WITH AID OF AN EXOPEPTIDASE

Field of the invention

The present invention relates to a process for preparing a fermented milk product. According to another aspect, the present invention relates to a fermented milk product and to a kit of parts. According to yet another aspect, the present invention relates to the use of enzyme having carboxypeptidase activity for accelerating the acidification speed of lactic acid bacteria.

Background

The food industry uses different bacteria, in the form of ferments, in particular lactic acid bacteria, in order to improve the taste and the texture of foods but also to extend the shelf life of these foods. In the case of the dairy industry, lactic acid bacteria are used intensively in order to bring about the acidification of milk substrate (by fermentation) but also in order to texturize the fermented milk product into which they are incorporated. Among the lactic acid bacteria used in the food industry, there can be mentioned the genera Streptococcus and Lactobacillus. The lactic acid bacterial species Streptococcus thermophilus and Lactobacillus delbrueckii ssp bulgaricus are used in particular in the formulation of the ferments used for the production of fermented milk products, for example yogurts.

Different lactic acid bacteria are generally combined to form a starter culture or starter culture composition (also referred to as starter). Such a starter culture is responsible for the acidification of the milk substrate and the taste and texture of the fermented milk product. To achieve a desired taste and texture, a starter culture usually comprises one or more strains having taste and/or texture providing properties. However, such strains might not be able to acidify the milk substrate to the desired degree in a time efficient manner because they are so called slow acidifiers. Therefore, starter cultures usually also comprise fast acidifiers, i.e. strain(s) having a strong acid generating property, to compensate for the slow acidifiers. A drawback of using an additional strain in a starter culture is that this dilutes the effect of the taste and/or texture providing strain(s), resulting in a higher amount of taste and/or texture providing strain(s) needed in the starter culture.

Therefore, there is a need in the art to improve the acid generating properties of slow acidifiers instead of compensating these slow acidifiers by incorporating fast acidifiers in the starter culture. In the industrial production of lactic acid bacteria, yeast extracts are commonly added to the growth medium to accelerate growth of the lactic acid bacteria. Particularly the growth of slow acidifiers is accelerated after addition of yeast extract. However, in the production of fermented milk products, it is not desired to add a non-dairy ingredient such as a yeast extract to the milk substrate because this would cause labelling issues and/or introduce yeasty notes to the fermented milk product. Therefore, there is a need in the art to improve the acid generating properties of slow acidifiers in processes for preparing fermented milk products. WO2017/009080 describes a process for preparing a fermented milk product which comprises 4 to 15 wt/wt% of protein by contacting milk with an enzyme having carboxypeptidase activity, Example 1 of WO2017/009080 uses yogurt starter culture TS-80J which is a so-called fast strain (as disclosed in example 1 of the present application). Example 2 of WO2017/009080 uses yogurt culture YS-131. YS-131 is a blend of cultures and is classified as a fast blend based on the fact that the time to reach pH 4.6 is 336 minutes using fresh milk and 42 degrees Celsius and based on the fact that the time to reach pH 4.6 is 394 minutes using fresh milk + SMP (4.2% protein) and 40 degrees Celsius (not exemplified herein).

Fatah Ahtesh et al (2016) (Journal of Food Science, vol. 81 , nr. 1 , pages M135-M143) describe incubation of 12% reconstituted skim milk with 1 % of L. helveticus strains in combination with Flavourzyme. Fig 1A (without Flavourzyme) shows that after 12 hours at 37 degrees Celsius none of the tested strains reached pH 4.6. Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. Detailed information about the composition of this mixture is described by Merz et al (2015, J Agric Food Chem, vol 63(23), pages 5682-5693). Flavourzyme comprises eight key enzymes: two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one o amylase.

WO2004/103082 describes that addition of yeast extract accelerates lactic acid bacteria fermentation.

WO2015/170985 describes a method for preparing yogurt using potato protein protease inhibitor to reduce the lag time of lactic acid bacteria fermentation.

WO2016/164096 describes a method for preparing a fermented milk product comprising treating a milk substrate having a protein content of 4% or higher with one or more proteases to improve an aspect of the fermented milk product. WO2016/164096 provides a list of such aspects, earlier conclusion of fermentation is an example thereof. WO2016/164096 also provides a list of proteases. An example of a protease is a protease of Enzyme class 3.4.17. WO2016/164096 further provides a list of cultures. Examples of cultures are YO-MIX 414, 532 and 860. Other examples of cultures are Probat 505, Choozit 220 or Choozit 230. The product documentation of the YO-MIX Yogurt cultures describes 4 different categories, one of them being "YO-MIX Real quick" and YO- Mix 860 is an example thereof.

WO2012/0221 12 describes the use of carboxypeptidase in the preparation of bean curd from soybean milk. Example 1 uses DELVO-TEC LL50-A. Examples 2 and 3 use DELVO-YOG SVV-21. These cultures are so-called fast cultures. DELVO-YOG SW-21 is used in yoghurt applications and has a short fermentation time of 250 minutes in 12% reconstituted milk when fermented at 40°C (not exemplified herein). LL-50A is a culture used in cheese applications and hence has a lower temperature optimum i.e. less than 38°C. The product documentation of DELVO- TEC LL-50 indicates a fermentation time of less than 7 hours and is promoted for its reliable acidification. Summary

This problem, amongst other problems, is solved by the present invention as defined in claim 1. More specifically, this problem, amongst other problems, is solved by providing a process for preparing a fermented milk product, comprising

a) contacting a milk substrate with an enzyme having exopeptidase activity; b) contacting a milk substrate with lactic acid bacteria;

wherein the lactic acid bacteria have a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 30% shorter in the presence 0.5% (w/w) of a yeast extract in the milk substrate than without the presence of the yeast extract in the solution of 12% skimmed milk powder and wherein said enzyme preparation is a substantially pure exopeptidase preparation or by providing a process for preparing a fermented milk product, comprising

a) contacting a milk substrate with an enzyme having exopeptidase activity; b) contacting a milk substrate with lactic acid bacteria;

wherein the lactic acid bacteria have a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder of more than 400 minutes and wherein said enzyme preparation is a substantially pure exopeptidase preparation.

According to another aspect, the present invention relates to a fermented milk product comprising an enzyme having exopeptidase activity and comprising lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 30% shorter in the presence 0.5% (w/w) of a yeast extract in the solution of 12% skimmed milk powder than without the presence of the yeast extract in the solution of 12% skimmed milk powder or lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder of more than 400 minutes, and wherein said enzyme preparation is a substantially pure exopeptidase preparation.

According to another aspect, the present invention relates to a kit of parts comprising an enzyme having exopeptidase activity and comprising lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 30% shorter in the presence 0.5% (w/w) of a yeast extract in the solution of 12% skimmed milk powder than without the presence of the yeast extract in the solution of 12% skimmed milk powder or comprising lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder of more than 400 minutes, and wherein said enzyme preparation is a substantially pure exopeptidase preparation.

According to yet another aspect, the present invention relates to the use of an enzyme having exopeptidase activity for accelerating the acidification speed of lactic acid bacteria, and wherein said enzyme preparation is a substantially pure exopeptidase preparation.

SEQ ID NO 1 is a protein sequence derived from Aspergillus niger.

Definitions

The term "milk substrate" as used in the present context, may be any raw and/ or processed milk material. Useful milk substrates may include solutions / suspensions of any milk or milk like products comprising lactose, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, solutions of dried milk, UHT milk, whey, whey permeate, acid whey or cream. Preferably the present milk substrate is milk or an aqueous solution of skim milk powder.

The term "milk" is intended to encompass milks from mammals and plant sources or mixtures thereof. Preferably, the milk is from a mammal source. Mammals sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, llama, mare and deer. In an embodiment, the milk is from a mammal selected from the group consisting of cow, sheep, goat, buffalo, camel, llama, mare and deer, and combinations thereof. Plant sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. Soy bean milk is preferred. In addition, the term "milk" refers to not only whole milk, but also skim milk or any liquid component derived thereof.

As used in this specification, the term "fermented dairy product" or "acidified dairy product" is intended to refer to products which are obtained by the multiplication of lactic acid bacteria in a milk base leading to a milk coagulum. The milk preparation used as raw material for the fermentation may be skimmed or non-skimmed milk, optionally concentrated or in the form of powder. Furthermore, this milk preparation may have been subjected to a thermal processing operation which is at least as efficient as pasteurization. The particular characteristics of the various fermented dairy products depend upon various factors, such as the composition of milk base, the incubation temperature, the lactic acid flora and/or non-lactic acid flora. Thus, fermented dairy products manufactured herein include, for instance, various types of regular yoghurt, low fat yogurt, non fat yoghurt, kefir, dahi, ymer, buttermilk, butterfat, sour cream and sour whipped cream as well as fresh cheeses such as quark and cottage cheese.

As used in the present specification, the term "yogurt" refers to products comprising "lactic acid bacteria such as Streptococcus salivarius thermophilus and Lactobacillus delbruekii subsp. bulgaricus, but also, optionally, other microorganisms such as Lactobacillus delbruekii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus casei, or any microorganism derived therefrom. The lactic acid strains other than Streptococcus salivarius thermophilus and Lactobacillus delbruekii subsp. bulgaricus, are intended to give the finished product various properties, such as the property of promoting the equilibrium of the flora.

As used herein, the term "yogurt" encompasses set yogurt, stirred yogurt, drinking yoghurt, Petit Suisse, heat treated yogurt and yogurt-like products. Preferably, the yogurt is a stirred yoghurt or a drinking yogurt. More preferably, the yogurt is a stirred yogurt. The term "yogurt" encompasses, but is not limited to, yoghurt as defined according to French and European regulations, e.g. coagulated dairy products obtained by lactic acid fermentation by means of specific thermophilic lactic acid bacteria only (i.e. Lactobacillus delbruekii subsp. bulgaricus and Streptococcus salivarius thermophilus) which are cultured simultaneously and are found to be live in the final product in an amount of at least 10 million CFU (colony-forming unit) / g. Preferably, the yogurt is not heat-treated after fermentation. Yogurts may optionally contain added dairy raw materials (e.g. cream) or other ingredients such as sugar or sweetening agents, one or more flavouring(s), fruit, cereals, or nutritional substances, especially vitamins, minerals and fibers. Such yogurt advantageously meets the specifications for fermented milks and yogurts of the AFNOR NF 04-600 standard and/or the codex StanA-lla-1975 standard. In order to satisfy the AFNOR NF 04-600 standard, the product must not have been heated after fermentation and the dairy raw materials must represent a minimum of 70% (m/m) of the finished product.

In the present context, the terms "fresh cheese", "unripened cheese", "curd cheese" and "curd-style cheese" are used interchangeably herein to refer to any kind of cheese such as natural cheese, cheese analogues and processed cheese in which the protein/ casein ratio does not exceed that of milk.

The term "starter" or "starter culture" as used herein refers to a culture of one or more food- grade micro-organisms, in particular lactic acid bacteria, which are responsible for the acidification of the milk base. Starter cultures may be fresh (liquid), frozen or freeze-dried. Freeze dried cultures need to be regenerated before use. For the production of a fermented dairy product, the starter is usually added in an amount from 0.01 to 3%, preferably from 0.01 and 0.02 % by weight of the total amount of milk base.

As used herein, the term "lactic acid bacteria" (LAB) or "lactic bacteria" refers to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, low-GC, acid tolerant, non- sporulating, non-respiring, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of lactose by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus responsible for the acidification of milk and for the texture of the dairy product. As used herein, the term "lactic acid bacteria" or "lactic bacteria" encompasses, but is not limited to, bacteria belonging to the genus of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbruekii subsp. bulgaricus, Streptococcus salivarius thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus and Bifidobacterium breve.

The term 'sequence identity' is defined herein that in order to determine the percentage of sequence identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. The percent sequence identity between two amino acid sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman- Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

The term 'time to reach pH 4.6' is defined as the time between start of fermentation of a milk substrate with lactic acid bacteria, or contacting a milk substrate with lactic acid bacteria, until a milk substrate with a pH of 4.6 is reached.

The term 'enzyme having exopeptidase activity' as used in the present context means an enzyme that catalyses the cleavage of the terminal peptide bond from a peptide chain, thereby releasing a single amino acid or a di or tripeptide.

Detailed description

The present invention relates to a process for preparing a fermented milk product, comprising

a) contacting a milk substrate with an enzyme having exopeptidase activity; b) contacting a milk substrate with lactic acid bacteria;

wherein the lactic acid bacteria have a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 30% shorter in the presence 0.5% (w/w) of a yeast extract in the solution of 12% skimmed milk powder than without the presence of the yeast extract in the solution of 12% skimmed milk powder, and wherein said enzyme preparation is a substantially pure exopeptidase preparation.

Alternatively, the present invention relates to a process for preparing a fermented milk product, comprising

a) contacting a milk substrate with an enzyme having exopeptidase activity; b) contacting a milk substrate with lactic acid bacteria;

wherein the lactic acid bacteria have a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder of more than 400 minutes, and wherein said enzyme preparation is a substantially pure exopeptidase preparation.

Surprisingly, the present inventors found that an enzyme having exopeptidase activity accelerates the acidification of slow acidifiers, or of lactic acid bacteria having a low acidification speed. This is advantageous because slow acidifiers having desired characteristics, like texturizing characteristics, can now be used in production of a fermented milk product. The enzyme having exopeptidase activity (for example a carboxypeptidase) does not have an effect on the lag time (i.e. the time needed for a pH change of 0.08), I.e. preferably, the enzyme having exopeptidase activity does not (or hardly not) reduce the lag time or, alternatively, the enzyme having exopeptidase activity does not (or hardly not) have an effect on the lag time. The enzyme having exopeptidase activity reduces the duration of the exponential growth phase of said lactic acid bacteria.

Lactic acid bacteria need (amongst other things) a carbohydrate as well as a protein source for their growth. The present application is directed to a method which improves the availability of the protein source by using an enzyme having exopeptidase activity that catalyses the cleavage of the terminal peptide bond from a protein within the milk substrate, thereby releasing a single amino acid or a di or tripeptide. Preferably, said enzyme having exopeptidase activity releases substantially amino acids only (i.e. at least 90%, more preferably at least 95% of the released products is an amino acid and only a very minor (at most 10 or preferably at most 5%) part is a di or tripeptide). Prior art documents describe the use of endopeptidases which results in the presence of larger peptides (of 4 or more amino acids) which can result in off flavour, textural defects or colour defects. The phrase "said enzyme preparation is a substantially pure exopeptidase preparation" refers to an enzyme preparation wherein the enzymatic activity is for at least 90% caused by a single enzyme. More preferably, at least 95% of the enzymatic activity is caused by a single enzyme.

Preferably, the present time to reach a pH of 4.6 of a solution of 12% skimmed milk powder is measured according to the standard ISO 26323|IDF 213, more preferably the first edition, dated 2009-07-01. To avoid any misunderstanding the starting material "a solution of 12% skimmed milk powder" does not comprises any exogenous enzyme preparation having exopeptidase activity.

More preferably, the present lactic acid bacteria are defined as lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 30% shorter in the presence 0.5% (w/w) of a yeast extract in the solution of 12% skimmed milk powder than without the presence of the yeast extract in the solution of 12% skimmed milk powder, in the following test:

-the milk substrate is 12% reconstituted skimmed milk powder which percentage is close to the milk used for desired applications such as yogurt;

-BD Bacto™ Yeast Extract, BD Bacto ^tm yeast extract or another yeast extract derived from Saccharomyces cerevisiae cells that have been autolyzed, or alternatively, another yeast extract, preferably a yeast extract commonly used for the growth of bacteria, might also be used, wherein the yeast extract is dosed to the milk substrate with an amount of 0.5 % (w/w) of the milk substrate;

-inoculation of milk substrate with an amount of lactic acid bacteria so that the equivalent of 0.1 % of an OD600 measurement of 1.2 was inoculated (200 μΙ_ in 200 ml milk substrate, per bottle in a 250 ml cylindrical bottle);

-wherein the time to reach a pH of 4.6 of a milk substrate is measured according to the standard ISO 26323|IDF 213, first edition, dated 2009-07-01 ;

-starting pH of the milk substrate is not adjusted; and

-fermentation temperature of 40°C. In a preferred embodiment, the present lactic acid bacteria are defined as lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 35%, more preferably at least 40%, even more preferably at least 45%, most preferably at least 50% shorter in the presence 0.5% (w/w) of a yeast extract in the solution of 12% skimmed milk powder than without the presence of the yeast extract in the solution of 12% skimmed milk powder. To avoid any misunderstanding the starting material "a solution of 12% skimmed milk powder" does not comprises any exogenous enzyme preparation having exopeptidase activity.

In a preferred embodiment, the lactic acid bacteria are defined as lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder of more than 400 minutes as determined in the following test:

-the milk substrate is 12% reconstituted skimmed milk powder which percentage is close to the milk used for desired applications such as yogurt;

-inoculation of milk substrate with an amount of lactic acid bacteria so that the equivalent of 0.1 % of an OD600 measurement of 1.2 was inoculated (200 μΙ_ in 200 ml milk substrate, per bottle in a 250 ml cylindrical bottle);

-wherein the time to reach a pH of 4.6 of a milk substrate is measured according to the standard ISO 26323|IDF 213, first edition, dated 2009-07-01 ;

-starting pH of the milk substrate is not adjusted; and

-fermentation temperature of 40°C.

In a preferred embodiment, the present lactic acid bacteria are defined as lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder of more than 450 minutes, more preferably more than 500 minutes, even more preferably more than 550 minutes, most preferably more than 600 minutes.

The inventors have further noted that slow and fast cultures react in a different way to increased levels of an enzyme preparation having exopeptidase activity. Slow strains show a dose- response of CGP, whereas the fast strains, although slightly stimulated, do not show this. The invention thus also provides a process for preparing a fermented milk product, comprising

a) contacting a milk substrate with an enzyme having exopeptidase activity; b) contacting a milk substrate with lactic acid bacteria;

wherein the lactic acid bacteria show a dose-response to an enzyme having exopeptidase activity and wherein said enzyme preparation is a substantially pure exopeptidase preparation.

Contacting a milk substrate with lactic acid bacteria as used in the present context is preferably carried out until the desired pH is reached. Alternatively, the present step of contacting a milk substrate with lactic acid bacteria is fermenting a milk substrate with lactic acid bacteria.

In a preferred embodiment, the present enzyme (preparation) having exopeptidase activity (EC.3.4.1 1-19) is an enzyme having carboxypeptidase activity or an enzyme having aminopeptidase activity. Preferably an enzyme having carboxypeptidase activity. More preferably, the enzyme having exopeptidase activity (EC.3.4.1 1-19) is a lactic acid bacteria exogenous enzyme. In other words, the enzyme having exopeptidase activity (EC.3.4.1 1-19) is not added to the milk substrate by expression of the lactic acid bacteria.

An enzyme having carboxypeptidase activity may be a carboxypeptidase, for instance a serine carboxypeptidase which has enzyme classification number EC 3.4.16. A carboxypeptidase may be derived from any suitable microorganism, for instance Aspergillus sp. such as Aspergillus niger, or Aspergillus oryzae. A carboxypeptidase may be carboxypeptidase I from Aspergillus niger as disclosed in Del Degan (1992) Applied Environmental Microbiology, 58, 2144-2152. A carboxypeptidase is a protease enzyme that hydrolyses a peptide bond at the carboxy- (C-) terminal end of a protein or peptide. The wording "derived" or "derivable" from with respect to the origin of a polypeptide as disclosed herein, means that when carrying out a BLAST search with a polypeptide as disclosed herein, the polypeptide may be derivable from a natural source, such as a microbial cell, of which an endogenous polypeptide shows the highest percentage homology or identity with the polypeptide as disclosed herein.

A carboxypeptidase as disclosed herein may be produced in any suitable host organism, for instance in fungi Aspergilli, e.g. Aspergillus niger or Aspergillus oryzae, Trichoderma, or the yeasts Saccharomyces, and Kluyveromyces or the bacteria of the genus Streptomyces, Escherichia, such as Escherichia coli or Bacilli, such as Bacillus subtilis by known methods in the art, for instance using standard molecular techniques as disclosed in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001.

In one embodiment, an enzyme having exopeptidase activity is a purified or a pure enzyme.

A pure or purified enzyme is an enzyme that may be at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% pure for instance as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.

In one embodiment, the enzyme having exopeptidase activity is a polypeptide having exopeptidase or carboxypeptidase activity which has at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature polypeptide sequence of SEQ ID NO: 1. The mature polypeptide sequence may comprise amino acids 53 to 520 of SEQ ID NO: 1 , wherein methionine at position 1 in SEQ ID NO: 1 is counted as number 1 .

In a preferred embodiment, the enzyme having exopeptidase activity is contacted with the milk substrate prior or during contacting the milk substrate with the lactic acid bacteria.

Preferably, the lactic acid bacteria belong to a genus chosen from the group consisting of Streptococcus spp., Lactobacillus spp., Bifidobacterium spp., Lactococcus spp., Streptococcus salivarius thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus and Bifidobacterium breve.

Preferably, the enzyme (preparation) having exopeptidase activity is used to reduce the fermentation time of a texturizing strain, i.e. a strain which increases viscosity. More preferably, the lactic acid bacteria are bacteria which are commercially used in the production of yogurt, i.e. the lactic acid bacteria are preferably Streptococcus spp bacteria or Streptococcus salivarius thermophilus bacteria.

The lactic acid bacteria can be added in different forms to the milk substrate. For example, the lactic acid bacteria can be added a frozen culture or as a freeze-dried culture. Preferably, the lactic acid bacteria are added as frozen pellets to the milk substrate.

Preferably, the milk substrate is contacted with the enzyme having exopeptidase activity in an amount of between 0.1 to 10 % (w/w) of the milk substrate, such as from 1 to 9 % (w/w), from 1 to 8 % (w/w), from 1 to 7 % (w/w), or from 1 to 6 % (w/w). Most preferably the amount is between 2 and 6 % (w/w), such as 2.5 or 5 % (w/w). More preferably, the enzymatic activity of enzyme having carboxypeptidase activity is more than 900 Carboxy Peptidase G unit (CPGU) per gram enzyme. More preferably, 1 CPGU can be defined as the amount of enzyme needed to decrease the optical density at 340 nm by 1 absorbance unit per minute (1 AU/min) in the conditions of the test (hydrolysis of 1.5 mM FA-Phe-Ala pH 4,5 at 37°C).

In a preferred embodiment, contacting the milk substrate with the enzyme having exopeptidase activity results in a time to reach pH 4.6 which is at least 10% shorter, such as least 20%, 30%, 35% or even at least 40% shorter. More preferably shorter compared to a time to reach pH 4.6 without the step of contacting the milk substrate with the enzyme having exopeptidase activity.

In a preferred embodiment, the present fermented milk product is chosen from the group consisting off yogurt, kefir, dahi, ymer, buttermilk, butterfat, sour cream, sour whipped cream, quark and cottage cheese. Preferably the present fermented milk product is yogurt.

In a more preferred embodiment, the present milk substrate or the present fermented milk product comprises less than 4 % (w/w) protein, such as less than 3.9, 3.8, 3.7, 3.6, 3.5., 3.4, 3.3, 3.2, 3.1 , 3.0, 2.9. 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1 , 2.0 % (w/w) or less than 1.0 % (w/w). The present inventors found that in milk substrates having a low amount of protein can still efficiently be fermented towards pH 4.6 by using an exopeptidase. This is unexpected since it is more difficult for lactic acid bacteria to release free amino acids if the amount of protein is lower.

According to another aspect, the present invention relates to a fermented milk product comprising an enzyme having exopeptidase activity and comprising lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 30% shorter in the presence 0.5% (w/w) of a yeast extract in the solution of 12% skimmed milk powder than without the presence of the yeast extract in the solution of 12% skimmed milk powder or lactic acid bacteria having a time to reach a pH of 4.6 of a milk substrate of more than 400 minutes, and wherein said enzyme preparation is a substantially pure exopeptidase preparation. The lactic acid bacteria are defined as above.

In a preferred embodiment, the present fermented milk product comprises an enzyme having exopeptidase activity in an amount of between 0.1 to 10 % (w/w), such as from 1 to 9 % (w/w), from 1 to 8 % (w/w), from 1 to 7 % (w/w), or from 1 to 6 % (w/w) of the fermented milk product. Most preferably the amount is between 2 and 6 % (w/w), such as 2.5 or 5 % (w/w) of the fermented milk product.

In a further preferred embodiment, the present fermented milk product comprises the enzyme having exopeptidase and / or carboxypeptidase which is a polypeptide having carboxypeptidase activity which has at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the mature polypeptide sequence of SEQ ID NO: 1. The mature polypeptide sequence may comprise amino acids 53 to 520 of SEQ ID NO: 1 , wherein methionine at position 1 in SEQ ID NO: 1 is counted as number 1 .

According to another aspect, the present invention relates to a kit of parts comprising an enzyme having exopeptidase activity and comprising lactic acid bacteria having a time to reach a pH of 4.6 of a solution of 12% skimmed milk powder which is at least 30% shorter in the presence 0.5% (w/w) of a yeast extract in the solution of 12% skimmed milk powder than without the presence of the yeast extract in the solution of 12% skimmed milk powder or comprising lactic acid bacteria having a time to reach a pH of 4.6 of a milk substrate of more than 400 minutes, and wherein said enzyme preparation is a substantially pure exopeptidase preparation. Preferably to a kit of parts comprising the present enzyme having exopeptidase activity or the present lactic acid bacteria. More preferably the kit of parts is a starter culture.

According to yet another aspect, the present invention relates to the use of an enzyme having exopeptidase and/or carboxypeptidase activity for production of fermented milk products, and wherein said enzyme preparation is a substantially pure exopeptidase preparation. Preferably fermented milk products comprising less than 4 % (w/w) protein, such as less than 3.9, 3.8, 3.7, 3.6, 3.5., 3.4, 3.3, 3.2, 3.1 , 3.0, 2.9. 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1 , 2.0 % (w/w) or less than 1.0 % (w/w).

According to another aspect, the present invention relates to the use of the present enzyme having carboxypeptidase activity for accelerating the acidification speed of lactic acid bacteria, more preferably for accelerating the acidification speed of lactic acid bacteria as defined herein.

EXAMPLES

Strains

Table 1

Culture Strain availability

Streptococcus thermophilus & commercially available from

DELVO®FRESH TT-50

Lactobacillus delbruekii subs p. DSM Food Specialties B.V., bulgaricus Delft, The Netherlands

CBS 140561 Streptococcus thermophilus deposited*

CBS 140557 Streptococcus thermophilus deposited*

BUC200 Streptococcus thermophilus DSM internal ref

Streptococcus thermophilus & commercially available from

DELVO®TEC TS-80J Lactobacillus delbruekii subs p. DSM Food Specialties B.V., bulgaricus Delft, The Netherlands

BUC300 Streptococcus thermophilus DSM internal ref *deposited at the Centraalbureau voor Schimmel-cultures (Fungal Biodiversity Centre),

Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands, The Netherlands, on September 29, 2015, under the provisions of the Budapest Treaty.

EXAMPLE 1

All strains were pre-cultured in Elliker Broth (Sigma-Aldrich) for 16 hours at 40°C. The cultures were then inoculated into milk so that the equivalent of 0.1 % of an OD600 measurement of 1 .2 was inoculated (200 μΙ_ in 200 ml milk).

Acidification performance was measured in 12% w/w reconstituted milk powder as per the protocol described in ISO 26323|IDF 213. The milk powder was from DariGold (SMP MH CDX) and the fermentation temperature was 40°C.

Acidification was measured in 12% reconstituted milk powder (RSM) with and without the addition of BD Bacto™ Yeast Extract (available from BD, New Jersey, US) which was added to an end concentration of 0.5% w/w.

The 6 strains tested could be grouped into 2 categories:

1. Fast strains with a time to reach pH 4.6 of less than 400 minutes for which the addition of yeast extract resulted in a decrease in the time to reach pH 4.6 of less than 30 % in comparison with the addition of yeast extract

2. Slow strains with a time to reach pH 4.6 of more than 400 minutes for which the addition of yeast extract resulted in a decrease in the time to reach pH 4.6 of more than 30%

Table 2

EXAMPLE 2

All strains were pre-cultured in Elliker Broth (Sigma-Aldrich) for 16 hours at 40°C. The cultures were then inoculated into milk so that the equivalent of 0.1 % of an OD600 measurement of 1 .2 was inoculated (200 μΙ_ in 200 ml milk).

Acidification performance was measured in 12% w/w reconstituted milk powder as per the protocol described in ISO 26323|IDF 213. The milk powder was from DariGold (SMP MH CDX) and the fermentation temperature was 40°C.

Acidification was measured in 12% reconstituted milk powder (RSM) with and without the addition of an enzyme having carboxypeptidase (Accelerzyme® CPG, DSM Food Specialties BV) which was added to an end concentration of 2.5% and 5% w/w.

Accelerzyme® CPG has activity of > 900 CPGU/g, where 1 CPGU can be defined as the amount of enzyme needed to decrease the optical density at 340 nm by 1 absorbance unit per minute (1 AU/min) in the conditions of the test (hydrolysis of 1.5 mM FA-Phe-Ala pH 4,5 at 37°C).

Table 3