阅读说明：本技术 性能改善的乳糖酶 (Lactase with improved performance ) 是由 H·拉杰 P·史密斯 T·埃克哈特 V·沃因诺维奇 C·E·G·苏勒 约翰尼斯·马腾·范丹 于 2018-04-11 设计创作，主要内容包括：本发明涉及表现出β-半乳糖苷酶活性的新的改善的肽或二聚肽,以及任选地在升高的温度下降低组合物中乳糖含量的改善的方法。(The present invention relates to new improved peptides or dimeric peptides exhibiting beta-galactosidase activity and an improved method for reducing the lactose content of a composition, optionally at elevated temperatures.)

1. 35, a peptide exhibiting enhanced β -galactosidase activity compared to the peptide of SEQ ID NO:

(a) the beta-galactosidase activity is determined by: incubating 13 μ L of a solution containing a known amount of purified lactase and 37 μ L of a solution containing 140mM lactose at pH6.7 and 37 ℃ for 10 minutes, terminating the lactase reaction by heating, determining the amount of glucose formed by incubating the reaction product with 80 μ L of a solution containing glucose oxidase (0.6g/L), 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (1.0g/L, ABTS) and horseradish peroxidase (0.02g/L) at 30 ℃ for 40 minutes, and determining the absorbance at 610nm using a fluorometer;

(b) 35, the increase in β -galactosidase activity is at least 20%.

2. The peptide according to claim 1, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 33, 14, 13, 19, 7, 9, 11, 26 and 27, 30 and 1, or has more than 85% amino acid sequence identity to any of these sequences.

3. A peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or an enzymatically active fragment thereof, or an amino acid sequence of any of said sequences having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions.

4. A dimeric peptide exhibiting beta-galactosidase activity, said dimeric peptide consisting of two peptides having the amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or an enzymatically active fragment thereof, or an amino acid sequence of any of said sequences having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid substitutions, additions, or deletions.

5. A method of reducing the lactose content of a lactose-containing composition, such as a dairy product, which composition comprises a milk-based matrix, which method comprises the step of contacting the composition with a peptide or dimeric peptide exhibiting β -galactosidase activity according to any one of claims 1-4 at a pH in the range 3-10 and a temperature of 0 ℃ -140 ℃; or with a host cell expressing either of the peptide or dimeric peptide.

6. Use of a peptide or dimeric peptide exhibiting beta-galactosidase activity according to any one of claims 1-4 for the production of a dairy product with a reduced lactose content.

7. A method of producing a dairy product, the method comprising the steps of:

a) providing a milk-based matrix comprising lactose;

b) adding to the lactose-containing milk-based matrix a peptide or dimeric peptide exhibiting beta-galactosidase activity of any one of claims 1-4; and

c) treating the milk-based substrate with the peptide or dimeric peptide exhibiting beta-galactosidase activity.

8. The method according to claim 7, wherein step c) is carried out at a pH in the range of 3-10, such as in the range of 3-9, such as in the range of 3-8, such as in the range of 3-7, such as in the range of 3-6, such as in the range of 3-5, such as in the range of 3-4, such as in the range of 4-10, such as in the range of 4-9, such as in the range of 4-8, such as in the range of 4-7, such as in the range of 4-6, such as in the range of 4-5, such as in the range of 5-10, such as in the range of 5-9, such as in the range of 5-8, such as in the range of 5-7, such as in the range of 5-6, for example in the range of 6-10, for example in the range of 6-9, for example in the range of 6-8, for example in the range of 6-7.

9. The method according to claim 7 or 8, wherein step c) or a part of step c) is carried out at a temperature of not more than about 25 ℃, such as not more than about 20 ℃, such as not more than about 18 ℃, such as not more than about 16 ℃, such as not more than about 14 ℃, such as not more than about 12 ℃, such as not more than about 10 ℃, such as not more than about 8 ℃, such as not more than about 7 ℃, such as not more than about 6 ℃, such as not more than about 5 ℃, such as not more than about 4 ℃, such as not more than about 3 ℃, such as not more than about 2 ℃.

10. The method according to any one of claims 7-9, wherein step c) or a part of step c) is carried out at a temperature of at least about 25 ℃, such as at least about 30 ℃, such as at least about 35 ℃, such as at least about 40 ℃, such as at least about 45 ℃, such as at least about 50 ℃, such as at least about 55 ℃, such as at least about 60 ℃, such as at least about 65 ℃, such as at least about 70 ℃, such as at least about 75 ℃, such as at least about 80 ℃, such as at least about 85 ℃, such as at least about 90 ℃, such as at least about 95 ℃, such as at least about 100 ℃, such as at least about 110 ℃, such as at least about 120 ℃, such as at least about 130 ℃, such as at least about 135 ℃, such as at least about 140 ℃.

11. The method of any one of claims 5-10, wherein:

(a) the peptides exhibiting beta-galactosidase activity have the amino acid sequences shown in SEQ ID NOs 22, 33, 14, 7, 26 and 27, 30 and 1, or have more than 85% amino acid sequence identity with any of these sequences; and

(b) 35, wherein:

(i) the beta-galactosidase activity is determined by: incubating 13 μ L of a solution containing a known amount of purified lactase and 37 μ L of a solution containing 140mM lactose at pH6.7 and 37 ℃ for 10 minutes, terminating the lactase reaction by heating, determining the amount of glucose formed by incubating the reaction product with 80 μ L of a solution containing glucose oxidase (0.6g/L), 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (1.0g/L, ABTS) and horseradish peroxidase (0.02g/L) at 30 ℃ for 40 minutes, and measuring the absorbance at 610nm using a spectrophotometer;

(ii) 35, an increase in β -galactosidase activity of at least 20% compared to the peptide of SEQ ID NO; and

(c) the step of contacting the lactose-containing composition with a peptide or dimeric peptide exhibiting beta-galactosidase activity is carried out at a temperature of 50 ℃ to 140 ℃.

12. The process according to any one of claims 5-11, wherein the process reduces the lactose concentration in the lactose-containing composition or in the milk-based base to less than 0.2%, preferably less than 0.1%.

13. The method according to any one of claims 5-12, obtaining a concentration of less than 0.2% lactose in the lactose-containing composition or the milk-based matrix 3-30 minutes, preferably 4-20 minutes, most preferably 4-10 minutes after addition of the peptide exhibiting beta-galactosidase activity.

14. Method according to any one of claims 5-13, wherein the peptide exhibiting β -galactosidase activity is added to the lactose-containing composition so as to obtain a mixture comprising lactase at a concentration of 0.001-0.2mg/ml, preferably 0.002-0.04 mg/ml.

15. The method according to any one of claims 5-14, wherein the step of contacting the lactose-containing composition with the peptide or dimeric peptide exhibiting β -galactosidase activity is performed at a temperature of 50-140 ℃ for a period of 4-20 minutes, and wherein the milk-based product is subsequently cooled and stored between 1 ℃ and room temperature, preferably at a temperature of 1-6 ℃, until further use.

Technical Field

The present invention relates to new improved peptides or dimeric peptides exhibiting beta-galactosidase activity and improved methods for reducing lactose content in compositions such as dairy products.

Background

Lactose hydrolysis is a good way for lactic acid bacteria to obtain glucose and galactose as carbon sources for growth in milk. Lactase (beta-galactosidase; EC 3.2.1.23) is an enzyme that hydrolyses the sugar lactose in milk to monosaccharides. A commercial use of lactase is to break down lactose in dairy products. Milk products with high lactose levels are difficult for lactose intolerant persons to digest. It is estimated that about 70% of all people worldwide have a limited ability to digest lactose. Thus, there is an increasing demand for dairy based food products containing no or only low levels of lactose.

Lactase has been isolated from a variety of organisms, including microorganisms of the genera Kluyveromyces (Kluyveromyces) and Bacillus (Bacillus). Kluyveromyces, in particular kluyveromyces fragilis (k.fragilis) and kluyveromyces lactis (k.lactis), and other fungi such as Candida (Candida), Torula (Torula) and Torulopsis (Torulopsis) are common sources of fungal lactase, while bacillus coagulans (b.coegulans) and bacillus circulans (b.circulans) are well known sources of bacterial lactase. Several commercial lactase preparations from these organisms are available, for example

(available from Novozymes, Denmark), HA-lactase (available from Chr. Hansen, Inc., Denmark) and

(available from DSM in the Netherlands), all of whichThe preparation is prepared from Kluyveromyces lactis. All these lactases are so-called neutral lactases with an optimum pH between pH6 and pH8 and an optimum temperature around 37 ℃. When such lactases are used for the production of e.g. low lactose yogurts, the enzyme treatment must be carried out in a separate step before fermentation, or high doses of enzymes must be used, since their activity decreases with decreasing pH during fermentation.

A typical method for producing pasteurized milk with reduced lactose comprises adding lactase to the milk followed by a long incubation time (10-48h (hours), usually 24h) at a temperature of about 6 ℃. Because of Ha-lactase andfit activity is 45-70. mu. mol per minute per mg of enzyme, so that to achieve the desired residual lactose level, enzyme doses of pasteurized milk are 55-70mg/L and 45-60mg/L, respectively. Ha-lactase and

the optimal temperature for the Fit enzyme is about 37 ℃. Incubation of the milk at 37 ℃ for longer will result in microbial growth.

Moreover, these lactases are not suitable for the hydrolysis of lactose in milk carried out at high temperatures, which in some cases is advantageous to keep the microbial count low and thus ensure high milk quality. Furthermore, the known lactase is not suitable for use in the required method for producing Ultra Heat Treated (UHT) milk, wherein the enzyme is added prior to the UHT treatment.

WO92/13068 relates to compositions comprising lactase activity obtained by sonication of microbial cells of bacteria or yeast. WO2010092057 and WO0104276 relate to cold active beta-galactosidases. WO07110619 relates to a β -galactosidase with high transgalactosylating activity, whereas WO2009071539 relates to a β -galactosidase with lower transgalactosylating activity.

Object of the Invention

It is an object of embodiments of the present invention to provide a beta-galactosidase with properties that enable the production of improved lactose-free or low-lactose products.

It is a further object of embodiments of the present invention to provide a beta-galactosidase with properties that enable improved production processes for reducing lactose in products, such as lactose-free products or lactose-low products, such as easier, faster, more reliable or cheaper production processes.

Disclosure of Invention

The present inventors have identified β -galactosidases having previously undescribed properties which enable the production of improved lactose-free or low lactose products and enable improved production processes for such lactose-free or low lactose products. In particular, these β -galactosidases have been shown to be very stable and have relatively high activity over a very wide temperature range and pH range. They can also be used at specific temperatures, for example at elevated temperatures and at pH values which are not normally seen with these enzymes. First, this enables the use of β -galactosidase at specific pH values and temperatures not known to be possible. It also enables the use of the same specific enzyme in several different applications, which is highly desirable in industry.

In a first aspect, the present invention provides a peptide exhibiting increased β -galactosidase activity compared to the peptide of SEQ ID NO:35, wherein:

(a) beta-galactosidase activity was determined by the following manner: incubating 13 μ L of a solution containing a known amount of purified lactase and 37 μ L of a solution containing 140mM lactose at pH6.7 and 37 ℃ for 10 minutes, terminating the lactase reaction by heating, determining the amount of glucose formed by incubating the reaction product with 80 μ L of a solution containing glucose oxidase (0.6g/L), 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (1.0g/L, ABTS) and horseradish peroxidase (0.02g/L) at 30 ℃ for 40 minutes, and determining the absorbance at 610nm using a fluorometer;

(b) 35, beta-galactosidase activity is increased by at least 20%.

Thus, the enzyme of the invention is characterized in that the enzymatic activity of beta-galactosidase is increased by at least 20%, but can be higher, and even significantly higher, compared to the peptide of SEQ ID NO: 35. Thus, the present invention provides peptides exhibiting an increase in beta-galactosidase activity of at least 40%, including at least 50% or at least 80%, compared to the peptide activity of SEQ ID NO: 35.

According to a preferred embodiment, the above-mentioned peptides are characterized by having an amino acid sequence selected from the group consisting of SEQ ID NO 22, 33, 14, 13, 19, 7, 9, 11, 26 and 27, 30 and 1 or a sequence having more than 85% amino acid sequence identity with any of these sequences. As shown in the examples of the present application, these peptides are observed to have a particularly advantageous beta-galactosidase activity, and according to a preferred embodiment of the present invention, the present invention thus provides peptides characterized by having an amino acid sequence selected from the group consisting of SEQ ID NOs 22, 33, 14, 13, 19, 7, 9, 11, 26 and 27, 30 and 1 or a sequence having more than 85% amino acid sequence identity to any of these sequences, and exhibiting a beta-galactosidase activity that is increased by at least 50% compared to the activity of the peptide of SEQ ID NO: 35.

In another embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ id No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or an amino acid sequence of the above amino acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions.

In yet another aspect, the present invention relates to dimeric peptides (dimeric peptides) exhibiting β -galactosidase activity consisting of two peptides having the amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29, or enzymatically active fragments thereof, or an amino acid sequence of any of the above sequences having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions.

The present invention relates to a nucleotide sequence encoding the above-described peptide exhibiting beta-galactosidase activity or dimeric peptide according to the present invention.

In another aspect, the present invention relates to a host cell comprising a nucleotide sequence encoding a peptide or dimeric peptide of the present invention exhibiting β -galactosidase activity.

In one aspect, the lactase of the invention is characterized by high specific activity. The production of 100-300. mu. mol glucose per minute per mg of the enzyme was observed. Thus, the novel lactase has a significantly higher activity than the enzymes of the prior art.

In another aspect, the present invention relates to a method of producing a peptide or dimeric peptide of the invention exhibiting β -galactosidase activity, said method comprising expressing in a suitable host cell a vector comprising a nucleotide sequence of the invention; and purifying the peptide or dimeric peptide from the expression product of the host cell.

In another aspect, the invention relates to a method of reducing the lactose content in a lactose-containing composition, such as a dairy product, comprising the step of contacting the composition at a pH of 3-10 and a temperature of 0 ℃ -140 ℃ with a peptide exhibiting β -galactosidase activity, said peptide having an amino acid sequence shown in SEQ ID NOs 1-33; or the dimeric peptides consist of two peptides having the amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences; or a step of contacting with a host cell expressing any of the peptides.

In another aspect, the invention relates to the use of a peptide exhibiting beta-galactosidase activity, said peptide having the amino acid sequence shown in SEQ ID NO 1-33, or of a dimeric peptide consisting of two peptides having the amino acid sequences shown in SEQ ID NO 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29, for the production of a milk product with a reduced lactose content; or a sequence having at least 80% sequence identity to any one of said sequences; or a host cell expressing any of said peptides, for use in the production of a milk product with a reduced lactose content.

In some embodiments, the lactose-containing composition or the dairy product is selected from the group consisting of: lactose-free milk, low lactose milk, yogurt, including unpasteurized and pre-and post-pasteurized yogurt, cheese, fermented milk products, dietary supplements and probiotic diet products. In some other embodiments, such a host cell is one selected from the group consisting of: bacteria of the genus Bifidobacterium (Bifidobacterium), such as Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium catenulatum (Bifidobacterium catenulatum), Bifidobacterium longum (Bifidobacterium longum), or Lactobacillus (Lactobacillus), such as Lactobacillus sake (l.sakei), Lactobacillus amylovorus (l.amyylovorus), Lactobacillus bulgaricus (l.delbrueckii. bulgaricus), Lactobacillus lactis (l.delbrueckii. subsp.lactis), Lactobacillus delbrueckii subspecies indiana (l.delbrueckii. byckii. inducicus), Lactobacillus crispatus (l.iscrispatus), Lactobacillus thermophilus (Lactobacillus), Lactobacillus helveticus (Lactobacillus), or Lactobacillus helveticus (Lactobacillus). In some other embodiments, the lactose concentration is reduced to less than about 1%, such as less than about 0.1% or less, such as less than about 0.01%.

In another aspect, the invention relates to a method of producing a dairy product, the method comprising the steps of:

a) providing a milk-based matrix comprising lactose;

b) adding to the lactose-containing milk-based matrix a peptide exhibiting beta-galactosidase activity, the peptide having an amino acid sequence shown in SEQ ID NO 1-33; the dimeric peptides consist of two peptides having the amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences; and

c) treating the milk-based substrate with the peptide or dimeric peptide exhibiting beta-galactosidase activity.

In one aspect, the present invention provides the method for producing a dairy product described above, wherein:

(a) peptides exhibiting beta-galactosidase activity have the amino acid sequences shown in SEQ ID NOs 22, 33, 14, 7, 26 and 27, 30 and 1, or have more than 85% amino acid sequence identity with any of these sequences; and

(b) the peptide exhibits increased beta-galactosidase activity compared to the peptide of SEQ ID NO:35, wherein:

(i) beta-galactosidase activity was determined by the following manner: incubating 13 μ L of a solution containing a known amount of purified lactase and 37 μ L of a solution containing 140mM lactose at pH6.7 and 37 ℃ for 10 minutes, terminating the lactase reaction by heating, determining the amount of glucose formed by incubating the reaction product with 80 μ L of a solution containing glucose oxidase (0.6g/L), 2' -azido-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (1.0g/L, ABTS) and horseradish peroxidase (0.02g/L) at 30 ℃ for 40 minutes, and measuring the absorbance at 610nm using a fluorometer;

(ii) 35, at least 20% more beta-galactosidase activity compared to the peptide of SEQ ID NO; and

(c) the step of contacting the lactose-containing composition with a peptide or dimeric peptide exhibiting beta-galactosidase activity is carried out at a temperature of 50 ℃ to 140 ℃.

The processes of the invention can reduce the lactose concentration in the lactose-containing composition or milk-based base to less than 0.2%, preferably less than 0.1%.

In one aspect, the method is designed to rapidly reduce lactose concentration. According to a preferred embodiment, the present invention therefore provides a method as described above, wherein the lactose concentration of the lactose-containing composition or the milk-based matrix obtained 3-30 minutes, preferably 4-20 minutes, most preferably 4-10 minutes after addition of the peptide exhibiting beta-galactosidase activity is less than 0.2%.

In a similar aspect, the present invention provides a method of using economically advantageous low concentrations of lactase. According to this aspect, the process of the invention as described above therefore adds a peptide exhibiting β -galactosidase activity to a composition containing lactose, so as to obtain a mixture comprising said peptide in a concentration of 0.001-0.2mg/ml, preferably 0.002-0.04 mg/ml.

In another embodiment, the above method provides a rapid decrease in lactose concentration at elevated temperatures followed by a further decrease at lower temperatures. According to this embodiment, a method is provided, wherein the step of contacting the lactose-containing composition with the peptide exhibiting β -galactosidase activity or dimeric peptide is performed at a temperature of 50 ℃ to 140 ℃ for a period of 4-20 minutes, wherein the milk-based product is subsequently cooled and stored between 1 ℃ to room temperature, preferably at a temperature of 1 ℃ to 6 ℃, until further use.

In another aspect, the invention relates to a dairy product prepared by the method of the invention. Accordingly, the present invention provides a milk product comprising peptides exhibiting beta-galactosidase activity, said peptides having the amino acid sequences shown in SEQ ID NO:22, 33, 14, 7, 26 and 27, 30 and 1, or having more than 85% amino acid sequence identity to any of these sequences.

In another aspect, the invention relates to a food product, such as a dairy product, comprising peptides exhibiting β -galactosidase activity, said peptides having the amino acid sequences shown in SEQ ID NOs 22, 33, 14, 7, 26 and 27, 30 and 1, or having more than 85% amino acid sequence identity to any of these sequences.

In general, the present invention relates to a food product, such as a dairy product, comprising a peptide exhibiting beta-galactosidase activity, said peptide having an amino acid sequence as shown in SEQ ID NOs 1-33, or said dimeric peptide consisting of two peptides having an amino acid sequence as shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences.

In another aspect, the invention relates to a food product, such as a dairy product, comprising a host cell expressing a peptide exhibiting β -galactosidase activity, said peptide having an amino acid sequence as set forth in SEQ ID NO 1-33, or said dimeric peptide consisting of two peptides having an amino acid sequence as set forth in SEQ ID NO 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences. In some embodiments, such food product is selected from the group consisting of beverages, baby food, cereals, bread, biscuits, candies, cakes, food supplements, dietary supplements, probiotic comestible products, prebiotic comestible products, animal feeds, poultry feeds, and pharmaceuticals, or from dairy products consisting of lactose-free milk, low lactose milk, milk powder, baby milk, yogurt, ice cream, cheese, fermented dairy products, dietary supplements, and probiotic diet products.

Drawings

FIG. 1 specific activity of purified enzyme, measured at pH6.7, 37 ℃ with lactose as substrate, is depicted as SUAL-1, discussed in example 6. The standard deviation measured under the given conditions was less than 6%.

FIG. 2 specific activity of purified enzyme in the presence of galactose, measured at pH6.7 at 37 deg.C, is described as SUAG, discussed in example 7. The standard deviation measured under the given conditions was less than 15%.

FIG. 3 specific activity of purified enzyme determined at pH6.7, 4 ℃ with lactose as substrate, depicted as SUAL-2, discussed in example 8. The standard deviation measured under the given conditions was less than 5%.

FIG. 4 specific activity of purified enzyme determined at pH6.7, 43 ℃ with lactose as substrate, depicted as SUAL-3, discussed in example 9. The standard deviation measured under the given conditions was less than 5%.

FIG. 5 specific activity of purified enzyme determined at pH5.5, 4 ℃ with lactose as substrate, depicted as SUAL-4, discussed in example 10. The standard deviation measured under the given conditions was less than 5%.

FIG. 6 specific activity of purified enzyme determined at pH5.5, 37 ℃ with lactose as substrate, depicted as SUAL-5, discussed in example 11. The standard deviation measured under the given conditions was less than 5%.

FIG. 7 specific activity of purified enzyme determined at pH5.5, 43 ℃ with lactose as substrate, depicted as SUAL-6, discussed in example 12. The standard deviation measured under the given conditions was less than 5%.

FIG. 8 specific activity of purified enzyme determined at pH4.5, 4 ℃ with lactose as substrate, depicted as SUAL-7, discussed in example 13. The standard deviation measured under the given conditions was less than 5%.

FIG. 9 specific activity of purified enzyme determined at pH4.5, 37 ℃ with lactose as substrate, depicted as SUAL-8, discussed in example 14. The standard deviation measured under the given conditions was less than 5%.

FIG. 10 specific activity of purified enzyme, measured at pH4.5, 43 ℃ with lactose as substrate, is depicted as SUAL-9, discussed in example 15. The standard deviation measured under the given conditions was less than 5%.

FIG. 11 percentage of lactose remaining in pasteurized milk after treatment with a fixed amount of enzyme at 5 ℃ for 24h, determined using HPLC.

FIG. 12 percentage of residual lactose in UHT milk determined by HPLC after treatment with a fixed amount of enzyme at 25 ℃ for 24 h.

FIG. 13 residual activity percentage of purified enzyme at elevated temperature determined with lactose as substrate. The activity at 37 ℃ at pH6.7 was taken as 100%.

FIG. 14 percentage residual lactose present in pasteurized milk after incubation with lactase at different temperatures of 37 ℃, 55 ℃ or 60 ℃. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

FIG. 15. percent residual lactose present in pasteurized milk after incubation with lactase at a concentration of 0.047 mg/ml. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

FIG. 16. percent residual lactose present in pasteurized milk incubated with lactase for different reaction times, i.e., 15 minutes or 30 minutes. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

Figure 17 pasteurized milk incubated with lactase at different enzyme doses, i.e. 0.047mg/ml or 0.024mg/mlPercent residual lactose present in the composition. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

Figure 18. percent residual lactose present in pasteurized milk incubated with lactase using different dosages and different reaction times. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

FIG. 19. percent residual lactose present in filtered milk incubated with lactase at 55 ℃. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

FIG. 20. percent residual lactose present in filtered milk incubated with lactase at 55 ℃ and different enzyme doses. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

FIG. 21. percent residual lactose present in filtered milk incubated with lactase at 55 ℃ for various reaction times. Used in the assay

The detection limit of the kit is 0.01-0.2% or 0.02-1.0% lactose.

FIG. 22 measured specific activities of the purified enzyme at different temperatures at pH 6.7. Specific activity values are defined as micromoles (μmole) of glucose formed per minute per mg of enzyme under given conditions. The standard deviation measured under the given conditions was between 5 and 11%.

FIG. 23 measured specific activities of the purified enzyme at different temperatures at pH 5.5. Specific activity values are defined as micromoles of glucose formed per minute per mg of enzyme under given conditions. The standard deviation measured under the given conditions was about 5%.

FIG. 24 measured specific activities of the purified enzyme at different temperatures at pH 4.5. Specific activity values are defined as micromoles of glucose formed per minute per mg of enzyme under given conditions. The standard deviation measured under the given conditions was about 5%.

Detailed Description

The present inventors have found that certain peptides and dimeric peptides exhibiting β -galactosidase activity are surprisingly stable under a number of different physical conditions, providing relatively high activity, beyond what is generally considered optimal for such enzymes.

Thus, these enzymes identified by the present inventors have a relatively high activity at about 4 ℃ or 5 ℃ and can therefore be used for lactose hydrolysis when producing e.g. fresh milk. Furthermore, these enzymes also have relatively high activity in the range of 10 ℃ to 25 ℃, so the very same enzymes can be used for lactose hydrolysis in UHT milk. This feasibility of enzymes even over a wide temperature range is highly relevant, since milk may be stored at room/ambient temperature, which may vary from region to region of the world and also from season to season. For UHT treatment, the temperature is typically about 135 ℃ or about 140 ℃. It is highly desirable that these enzymes are active at temperatures ranging up to 140 ℃ so that the enzymes can be added to raw milk prior to the UHT step. In current practice, the enzyme is added after the UHT step, since the functional activity of the enzyme known in the art is significantly reduced after the high temperature heat treatment step, e.g. down to a value below the measurable activity. While milk is stored at room temperature, it may vary widely in different parts of the world.

These novel improved peptides exhibiting beta-galactosidase activity were also found to be active in the temperature range typically used for pasteurization. Thus, these enzymes can be added to raw milk prior to pasteurization. It will be appreciated that after the pasteurisation step, the functional activity of the enzymes known in the art is significantly reduced, for example to a value below the measurable activity.

Another advantage of these novel improved peptides exhibiting β -galactosidase activity is that they have a relatively low degree of galactose inhibition. The lower galactose inhibition of these novel enzymes is of great significance for applications requiring very low lactose concentrations.

In terms of suitability for the fermentation product, it is highly advantageous that the enzymes described herein have a high β -galactosidase enzymatic activity over a relatively wide temperature range of 4 ℃ -43 ℃, e.g. at about 37 ℃, wherein fermentation is usually optimal, but this activity of β -galactosidase is also present at low pH values, e.g. as low as 4.5, or as low as 4.0, or as low as 3.5, or even as low as pH 3.

In summary, the present inventors have found that certain peptides exhibiting β -galactosidase activity are active over a wide temperature range, active over a wide pH range, have generally high hydrolytic activity without side activity, have no or little galactose inhibition (e.g., less than 60%), and are stable in long term storage.

The beta-galactosidase activity can be determined by measuring the amount of glucose released after incubation with lactose under defined conditions. The released glucose can be detected by a chromogenic reaction.

Definition of

The term "milk" as used herein and in the context of the present invention is to be understood as a milky secretion obtained by milking any mammal, such as a cow, sheep, goat, buffalo or camel.

As used herein, the term "lactose-containing composition" refers to any composition, e.g. any liquid, that contains a significant measurable degree of lactose, e.g. a lactose content above 0.002% (0.002g/100 ml). The term encompasses milk and milk-based substrates.

In the context of the present invention, the term "milk-based substrate" may be any raw milk material and/or processed milk material. Useful milk-based substrates include, but are not limited to, solutions/suspensions of any milk or milk-like product containing lactose, such as whole or low fat milk, skim milk, buttermilk, low lactose milk, reconstituted milk powder (reconstituted milk powder), condensed milk, milk powder solutions, UHT milk, whey permeate, acid whey, cream, solutions of fermented milk products, such as yogurt, cheese, dietary supplements, probiotic dietary products. Generally, the term milk-based substrate refers to raw or processed milk material that is further processed for the production of dairy products.

"pasteurization" as used herein refers to a process of reducing or eliminating the presence of living organisms, such as microorganisms, in a dairy-based substrate. Preferably, pasteurization is achieved by maintaining a specified temperature for a specified period of time. The specified temperature is usually reached by heating. The temperature and duration may be selected to kill or inactivate certain bacteria, such as harmful bacteria, and/or to inactivate enzymes in the milk. A rapid cooling step may then be performed.

The term "dairy product" as used herein may be any food product wherein one of the main ingredients is milk based. Typically, the main ingredient is milk-based, and in some embodiments, the main ingredient is a milk-based substrate that has been treated with an enzyme having β -galactosidase activity according to the method of the invention.

The dairy product of the invention may be, for example, skim milk, low fat milk, full fat milk, cream, UHT milk, milk with extended shelf life, fermented milk products, cheese, yoghurt, butter, milk spread (dairy spread), buttermilk (buttermilk), acidified milk drinks, sour cream (sour cream), whey based drinks, ice cream, condensed milk caramel sauce (dulce de leche) or flavoured milk drinks.

The dairy product may additionally comprise non-dairy ingredients, e.g. plant ingredients, such as vegetable oils, vegetable proteins and/or vegetable carbohydrates. The dairy product may further comprise other additives, such as enzymes, flavourings, microbial cultures (e.g. probiotic cultures), salts, sweeteners, sugars, acids, fruits, fruit preparations, fruit juices or any other ingredient known in the art, as an ingredient or additive to the dairy product.

The term "fermented dairy product" or "fermented dairy product" as used herein is to be understood as any dairy product of which any type of fermentation forms part of the production process. Examples of fermented dairy products are yoghurt, buttermilk, creme fraiche, quark and fromage frais, among others. The fermented dairy product may be produced by or include steps of any method known in the art.

The term "fermentation" as used herein refers to the conversion of carbohydrates to alcohols or acids by the action of microorganisms. In some embodiments, the fermentation of the present invention comprises converting lactose to lactic acid. In the context of the present invention, "microorganism" may comprise any bacterium or fungus capable of fermenting a milk substrate.

The term "increased beta-galactosidase activity" as used herein refers to a relatively high specific activity of beta-galactosidase compared to a reference sequence.

The term "peptide exhibiting beta-galactosidase activity" as used herein refers to any peptide having enzymatic activity that catalyzes the hydrolysis of the disaccharide lactose into its component monosaccharides, i.e. glucose and galactose. This peptide may also be referred to as lactase or simply as beta-galactosidase (EC: 3.2.1.23).

In a preferred embodiment, the β -galactosidase activity is determined by: mu.l of a solution containing a known amount of purified lactase was incubated with a solution containing 140mM lactose at pH6.7 and 37 ℃ for 10 minutes, and the lactase reaction was stopped by raising the temperature to 95 ℃ for 10 minutes. The amount of formed glucose was determined by incubating the reaction product with 80. mu.L of a solution of glucose oxidase (0.6g/L), 2' -azinobis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (1.0g/L ABTS), and horseradish peroxidase (0.02g/L) at 30 ℃ for 40 minutes, and measuring the absorbance at 610nm using a fluorescence photometer. The absorbance correlates with the glucose concentration formed per minute, and the maximum value determined (expressed in micromoles of glucose formed per minute) is determined as the lactase activity unit 1 (also referred to herein as UAL-1). Lactase specific activity at pH6.7, 37 ℃ (also referred to herein as SUAL-1) is defined as micromoles of glucose formed per minute per mg of enzyme and is determined by dividing UAL-1 by lactase protein concentration in milligrams. Example 6 illustrates the full details of a preferred alternative for performing this assay.

Although characterization of β -galactosidase activity with reference to the unit micromolar value of glucose formed per mg of enzyme per minute represents a standard method for determining activity, other units can be used to characterize lactase activity using the assays described above. Thus, some examples characterize lactase activity by reference to μ M glucose formed per μ M enzyme per second.

In alternative embodiments, the determination may be performed using different temperatures or different pH values for the lactase incubation.

The terms "peptide" and "oligopeptide" as used in the context of the present application should be considered synonymous (as generally recognized) and each term may be used interchangeably when the context requires a chain of at least two amino acids coupled by peptidyl linkages. The term "polypeptide" as used herein is used for chains containing more than ten amino acid residues. All peptide and polypeptide formulae or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. As used herein, "protein" refers to a peptide sequence produced by certain host organisms and may include post-translational modifications, such as added glycans.

The term "amino acid" or "amino acid sequence" as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of the above, as well as naturally occurring molecules or synthetic molecules. Herein, "fragment" refers to a fragment of a peptide exhibiting beta-galactosidase activity, which retains some enzymatic activity. Reference herein to "amino acid sequence" to refer to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms is not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the peptide molecule.

Exemplary peptides of the invention also include fragments of an enzyme or the full length of an enzyme that are at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or more residues in length. Thus, a "peptide fragment" or "enzymatically active fragment" of the present invention is a fragment that retains at least some functional enzymatic activity. Typically, the peptide fragments of the invention will still contain the functional catalytic domain or other necessary active site of the peptide exhibiting β -galactosidase activity. Other domains may be deleted.

The specific activity of beta-galactosidase will generally be measured and expressed as micromoles of glucose formed per minute per milligram of enzyme used. However, this ratio will vary depending on the conditions applied, such as temperature and pH. Thus, the value of β -galactosidase activity may also be referred to relative to a reference known enzyme, such as β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO: 35.

The term "sequence identity" as used herein with respect to amino acids means calculated as (n), unless otherwise indicated_ref-n_dif)·100/n_refIn which n is_difIs the total number of residues in the two sequences that are not identical when aligned, wherein n_refIs the number of residues in one of the sequences.

In some embodiments, sequence identity is determined by conventional methods, such as the similarity search method of Smith and Waterman,1981, adv.Appl.Math.2:482, Pearson & Lipman,1988, Proc.Natl.Acad.Sci.USA 85:2444, using the CLUSTAL W algorithm of Thompson et al, 1994, Nucleic Acids Res 22:467380, which are performed by Computer (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package of Genetics Computer Group). The BLAST algorithm (Altschul et al, 1990, mol. biol.215:403-10), for which the software is available through the national center for Biotechnology information www.ncbi.nlm.nih.gov/can also be used. When using any of the above algorithms, default parameters for "window" length, gap penalty, etc. will be used.

A peptide having a particular amino acid sequence described herein may differ from a reference peptide sequence by any one of amino acid substitution, addition/insertion, or deletion.

Some embodiments of the invention relate to the use of peptides having the amino acid sequences shown in SEQ ID NO 1-33 or sequences having at least 80% sequence identity to any of said sequences. In some embodiments, such sequence identity may be at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, such as a peptide having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid substitutions, additions, or deletions compared to any of the reference amino acid sequences set forth in SEQ ID NOs 1-33. The invention also features biologically active fragments of the peptides of the invention. Biologically active fragments of a peptide of the invention include peptides comprising an amino acid sequence substantially identical to or derived from the amino acid sequence of a peptide of the invention, which comprise fewer amino acids than a full-length protein, but exhibit the biological activity of a substantial portion of the corresponding full-length peptide. Typically, the biologically active fragment comprises a domain or motif having at least one activity of the variant protein of the invention. Biologically active fragments of the peptides of the invention may be, for example, peptides of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

As used herein, the term "host cell" includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding a peptide of the present invention. The host cell may be the cell type from which the particular enzyme is derived, or it may be an alternative cell type that is sensitive to the production of the particular enzyme. The term includes wild-type and attenuated strains.

Suitable host cells may be any bacteria, including lactic acid bacteria in the order Lactobacillales, including Lactococcus species (Lactococcus spp.), Streptococcus species (Streptococcus spp.), Lactobacillus species (Lactobacillus spp.), Leuconostoc species (Leuconostoc spp.), Pseudoleuconostoc species (Pseudoleuconostoc spp.), Pediococcus species (Pediococcus spp.), Brevibacterium species (Brevibacterium spp.), Enterococcus species (Enterococcus spp.), and Propionibacterium species (Propionibacterium spp.). Also included are lactic acid producing bacteria belonging to the anaerobic bacterium Bifidobacterium species (Bifidobacterium spp.) which are often used as food cultures alone or in combination with lactic acid bacteria. The definition also includes Lactococcus lactis (Lactobacillus lactis), Lactococcus lactis subsp (Lactococcus lactis subsp. cremoris), Leuconostoc lactis (Leuconostoc mesenteroides sp. cremoris), Leuconostoc Pseudoleuconostoc lactis (Leuconostoc mesenteroides sp. cremoris), Lactococcus lactis (Lactococcus lactis subsp. lactis. diacetyl), Lactobacillus casei (Lactobacillus casei subsp. lactis), and Lactobacillus paracasei (Lactobacillus paracasei. paracasei), including for example Lactobacillus thermophilus (Lactobacillus paracasei), Lactobacillus paracasei (Lactobacillus paracasei), Lactobacillus acidophilus (Lactobacillus paracasei). Other specific bacteria within this definition include bacteria of the family Bifidobacterium (Bifidobacterium), such as from the genus Bifidobacterium (Bifidobacterium), such as from Bifidobacterium animalis (Bifidobacterium animalis) or Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium catenulatum (Bifidobacterium catenulatum), Bifidobacterium infantis (Bifidobacterium infantis), or strains from the genus Lactobacillus (Lactobacillus), such as Lactobacillus sake, Lactobacillus acidophilus, Lactobacillus lactis, and Lactobacillus helveticus.

This definition of host cell also includes the following strains: mushrooms (Agaricus), e.g., Agaricus bisporus (a. bisporus); ascovaginospora; aspergillus (Aspergillus), such as Aspergillus niger (a. niger), Aspergillus awamori (a.awamori), Aspergillus foetidus (a.foetidus), Aspergillus japonicus (a.japonica), Aspergillus oryzae (a.oryzae); candida (Candida); chaetomium (Chaetomium); chaetotomastia; dictyostelium (dictyyostelium), such as Dictyostelium discodermatum (d.discoidea); kluyveromyces (Kluveromyces), such as kluyveromyces fragilis (k.fragilis), kluyveromyces lactis (k.lactis); mucor (Mucor), such as Mucor javanicus (m.javanicus), Mucor magnus (m.mucedo), Mucor mucedo (m.subtilissimas); neurospora (Neurospora), such as Neurospora crassa (n.crassa); rhizomucor (Rhizomucor), such as Rhizomucor pusillus (r.); rhizopus (Rhizopus), such as Rhizopus arrhizus (r), Rhizopus japonicus (r), Rhizopus stolonifer (r); sclerotinia (Sclerotinia), such as Sclerotinia glycines (s.libertiana); genus Torula (Torula); torulopsis (Torulopsis); trichophyton (Trichophyton), such as Trichophyton rubrum (Trichophyton rubrum); sclerotinia sclerotiorum (Whetzelinia), for example, sclerotinia sclerotiorum (w.sclerotiorum); bacillus (Bacillus), such as Bacillus coagulans (b.coengians), Bacillus circulans (b.circulans), Bacillus megaterium (b.megaterium), Bacillus fallow (b.novalis), Bacillus subtilis (b.subtilis), Bacillus pumilus (b.pumilus), Bacillus stearothermophilus (b.stearothermophilus), Bacillus thuringiensis (b.thuringiensis); bifidobacterium (bifidobacterium), such as bifidobacterium longum (b.longum), bifidobacterium bifidum (b.bifidum), bifidobacterium animalis (b.animalis); chryseobacterium (Chryseobacterium); citrobacter (Citrobacter), such as Citrobacter freundii (c.freundii); clostridium (Clostridium), such as Clostridium perfringens (c.perfringens); chromodiplospora (Diplodia), such as chromodiplospora gossypii (d.gossypina); enterobacter (Enterobacter), such as Enterobacter aerogenes (e.aerogenes), Enterobacter cloacae (e.cloacae); edwardsiella (edwards iella), Edwardsiella tarda (e.tarda); erwinia (Erwinia), such as Erwinia herbicola (e.herbicola); escherichia (Escherichia), such as Escherichia coli (e.coli); klebsiella (Klebsiella), such as Klebsiella pneumoniae (k.pneumoniae); miriococcus; myrothecium; mucor (Mucor); neurospora species (Neurospora), such as Neurospora crassa (n.crassa); proteobacteria (Proteus), such as proteobacteria vulgaris (p. vulgaris); providencia (Providencia), such as Providencia stuartii (p.stuartii); pycnoporus (Pycnoporus), such as Pycnoporus cinnabarinus (Pycnoporus cinnabrinus), Pycnoporus sanguineus (Pycnoporus sanguineus); ruminococcus (Ruminococcus), such as Ruminococcus streptococci (r.torques); salmonella (Salmonella), such as Salmonella typhimurium (s.typhimurium); serratia (Serratia), such as Serratia liquefaciens (s.liquefasciens), Serratia marcescens (s.marcocens); shigella (Shigella), e.g., Shigella flexneri(s); streptomyces species, such as Streptomyces antibioticus (s.antibioticus), Streptomyces castanea (s.canthaloglobiosporus), Streptomyces violaceus (s.violecorber); trametes (Trametes); trichoderma (Trichoderma), e.g., Trichoderma reesei (t.reesei), Trichoderma viride (t.viride); yersinia (Yersinia), for example Yersinia enterocolitica (y. enterocolitica).

Detailed description of the invention

As described above, the present invention relates to a peptide exhibiting β -galactosidase activity, which has an amino acid sequence represented by SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid substitutions, additions, or deletions.

Thus, in one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO. 1, or an amino acid sequence of the aforementioned sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 2, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 3, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ id No. 4, or an amino acid sequence of the above sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 5, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 6, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 7, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 8, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 9, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 10, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 11, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 12, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 13, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 14, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 15, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 16, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 17, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 18, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 19, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 20, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 21, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 22, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO. 23, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 24, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO. 25, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 26, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 27, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 28, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 29, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID No. 30, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO 31, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO:32, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions. In one embodiment, the invention relates to a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO. 33, or an amino acid sequence of the above sequence having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 8 described herein, at a temperature of about 4 ℃ and a pH of 6.7.

In some embodiments, the peptides of the invention have a beta-galactosidase activity, measured in micromoles (μmol) per mg of enzyme per minute of glucose formed at a temperature of about 4 ℃ and a pH of 6.7, which exceeds the beta-galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at μ Μ of glucose formed per mg of enzyme per minsec at a temperature of about 4 ℃ and pH6.7 that is higher than about 2 μ Μ glucose formed per μ Μ of enzyme per second, such as higher than about 4 μ Μ, such as higher than about 6 μ Μ, such as higher than about 8 μ Μ, such as higher than about 10 μ Μ, such as higher than about 12 μ Μ, such as higher than about 14 μ Μ, such as higher than about 16 μ Μ.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per mg of enzyme per min sec, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 10 described herein, at a temperature of about 4 ℃ and a pH of 5.5.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at a temperature of about 4 ℃ and at pH5.5 at μ M of glucose formed per μ M of enzyme per second, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 4 ℃ and pH5.5 at μ M of glucose formed per μ M enzyme per second which is higher than about 1 μ M glucose formed per μ M enzyme per second, such as higher than about 2 μ M, such as higher than about 3 μ M, such as higher than about 4 μ M, such as higher than about 5 μ M, such as higher than about 6 μ M, such as higher than about 7 μ M, such as higher than about 8 μ M.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, which exceeds the activity of β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 13 described herein, at a temperature of about 4 ℃ and a pH of 4.5.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at a temperature of about 4 ℃ and pH4.5 at μ M of glucose formed per μ M enzyme per second, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 4 ℃ and pH4.5 at μ M of glucose formed per μ M enzyme per second which is higher than about 0.5 μ M glucose formed per μ M enzyme per second, such as higher than about 1.0 μ M, such as higher than about 1.5 μ M, such as higher than about 2.0 μ M, such as higher than about 2.5 μ M, such as higher than about 3.0 μ M, such as higher than about 3.5 μ M, such as higher than about 4.0 μ M.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ M per second of glucose formed at a temperature of about 43 ℃ and pH6.7 under the conditions given in example 9 described herein, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at a temperature of about 43 ℃ and at pH6.7 at μ M of glucose formed per μ M enzyme per second, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 43 ℃ and pH6.7 at μ M of glucose formed per μ M enzyme per second which is higher than about 10 μ M glucose formed per μ M enzyme per second, such as higher than about 20 μ M, such as higher than about 40 μ M, such as higher than about 60 μ M, such as higher than about 80 μ M, such as higher than about 100 μ M, such as higher than about 120 μ M, such as higher than about 140 μ M, such as higher than about 160 μ M. In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 12 described herein, at a temperature of about 43 ℃ and a pH of 5.5.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ M of glucose formed per μ M of enzyme per second, at a temperature of about 43 ℃ and pH5.5, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 43 ℃ and pH5.5 at μ M of glucose formed per μ M enzyme per second which is higher than about 5 μ M glucose formed per μ M enzyme per second, such as higher than about 10 μ M, such as higher than about 15 μ M, such as higher than about 20 μ M, such as higher than about 25 μ M, such as higher than about 30 μ M, such as higher than about 35 μ M, such as higher than about 40 μ M, such as higher than about 45 μ M, such as higher than about 50 μ M, such as higher than about 55 μ M, such as higher than about 60 μ M.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 15 described herein, at a temperature of about 43 ℃ and at pH 4.5.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at a temperature of about 43 ℃ and pH4.5 at μ M of glucose formed per μ M enzyme per second, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 43 ℃ and pH4.5 at μ M of glucose formed per μ M enzyme per second which is higher than about 1 μ M glucose formed per μ M enzyme per second, such as higher than about 2 μ M, such as higher than about 3 μ M, such as higher than about 4 μ M, such as higher than about 5 μ M, such as higher than about 6 μ M, such as higher than about 7 μ M, such as higher than about 8 μ M, such as higher than about 9 μ M, such as higher than about 10 μ M, such as higher than about 11 μ M, such as higher than about 12 μ M.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, which exceeds the activity of β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 6 described herein, at a temperature of about 37 ℃ and a pH of 6.7.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at a temperature of about 37 ℃ and at pH6.7 at μ M of glucose formed per μ M enzyme per second, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 37 ℃ and pH6.7 at μ M of glucose formed per μ M enzyme per second which is higher than about 10 μ M glucose formed per μ M enzyme per second, such as higher than about 20 μ M, such as higher than about 30 μ M, such as higher than about 40 μ M, such as higher than about 50 μ M, such as higher than about 60 μ M, such as higher than about 70 μ M, such as higher than about 80 μ M, such as higher than about 90 μ M, such as higher than about 100 μ M, such as higher than about 110 μ M, such as higher than about 120 μ M, such as higher than about 130 μ M.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 11 described herein, at a temperature of about 37 ℃ and a pH of 5.5.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at a temperature of about 37 ℃ and at pH5.5 at μ M of glucose formed per μ M enzyme per second, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 37 ℃ and pH5.5 at μ M of glucose formed per μ M enzyme per second which is higher than about 5 μ M glucose formed per μ M enzyme per second, such as higher than about 10 μ M, such as higher than about 15 μ M, such as higher than about 20 μ M, such as higher than about 25 μ M, such as higher than about 30 μ M, such as higher than about 35 μ M, such as higher than about 40 μ M, such as higher than about 45 μ M, such as higher than about 50 μ M, such as higher than about 55 μ M, such as higher than about 60 μ M.

In some embodiments, the peptides of the invention have a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 14 described herein, at a temperature of about 37 ℃ and a pH of 4.5.

In some embodiments, the peptides of the invention have a measured beta-galactosidase activity at a temperature of about 37 ℃ and pH4.5 at μ M of glucose formed per μ M enzyme per second that exceeds the activity of beta-galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%. In some embodiments, the peptides of the invention have a β -galactosidase activity measured at a temperature of about 37 ℃ and pH4.5 at μ M of glucose formed per μ M enzyme per second which is higher than about 1 μ M glucose formed per μ M enzyme per second, such as higher than about 2 μ M, such as higher than about 3 μ M, such as higher than about 4 μ M, such as higher than about 5 μ M, such as higher than about 6 μ M, such as higher than about 7 μ M, such as higher than about 8 μ M, such as higher than about 9 μ M, such as higher than about 10 μ M, such as higher than about 11 μ M, such as higher than about 12 μ M, such as higher than about 13 μ M, such as higher than about 14 μ M, such as higher than about 15 μ M, such as higher than about 16 μ M, such as higher than about 17 μ M, such as higher than about 18 μ M.

In some embodiments, the peptide of the invention is derived from a bacterium of the genus bifidobacterium, such as bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium catenulatum, bifidobacterium longum, or from a bacterium of the genus lactobacillus, such as lactobacillus sake, lactobacillus amylovorus, lactobacillus delbrueckii subsp.

In some embodiments, the peptide according to the invention exhibits a galactose inhibition of less than 60%, such as less than 55%, such as less than 50%, such as less than about 45%, such as less than about 40%.

As mentioned above, part of the present invention relates to a method for producing a dairy product, the method comprising the steps of:

a) providing a milk-based matrix comprising lactose;

b) adding a peptide exhibiting beta-galactosidase activity and having the amino acid sequence shown in SEQ ID NOs 1-33 or a sequence having at least 80% sequence identity to any of said sequences to said lactose-containing milk-based matrix; and

c) treating said milk-based substrate with said peptide exhibiting beta-galactosidase activity.

In one aspect, these methods for producing a dairy product comprise the steps of:

a) providing a milk-based matrix comprising lactose;

b) adding a peptide exhibiting β -galactosidase activity and having the amino acid sequence shown in SEQ ID NOs 22, 33, 14, 7, 26 and 27, 30 and 1 (sequences G4, G16, G33, G40, G44, G66, G95, G158, G282 and G335) or having more than 85% amino acid sequence identity to any of these sequences to the lactose-containing milk-based matrix; and c) treating the milk-based substrate with the peptide exhibiting beta-galactosidase activity at a temperature of 50 ℃ to 140 ℃.

The process may be carried out in such a way as to reduce the concentration of lactose in the milk-based base to less than 0.2%, preferably less than 0.1%. In a preferred embodiment of this aspect, the method is performed such that the concentration of lactose in the milk-based matrix is less than 0.2% 3-30 minutes, preferably 4-20 minutes, most preferably 4-10 minutes after addition of the peptide exhibiting beta-galactosidase activity.

In one embodiment, the method uses a peptide exhibiting β -galactosidase activity having the amino acid sequence shown in SEQ ID NO:22, 33, 14 or 7 or having more than 85% amino acid sequence identity to any one of these sequences.

The method may utilize a low concentration of a peptide exhibiting beta-galactosidase activity and having the amino acid sequence shown in SEQ ID NO 22, 33, 14 or 7 or having more than 85% amino acid sequence identity to any of these sequences, e.g. at a concentration of 0.001-0.2mg/ml, preferably 0.002-0.04 mg/ml.

The incubation at high temperature is preferably limited to a short time. In a particularly preferred embodiment, the present invention provides a process as described above, wherein step c) is carried out at a temperature of 50 ℃ to 140 ℃ for a period of 4-20 minutes, followed by cooling and storing the dairy based product at a temperature of between 1 ℃ and room temperature, preferably between 1 ℃ and 6 ℃, until further use.

In some embodiments of the invention, such a peptide is derived from any one of the bacteria of the genus bifidobacterium, such as bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium catenulatum, bifidobacterium longum, or from the genus lactobacillus, such as lactobacillus sake, lactobacillus amylovorus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus delbrueckii subsp lactis, lactobacillus delbrueckii subsp india, lactobacillus crispatus, lactobacillus reuteri, lactobacillus helveticus, or from streptococcus thermophilus.

In some embodiments of the invention, step c) is carried out at a pH in the range of 3 to 10, such as in the range of 3 to 9, such as in the range of 3 to 8, such as in the range of 3 to 7, such as in the range of 3 to 6, such as in the range of 3 to 5, such as in the range of 3 to 4, such as in the range of 4 to 10, such as in the range of 4 to 9, such as in the range of 4 to 8, such as in the range of 4 to 7, such as in the range of 4 to 6, such as in the range of 4 to 5, such as in the range of 5 to 10, such as in the range of 5 to 9, such as in the range of 5 to 8, such as in the range of 5 to 7, such as in the range of 5 to 6, such as in the range of 6 to 10, for example in the range of 6-9, for example in the range of 6-8, for example in the range of 6-7.

In some embodiments of the invention, step c) or a portion of step c) is carried out at a temperature of no more than about 25 ℃, such as no more than about 20 ℃, such as no more than about 18 ℃, such as no more than about 16 ℃, such as no more than about 14 ℃, such as no more than about 12 ℃, such as no more than about 10 ℃, such as no more than about 8 ℃, such as no more than about 7 ℃, such as no more than about 6 ℃, such as no more than about 5 ℃, such as no more than about 4 ℃, such as no more than about 3 ℃, such as no more than about 2 ℃.

In some embodiments of the invention, the temperature at which step c) or a portion of step c) is carried out is at least about 25 ℃, such as at least about 30 ℃, such as at least about 35 ℃, such as at least about 40 ℃, such as at least about 45 ℃, such as at least about 50 ℃, such as at least about 55 ℃, such as at least about 60 ℃, such as at least about 65 ℃, such as at least about 70 ℃, such as at least about 75 ℃, such as at least about 80 ℃, such as at least about 85 ℃, such as at least about 90 ℃, such as at least about 95 ℃, such as at least about 100 ℃, such as at least about 110 ℃, such as at least about 120 ℃, such as at least about 130 ℃, such as at least about 135 ℃, such as at least about 140 ℃.

In some embodiments of the invention, the dairy product is selected from the group consisting of: lactose-free milk, low-lactose milk, yogurt, cheese, fermented dairy products, dietary supplements and probiotic dietary products.

In some embodiments of the invention, the milk-based substrate is selected from fresh or raw milk (obtained directly from the pasteurization step), milk obtained directly after the ultra-heat treatment (UHT) step or milk obtained directly after the fermentation step.

In some embodiments of the invention, the peptide used has a galactose inhibition of less than 60%, such as less than 55%, such as less than 50%, such as less than about 45%, such as less than about 40%.

In some embodiments of the invention, the dairy product is a fermented dairy product and said step b) is performed during or before fermentation.

In some embodiments of the invention, the process does not require the addition of additional enzymes after fermentation.

In some embodiments of the invention, the dairy product is a fermented dairy product and said step b) is performed immediately after fermentation.

In some embodiments of the invention, the milk product is fresh milk and said step b) is performed before, simultaneously with or immediately after the pasteurization step.

In some embodiments of the invention, the milk product is a Ultra Heat Treated (UHT) milk and said step b) is performed before, simultaneously with or immediately after the ultra heat treatment step.

In some embodiments of the invention, the temperature at which step c) is initiated is from 40 ℃ to 100 ℃, such as from 50 ℃ to 100 ℃, for example from 60 ℃ to 100 ℃, such as from 70 ℃ to 100 ℃, such as from 80 ℃ to 100 ℃, such as from 40 ℃ to 90 ℃, such as from 40 ℃ to 80 ℃, such as from 40 ℃ to 70 ℃, such as from 40 ℃ to 60 ℃, such as from 40 ℃ to 50 ℃.

In some embodiments of the invention, the ratio of lactase to transgalactosylase activity of the peptides is greater than 1:1 when hydrolyzing lactose in a milk-based substrate.

In some embodiments of the invention, when step c) is completed, less than 80% of the lactose has been hydrolyzed, and wherein more than 90% of the lactose has been hydrolyzed after one week.

Numbered embodiments

1. A peptide exhibiting β -galactosidase activity, said peptide having the amino acid sequence of the amino acid sequence shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or an enzymatically active fragment thereof, or an amino acid sequence of any of the above sequences having NO more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid substitutions, additions or deletions.

2. A dimeric peptide exhibiting beta-galactosidase activity, said dimeric peptide consisting of two peptides having the amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or an enzymatically active fragment thereof, or an amino acid sequence of any of the above sequences having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acid substitutions, additions, or deletions.

3. The peptide or dimeric peptide according to embodiment 1 or 2, which has a β -galactosidase activity, measured at μ Μ of glucose formed per μ Μ enzyme per second, which exceeds the activity of β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%, under the conditions given in example 8 described herein, at a temperature of about 4 ℃ and pH 6.7.

4. A peptide or dimeric peptide according to any one of embodiments 1-3, having a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme, at a temperature of about 4 ℃ and pH5.5 under the conditions given in example 10 described herein, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

5. The peptide or dimeric peptide according to any one of embodiments 1-4, having a β -galactosidase activity, measured at μ Μ of glucose formed per μ Μ enzyme per second, at a temperature of about 4 ℃ and pH4.5 under the conditions given in example 13 described herein, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

6. The peptide or dimeric peptide according to any one of embodiments 1-5, having a β -galactosidase activity, measured at μ Μ of glucose formed per μ Μ enzyme per second, at a temperature of about 43 ℃ and pH6.7 under the conditions given in example 9 described herein, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

7. The peptide or dimeric peptide according to any one of embodiments 1-6, having a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme at a temperature of about 43 ℃ and pH5.5 under the conditions given in example 12 described herein, which exceeds the activity of β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

8. The peptide or dimeric peptide according to any one of embodiments 1-7, having a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme at a temperature of about 43 ℃ and pH4.5 under the conditions given in example 15 described herein, which exceeds the activity of β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

9. The peptide or dimeric peptide according to any one of embodiments 1-8, having a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme at a temperature of about 37 ℃ and pH6.7 under the conditions given in example 6 described herein, which exceeds the activity of β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

10. The peptide or dimeric peptide according to any one of embodiments 1-9, having a β -galactosidase activity, measured at μ Μ of glucose formed per second per μ Μ enzyme at a temperature of about 37 ℃ and pH5.5 under the conditions given in example 11 described herein, which exceeds the activity of β -galactosidase defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

11. The peptide or dimeric peptide according to any one of embodiments 1-10, having a β -galactosidase activity, measured at μ Μ of glucose formed per μ Μ enzyme per second, at a temperature of about 37 ℃ and pH4.5 under the conditions given in example 14 described herein, which exceeds the β -galactosidase activity defined by SEQ ID NO:34 or SEQ ID NO:35 by at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 100%, such as at least about 200%, such as at least about 300%, such as at least about 400%, such as at least about 500%.

12. The peptide or dimeric peptide according to any one of embodiments 1 to 11, which is derived from a bacterium of the genus bifidobacterium, such as bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium catenulatum, bifidobacterium longum, or from a lactobacillus, such as lactobacillus sake, lactobacillus amylovorus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus delbrueckii subsp lactis, lactobacillus delbrueckii subsp indiae, lactobacillus crispatus, lactobacillus reuteri, lactobacillus helveticus, or from streptococcus thermophilus.

13. The peptide or dimeric peptide according to any one of embodiments 1-12, wherein the peptide or dimeric peptide exhibits galactose inhibition of less than 60%, such as less than 55%, such as less than 50%, such as less than about 45%, such as less than about 40%.

14. A nucleotide sequence encoding a peptide or dimeric peptide as defined in any one of embodiments 1 to 13.

15. A host cell comprising a nucleotide sequence as defined in embodiment 14.

16. A method for producing a peptide or dimeric peptide as defined in any one of embodiments 1 to 13, said method comprising expressing a vector comprising a nucleotide sequence as defined in embodiment 14 in a suitable host cell; and purifying the peptide or dimeric peptide from the expression product of the host cell.

17. A method of reducing the lactose content of a lactose-containing composition, such as a dairy product, comprising the step of contacting the composition at a pH of 3-10 and a temperature of 0 ℃ -140 ℃ with a peptide exhibiting β -galactosidase activity, or a dimeric peptide having the amino acid sequence shown in SEQ ID NOs 1-33, or the composition of two peptides having the amino acid sequence shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27, or 28 and 29; or an enzymatically active fragment thereof, or any sequence having at least 80% sequence identity to any one of said sequences or enzymatically active fragments; or with a host cell expressing either of the peptide or dimeric peptide.

18. The method of embodiment 17, wherein the composition is a dairy product selected from the group consisting of lactose-free milk, low lactose milk, yogurt, cheese, fermented dairy products, dietary supplements, and probiotic dietary products.

19. The method according to any one of embodiments 17-18, wherein the host cell is any one of the bacteria selected from the genus bifidobacterium, such as bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium catenulatum, bifidobacterium longum, or any one of the genera lactobacillus, such as lactobacillus sake, lactobacillus amylovorus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus delbrueckii subsp lactis, lactobacillus delbrueckii subsp indiae, lactobacillus crispatus, lactobacillus reuteri, lactobacillus helveticus, or is selected from streptococcus thermophilus.

20. The method according to any one of embodiments 17-19, wherein the lactose concentration is reduced to less than about 1%, such as to less than about 0.1% or less, such as to less than about 0.01%.

21. A peptide exhibiting beta-galactosidase activity, said peptide having an amino acid sequence shown in SEQ ID NOs 1-33, or said dimeric peptide consisting of two peptides having amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences; or a host cell expressing either of said peptides or dimeric peptides, for use in the production of a dairy product with a reduced lactose content.

22. The use according to embodiment 21, wherein the dairy product is selected from the group consisting of lactose-free milk, low lactose milk, yoghurt, cheese, fermented dairy products, dietary supplements and probiotic dietary products.

23. The use according to any one of embodiments 21-22, wherein the host cell is any one selected from the group consisting of: bacteria of the genus Bifidobacterium, such as Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium longum, or Lactobacillus, such as Lactobacillus sake, Lactobacillus amylovorus, Lactobacillus delbrueckii subsp.

24. A method of producing a dairy product, the method comprising the steps of:

a) providing a milk-based matrix comprising lactose;

b) adding to the lactose-containing milk-based matrix a peptide exhibiting beta-galactosidase activity, the peptide having an amino acid sequence shown in SEQ ID NO 1-33; or the dimeric peptides consist of two peptides having the amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences; and

c) treating the milk-based substrate with the peptide or dimeric peptide exhibiting beta-galactosidase activity.

25. The method according to embodiment 24, wherein the peptide or dimeric peptide is derived from any one of the bacteria of the genus bifidobacterium, such as bifidobacterium adolescentis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium catenulatum, bifidobacterium longum, or from the genus lactobacillus, such as lactobacillus sake, lactobacillus amylovorus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus delbrueckii subsp lactis, lactobacillus delbrueckii subsp indinavis, lactobacillus crispatus, lactobacillus reuteri, lactobacillus helveticus, or from streptococcus thermophilus.

26. The process according to any of embodiments 24 to 25, wherein step c) is carried out at a pH in the range of from 3 to 10, such as in the range of from 3 to 9, such as in the range of from 3 to 8, such as in the range of from 3 to 7, such as in the range of from 3 to 6, such as in the range of from 3 to 5, such as in the range of from 3 to 4, such as in the range of from 4 to 10, such as in the range of from 4 to 9, such as in the range of from 4 to 8, such as in the range of from 4 to 7, such as in the range of from 4 to 6, such as in the range of from 4 to 5, such as in the range of from 5 to 10, such as in the range of from 5 to 9, such as in the range of from 5 to 8, such as in the range of from 5 to 7, such as in the range of from 5 to 6, for example in the range of 6-10, for example in the range of 6-9, for example in the range of 6-8, for example in the range of 6-7.

27. The method of any one of embodiments 24-26, wherein step c) or a portion of step c) is carried out at a temperature of no more than about 25 ℃, such as no more than about 20 ℃, such as no more than about 18 ℃, such as no more than about 16 ℃, such as no more than about 14 ℃, such as no more than about 12 ℃, such as no more than about 10 ℃, such as no more than about 8 ℃, such as no more than about 7 ℃, such as no more than about 6 ℃, such as no more than about 5 ℃, such as no more than about 4 ℃, such as no more than about 3 ℃, such as no more than about 2 ℃.

28. The method according to any one of embodiments 24-27, wherein step c) or a portion of step c) is carried out at a temperature of at least about 25 ℃, such as at least about 30 ℃, such as at least about 35 ℃, such as at least about 40 ℃, such as at least about 45 ℃, such as at least about 50 ℃, such as at least about 55 ℃, such as at least about 60 ℃, such as at least about 65 ℃, such as at least about 70 ℃, such as at least about 75 ℃, such as at least about 80 ℃, such as at least about 85 ℃, such as at least about 90 ℃, such as at least about 95 ℃, such as at least about 100 ℃, such as at least about 110 ℃, such as at least about 120 ℃, such as at least about 130 ℃, such as at least about 135 ℃, such as at least about 140 ℃.

29. The method of any of embodiments 24-28, wherein the dairy product is selected from the group consisting of: lactose-free milk, low-lactose milk, yogurt, cheese, fermented dairy products, dietary supplements and probiotic dietary products.

30. The method according to any one of embodiments 24-29, wherein the milk-based substrate is selected from fresh or raw milk (obtained directly from a pasteurization step), milk obtained directly after an ultra-heat treatment (UHT) step or milk obtained directly after a fermentation step.

31. The method according to any one of embodiments 24-30, wherein the peptide or dimeric peptide has a galactose inhibition of less than 60%, such as less than 55%, such as less than 50%, such as less than about 45%, such as less than about 40%.

32. The method according to any one of embodiments 24-31, wherein the dairy product is a fermented dairy product and said step b) is performed during or before fermentation.

33. The method of embodiment 32, which does not require the addition of additional enzymes after fermentation.

34. The method according to any one of embodiments 24-31, wherein the dairy product is a fermented dairy product and said step b) is performed immediately after fermentation.

35. The method according to any one of embodiments 24-31, wherein the milk product is fresh milk and the step b) is performed before, simultaneously with or immediately after the pasteurization step.

36. The process according to any of embodiments 24-31, wherein the milk product is a ultra-heat treated (UHT) milk and step b) is performed before, simultaneously with or immediately after the ultra-heat step treatment.

37. The method according to any one of embodiments 24-36, wherein the temperature at which step c) is initiated is from 40 ℃ to 100 ℃, such as from 50 ℃ to 100 ℃, such as from 60 ℃ to 100 ℃, such as from 70 ℃ to 100 ℃, such as from 80 ℃ to 100 ℃, such as from 40 ℃ to 90 ℃, such as from 40 ℃ to 80 ℃, such as from 40 ℃ to 70 ℃, such as from 40 ℃ to 60 ℃, such as from 40 ℃ to 50 ℃.

38. The method according to any one of embodiments 24-37, wherein the ratio of lactase to transgalactosylase activity of the peptide or dimeric peptide is greater than 1:1 when hydrolyzing lactose in a milk-based substrate.

39. The method according to any one of embodiments 24-38, wherein when step c) is completed, less than 80% of the lactose has been hydrolyzed, and wherein more than 90% of the lactose has been hydrolyzed after one week.

40. A dairy product prepared by the method defined in any one of embodiments 24-39.

41. Food product, such as a dairy product, comprising a peptide exhibiting beta-galactosidase activity, said peptide having an amino acid sequence shown in SEQ ID NOs 1-33, or said dimeric peptide consisting of two peptides having amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences.

42. Food product, such as a dairy product, comprising a host cell expressing a peptide exhibiting beta-galactosidase activity, said peptide having the amino acid sequence shown in SEQ ID NOs 1-33, or said dimeric peptide consisting of two peptides having the amino acid sequences shown in SEQ ID NOs 2 and 3, 5 and 6, 20 and 21, 23 and 24, 26 and 27 or 28 and 29; or a sequence having at least 80% sequence identity to any one of said sequences.

43. The food product according to embodiment 42, selected from the group consisting of a beverage, a baby food, a cereal, a bread, a biscuit, a candy, a cake, a food supplement, a dietary supplement, a probiotic comestible product, a prebiotic comestible product, an animal feed, a poultry feed and a medicament, or a dairy product selected from the group consisting of lactose-free milk, low lactose milk, milk powder, baby milk, yogurt, ice cream, cheese, fermented dairy products, dietary supplements and probiotic dietary products.

TABLE 1 Gene number and corresponding sequence identification number.