JP7316758B2 - Lactase preparations that can reduce the risk of off-flavours - Google Patents

Description

The present invention relates to lactase preparations that can reduce the risk of off-flavours. More specifically, the present invention relates to a lactase preparation which, when acted on raw milk, suppresses the amount of isovaleric acid generated compared to conventional preparations.

Lactose is present in dairy raw materials such as milk and dairy products produced therefrom. Due to the presence of lactase in the small intestine of most humans, lactose in dairy products is broken down into glucose and galactose in the small intestine. However, in some humans where lactase does not act sufficiently, lactose is not sufficiently degraded and moves to the large intestine. Bacteria in the large intestine degrade lactose, causing diarrhea and indigestion (lactose intolerance). In order to prevent such symptoms, lactose is preliminarily decomposed by lactase to produce dairy products.

At the current state of the art, it is extremely difficult to chemically synthesize lactase. Therefore, lactase is produced using microorganisms such as yeast, fungi, and bacteria. However, since the microorganism also produces substances other than lactase, lactase preparations used industrially usually contain substances other than lactase as contaminants. Such contaminants can cause problems, one of which is off-flavour.

For example, Patent Document 1 reports that when arylsulfatase is contaminated, p-cresol is released from a conjugate of p-cresol (4-methylphenol) and sulfuric acid contained in milk, and lactase with reduced arylsulfatase activity It proposes a method of producing dairy products in the formulation.

Patent No. 5544088

Since p-cresol is a so-called chemical-like odorant, it is true that release of p-cresol by contamination with arylsulfatase can cause off-flavors in dairy products. However, even with reduced arylsulfatase, off-flavours could still occur. In other words, other contaminants may also generate some odorous substances and cause off-flavours.

Based on this background, the present application aims to identify contaminants that can cause off-flavours, and to provide a lactase preparation and a method for producing the same that can reduce the risk of off-flavours more than before.

The present inventors have completed the present invention as a result of earnest research on the above problems.

Thus, a first aspect of the present invention is a lactase preparation having a β-glucuronidase activity of 2,400 U/g or less per 100 U/g of lactase activity.

Also, certain embodiments of the second aspect of the present invention are methods of producing a lactase preparation from a microorganism capable of producing lactase, wherein the microorganism naturally produces β-glucuronidase activity, and β - A method for producing a lactase preparation comprising a treatment such that the amount and/or activity of glucuronidase activity is limited compared to the wild strain.

The microorganism may be a microorganism belonging to the genus Kluyveromyces, which naturally has on its genome a structural gene encoding the enzyme having the amino acid sequence represented by SEQ ID NO: 1 or a family enzyme thereof.

The treatment may be an operation of adding, deleting, substituting or altering one or more of the genes so as to suppress the expression of these structural genes.

Yet another embodiment of the second aspect of the present invention is a method of producing a lactase preparation from a microorganism capable of producing lactase, wherein the activity of β-glucuronidase in the lactase preparation is measured one or more times. A method for producing a preparation.

The method may include comparing the measured β-glucuronidase activity to a predetermined reference value. This method may also include purifying the measured β-glucuronidase activity to a predetermined reference value or less when the measured β-glucuronidase activity exceeds the predetermined reference value.

A lactase preparation is provided by the method for producing a lactase preparation of the second aspect.

A third aspect of the present invention is a mixture comprising raw milk and the lactase preparation of the first aspect and/or the lactase preparation produced by the second aspect.

Furthermore, in the fourth aspect of the present invention, the β-glucuronidase activity contained in the lactase preparation is used as an indicator, and the amount of isovaleric acid produced and/or the risk of off-flavor when the lactase preparation is allowed to act on raw milk. is a method of predicting

According to the present invention, a lactase preparation that can reduce the risk of off-flavor based on isovaleric acid than before, a method for producing the same, and milk with reduced risk of off-flavor produced using such a lactase preparation product can be provided.

Data showing that β-glucuronidase activity causes isovaleric acid-based off-flavours.

The present invention will be described in detail below.

<First aspect: lactase preparation>
According to a first aspect of the present invention there is provided a lactase preparation capable of reducing the risk of off-flavours based on isovaleric acid.

A lactase preparation is a solution or solid containing lactase as the main component. The lactase preparation may contain optional additives. When the lactase preparation is a solution, it may be dissolved in any solvent as long as it does not inactivate the lactase.

Lactose is a disaccharide formed by a glycosidic bond between the hydroxyl group at the 1-position carbon of β-galactose and the hydroxyl group at the 4-position carbon of glucose.

Lactase is an enzyme that hydrolyzes lactose into glucose and galactose, and is a type of β-galactosidase. Lactase is roughly divided into neutral lactase and acid lactase.

A lactase preparation of the invention may contain two or more different lactases. In this case, two or more neutral lactases may be contained, two or more acid lactases may be contained, or one or more neutral lactases and one or more acid lactases may be contained. You may have In addition, one or more different enzymes other than lactase may be mixed as long as it does not contradict the purpose of the present invention.

Neutral lactase has an optimum pH for activity in the neutral region, and can decompose lactose in this active state. The optimum pH for neutral lactase activity is 6.0-7.5. Moreover, those having the property of being deactivated in an acidic region are preferable. More specifically, the inactivation pH is preferably 4.0 to 6.0.

Acid lactase has an optimum pH for activity in the acidic region, and can decompose lactose in this active state. The optimum pH for acid lactase activity is 3.0-5.9.

In the present invention, the pH range of 6 to 8 is expressed as the neutral region, and the pH range of less than 6 is expressed as the acidic region.

The neutral lactase activity of the lactase preparation of the present invention is not particularly limited and can be adjusted as appropriate. Although not limited, it may have a neutral lactase activity of 0 NLU/g or more, 10 NLU/g or more, 100 NLU/g or 1,000 NLU/g or more. It may also have a neutral lactase activity of 100,000 NLU/g or less, 90,000 NLU/g or less, or 80,000 NLU/g or less.

Here, the unit of neutral lactase activity "NLU" is an abbreviation for Neutral Lactase Unit.

Neutral lactase activity can be measured, for example, by analyzing the reaction in which neutral lactase hydrolyzes the substrate o-nitrophenyl-β-galactopyranoside (ONPG) into o-nitrophenyl and galactose. be. The reaction can be terminated by the addition of sodium carbonate. The degradation product, o-nitrophenyl, exhibits a yellow color in alkaline media, and the neutral lactase activity (NLU/g) can be measured by the change in absorbance. A specific procedure is published in the Food Chemicals Codex (FCC) 4th Edition, July 1, 1996, pp. 801-802/lactase (neutral) (β-galactosidase) activity. ing.

The acid lactase activity of the lactase preparation of the present invention is not particularly limited and can be adjusted as appropriate. Although not limited, it may have an acid lactase activity of 0 ALU/g or more, 10 ALU/g or more, 100 ALU/g or 1,000 ALU/g or more. It may also have an acid lactase activity of 1,000,000 ALU/g or less, 900,000 ALU/g or less, or 800,000 ALU/g or less.

Here, the unit of acid lactase activity "ALU" is an abbreviation for Acid Lactase Unit.

Acid lactase activity can be measured, for example, by analyzing the reaction in which acid lactase hydrolyzes the substrate o-nitrophenyl-β-galactopyranoside (ONPG) into o-nitrophenyl and galactose. The reaction can be terminated by the addition of sodium carbonate. The degradation product, o-nitrophenyl, exhibits a yellow color in an alkaline medium, and the acid lactase activity (ALU/g) can be measured by the change in absorbance. A specific procedure is published in the Food Chemicals Codex (FCC) 4th Edition, July 1, 1996, pages 802-803/lactase (acidic) (β-galactosidase) activity. there is

The total lactase activity (LU/g) of neutral lactase and acid lactase in the lactase preparation of the present invention is not particularly limited and can be adjusted as appropriate. Although not limited, it may have a lactase activity of 10 LU/g or more, 100 LU/g, or 1,000 LU/g or more. It may also have a lactase activity of 1,100,000 LU/g or less, 1,000,000 LU/g or less, or 900,000 LU/g or less.

Here, the unit of lactase activity “LU” is an abbreviation for Lactase Unit.

As noted above, lactase preparations have been utilized to produce lactose-free or low-lactose processed dairy products. The inventors have found that when conventional lactase preparations are used to produce dairy products, off-flavours derived from isovaleric acid (3-methylbutanoic acid) are produced. Isovaleric acid is a sour-smelling substance that causes discomfort. In fact, it is designated as a specific offensive odor substance under the Offensive Odor Control Law in Japan.

In addition, p-cresol can also be generated by contaminants when a conventional lactase preparation is applied to raw milk. In this case, the stimuli of both isovaleric acid and p-cresol are simultaneously received. Furthermore, the co-existence of isovaleric acid and p-cresol can increase the volatility of one or both of them, which can increase the perceived discomfort. Thus, prevention of generation and/or release of isovaleric acid is important from the viewpoint of off-flavor prevention.

The present inventors have also found that a protein having the amino acid sequence shown in SEQ ID NO: 1, which is contained as a minor contaminant in conventional lactase preparations, causes the generation/release of isovaleric acid. Further research is needed to determine what substrates this protein acts on to generate and/or release isovaleric acid.

A protein having the amino acid sequence shown in SEQ ID NO: 1 is a β-glucosidase produced by Kluyveromyces lactis. This β-glucosidase had β-glucuronidase activity along with β-glucosidase activity. Since β-glucuronidase is an enzyme that hydrolyzes glycosides in which glucuronic acid and aglycone are linked to β-glucuronide, the leading hypothesis at present is that glycosides of glucuronic acid and isovaleric acid are present in milk. It is assumed that Therefore, β-glucosidases exhibiting β-glucuronidase activity in the present invention refer to all those derived from prokaryotes, archaea, and eukaryotes. Among these, it particularly refers to eukaryote-derived β-glucosidase, yeast-derived β-glucosidase, yeast-derived β-glucosidase belonging to the genus Kluyveromyces, and K. lactis-derived β-glucosidase.

Of course, this hypothesis does not exclude the possibility of generation/release of isovaleric acid from other substrates or mechanisms.

When the lactase activity in the lactase preparation of the present invention is converted to 100 LU/g, the β-glucuronidase activity is 2,400 U/g or less, preferably 240 U/g or less, more preferably 24 U/g or less, and 0 U/g. g is most preferred. Above this upper limit of β-glucuronidase activity relative to lactase activity, a high probability of off-flavors can occur when the lactase preparation is added to raw milk to obtain the desired lactase action.

The β-glucuronidase activity of the lactase preparation of the present invention is not particularly limited, but is preferably 120,000 U/g or less, preferably 1,200 U/g or less, most preferably 0 U/g, in consideration of the balance with lactase activity. .

The enzymatic activity (U/g) of β-glucuronidase that can be contained in the lactase preparation is quantified by the following method using, for example, 4-methylumbelliferyl-β-D-glucuronide hydrate (MUG) as a substrate. may β-glucuronidase hydrolyzes MUG to glucuronic acid and 4-methylumbelliferone. The concentration of 4-methylumbelliferone can be easily determined from fluorescence intensity at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.

The lactase preparation itself or the one obtained by appropriately diluting it with pure water is used as a sample for evaluation.

MUG is dissolved in pure water to prepare an aqueous solution with a concentration of 2 mM, and 0.5 mL of this MUG aqueous solution and a predetermined buffer solution (for example, 100 mM potassium phosphate buffer (pH 6.5) containing 500 mM potassium chloride) are added to 0.25 mL. After mixing, the liquid temperature is adjusted to a predetermined temperature (for example, 37°C).

0.25 mL of the sample for evaluation is mixed with this MUG aqueous solution, and reacted at the predetermined temperature for a predetermined time (for example, 60 minutes). After the predetermined time has passed, a predetermined alkaline solution (for example, 1.0 mL of 0.1 M sodium hydroxide solution) is quickly added to stop the reaction. Fluorescence intensity is measured for these at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.

A plurality of 4-methylumbelliferones adjusted to a predetermined concentration were prepared, and the fluorescence intensity was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm to prepare a calibration curve, and a measurement sample was prepared based on this calibration curve. Calculate the amount of 4-methylumbelliferone produced in . 1 U (Unit) can be evaluated when the amount of 4-methylumbelliferone produced per hour of the reaction between the substrate and the enzyme is 1 nmol. Thus, the activity per gram (U/g) can be obtained.

As described above, the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of Kluyveromyces lactis β-glucosidase. Among the enzymes that function as β-glucuronidase activators, the β-glucosidase family enzyme having the amino acid sequence shown in SEQ ID NO: 1 is speculated to produce/release isovaleric acid.

An enzyme E1 belongs to another enzyme E2 family when the homology between E1 and E2 based on the amino acid sequences is 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more means that Amino acid sequence homology can be easily calculated using known software.

Additionally, that an enzyme E1 is in the family of another enzyme E2 means that the enzyme E1 can perform the same function as that of the enzyme E2. Although there is no particular limitation on the activity ratio in this function, for example, when the activity of the highly active enzyme is 100%, the activity of the other enzyme is 1% or more, 5% or more, 10% or more, 20% or more, 30% or more. % or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

Based on this, it is determined that the enzyme having the above-described homology with the amino acid sequence shown in SEQ ID NO:1 and capable of exhibiting the function as a β-glucuronidase activator is a member of the β-glucosidase family enzyme shown in SEQ ID NO:1. Since the β-glucosidase having the amino acid sequence shown in SEQ ID NO: 1 can hydrolyze MUG, this family enzyme is presumed to have hydrolytic activity against MUG.

Microorganisms belonging to the genus Kluyveromyces (yeast) are speculated to have a family of enzymes, β-glucosidases, having the amino acid sequence shown in SEQ ID NO:1. Of course, it is not intended to limit the invention.

Thus, the lactase preparations of the present invention are in particular those of the genus Kluyveromyces having a structural gene encoding a β-glucosidase (also the glucuronidase activity) having the amino acid sequence shown in SEQ ID NO: 1 or a β-glucosidase of the family. is a lactase preparation that can be produced from a microorganism belonging to

Various other ingredients may be added to the lactase preparation of the present invention, if desired. For example, additives such as stabilizers that contribute to stabilization of lactase may be added.

Specific examples of stabilizers include trehalose, sorbitol, glycerol, glycerol trimethylolpropane, neopentyl glycol, triethanolamine, glycol, diglycol, triethylene glycol, glycol, glycine, sodium chloride, and lactate. is less than 1000. Stabilizers may be used singly or in combination.

<Second aspect: method for producing lactase preparation>
According to a second aspect of the invention there is provided a method for producing a lactase preparation.

Chemical synthesis of lactase is extremely difficult with current technology. Lactase is thus typically obtained by culturing a lactase-producing microorganism and purifying the lactase from its liquid or solid medium.

Microorganisms that produce lactase are not particularly limited, and any microorganism (cell) that can produce lactase can be used. It may be prokaryotic, eukaryotic, or archaeal. Microorganisms also include cells isolated from multicellular organisms.

Currently used lactase-producing microorganisms include various organisms such as yeast, fungi, and bacteria. Typical examples include microorganisms belonging to the genus Kluyveromyces, Aspergillus, Bacillus or Penicillium. Among these, microorganisms belonging to the genus Kluyveromyces are preferred. Microorganisms may be in a haploid state as well as a diploid or higher polyploid state. A haploid has an advantage of easy genetic manipulation, and a polyploid has an advantage of easy continuation of gene mutation for a long period of time.

Examples of microorganisms belonging to the genus Kluyveromyces include K. fragilis, K. marxianus, and K. lactis. and most preferred is Kluyveromyces lactis.

Examples of microorganisms belonging to the genus Bacillus include B. subtilis, B. licheniformis, B. lentus, B. brevis, and Bacillus stearo. B. stearothermophilus, B. alkalophllus, B. amyloliquefaciens, B. coagulans, B. circulans, Bacillus • B. lautus, B. megaterium, or B. thuringiens.

Microorganisms of the genus Aspergillus include, for example, Aspergillus oryzae (A. oryzae) and Aspergillus niger (A. niger).

Microorganisms belonging to the genus Penicillium include, for example, Penicillium multicolor (P. multicolor).

The lactase produced by these microorganisms is usually the lactase that the microorganism can naturally produce. Thus, lactases typically include those derived from microorganisms belonging to the genera Kluyveromyces, Aspergillus, Bacillus or Penicillium.

However, it goes without saying that a mutated lactase may be produced by altering or substituting the lactase gene in a microorganism that naturally produces lactase. The lactase gene of another organism may be donated to produce the lactase of another organism.

Alternatively, a lactase-producing microorganism may be obtained by imparting a lactase gene to a microorganism that does not originally produce lactase.

Methods for imparting a gene include, for example, a method of introducing a vector containing the target gene. Any vectors can be used, including plasmids and viral DNAs. Naturally, the vector may also contain operator sequences for transcription control, antibiotic resistance genes for selection, and the like.

Here, the lactase gene to be imparted may be the lactase gene of any organism. As described above, typical examples include lactase genes derived from microorganisms belonging to the genera Kluyveromyces, Aspergillus, Bacillus, or Penicillium. Also, the lactase gene may be of not only wild type but also mutant type.

Any method for producing a lactase preparation from a lactase-producing microorganism can be used. Examples thereof include the method disclosed in International Publication No. 2014-185364.

A method for producing a lactase preparation from a lactase-producing microorganism mainly comprises the steps of culturing the lactase-producing microorganism, causing the microorganism to produce lactase simultaneously with and/or after this culturing, and purifying the lactase preparation from the lactase-containing feedstock.

Any technique for culturing the lactase-producing microorganism can be used. For example, it may be cultured at 20-40° C. for 24-240 hours in a medium containing lactose and a nitrogen source and having a pH of 3-10.

Any technique can be used to cause the lactase-producing microorganism to produce lactase. Microorganisms may be induced to produce lactase by adding any compound or applying a stimulus such as heat shock. Alternatively, it may be naturally produced by microorganisms.

The method of collecting the lactase-containing raw material from the obtained culture is also arbitrary. For example, it may be extracted from collected cells, or mutated cells that are excreted outside the cells may be used. When cultured in a liquid medium, the culture solution itself may be used.

The lactase-containing raw material may be a liquid material including a culture medium or a solid material, but is preferably a liquid material.

Any technique can be used to purify lactase from a lactase-containing raw material.

For example, it is purified by affinity chromatography using a column with high affinity for lactase and low affinity for contaminants (eg, β-glucuronidase activity). The column may contain, for example, an antibody that specifically acts on lactase.

Additionally/alternatively, size exclusion chromatography or the like may be used to separate lactase from contaminants (eg, β-glucuronidase activity).

In addition/apart from this, an inhibitor, a scavenger (such as an antibody) or a degrading agent (such as a protease) that specifically acts on the contaminant (e.g., β-glucuronidase activity) is added to remove the contaminant. Amount and/or activity may be reduced.

(First embodiment)
In the method for producing a lactase preparation of the first embodiment, a lactase preparation is produced from a lactase-producing microorganism capable of naturally producing β-glucuronidase activity, and the amount of β-glucuronidase activity in the production method is and/or treatment is performed such that the activity is reduced relative to the wild-type strain.

The aforementioned microorganisms belonging to the genera Kluyveromyces, Aspergillus, Bacillus or Penicillium are typical examples of such microorganisms.

Here, reduction is sufficient as long as the amount/activity of β-glucosidase is lower than that of the wild type, and is not limited to the form of complete elimination.

This treatment may be by spontaneous mutation or mutagenesis, or may involve genetic manipulation of the microorganism. If such a microorganism is prepared, a suitable lactase preparation may be purified from passages thereof. As a result, it is considered that the number of steps in the manufacturing method is unlikely to increase, and the time and economic costs can be suppressed. In addition, adverse effects such as deactivation of lactase activity due to an increase in the number of steps can be avoided.

Genetic manipulations include manipulations to impart genes that are not naturally present in microorganisms, manipulations to delete genes that are naturally present in microorganisms, manipulations that modify at least part of genes that are naturally present in microorganisms, manipulations that are naturally possessed, Examples include an operation of replacing a gene with another gene.

Techniques for completely erasing the expression of the β-glucuronidase activity include, for example, techniques for deleting (knocking out) the gene for the β-glucuronidase activity from the genome of the lactase-producing microorganism through homologous recombination.

Objects to be deleted include not only the genomic region (structural gene) that encodes the β-glucuronidase activity itself, but also the genomic region (expression regulation gene) that regulates the expression of this structural gene. An expression control gene is a region that functions specifically to synthesize the β-glucuronidase activity (structural gene is expressed). More specifically, it includes regulation of transcription from DNA to RNA, regulation of translation from mRNA to protein, and the like. Removal or disruption of these can be manipulated so that the β-glucuronidase activity is not completely synthesized. Such a completely deficient strain is advantageous in that contamination with β-glucuronidase activity can be completely prevented.

Alternatively, a method of replacing or modifying the wild-type structural gene of the genome with a gene encoding a mutant β-glucuronidase activity that has completely lost enzymatic activity or a completely different enzyme is also conceivable.

Again, the present invention is not limited to embodiments that completely abolish synthesis of β-glucuronidase activity, as described above. Also within the scope of the invention are embodiments that reduce the amount and/or activity of β-glucuronidase activity relative to the wild type strain. In particular, such a technique is effective when the β-glucuronidase activator is an essential gene and it is difficult to completely delete it.

Techniques for reducing the amount of β-glucuronidase activity compared to the wild strain include, for example, reducing the amount of RNA transcribed from the structural gene, inhibiting RNA processing, increasing the amount of degradation of these RNAs, and increasing the amount of RNA translated from mRNA. decreased protein production and increased degradation of this protein.

For example, by substituting or modifying an expression control gene, a method of making the amount of transcription or translation lower than that of a wild strain can be conceived. At this time, it may be substituted or modified so that the amount of transcription or translation can be artificially controlled to be low, or substituted or modified so that the amount of transcription or translation is naturally reduced under normal growth conditions of microorganisms. good too.

Alternatively/additionally, a defective strain completely lacking the β-glucuronidase activator gene is introduced with a vector containing the structural gene for the β-glucuronidase activator, resulting in less expression than the wild-type strain. It is also possible to consider an adjustment method. For example, a vector containing the structural gene of the β-glucuronidase activity may be introduced in a state in which the expression level is controllable, and the synthesis of the β-glucuronidase activity may be controlled to be lower than the normal expression level. .

Alternatively/additionally, it is also contemplated to replace or modify the wild-type gene with a mutant gene, such as one encoding a readily degradable mutant β-glucuronidase activity. This degradation includes not only degradation at the protein level but also degradation at the mRNA level. That is, the finally produced protein may be easily degraded by protease or the like and the amount may be reduced compared to the wild type, and/or the mRNA may be easily degraded compared to the wild type and the amount of mRNA may be reduced compared to the wild type. As a result, the amount of protein translation may be reduced. Naturally, this manipulation not only replaces or modifies the wild-type structural gene of the genome with the mutant gene, but also completely deletes the wild-type structural gene of the genome and generates a vector containing such a mutant gene. may be introduced.

For example, RNA interference may be performed on the mRNA of the β-glucuronidase activity to reduce the amount of mRNA and the amount of protein relative to the wild-type strain. The RNA fragment used for RNA interference may be synthesized RNA, which may be appropriately added during culturing, or may be produced by introducing a vector or inserting it into the genome so that the microorganism itself produces it.

Alternatively/in addition, if the lactase-producing microorganism is a polyploid microorganism (e.g. diploid yeast), the β-glucuronidase activator gene is retained on at least one chromosome and the The gene may be deleted so that the β-glucuronidase activity is not synthesized. For example, if the lactase-producing microorganism is a diploid yeast and both chromosomes have a gene for β-glucuronidase activity, β-glucuronidase can be produced by deletion of one of the genes for β-glucuronidase activity (hetero deletion). The expression level of the active form may be halved. Naturally, the target to be deleted may be either a structural gene or an expression-regulating gene, as described above.

Alternatively/in addition to this, the expression level of β-glucuronidase activity may be directly controlled as described above, but the expression of other genes that contribute to the expression level of β-glucuronidase activity may be similarly controlled. may be achieved to reduce the amount of β-glucuronidase activity present. That is, other genes that function to synthesize β-glucuronidase activators and/or prevent them from being decomposed may be controlled to suppress their expression, or β-glucuronidase activators may be synthesized. Other genes that function to prevent and/or be degraded may be controlled to promote their expression.

Alternatively/in addition, the β-glucuronidase activity may be weaker than the wild type.

For example, it is conceivable to generate a mutant gene that encodes a mutant with weakened activity and replace or modify the wild-type structural gene of the genome with this mutant gene. In addition to replacing or modifying the wild-type structural gene in the genome with the mutant gene, the wild-type structural gene in the genome may be completely deleted and then a vector containing the mutant gene may be introduced.

Alternatively/in addition to this, post-translational modification (for example, sugar chain modification) may be controlled to reduce the activity.

As a matter of course, a plurality of these techniques may be combined and executed.

A typical example of the present invention is the purification by affinity chromatography of a lactase preparation from a microorganism belonging to the genus Kluyveromyces, Aspergillus, Bacillus or Penicillium that lacks the structural gene for the β-glucuronidase activity. method. For polyploid microorganisms such as diploid, all structural genes may be deleted.

In particular, when the lactase-producing microorganism is yeast belonging to the genus Kluyveromyces, it is speculated that the genome of this microorganism contains a structural gene encoding the enzyme having the amino acid sequence represented by SEQ ID NO: 1 or a family enzyme thereof. be. The above treatment may be performed so that the expression of these genes is suppressed. In particular, at least one of these structural genes may be deleted, replaced or altered. Thus, lactase preparations may be produced from haploid and/or diploid Kluyveromyces yeast (especially Kluyveromyces lactis) in which some or all of these genes have been deleted, replaced or altered. Producing a lactase preparation from a microorganism of the genus Kluyveromyces in which the structural gene encoding the enzyme having the amino acid sequence represented by SEQ ID NO: 1 or a family enzyme thereof has been deleted, replaced or modified. is a typical example of the first embodiment.

(Second embodiment)
In the method for producing a lactase preparation of the second embodiment, a lactase preparation is produced from a microorganism capable of producing lactase, and the β-glucuronidase activity of the lactase preparation is measured one or more times during the production method.

The β-glucuronidase activity of the lactase preparation is determined, for example, by activity evaluation by MUG as described above. At this time, the lactase activity of this lactase preparation may be assessed.

It may further comprise comparing the measured β-glucuronidase activity to a predetermined reference value. This criterion may be the β-glucuronidase activity itself or its ratio to the lactase activity. The reference value can be arbitrarily set by the practitioner of the manufacturing method.

In addition, when the measured β-glucuronidase activity exceeds a predetermined reference value, additional purification may be performed so that it becomes equal to or less than the predetermined reference value. The additional purification step means either transferring to another purification step independent of the already performed purification step or returning to the already performed purification step.

As an independent and separate purification step, a step dedicated to separating lactase and β-glucuronidase activity may be provided. For example, purification steps such as size exclusion chromatography targeting β-glucuronidase activity may be performed.

Alternatively/in addition, a step of inactivating β-glucuronidase activity with an inhibitor that specifically deactivates β-glucuronidase activity may be performed. Alternatively/in addition, a step of removing β-glucuronidase activity using a capture agent (eg, an antibody) that specifically captures β-glucuronidase activity may be performed. Alternatively/in addition, a step of degrading β-glucuronidase activity using a degrading agent (eg, a protease) that specifically degrades β-glucuronidase activity may be performed. Naturally, a purification step may be performed after these steps.

Of course, the first embodiment may be combined with the second embodiment to produce a lactase preparation. Thus, a lactase preparation may be produced according to the third embodiment below.

(Third embodiment)
In the method for producing a lactase preparation of the third embodiment, a lactase preparation is produced from a lactase-producing microorganism capable of naturally producing β-glucuronidase activity, and in the production method, the amount of β-glucuronidase activity is and/or treatment is performed such that the activity is reduced relative to the wild-type strain, and the activity of β-glucuronidase activity of the lactase preparation is measured one or more times.

<Third aspect: mixture containing lactase preparation>
According to a third aspect of the invention there is provided a mixture comprising raw milk and a lactase preparation as described above.

The mixture obtained by mixing the lactase preparation of the present invention with raw material milk includes not only a simple mixture, but also dairy products produced by processing this mixture.

Raw material milk is not particularly limited, and examples thereof include milk of animals such as cows, sheep, and goats.

Examples of dairy products produced from a mixture of a lactase preparation and raw material milk include milk drinks such as cow's milk, fermented milk, ice cream, milk jam, and the like.

Among other things, long-life milk is produced by the lactase preparation of the invention. Long-life milk is milk intended for long-term storage (for example, refrigerated storage at 10° C. or less for several months). The risk of off-flavours is particularly great as lactase preparations can act over long periods of time. It is therefore dairy products that particularly benefit from the lactase preparations of the invention.

<Fourth aspect: off-flavor prediction method>
According to the fourth aspect of the present invention, the β-glucuronidase activity contained in the lactase preparation is used as an index, and the amount of isovaleric acid produced and/or the risk of off-flavor when the lactase preparation is allowed to act on raw milk. A method for predicting is provided.

Naturally, the higher the β-glucuronidase activity contained in the lactase preparation, the greater the generation/release of isovaleric acid. When it exceeds a certain threshold, it is observed as an off-flavour.

Therefore, when a lactase preparation is added to raw milk, the β-glucuronidase activity contained therein can be used as an index to predict the production amount of isovaleric acid and/or the risk of off-flavours.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples.

[Identification of off-flavour factors]
The present inventors have confirmed that off-flavours are produced by the techniques described below for several factors. Among these factors, one of the factors confirmed to generate off-flavour was β-glucosidase having β-glucuronidase activity of K. lactis. The technique using β-glucosidase, which is a β-glucuronidase activator, as a representative example of the off-flavour factor is specifically described below.

First, a large amount of β-glucuronidase activity was prepared in order to analyze whether it is an off-flavour factor. A plasmid was prepared by inserting the K. lactis β-glucosidase gene into the multicopy plasmid pRS426. The prepared plasmid was introduced into the budding yeast BY4741 strain to produce a large amount of β-glucosidase.

After producing β-glucosidase, this mutant yeast was extracted with Zymolyase (registered trademark) to obtain a yeast extract. Using this yeast extract as a starting sample, β-glucosidase was purified by column chromatography. The columns used were DEAE-Toyopearl column, Butyl-Toyopearl column and Giga Cap Q-Toyopearl column. The purified fraction obtained exhibited sufficient β-glucuronidase activity. On the other hand, the activity of arylsulfatase, which had already been identified as an off-flavour factor, was below the detection limit. As shown in Table 1, the degree of purification of β-glucosidase as β-glucuronidase activity was about 6 times, and the recovery rate of activity was 42%. After adding 1% lactose as an excipient to this purified solution, it was freeze-dried to obtain a β-glucuronidase active powder.

The following accelerated test using a large amount of enzyme was performed in order to analyze whether off-flavours were caused.

(Sample 1)
As a control, a sample was used in which 12 g of pure water was added to 88 g of non-fat milk (manufactured by Koiwai Milk Products Co., Ltd.).

(Sample 2)
A β-glucuronidase active solution was prepared by dissolving the purified β-glucuronidase active powder. A sample was obtained by adding 12 g of a β-glucuronidase activator solution (120,000 U) to 88 g of non-fat milk (manufactured by Koiwai Dairy Co., Ltd.) through a 0.22 μm filter.

(Sample 3)
A sample was obtained in the same manner as in sample 2, except that a commercially available E. coli-derived glucuronidase (manufactured by Merck Ltd.) was added instead of the K. lactis-derived β-glucuronidase activity.

The three types of samples thus obtained were allowed to stand at 12° C. for 5 days, and then subjected to odor analysis.

The following evaluation was performed by Daiwa Service Co., Ltd. First, a sensory test was conducted by a panelist. As a result of smelling the three kinds of samples, an unpleasant sour odor was observed in sample 2. Also, in sample 3, a so-called chemical odor was observed. On the other hand, no offensive odor was observed in sample 1.

In order to analyze the odorous components, gas chromatography-mass spectrometry (GC-MS) was performed to identify the odorous components. The results are shown in FIG.

In sample 3 (glucuronidase added), p-cresol was mainly detected. It is believed that p-cresol was liberated from the substrate contained in the raw milk. This analysis result agrees with the analysis of the panelists who felt a chemical odor. On the other hand, isovaleric acid was mainly detected in sample 2 (β-glucuronidase activity added). Isovaleric acid is a substance that exhibits a pungent and sour odor accompanied by discomfort. This analysis result also agrees with the analysis of the panelists who felt a sour odor.

While the p-cresol threshold in milk is typically 10 ppb, the results of sample 3 showed that the presence of isovaleric acid in the milk lowered the p-cresol threshold. Given the presence of substrates for p-cresol and isovaleric acid in milk, even low β-glucuronidase activity would increase the risk of developing off-flavours over time. In particular, dairy products that are stored for one month or more (eg, long-life milk) tend to cause off-flavour problems, and the present invention can be applied to such dairy products.

Arylsulfatases and glucuronidases liberate p-cresol from their substrates, causing off-flavours. This is well known to those skilled in the art. On the other hand, it is not well known that isovaleric acid is a causative odorant for the off-flavours of dairy products and that β-glucuronidase activity is involved in it. Furthermore, it was not well known, nor could it possibly be assumed, that β-glucosidase acts as a β-glucuronidase activator. These facts are reported by the present inventors for the first time.

Based on these results, by reducing the amount/activity of the β-glucuronidase active form contained in the lactase preparation, it is possible to reduce the generation/release of isovaleric acid compared to conventional methods, thereby reducing the risk of off-flavor generation. It turned out that it is possible.

[Preparation of β-glucosidase-deficient strain]
K. lactis lacking at least one structural gene for the β-glucuronidase activity (enzyme of SEQ ID NO: 1) was prepared by the following method.

As a method of deleting the β-glucuronidase activator gene, a method of transforming a linear DNA double strand having a homologous region of genomic DNA was used. Electroporation and lithium acetate methods were used for gene disruption in yeast chromosomes.

More specifically,
(1): A linear double-stranded DNA having a fusion sequence flanking a drug resistance gene such as an antibiotic called a marker gene between two base sequences of 1000 bp at both ends of the β-glucuronidase activator gene of SEQ ID NO: 1, It was synthesized using a PCR (polymerase chain reaction) method.
(2): The DNA sequence constructed in (1) was transformed into a host organism.
(3): Using a medium for host cell culture supplemented with drugs such as antibiotics, only transformants in which the DNA sequence for transformation was inserted into the chromosome were grown.
(4): The PCR method was used to confirm whether the target gene in the chromosome in the host cell was accurately replaced by the marker gene, that is, whether the target gene was disrupted.

A target gene-disrupted strain was prepared by the above methods (1) to (4). G418 resistance, hygromycin B resistance and the like are used as the drug resistance used in the step (1). Such drug-resistant microorganisms can be produced by transformation. The drug resistance gene used in the step (1) includes the kan gene for the antibiotic G418 and the hph gene for hygromycin B.

Separately, after treating yeast cells that produce β-glucuronidase activity with UV, γ-rays, hydrogen peroxide, NTG, EMS, etc., confirming the decrease in the activity from the growing cells. could also be obtained by

Claims (3)

A method for producing a lactase preparation from a microorganism belonging to the genus Kluyveromyces capable of producing lactase, comprising:
The microorganism is a microorganism that naturally produces β-glucosidase, which is a β-glucuronidase activity,
A method for producing a lactase preparation comprising a treatment such that the amount and/or activity of said β-glucosidase is limited compared to the wild type strain. The microorganism is an enzyme having the amino acid sequence represented by SEQ ID NO: 1, or an enzyme having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having β-glucosidase activity and β-glucuronidase activity. A microorganism that naturally has on its genome a structural gene that encodes
The treatment is an operation to add, delete, replace or modify one or more of the genes so as to suppress the expression of these structural genes.
A method for producing a lactase preparation according to claim 1, characterized in that: A method for producing a lactase preparation according to claim 1, wherein the β-glucuronidase activity of the lactase preparation is measured one or more times.

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