Selection of lactic acid bacteria for storage at ambient temperatures

Abstract

The present disclosure relates to method for selection of lactic acid bacteria for storage at ambient temperature, compositions comprising these, and fermented products made using these.

Description

[0001] TITLE: Selection of Lactic acid bacteria for storage at ambient temperatures

[0002] BACKGROUND

[0003] Industrially produced lactic acid bacteria are widely used for production of fermented products. It is well known that cultures of lactic acid bacteria are susceptible to damage caused elevated temperature and humidity. Efforts to avoid such damage have traditionally on cold preservation of lactic acid bacteria up until use of the lactic acid bacteria in a fermentation process or as a food additive.

[0004] However, little to no effort has been made in optimizing stability and acidification activity of dried lactic acid bacteria prior to addition to a food product or fermentation process. Prior to use in food and dairy products, cultures of lactic acid bacteria are often produced in dried formats, such as freeze-dried or spray dried granulates or powders. Commonly, these dried lactic acid bacteria are produced in one location and distribution to a wide range of remote sites. This often necessitates long transport periods before the dried lactic acid bacteria reaches the remote site where they are intended for use and may often also necessitates on site storage, while the remote site prepares for the next production cycle.

[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 20°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.

[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] Accordingly, there is a growing need for lactic acid bacteria that can remain stable for increased periods of time when stored in a dried format, e.g., which maintain viability and acidification activity following longer periods of shipping and / or storage at elevated temperatures, without having to rely on the use of a cold chain or cold storage.

[0008] 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. There are also biological factors that influence the stability of lactic acid bacteria, namely phages, which can cause significant economic damage in infected batches and sometimes even lead to complete loss of dairy productions relying on fermentation processes caused by lactic acid bacteria.

[0009] Some probiotic lactic acid bacteria products are particularly vulnerable to phages, as the probiotics rely on providing live lactic acid bacteria to the person ingesting the product, and phage activity can severely diminish the number of live probiotic bacteria remaining in the probiotic product when stored at higher temperatures that allow the phages to replicate and prey on the lactic acid bacteria.

[0010] While methods for obtaining phage-resistant lactic acid bacteria are generally known in the prior art and for example from WO 2023 / 222575 (CHR HANSEN), the prior art is silent on how to overcome the issue of lactic acid bacteria susceptibility toward higher temperatures.

[0011] Thus, there is a growing need for ways to develop lactic acid bacteria that can address both the abiotic stress of regions with warmer climates, and the biotic stress caused by phages.

[0012] SUMMARY

[0013] In a first aspect, the present disclosure relates to a method for selecting lactic acid bacteria for dry storage at ambient temperature, the method comprising ai) providing one or more dried phage-resistant lactic acid bacteria, or aii) providing a library of dried phage-resistant lactic acid bacteria, b) storing the dried phage-resistant lactic acid bacteria of ai) or aii) at a test storage temperature for a time period sufficient for a change in one or more parameters representative of the acidification activity can be measured, such as for at least 2 days, such as at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, 3 months or such as at least 6 months thereby providing stored dried phage-resistant lactic acid, c) incubating the stored dried phage-resistant lactic acid bacteria of step b) for at least two hours in a liquid sugar-based or milk-based incubation medium to obtain incubated phage-resistant lactic acid bacteria, d) determining a value for one or more parameters representative of the acidification activity of the incubated phage-resistant lactic acid bacteria c), thereby obtaining a determined value, e) comparing the determined value in step d) to a reference value, and f) selecting any incubated phage-resistant lactic acid bacteria for which the determined value obtained in step d) corresponds to an improved acidification activity when compared to the reference value, thereby providing selected phage resistant lactic acid bacteria.

[0014] In a second aspect, the present disclosure relates to compositions comprising phage resistant lactic acid bacteria selected according to the method as disclosed herein.

[0015] In a third aspect, the present disclosure relates to the use of compositions as defined herein and / or phage resistant lactic acid bacteria selected according to the method discloses herein for producing a lactic acid bacteria containing product.

[0016] DETAILED DESCRIPTION

[0017] The present disclosure relates to methods for assessing and selecting lactic acid bacteria that are phage-resistant and which remain active following storage at ambient temperatures in dried format. For many years, the main focus of research has been to either improve ph age- resista nee in bacteria in general or to improve quality of food products per se.

[0018] However, it was found in the present disclosure that other traits and benefits can also arise from a phage-resistance protocol in addition to phage-resistance. For example, in figures 1-4, it is shown that prior to storage, the acidification activity as represented by the time to reduce pH by 0.08 (ta) and pH measured following 6 hours of incubation for a wild type strain(DSM22589) and the phageresistant strain (DSM34235), were nearly identical, with the wild type strain having a slightly lower pH (6h)prior to storage. However, following dry storage for at least 14 days at 37°C, this picture starts to change and is fully reversed following a month of storage. After a month or more of storage, the phage-resistant strain DSM34235 clearly maintains a better acidification activity and the pH in the incubation medium after 6 hours incubation is lower than that of the wild type strain. This improvement in the phage-resistant strain acidification activity and pH is maintained all the way up to storage for 6 months, where the phage resistant strain maintains nearly twice as high acidification activity as the wild type strain.

[0019] It is clear from these results that not only did the phage-resistance protocol lead to phageresistance in DSM34235, but the phage-resistance protocol also induced an improved ability in DSM34235 to recover from long term storage and maintain a higher acidification activity following long term storage in a dried format, when compared to the wild type strain.

[0020] It is currently believed that the process by which other traits and benefits emerge from phageresistance protocols, is a random process. As such it is difficult, if not impossible, to predict which other traits that may coincide with phage-resistance following a phage resistance protocol. Additionally, any trait that appears during such a process may prove to be beneficial in one situation, but prove to be disadvantageous in another situation. Therefore, it is necessary to establish routines for testing phage-resistant bacteria for specific traits and establishing criteria for when to use these for specific purposes.

[0021] Accordingly, the present disclosure relates to a method for selection of phage-resistant lactic acid bacteria that have improved acidification activity when stored at ambient temperature. The method comprises providing one or more dried phage-resistant lactic acid bacteria, measuring their acidification activity and or pH of the incubation medium following storage at ambient temperature, comparing the measured value to a reference value, and selecting the dried phage-resistant lactic acid bacteria that have a measured value that is better than the reference value, such as selecting the dried phage-resistant lactic acid bacteria that has minimum loss during storage compared to the initial ta or pH(6h)..

[0022] Thus, such a method for selection of phage-resistant lactic acid bacteria that are stable when stored in a dried format at ambient temperatures, is a method comprising ai) providing one or more dried phage-resistant lactic acid bacteria, or aii) providing a library of dried phage-resistant lactic acid bacteria, b) storing the dried phage-resistant lactic acid bacteria of ai) or aii) at a test storage temperature for at least 2 days, such as at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, or such as at least 3 months to provide stored dried phage-resistant lactic acid, c) incubating the stored dried phage-resistant lactic acid bacteria of step b) for at least two hours in a liquid sugar-based or milk-based incubation medium to obtain incubated phage-resistant lactic acid bacteria, d) determining a value for one or more parameters representative of the acidification activity of the incubated phage-resistant lactic acid bacteria of c), e) comparing the one or more parameters determined in step d) to a reference value, and f) selecting any incubated phage-resistant lactic acid bacteria for which the determined value of one or more parameters in step d) corresponds to an improved acidification activity compared to the reference value, thereby providing selected phage resistant lactic acid bacteria.

[0023] Providing dried phage-resistant lactic acid bacteria

[0024] The providing of dried phage-resistant lactic acid bacteria referred to in ai) and aii) can be achieved in several ways. For example, dried phage-resistant lactic acid bacteria can be prepared from available phage-resistant lactic acid bacteria by culturing of these, followed by at least a drying process. Alternatively, phage-resistant lactic acid bacteria that are already available in a dried format can also be used.

[0025] However, phage-resistant lactic acid bacteria can also be provided by obtaining them from lactic acid bacteria that are not phage-resistant, by subjecting these lactic acid bacteria to a phage resistance protocol, selecting the surviving strains, followed by culturing, formulating, and drying of the surviving strains to provide dried phage-resistant lactic acid bacteria.

[0026] In the case of providing dried phage-resistant lactic acid bacteria from lactic acid bacteria that are not already known to be phage-resistant, the steps of ai) and / or aii) further comprises the steps xi) subjecting one or more lactic acid bacteria wild type strain(s) or a library of lactic acid bacteria wild type strains to a phage-hardening / phage-resistance protocol, xii) selecting the lactic acid bacteria that survives the phage-hardening / phage- resistance protocol, thereby providing one or more phage-resistant lactic acid bacteria or a library of phage-resistant lactic acid bacteria, and xiii) drying the one or more phage-resistant lactic acid bacteria or drying the library of phage-resistant lactic acid bacteria, thereby providing one or more dried phage-resistant lactic acid bacteria, or a library of dried phageresistant lactic acid bacteria. However, it should be noted that lactic acid bacteria that are already known to be phage-resistant may also be subjected to the steps of xi), xii), and xiii).

[0027] The providing of phage-resistant lactic acid bacteria may be fulfilled by either providing one or more dried phage-resistant lactic acid bacteria, or by providing a library of dried phage resistant lactic acid bacteria in any one of the ways disclosed herein.

[0028] Test storage and test storage temperature

[0029] During step b) the dried phage resistant lactic acid bacteria are stored at a test storage temperature to mimic conditions of storage at ambient temperature and help assess how well the dried phage resistant lactic acid bacteria can deal with the negative impacts of storage at elevated temperatures. However, it is also important to assess how well the dried phage resistant lactic acid bacteria deal with the negative impact of ambient temperature storage over time, as each strain may differ in how well it deals with short-term storage and long-term storage at ambient temperatures. Therefore, a series of different test storage durations should be applied for a given test storage temperature. In this way, it is possible to assess and evaluate which species and strains of lactic acid bacteria that should be used for applications that demand varying storage durations at ambient temperature. After the dried phage resistant lactic acid bacteria have undergone pre-determined periods of storage at a predetermined test storage temperature, they are removed from storage and subjected to an incubation step, step c).

[0030] Test storage duration

[0031] A test storage duration in the present context is the period of time that dried phage resistant lactic acid bacteria are stored in step b) before transfer to the incubation in step c). There is no particular upper limit for how long the dried phage resistant lactic acid bacteria can be stored at a test storage temperature in step b). However, usually the test storage duration is a period of from as low as 2 days and all the way up to 6 months. Thus, dried phage resistant lactic acid bacteria may be stored at a test storage temperature for at least 2 days, such as at least 3-7 days, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months at a test storage temperature. In one or more exemplary embodiments, step b) comprises storing the dried phage resistant lactic acid bacteria for 3-7 days at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 2 weeks at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 3 weeks at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 1 month at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 2 months at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 3 months at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 4 months at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 5 months at a test storage temperature. In one or more exemplary embodiments, step b comprises storing the dried phage resistant lactic acid bacteria for 6 months at a test storage temperature.

[0032] Test storage temperature

[0033] In the present context, a test storage temperature, is a temperature at which a storage test can be used to estimate how well a given lactic acid bacteria can tolerate storage at ambient temperatures. Generally speaking, any ambient temperature can be used to estimate storage tolerance of lactic acid bacteria. Because the negative impacts of storing dried lactic acid bacteria increases significantly from around 20°C and up to the lethal temperature for lactic acid bacteria, then an advantageous test storage temperature, is a temperature in the range of 20-53°C, or such as a temperature in the range of 25-43°C, 20-40°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, 35-43°C, 37-40°C, or a temperature in the range of 37-43°C.

[0034] In one or more embodiments the test storage temperature of step b) is a temperature in the range of 20-43°C, or such as a temperature in the range of 25-43°C, 20-40°C, 25-40°C, 30-40°C, 30- 43°C, 35-40°C, 35-43 °C, or a temperature in the range of 37-43°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 25-40°C, 30-40°C, 35-40 °C, or a temperature in the range of 37-40°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 25-43°C, 30-43°C, 35-43 °C or a temperature in the range of 37-43°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 25-43°C, 30-43°C, 35-43°C, 37-43°C, 40-48 °C, 45-53 °C or a temperature in the range of 48-53°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 30-40°C, 35-40°C or a temperature in the range of 37-40°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 30-43°C, 35-43°C or a temperature in the range of 37-43°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 35- 40°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 35-43°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 37-43°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 37-43°C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 40-48 °C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 45-53 °C. In one or more embodiments, the test storage temperature of step b), is a temperature in the range of 48-53°C.

[0035] In one or more embodiments, the test storage temperature, is a temperature of 37°C. Particularly interesting test storage temperatures include temperatures of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, or 43°C.

[0036] Phage resistant lactic acid bacteria, which have undergone storage at a test storage temperature for a test storage duration as defined in step b) are referred to herein as stored dried phage resistant lactic acid bacteria.

[0037] Incubation step

[0038] Following storage, the stored dried phage resistant lactic acid bacteria must be allowed to revive for a short while in a liquid incubation medium, before it is possible to assess how many of the bacteria survived storage and / or how metabolically active the surviving bacteria are.

[0039] To allow for this assessment, the stored dried phage resistant lactic acid bacteria are transferred from storage conditions under step b) to an incubation medium for incubation in step c).

[0040] The incubation step, step c), is generally performed by transferring the stored dried phage resistant lactic acid bacteria from storage into a liquid growth medium referred to herein as the incubation medium. Suitable incubation media include broths based on sugar(s) that is / are fermentable by the lactic acid bacteria, milk based media, such as bovine milk, and plant-based milk based media. Preferably, the incubation medium comprises a milk component and may be based on reconstituted milk, such as reconstituted skimmed milk. A particularly desirable incubation medium is a sterilized reconstituted skimmed milk (RSM) medium comprising 9,5% dry matter content.

[0041] Ideally, the incubation step is performed at a temperature that is the temperature optimum of the stored dried lactic acid bacteria species or strain, to allow for the best possible post storage recovery conditions for the lactic acid bacteria. However, the incubation step may also be performed at the same temperature as the test storage temperature. In general, a desirable incubation temperature is a temperature in a range of 20-30°C, 20-40°C, 25-40°C, 30-40°C, 30- 43°C, 35-40°C, 35-43°C, 37-43°C, or a temperature in the range of 37-43°C. A desirable incubation temperature may be selected as one from the list consisting of 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, and 43°C. More particularly desirable incubation temperatures are temperatures selected as one of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, and 43°C.

[0042] The dried phage resistant lactic acid bacteria are incubated for a period of time (incubation time) that is long enough that a difference in acidification activity and / or pH of the incubation medium can be measured and / or detected between different strains and species. There are many ways of detecting such changes, and depending on the chosen approach, the incubation time may vary. Generally, the incubation time will be a period of between 2 and 24 hours, such as between 2-20, 2-16, 2-10, 2-8, 3-20, 3-16, 3-10, 3-8, 4-20, 4-16, 4-10, or between 4-8 hours.

[0043] The stored dried phage resistant lactic acid bacteria are incubated for at least 2 hours to obtain incubated phage resistant lactic acid bacteria. The incubated phage resistant lactic acid bacteria are then used in step d) to infer information about how well the incubated phage resistant lactic acid bacteria dealt with the storage conditions of step b).

[0044] When determining the pH of the incubation medium after a period of time (6 hours in example 1) , there is in theoretical no hard limit for how long the stored phage resistant lactic acid bacteria can be incubated, but a standard incubation period of the incubation step c) is somewhere within the interval of 2-24, 2-20, 2-16, 2-10 or 2-8 hours. It is particularly desirable that the incubation period is 2-8 hours, 3-8 hours, 4-8 hours, 3-5 hours or 3-6 hours long. The incubation may also be a period of 2, 3, 4, 5, 6, 7, or 8 hours long. In one or more exemplary embodiments, the stored dried phage resistant lactic acid bacteria are incubated in step c) for 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 2-8 hours, 3-8 hours, 4-8 hours, 3-5 hours or 3-6 hours. In one or more exemplary embodiments, the stored dried phage resistant lactic acid bacteria are incubated in step c) for 4-8 hours, such as 4, 5, 6, 7, or 8 hours. In one or more exemplary embodiments, the stored dried phage resistant lactic acid bacteria are incubated in step c) for 6 hours.

[0045] The incubation step may be performed at the optimum growth temperature of the lactic acid bacteria strain, at the same temperature as the storage temperature, or it may be performed at a higher temperature than the storage temperature. It is particularly desirable that the temperature during the incubation in step c) is a temperature selected from 20-30°C, 20-40°C, 25-40°C, 30- 40°C, 30-43°C, 35-40°C, 35-43°C, 37-43°C, or a temperature in the range of 37-43°C. Particularly interesting incubation temperatures include temperatures of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, or 43°C. Acidification activity

[0046] In the present context, acidification activity refers to the rate at which lactic acid bacteria produce acids, mainly lactic acid, and thereby influences the pH of the immediate environment, such as for example of a fermentation medium, incubation medium, dairy product, food product and / or fermented product. Thus, the acidification activity is related to the metabolism and metabolic fitness of the lactic acid bacteria and can therefore also, when determined after a storage period and compared to a determined value prior to storage, be used as an indication of the metabolic activity, metabolic fitness and loss of acidification activity of the lactic acid bacteria post storage. Thus, in the present context a phage resistant lactic acid bacteria strain is selected for dry storage when an improvement in one or more parameters representative of acidification activity can be detected for the strain. However, there are many ways of determining a value of one or more parameters that is representative of the acidification activity of lactic acid bacteria and only a few of the most useful are exemplified in the present disclosure. Generally, the skilled person will understand which parameters to look for and whether a change in parameter value represents an improvement or a decline of acidification activity. In the present context, particularly useful ways of determining a value that is representative of the acidification activity include determining ta, tm, pH of the incubation medium, A pH, or acidification loss. Each of these parameters are described in detail below.

[0047] Determination of ta- time to obtain a reduction in pH of 0.08

[0048] 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.

[0049] Thus, for selection based on tain step f), strains with a determined value corresponding to improved acidification activity compared to a reference value, are strains that have a numerically lower determined tavalue than the tareference value. pH of the incubation medium

[0050] 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-meterto 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 a lower pH, when compared to a reference value. This 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 at ambient temperature.

[0051] Thus, for selection based on pH of the incubation medium in step f), strains with a determined value corresponding to an improved acidification activity compared to a reference value, are strains that have a numerically lower determined value of pH in the incubation medium than the pH in the incubation medium-reference value.

[0052] A pH

[0053] In the present disclosure, A pH, refers to the pH measured in the incubation medium at a specific time for a sample that has undergone storage minus the pH measured in the incubation medium following the same specific time, but prior to storage. This way, the A pH parameter is a direct indicator of the change of pH following incubation, that has been induced by storage of the sample.

[0054] In the present context, it is desirable for the lactic acid bacteria to be metabolically active and reduce pH as much as possible and as fast as possible, post storage incubation. Therefore, for example a strain that post storage reduced the pH of the incubation medium less, would lead to a positive A pH, and the less capable of reducing pH post storage the strain would be, the higher the A pH would become. Conversely, for example a strain reduced the pH of the incubation medium more post storage than prior to storage, would yield a negative A pH. An example of determination of A pH values and comparison between A pH values for S. thermophilus strains DSM 22589 (wild type) and DSM 34235 (phage resistant strain) is shown in table 4.

[0055] Thus, for selection based on A pH in step f), strains with a determined value corresponding to an improved acidification activity compared to a reference value, are strains that have a numerically lower determined value of A pH than the A pH reference value. Time to reach maximum acidification rate - tm

[0056] In the present context, the tmvalue relates to the time it takes for the lactic acid bacteria strain that is inoculated into the incubation to reach the maximum acidification rate. For lactic acid bacteria, the maximum acidification rate is reached when the lactic acid bacteria have recovered from the transition from dry storage to incubation medium and the lactic acid bacteria enter the exponential growth phase. The tmvalue is determined by measuring the pH of the incubation medium for the whole period of the incubation step and making a pH curve plotting pH against time. This curve is then subjected to derivative analysis to identify the time point at which the maximum acidification rate was reached, i.e. the tmvalue. A tmvalue can be used for comparison against a reference value alone or as part of a series. The difference in the timing of when the tmoccurs following dry storage for different periods of time, can then be used to estimate how well a given lactic acid bacteria can tolerate dry storage. In the present context, tmis measured in terms of minutes unless otherwise specified.

[0057] The timing of when the maximum acidification rate is reached, can be affected by how well the lactic acid bacteria can recover from storage. Shorter time to reach the maximum acidification, indicates a faster recovery and faster transition from storage into exponential growth and a longer time to reach maximum acidification rate indicates a slower recovery and slower transition into exponential growth. An example of determination of tmvalues and comparison between tmvalues for S. thermophilus strains DSM 22589 (wild type) and DSM 34235 is discussed in example 2 and shown in table 5 and figures 3-4.

[0058] Thus, for selection based on tmin step f), strains with a determined value corresponding to an improved acidification activity compared to a reference value, are strains that have a numerically lower determined value of tmthan the tmreference value.

[0059] Rate of loss of acidification activity

[0060] The rate of loss of acidification activity, also referred to in short herein as acidification loss, as used in the present context, refers to the loss of acidification activity of lactic acid bacteria during dry storage compared to the acidification activity of the lactic acid bacteria prior to storage. The consequence of a loss of acidification activity of lactic acid bacteria following storage is that it takes longer time for the lactic acid bacteria to reach the maximum acidification rate (tm). The acidification loss is quantified mathematically through linear regression of a series of tmvalues determined for a lactic acid bacteria strain following different lengths of storage. The acidification loss is represented by the value of the slope of the linear function that is obtained through linear regression of the tmvalue data series vs storage time and is measured in minutes per month of storage. Examples of such linear regression and linear functions are exemplified in example 3 and the linear functions are shown in figures 3 and 4.

[0061] The number of minutes per months signifies the added time it will take for the lactic acid bacteria to reach a maximum acidification time, and thus directly corresponds to the acidification loss for the lactic acid bacteria strain for each month of storage.

[0062] Thus, for selection based on acidification loss in step f), strains with a determined value corresponding to an improved acidification activity compared to a reference value, are strains that have a numerically lower determined value of acidification loss, i.e. lower number of minutes per month than the number of minutes per month of the tmreference value.

[0063] Additionally, the unit used to measure acidification loss in present context, minutes per month, may be converted into minutes per days instead, by dividing the value of the slope of the linear function by 30, wherein 30 is used to represent the number of days per month. When acidification loss is represented by minutes per day instead, comparisons to acidification loss, can be measured as soon as a function can be derived from the data series. It is important to note that because of this possibility to convert minutes per month into minutes per day, a reference value as used herein, which indicates acidification loss in terms of minutes per month, is also considered to represent the corresponding reference value in terms of minutes per day.

[0064] For instance, because the linear regression is performed on derivatives of the time to maximum acidification (tm), instead of waiting a month or longer to determine the acidification loss, a data series for (tm) can be obtained in a matter of days, which allows for quick and robust determination of the acidification loss as defined herein, providing a means for fast comparison of different strains in terms of acidification loss.

[0065] When making the selection in step f) based on the comparison of a determined value (e.g. a determined value of ta, pH of incubation medium, A pH, tmand / or acidification loss) to a reference value, a numerically lower determined value is any value that is at least 5% lower than the reference value. I.e. for a A pH of 0.1 , a numerically lower value would be a value of 0.095 (0.1x5 / 100). This measure is used for any reference value that is measured in terms of pH or A pH. For data or data series that are measured in terms of minutes (e.g. minutes per month for acidification loss), a numerically lower determined value is a value that is lower by 5% of a single minute, i.e. for a reference value of 40 minutes per month, a numerically lower value would be 39,095 minutes, or 39 minutes and 54,3 seconds.

[0066] In cases where such calculation cannot be meaningfully made and rounding is applied instead, any value that would be rounded up to the reference value, is considered to full fill the requirement of being numerically lower than the reference value. Any phage resistant lactic acid bacteria selected in step f) may be subjected to at least one additional cycle of storage, incubation and selection as defined by steps b)-f) of the present method. Additionally, any phage-resistant lactic acid bacteria selected in step f) may also be subjected to at least one additional cycle through a phage resistance protocol, before going through an additional cycle of storage, incubation and selection as defined by steps b)-f) of the present method.

[0067] Any of the dried phage resistant lactic acid bacteria selected in step f) of the method disclosed herein may further be used for production of a fermented lactic acid bacteria product at a remote facility following a period of storage at ambient temperature.

[0068] Such use comprises firstly providing one or more phage-resistant lactic acid bacteria selected in step f) as defined herein in a dried format. Secondly, such use further comprises keeping the one or more dried phage-resistant lactic acid bacteria at ambient temperature during transport to the remote site and / or keeping the one or more phage-resistant lactic acid bacteria in a dried format at ambient temperature during storage at the remote site. Lastly, after transport and / or storage at ambient temperature, a fermentation process is run using the one or more phage resistant lactic acid bacteria, thereby producing a fermented lactic acid product at a remote site.

[0069] Reference value

[0070] A reference value within the present context is understood as a value of a parameter that is representative for the acidification activity of a wild type lactic acid bacteria strain.

[0071] These reference values are criteria that are used to guide selection of phage-resistant lactic acid bacteria in step f) of the method as disclosed herein. The chosen reference values are helpful in determining which phage resistant lactic acid bacteria that have properties which either makes them suitable for use in applications which require or are intended to include prolonged dry storage at elevated temperatures, or which makes them useful for further development of phage resistant lactic acid bacteria strains that are well suited for prolonged dry storage at elevated temperatures.

[0072] Particularly interesting reference values include values of ta, tm, acidification loss, pH of incubation medium, and A pH. The reference values may in particular be values of ta, tm, acidification loss, pH of incubation medium, and A pH that were determined following dry storage of one or more lactic acid bacteria wild type strains, such as for example DSM22589.

[0073] In one particular embodiment, step f) of the method as disclosed herein relates to selection of phage resistant lactic acid bacteria having an improved determined value relative to a reference value for one or more of the parameters selected from the list consisting of ta, tm, acidification loss, pH of incubation medium, and A pH. It is to be understood that comparisons of determined values to reference values can only be made for determined values and reference values obtained under the same conditions, i.e. the same storage period, storage temperature, incubation period, incubation temperature, and incubation medium.

[0074] In the following section a number of exemplary reference values are defined, which may be used in the comparison of a determined value to a reference value in step f) of the method disclosed herein

[0075] Reference value 1 (Rv1): A ta, tm, acidification loss, pH of incubation medium, or A pH determined for one or more lactic acid bacteria wild type strains following dry storage and incubation under the same conditions as the phage resistant lactic acid bacteria of ai) or aii) was subjected to in steps b) and c).

[0076] Reference value 2 (Rv2): A ta, tm, acidification loss, pH of incubation medium, or A pH determined for DSM22589 following dry storage and incubation under the same conditions as the phage resistant lactic acid bacteria of ai) or aii) was subjected to in steps b) and c).

[0077] Reference value 3 (Rv3): A ta, tm, acidification loss, pH of incubation medium, or A pH determined for DSM34235 following dry storage and incubation under the same conditions as the phage resistant lactic acid bacteria of ai) or aii) was subjected to in steps b) and c).

[0078] Reference values based on determination of tathat are useful for making comparison and selection of phage resistant lactic acid bacteria that are suitable for storage at ambient temperature include the following:

[0079] Reference value 4 (Rv4): A taof 80 minutes following storage at the test storage temperature of b) for 2 weeks.

[0080] Reference value 5 (Rv5): A taof 85 minutes following storage at the test storage temperature of b) for 2 weeks.

[0081] Reference value 6 (Rv6): A taof 100 minutes following storage at the test storage temperature of b) for 1 month.

[0082] Reference value 7 (Rv7): A taof 120 minutes following storage at the test storage temperature of b) for 2 months.

[0083] Reference value 8 (Rv8): A taof 130 minutes following storage at the test storage temperature of b) for 3 months.

[0084] Reference value 9 (Rv9): A taof 140 minutes following storage at the test storage temperature of b) for 3 months.

[0085] Reference value 10 (Rv10): A taas defined in one or more of Rv4-Rv9. Reference value 11 (Rv11): A taas defined all of Rv4 and Rv6-Rv8.

[0086] Reference value 12 (Rv12): A taas defined in all of Rv4, Rv6-Rv7 and Rv9.

[0087] Reference values based on determination of tmthat are useful for making comparison and selection of phage resistant lactic acid bacteria that are suitable for storage at ambient temperature include the following:

[0088] Reference value 13 (Rv13): A tmof 300 minutes following storage at the test storage temperature of b) for 1 month.

[0089] Reference value 14 (Rv14): A tmof 350 minutes following storage at the test storage temperature of b) for 2 months.

[0090] Reference value 15 (Rv15): A tm of 375 minutes following storage at the test storage temperature of b) for 3 months.

[0091] Reference value 16 (Rv16): A tmof 600 minutes following storage at the test storage temperature of b) for 6 months.

[0092] Reference value 17 (Rv17): A tmof 650 minutes following storage at the test storage temperature of b) for 6 months.

[0093] Reference value 18 (Rv18): A tmas defined in one or more of Rv13-Rv17.

[0094] Reference value 19 (Rv19): A tmas defined in all of Rv13-Rv16.

[0095] Reference value 20 (Rv20): A tmas defined in all of Rv13-Rv15 and Rv 16.

[0096] Reference values based on determination of acidification loss that are useful for making comparison and selection of phage resistant lactic acid bacteria that are suitable for storage at ambient temperature include the following:

[0097] Reference value 21 (Rv21): An acidification loss of 60 minutes per month

[0098] Reference value 22 (Rv22): An acidification loss of 70 minutes per month

[0099] Reference value 23 (Rv23): An acidification loss of 80 minutes per month

[0100] Reference values based on determination of pH in the incubation medium that are useful for comparison and selection of lactic acid bacteria that are suitable for storage at ambient temperature include the following:

[0101] Reference value 24 (Rv24): A pH of 5.5 after 6 hours of incubation following storage at the test storage temperature of b) for 1 month.

[0102] Reference value 25 (Rv25): A pH of 5.7 after 6 hours of incubation following storage at the test storage temperature of b) for 2 months. Reference value 26 (Rv26): A pH of 5.8 after 6 hours of incubation following storage at the test storage temperature of b) for 2 months.

[0103] Reference value 27 (Rv27): A pH of 5.9 after 6 hours of incubation following storage at the test storage temperature of b) for 3 months.

[0104] Reference value 28 (Rv28): A pH of 6.0 after 6 hours of incubation following storage at the test storage temperature of b) for 3 months.

[0105] Reference value 29 (Rv29): A pH of 6.2 after 6 hours of incubation following storage at the test storage temperature of b) for 6 months.

[0106] Reference value 30 (Rv30): A pH of 6.3 after 6 hours of incubation following storage at the test storage temperature of b) for 6 months.

[0107] Reference value 31 (Rv31): A pH as defined in one or more of Rv24-Rv30.

[0108] Reference value 32 (Rv32): A pH as defined in all of Rv24, Rv25, Rv27 and Rv29.

[0109] Reference value 33 (Rv33): A pH as defined in all of Rv24, Rv26, Rv28 and Rv30.

[0110] Reference values based on determination of A pH that are useful for making comparison and selection of phage resistant lactic acid bacteria that are suitable for storage at ambient temperature include the following:

[0111] Reference value 34 (Rv34): A A pH of 0.3 following storage at the test storage temperature of b) for 1 month.

[0112] Reference value 35 (Rv35): A A pH of 0.4 following storage at the test storage temperature of b) for 1 month.

[0113] Reference value 36 (Rv36): A A pH of 0.5 following storage at the test storage temperature of b) for 2 months.

[0114] Reference value 37 (Rv37): A A pH of 0.6 following storage at the test storage temperature of b) for 2 months.

[0115] Reference value 38 (Rv38): A A pH of 0.7 following storage at the test storage temperature of b) for 3 months.

[0116] Reference value 39 (Rv39): A A pH of 0.8 following storage at the test storage temperature of b) for 3 months.

[0117] Reference value 40 (Rv40): A A pH of 0.9 following storage at the test storage temperature of b) for 3 months. Reference value 41 (Rv41): A A pH of 1 following storage at the test storage temperature of b) for 6 months.

[0118] Reference value 42 (Rv42): A A pH of 1 .1 following storage at the test storage temperature of b) for 6 months.

[0119] Reference value 43 (Rv43): A A pH of 1 .2 following storage at the test storage temperature of b) for 6 months.

[0120] Reference value 44 (Rv44): A A pH as defined in one or more of Rv34-Rv43.

[0121] Reference value 45 (Rv45): A A pH as defined in one or more of Rv34-Rv40 and Rv41-Rv43.

[0122] Reference value 46 (Rv46): A A pH as defined in all of Rv34, Rv36, Rv38 and Rv41 .

[0123] Reference value 47 (Rv47): A A pH as defined in all of Rv34, Rv36, Rv38 and Rv42.

[0124] Reference value 48 (Rv48): A A pH as defined in all of Rv35, Rv37, Rv39 and Rv42.

[0125] Reference value 49 (Rv49): A A pH as defined in all of Rv35, Rv37, Rv40 and Rv43.

[0126] Thus, a reference value as referred to in step e) and f) of the present method may be selected as one or more from the list consisting of Rv1-Rv49. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1- Rv49. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv4-Rv40, Rv42-Rv43, Rv45, and Rv47- Rv49. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv4-Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv13-Rv23, Rv34-Rv40, Rv42- Rv43, Rv45, and Rv47-Rv49. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv21-Rv23, Rv34- Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv21- Rv23, Rv34-Rv40, and Rv42-Rv43. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv21-Rv23, and Rv34-Rv40.ln one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv6, Rv13, Rv25, and Rv34. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv8, Rv15, Rv27, and Rv38. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, Rv8, Rv16, Rv29, and Rv42. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, and Rv21-Rv23. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1-Rv2, and Rv34-Rv40. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv1 and Rv2. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv21-Rv23. In one or more exemplary embodiments, the reference value (Rv) is selected as one or more reference value(s) from the list consisting of Rv34-Rv40.

[0127] Generally, the test storage temperature for any one of Rv1-Rv49 is a temperature in the range of 20-43°C, such as a temperature in a range of 25-40°C, 25-43°C, 30-40°C, 30-43°C, 35-40°C, 35- 43°C, 37-43°C, 37-43°C, or a temperature of 37°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-Rv49 is a temperature in the range of 20-43°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-Rv49 is a temperature in the range of 25-40°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-49 is a temperature in the range of 25-43°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-49 is a temperature in the range of 30-40°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-49 is a temperature in the range of 30-43°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-49 is a temperature in the range of 35-40°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-49 is a temperature in the range of 35-43°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1 -49 is a temperature in the range of 37-40°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-49 is a temperature in the range of 37-43°C. In one or more exemplary embodiments the test storage temperature for any one of Rv1-49 is a temperature is 37°C.

[0128] While any of these reference values may be useful in the context of the method as disclosed herein, the two parameters representative of the acidification activity "Time to reach maximum acidification activity" (tm) and "Rate of loss of acidification activity" (acidification loss) are considered particular useful, as the tm value represents a very specific time point during the incubation step for each sample and the acidification loss determined from a series of tmvalues plotted against time, converts this time point into a general trend defined by a linear function, which defines the acidification loss overtime across the whole incubation period in a single metric, i.e. , by the slope of the linear function.

[0129] In one or more particularly interesting embodiments, the one or more parameters representative of the acidification activity is selected as "Time to reach maximum acidification activity" (tm) and / or "Rate of loss of acidification activity" (acidification loss). In one or more particularly interesting embodiments, the one or more parameters representative of the acidification activity is selected as "Time to reach maximum acidification activity" (tm). In one or more particularly interesting embodiments, the one or more parameters representative of the acidification activity is selected as "Rate of loss of acidification activity" (acidification loss). In one or more particularly interesting embodiments, the reference value is selected as one or more reference value(s) from the list consisting of Rv13-23. In one or more particularly interesting embodiments, the reference value is selected as one or more reference value(s) from the list consisting of Rv13-17. In one or more particularly interesting embodiments, the reference value is selected as one or more reference value(s) from the list consisting of Rv18-20. In one or more particularly interesting embodiments, the reference value is selected as one or more reference value(s) from the list consisting of Rv21- 23.

[0130] Comparison to a reference value

[0131] In the present context, comparison to a reference value means making a comparison of a determined value for one or more parameters representative of the acidification activity obtained in step d) to a reference value. In some cases, the one or more parameters representative of the acidification activity may be a measurement of pH or a function derived from measurements of pH. In particular, a reference value as used in the present context is either a value of ta, tm, acidification loss, pH of incubation medium, or A pH as defined herein. When comparison is made between a determined value obtained in step d) and a reference value measured for a strain of lactic acid bacteria, such as e.g. the DSM22589 strain, the reference value must have been determined under the same conditions under steps b) and c) as the determined value.

[0132] Additionally, when making comparisons between a reference value and a determined value obtained from samples of lactic acid bacteria strains according to the method as defined herein, the reference value and the determined value must both originate from lactic acid bacteria strains belonging to the same species of lactic acid bacteria.

[0133] A reference value can be established by individual measurements made following regular intervals of storage, or it can be derived mathematically by functional analysis from a series of measurements made for samples of a specific strain that were stored for different periods of time (see e.g. DSM22589 in example 3), or it can be established as a target value for a parameter that indicates a desired minimum ability to tolerate storage in a dried format for a specific period of time.

[0134] Specific strains that are useful to measure reference values for include the wild type strain that gave rise to a given phage-resistant strain to be tested, and the wild type DSM22589 strain. For future testing, the DSM34235 strain may also be used to establish reference values for further improvements of acidification activity. The reference value may be derived from the mother strain of the dried phage-resistant lactic acid bacteria.

[0135] Beneficial use of stable phage resistant lactic acid bacteria

[0136] The bacteria and compositions as defined herein may be used in the production of dried format inoculants, starter cultures and direct vat sets (DVS). These dried format inoculants, starter cultures, and direct vat sets (DVS) will, as previously discussed, have retained improved metabolic activity following storage at ambient temperatures. This improved metabolic activity, results in these dried format inoculants, starter cultures, and direct vat sets (DVS) having an improved shelf life when stored at ambient temperatures. Therefore, these inoculants, starter cultures, and direct vat sets (DVS) are particularly well suited for use following long term storage at ambient temperatures. Such use may be preferable for general distribution to regions where there is limited cold storage availability and / or during transport to regions with a warmer climate, which will increase shelf time locally and reduce reliance on cold chain logistics. These improvements will also help improve food security in regions where electricity, and cold storage may in short supply or only intermittently available. One specific use of these inoculants, starter cultures, and direct vat sets, relates to their use in production of fermented lactic acid bacteria products, following storage at ambient temperatures. These fermented lactic acid bacteria products can for instance be dairy products, such as yogurt, buttermilk, kefir, quark, tvorog, creme fraiche, sour cream or cheese.

[0137] Thus, in one or more exemplary embodiments, the present disclosure relates to the use of a composition as defined herein or a phage resistant lactic acid bacteria as selected according to step f) of the method disclosed herein, for production of a fermented lactic acid bacteria product, after storage at ambient temperature in a dried format. Thus, in one or more exemplary embodiments, the present disclosure relates to the use of a composition as defined herein or a phage resistant lactic acid bacteria as selected according to step f) of the method disclosed herein, for production of a fermented lactic acid bacteria product, after storage for 14 days, 1 month, 2 months or 3 months at ambient temperature in a dried format.

[0138] In one or more exemplary embodiments, the present disclosure relates to the use of a composition as defined herein or a phage resistant lactic acid bacteria as selected according to f) of the method disclosed herein, for production of a fermented lactic acid bacteria product, after storage for 1 month at ambient temperature in a dried format.

[0139] In one or more exemplary embodiments, the present disclosure relates to the use of a composition as defined herein or a phage resistant lactic acid bacteria as selected according to f) of the method disclosed herein, for production of a fermented lactic acid bacteria product after storage for 1 month at ambient temperature in a dried format, and wherein the lactic acid bacteria of the composition or the selected lactic acid bacteria have an acidification loss of loss of less than 60 minutes per month, less than 70 minutes per month, or less than 80 minutes per month following storage at ambient temperature in a dried format.

[0140] In particular, these inoculants, starter cultures, and direct vat sets (DVS) are also highly suited for transportation and / or storage to a remote site prior to their use in production of lactic acid bacteria containing products and fermented lactic acid bacteria products, such as dairy products.

[0141] Thus, in one or more embodiments, the inoculants, starter cultures, and direct vat sets (DVS) disclosed herein, are used for producing a fermented lactic acid bacteria product at a remote site following storage at ambient temperature during transportation to the remote site and / or storage at ambient temperature at the remote site.

[0142] Remote site use

[0143] In the present context a remote site is a site that is in a different location than the production site where lactic acid bacteria selected 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 lactic acid bacteria 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 was a need for cold storage facilities and / or a cold chain for preservation of the lactic acid bacteria during transport in order to preserve the metabolic activity and or viability of the lactic acid bacteria. However, using phage resistant lactic acid bacteria selected in step f) of the method disclosed herein in dry format, the dependency for such cold storage and / or cold chain is drastically reduced, allowing for storage of the lactic acid bacteria at ambient temperatures during transport to the remote site and / or storage at the remote site following transport, without compromising the usefulness (i.e. metabolic activity and / or viability) of the lactic acid bacteria.

[0144] Therefore, in one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) of the method disclosed herein, for the preparation of a lactic acid bacteria product following storage at ambient temperature of the dried phage-resistant lactic acid bacteria.

[0145] In one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) and in a dried format for the preparation of a lactic acid bacteria product, following storage for 14 days, 1 month, 2 moths or 3 months at ambient temperature of the dried phage-resistant lactic acid bacteria.

[0146] Therefore, in one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) and in a dried format for the preparation of a lactic acid bacteria product, following storage at ambient temperature during transportation to a remote site and / or storage at the remote site at ambient temperature of the dried phage-resistant lactic acid bacteria.

[0147] Therefore, in one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) and in a dried format for the preparation of a lactic acid bacteria product, following storage at ambient temperature during transportation to a remote site and / or storage at the remote site at ambient temperature for at least 2 weeks.

[0148] Therefore, in one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) and in a dried format for the preparation of a lactic acid bacteria product, following storage at ambient temperature during transportation to a remote site and / or storage at the remote site at ambient temperature for at least 3 weeks.

[0149] Therefore, in one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) and in a dried format for the preparation of a lactic acid bacteria product, following storage at ambient temperature during transportation to a remote site and / or storage at the remote site at ambient temperature for at least 1 month.

[0150] Therefore, in one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) and in a dried format for the preparation of a lactic acid bacteria product, following storage at ambient temperature during transportation to a remote site and / or storage at the remote site at ambient temperature for at least 2 months.

[0151] Therefore, in one or more embodiments, the present disclosure relates to the use of phage resistant lactic acid bacteria selected according to step f) and in a dried format for the preparation of a lactic acid bacteria product, following storage at ambient temperature during transportation to a remote site and / or storage at the remote site at ambient temperature for at least 3 months.

[0152] Therefore, in one or more embodiments, remote may mean that there is a physical distance between the production site and a site, where the product is used, i.e. such as for dairy or cheese. Thus, it is necessary to pack the product at production site and ship it to the site of use. The distance can be 1 km, 50 km, 100 km, 1000 km, 3000 km, 5000 km, 10000 km, 12000 km. In absence of cold chain the product can be exposed to various ambient temperatures.

[0153] Compositions

[0154] The phage resistant lactic acid bacteria that are selected for storage at ambient temperature in step f) of the method disclosed, may be used for making compositions containing selected phage resistant lactic acid bacteria. In particular, the selected phage resistant lactic acid bacteria herein, may be dried and used for making dry compositions of selected phage resistant lactic acid bacteria. In particular, the dry compositions of selected phage resistant lactic acid bacteria disclosed herein are intended for use following prolonged storage at ambient temperature.

[0155] Because of the selection method as defined herein, the selected lactic acid bacteria have improved acidification activity following storage at ambient temperature in a dried format. The use of these selected phage resistant lactic acid bacteria having this improved acidification activity for making dry compositions, result in dry compositions comprising selected phage resistant lactic acid bacteria in a dried format, that have improved shelf-life following storage at ambient temperatures.

[0156] Thus, in one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity compared to Rv1 and / or Rv2.

[0157] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity compared to Rv1 .

[0158] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity compared to Rv2.

[0159] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity compared to Rv3.

[0160] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv49.

[0161] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one reference values selected from the list consisting of Rv1-Rv49.

[0162] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv4- Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0163] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv4- Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0164] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv4- Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0165] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv13-Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0166] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv21-Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0167] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv21-Rv23, Rv34-Rv40, and Rv42-Rv43.

[0168] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv21-Rv23, and Rv34-Rv40.

[0169] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv6, Rv13, Rv25, and Rv34.

[0170] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv8, Rv15, Rv27, and Rv38.

[0171] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv8, Rv16, Rv29, and Rv42. In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, and Rv21-Rv23.

[0172] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv13-Rv23.

[0173] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, and Rv34-Rv40.

[0174] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising a selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1 and Rv2. In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv13-Rv23. In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv13-Rv20. In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv13-Rv17. In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv18-Rv20. In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv21-Rv23.

[0175] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv34-Rv40.

[0176] Any one of the dry compositions as defined herein, may in addition to phage resistant lactic acid bacteria selected according to step f) of the method as disclosed herein, further comprise one or more additional lactic acid bacteria species and / or strains. In some cases, the one or more additional lactic acid bacteria may comprise wild type lactic acid bacteria and / or other phage resistant lactic acid bacteria.

[0177] Any one of the dry compositions as defined herein may be used as a starter culture or direct vat set (DVS). Any one of the dry compositions as defined herein may be used to produce a starter culture or direct vat set (DVS).

[0178] In one or more exemplary embodiments, a starter culture or direct vat set (DVS) comprises dried selected lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv49.

[0179] In one or more exemplary embodiments, a starter culture or direct vat set (DVS) comprises dried selected lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv21-Rv23, Rv34-Rv40, Rv42- Rv43, Rv45, and Rv47-Rv49.

[0180] In one or more exemplary embodiments, a starter culture or direct vat set (DVS) comprises dried selected lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv1-Rv2, Rv21-Rv23, and Rv34-Rv40.

[0181] In one or more exemplary embodiments, a starter culture or direct vat set (DVS) comprises dried selected lactic acid bacteria having an improved acidification activity when compared to one or more reference values selected from the list consisting of Rv21-Rv23.

[0182] In one or more exemplary embodiments, a starter culture or direct vat set (DVS) comprises dried selected lactic acid bacteria having an acidification loss of less than 60 minutes per month, less than 70 minutes per month, or less than 80 minutes per month following storage in a dried format at ambient temperature.

[0183] In one or more exemplary embodiments, a starter culture or direct vat set (DVS) comprises dried selected lactic acid bacteria having an acidification loss of less than 60 minutes per month following storage in a dried format at ambient temperature.

[0184] In interesting embodiments, a dry composition, starter culture and / or direct vat set as defined herein, is used for producing a fermented product at a remote site following storage of the dry composition, dried starter culture and / or dried direct vat set (DVS) at ambient temperature during transportation to the remote site and / or following storage at ambient temperature at the remote site. In particularly interesting embodiments, the transportation period and / or storage period at ambient temperature exceeds 1 month. In other particularly interesting embodiments, the transportation period and / or storage period at ambient temperature exceeds 3 months. In more particularly interesting embodiments, the transportation period and / or storage period at ambient temperature exceeds 6 months. The fermented product produced using the dry composition, starter culture, and / or direct vat set (DVS) as defined herein, may be a fermented dairy product. A fermented dairy product may be selected as one or more of yogurt, buttermilk, kefir, quark, tvorog, creme fraiche, sour cream and / or cheese.

[0185] In one or more exemplary embodiments, the dry composition, starter culture and / or direct vat set as defined herein, is used to produce a fermented product with increased stability during storage at ambient temperature. In one or more exemplary embodiments, the fermented product with increased stability during storage at ambient temperature is a dairy product. In one or more exemplary embodiments, the dairy product with increased stability during storage at ambient temperature is selected as one or more from the list consisting of yogurt, buttermilk, kefir, quark, tvorog, creme fraiche, sour cream and / or cheese.

[0186] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria selected according to the method disclosed herein, wherein the composition has an improved shelf life when stored at ambient temperature.

[0187] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria selected according to the method disclosed herein, wherein the composition has an improved shelf life for up to 2 years when stored at ambient temperature.

[0188] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria selected according to the method disclosed herein, in a dried format, wherein the composition is an inoculant, a starter culture, or a direct vat set (DVS).

[0189] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria selected according to the method disclosed herein, in a dried format, wherein the composition is used for producing a dried format inoculant, a starter culture, or a direct vat set (DVS).

[0190] In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria selected according to the method disclosed herein, wherein the selected phage resistant lactic acid bacteria are of the S. thermophilus strain deposited under DSM deposit number DSM34235. In one or more exemplary embodiments, the present disclosure relates to a dry composition comprising selected phage resistant lactic acid bacteria selected according to the method disclosed herein, wherein the phage resistant lactic acid bacteria are not of the S. thermophilus strain deposited under DSM deposit number DSM34235.

[0191] Ambient temperature

[0192] In the present disclosure, the expression “ambient temperature” means the temperature of the surroundings, e.g. room temperature. The ambient temperature may be a temperature above 20°C. An ambient temperature may also be a temperature in a range of 20-53°C, such as a temperature in a range of 25-43°C, 20-40°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. The ambient temperature 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). In particularly relevant cases, an ambient temperatures is a temperature selected as one of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C or 43°C. In other relevant cases, an ambient temperature is a temperature selected as one of 30, 31 , 32, 33, 34, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C or 43°C.

[0193] In one or more embodiments, an ambient temperature is the temperature of the surroundings or room temperature. In one or more embodiments, an ambient temperature is the temperature of the surroundings. In one or more embodiments, an ambient temperature is room temperature. In one or more embodiments, an ambient temperature is a temperature above 20°C. In one or more embodiments, an ambient temperature is a temperature in the range of 20-53°C. In one or more embodiments, an ambient temperature is a temperature in the range of 20-43°C, or such as a temperature in the range of 25-43°C, 20-40°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, 35-43°C, 37- 40°C, or a temperature in the range of 37-43°C. In one or more embodiments, an ambient temperature is a temperature in the range of 20-40°C, or such as a temperature in the range of 25- 40°C, 30-40°C, 35-40°C, or a temperature in the range of 37-40°C. In one or more embodiments, an ambient temperature is a temperature in the range of 25-40°C, 30-40°C, 35-40°C, or a temperature in the range of 37-40°C. In one or more embodiments, an ambient temperature is a temperature in the range of 25-40°C, 30-40°C, 35-40°C, or a temperature in the range of 37-43°C. In one or more embodiments, an ambient temperature is a temperature in the range of 25-40°C, 30-43°C, or a temperature in the range of 35-43°C. In one or more embodiments, an ambient temperature is a temperature in the range of 25-40°C. In one or more embodiments, an ambient temperature is a temperature in the range of 30-40°C. In one or more embodiments, an ambient temperature is a temperature in the range of 35-40°C. In one or more embodiments, an ambient temperature is a temperature in the range of 37-40°C.ln one or more embodiments, an ambient temperature is a temperature in the range of 25-43°C. In one or more embodiments, an ambient temperature is a temperature in the range of 30-43°C. In one or more embodiments, an ambient temperature is a temperature in the range of 35-43°C. In one or more embodiments, an ambient temperature is a temperature in the range of 37-43°C. In one or more embodiments, an ambient temperature is a temperature in the range of 40-48°C. In one or more embodiments, an ambient temperature is a temperature in the range of 45-53°C. In one or more embodiments, an ambient temperature is a temperature in the range of 48-53°C. In one or more embodiments, an ambient temperature is a temperature of 37°C.

[0194] Lactic acid bacteria

[0195] 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.

[0196] 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, Loigolactobacilus, 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.

[0197] 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.

[0198] Thus, in one or more embodiments, a lactic acid bacteria are bacteria belonging to a genus selected from the group consisting of Lactococcus , Streptococcus., Lactobacillusthe the latter now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, 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, Bifidobacterium and combinations thereof.

[0199] In one or more embodiments, the lactic acid bacteria are bacteria that belong to the Streptococcus genus. In one or more embodiments, the lactic acid bacteria are bacteria that belong to the species Streptococcus thermophilus. In one or more embodiments, the lactic acid bacteria are of the strain of Streptococcus thermophilus deposited with DSMZ under the accession number DSM34235.

[0200] In one or more embodiments, the lactic acid bacteria are bacteria belonging to a genus selected from the group consisting of Lactococcus, Streptococcus, Lactobacillus the latter now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, 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, Bifidobacterium and combinations thereof, but is not the strain of Streptococcus thermophilus deposited with DSMZ under the accession number DSM34235.

[0201] In one or more embodiments, the lactic acid bacteria are bacteria that belong to the species Streptococcus thermophilus, but is not the strain of Streptococcus thermophilus deposited with DSMZ under the accession number DSM34235.

[0202] In one or more embodiments, the lactic acid bacteria are bacteria that belong to the Streptococcus genus, but is not the strain of Streptococcus thermophilus deposited with DSMZ under the accession number DSM34235.

[0203] Dried lactic acid bacteria

[0204] Dried lactic acid bacteria, is a term that refers to cultures of lactic acid bacteria that has undergone at least one drying step. Any one of several commonly known methods for drying bacteria can be used for producing dried lactic acid bacteria. Such methods include freeze-drying, spray-drying and vacuum drying. Generally, freeze-drying is considered a 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.

[0205] Thus, in one or more embodiments, the dried lactic acid bacteria are dried by freeze-drying, spraydrying or vacuum drying. In one or more embodiments, the dried lactic acid bacteria are dried by spray drying or freeze drying. In one or more embodiments, the dried lactic acid bacteria are dried by spray drying. In one or more embodiments, the dried lactic acid bacteria are dried by freeze drying.

[0206] Dried lactic acid bacteria are also referred to herein as lactic acid bacteria in a dried format. The phrase dry compositions comprising lactic acid bacteria also refers to dry compositions comprising dried lactic acid bacteria. Such compositions may be produced by any of the drying methods as discussed above.

[0207] Selected phage resistant lactic acid bacteria

[0208] The term selected phage resistant lactic acid bacteria as defined herein, refers to phage resistant lactic acid bacteria that has been tested according to the method as disclosed herein and selected in step f). A selected phage resistant lactic acid bacteria are also referred to herein as selected lactic acid bacteria, lactic acid bacteria selected in / according to step f), and / or referred to as phage resistant lactic acid bacteria selected in / according to step f).

[0209] DVS sets and starter cultures

[0210] A Direct Vat Set (DVS) as used in the present context refers to blends of lactic acid bacteria comprising more than one species and / or strains of lactic acid bacteria intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product. In some cases, DVS cultures as referred to herein may comprise both phage resistant lactic acid bacteria selected according to the method as disclosed herein, and wild type lactic acid bacteria strains and.

[0211] Starter cultures as used herein refers to inoculates or bulk starter cultures comprising one or more phage resistant lactic acid bacteria species and / or strains that are intended for use in bulk starter propagation prior to the actual fermentation process. Starter cultures as referred to herein may comprise both comprise both phage resistant lactic acid bacteria selected according to the method as disclosed herein, and wild type lactic acid bacteria. Wild type

[0212] As used within the present context, the term "wild type" refers to a non-GMO lactic acid bacteria strain that has not previously been subjected to a phage resistance protocol. Such a wild type strain may either be a naturally occurring lactic acid bacteria strain that has not undergone selection in a laboratory or industrial selective process, or it may be a mother strain (i.e. the strain that was subjected to a phage resistance protocol to give rise to the phage resistant strain) of a phage resistant lactic acid bacteria strain tested in the method as disclosed herein.

[0213] Phages

[0214] Bacteriophages, herein referred to as phages, are viruses that infect and replicate in bacteria. These phages are composed of proteins that encapsulate a DNA or RNA genome and they replicate within the bacteria following injection of their genome into the cytoplasm of the bacteria.

[0215] Phages are known to impact dairy production and may in severe cases completely terminate the fermentation processes necessary for producing the dairy product. These phages include the pac type bacteriophage and the cos type bacteriophage. Examples of strains belonging to these types of bacteriophages are listed in table 1 .

[0216] Phage-resistant lactic acid bacteria

[0217] The lytic development of bacteriophages involves adsorption of the phages to the host cell surface, injection of phage DNA into the cell, synthesis of phage proteins, replication of phage DNA, assembly of progeny phages and release of progeny from the host. Cell-mediated mechanisms of interference with any of these events can prevent a phage infection. The ability of bacterial cultures to resist bacteriophage infection during industrial use depends on host strain characteristics affecting one or more of the above mechanisms.

[0218] Phage-resistant lactic acid bacteria strains may be generated by exposing a phage-sensitive wild type strain to the desired phage or phages, e.g. by growing the phage-sensitive wild type strain in the presence of a concentration of the desired phage(s) sufficient to induce a selection pressure on said wild type strain. Surviving bacteria can then be selected and their phage-resistance assayed by standard plaque-assays. Methods for generating phage-resistant lactic acid bacteria are generally known in the prior art and are referred to herein as ph age- resista nee protocols.

[0219] It will be appreciated that phage-resistance may be obtained with strains originating from many species. For example, any lactic acid bacteria may be used as a wild type strain and any Streptococcus species, such as Streptococcus thermophilus, may be used as a wild type strain. In one or more embodiments, a phage-resistant lactic acid bacteria derived from the wild type strain may be any mutant with improved phage-resistance as compared to the wild type strain.

[0220] In one or more embodiment, the phage-resistant lactic acid bacteria belong to the species Streptococcus thermophilus.

[0221] In one or more embodiments, the phage-resistant Streptococcus species has increased phage resistance towards the pac type bacteriophage deposited under the reference number DSM 34256 and / or the cos type bacteriophage deposited under the reference number DSM 34257 as compared to its wild type strain.

[0222] In the present context, the term "increased phage-resistance", phage-resistance or "phageresistant" means that the lactic acid bacteria strain when tested in a plaque assay have an increase phage resistance to at least one phage expressed as the difference in pfu / mL (plaque forming unit per mL) obtainable with said at least one bacteriophage on the given strain, compared to the pfu / mL obtainable with the same bacteriophage on the wild type strain. A strain with improved resistance to a bacteriophage preferably show a reduction of pfu / mL of a factor of at least 50, of at least 100, of at least 500, of at least 1000, of at least 5000, of at least 10000 or more.

[0223] Preparation of phage-resistant Lactic acid bacteria

[0224] Ph age- resista nee can be routinely tested for in standard plaque assays, which are commonly known in the prior art. One example of such a method is described in example 1 of WO2023 / 222575.

[0225] The method generally comprises plating out a 0.1 mL of a wild type overnight culture together with 0.1 mL of a suitable phage (e.g. DSM 34256) in a dose of 109phage particles per mL, followed by incubation overnight. Suitable plates include, but are not limited to M17-2% lactose agar plates with 10mM MgCL / CaCL.

[0226] Phage resistant mutants may then be selected from the bacterial colonies that show up on the following day, and to verify the emergence of phage resistance, the bacterial colonies selected should be three times colony purified and retested in a plaque assay the same phage strain, e.g. DSM 34256. Phage resistance can be confirmed by no observation of plaques during the plaque assay. Strain deposits and expert solution

[0227] The applicant 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.

[0228] Table 1. Deposits made at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ- German Colection of Microorganisms and Cel Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany. All strains were disclosed in previous applications mentioned in the table below.

[0229] Table 1 : Deposited strains referenced in the present disclosure.

[0230] Items

[0231] 1 . A method for selecting lactic acid bacteria for dry storage at ambient temperature, the method comprising ai) providing one or more dried phage-resistant lactic acid bacteria, or aii) providing a library of dried phage-resistant lactic acid bacteria, b) storing the dried phage-resistant lactic acid bacteria of ai) or aii) at a test storage temperature for a time period sufficient for a change in one or more parameters representative of the acidification activity can be measured, such as for at least 2 days, such as at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, 3 months or such as at least 6 months thereby providing stored dried phage-resistant lactic acid, c) incubating the stored dried phage-resistant lactic acid bacteria of step b) for at least two hours in a liquid sugar-based or milk-based incubation medium to obtain incubated phage-resistant lactic acid bacteria, d) determining a value for one or more parameters representative of the acidification activity of the incubated phage-resistant lactic acid bacteria c), thereby obtaining a determined value, e) comparing the determined value in step d) to a reference value, and f) selecting any incubated phage-resistant lactic acid bacteria for which the determined value obtained in step d) corresponds to an improved acidification activity when compared to the reference value, thereby providing selected phage resistant lactic acid bacteria. The method according to item 1 , determining a value for one or more parameters representative of the acidification activity of the dried phage-resistant lactic acid bacteria of step ai) or aii) immediately after freeze-drying, thereby obtaining an initial acidification value. The method according to items 1 or 2, wherein the one or more parameters representative of the acidification activity of the incubated phage resistant lactic acid bacteria in step d) and the reference value in step e) is selected as one or more of ta, A pH, pH of the incubation medium, tm, or acidification loss. The method according to any of the preceding items, wherein the phage-resistant lactic acid bacteria are resistant to PAC- or COS-type bacteriophages. The method according to any of the preceding items, the method further comprises: a. selecting dried phage-resistant lactic acid bacteria that exhibit resistance to one or more PAC- or COS -type bacteriophages, wherein the resistance is determined by exposing the lactic acid bacteria to PAC- or COS-type bacteriophages under test conditions and assessing bacterial viability or growth inhibition, b. storing the selected dried PAC- or COS-type phage-resistant lactic acid bacteria at ambient temperature as per step b) of items 1 , c) performing steps c) through f) of any of items 1 to identify and select PAC- or COS-type phage-resistant lactic acid bacteria with improved acidification activity.

[0232] 6. The method according to any of the preceding claims, wherein the PAC- or COS-type bacteriophages are DSM 34257 or DSM 34257, respectively.

[0233] 7. The method according any of the preceding items, wherein the one or more parameters representative of the acidification activity of the incubated phage resistant lactic acid bacteria in step d) and the reference value in step e) is selected as one or more of Tm, and / or acidification loss.

[0234] 8. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv49.

[0235] 9. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv2, Rv4-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0236] 10. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv2, Rv4-Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0237] 11 . The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1 -Rv2, Rv13-Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0238] 12. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1 -Rv2, Rv21 -Rv23, Rv34-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49. 13. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1 -Rv2, Rv21 -Rv23, Rv34-Rv40, and Rv42-Rv43.

[0239] 14. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1 -Rv2, Rv21 -Rv23, and Rv34-Rv40.

[0240] 15. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv2, Rv6, Rv13, Rv25, and Rv34.

[0241] 16. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv2, Rv8, Rv15, Rv27, and Rv38.

[0242] 17. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv2, Rv8, Rv16, Rv29, and Rv42.

[0243] 18. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1 -Rv2, and Rv21 -Rv23.

[0244] 19. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv2, and Rv34-Rv40.

[0245] 20. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more of Rv1 and Rv2.

[0246] 21 . The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more of Rv21-Rv23.

[0247] 22. The method according to any one of the preceding items, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv34-Rv40. 23. The method according to any one of the preceding items, wherein step ai) and / or step aii) further comprises xi) subjecting one or more lactic acid bacteria wild type strain(s) or a library of lactic acid bacteria wild type strains to a phage-hardening / phage- resistance protocol, xii) selecting the lactic acid bacteria that survives the phage- hardening / phage-resistance protocol, thereby providing one or more phageresistant lactic acid bacteria or a library of phage-resistant lactic acid bacteria, and xiii) drying the one or more phage-resistant lactic acid bacteria or drying the library of phage-resistant lactic acid bacteria, thereby providing one or more dried phage-resistant lactic acid bacteria, or a library of dried phage-resistant lactic acid bacteria.

[0248] 24. The method according to any one of the preceding items, wherein the phage resistant lactic acid bacteria selected in step f) is subjected to at least one additional cycle of storage, incubation and selection as defined in steps b)-f).

[0249] 25. The method according to any one of the preceding items, wherein the phage-resistant dried lactic acid bacteria selected in step f) is subjected to at least one additional cycle of a phage resistance protocol followed by storage, cultivation and selection according to the method as defined in steps a)-f).

[0250] 26. The method according to any one of the preceding items, wherein a) comprises only ai) and not aii).

[0251] 27. The method according to any one of the preceding items, wherein a) comprises only aii) but not ai).

[0252] 28. The method according to any one of the preceding items, wherein the dried lactic acid bacteria are dried by freeze-drying, vacuum drying or spray-drying. 29. The method according to any one of the preceding items, wherein the dried lactic acid bacteria are dried by spray drying or freeze drying.

[0253] 30. The method according to any one of the preceding items, wherein the dried lactic acid bacteria are dried by spray drying.

[0254] 31 . The method according to any one of the preceding items, wherein the dried lactic acid bacteria are dried by freeze drying.

[0255] 32. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 20-43°C, such as a temperature in the range of 25-43°C, 20- 40°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, 35-43°C, 37-40°C, or a temperature in the range of 37-43°C.

[0256] 33. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 20-40°C, or such as a temperature in the range of 25-40°C, 30-40°C, 35-40°C, or a temperature in the range of 37-40°C.

[0257] 34. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 25-40°C, 30-40°C, or a temperature in the range of 35-40°C.

[0258] 35. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 30-40°C, or a temperature in the range of 35-40°C.

[0259] 36. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 30-43°C, or a temperature in the range of 35-43°C.

[0260] 37. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 35-40°C.

[0261] 38. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 35-43°C.

[0262] 39. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 37-40°C. 40. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 37-43°C.

[0263] 41 . The method according to any one of the preceding items, wherein ambient temperature is a temperature of 37°C.

[0264] 42. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature in the range of 20-43°C, such as a temperature in the range of 25-43°C, 20-40°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, 35-43°C, 37-40°C, or a temperature in the range of 37-43°C.

[0265] 43. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature in the range of 25-43°C, or such as a temperature in the range of 25-40°C, 30-43°C 30-40°C, 35-43°C, 35-40°C, 37-43°C, or a temperature in the range of 37-40°C.

[0266] 44. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature in the range of 30-40°C, 35-40°C or a temperature in the range of 37-40°C.

[0267] 45. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature in the range of 30-43°C, 35-43°C, or a temperature in the range of 37-43°C.

[0268] 46. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature in the range of 35-40°C.

[0269] 47. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature in the range of 35-43°C.

[0270] 48. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature in the range of 37-40°C. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature of 37-43°C. The method according to any one of the preceding items, wherein a test storage temperature, is a temperature of 37°C. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 20-43°C, such as a temperature in the range of 25-43°C, 20- 40°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, or a temperature in the range of 35-43°C and wherein a test storage temperature, is a temperature in the range of 30-40°C, or a temperature in the range of 35-40°C. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 20-43°C, such as a temperature in the range of 25-43°C, 20- 40°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, or a temperature in the range of 35-43°C and wherein a test storage temperature, is a temperature in the range of 35-40°C. The method according to any one of the preceding items, wherein ambient temperature is a temperature in the range of 20-43°C, such as a temperature in the range of 25-43°C, 20- 40°C, 25-40°C, 30-40°C, 30-43°C, 35-40°C, or a temperature in the range of 35-43°C and wherein a test storage temperature, is a temperature of 37°C. The method according to any one of the preceding items, wherein b) comprises storing the dried phage-resistant lactic acid bacteria for 3-7 days. The method according to any one of the preceding items, wherein b) comprises storing the dried phage-resistant lactic acid bacteria for 2 weeks. The method according to any one of the preceding items, wherein b) comprises storing the dried phage-resistant lactic acid bacteria for 1 month. The method according to any one of the preceding items, wherein b) comprises storing the dried phage-resistant lactic acid bacteria for 2 months. The method according to any one of the preceding items, wherein b) comprises storing the dried phage-resistant lactic acid bacteria for 3 months. The method according to any one of the preceding items, wherein b) comprises storing the dried phage-resistant lactic acid bacteria for 6 months. The method according to any one of the preceding items, wherein the incubating for at least 2 hours in c) is an incubation period of between 2 and 24 hours, such as between 2- 20, 2-16, 2-10, 2-8, 3-20, 3-16, 3-10, 3-8, 4-20, 4-16, 4-10, or between 4-8 hours. The method according to any one of the preceding items, wherein the incubating for at least 2 hours in c) is an incubation period of 14-18 hours. The method according to any one of the preceding items, wherein the incubating for at least 2 hours in c) is an incubation period of 16 hours. The method according to any one of the preceding items, further comprising producing a fermented product at a remote site by

[0271] -providing one or more phage-resistant lactic acid bacteria selected as defined in a method according to any one of the preceding items in a dried format,

[0272] -keeping the one or more selected phage-resistant lactic acid bacteria in a dried format at ambient temperature during transport to the remote site and / or keeping the one or more selected phage-resistant lactic acid bacteria in a dried format at ambient temperature during storage at the remote site, and

[0273] -running a fermentation process comprising the one or more selected phageresistant lactic acid bacteria after transport and / or storage at ambient temperature at the remote site, thereby producing a fermented lactic acid product at a remote site. A method according to any one of items 1-65, the method further comprising producing a fermented lactic acid bacteria product at a remote site, the method comprising -providing one or more selected phage-resistant lactic acid bacteria selected as defined in a method according to any one of items in a dried format,

[0274] -keeping the one or more dried selected phage-resistant lactic acid bacteria at ambient temperature during transport to the remote site and / or keeping the one or more dried selected phage-resistant lactic acid bacteria at ambient temperature during storage at the remote site, and

[0275] -running a fermentation process comprising the one or more dried selected phageresistant lactic acid bacteria after transport and / or storage at ambient temperature at the remote site, thereby procuring a fermented lactic acid product at a remote site.

[0276] 65. The method according to any one of the preceding items, wherein the phage-resistant lactic acid bacteria are bacteria belonging to a genus selected from the list consisting of Lactococcus , Streptococcus, Lactobacillus the latter now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, 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.

[0277] 66. The method according to any one of the preceding items, wherein the phage-resistant lactic acid bacteria belong to the Streptococcus genus.

[0278] 67. The method according to any one of the preceding items, wherein the phage-resistant lactic acid bacteria belong to the species Streptococcus thermophilus.

[0279] 68. The method according to any one of the preceding items, wherein the phage resistant lactic acid bacteria are of the Streptococcus thermophilus strain deposited under DSM deposit number DSM34235, and mutants and variants thereof. 69. The method according to any one of the preceding items, wherein the phage resistant lactic acid bacteria are not of the Streptococcus thermophilus strain deposited under DSM deposit number DSM34235.

[0280] 70. A composition comprising a phage-resistant lactic acid bacteria selected according to the method defined in any one of items 1-65 in a dried format.

[0281] 71 . The composition according to item 66, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv49.

[0282] 72. The composition according to any one of items 66-67, wherein the selected phage resistant lactic acid has an improved acidification activity compared to a reference value (Rv) selected from the list consisting of Rv1-Rv49.

[0283] 73. The composition according to any one of items 66-68, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv4-Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0284] 74. The composition according to any one of items 66-69, wherein the selected phage resistant lactic acid has an improved acidification activity than one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv4-Rv23, Rv34-Rv40, Rv42- Rv43, Rv45, and Rv47-Rv49.

[0285] 75. The composition according to any one of items 66-70, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv13-Rv23, Rv34- Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49.

[0286] 76. The composition according to any one of items 66-71 , wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv21-Rv23, Rv34- Rv40, Rv42-Rv43, Rv45, and Rv47-Rv49. The composition according to any one of items 66-72, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv21-Rv23, Rv34- Rv40, and Rv42-Rv43. The composition according to any one of items 66-73, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv21-Rv23, and Rv34- Rv40. The composition according to any one of items 66-74, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv6, Rv13, Rv25, and Rv34. The composition according to any one of items 66-75, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv8, Rv15, Rv27, and Rv38. The composition according to any one of items 66-76, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, Rv8, Rv16, Rv29, and Rv42. The composition according to any one of items 66-77, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, and Rv21-Rv23. The composition according to any one of items 66-78, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv2, and Rv34-Rv40. The composition according to any one of items 66-79, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from Rv1 and Rv2. 85. The composition according to any one of items 66-80, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from Rv21-Rv23.

[0287] 86. The composition according to any one of items 66-81 , wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from Rv34-Rv40.

[0288] 87. Use of a phage-resistant lactic acid bacteria selected according to the method as defined in any one of items 1-65 or a composition as defined in any one of 66-82, for producing a lactic acid bacteria containing product.

[0289] 88. Use according to item 83, wherein the composition or dried phage-resistant lactic acid bacteria are stored for at least 2 days at ambient temperature prior to preparation of the lactic acid bacteria product.

[0290] 89. The use according to any one of items 83-84, wherein the lactic acid product is a dried starter culture or dried direct vat set (DVS).

[0291] 90. The use according to item 85, wherein the dried starter culture or dried direct vat set (DVS) has an acidification loss of less than 60 minutes per month, less than 70 minutes per month, or less than 80 minutes per month following storage in a dried format at ambient temperature.

[0292] 91 . Use according to any one of items 85-86, wherein the dried starter culture or dried direct vat set (DVS) has an acidification loss of less than 60 minutes per month following storage in a dried format at ambient temperature.

[0293] 92. The use according to any one of items 83-87, wherein the lactic acid product is a dried starter culture or dried direct vat set (DVS) that has improved shelf life during storage at ambient temperatures. 93. The use according to any one of items 83-88, wherein the lactic acid bacteria containing product is a dried starter culture or dried direct vat set that has an acidification activity of below 100 minutes following storage at ambient temperature.

[0294] 94. The use according to any one of items 83-89, wherein the lactic acid bacteria containing product is a dried starter culture or dried direct vat set that has an acidification activity of below 100 minutes following storage for 1 month at ambient temperature.

[0295] 95. The use of a lactic acid bacteria containing product, dried starter culture and / or dried direct vat set (DVS) according to any one of items 83-90, for producing a fermented product.

[0296] 96. The use according to item 91 , for producing a fermented product at a remote site, following storage of the lactic acid bacteria containing product, dried starter culture and / or dried direct vat set (DVS) at ambient temperature during transportation to the remote site and / or following storage at ambient temperature at the remote site.

[0297] 97. The use according to item 92, wherein the fermented product is dairy product.

[0298] 98. The use according to item 93, wherein the dairy product is yogurt, buttermilk, kefir, quark, tvorog, creme fraiche, sour cream or cheese.

[0299] 99. The use according to any one of items 83-94 for providing a fermented product with increased stability during storage at ambient temperature.

[0300] 100. The use according to any one of items 83-95, for producing a fermented lactic acid bacteria product, wherein the phage-resistant lactic acid bacteria have an acidification loss of less than 60 minutes per month, less than 70 minutes per month, or less than 80 minutes per month following storage in a dried format at ambient temperature.

[0301] 101 . Use according to any one of items 83-96, for producing a lactic acid bacteria containing product with improved stability when stored at ambient temperature, wherein the phage-resistant lactic acid bacteria have an acidification loss of less than 60 minutes per month during storage in a dried format at ambient temperature. 102. The compositions according to any one of items 66-82, and / or the use according to any one of items 83-97, wherein the phage-resistant lactic acid bacteria are bacteria belonging to a genus selected from the list consisting of Lactococcus, Streptococcus, Lactobacillus now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, 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.

[0302] 103. The compositions according to any one of items 66-82, and / or the use according to any one of items 83-97, wherein the phage-resistant lactic acid bacteria belong to the Streptococcus genus.

[0303] 104. The compositions according to any one of items 66-82, and / or the use according to any one of items 83-97, wherein the phage-resistant lactic acid bacteria belong to the species Streptococcus thermophilus.

[0304] 105. The compositions according to any one of items 66-82, and / or the use according to any one of items 83-97, wherein the phage resistant lactic acid bacteria are of the Streptococcus thermophilus strain deposited under DSM deposit number DSM34235.

[0305] 106. The compositions according to any one of items 66-82, and / or the use according to any one of items 83-97, wherein the phage resistant lactic acid bacteria are not of the Streptococcus thermophilus strain deposited under DSM deposit number DSM34235.

[0306] BRIEF DESCRIPTION OF THE FIGURES

[0307] Figure 1

[0308] Figure 1 shows the time to observe a change in pH of 0.08 (ta) in RSM medium at 43°C following inoculation of the RSM medium with freeze-dried Streptococcus thermophilus DSM22589 (wild type strain)(black filled circles) or freeze-dried Streptococcus thermophilus DSM34235 (phage resistant strain) (white filled circles) following storage at 37°C for 0 days, 0.5 month, 1 month, 2 months, 3 months and 6 months. Figure 2

[0309] Figure 2 shows pH measured in RSM medium at 43°C, 6 hours following inoculation of the RSM medium with freeze-dried Streptococcus thermophilus DSM22589 (wild type strain)(black filled circles) or freeze-dried Streptococcus thermophilus DSM34235 (phage resistant strain) (white filled circles) following storage at 37°C for 0 days, 0.5 month, 1 month, 2 months, 3 months and 6 months.

[0310] Figure 3 Figure 3 shows a plot of a data series of time from start of incubation to max acidification rate (tm) against storage time for DSM22589 (example 2) and a linear function derived from the data series (example 3).

[0311] Figure 4 Figure 3 shows a plot of a data series of time from start of incubation to max acidification rate (tm) against storage time for DSM34235 (example 2) and a linear function derived from the data series (example 3).

[0312] EXAMPLES

[0313] Example 1:

[0314] Industrial freeze-dried products, FD-DVS DSM22589 (wild type strain), batch 3637684 and FD- DVS DSM34235 (phage resistant strain), batch 3608901 , were aliquoted in aluminium pouches, sealed and set in storage stability test at 37°C for 0 days, 0.5 months, 1 month, 2 months, 3 months and 6 months. Following storage, the cultures were incubated in sterilized reconstituted skimmed milk (RSM) with 9,5% dry matter content at 43°C for 16 hours. A series of freeze dried DSM22589 samples were inoculated in RSM at 0,0015% (w / w) following respectively 0, 0.5, 1 , 2, 3 and 6 months of storage at 37°C and a series of freeze dried DSM34235 samples were inoculated in RSM at 0,0025 % (w / w) following respectively 0, 0.5, 1 , 2, 3 and 6 months of storage at 37°C. pH was measured continuously and the acidification rate was calculated throughout the 16 hours of incubation using the iCinac system (KPM; AMS Alliance). From these data, the time it took for each sample to obtain a pH change of 0.08 was calculated (ta) and this data is shown in table 2 and figure 1 .

[0315] Table 2: Time to a change in pH of 0.08 (ta) following incubation in RSM of freeze-dried DSM22589 or DSM34235 that was stored for 0, 0.5, 1 , 2, 3, or 6 months at 37°C.

[0316] As can be seen from data in table 2 and figure 1 , the two cultures have similar taprior to storage (time 0), whereas the DSM34235 culture performs increasingly better than DSM22589 in terms of taas storage time increases. As can be seen from the data in figure 1 , DSM34235 maintains a tafollowing 1 month of storage that is nearly as low as the taof DSM22589 following 14 days of storage. DSM34235 also shows a much lower tacompared to DSM22589 following both 2-, 3- and 6 months storage, clearly indicating that DSM34235 recovers faster from long term storage at 37°C, thus being more stable during storage at 37°C. Additionally, the pH values of each sample for each strain following 6 hours of incubation were measured (see table 3 and figure 2) and the difference in pH (A pH) calculated by determining the pH for a sample stored for a period of time at 37°C (e.g. 1 month) and subtracting the pH value measured for the same strain at day 0 (prior to storage at 37°C). This difference in pH (A pH (6h)) is shown in table 4.

[0317] Table 3:

[0318] As can be seen in table 3, the wild type strain and the phage resistant strain of lactic acid bacteria. have similar capacity for reducing the pH of the incubation medium, priori to dry storage at ambient temperature. However, as the strains are stored for about 1 month, the phage resist strain (DSM34235) shows a greater ability to reduce the pH of the incubation medium, following dry storage at ambient temperature. This trend persists 3 months and even following 6 months of dry storage at ambient temperature.

[0319] Table 4: A pH following 6 hours of incubation in RSM of freeze-dried DSM22589 or DSM34235 that was stored for 0, 0.5, 1 , 2, 3, or 6 months at 37°C.

[0320] In this context, an increase in A pH indicates that a sample is slower, i.e. less capable of acidifying the incubation medium (in this case RSM). Thus, a sample that has been stored for a period of time and has a positive A pH (6h), is less capable and less efficient in acidifying the incubation medium. This loss in acidification activity following storage, is an indication that the sample has lost metabolic fitness and viability during the storage period.

[0321] However, the loss of acidification activity indicated by the A pH value can also be used to estimate which strains and / or species of lactic acid bacteria that are best able to withstand the negative impact of the storage period, i.e. which strain or species that remains more stable during storage.

[0322] When analyzing the data of table 2 and figure 2, it is clear that while the two strains appear to deal equally well with short term storage, i.e. for half a month (A pH 0.23 vs 0.26), the longer storage the strains are subjected to, there is a clear trend that the phage resistant strain (DSM34235) has a lower A pH and therefore is better able to deal with storage at 37°C than the wild type strain (DSM22459) and thus appears to remain more stable following long term storage at 37°C.

[0323] Example 2 - determining a time to reach maximum acidification rate (tm)

[0324] Acidification activity of freeze-dried strains was measured by iCinac system (KPM; AMS Alliance). Sterilized reconstituted skimmed milk (RSM) with 9,5% dry matter content was used as a substrate. Freeze-dried strains were inoculated in 200 ml of RSM. A series of freeze dried DSM22589 samples were inoculated in RSM at 0,0015% (w / w) following respectively 0, 0.5, 1 , 2, 3 and 6 months of storage at 37°C and a series of freeze dried DSM34235 samples were inoculated at 0,0025 % (w / w) following respectively 0, 0.5, 1 , 2, 3 and 6 months of storage at 37°C. Bottles containing RSM inoculated with S. thermophilus DSM22589 samples or inoculated S. thermophilus DSM34235 samples were incubated at 43°C for 16 hours. The iCinac performed continuous measurement of pH and the acidification rate was calculated for each sample by the iCinac software.

[0325] Following incubation each of the acidification activity curves measured overtime, was subject to derivative analysis to identify the amount of time measured in minutes (tm) that had passed for each sample, before the maximum acidification rate was attained. The values for tm are shown in table 5 as minutes against storage time in months and plotted in figures 3 (DSM22589) and figure 4 (DSM34235). The mathematical method and reasoning behind this analytical approach is explained in Fonseca et al., Journal of Dairy Research, 2000, 67: 83-90.

[0326] Table 5: Table 5 shows the incubation passed before max acidification rate is achieved for samples of DSM22589 or DSM34235 following storage for 0, 0.5, 1 , 2, 3, or 6 months respectively.

[0327] Example 3 - Determining the rate of loss of acidification activity by functional analysis

[0328] Based on the measured time to maximum acidification rate (tm) determined in example 2 and plotted against storage time (ts) as shown in figure 3 and figure 4, a linear function was derived from the plotted data points. It has been found that the rate of loss of acidification activity over time (acidification loss) corresponds to the slope of the linear function derived from a series of tmvalues plotted against the time passed on the x axis, in this case storage time passed at 37°C. The mathematical method and reasoning behind this analytical approach is explained in Fonseca et al., Journal of Dairy Research, 2000, 67: 83-90.

[0329] Linear regression of the tmdata points for DSM22589 against storage time (see table 5; figure 3) produces the equation: tm= 90,21 * ts+ 206,4. Thus, in the case of DSM22589, the acidification loss can be determined as 90 (90,21) minutes / month and the initial tm(tm prior to storage) is determined as 206 (206,4) minutes. An acidification loss of 90 minutes for DSM22589 is an indication that the time until the maximum acidification rate is reached (tm) is extended by 90 minutes for each additional month DSM22589 freeze dried samples are stored at 37°C.

[0330] Linear regression of the tmdata points for DSM34235 against storage time (see table 5; figure 3) produces the equation: tm= 55,93 * ts+ 239,5. Thus, in the case of DSM34235, the acidification loss can be determined as 56 (55,93) minutes / month and the initial tm(tm prior to storage) is determined as 240 (239,5) minutes. An acidification loss of 56 (55,93) minutes / month for DSM34235 is an indication that the time until the maximum acidification rate is reached (tm) is extended by 56 minutes for each additional month DSM34235 freeze dried samples are stored at 37°C.

[0331] It is clear from comparison of the acidification loss determined through linear regression, that the phage resistant S. thermophilus strain DSM34235 (56 minutes / month) deals significantly better with dry storage at 37°C than the wild type S. thermophilus strain DSM22589 (90 minutes / month) and that DSM34235 is clearly superior to DSM22589 in applications that would require prolonged periods of dry storage at 37°C, i.e. during long transportation distances, during on site storage or during storage at a remote site prior to use in downstream applications.

[0332] Example 4 - Determining the acidification activity of freeze-dried products

[0333] The acidification activities of DSM 22589 and DSM 34235 were evaluated immediately following freeze-drying (lyophilization) using the same method and conditions described in Example 1 . The results of the analyses are presented below:

[0334] After freeze-drying, the lyophilized products were stored at a subzero temperature of -55°C until the storage test began. Storage at this temperature minimizes the loss of viability over time.

[0335] When the acidification parameters ta and pH (6h) were compared between the post-freeze-drying measurements and the start of the storage test, DSM 34235 showed nearly identical values. This result demonstrates that both the viability and acidification activity of the strain were successfully preserved during storage at -55°C.

[0336] For DSM 22589, a slight improvement in acidification speed was observed at the start of the storage trial compared to immediately after production, as indicated by a lower pH (6h), i.e. pH= 5.09 at the start of storage test versus pH = 5.22 in the freeze-dried product. This variation can be attributed to the inherent characteristics of biological materials and batch-to-batch differences in the milk used for analysis. Importantly, these findings confirm that there was no loss in acidification activity for DSM 22589 during storage at -55°C.

[0337] Including a step to assess viability immediately after freeze-drying ensures that the effects of lyophilization can be distinguished from any changes occurring during subsequent storage. For all examples 1 to 4 the incubation stopped after 16 hours.

[0338] Discussion

[0339] A delay in the timing of when a given sample reaches the maximum acidification rate (tm) in the incubation step is indicative of a reduction in the samples ability to recover from the storage conditions and return to a normal metabolic function post-storage. Such a reduction is a direct indication that the storage period caused harm to the general metabolic fitness of the lactic acid bacteria undergoing storage.

[0340] The metabolic fitness of the lactic acid bacteria post-storage is important, as in many dairy products, the lactic acid bacteria are an important factor in conservation of the product, as the usual rapid decline in pH caused by the lactic acid bacteria, impairs contamination by other undesired species, such as bacteria, phages and / or fungi that cause spoilage. Thus, once the fermentation process is started in dairy practices, a more rapid recovery by the lactic acid bacteria, e.g. an FD DVS inoculum, reduces the time frame in which undesirable microorganisms can infect the resulting dairy product and consequently both reduces the risk of spoilage, and increases the quality of the product.

[0341] As can be generally seen from the data presented in examples 1-3, the phage resistant strain DSM34235 performs markedly better in terms of maintaining acidification activity over prolonged periods of dry storage at 37°C than the wild type strain DSM22589.

[0342] Thus, the selection method disclosed herein allows for systematic selection of phage resistant lactic acid bacteria strains that remain more stable during dry storage at ambient temperatures. The selection method disclosed herein allows for making incremental improvements, i.e. selecting any phage resistant strain that has an improved acidification activity following storage compared to a wild type strain. However, the selection method also allows for a more targeted approach, where only phage resistant strains that maintain particularly desirable acidification activities following dry storage at ambient temperatures.

[0343] Approaching the problem of dry storage stability of lactic acid bacteria systematically as disclosed herein will help reduce reliance on cold storage chains and cold storage rooms, and help improving food security and dairy product quality in regions around the world, where temperatures, either due to the local climate or due to climate change, commonly exceeds 25°C, 30°C and even 35°C, thus causing severe harm to lactic acid bacteria that are stored in dry formats prior to use in e.g. dairy production.

Claims

CLAIMS1 . A method for selecting lactic acid bacteria for dry storage at ambient temperature, the method comprising ai) providing one or more dried phage-resistant lactic acid bacteria, or aii) providing a library of dried phage-resistant lactic acid bacteria, b) storing the dried phage-resistant lactic acid bacteria of ai) or aii) at a test storage temperature for a time period sufficient for a change in one or more parameters representative of the acidification activity can be measured, such as for at least 2 days, such as at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, 3 months or such as at least 6 months thereby providing stored dried phage-resistant lactic acid, c) incubating the stored dried phage-resistant lactic acid bacteria of step b) for at least two hours in a liquid sugar-based or milk-based incubation medium to obtain incubated phage-resistant lactic acid bacteria, d) determining a value for one or more parameters representative of the acidification activity of the incubated phage-resistant lactic acid bacteria c), thereby obtaining a determined value, e) comparing the determined value in step d) to a reference value, and f) selecting any incubated phage-resistant lactic acid bacteria for which the determined value obtained in step d) corresponds to an improved acidification activity when compared to the reference value, thereby providing selected phage resistant lactic acid bacteria.

2. The method according to claim 1 , wherein the one or more parameters representative of the acidification activity of the incubated phage resistant lactic acid bacteria in step d) and the reference value in step e) is selected as one or more of ta, A pH, pH of the incubation medium, tm, and / or acidification loss.

3. The method according to claim 2, wherein the one or more parameters representative of the acidification activity of the incubated phage resistant lactic acid bacteria in step d) and the reference value in step e) is selected as one or more of tm, and / or acidification loss.

4. The method according to any one of the preceding claims, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv49.

5. The method according to any one of the preceding claims, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv1-Rv2 and Rv13- Rv23.

6. The method according to any one of the preceding claims, wherein the reference value (Rv) of e) and f) is selected as one or more from the list consisting of Rv13-Rv23.

7. The method according to any one of the preceding claims, wherein ambient temperature is a temperature in the range of 30-43°C, or a temperature in the range of 35-43°C.

8. The method according to any one of the preceding claims, wherein a test storage temperature, is a temperature in the range of 30-43°C, 35-43°C, or a temperature in the range of 37-43°C.

9. The method according to any one of the preceding claims, wherein the method further comprises producing a fermented lactic acid bacteria product at a remote site, the method comprising-providing one or more selected phage-resistant lactic acid bacteria selected as defined in a method according to any one of claims in a dried format,-keeping the one or more dried selected phage-resistant lactic acid bacteria at ambient temperature during transport to the remote site and / or keeping the one or more dried selected phage-resistant lactic acid bacteria at ambient temperature during storage at the remote site, and-running a fermentation process comprising the one or more dried selected phageresistant lactic acid bacteria after transport and / or storage at ambient temperature at the remote site, thereby procuring a fermented lactic acid product at a remote site.

10. The method according to any one of the preceding claims, wherein the phage-resistant lactic acid bacteria are bacteria belonging to a genus selected from the list consisting of Lactococcus , Streptococcus, Lactobacillus the latter now known as Ligilactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, 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.11 . The method according to any one of the preceding claims, wherein the phage-resistant lactic acid bacteria belong to the Streptococcus genus.

12. The method according to any one of the preceding claims, wherein the phage resistant lactic acid bacteria are of the Streptococcus thermophilus strain deposited under DSM deposit number DSM34235.

13. A composition comprising selected phage-resistant lactic acid bacteria selected according to the method defined in any one of claims 1-12 in a dried format.

14. The composition according to claim 13, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from the list consisting of Rv1-Rv49.

15. The composition according to any one of claims 13-14, wherein the selected phage resistant lactic acid has an improved acidification activity compared to one or more reference values (Rv) selected from Rv21-Rv23.

16. Use of a selected phage resistant lactic acid bacteria selected according to the method as defined in any one of claims 1 -12 or a composition as defined in any one of 13-14, for producing a lactic acid bacteria containing product.

17. The use according to claim 16, wherein the selected phage resistant lactic acid bacteria containing product is a dried starter culture or dried direct vat set (DVS).

18. The use according to any one of claims 16-17, wherein the dried starter culture or dried direct vat set (DVS) has an acidification loss of less than 60 minutes per month, less than 70 minutes per month, or less than 80 minutes per month following storage in a dried format at ambient temperature.

19. The use according to any one of claims 16-18, wherein the dried starter culture or dried direct vat set (DVS) has an acidification loss of less than 60 minutes per month following storage in a dried format at ambient temperature.

20. The use according to any one of claims 16-19, wherein the selected phage resistant lactic acid bacteria product is a dried starter culture or dried direct vat set (DVS) that has improved shelf life during storage at ambient temperatures.21 . The use of a selected phage resistant lactic acid bacteria containing product, dried starter culture and / or dried direct vat set (DVS) according to any one of claims 16-20, for producing a fermented product.

22. The use according to claim 21 , for producing a fermented product at a remote site, following storage of the selected lactic acid bacteria containing product, dried starter culture and / or dried direct vat set (DVS) at ambient temperature during transportation to the remote site and / or following storage at ambient temperature at the remote site.

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