Patent Description:
Traditional starter cultures for fermented milk have been mainly developed for western countries (Europe, North America). The fermentative pathway is typically carried out by two different bacteria: Streptococcus thermophilus (ST) and Lactobacillus delbruekii subsp. bulgaricus.

Texture is a very important quality parameter for fermented milks and consumers continuously request fermented milk products having new taste and/or new texture. Among important texture descriptors for fermented products, high mouthfeel, long texture and smoothness are well liked by many consumers. There is therefore a need to improve the rheological properties of fermented milks.

Improvement of rheological properties of fermented milks can be obtained by the use of thickening agents, such as alginates. However, there is trend for more natural products perceived as healthy by the consumers, with a minimal of added ingredients (additive-free yoghurts).

<CIT> and <CIT> applications describe cultures comprising respectively Streptococcus thermophilus and Lactobacillus fermentum strains, and Streptococcus thermophilus and Lactobacillus johnsonii strains, and their use in the manufacture of low-fat fermented milk products. According to these applications, the replacement of the conventionally used Lactobacillus delbruekii subsp. bulgaricus by either Lactobacillus fermentum or Lactobacillus johnsonii - in combination with Streptococcus thermophilus strain(s) - would lead to low-fat fermented milk products having improved rheological properties (higher viscosity) and lower post-acidification.

<NPL> discloses two strains of L plantarum, namely SF2A35B and Lp90, showing a ropy phenotype. The ropy phenotype was shown to be related to a high production of exopolysaccharides. The strains were not used in yogurt production. <CIT> discloses that strain SF2A35B is identical to <NUM> deposited under CBS125104. The strain was used in the preparation of fermented soy products.

NACHTIGALL, CARSTEN ET AL (<NUM>-<NUM>-<NUM>), discloses that a lactic acid bacterium producing exopolysaccharides does not automatically produce a ropy texture.

There is still a need for new means to manufacture fermented products with differentiation in texture, in particular high mouth and spoon thickness and high stickiness, while limiting the presence of additives in the fermented products.

A first aspect of the invention relates to a Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April 26th, <NUM>, which - when inoculated into milk - generates a fermented milk presenting a high thickness and a high ropiness and a high thickness in mouth, in particular presenting three of the following rheological features, as assayed by test A: a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa; b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>.

A second aspect of the invention relates to a Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April 26th, <NUM> which - when inoculated into milk - generates a fermented milk presenting a high thickness and a high ropiness and high thickness in mouth, and further characterized by being a low post acidification strain at the temperature of fermentation.

A third aspect of the invention relates to a bacterial composition comprising or consisting of a Lactobacillus plantarum strain of the invention, in particular in combination with at least another lactic acid bacteria.

A fourth aspect of the invention relates to a composition comprising or consisting of the L. plantarum strain of the invention or the bacterial composition of the invention and a booster.

A fifth aspect of the invention relates to a kit-of-part comprising or consisting of the L. plantarum strain of the invention or the bacterial composition of the invention and a booster.

A sixth aspect of the invention relates to a method for manufacturing a fermented product, comprising inoculating a substrate with the L. plantarum strain of the invention, the bacterial composition of the invention, the composition of the invention or the kit-of part of the invention, and fermenting said inoculated substrate, to obtain a fermented product.

A seventh aspect of the invention relates to a fermented product comprising the L. plantarum strain of the invention.

An eight aspect of the invention relates to the use of the L. plantarum strain of the invention, the bacterial composition of the invention, the composition of the invention or the kit-of part of the invention, in the manufacture of a fermented dairy product.

The inventors have surprisingly identified a lactic acid bacterium which can be used to obtain a fermented milk with unique texture and rheological properties. This lactic acid bacterium is a Lactobacillus plantarum strain as claimed (L. plantarum), and can be used to provide a fermented milk with high thickness and high ropiness and high thickness in mouth.

The invention is directed to a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting three of the following rheological features, as assayed by test A:.

For the avoidance of doubt, a Lactobacillus plantarum strain is defined herein as described in the literature, in particular in <NPL>.

The rheological parameters described herein have been assayed using the following Test A (also described in example <NUM>). The shear stress value measured at shear rate <NUM>-<NUM> as defined herein and the shear stress value measured at shear rate <NUM>-<NUM> as defined herein are - as calculated by Test A - with a standard deviation of ± <NUM> Pa, between replicates within the same experiment. The difference value of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> as defined herein is - as calculated by Test A - with a standard deviation of ± <NUM>, between replicates within the same experiment.

Thus, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> as defined herein and a shear stress measured at shear rate <NUM>-<NUM> as defined herein and a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> as defined herein, measured under the following conditions:.

<NUM> UHT milk (<NUM>% protein, <NUM>% fat) added with <NUM>% sucrose and <NUM>% of yeast extract powder (LP0021) from Oxoid™ is heat treated at <NUM> for <NUM> and cooled down to room temperature. A culture of each strain to be tested is inoculated into milk at <NUM><NUM> CFU/ mL. The inoculated milk is then fermented at <NUM> to reach pH <NUM> (typically about <NUM>-<NUM>). After fermentation, the sample is stirred by a rotator (RZR <NUM> control, Heidolph) at <NUM> rpm for <NUM> and stored at <NUM> overnight, before measurement by rheometer. The used tank and bladder are as disclosed in <FIG>. The sample is assessed by a rheometer (Anton Paar MCR <NUM>, CC27-SN27450, Austria) using a coaxial cylinder C-CC27-T200/SS and a bob-cup. The rheometer is set to a constant temperature of <NUM> during the measurement. Settings are as follows:.

The shear stress measured at shear rate <NUM>-<NUM> represents the thickness of the fermented milk. According to the invention, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, as assayed by test A described herein. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa. In an embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa. The values or ranges of shear stress measured at shear rate <NUM>-<NUM> as claimed are disclosed in combination with any values or ranges of shear stress measured at shear rate <NUM>-<NUM> claimed and any values or ranges of Δ of the shear stress measured at <NUM>-<NUM> - the shear stress measured at <NUM>-<NUM> claimed. These values and ranges are assayed by test A described herein.

The shear stress measured at shear rate <NUM>-<NUM> represents the coating or the mouth thickness (also called the thickness in mouth) of the fermented milk. According to the invention, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa as assayed by test A described herein. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa. In an embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa. The values or ranges of shear stress measured at shear rate <NUM>-<NUM> claimed are disclosed in combination with any values or ranges of shear stress measured at shear rate <NUM>-<NUM> as claimed and any values or ranges of Δ of the shear stress measured at <NUM>-<NUM> - the shear stress measured at <NUM>-<NUM> claimed.

These values and ranges are assayed by test A described herein.

The difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> (Δ shear stress <NUM>-<NUM> - <NUM>-<NUM>) represents the ropiness or stickiness of the fermented milk. According to the invention, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> higher than <NUM>. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> higher than <NUM>. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> higher than <NUM>. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> higher than <NUM>. In an embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> in the range <NUM>-<NUM>. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> in the range <NUM>-<NUM>. In a particular embodiment, the claimed Lactobacillus plantarum strain has the ability to generate a fermented milk presenting a Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> in the range <NUM>-<NUM>. The values or ranges of Δ shear stress <NUM>-<NUM> - <NUM>-<NUM> as claimed are disclosed in combination with any values or ranges of shear stress measured at shear rate <NUM>-<NUM> as claimed and any values or ranges of shear stress measured at shear rate <NUM>-<NUM> as claimed. These values and ranges are assayed by test A described herein.

In an embodiment, the invention concerns a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa.

In an embodiment, the invention concerns a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa.

In an embodiment, the invention concerns a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>, in particular higher than <NUM>, in particular higher than <NUM>, or in the range <NUM>-<NUM>, in particular <NUM>-<NUM>, in particular <NUM>-<NUM>.

In an embodiment, the invention concerns a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa, or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa; and b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa. In a particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa and b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa. In another particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa and b) a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa.

In an embodiment, the invention concerns a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa, or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>, in particular higher than <NUM>, in particular higher than <NUM>, in particular higher than <NUM>, or in the range <NUM>-<NUM>, in particular <NUM>-<NUM>, in particular <NUM>-<NUM>. In a particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>. In another particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> in the range <NUM>-<NUM>.

In an embodiment, the invention concerns a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>, in particular higher than <NUM>, in particular higher than <NUM>, in particular higher than <NUM>, or in the range <NUM>-<NUM>, in particular <NUM>-<NUM>, in particular <NUM>-<NUM>. In a particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>. In another particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting b) a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> in the range <NUM>-<NUM>.

The invention concerns a Lactobacillus plantarum strain as claimed which - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa, or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa; b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa, in particular higher than <NUM> Pa or in the range <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa, in particular <NUM>-<NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>, in particular higher than <NUM>, in particular higher than <NUM>, in particular higher than <NUM>, or in the range <NUM>-<NUM>, in particular <NUM>-<NUM>, in particular <NUM>-<NUM>. In a particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa; b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>. In another particular embodiment, said Lactobacillus plantarum strain as claimed - when inoculated into milk - generates a fermented milk presenting a) a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa; b) a shear stress measured at shear rate <NUM>-<NUM> in the range <NUM>-<NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> in the range <NUM>-<NUM>.

Any Lactobacillus plantarum strain as claimed fulfilling the three rheological feature(s) defined in the claims is part of the invention. The invention has been exemplified with the DSM32493 strain, deposited at the DSMZ. There is no reason to doubt that other L. plantarum strains sharing the three rheological feature (s) defined herein with the DSM32493 strain exist, with the possibility of using the DSM32493 strain as a positive control.

In a particular embodiment, the invention is directed to the Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April <NUM>th, <NUM>.

In a particular embodiment, the invention also concerns a variant of DSM32493 as claimed. By "variant of DSM32493 strain", it means a Lactobacillus plantarum strain derived from the DSM32493 strain and which generates a fermented milk presenting the three rheological features described herein (as assayed by test A), i.e., the three rheological features: a) a shear stress measured at shear rate <NUM> as claimed and b) a shear stress measured at shear rate <NUM>-<NUM> as claimed and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> as claimed. The DSM32493 strain variant as claimed is able to generate a fermented milk presenting, as assayed by test A, a) a shear stress measured at shear rate <NUM>-<NUM> (thickness) higher than <NUM> Pa; b) a shear stress measured at shear rate <NUM>-<NUM> (coating/mouth thickness) higher than <NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> (ropiness/stickiness) higher than <NUM>. In a particular embodiment, the DSM32493 strain variant as claimed is able to generate a fermented milk presenting, as assayed by test A, a) a shear stress measured at shear rate <NUM>-<NUM> (thickness) higher than <NUM> Pa; b) a shear stress measured at shear rate <NUM>-<NUM> (coating/mouth thickness) higher than <NUM> Pa; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> (ropiness/stickiness) higher than <NUM>. In a particular embodiment, the DSM32493 strain variant keeps the properties of the DSM32493 strain, with respect to generating a fermented milk presenting the three rheological features, as assayed by test A, a) a shear stress measured at shear rate <NUM>-<NUM> as claimed b) a shear stress measured at shear rate <NUM>-<NUM> as claimed and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> as claimed. By "keep the properties", it is meant that the values of the rheological features obtained using the DSM32493 variant are at least the values obtained using the DSM32493 strain. The definitions and specific embodiments detailed for the rheological features under the L. plantarum strain characterization apply similarly in the context of any variant of DSM32493 strain claimed, in particular for the combination of the rheological features of the minimal values and ranges of the shear stress measured at shear rate <NUM>-<NUM>, the minimal values and ranges of the shear stress measured at shear rate <NUM>-<NUM>, the minimal values and ranges of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> claimed.

A variant of DSM32493 as claimed is a Lactobacillus plantarum strain presenting at least one mutation, such as the addition, deletion, insertion and/or substitution of at least one nucleotide in its genome as compared to the DSM32493 strain, wherein the genome sequence of the variant has an identity of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% to the genome sequence of the DSM32493 strain and wherein the variant is.

The invention is also directed to a Lactobacillus plantarum as defined herein (including variant of the DSM32493 strain), which - in addition to provide a fermented milk presenting the three of the following rheological features, as assayed by test A, a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa as defined herein; b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa as defined herein; c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM> as defined herein - is further characterized by being a low post-acidification strain at fermentation temperature.

By "low post acidification strain at fermentation temperature", it is meant a L. plantarum strain of the invention which - when inoculated into milk - generates a fermented milk, the pH of which - after fermentation - does not fluctuate more than <NUM> unit when the fermented milk is kept at the fermentation temperature as assayed by test B (see below). The acidification kinetics is determined by the continuous recording of the pH as a function of time, for example using a Cinac system (<NPL>).

The acidification kinetics of milk inoculated with the low post-acidification L. plantarum strain of the invention and brought at the fermentation temperature follows a two-phase profile, comprising an initial period of sigmoidal pH decrease down to a pH value between <NUM> and <NUM> (to generate the fermented milk), followed by a second period in which the pH value does not fluctuate more than <NUM> unit (when the fermented milk is kept at the fermentation temperature). To characterize the low post acidifying feature of the L. plantarum strain of the invention, the pH is measured once the fermentation step terminates (end of the initial period), the fermented milk is kept at the fermentation temperature, the pH is measured at least <NUM> hours later, and the evolution of the pH (difference) is measured. In a particular embodiment, the fermentation temperature is <NUM>.

In an embodiment, the pH obtained at the end of the initial period is comprised between <NUM> and <NUM>. In a particular embodiment, the pH obtained at the end of the initial period is comprised between <NUM> and <NUM>. In a particular embodiment, the pH obtained at the end of the initial period is comprised between <NUM> and <NUM>. In a particular embodiment, the pH obtained at the end of the initial period is comprised between <NUM> and <NUM>.

In a particular embodiment, the low post-acidification strain of the invention is able to generate a fermented milk, the pH of which does not fluctuate more than <NUM> unit, in particular not more than <NUM> unit, in particular not more than <NUM> unit, when kept at the fermentation temperature for a period of <NUM> hours after the end of the initial period.

In a particular embodiment, the low post acidifying feature characterization is carried out, by assaying Test B described below (see also example <NUM>):.

UHT milk (<NUM>% protein, <NUM>% fat) added with <NUM>% sucrose and <NUM>% of yeast extract powder (LP0021) from Oxoid™ is heat treated at <NUM> for <NUM> and cooled down to room temperature. A culture of each strain to be tested is inoculated into milk at <NUM><NUM> CFU/ mL and the inoculated milk placed at <NUM> (t=<NUM>). The inoculated milk is fermented until a pH of <NUM> is reached (tpH <NUM>) and kept at <NUM> for at least <NUM> hours, and the pH of the milk continuously monitored. The delta pH (ΔpH = pH at (tpH4. <NUM>+<NUM>) - pH <NUM>) is used to represent the post-acidification at fermentation temperature.

In a particular embodiment of a low post-acidification strain of the invention is a L. plantarum strain having at least one mutation in its ATP-synthase operon (for example point mutation, deletion, insertion,. ), such that the strain has a reduced H+-ATPase activity. The wild-type sequence of the ATP-synthase operon is as set forth in SEQ ID NO:<NUM>. The person skilled in the art knows how to determine whether this operon is mutated and how to measure the H+-ATPase activity of a bacterium [see for example <NPL>]. In a particular embodiment, a low post-acidification L. plantarum strain of the invention as defined herein has at least one mutation in the ATP synthase alpha subunit gene of the ATP-synthase operon (herein referred as "the ATP synthase alpha subunit gene"). In a particular embodiment, a low post-acidification L. plantarum strain of the invention as defined herein has at least one mutation in the ATP synthase alpha subunit gene of the ATP-synthase operon as defined in SEQ ID NO:<NUM>. In a particular embodiment, in combination with the previous embodiment on SEQ ID NO:<NUM> or not, the ATP synthase alpha subunit gene of the low post-acidification L. plantarum strain of the invention as defined herein encodes a ATP synthase alpha subunit protein having an aspartic acid residue at position <NUM>. In a particular embodiment, the ATP synthase alpha subunit protein of the low post-acidification L. plantarum strain of the invention as defined herein is as defined in SEQ ID NO:<NUM>. In a particular embodiment, in combination with the previous embodiment on SEQ ID NO:<NUM> or not, the ATP synthase alpha subunit gene of the low post-acidification L. plantarum strain of the invention as defined herein bears the mutation G to A at its position <NUM> (changing the glycine residue at position <NUM> by an aspartic acid residue). In a particular embodiment, the ATP synthase alpha subunit gene of the low post-acidification L. plantarum strain of the invention as defined herein is as defined in SEQ ID NO:<NUM> (wherein the codon GGT at positions <NUM>-<NUM> is changed to GAT).

In an embodiment, the invention is directed to a low post-acidification variant as claimed, which is a variant of the Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April <NUM>th, <NUM>. In a particular embodiment, the invention is directed to a low post-acidification variant of the Lactobacillus plantarum strain DSM32493 as claimed, wherein said variant bears a mutation in the ATP synthase alpha subunit gene (as compared to the DSM32493 strain), in particular bears the mutation G to A at position <NUM>.

The definitions and specific embodiments detailed for the rheological features under the L. plantarum strain characterization (including the variant of DSM32493 strain) apply similarly in the context of the low post-acidification variant, in particular for but not limited to, the minimal values and ranges of the shear stress measured at shear rate <NUM>-<NUM>, the minimal values and ranges of the shear stress measured at shear rate <NUM>-<NUM>, the minimal values and ranges of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> claimed.

The invention is also directed to a bacterial composition comprising or consisting of a L. plantarum strain of the invention (in particular a low post-acidification L. plantarum strain of the invention). In a particular embodiment, the bacterial composition is a pure culture, i.e., comprises or consists of a single bacterium strain. In another embodiment, the bacterial composition is a mixed culture, i.e., comprises or consists of the L. plantarum strain of the invention and at least one other bacterium strain. Thus, in an embodiment, a bacterial composition of the invention comprises or consists of a L. plantarum strain of the invention and at least one lactic acid bacterium, in particular at least another Lactobacillus plantarum strain and/or a Lactobacillus delbrueckii subsp bulgaricus strain. By "at least" (in reference to bacterium strain, lactic acid bacterium or another Lactobacillus plantarum strain), it is meant <NUM> or more, and in particular <NUM>, <NUM>, <NUM>, <NUM> or <NUM> strains. Thus, in an embodiment, the composition of the invention comprises or consists of - in addition to the L. plantarum strain of the invention, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> strains, in particular <NUM>, <NUM>, <NUM>, <NUM> or <NUM> lactic acid bacteria strains. In a particular embodiment, the bacterial composition of the invention does not contain Streptococcus thermophilus strain(s).

In a particular embodiment, the bacterial composition as defined herein, either as a pure or mixed culture as defined above is under frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder. In a particular embodiment, the bacterial composition of the invention is in a frozen format or in the form of pellets or frozen pellets, in particular contained into one or more box or sachet. In another embodiment, the bacterial composition as defined herein is under a powder form, such as a dried or freeze-dried powder, in particular contained into one or more box or sachet.

In a particular embodiment, the bacterial composition of the invention, either as a pure culture or mixed culture as defined above, and whatever the format (frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder) comprises the L. plantarum strain of the invention in a concentration comprised in the range of <NUM><NUM> to <NUM><NUM> cfu (colony forming units) per gram of the bacterial composition. In a particular embodiment, the concentration of the L. plantarum within the bacterial composition of the invention is in the range of <NUM><NUM> to <NUM><NUM> cfu per gram of the bacterial composition, and in particular at least <NUM><NUM>, at least <NUM><NUM>, at least <NUM><NUM>, at least <NUM><NUM> or at least <NUM><NUM> CFU/g of the bacterial composition. In a particular embodiment, when in the form of frozen or dried concentrate, the concentration of the Lactobacillus plantarum strain - as pure culture or as a mixed culture - within the bacterial composition is in the range of <NUM><NUM> to <NUM><NUM> cfu/g of frozen concentrate or dried concentrate, and more preferably at least <NUM><NUM>, at least <NUM><NUM>, at least <NUM><NUM>, at least <NUM><NUM> or at least <NUM><NUM> cfu/g of frozen concentrate or dried concentrate.

The invention is also directed to a composition or a kit-of-part comprising or consisting of a L. plantarum strain of the invention (in particular a low post-acidification L. plantarum strain of the invention) or a bacterial composition as defined herein (such as pure or mixed culture) and a booster. In a particular embodiment, said booster is a yeast extract or an amino-acid containing composition. In a particular embodiment, the booster is a yeast extract, such as a yeast extract powder. Any yeast extract (or powder) can be used, for example but not limited to the yeast extract powder LP0021 from Oxoid™.

The expression "A composition comprising or consisting of a L. plantarum strain of the invention or a bacterial composition of the invention and a booster" means that the L. plantarum or the bacterial composition and the booster are physically mixed together. The booster amount within the composition is such that the booster is inoculated into milk in the range of <NUM>% to <NUM>%. In an embodiment, the composition is under frozen format or in the form of frozen pellets. In another embodiment, the composition is under dried or freeze-dried format or in the form of a powder or dried powder. In contrast, the expression "A kit-of-part comprising or consisting of a L. plantarum strain of the invention or a bacterial composition of the invention and a booster" means that the L. plantarum or the bacterial composition and the booster are physically separated but intended to be used together. Thus, the L. plantarum or the bacterial composition and the booster are in different boxes or sachets. In an embodiment, the L. plantarum or the bacterial composition and the booster are both under frozen format or in the form of frozen pellets. In another embodiment, the L. plantarum or the bacterial composition and the booster are both under dried or freeze-dried format or in the form of a powder or dried powder.

The invention also concerns a method for manufacturing a fermented product, comprising a) inoculating a substrate with a L. plantarum strain of the invention (in particular a low post-acidification L. plantarum strain of the invention) and b) fermenting said inoculated substrate, to obtain a fermented product. In a particular embodiment, the L. plantarum strain of the invention is inoculated as a bacterial composition as defined herein, such as a pure culture or a mixed culture. In an embodiment, the L. plantarum strain of the invention is inoculated as a composition as defined herein, i.e., step a) comprises or consists in inoculating the bacterial composition as defined herein. In another embodiment, the L. plantarum strain of the invention is inoculated as a kit-of-part as defined herein i.e., step a) comprises or consists in a1) inoculating the L. plantarum strain (or the bacterial composition) and a2) inoculating the booster, wherein the L. plantarum strain (or the bacterial composition) and the booster are inoculated simultaneously, or wherein the L. plantarum strain (or the bacterial composition) is inoculated before the booster or wherein the booster is inoculated before the L. plantarum strain (or the bacterial composition). In a particular embodiment, the method of the invention does not comprise inoculation of Streptococcus thermophilus strain(s). In a particular and preferred embodiment, the substrate is inoculated in step a) with a pure culture of the L. plantarum strain of the invention.

When booster is used, the booster is inoculated into milk in the range of <NUM>% to <NUM>%. In a particular embodiment, the booster is inoculated into milk in the range of <NUM> to <NUM>%. In a particular embodiment, the booster is inoculated into milk in the range of <NUM> to <NUM>%.

The fermentation time and temperature are parameters which are dependent upon the wanted final fermented product. In an embodiment, the fermentation temperature is comprised between <NUM> and <NUM>, in particular <NUM> and <NUM>, in particular between <NUM> and <NUM>. The time of fermentation is determined according to the final pH desired at the end of the fermentation, and is comprised between <NUM> and <NUM> hours, in particular between <NUM> and <NUM> hours. Thus, in an embodiment, the fermentation is carried out until a pH comprised between <NUM> and <NUM>, in particular between <NUM> and <NUM>, in particular between <NUM> and <NUM>, in particular between <NUM> and <NUM>, is obtained.

Any substrate can be used in the method of the invention. Thus, in a particular embodiment, the substrate is selected in the group consisting of milk, milk of vegetal origin (such as soy milk) or cereal flour.

In a particular embodiment, the substrate used in the method of the invention is milk substrate. Thus, in an embodiment, the invention is directed to a method for manufacturing a fermented milk product comprising a) inoculating a milk substrate with a L. plantarum strain of the invention (in particular a low post-acidification L. plantarum strain of the invention) or a bacterial composition as defined herein or a composition as defined herein or a kit-of-part as defined herein; and b) fermenting said inoculated milk substrate, to obtain a fermented milk product. By "milk substrate", it is meant milk of animal origin. In a particular embodiment, the milk substrate originates from cow, goat, sheep, buffalo, zebra, horse, donkey, or camel, and the like. The milk may be in the native state, a reconstituted milk or a skimmed milk.

In another embodiment, the substrate is of plant origin, and can be obtained from extracts of plant material. In a particular embodiment, the substrate is soy, such as soy milk. In another embodiment, the substrate is cereal flour.

The invention is also directed to a fermented product, which is obtained using a L. plantarum strain of the invention (in particular a low post-acidification L. plantarum strain of the invention) or a bacterial composition of the invention or a composition of the invention or a kit-of-part of the invention, in particular obtained or obtainable by the method of the invention. Thus, the invention is directed to a fermented product comprising the L. plantarum strain of the invention (in particular a low post-acidification L. plantarum strain of the invention). In a particular embodiment, the L. plantarum of the invention is the only bacterium found in the fermented product of the invention. In a particular embodiment, the L. plantarum of the invention is the only acidifying bacterium found in the fermented product of the invention.

In a particular embodiment, the fermented product of the invention is a fermented dairy product, in particular a fermented dairy food product or a fermented dairy feed product. In a particular embodiment, the fermented dairy food product of the invention is fresh fermented milk.

In a particular embodiment, the fermented product of the invention is a fermented soy product. In a particular embodiment, the fermented product of the invention is a fermented cereal product.

In a particular embodiment, the fermented product of the invention - in particular the fermented dairy food product as defined herein - contains the DSM32493 strain deposited at the DSMZ on April <NUM>th, <NUM> or any variant thereof as claimed. In a particular embodiment, the fermented product of the invention - in particular the fermented dairy food product as defined herein - contains any L. plantarum variant of the DSM32493 strain able to generate a fermented milk presenting, as assayed by test A, a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa as defined herein; b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa as defined herein; and c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM> as defined herein. In a particular embodiment, the fermented product of the invention - in particular the fermented dairy food product as defined herein - contains any L. plantarum variant as claimed of the DSM32493 strain keeping all three claimed rheological features of the DSM32493 strain. In a particular embodiment, the fermented product of the invention - in particular the fermented dairy food product as defined herein - contains any L. plantarum variant of the DSM32493 strain claimed keeping the rheological features of the DSM32493 strain. In a particular embodiment, the fermented product of the invention - in particular the fermented dairy food product as claimed - contains a low post-acidification variant as defined herein of the Lactobacillus plantarum strain DSM32493. In a particular embodiment, the fermented product of the invention - in particular the fermented dairy food product as defined herein - contains a low post-acidification variant of the Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April <NUM>th, <NUM>, wherein said variant bears a mutation in the ATP synthase alpha subunit gene (as compared to the DSM32493 strain), in particular bears the mutation G to A at its position <NUM>, in particular wherein its ATP synthase alpha subunit gene is as defined in SEQ ID NO:<NUM>.

The definitions and specific embodiments detailed for the rheological features under the L. plantarum strain characterization apply similarly in the context of the fermented dairy product of the invention, in particular for the minimal values and ranges of the shear stress measured at shear rate <NUM>-<NUM>, the minimal values and ranges of the shear stress measured at shear rate <NUM>-<NUM>, the minimal values and ranges of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> claimed. Similarly, the definitions and specific embodiments detailed for the L. plantarum strain characterization apply similarly in the context of the fermented dairy product of the invention, in particular for but not limited to, the DSM32493 variant claimed, the low post-acidification variant and the mutation of the ATP synthase alpha subunit gene, in particular the mutation G to A at its position <NUM>, in particular the ATP synthase alpha subunit gene as defined in SEQ ID NO:<NUM>.

The invention is also directed to the use of the L. plantarum strain of the invention (in particular a low post-acidification L. plantarum strain of the invention) or the bacterial composition of the invention or the composition or the kit-of part of the invention, in the manufacture of a fermented dairy product.

In a particular embodiment, the L. plantarum strain is the DSM32493 strain deposited at the DSMZ on April <NUM>th, <NUM> or any variant thereof as claimed, in particular a low post-acidification variant of the Lactobacillus plantarum strain DSM32493 as claimed, particular a low post-acidification variant of the Lactobacillus plantarum strain DSM32493 bearing a mutation in the ATP synthase alpha subunit gene. The definitions and specific embodiments detailed for the L. plantarum strain characterization apply similarly in the context of the use of the invention, in particular for but not limited to, the DSM32493 variant, the low post-acidification L. plantarum and the mutation of the ATP synthase alpha subunit gene, in particular the mutation G to A at its position <NUM>, in particular the ATP synthase alpha subunit gene as defined in SEQ ID NO:<NUM>.

The following deposit was made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

It is requested that the biological material shall be made available only by the issue of a sample to an expert nominated by the requester. In respect to those designations in which a European Patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample, and approved either i) by the Applicant and/or ii) by the European Patent Office, whichever applies (Rule <NUM> EPC).

Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

The following method was used for screening Lactobacillus plantarum strains of the DuPont collection. Thus, <NUM> Lactobacillus plantarum isolated from various sources were screened as follows:.

From the <NUM> tested strains, the following classification was obtained:.

plantarum strain representative of each class was then assayed for rheological and sensory analyses.

plantarum strains selected from example <NUM> were used to ferment milk and the rheological properties of the obtained fermented milks were determined.

Strains: The following strains were used:.

The flow curves of fermented milks obtained using either the DSM32493 strain, the LP12111 strain, the LP12428 strain or the Lp115 strain were obtained (<FIG>). As show in <FIG>, as compared to the LP12111, LP12428 and Lp115 strains which share close flow curves, the flow curve of the DSM32493 strain is atypical due to its significantly higher thickness and ropiness.

For each fermented milk, the shear stress at <NUM><NUM>/s, the shear stress at <NUM><NUM>/s and the difference between the shear stress at <NUM><NUM>/s and the shear stress at <NUM><NUM>/s were then calculated (Table <NUM>). The shear stress at <NUM><NUM>/s was correlated to sensory thickness, the shear stress at <NUM><NUM>/s was correlated to mouth-thickness and the difference between the shear stress at <NUM><NUM>/s and the shear stress at <NUM><NUM>/s was correlated to ropiness.

As detailed in Table <NUM>, the fermented milk obtained using DSM32493 shows high values of thickness, mouth thickness and ropiness, almost double those obtained using LP12111, LP12428 and Lp115 strains.

Thus, these results show that a fermented milk having high values of thickness, mouth thickness and ropiness (as defined herein) can be obtained using a L. plantarum of the invention (in particular the DSM32493 strain).

Interestingly and as an advantage of the invention, these results also show that a fermented milk having high values of thickness, mouth thickness and ropiness (as defined herein) can be obtained using a pure culture of L. plantarum of the invention (in particular a pure culture of the DSM32493 strain). This is in contrast with fermented milk obtained with compositions described in the literature which comprise several strains (of which at least a Streptococcus thermophilus strain).

An expert panel comprised of five culture/dairy application specialists evaluated the sensory properties of each fermented milk obtained using the DSM32493 strain, the LP12111 strain, the LP12428 strain or the Lp115 strain. Seven flavour and texture descriptors were evaluated and reported on a spider chart (<FIG>). Whereas the fermented milks obtained using LP12111, LP12428 and Lp115 strains show comparable charts, the fermented milk obtained using the DSM32493 strain shows significant differences in several of these descriptors. Thus, fermented milks obtained using one of LP12111, LP12428 and Lp115 strains show grainy texture (so a non-shining appearance), low ropiness and smoothness, and low to medium thickness in mouth and spoon. In contrast, fermented milk obtained using the DSM32493 strain shows very high thickness in mouth and spoon, a very long texture, high ropiness and smoothness and has a shining appearance. Appearance of fermented milks obtained using either the DSM32493 strain, the LP12111 strain, the LP12428 strain or the Lp115 strain is shown in <FIG>.

The rheological properties of a fermented milk obtained using DSM32493 was then compared with the ones of a milk fermented using a commercial yogurt starter culture with strong texturing property (Danisco; YO-MIX® <NUM>) consisting of a combination of Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus strains. Test A as described in example <NUM> was carried out, with inoculation of either the DSM32493 strain or the YO-MIX® <NUM> culture, and the flow curve of the fermented milks were obtained (<FIG>). For each fermented milk, the shear stress at <NUM><NUM>/s, the shear stress at <NUM><NUM>/s and the difference between the shear stress at <NUM><NUM>/s and the shear stress at <NUM><NUM>/s were then calculated (Table <NUM>).

As show in <FIG> and detailed in Table <NUM>, the DSM32493 strain alone is sufficient to manufacture a fermented milk with a thickness and ropiness as good as the ones obtained using a traditional culture of S. thermophilus and L. bulgaricus. Moreover, this comparative example confirms the uniqueness of the DSM32493 strain to give a fermented milk with a high mouth-thickness.

This high mouth-thickness value provided by the DSM32493 strain is confirmed by the long texture clearly visible on the left photograph of <FIG> as compared to the right photograph [obtained using YO-MIX® <NUM>].

These results confirm that the DSM32493 is industrially interesting to obtain fermented milk presenting a high thickness, a high ropiness and a high mouth-thickness.

The DSM32493 strain was streaked on MRS agar plate and anaerobically incubated at <NUM> for <NUM> days. A single colony was picked up and grew in <NUM> MRS broth <NUM> overnight. 200µL of fresh culture (O. D = <NUM>) was inoculated into <NUM> of <NUM>. 5x MRS containing 700µg/mL of neomycin, and then incubated at <NUM> for <NUM> days. A serial dilution of the fresh culture was prepared and plated on <NUM>. 5x MRS agar plate with 700µg/mL of neomycin and incubated at <NUM> for <NUM> days. Single colonies were picked up, and each inoculated into both of <NUM>. 5x MRS (pH <NUM>) and <NUM>. 5x MRS (pH adjusted to pH <NUM>) in <NUM>-well plates and incubated at <NUM> for <NUM> days. Colonies which grew in <NUM>. 5x MRS (pH <NUM>) but not <NUM>. 5x MRS (pH <NUM>) were selected. The acidification curve of each selected colony in MRS at <NUM> was monitored using iCinac for <NUM> days. Colonies with significant higher end pH (and considered as low post acidification mutants at fermentation temperature) were selected and their DNA sequenced.

One mutant (Lp12733) selected by the protocol described above was shown to contain the mutation G to A in position <NUM> of the ATP synthase alpha subunit gene of the ATP-synthase operon, resulting in the substitution of the glycine residue at position <NUM> by an aspartic acid residue. Presence of this mutation can be checked by PCR using the primers as defined in SEQ ID NOs: <NUM> and <NUM>, and then DNA sequencing using the primer as defined in SEQ ID NO:<NUM>.

The DSM32493 strain and its low post acidification mutant Lp12733 were used to ferment milk and the pH was recorded over time, using Test B described herein. Cinac curves were obtained and are shown in <FIG>.

Milk: UHT milk (<NUM>% protein, <NUM>% fat) added with <NUM>% sucrose and <NUM>% of yeast extract powder (LP0021) from Oxoid™ was heat treated at <NUM> for <NUM> and cooled down to room temperature.

Fermentation: For each strain, a freeze-dried culture was inoculated into milk at <NUM><NUM> CFU/ mL and the inoculated milk placed at <NUM> (t=<NUM>). The inoculated milk was fermented until a pH of <NUM> (tpH4. <NUM>) is reached and kept at <NUM> for at least <NUM> hours, and the pH of the milk continuously monitored.

Post-acidification measurements: The delta pH (ΔpH = pH at (tpH45+<NUM>) - pH <NUM>) was used to represent the post-acidification at fermentation temperature.

As shown in <FIG>, the pH of fermented milk obtained using the DSM32493 strain at tpH4. <NUM>+<NUM> hours is <NUM> whereas the pH of the fermented milk obtained using the Lp12733 strain (low post acidification mutant of the DSM32493 strain) is <NUM> (i.e. the pH of the fermented milk is steady at pH <NUM>). Thus, the ΔpH of the DSM32493 strain is about <NUM>, whereas the ΔpH of the Lp12733 strain is <NUM>.

Claim 1:
A Lactobacillus plantarum strain which is the strain DSM32493 deposited at the DSMZ on April 26th, <NUM>, or a variant of said DSM32493 strain with a genome sequence identity of at least <NUM>% to the genome sequence of the
DSM32493 strain, which variant is produced by spontaneous mutation or genetic engineering of DSM32493, which when inoculated into milk - generates a fermented milk presenting the following rheological features, as assayed by test A as described in the description:
a) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa;
b) a shear stress measured at shear rate <NUM>-<NUM> higher than <NUM> Pa; and
c) a difference of the shear stress measured at <NUM>-<NUM> minus the shear stress measured at <NUM>-<NUM> higher than <NUM>.