Storage stability of Lacticacea rhamnosus

Abstract

This disclosure or application provides the bacterium Lacticaseibacillus (L.) rhamnosus DSM34194. This bacterium is characterized by its ability to maintain high viability in fermented dairy products even when stored at room temperature, and further by its low post-acidification activity. This disclosure or application further provides compositions or starter cultures containing the bacterium, methods for producing fermented dairy products using the bacterium or starter culture, and fermented dairy products obtained by these methods.

Description

[Technical Field]

[0001] This disclosure or application relates to a bacterium of the species Lacticaseibacillus rhamnosus (L. rhamnosus) that maintains high viability in fermented dairy products even during storage at room temperature. The bacterium of this disclosure or application is further characterized by low post-acidification activity. Loss of viability and post-acidification are effects frequently observed in fermented dairy products, particularly when stored at room temperature, i.e., when the cold chain is interrupted. This disclosure or application provides a bacterium of the species Lacticaseibacillus rhamnosus, a starter culture containing the bacterium, a method for producing a fermented dairy product using the bacterium or starter culture, and a fermented dairy product obtained by these methods. [Background technology]

[0002] Fermented dairy products are nutritious and flavorful dairy foods produced and consumed worldwide. The bacterial cultures commonly used in milk fermentation and dairy food production include lactic acid bacteria such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These bacteria produce lactic acid and other compounds, particularly numerous additional organic acids, which lower the pH, resulting in a delicate tartness and a creamy, rich texture. Consumers appreciate the balanced flavor and the benefits associated with consuming yogurt containing live, active bacterial cultures.

[0003] The number of surviving lactic acid bacteria in fermented dairy products is routinely determined for each product. In many countries, there are also minimum total bacterial count requirements for surviving lactic acid bacteria in fermented dairy products. For example, in China, fermented milk must have at least 1 × 10⁶ bacteria during its shelf life. 6The total number of lactic acid bacteria (CFU / g) must be maintained.

[0004] This can be problematic because Lactobacillus delbrueckii and Streptococcus thermophilus, the most frequently used strains in the production of fermented dairy products, are unstable when stored at room temperature and, to a lesser extent, still unstable even when stored at around 4°C. Furthermore, Lactobacillus delbrueckii and Streptococcus thermophilus may behave differently depending on the milk composition.

[0005] Maintaining the viability of lactic acid bacteria in fermented dairy products is generally achieved by cooling the product. For example, in the production of many dairy products such as yogurt, fermentation of the product is stopped by cooling it to a desired pH or total acidity (TA). Continuous cooling maintains viability but does not completely inhibit the metabolic activity of bacteria during storage.

[0006] In many countries, the distribution and sales conditions for fermented dairy products are not ideal. This is due to the fact that refrigeration temperatures are not maintained throughout the entire shelf life of the product. As a result of the loss of protection from the cold chain, surviving lactic acid bacteria exhibit increased metabolic activity, which leads to further acidification of the fermented dairy product, also known as post-acidification, and loss of the lactic acid bacteria's ability to survive. Fermented dairy products stored at temperatures above 25°C for more than 14 days typically contain a total number of lactic acid bacteria of 1 × 10⁶. 6 The result is less than CFU / g, and often 1 × 10⁻⁶ 6 The CFU / g level becomes significantly lower. Such products no longer meet the viable bacterial count thresholds required in many countries. Furthermore, the quality of the fermented dairy product deteriorates, resulting in an undesirable experience for consumers. These problems of loss of viability and flavor are particularly pronounced in products fermented with Lactobacillus delbruickii subspecies bulgaricus and Streptococcus thermophilus.

[0007] To solve these problems, lactic acid bacteria with strong viability, low or no additional post-acidification activity, and little sensory impact on fermented dairy products are needed. In particular, each bacterium must maintain its viability even when stored under normal temperature conditions, and at the same time, it must not cause undesirable flavors, further pH reduction, or gas production.

[0008] Bacterial species such as Lactobacillus paracasei, Lactobacillus plantarum, and Lactobacillus rhamnosus are known to survive well at normal temperature. However, strains of these species tend to contribute significantly to post-acidification when their metabolic activity increases under normal temperature.

[0009] For example, International Publication No. 2019 / 081577 pamphlet of Chr. Hansen A / S discloses Lactiplantibacillus rhamnosus strain DSM32666 and its mutants. The International Publication No. 2019 / 081577 pamphlet teaches that by using CBS141584 as part of the starter culture, an enhanced creamy flavor can be provided to the fermented product. However, the impact of post-acidification is not mentioned. Similarly, the International Publication No. 2021 / 239574 pamphlet of Chr. Hansen A / S discloses Lactiplantibacillus rhamnosus strain DSM33515 and its mutants showing low post-acidification activity.

[0010] It is also desirable to provide a starter culture that addresses the issues of viability and post-acidification in fermented dairy products, preferably those prepared from a milk base containing sugars such as sucrose or fructose. SUMMARY OF THE INVENTION

[0011] The above problem is thus resolved by the bacteria of the species Lacticaseibacillus rhamnosus and its variants, deposited as DSM34194.

[0012] This disclosure or application provides a solution to the above-mentioned problems by offering a novel, improved, and robust strain of Lacticaseibacillus rhamnosus that maintains high viability even at room temperature and exhibits very low post-acidification activity.

[0013] This disclosure or application also relates to a bacterium of the species Lacticaseibacillus rhamnosus, the bacterium being a mutant strain of the bacterium deposited as DSM34194, the mutant strain maintaining viability during storage at 25°C, and tests relating to maintaining viability have been conducted to ensure at least 1 × 10⁶ before storage. 7 The process involves preserving a fermented dairy product containing a mutant strain of CFU / g, wherein the fermented dairy product is stored at 25°C for 21 days and then has at least 1 × 10⁻¹⁴ units of the mutant strain. 6 This provides bacteria, including mutant strains with CFU / g.

[0014] This disclosure or application also provides compositions comprising bacteria of the species Lacticaseibacillus rhamnosus as described above. In one embodiment, the composition comprises bacteria of the species Lacticaseibacillus rhamnosus as described above, at least 1 × 10⁻⁶. 9 It contains at a concentration of CFU / g. In a preferred embodiment, the composition contains at least 1 × 10⁶ bacteria of the species Lacticaseibacillus rhamnosus. 10 The composition contains bacteria of the species Lacticaseibacillus rhamnosus at a concentration of CFU / g. In a more preferred embodiment, the composition contains at least 1 × 10⁶ bacteria of the species Lacticaseibacillus rhamnosus. 11 Contains at a concentration of CFU / g.

[0015] In further embodiments, the above composition includes a starter culture. In another embodiment, the starter culture includes bacteria of Lactobacillus delbruickii subspecies bulgaricus and / or Streptococcus thermophilus.

[0016] The compositions of the present disclosure or the present application may additionally contain cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorings, flavors, or mixtures thereof. The composition may be in a frozen or lyophilized form.

[0017] In related embodiments, the present disclosure or the present application also provides a method for producing a fermented dairy product, comprising adding bacteria of the Lactobacillus rhamnosus species as described above or a composition as described above to a milk base, and fermenting the milk base at a temperature between 22°C and about 43°C until a pH of 4.5 or less or a total acidity (TA) of 70 or less is achieved, or until a pH of 4.7 or less, for example 4.55 or less, is achieved.

[0018] The present disclosure or the present application also provides a fermented dairy product containing bacteria of the Lactobacillus rhamnosus species as described above. In one embodiment, the dairy product contains bacteria of the Lactobacillus rhamnosus species as described above at a concentration of at least 1×10 6 CFU / g. The dairy product can be obtained by the method as described above.

[0019] In another embodiment, the fermented dairy product can maintain a pH above 3.5 when stored at 25°C for at least 21 days. In a preferred embodiment, the fermented dairy product maintains a viable lactic acid bacteria count of at least 1×10 6 CFU / g when stored at 25°C for at least 21 days.

[0020] The present disclosure or the present application also provides a fermented dairy product as described above, which is Yakult, cheese, yogurt, fruit yogurt, yogurt drink, strained yogurt (Greek yogurt, Labneh, Skyr), quark, Fromage frais, sour cream, buttermilk, white blind cheese, UF feta and cottage cheese, or cream cheese.

[0021] This disclosure or application also provides fermented dairy products as described above, prepared from a milk base containing sucrose or fructose, such as syrup. [Modes for carrying out the invention]

[0022] The inventors have found that, surprisingly, the Lacticaseibacillus rhamnosus strain deposited as DSM34194 and / or its mutants possess both high viability and low post-acidification activity, even when stored for extended periods under room temperature conditions, making them advantageous for use in combination with common starter cultures for producing fermented dairy products. In one embodiment, the Lacticaseibacillus rhamnosus strain and / or its mutants deposited as DSM34194 can be characterized by maintaining viability during storage at 25°C, and tests relating to maintaining viability have shown that at least 1 × 10⁶ cells were present before storage. 7 The process involves preserving a fermented dairy product containing deposited bacteria and / or mutant strains at a concentration of CFU / g, wherein the fermented dairy product is stored at 25°C for 21 days and then has a concentration of at least 1 × 10⁶. 6 The deposited bacteria and / or mutants are contained in a CFU / g quantity. In a preferred embodiment, the bacteria and / or mutants of the species Lacticaseibacillus rhamnosus deposited as DSM34194 can be characterized as maintaining viability during storage at 25°C, and the test for maintaining viability is performed at least 1 × 10 before storage. 8 CFU / g, more preferably at least 1 × 10⁻¹⁶ 9 The process involves preserving a fermented dairy product containing deposited bacteria and / or their mutant strains in a quantity of CFU / g, wherein the fermented dairy product is stored at 25°C for 21 days and then has a capacity of at least 1 × 10⁶. 6 Contains deposited bacteria or mutant strains in CFU / g. In a particularly preferred embodiment, the storage stability test used to determine the viability after storage according to all embodiments of this disclosure or this application is carried out using stirred yogurt as the fermented dairy product.

[0023] DSM34194 was identified through extensive screening of 16,000 mutant strains of Lactobacillus rhamnosus. DSM34194 was then selected by high-throughput screening from a pool of mutants generated using ethyl methanesulfonate (EMS) mutagenesis, based on its low acidification. The high-throughput screening included picking hit mutants from agar, growing the mutants in broth, acidifying the milk at 40°C, acidifying one replica at room temperature for 12 days, acidifying the other replica at 17°C for 2 weeks, and selecting mutants with low post-acidification at 17°C.

[0024] In the context of this disclosure or this application, the terms “lactic acid bacteria” or “LAB” refer to food-grade bacteria that produce lactic acid as the primary metabolic end product of carbohydrate fermentation. These bacteria are associated by their common metabolic and physiological characteristics and are typically Gram-positive, low-GC, acid-tolerant, non-spore-forming, non-respiratory, rod-shaped, or cocci. During the fermentation process, the consumption of lactose by these bacteria leads to lactic acid formation, which lowers the pH and results in the formation of protein coagulations. These bacteria thus contribute to the acidification of milk and the texture of dairy products. As used herein, the term "lactic acid bacteria" refers to bacteria belonging to the genera Lactobacillus, Bifidobacterium, Streptococcus, Lacticaseibacillus, and Lactococcus, such as Lactobacillus delbruickii subspecies bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, and Bifidobacterium breve. This includes, but is not limited to, species of the genus Leuconostoc (breve) and other fungal species.

[0025] Lacticaseibacillus rhamnosus is a lactic acid bacterium that was formerly known as Lactobacillus rhamnosus and has been renamed (Zheng et al., Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107).

[0026] In the context of this disclosure or application, the term “mutant” refers to strains of the species Lacticaseibacillus rhamnosus that can be obtained from the deposited strain as DSM34194, for example, by genetic engineering, radiation, and / or chemical treatment. The mutant strains are preferably functionally equivalent mutants, for example, having substantially the same or improved characteristics as the deposited strain, particularly with respect to the effect of maintaining viability and / or inhibiting post-acidification. Each mutant strain represents an embodiment of this disclosure or application. The term “mutant” specifically refers to strains obtained by treating the strains of this disclosure or application with chemical mutagens such as ethanemethanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), any conventionally used mutagenic treatment including UV light, or naturally occurring mutants. The mutant strain may be subjected to several mutagenesis processes (a single process should be understood as one mutagenesis step followed by a screening / selection step), but currently it is preferable that no more than 20 processes, no more than 10 processes, or no more than 5 processes (or screening / selection steps) are performed. In the preferred mutant strain, less than 5%, less than 1%, or even less than 0.1% of the nucleotides in the bacterial genome are substituted or deleted by other nucleotides compared to the deposited strain.

[0027] LAB is generally added to milk in the form of a starter culture. As used herein, the terms “starter” or “starter culture” refer to a culture of one or more food-grade microorganisms, particularly lactic acid bacteria, that are involved in the acidification of the milk base. The “starter culture” may be Streptococcus thermophilus and / or Lactobacillus delbruickii subspecies bulgaricus. The starter culture may be fresh, but most often it is frozen or freeze-dried. These products are also known as “direct vat set” (DVS) cultures and are prepared for direct inoculation into fermentation vessels or fermentation vats for the production of fermented dairy products or dairy products such as cheese. Each starter culture is commercially available from many sources, including Premium 1.0, F-DBA YoFlex Mild 2.0, F-DVS YF-L901, YF-907, FD-DVS CH-1, and four cultures containing a mixture of Streptococcus thermophilus and Lactobacillus delbruckii subspecies bulgaricus, commercially available from Christian Hansen.

[0028] In the context of this disclosure or application, the term “milk” is used broadly in its general sense to refer to the liquid produced by the mammary glands of animals or by plants. According to this disclosure or application, milk may be processed, and the term “milk” includes whole milk, skim milk, non-fat milk, low-fat milk, whole-fat milk, lactose-reduced milk, or concentrated milk. Non-fat milk is a non-fat dairy product or skim milk product. Low-fat milk is typically defined as milk containing about 1% to about 2% fat. Whole-fat milk often contains 2% or more fat. The term “milk” is intended to include milk from different mammalian and plant sources. Mammalian sources of milk include, but are not limited to, cattle, sheep, goats, buffalo, camels, llamas, horses, and deer. Examples of plant sources of milk include, but are not limited to, milk extracted from soybeans, peas, peanuts, barley, rice, oats, quinoa, almonds, cashews, coconuts, hazelnuts, hemp, sesame seeds, and sunflower seeds. In the methods and products of this disclosure or application, bovine milk is most preferably used as a starting material for fermentation.

[0029] The term "milk" also includes dairy products that have been fat-reduced and / or lactose-reduced. Each of these products can be prepared using methods well known in the art and are commercially available. Lactose-reduced milk can be produced by any method known in the art, which includes hydrolyzing lactose to glucose and galactose with the enzyme lactase, or by nanofiltration, electrodialysis, ion exchange chromatography, and centrifugation.

[0030] The terms “dairy products” or “milk base” are used broadly in this disclosure or application to refer to compositions based on milk or milk components that can be used as a culture medium for growing and fermenting LABs. Dairy products or bases include milk-derived components and any other components that can be used for the purpose of growing or fermenting LABs.

[0031] Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art. As used herein, “homogenization” means vigorous mixing to obtain a soluble suspension or emulsion. When performed prior to fermentation, homogenization may be carried out to break down the milk fat into smaller pieces so that it does not separate from the milk. This can be achieved by pumping the milk under high pressure through a small orifice. As used herein, “pasteurization” means treatment of the milk substrate to reduce or remove the presence of viable organisms such as microorganisms. Preferably, pasteurization is achieved by maintaining a specific temperature for a specific period of time. The specific temperature is usually achieved by heating. The temperature and period may be selected to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0032] The terms “fermented dairy products,” “food products,” or “feed products” refer to products obtainable by the fermentation methods of this disclosure or application, and include Yakult, cheese, yogurt, fruit yogurt, yogurt drinks, strained yogurt (Greek yogurt, labneh, skyr), quark, fromage frais, sour cream, buttermilk, white blind cheese, UF feta and cottage cheese, and cream cheese. The term “food products” further includes other fermented foods, including fermented meat products such as fermented sausages and fermented fish products.

[0033] The term “cheese” is understood to encompass any cheese, including hard cheeses, semi-hard cheeses, and soft cheeses, for example, the following types of cheese: cottage cheese, feta, cheddar, parmesan, mozzarella, Emmental, Dumbo, Gouda, Edam, feta-type cheeses, blue cheese, brine cheese, Camembert, and Brie. Methods for converting coagulated substances into cheese are known to those skilled in the art and can be found in the literature, for example, see Kosikowski, FV, and VV Mistry, “Cheese and Fermented Milk Foods”, 1997, 3rd Ed. FV Kosikowski, LLC Westport, CT. As used herein, cheeses with an NaCl concentration of less than 1.7% (w / w) are referred to as “low-salt cheeses.”

[0034] In the context of this disclosure or this application, the term “yogurt” means products containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus, and Lactobacillus paracasei, or any microorganisms derived therefrom. Lactic acid bacteria strains other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are included to impart various properties to the final product, such as properties that promote flora balance. As used herein, the term "yogurt" includes set yogurt, stirred yogurt, drinkable yogurt, Petit Suisse, heat-treated yogurt, strained or Greek-style yogurt characterized by high protein levels, and yogurt-like products.

[0035] In particular, the term "yogurt" includes, but is not limited to, yogurt as defined in accordance with French and European regulations, for example, coagulated dairy products obtained by lactic acid fermentation using only certain thermophilic lactic acid bacteria (i.e., Lactobacillus delbruickii subsp. bulgaricus and Streptococcus thermophilus) that are cultured together and found to be viable in the final product at a concentration of at least 10 million CFU (colony-forming units) / g. Yogurt may optionally contain added dairy ingredients (e.g., cream) or other components, such as sugar or sweeteners, one or more flavorings, fruits, grains, or nutrients, especially vitamins, minerals, and fiber, as well as stabilizers and thickeners. Optionally, yogurt may meet the specifications for fermented milk and yogurt of AFNOR NF 04-600 and / or Codex Stan A-IIa-1975. To meet the AFNOR NF 04-600 standard, the product must not be heated after fermentation, and dairy ingredients must constitute at least 70% (m / m) of the final product.

[0036] In the context of this disclosure or application, “ambient temperature” is defined herein as an air temperature range of 15 to 37°C, preferably 15 to 30°C, more preferably 18 to 25°C, or 25°C, all of which are under 1 atmosphere.

[0037] In the context describing this disclosure or this application (particularly in the context of the claims), the use of "a," "an," and "the" and similar reference subjects should be interpreted as encompassing both singular and plural, unless otherwise indicated herein or unless it is clearly inconsistent with the context.

[0038] In another embodiment, the present disclosure or application provides a bacterium of the species Lacticaseibacillus rhamnosus deposited as DSM34194 or a mutant thereof, wherein the bacterium maintains a higher total lactic acid bacteria count in fermented dairy products containing the deposited bacterium or mutant thereof during post-fermentation storage compared with dairy products containing Lacticaseibacillus rhamnosus deposited as DSM33515, the higher total lactic acid bacteria count being determined after storing the product fermented with the starter culture and Lacticaseibacillus rhamnosus strain at 25°C for 21 days.

[0039] In yet another embodiment, the present disclosure or application provides a bacterium of the species Lacticaseibacillus rhamnosus deposited as DSM34194 or a mutant thereof, the bacterium reducing post-acidification of fermented dairy products containing the deposited bacterium or mutant thereof during post-fermentation storage compared to dairy products containing Lacticaseibacillus rhamnosus deposited as DSM33515, the reduction in post-acidification being determined after storing the product fermented with the starter culture and Lacticaseibacillus rhamnosus strain at 25°C for 21 days.

[0040] In a related aspect, the bacteria of the species Lacticaseibacillus rhamnosus or its variants deposited as DSM34194 exhibit the above functional characteristics, which include: (a) Maintaining viability during storage at 25°C, and testing relating to maintaining viability shall be performed at least 1 × 10 before storage. 7 The process involves storing fermented dairy products containing bacteria CFU / g, and the fermented dairy products must contain at least 1 × 10¹⁶ bacteria after storage at 25°C for 21 days. 6 Contains bacteria CFU / g; (b) To maintain a higher total lactic acid bacteria count in fermented dairy products containing deposited bacteria or their variants during post-fermentation storage compared to dairy products containing Lacticaseibacillus rhamnosus deposited as DSM33515, the higher total lactic acid bacteria count being determined after storing the product fermented with the starter culture and Lacticaseibacillus rhamnosus strain at 25°C for 21 days; and / or (c) Compared to dairy products containing Lacticaseibacillus rhamnosus deposited as DSM33515, the objective is to reduce post-acidification of fermented dairy products containing the deposited bacteria or its mutant strains during post-fermentation storage, the reduction in post-acidification being determined after storing the product fermented with the starter culture and Lacticaseibacillus rhamnosus strain at 25°C for 21 days.

[0041] In further embodiments, the present disclosure or application provides a composition comprising a bacterium of the species Lacticaseibacillus rhamnosus or a mutant thereof as described above. The composition may also contain other lactic acid bacteria, organic compounds, and / or inorganic compounds. In one alternative example, the composition comprises the bacterium and / or mutant thereof in at least 10 9 At a concentration of CFU / g, or at least 10 10 At a concentration of CFU / g, or at least 10 11 Contains at a concentration of CFU / g.

[0042] Each composition can take the form of a starter culture. As described above, a starter culture contains lactic acid bacteria and is commonly used to inoculate milk for the production of fermented dairy products. A starter culture can contain bacteria of the Lactobacillus and / or Streptococcus genera, such as Lactobacillus delbruickii subspecies bulgaricus and / or Streptococcus thermophilus.

[0043] The compositions of this disclosure or application may further comprise cryoprotectants, freeze-drying protectants, antioxidants, nutrients, fillers, flavorings, flavors, and / or mixtures thereof. Each compound is often added to starter culture compositions to stabilize lactic acid bacteria during storage. The compositions of this disclosure or application may be maintained in a frozen or freeze-dried state. Methods for producing freeze- or freeze-dried compositions containing viable lactic acid bacteria are well known in the art.

[0044] In particular, the use of protective agents such as cryoprotectants and freeze-drying protective agents to improve the viability of lactic acid bacteria is well known to those skilled in the art. Suitable cryoprotectants or freeze-drying protective agents include monosaccharides, disaccharides, trisaccharides, and polysaccharides (glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch, and acacia gum, etc.), polyols (erythritol, glycerol, inositol, mannitol, sorbitol, treitol, xylitol, etc.), amino acids (proline, glutamic acid, etc.), and complex substances (skim milk, peptone, gelatin, yeast extract, etc.). Examples include inorganic compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and its salts, gallate, cysteine, sorbitol, mannitol, and maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, and vitamins (such as B vitamins and vitamin C). The above composition may optionally contain further substances including fillers (such as lactose and maltodextrin) and / or flavorings.

[0045] In one embodiment, the present disclosure or application therefore provides for at least 10 per gram of frozen material. 9 In terms of colony-forming unit (CFU) concentration, or at least 10 per gram of frozen material. 10 In terms of CFU concentration, or at least 10 per gram of frozen material. 11 The present invention provides a composition in the form of a solid, freeze-dried or freeze-dried starter culture containing lactic acid bacteria at a CFU concentration.

[0046] In further embodiments, the present disclosure or application provides a method for producing a fermented dairy product, comprising adding a bacterium of the species Lacticaseibacillus rhamnosus or a mutant thereof, or a composition thereof, to a milk base, and fermenting the milk base at a temperature between 22°C and about 43°C until a pH of 4.5 or less is achieved, or until a pH of 4.7 or less, for example, 4.55 or less is achieved, or alternatively, until a total acidity (TA) of 70 or less is achieved. Methods for fermenting dairy products are well known in the art, and those skilled in the art will be able to carry out each of these methods using the bacteria and compositions of the present application.

[0047] In one preferred embodiment, the present disclosure or application provides a method for producing a fermented dairy product, comprising adding a bacterium of the species Lacticaseibacillus rhamnosus or a mutant thereof to a milk base containing a sugar such as sucrose, fructose, or glucose, and fermenting the milk base at a temperature between 22°C and about 43°C until a pH of 4.5 or less is achieved, or until a pH of 4.7 or less, for example, 4.55 or less is achieved.

[0048] This disclosure or application also provides fermented dairy products containing the above-described bacteria of the species Lacticaseibacillus rhamnosus or a mutant thereof. The fermented dairy products preferably contain at least 1 × 10⁻¹⁶ of the above-described bacteria or mutant thereof. 6 It is contained at a concentration of CFU / g. Fermented dairy products can be obtained by the method described above.

[0049] In a particularly preferred embodiment, the fermented dairy product does not exhibit any change in pH or a significant decrease in the number of bacteria of the present disclosure or application during storage, even when stored at room temperature. The fermented dairy product may be characterized in that, for example, when stored at 25°C for at least 21 days, the product maintains a pH greater than 3.5. Alternatively or additionally, the fermented dairy product, when stored at 25°C for at least 21 days, exhibits at least 1 × 10⁻¹⁶ 6 It can be characterized in that it maintains a CFU / g number of viable lactic acid bacteria.

[0050] This disclosure or application includes any fermented dairy product containing the bacteria of this disclosure or application. Preferably, the fermented dairy product is selected from Yakult, cheese, yogurt, fruit yogurt, yogurt beverage, strained yogurt (Greek yogurt, labneh, skyr), quark, fromage frais, sour cream, buttermilk, white blind cheese, UF feta and cottage cheese, and / or cream cheese. [Examples]

[0051] Example 1: Use of DSM34194 in stirred yogurt In this example, fresh, stirred yogurt containing high-fructose corn syrup HFCS55 as an added sugar was used to demonstrate the effect of DSM34194. This type of recipe is widely used in low-priced yogurts. In the reference sample, a common yogurt starter culture (Streptococcus thermophilus, Lactobacillus bulgaricus) was used as the starter culture for yogurt fermentation. In the test sample, Lacticaseibacillus rhamnosus strain DSM34194 was added to the yogurt starter culture to produce yogurt. The effect of DSM34194 was evaluated by measuring post-acidification and bacterial count.

[0052] sample: Reference sample: YF-L907 (Streptococcus thermophilus, Lactobacillus bulgaricus), Christian Hansen Test sample: YF-L907+DSM34194

[0053] The milk base was prepared according to Table 1 and pasteurized at 95°C for 300 seconds. [Table 1]

[0054] As a reference sample, YF-L907 was inoculated into a milk base at 100 u / T (units / ton) and fermented at 42°C until the target pH of 4.50 was reached. As test samples, YF-L907 and DSM34194 were inoculated at 100 u / T and 2.0 × 10⁻⁶, respectively. 6 The yogurt was inoculated into a milk base at CFU / g and fermented at 42°C until the target pH of 4.50 was reached, after which the curd was broken up. The resulting yogurt was cooled to 25°C and uniformly textured under a pressure of 2 bar. The yogurt was aseptically filled into individual cups and stored overnight at 4-6°C.

[0055] To simulate storage in a broken cold chain, matured yogurt was stored at 25°C for the entire storage period. pH, total acidity (TA), and total lactic acid bacteria count were monitored every 7 days. The total lactic acid bacteria count was determined by anaerobic incubation at 37°C for 3 days using Difco MRS agar and the pour plate method. The number of Lactobacillus rhamnosus bacteria was determined by anaerobic incubation at 37°C for 3 days using Difco MRS + vancomycin (50 mg / L) agar and the pour plate method. MRS + vancomycin is a selective agar that allows the growth of Lactobacillus rhamnosus but not Streptococcus thermophilus and Lactobacillus bulgaricus. pH was measured at room temperature using a Mettler-Toledo pH meter (mode: SG2), and the pH meter was calibrated using the two-point method (pH 4.01, pH 7.00) before use. Total acidity (TA) was measured at room temperature according to the GB5413.34 method (National food safety standard Determination of acidity in milk and milk products, China).

[0056] result [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0057] At 25°C, both the reference sample and the test sample showed a decrease in pH and an increase in TA. The test sample maintained similar values ​​for both pH and TA as the reference sample, indicating that DSM34194 caused little to no acidification. Therefore, the test sample showed similar post-acidification as the reference sample.

[0058] At 25°C, the total number of lactic acid bacteria in the reference sample was 1.0 × 10⁶ after 14 days. 6 The CFU / g level was below 10, and it decreased sharply by day 21. The measured bacterial count was 0.00 × 10⁶ on day 21. 0 The value was CFU / g (single-step dilution was used for measurement; the actual value was 0.00 × 10⁻⁶). 0 It may be slightly higher than CFU / g, but it is still 1.0 × 10⁻⁶ 6 (Much lower than CFU / g). This meant that under harsh storage conditions, the yogurt starter culture, Streptococcus thermophilus and Lactobacillus bulgaricus, rapidly decreased. Meanwhile, in the test samples, the total lactic acid bacteria count was 1.0 × 10⁶ over 21 days. 6 The CFU / g level remained above the limit, fully meeting the requirements of the Chinese national standard. Bacterial counts in the reference and test samples were confirmed using MRS + vancomycin agar. No bacteria were found in the reference sample, meaning that all Streptococcus thermophilus and Lactobacillus bulgaricus were inhibited by vancomycin; however, in the test sample, the bacterial count was 1.0 × 10⁶ over 21 days. 6 The CFU / g level was above average, and the bacterial count was close to the value in the reference sample.

[0059] This demonstrates that DSM34194 survives well at room temperature and contributes to the total lactic acid bacteria count with little to no effect on post-acidification. In addition, DSM34194 can be advantageously used in formulations of syrup-containing fermented dairy products.

[0060] Example 2 Use of DSM34194 in White Yakult In this example, a white Yakult-like product was used to demonstrate the effects of DSM34194. White Yakult is a two-step process beverage, where a fermented milk base is produced in the first step and diluted with pasteurized syrup in the second step. A standard yogurt culture (Streptococcus thermophilus, Lactobacillus delbruickii subsp. bulgaricus) was used as a reference sample and a starter culture for fermentation. For the test sample, Lacticaseibacillus rhamnosus strain DSM34194 was added to the standard yogurt culture. The effects of DSM34194 were evaluated by measuring post-acidification and bacterial count.

[0061] sample: Reference sample: Premium 1.0, Christian Hansen Test sample: Premium1.0+DSM34194

[0062] The milk base was prepared according to Table 7 and pasteurized at 95°C for 300 seconds. [Table 7]

[0063] As a reference sample, Premium 1.0 was inoculated into a milk base at 100 u / T (units / ton) and fermented at 42°C until the target pH of 4.30 was reached. As test samples, Premium 1.0 and DSM34194 were inoculated at 100 u / T and 5.0 × 10⁻⁶, respectively. 6The milk base was inoculated at CFU / g and fermented at 42°C until the target pH of 4.30 was reached, after which the curd was broken up. The resulting yogurt base was cooled to below 15°C. Syrup water was prepared according to the formulation in Table 8 and pasteurized at 95°C for 300 seconds. The pasteurized syrup water was cooled to below 15°C. The fermented milk base was mixed with syrup water according to the formulation in Table 9 and then homogenized at below 15°C at 50 bar, and then at 150 bar. The finished beverage was then filled into individual bottles and stored overnight at 4-6°C. [Table 8] [Table 9]

[0064] To simulate storage conditions during a cold chain disruption, a white Yakult-like beverage was stored at 25°C. pH, total acidity (TA), and total lactic acid bacteria count were monitored every 7 days.

[0065] The total lactic acid bacteria count was determined by anaerobic incubation at 37°C for 3 days using Difco MRS agar and the pour plate method. The Lacticaseibacillus rhamnosus count was determined by anaerobic incubation at 37°C for 3 days using Difco MRS + vancomycin (50 mg / L) agar and the pour plate method. pH was measured at room temperature using a Mettler-Toledo pH meter (mode: SG2). The pH meter was calibrated using the two-point method (pH 4.01, pH 7.00) before use. Total acidity (TA) was measured at room temperature according to GB5413.34 (China National Food Safety Standard, Determination of Acidity in Milk and Dairy Products).

[0066] result [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0067] At 25°C, both the reference sample and the test sample showed a decrease in pH and an increase in TA. The test sample maintained similar values ​​for both pH and TA as the reference sample, indicating that DSM34194 caused little to no acidification. Therefore, the test sample showed similar post-acidification as the reference sample.

[0068] At 25°C, the total number of lactic acid bacteria in the reference sample decreased sharply after 14 days, exceeding, for example, the 1.0 × 10⁶ required by Chinese national standards. 6 The CFU / g requirement could not be met. The measured bacterial count was 0.00 × 10⁶ on days 14 and 21. 0 The value was CFU / g (single-step dilution was used for measurement; the actual value was 0.00 × 10⁻⁶). 0 It may be slightly higher than CFU / g, but it is still 1.0 × 10⁻⁶ 6 (Much lower than CFU / g). On the other hand, in the test samples to which DSM34194 was added, the total number of lactic acid bacteria was 1.0 × 10 over 21 days. 6 The bacterial count remained above CFU / g, fully meeting the requirements of the Chinese national standard. The bacterial count in the reference sample was confirmed using MRS + vancomycin agar. No bacteria were found in the reference sample, meaning that all Streptococcus thermophilus and Lactobacillus delbruickii subspecies bulgaricus were inhibited by vancomycin. In the test sample, the bacterial count was 1.0 × 10⁶ for 21 days in both MRS agar and MRS + vancomycin agar. 6 The CFU / g value was above this, meaning that after 14 days of storage, DSM34194 was still surviving in the beverage.

[0069] This demonstrates that DSM34194 survives well at room temperature and contributes to the total lactic acid bacteria count with little effect on post-acidification.

[0070] Example 3 Comparison of DSM34194 and Lactobacillus rhamnosus strain LGG (registered trademark) deposited as ATCC53103. In this example, a comparison was made between different Lactobacillus rhamnosus strains, particularly Lactobacillus rhamnosus strain LGG® (registered trademark, see U.S. Patent No. 10072310) deposited as ATCC53103, and Lactobacillus rhamnosus DSM34194, regarding post-acidification and bacterial count stability during long-term storage at 25°C. This comparison was conducted using lactose-deficient cultures (e.g., YoFlex Acidifix 1.0) and lactose-positive cultures (e.g., YoFlex Premium 1.0), in yogurt prepared with 8% sucrose and stored at 25°C.

[0071] sample: Reference sample: YoFlex Acidifix 1.0 (lactose-deficient culture containing lactose-deficient Streptococcus thermophilus strain and lactose-deficient Lactobacillus delbruckii subsp. bulgaricus strain), Christian Hansen. Test samples: YoFlex Acidifix 1.0 + LGG (registered trademark) (Lactobacillus rhamnosus strain LGG (registered trademark), deposited as ATCC53103), both commercially available from Christian Hansen. Test sample: YoFlex Acidifix 1.0 + DSM34194 Reference sample: YoFlex Premium 1.0 (lactose-positive culture containing lactose-deficient Streptococcus thermophilus strain and lactose-positive Lactobacillus delbruckii subsp. bulgaricus strain), Christian Hansen.

[0072] Test sample: YoFlex Premium 1.0 + LGG (registered trademark) (Lactobacillus rhamnosus strain LGG (registered trademark), deposited as ATCC53103), both from Christian Hansen. Test sample: YoFlex Premium 1.0+DSM34194

[0073] The milk base was prepared according to Table 16 and pasteurized at 95°C for 300 seconds. [Table 16]

[0074] Add the reference sample (500U / 2500L YoFlex Acidifix 1.0 or 250U / 2500L YoFlex Premium 1.0) to the milk base in 4 x 10 6 The samples were inoculated with or without LGG® or DSM34194 at a concentration of CFU / g. Yogurt was produced by fermenting milk in 200mL bottles at 43°C until a final pH of 4.55 was reached. After fermentation, the yogurt was stored at 25°C. The pH was measured after 1 day, 14 days, and 28 days.

[0075] Bacterial counts were assessed at 1 day, 14 days, and 28 days after the start of the experiment by smearing yogurt onto MRS with vancomycin and vancomycin-free MRS plates, and then incubating the plates anaerobically at 37°C.

[0076] result [Table 17]

[0077] As shown in Table 17, DSM34194, in both yogurts produced using the starter culture Acidifix 1.0 and yogurts produced using Premium 1.0, exhibits a significantly higher pH and provides a more stable pH when stored at 25°C compared to yogurt produced using LGG® or yogurt produced without a culture with a stable bacterial count (reference sample). Therefore, DSM34194 has a significantly less impact on post-acidification than LGG®, preferably from day 1 onwards, for example, between days 2 and 28 or 14 and 28.

[0078] In addition, the bacterial count was also determined. [Table 18]

[0079] In yogurt produced using Premium without a culture with a stable bacterial count, the bacterial count was 1 × 10⁶ after 28 days. 6 The number of bacteria had decreased to less than CFU / g. In samples using LGG® or DSM34194, the bacterial count remained at 1 × 10⁶ after 28 days. 6 The CFU / g ratio was above average. [Table 19]

[0080] In reference samples smeared with MRS+vancomycin, no growth was detected, confirming that all Streptococcus thermophilus and Lactobacillus delbruickii subspecies bulgaricus were inhibited by vancomycin. In test samples treated with DSM34194 or LGG®, bacterial counts were 1 × 10⁶ for up to 28 days in both MRS agar and MRS+vancomycin agar. 6 The CFU / g ratio was above average.

[0081] In conclusion, Examples 1 and 2 demonstrate that DSM34194 survives well at room temperature, contributes to the total lactic acid bacteria count, and has little to no effect on post-acidification, regardless of the starter culture used (YF-L907 or Premium 1.0, both manufactured by Christian Hansen) or the fermented dairy product produced (stirred yogurt or white Yakult). Example 3 demonstrates an unexpected technical effect of DSM34194 compared to LGG®. Although both DSM34194 and LGG® are Lactobacillus rhamnosus strains, Example 3 shows that DSM34194 has a different and desirable post-acidification profile than LGG®, and therefore maintains a high bacterial count (≧1×10⁻⁶) in fermented dairy products even during storage at room temperature. 6 Having CFU / g) and simultaneously possessing low post-acidification activity is clearly indicated as favorable for the purposes of this disclosure or the present invention.

[0082] Deposit and Expert Solutions The applicant has made the following deposit to the depositary institution that has acquired the status of an international depositary authority under the Budapest Convention on the International Recognition of the Deposit of Microorganisms in Patent Procedure: Leibniz Institute DSMZ - German Microbial and Cell Culture Collection (Inhoffenstr. 7B, 38124 Braunschweig, Germany). [Table 20]

[0083] The applicant requests that the samples of the deposited microorganisms described below be made available only to experts until the date the patent is granted, in accordance with the available regulations governed by the Industrial Property Office of the Budapest Convention Contracting Parties.

[0084] List of embodiments in claim form 1. A bacterium of the species Lacticaseibacillus rhamnosus, or a variant thereof, deposited as DSM34194. 2. The bacterium is a mutant strain of the bacterium *Lacticaseibacillus rhamnosus* deposited as DSM34194, and the mutant strain maintains viability during storage at 25°C, and tests for maintaining viability were conducted at least 1 × 10⁶ before storage. 7 The process involves preserving a fermented dairy product containing a mutant strain of CFU / g, wherein the fermented dairy product is stored at 25°C for 21 days and then has at least 1 × 10⁻¹⁴ units of the mutant strain. 6 A bacterium of the species Lacticaseibacillus rhamnosus according to Embodiment 1, including a mutant strain of CFU / g. 3. A composition comprising a bacterium of the species Lacticaseibacillus rhamnosus or a mutant thereof as described in Embodiment 1 or Embodiment 2. 4. The bacteria or mutant strain is at least 10 9 A concentration of CFU / g, or at least 10 10 A concentration of CFU / g, or at least 10 11 The composition according to Embodiment 3, having a concentration of CFU / g. 5. The composition according to Embodiment 3 or Embodiment 4, wherein the composition further comprises a starter culture. 6. The composition according to any one of Embodiments 3 to 5, wherein the starter culture contains bacteria of the species Lactobacillus delbrueckii subsp. bulgaricus and / or Streptococcus thermophilus. 7. The composition according to any one of embodiments 3 to 6, wherein the composition further comprises a freeze-protecting agent, a freeze-drying protective agent, an antioxidant and / or nutrients. 8. The composition according to any one of Embodiments 3 to 7, which is frozen or freeze-dried. 9. Adding a bacterium of the species Lacticaseibacillus rhamnosus or a mutant thereof described in any of Embodiments 1 to 4, or a composition described in any of Embodiments 5 to 8, to a milk base. Ferment the milk base at a temperature between 22°C and approximately 43°C until a pH of 4.5 or less or a total acidity (TA) of 70 or less is achieved, or until a pH of 4.7 or less, for example, 4.55 or less, is achieved, thereby obtaining a fermented dairy product. A method for producing fermented dairy products, including 10. The method according to Embodiment 9, wherein the milk base contains 4-12% (w / w) sucrose, preferably 6-10% (w / w) or 8-9% (w / w) sucrose. 11. A bacterium of the species Lacticaseibacillus rhamnosus or a mutant thereof described in any of Embodiments 1 to 4, or a composition described in any of Embodiments 5 to 8, contains at least 1 × 10 6 CFU / g, preferably 1 × 10⁻⁶ 6 CFU / g ~7 x 10 6 CFU / g, more preferably 2×10 6 CFU / g ~6 x 10 6 CFU / g or 4 x 10 6 CFU / g ~ 5 x 10 6 The method according to either Embodiment 9 or 10, wherein the substance is added at a concentration of CFU / g. 12. A fermented dairy product containing the bacteria of the species Lacticaseibacillus rhamnosus described in Embodiment 1 or Embodiment 2, or a mutant thereof. 13. Bacteria or their mutant strains are at least 10 6 Concentration of CFU / g, preferably 10 6 CFU / g~10 9 CFU / g, or 10 6 CFU / g~10 8 CFU / g, or 10 6 CFU / g~10 7 A fermented dairy product according to Embodiment 12, present at a concentration of CFU / g. 14. A fermented dairy product according to Embodiment 12 or Embodiment 13, which can be obtained by any of the methods in Embodiments 9 to 11. 15. A fermented dairy product according to any one of embodiments 12 to 14, which maintains a pH greater than 3.5, preferably greater than 3.8, when stored at 25°C for at least 21 days. 16. If stored at 25°C for at least 21 days, at least 10 6 A fermented dairy product according to any one of embodiments 12 to 15, which maintains a number of viable lactic acid bacteria per CFU / g. 17. A fermented dairy product according to any one of embodiments 12 to 16, which is Yakult, cheese, yogurt, fruit yogurt, yogurt drink, strained yogurt (e.g., Greek yogurt or labneh), quark, fromage frais, or cream cheese.

Claims

1. A bacterium of the species Lacticaseebacillus (L.) rhamnosus, deposited as DSM34194.

2. A composition comprising the bacterium of the species Lacticaseibacillus rhamnosus as described in claim 1.

3. The bacteria at least 10 ９ A concentration of CFU / g, or at least 10 １０ A concentration of CFU / g, or at least 10 １１ The composition according to claim 2, wherein the concentration is CFU / g.

4. The composition according to claim 2 or 3, further comprising a starter culture, wherein the starter culture comprises bacteria of the species Lactobacillus delbrueckii subsp. bulgaricus and / or Streptococcus thermophilus.

5. The composition according to any one of claims 2 to 4, wherein the composition further comprises a freeze-protecting agent, a freeze-drying protective agent, an antioxidant, and / or nutrients.

6. The composition according to any one of claims 2 to 5, which is frozen or freeze-dried.

7. Adding the bacteria of the species Lacticaseibacillus rhamnosus described in claim 1, or the composition described in any one of claims 2 to 6, to a milk base. Ferment the milk base at a temperature between 22°C and approximately 43°C until a pH of 4.5 or less or a total acidity (TA) of 70 or less is achieved, or until a pH of 4.7 or less, for example, 4.55 or less is achieved. A method for producing fermented dairy products, including

8. The method according to claim 7, wherein the milk base contains 4-12% (w / w) sucrose, preferably 6-10% (w / w) or 8-9% (w / w) sucrose.

9. The bacterium of the Lactobacillus rhamnosus species according to claim 1, or the composition according to any one of claims 2 to 6, is at least 1×10 ６ CFU / g, preferably 1×10 ６ CFU / g to 7×10 ６ CFU / g, more preferably 2×10 ６ CFU / g to 6×10 ６ CFU / g or 4×10 ６ CFU / g to 5×10 ６ The method according to any one of claims 7 or 8, which is added at a concentration of CFU / g.

10. A fermented dairy product comprising the bacteria of the species Lacticaseibacillus rhamnosus as described in claim 1.

11. The bacteria at least 10 ６ CFU / g concentration, preferably 10 ６ CFU / g ~ 10 ９ CFU / g, or 10 ６ CFU / g ~ 10 ８ CFU / g, or 10 ６ CFU / g ~ 10 ７ The fermented dairy product according to claim 10, which is present at a concentration of CFU / g.

12. A fermented dairy product according to claim 10 or claim 11, obtainable by any of the methods of claims 7 to 9.

13. A fermented dairy product according to any one of claims 10 to 12, which maintains a pH greater than 3.5, preferably greater than 3.8, when stored at 25°C for at least 21 days.

14. If stored at 25°C for at least 21 days, at least 10 ６ A fermented dairy product according to any one of claims 10 to 13, which maintains a number of viable lactic acid bacteria of CFU / g.

15. A fermented dairy product according to any one of claims 10 to 14, which is Yakult, cheese, yogurt, fruit yogurt, yogurt drink, strained yogurt (e.g., Greek yogurt or labneh), quark, fromage frais, or cream cheese.

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