the lactase is generally derived from a cytoplasm of a microorganism. As a specific method for producing a solution of intracellular lactase, there can be a method in which microorganisms are first cultured and a lactase is separated therefrom. For this method, it is necessary to disrupt a cell wall to release the enzyme from a cytoplasm. For cell lysis, a technique such as permeation of a cell wall using an organic solvent such as octanol, ultrasonic degradation, or French pressing is used. The lactase can be separated and recovered from a lysed cell by using a well known method. In the case of the latter, disruption of a cell wall is not necessary, and based on centrifuge or filtering after culture, for example, cells in the culture solution can be removed. After termination of fermentation or removal of cells, the lactase is recovered from the culture solution. In any of those cases, the lactase can be isolated and purified by a conventionally known means.

The lactase solution of the present invention contains an aggregation inhibitor for preventing aggregation of a lactase and other proteins contained in the lactase solution, wherein the aggregation inhibitor is at least one selected from a surfactant with HLB of <NUM> to <NUM>, polyethylene glycol, and a Mg ion or a salt thereof. The aggregation inhibitor which is used in the present invention can be selected to be a (Type <NUM>) surfactant with HLB of <NUM> to <NUM>. In addition, there are mentioned a (Type <NUM>) lipotropic surfactant, (Type <NUM>) non-ionic lipotropic surfactant, (Type <NUM>) non-ionic surfactant, and (Type <NUM>) natural product-based surfactant. Preferably, it is a non-ionic surfactant with HLB of <NUM> to <NUM>. It is understood in this regard that, as those aggregation inhibitors are present in a system, the hydrophobic interaction between proteins is either inhibited or prevented, and as a result, forming of a clogging material caused by aggregation is prevented even under stirring or shaking for a long period of time. From the viewpoint of emulsion stability and degree of the effect of dispersing a hydrophobic substance in an aqueous solution, a surfactant with HLB of <NUM> to <NUM> is used. Hereinbelow, those surfactants are described in detail. Meanwhile, the types are classified depending on physical properties, origin, or the like, and a component belonging to one type may also belong to other type. Furthermore, it is also possible that plural kinds of surfactants of the same type may be used in combination, or plural kinds of surfactants of the different type may be used in combination.

First, "HLB" of the surfactant with HLB of <NUM> to <NUM> is a value which is calculated by Griffin method. Furthermore, more preferable HLB is <NUM> to <NUM>. As for the surfactant of Type <NUM>, a below-mentioned non-ionic surfactant can be also used in addition to an anionic surfactant and an amphoteric surfactant.

Next, examples of the lipotropic surfactant include a reaction product between alcohol, polyoxyethylene alkyl ether, fatty acid, bile acid, glycerin fatty acid ester, acetylated glycerin fatty acid ester, lower alcohol fatty acid ester, polyethylene glycol fatty acid ester, polyethylene glycol glycerin fatty acid ester, polypropylene glycol fatty acid ester, sucrose fatty acid ester, polyethylene fatty acid ester, polyoxyethylene glyceride, a lactic acid derivative of mono/diglyceride, propylene glycol diglyceride, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene - polyoxypropylene block copolymer, transesterified vegetable oil, sterol, a sterol derivative, sugar ester, sugar ether, sucroglyceride, polyoxyethylene vegetable oil, polyoxyethylene hydrogenated vegetable oil, or polyhydric alcohol, and at least one from a group consisting of fatty acid, glyceride, vegetable oil, hydrogenated vegetable oil, and sterol, and a mixture thereof.

Furthermore, examples of the non-ionic lipotropic surfactant include a reaction product between alkyl glucoside, alkyl maltoside, alkylthioglcoside, lauryl macrogol glyceride, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenol, polyethylene glycol fatty acid ester, polyethylene glycol glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene-polyoxypropylene block copolymer, polyglycerin fatty acid ester, polyoxyethylene glyceride, polyoxyethylene sterol, a derivative and a homologue thereof, polyoxyethylene vegetable oil, polyoxyethylene hydrogenated vegetable oil, or polyhydric alcohol, and at least one from a group consisting of fatty acid, glyceride, vegetable oil, hydrogenated vegetable oil, and sterol, tocopherol polyethylene glycol succinate, sugar ester, sugar ether, sucroglyceride, and a mixture thereof.

Furthermore, examples of the non-ionic surfactant include n-alkyl PEO monoether such as Brij35 or polyoxyethylene (<NUM>) cetyl ether, n-alkyl phenyl PEO such as Lubrol PX, Lubrol WX, nonidet P-<NUM>, octylphenol poly(ethylene glycol ether)n10 or octylphenol poly(ethylene glycol ether)n7, tetramethyl butylphenyl PEO, n-octylglycoside, octyl-thioglycopyranoside, tween-<NUM>, tween-<NUM>, tween-<NUM>, tween-<NUM>, tween-<NUM>, tween-<NUM>, alkylaryl polyether alcohol (TritonX-<NUM>), and polyethylene glycol tert-octyl phenyl ether (TritonX-<NUM>).

Furthermore, examples of the natural product-derived surfactant include sucrose fatty acid ester, polyethylene fatty acid ester, lecithin, lysolecithin, and Quillaja saponin.

Among them, the most preferred aggregation inhibitor I is at least one selected from a group consisting of polyoxyethylene sorbitan fatty acid ester (for example, Polysorbate <NUM>), alkylaryl polyether alcohol (for example, TritonX-<NUM>), and glycerin fatty acid ester.

As an aggregation inhibitor for preventing aggregation of a lactase and other proteins contained in the lactase solution of the present invention, a protective agent having an activity of covering a surface of a lactase and other proteins may be used. As a protective agent, a polyethylene glycol is used.

Due to the reason of effective protection of proteins by having a high hydration property and a very flexible structural skeleton, polyethylene glycol is used. Number average molecular weight of polyethylene glycol is preferably <NUM> to <NUM>, and more preferably <NUM> to <NUM>.

It is understood that, because polyether also has a hydrophilic part and a hydrophobic part, the hydrophobic interaction between a lactase and other proteins contained in the lactase solution can be prevented by it, and as a result, it has an effect of lowering the forming of clogging materials. Furthermore, it is also possible that plural kinds of polyether of the same type may be used in combination, or plural kinds of polyether of the different type may be used in combination.

As for the thickening polysaccharides which may be used include gellan gum, carageenan, xanthan gum, locust bean gum, guar gum, pectin, agar, gelatin, gum Arabic, glucomanna, tara gum, pulluran, polysaccharides of tamarind seed, tragacanth gum, karaya gum, alginic acid, sodium alginate, macrophomopsis gum, starch such as corn starch and tapioca starch, dextrin, and cyclodextrin.

Furthermore, as an aggregation inhibitor for preventing aggregation of a lactase and other proteins contained in the lactase solution, a metal ion or a salt thereof which has a salt-dissolving effect can be mentioned. From the viewpoint of easily obtaining suitable ionic strength of a lactase solution, a Mg ion, or a salt thereof is used.

By adding a metal ion or a salt thereof, suitable ionic strength of the solution is obtained, and thus the hydrophobic protein interaction between a lactase and other proteins contained in the lactase solution is lowered. As a result, it is understood that, as the proteins are not likely to aggregate, it has an effect of lowering the forming of clogging materials. Furthermore, it is also possible that plural kinds of a metal ion of the same type or a salt thereof may be used in combination, or plural kinds of a metal ion of the different type or a salt thereof may be used in combination.

Those aggregation inhibitors I to III may be used singly, or the aggregation inhibitors of different type may be used in combination. For example, it is also possible to use in combination the aggregation inhibitors I and II, the aggregation inhibitors II and III, the aggregation inhibitors I and III, and the aggregation inhibitors I, II and III. Furthermore, as the effect is minor with the aggregation inhibitor III alone, it is preferable to use in combination the aggregation inhibitors I and III, and the aggregation inhibitors II and III.

The lactase solution of the present invention may contain various components, if necessary. Specific examples thereof include metal salts, various saccharides, ascorbic acid, glycerin, or the like which contribute to stabilization of a lactase, starch and dextrin as a vehicle for easy use, and inorganic salts having a buffering activity.

The aggregation inhibitor I is added within a range of <NUM>% by mass to <NUM>% by mass, preferably <NUM>% by mass to <NUM>% by mass, and more preferably <NUM>% by mass to <NUM>% by mass on the basis of the total mass of the lactase solution.

The aggregation inhibitor II is added within a range of <NUM>% by mass to <NUM>% by mass, preferably <NUM>% by mass to <NUM>% by mass, and more preferably <NUM>% by mass to <NUM>% by mass on the basis of the total mass of the lactase solution.

The aggregation inhibitor III has a concentration of <NUM> or more and <NUM> or more less, preferably <NUM> or more and <NUM> or more less, and more preferably <NUM> or more and <NUM> or less as a metal component in the lactase solution. It most preferably has the concentration of <NUM> or more and <NUM> or less. In the order of having higher salt-dissolving effect, a magnesium ion is used.

The lactase solution of the present invention preferably has a neutral lactase activity of <NUM> to <NUM>,<NUM> NLU/g. More preferably, it has the activity of <NUM> to <NUM>,<NUM> NLU/g, and even more preferably it has the activity of <NUM>,<NUM> to <NUM>,<NUM> NLU/g. "NLU" represents Neutral Lactase Unit. A method for measuring the activity is as described below. The measurement is carried out based on hydrolysis of a substrate o-nitrophenyl-β-galactopyranoside (ONPG) to o-nitrophenyl and galactose. The reaction is terminated by addition of sodium carbonate. Thus formed o-nitrophenyl exhibits yellow color in an alkali medium, and a change in absorbance is used for the measurement of enzyme activity (expressed in NLU/g). This order is disclosed in <NPL>/lactase (neutral) (β-galactosidase) activity.

The lactase solution of the present invention preferably has an acidic lactase activity of <NUM> to <NUM>,<NUM> ALU/g. More preferably, it has the activity of <NUM> to <NUM>,<NUM> ALU/g, and even more preferably it has the activity of <NUM>,<NUM> to <NUM>,<NUM> ALU/g. "ALU" represents Acid Lactase Unit. A method for measuring the activity is as described below. The measurement is carried out based on hydrolysis of a substrate o-nitrophenyl-β-galactopyranoside (ONPG) to o-nitrophenyl and galactose. The reaction is terminated by addition of sodium carbonate. Thus formed o-nitrophenyl exhibits yellow color in an alkali medium, and a change in absorbance is used for the measurement of enzyme activity (expressed in ALU/g). This order is disclosed in <NPL>/lactase (acidic) (β-galactosidase) activity.

The lactase solution of the present invention may be any one of a neutral lactase solution and an acidic lactase solution, and it may be also a lactase solution in which both are mixed to function in a neutral to acidic condition.

The determination can be made by subjecting the lactase solution of the present invention to a stirring treatment at <NUM> rpm, <NUM> for <NUM> hours with a concentration for having the activity of <NUM>,<NUM> NLU/g or <NUM>,<NUM> ALU/g and calculating a decrease amount of the permeation rate at the time of terminating the permeation test (that is, permeation of <NUM>) when the permeation rate (Flux) immediately after starting the permeation test at the following conditions (that is, permeation of <NUM>) is <NUM>%. It is more preferable to have a higher permeation rate at the time of terminating the permeation test. It is preferably <NUM>% or more, more preferably <NUM>% or more, and even more preferably <NUM>% or more. The upper limit is <NUM>%, for example.

Activity of the lactase solution can be adjusted by dilution with ion exchange water or concentration by ultrafiltration.

The lactase solution of the present invention is characterized in that the filter permeation rate which is measured at the following conditions is <NUM>/min × m<NUM> or more after the lactase solution is subjected to a stirring treatment at <NUM> rpm, <NUM> for <NUM> hours with a concentration for having the activity of <NUM>,<NUM> NLU/g or <NUM>,<NUM> ALU/g. The filter permeation rate is preferably <NUM>/min × m<NUM> or more, more preferably <NUM>/min × m<NUM> or more, and most preferably <NUM>/min × m<NUM> or more.

The filter clogging phenomenon of a lactase can be reproduced by carrying out a reciprocal shaking or stirring operation of a lactase solution. Time for carrying out a reciprocal shaking or stirring operation can be set between <NUM> minutes and <NUM> days, although it may vary depending on the heating temperature and activity value of a lactase solution. It is preferably between <NUM> hour to <NUM> days, and more preferably between <NUM> hours and <NUM> days. By carrying out heating of a lactase solution while performing a reciprocal shaking or stirring operation, a clogging phenomenon can be promoted.

The reciprocal shaking of a lactase solution is preferably <NUM> spm (strokes/minute) to <NUM> spm (strokes/minute), more preferably <NUM> spm (strokes/minute) to <NUM> spm (strokes/minute), and even more preferably <NUM> spm (strokes/minute) to <NUM> spm (strokes/minute).

The stirring operation of a lactase solution is preferably <NUM> rpm (rotation/minute) to <NUM> rpm (rotation/minute), more preferably <NUM> rpm (rotation/minute) to <NUM> rpm (rotation/minute), and even more preferably <NUM> rpm (rotation/minute) to <NUM> rpm (rotation/minute).

Temperature for heating a lactase solution is preferably <NUM> to <NUM> or so, more preferably <NUM> to <NUM>, and even more preferably <NUM> to <NUM>. As the temperature increases, it is easier to have an occurrence of a clogging phenomenon.

It is sufficient that the lactase activity of a lactase solution is within a range of <NUM> to <NUM>,<NUM> NLU/g, or a range of <NUM> to <NUM>,<NUM> ALU/g. As the activity value increases, it is easier to have an occurrence of a clogging phenomenon.

The filter permeability can be evaluated, in a low temperature environment, by having a sample which has been adjusted to have lactase content to maintain a constant activity permeated through a certain filter with certain membrane area under pressure, and measuring the rate of the permeation. Specifically, the evaluation can be carried out as described below.

The following operation is carried out in a low temperature to room temperature (for example, <NUM> to <NUM>, preferably <NUM> to <NUM>, and more preferably <NUM> to <NUM>) environment.

The measurement device, lactase solution sample, and ion exchange water are cooled to low temperature to room temperature (for example, <NUM> to <NUM>, preferably <NUM> to <NUM>, and more preferably <NUM> to <NUM>).

The lactase activity of a lactase solution sample is adjusted to a constant level by dilution with ion exchange water or concentration by ultrafiltration followed by thorough mixing. Meanwhile, the lactase activity may be adjusted to any one of <NUM> to <NUM>,<NUM> NLU/g and <NUM> to <NUM>,<NUM> ALU/g or so depending on the viscosity of a lactase solution.

As for the test device, a filter holder mounted with a pressurizable tank is used as an inlet for measurement sample, and an additional separate filter holder may be connected thereto and used. The filter holder mounted with a pressurizable tank can be used by itself. However, to have a condition for allowing clogging of a filter, the former is preferable, and the diameter (ϕ) thereof is preferably larger than the ϕ of a filter holder to be connected.

In general, the sample permeation part in a filter holder has a configuration in which, from the opening side of a measurement sample, an O-ring, a membrane, and a support screen are provided, but it is not limited thereto. The membrane has, although not particularly limited, pore diameter of preferably <NUM> to <NUM>, and more preferably <NUM> to <NUM>. Furthermore, it may be hydrophilic or hydrophobic. More preferably, it is hydrophilic. ϕ is not particularly limited as long as the effective membrane area can be adjusted with it. The effective membrane area can be adjusted by sealing with a liquid-impermeable label or the like, and it is <NUM> to <NUM><NUM>, for example, and preferably <NUM> to <NUM><NUM>.

When a lactase solution of a prior art (that is, not containing an aggregation inhibitor) is used as a lactase solution according to the above order of measuring filter permeability, the permeation rate is greatly lowered at the time of terminating the permeation test. If the permeation rate is higher than the permeation rate of a lactase solution of a related art, it can be determined that the substance added as an aggregation inhibitor has a clogging inhibiting effect.

The specific method for preparing a lactase of the present invention is as described above. Although the descriptions are omitted herein, the method includes (<NUM>) a step of extracting a lactase accompanied with disruption of a cell wall after performing yeast culture, and (<NUM>) a purification step for removing impurities derived from a culture product from the extracted lactase. The method for preparing a lactase solution of the present invention includes (<NUM>) a step of adding an aggregation inhibitor (+ other additives, if necessary) to the above lactase (which may be freshly prepared product or a commercially available product), and (<NUM>) a step of filtering for sterilization.

With regard to a specific mode of using the lactase solution, the lactase solution is used for production of fermented milk, for example. Examples of a method for producing fermented milk based on decomposition of lactose include the following:.

Furthermore, as a specific mode of using the lactase solution of the present invention, the lactase solution can be used for production of long-life milk. Long-life milk is milk for long term storage, and the process for producing it includes a sterilization step and a continuous aseptic packaging step. In general, milk is treated by ultra-high temperature short-time sterilization at <NUM> to <NUM> for several seconds, and filled by a step by which a paper pack sterilized in advance with hydrogen peroxide is aseptically packaged.

The lactase solution added to long-life milk is generally added after filtering sterilization when milk after ultra-high temperature short-time sterilization is filled.

The lactase solution according to the present invention is particularly suitable for use in production of a dairy product. As described herein, the dairy product indicates ice, milk products such as long-life milk, yoghurt, raw cream, sour cream, cheese, or the like. The lactase solution of the present invention is particularly useful for production of long-life milk.

When the use of a lactase is considered, two main categories are present depending on whether the lactase is a neutral lactase or an acidic lactase. That is due to the pH profile of each use. It can be said that, for a use in neutral pH, a neutral lactase is generally preferable while an acidic lactase is more suitable for a use in an acidic range.

Herein, the aforementioned filter permeability test <NUM> can be applied not only to a lactase solution but also to a protein and a peptide. That is because, as described below, an aggregated product is believed to be formed as hydrophobic parts in a molecule contact each other to yield an adhesion.

Hereinbelow, detailed descriptions are given for the filter permeability test.

The present test method is a test method for evaluating clogging characterized in that,.

According to the test method of the present invention, a phenomenon of clogging of a membrane filter can be reproduced so that a determination test regarding solving means or the like can be briefly carried out.

By using YNL (manufactured by GODO SHUSEI CO. , product name: GODO-YNL) as a lactase solution, conditions for having clogging were investigated. Meanwhile, GODO-YNL which has been used is a neutral lactase derived from Kluyveromyces, and it has the activity of <NUM>,<NUM> NLU/g.

<NUM> of YNL was added to a <NUM> Aiboi (wide mouth (manufactured by AS ONE Corporation. , product number of <NUM>-<NUM>-<NUM>)), and subjected to reciprocal shaking (<NUM> spm (strokes/minute)) or standing for <NUM>, <NUM>, <NUM>, or <NUM> days at <NUM>, <NUM>, or <NUM> by using a shaking device (manufactured by Taiyo Kagaku Co. , product name: TAIYO INCUBATOR PERSONAL, stroke of <NUM>). Each YNL was weighed in an amount of <NUM>, and then by using a sample of which activity is adjusted to <NUM>,<NUM> NLU/g according to dilution of <NUM> times with ion exchange water, the filter permeability was measured.

Detailed conditions of the filter permeability test <NUM> for the present example are described in detail hereinbelow.

The following operations were performed in an environment of <NUM>.

The results are shown in <FIG>. Panel A indicates the result obtained after standing or reciprocal shaking at <NUM>, panel B indicates the result at <NUM>, and panel C indicates the result at <NUM>. At each temperature, all the samples which have been allowed to stand exhibited a small decrease in the permeation rate regardless of the days of standing. On the other hand, the permeation amount from a sample after reciprocal shaking exhibited a dramatically reduced permeation rate in accordance with an increase of the days of shaking. The measurement cannot be made for the sample shaken for <NUM> days at <NUM> or <NUM> due to clogging. As such, it is considered that clogging has mainly occurred due to forming of an aggregate as caused by reciprocal shaking treatment. Meanwhile, the sample after reciprocal shaking at <NUM> and the sample after reciprocal shaking at <NUM> exhibited slight turbidity after <NUM> days, but no turbidity was measured from the sample which has been allowed to stand. However, in both of the sample after reciprocal shaking and the sample after standing, there was a tendency that the permeation amount decreases as the temperature increases. It was found that the increased temperature accelerates forming of an aggregate which is generated by shaking.

As shown in panel D, the lactase activity hardly changed in the case of having shaking or standing at each temperature. As such, it was found that the forming of an aggregate which causes clogging hardly has any effect on the lactase activity.

It was also investigated whether or not the same phenomenon as above is observed with stirring instead of reciprocal shaking. <NUM> of YNL was added to a <NUM> beaker and subjected to stirring for <NUM> hours at <NUM> at each stirring rate (minimum stirring rate: <NUM> rpm, rate <NUM>: <NUM> rpm, rate <NUM>: <NUM> rpm, and rate <NUM>: <NUM> rpm)) by using a stirring device (manufactured by AS ONE Corporation, product name of HS-<NUM>). After that, the sample was prepared in the same manner as above, and the filter permeability was measured in the same manner as the filter permeability test above.

The results are shown in <FIG>. Compared to those having no treatment, a significant deterioration in permeability was observed according to a stirring operation.

From the above results, it was found that the physical stimulation such as shaking or stirring is the main reason of forming an aggregate which yields an occurrence of clogging. As such, it is believed that an aggregate is also formed by vibration or the like which is generated during transport after production of a lactase solution.

Meanwhile, although not illustrated, when the above stirring treatment was carried out by using those in which content of polysaccharides and oligosaccharides included in a lactase solution was less than <NUM>/kg, or more than <NUM>/kg, and the above stirring treatment was carried out, the aggregate was also formed in any lactase solution at the same level as above. Namely, it was demonstrated that the occurrence of an aggregate is hardly affected by polysaccharides and oligosaccharides included in a lactase solution.

In the following determinations, the test was carried out while the condition for reproducing the aggregate forming to cause a clogging phenomenon includes stirring at <NUM> rpm, <NUM> for <NUM> hours.

The mechanism for having an occurrence of a clogging material (that is, aggregate) caused by shaking or temperature is believed to be as follows, although it remains unclear. According to physical stimulation such as shaking, collision between a protein molecule and a protein molecule is caused. At that time, according to contact and adhesion between hydrophobic parts in the molecule, an aggregate is formed. As such, the present inventors considered the possibility of preventing, by addition of a component capable of lowering hydrophobic interactions, forming of an aggregate which causes clogging, and they investigated the effects of addition of various components.

As a surfactant, Tween80 (HLB <NUM>), TritonX-<NUM> (Tx100, HLB <NUM>) and RYOTO polyglyester (HLB <NUM>) were added, each at a concentration of <NUM>% by mass, to YNL (in <NUM> beaker, <NUM>), and according to a stirring treatment, an aggregate causing a clogging phenomenon was formed. After that, a sample was prepared in the same manner as above, and the filter permeability was measured by the above filter permeability test. By using a control in which a stirring treatment has been carried out without addition of an additive, the filter permeability was also measured. The results are shown in <FIG>. Among the added materials, TritonX-<NUM> and Tween80 were particularly effective as an aggregation inhibitor.

Herein, specific determination of clogging was performed by calculating the decrease (in %) at the time of end of the test (that is, permeation of <NUM>) (that is, relative permeation rate (%) at permeation of <NUM>) compared to Flux immediately after start of the test (that is, permeation of <NUM>). With regard to the determination of clogging inhibition effect of an additive, it can be found to be effective when the above calculation result is higher than the control (<NUM>%). Because the calculation value of a sample with no stirring was about <NUM>%, this value was taken as the maximum value. The result of not less than <NUM>% was evaluated as ⊚, the result of <NUM> to less than <NUM>% was evaluated as ∘, the result of <NUM> to less than <NUM>% was evaluated as ×, and the result showing impermeability for <NUM> was evaluated as ××. The results are shown in Table <NUM>.

The filter permeability was measured in the same manner as the surfactant except that, as polyalkylene glycol, polyethylene glycol (PEG200 (manufactured by Wako Pure Chemical Industries, Ltd. ), PEG400 (manufactured by KANTO CHEMICAL CO. ), PEG4000 (manufactured by KANTO CHEMICAL CO. ), PEG6000 (manufactured by KANTO CHEMICAL CO. ), and PEG200000 (manufactured by Wako Pure Chemical Industries, Ltd. )) were used at <NUM>% by mass. The results are shown in <FIG> and Table <NUM>.

The filter permeability was measured in the same manner as the surfactant except that, as a metal salt or a salt thereof, MgSO<NUM> was added as a metal component to have pre-determined concentration. The results are shown in <FIG> and Table <NUM> together with the determination of the addition concentration which is described below.

With TritonX-<NUM> and Tween80 which were found to be particularly effective in the above test, the same test as above was carried out with modification of an addition amount to determine the effective addition concentration. The results are shown in <FIG> and Table <NUM>.

Under the conditions of this test, the effect was observed when TritonX-<NUM> (panel A) is in a range of <NUM> to <NUM>% and Tween80 (panel B) is in a range of <NUM> to <NUM>%.

Various polyethylene glycols were used as polyalkylene glycol and, with modification of the addition concentration, the same test as the surfactant was performed and the effective addition concentration was determined. The results are shown in <FIG>, and Table <NUM> and Table <NUM>.

MgSO<NUM> was used as metal salt and, with modification of the addition concentration (<NUM>, <NUM>, and <NUM>), the same test as the surfactant was performed and the effective addition concentration was determined. The results are shown in <FIG> and Table <NUM>.

Claim 1:
A lactase solution of which a permeation rate is <NUM>/min × m<NUM> or more upon measuring at the time of permeation of <NUM>/m<NUM> through a membrane filter with a pore diameter of <NUM> at a concentration for having an activity of <NUM>,<NUM> NLU/g or <NUM>,<NUM> ALU/g, after being subjected to a stirring treatment at <NUM> rpm, <NUM> for <NUM> hours with a concentration for having an activity of <NUM>,<NUM> NLU/g or <NUM>,<NUM> ALU/g,
wherein the lactase solution comprises an aggregation inhibitor,
wherein the aggregation inhibitor is at least one selected from a surfactant with HLB of <NUM> to <NUM>, polyethylene glycol, and a Mg ion or a salt thereof, wherein a concentration of the surfactant with HLB of <NUM> to <NUM> is <NUM>% by mass to <NUM>% by mass, a concentration of the polyethylene glycol is <NUM>% by mass to <NUM>% by mass, and a concentration of the Mg ion or the salt thereof is <NUM> to <NUM>.