YOGURT WITH REDUCED RIPENING

Abstract

The present invention relates to a process for the production of a fermented milk product, preferably yogurt, comprising fermenting milk with lactic acid bacteria and contacting the milk with a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin.

Claims

1. A process for the production of yogurt, comprising fermenting milk with lactic acid bacteria and contacting milk with a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin.

2. Process according to claim 1, wherein the polypeptide having chymosin activity is capable of hydrolysing bovine alpha s1-casein at position F23F24 so as to form s1-I CN (f24-199) more rapidly than camel chymosin.

3. Process according to claim 1, wherein the C/P ratio is 2, or optionally 5, or optionally 10 times higher than the C/P ratio of bovine chymosin.

4. Process according to claim 1, wherein the polypeptide having chymosin activity has an amino acid sequence which; a. when aligned with the chymosin comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue corresponding to amino acid 51 and/or 221; and/or b. when aligned with SEQ ID NO: 4 comprises at least one substitution of an amino acid residue located in the S2 binding pocket.

5. Process according to claim 4, wherein an amino acid residue located in the S2 binding pocket corresponds to position 219, 223, 288, 290, 295 or 297 when aligned with SEQ ID NO: 4.

6. Process according to claim 4, wherein the polypeptide having chymosin activity shares at least about 65% sequence identity with a polypeptide according to SEQ ID NO: 2 or 4.

7. Process according to claim 4, wherein the polypeptide having chymosin activity comprises the sequence set out in SEQ ID NO: 2 carrying one of the following mutations or combinations of mutations: A51V; K221L; K221M; K221V; V223Q; A51V and K221V; A51V and K221M; A51V, K221V, S135T, A126G, S273Y and Q240E; A51I and K221T; or A51V, K221V, N50D, N144H, N160D, S201D, Q242E, M267E and Q280E.

8. A process according to claim 4, wherein the polypeptide having chymosin activity comprises the sequence set out in SEQ ID NO: 4 carrying one of the following mutations: F223C, F223D, F223E, F223L, F223M, F223N, F223Q, F223V, F223Y, F223I, Q288G, Q288H, Q288N, Q288R, Q288S, D290A, D290G, D290L, D290M, D290Q, D290S, D290T, L295F, L295I, L295K, L295M, L295R, L295T, L295Y, L295W, I297T or I297V.

9. Process according to claim 1, wherein the polypeptide having chymosin activity is derived from a mammal.

10. Process according to claim 1, wherein the polypeptide having chymosin activity is derived from a cow.

11. Process according to claim 1, wherein the polypeptide having chymosin activity is derived from a camel.

12. Yogurt having a reduced ripening, comprising lactic acid bacteria and a polypeptide having chymosin activity as defined in claim 1.

13. A kit of parts comprising lactic acid bacteria and a polypeptide having chymosin activity as defined in claim 1.

14. A polypeptide having chymosin activity as defined in claim 1 for the production of yogurt.

15. A polypeptide having chymosin activity as defined in claim 1 for reducing the ripening in yogurt or for increasing the shelf life of yogurt.

Description

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

[0026] The present invention relates to a process for the production of a fermented milk product, preferably yogurt, comprising fermenting milk with lactic acid bacteria and contacting the milk with a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin.

[0027] The inventors of the present invention found that using a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin in the production of fermented milk products results in fermented milk products, such as yogurt, having a reduced ripening.

[0028] In a preferred embodiment, the present the polypeptide having chymosin activity is capable of hydrolysing bovine alpha s1-casein at position F23F24 so as to form s1-I CN (f24-199) more rapidly than camel chymosin.

[0029] In another preferred embodiment, the C/P ratio is 2, for example 5, for example 10, such as 10 times higher than the C/P ratio of bovine chymosin. A polypeptide having chymosin activity of the invention may typically have a high specific milk clotting activity (C) and a low general, i. e. non-specific, proteolytic activity (P) with regard to milk proteins. Accordingly, the C/P ratio should preferably be as high as possible, as a relatively high P-value, during the yogurt manufacturing process and during shelf life of the yogurt will lead to the formation of low molecular peptides and free amino acids, which in turn may confer to the yogurt an undesirable bitter taste. C/P ratio may be expressed as a relative C/P ratio, for example in relation to a chymosin such as the bovine chymosin of SEQ ID NO: 2.

[0030] In a preferred embodiment, the present polypeptide having chymosin activity has an amino acid sequence which; [0031] a. when aligned with the chymosin comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue corresponding to amino acid 51 and/or 221; and/or [0032] b. when aligned with SEQ ID NO: 4 comprises at least one substitution of an amino acid residue located in the S2 binding pocket, for example at a position corresponding to amino acid 223.

[0033] Such a protein will typically be: capable of hydrolysing bovine alpha s1-casein at position F23F24 so as to form s1-I CN (f24-199) more rapidly than camel chymosin; and have a C/P ratio higher than the C/P ratio of bovine chymosin.

[0034] Thus, at a position corresponding to amino acid 51 and/or 221 as defined with reference to SEQ ID NO: 2, a different amino acid may be present than is present at amino acid 51 and/or 221 in SEQ ID NO: 2. Thus, at a position corresponding to an amino acid in the S2 binding pocket as defined with reference to SEQ ID NO: 4, a different amino acid may be present than is present at that position within the S2 binding pocket in SEQ ID NO: 4.

[0035] Typically, a polypeptide having chymosin activity in the invention is a polypeptide having at least about 65% homology with SEQ ID NO: 2 or SEQ ID NO: 4, for example at least about 70% homology with SEQ ID NO: 2 or SEQ ID NO: 4, such as a least about 75% homology with SEQ ID NO: 2 or SEQ ID NO: 4, such as at least about 80% homology with SEQ ID NO: 2 or SEQ ID NO: 4, for example at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% homology with SEQ ID NO: 2 or SEQ ID NO: 4.

[0036] Increased early development of fermented milk is related to the affinity of a specific region of alpha s1-casein to the different binding pockets in the peptide-binding groove of the coagulant. Accordingly, herein are described amino acids in the S2 binding pocket (Schechter en Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162) relevant for this affinity and this affinity may be modulated by altering amino acids in this pocket in order to increase the rate of hydrolysis of bovine alpha s1-casein at position F23F24 so as to form s1-I CN (f24-199).

[0037] For example, introduction of a different amino acid side chain at position V223, as defined with reference to the sequence of the bovine chymosin, leads to a reduced first cut in alpha s1-casein. However, any chymosin with an amino acid change at the corresponding position, and other neighboring positions in the S2 binding pocket may lead to an altered affinity of the alpha s1-casein for the chymosin and a change in the kinetics of the first cut in alpha s1-casein.

[0038] Amino acid changes at the positions that are part of the S2 binding pocket are, most notably, T219, F223, Q288, D290, L295 and 1297 in camel chymosin or amino acids at equivalent positions in other mammalian chymosins (e.g. T219, V223, Q288, E290, K295 and 1297 in bovine chymosin), may also modulate the affinity of the alpha s1-casein for the chymosin, and thus alter the kinetics of the first cut in alpha s1 casein.

[0039] Accordingly, preferred polypeptides having chymosin activity of the invention may comprise the sequence set out in SEQ ID NO: 4 carrying one of the following mutations:

[0040] F223C, F223D, F223E, F223L, F223M, F223N, F223Q, F223V, F223Y, F223I

[0041] Q288G, Q288H, Q288N, Q288R, Q288S

[0042] D290A, D290G, D290L, D290M, D290Q, D290S, D290T

[0043] L295F, L295I, L295K, L295M, L295R, L295T, L295Y, L295W

[0044] I297T, 1297V

[0045] Combinations of such mutations at different positions may be used.

[0046] We describe that changes in amino acids A51 and K221 within the bovine chymosin amino acid sequence are important for an increased C/P. Introduction of these changes in a coagulant of choice will lead to a higher C/P value and therefore increased storage stability of the made yogurt.

[0047] Other positions that may be substituted (as defined with reference to the bovine sequence of SEQ ID NO: 2) are 48, 50, 61, 62, 109, 117, 126, 135, 144, 160, 161, 201, 202, 203, 221, 240, 242, 244, 254, 267, 280, 292 or 295. One or more of these positions may be changed so that it is different from the amino acid at that position as defined with reference to the bovine sequence of SEQ ID NO: 2.

[0048] Preferred chymosin polypeptides of the invention may comprise the sequence set out in SEQ ID NO: 2 carrying one of the following mutations or combinations of mutations:

[0049] A51V;

[0050] K221L;

[0051] K221M;

[0052] K221V;

[0053] V223Q;

[0054] A51V and K221V;

[0055] A51V and K221M;

[0056] A51V, K221V, S135T, A126G, S273Y and Q240E;

[0057] A51I and K221T; or

[0058] A51V, K221V, N50D, N144H, N160D, S201D, Q242E, M267E and Q280E

[0059] According to the invention, two properties are combined that permits, for example, the construction of a calf chymosin variant having higher C/P value useful or the construction of a camel chymosin variant having capable of more rapidly hydrolysing bovine alpha s1-casein at position F23F24 so as to form s1-1 CN.

[0060] Preferably, preferred polypeptides having chymosin activity of the invention are derived from a mammal, more preferably from a cow or from a camel.

[0061] Fermenting milk with lactic acid bacteria and contacting the milk with a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin may be carried at the same time. Alternatively, the milk is first contacted with the polypeptide having chymosin activity, and thereafter fermented with lactic acid bacteria. Vice versa, the milk is first fermented with lactic acid bacteria, and thereafter contacted with the polypeptide having chymosin activity. Alternatively, a part of the milk is contacted with a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin while another part of the milk is fermented with lactic acid bacteria, where after both parts can be combined to provide the fermented milk product, preferably yogurt.

[0062] Preferably the present lactic acid bacteria selected from the group consisting of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbruekii subsp. bulgaricus, Streptococcus thermophilus or Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus and Bifidobacterium breve. More preferably the present lactic acid bacteria comprise Streptococcus thermophilus and Lactobacillus delbruekii subsp. Bulgaricus. This combination of strains is advantageous in for instance a process for the production of a fermented milk product such as yogurt or in the final properties of the fermented milk product such as yogurt. These strains may for instance further improve the acidification speed or they may confer certain flavours.

[0063] Lactobacillus delbrueckii ssp. bulgaricus, when present in the method of the invention, may constitute between 0.1% and 10% of the total cfu's of the present lactic acid bacteria, preferably between 0.2% and 5%, more preferably between 0.5% and 2%, more preferably between 0.8 and 1.2%, most preferably 1%.

[0064] In one embodiment, the process of the invention provides a fermented milk product wherein one or more textural attributes selected from the group consisting of rheology, appearance, the structure, mouthfeel, the after feel of the fermented milk product, preferably yogurt, has been improved. Most preferably, the invention provides a process wherein the rheology attributes of the fermented milk product, preferably yogurt, more preferably the Brookfield and/or the shear stress has been improved. In another preferred embodiment, the invention provides a process wherein the appearance of the fermented milk product, preferably yogurt, more preferably the shininess and/or the whiteness has been improved. In yet another preferred embodiment, the invention provides a process wherein the structure of the fermented milk product, preferably yogurt, more preferably the visual aspects such as ropiness and/or visual thickness and/or smoothness has been improved. In another preferred embodiment, the invention provides a process wherein the mouthfeel of the fermented milk product, preferably yogurt, more preferably the thickness and/or the creaminess and/or the sliminess and/or the melting and/or the astringency has been improved. In another preferred embodiment, the invention provides a process wherein the after feel of the fermented milk product, preferably yogurt, more preferably the astringency and/or the pungency and/or the fat coating has been improved.

[0065] Highly preferably, the invention provides a process of the invention wherein two or more, preferably three or more, more preferably four or more textural attributes selected from the group consisting of Brookfield and the shear stress and the shininess and the whiteness and ropiness and visual thickness and smoothness and the thickness and the creaminess and the astringency and the pungency and the fat coating has been improved.

[0066] The most preferred fermented milk product that is produced by the process of the invention is yogurt as defined hereinbefore.

[0067] The milk that may be used in the process of the invention, may be any milk suitable for the production of a fermented milk product, such as yogurt. Milk has been defined hereinbefore and may encompass milks from mammals and plant sources or mixtures thereof. Preferably, the milk is from a mammal source. Mammal 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. In addition, the term milk refers to not only whole milk, but also skim milk or any liquid component derived thereof. The fat content in the milk and in the subsequent fermented milk product, such as yogurt, may be as is known in the prior and as is referred in the background of the invention.

[0068] In a preferred embodiment, the present process comprising a step of inactivating the polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin. For example inactivating by heat treatment.

[0069] According to another aspect, the present invention relates to yogurt, preferably yogurt having a reduced ripening or having a reduced proteolysis, comprising lactic acid bacteria and comprising a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin as defined before. Preferably, the present yogurt has a reduced ripening in comparison with a similar yogurt which comprises bovine chymosin. More preferably, the present yogurt has an increased texture shelf stability in comparison with a similar yogurt which comprises bovine chymosin.

[0070] According to another aspect, the present invention relates to a kit of parts comprising lactic acid bacteria and comprising a polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin as defined before.

[0071] According to yet another aspect, the present invention relates to the use of the present polypeptide having chymosin activity having a C/P ratio higher than the C/P ratio of bovine chymosin as defined before producing yogurt, preferably for reducing the ripening in yogurt or for increasing the shelf life of yogurt such as for increasing the texture shelf stability of yogurt. More preferably the present invention relates to the use of the present polypeptide for producing a yogurt having a shelf life of more than 4, more than 5, or more than 6 weeks. Preferably more than 4, more than 5, or more than 6 weeks when stored under standard conditions in a refrigerator. Alternatively, the present invention relates to the use of the present polypetide for increasing the shelf life of yogurt with at least 1, 2, 3, 4 or 5 weeks. More specifically, the present invention relates to the use of the present polypeptide for increasing the shelf life of yogurt with at least 1, 2, 3, 4 or 5 weeks in comparison with yogurt prepared with cow chymosin, preferably cow chymosin according to the amino acid sequence as shown in SEQ ID No 2.

Materials and Methods

1. Bacterial Strains.

[0072] Starter culture Delvo Yog for stirred yogurt was used as direct inoculation starter, obtained from DSM Food Specialties B.V.

2. Chymosin.

[0073] Maxiren calf chymosin obtained from DSM Food Specialties B.V, and the polypeptides of the invention made as disclosed in WO2013164481.

3. Yogurt Preparation

[0074] Stirred yogurt was made using a flow pasteurizer and a smoothener with back cooling. The milk composition was 3.4% fat and 3.5% protein and the starter culture was used. The rennets were added alongside the starter culture with a dosage of 0.0002% based on a formulation of 180 IMCU/mls. The fermentation temperature was 42 C. The acidification was monitored using Cinac at the same temperature. Once a pH of 4.6 was reached, the yogurts were smoothened and filled out in suitable containers. The containers were then stored at 4 C.

4. Sensory Analysis of Yogurts

[0075] The sensory panel consisted of 8 members who had a specific training in sensory evaluation of yogurts. Products were presented in 3-digit coded, white plastic isothermal cups stored at 4 C. The samples were approximately 10 C. when they were tested. Panellists were provided with mineral water and plain crackers for palate cleaning between samples. The sessions were carried out in a temperature controlled room at 20 C. under white lighting in individual booths.

[0076] Data acquisition was assisted by FIZZ Sensory Analysis Software. Both monadic and hedonic scales are being used to rate the flavour and the texture attributes of products. The attributes were evaluated in the following order: visual texture with a spoon, texture-in-mouth, taste and aroma.

5. Sensory Analysis of Yogurts QDA

[0077] Descriptive sensory analysis was done by using the Quantitative Descriptive Analysis Method (Stone, H. and Sidel, J. L. Sensory Evaluation Practises 3.sup.rd dition, 2004). First, the panellists developed a list of attributes including definitions by means of evaluating a wide variety of references and a wide array of yogurts. Secondly, training sessions were organized to enable panellists to learn to consistently differentiate and replicate the yogurt samples. During the actual QDA measurements the intensities of the selected attributes were obtained per product by the FIZZ (Biosystems; France) sensory data acquisition system, using unstructured line scales ranging from 0-100. The products were offered semi-monadically and evaluated twice by the panellists (n=14) by means of a Balanced Complete Block design to avoid sequence effects. Statistical analysis of the data was done by analysis of variance with Fisher's least significant difference (LSD) as a post hoc test (SenPaq) and modelled using Principle Component analysis (PCA) (SenPaq).

6. Shear Stress of Yogurt

[0078] The samples were measured using a Physica MCR501 rheometer equipped with a concentric cylinder measurement system (CC-27). A solvent trap was used to prevent evaporation of water as much as possible. The samples were slightly stirred with a spoon before loading into the rheometer. Before measurement the samples were allowed to rest and heat/cool to the measuring temperature (25 C.) for 5 minutes. A standard experimental protocol was applied consisting out of the following two measuring sequences: [0079] 1. A strain sweep to determine the initial gel strength (dynamic shear modulus): this is an oscillatory test where at a fixed angular frequency (omega=10 rad/s) an increasing amplitude is applied: on a logarithmic scale the amplitude is increased from 0.01 to 100% with 5 measuring points per decade. [0080] 2. After the strain sweep the yogurts are allowed to rest for 30 seconds in the rheometer and subsequently a shear rate sweep is applied to determine the shear stress in mouth: This consists of applying an increasing shear rate to the yogurts ranging from 0.001 to 1000 s.sup.1 on a logarithmic scale with 3 measuring points per decade (no fixed time setting: the rheometer software determines the required shearing time per measuring point).

[0081] This experiment gives a flow curve whereby the measured stress is plotted as a function of the applied shear rate. This curve can then be combined with literature data to determine the relevant shear stress in the mouth as explained in the following.

[0082] By sensory panelling of various food products Shama and Sherman identified windows of instrumental shear stresses and shear rates corresponding to products with similar thickness ratings but different shear-thinning behavior. These windows correspond to the rheological regimes applied in the mouth during thickness rating. The governing shear rate was shown to be dependent on the viscosity of the product itself. (see FIG. 1 from Shama, F. and Sherman, P. Journal of Texture Studies, 4, 111-118. (1973), Identification of stimuli controlling the sensory evaluation of viscosity II oral methods).

[0083] For the yogurts of the example below the (predicted) shear stress in the mouth is determined by plotting the experimentally measured flow curves (measured shear stress in function of applied shear rate of the shear rate sweep experiment described above) onto the aforementioned FIG. 1 from Shama and Sherman. The predicted shear stress in the mouth is defined as the cross-over between the measured flow curves and the upper bound of the shear rateshear stress windows of FIG. 1 of Shama and Sherman. In FIG. 2 the authors give examples for various food stuffs. The thus derived shear stress gave a good correlation with the sensory perception of thickness in the mouth.

7. Brookfield

[0084] Viscosity measurements were performed using a Brookfield RVDVII+Viscometer, which allows viscosity measurement on an undisturbed product (directly in the pot). The Brookfield Viscometer determines viscosity by measuring the force required to turn the spindle into the product at a given rate. The Helipath system with a T-C spindle was used as it is designed for non-flowing thixotropic material (gels, cream). It slowly lowers or raises a rotating T-bar spindle into the sample so that not always the same region of the sample is sheared (helical path). Thus, the viscometer measures constantly the viscosity in fresh material and is thus considered to be the most suitable for measuring stirred yogurt viscosity. A speed of 30 rpm was used for 31 measuring points, at an interval of 3 seconds. The averages of the values between 60 and 90 seconds are reported.

EXAMPLES

Example 1

[0085] Yogurt was prepared as in the materials and methods with a polypeptide of the invention and the results are shown in table 1 below.

TABLE-US-00001 Polypeptide of the Maxiren invention Predicted shear stress in the mouth (Pa) 18.95 19.08 Quality of yogurt after 4 weeks Good Good Quality of yogurt after 5 weeks Overripe Good Quality of yogurt after 6 weeks Overripe Good

[0086] The above results show that the predicted shears stress is more or less comparable, whereas the yogurt produced with the present polypeptide provides a reduced ripening of the yogurt.