CN117980483A

CN117980483A - Lactase, lactase preparation, gene, recombinant vector and transformant

Info

Publication number: CN117980483A
Application number: CN202280059879.XA
Authority: CN
Inventors: 尾高伶; 佐野凉子; 岩崎卓己; 马场将弘; 佐分利亘; 森春英
Original assignee: Godo Shusei KK
Current assignee: Godo Shusei KK
Priority date: 2021-09-07
Filing date: 2022-09-07
Publication date: 2024-05-03

Abstract

A lactase preparation having excellent lactose decomposition in milk is provided. The lactase of one embodiment of the present invention has, as lactase activity, any one or a combination of a plurality of activities described in the following (1) to (6). (1) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.1% or less after 16 hours at 10 ℃, is 15.1mg/L or less. (2) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.01% or less after 16 hours at 10 ℃, is 30.2mg/L or less. (3) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.1% or less after 24 hours at 10 ℃, is 10.1mg/L or less. (4) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.01% or less after 24 hours at 10 ℃, is 15.1mg/L or less. (5) When the final concentration of milk was 1.4LYU/ml, the residual lactose concentration was 0.1% or less after 24 hours at 10 ℃. (6) When the final concentration of milk was 2.1LYU/ml, the residual lactose concentration was 0.01% or less after 24 hours at 10 ℃.

Description

Lactase, lactase preparation, gene, recombinant vector and transformant

Technical Field

The present invention relates to lactase, lactase preparations, genes, recombinant vectors and transformants.

Background

Since ancient times, milk has been used for a long time as a nutritious beneficial food. Milk contains lactose, which is one of the sugars. Lactose is decomposed in the intestines by lactase, but in some people, a large amount of milk and processed milk products (hereinafter, collectively referred to as "milk products") are ingested due to a decrease in the secretion amount of lactase in the growth intestines, which causes so-called lactose intolerance such as abdominal pain and diarrhea. This is one of the reasons for preventing the wide-range intake of the food rich in nutrition.

In recent years, dairy products with a previously reduced lactose have been provided. In the case of such a dairy product, even lactose intolerant people can ingest the dairy product without any problem.

Lactose reduction can be performed by various methods. For example, a method of hydrolyzing lactose in milk by treating milk before preparing milk with a lactase preparation is disclosed (for example, refer to patent document 1).

Prior art literature

Patent literature

Patent document 1: japanese patent application laid-open No. 2004-534527

Disclosure of Invention

Problems to be solved by the invention

The problem with existing lactase preparations is that lactose decomposition ceases as lactose decomposition proceeds in the milk. Therefore, in order to achieve lactose-free (lactose concentration 0.1 mass% or 0.01 mass%) milk, it is necessary to increase lactose decomposition.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a lactase and a lactase preparation which are excellent in lactose decomposition in milk (particularly in milk).

Means for solving the problems

One mode of the invention is lactase. The lactase has any one or a combination of the activities described in the following (1) to (6) as lactase activity.

(1) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.1% or less after 16 hours at 10 ℃, is 15.1mg/L or less.

(2) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.01% or less after 16 hours at 10 ℃, is 30.2mg/L or less.

(3) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.1% or less after 24 hours at 10 ℃, is 10.1mg/L or less.

(4) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.01% or less after 24 hours at 10 ℃, is 15.1mg/L or less.

(5) When the final concentration of milk was 1.4LYU/ml, the residual lactose concentration was 0.1% or less after 24 hours at 10 ℃.

(6) When the final concentration of milk was 2.1LYU/ml, the residual lactose concentration was 0.01% or less after 24 hours at 10 ℃.

Lactase of the above mode may be selected from any one of the following (i) to (iii).

(I) A protein consisting of the amino acid sequence shown in SEQ ID No. 1,

(Ii) A protein having lactase activity, which comprises an amino acid sequence in which 1 to several amino acid residues of the amino acid sequence shown in SEQ ID No. 1 are deleted, substituted or inserted,

(Iii) A protein having lactase activity, which comprises an amino acid sequence having an amino acid sequence represented by SEQ ID No.1 and having an identity of 70% or more.

In the lactase of the above aspect, the milk may satisfy one or more of the following conditions.

(A) The amount of solids in the milk is 0.1 to 30 mass%,

(B) The lactose content in the milk is 0.1 mass% or more and 30 mass% or less,

(C) The amount of protein in milk is 0.1 mass% or more and 30 mass% or less,

(D) The fat content in the milk is 0.1 mass% or more and 30 mass% or less.

Other modes of the invention are lactase formulations. The lactase preparation contains:

The lactase and

At least one selected from water, salt, excipient, suspending agent, buffer, stabilizer, preservative and physiological saline. The above salt may be one or more selected from the group consisting of magnesium chloride, sodium chloride, potassium chloride, calcium chloride and manganese chloride. In addition, the lactase formulation may contain a lactase as described above and a second lactase of a different origin or nature than the lactase.

Another mode of the present invention is a gene encoding a protein of the following (i), (ii) or (iii).

(I) A protein consisting of the amino acid sequence shown in SEQ ID No. 1,

The gene of the above-described mode may be composed of any one of the DNAs of the following (a) to (d).

(A) DNA composed of the base sequence shown in SEQ ID No. 2,

(B) A DNA which is composed of a base sequence in which 1 to several bases of the base sequence shown in SEQ ID No.2 are deleted, substituted or added and which encodes the above protein having lactase activity,

(C) A DNA which comprises a nucleotide sequence having a nucleotide sequence of 70% or more identity with the nucleotide sequence shown in SEQ ID No. 2 and which encodes the above protein having lactase activity,

(D) DNA which hybridizes with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID No. 2 under stringent conditions and encodes the above protein having lactase activity.

Further, SEQ ID NO.2 is a base sequence including a stop codon.

Yet another embodiment of the present invention is a recombinant vector having the above-described gene.

Still another embodiment of the present invention is a transformant transformed with the recombinant vector described above.

Yet another aspect of the present invention is a method for producing lactose-decomposed milk. The manufacturing method of lactose decomposed milk comprises the following steps: the step of adding the lactase or the lactase preparation to raw milk, and the step of allowing the lactase contained in the lactase or the lactase preparation to react with the raw milk for only a predetermined period of time.

Effects of the invention

According to the present invention, a technique related to lactase and lactase preparations excellent in lactose decomposition in milk (particularly in milk) can be provided.

Drawings

FIG. 1A-1453 and other lactase preparations were added in an amount of about 15.1. Mu.g relative to 1mL of milk, showing the change in residual lactose concentration at 10 ℃.

FIG. 2A magnified view of the range of the horizontal axis (10-50 hours) and the vertical axis (0-0.20% lactose concentration in milk) of FIG. 1 is shown in FIG. 2.

FIG. 3A-1453 shows the lactose decomposition rate (reaction rate) when various salts were added to the purified enzyme solution of BL 105A-1453.

FIG. 4 is a schematic diagram showing the optimum pH of the purified enzyme solution BL 105A-1453.

FIG. 5 is a schematic diagram showing the pH stability of the purified BL 105A-1453 enzyme solution.

FIG. 6 is a schematic diagram showing the optimum temperature of the purified enzyme solution BL 105A-1453.

FIG. 7 is a schematic diagram showing the temperature stability of the purified BL 105A-1453 enzyme solution.

FIG. 8 is a schematic diagram showing the main structure of the product analyzed by HPAEC-PAD.

FIG. 9A schematic diagram showing the transition of lactose concentration in fermentation stage and storage when lactase and primer (starter) are added simultaneously to milk.

FIG. 10 is a schematic diagram showing the transition of lactose concentration when YNL and BL105A_1453 are added to milk alone or after mixing.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail. In the present specification, the expression "a-b" in the description of the numerical range is represented as a or more and b or less unless otherwise specified.

< Lactase >

The lactase according to the embodiment preferably has 2 or more, more preferably 3 or more, still more preferably 4 or more, and particularly preferably 5 or more, of any one or more of the activities described in the following (1) to (6) as lactase activity. Most preferably all of (1) - (6) are satisfied.

(1) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.1% or less after 16 hours at 10 ℃, is 15.1mg/L or less,

(2) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.01% or less after 16 hours at 10 ℃, is 30.2mg/L or less,

(3) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.1% or less after 24 hours at 10 ℃, is 10.1mg/L or less,

(4) When the lactase is added to milk, the protein content of the lactase, which has a residual lactose concentration of 0.01% or less after 24 hours at 10 ℃, is 15.1mg/L or less,

(5) When the final concentration of milk was 1.4LYU/ml, the residual lactose concentration was 0.1% or less after 24 hours at 10 ℃,

(6) When the final concentration of milk was 2.1LYU/ml, the residual lactose concentration was 0.01% or less after 24 hours at 10 ℃,

And, the lactase amounts of (1) - (4) are values calculated by Bradford method.

In addition, the lactase of the embodiment is selected from any one of the following (i) to (iii).

(I) A protein consisting of the amino acid sequence shown in SEQ ID No. 1,

The above (i) is a protein having lactase activity as described below.

The method for measuring lactase activity is not particularly limited, and may be carried out by any known method. Lactase activity can be measured, for example, by the method described below. In the present embodiment, having lactase activity means that the lactase activity in the method described below is 1LYU/mg-protein or more when the amount of protein contained in the measurement object is 1 mg.

The protein consisting of the amino acid sequence shown in SEQ ID No.1 (hereinafter, referred to as BL 105A-1543 or 1453) is a lactase derived from Bifidobacterium longum (Bifidobacterium longum) 105-A strain. The lactase has a very high lactose decomposition effect, and is therefore exemplified as a preferable example.

In the amino acid sequence in which 1 to several amino acid residues of the amino acid sequence shown in SEQ ID No. 1 are deleted, substituted or inserted, the number of amino acid residues deleted, substituted or inserted (hereinafter, may be referred to as the number of deletions or the like) is preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 8. The number of deletions or the like of the amino acid residues in the amino acid sequence shown in SEQ ID No. 1 is preferably the number that shows the same enzymatic activity as lactase composed of the amino acid sequence shown in SEQ ID No. 1.

In this embodiment, the sequence identity with the amino acid sequence shown in SEQ ID NO. 1 is preferably 70% or more, more preferably 80% or more, further preferably 85% or more, particularly preferably 90% or more, particularly preferably 95% or more, and most preferably 99% or more. The percentage identity of such sequences may be calculated using published or commercially available software with algorithms to compare the reference sequences as query sequences. For example, BLASR, FASTA, GENETYX (manufactured by GENETYX Co., ltd.) or the like can be used.

The lactase according to this embodiment is preferably a protein having lactase activity, which is composed of an amino acid sequence having any of the above sequence identities.

< PH Activity >

The protein consisting of the amino acid sequence shown in SEQ ID No.1 shows lactase activity in the range of pH5.0-8.5, optimally in the range of 5.5-6.5. The optimum pH was 6.0.

< PH stability >

The protein consisting of the amino acid sequence shown in SEQ ID No.1 is lactase stable in the pH range of 5.0-9.0. Preferably pH5.2-8.4 is more stable.

< Optimum temperature >

The protein composed of the amino acid sequence shown in the sequence number 1 has lactase activity in the range of more than 0 ℃ to 60 ℃, has higher lactase activity in the range of 20 ℃ to 60 ℃, and has an optimal temperature range of 40 ℃ to 55 ℃ and an optimal temperature of 50 ℃.

< Temperature stability >

The protein consisting of the amino acid sequence shown in SEQ ID No. 1 is lactase which shows thermostability to 50 ℃. The protein showed 100% lactase residual activity even after being kept at 50℃for 1 hour.

(Gene encoding lactase)

The gene according to the embodiment is a gene encoding the protein having lactase activity, preferably a gene encoding any one of the amino acid sequences (i) to (iii) described above, more preferably a gene composed of any one of the DNAs (a) to (d) described below.

(A) DNA composed of the base sequence shown in SEQ ID No. 2,

(B) A DNA which is composed of a base sequence in which 1 to several bases of the base sequence shown in SEQ ID No. 2 are deleted, substituted or added and which encodes a protein having lactase activity,

(C) A DNA which comprises a nucleotide sequence having a nucleotide sequence of 70% or more identity with the nucleotide sequence shown in SEQ ID No. 2 and which encodes a protein having lactase activity,

(D) DNA which hybridizes with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID No. 2 under stringent conditions and encodes a protein having lactase activity.

Among the base sequences in which 1 to several bases of the base sequence represented by SEQ ID No. 2 are deleted, substituted or inserted, 1 to several bases are preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3, still more preferably 1 or 2. The deletion of a base means the removal or disappearance of the base, the substitution of a base means the substitution of a base with another base, and the insertion of a base means the addition of a base. "inserting" includes inserting bases at one or both ends of a sequence, and inserting additional bases between bases in the sequence.

In the gene of the present embodiment, the nucleotide sequence identity is preferably 70% or more, more preferably 80% or more, further preferably 85% or more, particularly preferably 90% or more, particularly preferably 95% or more, and most preferably 99% or more.

The sequence identity of a base sequence can be determined using the algorithm BLAST of KARLIN AND Altschul (Pro. Natl. Acad. Sci. USA,1993, 90:5873-5877). Based on this algorithm BLAST, programs called BLASTN and BLASTX were developed (J.mol. Biol.,1990,215, p.403-410). In addition, a homology analysis (Search homology) program of genetic information processing software Genetyx may also be used. The specific procedures for these analytical methods are well known (see www.ncbi.nlm.nih.gov).

Stringent conditions are conditions under which base sequences having high identity hybridize to each other, and base sequences having lower identity to each other than the base sequences do not hybridize to each other. The "stringent conditions" may be changed according to the degree of identity obtained. The more stringent the conditions, the more identical the sequences will hybridize. For example, stringent conditions include those described in Molecular Cloning: ALaboratory Manual (Second Edition, J.Sambrook et al, 1989). Specifically, hybridization conditions are exemplified by conditions under which hybridization is carried out in a solution containing 6 XSSC (composition of 1 XSSC: 0.15M sodium chloride solution, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 XDenhardt's solution and 100mg/mL herring sperm DNA together with the probe at 65℃for 8 to 16 hours.

The protein having lactase activity (lactase) of the present embodiment can be obtained by introducing a vector containing the gene encoding lactase into a microorganism according to a known method. The transformant may be maintained in the form of a vector, or the gene may be maintained in the genome. That is, when the transformant is cultured in an appropriate medium, the vector contained in the transformant or the gene encoded on the genome is expressed, and the protein having lactase activity (lactase) of the present embodiment is produced. The lactase thus produced is isolated and purified from the culture, whereby lactase can be obtained.

The gene encoding the lactase of this embodiment may be isolated from strain Bifidobacterium longum (bifidobacterium longum) 105-a using any method used in the art. For example, the gene can be obtained by extracting the whole genome DNA of Bifidobacterium longum (Bifidobacterium longum) 105-A strain, then selectively amplifying the target gene by PCR using primers designed based on the gene sequences upstream and downstream of the base sequence of SEQ ID No. 2, and purifying the amplified gene.

The type of the vector containing the gene encoding lactase of the present embodiment is not particularly limited, and examples thereof include vectors commonly used for producing proteins, such as plasmids, cosmids, phages, viruses, YACs, and BACs. Among them, plasmid vectors are preferable, and commercially available plasmid vectors for protein expression, such as pET and pBIC, can be suitably used. Procedures for introducing genes into plasmid vectors are well known in the art.

Examples of the microorganisms used for introducing the constructed vector include bacteria belonging to the genus Streptomyces, the genus Brevibacterium, the genus Staphylococcus, the genus Enterococcus, the genus Listeria, the genus Bacillus, the genus Escherichia, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces kluyveromyces, the genus Aspergillus, the genus Penicillium, the genus Trichoderma, and eukaryotic microorganisms. As a method of introduction, a method generally used in the art can be used.

If the transformant obtained is cultured in an appropriate medium, the lactase of the present embodiment is produced by expressing the gene introduced into the transformant. The medium used in the culture can be appropriately selected by those skilled in the art depending on the kind of transformant. The target lactase thus produced can be isolated from the culture by a usual method and purified by a known purification method.

Lactase derived from Bifidobacterium longum (bifidobacterium longum) 105-a strain has high lactose decomposition in milk as shown in examples described below.

< Lactase preparation >

The lactase preparation (lactase composition) of the present embodiment may contain, for example, water, salt, excipient, suspending agent, buffer, stabilizer, preservative, physiological saline, and the like, in addition to the active ingredient (lactase).

The lactase may be used in an amount of 1, or may be used in combination of two or more. By mixing lactases having different lactose decomposition effects for lactose-containing subjects, it becomes easy to enjoy the advantages of each lactase. For example, when a combination of a plurality of lactases having different sources is used, a combination of a lactase having lactose decomposition in the neutral region and a lactase having lactose decomposition in the acidic region is used, and a combination of a lactase having a high initial lactose decomposition rate but a final lactose decomposition rate of less than 99.9% and a lactase having a final lactose decomposition rate of 99.9% or more is used.

The first lactase and the second lactase having a different source or property from the first lactase may be used as lactase preparations in combination with each other according to the desired characteristics. The number of the third lactase, the fourth lactase, etc. to be mixed is not limited. When BL 105A-1453 is used as the primary lactase, the source of the lactase is Bifidobacterium longum-105-A, and the final lactose decomposition rate is more than 99.9%. As the enzyme, lactase having a high initial decomposition rate such as lactase derived from different sources and milk can be used as the second lactase. For example, lactase derived from yeast and lactase derived from mold, lactase derived from lactic acid bacteria may be used. More specifically lactase derived from Kluyveromyces lactis (Kluyveromyces lactis) and Kluyveromyces marxianus (Kluyveromyces marxianus), lactase derived from Aspergillus oryzae (Aspergillus oryzae), lactase derived from Lactobacillus delbrueckii (Lactobacillus delbrueckii).

In the case where a plurality of lactases are contained in the lactase preparation, the amount of lactase protein may be based on the mass of lactase protein. In the case of the first lactase and the second lactase, the mass ratio may be in the range of 1:99-99:1, preferably in the range of 10:90-90:10, more preferably in the range of 20:80-80:20, most preferably in the range of 25:75-50:50. When the amount is within these ranges, lactase preparations having various characteristics can be easily provided.

The lactase preparation of the present embodiment has an improved lactase activity by containing a salt or an ion thereof. Examples of the salt include magnesium chloride, sodium chloride, potassium chloride, calcium chloride, and manganese chloride. Examples of the ion include monovalent cations such as sodium and potassium, and divalent cations such as magnesium, manganese and calcium. In particular, the lactase preparation of the present embodiment has significantly improved lactase activity by having 2 or more salts selected from the group consisting of magnesium chloride, sodium chloride, potassium chloride, calcium chloride, and manganese chloride or ions thereof.

The lactase preparation containing salt or ions thereof may be used for promoting lactose decomposition, and may be added to or contained in an aqueous lactose solution in addition to raw milk such as cow's milk. Even in the case where the lactase preparation does not contain the above-mentioned salt or ion thereof, if the reaction system contains the above-mentioned salt or ion thereof, the lactose decomposition rate in the reaction system can be increased. For example, the reaction system in which the active ingredient (lactase) is added to the lactose aqueous solution can be further added with the above salt to cause the reaction system to contain ions, thereby increasing the lactose decomposition rate.

The lower limit of the content of the salt contained in the lactase preparation is preferably 0.1mM or more, more preferably 0.5mM or more, but also 1mM or more, but also 10mM or more, more preferably 40mM or more, from the viewpoint of increasing the lactose decomposition rate.

The upper limit of the content of the salt is preferably 1000M or less, more preferably 500M or less, or 100M or less, more preferably 50M or less. The upper and lower values of the salt content in the lactase preparation may be appropriately combined with each other.

The lower limit of monovalent cations contained in the lactase preparation is preferably 0.1mM or more, more preferably 1mM or more, and may be 10mM or more, or 100mM or more, more preferably 500mM or more. The upper limit of the monovalent cation is preferably 1000M or less, more preferably 500M or less, or 100M or less, and still more preferably 50M or less. The upper and lower values of monovalent cations contained in the lactase formulation may be appropriately combined in the above ranges.

The lower limit of the divalent cation contained in the lactase preparation is preferably 0.1mM or more, more preferably 1mM or more, but also 10mM or more, more preferably 40mM or more. The upper limit of the divalent cation is preferably 100M or less, more preferably 20M or less, but may be 10M or less, more preferably 2M or less. The upper and lower limit values of the divalent cations contained in the lactase preparation may be appropriately combined with the above-described ranges.

The pH of the lactose aqueous solution is typically 4.5 to 9.0 in terms of obtaining a sufficient lactose decomposition rate by salt.

The shape of the lactase formulation is not limited. The material may be solid or liquid at room temperature. In the case of a solid, the lactase formulation is preferably solid, and in the case of a liquid (e.g. milk), the lactase formulation is preferably liquid.

(Protein quality)

The lower limit of the content of the protein in the lactase preparation of the present embodiment is preferably 0.072mg/mL or more in the case of a liquid lactase preparation, and preferably 0.72mg/g or more in the case of a solid lactase preparation. On the other hand, the upper limit of the content of the protein is not particularly limited, and is preferably 720mg/mL or less, for example.

By setting the protein content to a lower limit or more, a desired activity can be ensured even if the storage time is prolonged.

The amount of protein contained in the lactase preparation of this embodiment can be measured by Bradfor method.

(Activity value)

The lactase formulation of this embodiment desirably has a lactase activity of 10-100,000LYU/mL, more desirably has an activity of 100-90,000LYU/mL, and still more desirably has an activity of 1,000-80,000LYU/mL.

The measurement method of the activity is as follows. To a lactose solution (final concentration 10%) dissolved in a 0.1M phosphate buffer (pH 6.5) containing a 0.1mM manganese chloride solution, a lactase preparation (enzyme solution) to be measured was added, and the mixture was kept at pH6.5 for 10 minutes at 37 ℃. The reaction was terminated by adding 1.5N sodium hydroxide solution. The reaction solution was cooled to room temperature, and the solution was neutralized by adding 1.5N hydrochloric acid. Using Glucose CII Test Wako (manufactured by Wako pure chemical industries, ltd.), the amount of free glucose in the reaction solution was determined. The amount of enzyme that produced 1. Mu. Mol of glucose under the present conditions for 1 minute was defined as 1LYU.

(Stabilizers)

In the lactase preparation of the present embodiment, the content of the stabilizer is preferably 10 to 90% by mass, more preferably 20 to 80% by mass, and still more preferably 30 to 70% by mass.

As the stabilizer, sorbitol, glycerin and the like can be mentioned.

< Method for producing lactose-decomposed milk >

(Raw milk)

The milk using the lactase or lactase preparation of the present embodiment is not particularly limited as long as it contains lactose. Examples of milk sources include cows, goats, sheep, and the like. Of these, milk derived from cow is preferably used.

The milk can be directly used, or added with water, whole milk powder, skimmed milk powder, cream powder, whey powder, protein concentrated whey powder, buttermilk powder, sweetened milk powder, formula milk powder, etc. as required. The amount of solids, lactose, etc., which will be described later, can also be adjusted. In this embodiment, the above-described milk to which substances other than enzymes are added is referred to as raw milk.

The amount of solids contained in the raw milk may be 0.1% by mass or more and 30% by mass or less, 0.5% by mass or more and 20% by mass or less, 1% by mass or more and 10% by mass or less, 2% by mass or more and 8% by mass or less, preferably 4.0% by mass or more, more preferably 6.0% by mass or more, and even more preferably 8.0% by mass or more. The upper limit of the amount of solids contained in the original milk is not particularly limited, but is, for example, 30% by mass.

The amount of lactose contained in the raw milk may be 0.1% by mass or more and 30% by mass or less, 0.5% by mass or more and 20% by mass or less, 1% by mass or more and 10% by mass or less, 2% by mass or more and 8% by mass or less, preferably 4.0% by mass or more and 5.8% by mass or less, preferably 4.4% by mass or more and 5.4% by mass or less, and more preferably 4.8% by mass or more and 5.0% by mass or less.

The amount of protein contained in the raw milk may be 0.1% by mass or more and 30% by mass or less, 0.5% by mass or more and 20% by mass or less, 1% by mass or more and 10% by mass or less, 2% by mass or more and 8% by mass or less, preferably 2.0% by mass or more and 4.8% by mass or less, more preferably 2.6% by mass or more and 4.2% by mass or less, and still more preferably 3.2% by mass or more and 3.6% by mass or less.

The amount of fat contained in the raw milk may be 0.1% by mass or more and 30% by mass or less, 0.5% by mass or more and 20% by mass or less, 1% by mass or more and 10% by mass or less, 2% by mass or more and 8% by mass or less, preferably 1.5% by mass or more, more preferably 2.5% by mass or more, and even more preferably 3.5% by mass or more.

The method of sterilizing the raw milk is not particularly limited, and examples thereof include a low temperature long time sterilization (LTLT), a high temperature long time sterilization (HTLT) and an ultra high temperature instantaneous sterilization (UHT) in this order of low heat history. Of these sterilization methods, UHT is preferably applied to sterilization of milk. The raw milk may be raw milk which is not sterilized.

In the above raw milk, lactose or a lactase preparation of the present embodiment is added at a predetermined concentration and then reacted at a predetermined temperature for a predetermined period of time, whereby lactose-decomposed milk having a high lactose decomposition rate can be produced. Milk is preferably used as raw milk.

The reaction time is, for example, 1 hour to 48 hours, preferably 24 hours to 48 hours.

The reaction temperature is, for example, in the range of more than 0℃to 40℃and preferably 1℃to 25℃and 1℃to 10 ℃.

The lactose concentration in the milk was about 4.7 mass%. The amount of lactose contained in lactose-decomposed milk is preferably 0.1% by mass or less (lactose-decomposed rate 97.87% or more), more preferably 0.01% by mass or less (lactose-decomposed rate 99.79% or more). Thus, it can be sold as lactose-free milk.

Lactose decomposed milk having a lactose content of 0.1 mass% or less (lactose decomposition rate 97.87% or more) is easily obtained if lactase in the raw milk is 1.4LYU/mL or more, and is preferable.

Lactose decomposed milk having a lactose content of 0.01 mass% or less (lactose decomposition rate 99.79% or more) is easily obtained if lactase in the raw milk is 2.1LYU/mL or more, and is preferable.

The embodiments of the present invention have been described above, and these are examples of the present invention, and various configurations other than the above may be adopted.

Examples

The present invention will be described below with reference to examples and comparative examples, but the present invention is not limited thereto.

< Use of enzyme >

Lactase for evaluation is shown below.

BL105A_1453: [ purified enzyme expressed by E.coli recombinant expression derived from Bifidobacterium longum-A strain ]

YNL 2: [ from Kluyveromyces lactis, contract alcohol Co., ltd., #21006]

YNL 2LS: [ from Kluyveromyces lactis, contract alcohol Co., ltd., #21002]

Lactase a: [ derived from Kluyveromyces lactis ]

Lactase B: [ derived from Bifidobacterium bifidum ]

Lactase C: [ derived from Lactobacillus delbrueckii ssp. Bulgaricicus ]

< Production of crude enzyme solution of BL 105A-1453 >

E.coli BL21 (DE 3) transformant strain into which BL105A_1453 expression plasmid pET23a-BL105A_1453 was introduced was cultured overnight at 18℃in a test tube containing 5mL of LB Amp medium (50. Mu.g/mL). mu.L of the culture medium was inoculated into a 100 mL-capacity Erlenmeyer flask containing 25mL of TB Amp medium (50. Mu.g/mL), and cultured at 18℃with shaking. This step was performed for 10 minutes. Using an absorptiometer, IPTG (isopropyl-. Beta. -thiogalactoside) was added at a final concentration of 0.5mM at a point of time when absorbance A ₆₁₀ reached 0.5, to induce the production of recombinant proteins. Induction culture was carried out at 18℃for 24 hours. The cells were recovered from the culture broth by centrifugation (8,000 rpm,10 minutes, 4 ℃) and suspended in 10mL of a binding buffer for nickel column chromatography (20 mM sodium phosphate buffer, pH7.5, 500mM sodium chloride), and the cells were disrupted by ultrasonic treatment (URTRA SONIC DISRUPTOR UR-200P, manufactured by TOMY Seisakusho). The supernatant obtained by centrifugation (8,000 rpm,10 minutes, 4 ℃) was used as a crude enzyme solution containing BL 105A-1453.

< Production of purified enzyme solution of BL 105A-1453 >

The crude enzyme solution obtained above was supplied to a Ni-NTA Agarose column (. Phi.1.0 cm. Times.4.0 cm; QIAGEN) equilibrated with a binding buffer. The unadsorbed protein was removed with wash buffer (20 mM sodium phosphate buffer, pH7.5, 500mM sodium chloride, 50mM imidazole) and the adsorbed protein was eluted by a linear concentration gradient of 20-500mM imidazole (elution buffer A,20mM sodium phosphate buffer, pH7.5, 20mM imidazole; elution buffer B,20mM sodium phosphate buffer, pH7.5, 500mM imidazole; 60mL each). The amount of the fraction was 8mL in the washing and 3mL in the elution. Fractions whose activity was confirmed by detection of ONPG decomposition active plate (eluted fra.7-14) were recovered. The recovered fractions were dialyzed 1 time against 10mM sodium phosphate buffer (pH 6.5) using a dialysis membrane (cellulose tube 36/32;Viskase Companies,Inc.) and further against the same buffer 5L for 2 times. After dialysis, the solution was concentrated to 1.5mL with Amicon Ultra-15 (MWCO, 100,000;Merck Millipore). This was used as BL 105A-1453 purified enzyme solution. The resulting purified enzyme solution was added with glycerol at a final concentration of 50%, and stored at-80 ℃. The purified enzyme solution was subjected to SDS-PAGE to confirm a single band.

< Time-dependent decomposition of lactose in milk >

Lactose decomposition tests were performed on the above-prepared BL 105A-1453 purified enzyme solution and other lactase preparations (enzyme solutions). A1 mL portion of commercially available milk (lactose content: 4.7% by mass, manufactured by Ming's Co., ltd.) was injected into a 1.5mL Eppendorf tube, and the mixture was pre-incubated at 10℃for 10 minutes. The final concentration of each lactase was adjusted to 0.01, 0.02, 0.03, 0.04, 0.06, 0.08, 012 and 0.16v/v% by adding thereto 20. Mu.L of each enzyme solution, and stored at 10℃for 48 hours. As a blank, a substance in which 20. Mu.L of buffer was added to 1mL of milk was used. For BL 105A-1453, the preparation (manufacturing) activity was assumed to be 3500LYU/mL. The reaction was stopped by adding 1/20 of 20% sulfosalicylic acid. As the buffer, a 0.1M phosphate buffer (pH 6.5) containing 0.1mM manganese chloride was used.

< Method for calculating lactose decomposition Rate >

Sugar analysis was performed on each sample using HPLC and HPAEC-PAD according to the following method.

HPLC analysis

The sample diluted 5 times with ultrapure water was filtered through a filter having a pore size of 0.22 μm, and the filtrate was analyzed by HPLC. Conditions at the time of HPLC analysis are shown below.

A main body: high-speed liquid chromatography (HPLC) Waters2696

Column: TRANSGENOMIC, manufactured by CARBOSep CHO-620CA85℃

Mobile phase: water and its preparation method

Flow rate: 0.4 mL/min

A detector: RI (RI)

Sample injection amount: 5 mu L

HPAEC-PAD analysis

The 250-fold diluted sample was filtered through a filter having a pore size of 0.22 μm, and the filtrate was analyzed using HPAEC-PAD. The conditions for HPAEC-PAD analysis are shown below.

A main body: dionex corporation, ICS-6000 ⁺, AS-Ap autosampler

Column: carboPac PA1 manufactured by Dionex Co., ltd20℃

Mobile phase: the following 4 liquid gradients

A:100mM sodium hydroxide solution

B: 600mM sodium acetate solution containing 100mM sodium hydroxide

C: water and its preparation method

D:50mM sodium acetate solution

Flow rate: 1.0 mL/min

A detector: pulse ampere detector, 30 DEG C

Sample injection amount: 25 mu L

For samples in which about 50% or more of lactose was decomposed by HPLC analysis, the analysis value obtained by HPAEC-PAD analysis was used for calculation of lactose decomposition rate. Lactose decomposition rate was calculated by comparing the residual lactose concentration of each sample relative to the lactose concentration of milk analyzed under the same conditions.

< Lactose concentration in milk reaching 0.1% by mass or 0.01% by mass lactase Activity (LYU/mL) >)

Lactose concentration in milk at 10℃to 0.1% or 0.01% by mass lactase activity (LYU/mL) at each reaction time is shown in Table 1. The lactase activity represents the final concentration in milk.

TABLE 1

< Lactose decomposition energy corresponding to the amount of protein contained in the measurement object at 10 >

In order to compare lactose decomposition energy corresponding to the amount of protein contained in the object to be measured, specific activity was calculated using LYU activity as an index. The protein quantification of the BL 105A-1453 enzyme solution and other lactase preparations was performed according to the Bradford method.

By combining the obtained specific activity values with the results in Table 1, the amounts of protein required for the lactose concentration to be 0.1 mass% and 0.01 mass% were calculated. BL 105A-1453 was observed to be most advantageous in this comparison of lactase in the 16-48 hour reaction (see Table 2).

TABLE 2

FIGS. 1 and 2 (partially enlarged view of FIG. 1) are schematic diagrams showing the transition of residual lactose concentration at 10℃when BL 105A-1453 and other lactase proteins were added in an amount of about 15.1. Mu.g relative to 1mL of milk. As shown in fig. 1 and 2, the residual lactose concentration of the BL105a_1453 purified enzyme solution after 16 or 24 hours at 10 ℃ was 0.1% or less and 0.01% or less, respectively, relative to milk, and the lactose decomposition rate was higher than that of the other lactase preparations prepared.

Analysis of activating factors in milk

For YNL 2 purified enzyme solution and BL105A_1453 purified enzyme solution, milk was analyzed for activating factors.

Specifically, 10. Mu.L of an enzyme solution was added to 190. Mu.L of a solution obtained under the following conditions, and the mixture was kept at 10℃or 37℃for 10 minutes, whereby the lactose decomposition rate (reaction rate) in the reaction system was calculated.

Condition 1:139mM lactose, 0.5mM MES-NaOH (pH=6.5)

Condition 2: contains 139mM lactose (Na ⁺：18.4mM、K⁺：39.6mM、Mg²⁺:4.25 mM) as mixed salt (NaCl, KCl, mgCl ₂)

Condition 3: milk

Condition 4: graded milk

Condition 5: desalted fractionated milk-1 (removal of charged components having a molecular weight of < 100 in condition 4 by electrodialysis)

Condition 6: desalted fractionated milk-2 (removal of charged component in condition 4 by ion exchange resin)

Lactose decomposition rate was calculated according to the following procedure.

After adding 10. Mu.L of the enzyme solution to 190. Mu.L of the solution obtained under each of the above conditions, the reaction was stopped by adding 10. Mu.L of 21% sulfosalicylic acid after holding at 10℃or 37℃for 10 minutes. To 50. Mu.L of a 10-fold dilution of the sample supernatant recovered by centrifugation with ultrapure water, 100. Mu.L (pH 7.0) of 2M Tris-HCl buffer and 20. Mu.L of glucose-quantifying reagent (Glucose CII Test Wako) were added, and the mixture was kept at 37℃for 1 hour to measure A ₅₀₅. Glucose concentrations were calculated from A ₅₀₅ based on standard curves (0-600. Mu.M glucose and A ₅₀₅). Also, a mixed solution of glucose aqueous solution: milk supernatant=9:1 was used in the standard curve. The amount of enzyme that hydrolyzes 1. Mu. Mol of lactose for 1 minute under the present condition was defined as 1 unit. The results obtained are shown in tables 3 and 4.

TABLE 3

TABLE 4

Regarding YNL 2, by electrodialysis of fractionated milk, the activity was reduced to a level of about 139mM lactose, and by addition of mixed salts, the activity exceeded the level in milk, thus judging that the salts in milk contributed significantly to the activation of enzymes.

Regarding BL 105A-1453, in the desalting of fractionated milk by ion exchange resin, the activity was reduced to a level of 139mM lactose, but in the desalting by electrodialysis, the activity reduction was smoothed. The addition of the mixed salt is considered to be an active level in milk, and thus the salt in milk is considered to act on activation, but it is considered that components which have not been removed in electrodialysis may act instead of the salt.

Evaluation of the Effect of Metal salts on BL 105A-1453

The o-nitrobenzene-. Beta. -D-galactopyranoside (ONPG) decomposition rate (reaction rate) at 37℃in each of the following reaction systems was calculated.

Reaction system 1: (Na ⁺) (50. Mu.L)

2MM ONPG, 10mM MES-NaOH (pH 6.0, containing 5.3mM Na ⁺), 0-50mM NaCl, enzyme

Reaction system 2: (K ⁺、Mg²⁺、Mn²⁺、Ca²⁺) (50. Mu.L)

2MM ONPG, 10mM MES-NaOH (pH 6.0, containing 6mM K ⁺)、0-50mM KCl、0-10mM MgCl₂、0-0.1mM MnCl₂ or 0-50mM CaCl ₂, enzyme)

The ONPG decomposition rate was calculated as follows.

After the reaction system 1 or 2 was reacted at 37℃for 10 minutes, 100. Mu.L of a 1M sodium carbonate solution was added thereto to measure A ₄₀₀. The amount of enzyme that breaks down 1. Mu. Mol of ONPG under the present conditions for 1 minute was defined as 1 unit. The results obtained are shown in fig. 3.

< Properties of the enzymology of BL 105A-1453 >

Optimum pH

The optimum pH of lactase was determined by the following method.

BL 105A-1453 enzyme solution diluted with buffer (0.1M sodium phosphate buffer containing 0.1mM manganese chloride, pH 4.5-9.5) adjusted to each pH was collected in a 1.5mL centrifuge tube at 150. Mu.L, mixed with 150. Mu.L of the buffer, and pre-incubated at 37℃for 3 minutes. With 300. Mu.L of the substrate (ONPG 100 mg/distilled water 100 mL) at 37℃for 1 minute, 600. Mu.L of 0.2M sodium carbonate was added, and the reaction was stopped. The lactase activity was calculated from the absorbance of the reaction solution OD ₄₂₀. Lactase activity 1 unit is defined as the amount of enzyme required to change the measured value to 1 within 10 minutes.

The results obtained are shown in fig. 4. The optimum pH of BL105A_1453 is 6.0.

PH stability

The pH stability of lactase was determined by the following method.

BL 105A-1453 enzyme solution was diluted 10-fold with each buffer (pH 3.0-10.5) and incubated at 37℃for 30 minutes. Cooled on ice for 3 minutes and further diluted 200-fold with buffer at pH 6.0. 300. Mu.L of the diluted enzyme solution was collected in a 1.5mL centrifuge tube, mixed with 300. Mu.L of buffer, and pre-incubated at 37℃for 3 minutes. With 600. Mu.L of matrix (ONPG 100 mg/distilled water 100 mL) at 37℃for 1 min, 0.2M sodium carbonate 1200. Mu.L was added and the reaction was stopped. The lactase activity was calculated from the absorbance of the reaction solution OD ₄₂₀. Lactase activity 1 unit is defined as the amount of enzyme required to change the measured value to 1 within 10 minutes.

The pH stability of lactase can be determined by the same method as shown in the method shown in the optimum pH of lactase except that a buffer is used and incubated at 37℃for 30 minutes.

The following buffers were used for the buffers at each pH, and the pH was plotted as measured at the time of enzyme dilution.

·pH3.5-5.5

: 0.1M sodium acetate buffer containing 0.1mM manganese chloride

·pH5.5-9.0

: 0.1M sodium phosphate buffer containing 0.1mM manganese chloride

·pH8-10.5

: 0.1M Glycine-sodium hydroxide buffer containing 0.1mM manganese chloride

The results obtained are shown in fig. 5. The stable interval of BL105A_1453 is pH5.2-8.4.

Optimum temperature

The optimum temperature of lactase was determined by the following method.

300. Mu.L of BL 105A-1453 enzyme solution diluted 2000-fold with sodium phosphate buffer at pH6.0 was collected, mixed with 300. Mu.L of the buffer, and incubated at 4, 10, 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60℃for 3 minutes. Thereafter, activity measurement was performed at each temperature in the same manner as the pH stability test.

The results obtained are shown in fig. 6. In FIG. 6, the activity at 37℃is used as a reference (relative activity 100%). The optimum temperature of BL105A_1453 is 50 ℃.

Temperature stability

The temperature stability of lactase was determined by the following method.

BL 105A-1453 enzyme solution was diluted 10-fold with buffer pH6.0 and incubated at 37, 50, 55, 60, 65, 70℃for 0, 5, 10, 30, 60 minutes. Cooled on ice for 3 minutes and further diluted 200-fold with buffer at pH 6.0. The diluted enzyme solution was collected in a tube at 300. Mu.L, mixed with 300. Mu.L of buffer, and pre-incubated at 37℃for 3 minutes. Thereafter, the same activity measurement as that of the pH stability test was performed.

The results obtained are shown in fig. 7. BL 105A-1453 showed 100% lactase residual activity even after 1 hour of reaction at 50 ℃.

< Galactooligosaccharide production >

When BL 105A-1453 was allowed to act on each of the substrates in the presence of 30 or 50w/v% lactose, it was confirmed that a maximum of 20% of galactooligosaccharides having 3 or more sugars were produced (as the yield of galactooligosaccharides). The primary structure of the galactooligosaccharides produced by BL 105A-1453 is 3' -galactosyl lactose (FIG. 8, lower panel). As shown in the upper part of FIG. 8, the main structure of the galactooligosaccharide-producing enzyme (trade name: BIOLACTA) derived from Bacillus circulans (Bacillus circulans) was 4' -galactosyl lactose, and therefore, it was confirmed that the types of galactooligosaccharides produced were different.

In addition, 3 '-galactosyl lactose is a substance expected to have a bifidobacterium proliferation effect in the human intestine, similarly to 4' -galactosyl lactose.

Here, in the case of producing 3-sugar galactooligosaccharides, 1 molecule of glucose and 1 molecule of 3-sugar galactooligosaccharides are produced from 2 molecules of lactose. The theoretical yield of galactooligosaccharide at this time was 74 mass%. In the case of producing 4-sugar galactooligosaccharides, 2 molecules of glucose and 1 molecule of 4-sugar galactooligosaccharides are produced from 3 molecules of lactose. The theoretical yield of galactooligosaccharide at this time was 65 mass%. Therefore, in the case of producing galactooligosaccharides from lactose, galactooligosaccharides that produce only 3 sugars are most efficient in terms of yield. The yield (mass%) of galactooligosaccharide herein means a percentage of "the produced amount of galactooligosaccharide" divided by "the consumed amount of lactose". The resulting galactooligosaccharides can be determined by HPLC. Specifically, the method described in WO2019/194062 can be employed.

< Production of fermented milk >

A primer (10 mg/100g of lactic acid bacteria YF-L812 strain and 5mg/100g of bifidobacterium BB-12 strain) was added to commercially available milk (manufactured by Ming's solution Co., ltd.) and the mixture was stirred and dispensed, and then BL 105A-1453 enzyme solution was added so that the product activity became 5200LYU/mL and 0.02% and 0.06%. As a control, YNL 2 0.02% addition region and lactase B (derived from Bifidobacterium bifidum enzyme) 0.02% addition region were set. Samples were taken 0, 2, and 4 hours after the start of fermentation, and the residual lactose concentration (% by mass) was calculated by sugar analysis. However, since the additional zone of BL 105A-1453 was rapid in fermentation progress, sampling was also performed over 3 hours. After the fermentation, the sample was transferred to 10℃and the residual lactose concentration was calculated after 24, 48 and 72 hours. The samples for sugar analysis were all diluted 3-fold with water and deproteinized by sulfosalicylic acid. Other conditions for sugar analysis were as described above in HPLC analysis.

The passage of lactose concentration in the fermented milk obtained as described above is shown in fig. 9. When compared with the same addition (0.02% addition zone), BL 105A-1453 reached a decomposition rate equal to that of lactase B at the end of fermentation (after 4 hours), and the lactose concentration in the sample was shown to be 0.4%. On the other hand, when 0.06% of BL 105A-1453 was added, the lactose concentration at 2 hours of fermentation became 0.1% or less. Only lactase B reacted even at 10 ℃ and low pH after the end of fermentation, and after 48 hours from the start of fermentation, lactose concentration reached 0.1% or less. The activity of BL 105A-1453 was decreased when the pH was 4.7 or less in the above-mentioned pH stability test, and it was considered that the inactivation was started at the end of fermentation.

According to the above results, lactose can be efficiently decomposed in the fermentation stage by the method of obtaining fermented milk by adding BL105A_1453 together with the primer. Thus, lactose-free fermented milk can be stably produced without requiring an additional process in the fermented milk production stage.

< Lactose decomposition test in milk (10 ℃ C.) combining YNL 2 and BL 105A-1453 >

Lactose decomposition test in milk was performed by combining the preparation of BL 105A-1453 and YNL 2. To milk, 20. Mu.L of each enzyme was added so that the final concentration became 0.04% (total of YNL 2 and BL 105A-1453: 0.04%), and the mixture was kept at 10℃for 48 hours. At this time, the activity of the preparation of BL105A_1453 was assumed to be 5,200LYU/mL. Other conditions and sugar analysis were carried out in accordance with the above-mentioned < time-dependent change of lactose decomposition in milk >, < method of calculating lactose decomposition rate >, "HPLC analysis", and "HPAEC-PAD analysis".

The results obtained are shown in fig. 10. The initial rate of lactose decomposition becomes higher depending on the YNL 2 concentration, and the final rate of lactose decomposition becomes higher depending on the BL 105A-1453 concentration. In fig. 10, the lactose concentration of less than 0.01% is in the lactose-free region.

From the above results, it was confirmed that by using a plurality of enzymes as lactase, a desired result was easily obtained.

Claims

1. A lactase having, as lactase activity, any one or a combination of the activities described in the following (1) to (6):

(1) When the lactase is added into milk, the protein content of the lactase with the residual lactose concentration below 0.1% after 16 hours at 10 ℃ is below 15.1mg/L,

(2) When the lactase is added into milk, the protein content of the lactase with the residual lactose concentration of less than 0.01% is less than 30.2mg/L after 16 hours at 10 ℃,

(3) When the lactase is added into milk, the protein content of the lactase with the residual lactose concentration below 0.1% after 24 hours at 10 ℃ is below 10.1mg/L,

(4) When the lactase is added into milk, the protein content of the lactase with the residual lactose concentration of less than 0.01% is less than 15.1mg/L after 24 hours at 10 ℃,

2. The lactase of claim 1, selected from any one of the following (i) - (iii):

(i) A protein consisting of the amino acid sequence shown in SEQ ID No. 1,

3. Lactase according to claim 1 or2, wherein the milk of claim 1 meets one or more of the following conditions:

(A) The amount of solids in the milk is 0.1 to 30 mass%,

(B) The lactose content in the milk is 0.1 mass% or more and 30 mass% or less,

(C) The amount of protein in milk is 0.1 mass% or more and 30 mass% or less,

(D) The fat content in the milk is 0.1 mass% or more and 30 mass% or less.

4. A lactase formulation comprising:

a lactase as claimed in any one of claims 1 to 3, and

At least one selected from water, salt, excipient, suspending agent, buffer, stabilizer, preservative and physiological saline.

5. The lactase formulation of claim 4, wherein the salt is one or more selected from the group consisting of magnesium chloride, sodium chloride, potassium chloride and calcium chloride.

6. A lactase formulation comprising the lactase of any one of claims 1-3 and a second lactase of a different source or nature than the lactase.

7. A gene encoding a protein of the following (i), (ii) or (iii):

(i) A protein consisting of the amino acid sequence shown in SEQ ID No. 1,

8. The gene according to claim 7, which is a gene composed of any one of the following DNAs (a) to (d):

(a) DNA composed of the base sequence shown in SEQ ID No. 2,

(B) A DNA which is composed of a base sequence in which 1 to several bases of the base sequence shown in SEQ ID No.2 are deleted, substituted or added and which encodes the protein having lactase activity,

(C) Consists of a nucleotide sequence having a nucleotide sequence of 70% or more identity with the nucleotide sequence shown in SEQ ID No. 2 and encoding the protein having lactase activity,

(D) DNA which hybridizes with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID No. 2 under stringent conditions and which codes for the protein having lactase activity.

9. A recombinant vector having the gene of claim 7 or 8.

10. A transformant transformed with the recombinant vector of claim 9.

11. A method of manufacturing lactose decomposed milk, comprising:

A process of adding the lactase of any one of claims 1-3 or the lactase preparation of claim 4 or 5 to raw milk, and

And a step of allowing lactase contained in the lactase or the lactase preparation to react with the raw milk for only a predetermined period of time.