WO2023060509A1

WO2023060509A1 - New food products with improved properties

Info

Publication number: WO2023060509A1
Application number: PCT/CN2021/123842
Authority: WO
Inventors: Yuejian MAO
Original assignee: Dupont Nutrition Biosciences Aps
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2023-04-20
Also published as: CN118434286A; WO2023061994A1

Abstract

Process for manufacturing a food product based on the inoculation of a food product with stable lactic acid bacteria able to maintain viability and to slightly decrease pH when stored at ambient temperature in combination with erythritol. Use of these stable lactic acid bacteria in combination with erythritol for inoculation in a food product.

Description

NEW FOOD PRODUCTS WITH IMPROVED PROPERTIES

FIELD OF THE INVENTION

The application is directed to a process for manufacturing a food product based on the inoculation of a food product with one or more stable lactic acid bacteria able to maintain viability and to slightly decrease pH when stored at ambient temperature. The invention is also directed to the use of these one or more stable lactic acid bacteria in combination with a sugar alcohol for inoculation in a food product.

BACKGROUND TO THE INVENTION

These last years, a trend has emerged for food products, in particular dairy products, containing high level of live bacteria (for their health benefits) , which at the same time can be stored at ambient temperature. Indeed, consumers are looking for healthy food products which are easy to be consumed, i.e., easy to be transported and to be stored. Such ambient products are also advantageous in countries where the cold chain during the distribution and storage of food products containing high level of live bacteria is complex, not to say economically or technically impossible.

The two major issues resulting from the storage at ambient temperature of food products containing high level of live bacteria are: (1) the multiplication of the live bacteria in the food product resulting in the production of undesired metabolites which finally impact the quality of the food product (as an example, lactic acid bacteria are able to produce lactic acid at ambient temperature resulting in a non-acceptable pH decrease of the final product such as dairy product) , and then (2) the death of the bacteria which are not able to survive in the food matrix at ambient temperature, resulting in the loss of the benefits associated with the bacteria.

Application WO2017/194650 describes Lactobacillus strains of the species paracasei, rhamnosus, fermentum or delbrueckii subsp bulgaricus capable of retaining viability in an amount of at least 10 ³ cfu/g (starting from a level of 2.5x10 ⁷ cfu/g) and not decreasing the pH of a test product more than 0.8 units, after storage for 150 days (5 months) at 25℃.

However, food producers are looking for means for constantly improving the level of lactic acid bacteria (in particular by reducing the level of viability decrease as compared to the inoculation level) and for reducing the pH decrease in food products, in order to guarantee their customers acceptable bacteria health benefits and a constant food product quality, in particular when the food product is stored at ambient temperature. Moreover, in some countries, such as China, there is a requirement both from producers and consumers to be able to store food products for at least 6 months at ambient temperature, without impacting the characteristics of the food product.

Therefore, there is still a need to identify bacteria which are -when inoculated in a food product –both able to retain a high viability and able to lead to a lesser pH decrease, after storage of this food product at ambient temperature in stricter conditions (at least 6 months) .

DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the viability (in log cfu) of 80 strains (representing 33 Lactobacillus species) in a test yoghurt after storage for 30 days at 37℃ by assay A.

Figure 2 is a graph showing the pH of a test yoghurt inoculated with one of 80 strains (33 Lactobacillus species) after storage for 30 days at 37℃ by assay A.

Figure 3 is a graph showing the pH of a test yoghurt inoculated with one of the 20 tested strains (grey bars) and showing the viability (in log cfu) of the tested 20 strains in a test yoghurt (black dots) , after storage for 30 days at 37℃ by assay A.

Figure 4 is a graph showing (A) the evolution of viability (in log cfu) of the DSM32493 strain in a yoghurt and (B) the evolution of the pH of the yoghurt, during storage for 180 days at 25℃.

Figure 5 is a graph showing (A) the evolution of viability (in log cfu) of the DSM32493v strain in a yoghurt and (B) the evolution of the pH of the yoghurt, during storage for 180 days at 25℃.

Figure 6 is a graph showing (A) the evolution of viability (in log cfu) of the DSM33120 strain in a yoghurt and (B) the evolution of the pH of the yoghurt, during storage for 180 days at 25℃.

Figure 7 is a graph showing (A) the evolution of viability (in log cfu) of the DSM33121 strain in a yoghurt and (B) the evolution of the pH of the yoghurt, during storage for 180 days at 25℃.

Figure 8 is a graph showing the evolution of viability (in log cfu) of the DSM 33120 strain in five yoghurt, during storage for 30 days at 25℃.

Figure 9 is a graph showing the evolution of the pH of five test yoghurt inoculated with DSM 33120 strains after storage for 30 days at 25℃.

DETAILED DESCRIPTION

The inventors have surprisingly identified strains of the Lactobacillus genus, which can be added to a food product, such that both the viability of these strains in the food product and the pH of this food product are acceptably decreased, such as when stored at ambient temperature. Thus, this food product contains a high level of bacteria and has an acceptable pH, also when stored at ambient temperature, for at least 6 months.

The invention is directed to a process for manufacturing a food product, said process comprising:

1) providing an initial food product with a pH of between 3.4 and 4.6, in particular an initial low bacteria-containing food product with a pH of between 3.4 and 4.6 containing a level of bacteria which is no more than 1x10 ² CFU per g of said initial low bacteria-containing food product; which food product contains erythritol in an amount of from about 0.1%to about 15%;

2) adding to the initial food product, in particular to the initial low bacteria-containing food product, one or more stable lactic acid bacteria in a total amount of at least 1.0x10 ⁵ CFU per g, to obtain a food product,characterized in that:

(i) each of said one or more stable lactic acid bacterium is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus (also known as Lacticaseibacillus rhamnosus) , Bifidobacterium lactis, Lactobacillus plantarum (also known as Lactiplantibacillus plantarum) , Lactobacillus zymae (also known as Levilactobacillus zymae) , Lactobacillus rossiae (also known as Furfurilactobacillus rossiae) , Lactobacillus collinoides (also known as Secundilactobacillus collinoides) , Lactobacillus similis (also known as Secundilactobacillus similis) , Lactobacillus versmoldensis (also known as Companilactobacillus versmoldensis) , Lactobacillus acidipiscis (also known as Ligilactobacillus acidipiscis) , Lactobacillus hammesii (also known as Levilactobacillus hammesii) , Lactobacillus namurensis (also known as Levilactobacillus namurensis) , Lactobacillus nodensis (also known as Companilactobacillus nodensis) and Lactobacillus tucceti (also known as Companilactobacillus tucceti) ; and

(ii) each of said one or more stable lactic acid bacterium, when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds:

a) retains viability in an amount of at least 1x10 ⁶CFU/g after storing said test yoghurt 30 days at a temperature of 25 ℃; and

b) decreases the pH of said test yoghurt of at most 0.3 units after storing said test yoghurt 30 days at a temperature of 25 ℃.

The invention also relates to the use of one or more stable lactic acid bacteria for inoculation in an initial food product, in particular an initial low bacteria-containing food product, with a pH of between 3.4 and 4.6 in combination with erythritol in an amount of from about 0.1%to about 15%, wherein

(i) said stable lactic acid bacterium is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti; and

(ii) said stable lactic acid bacterium, when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds:

Initial food product

An initial food product with a pH of between 3.4 and 4.6 is provided in step 1) of the process of the invention or provided in the use of the invention

By “food product” , it is meant any product which is intended for human consumption. According to the invention (and in particular step 2 of the process) , the initial food product must be suitable for being inoculated with the one or more stable lactic acid bacteria. By “initial food product” , it is meant a food product before addition of the one or more stable lactic acid bacteria, and therefore which does not contain stable lactic acid bacteria as defined herein. The initial food product must be distinguished from the “food product stable at ambient temperature” which contains stable lactic acid bacteria as defined herein.

In an embodiment, said initial food product is a fermented food product. Fermentation is carried out through the action of a bacteria starter by conversion of carbohydrates into acid. A “bacteria starter” is defined as a composition comprising or consisting of one or more bacteria which is able to start and perform the fermentation of a substrate. In a particular embodiment, said initial food product is an acetic acid-fermented food product, meaning that the fermentation is carried out through the action of acetic acid bacteria starter by conversion of carbohydrates into acetic acid. In an embodiment, said initial food product is a lactic acid-fermented food product, meaning that the fermentation is carried out through the action of lactic acid bacteria starter by conversion of carbohydrates into lactic acid. The expression “lactic acid bacteria” (LAB) relates to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. Lactic acid bacteria are well known in the art, and include strains of the Lactococcus genus, of the Streptococcus genus, of the Lactobacillus genus, of the Bifidobacterium genus, of the Leuconostoc genus, of the Enterococcus genus, of the Pediococcus genus, of the Brevibacterium genus and of the Propinibacterium genus.

In an embodiment, said initial food product of step 1) is selected from the group consisting of a milk-based product, a fruit-based product such as fruit-based beverage, a vegetable-based product such as a vegetable-based beverage, a cereal-based product such as a cereal-based beverage, a rice-based product such as rice-based beverage, a nut-based product such as nut-based beverage, a soy-based product and any mixture thereof. By “milk-based product” , “fruit-based product or beverage” , “vegetable-based product or beverage” , “cereal-based product or beverage” , “rice-based product or beverage” , “nut-based product or beverage” and “soy-based product” , it is meant that the main component of the initial food product is respectively milk, fruit, vegetable, cereal, rice, nut and soy. In an embodiment, milk, fruit, vegetable, cereal, rice, nut and soy are the only component used as substrate to manufacture the milk-based product, fruit-based product or beverage, vegetable-based product or beverage, cereal-based product or beverage, rice-based product or beverage, nut-based product or beverage and soy-based product (as initial food products) respectively. The term “beverage” is defined in this application as a liquid food product.

In an embodiment, the milk-based product (as initial food product) is a fermented dairy product or a chemically-acidified dairy product. In an embodiment, a fermented dairy product, is selected from the group consisting of a fermented milk, a yoghurt, a cheese, sour cream, buttermilk and fermented whey. Fermented dairy products are well known in the art and are manufactured through the action of a lactic acid bacteria starter (as defined herein) on a milk substrate (the pH of milk substrate is around 6.5 to 7) . A “milk substrate” is defined herein as any milk of mammal origin, including but not limited to, cow, sheep and goat milk. The milk may be in the native state, a reconstituted milk or a skimmed milk. The milk substrate, in particular the milk, is typically previously treated, in particular by standardization, addition of additives [e.g., sugar, sweeteners and/or stabilisers] , homogenization and/or heat-treatment [e.g., pasteurization] . In a particular embodiment, the fermented milk is obtained by fermentation of milk with a lactic acid bacteria starter selected from the group consisting of a starter comprising a Streptococcus thermophilus strain, a starter comprising a strain from the Lactobacillus genus and a starter comprising a Lactococcus lactis strain. In a particular embodiment, said fermented milk is obtained by fermentation of milk with a lactic acid bacteria starter selected from the group consisting of a starter comprising or consisting of Streptococcus thermophilus and Lactobacillus bulgaricus, a starter comprising or consisting of Streptococcus thermophilus and Lactobacillus johnsonii and a starter comprising or consisting of Streptococcus thermophilus and Lactobacillus fermentum. In a particular embodiment, said fermented milk is a yoghurt.

In an embodiment, the fruit-based product (as initial food product) is a fruit-based beverage. In a particular embodiment, the fruit-based product is a fruit juice or a fermented fruit juice.

In an embodiment, the vegetable-based product (as initial food product) is a vegetable-based beverage. In a particular embodiment, the vegetable-based product is a vegetable juice or a fermented vegetable juice.

In an embodiment, the cereal-based product (as initial food product) is a cereal-based beverage. In a particular embodiment, the cereal-based product is a chemically-acidified cereal product, a fermented cereal product, a chemically-acidified cereal beverage or a fermented cereal beverage.

In an embodiment, the rice-based product (as initial food product) is a rice-based beverage. In a particular embodiment, the rice-based product is a chemically-acidified rice product, a fermented rice product, a chemically-acidified rice beverage or a fermented rice beverage.

In an embodiment, the nut-based product is a nut-based beverage. In a particular embodiment, the nut-based product is a chemically-acidified nut product, a fermented nut product, a chemically-acidified nut beverage or a fermented nut beverage. In a particular embodiment of any nut-based product described herein, the food product is a walnut-based product.

In an embodiment, the soy-based product (as initial food product) is a soy-based beverage. In a particular embodiment, the soy-based product is a fermented soy milk product.

In an embodiment, the term “initial food product” also cover any mixture of milk-based product, fruit-based product or beverage, vegetable-based product or beverage, cereal-based product or beverage, rice-based product or beverage, nut-based product or beverage and soy-based product as defined herein, such as, for example but not limited to, a mixture of a milk-based product and cereal-based beverage, or a mixture of a milk-based product and fruit-based beverage.

In an embodiment, said initial food product is an initial “low bacteria-containing” food product with a pH of between 3.4 and 4.6, i.e. is an initial food product as defined herein with a level of bacteria which is no more than 1x10 ² CFU per g of said initial low bacteria-containing food product. By “level of bacteria” used herein, it is meant the total amount of bacteria as calculated as cfu/g of product. The cfu count can be measured by plating dilution (s) of the product to be tested on MRS/M17/PCA agar [Atlas, 2010 Handbook of Microbiological Media, Fourth Edition, pages 986, 1231 and 1402] .

Any initial low bacteria-containing food product can be used in step 1) of the process of the invention or in the use of the invention. According to the invention (and in particular step 2 of the process) , the initial low bacteria-containing food product must be suitable for being inoculated with the stable bacteria.

In an embodiment, the initial food product naturally has a level of bacteria which is no more than 1x10 ² CFU per g of food product.

In another embodiment, the initial food product has a level of bacteria, other than the stable lactic acid bacteria as defined herein, which is more than 1x10 ² CFU per g of food product. The presence of bacteria, in particular lactic acid bacteria, can result from the use of these microorganisms (in particular as starter) during the manufacture of the initial food product, for example when the initial food product results from fermentation of a substrate (as explained above) .

In an optional embodiment of the invention, the initial food product is therefore treated, previously to the inoculation of the stable LAB, so as to obtain an initial low bacteria-containing food product. By “treating” , it is meant any treatment which inactivates the bacteria contained in the initial food product (e.g. which inhibits or reduces the bacteria growth or kills bacteria) , so as to reduce the level of bacteria to no more than 1x10 ² CFU per g of the low bacteria-containing food product. Treatment means are well known in the art. In an embodiment, the initial food product is treated using means selected from the group consisting of high-pressure sterilization, irradiation, ultra-filtration and heat-treating. In a particular embodiment, the initial food product is heat-treated so as to reduce the level of bacteria to no more than 1x10 ² CFU per g of the low bacteria-containing food product.

By “heat-treating” , it is meant any treatment based on temperature which inactivates the bacteria contained in the initial food product (e.g. which inhibits or reduces the bacteria growth or kills bacteria) , so as to reduce the level of bacteria in the low bacteria-containing food product to no more than 1x10 ² CFU per g of the low bacteria-containing food product.

Thus, in an embodiment, the invention is directed to a process for manufacturing a food product, said process comprising:

1) providing an initial food product with a pH of between 3.4 and 4.6, which food product contains erythritol in an amount of from about 0.1%to about 15%;

1b) treating the initial food product so as to obtain a level of bacteria which is no more than 1x10 ² CFU per g [of the resulting initial low bacteria-containing food product] , in particular by heat-treating said initial food product; and

2) adding to the initial low bacteria-containing food product, one or more stable lactic acid bacteria in a total amount of at least 1.0x10 ⁵ CFU per g, to obtain a food product, characterized in that:

(i) each of said one or more stable lactic acid bacterium is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti; and

(ii) each of said one or more stable lactic acid bacterium, when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃for 25 seconds:

In an embodiment, the process of the invention is carried out in a fermented milk as defined herein, in particular a yoghurt as defined herein. Thus, the invention is directed to a process for manufacturing a fermented milk, in particular a yoghurt, said process comprising

1a) producing an initial fermented milk, in particular an initial yoghurt, with a pH of between 3.4 and 4.6 by fermentation of a milk substrate which milk substrate contains erythritol in an amount of from about 0.1%to about 15%;

1b) treating, in particular heat-treating, said initial fermented milk, in particular said initial yoghurt, so as to obtain an initial low bacteria-containing fermented milk, in particular an initial low bacteria-containing yoghurt containing a level of bacteria which is no more than 1x10 ² CFU per g; and

2) adding to the initial low bacteria-containing fermented milk, in particular to the initial low bacteria-containing yoghurt, one or more of stable lactic acid bacteria strains in a total amount of at least 1.0x10 ⁵ CFU per g to obtain a fermented milk, in particular a yoghurt,

characterized in that:

(i) each of one or more said stable lactic acid bacterium is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, , Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti; and

(ii) each of one or more said stable lactic acid bacterium, when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds:

The initial, optionally low bacteria-containing, food product [in particular provided as such (step 1) or after treatment (step 1 b) or after production by fermentation and treatment (step 1b) ] must be suitable to reach the goal of the invention, i.e., to manufacture a food product.

In an embodiment, the pH of the initial, optionally low bacteria-containing, food product is between 3.4 and 4.6. In an embodiment, the pH of the initial, optionally low bacteria-containing, food product is between 3.4 and 4.0. In an embodiment, the pH of the initial, optionally low bacteria-containing, food product is between 4.0 and 4.6. In an embodiment, the pH of the initial, optionally low bacteria-containing, food product is between 3.6 and 4.2. The pH can be determined by using any pH meter.

In an embodiment, optionally in combination with any embodiment of the previous paragraph, the sugar content of said initial, optionally low bacteria-containing, food product is between 0 and 13%. By “sugar content” , it is meant the total content of sugar in the initial, optionally low bacteria-containing, food product, whether it is the sugar originally contained in the initial food product, sugar added into the initial food product or a combination of the sugar originally contained in the initial food product and sugar added into the initial food product. It is to be understood that sugar in the context of “sugar content” does not include sugar alcohols, such as erythritol. In an embodiment, the sugar content of said initial, optionally low bacteria-containing, food product is between 4 and 10%. In an embodiment, the sugar content of said initial, optionally low bacteria-containing, food product is between 6 and 9%. In a particular content, the sucrose content of said initial, optionally low bacteria-containing, food product is between 0 and 8%. In an embodiment, the sucrose content of said initial, optionally low bacteria-containing, food product is between 5 and 8%.

In some embodiments, the process of the invention provides for the manufacturing of a food product stable at ambient temperature.

In some embodiments, the erythritol used in the methods of the present invention is present in an amount of from about 0.1%to about 14%, such as in an amount of from about 0.1%to about 12%, such as in an amount of from about 0.1%to about 10%, such as in an amount of from about 0.1%to about 8%, such as in an amount of from about 0.1%to about 6%, such as in an amount of from about 0.1%to about 4%, such as in an amount of from about 0.5%to about 15%, such as in an amount of from about 1%to about 15%, such as in an amount of from about 2%to about 15%, such as in an amount of from about 4%to about 15%, such as in an amount of from about 6%to about 15%, such as in an amount of from about 8%to about 15%, such as in an amount of from about 10%to about 15%, such as in an amount of from about 1%to about 8%, such as in an amount of from about 2%to about 6%, such as in an amount of from about 3%to about 6%.

As used herein “erythritol” refers to the sugar alcohol with the formula (2R, 3S) -Butane-1, 2, 3, 4-tetrol. Erythritol may be obtained naturally from fruit and fermented foods. Alternatively, it may be obtained from glucose by fermentation with a yeast, such as Moniliella pollinis.

Adding/inoculating one or more stable lactic acid bacteria to the initial food product

The initial food product as defined herein, in particular to the initial low bacteria-containing food product as defined herein, is inoculated with one or more stable lactic acid bacteria in a total amount of at least 1.0x10 ⁵ CFU per g (step 2 of the process of the invention or use of the invention) .

Within the context of the invention, “adding” is used interchangeably with “inoculating” (as well as “added” and “inoculated” ) and means that the one or more stable lactic acid bacteria (as defined herein) are put in contact with the initial food product. By “one or more” , it is meant at least one lactic acid bacterium (LAB) . In an embodiment, the number of LABs added to the food product is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In an embodiment, 1 stable LAB is added to the food product. In an embodiment, 2 stable LABs are added to the food product. In an embodiment, 3 stable LABs are added to the food product. In an embodiment, 4 stable LABs are added to the food product. In an embodiment, 5 stable LABs are added to the food product.

The one or more stable LABs are added to the initial food product, in particular to the initial low bacteria-containing food product, in a total amount of at least 1x10 ⁵ cfu per g of food product. When several (i.e., at least 2) stable LABs are added, “total amount” means the sum of each individual amount of inoculated stable LAB (as an example, addition of a first stable LAB at 3x10 ⁵ cfu/g and of a second stable LAB at 7x10 ⁵ cfu/g leads to a total amount of 1x10 ⁶ cfu/g) . In an embodiment, the one or more stable LABs are added to the initial food product, in particular to the initial low bacteria-containing food product, in a total amount selected from the group consisting of at least 5x10 ⁵ CFU per g, at least 1x10 ⁶ CFU per g, at least 5x10 ⁶ CFU per g or at least 1x10 ⁷ CFU per g of the initial food product. In an embodiment, the one or more stable LABs are added to the initial food product, in particular to the initial low bacteria-containing food product, in a total amount range selected from the group consisting of from 1x10 ⁵ to 1x10 ⁸ cfu per g, from 1x10 ⁶ to 1x10 ⁸ cfu per g and from 5x10 ⁶ to 1x10 ⁸ cfu per g.

The one or more stable LABs can be inoculated into the initial food product under any form, such as under frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in the form of a powder or dried powder. In an embodiment, the one or more stable LABs are added to the initial food product, under liquid form, for example as bulk starter [i.e., a LAB culture previously propagated in a growth medium to obtain the required concentration of inoculation] . In an embodiment, the one or more stable LABs are directly added to the initial food product under the form of concentrates, for example frozen or dried concentrates. In an embodiment the one or more stable LABs are added to the food product under liquid form as a dilution [e.g. in water or saline solution] of concentrates, such as of frozen or dried concentrates. The expression “directly inoculated” means that the one or more stable LABs are added into the initial food product without previous propagation. The direct inoculation requires that the concentration of the one or more stable LABs be high enough. Thus, the concentration of stable LABs in the frozen or dried concentrate is in the range of 10 ⁸ to 10 ¹² cfu per g of concentrate, and more preferably at least 10 ⁸, at least 10 ⁹, at least 10 ¹⁰, at least 10 ¹¹ or at least 10 ¹² cfu/g of concentrate.

In an embodiment, said one or more strains are aseptically added to the initial food product. By “aseptically” , it is meant that no other microorganisms than the one or more stable lactic acid bacteria are added to the food product, e.g. by using Tetra FlexDos ^TM aseptic in-line inoculation system.

The stable lactic acid bacteria (stable LABs)

The one or more stable lactic acid bacteria (LABs) added to the initial food product (in step 2) of the claimed process of the invention or in the use of the invention) are characterized by the 2 following features:

(i) the one or more stable lactic acid bacteria strain is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti.

In an embodiment, the one or more stable lactic acid bacteria strain is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus versmoldensis and Lactobacillus namurensis. In an embodiment, the one or more stable lactic acid bacteria strain is selected from the group consisting of strains of species Lactobacillus plantarum and Lactobacillus zymae.

In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus plantarum. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus zymae. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus rossiae. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus collinoides. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus similis. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus versmoldensis. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus acidipiscis. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus hammesii. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus namurensis. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus nodensis. In an embodiment, the one or more stable lactic acid bacteria strain is of species Lactobacillus tucceti.

For the avoidance of doubt, the Lactobacillus species described herein are as defined in the literature, in particular in Salvetti et al. 2012 Probiotics &Antimicro. Prot. 4 (4) : 217-226, and Cay et al. 2012 Int. J. Syst. Evol. Microbiol. 62: 1140-1144. In case new nomenclature was used, they are described in Zheng et al., Int. J. Syst. Evol. Microbiol. DOI 10. 1099/ijsem. 0.004107.

(ii) each of the one or more stable lactic acid bacteria strain remains viable in, and do not decrease significantly the pH of, a heat-treated yogurt stored 30 days at a temperature of 37℃ (i.e. under strict conditions, ) .

Thus, in an embodiment, said stable lactic acid bacteria strain, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds:

a) retains viability in an amount of at least 5.0x10 ³ CFU/g after storing said test yoghurt 30 days at a temperature of 37 ℃; and

b) decreases the pH of said test yoghurt of at most 0.6 units after storing said test yoghurt 30 days at a temperature of 37 ℃.

In an embodiment, said stable lactic acid bacteria strain, when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds:

a) retains viability in an amount of at least 1x10 ⁶ CFU/g after storing said test yoghurt 30 days at a temperature of 25 ℃; and

b) decreases the pH of said test yoghurt of at most. 0.3 units after storing said test yoghurt 30 days at a temperature of 25 ℃.

In an embodiment, the sugar content of said test yogurt is between 0 and 13%. In an embodiment, the sugar content of said test yogurt is between 4 and 10%. In an embodiment, the sugar content of said test yogurt is between 6 and 9%. In a particular content, the sucrose content of said test yogurt is between 0 and 8%. In an embodiment, the sucrose content of said test yogurt is between 5 and 8%. In an embodiment, the sugar content of said test yogurt is between 12 and 13%, including a sucrose content between 7 and 9%.

In an embodiment, feature (ii) is tested applying assay A as described below:

- the inoculum of the LAB to be tested is prepared as follows: a culture of the LAB at 10 ⁶ cfu/ml is cultured in 10mL MRS/M17 broth overnight at 37 ℃; after 2h at 4℃, the culture is centrifuged at 4000rpm for 10min; the pellet is resuspended in 10mL sterile saline; the centrifugation/resuspension step is repeated a second time, to give the inoculum

- after standardization of the inoculum at around 1x10 ⁹ CFU/mL, 0.4 mL of the inoculum was added into 40mL of heat-treated yoghurt (2.8%protein, 3%fat, 12.5%of total sugar including 8%sucrose; pH 4.3) and well mixed [final stable LAB concentration in heat-treated yoghourt is around 1x10 ⁷ CFU/g of yoghurt] ; the tube is then sealed.

- the inoculated yoghurt is incubated at 37℃ for 30 days.

- after 30 days, the pH is determined by pH meter (Mettler Toledo, SevenEasy) ; the pH at day 30 is then compared to the pH of the heat-treated yoghurt at the time of LAB addition

- after 30 days, the CFU count is determined by plating on MRS/M17 agar as follows: 1mL of yogurt sample was serial diluted by sterile saline to 10 ^-7; MRS/M17 agar (1.5%) was melted and maintained at 48℃ in water bath; 1mL of 10 ^-1 to 10 ^-7 dilution was added to petri dish and poured with 25mL of the MRS/M17 agar; the plates were incubated at 37℃ anaerobically for 2 days for counting; the amount of LABs at day 30 is then compared to the amount of LAB added to the heat-treated yoghurt.

In an embodiment of the feature a) , said stable lactic acid bacteria strain, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) , retains viability in an amount of at least 5x10 ³ CFU/g, at least 1x10 ⁴ CFU/g, at least 5x10 ⁴ CFU/g, at least 1x10 ⁵ CFU/g, at least 5x10 ⁵ CFU/g or at least 1x10 ⁶ CFU/g, after storing said test yoghurt 30 days at a temperature of 37℃.

In an embodiment of the feature b) , considered individually or in combination with the embodiment of the previous paragraph [feature a] , said stable lactic acid bacteria strain, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) , decreases the pH of said test yoghurt of at most 0.6 units, at most 0.5 units, at most 0.4 units or at most 0.3 units, after storing said test yoghurt 30 days at a temperature of 37 ℃.

In an embodiment, said stable lactic acid bacteria strain, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

a) retains viability in an amount selected from the group consisting of at least 5.0x10 ³ CFU/g, at least 1.0x10 ⁴ CFU/g, at least 5.0x10 ⁴ CFU/g, at least 1.0x10 ⁵ CFU/g, at least 5.0x10 ⁵ CFU/g or at least 1.0x10 ⁶ CFU/g; and

b) decreases the pH of said test yoghurt of at most 0.6 units, at most 0.5 units, of at most 0.4 units or at most 0.3 units, after storing said test yoghurt 30 days at a temperature of 37 ℃.

In a particular embodiment, said stable lactic acid bacteria strain, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

a) retains viability in an amount selected from the group consisting of at least 1x10 ⁴ CFU/g, at least 5x10 ⁴ CFU/g, at least 1x10 ⁵ CFU/g, at least 5x10 ⁵ CFU/g or at least 1x10 ⁶ CFU/g; and

a) retains viability in an amount selected from the group consisting of at least 5x10 ³ CFU/g, at least 1x10 ⁴ CFU/g, at least 5x10 ⁴ CFU/g, at least 1x10 ⁵ CFU/g, at least 5x10 ⁵ CFU/g or at least 1.0x10 ⁶ CFU/g; and

b) decreases the pH of said test yoghurt of at most 0.5 units, of at most 0.4 units or at most 0.3 units, after storing said test yoghurt 30 days at a temperature of 37 ℃.

b) decreases the pH of said test yoghurt of most 0.5 units, of at most 0.4 units or at most 0.3 units, after storing said test yoghurt 30 days at a temperature of 37 ℃.

a) retains viability in an amount selected from the group consisting of at least 1x10 ⁵ CFU/g, at least 5x10 ⁵ CFU/g or at least 1x10 ⁶ CFU/g; and

b) decreases the pH of said test yoghurt of at most 0.3 units, after storing said test yoghurt 30 days at a temperature of 37 ℃.

Any Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti strain fulfilling feature (ii) as defined herein, in particular when assessed by test A, can be used in the process of the invention or the use of the invention.

In an embodiment, said stable lactic acid bacteria strain is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti, and, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

a) retains viability in an amount selected from the group consisting of at least 5x10 ³ CFU/g, at least 1x10 ⁴ CFU/g, at least 5x10 ⁴ CFU/g, at least 1x10 ⁵ CFU/g, at least 5.0x10 ⁵ CFU/g or at least 1x10 ⁶ CFU/g; and

In an embodiment, said stable lactic acid bacteria strain is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus versmoldensis, Lactobacillus hammesii, Lactobacillus similis, Lactobacillus nodensis, Lactobacillus tucceti and Lactobacillus namurensis, and, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

In an embodiment, said stable lactic acid bacteria strain is selected from the group consisting of strains of species Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus versmoldensis, Lactobacillus similis and Lactobacillus namurensis, and, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

a) retains viability in an amount selected from the group consisting of at least 5x10 ³ CFU/g, at least 1x10 ⁴ CFU/g, at least 5x10 ⁴ CFU/g, at least 1x10 ⁵ CFU/g, at least 5x10 ⁵ CFU/g or at least 1x10 ⁶ CFU/g; and

In an embodiment, said stable lactic acid bacteria strain is selected from the group consisting of strains of species Lactobacillus plantarum and Lactobacillus zymae, and, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

In an embodiment, said stable lactic acid bacteria strain is of species Lactobacillus plantarum, and, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

In an embodiment, the one or more stable LABs, to be added to step 2) of the process of the invention or the be used in the use of the invention, is selected from the group consisting of the Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April 26 ^th, 2017, a variant of the DSM32493 strain, the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019, a variant of the DSM33120 strain, the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 and a variant of the DSM33121 strain.

In an embodiment, the one or more stable LABs, to be added to step 2) of the process of the invention or the be used in the use of the invention, is the Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April 26 ^th, 2017, or a variant of the DSM32493 strain.

In an embodiment, the one or more stable lactic acid bacteria, to be added to step 2) of the process of the invention or to be used in the use of the invention, is a variant of the Lactobacillus plantarum strain DSM32493 deposited at the DSMZ on April 26 ^th, 2017, wherein said variant bears a mutation (for example point mutation, deletion, insertion, …) in the ATP synthase alpha subunit gene (as compared to the DSM32493 strain) . The wild-type sequence of the ATP-synthase operon is as set forth in SEQ ID NO: 1. 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 Jaichumjai et al. 2010; Food Microbiology 27 (2010) 741-748] . In an embodiment, the one or more stable lactic acid bacteria is a variant of DSM32493, wherein said variant 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, the one or more stable lactic acid bacteria is a variant of DSM32493, wherein the ATP synthase alpha subunit gene of said variant of DSM32493 as defined herein encodes a ATP synthase alpha subunit protein having an aspartic acid residue at position 169. In a particular embodiment, the one or more stable lactic acid bacteria is a variant of DSM32493, wherein said variant has at least one mutation in the ATP synthase alpha subunit gene of the ATP-synthase operon as defined in SEQ ID NO: 2. In a particular embodiment, in combination with the previous embodiment on SEQ ID NO: 2 or not, the one or more stable lactic acid bacteria is a variant of DSM32493 as defined herein, wherein said variant bears the mutation G to A at its position 506 of the ATP synthase alpha subunit gene (as compared to the DSM32493 strain) . In a particular embodiment, the one or more stable lactic acid bacteria is a variant of DSM32493 as defined herein, wherein the ATP synthase alpha subunit gene of said variant is as defined in SEQ ID NO: 4 (wherein the codon GGT at positions 505-507 is changed to GAT) . In a particular embodiment, the one or more stable lactic acid bacteria, to be added to step 2) of the process of the invention, is a variant of DSM32493, wherein the ATP synthase alpha subunit gene of said variant of DSM32493 as defined herein encodes an ATP synthase alpha subunit protein as defined in SEQ ID NO: 5.

In an embodiment, the one or more stable LABs, to be added to step 2) of the process of the invention or the be used in the use of the invention, is the Lactobacillus plantarum strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019 or a variant of the DSM33120 strain.

In an embodiment, the one or more stable LABs, to be added to step 2) of the process of the invention or the be used in the use of the invention, is the Lactobacillus plantarum strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 or a variant of the DSM33121 strain.

Lactobacillus plantarum strains

The invention is also directed to a Lactobacillus plantarum strain selected from the group consisting of the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019, a variant as defined herein of the DSM33120 strain, the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 and a variant as defined herein of the DSM33121 strain.

In an embodiment, the invention is directed to a Lactobacillus plantarum strain selected from the group consisting of the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019 or a variant as defined herein of the DSM33120 strain. In an embodiment, the invention is directed to a Lactobacillus plantarum strain selected from the group consisting of the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 or a variant as defined herein of the DSM33121 strain.

Bacterial compositions

The invention is also directed to a bacterial composition comprising or consisting of a Lactobacillus plantarum strain selected from the group consisting of the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019, a variant as defined herein of the DSM33120 strain, the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 and a variant as defined herein of the DSM33121 strain.

In a particular embodiment, the bacterial composition is a pure culture, i.e., comprises or consists of a single Lactobacillus plantarum strain of the invention. In another embodiment, the bacterial composition is a mixed culture, i.e., comprises or consists of a Lactobacillus plantarum strain of the invention and at least one other bacterium strain, in particular one other lactic acid bacterium.

In an embodiment, the bacterial composition, either as a pure or mixed culture as defined above, further comprises a food acceptable ingredient.

In a particular embodiment, the bacterial composition, 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 Lactobacillus plantarum strain of the invention in a concentration comprised in the range of 10 ⁵ to 10 ¹² cfu (colony forming units) per gram of the bacterial composition. In a particular embodiment, the concentration of the Lactobacillus plantarum strain within the bacterial composition of the invention is in the range of 10 ⁷ to 10 ¹² cfu per gram of the bacterial composition, and in particular at least 10 ⁷, at least 10 ⁸, at least 10 ⁹, at least 10 ¹⁰ or at least 10 ¹¹ 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 of the invention -as pure culture or as a mixed culture -within the bacterial composition is in the range of 10 ⁸ to 10 ¹² cfu/g of frozen concentrate or dried concentrate, and more preferably at least 10 ⁸, at least 10 ⁹, at least 10 ¹⁰, at least 10 ¹¹ or at least 10 ¹² cfu/g of frozen concentrate or dried concentrate.

Variants of Lactobacillus plantarum strains

The features detailed herein for the variants of the deposited Lactobacillus plantarum strains apply to the L. plantarum strains added within the method of the invention, to the L. plantarum strains as such and the L. plantarum strains as part of the bacterial composition.

A variant of the DSM32493, DSM33120 or DSM33121 strain is herein defined as 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, DSM33120 or DSM33121 strain respectively. In a particular embodiment, the genome sequence of the variant has an identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96%, at least 99.98%, or at least 99.99%to the genome sequence of the DSM32493, DSM33120 or DSM33121 strain respectively. Such a variant can be for example:

- a natural variant obtained spontaneously from the DSM32493, DSM33120 or DSM33121 strain after incubation in a selection medium. A natural variant is thus obtained without any genetic manipulation but only by spontaneous mutation of the strain and selection of the strain in an appropriate medium; an example of protocol used to select particular mutants of the DSM32493, DSM33120 or DSM33121 strain is disclosed in example 5; or

- a variant comprising at least one mutation in its genome, said mutation being induced by genetic engineering, for instance by directed mutagenesis or random mutagenesis. Random mutagenesis can be performed with UV radiations or mutagenic compounds such as nitrous acid, ethyl-methanesulfonate, NMethyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea, acridine orange, proflavine.

In an embodiment, said variant of the DSM32493, DSM33120 or DSM33121 strain (as defined herein) , when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds (such as by applying assay A) :

a) retains viability in an amount selected from the group consisting of at least 5x10 ³ CFU/g, at least 1x10 ⁴ CFU/g, at least 5x10 ⁴ CFU/g, at least 1x10 ⁵ CFU/g, at least 5x10 ⁵ CFU/g or at least 1x. 10 ⁶ CFU/g; and

b) decreases the pH of said test yoghurt of at most 0.6 units, at most 0.5 units, of at most 0.4 units or at most 0.3 units,

after storing said test yoghurt 30 days at a temperature of 37 ℃.

In an embodiment, said variant of the DSM32493, DSM33120 or DSM33121 strain (as defined herein) keeps the at least same viability and the at most same pH decrease as the DSM32493, DSM33120 or DSM33121 strain respectively (when each added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds, such as by applying assay A) , i.e. said variant of the DSM32493, DSM33120 or DSM33121 strain:

a) retains a viability identical to the viability retention of the DSM32493, DSM33120 or DSM33121 strain respectively or retains a higher viability than the viability retention of the DSM32493, DSM33120 or DSM33121 strain respectively (calculated in cfu/g) ; and

b) decreases the pH of the test yoghurt identically to the pH decrease of the DSM32493, DSM33120 or DSM33121 strain respectively or decreases the pH less than the pH decrease of the DSM32493, DSM33120 or DSM33121 strain respectively (calculated in pH unit) .

A food product stable at ambient temperature

The aim of the process is to manufacture a food product, such as one stable at ambient temperature. The expression “stable at ambient temperature” when referring to a food product means a food product containing one or more stable lactic acid bacteria as defined herein, and for which both the amount of stable lactic acid bacteria and the pH is not significantly decreased when stored at ambient temperature.

Thus, a food product, as manufactured by the process of the invention, is considered stable when after storing this product for 180 days at a temperature of 25 ℃:

- its pH is not decreased more than 0.7 unit; and

- the amount of stable lactic acid bacteria it contains is not decreased more than 3 log and/or is at least 1x10 ³ CFU/g.

Thus, the food product is stored for 180 days at 25℃, from the day where the one or more stable lactic acid bacteria as defined herein are added to the initial food product containing erythritol in an amount of from about 0.1%to about 15% (day 0) . In an embodiment, the food product is stored under a sealed format (i.e., in closed sterile container) .

After 180 days, the pH is determined by pH meter (Mettler Toledo, SevenEasy) and compared to the pH of the food product at day 0. Thus, in an embodiment, the pH of the food product at 180 days is not decreased more than 0.6 unit, more than 0.5 unit or more than 0.4 units (as compared to the pH at day 0) .

After 180 days, the CFU count is determined as described in assay A detailed herein and compared with the amount of one or more stable lactic acid bacteria as defined herein added at day 0. Thus, in an embodiment, the amount of one or more stable lactic acid bacteria it contains is at least 1x10 ³ CFU/g (as compared to the amount added at day 0) , whatever the level of addition in step 2) (which is at least 1x10 ⁵ CFU) . In an embodiment, the amount of one or more stable lactic acid bacteria it contains is not decreased more than 3 log (as compared to amount added at day 0) . In an embodiment, the amount of one or more stable lactic acid bacteria it contains is not decreased more than 2 log (as compared to amount added at day 0) . In an embodiment, the amount of one or more stable lactic acid bacteria it contains is not decreased more than 3 log and is at least 1x10 ³ CFU/g (as compared to the amount added at day 0) . In an embodiment, the amount of one or more stable lactic acid bacteria it contains is not decreased more than 2 log and is at least 1x10 ³ CFU/g (as compared to the amount added at day 0) .

The invention also relates to a food product, such as a food products stable at ambient temperature, as defined herein or as obtained by the process of the invention, and containing one or more stable lactic acid bacteria as defined herein.

In an embodiment, the food product, as defined herein or as obtained by the process of the invention, contains a Lactobacillus plantarum strain selected from the group consisting of the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019, a variant as defined herein of the DSM33120 strain, the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 and a variant as defined herein of the DSM33121 strain.

In an embodiment, the food product, as defined herein or as obtained by the process of the invention, contains a Lactobacillus plantarum strain selected from the group consisting of the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019 or a variant as defined herein of the DSM33120 strain. In an embodiment, the food product, as defined herein or as obtained by the process of the invention, contains a Lactobacillus plantarum strain selected from the group consisting of the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 or a variant as defined herein of the DSM33121 strain.

In an embodiment, the food product of the invention (as such or as obtained by the process of the invention) has its pH which is not decreased more than 0.7 unit, and has the amount of stable lactic acid bacteria it contains not decreased more than 3 log and/or is at least 1x10 ³ CFU/g, after storing this product for 180 days at a temperature of 25 ℃.

In an embodiment, the food product of the invention (as such or as obtained by the process of the invention) is selected from the group consisting of a milk-based food product, a fruit-based food product such as fruit-based food beverage, a vegetable-based food product such as a vegetable-based food beverage, a cereal-based food product such as a cereal-based food beverage, a rice-based food product such as rice-based food beverage, a nut-based food product such as nut-based food beverage, a soy-based food product and any mixture thereof. In an embodiment, the milk-based food product food product is a fermented dairy product or a chemically-acidified dairy product. In an embodiment, a fermented dairy product, is selected from the group consisting of a fermented milk, a yoghurt, a cheese, sour cream, buttermilk and fermented whey. In an embodiment, the milk-based food product is a fermented milk.

In some embodiments, the food product prepared according to the present invention is a fermented food product, such as a fermented dairy product, a fermented juice, a fermented vegetable juice, a fermented cereal product, a fermented rice product, a fermented nut product, a fermented soy milk product and any mixture thereof, which fermented product is fermented with lactic acid bacteria starter, such as culture comprising the DSM 33849 strain and/or the DSM 32823 strain.

In an embodiment, the food product of the invention -in particular the fermented dairy food product as defined herein -contains one or more said stable lactic acid bacterium selected from the group consisting of strains of species Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti, wherein each of one or more said stable lactic acid bacterium, when added in an amount of 1x10 ⁷ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds: a) retains viability in an amount of at least 5.0x10 ³ CFU/g after storing said test yoghurt 30 days at a temperature of 37 ℃; and b) decreases the pH of said test yoghurt of at most 0.6 units after storing said test yoghurt 30 days at a temperature of 37 ℃. In a particular embodiment, said one or more stable lactic acid bacteria strain are selected from the group consisting of strains of species Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus versmoldensis and Lactobacillus namurensis. In a particular embodiment, said one or more stable lactic acid bacteria strain are selected from the group consisting of strains of species Lactobacillus plantarum and Lactobacillus zymae. In a particular embodiment, said one or more stable lactic acid bacteria strain are of the species Lactobacillus plantarum. In a particular embodiment, said one or more stable lactic acid bacteria strain is DSM32493 strain deposited at the DSMZ on April 26 ^th, 2017 or any variant thereof as defined herein.

The definitions and specific embodiments detailed for the process of manufacture of the invention apply similarly in the context of the food product of the invention, in particular for but not limited to, the lactic acid bacteria species, the number of lactic acid bacteria, the pH decrease feature after storing for 180 days at a temperature of 25 ℃ of the LAB to be added, the LAB viability retention feature after storing for 180 days at a temperature of 25 ℃ of the LAB to be added, any combination of this pH decrease and LAB viability retention features, the type of food product (such as beverage) and the nature of food product (such as milk-based, fruit-based, vegetable-based, cereal-based, rice-based, nut-based and soy-based food product and any mixture thereof) .

DEPOSIT and EXPERT SOLUTION

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

- Lactobacillus plantarum strain DGCC12411 deposited by DuPont Nutrition Biosciences ApS under accession number DSM32493 on April 26 ^th, 2017, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig -Germany] ;

- Lactobacillus plantarum strain DGCC12119 deposited by DuPont Nutrition Biosciences ApS under accession number DSM33120 on May 22 ^nd, 2019, at the DSMZ; and

- Lactobacillus plantarum strain DGCC12480 deposited by DuPont Nutrition Biosciences ApS under accession number DSM33121 on May 22 ^nd, 2019, at the DSMZ.

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 32 EPC) .

SEQUENCES

SEQ ID NO: 1: L. plantarum ATP-synthase operon

SEQ ID NO: 2: ATP synthase alpha subunit gene of the DSM32493 strain

SEQ ID NO: 3: ATP synthase alpha subunit protein of the DSM32493 strain

SEQ ID NO: 4: ATP synthase alpha subunit gene of a variant of DSM32493 strain

SEQ ID NO: 5: ATP synthase alpha subunit protein of a variant of DSM32493 strain

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

EXAMPLES

Example 1: screening of stable lactic acid bacteria (species)

Stable lactic acid bacteria were selected using assay A as described below:

Inoculum preparation

Each LAB to be tested was prepared as follows: a culture of the LAB at 10 ⁶ cfu/ml was cultured in 10mL MRS/M17 broth overnight at 37 ℃; after 2h at 4℃, the culture was centrifuged at 4000rpm for 10min; the pellet was resuspended in 10mL sterile saline; the centrifugation/resuspension step was repeated a second time. The inoculum was standardized at an amount of around 1x10 ⁹ CFU/ml. The Lactobacillus species listed in Table 1 were tested.

Test yoghurt

A yoghurt having the following features -2.8%protein, 3%fat, 12.5%of total sugar including 8%sucrose; pH 4.3 -was heat-treated to reduce the level of bacteria to less than 1x10 ² CFU per g.

Addition/Inoculation

0.4mL of the prepared inoculum was added into 40mL of the heat-treated yoghurt (in tube) and well mixed. The tube was then sealed. The concentration of stable LAB added into the heat-treated yoghurt (day 0) was around 1x10 ⁷ CFU/g of yoghurt.

Storage

The sealed tube was then stored at 37℃ for 30 days. These conditions were considered to represent an accelerated model of storage at ambient temperature.

At day 30, the pH and the amount of stable LAB (cell count) were determined, and the difference with respectively the pH and amount of added stable LAB at day 0 was calculated.

pH and cell count measurement

The pH was determined by pH meter (Mettler Toledo, SevenEasy)

The CFU count was determined by plating on MRS/M17 agar as follows: 1mL of yogurt sample (day 30) was serial diluted by sterile saline to 10 ^-7; MRS/M17 agar (1.5%) was melted and maintained at 48℃ in water bath; 1mL of 10 ^-1 to 10 ^-7 dilution was added to petri dish and poured with 25mL of the MRS/M17 agar; the plates were incubated at 37℃ anaerobically for 2 days for counting.

Selection

The two following features were considered for selecting stable LABs:

- an amount of LAB of at least 5x10 ³ cfu (3.69 log ₁₀ cfu) ; and

- a pH decrease of at most 0.6 unit (i.e., a pH of at least 3.7) .

Results

80 strains, representative of 33 species, were tested and selected by assay A. The level of strains (cfu) in the yoghurt and the pH of the yoghurt, after storage at 37℃ for 30 days is summarized in Table 1, and represented in Figure 1 (log cfu) and Figure 2 (pH) .

Table 1: log cfu and pH after storage at 37℃ for 30 days obtained by assay A using strains of 33 species of Lactobacillus; STD: standard deviation; n.a.: not applicable

As shown in Table 1 and Figures 1 and 2, strains of 11 Lactobacillus species fulfilled the two parameters defined for selection, i.e., both a viability of at least 5x10 ³ cfu (3.69 log ₁₀ cfu) and a pH decrease of at most 0.6 unit (i.e., a pH of at least 3.7) after storage at 37℃ for 30 days: Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti.

Example 2: screening of stable lactic acid bacteria (strains)

A more in-depth study of the strains encompassed by these 11 Lactobacillus species was carried out, by assay A. Strains were then classified in 4 categories according to their viability and the pH of the yoghurt, after storage at 37℃ for 30 days (Table 2) .

Table 2: classification of strains with respect to their viability and ability to decrease pH

Results obtained by assay A on 20 strains is summarized in Table 3 and Figure 3.

Strain	log CFU	pH	classification
L. acidipiscis B6	3.8	3.79	4
L. plantarum DSM32493	3.8	3.99	3
L. namurensis C8	4.0	3.92	2
L. namurensis F12	4.8	3.96	2
L. namurensis H9	4.8	3.78	3
L. versmoldensis F7	5.0	3.85	2
L. namurensis G1	5.1	3.80	2
L. similis E11	5.4	3.91	3
L. zymae F4	5.5	3.87	2
L. plantarum DSM32493v*	5.5	4.10	1
L. versmoldensis H1	6.1	3.77	3
L. rossiae E5	6.1	3.98	2
L. collinoides F9	6.1	3.92	2
L. zymae C8	6.4	4.05	1
L. hammesii F3	6.5	3.75	3
L. versmoldensis D9	6.5	3.79	3
L. versmoldensis D1	6.5	3.86	2
L. zymae E12	6.9	4.06	1
L. nodensis F11	7.3	3.79	3
L. tucceti G4	7.3	3.78	3

Table 3: log CFU and pH obtained using stable lactic acid bacteria after storage 30 days at 37℃ (*: DSM32493v is a variant of DSM32493 bearing the mutation G to A at position 506 of its ATP synthase alpha subunit gene as compared to DSM32493 and producing an ATP synthase alpha subunit protein as defined in SEQ ID NO: 5)

Thus, the 20 strains were classified as follows:

- 3 strains in category 1 (exceptionally high viability and exceptionally low pH decrease, after storage)

- 8 strains in category 2 (very high viability and very low pH decrease, after storage)

- 8 strains in category 3 (very high viability and low pH decrease, or high viability and very low pH decrease, after storage)

- 1 strain in category 4 (high viability and low pH decrease, after storage) .

These results show that the assay A described herein enables to select strains not only maintaining a high viability in yoghurt after storage at 37℃ for 30 days, but also strains slightly decreasing the pH of this yoghurt after storage. These 20 strains are stable lactic acid bacteria suitable to manufacture a food product of the invention.

Example 3: manufacture of a food product stable at ambient temperature with L. plantarum DSM32493

A yoghurt having the following features -2.8%protein, 3%fat, 8%sucrose; pH 4.3 -was heat-treated to reduce the level of bacteria to less than 1x10 ² CFU per g. The DSM32493 strain (classified in category 3 according to example 2) was inoculated at a level of 1x10 ⁷ cfu/ml of yoghurt.

The inoculated yoghurt was mixed, sealed and stored at 25℃ for 180 days. These conditions represent average ambient storage conditions, when food products are stored out of the fridge or out of cold rooms.

The pH and the amount of stable LAB (cell count) were determined as described for assay A above, at

days

90, 120, 150 and 180. Strain viability and pH over time are represented in Figures 4A and 4B respectively.

The amount of DSM32493 strain after 180 days at 25℃ is 4.8 log10 CFU, i.e., was above 1x10 ⁴ cfu/g of product. This represented a decrease in the amount of bacteria which is less than 3 log, confirming that the DSM32493 can maintain a high viability after 6-month storage at ambient temperature. Interestingly, the maximal amount decrease was obtained at 150 days and slightly increased between day 150 and day 180.

The pH of the product after 180 days at 25℃ was 3.67, i.e., representing a pH decrease which is less than 0.7 unit. Interestingly, the maximal pH decrease was reached at 90 days, and was stable between day 90 and day 180.

These data confirm that the DSM32493 strain is a suitable lactic acid bacterium to manufacture food product stable at ambient temperature. This also confirm more generally that lactic acid bacteria able either to maintain a viability of 5x10 ³ cfu/g together with a pH decrease of at most 0.5 or to maintain a viability of 1x10 ⁴ cfu/g together with a pH decrease of at most 0.6 (i.e., classified in category 3) when selected by assay A, are suitable stable lactic acid bacteria to manufacture food product stable at ambient temperature

Example 4: manufacture of a food product stable at ambient temperature with a variant of L. plantarum DSM32493 (DSM32493v)

A yoghurt having the following features -2.8%protein, 3%fat, 8%sucrose; pH 4.3 -was heat-treated to reduce the level of bacteria to less than 1x10 ² CFU per g. A variant of the DSM32493 strain, DSM32493v (classified in category 1 according to example 2) , was inoculated at a level of 1x10 ⁷ cfu/ml of yoghurt. The inoculated yoghurt was mixed, sealed and stored at 25℃ for 180 days.

days

90, 120, 150 and 180. Strain viability and pH over time are represented in Figures 5A and 5B respectively.

The amount of DSM32493v strain after 180 days at 25℃ is 5.3 log10 CFU, i.e., was above 1x10 ⁵ cfu/g of product. This represented a decrease in the amount of bacteria which is less than 2 log, confirming that the DSM32493v can maintain a very high viability after 6-month storage at ambient temperature.

The pH of the product after 180 days at 25℃ was 3.77, i.e., representing a pH decrease which is less than 0.6 unit. Interestingly, the maximal pH decrease was reached at 90 days, and was stable between day 90 and day 180.

These data confirm that the DSM32493v strain is a suitable lactic acid bacterium to manufacture food product stable at ambient temperature. This also confirm more generally that lactic acid bacteria able to maintain a viability of 1x10 ⁵ cfu/g together with a pH decrease of at most 0.3 (i.e., classified in category 1) when selected by assay A, are suitable stable lactic acid bacteria to manufacture food product stable at ambient temperature.

Altogether these data show that strains selected according to assay A as described herein are confirmed to be suitable for the manufacture of a food product stable at ambient temperature.

Example 5: identification of further stable Lactobacillus plantarum strains

Further L. plantarum strains of the Dupont Danisco collection were tested by assay A, and their viability and the pH of the yoghurt, after storage at 37℃ for 30 days determined. Results for 2 L. plantarum strains are described in Table 4.

Strain	log CFU	pH	classification
L. plantarum DSM33120	5.4	4.11	1
L. plantarum DSM33121	5.7	4.08	1

Table 4: log CFU and pH obtained using stable L. plantarum strains after storage 30 days at 37℃

The two L. plantarum strains identified (DSM33120 and DSM33121) show an exceptional high viability and exceptional low pH decrease, after storage, when tested by assay A, and were classified in category 1. These 2 new strains are stable lactic acid bacteria suitable to manufacture a food product stable at ambient temperature.

These results confirm that assay A described herein enables to select strains not only maintaining a high viability in yoghurt after storage at 37℃ for 30 days, but also strains slightly decreasing the pH of this yoghurt after storage.

Example 6: manufacture of a food product stable at ambient temperature with L. plantarum DSM33120 or DSM33121 strain

A yoghurt having the following features -2.8%protein, 3%fat, 8%sucrose; pH 4.3 -was heat-treated to reduce the level of bacteria to less than 1x10 ² CFU per g. The DSM33120 or DSM33121 strain (classified in category 1 according to example 5) was inoculated at a level of 1x10 ⁷ cfu/ml of yoghurt.

The inoculated yoghurt was mixed, sealed and stored at 25℃ for 180 days. These conditions represent average ambient storage conditions, when food products are stored out of the fridge or out of cold rooms. The pH and the amount of stable LAB (cell count) were determined as described for assay A above, at

days

90, 120, 150 and 180.

Strain viability and pH over time for the DSM33120 strain are represented in Figures 6A and 6B respectively. The amount of DSM33120 strain after 180 days at 25℃ is 5.96 log10 CFU, i.e., was above 9x10 ⁵ cfu/g of product. This represented a decrease in the amount of bacteria of about 1 log, confirming that the DSM33120 can maintain a very high viability after 6-month storage at ambient temperature. The pH of the product after 180 days at 25℃ was 3.75, i.e., representing a pH decrease which is less than 0.5 unit.

Strain viability and pH over time for the DSM33121 strain are represented in Figures 7A and 7B respectively. The amount of DSM33121 strain after 180 days at 25℃ is 6.35 log10 CFU, i.e., was above 2x10 ⁶ cfu/g of product. This represented a decrease in the amount of bacteria which is less than 0.7 log, confirming that the DSM333121 can maintain a very high viability after 6-month storage at ambient temperature. The pH of the product after 180 days at 25℃ was 3.87, i.e., representing a pH decrease which is less than 0.4 unit.

These data confirm that the DSM33120 and DSM33121 strains are suitable lactic acid bacteria to manufacture food product stable at ambient temperature. This also confirm more generally that lactic acid bacteria able either to maintain a viability of 1x10 ⁵ cfu/g together with a pH decrease of at most 0.3 (i.e., classified in category 1) when selected by assay A, are suitable stable lactic acid bacteria to manufacture food product stable at ambient temperature.

Example 7:

Strains used

The Lactiplantibacillus plantarum (also known as Lactobacillus plantarum) strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019. The Streptococcus thermophilus strain DSM 32823 deposited at the DSMZ on May 29 ^th, 2018. The Streptococcus thermophilus strain DSM 33849 deposited at the DSMZ on Apr 20 ^th, 2021.

Manufacture sugar free ambient yogurt with live culture

Five sugar free ambient yoghurt recipes were described in Table 5.

Table 5. detailed recipes of five ambient yoghurt recipes. -, not added.

The sugar free ambient yoghurt was manufacture by following process:

- WPC hydration: heat the raw milk to 45-55℃, add WPC80 and stir 10-15min for dispersion, thereafter hydration for 40～60min without stirring

- Pasteurization: Heat to 60℃, add stabilizer and erythritol, stir and homogenization (at 180 bar) and then pasteurize at 95℃ for 5min.

- Fermentation: Cool the pasteurized milk to 43℃, inoculate the starter culture (e.g. DSM 32823, or DSM 33849, or Yo-Mix 883) and ferment at 43℃ till pH4.

- Cool down: Stir at 150 RPM for 3 min, and cool down to 20℃

- Pasteurization: Pasteurize at 75℃ for 25s

- Aseptic inoculation live culture: Inoculate the 5x10 ⁶ CFU/mL live culture DSM 33120 aseptically, and mix it well

The inoculated yoghurt was sealed and stored at 25℃ for 30 days. These conditions represent average ambient storage conditions, when food products are stored out of the fridge or out of cold rooms.

pH and cell count measurement

The pH was determined by pH meter (Mettler Toledo, SevenEasy) . The CFU count was determined by plating on MRS agar as follows: 1mL of yogurt sample (

day

0, 15, 30) was serial diluted by sterile saline to 10 ^-7; MRS agar (1.5%) was melted and maintained at 48℃ in water bath; 1mL of 10 ^-1 to 10 ^-7 dilution was added to petri dish and poured with 25mL of the MRS agar; the plates were incubated at 37℃ anaerobically for 2 days for counting.

Results

pH and strain viability over time are represented in Figures 8 and 9 respectively.

Recipe 1#is a sugar free recipe fermented by DSM 32823. It’s initial pH at day 0 is 4.37±0.01, which dropped to 3.98 ± 0.01 after 30 days storage at 25℃. The pH decline ΔpH=0.39±0.01. It’s initial logCFU at day 0 is 6.61±0.02, which increased to 7.44±0.05 after 30 days storage at 25℃. The logCFU increase Δ logCFU =0.84±0.06.

Recipe 2#is a sugar free recipe contains 5%erythritol fermented by DSM 32823. It’s initial pH at day 0 is 4.31±0.00, which dropped to 4.21 ± 0.01 after 30 days storage at 25℃. The pH decline ΔpH=0.10±0.01. It’s initial logCFU at day 0 is 6.75±0.02, which increased to 7.70±0.11 afte 30 days storage at 25℃. The logCFU increase Δ logCFU =0.95±0.11.

Recipe 3#is a sugar free recipe fermented by DSM 33849. It’s initial pH at day 0 is 4.44±0.00, which dropped to 4.18 ± 0.01 after 30 days storage at 25℃. The pH decline ΔpH=0.26±0.01. It’s initial logCFU at day 0 is 6.72±0.03, which increased to 7.38±0.01 after 30 days storage at 25℃. The logCFU increase Δ logCFU =0.66±0.03.

Recipe 4#is a sugar free recipe contains 5%erythritol fermented by DSM 33849. It’s initial pH at day 0 is 4.35±0.00, which dropped to 4.31 ± 0.02 after 30 days storage at 25℃. The pH decline ΔpH=0.04±0.01. It’s initial logCFU at day 0 is 6.78±0.06, which increased to 7.48±0.06 after 30 days storage at 25℃. The logCFU increase Δ logCFU =0.70±0.11.

Recipe 5#is a commercial ambient yogurt contains 7.5%sucrose which fermented by starter culture Yo-Mix 883. It was used as reference. It’s initial pH at day 0 is 4.29±0.00, which dropped to 3.89 ± 0.01 after 30 days storage at 25℃. The pH decline ΔpH=0.40±0.01. It’s initial logCFU at day 0 is 6.66±0.03, which increased to 6.69±0.09 after 30 days storage at 25℃. The logCFU increase Δ logCFU =0.03±0.08.

Recipe no.	pH drop (ΔpH)	logCFU change
1#	0.39 ± 0.01	0.84 ± 0.06
2#	0.10 ± 0.01	0.95 ± 0.11
3#	0.26 ± 0.01	0.66 ± 0.03
4#	0.04 ± 0.01	0.70 ± 0.11
5#	0.40 ± 0.01	0.03 ± 0.08

Table 6. pH drop and logCFU change of DSM 33120 in five ambient yoghurt recipes stored 25℃ for 30 days.

Reduction of pH decline by erythritol

In comparison to 1# (ΔpH=0.39±0.01) , adding 5%erythritol (2#recipe, ΔpH=0.10±0.01) , the pH drop (ΔpH) was reduced 74.4%in the ambient shelf-life assessment at 25℃ for 30 days. In comparison to 3# (ΔpH=0.26±0.01) , add adding 5%erythritol (4#recipe) , (ΔpH=0.04±0.01) , the pH drop (ΔpH) was reduced 84.6%in the ambient shelf-life assessment at 25℃ for 30 days. In comparison to commercial ambient yogurt recipe (5#, ΔpH=0.40±0.01) , ΔpH of sugar free recipe 1#was very close, while 2# (ΔpH=0.10±0.01) and 4# (ΔpH=0.04±0.01) were significantly lower. Both results showed that adding erythritol to the yogurt recipe can significantly reduce the pH decline in shelf-life storage.

Reduction of pH decline by using DSM 33849 as starter culture

In comparison to 1# (ΔpH=0.39±0.01) , replacing the starter culture DSM 32823 by DSM 33849 in yogurt fermentation, the pH drop of 3# (ΔpH=0.26±0.01) was reduced 33.3%in the ambient shelf-life assessment at 25℃ for 30 days. In comparison to 2# (ΔpH=0.10±0.01) , replacing the starter culture DSM 32823 by DSM 33849 in yogurt fermentation, the pH drop of 4# (ΔpH=0.04±0.01) was reduced 60%in the ambient shelf-life assessment at 25℃ for 30 days. In comparison to commercial ambient yogurt recipe (5#, ΔpH=0.40±0.01) , ΔpH of sugar free recipe 1#was very close, while 3# (ΔpH=0.26±0.01) was significantly lower. These results showed that using DSM 33849 as starter culture in yogurt fermentation can significantly reduce the pH decline in shelf-life storage.

Best pH stability in yogurt contains erythritol and fermented with DSM 33849

In comparison to commercial ambient yogurt recipe (5#, ΔpH=0.40±0.01) , ΔpH of sugar free recipe 1# (ΔpH=0.39±0.01) was very close. Replacing the starter culture DSM 32823 by DSM 33849 and adding 5%erythritol, 4#showed the best pH stability in the ambient shelf-life assessment at 25℃ for 30 days. The pH drop of 4#was 89.7%reduced in comparison to 1#after storage 30 days at 25℃.

Slightly increased live culture CFU level by erythritol

In comparison to commercial ambient yogurt recipe (5#, ΔlogCFU =0.03 ± 0.08) , the live culture level increase of #1 sugar recipe (ΔlogCFU= 0.84 ± 0.06) was significantly increased 27-folds. In comparison to 1# (ΔlogCFU= 0.84 ± 0.06) , adding 5%erythritol (2#recipe, ΔlogCFU=0.95 ± 0.11) , the live culture level increase (ΔlogCFU) was slightly increased (11.6%) in the ambient shelf-life assessment at 25℃ for 30 days. In comparison to 3#(ΔlogCFU=0.66 ± 0.03) , add adding 5%erythritol (4#recipe, ΔlogCFU=0.70 ± 0.11) , the live culture level increase (ΔlogCFU) was also slightly increased (6%) in the ambient shelf-life assessment at 25℃ for 30 days. Both results showed that adding erythritol to the yogurt recipe can slightly increase the viability of DSM 33120 in shelf-life storage.

Slightly declined live culture CFU level by using DSM 33849 as starter culture

In comparison to 1# (ΔlogCFU= 0.84 ± 0.06) , replacing the starter culture DSM 32823 by DSM 33849 in yogurt fermentation, the live culture level increase (ΔlogCFU) of 3#(ΔlogCFU= 0.66 ± 0.03) was slightly decresed (21.4%) in the ambient shelf-life assessment at 25℃ for 30 days. In comparison to 2# (ΔlogCFU=0.95 ± 0.11) , replacing the starter culture DSM 32823 by DSM 33849 in yogurt fermentation, the live culture level increase (ΔlogCFU) of 4# ( (ΔlogCFU=0.70 ± 0.11) was also slightly decreased (26.3%) in the ambient shelf-life assessment at 25℃ for 30 days. However, the CFU level of both 3#and 4#were still much higher than commercial ambient yogurt recipe (5#, ΔlogCFU =0.03 ± 0.08) .

Highest live culture CFU level recipe contains erythritol and fermented with DSM 32823

Recipe 2#which contains 5%erythritol and fermented by DSM 32823 showed the highest viable live culture (logCFU=7.70±0.11) after storage at 25℃ for 30 days. Meanwhile, it’s pH drop is also pretty low (ΔpH=0.10±0.01) .

These data confirm that adding erythritol to the yogurt recipe can significantly improve the post-acidification (pH drop) of yogurt even stored at ambient condition for up to 30 days. In addition, the CFU levels of live culture L. plantarum DSM 33120 were increased in all four sugar free recipes 1# (ΔlogCFU=0.84 ± 0.06) , 2# (ΔlogCFU=0.95 ± 0.11) , 3# (ΔlogCFU=0.66 ± 0.03) , and 4# (ΔlogCFU=0.70 ± 0.11) in comparison to commercial ambient yogurt recipe 5# (ΔlogCFU=0.03 ± 0.08) . This result confirmed both adding erythritol benefit both of post-acidification (pH drop) and live culture level in yogurt shelf-life storage. This result also confirmed that the reduction of pH drop by erythritol and/or replacing starter culture by DSM 33849 is not due to reduce the viability of live culture DSM 33120.

Claims

A process for manufacturing a food product, said process comprising:

1) providing an initial food product with a pH of between 3.4 and 4.6, in particular an initial low bacteria-containing food product with a pH of between 3.4 and 4.6 containing a level of bacteria which is no more than 1x10 ² CFU per g; which food product contains erythritol in an amount of from about 0.1%to about 15%.

2) adding to the initial food product, in particular to the initial low bacteria-containing food product, one or more stable lactic acid bacteria in a total amount of at least 1x10 ⁵ CFU per g, to obtain a food product stable at ambient temperature,

characterized in that:

(i) each of said one or more stable lactic acid bacterium is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti; and

(ii) each of said one or more stable lactic acid bacterium, when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds:

a) retains viability in an amount of at least 1x10 ⁶ CFU/g after storing said test yoghurt 30 days at a temperature of 25 ℃; and

b) decreases the pH of said test yoghurt of at most. 0.3 units after storing said test yoghurt 30 days at a temperature of 25 ℃.
The process according to claim 1, wherein said stable lactic acid bacterium when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds, retains viability in an amount of at least 1x10 ⁴ CFU/g, at least 5x10 ⁴ CFU/g at least 1x10 ⁵ CFU/g, at least 5x10 ⁵ CFU/g or at least 1x10 ⁶ CFU/g, after storing said test yoghurt 30 days at a temperature of 25 ℃.
The process according to claim 1 or 2, wherein said stable lactic acid bacterium, when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds decreases the pH of said test yoghurt of at most 0.3 units, at most 0.2 units or at most 0.1 units, after storing said test yoghurt 30 days at a temperature of 25 ℃.
The process according to any one of claims 1 to 3, which process provides for the manufacturing of a food product stable at ambient temperature.
The process according to any one of claims 1 to 4, wherein said initial food product with a pH of between 3.4 and 4.6 is selected from the group consisting of a milk-based product such as a fermented dairy product or a chemically-acidified dairy product, a fruit-based product such as a fruit juice or a fermented juice, a vegetable-based product such as a vegetable juice or a fermented vegetable juice, a cereal-based product such as a chemically-acidified cereal product or a fermented cereal product, a rice-based product such as a chemically-acidified rice product or a fermented rice product, a nut-based product such as a chemically-acidified nut product or a fermented nut product a soy-based product such as a fermented soy milk product and any mixture thereof.
The process according to any one of claims 1 to 5, wherein said initial food product with a pH of between 3.4 and 4.6 is a dairy food product, in particular a fermented milk product, more particularly a yoghurt.
The process according to any one of claims 1 to 6, wherein said initial low-bacteria containing food product with a pH of between 3.4 and 4.6 is an initial food product treated so as to obtain a level of bacteria which is no more than 1x10 ² CFU per g of said initial low bacteria-containing food product, in particular an initial heat-treated food product.
A process according to any one of claims 1 to 7, comprising:

1) providing an initial food product with a pH of between 3.4 and 4.6;

1b) treating the initial food product so as to obtain a level of bacteria which is no more than 1x10 ² CFU per g of said initial low bacteria-containing food product, in particular by heat-treating said initial food product; and

2) adding to the initial low bacteria-containing food product one or more stable lactic acid bacteria in a total amount of at least 1x10 ⁵ CFU per g, to obtain a food product stable at ambient temperature.
The process according to any one of claims 1 to 8, wherein said initial low-bacteria containing food product is a treated or heat-treated dairy food product, in particular a treated or heat-treated fermented milk product, more particularly a treated or heat-treated yoghurt.
The process according to any one of claims 1 to 9, said process comprising:

1a) producing an initial fermented milk, in particular an initial yoghurt, with a pH of between 3.4 and 4.6 by fermentation of a milk substrate;

1b) treating, in particular heat-treating, said initial fermented milk, in particular said initial yoghurt, so as to obtain an initial low bacteria-containing fermented milk, in particular an initial low bacteria-containing yoghurt containing a level of bacteria which is no more than 1x10 ² CFU per g; and

2) adding to the initial low bacteria-containing fermented milk, in particular to the initial low bacteria-containing yoghurt, one or more of stable lactic acid bacteria strains in a total amount of at least 1x10 ⁵ CFU per g to obtain a fermented milk, in particular a yoghurt, stable at ambient temperature.
The process according to any one of claims 1 to 10, wherein the pH of said initial food product, in particular of the initial low bacteria-containing food product, is between 3.4 and 4.0, between 4.0 and 4.6 or between 3.6 and 4.2.
The process according to any one of claims 1 to 11, wherein said one or more of stable lactic acid bacteria are added to the initial food product, in particular to the initial low bacteria-containing food product, in a total amount of at least 5x10 ⁵ per g, at least 1x10 ⁶ per g, at least 5x10 ⁶ per g or at least 1x10 ⁷ CFU per g.
The process according to any one of claims 1 to 12, wherein said one or more stable lactic acid bacteria are added aseptically to the initial food product, in particular to the initial low bacteria-containing food product.
The process according to any one of claims 1 to 13, wherein said one or more of stable lactic acid bacteria is of the species Lactobacillus plantarum.
The process according to claim 14, wherein said one or more of stable lactic acid bacteria is selected from the group consisting of the strain DSM32493 deposited at the DSMZ on April 26th, 2017, a variant of the DSM32493 strain, the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019, a variant of the DSM33120 strain, the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 and a variant of the DSM33121 strain, wherein said variant -when added in an amount of 5x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds -a) retains viability in an amount of at least 1x10 ⁶ CFU/g after storing said test yoghurt 30 days at a temperature of 25 ℃; and b) decreases the pH of said test yoghurt of at most 0.2 units after storing said test yoghurt 30 days at a temperature of 25 ℃.
The process according to claim 15, wherein said variant of DSM32493 is the DSM32493 strain into which the ATP synthase alpha subunit gene of the ATP-synthase operon bears the mutation G to A at its position 506.
The process according to any one of claims 1 to 16, wherein a food product is stable at ambient temperature when, after storing it for 180 days at a temperature of 25 ℃:

- its pH is not decreased more than 0.7 unit; and

- the amount of stable lactic acid bacteria it contains is at least 1x10 ³ CFU/g and/or is not decreased more than 3 log.
The process according to any one of claims 1 to 17, wherein said erythritol is present in an amount of from about 0.1%to about 14%, such as in an amount of from about 0.1%to about 12%, such as in an amount of from about 0.1%to about 10%, such as in an amount of from about 0.1%to about 8%, such as in an amount of from about 0.1%to about 6%, such as in an amount of from about 0.1%to about 4%, such as in an amount of from about 0.5%to about 15%, such as in an amount of from about 1%to about 15%, such as in an amount of from about 2%to about 15%, such as in an amount of from about 4%to about 15%, such as in an amount of from about 6%to about 15%, such as in an amount of from about 8%to about 15%, such as in an amount of from about 10%to about 15%, such as in an amount of from about 1%to about 8%, such as in an amount of from about 2%to about 6%, such as in an amount of from about 3%to about 6%.
The process according to any one of claims 1 to 17, wherein said food product is a fermented food product, such as a fermented dairy product, a fermented juice, a fermented vegetable juice, a fermented cereal product, a fermented rice product, a fermented nut product, a fermented soy milk product and any mixture thereof, which fermented product is fermented with lactic acid bacteria starter, such as culture comprising the DSM 33849 strain and/or the DSM 32823 strain.
A food product, obtained by the process of any one of claims 1 to 19.
Use of one or more stable lactic acid bacteria for inoculation in a food product, in particular an initial low bacteria-containing food product, with a pH of between 3.4 and 4.6 in combination with erythritol in an amount of from about 0.1%to about 15%, wherein

(i) each of said one or more stable lactic acid bacterium is selected from the group consisting of strains of species Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus zymae, Lactobacillus rossiae, Lactobacillus collinoides, Lactobacillus similis, Lactobacillus versmoldensis, Lactobacillus acidipiscis, Lactobacillus hammesii, Lactobacillus namurensis, Lactobacillus nodensis and Lactobacillus tucceti; and

(ii) each of said one or more stable lactic acid bacterium, when added in an amount of 1x10 ⁶ CFU per g to a test yogurt having a pH of 4.3, previously heat-treated at 75 ℃for 25 seconds:

a) retains viability in an amount of at least 1x10 ⁶ CFU/g after storing said test yoghurt 30 days at a temperature of 25 ℃; and

b) decreases the pH of said test yoghurt of at most 0.3 units after storing said test yoghurt 30 days at a temperature of 25 ℃.
A Lactobacillus plantarum strain selected from the group consisting of the strain DSM33120 deposited at the DSMZ on May 22 ^nd, 2019, a variant of the DSM33120 strain, the strain DSM33121 deposited at the DSMZ on May 22 ^nd, 2019 and a variant of the DSM33121 strain, in combination with erythritol in an amount of from about 0.1%to about 15%, wherein said variant -when added in an amount of 5x10 ⁶ CFU per g to a test yogurt which is fermented by DSM 33849 having a pH of 4.3, previously heat-treated at 75 ℃ for 25 seconds -a) retains viability in an amount of at least 1x10 ⁶ CFU/g after storing said test yoghurt 30 days at a temperature of 25℃; and b) decreases the pH of said test yoghurt of at most 0.3 units after storing said test yoghurt 30 days at a temperature of 25℃.