Use of glycosidase in preparation of a milk product

Abstract

A method for making a milk product (e.g. a yogurt) comprising adding an effective amount of an N-linked glycosidase and/or an O-linked glycosidase to milk.

Claims

1. A method for producing a fermented milk yogurt product comprising: (a) treating a milk substrate with an enzyme having O-linked glycosidase activity, and (b) fermenting the milk substrate with lactic acid bacteria, wherein the fermented milk yogurt product that has increased gel firmness relative to a fermented milk yogurt product produced with a comparable method but without the enzyme having O-linked glycosidase activity, wherein treating the milk substrate with the enzyme having O-linked glycosidase activity does not increase syneresis in the fermented milk product relative to a fermented milk yogurt product produced with a comparable method but without the enzyme having O-linked glycosidase activity, and wherein the enzyme having O-linked glycosidase activity is selected from -N-acetyl-galactosaminidase (EC number: 3.2.1.49); -galactosidase (EC number: 3.2.1.22); neuraminidase (EC number: 3.2.1.18); and combinations thereof.

2. The method of claim 1, wherein step (a) is performed before or during step (b).

3. The method of claim 1, wherein the milk substrate is selected from the group consisting of milk from animals and milk of plant origin.

4. The method of claim 3, wherein the milk from animals is from cows, sheep, ewes, goats, buffaloes, or camels.

5. The method of claim 3, wherein the milk of plant origin is soy milk, oak milk, rice milk, or almond milk.

6. The method of claim 1, wherein the the lactic acid bacteria belongs to a species selected from the group consisting of: Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, Bifidobacterium bifidum, and Bifidobacterium longum.

7. The method of claim 1, wherein 10.sup.5 to 10.sup.13 CFU/g of the lactic acid bacteria are added to the milk substrate.

8. A yogurt product obtained by the method of claim 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Dairy Animal Milk Product

(2) Generally speakingthe pH of a herein preferred milk product, including a dairy animal milk product (i.e. a product based on animal milk), is a pH from pH 3 to pH 6.5more preferably from pH 3.5 to pH 5.75.

(3) As know to the skilled personone may get the relevant pH of a milk product by e.g. fermenting with a suitable lactic acid bacteria culture.

(4) However, as known to the skilled person one may simply add a suitable acid (such as lactic acid) to get the required pH.

(5) Alternative one may add a lactone (e.g. GDL lactone) to get the required pH or use other suitable known methods (e.g. enzymatic methods or pressureatiation with carbon dioxide) to get the required pH.

(6) As discussed abovethe use of a glycosidase to improve gel firmness as described herein may be particular useful in relation to so-called low fat milk products.

(7) Accordingly, in a preferred embodiment the milk substrate used in step (i) of the method of the first aspect is a milk substrate with a low fat contenti.e. with a fat content of less than 3.5% fat, more preferably with a fat content of less than 1.5% and even more preferably with a fat content of less than 0.75%.

(8) As discussed abovean advantage of the use of a glycosidase to improve gel firmness as described may be that one does not need to increase the protein content of e.g. a low fat milk product in order to get sufficient adequate gel firmness and thereby further minimizing the total calorific/energy content of the final low fat milk product (e.g. a low fat yogurt).

(9) Accordingly, in a preferred embodiment the final milk product (e.g. yogurt) has a total calorific/energy content of less than 150 kilo calories per 100 g of milk product, more preferably the final milk product (e.g. yogurt) has a total calorific/energy content of less than 100 kilo calories per 100 g of the milk product.

(10) As knownit is routine work for the skilled person to determine the calories content of a milk product of interest.

(11) A suitable example of a milk product is a fermented milk product or a cheese.

(12) In a preferred embodiment, the milk product is a fermented milk product.

(13) In a preferred embodimentthe fermented milk product is at least one fermented milk product selected from the group consisting of: yogurt, alternate culture yogurt, butter milk, acidophilus milk, kefir, kumys and quark. Most preferably, fermented milk product is a yogurt.

(14) In the present contextthe terms yogurt and fermented milk have their usual meanings. In US2005/0095316A1 and US2005/0095317A1 (both Danone) are these terms defined in accordance with a relevant official decree/regulation in Francebelow is essentially referred to the same standard known definitions of these terms.

(15) As known to the skilled personto obtain a yogurt or fermented milk product it is in particular recalled that there must not be a significant elimination of milk serum and that there must be a heat treatment at least equivalent to pasteurization.

(16) A suitable relevant heat treatment for making a fermented milk product such as e.g. a yogurt is, for example, a heat treatment of from 85 to 98 C. for 15 seconds to 30 minutes.

(17) Because of the application of a heat treatment which is at least equivalent to standard pasteurization, milk serum proteins of the milk substrate are denaturated more or less (from 25 to 99% of them, approximately).

(18) As evident to a skilled personsince there for a yogurt or fermented milk product must not be a significant elimination of milk seruma yogurt or fermented milk product is not a cheese product as described in U.S. Pat. No. 7,560,127B2 (see above).

(19) The term fermented milk relates to dairy product prepared with skimmed or unskimmed milks or skimmed or unskimmed, concentrated or powdered milks, enriched or not enriched with milk constituents, which has been subjected to heat treatment at least equivalent to pasteurization, inoculated with microorganisms belonging to the species that is or are characteristic of each product.

(20) The amount of free lactic acid which they contain should preferably not be less than 0.6 gram per 100 grams at the time of sale to the consumer.

(21) Fermented milks should preferably be kept, up to the time of sale to the consumer, at a temperature capable of preventing them spoiling.

(22) The term yogurt denotes fermented milk obtained, according to fair and traditional practices, preferably by the development of specific thermophilic lactic acid bacteria only, such as e.g. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, which preferably should be inoculated simultaneously and preferably be live in the finished product, at a rate of preferably at least 10 million bacteria per gram expressed in relation to the milk-containing portion.

(23) A fermented milk product is normally obtained by

(24) (A): inoculating from 10.sup.5 to 10.sup.13 cfu/ml (preferably 10.sup.6 to 10.sup.11 cfu/ml) of lactic acid bacteria (LAB) culture to the animal milk substrate; and

(25) (B): fermenting the milk substrate from 2 to 120 hours at a temperature from 10 C. to 55 C.

(26) As known to the skilled personsuitable species of lactic acid bacteria include Bifidobacterium, Lactobacillus (such as Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus casei or Lactobacillus helveticus), Streptococcus (such as Streptococcus thermophilus), Lactococcus (such as Lactococcus lactis), Leuconostoc (such as Leuconostoc lactis, Leuconostoc mesenteroides).

(27) When the milk substrate is inoculated with a ferment made up of strains of Lactobacillus delbrueckii subsp. bulgaricus and of Streptococcus thermophilus, the product is generally understood to be a yogurt.

(28) As known in the artthe term alternate culture yogurt refers to a fermented milk product made by using cultures of Streptococcus thermophilus and any Lactobacillus species.

(29) As known in the artthe term acidophilus milk refers to a fermented milk product made by using culture of Lactobacillus acidophilus.

(30) As known in the artthe term Kefir refers to a fermented milk product made by using starter culture prepared from kefir grains, Lactobacillus kefiri, species of the genera Leuconostoc, Lactococcus and Acetobacter growing in a strong specific relationship. Kefir grains constitute both lactose fermenting yeasts (Kuyveromyces marxianus) and non-lactose-fermenting yeasts (Saccharomyces unisporus, Saccharomyces cerevisiae and Saccharomyces exiguus).

(31) As known in the artthe term Kumys refers to a fermented milk product made by using cultures of Lactobacillus delbrueckii subsp. bulgaricus and Kluyveromyces marxianus.

(32) In a preferred embodimentthe milk product is a yogurt, wherein the yogurt is made by inoculation with a yogurt lactic acid bacteria culture that comprises Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus capable of synthesizing extracellular polysaccharides (EPS)this preferred embodiment may be particular relevant if the yogurt is a low fat yogurt, i.e. wherein the milk substrate used in step (i) of the method of the first aspect is a milk substrate with a low fat contenti.e. with a fat content of less than 3.5% fat, more preferably with a fat content of less than 1.5% and even more preferably with a fat content of less than 0.75%.

(33) As discussed abovethe prior art describes a number of such strains of Streptococcus thermophilus that produce EPSsee e.g. WO2007/095958A1 (Chr. Hansen A/S).

(34) Accordingly, the skilled person can routinely identify a number of such EPS producing strains and he can also routine identify if a specific strain of interest is capable of synthesizing EPS or not.

(35) An example of a herein possible relevant theoretical business scenario could be that a company makes a milk concentrate/powder by use of a glycosidase as described herein and then sells this milk concentrate/powder to e.g. a yogurt producer that use this in their yogurt production to get a yogurt with improved/increased gel firmnessi.e. they may get the improved gel firmness without any extra addition as such of glycosidase during the yogurt production as such.

(36) As understood by the skilled personsuch a theoretical business scenario would be an example of a method within the scope of the method of the first aspect as discussed herein. The milk concentrate/powder may be seen as an example of a dairy animal milk product of the method of the first aspect. Further, as understood by the skilled person in the present contextthe final yogurt will have the improved/increased gel firmness due to the previous addition of the glycosidase to the milki.e. the yogurt producer will also perform actions within the scope of the method of the first aspect as discussed herein.

(37) Glycosidase

(38) As discussed abovethe term glycosidase (also called glycoside hydrolase) refers to an enzyme that catalyzes the hydrolysis of the glycosidic linkage/bonda glycosidic bond is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate.

(39) As described abovea glycosidase may herein also be termed a deglycosylation enzyme.

(40) The glycosidase may be a natural glycosidase or it may be a variant/mutated of a natural glycosidaseas known to the skilled person, one may make mutated variants of a enzyme of interest (here a glycosidase) to e.g. improve the stability of the enzyme while maintaining the key enzymatic activity (here glycosidase activity) of the enzyme.

(41) In order to e.g. get a minimum of unwanted syneresis (in particular if the milk product is a fermented milk product such as a yogurt)it may be preferred that the glycosidase is an N-linked glycosidase.

(42) As discussed above, the term N-linked glycosidase is a well defined term in the art and the skilled person knows if a specific glycosidase of interests is a N-linked glycosidase or not. Further the prior art describes a number of different herein suitable N-linked glycosidases.

(43) Examples of a herein suitable N-linked glycosidase may be at least one glycosidase selected from the group consisting of: Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase (EC number: 3.5.1.52; alternative names: N-Glycosidase-F or PNGase-F) and Endo--N-acetylglucosaminidase H (EC number: 3.2.1.96; alternative name ENDO-H).

(44) The immediately above described N-linked glycosidases may in the present context be described as glycosidases that have N-linked glycosidase activity and no herein significant O-linked glycosidase activity.

(45) Accordingly, it may herein be preferred that the N-linked glycosidase is an N-linked glycosidase that have no herein relevant O-linked glycosidase activity (such as no O-linked glycosidase activity).

(46) The N-Glycosidase-F, also known as PNGase-F, used in the process, is an asparagine amidase (EC 3.5.1.52) that may be derived from Flavobacterium mesingosepticum. It catalyses the complete and intact cleavage of N-linked oligosaccaharides from glycoproteins. It may be derived as a commercial product from New England Biolabs Inc. under the name PNGase-F or produced recombinantly in a strain like Escherichia coli as we have done using the plasmid and method described in working Example 1 herein.

(47) Endo--N-acetylglucosaminidase H (EC 3.2.1.96), also known as ENDO-H, may be derived from Streptomyces plicatus. ENDO-H catalyses the hydrolysis of the glycosidic bond between the two N-acetylglycosamines of N-linked glycosylations. It may be derived as a commercial product from New England Biolabs Inc. under the name ENDO-H.

(48) Examples of a herein suitable O-linked glycosidase may be at least one glycosidase selected from the group consisting of: -N-acetyl-galactosaminidase (EC number: 3.2.1.49; alternative name: GalNAC); -galactosidase (EC number: 3.2.1.22); and neuraminidase (EC number: 3.2.1.18).

(49) GalNAC is a highly specific exoglycosidase that catalyzes the hydrolysis of -linked D-N-acetyl-galactosamine residues from. It may be derived as a commercial product from New England Biolabs Inc.

(50) As discussed above, the term O-linked glycosidase is a well defined term in the art and the skilled person knows if a specific glycosidase of interests is a O-linked glycosidase or not. Further the prior art describes a number of different herein suitable O-linked glycosidases.

(51) The effective amount/activity of a glycosidase is herein determined according to the art.

(52) According to the artfor a N-linked glycosidase (such as e.g. PNGase-F and Endo-H) one activity unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10 g of denatured RNase-B in 1 hour at 37 C. in a total reaction volume of 10 l.

(53) For GalNAC (an O-linked glycosidase) one activity unit is defined as the amount of enzyme required to cleave >95% of the terminal -D-N-acetyl-galactosamine from 1 nmol (GalNAc1-3)(Fuc1-2)Gal1-4Glc-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37 C. in a total reaction volume of 10 l.

(54) A number of herein relevant glycosidase enzymes are commercially available from the company New England Biolabsreference is also made to the product catalogue New England Biolabs (as e.g. available on-line on their web-page) for further details in relation to specific standard definitions of herein relevant glycosidase activity units.

(55) Step (i) of the Method of First Aspect

(56) Step (i) of the herein described method of the first aspect reads: (i): adding an effective amount of an N-linked glycosidase and/or an O-linked glycosidase to an animal milk substrate.

(57) As understood by the skilled person in the present contextthe N-linked glycosidase and/or an O-linked glycosidase is normally added to the animal milk substrate as a substantial pure glycosidase compositione.g. a glycosidase composition, wherein the glycosidase activity represents at least 5% of the total enzymatic activity of the glycosidase composition as such.

(58) It may be a glycosidase composition, wherein the glycosidase activity represents at least 25% of the total enzymatic activity of the glycosidase composition or a glycosidase composition, wherein the glycosidase activity represent at least 50% of the total enzymatic activity of the glycosidase composition.

(59) Many times if would be preferred that such a substantial pure glycosidase composition is a glycosidase composition, wherein the glycosidase activity represent at least 90% of the total enzymatic activity of the glycosidase composition.

(60) An effective amount of a glycosidase may be one specific type of a glycosidase (e.g. PNGase-F) or be a mixture of herein relevant glycosidase enzymes (e.g. two different N-linked glycosidases or one N-linked and one O-linked glycosidase).

(61) When there herein is said that it may be preferred that the glycosidase added in step (i) of the method of the first aspect is a N-linked glycosidase it of course means that there must be added an effective amount of a N-linked glycosidase in step (i) and this N-linked glycosidase givesas a result of its presenceimproved gel firmness to the dairy milk product.

(62) However, when said that N-linked glycosidase is preferred it does of course not mean that there must not be added any O-linked glycosidase in step (i).

(63) The same applies when herein is said that O-linked glycosidase is preferredi.e. here there must be added O-linked glycosidase in step (i)but there may also be added N-linked glycosidase.

(64) To the contrary and as evident to the skilled personwhen there herein is said that the glycosidase is only an O-linked glycosidase then there must not be added N-linked glycosidase in step (i) of the first aspect. The same applies when herein is said the glycosidase is only an N-linked glycosidase then there must not be added O-linked glycosidase in step (i) of the first aspect.

(65) As known in the arta dairy milk product is generally given a heat treatment. As known in the artheat treatment typically uses temperatures below boiling since at very high temperatures, casein micelles will irreversibly aggregate (or curdle).

(66) In the present contextthe term pasteurized in relation to a pasteurized dairy animal milk product refers to a standard pasteurization step (i.e. involving a suitable heat treatment of the milk). In the present contextit is evident that the skilled person knows if a specific milk product of interest is a pasteurized dairy animal milk product or not.

(67) As understood by the skilled persona standard pasteurization step may be heat treatment of around 71-72 C. for around 15-20 seconds

(68) As discussed aboveto obtain a yogurt or fermented milk product it is in particular recalled that there must not be a significant elimination of milk serum and that there must be a heat treatment at least equivalent to pasteurization.

(69) A suitable relevant heat treatment for making a fermented milk product such as e.g. a yogurt is, for example, a heat treatment of from 85 to 98 C. for 15 seconds to 30 minutes.

(70) Depending on the type of heat treatment usedthe heat treatment may e.g. be a heat treatment of the milk by using a temperature from 65 to 150 C. for a fraction of a second to 30 minutes.

(71) As discussed abovewhen the milk product is a fermented milk product such as e.g. a yogurt there must be a heat treatment at least equivalent to pasteurization.

(72) The glycosidase may in step (i) be added before or after the heat treatment stepi.e. the heat treatment of the milk by using a temperature from 65 to 150 C. for a fraction of a second to 30 minutes.

(73) In some cases it may be preferred that that glycosidase is added to already heat treated milk. As discussed abovewhen the milk product is a fermented milk product the milk substrate is inoculated and fermented with a relevant lactic acid bacteria (LAB) culture.

(74) When the milk product is a fermented milk productthe glycosidase may be added before, together or after the inoculation of the milk substrate with the lactic acid bacteria (LAB) culture.

(75) In relation to milk product in generalit may preferred that the glycosidase is added before the pH of the milk substrate gets below pH 6.

(76) In relation to a fermented milk productit is preferred that the glycosidase is added before the relevant lactic acid bacteria fermentation process has endedi.e. preferably before the pH of the milk substrate gets below pH 6.

(77) It is herein believed that addition of from 10 activity units per ml milk to 1000 activity units per ml milk of glycosidase is enough to get a herein relevant effective amount of a glycosidasei.e. enough to get a herein relevant improved gel firmness.

(78) If relevant for a specific purposeone may add more glycosidase e.g. up to 20000 activity units per ml milk of glycosidase.

(79) As discussed abovethe effective amount/activity of a glycosidase is herein determined according to the art.

(80) Step (ii) of the Method of First Aspect

(81) Step (ii) of the herein described method of the first aspect reads: (ii): performing adequate step(s) to get a dairy animal milk product, wherein the adequate step(s) is/are performed under conditions, wherein the effective amount of the glycosidase givesas a result of its presenceimproved gel firmness to the dairy milk product

(82) As discussed aboveperforming adequate step(s) to get a dairy animal milk product of interest is routine work for the skilled persone.g. if the milk product is e.g. a yogurt, the skilled person of course knows the adequate step(s) to get yogurt of interest.

(83) In relation to the conditions, wherein the effective amount of the glycosidase gives improved gel firmness to the dairy milk productit is evident that these conditions shall be conditions, wherein the glycosidase enzyme as such is active during a sufficient time period.

(84) As shown in the working Examples hereinthe present inventors have identified that a number of different glycosidase enzymes have a suitable activity during normal suitable conditions (e.g. temperature, pH etc) for making a herein relevant milk product such as a yogurt.

(85) In short and as understood by the skilled personthe preferred conditions to get preferred herein relevant glycosidase activity will depend on the specific glycosidase enzyme(s) used (e.g. PNGase-F) and the specific milk product to be made (e.g. a yogurt).

(86) In the present contextexamples of suitable reaction conditions for the glycosidase to give the herein relevant improved gel firmness could be: Temperature from 10 to 50 C. (such as from 20 to 40 C.); pH from pH 3 to pH 9 (such as from pH 4 to pH 7.5, from 3 to 7 or from 3 to 6.5); a time period from 10 minutes to 120 hours (such as from 1 hour to 120 hours, or from 0.5 to 5 hours)

(87) In a preferred embodiment, the presence of the glycosidase in step (ii) gives a 1.25 times improved gel firmness to the dairy milk product; more preferably a 1.50 times improved gel firmness to the dairy milk product and even more preferably the presence of the glycosidase in step (ii) gives a 1.7 times improved gel firmness to the dairy milk product.

(88) As discussed abovethe present inventors identified that use of the glycosidase does not negatively affect the viscosity of the final milk product.

(89) Accordingly, in a preferred embodimentstep (ii) is performed under conditions, wherein the effective amount of the glycosidase givesas a result of its presenceno negative effect on the viscosity to the dairy milk product.

(90) It is routine work for the skilled person to measure viscosity of a milk product of interest (e.g. a yogurt).

(91) In working Example 2 herein is provided a suitable standard method for measurement of viscosity of a milk product of interestpreferably the herein relevant viscosity is measured according to the method of this Example 2.

(92) The article of A. N. Hassan et al (J. Dairy Sci: 86:1632-1638; 2003) discussed above also describes a suitable standard method for measurement of viscosity.

(93) See e.g. the materials and method section and FIG. 1 of the article, where shear stress is determinedas can be seen in Example 2 herein, the parameter shear stress is used as a measure of viscosity.

(94) Since it is easy for the skilled person to measure viscosity of a milk product of interestit is of course also easy for the skilled person to determine the preferred embodiment of step (ii) of the method of the first aspect relating to if:

(95) step (ii) is performed under conditions, wherein the effective amount of the glycosidase givesas a result of its presenceno negative effect on the viscosity to the dairy milk product.

(96) In order to determine this requirementthe skilled person shall simply perform the adequate step(s) to get a milk product of step (ii) with and without presence of the effective amount of the glycosidaseand then determine the herein relevant viscosity effect of the presence of the added amount of glycosidase.

(97) As can be seen in the working Examples hereinone may actually get an improved viscosity by using a glycosidase as described herein.

(98) Accordingly, in a preferred embodiment, the presence of the glycosidase in step (ii) gives a 1.10 times increased viscosity to the dairy milk product; more preferably a 1.20 times increased viscosity to the dairy milk product.

(99) As understood by the skilled personthe method to measure viscosity as e.g. described in working Example 2 herein and the article of A. N. Hassan et al will (within relatively minor measurement uncertainties) give the same relative resultsi.e. independently of the method used one will get the same result with respect to the relative improvement of the viscosity.

(100) As discussed abovein working Examples herein was demonstrated that both N-linked and O-linked glycosidase gave no herein unwanted syneresis effect in relation to making a yogurt.

(101) Accordingly, in a preferred embodiment step (ii) is performed under conditions, wherein the effective amount of the glycosidase givesas a result of its presenceno increase in the syneresis effect to the dairy milk product.

(102) Thisno increase in the syneresis effectis particular relevant when the dairy milk product is a fermented milk product such as e.g. a yogurt.

(103) It is routine work for the skilled person to measure syneresis effect of a milk product of interest (e.g. a yogurt).

(104) In working Example 3 herein is provided a suitable standard method for measurement of syneresis of a milk product of interestpreferably the herein relevant syneresis is measured in accordance with the method of this Example 3.

(105) Essentially, the standard method to measure syneresis effect of Example 3 is based on a proper relevant storage of a milk product of interest and measurement of the amount of whey on top of the milk product of interest.

(106) Since it is easy for the skilled person to measure syneresis of a milk product of interestit is of course also easy for the skilled person to determine the preferred embodiment of step (ii) of the method of the first aspect relating to if:

(107) step (ii) is performed under conditions, wherein the effective amount of the glycosidase givesas a result of its presenceno increase in the syneresis effect to the dairy milk product.

(108) In order to determine this requirementthe skilled person shall simply perform the adequate step(s) to get a milk product of step (ii) with and without presence of the effective amount of the glycosidaseand then determine the herein relevant syneresis effect of the presence of the added amount of glycosidase.

(109) Also, the present invention relates to a method for producing a milk product, said method comprising:

(110) a) providing a milk substrate;

(111) b) treating the milk substrate with an enzyme having N-linked glycosidase activity; and

(112) c) optionally fermenting the milk substrate with a microorganism, such as a lactic bacterium.

(113) In an interesting embodiment, step b) is performed before or during step c).

(114) As discussed above, the milk substrate may origin from any animal or non-animal source.

(115) The milk product is preferably produced substantially without, or completely without any addition of a thickener and/or stabilizer, such as pectin, gelatin, starch, modified starch, carrageenan, alginate, and guar gum.

(116) In an interesting embodiment, the microorganism is a lactic acid bacterium and/or a microorganism which produces a polysaccharide, such as an exopolysaccharide (EPS).

(117) The microorganism may be a lactic acid bacterium, and preferably belong to a species selected from the group consisting of: Streptococcus thermophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. Casei, Lactobacillus paracasei subsp. Paracasei, Bifidobacterium bifidum, and Bifidobacterium longum.

(118) Interesting embodiments of any method of the present invention are: A method wherein the milk substrate is subjected to pasteurization before acidification and the enzyme treatment is performed before pasteurization; A method wherein the milk substrate is subjected to heat treatment prior to treatment with the enzyme having N-linked glycosidase activity; A method wherein the milk product is selected from the group consisting of: a set-type fermented milk product, a drinkable fermented milk product, and a spoonable fermented milk product; A method wherein the milk product has a milk solid non-fat content of less than 8%; A method wherein the milk product has a fat content of less than 2%; A method wherein the milk product has a fat content of less than 0.5%; A method wherein the milk product is packaged (ie the method comprises packaging); and/or A method wherein the glycosidase is selected from the group consisting of: -N-acetyl-galactosaminidase; GalNAC); -galactosidase; neuraminidase; Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase; N-Glycosidase-F; PNGase-F; Endo--N-acetylglucosaminidase H; ENDO-H and any enzyme classified in EC 3.2.1.49, EC 3.2.1.18, EC 3.2.1.22, EC 3.5.1.52, or EC 3.2.1.96.

(119) In the present context, the term packaging (a suitable amount of) the product in a suitable package relates to the final packaging of the product to obtain a product in distributable form so that the product can be ingested by e.g. a person or a group of persons. A suitable package may thus be a bottle, container, package or similar, and a suitable amount may be e.g. 10 ml to 5000 ml, but it is presently preferred that the amount in a package is from 50 ml to 1000 ml. Such a packaged product is a part of the present invention.

(120) In a further aspect, the present invention also relates to a milk product obtainable by a method of the present invention.

(121) In yet a further aspect, the present invention relates to a milk product which is obtainable by a method comprising adding an N-linked glycosidase and/or a O-linked glycosidase to a milk substrate. The glycosidase should be added in an effective amount to give the desired gel stiffness.

(122) The milk products of the invention may have been fermented by inoculation with a lactic acid bacteria culture, prior to and/or during and/or after the treatment with the glycosidase.

(123) The milk product may be packaged, e.g. in a sealed container having a volume in the range of 10 ml to 5000 ml, such as in a container having a volume of 25 ml to 1500 ml or a volume of 50 ml to 1000 ml.

(124) In a further aspect, the present invention relates to the use of an N-linked glucosidase in a method for preparation of a milk product, such as a fermented milk product (such as yoghurt) or cheese (such as fresh cheese, fromage frais, quark, etc).

(125) Also, the present invention relates to the use of an N-linked glucosidase in a method for improving the texture (such as gel firmness or stiffness) of a milk product, such as a fermented milk product (such as yoghurt) or cheese (such as fresh cheese, fromage frais, quark, etc).

(126) In a presently preferred embodiment, the invention relates to the use of an enzyme having N-linked and/or O-linked glycosidase activity for improving the mouth feel of a milk product, such as yoghurt.

(127) The use may comprise that the milk product has been produced using a lactic acid bacterium which produces a polysaccharide, such as an exopolysaccharide (EPS).

(128) The use of the terms a and an and the and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms comprising, having, including and containing are to be construed as open-ended terms (i.e., meaning including, but not limited to,) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

EXAMPLES

Example 1: Glycosidases

(129) The examples of glycosidases used in the method of as described herein is an asparagine amidase, an acetylglucosaminidase or a galactosaminidase.

(130) The N-Glycosidase-F, also known as PNGase-F, used in the process, is an asparagine amidase (EC 3.5.1.52) which may be derived from Flavobacterium mesingosepticum. It catalyses the complete and intact cleavage of N-linked oligosaccaharides from glycoproteins. It may be obtained as a commercial product from New England Biolabs Inc. under the name PNGase-F or produced recombinantly in a strain like Escherichia coli as we have done using the plasmid and method described in Loo et at (Protein Expression and Purification 24, 90-98, 2002).

(131) Endo--N-acetylglucosaminidase H (EC 3.2.1.96), also known as ENDO-H, may be derived from e.g. Streptomyces plicatus. ENDO-H catalyses the hydrolysis of the glycosidic bond between the two N-acetylglycosamines of N-linked glycosylations. It may be obtained as a commercial product from New England Biolabs Inc. under the name ENDO-H.

(132) -N-acetyl-galactosaminidase (EC 3.2.1.49) is a highly specific exoglycosidase that catalyzes the hydrolysis of -linked D-N-acetyl-galactosamine residues from threonines or serines. It may be obtained as a commercial product from New England Biolabs Inc. under the name the name -N-acetyl-galactosaminidase.

(133) For the in-house produced PNGase-F we have confirmed a high grade (>95%) of purity using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie brilliant blue (CBB) staining. To further confirm the purity the pooled enzyme was separated by SDS-PAGE and stained using silver. Appearing bands were next analyzed by mass-spec analysis which confirmed the presence of PNGase-F and only PNGase-F.

(134) Potential a-specific proteolytic activity of the in-house purified PNGase-F was evaluated in a proteolytic assay using bovine-kappa-casein (Sigma-Aldrich) as a substrate. In this assay we tested the proteolytic activity at various pH values resembling the natural pH drop occurring in yogurt fermentation. To challenge the system we utilized a 20 fold higher enzyme concentration than in the process of the invention and incubated four hours at each pH-value. Under these conditions we did not find any evidence of a-specific proteolytic activity.

(135) Conclusions:

(136) Based on the results discussed aboveit was clear that the so-called in-house produced PNGase-F was free of contaminants.

(137) The other commercially available glycosidases discussed above were also free of contaminants as described by the supplier.

Example 2: Method for Measuring Gel Firmness and Viscosity

(138) Gel firmness was measured by the use of an Anton Paar rheometer with an automatic sample changer (Physica DSR Rheometer+ASC). The measuring bob was placed in the measuring cup containing 20 ml sample, which had been stirred by the hand and heated to 13 C. After the bob had been placed in the sample a wait time was applied. Next, gel firmness was measured by oscillation. Here the strain was kept constant at 0.3% and the frequency was increased from 0.5 Hz to 30 Hz. From the measurement the elastic modulus (G) and the viscous modulus (G) could be calculated, and from these the complex modulus (G*) was obtained:
G*={square root over (G.sup.2+G.sup.2)}

(139) G* at 1 Hz was then used as a measure of the gel firmness and used for comparison of the different samples.

(140) Using the same equipment (Anton Paar rheometer) the viscosity was measured by increasing the shear rate from 0.2707 l/s to 300 l/s with measuring points (shear stress) every 10 s. The shear rate was then decreased from 275 l/s to 0.2707 l/s with measuring points every 10 s. Shear stress at 300 l/s was then used as a measure of the viscosity of the product.

(141) Conclusions:

(142) Based on above standard methods for measuring gel firmness and viscosityit is routine work for the skilled person to determine if there has been an improvement of the gel firmness and/or viscosity to a dairy milk product of interests by addition of a glycosidase according the method for making a dairy animal milk product as described herein.

Example 3: Methods for Measuring Syneresis

(143) Two different methods for measuring the amount of syneresis have been used in the invention:

(144) Method 1:

(145) 50 ml yogurt (produced like in example 4) is put into an Eppendorf tube and placed in cold storage (5 C.) for 14 days, after which the amount of whey on top of the yogurt is measured with a ruler (in mm).

(146) As evident to the skilled personone may also measure the syneresis effect of another milk product than yogurt by storage of a milk product of interest and measure the amount of whey on top of the milk product of interest.

(147) Method 2:

(148) Skim milk was fortified with 2% skim milk powder (SMP, producer?) and placed in the refrigerator overnight. The batch was heat treated for 20 minutes at 90 C. 75 ml milk solution was put into a 100 ml volumetric flask together with 0.02% YoFlex Advance 2.0 yogurt culture (may be obtained from Chr. Hansen A/S, Denmark) and enzyme (either PNGase or GalNAC). The total weight of the milk, culture and enzyme was noted. The solution was heated to 43 C. and fermented to pH 4.55 after which the flasks were put in cold storage for 7 days. After 7 days the whey on top of the yogurt was poured of and weighed. The amount of syneresis can hereafter be calculated as a percentage.

(149) As evident to the skilled personone may also measure the syneresis effect of another milk product than yogurt by e.g. fermenting with another not yogurt culture of interest, storage and measure the amount of whey on top of the milk product of interest.

(150) Conclusions:

(151) Based on above standard methods for measuring syneresis effectit is routine work for the skilled person to determine if there has been a significant syneresis effect to a dairy milk product of interests by addition of a glycosidase according the method for making a dairy animal milk product as described herein.

Example 4: Effect of PNGaseF on Gel Firmness (2 l Scale)

(152) 2 liters of skim milk (0.1% fat, Arla Express, Slagelse) was fortified with 1.6% skim milk powder (SMP, producer?) and placed in the refrigerator overnight. The solution was heat treated for 20 minutes at 90 C., cooled down to 43 C., inoculated with 0.02% YoFlex Advance 2.0 (obtained from Chr. Hansen A/S, Denmark) and either 10 ml PNGase-F or in the reference sample 10 ml 20 mM Na-phosphate buffer pH 7.5 containing 50 mM NaCl and fermented to pH 4.55. When pH 4.55 was reached, the yogurt was stirred and passed through a post treatment unit (PTU), which subjects the yogurt to a back pressure of 2 bars at 25 C. The final product was subjected to rheometer analysis on day 5.

(153) TABLE-US-00001 TABLE 1 Experimental results of a 2 I scale experiment of the preparation of a low fat yogurt in the absence and presence of in-house PNGase-F Complex Shear stress at Syneresis Modulus (Pa) (300 1/s) (mm) Advance 2.0 29.2 0.86 63.7 0.61 8 Advance 2.0 + PNGase-F 51.0 0.57 70.7 0.2 4 (250 U/ml milk)

(154) For the N-glycosidases one activity unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10 g of denatured RNase-B in 1 hour at 37 C. in a total reaction volume of 10 l

(155) The numbers in table 1 show that PNGase-F increases the gel firmness of a low fat yogurt. Under these conditions with 75% compared to the reference. The viscosity obtained in the presence of PNGase-F is comparable or slightly better than the reference sample. In data not shown here we found that the increase in gel firmness in response to PNGase-F treatment was dose dependent. We also evaluated syneresis using method 1 from Example 3 and found that the syneresis was reduced in response to PNGase-F treatment.

(156) Conclusions:

(157) The results above demonstrated that PNGase-F increases the gel firmness of a low fat yogurt. Under the conditions of this experiment with 75% (i.e. 1.75 times) compared to the reference. Further was found that the syneresis was reduced in response to PNGase-F treatment.

(158) The viscosity obtained in the presence of PNGase-F is comparable or slightly better than the reference sample.

Example 5: Testing of Different Glycosidases in Low Fat Yogurt

(159) Yogurts were made directly in 20 ml rheometer cups. Hereby a set-yogurt was obtained. Skim milk (0.1% fat, Arla Express, Slagelse) was fortified with 2% skim milk powder (SMP) and placed in the refrigerator overnight. The solution was heat treated for 20 minutes at 90 C., cooled down to 43 C., inoculated with 0.02% YoFlex Advance 2.0 and different deglycosidases and fermented (Table 1)

(160) The samples were then placed in the refrigerator and the rheology was measured at day 3 according to example 2.

(161) TABLE-US-00002 TABLE 2 Experimental result of a 20 ml cup lab scale experiment of the preparation of a low fat yogurt in the presence and absence of commercially obtained deglycosidases. All tested enzymes in this table were obtained from New England Biolabs Inc. Concentration Gel firmness (U/ml milk) (Pa) Advance 2.0 + PNGase-F 250 608 32.5 Advance 2.0 + Endo-H 250 564 .sup. Advance 2.0 + -N-Acetyl 50 482 19.8 galactosaminidase Advance 2.0 0 391 7.5

(162) For N-glycosidases PNGase-F and Endo-H one unit is defined as in Table 1. For -N-Acetyl-galactosaminidase, an O-glucosidase, one unit is defined as the amount of enzyme required to cleave >95% of the terminal -D-N-acetyl-galactosamine from 1 nmol (GalNAca1-3)(Fuc1-2)Gal1-4Glc-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37 C. in a total reaction volume of 10 l.

(163) For all tested glycosidases, N-glucosidases as well as the O-glucosidase GalNAC, the gel firmness was found to be improved markedly. At the tested concentrations PNGase-F obtained commercially was found to have the most potent effect on the gel firmness of the low fat yogurt.

(164) Conclusions:

(165) The results above demonstrated that for all tested glycosidases, N-glucosidases as well as the O-glucosidase GalNAC, the gel firmness was found to be improved markedly.

Example 6: Syneresis Experiment Using a Glycosidase Working on O-Linked Glycosylations

(166) In U.S. Pat. No. 7,560,127 it was shown that the -glucosidase GalNAC could lead to cheese curd/clotting formation. We here analyzed what effect GalNAC had on syneresis formation in low fat yogurt. The experiment was conducted as described in Example 3 Method 2. Yogurts were made directly in 100 ml rheometer cups. Hereby a set-yogurt was obtained. Skim milk (0.1% fat, Arla Express, Slagelse) was fortified with 2% skim milk powder (SMP, Aria Express, Slagelse) and placed in the refrigerator overnight. The solution was heat treated for 20 minutes at 90 C., cooled down to 43 C., inoculated with 0.02% YoFlex Advance 2.0 and GalNAC at a concentration of 100 U/ml of milk. For the reference sample without enzyme the percentage of whey after 21 days was 0.30.04% whereas the whey fraction for the enzyme treated sample was 0.20.08%. Surprisingly, it was therefore found that treatment with GalNAC did not increase the syneresis when compared to a reference sample fermented with the same culture. This result was unexpected since U.S. Pat. No. 7,560,127 would indicate that removal of O-linked glycans leads to curd formation and thus separation of the whey.

(167) Conclusions:

(168) The results above demonstrated that the tested O-linked glycosidase did not increase the syneresis during the production of the yogurt.

Example 7: LAB and Chemically Acidified Milk

(169) In the current example we wanted to assess whether or not the effect of PNGase-F was dependent on the mechanism of acidification. Experimentally this was addressed by comparing the effect of PNGase-F on a fermented yogurt and a yogurt acidified chemically with Glucono--lactone (GDL).

(170) A milk consisting of 9.5% dry matter, was inoculated with either YoFlex Advance 2.0 or Glucono--lactone (GDL) and PNGase-F as indicated in Table 3, heated to 43 C. and fermented to pH 4.55. After samples had been stored for 3 days at 5 C., the samples were stirred with a stirrer and poured into the rheometer cups and the rheology was measured according to example 2 and 3.

(171) The results presented in table 3 confirmed that the effect of PNGase-F may be described as a physical phenomenon. The chemically acidified yogurt generates a much firmer gel than the Advance 2.0 culture. However, for both methods of acidification it is evident that the addition of PNGase-F improves the gel firmness.

(172) TABLE-US-00003 TABLE 3 Experimental result of a 200 ml lab scale experiment of the preparation of a low fat yogurt in the presence of PNGase-F using either Advance 2.0 or GDL for acidification Concentration Gel Firmness (U/ml milk) (Pa) Advance 2.0 + PNGase-F 250 35.3 7.8 Advance 2.0 0 24.7 0.9 GDL + PNGase-F 250 148.0 7.0 GDL 0 121.5 6.4
Conclusions:

(173) The results above demonstrated for both methods of acidification it is evident that the addition of PNGase-F improved the gel firmness.

(174) Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

(175) 1. US2005/0095316A1 (Danone) 2. US2005/0095317A1 (Danone) 3. WO2007/095958A1 (Chr. Hansen A/S) 4. A. N. Hassan et al (J. Dairy Sci: 86:1632-1638; 2003) 5. U.S. Pat. No. 7,560,127B2 (DSM) 6. E. Cases et al (Journal of Food Science; Vol. 68, Nr. 8, 2003, Pages 2406-2410) 7. EP1489135A1 8. R. Scott, (1986), Cheesemaking process, second ed., Elsevier Applied Science Publishers, London and New York. 9. G. Bylund, (1995), Dairy processing handbook, Tetra Pak Processing Systems, Lund, Sweden 10. F. Kosikowski, (1982), Cheese and fermented milk foods, second ed., Kosikowski & Associates, New York

(176) All references cited in this patent document are hereby incorporated herein in their entirety by reference.