Methods for improving stability of lactic acid bacteria during storage

Abstract

Disclosed herein are methods and compositions for improving storage stability of dry lactic acid bacteria compositions.

Description

[0001] TITLE: Methods for improving stability of lactic acid bacteria during storage

[0002] The present invention relates to novel methods for providing lactic acid bacteria that have improved stability when undergoing storage.

[0003] BACKGROUND

[0004] Industrially produced lactic acid bacteria are widely used for production of fermented products. Cultures of lactic acid bacteria are often formulated as frozen- or in dry-formats, such as freeze-dried granulates or powders. It is well known that dry compositions of lactic acid bacteria are susceptible to elevated temperature and humidity, and that lack of cold chain during shipment and storage can have serious, negative impact on the viability and acidification activity of the bacterial cultures.

[0005] Unfortunately, the negative impacts from storage at elevated temperatures are particularly harsh on the dried lactic acid bacteria when they are stored at temperatures of 25°C or higher. The traditional solution is to store the dried lactic acid bacteria in cold storage, such as a fridge or a freezer to maintain viability and acidification activity of the lactic acid bacteria. However, providing cold storage or a cold chain for the lactic acid bacteria during transport or dedicated cold facilities for storage in regions with warmer climates can be both challenging, cumbersome, and expensive due to restrictions in availability, power requirements and logistics capacity of the remote site. At the same time, demand for lactic acid bacteria containing products, such as for example dairy products, is increasing in many regions around the world with warmer climates.

[0006] Therefore, with increasing demand for lactic acid bacteria derived products, for example dairy products, in regions around the world with a warm climate and regions experiencing increasingly warmer climates due to climate change, there is a need for an alternative solution that does not dependent on the availability of cold storage facilities and cold chains, which further depends on logistics, and requires a constant supply of electricity.

[0007] Meeting this need will help facilitate dairy production in warmer climates and regions experiencing warming climates due to climate change, as well as generally increasing the robustness of the supply chain. This will ultimately also lead to improved food availability and food security as well as better food and dairy products in regions with warmer climates.

[0008] It is an object of the present disclosure to overcome these issues and improve food safety and food supply in regions with warmer climates. SUMMARY

[0009] The present disclosure relates to dry compositions of lactic acid bacteria that show an increased stability of the lactic acid bacteria after storage at temperatures of above 25°C, such as 37°C. It has been found that such an increased stability can be obtained by controlling the amount of reducing sugar in the dry lactic acid bacteria compositions.

[0010] Thus, a first aspect of the present disclosure relates to a dry lactic acid bacteria composition comprising

[0011] -lactic acid bacteria

[0012] -a total concentration concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols, and

[0013] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0014] The present disclosure also relates to a method for producing such a dry lactic acid bacteria composition.

[0015] Thus, in a second aspect, the present disclosure relates to a method for producing a dry lactic acid bacteria composition comprising the steps

[0016] xi) providing a liquid lactic acid bacteria concentrate,

[0017] xii) mixing the liquid lactic acid bacteria concentrate of xi) with a cryoprotectant to form a liquid lactic acid bacteria composition comprising

[0018] -lactic acid bacteria

[0019] -a total dry weight concentration of 1-50% of one or more nonreducing sugars and / or sugar alcohols in the liquid lactic acid bacteria composition w / w, and

[0020] -a total dry weight concentration of less than 4% w / w of reducing sugar in the liquid lactic acid bacteria composition,

[0021] and xiii) subjecting the liquid lactic acid bacteria composition of xii) to one or more drying steps, thereby obtaining a dry lactic acid bacteria composition comprising -lactic acid bacteria

[0022] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, and

[0023] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0024] In a third aspect, such dry lactic acid bacteria compositions are used for producing a starter culture, a direct vat set (DVS) or an inoculant.

[0025] In a fourth aspect, such dry lactic acid bacteria compositions are used for providing a starter culture, direct vat set (DVS) or inoculant, which has improved stability following storage at temperatures above 25°C for at least two weeks, 4 weeks, 8 weeks, or 12 weeks.

[0026] In a fifth aspect, such dry lactic acid bacteria compositions, starter cultures, direct vat sets (DVS), or inoculants are used for producing a fermented product, a dairy product or a food product.

[0027] DETAILED DESCRIPTION

[0028] The present disclosure relates to dry compositions of lactic acid bacteria and methods for formulation of dry lactic acid bacteria with increased the stability in dry lactic acid bacteria compositions, when such dry lactic acid bacteria are stored at temperatures above 25°C.

[0029] As shown in examples 1-4 and figures 1-9 of the present disclosure, the content of reducing sugars in dry compositions of lactic acid bacteria is connected to the stability of lactic acid bacteria in dry composition and their metabolic activity as measured by determining acidification activity (ta) for compositions of dried lactic acid bacteria incubated following storage at 37°C. Lower amounts of reducing sugars in the dry lactic acid bacteria compositions lead to improved stability and acidification activity of the lactic acid bacteria in the dry compositions as shown in figures 1-3 and 5-7 by an increased ability to retain acidification activity during storage and overtime. From these results it is clear storage stability and shelf life of dry lactic acid bacteria compositions at temperatures above 25°C can be improved vastly by limitation of reducing sugar content in the dry lactic acid bacteria compositions.

[0030] Thus, a first aspect of the present disclosure relates to a dry lactic acid bacteria composition comprising

[0031] -lactic acid bacteria

[0032] -a total concentration concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols, and

[0033] - a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0034] As demonstrated in example 1c it appears that dry compositions of lactic acid bacteria compositions participate in the Maillard reaction as indicated by browning when stored at temperatures higher than 25°C, e.g., temperatures of 37°C, leading to accelerated degradation the lactic acid bacteria in the dry composition. As the Maillard reaction requires the presence of a reducing agent, such as a reducing sugar, it was found herein that the stability during storage of dry lactic acid bacteria compositions at higher temperatures, could be improved by limitation and / or replacement of reducing sugar content in the fermentation medium, the fermentation broth at end of fermentation, and in downstream ingredients, such as cryoprotectants in the case of freeze drying. It was further found that suitable replacements for reducing sugars, include non-reducing sugars and sugar alcohols, as neither of these easily participate in the Maillard reaction.

[0035] For these reasons, a dry lactic acid bacteria composition according to the present disclosure comprises less than 4% w / w reducing sugar and one or more non-reducing sugars and / sugar alcohols in a total concentration of 1-50% w / w.

[0036] Although having reducing sugar present in a total concentration of less than 4% w / w is considered well suitable for improving storage stability of dry lactic acid bacteria compositions, because any amount of reducing sugar may participate in the Maillard reaction, preferably reducing sugars is present in a total concentration of less than 3.5% w / w, more preferably reducing sugars is present in a total concentration of less than 3% w / w, yet more preferably reducing sugars is present in a total concentration of less than 2.5% w / w, yet more preferably reducing sugars is present in a total concentration of less than 2% w / w, yet more preferably reducing sugars is present in a total concentration of less than 1.5% w / w, yet more preferably reducing sugars is present in a total concentration of less than 1.0% w / w, yet more preferably reducing sugars is present in a total concentration of less than 0.5% w / w, yet more preferably reducing sugars is present in a total concentration of less than 0.1% w / w, or yet more preferably no reducing sugars are present.

[0037] Commonly, reducing sugars such as lactose and glucose are used for stabilizing microorganisms through drying processes, formulation and / or within dry compositions. However, when formulating dry compositions comprising lactic acid bacteria intended for storage at higher temperatures, i.e. temperatures above 25°C, reducing sugars instead become detrimental to the stability of the lactic acid bacteria in dry composition. The stabilizing effect that reducing sugars generally have on lactic acid bacteria in dry composition when stored frozen or in cold storage, can instead obtained for lactic acid bacteria in dry compositions stored at temperatures above 25°C by using non-reducing sugars and / or sugar alcohols in the place of reducing sugars. In the present context, such stabilizing effects on lactic acid bacteria in dry compositions is obtained using a total concentration of 1-50% of one or more non-reducing sugars and / or sugar alcohols. However, the total concentration of one or more non-reducing sugars and / or sugar alcohols may also be a total concentration selected from 1-40% w / w, 1-30% w / w, 1-20% w / w, 5-10% w / w, 2-10% w / w, 1.5-10% w / w, 1-10% w / w, 2-5% w / w, 1.5-5% w / w w / w, and 1-5% w / w.

[0038] An example of the stabilizing effects of non-reducing sugars is found in example 3 (and shown in figure 6), where the use of a cryoprotectant comprising a non-reducing sugar (sucrose) as the only sugar resulted in a lower loss of CFU count of DSM 15954 (higher viability) following storage at 37°C, compared to when two cryoprotectants comprising a mix of reducing and non-reducing sugars (sucrose+fructose and sucrose+galactose) were used.

[0039] Thus, it is clear from these data that to improve the stability of dry lactic acid bacteria compositions, one must also consider which cryoprotectant to use and seek to minimize the cryoprotectant content of reducing sugars.

[0040] In general, any cryoprotectant that is suitable for human consumption and which does not comprise reducing sugars can be contemplated for use with the dry lactic acid bacteria composition as defined herein.

[0041] Thus, in one or more embodiments, a dry lactic acid bacteria composition comprises

[0042] -lactic acid bacteria

[0043] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition, and

[0044] -a cryoprotectant free of reducing sugars.

[0045] In one or more embodiments, a dry lactic acid bacteria composition comprises

[0046] -lactic acid bacteria

[0047] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0048] -a total concentration of less than 3.5% w / w of reducing sugar in the dry lactic acid bacteria composition, and

[0049] -a cryoprotectant free of reducing sugars.

[0050] In one or more embodiments, a dry lactic acid bacteria composition comprises

[0051] -lactic acid bacteria

[0052] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0053] -a total concentration of less than 3% w / w of reducing sugar in the dry lactic acid bacteria composition, and

[0054] -a cryoprotectant free of reducing sugars.

[0055] In one or more embodiments, a dry lactic acid bacteria composition comprises

[0056] -lactic acid bacteria

[0057] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0058] -a total concentration of less than 2.5% w / w of reducing sugar in the dry lactic acid bacteria composition, and

[0059] -a cryoprotectant free of reducing sugars.

[0060] In one or more embodiments, a dry lactic acid bacteria composition comprises

[0061] -lactic acid bacteria -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0062] -a total concentration of less than 2% w / w of reducing sugar in the dry lactic acid bacteria composition, and

[0063] -a cryoprotectant free of reducing sugars.

[0064] Within the context of the present disclosure, it was found that a combination of one or more nonreducing sugars and / or sugar alcohols, an amino acid, an organic acid, and a structural enhancer, is particularly useful for improving the stability of lactic acid bacteria in dry compositions when stored at temperatures above 25°C.

[0065] Thus, in one or more exemplary embodiments, a dry lactic acid bacteria composition comprises -lactic acid bacteria

[0066] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0067] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0068] -one or more amino acids,

[0069] -at least one antioxidant, and

[0070] -at least one structural enhancer.

[0071] Thus, in one or more exemplary embodiments, a dry lactic acid bacteria composition comprises -lactic acid bacteria

[0072] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0073] -a total concentration of less than 3.5% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0074] -one or more amino acids,

[0075] -at least one antioxidant, and

[0076] -at least one structural enhancer.

[0077] Thus, in one or more exemplary embodiments, a dry lactic acid bacteria composition comprises -lactic acid bacteria

[0078] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0079] -a total concentration of less than 3% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0080] -one or more amino acids,

[0081] -at least one antioxidant, and

[0082] -at least one structural enhancer.

[0083] Thus, in one or more exemplary embodiments, a dry lactic acid bacteria composition comprises -lactic acid bacteria

[0084] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0085] -a total concentration of less than 2.5% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0086] -one or more amino acids,

[0087] -at least one antioxidant, and

[0088] -at least one structural enhancer.

[0089] In one or more exemplary embodiments, a dry lactic acid bacteria composition comprises -lactic acid bacteria

[0090] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0091] -a total concentration of less than 2% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0092] -one or more amino acids,

[0093] -at least one antioxidant, and

[0094] -at least one structural enhancer.

[0095] The one or more amino acids, at least one antioxidant, and the at least one structural enhancer may be added in particularly desirable concentrations. Thus, in one or more embodiments, the dry lactic acid bacteria composition as defined herein comprises one or more amino acids in a total concentration of 1-20% w / w, at least one antioxidant in a total concentration of 1-20% w / w, and at least one structural enhancer in a total concentration of 1-30% w / w.

[0096] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids in a total concentration of 5-15% w / w, at least one antioxidant in a total concentration of 5-18% w / w, and at least one structural enhancer in a total concentration of 5-18% w / w.

[0097] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids in a total concentration of 8-12% w / w, at least one antioxidant in a total concentration of 12-16% w / w, and at least one structural enhancer in a total concentration of 12-16% w / w.

[0098] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids, and at least one structural enhancer in a ratio of 2-3 parts amino acids, 3-4 parts antioxidant and 3-4 parts structural enhancer.

[0099] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids, and at least one structural enhancer in a ratio of 2 parts amino acids, 3 parts antioxidant and 3 parts structural enhancer.

[0100] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids, and at least one structural enhancer in a ratio of 3 parts amino acids, 4 parts antioxidant and 4 parts structural enhancer.

[0101] Preferably, the one or more amino acids of the dry lactic acid bacteria composition as defined herein are selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof.

[0102] Preferably, the at least one antioxidant of the dry lactic acid bacteria composition as defined herein is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof. Preferably, the at least one structural enhancer of the dry lactic acid bacteria as defined herein is selected from caseinate, starch, spray gum, and pea protein isolate.

[0103] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, at least one antioxidant selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, and, at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate. In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, in a total concentration of 1-20% w / w, the at least one antioxidant is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, in a total concentration of 1-20% w / w, and at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate, in a total concentration of 1-30% w / w.

[0104] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, in a total concentration of 5-15% w / w, the at least one antioxidant is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, in a total concentration of 5-18% w / w, and at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate, in a total concentration of 5-18% w / w.

[0105] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, in a total concentration of 8-12% w / w, the at least one antioxidant is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, in a total concentration of 12-16% w / w, and at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate, in a total concentration of 12-16% w / w.

[0106] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected as glutamic acid or a salt thereof, at least one antioxidant selected as ascorbic acid or a salt thereof, and at least one structural enhancer selected as caseinate.

[0107] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises glutamic acid or a salt thereof in a total concentration of 1-20% w / w, ascorbic acid or a salt thereof in a total concentration of 1-20% w / w, and caseinate in a total concentration of 1-30% w / w.

[0108] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises glutamic acid or a salt thereof in a total concentration of 5-15% w / w, ascorbic acid or a salt thereof in a total concentration of 5-18% w / w, and caseinate in a total concentration of 5-18% w / w.

[0109] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises glutamic acid or a salt thereof in a total concentration of 8-12% w / w, ascorbic acid or a salt thereof in a total concentration of 12-16% w / w, and caseinate in a total concentration of 12-16% w / w.

[0110] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, in a total concentration of 5-12% w / w, the at least one antioxidant is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, in a total concentration of 5-16% w / w, and at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate, in a total concentration of 5-16% w / w.

[0111] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, at least one antioxidant is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, and at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate, in a ratio of 2-3 parts amino acids, 3-4 parts antioxidant and 3-4 parts structural enhancer.

[0112] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, at least one antioxidant is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, and at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate, in a ratio of 2 parts amino acids, 3 parts antioxidant and 3 parts structural enhancer.

[0113] In one or more embodiments, the dry lactic acid bacteria composition as defined herein further comprises one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof, at least one antioxidant is selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, and at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate, in a ratio of 3 parts amino acids, 4 parts antioxidant and 4 parts structural enhancer.

[0114] In some cases, the dry lactic acid bacteria composition further comprises fermentation broth residues and lactic acid.

[0115] The dry lactic acid bacteria composition as defined herein comprises lactic acid bacteria in an amount of about 106-1013CFU. Thus, in one or more embodiments, a dry lactic acid bacteria composition comprises at least 106, 107, 108, 109, 1010, 1011, 1012, or at least 1013CFU lactic acid bacteria. In one or more exemplary embodiments, a dry lactic acid bacteria composition comprises at least 106CFU lactic acid bacteria. In one or more exemplary embodiments, a dry lactic acid bacteria composition comprises at least 106CFU lactic acid bacteria, preferably at least 107CFU lactic acid bacteria, more preferably at least 108CFU lactic acid bacteria, or yet more preferably at least 109CFU lactic acid bacteria, yet more preferably at least 1010CFU lactic acid bacteria, yet more preferably at least 1011CFU lactic acid bacteria, yet more preferably at least 1012CFU lactic acid bacteria, or yet more preferably at least 1013CFU lactic acid bacteria.

[0116] In some cases, the dry lactic acid bacteria composition may comprise maltodextrin as an additive. When maltodextrin is used an additive, only maltodextrins with a low number of free chain ends are used, i.e., maltodextrins with a dextrose equivalent of between 2-12.

[0117] Thus, in one or more embodiments, the dry lactic acid bacteria compositions defined as herein may comprise up to a total concentration of 1% w / w maltodextrin with a dextrose equivalent of 2-12.

[0118] Methods for producing dry lactic acid compositions according to the present disclosure

[0119] The dry lactic acid compositions of the present disclosure are produced by the mixing of a lactic acid bacteria concentrate with a cryoprotectant, followed by a drying step to produce the dry lactic acid bacteria composition.

[0120] The lactic acid bacteria concentrate is mixed with cryoprotectant in a ratio that following the drying step results in a final concentration in the dry lactic acid bacteria composition of less than 4% w / w, preferably less than 3.5% w / w, more preferably less than 3% w / w, yet more preferably less than 2.5% w / w, yet more preferably less than 2% w / w, yet more preferably less than 1.5% w / w, yet more preferably less than 1% w / w, yet more preferably less than 0.5% w / w, yet more preferably less than 0.1% w / w reducing sugar, and a final concentration of non-reducing sugars and / or sugar alcohols is in the range 1-50% w / w.

[0121] A method for producing a dry composition according to the present disclosure can therefore be described as a method comprising the steps

[0122] xi) providing a liquid lactic acid bacteria concentrate,

[0123] xii) mixing the liquid lactic acid bacteria concentrate of xi) with a cryoprotectant to form a liquid lactic acid bacteria composition comprising

[0124] -lactic acid bacteria -a total dry weight concentration of 1-50% of one or more nonreducing sugars and / or sugar alcohols in the liquid lactic acid bacteria composition w / w, and

[0125] -a total dry weight concentration of less than 4% w / w of reducing sugar in the liquid lactic acid bacteria composition,

[0126] and

[0127] xiii) subjecting the liquid lactic acid bacteria composition of xii) to one or more drying steps, thereby obtaining a dry lactic acid bacteria composition comprising -lactic acid bacteria

[0128] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, and

[0129] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0130] In one or more embodiments, the method comprises a total dry weight concentration of less than 3.5% w / w of reducing sugar in step xii) and step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar as defined in step xii) and step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2.5% w / w of reducing sugar as defined in step xii) and step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar as defined in step xii) and step xiii).

[0131] In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 4% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 3.5% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 3% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 2.5% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 2% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 1.5% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 1% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 3% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 0.5% w / w of reducing sugar step xiii).

[0132] In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 4% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 3.5% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 3% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 2.5% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 2% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 1.5% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 1% w / w of reducing sugar step xiii). In one or more embodiments, the method comprises a total dry weight concentration of less than 2% w / w of reducing sugar in step xii) and a total dry weight concentration of less than 0.5% w / w of reducing sugar step xiii).

[0133] A liquid lactic acid bacteria composition as referred to in step xii) may comprise up to about 80% w / w water.

[0134] In order to achieve such a low concentration of reducing sugars in the dry lactic acid bacteria composition, it is necessary to manage the amount of reducing sugars used in the fermentation process as part of the fermentation medium. As can be seen in particular in example 1a the fermentation of lactic acid bacteria composition designated FD-DVS 2 in 4% w / w lactose fermentation medium, led to significant accumulation of the reducing sugar galactose, which made up to 17.5% w / w of the dry composition FD-DVS 2 This accumulation of galactose led to a significantly worse storage stability over time as shown in table 2 and figure 4 and a reduced acidification activity following storage at 37°C.

[0135] Accordingly, the fermentation medium and fermentation process should ideally only comprise the use of reducing sugars that are fully metabolizable by the lactic acid bacteria cultured in a given fermentation process. Similarly, the fermentation medium should comprise only either non-reducing sugars which are fully metabolizable by a given lactic acid bacteria strain, or non-reducing sugars, where any reducing sugars that may be released due to metabolization of the non-reducing sugars, are reducing sugars that are fully metabolizable by a given lactic acid bacteria.

[0136] In one or more exemplary embodiments, the method for producing a dry lactic acid bacteria composition as disclosed herein further comprises;

[0137] -culturing lactic acid bacteria in a fermentation process, wherein the fermentation process is carried out by;

[0138] -using only reducing sugars and / or non-reducing sugars that are fully metabolizable by the lactic acid bacteria in the fermentation medium, and / or

[0139] -using only partially metabolizable reducing sugars and / or non-reducing sugars that when metabolized only release non-reducing sugars or release reducing sugars that are fully metabolizable by the lactic acid bacteria cultured in the fermentation process, and

[0140] -at the end of the fermentation process, concentrating the fermentation broth to produce the lactic acid bacteria concentrate used in step xi).

[0141] To improve dry storage stability at e.g. 37°C for dry compositions of lactic acid bacteria of the S. thermophilus strain, it is highly important that the fermentation medium does not contain galactose and does not contain a sugar that is only partially metabolized by S. thermophilus, (i.e. lactose, which is only partially metabolized, leading to accumulation of galactose in the fermentation broth).

[0142] In one or more exemplary embodiments, the method for producing a dry lactic acid bacteria composition comprising an S. Thermophilus strain incapable of metabolizing galactose (e.g. DSM35049) further comprises; -culturing lactic acid bacteria belonging to an S. Thermophilus strain in a fermentation process, wherein the fermentation process is carried out without using galactose and without using reducing sugars and / or non-reducing sugars that contain galactose and / or release galactose when metabolized during the fermentation process, and

[0143] -at the end of the fermentation process, concentrating the fermentation broth to produce the lactic acid bacteria concentrate used in step xi).

[0144] Generally speaking, the knowledge of which reducing and non-reducing sugars that are fully metabolizable, partially metabolizable and non-metabolizable by specific species and strains of lactic acid bacteria is considered to be textbook knowledge and would be known to the person skilled in the art.

[0145] A non-limiting list of examples are listed in the table below to illustrate the process by which such choices of sugars may be made.

[0146] Commonly used cryoprotectants within the field of lactic acid bacteria may comprise reducing sugars such as lactose (e.g. in the form of milk / skimmed milk powder), as they are intended for use with dry compositions of lactic acid bacteria that are intended to be stored at cold storage temperatures and below 0°C. At these temperatures, the impact of reducing sugars on the stability of the lactic acid bacteria is negligible. However, if these compositions were intended for storage at higher temperatures, the reducing sugars in such cryoprotectants would become a problem.

[0147] In the present context, to improve storage stability of lactic acid bacteria in dry lactic acid bacteria compositions during storage at temperatures above 25°C, ideally the cryoprotectant should comprise as little reducing sugars as possible. One way of ensuring this is the case, is to use cryoprotectants which comprises non-reducing sugars and / or sugar alcohols in place of the reducing sugar component.

[0148] In one or more embodiments, the cryoprotectant comprises one or more non-reducing sugars and / or sugar alcohols, and is essentially free of reducing sugars.

[0149] However, the cryoprotectant used in the methods herein and referred to in stepxii) is not particularly limited as long as it is suitable for human dietary use and only comprises reducing sugars in a concentration that results in a concentration of reducing sugars lower than 4% w / w, preferably lower than 3.5% w / w, more preferably lower than 3% w / w, even more preferably lower than 2.5% w / w, or yet more preferably 2% w / w, yet even more preferably lower than 1.5% w / w, yet even more preferably lower than 1% w / w, yet even more preferably lower than 0.5% w / w, yet even more preferably lower than 0.1% w / w, in the dry lactic acid bacteria composition obtained in step xiii) after mixing with a lactic acid bacteria concentrate and drying. In one or more embodiments, the cryoprotectant used in the methods herein and referred to in step xii) is not particularly limited as long as it is suitable for human dietary use and only comprises reducing sugars in a concentration that results in a concentration of reducing sugars lower than 2% w / w, preferably lower than 1.5% w / w, more preferably lower than 1% w / w, even more preferably lower than 0.5% w / w, or yet more preferably 0.1% w / w in the dry lactic acid bacteria composition obtained in step xiii) after mixing with a lactic acid bacteria concentrate and drying.

[0150] In one or more embodiments, a desirable cryoprotectant comprises one or more non-reducing sugars and / or sugar alcohols, one or more amino acids, at least one antioxidant, and at least one structural enhancer.

[0151] In one or more embodiments, the desirable cryoprotectant comprises one or more non-reducing sugars and / or sugar alcohols in a concentration that results in a concentration of one or more nonreducing sugars and / or sugar alcohols of 1-50% w / w in a dry lactic acid bacteria composition obtained in step xiii), after mixing with a lactic acid bacteria concentrate and drying.

[0152] In one or more embodiments, a desirable cryoprotectant is a cryoprotectant, which comprises only ingredients that are isolated from plants.

[0153] In one or more embodiments, a particularly desirable cryoprotectant comprises

[0154] -one or more non-reducing sugars and / or sugar alcohols,

[0155] -one or more amino acids selected from the list consisting of glutamic acid or a salt thereof, and, aspartic acid and a salt thereof,

[0156] -at least one antioxidant selected from the list consisting of ascorbic acid or a salt thereof, and, citric acid or a salt thereof,

[0157] -at least one structural enhancer selected from the list consisting of caseinate, starch, spray gum, pea protein isolate, and

[0158] wherein the cryoprotectant is essentially free of reducing sugar.

[0159] In one or more embodiments, the one or more amino acids are selected as glutamic acid or a salt thereof, the at least one antioxidant selected as ascorbic acid, and the at least one structural enhancer selected as caseinate.

[0160] The one or more amino acids may be present in the cryoprotectant used in the method as defined herein in a concentration of that would yield a total concentration of 1-20% w / w, 5-15% w / w, 5-12% w / w or 8-12% w / w in the dry lactic acid composition as produced by the method disclosed herein. The at least one antioxidant may be present in the cryoprotectant used in the method as defined herein in a concentration of that would yield a total concentration of 1-20% w / w, 5-18% w / w, 5-16% w / w or 12-16% w / w in the dry lactic acid composition as produced by the method disclosed herein. The at least one structural enhancer may be present in the cryoprotectant used in the method as defined herein in a concentration of that would yield a total concentration of 1-30% w / w, 5-18% w / w, 5-16% w / w or 12-16% w / w in the dry lactic acid composition as produced by the method disclosed herein.

[0161] The calculations, measurements and weighing of ingredients necessary for matching concentrations of ingredients in the lactic acid bacteria concentrate with ingredients in the cryoprotectant and obtaining the desired concentrations in the dry lactic acid bacteria concentration are routine adjustments and tasks only, that would be obvious to the person skilled in the art without exercise of inventive skills.

[0162] Drying of lactic acid bacteria compositions

[0163] Any one of several commonly known methods for drying bacteria can be used for producing dried compositions comprising lactic acid bacteria. Thus, the one or more drying steps of stepxiii) may include freeze-drying, spray-drying and vacuum drying of a lactic acid bacteria composition, culture or concentrate. Generally, freeze-drying is considered as the more gentle drying method, which preserves a higher number of viable bacteria, while spray-drying is considered the more convenient drying method, being easy to use and having a low process cost.

[0164] Thus, in one or more embodiments, the dry lactic acid bacteria composition is obtained by a method as defined herein where the one or more drying steps of step xiii) comprise one of freeze-drying, spray-drying and vacuum drying of the liquid lactic acid bacteria composition formed in step xii).

[0165] Thus, in one or more embodiments, the dry lactic acid bacteria composition is obtained by a method as defined herein where the one or more drying steps of step xiii) comprise one of spray drying or freeze drying of the liquid lactic acid bacteria composition formed in step xii).

[0166] Thus, in one or more embodiments, the dry lactic acid bacteria composition is obtained by a method as defined herein where the one or more drying steps of step xiii) comprise spray drying of of the liquid lactic acid bacteria composition formed in step xii). Thus, in one or more embodiments, the dry lactic acid bacteria composition is obtained by a method as defined herein where the one or more drying steps of step xiii) comprise spray drying of of the liquid lactic acid bacteria composition formed in stepxii).

[0167] Thus, in one or more embodiments, the dry lactic acid bacteria composition is obtained by a method as defined herein where the one or more drying steps of step xiii) comprise freeze drying of the liquid lactic acid bacteria composition formed in stepxii).

[0168] In one or more embodiments, when the one or more drying steps of xiii) comprise freeze-drying, the one or more drying steps comprise

[0169] xiii-a) freezing the liquid lactic acid bacteria composition formed in step xii) to produce a frozen lactic acid bacteria composition, and

[0170] xiii-b) exposing the frozen lactic acid bacteria produced in step xiii-a) to a lowered pressure, removing the frozen water by sublimation and thereby obtaining a dry lactic acid bacteria composition.

[0171] In one or more embodiments, when the one or more drying steps of xiii) comprise freeze-drying, the one or more drying steps comprise

[0172] xiii-a1) freezing the liquid lactic acid bacteria composition formed in stepxii) by exposure to a cryogenic liquid, thereby producing a frozen lactic acid bacteria composition, and

[0173] xiii-b1) exposing the frozen lactic acid bacteria produced in step xiii-a) to a vacuum, removing the frozen water by sublimation and thereby obtaining a dry lactic acid bacteria composition.

[0174] In one or more embodiments, the cryogenic liquid used for freezing the liquid lactic acid bacteria in step xiii-a1 ) is liquid nitrogen.

[0175] A dry lactic acid bacteria composition as referred to herein generally has a water activity of between 0.1 to 0.20 awafter completion of the drying process. Thus, in one or more embodiments, the drying step may be performed on a liquid lactic acid bacteria composition until the water activity awof the liquid lactic acid bacteria composition is reduced to between about 0.1 to 0.20. DEFINITIONS

[0176] Fermentation medium

[0177] A fermentation medium as used herein is a composition that is designed to meet the nutritional requirements of a microorganism, i.e. in this case lactic acid bacteria, and thereby facilitate the growth of these lactic acid bacteria in a fermentation process. In order to meet these nutritional requirements, the fermentation medium contains at least one or more carbon source(s), one or more nitrogen source(s), minerals, and some growth factors, such as essential amino acids, fatty acids and vitamins. The fermentation medium can further be supplemented with various additional components if specific effects or conditions are desired during the fermentation process.

[0178] Fermentation broth

[0179] A fermentation broth as used herein refers to the liquid phase of a fermentation process. This liquid phase comprises both ingredients originating from the fermentation medium, byproducts and / or breakdown products resulting from fermentation of the fermentation medium, and residues, molecules and / or cellular debris originating from the microorganism(s) performing the fermentation process.

[0180] Reducing sugars

[0181] In the present context a reducing sugar is any sugar that is capable of acting as a reducing agent. All monosaccharides, including the common dietary monosaccharides glucose, fructose and galactose are reducing sugars. The reducing sugars can be divided into two groups, the aldoses, which have an aldehyde group, and the ketoses, which have a ketone group. Ketoses must first tautomerize to form aldoses via an enediol intermediate before they can act as reducing sugars. The aldehyde group of the aldose can then be oxidized by interaction with an oxidizing agent to form a carboxylic acid, reducing the oxidizing agent in the reduction process.

[0182] The cyclic hemiacetal forms of aldoses can open to reveal an aldehyde, and certain ketoses can undergo tautomerization to become aldoses. However, acetals, including those found in polysaccharide linkages, cannot easily become free aldehydes, and can therefore also act as reducing agents unless conditions are extreme. Thus, only disaccharides and polysaccharides comprising hemiacetal groups are considered reducing sugars. Examples of reducing disaccharides include lactose, maltose and cellobiose.

[0183] As discussed reducing sugars are known to oxidise and are notoriously known fortheir participation in the Maillard reaction, wherein the reducing sugars of a given product reacts with e.g. amino acids, releasing water in the process and leading to a non-enzymatic browning of the product. The speed of the Maillard reaction is heat dependent and known to proceed rapidly in for example bread baked at e.g. temperatures of 140-165°C. Although, the Maillard reaction does not proceed as rapidly at temperatures such as 25°C, the Maillard reaction is still relevant for products stored at 25°C for any amount of time, as the process still occurs as time passes by, although at a lower pace.

[0184] In alkaline solutions reducing sugars are present predominantly in the aldehyde or ketone form, which allows the the reducing sugars to act as reducing agents. Thus, it can be tested whether a given sugar is reducing or not, by testing the reducing sugar in an alkaline solution.

[0185] Non-reducing sugars

[0186] Non-reducing sugars are sugars which do not easily act as reducing agents because they do not have a free aldehyde or ketone group that can participate in such reduction reaction.

[0187] Non-reducing sugars can be found as disaccharides, oligosaccharides or polysaccharides. Nonreducing disaccharides like sucrose and trehalose have glycosidic bonds between their anomeric carbons and thus cannot convert to an open-chain form with an aldehyde group and they are stuck in the cyclic form.

[0188] Examples of reducing sugars include sucrose, trehalose, raffinose, stachyose, and verbascose. In one or more embodiments, a non-reducing sugar is selected as one or more from the list consisting of sucrose, trehalose, raffinose, stachyose, and verbascose.

[0189] In more particular embodiments, a non-reducing sugars is selected as one or more from the list consisting of sucrose and trehalose. In more particular embodiments, a non-reducing sugars is selected as sucrose. In more particular embodiments, a non-reducing sugars is selected as trehalose.

[0190] Sugar alcohols

[0191] Sugar alcohols as used herein are organic carbon compounds which have a single -OH attached to each carbon. Linear sugar alcohols are compounds that fulfill the general chemical formula HOCH2(CHOH)nCH2OH, wherein n is a positive natural number. Sugar alcohols occur naturally but can also be obtained as products of fermentation processes or as products of industrial hydrogenation of sugars and in particular by hydrogenation of pentoses and hexoses. Examples of sugar alcohols include glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol and polyglycitol.

[0192] In one or more exemplary embodiments, a sugar alcohol as referred to herein is a sugar alcohol comprising n carbon atoms, wherein n is selected as a natural number in the range of 2-24, 2-18, 2-12, 2-6, 2-5, or 2-4. In one or more exemplary embodiments, a sugar alcohol comprising n carbon atoms, wherein n is selected as a natural number in the range of 2-24. In one or more exemplary embodiments, a sugar alcohol comprising n carbon atoms, wherein n is selected as a natural number in the range of 2-18. In one or more exemplary embodiments, a sugar alcohol comprising n carbon atoms, wherein n is selected as a natural number in the range of 2-12. In one or more exemplary embodiments, a sugar alcohol comprising n carbon atoms, wherein n is selected as a natural number in the range of 2-6. In one or more exemplary embodiments, a sugar alcohol comprising n carbon atoms, wherein n is selected as 3, 4, 5 and / or 6. In one or more exemplary embodiments, a sugar alcohol comprising n carbon atoms, wherein n is selected as 4, 5 and / or 6.

[0193] In one or more exemplary embodiments, the sugar alcohol is selected as one of glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol and polyglycitol.

[0194] In one or more exemplary embodiments, the sugar alcohol is selected as one of glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, and lactitol.

[0195] In one or more exemplary embodiments, the sugar alcohol is selected as one of glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, and inositol.

[0196] In one or more exemplary embodiments, the sugar alcohol is selected as one of erythritol, inositol, arabitol, mannitol, lactitol, maltitol, and isomalt.

[0197] In one or more exemplary embodiments, the sugar alcohol is selected as one of inositol, xylitol, and sorbitol.

[0198] Non-reducing sugars and sugar alcohols in dry compositions of lactic acid bacteria

[0199] The dry lactic acid bacteria composition as disclosed herein may comprise one or more nonreducing sugars, one or more sugar alcohols, or a combination thereof. There are no particular limitations on the type of non-reducing sugar or sugar alcohol as long as the non-reducing sugar and / or sugar alcohol are suitable for human and / or animal consumption. In one or more embodiments, the one or more non-reducing sugar and / or one or more sugar alcohol are selected as non-reducing sugars and / or sugar alcohols with a total number of 30 or less carbon atoms

[0200] In one or more embodiments, the one or more non-reducing sugars are selected as non-reducing disaccharide(s).

[0201] In one or more embodiments, the non-reducing sugar is selected as non-reducing disaccharide(s) and / or the sugar alcohol is selected as a sugar alcohol with a total number 30 or less carbon atoms.

[0202] In one or more embodiments, the non-reducing sugar and / or sugar alcohol is selected as one or more from the list consisting of sucrose, trehalose, erythritol, raffinose, stachyose, verbascose, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, isomalt, maltotriitol, and maltotetraitol. In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as one or more from the list consisting of sucrose, trehalose, erythritol, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol and isomalt.

[0203] In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as one or more from the list consisting of sucrose, trehalose, erythritol, inositol, xylitol, sorbitol, maltitol and isomalt.

[0204] In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as one or more from the list consisting of sucrose, trehalose, erythritol, inositol, arabitol, mannitol, lactitol, maltitol, isomalt.

[0205] In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as one or more from the list consisting of sucrose, trehalose, inositol, xylitol and sorbitol.

[0206] In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as one or more from the list consisting of trehalose, sucrose and inositol.

[0207] In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as sucrose and one of trehalose and inositol.

[0208] In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as inositol and one of trehalose and sucrose.

[0209] In one or more embodiments, the non-reducing sugars and / or sugar alcohols are selected as trehalose and one of inositol and sucrose.

[0210] In one or more embodiments, the one or more non-reducing sugars and / or sugar alcohols are selected as inositol.

[0211] In one or more embodiments, the one or more non-reducing sugars and / or sugar alcohols are selected as trehalose.

[0212] In one or more embodiments, one or more non-reducing sugars and / or sugar alcohols are selected as sucrose. Fully metabolizable sugars

[0213] Fully metabolizable sugars in the present context are sugars which can be completely consumed by a bacteria of interest during fermentation. In the present context a bacteria of interest is a lactic acid bacteria as defined herein. For monosaccharides, this condition is fulfilled if the monosaccharide is fermented, leading to release of an acid, which is predominantly lactic acid in the case of lactic acid bacteria. Di- and polysaccharides are considered fully metabolizable, when the di- and polysaccharides can be enzymatically digested to release their component sugars, and all of these component monosaccharide sugars can be consumed by fermentation. For example, the disaccharide trehalose can be enzymatically digested leading to release of two glucose residues. Thus for a bacteria capable of digesting trehalose and utilizing glucose, both trehalose and glucose would be considered fully metabolizable. In the present context lactose would only be considered fully metabolizable for a given bacteria which is capable of metabolizing lactose and the glucose and galactose residues resulting from lactose breakdown. Similarly, polysaccharides are only considered fully metabolizable if a given bacteria is capable of cleaving it, first into shorter chain polysaccharides, such as disaccharides, and then into its component monosaccharides, and finally, if the bacteria is capable of utilizing these monosaccharides.

[0214] Partially metabolizable sugars

[0215] Partially metabolizable sugars are within the present context di-and polysaccharides that contain monosaccharides, which cannot be utilized by a given bacteria in a fermentation process. In the above example with lactose, if a given bacteria is able to utilize glucose, but unable to utilize galactose in a fermentation process, then forthat bacteria, lactose would be considered a partially metabolizable sugar.

[0216] Non-metabolizable sugars

[0217] Non-metabolizable sugars are within the present context for example monosaccharides that cannot be utilized in a fermentation process by a given bacteria. Non-metabolizable sugars within the present context are also di- or polysaccharides, which either cannot be broken down to such an extent that some of its constituent saccharides can be utilized by a given bacteria in a fermentation process, or di- or polysaccharides, which cannot be broken down by a given bacteria at all.

[0218] Thus, to sum up, for a bacteria capable of breaking lactose down to galactose and glucose and capable of utilizing glucose, but not galactose in a fermentation process, glucose is considered a fully metabolizable sugar, while galactose is considered a non-metabolizable sugar, and lactose would be considered a partially metabolizable sugar.

[0219] Slow turnover sugars

[0220] When dealing with lactic acid bacteria and bacteria in general, it is also important to note that sugar utilization in fermentation media containing more than one types of sugar may occur in a preferential manner, leading to complete metabolization of one sugar species (e.g. glucose) and a slower metabolization of another sugar (e.g. fructose) leading to an accumulation of the slower metabolized sugar species in the fermentation medium.

[0221] The term 'slow turnover sugars' as used in the present disclosure refers to these sugars that are metabolized more slowly.

[0222] This slower metabolism can result from factors such as low enzymatic specificity, preferential expression of enzymes for utilizing other sugars, or operon-controlled regulation, which either delays or suppresses the expression of enzymes required for utilizing the slow turnover sugar.

[0223] Examples of metabolic sugar utilization in lactic acid bacteria

[0224] The most commonly used lactic acid bacteria in industrial production of fermented milk products comprise species Streptocccus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactococcus lactis, Lactobacillus helveticus and Leuconostoc mesenteroides.

[0225] Species Streptocccus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus differentiate from the other mentioned species by the inability to utilize the whole lactose molecule. Vast majority of Streptocccus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus do not efficiently, or not at all, utilize the galactose moiety of lactose. Consequently, galactose is excreted by the cells to surrounding environment and significant amounts of galactose can be detected in lactose-containing fermentation broths or in fermented milk. The Streptococcus thermophilus strains are further known to utilize sucrose, both alone or in combination with lactose. Utilization of monosaccharides, such as fructose, galactose and glucose, was found to be highly variable (Gasser et al., Food Microbiology, Vol 107, 2022;

[0226] https: / / doi. Org / 10.1016 / j.fm.2O22.104080).

[0227] Lactobacillus delbrueckii subsp. bulgaricus has a narrow spectrum of fermentable carbohydrates. Beside lactose, fructose, glucose and mannose were reported to be utilized by strain of this species (Carvalho et al. 2004. Biotechnol. Prog. 2004, 20, 248-254;

[0228]

[0229] t / / www. biomedcentral, com / 1471. Sucrose is not utilized (DOI 10.1099 / ijsem.0.004107).

[0230] Lactobacillus delbrueckii subsp. lactis has a broader spectrum of fermentable carbohydrates, including cellobiose, fructose, galactose, glucose, lactose, mannose, sucrose and trehalose Carvalho et al. 2004. Biotechnol. Prog. 2004, 20, 248-254 (htp: / / www. biomedcentral. com / 1471 -

[0231] The spectrum of fermentable carbohydrates of Lactococus lactis comprise of cellobiose, fructose, galactose, glucose, lactose, maltose, mannose and trehalose (doi: 10.1093 / femsre / fuaa033; Andersson et al. 2001. The Journal of Biological Chemistry 276 (46), Nov 16:42707-42713). Thus, for the lactic acid bacteria species mentioned above, it should be avoided using sugars that are either not fully metabolizable by the bacteria or which are only partially metabolizable by the lactic acid bacteria and which lead to accumulation of an unmetabolizable reducing sugar, e.g. use of lactose in the fermentation medium leading to accumulation of galactose in a fermentation process comprising lactic acid bacteria of Streptococcus thermophilus or the subspecies Lactobacillus delbrueckii subsp. bulgaricus. Similarly, to the extent it is possible, it should be avoided to introduce slow turnover sugars or partially metabolizable sugars that lead to release of slow turnover sugars when broken down in the fermentation medium.

[0232] Generally, the sugar metabolism of individual species of lactic acid bacteria is text book knowledge and with the directions provided herein, it will be a routine task only for the skilled person to adapt a fermentation medium to a species or strain of lactic acid bacteria and avoid build-up of reducing sugars in the fermentation medium.

[0233] Lactic acid bacteria concentrate

[0234] The terms cell concentrate, concentrate, lactic acid bacteria cell concentrate, lactic acid bacteria concentrate, concentrate of lactic acid bacteria, and cell concentrate of lactic acid bacteria are used herein to refer to the solid matter parts of a lactic acid bacteria fermentation broth, which has been spun down or centrifuged to separate solid parts (including lactic acid bacteria) of the fermentation broth from the liquid parts of the fermentation broth. The solid parts, including the lactic acid bacteria that remains after removal of the liquid parts of the fermentation broth is the lactic acid bacteria concentrate.

[0235] Formulation of lactic acid bacteria concentrate in preparation for drying Before drying, a lactic acid bacteria concentrate as defined herein is formulated with a cryoprotectant. The cryoprotectant is typically a sterilized water solution of ingredients with cryo-and lyo- functionality, which protect cells and maintain their viability and activity during freezing, freeze-drying or drying.

[0236] Encapsulation index

[0237] In the present disclosure an encapsulation index (El) refers to mixing liquid cryoprotectant with liquid cell concentrate on the dry weight basis and corresponds to the ratio of the dry weight of cryoprotectant to the dry weight of lactic acid bacteria concentrate used in producing a dry lactic acid bacteria composition.

[0238] Accordingly, in the case of an encapsulation index of 1, an equal dry weight of cryoprotectant and lactic acid bacteria concentrate is mixed, e.g. 1 g dry weight of cryoprotectant is mixed with 1 g dry weight of lactic acid bacteria concentrate. For an encapsulation index of 0.5, 0,5 g dry weight of cryoprotectant is mixed with 1 g dry weight of lactic acid bacteria concentrate.

[0239] Dry lactic acid bacteria compositions

[0240] In this section, the content of specific dry lactic acid bacteria compositions of the present disclosure are described in further details.

[0241] Composition A

[0242] A dry lactic acid bacteria composition comprising

[0243] -lactic acid bacteria

[0244] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, and

[0245] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0246] Composition B

[0247] A dry lactic acid bacteria composition comprising

[0248] -lactic acid bacteria -a total concentration of 1-50% w / w of one or more one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, erythritol, raffinose, stachyose, verbascose, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, isomalt, maltotriitol, and maltotetraitol, in the dry lactic acid bacteria composition, and

[0249] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0250] Composition C

[0251] A dry lactic acid bacteria composition comprising

[0252] -lactic acid bacteria

[0253] -a total concentration of 1-50% w / w of one or more one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, erythritol, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, and isomalt, in the dry lactic acid bacteria composition, and

[0254] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0255] Composition D

[0256] A dry lactic acid bacteria composition comprising

[0257] -lactic acid bacteria

[0258] -a total concentration of 1-50% w / w of one or more one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, inositol, xylitol, and sorbitol, in the dry lactic acid bacteria composition, and -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0259] Composition E

[0260] A dry lactic acid bacteria composition comprising

[0261] -lactic acid bacteria - a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, and inositol, in the dry lactic acid bacteria composition, and

[0262] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0263] In one or more embodiments, the dry lactic acid bacteria composition as disclosed herein, further comprises one or more amino acids, at least one antioxidant, and at least one structural enhancer. The one or more amino acids may be selected as one or more from glutamic acid or salts thereof, or aspartic acid or salts thereof, the at least one antioxidant may be selected as one or more of ascorbic acid or a salt thereof, or citric acid or a salt thereof, and the at least one structural enhancer may be selected as one or more of caseinate, starch, spray gum, pea protein isolate. Such compositions are described further in composition F-N below.

[0264] Composition F

[0265] A dry lactic acid bacteria composition comprising

[0266] -lactic acid bacteria

[0267] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, erythritol, raffinose, stachyose, verbascose, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, isomalt, maltotriitol, and maltotetraitol, in the dry lactic acid bacteria composition,

[0268] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0269] -one or more amino acids,

[0270] -at least one antioxidant, and

[0271] -at least one structural enhancer.

[0272] Composition G:

[0273] A dry lactic acid bacteria composition comprising

[0274] -lactic acid bacteria -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, erythritol, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol and isomalt, in the dry lactic acid bacteria composition,

[0275] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0276] -one or more amino acids,

[0277] -at least one antioxidant, and

[0278] -at least one structural enhancer.

[0279] Composition H:

[0280] A dry lactic acid bacteria composition comprising

[0281] -lactic acid bacteria

[0282] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, inositol, xylitol, and sorbitol, in the dry lactic acid bacteria composition,

[0283] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0284] -one or more amino acids,

[0285] -at least one antioxidant, and

[0286] -at least one structural enhancer.

[0287] Composition I:

[0288] A dry lactic acid bacteria composition comprising

[0289] -lactic acid bacteria

[0290] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, and inositol, in the dry lactic acid bacteria composition,

[0291] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition, -one or more amino acids,

[0292] -at least one antioxidant, and

[0293] -at least one structural enhancer.

[0294] Composition J:

[0295] A dry lactic acid bacteria composition comprising

[0296] -lactic acid bacteria

[0297] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0298] -a total concentration of reducing sugar in the dry lactic acid bacteria composition of less than reducing sugar in a total concentration of less than 4% w / w, -one or more amino acids selected from glutamic acid or a salt thereof, and aspartic acid or salt thereof,

[0299] -at least one antioxidant selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, and

[0300] -at least one structural enhancer selected from caseinate, starch, spray gum, pea protein isolate.

[0301] Composition K:

[0302] A dry lactic acid bacteria composition comprising

[0303] -lactic acid bacteria

[0304] - a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0305] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0306] -glutamic acid or a salt thereof,

[0307] -ascorbic acid or a salt thereof, and

[0308] -Caseinate or a salt thereof. Composition L:

[0309] A dry lactic acid bacteria composition comprising

[0310] -lactic acid bacteria

[0311] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0312] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0313] -one or more amino acids selected from glutamic acid or a salt thereof and aspartic acid or salt thereof, in a total concentration of 1-20% w / w,

[0314] -at least one antioxidant selected from ascorbic acid or a salt thereof, and citrate or a salt thereof, in a total concentration of 1-20% w / w, and

[0315] -at least one structural enhancer selected from caseinate, starch, spray gum and pea protein isolate, in a total concentration of 1-30% w / w.

[0316] Composition M:

[0317] A dry lactic acid bacteria composition comprising

[0318] -lactic acid bacteria

[0319] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0320] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0321] -glutamic acid or a salt thereof in a total concentration of 5-15% w / w, -ascorbic acid or a salt thereof in a total concentration of 5-18% w / w, and -caseinate or a salt thereof in a total concentration of 5-18% w / w.

[0322] Composition N:

[0323] A dry lactic acid bacteria composition comprising

[0324] -lactic acid bacteria -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition,

[0325] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition,

[0326] -glutamic acid or a salt thereof in a total concentration of 8-12% w / w, -ascorbic acid or a salt thereof in a total concentration of 12-16% w / w, and -caseinate or a salt thereof in a total concentration of 12-16% w / w.

[0327] For the compositions A-N it has been found advantageous that the total concentration of reducing sugars in the compositions A-N described above, is less than 4% w / w as the total concentration of reducing sugars also defines how much reducing sugar that can participate in the maillard reaction and contribute to degradation of the dry lactic acid bacteria composition. However, for the same reason, it may also be advantageous to provide dry lactic acid bacteria compositions with even lower concentrations of reducing sugars, such as a total concentration of reducing sugars less than or equal to 3.5% w / w, 3% w / w, 2.5% w / w, 2% w / w, 1.5% w / w, less than or equal to 1% w / w, less than or equal to 0.5% w / w, or less than or equal to 0.1% w / w.

[0328] Thus, in embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below 4% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 3.5% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 3.0% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 2.5% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 2% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 1.5% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 1% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 0.5% w / w. In one or more embodiments, the dry lactic acid bacteria composition disclosed in composition A-N comprises reducing sugars in a total concentration of below or equal to 0.1% w / w.

[0329] In some advantageous embodiments, it is particularly important that the total concentration of reducing sugars in the compositions A-N described above, is less than 2% w / w as the total concentration of reducing sugars also defines how much reducing sugar that can participate in the maillard reaction and contribute to degradation of the dry lactic acid bacteria composition.

[0330] However, for the same reason, it may also be advantageous to provide dry lactic acid bacteria compositions with even lower concentrations of reducing sugars, such as a total concentration of reducing sugars less than 1.5% w / w, less than 1% w / w, less than 0.5% w / w, or less than 0.1% w / w.

[0331] Thus, in embodiments, the dry lactic acid bacteria composition comprises reducing sugars in a total concentration of below 2% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises reducing sugars in a total concentration of below 1.5% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises reducing sugars in a total concentration of below 1% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises reducing sugars in a total concentration of below 0.5% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises reducing sugars in a total concentration of below 0.1% w / w.

[0332] In advantageous embodiments, the total concentration of reducing sugars in compositions A-N is a total concentration of reducing sugars below 1.5% w / w. In more advantageous embodiments, the total concentration of reducing sugars in compositions A-N is a total concentration of reducing sugars below 1.0% w / w. In yet more advantageous embodiments, the total concentration of reducing sugars in compositions A-N is a total concentration of reducing sugars below 0.5% w / w. In particularly advantageous embodiments, the total concentration of reducing sugars in compositions A-N is a total concentration of reducing sugars below 0.1% w / w.

[0333] Similarly, it may also prove advantageous that the dry lactic acid bacteria composition comprises less than 50% w / w non-reducing sugars and / or sugar alcohols. It may be desirable that the dry lactic acid bacteria composition comprises a total concentration of less than 50% non-reducing sugars and / or sugar alcohols, wherein the total concentration of reducing sugars and / or sugar alcohols in the range of 1-50% w / w, 1-40% w / w, 1-30% w / w, 1-20% w / w, 5-10% w / w, 2-10% w / w, 1.5-10% w / w, 1-10% w / w, 2-5% w / w, 1.5-5% w / w, 1-5% w / w.

[0334] In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-50% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of nonreducing sugars and / or sugar alcohols in the range of 1-40% w / w. In one or more embodiments, the dry compositions of lactic acid bacteria comprise a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-30% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-20% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 5-10%. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 2-10% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of nonreducing sugars and / or sugar alcohols in the range of 1.5-10% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-10% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 2-5% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1.5-5% w / w. In one or more embodiments, the dry lactic acid bacteria composition comprises a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-5% w / w.

[0335] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-40% w / w. In more advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of nonreducing sugars and / or sugar alcohols in the range of 1-30% w / w. In yet more advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-20% w / w. In one or more particularly advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 5-10% w / w. In more particularly advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 2-10% w / w. In yet more particularly advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1.5-10% w / w. In yet more particularly advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-10% w / w. In yet more particularly advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 2-5% w / w. In yet more particularly advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of nonreducing sugars and / or sugar alcohols in the range of 1.5-5% w / w. In yet more particularly advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1.0-5% w / w.

[0336] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-40% w / w and a total concentration of reducing sugar less than 1.5% w / w.

[0337] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-40% w / w and a total concentration of reducing sugar less than 1.0% w / w.

[0338] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-40% w / w and a total concentration of reducing sugar less than 0.5% w / w.

[0339] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-40% w / w and a total concentration of reducing sugar less than 0.1% w / w.

[0340] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-40% w / w and a total concentration of reducing sugar of less than 0.05% w / w. In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-20% w / w and a total concentration of reducing sugar less than 1.5% w / w.

[0341] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-20% w / w and a total concentration of reducing sugar less than 1.0% w / w.

[0342] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-20% w / w and a total concentration of reducing sugar less than 0.5% w / w.

[0343] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-20% w / w and a total concentration of reducing sugar less than 0.1% w / w.

[0344] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-20% w / w and a total concentration of reducing sugar of 0% w / w.

[0345] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-10% w / w and a total concentration of reducing sugar less than 1.5% w / w.

[0346] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-10% w / w and a total concentration of reducing sugar less than 1.0% w / w.

[0347] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-10% w / w and a total concentration of reducing sugar less than 0.5% w / w.

[0348] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-10% w / w and a total concentration of reducing sugar less than 0.1% w / w.

[0349] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-10% w / w and a total concentration of reducing sugar of 0% w / w.

[0350] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-5% w / w and a total concentration of reducing sugar less than 1.5% w / w.

[0351] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-5% w / w and a total concentration of reducing sugar less than 1.0% w / w.

[0352] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-5% and a total concentration of reducing sugar less than 0.5% w / w.

[0353] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-5% and a total concentration of reducing sugar less than 0.1% w / w.

[0354] In advantageous embodiments, the total concentration of non-reducing sugars and / or sugar alcohols and total concentration of reducing sugars of the compositions A-N is a total concentration of non-reducing sugars and / or sugar alcohols in the range of 1-5% w / w and a total concentration of reducing sugar of 0.05% w / w.

[0355] In some cases, the dry lactic acid bacteria composition further comprises residues from the fermentation broth resulting from the lactic acid bacteria fermentation process in which the lactic acid bacteria contained in the dry composition was cultured. Thus, in one or more embodiments, the compositions A-N further comprise fermentation broth residues and lactic acid.

[0356] The dry lactic acid bacteria composition as defined herein comprises lactic acid bacteria in an amount of about 106-1013CFU / g. Thus, in one or more embodiments, a dry lactic acid bacteria composition comprises at least 106, 107, 108, 109, 1010, 1011, 1012or 1013CFU / g lactic acid bacteria. In one or more exemplary embodiments, a dry lactic acid bacteria composition comprises at least 106CFU / g lactic acid bacteria. In one or more exemplary embodiments, a dry lactic acid bacteria composition comprises at least 106CFU / g lactic acid bacteria, preferably at least 107CFU / g lactic acid bacteria, more preferably at least 108CFU / g lactic acid bacteria, or yet more preferably at least 109CFU / g lactic acid bacteria, yet more preferably at least 1010CFU / g lactic acid bacteria, yet more preferably at least 1011CFU / g lactic acid bacteria, yet more preferably at least 1012CFU / g lactic acid bacteria, or yet more preferably at least 1013CFU / g lactic acid bacteria.

[0357] Use of dry lactic acid bacteria compositions

[0358] The dry lactic acid bacteria compositions are particularly useful for providing starter cultures, direct vat sets (DVS) and / or inoculants. As demonstrated herein the dry lactic bacteria compositions have been shown to remain highly stable when stored at temperatures above 25°C.

[0359] Thus, in one or more embodiments, the dry lactic acid compositions disclosed herein are used for providing a starter culture, direct vat set (DVS) or inoculant, which has improved stability following storage at temperatures above 25°C for up to at least two weeks, 4 weeks, 8 weeks, or 12 weeks. In particular, the dry lactic acid bacteria compositions disclosed herein, are useful for use as starter culture, direct vat set (DVS) or inoculant following storage at temperatures above 25°C for at least two weeks. Thus, in one or more embodiments, the dry lactic acid compositions disclosed herein are intended for use as starter cultures, direct vat set (DVS) or inoculants, following storage at temperatures above 25°C for two weeks.

[0360] The starter cultures, starter cultures and direct vat sets (DVS) produced using the dry lactic acid bacteria compositions disclosed herein are further useful for producing a fermented product, and more particularly, highly useful for producing a fermented product following storage of the starter cultures, starter cultures and direct vat sets (DVS) at 25°C.

[0361] Thus, in one or more embodiments, a starter culture, starter culture or direct vat set (DVS) produced using the dry lactic acid bacteria compositions disclosed herein is used for producing a fermented product following storage at 25°C. In one or more embodiments, a starter culture, starter culture or direct vat set (DVS) produced using the dry lactic acid bacteria compositions disclosed herein is used for producing a fermented product following storage at 25°C for up to at least 2 weeks, 4 weeks, 8 weeks, 12 weeks. In one or more embodiments, a starter culture, starter culture or direct vat set (DVS) produced using the dry lactic acid bacteria compositions disclosed herein is used for producing a fermented product following storage at 25°C for up to at least 2 weeks, 4 weeks, 8 weeks, 12 weeks, or 16, 20 weeks or 24 weeks. The dry lactic acid bacteria compositions as disclosed herein and / or starter cultures, DVS and inoculants comprising the dry lactic acid bacteria compositions as disclosed herein are also useful for producing a product or fermented product at a remote site.

[0362] In the present context a remote site is a site that is in a different location than the production site where dry lactic acid bacteria composition disclosed herein is produced. Such a location may be close by, but it may also be a significant distance away from the production site. The distance between production site and remote site may be so great that transport by e.g. ship, cargo truck or airplane is required. Transporting dry lactic acid bacteria compositions from the production site to the remote site may therefore take a significant amount of time, such as a week, several weeks or maybe even months. Additionally, after arrival at the remote site, there may be a need for further storage, while the remote site prepares their next production cycle to make use of the lactic acid bacteria. Traditionally, during all this time, there is traditionally a need for cold storage facilities and / or a cold chain for preservation of the lactic acid bacteria during transport.

[0363] However, using the dry lactic acid bacteria compositions as disclosed herein, the dependency on such cold storage and / or cold chain is drastically reduced, allowing for storage of the lactic acid bacteria at temperatures above 25°C during transport to the remote site and / or storage at the remote site following transport, without compromising the usefulness (i.e. stability, metabolic activity and / or viability) of the lactic acid bacteria. This improvement is a great step forward in terms of food security in regions with prolonged periods of temperatures above 25°C and where cold storage facilities may be scarce and electricity sometimes unavailable. Additionally, not relying on such features will also lead to reductions in cost and increases in profitability of such starter cultures, DVS and inoculants.

[0364] Thus, in one or more embodiments, the dry lactic acid bacteria compositions, starter cultures, DVS and inoculants are intended for use in a production a fermented product at a remote site, following storage of dry lactic acid bacteria composition at temperatures above 25°C during transportation to the remote site and / or following storage at temperatures above 25°C at the remote site.

[0365] In one or more embodiments, the dry lactic acid bacteria compositions disclosed herein and starter cultures, DVS and inoculants comprising the dry lactic acid bacteria compositions disclosed are also intended for use in producing dairy products, e.g., yogurt, buttermilk, kefir, quark, tvorog, creme fraiche, sour cream or cheese. In one or more embodiments, such a dairy product may be a vegetable based dairy product, such as a vegurt.

[0366] In one or more embodiments an inoculant comprising an inoculant comprising dry lactic acid bacteria composition as defined herein is used as a dietary supplement. In one or more embodiments an inoculant comprising the dry lactic acid bacteria composition as defined herein is used as a probiotic ingredient. Temperatures above 25°C

[0367] Storage at a temperature or elevated temperature as referred to in the present disclosure relates to the temperature of the surrounding environment of the dry lactic acid bacteria. The temperature of the surrounding environment may be controlled, i.e. the temperature is regulated to be the same over the course of one full day (24 hours), or it may be uncontrolled, i.e. it varies over the course of one full day (24 hours). When the temperature is uncontrolled, it may vary with the temperature of the surrounding environment and may be influenced by general weather conditions and by the day / night cycle. Storage at an elevated temperature is in the present context considered to be a temperature above 25°C.

[0368] In one or more embodiments, a temperature above 25°C refers to the temperature of the surroundings or the temperature of a room. In one or more embodiments, a temperature above 25°C refers to the temperature of the surroundings. In one or more embodiments, a temperature above 25°C refers to the temperature of a room.

[0369] In some cases, a temperature above 25° as referred to herein is a temperature in the range of 25-55°C, However a temperature above 25°C may also be a temperature in a range of 25-43°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, 35-43°C, 37-40°C, 37-43°C 40-48°C, 45-53°C, or a temperature in the range of 48-53°C.

[0370] In one or more embodiments, a temperature above 25° is a temperature in the range of 25-55°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 25-53°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 25-48°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 25-43°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 25-40°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 30-40°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 30-43°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 30-55°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 30-50°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 35-40°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 35-43°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 37-40°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 37-43°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 40-48°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 45-53°C. In one or more embodiments, a temperature above 25° is a temperature in the range of 48-53°C. Water activity

[0371] Water activity, aw, is the partial vapor pressure of water in a substance divided by the standard state partial vapor pressure of water. awmeasurements of freeze-dried materials were done at room temperature using an Rototronic Hygrolab.

[0372] Contents of dry composition in percent

[0373] In the present context, when concentrations of various ingredients are given in percentage for the dry lactic acid compositions defined herein, the indication of percentage is given in weight / weight percentage, meaning that a designation of 1% is intended to mean 1% w / w, i.e., 10 g per kg in terms of concentration.

[0374] Measurement of sugar and sugar alcohol contents

[0375] Several methods for detection of sugars and sugar alcohols are available in the prior art. One example of such methods is the use of HPLC for detection of sugars in a given composition or solution. In the context of the present disclosure, reducing sugar content, non-reducing sugar content and / or sugar alcohol content can be measured using HPLC in e.g. a fermentation medium, a cell concentrate, a cryoprotective composition, and / or a dry lactic acid bacteria composition as disclosed herein. Such methods for HPLC detection of sugars and / or sugar alcohols are well known in the prior art would only involve routine tasks for the skilled person.

[0376] Determination of ta- time to obtain a reduction in pH of 0.08

[0377] In the present disclosure, the tais measured in terms of the number of minutes it takes to lower the pH of the incubation medium by 0.08. Thus, a higher number of minutes, i.e. a higher ta, represents a lower acidification activity, as it takes longer for the lactic acid bacteria to reduce pH by 0.08 points. Conversely, a lower number of minutes, i.e. a lower tarepresents a higher acidification activity, as the lactic acid bacteria takes less time to reduce pH by 0.08 points. tacan for example be measured using an iCinac system (KPM; AMS Alliance), but generally any piece of equipment that is capable of accurately measuring pH in a liquid medium may be used. In the present context tais measured in terms of minutes unless otherwise specified. pH of the incubation medium

[0378] Methods for measuring the pH of a solution are generally known to the skilled person and can be easily applied for measuring the pH in the incubation medium of the incubation step c). A nonlimiting example of this is the use of a pH-meter to measure the pH of the incubation medium at various intervals. The iCinac system (KPM; AMS Alliance) can also be applied for this purpose. pH is determined by performing these standard assays of incubated dried lactic acid bacteria prior to and following storage. Like the acidification activity, these pH measurements correspond directly to the metabolic activity of the lactic acid bacteria, and can then be used to determine if the pH of the incubation medium is the same, a higher or a lower pH, when compared to a reference value. As used in the present context, such a reference value could for example be the pH of the incubation medium measured for an incubated dry composition of lactic acid bacteria prior to storage. This comparative difference in pH (if any) is a direct measure of the difference in metabolic activity of the lactic acid bacteria prior to and following storage.

[0379] Lactic acid bacteria

[0380] As used herein the term lactic acid bacteria (LAB) designates a gram-positive, microaerophilic or anaerobic bacterium which ferments sugars and produce acids including lactic acid (as the predominantly produced acid) and acetic acid.

[0381] The industrially most useful lactic acid bacteria are found in the genera Lactococcus, Streptococcus., Lactobacillus the latter now known as Lactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Ligilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107, Leuconostoc., Oenococcus, Weissella, Pediococcus, and Enterococcus. Additionally, lactic acid producing bacteria belonging to the group of the strict anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp., are generally included in the group of lactic acid bacteria. These are frequently used as food cultures alone or in combination with other lactic acid bacteria.

[0382] Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and Streptococcus thermophilus, are normally supplied to the dairy industry either as cultures for bulk starter propagation or as so-called " Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product. Such cultures are in general referred to as "starter cultures" or "starters". In the present context such starters comprise dried lactic acid bacteria selected according to the method as defined herein.

[0383] Thus, in one or more embodiments, a lactic acid bacteria is a bacteria belonging to a genus selected from the group consisting of Lactococcus, Streptococcus, Lactobacillus now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107, Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, and Bifidobacterium.

[0384] In one or more embodiments, a lactic acid bacteria is a bacteria belonging to the Streptococcus genus. In one or more embodiments, a lactic acid bacteria is a bacteria belonging to the Streptococcus thermophilus species. In one or more embodiments, a lactic acid bacteria is a bacteria of the Streptococcus thermophilus strain DSM 35049. In one or more embodiments, a lactic acid bacteria is a bacteria belonging to the Bifidobacterium genus. In one or more embodiments, a lactic acid bacteria is a bacteria belonging to the Bifidobacterium animalis subsp. lactis species. In one or more embodiments, a lactic acid bacteria is a bacteria belonging to the Bifidobacterium animalis subsp. lactis strain DSM 15954.

[0385] Starter cultures and Direct vat set (DVS) compositions for use in food products.

[0386] Starter cultures and DVS cultures comprising dry lactic acid bacteria compositions as defined herein may be used as ingredients in the production of food products, for production of fermented products, for production of dairy products, and as ingredients in plant based food products and production of fermented plant based products.

[0387] Inoculum for use in making a probiotic product or dietary supplement

[0388] An inoculum comprising a dry lactic acid bacteria composition as referred to herein may be used as an ingredient in a probiotic product or be used as ingredient in production of a dietary supplement. Thus, in one or more embodiments, an inoculum as disclosed herein is used as ingredient in a probiotic product or as ingredient in a composition of dietary supplement. FD-DVS - Freeze dried direct vat set

[0389] The term FD-DVS as used herein refers a dry composition of lactic acid bacteria that has been formulated with a cryoprotectant and dried by freeze-drying (FD). These compositions are suitable for direct addition to a reactor or vat and are therefore referred to as direct vat sets (DVS).

[0390] DSM 35049 strain

[0391] The DSM 35049 is a S. thermophilus strain that is particularly susceptible to temperatures above 25°C. As a consequence, this strain is also an ideal model bacteria fortesting new ways for mitigating loss of stability / acidification activity of lactic acid bacteria post dry storage at elevated temperatures, i.e., temperatures above 25°C.

[0392] Strain deposits and expert solution

[0393] The applicant, Chr. Hansen (part of Novonesis A / S group) Denmark, requests that a sample of the deposited microorganisms stated in the table below may only be made available to an expert, until the date on which the patent is granted.

[0394] Table 6. Deposits made at a Depositary institution having acquired the status of international depositary authority under the Budapest T reaty 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.

[0395] T able 6: Overview of deposited strains used in the context of the present disclosure Deposited microorganism DSM accession Deposit date Disclosure No.

[0396] Streptococcus thermophilus DSM 35049 11th June 2024 - Bifidobacterium animalis DSM 15954 30 September 2003 W02005 / 060937A1 subsp. lactis

[0397] Lactococcus lactis subsp. DSM 35184 25 September 2024 - cremoris

[0398] Streptococcus thermophilus DSM 34235 7 April 2022 WO2023 / 222575A1

[0399]

[0400] Items

[0401] 1. A dry lactic acid bacteria composition comprising

[0402] -lactic acid bacteria

[0403] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, and

[0404] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0405] 2. The dry lactic acid bacteria composition according to item 1, comprising a total concentration of less than 3.5% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0406] 3. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of less than or equal to 3% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0407] 4. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of less than or equal to 2.5% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0408] 5. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of less than or equal to 2% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0409] 6. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of less than or equal to 1.5% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0410] 7. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of less than or equal to 1.0% w / w of reducing sugar in the dry lactic acid bacteria composition. 8. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of less than or equal to 0.5% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0411] 9. The dry lactic acid bacteria composition according to any one of the preceding items comprising a total concentration of less than or equal to 0.1% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0412] 10. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of less than or equal to 0.05% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0413] 11. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 1-40% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

[0414] 12. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 1-30% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

[0415] 13. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 1-20% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

[0416] 14. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 1-10% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

[0417] 15. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 1.5-10% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

[0418] 16. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 2-10% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition. 17. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 1.5-5% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

[0419] 18. The dry lactic acid bacteria composition according to any one of the preceding items, comprising a total concentration of 2-5% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

[0420] 19. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of sucrose, trehalose, erythritol, raffinose, stachyose, verbascose, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, isomalt, maltotriitol, and maltotetraitol.

[0421] 20. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars are mono and / or disaccharides, and the one or more sugar alcohols are sugar alcohols with a carbon count in the range of 3-16 carbon atoms.

[0422] 21. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of sucrose, trehalose, erythritol, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol and isomalt.

[0423] 22. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of sucrose, trehalose, erythritol, inositol, xylitol, sorbitol, maltitol and isomalt.

[0424] 23. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of sucrose, trehalose, erythritol, inositol, arabitol, mannitol, lactitol, maltitol, isomalt.

[0425] 24. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of sucrose, trehalose, inositol, xylitol, sorbitol. 25. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of trehalose, sucrose and inositol.

[0426] 26. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected as sucrose and one of trehalose, sorbitol and inositol.

[0427] 27. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected as sucrose and one of trehalose and inositol.

[0428] 28. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected as inositol and one of trehalose and sucrose.

[0429] 29. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected as trehalose and one of inositol and sucrose.

[0430] 30. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected as inositol.

[0431] 31. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected as trehalose.

[0432] 32. The dry lactic acid bacteria composition according to any one of the preceding items, wherein the one or more non-reducing sugars and / or sugar alcohols are selected as sucrose.

[0433] 33. The dry lactic acid bacteria composition according to any one of the preceding items, further comprising -one or more amino acid selected from the list consisting of glutamic acid or a salt thereof, and, aspartic acid and a salt thereof

[0434] -at least one antioxidant selected from the list consisting of ascorbic acid or a salt thereof, and, citric acid or a salt thereof

[0435] -at least one structural enhancer selected from the list consisting of caseinate, starch, spray gum, pea protein isolate.

[0436] 34. The dry lactic acid bacteria composition according to any one of the preceding items, further comprising

[0437] -one or more amino acids in a total concentration of 1-20% w / w, -at least one antioxidant in a total concentration of 1-20% w / w, and -at least one structural enhancer in a total concentration of 1-30% w / w.

[0438] 35. The dry lactic acid bacteria composition according to any one of the preceding items, further comprising

[0439] -one or more amino acids in a total concentration of 5-15% w / w, -at least one antioxidant in a total concentration of 5-18% w / w, and -at least one structural enhancer in a total concentration of 5-18% w / w.

[0440] 36. The dry lactic acid bacteria composition according to any one of the preceding items, further comprising

[0441] -one or more amino acids in a total concentration of 5-12% w / w, -at least one antioxidant in a total concentration of 5-16% w / w, and -at least one structural enhancer in a total concentration of 5-16% w / w.

[0442] 37. The dry lactic acid bacteria composition according to any one of the preceding items further comprising

[0443] -one or more amino acids in a total concentration of 8-12% w / w, -at least one antioxidant in a total concentration of 12-16% w / w, and -at least one structural enhancer in a total concentration 12-16% w / w. The dry lactic acid bacteria composition according to any one of the preceding items, wherein

[0444] -the amino acid is glutamic acid or a salt thereof, -the antioxidant is ascorbic acid or a salt thereof, and

[0445] -the structural enhancer is caseinate.

[0446] The dry lactic acid bacteria composition according to any one of the preceding items, wherein

[0447] -the one or more non-reducing sugars and / or sugar alcohols is selected from sucrose, inositol and / or trehalose, -the amino acid is selected as glutamic acid or a salt thereof, -the antioxidant is selected as ascorbic acid or a salt thereof, and -the structural enhancer is selected as caseinate.

[0448] The dry lactic acid bacteria composition according to any one of the preceding items, further comprising

[0449] -lactic acid

[0450] -fermentation broth residues.

[0451] The dry lactic acid bacteria composition according to any one of the preceding items, wherein the lactic acid bacteria of the dry lactic acid bacteria composition have an improved shelf life when stored at temperatures above 25°C.

[0452] A method for producing the dry lactic acid bacteria composition as defined in items 1-41, the method comprising

[0453] xi) providing a lactic acid bacteria concentrate,

[0454] xii) mixing the lactic acid bacteria concentrate ofxi) with a cryoprotectant to form a liquid lactic acid bacteria composition comprising -lactic acid bacteria

[0455] -a dry weight total concentration of 1-50% w / w of one or more nonreducing sugars and / or sugar alcohols in the liquid lactic acid bacteria composition, and

[0456] -a total dry weight total concentration of less than 4% w / w reducing sugar in the liquid lactic acid bacteria composition,

[0457] and

[0458] xiii) subjecting the liquid lactic acid bacteria composition of xii) to one or more drying steps, thereby obtaining a dry lactic acid bacteria composition comprising

[0459] -lactic acid bacteria

[0460] -a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, and

[0461] -a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

[0462] 43. The method according to item 42, wherein the reducing sugar total concentration in the liquid lactic acid bacteria composition is a total dry weight concentration less than 4% w / w, preferably less than 3.5% w / w, more preferably less than 3% w / w, yet more preferably less than 2.5% w / w, yet more preferably less than 2% w / w, yet more preferably less than 1.5% w / w, yet more preferably less than 1%. yet more preferably less than 0.5%, yet more preferably less than 0.1% w / w, yet more preferably less than 0.05% w / w and the reducing sugar total concentration in the dry lactic acid bacteria composition is less than 4% w / w, preferably less than 3.5% w / w, more preferably less than 3% w / w, yet more preferably less than 2.5% w / w, yet more preferably less than 2% w / w, preferably less than 1.5% w / w, more preferably less than 1% w / w, yet more preferably less than 0.5% w / w, yet more preferably less than 0.1% w / w, yet more preferably less than 0.05% w / w.

[0463] 44. The method according to any one of items 42-43, wherein the total concentration of one or more non-reducing sugars and / or sugar alcohols in the liquid lactic acid bacteria composition is a total dry weight concentration of 1-50% w / w, 1-40% w / w, 1-30% w / w, 1- 20% w / w, 1-10% w / w, 1.5-10% w / w, 2-10% w / w, 1.5-5% w / w, or 2-5% w / w and the total concentration of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition is a total concentration of 1-50% w / w, 1-40% w / w, 1-30% w / w, 1-20% w / w, 1-10% w / w, 1.5-10% w / w, 2-10% w / w, 1.5-5% w / w, or 2-5% w / w.

[0464] The method according to item 42-44, wherein the one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, erythritol, raffinose, stachyose, verbascose, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, isomalt, maltotriitol, and maltotetraitol.

[0465] The method according to item 42-45, wherein the cryoprotectant comprises

[0466] a) one or more non-reducing sugars and / or sugar alcohols, and b) is essentially free of reducing sugars.

[0467] The method according to item 42-46, wherein the cryoprotectant comprises one or more non-reducing sugars and / or sugar alcohols selected from the list consisting of sucrose, trehalose, erythritol, raffinose, stachyose, verbascose, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, isomalt, maltotriitol, and maltotetraitol.

[0468] The method according to item 42-47, wherein the cryoprotectant comprises one or more non-reducing sugars and / or sugar alcohols selected from sucrose, trehalose and / or inositol.

[0469] The method according to item 42-48, wherein the cryoprotectant further comprises

[0470] -one or more amino acids selected from the list consisting of glutamic acid or a salt thereof, and, aspartic acid and a salt thereof,

[0471] -at least one antioxidant selected from the list consisting of ascorbic acid or a salt thereof, and, citric acid or a salt thereof, and

[0472] -at least one structural enhancer selected from the list consisting of caseinate, starch, spray gum, pea protein isolate.

[0473] The method according to item 42-49, wherein the cryoprotectant further comprises -one or more amino acids in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 1-20% w / w,

[0474] -at least one antioxidant in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 1-20% w / w, and

[0475] -at least one structural enhancer in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 1 -30% w / w.

[0476] The method according to item 42-50, wherein the cryoprotectant further comprises

[0477] -one or more amino acids in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 5-15% w / w,

[0478] -at least one antioxidant in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 5-18% w / w, and

[0479] -at least one structural enhancer in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 5-18% w / w.

[0480] The method according to item 42-51, wherein the cryoprotectant further comprises

[0481] -one or more amino acids in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 5- 12% w / w,

[0482] -at least one antioxidant in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 5- 16% w / w, and

[0483] -a structural enhancers in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 5- 16% w / w. 53. The method according to item 42-52, wherein the cryoprotectant further comprises

[0484] -one or more amino acid in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 8- 12% w / w,

[0485] -at least one antioxidant in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 12- 16% w / w, and

[0486] -at least one structural enhancer in a concentration resulting in a dry weight total concentration in the dry lactic acid bacteria composition of 12-16% w / w.

[0487] 54. The method according to item 42-53, wherein

[0488] -the one or more amino acids are selected as glutamic acid or a salt thereof,

[0489] -the at least one antioxidant is selected as ascorbic acid or a salt thereof, and

[0490] -the at least one structural enhancer is selected as caseinate.

[0491] 55. The method according to any one of items 42-54, wherein the one or more drying steps are selected from freeze-drying, spray-drying and vacuum drying.

[0492] 56. The method according to any one of items 42-55, wherein the one or more drying steps are freeze-drying steps.

[0493] 57. The dry lactic acid bacteria composition according to any one of items 1-41, or the dry lactic acid bacteria composition produced according to the method as defined in any one of items 38-56, wherein the lactic acid bacteria of the dry lactic acid bacteria composition are present in a concentration of at least 106CFU / g of the dry lactic acid bacteria composition. The dry lactic acid bacteria composition according to any one of items 1-41, or the dry lactic acid bacteria composition produced according to the method as defined in any one of items 38-56, wherein the lactic acid bacteria of the dry lactic acid bacteria composition are present in a concentration of 106-1013CFU / g of the dry lactic acid bacteria composition.

[0494] The dry lactic acid bacteria composition according to any one of items 1-41, or the dry lactic acid bacteria composition produced according to the method as defined in any one of items 42-56, wherein the lactic acid bacteria of the dry lactic acid bacteria composition are present in a concentration of at least 109CFU / g of the dry lactic acid bacteria composition.

[0495] The dry lactic acid bacteria composition according to any one of items 1-41, or the dry lactic acid bacteria composition produced according to the method as defined in any one of items 42-56, wherein the lactic acid bacteria of the dry lactic acid bacteria composition are present in a concentration of 109-1013CFU / g of the dry lactic acid bacteria composition.

[0496] The dry lactic acid bacteria composition according to any one of items 1-41, or the dry lactic acid bacteria composition produced according to the method as defined in any one of items 42-56, wherein the lactic acid bacteria is a bacteria belonging to a genus selected from the group consisting of Lactococcus, Streptococcus., Lactobacillus now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107, Leuconostoc., Oenococcus, Weissella, Pediococcus, Enterococcus, and Bifidobacterium.

[0497] The dry lactic acid bacteria composition according to any one of items 1-41, or the dry lactic acid bacteria composition produced according to the method as defined in any one of items 42-56, wherein the lactic acid bacteria is a bacteria belonging to the Streptococcus genus.

[0498] The dry lactic acid bacteria composition according to any one of items 1-41, or the dry lactic acid bacteria composition produced according to the method as defined in any one of items 42-56, wherein the lactic acid bacteria is a bacteria belonging to the Streptococcus thermophilus species. 64. Use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56 as a starter culture, direct vat set (DVS) or inoculant.

[0499] 65. Use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56 as a starter culture, direct vat set (DVS) or inoculant following storage at temperatures above 25°C.

[0500] 66. Use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56, for providing a starter culture, direct vat set (DVS) or inoculant, which has improved stability following storage at temperatures above 25°C for at least two weeks, 4 weeks, 8 weeks, or 12 weeks.

[0501] 67. Use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56 as a starter culture, direct vat set (DVS) or inoculant following storage at temperatures above 25°C for at least two weeks.

[0502] 68. Use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56 for storage at temperatures above 25°C.

[0503] 69. Use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56 for storage at temperatures above 25°C.

[0504] 70. Use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56 for storage at temperatures above 25°C. 71. The use of a dry lactic acid bacteria composition according to any one of items 1-41 and 57-62, or a dry lactic acid bacteria composition produced in a method according to any one of items 42-56, for producing a fermented product.

[0505] 72. The use according to item 71, for producing a fermented product at a remote site, following storage of dry lactic acid bacteria composition at temperatures above 25°C during transportation to the remote site and / or following storage at ambient temperature at the remote site.

[0506] 73. The use according to any one of items 64-72, for producing a dairy product.

[0507] 74. The use according to any one of items 73, wherein the dairy product is yogurt, vegurt, buttermilk, kefir, quark, tvorog, creme fraiche, sour cream or cheese.

[0508] BRIEF DESCRIPTION OF THE FIGURES

[0509] Figure 1

[0510] Fig. 1 shows a bar plot of the sugar content of dry lactic acid FD-DVS product compositions comprising lactic acid bacteria belonging to the S. thermophilus strain DSM 35049.

[0511] Figure 2

[0512] Fig. 2. Acidification activities of FD-DVS DSM 35049 products during storage measured by taCorrelation of FD-DVS products to fermentation processes with initial sugar concentrations are as follows: (1) 2% lactose; (2) 4% lactose; (3) 2% sucrose; (4) 4% sucrose; (5) 2% glucose; (6) 4% glucose; (7) 2% lactose and 2% sucrose; (8) 4% lactose and 4% sucrose.

[0513] Figure 3

[0514] Fig. 3. Acidification activities of FD-DVS DSM 35049 products during storage measured by pH (4h). Correlation of FD-DVS products to fermentation processes with initial sugar concentrations are as follows: (1) 2% lactose; (2) 4% lactose; (3) 2% sucrose; (4) 4% sucrose; (5) 2% glucose; (6) 4% glucose; (7) 2% lactose and 2% sucrose; (8) 4% lactose and 4% sucrose. Figure 4

[0515] Fig. 3. Formation of free water measured by awin FD-DVS DSM35049 products during storage. Correlations of FD-DVS to fermentation processes with initial sugar concentrations are as follows: (1) 2% lactose; (2) 4% lactose; (3) 2% sucrose; (4) 4% sucrose; (5) 2% glucose; (6) 4% glucose; (7) 2% lactose and 2% sucrose; (8) 4% lactose and 4% sucrose.

[0516] Figure 5

[0517] Fig. 5 shows the acidification activity (fig. 5A) and incubation medium pH (4h) (fig. 5B) following storage from 0 to 12 weeks at 37°C of two dry compositions comprising S. thermophilus strain DSM 35049 obtained from the same fermentation process, but with different cryoprotectant compositions. Black-filled diamonds: Cryoprotectant with reducing sugar; White-filled circles: Cryoprotectant without reducing sugar.

[0518] Figure 6

[0519] Fig. 6 shows log (CFU loss) following from 0 to 25 weeks of storage at 37°C of three dry compositions comprising the Bifidobacterium animalis subsp. lactis deposited as DSM 15954 made from the same fermentation process but formulated with three different cryoprotectants. Black-filled circle (product 01): Cryoprotectant comprising sucrose and without reducing sugar; white filled circle (product 02): Cryoprotectant comprising sucrose and fructose; white filled square (product 03): Cryoprotectant comprising sucrose and galactose.

[0520] Figure 7

[0521] Figure 7 shows the acidification activity by parameter Ta (fig. 7A) and incubation medium pH (6 hours) (fig. 7B) following storage from 0 to 12 weeks at 37°C of Lactococcus lactis subsp. cremoris DSM 35184 formulated with;

[0522] Cryoprotectant A: at Encapsulation index = 0,5 (white circles and dotted line) and at Encapsulation index = 1 (white circles and full line); and,

[0523] Cryoprotectant B: at Encapsulation index = 0,5 (black circles and dotted line) and at Encapsulation index = 1 (black-filled circles and full line). Figure 8

[0524] Figure 8 shows the water activity of dry lactic acid bacteria compositions comprising Lactococcus lactis subsp. cremoris DSM 35184 following storage from 0 to 12 weeks at 37°C and formulated with cryoprotectant A at Encapsulation index = 0,5 (white circles and dotted line) and at Encapsulation index = 1 (white circles and full line); and with cryoprotectant B at Encapsulation index = 0,5 (black circles and dotted line) and at encapsulation index = 1 (black-filled circles and full line).

[0525] Figure 9

[0526] Figure 9 shows the color of the FD-DVS DSM 35049 products at the time 0 of the storage stability test (upper row) and after 31 days of the storage stability (lower row). Correlation of FD-DVS products 1-8 from example 1 to fermentation processes with initial sugar concentrations are as follows: (1) 2% lactose; (2) 4% lactose; (3) 2% sucrose; (4) 4% sucrose; (5) 2% glucose; (6) 4% glucose; (7) 2% lactose and 2% sucrose; (8) 4% lactose and 4% sucrose.

[0527] Figure 10

[0528] Figure 10 shows acidification activities of two dry compositions comprising S. thermophilus strain DSM 34235, measured by parameter ta (figure 10A) and by parameter pH (6h) (figure 10B) following storage from 0 to 12 weeks at 37°C. White filled circle: Product FD-DVS 1 without reducing sugars in composition; black filled circle: product FD-DVS 2 with reducing sugars in composition. EXAMPLES

[0529] A series of experiments were performed to evaluate the impact of sugar in fermentation process and in dry lactic acid bacteria composition on the stability of the dry lactic acid bacteria compositions following storage at 37°C. The fermentation processes used in the following examples were performed according to the process as outlined below.

[0530] Industrial production of lactic acid bacteria starts with a fermentation process. Lactic acid bacteria are grown in a fermenter in a seeded, complex fermentation medium, containing various nutrients and one- or more sugars. After that the bacterial biomass is produced to a sufficient amount and the bacteria reached the early stationary growth phase, fermentation is terminated. Fermentation broth is processed by separation, i.e. centrifugation, and the bacterial biomass is recovered as cell concentrate. The cell concentrate is formulated with a cryoprotectant. In subsequent steps, the formulated cell concentrate is frozen by pelletizing in liquid nitrogen and the frozen pellets are freeze-dried to final product, which is a freeze-dried granulate. (Hoier et al. 2010, Technology of Cheesemaking, Second Edition, Eds. Barry A. Law and A. Y. Tamime).

[0531] Example 1 - Assessing impact of sugars on stability of dry lactic acid bacteria compositions Correlation between sugars used as a substrate in fermentation process for production of lactic acid bacteria and storage stability of freeze-dried lactic acid bacteria has been investigated.

[0532] Streptococcus thermophilus DSM 35049 has been grown in a complex growth medium, containing varying concentrations of one or two sugars. Compositions of fermentation media are specified in Table 1. Fermentations were conducted in lab-scale, with 2-L fermentation media in fermenters with agitation, under nitrogen blanket and with pH control by ammonia water. DSM35049 was grown under optimal growth conditions with respect to the temperature and pH. Fermentations were terminated in stationary growth phase after stop of lactic acid production. Fermentation broths were analyzed by HPLC for sugars at the start and end of the fermentations. As it appears from results in Table 1, varying concentrations of residual sugars were detected in fermentation broths. Galactose was the most abundant residual sugar from lactose-based fermentations, fructose was present in lower concentrations as residue from sucrose-based fermentations. If sugars were served at concentrations exceeding metabolic capacity of the strain, as in fermentation 8, then beside galactose and fructose, sucrose was detected in fermentation broth at 25% (w / w) of its initial concentration.

[0533] Fermentation Substrate Start of fermentation End of fermentation

[0534]

[0535] 1 2% lactose 1,9 % lactose 1,0% galactose

[0536] 2 4% lactose 3,9 % lactose 1,9% galactose

[0537] 3 2% sucrose 2,1% sucrose 0,4% fructose

[0538] 4 4% sucrose 4,3% sucrose 0,5% fructose

[0539] 5 2% glucose 1,9% glucose 0,1% glucose

[0540] 0,2% fructose 0,2% fructose

[0541] 6 4% glucose 3,8% glucose 0,2% fructose

[0542] 0,4% fructose

[0543] 7 2% lactose 1,9% lactose 0,7% galactose

[0544] 2% sucrose 2,1% sucrose 0,3% fructose 8 4% lactose 3,8% lactose 1,0% sucrose

[0545] 4% sucrose 4,3% sucrose 1,3% galactose

[0546] 0,5% fructose

[0547]

[0548] Table 1. Experimental set-up for DSM35049 lab-scale fermentations, actual concentrations of initial sugars and final metabolites (% w / w) in fermentation broths.

[0549] Biomass was harvested from each fermenter by centrifugation at +4°C. Concentration factor from a range 10 - 14 was applied. Cell concentrates with dry matter content of 4% (w / w) to 8% (w / w) were produced. Cryoprotectant A, comprising on the dry weight basis (w / w) 7% Na-caseinate, 4,7% Inositol, 4,7% monosodium glutamate and 7,2% Na-ascorbate, was mixed with cell concentrates at Encapsulation Index = 1 (i.e. 1 g dry weight of cryoprotectant was dosed per 1 g dry weight of cell concentrate). Formulated cell concentrates were kept on ice for 15 minutes and subsequently frozen in liquid nitrogen in form of pellets. Frozen pellets were freeze-dried at pressure of 0,3 mbar to freeze-dried granulates. In total, eight different FD-DVS prototypes were produced and numbered as FD-DVS 1-8 (see e.g. table 2).

[0550] Concentrations of sugars in lab-scale FD-DVS products were determined by HPLC analysis, and the results are shown in Figure 1. Beside lactic acid, the matrix of FD-DVS products contained high amount of galactose, when the biomass was grown on lactose, and lower amount of fructose, when the biomass was grown on sucrose or glucose. A summary of concentrations of fermentation metabolites in FD-DVS compositions, categorized as reducing sugars and non-reducing sugars, is presented in Table 2.

[0551] Fermentation FD-DVS Reducing Non-reducing

[0552] sugars (% w / w) sugars (% w / w)

[0553] 1 1 8,0 0

[0554] 2 2 18,1 0

[0555] 3 3 3,5 0

[0556] 4 4 2,5 0

[0557] 5 5 1,7 0

[0558] 6 6 1,6 0

[0559] 7 7 7,8 0

[0560] 8 8 9,1 5,3 (sucrose

[0561] content)

[0562]

[0563] Table 2: Generalized sugar content of FD-DVS DSM35049 products.

[0564] FD-DVS prototypes were analyzed for active cells counts by flowcytometry and for water activity aw(Table 3). Water activities varied by factor 3 between the driest product, FD-DVS 1, and FD-DVS 7, which had the highest amount of free water. Higher water activity correlated with higher concentrations of residual reducing sugars in final fermentation broth.

[0565] FD-DVS Active cells Zg FD-DVS aw

[0566] 1 I 7.0E+10 | 0,055

[0567]

[0568]

[0569] 2 7.8E+10 0,161

[0570] 3 1.0E+11 0,072

[0571] 4 8.5E+10 0,092

[0572] 5 1.9E+11 0,146

[0573]

[0574] 6 2.9E+11 0,141

[0575] 7 4.1E+10 0,170

[0576] 8 9.4E+10 0,163

[0577]

[0578] Table 2. Overview of FD-DVS DSM35049 prototypes with active cell counts and aw.

[0579] Example 1a: Acidification activity and pH before and after storage

[0580] The FD-DVS granulates were distributed in three grams portions in aluminium bags. The gas phase in bags was of atmospheric air. The bags were sealed and subjected to storage stability test for 31 day at 37°C. Sampling was done at time 0-, 7-, 14- and 31 days. The samples were tested in an acidification assay either before storage or following a period of storage. The lactic acid bacteria were inoculated with inoculum from range 0,001% - 0,009% in sterilized reconstituted skimmed milk (RSM) with 9,5% dry matter content and incubated at 37°C for 16 hours. pH was measured continuously, and the parameters of acidification activity were monitored and calculated by the iCinac system (KPM; AMS Alliance).

[0581] For comparison of the acidification performances, a standardization of the doses of FD-DVS was made, corresponding to the inoculum with 3E+06 active cells / g milk at the start of stability study and following performance of this dose during stability test. Acidification activity was assessed by parameters taand pH. The pH values shown in table 4 are the pH measurements recorded after 4 hours of incubation. Results of acidification activities at time zero of the stability study, i.e. prior to storage, are presented in Table 4.

[0582] Product ta (min) pH (4h)

[0583] FD-DVS 1 62 4,89

[0584] FD-DVS 2 103 5,33

[0585] FD-DVS 3 65 4,94

[0586] FD-DVS 4 71 4,98

[0587] FD-DVS 5 93 5,19

[0588] FD-DVS 6 115 5,50

[0589]

[0590] FD-DVS 7 71 5,04

[0591] FD-DVS 8 117 5,51

[0592]

[0593] Table 3. Acidification activity of FD-DVS DSM35049 products at time 0 of the stability test.

[0594] The eight FD-DVS products showed a significant difference in acidification activities. Products FD-DVS 1, FD-DVS 3, FD-DVS 4, FD-DVS 5 and FD-DVS 7, were acidifying faster than FD-DVS 2, FD-DVS 6 and FD-DVS 8.

[0595] FD-DVS 2, FD-DVS 6 and FD-DVS 8 showed the poorest acidification activities before storage and all of these samples deteriorated quickly with storage time (table 5), showing significant lower acidification activity already after 7 days of storage. FD-DVS 7 had a decent acidification activity before storage, but this sample also deteriorated rapidly with storage time, losing as much acidification activity as samples FD-DVS 2, FD-DVS 6 and FD-DVS 8.

[0596] The FD-DVS 2 had a very poor performance during storage compared to the other samples, with a very rapid drop off of acidification activity already after 7 days of storage. As the FD-DVS 2 product was differentiating from all other seven FD-DVS products by having a high concentration of reducing sugars, (18,1% (w / w) as indicated in table 2) in the FD-DVS, with particularly galactose constituting 17,5% (w / w) of the FD-DVS composition.

[0597] An overview of acidification parameters taand pH (4h) from the stability study is given in T able 5. A graphic presentation of the results is shown in figures 2 and 3.

[0598] 0 days 7 days 14 days 31 days FD-DVS ta(min) pH (4h) ta(min) pH (4h) ta (min) pH (4h) ta (min) pH (4h) 1 62 4,89 86 5,16 113 5,52 173 6,15 2 103 5,33 431 6,56 556 6,55 529 6,54 3 65 4,94 88 5,17 100 5,31 113 5,45 4 71 4,98 87 5,13 93 5,25 112 5,42 5 93 5,19 105 5,39 110 5,51 136 5,78 6 115 5,50 155 6,09 203 6,38 372 6,54 7 71 5,04 202 6,37 319 6,53 545 6,53

[0599]

[0600] 8 117 5,51 253 6,48 452 6,54 558 6,52

[0601]

[0602] Table 4. Acidification parameters of FD-DVS DSM35049 products during 1 month of storage at 37°C.

[0603] The most stable products, which maintained the acidification activities best during storage at 37°C were FD-DVS 3, FD-DVS 4, FD-DVS 5. These products were characterized by having reducing sugar concentrations below 4% (w / w), with FD-DVS-3 having the higher content of the three at 3.5% (w / w) (see table 2). The most unstable products were FD-DVS 2, FD-DVS 7, and FD-DVS 8, which contained 7,8% (w / w) - 18,1% (w / w) reducing sugars in FD-DVS, with galactose in range 5,5% (w / w) - 17,6% (w / w) of FD-DVS. FD-DVS 6 performed also poorly in stability despite of galactose absence and a low fructose content (1,5% (w / w) in the FD-DVS composition.

[0604] FD-DVS 1 had one of the highest levels of reducing sugars at 8,0 % (w / w) despite originating from a fermentation process containing only 2% lactose. The most prevalent reducing sugar was galactose, making up 95% of the total reducing sugar concentration in FD-DVS-1. This high concentration of galactose did not show a negative impact on the short-term stability of FD-DVS DSM35049 up to 14 days. However, when the exposure of FD-DVS 1 to 37°C was prolonged to 31 days, the loss of acidification activity accelerated and exceeded the losses in the three, best performing FD-DVS products, FD-DVS 3, FD-DVS 4 and FD-DVS 5.

[0605] Example 1b: Water activity

[0606] Water activities of FD-DVS products (FD-DVS 1-8) were followed during stability test (Figure 4). The highest increase in water activity was observed in FD-DVS 2, FD-DVS 7 and FD-DVS 8, which all had the common factors of containing high concentrations of galactose and having been fermented in fermentation medium containing at least 4% total sugars. These three products had highest initial awand highest increase of awto aw= 0,24 - 0,34 over the time of stability test. By example with these three FD-DVS products it was demonstrated that formation of free water correlated with destabilization and loss of acidification activity of the FD-DVS products.

[0607] Example 1c: Visual inspection and the Maillard reaction during storage at 37°C

[0608] The eight dry lactic acid bacteria compositions (FD-FVS 1-8) were further examined by visual inspection before entering storage and following 31 days of storage at 37°C. Pictures of each of the samples were taken before and after storage and the images are shown in figure 9.

[0609] It is particularly clear from figure 9 that the FD-DVS samples FD-DVS 2, FD-DVS 7 and FD-DVS 8 which were all obtained by fermentation in fermentation medium that had at least 4% w / w sugar content and having particularly high sugar concentrations of about 8% w / w following formulation with a cryoprotectant and freeze-drying showed clear signs of browning following 31 days of storage at 37°C, whereas the FD-DVS samples FD-DVS 3, FD-DVS 4 and FD-DVS 5 which where fermented in fermentation medium containing 2%- 4% w / w sugar and had a total reducing sugar content of less than 4% w / w showed significantly reduced browning compared to FD-DVS 2, FD-DVS 7 and FD-DVS 8.

[0610] The browning of these samples indicate that Maillard products were being formed during storage at 37°C and further suggest that residual reducing sugars from the fermentation broth participate in the Maillard reactions during storage at 37°C and thereby play a significant role in reducing the storage stability of dry lactic acid bacteria compositions.

[0611] Example 2 - Impact of cryoprotectant on lactic acid bacteria stability in dry compositions

[0612] A galactose-free concentrate of lactic acid bacteria belonging to the S. thermophilus strain DSM 35049 was produced in pilot-scale. The concentrate was divided in aliquots and these were formulated with following cryoprotectants at Encapsulation Index = 0,9:

[0613] 1) Cryoprotectant A (no reducing sugars):

[0614] Cryoprotectant composition on the dry weight basis (w / w), comprising 7% Na-caseinate, 4,7% Inositol, 4,7% monosodium glutamate and 7,2% Na-ascorbate.

[0615] 2) Cryoprotectant B (skimmed milk powder containing lactose):

[0616] Cryoprotectant composition on the dry weight basis, comprising 6% Skim Milk Powder, 2,6% inositol, 2,6% monosodium glutamate and 4,7% Na-ascorbate;

[0617] Formulated cell concentrates with cryoprotectants were pelletized in liquid nitrogen, freeze-dried at the pressure of 0,5 mbar and subjected to a storage stability study where they were stored at 37°C for 12 weeks. Acidification activities were measured as described in Example 1 with 0,003% inoculum.

[0618] The storage test shows that compositions made using the Cryoprotectant A which did not comprise any reducing sugars exhibited superior stabilty over the cryoprotectant Cryoprotectant B which comprised skimmed milk powder and therefore also contained the reducing sugar lactose which is a component of skimmed milk powder (Figure 5). Example 3 - Impact of reducing sugars in cryoprotectant formulations

[0619] To assess the impact of reducing sugars in cryoprotectants used with lactic acid bacteria concentrates to produce dry lactic acid bacteria compositions by freeze drying, a series of experiments were set up using a cryoprotectant comprising sucrose without reducing sugar (product 01), a cryoprotectant comprising sucrose and fructose (product 02) and a cryoprotectant comprising sucrose and galactose (product 03). Beside these sugars, the cryoprotectants contained maltodextrin and sodium ascorbate.

[0620] Bacteria belonging to the strain of Bifidobacterium animalis subsp. lactis (DSM 15954), were grown in a 350-L pilot scale fermenter in a complex fermentation medium and the biomass was harvested by centrifugation and divided into three aliquots. Each of the three aliquots were formulated with each one of the cryoprotectants discussed above yielding product 01 (figure 6, black circles), product 02 (figure 6, white circles) and product 03 (white squares), pelletized in liquid nitrogen and subsequently freeze dried at pressure 0,3 mbar. The freeze dried granulates were ground and powders were blended with microcrystalline cellulose. The initial potencies of these blends were 2E+10 CFU / g blend and the water activities were in the range of 0.22-0.25.

[0621] A storage stability assay was conducted by storing these blends in aluminum pouches at 37°C over a period of 24 weeks. The stability of the DSM 15954 cells was by determined by measuring the viable DSM 15954 cells through the CFU method.

[0622] As can be seen in figure 6, the stability of the DSM15954 cells in dry composition at 37°C was highest for the blend comprising the reducing sugar free sucrose cryoprotectant, with stability of the DSM 15954 cells being the lowest in the blend comprising the sucrose + galactose cryoprotectant. These experiments further confirm that reducing sugars are detrimental to the stability of lactic acid bacteria in dry composition when stored at temperatures above 25°C.

[0623] Additionally, these experiments demonstrate that this detrimental effect is not unique to dry lactic bacteria compositions containing lactic acid bacteria of the Streptococcus genus, or the S. thermophilus species.

[0624] Example 4 - Lactococcus fermentation in trehalose-based medium and impact of reducing sugar content of cryoprotectant

[0625] Lactococcus lactis subsp. cremoris DSM 35184 was grown in a fermentation medium with trehalose. The cells were harvested and cell concentrate with 19,4% dry matter content was produced. The cell concentrate was divided into four aliquots and formulated in following way:

[0626] 1. Cryoprotectant A (no reducing sugars) Cryoprotectant composition on the dry weight basis (w / w), comprising 5,6% Na-caseinate, 3,7% Inositol, 3,7% Monosodium glutamate and 5,5% Sodium ascorbate. The dry matter content of this cryoprotectant was measured to 18,3% (w / w). First aliquot of cell concentrate was formulated with cryoprotectant dose corresponding to Encapsulation Index = 0,5 (Formulation 01, figures 7-8 white circles and dotted lines), while the second aliquot of cell concentrate was formulated with cryoprotectant dose corresponding to Encapsulation Index = 1 (Formulation 02, figures 7-8 white circles and full lines).

[0627] 2. Cryoprotectant B (with reducing sugar lactose)

[0628] Cryoprotectant composition on the dry weight basis (w / w), comprising 7,3% Skim milk powder, 3% Inositol, 3% Monosodium glutamate and 5,7% Sodium ascorbate. The dry matter content of this cryoprotectant was measured to 16,5% (w / w). Third aliquot of cell concentrate was formulated with cryoprotectant B at a dose corresponding to Encapsulation Index = 0,5 (Formulation 03, figures 7-8 black circles and dotted lines), and the fourth aliquot of cell concentrate was formulated with cryoprotectant B at a dose corresponding to Encapsulation Index = 1 (Formulation 04, figures 7-8 black circles and full line).

[0629] Formulated cell concentrates were pelletized in liquid nitrogen, freeze-dried at a pressure of 0,65 mbar and subjected to a storage stability study at 37°C for 12 weeks (Figure 7). Acidification activity was measured in sterilized RSM milk, as described in Example 1, inoculated with 0,0024% inoculum, at 30°C for 16 hours. Parameters Taand pH (6h) were followed. The storage test shows that compositions made with cryoprotectant A without reducing sugars, FD-DVS 01 and FD-DVS 02, exhibited very similar stability with the low- and high- dose of cryoprotectant A. Stability of these two formulations was superior to the stability of formulations FD-DVS 03 and FD-DVS 04, made with skim milk and lactose in the cryoprotectant. Comparison of the two doses of cryoprotectant B in formulations also revealed that the stability of bacteria was strongly affected by the dose of cryoprotectant. By formulation FD-DVS 04 it was demonstrated that the higher the dose of cryoprotectant B, the larger the loss of acidification activity and poorer stability of freeze-dried product.

[0630] All four FD-DVS products were during the 12 weeks storage at 37°C tested for water activity, which is shown in figure 8. At the start of the trial were all four products dry and had water activities in range 0,023 - 0,071. During storage at 37°C, increase of the water activity in all four formulations was measured and reflected a progressing formation of water. While formulation FD-DVS 01 and FD-DVS 02 showed modest and very similar increase of water activity levelling out at around aw = 0,07 after 12 weeks storage in both products, pronounced increase of water activities was detected in FD-DVS 03 and FD-DVS 04, with aw= 0,12 and aw= 0,22, respectively, at the end of the storage period. The FD-DVS 04 with the highest increase of water activity correlated with the lowest FD-DVS stability.

[0631] Example 5 - Storage stability of Streptococcus thermophilus DSM 34235 grown in fermentation with glucose

[0632] Streptococcus thermophilus DSM 34235 was grown by a fermentation in a complex fermentation medium supplemented with one fermentable sugar, i.e. with glucose at a concentration 3% (w / w). Fermentation was conducted in a 35-L fermenter with agitation, under nitrogen blanket and with pH control by ammonia water. The strain was grown under optimal growth conditions with respect to the temperature and pH. Fermentation was terminated in the stationary growth phase, after glucose depletion from the fermentation media and stop of the lactic acid production. There were no residues of glucose in fermentation broth at the end of fermentation. The biomass was harvested at 10°C by centrifugation, producing a cell concentrate with 9,8% (w / w) dry matter content. The cell concentrate was formulated with cryoprotectant A, which comprised, on a dry weight basis (w / w) 7% Na-caseinate, 4,7% Inositol, 4,7% monosodium glutamate and 7,2% Na-ascorbate. The cryoprotectant was mixed with cell concentrate at an Encapsulation Index = 0,5. The formulated cell concentrate was frozen in liquid nitrogen in form of pellets, which were subsequently freeze-dried at a pressure of 0,5 mbar to freeze-dried granulate. The product obtained through this process is referred to as FD-DVS 1 and did not contain any reducing sugar in its final freeze-dried composition.

[0633] The FD-DVS 1 granulate was packaged in 10-g portions in aluminium bags. The gas phase in bags was atmospheric air. The bags were sealed and subjected to a storage stability test at 37°C for 12 weeks. Sampling was performed at time points 0, 2, 4, 8 and 12 weeks during the storage test.

[0634] Acidification activity was measured in sterilized RSM milk, as described in Example 1, using 0,0025% (w / w) inoculum, at 43°C for 16 hours. Parameters ta(min) and pH (6h), i.e. the pH after 6 hours of fermentation of the milk, were monitored. The set-up for the storage stability test included, alongside FD-DVS 1, a benchmark product FD-DVS 2, which was a FD-DVS granulate of DSM 34235 containing more than 4% (w / w) reducing sugars in the freeze-dried composition.

[0635] Acidification activity of FD-DVS 2 was measured under the same storage conditions and with the same assay as described for FD-DVS 1.

[0636] An overview of acidification parameters ta (min) and pH (6h) during the storage stability study is provided in Table 6. A graphic presentation of the results is shown in figure 10. 0 weeks 2 weeks 4 weeks 8 weeks 12 weeks FD-DVS ta pH ta pH ta pH ta pH ta pH (min) (6h) (min) (6h) (min) (6h) (min) (6h) (min) (6h) 1 93 5,24 108 5,29 111 5,34 122 5,51 130 5,56 2 66 5,15 86 5,41 99 5,43 123 5,63 143 5,84

[0637]

[0638] Table 6. Acidification parameters ta and pH (6h) of DSM 34235 in FD-DVS 1 and FD-DVS 2 during 12 weeks storage at 37°C.

[0639] At the start of the storage test (time 0), FD-DVS 1 exhibited a longer ta and a slightly higher pH (6h) compared to FD-DVS 2, indicating a slightly slower activity for FD-DVS 1 relative to FD-DVS 2. Exposure of both products to 37°C during the storage test negatively affected their acidification performances, as reflected by the increased ta and pH (6h) values over time. However, the overall loss of activity, as measured by ta and pH (6h), was significantly more pronounced in FD-DVS 2 than in FD-DVS 1.

[0640] This study demonstrated that the freeze-dried formulation of DSM 34235 without reducing sugars, FD-DVS 1, exhibited superior stability over time compared to FD-DVS 2, which contained reducing sugars. BUDAPEST TREATY ON THE INTERNATIONAL

[0641] RECOGNITION OF THE DEPOSIT OF MICROORGANISMS Deutsche Sammlung von FOR THE PURPOSES OF PATENT PROCEDURE Mikroorganismen und Zellkulturen GmbH

[0642] INTERNATIONAL FORM

[0643] Chr. Hansen A / S

[0644] Boege Alle 10-12

[0645] DK-2970 Hoersholm RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT

[0646] issued pursuant to Rule 7.1 by the

[0647] INTERNATIONAL DEPOSITARY AUTHORITY

[0648] identified at the bottom of this page

[0649] I. IDENTIFICATION OF THE MICROORGANISM

[0650] Identification reference given by the DEPOSITOR: Accession number given by the

[0651] CHCC5445 INTERNATIONAL DEPOSITARY AUTHORITY:

[0652] DSM 15954

[0653] II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC DESIGNATION

[0654] The microorganism identified under I above was accompanied by:

[0655] ( ) a scientific description

[0656] ( ^) a proposed taxonomic designation

[0657] (Mark with a cross where applicable).

[0658] III. RECEIPT AND ACCEPTANCE

[0659] This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2003-09-30

[0660] (Date of the original deposit)'.

[0661] IV. RECEIPT OF REQUEST FOR CONVERSION

[0662] The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

[0663] V. INTERNATIONAL DEPOSITARY AUTHORITY

[0664] Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of person(s) having the power to represent the MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized official(s):

[0665] Address: Mascheroder Weg lb

[0666] D-38124 Braunschweig

[0667] Date: 2003-10-13

[0668]

[0669] ‘ Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired.

[0670] Form DSMZ-BP / 4 (sole page) 12 / 2001

Claims

CLAIMS1. A dry lactic acid bacteria composition comprising-lactic acid bacteria-a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, and-a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

2. The dry lactic acid bacteria composition according to claim 1, comprising a total concentration of less than or equal to 3% reducing sugar in the dry lactic acid bacteria composition.

3. The dry lactic acid bacteria composition according to any one of the preceding claims, comprising a total concentration of less than or equal to 2% reducing sugar in the dry lactic acid bacteria composition.

4. The dry lactic acid bacteria composition according to any one of the preceding claims, comprising a total concentration of less than or equal to 1% reducing sugar in the dry lactic acid bacteria composition.

5. The dry lactic acid bacteria composition according to any one of the preceding claims, comprising a total concentration of 1-30% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

6. The dry lactic acid bacteria composition according to any one of the preceding claims, a comprising total concentration of 2-10% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition.

7. The dry lactic acid bacteria composition according to any one of the preceding claims, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of sucrose, trehalose, erythritol, raffinose, stachyose, verbascose, inositol, xylitol, sorbitol, arabitol, mannitol, lactitol, maltitol, isomalt, maltotriitol, and maltotetraitol.

8. The dry lactic acid bacteria composition according to any one of the preceding claims, wherein the one or more non-reducing sugars and / or sugar alcohols are selected from a list consisting of trehalose, sucrose and inositol.

9. The dry lactic acid bacteria composition according to any one of the preceding claims, further comprising-one or more amino acids,-at least one antioxidant, and-a structural enhancer.

10. The dry lactic acid bacteria composition according to any one of the preceding claims, further comprising-one or more amino acids in a total concentration of 1-15% w / w, -at least one antioxidant in a total concentration of 1-20% w / w, and -a structural enhancer in a total concentration of 1-30% w / w.

11. The dry lactic acid bacteria composition according to any one of claims 9-10 wherein-the one or more amino acid is selected from glutamic acid or a salt thereof, and aspartic acid or a salt thereof,-the at least one antioxidant is selected from ascorbic acid or a salt thereof, and citric acid or a salt thereof, and- the at least one structural enhancer is selected from caseinate, starch, spray gum, and pea protein isolate.

12. The dry lactic acid bacteria composition according to any one of the preceding claims, wherein the lactic acid bacteria of the dry lactic acid bacteria composition are present in a concentration of 106-1013CFU / g.

13. The dry lactic acid bacteria composition according to any one of the preceding claims, wherein the lactic acid bacteria of the dry lactic acid bacteria composition have an improved shelf life when stored at temperatures above 25°C.

14. A method for producing the dry lactic acid bacteria composition as defined in claims 1-13, the method comprisingxi) providing a lactic acid bacteria concentrate,xii) mixing the lactic acid bacteria concentrate ofxi) with a cryoprotectant to form a liquid lactic acid bacteria composition comprising-lactic acid bacteria-a total dry weight concentration of 1-50% w / w of one or more nonreducing sugars and / or sugar alcohols in the liquid lactic acid bacteria composition, and-a total dry weight concentration of less than 4% w / w of reducing sugar in the liquid lactic acid bacteria composition,andxiii) subjecting the liquid lactic acid bacteria composition of xii) to one or more drying steps, thereby obtaining a dry lactic acid bacteria composition comprising-lactic acid bacteria-a total concentration of 1-50% w / w of one or more non-reducing sugars and / or sugar alcohols in the dry lactic acid bacteria composition, and-a total concentration of less than 4% w / w of reducing sugar in the dry lactic acid bacteria composition.

15. The dry lactic acid bacteria composition according to any one of claims 1-13, or a dry lactic acid bacteria composition according to the method of claim 14, wherein the lactic acid bacteria is a bacteria belonging to a genus selected from the group consisting of Lactococcus, Streptococcus., Lactobacillus now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus,Agrilactobacillus, Schleiferilactobacillus, Loigolactobacillus, Lacticaseibacillus, Latilactobacillus, Dellaglioa, Liquorilactobacillus, Lactiplantibacillus, Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus and Lentilactobacillus as described in Zheng et al, Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107, Leuconostoc, Oenococcus, Weissella, Pediococcus, Enterococcus, and Bifidobacterium.

16. Use of the dry lactic acid bacteria composition according to any one of claims 1-13, or the dry lactic acid bacteria composition produced in a method according to any one of claims 14-15 as a starter culture, direct vat set (DVS) or inoculant.

17. Use of the dry lactic acid bacteria composition according to any one of claims 1-13, or the dry lactic acid bacteria composition produced in a method according to any one of claims 14-15, for providing a starter culture, direct vat set (DVS) or inoculant, which has improved stability following storage at temperatures above 25°C for at least two weeks, 4 weeks, 8 weeks, or 12 weeks.

18. The use of a starter culture, direct vat set (DVS) or an inoculant according to any one of claims 16-17, for producing a fermented product, a dairy product or a food product.

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