Lactic acid bacteria for use in the production of yoghurt analogs

Abstract

The present disclosure generally relates to the field of starter cultures for dairy alternatives, more specifically yoghurt analogs. In particular, the present disclosure relates to a Streptococcus thermophilus strain, a method for producing a fermented plant-based composition using the Streptococcus thermophilus strain, and the fermented plant-based composition obtained by such a method.

Description

[0001] P8145EP00

[0002] 1

[0003] TITLE

[0004] Lactic acid bacteria for use in the production of yoghurt analogs

[0005] FIELD

[0006] The present disclosure generally relates to the field of starter cultures for dairy alternatives, more specifically yoghurt analogs. In particular, the present disclosure relates to a Streptococcus thermophilus strain, a method for producing a fermented plant-based composition using the Streptococcus thermophilus strain, and the fermented plant-based composition obtained by such a method.

[0007] BACKGROUND

[0008] In the manufacture of yoghurt analogs, plant bases are fermented by lactic acid bacteria (LAB) to provide flavor, texture, and acidification. From a food safety point of view, it is desirable to ferment plant bases to a pH of 4.6 or lower within 10 hours, however, these acidification targets cannot be met for some plant bases without the addition of sugar to the base. The necessity to add sugar adds to the costs of manufacturing yoghurt analogs and conflicts with manufacturers wish to produce healthier products containing less sugar.

[0009] Yoghurt analogs may be produced from a plant base, or a mixture of a plant base and milk obtained from mammals, such as cow's milk. Analogs manufactured from mixtures of plant bases and milk obtained from mammals are often referred to as hybrid products. Various plant bases are commercially available, such as almond, rice, pea, fava, oat, and soy bases. Conventionally, combinations of one or more Streptococcus thermophilus (S. thermophilus') strains and one or more Lactobacillus delbrueckii subsp. bulgaricus {Lb. bulgaricus) strains are inoculated into the plant base and allowed to ferment the base at a suitable temperature to provide the yoghurt analog. Other bacterial strains, sometimes referred to as adjunct strains or adjunct cultures, can be added to facilitate faster fermentation, however, yoghurt analogs obtained with such strains not belonging to S. thermophilus or Lb. bulgaricus are generally regarded as non-yoghurt products and are less desired by consumers. Indeed, in many jurisdictions, a product cannot be labelled as a yoghurt if it does not contain significant amounts of S. thermophilus and Lb. bulgaricus, some jurisdictions even requiring a yoghurt to essentially only contain these two bacterial species. Furthermore, adjunct cultures add costs and complexity to the manufacturing process, and yoghurt manufacturers thus prefer to use only the conventional bacterial species.P8145EP00

[0010] 2

[0011] Soy bases are currently the most popular plant base used in the manufacture of yoghurt analogs. The main fermentable sugar in this base is sucrose, which means that Lb. bulgaricus, which is known to not ferment on sucrose, essentially does not contribute to the acidification. Thus, when using conventional S. thermophilus and Lb. bulgaricus strains in the fermentation of soy bases, it is essentially only S. thermophilus that provides acidification. Typically, soy bases contain in the range of 0.6-0.9 wt% sucrose, however, this concentration of sucrose is insufficient for reaching the acidification target (pH 4.6 within 10 hours) when using conventional S. thermophilus. Examples of insufficient acidifications of soy bases with conventional cultures are shown in figure 1. Here, acidification curves of two commercially available soy bases, which were fermented with a commercially available starter culture containing S. thermophilus and Lb. bulgaricus, are shown. The first soy base is acidified from a pH of 7.2 to a pH of 5.3 at 10 hours, whereas the second soy base is acidified from a pH of about 7 to a pH of 4.7 at 10 hours. To aid the fermentation such that the acidification target can be reached, additional sucrose is commonly added to the soy base, the specific amount of sucrose depending on the soy base's natural sucrose content, start pH, and buffering capacity. Manufacturers, however, wish to reduce the amount of added sugars to meet consumer's demand for healthier products and to reduce costs.

[0012] Hence, there remains a need for a method for fermenting a plant base to food safety acidification targets using lower amounts of added sugar, preferably no added sugars, when fermenting with S. thermophilus and Lb. bulgaricus.

[0013] SUMMARY

[0014] When S. thermophilus is fermenting in soy base, sucrose is taken up by the bacterium via a PTS system, phosphorylated and then split into glucose-6-P and fructose. Glucose is then used for growth and some of the fructose is excreted into the soy base.

[0015] Surprisingly, novel S. thermophilus strains identified by the inventors of the present invention were found to not leave any fructose in a soy base after fermentation. Further investigation showed that these S. thermophilus strains can acidify soy bases down to the target pH of 4.6 or lower within 10 hours. Also, media containing fructose or sucrose as the only carbon source were fermented with the strains, and the acidification curves of the fermentations are shown in figures 3 and 4, respectively. The results indicate that the strains can utilize both the glucose-6-P and the fructose part from sucrose for growth, and that the utilization of both monosaccharides of the sucrose molecule is the reason for the deeper acidification. As shown in figure 2, sequencing of strains revealed that the genomes of the strains of the present invention encode an intact gene forP8145EP00

[0016] 3

[0017] Fructose PTS subunit IIC, whereas prior art strains contain a frameshift in the same gene. It is thought that the frameshift mutation renders the gene non-functional in the prior art strains. The ubiquitousness of this frameshift mutation in conventional S. thermophilus may be explained by the fact that there has been no selective pressure for conserving Fructose PTS functionality throughout the hundreds of years of continuous selection of S. thermophilus strains for producing yoghurt, since mammalian milk does not contain fructose. Without being bound by theory, it is believed that the fructose excreted by S. thermophilus is taken up and 1-phosphorylated by the Fructose PTS, the fructose-l-P in turn entering the glycolysis pathway. On the other hand, it is believed that strains that do not possess an intact Fructose PTS substantially do not reuptake the excreted fructose.

[0018] Accordingly, the present disclosure provides in a first aspect a Streptococcus thermophilus strain, wherein the Streptococcus thermophilus strain comprises a first nucleotide encoding a first polypeptide, the first polypeptide having at least 90%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with the polypeptide of SEQ ID NO:1.

[0019] The polypeptide of SEQ ID NO:1 is polypeptide encoded by the intact Fructose PTS subunit IIC. The advantageous deeper acidification provided by the S. thermophilus strain possessing this intact Fructose PTS subunit IIC is evident from figure 5, which show that the strain can be used to ferment a soy base to a pH of 4.6 in less than 7 hours.

[0020] Definitions

[0021] As used herein, the term "wt%" means weight percent, i.e., "g per 100 g".

[0022] As used herein, the terms such as "2 % sucrose" and "2 % fructose" refer to 2% weight / volume solution, i.e., "g per 100 mL".

[0023] The terms "yoghurt analog" or "plant-based composition" used herein are meant to refer to dairy-like products, which are products used as culinary replacements for dairy products, prepared where one or more mammalian milk constituents have been replaced with plant material and the resulting food resembles the original product. The mammalian milk constituents are replaced completely or at least 30% by dry weight with plant material, for example, using planted-based milks derived from legumes (such as soybeans, pea, lentils or chickpeas), nuts (such as coconut), cereals (such as oat).P8145EP00

[0024] 4

[0025] As used herein, the adjective "dairy" shall be taken to mean a composition or product comprises or consists of mammalian milk matter, i.e. the lacteal secretion obtainable by milking.

[0026] As used herein, the term "free from" should be understood as a composition or product which does not contain a given substance but where trace amounts or contaminants thereof may be present.

[0027] As used herein, the term "added sugar" shall refer to sugars that are added during the processing of foods (e.g. refined sugars that may be added to a plant base of processed plant matter) as opposed to sugars naturally occurring in said foods. Added sugars include sugars (free, mono- and disaccharides), sugars from syrups and honey, and sugars from concentrated fruit or vegetable juices that are in excess of what would be expected from the same volume of 100 percent fruit or vegetable juice of the same type.

[0028] As used herein, the term "fermentation" or "fermenting" refers to a process wherein carbohydrates are transformed into a range of metabolites through chemical reactions carried out by one or more microorganisms.

[0029] The term "lactic acid bacteria" ("LAB") designates food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, low-GC, acid tolerant, non-sporulating, non-respiring, rodshaped bacilli or cocci.

[0030] During the fermentation stage, the consumption of carbohydrate by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum.

[0031] The term "plant base", is used to describe the plant material used as a base for fermentation. As an example, the plant base may be a soy base. Such a soy base may e.g., be obtained by soaking and grinding soybeans, boiling the mixture, and filtering out remaining particulates. Soy base can, e.g., be a soy milk, which is a plant-based drink.P8145EP00

[0032] 5

[0033] The relatedness between two amino acid sequences or between two nucleotide sequences is described herein by the parameter "sequence identity".

[0034] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

[0035] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0036] In the context of the present application, "M17 medium" means a cell culture medium with the following composition (per liter H2O):

[0037] ascorbic acid 0.5 g, magnesium sulfate 0.25 g, meat extract 5 g, meat peptone (peptic) 2.5 g, sodium glycerophosphate 19 g, soya peptone (papainic) 5 g, tryptone 2.5 g, yeast extract 2.5 g, and wherein the medium has final pH 7.0±0.2 (25°C).

[0038] 2 % fructose M17 is an "M17 medium" which furthermore contains 20 g fructose / 1 iter H2O. 2 % sucrose M17 is an "M17 medium" which furthermore contains 20 g sucrose / liter H2O.

[0039] As known to the skilled person, the M17 medium is a medium that is well-known to be suitable for growth of Streptococcus thermophilus.

[0040] BRIEF DESCRIPTION OF THE FIGURES

[0041] Figures 1 shows an acidification curve of two soy bases with a culture containing S. thermophilus according to the prior art.

[0042] Figure 2 shows a nucleic acid alignment between an intact gene encoding Fructose PTS subunit IIC from a strain according to an embodiment of the invention and a truncated Fructose PTS subunit IIC according to the prior art.

[0043] Figure 3 shows an acidification curve of the fermentation of 2 % fructose M17 with strains according to embodiments of the invention.P8145EP00

[0044] 6

[0045] Figure 4 shows an acidification curve of the fermentation of 2 % sucrose M17 with strains according to embodiments of the invention.

[0046] Figure 5 shows an acidification curve of the fermentation of a soy base with strains according to embodiments of the invention.

[0047] Detailed description

[0048] In an embodiment, the Streptococcus thermophilus strain has a maximum acidification speed in a 2 % fructose M17 medium of at least 0.5 pH units / hour, preferably at least 0.6 pH units / hour, most preferably at least 0.7 pH units / hour, wherein the maximal acidification speed is determined by:

[0049] i) providing a 2 % sucrose M17 medium and a 2 % fructose M17 medium ii) inoculating the Streptococcus thermophilus strain in the 2 wt% sucrose M17 medium thereby providing a first suspension,

[0050] iii) allowing the Streptococcus thermophilus strain to drive a fermentation in the first suspension for 15 hours at 40°C, thereby providing a fermentate, iv) heating the 2 % fructose M17 medium to 40°C,

[0051] v) inoculating the fermentate in the 2 % fructose M17 medium at an inoculation rate of 1 wt% thereby obtaining a second suspension,

[0052] vi) keeping the second suspension at a temperature of 40°C for a fermentation period of 15 hours and measuring the pH of the suspension every minute during the fermentation period,

[0053] vii) determining, from the pH measured in vi) the maximal drop in pH that occurred during an hour of the fermentation period.

[0054] In a specific version of the previous embodiment, the maximum acidification speed is in the range of 0.7-1.3 pH units / hour.

[0055] In an embodiment, the Streptococcus thermophilus strain is selected from Streptococcus thermophilus DSM 35267 and Streptococcus thermophilus DSM 35268.

[0056] The inventors of the present invention have furthermore found that when inoculating a soy base with a culture of S. thermophilus and Lb. bulgaricus, wherein the culture contains up to 50 wt% of Lb. bulgaricus, the advantageous deeper fermentation provided by the S. thermophilus is not affected.

[0057] Accordingly, the present disclosure provides in a second aspect a culture comprising the Streptococcus thermophilus strain of the first aspect, wherein the culture furthermore comprises a Lactobacillus delbrueckii subsp. bulgaricus strain.P8145EP00

[0058] 7

[0059] In an embodiment the culture comprises up to 50 wt% Lb. bulgaricus, such as in the range of 0.1-50 wt% of Lb. bulgaricus.

[0060] The present disclosure provides in a third aspect a method for producing a fermented plant-based composition, the method comprising the steps of:

[0061] i) providing a plant base,

[0062] ii) inoculating the Streptococcus thermophilus strain according to the first aspect or the culture according to the second aspect into the plant base, and

[0063] iii) allowing the Streptococcus thermophilus strain to acidify the plant base thereby obtaining the fermented plant-based composition.

[0064] In an embodiment, the Streptococcus thermophilus strain is allowed in the step iii) to acidify the plant base to a pH of 4.6 or lower. In a specific version of this embodiment, the Streptococcus thermophilus is allowed in the step of iii) to acidify the plant base to a pH in the range of 4.5 to 4.6.

[0065] In an embodiment, the step iii) is carried out within 10 hours.

[0066] In an embodiment, the plant base is kept at a temperature in the range of 37-44 °C during the step of iii).

[0067] The present disclosure provides in a fourth aspect a use of the S. thermophilus strain of according to the first aspect 1 or the culture of the second aspect for fermenting a plant base.

[0068] In an embodiment, the plant base is substantially free from added sugars.

[0069] In an embodiment, the plant base has a natural sucrose content of at most 1.5 wt%, such as 0.3 to 1.5 wt%, preferably at most 1.0 wt%, such as 0.3 to 1.0 wt%, more preferably at most 0.9 wt%, such as 0.3 to 0.9 wt%.

[0070] In an embodiment, wherein the plant base is selected from almond base, rice base, and soy base. Preferably, the plant base is a soy base.

[0071] In an embodiment, the plant base has a pH in the range of 6.5 to 7.3, such as in the range of 6.7 to 7.2.P8145EP00

[0072] 8

[0073] In an embodiment, the plant base has a protein content in the range of 3.6 to 6.2 wt%, such as in the range of 3.8 to 5.5 wt%, such as in the range of 3.9 to 5.1 wt%.

[0074] In an embodiment, the plant base has a pH in the range of 6.5 to 7.3, and a protein content in the range of 3.6 to 6.2 wt%. In another version of this embodiment, the plant base has a pH in the range of 6.5 to 7.3, and a protein content in the range of 4 to 5 wt%.

[0075] In an embodiment of the method, the plant base has a pH in the range of 6.5 to 7.3, a protein content in the range of 3.6 to 6.2 wt%, and is substantially free from added sugar.

[0076] DEPOSIT AND EXPERT SOLUTION

[0077] The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0078] Table 1: The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

[0079]

[0080] EXAMPLES

[0081] Example 1 :

[0082] Screening of fructoohilic strains of Streptococcus thermoohilus from CHCC.

[0083] Streptococcus thermophilus strains from Chr. Hansen Culture Collection (CHCC) were screened for the ability to grow in M17 broth added 2 % fructose at 40°C under anaerobic conditions. Initial screening was performed in microtiter plates format (200 pl). The optical density was measured in a BioTek LogPhase 600 Microbiology ReaderP8145EP00

[0084] 9

[0085] From the best growing leads, strains DSM 35267 and DSM 35268 were picked and further tested in a 10ml tube containing the same broth. The two strains were capable of reaching an optical density at 600nm (OD600) at 2 or more after overnight incubation at 40°C under anaerobic conditions whereas other strains did not grow and grew only to a very low OD.

[0086] In addition, DSM 35267, DSM 35268, and other S. thermophilus strains were analyzed for their ability to grow in 2 % sucrose M17, 2 % fructose M17 and in soy base (Provamel, Organic Soya Drink unsweetened, Alpro C.V.A., Vlamingstraat 28, BE-8560 Wevelgem; Naturli', Organic Soy drink, Unsweetened, Naturli' Foods A / S, Gronvangsalle 2a, DK-6600 Vejen).

[0087] Strains were grown anaerobically overnight at 40 C in a 10 ml containing M17 + 2 % lactose. Two ml of these cultures (1%) was used to inoculate 200 ml baby bottles with each of the four media. Development of pH was followed using a iCINAC pH logger (iCinac, AMS Alliance, France) to provide the graphs shown in figs 3-5.

[0088] Finally, a starter culture containing S. thermophilus and Lb. bulgaricus (Vega Premium, Chr. Hansen A / S) was used in a fermentation of two soy bases (Provamel, Organic Soya Drink unsweetened, Alpro C.V.A., Vlamingstraat 28, BE-8560 Wevelgem; Naturli', Organic Soy drink, Unsweetened, Naturli' Foods A / S, Gronvangsalle 2a, DK-6600 Vejen). The acidification curves of the fermentations were logged via iCINAC and are shown in fig 1.

[0089] Conclusion

[0090] Strains DSM 35267 and DSM 35268 both possessing an intact gene for Fructose PTS subunit IIC were found to ferment on both sucrose and fructose. Furthermore, as shown in figs 1 and 5, the two strains DSM 35267 and DSM 35268 fermented Provamel soy base to a lower pH (deeper) than prior S. thermophilus strains not possessing an intact gene for Fructose PTS subunit IIC. Specifically, DSM 35267 and DSM 35268 reached pH 4.6 at around 7 hours and continued to acidify to a pH of 4.5, whereas the fermentation with prior art strains did not go below pH 4.7.

Claims

P8145EP0010CLAIMS1. A Streptococcus thermophilus strain, wherein the Streptococcus thermophilus strain comprises a first nucleotide encoding a first polypeptide, the first polypeptide having at least 90%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% amino acid identity with the polypeptide of SEQ ID NO:1.

2. The Streptococcus thermophilus strain of claim 1, wherein the Streptococcus thermophilus strain has a maximum acidification speed in a 2 % fructose M17 medium of at least 0.5 pH units / hour, preferably at least 0.6 pH units / hour, most preferably at least 0.7 pH units / hour, wherein the maximal acidification speed is determined by:i) providing a 2 % sucrose M17 medium and a 2 % fructose M17 medium ii) inoculating the Streptococcus thermophilus strain in the 2 wt% sucrose M17 medium thereby providing a first suspension,iii) allowing the Streptococcus thermophilus strain to drive a fermentation in the first suspension for 15 hours at 40°C, thereby providing a fermentate, iv) heating the 2 % fructose M17 medium to 40°C,v) inoculating the fermentate in the 2 % fructose M17 medium at an inoculation rate of 1 wt% thereby obtaining a second suspension,vi) keeping the second suspension at a temperature of 40°C for a fermentation period of 15 hours and measuring the pH of the suspension every minute during the fermentation period,vii) determining, from the pH measured in vi) the maximal drop in pH that occurred during an hour of the fermentation period.

3. The Streptococcus thermophilus strain of claim 1 or 2, wherein the Streptococcus thermophilus strain is selected from Streptococcus thermophilus DSM 35267 and Streptococcus thermophilus DSM 35268.

4. A culture comprising the Streptococcus thermophilus strain of any one of the preceding claims, wherein the culture furthermore comprises a Lactobacillus delbrueckii subsp. bulgaricus strain.

5. A method for producing a fermented plant-based composition, the method comprising the steps of:i) providing a plant base,ii) inoculating the Streptococcus thermophilus strain according to any one of the claims 1-3 or the culture of claim 4 into the plant base, andiii) allowing the Streptococcus thermophilus strain to acidify the plant base thereby obtaining the fermented plant-based composition.P8145EP00116. The method of claim 5, wherein in step iii) the Streptococcus thermophilus strain is allowed to acidify the plant base to a pH of 4.6 or lower.

7. The method of claim 5 or 6, wherein step iii) is carried out within 10 hours.

8. The method of any one of the claims 5-7, wherein the plant base is substantially free from added sugars.

9. The method of any one of the claims 5-7, wherein the plant base has a natural sucrose content of at most 1.5 wt%, preferably at most 1.0 wt%, more preferably at most 0.9 wt%.

10. The method of any one of the claims 5-8, wherein the plant base is selected from almond base, rice base, and soy base.

11. Use of a Streptococcus thermophilus strain according to any one of the claims 1-3 or the culture of claim 4 for fermenting a plant base.

12. The use according to claim 11, wherein the plant base is substantially free from added sugars.

13. The use according to claim 11 or 12, wherein the plant base has a natural sucrose content of at most 1.5 wt%, preferably at most 1.0 wt%, more preferably at most 0.9 wt%.

14. The use according to any one of the claims 11-13, wherein the plant base is selected from an almond base, a rice base, and a soy base.

15. The use according to claim 14, wherein the plant base is a soy base.

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