Lactobacillus for biological control with low post-acidification

Abstract

The present invention is in the field of dairy technology. It relates to a method for producing a fermented milk product, characterized in that the bacterium Lactobacillus rhamnosus DSM 33515 or a mutant strain obtainable therefrom is used. Lactobacillus rhamnosus DSM 33515 has low post-acidification in fermented milk products and can provide antibacterial effects. The present invention also provides Lactobacillus rhamnosus DSM 33515 or a mutant strain obtainable therefrom, as well as related compositions.

Description

[Technical Field]

[0001] Field of the present invention This invention relates to the bacterium Lactobacillus rhamnosus, which can reduce post-acidification in fermented dairy products and provide antibacterial activity. Post-acidification and microbial contamination are effects frequently observed in fermented dairy products stored at temperatures higher than refrigeration temperatures. This invention further provides a starter culture containing the bacterium, a method for producing fermented dairy products using the bacterium or culture thereof, and fermented dairy products obtained thereby, including food, feed, and pharmaceutical products. [Background technology]

[0002] Background of the present invention Lactic acid bacteria (LABs) have been used for decades to extend the shelf life of food products. During fermentation, lactic acid bacteria produce lactic acid and other organic compounds, which lowers the pH of the food product, making it unfavorable for the growth of undesirable microorganisms such as yeast and mold.

[0003] Biocontrol is defined as extending shelf life and improving food safety using natural or controlled antimicrobial compounds. In dairy products, spoilage by mold and yeast cells is a significant problem that negatively impacts shelf life. Over the past decade, considerable effort has been invested in investigating the biocontrollability of LABs, identifying new strains with biocontrollable properties from various food sources, and elucidating the hidden mechanisms of observed physiological activity. Numerous metabolites produced by LABs have been identified as possessing antifungal activity.

[0004] Further research identified competitive exclusion of limited resources by other organisms as the primary mechanism of fungal growth inhibition by lactic acid bacteria. In particular, the loss of the essential trace element manganese is a major bioprotective mechanism for lactic acid bacteria in dairy products. It was also shown that manganese capture is an active mechanism and that energy is required to maintain a high manganese gradient (Siedler et al. "Competitive exclusion is a major bioprotective mechanism of lactobacilli against fungal spoilage in fermented milk products." Applied and environmental microbiology 86.7 (2020)).

[0005] Simultaneously, it was found that the high antifungal activity of the biological control strains usually occurs in conjunction with high activity, and that this leads to post-acidification, i.e., continued acidification after the completion of fermentation. The production of biological control compounds in LABs usually exhibits growth-related dynamics, and therefore is expected to stop when growth slows down (Lv et al. "Modelling the production of nisin by Lactococcus lactis in fed-batch culture." Applied microbiology and biotechnology 68.3 (2005): 322-326).

[0006] Since milk acidification is typically associated with growth (Dandoy et al. "The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS." Microbial cell factories. Vol. 10. No. S1. BioMed Central, 2011), strains that exhibit reduced post-acidification are also expected to have reduced biological control effects.

[0007] European patent EP16182341B1 discloses Lactobacillus rhamnosus strain CBS141584, which has antibacterial properties. However, it did not mention low post-acidification. Therefore, the challenge lies in developing new biological control strains that offer a combination of low post-acidification and high biological control efficacy. [Overview of the project]

[0008] Therefore, the present invention provides bacteria such as the species Lactobacillus rhamnosus deposited as DSM33515 or mutant Lactobacillus rhamnosus available from the deposited bacteria.

[0009] The deposited bacteria or the mutant strains available therefrom are as follows: (a) Increase the pH of a fermented dairy product containing the deposited bacteria or mutant strain during post-fermentation storage compared to a dairy product containing Lactobacillus rhamnosus deposited as DSM32092, wherein the pH increase is at least 0.1; and wherein the pH increase is at least 10% compared to the starter culture. 7 The product fermented using Lactobacillus rhamnosus strains at a concentration of CFU / g was stored at 25°C for 28 days and then measured, and (b) Reduce the growth of mold in fermented dairy products containing deposited bacteria or mutants during post-fermentation storage compared to dairy products that do not contain deposited bacteria or mutants, wherein the reduction in mold growth is at least 10% compared to the starter culture. 7 The product fermented using the aforementioned mutant strain with a CFU / g concentration is stored at 7°C for 28 days and then measured. This is possible.

[0010] In a further embodiment, the present invention provides a bacterium such as Lactobacillus rhamnosus deposited as DSM33515 or a mutant Lactobacillus rhamnosus available from the deposited bacteria, wherein the deposited bacteria or mutant strain increases the pH of the fermented dairy product containing the deposited bacteria or mutant strain during post-fermentation storage compared to a dairy product containing Lactobacillus rhamnosus deposited as DSM32092, wherein the pH increase is at least 0.1, and wherein the pH increase is at least 10% of the starter culture. 7 The product, fermented using Lactobacillus rhamnosus strains at a concentration of CFU / g, is stored at 25°C for 28 days before being measured.

[0011] In a further aspect, the present invention provides a bacterium such as the deposited species Lactobacillus rhamnosus as DSM33515 or a mutant Lactobacillus rhamnosus available from the deposited bacterium, wherein the deposited bacterium and mutant strain reduce mold growth in fermented dairy products containing the deposited bacterium or mutant strain during post-fermentation storage compared to dairy products without the deposited bacterium or mutant strain, wherein the reduction in mold growth is observed between the starter culture and at least 10 7 The CFU / g concentration is measured after the product, fermented using deposited bacteria or mutant strains, is stored at 7°C for 28 days.

[0012] The present invention further provides a composition comprising bacteria such as the species Lactobacillus rhamnosus described above. In one embodiment, the composition further comprises a starter culture. In another embodiment, the composition further comprises at least one cryoprotectant. In a preferred embodiment, the composition is cryogenically or freeze-dried.

[0013] The present invention further provides a method for producing a fermented dairy product, comprising adding bacteria such as the previously described Lactobacillus rhamnosus strain or a composition containing them to milk or a dairy product, and fermenting the mixture at a temperature of about 22°C to about 43°C until a pH of 4.6 or less is achieved.

[0014] The present invention provides a fermented dairy product containing bacteria such as the species Lactobacillus rhamnosus described above. Preferably, the fermented dairy product is obtained by the method described above. In a further embodiment, the fermented dairy product maintains a pH greater than 3.8 when stored at 25°C for at least 28 days. In another embodiment, bacteria such as the species Lactobacillus rhamnosus are present for at least 10 7 It exists at a concentration of CFU / g.

[0015] In addition, the present invention provides a food, feed, or pharmaceutical product containing bacteria such as the previously described species Lactobacillus rhamnosus, or a composition containing them. In a preferred embodiment, the food, feed, or pharmaceutical product is available by the method described above. [Brief explanation of the drawing]

[0016] [Figure 1]Figure 1 shows the pH change over time in a fermented dairy product when stored at 25 ± 1°C for 28 days. The product was fermented using only the starter culture (containing FD-DVS YF-L812, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) (reference; △), or using the starter culture (□) combined with the bacteria deposited as DSM33515, or using the starter culture (○) combined with the bacteria deposited as DSM32092. [Figure 2] Figure 2 shows the pH change over time in a fermented dairy product when stored at 25 ± 1°C for 28 days. The product was fermented using only the starter culture (containing FD-DVS Premium5.0, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) (reference, △), or using the starter culture (□) combined with the bacteria deposited as DSM33515, or using the starter culture (○) combined with the bacteria deposited as DSM32092. [Figure 3] Figure 3 shows the mold growth on plates prepared from milk fermented using only the starter culture (reference, first column), or using the starter culture combined with Lb. rhamnosus bacteria deposited as DSM33515 (second column), or using the starter culture combined with Lb. rhamnosus bacteria deposited as DSM32092 (third column). Target contaminating fungi: (A) P. brevicompactum, (B) P. crustosum, (C) P. solitum, (D) P. carneum, (E) P. paneum and (F) P. roqueforti were added at a concentration of 500 spores / spot. The plates were incubated at 7 ± 1°C for 28 days. [Figure 4]Figure 4 shows the pH change over time in a fermented dairy product when stored at 7 ± 1 °C for 28 days. The product was fermented using only the starter culture (reference, ○), or a starter culture combined with the bacteria deposited as DSM33515 (□), or a starter culture combined with CBS141584 (△). [Figure 5] Figure 5 shows the pH change over time in a fermented dairy product when stored at 25 ± 1 °C for 28 days. The product was fermented using only the starter culture (reference, ○), or a starter culture combined with the bacteria deposited as DSM33515 (□), or a starter culture combined with the Lb. rhamnosus strain CBS141584 (△).

Mode for Carrying Out the Invention

[0017] Detailed Description of the Invention Food cultures using the biocontrol effect to provide a safe additional solution for traditional fermented products, including Lactobacillus rhamnosus culture DSM32092, are available. These biocontrol strains are used in combination with a normal starter culture to co-ferment milk into a fermented product. During fermentation, DSM32092 exhibits a biocontrol effect, thereby providing an extended shelf life of the fermented product against mold and yeast. The fermentation of many dairy products such as yogurt is stopped, and the product is cooled at a specific pH. After fermentation, the bacteria are often active even during storage. This results in the production of additional lactic acid, and the process is known as post-acidification. The resulting low pH of the final product is undesirable as it has a negative sensory impact on the product. The invention described herein involves the development of a novel and improved biocontrol strain showing a combination of reduced post-acidification and high biocontrol effect. A large-scale screening of more than 11,000 mutants of DSM32092 led to the identification of the strain DSM33515. This strain provides low post-acidification and high antifungal activity.

[0018] Accordingly, the Lactobacillus rhamnosus strains of the present invention, i.e., the strains and mutants deposited as DSM33515 that maintain advantageous properties, are characterized in that when stored at 25°C for at least 28 days, a fermented dairy product containing the strain maintains a pH greater than 3.8, wherein the fermented dairy product is obtained by a method comprising adding the Lactobacillus rhamnosus strain or a composition containing them to milk or dairy products as previously described, fermenting the mixture at a temperature of about 22°C to about 43°C until a pH of 4.6 or less is achieved, shaking the fermented product, and then cooling it. It should be understood that the characteristic of the Lactobacillus rhamnosus strains of the present invention being able to maintain a pH greater than 3.8 when stored at 25°C for at least 28 days merely characterizes the assays commonly used to measure their effect. The Lactobacillus rhamnosus strains of the present invention, including food or feed products, and compositions containing them, do not actually need to be stored under these conditions, nor do they require them.

[0019] The Lactobacillus rhamnosus strain of the present invention was produced as follows: A pool of mutant strains was obtained from the parent strain DSM32092 using ethyl methanesulfonate (EMS) mutagenesis. Preliminary experiments were conducted to establish the efficacy and sterilization rate of EMS for this strain. A sterilization rate of ≥95% was targeted. Based on this, 15 μl of EMS was added to 1 ml of overnight culture (OD). 620 Approximately 3-4). Next, incubate the culture at 37°C for 4 hours, then 10 -2 ~10 -6The cultures were diluted to a range of values. Subsequently, the diluted cultures were spread onto MRS plates and tested for cell count. The mutant pool was spread onto MRS-Difco agar using sterile glass beads and incubated anaerobically at 37°C for 2 days. Approximately 11,000 single colonies were picked using a colony-picking robot and then inoculated into 200 μl of MRS-Difco broth in a 96-shallow-well microtiter plate, and incubated anaerobically overnight at 37°C. 20 μl of the volume was used for post-acidification of the milk, and the remaining volume was fortified with glycerol (20%), frozen, and stored. 20 μl of the volume was transferred to a 96-deep-well plate containing 1980 μl of UHT skim milk fortified with 2% sucrose and pH color indicator (1% inoculum) and incubated at 40°C for 40 hours. UHT skim milk was prepared by reconstituting skim milk containing 38% protein, 53% lactose, <1.25% lipids, and 3.9% moisture (Arla Foods amba, Denmark) to a level of 9.5% dry matter, and then pasteurized at 99°C for 30 minutes, followed by cooling to 40°C. Plates containing the acidified milk were scanned from the bottom using the pH colorimetric method described in Poulsen et al. 2019 (Poulsen, VK, Derkx, P., Oregaard, G. (2019): "High-Throughput Screening for Texturing Lactococcus Strains". FEMS Microbiological Letters), where the color (hue) values ​​were converted to pH values. 462 mutant strains exhibiting better growth and a higher final pH in MRS-Difco broth compared to the parent strain were collected in five 96-well microtiter plates and used to acidify milk in or out of the starter culture YF-L812 (Lactobacillus del brueckii subspecies bulgaricus and Streptococcus thermophilus).Inoculated pH-color-indicating milk samples were incubated on a flatbed scanner (HP ScanJet G4010) equipped with a temperature-controlled hood set to 40°C for 20 hours with a starter culture or 40 hours without. 462 fermented milk samples and controls (milk uninoculated with any strain or the parent strain DSM32092) were evaluated for their ability to inhibit yeast strains from Debaryomyces hansenii, a species previously isolated from yogurt as a spoilage strain. 150 μl of fermented milk was transferred to individual wells of a 96-well plate, and approximately 20 cells of the D. hansenii strain were inoculated into each well. Four days after incubation at 17°C, dilution rows were spotted onto selective YGC agar plates and yeast growth was analyzed by optical inspection. Lead strains with a higher final pH compared to the parent strain (at least 0.2 units) (which can inhibit D. hansenii), as well as the parent strain. The strains underwent three rounds of single-colony purification, followed by characterization of their post-acidification properties and yeast inhibitory activity in baby bottles (200 ml scale). Interestingly, the majority of lead strains that did not undergo post-acidification lost their ability to inhibit yeast. Only about 1% of the 462 lead strains retained their physiological activity, i.e., they did not undergo post-acidification while simultaneously possessing physiological activity similar to the parent strain. From these, DSM33515 was selected as the best-functioning strain with very good functional properties.

[0020] The Lactobacillus rhamnosus strain of the present invention has the specific advantage of reducing the risk of post-acidification while maintaining antifungal activity, thereby improving the storage stability of food products made using these bacteria, particularly storage stability at temperatures above refrigeration.

[0021] Compared to the Lactobacillus rhamnosus strain deposited as DSM32092, the pH increase caused by the mutant strain reaches at least 0.1. This increase is measured after storing the fermented product at 25°C for 28 days.

[0022] In the context of this application, the term "lactic acid bacteria" or "LAB" refers to food-grade bacteria that produce lactic acid as the main metabolic end product of sugar fermentation. These bacteria share common metabolic and physiological characteristics and are typically Gram-positive, low GC, acid-tolerant, non-spore-forming, non-respiratory, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of lactose by these bacteria triggers lactic acid formation, lowering the pH and leading to the formation of protein aggregates. Thus, these bacteria are involved in the acidification of milk and the texture of dairy products. As used herein, the term "lactic acid bacteria" refers to bacteria of genera such as Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., and Lactococcus spp., such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, and Lactobacillus paracasei. This includes, but is not limited to, bacteria belonging to the genus Leuconostoc, such as *Lactobacillus paracasei*, *Lactobacillus plantarum*, *Lactobacillus helveticus*, *Lactobacillus acidophilus*, *Bifidobacterium breve*, and species of the genus *Leuconostoc*.

[0023] "Mold" refers to fungi that grow in the form of multicellular filaments called hyphae. With respect to mold, the term "inhibit" means, for example, that the growth, spore formation, spore number or concentration of mold inside and / or on the surface of a food product containing the Lactobacillus fermentum strain of the present invention is reduced compared to a food product that does not contain such bacteria. The degree of inhibition brought about by the Lactobacillus rhamnosus strain of the present invention is preferably determined by growth in agar-solidified fermented milk in the presence and absence of Lactobacillus rhamnosus. Examples of molds include members of the genus Penicillium such as Penicillium solitum, Penicillium brevicompactum, Penicillium crustosum, Penicillium roqueforti, Penicillium paneum and Penicillium carneum.

[0024] Yeast is a fungus that grows as single cells. The Lactobacillus rhamnosus strain of the present invention, i.e., the strain deposited as DSM33515 and mutants that maintain advantageous properties, can inhibit the growth of mold and further inhibit the growth of yeast. In connection with the growth of yeast, the term "inhibit" also means, for example, a decrease in the growth or a decrease in the number or concentration of yeast in and / or on the surface of a food product containing the bacteria of the present invention compared to a food product that does not contain such bacteria. Also in this case, as before, the degree of inhibition provided by the Lactobacillus rhamnosus strain of the present invention is preferably measured by growth on agar-solidified fermented milk in the presence and absence of Lactobacillus rhamnosus.

[0025] An assay for measuring the decrease in the growth of mold or yeast in a fermented milk product is carried out with the starter culture alone, as well as with the starter culture and at least 10 7The present invention is preferably carried out by inoculating milk with Lactobacillus rhamnosus at a concentration of CFU / g, fermenting the milk to a pH of 4.6, mixing the fermented milk with agar, filling the mixture into an agar plate, adding target mold and / or yeast contaminants at a concentration of 500 spores / spot, storing the plate at 7°C for 28 days, and comparing the growth of mold and / or yeast on the plate containing Lactobacillus rhamnosus with that of a plate containing only a commercially available starter culture. The entire procedure for each assay is provided in Example 2.

[0026] In the context of this application, the term “mutant” should be understood as a strain derived from the strain of the present invention produced by means such as genetic engineering, irradiation and / or chemical treatment. A mutant is a mutant that is functionally equivalent to the deposited strain of the present invention, for example, having substantially identical or improved characteristics, particularly with respect to the effect on post-acidification and / or inhibition of biological control. Each mutant represents an embodiment of the present invention. The term “mutant” specifically refers to a strain obtained by subjecting the strain of the present invention to any commonly used mutagenesis treatment, including treatment with chemical mutagens such as ethanemethanesulfonic acid (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG) or UV light, or a naturally occurring mutant. A mutant may be subjected to multiple mutagenesis treatments (each treatment should be understood as one mutagenesis treatment followed by a screening / selection step), but preferably, it is desirable that 20 or fewer, or 10 or fewer, or 5 or fewer treatments (or screening / selection steps) are performed. In preferred mutant strains, less than 5%, less than 1%, or less than 0.1% of nucleotides in the bacterial genome are shifted by or deleted by other nucleotides compared to the parent strain.

[0027] In the context describing the present invention (particularly in the context of the following claims), the terms “a,” “an,” and “the,” and similar references, should be interpreted as encompassing both singular and plural forms unless otherwise specifically indicated herein or unless otherwise clearly contradicted by the context.

[0028] Each composition may contain numerous additional bacteria, including LAB. Preferred compositions of the present invention are characterized by further comprising at least one bacterium selected from one or more species of the genera Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, for example, Lactobacillus delbruckii subspecies bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, Bifidobacterium breve, and Leuconostoc.

[0029] In a particularly preferred embodiment, the composition of the present invention comprises bacteria such as the species Lactobacillus rhamnosus deposited as DSM33515 or mutant Lactobacillus rhamnosus available from the deposited bacteria, and one or more further bacteria. In one embodiment, several different strains of Lactobacillus rhamnosus are combined.

[0030] The composition of the present invention may further contain cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorings, or mixtures thereof. The composition may be in frozen or lyophilized form. Preferably, the composition contains one or more of cryoprotectants, lyoprotectants, antioxidants, and / or nutrients; more preferably, a cryoprotectant, a lyoprotectant, and / or antioxidant; most preferably, a cryoprotectant or a lyoprotectant, or both. The use of protective agents such as cryoprotectants and lyoprotectants is known to those skilled in the art. Suitable cryoprotectants or freeze-drying protectants include monosaccharides, disaccharides, trisaccharides and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and acacia gum), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, treitol, and xylitol), amino acids (such as proline and glutamic acid), complex substances (such as skim milk, peptone, gelatin, and yeast extract), and 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 the B vitamin family and vitamin C). The composition may optionally contain further substances, including bulking agents (such as lactose and maltodextrin) and / or flavorings.

[0031] LAB is most commonly added to milk in the form of a starter culture. In this context, the terms "starter" or "starter culture" refer to a culture of one or more food-grade microorganisms, particularly lactic acid bacteria, involved in the acidification of a milk base. Starter cultures may be fresh, but are most frequently found frozen or freeze-dried. These products are also known as "Direct Vat Set" (DVS) cultures and are produced for direct inoculation into fermentation vessels or vats for the production of fermented dairy products or dairy products such as cheese. Each starter culture is commercially available from numerous manufacturers, including Premium 5.0, YF-L812, F-DBA YoFlex Mild 2.0, F-DVS YF-L901, and FD-DVS CH-1. These four cultures, containing a mixture of Streptococcus thermophilus and Lactobacillus delbruckii subspecies bulgaricus, are commercially available from Chr. Hansen.

[0032] Therefore, in one aspect of the present invention, a bacterium of the Lactobacillus fermentum species having the above-described antifungal activity is provided, with at least 10 per gram of frozen material. 9 Concentration of colony-forming units (CFU), or at least 10 per gram of frozen material. 10 Concentration of CFU / g, or at least 10 per gram of frozen material. 11 The present invention provides compositions in the form of solid-frozen or freeze-dried starter cultures containing lactic acid bacteria at a concentration of CFU / g. These starter cultures further contain bacteria such as Lactobacillus rhamnosus, deposited as DSM33515, or mutant Lactobacillus rhamnosus available from deposited bacteria.

[0033] A strain of Lactobacillus rhamnosus is also described, characterized in that this bacterium increases the pH of fermented dairy products containing the Lactobacillus rhamnosus strain during post-fermentation storage compared to dairy products fermented using the same starter culture containing Lactobacillus rhamnosus deposited as DSM32092, wherein the pH increase is at least 0.1, and wherein the pH increase is at least 10% compared to the starter culture. 7 The product, fermented using Lactobacillus rhamnosus at a concentration of CFU / g, is measured after being stored at 25°C for 28 days.

[0034] In a further embodiment, the present invention provides a method for producing a fermented dairy product, comprising adding the Lactobacillus rhamnosus strain of the present invention or a composition containing them to milk or a dairy product, and fermenting the mixture at a temperature of about 22°C to about 43°C until the pH reaches 4.6 or less.

[0035] In the context of this application, the term “milk” is used broadly in a general sense to refer to the liquid produced by the mammary glands of animals or by plants. According to the present invention, milk may be processed, and the term “milk” includes whole milk, skim milk, non-fat milk, low-fat milk, whole-fat milk, low-lactose milk, or concentrated milk. Non-fat milk is a non-fat 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 encompass milk from various mammalian and plant sources. Mammalian sources of milk include, but are not limited to, cows, sheep, goats, buffalo, camels, llamas, mere, and deer. 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 method and product of the present invention, cow's milk is most preferably used as a starting material for fermentation.

[0036] The term “milk” also includes dairy products with reduced fat and / or reduced lactose. Each of these products can be prepared using methods well known in the art and is commercially available. Reduced lactose milk can be produced according to any method known in the art, including hydrolysis of lactose to glucose or galactose by lactose enzymes, or by nanofiltration, electrodialysis, ion exchange chromatography and centrifugation.

[0037] The terms “dairy products” or “milk-based” are used broadly in this application to refer to milk or milk component-based compositions that can be used as a culture medium for the growth and fermentation of LABs. Dairy products or milk-based compositions include milk-derived components and other components that can be used for the purpose of growing or fermenting LABs.

[0038] Prior to fermentation, the milk substrate may be homogenized or sterilized according to methods known in the art. In this specification, “homogenization” means vigorous mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, it may be done so that the milk fat is dispersed to a size so small that it does not separate from the milk. This can be achieved by extruding the milk under high pressure through a small orifice. In this specification, “sterilization” means treating the milk substrate to reduce or kill living organisms, such as microorganisms. Preferably, sterilization is achieved by maintaining the milk substrate at a specific temperature for a certain period of time. The specific temperature is usually achieved by heating. The temperature and time may be selected to kill or inactivate specific bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0039] The present invention further provides a method in which the fermented product is stored at a temperature above 7°C, preferably between 7°C and 25°C. The product may be stored at any time, but preferably for a period of at least 14 days, and the pH of the fermented dairy product is maintained above pH 4.0 during storage.

[0040] The present invention further provides a method for producing food, feed, or pharmaceutical products, including a method for producing the fermented dairy product described above, and food, feed, or pharmaceutical products obtained by this method.

[0041] Fermentation is carried out to produce food products, feed products, or pharmaceuticals. The terms “fermented dairy products,” “food,” or “feed” products refer to products obtained by the fermentation method of the present invention, and include cheese, yogurt, fruit yogurt, yogurt drinks, strained yogurt (Greek yogurt, labneh), quark, fromage frais, and cream cheese. The term “food” further encompasses other fermented foods, including fermented meat and fermented fish products such as fermented sausages.

[0042] The term “cheese” is understood to encompass all types of cheese, including hard, semi-hard, and soft cheeses such as cottage cheese, feta, cheddar, parmesan, mozzarella, Emmental, Dumbo, Gouda, Edam, feta-type cheeses, blue cheese, fermented cheeses, Camembert, and Brie. Those skilled in the art know how to convert coagulations into cheese, and the methods are found in the respective literature, see, for example, 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.”

[0043] In the context of this application, the term “yogurt” refers to a product containing Streptococcus thermophilus and Lactobacillus delbruckii subspecies bulgaricus, and optionally other microorganisms, such as Lactobacillus delbruckii subspecies lactis, Bifidobacterium animalis subspecies lactis, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus paracasei, or any microorganism derived therefrom. Other lactic acid bacteria strains besides Streptococcus thermophilus and Lactobacillus delbruckii subspecies bulgaricus are also included to impart various properties to the final product, such as properties that promote the balance of the intestinal flora. As used herein, the term “yogurt” includes set yogurt, starched yogurt, drinking yogurt, Petit Suisse, heat-treated yogurt, strained yogurt, or Greek-style yogurt characterized by being a high-protein and yogurt-like product.

[0044] In particular, the term "yogurt" is defined in accordance with French and European regulations as a coagulated dairy product obtained by lactic acid fermentation using only specific thermophilic lactic acid bacteria (i.e., Lactobacillus delbruckii subspecies bulgaricus and Streptococcus thermophilus), in which these bacteria are cultured together and contain at least 10 × 10⁻¹⁰ bacteria in the final product. 6 This includes, but is not limited to, yogurt in which organisms are viable in terms of CFU (colony-forming units) / g. Yogurt may, as it may, contain dairy ingredients (e.g., cream), or other ingredients such as sugar or sweeteners, one or more flavorings, fruits, grains, or nutrients, especially vitamins, minerals and fiber, as well as stabilizers and thickeners. Where necessary, yogurt shall meet the fermented milk and yogurt standards of AFNOR NF 04-600 and / or codex StanA-lla-1975. To meet the AFNOR NF 04-600 standard, the product shall not be heated after fermentation, and dairy ingredients shall constitute at least 70% (m / m) of the finished product.

[0045] Deposited shares and sales to specialists only The applicant requests that, until the date the patent is granted, the deposited microbial samples described below be made available only to experts, subject to the availability provisions controlled by the Industrial Property Offices of the Contracting Parties to the Budapest Convention.

[0046] The applicant has identified Lactobacillus rhamnosus strain DSM32092 as July 16, 2015 It was deposited with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig, and was assigned depositation number: DSM32092. The applicant deposited Penicillium solitum DSM32093 with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig on July 16, 2015, and was assigned deposit number: DSM32093. The applicant deposited Penicillium brevicompactum DSM32094 with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig on July 16, 2015, and was assigned deposit number: DSM32094. The applicant deposited Lactobacillus rhamnosus DSM33515 with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig on May 5, 2020, and was assigned deposit number: DSM33515. The applicant deposited Penicillium crustusum DSM33517 with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig on May 5, 2020, and was assigned deposit number: DSM33517. The applicant deposited Penicillium rokforti DSM33518 with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig on May 5, 2020, and was assigned deposit number DSM33518. The applicant deposited Penicillium paneum DSM33519 with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig on May 5, 2020, and was assigned deposit number: DSM33519. The applicant deposited Penicillium carneum DSM33520 with the German Collection of Microbial Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr.7B, D-38124 Braunschweig on May 5, 2020, and was assigned deposit number DSM33520. [Examples]

[0047] Example 1 The biological control strain of Lb. rhamnosus deposited as DSM33515 - slight effect on post-acidification. The effect of the Lb. rhamnosus strain deposited as DSM33515 on post-acidification was tested compared to the starter culture alone and the parent strain deposited as DSM32092.

[0048] For this purpose, a homogenized milk base consisting of 2.8% protein, 1.2% lipid, and 10% sucrose was heat-treated at 95±1°C for 5 minutes and then rapidly cooled. A commercially available starter culture (FD-DVS YF-L812 or FD-DVS Premium 5.0) was inoculated at 0.02% (v / w), and the inoculated milk was distributed into 3-liter buckets. Each bucket contained 1 × 10⁶ units. 7 Lb. rhamnosus bacteria deposited as DSM33515 were inoculated at a total concentration of CFU / g, with 1 × 10⁶ cells per bucket. 7 Lb. rhamnosus deposited as DSM32092 was inoculated at a total CFU / g concentration, and one bucket was used as a reference, inoculated with only the starter culture. All bottles were incubated in a water bath at 43±1°C and fermented in this state until the pH reached 4.60±0.1. After fermentation, the bottles were vigorously agitated to break up any clumps, dispensed into 50 ml cups, and rapidly cooled on ice.

[0049] To observe the effect on post-acidification, three types of fermented milk samples (starter only, starter + bacteria deposited as DSM33515, and starter + bacteria deposited as DSM32092) were stored at 7±1℃, 12±1℃, and 25±1℃ for 28 days, and at 37±1℃ for 7 days, and the pH was measured on days 1, 7, 14, 21, and 28.

[0050] Figures 1 and 2 illustrate the effects on post-acidification when combined with FD-DVS YF-L812 and FD-DVS Premium 5.0, respectively, showing that the addition of Lb. rhamnosus deposited as DSM33515 caused less post-acidification compared to Lb. rhamnosus deposited as DSM32092.

[0051] Example 2 Lb. rhamnosus strain DSM33515 possesses both low resistance to post-acidification and high antifungal properties. To analyze the inhibitory effect of Lb. rhamnosus deposited as DSM33515, a quasi-quantitative agar assay similar to the yogurt manufacturing process and product was used:

[0052] A homogenized milk base consisting of 2.8% protein, 1.2% lipid, and 10% sucrose was heat-treated at 95±1°C for 5 minutes and then rapidly cooled. A commercially available starter culture (FD-DVS YF-L812) was inoculated at 0.02% (v / w), and the inoculated milk was distributed into 3L buckets. Each bucket contained 1 × 10⁶ units. 7 Lb. rhamnosus, deposited as DSM33515, was inoculated at a total concentration of CFU / g, and 1 × 10⁶ of cells were placed in a separate bucket. 7 Lb. rhamnosus DSM32092 was inoculated at a total CFU / g concentration, and one bucket was used as a reference, inoculated with only the starter culture. All buckets were incubated in a water bath at 43±1°C and fermented in this state until a pH of 4.60±0.1 was reached. After fermentation, the buckets were vigorously agitated to break up any clumps, dispensed into 200 ml cups, and rapidly cooled in a cooling chamber. Next, 40 ml of 5% sterile agar solution, which had been warmed to 40°C, dissolved, and then cooled to 60°C, was added to the fermented milk. Then, this fermented milk and agar solution was poured into sterile petri dishes, and the plates were dried in an LAF bench for 30 minutes.

[0053] The following six different fungal spore suspensions were spotted onto agar plates at a concentration of 500 spores / spot: P. brevicompactum (deposited as DSM32094), P. crustusum (deposited as DSM33517), P. solitum (deposited as DSM32093), P. carneum (deposited as DSM33520), P. paneum (deposited as DSM33519), and P. roqueforti (deposited as DSM33518). Three fungal species were spotted onto each plate, and target contaminants were added at a concentration of 500 spores / spot. The plates were incubated at 7±1°C for 28 days, and fungal growth was monitored periodically.

[0054] The results of the agar assay are shown in Figure 3, and they showed that all the tested molds grew very well on agar plates made from milk fermented using only the starter culture (reference). However, when Lb. rhamnosus, deposited as DSM33515, was present during milk fermentation, the resulting plates inhibited the growth of the six Penicillium species tested. The effect of Lb. rhamnosus was at a similar level to that of Lb. rhamnosus DSM32092, which is known to cause more post-acidification.

[0055] Example 3 Sensory evaluation The biological control strain Lb. rhamnosus, deposited as DSM33515, was used to test the sensory effects of fermented dairy products stored for two weeks at a slightly accelerated temperature (12°C) or 25°C, compared to starter cultures alone and the parent strain DSM32092.

[0056] A homogenized milk base consisting of 2.8% protein, 1.2% lipid, and 10% sucrose was heat-treated at 95±1°C for 5 minutes and then rapidly cooled. A commercially available starter culture (FD-DVS YF-L812) was inoculated at 0.02% (v / w), and the inoculated milk was distributed into 3L buckets. Each bucket contained 1 × 10⁶ units. 7Lb. rhamnosus bacteria deposited as DSM33515 were inoculated at a total concentration of CFU / g, with 1 × 10⁶ cells per bucket. 7 Lb. rhamnosus deposited as DSM32092 was inoculated at a total CFU / g concentration, and one bucket was used as a reference, inoculated with only the starter culture. All buckets were incubated in a water bath at 43±1°C and fermented in this state until a pH of 4.60±0.1 was reached. After fermentation, the buckets were vigorously agitated to break up any clumps, dispensed into 200 mL cups, and rapidly cooled in a cooling chamber. One set of samples was stored at 12°C for two weeks, and the other set of samples was stored at 25°C for two weeks.

[0057] In explanatory analysis, a trained panel evaluates the designated properties of products based on the perceived intensity levels. These quantitative ratings are then used to explain the similarities and differences between products in the evaluated set (Lawless, HT, & Heymann, H. (2010). Sensory evaluation of food: principles and practices. Springer Science & Business Media):

[0058] Twelve trained judges participated in the study and evaluated six samples using only three relevance criteria as defined herein. The characteristic list was based on evaluations of the same samples stored at 25°C. For evaluation, the samples were presented to the judges in a randomized order, i.e., a total of 12 samples, according to a double-row Latin square grid. Characteristic intensity was graded using a systematic linear scale with five divisions labeled "none" on the left and "strong" on the right.

[0059] For statistical evaluation of the results for intensity assessment, three-way multivariate analysis of variance (MANOVA) using Wilks' test to confirm overall sample differences, and analysis of variance (ANOVA) to identify which traits had significant differences by considering the factors—product, judge, and replication, as well as their bidirectional correlations. A minimum significance (LSD) test was used to detect significant differences between product samples when the trait had a significant product effect. A significance level of α = 0.05 was selected for the test.

[0060] Tables 1 and 2 show the results of sensory evaluations conducted on samples stored at 12°C and 25°C for 14 days, respectively. These results indicate that the addition of Lb. rhamnosus, deposited as DSM33515 together with the starter culture, improved the sensory characteristics of the product when stored at 12°C or 25°C for 14 days, compared to the parent strain DSM3351. Specifically, in the sample using DSM32092, only a slight buttery aroma was detected in the sample stored at 12°C for 14 days compared to the baseline. When the sample was stored at 25°C for 14 days, Lb. rhamnosus DSM32092 resulted in more acidity but only a slight sweetness and milky aroma compared to the sample inoculated with only the starter culture.

[0061] Table 1: Results of sensory evaluation conducted on samples stored at 12°C for 14 days. The characteristic list includes the corresponding p-values ​​for each characteristic, mean, and group sample based on the least significant difference (LSD) test for characteristics showing significant differences between products, as well as F-values ​​for factor products from a three-way ANOVA of the significant samples. Different letters indicate a significant difference at p<0.05. [Table 1]

[0062] Table 2: Results of sensory evaluation conducted on samples stored at 25°C for 14 days. The characteristic list includes the corresponding p-values ​​for each characteristic, mean, and group sample based on the least significant difference (LSD) test for characteristics showing significant differences between products, as well as F-values ​​for factor products from a three-way ANOVA of the significant samples. Different letters indicate a significant difference at p<0.05. [Table 2]

[0063] The table shows that the samples prepared using DSM33515 appeared to have a stronger milky and sweeter taste, and less acidity, compared to the samples prepared using DSM32092.

[0064] Example 4 Lb. rhamnosus DSM33515 shows only a slight effect on post-acidification compared to CBS141584. Lb. rhamnosus DSM33515 was tested for its effect on post-acidification compared to starter cultures alone and the strain deposited as CBS141584 disclosed in European Patent EP16182341.

[0065] For this purpose, a homogenized milk base consisting of 2.8% protein, 1.2% lipid, and 10% sucrose was heat-treated at 95±1°C for 5 minutes and then rapidly cooled. A commercially available starter culture (FD-DVS YF-L812, Chr. Hansen A / S Denmark, containing Lactobacillus delbruecki subspecies bulgaricus and Streptococcus thermophilus) was inoculated at 0.02% (v / w), and the inoculated milk was distributed into 3-liter buckets. Each bucket contained 1 × 10⁶ 7 Lb. rhamnosus, deposited as DSM33515, was inoculated at a total concentration of CFU / g, with 1 × 10⁶ cells per bucket. 7CBS141584 was inoculated at a total concentration of CFU / g, and one bucket was used as a reference, inoculated with only the starter culture. All bottles were incubated in a water bath at 43±1°C and fermented in this state until a pH of 4.60±0.1 was reached. After fermentation, the bottles were vigorously agitated to break up any clumps, dispensed into 50 ml cups, and rapidly cooled on ice.

[0066] To observe the effect on post-acidification, three types of fermented milk samples (starter only, starter + bacteria deposited as DSM33515, and starter + bacteria deposited as CBS141584) were stored at 7±1℃ and 25±1℃ for 28 days, and the pH was measured on days 1, 7, 14, 21, and 28.

[0067] Figures 4 and 5 show the effects of DSM33515 and CBS141584 on post-acidification when combined with starter cultures. Clearly, the addition of Lb. rhamnosus DSM33515 caused less post-acidification compared to CBS141584.

Claims

1. A bacterium deposited as DSM33515, consisting of Lactobacillus rhamnosus strain or a Lactobacillus rhamnosus mutant strain derived therefrom: The Lactobacillus rhamnosus strain deposited as DSM33515, or a Lactobacillus rhamnosus mutant strain derived therefrom, (a) During storage after fermentation, the pH of the fermented dairy product containing Lactobacillus rhamnosus strain deposited as DSM33515 or a Lactobacillus rhamnosus mutant strain derived therefrom is increased compared to the dairy product containing Lactobacillus rhamnosus strain deposited as DSM32092, wherein the increase in pH is at least 0.1; and (b) During storage after fermentation, reduce the growth of mold in fermented dairy products containing Lactobacillus rhamnosus strain deposited as DSM33515 or Lactobacillus rhamnosus mutant strains derived therefrom, compared to dairy products that do not contain it. Here, the increase in pH or decrease in mold growth in fermented dairy products containing the aforementioned Lactobacillus rhamnosus mutant strain is no less significant than that observed in dairy products containing the Lactobacillus rhamnosus strain deposited as DSM33515. The aforementioned increase in pH is due to the starter culture being at least 10 7 The product fermented using Lactobacillus rhamnosus strain at a concentration of CFU / g was stored at 25°C for 28 days and then measured. The reduction in the growth of the mold was compared to the starter culture by at least 10%. 7 The product, fermented using Lactobacillus rhamnosus strain at a concentration of CFU / g, is measured after being stored at 7°C for 28 days. The aforementioned bacteria.

2. A bacterium deposited as DSM33515, specifically a Lactobacillus rhamnosus strain or a Lactobacillus rhamnosus mutant strain derived therefrom, The Lactobacillus rhamnosus strain deposited as DSM33515 or a Lactobacillus rhamnosus mutant strain derived therefrom increases the pH of the fermented dairy product containing the Lactobacillus rhamnosus strain deposited as DSM33515 or a Lactobacillus rhamnosus mutant strain derived therefrom during storage after fermentation, compared to the dairy product containing the Lactobacillus rhamnosus strain deposited as DSM32092, where the increase in pH is at least 0.

1. Here, the increase in pH of the fermented dairy product containing the aforementioned Lactobacillus rhamnosus mutant strain was no less significant than that of the dairy product containing the Lactobacillus rhamnosus strain deposited as DSM33515. The aforementioned increase in pH is due to the starter culture being at least 10 7 The bacterium is measured after the product, which is fermented using Lactobacillus rhamnosus strain at a concentration of CFU / g, is stored at 25°C for 28 days.

3. The bacterium Lactobacillus rhamnosus, deposited as DSM33515, The Lactobacillus rhamnosus strain deposited as DSM33515 reduces the growth of mold in fermented dairy products containing the Lactobacillus rhamnosus strain during storage after fermentation, compared to dairy products without it. Here, the reduction in the growth of the aforementioned mold is observed in the starter culture and at least 10 7 The product, fermented using Lactobacillus rhamnosus strain at a concentration of CFU / g, is measured after being stored at 7°C for 28 days. The aforementioned bacteria.

4. The Lactobacillus rhamnosus strain deposited as DSM33515 or a Lactobacillus rhamnosus mutant strain derived therefrom reduces the growth of yeast in fermented dairy products containing the Lactobacillus rhamnosus strain deposited as DSM33515 or a Lactobacillus rhamnosus mutant strain derived therefrom during storage after fermentation, compared to dairy products without it. Here, the decrease in yeast growth in fermented dairy products containing the aforementioned Lactobacillus rhamnosus mutant strain is no less significant than that in dairy products containing the Lactobacillus rhamnosus strain deposited as DSM33515. The aforementioned decrease in yeast growth is due to the starter culture and at least 10 7 The bacteria according to any one of claims 1 to 3, wherein the product fermented using Lactobacillus rhamnosus strain at a concentration of CFU / g is stored at 7°C for 28 days and then measured.

5. A composition comprising the bacteria described in any one of claims 1 to 4.

6. The composition according to claim 5, wherein the composition further comprises a starter culture.

7. The composition according to claim 5 or 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 claim 5 or 6, wherein the composition is frozen or freeze-dried.

9. A method for producing a fermented dairy product, comprising adding a bacterium according to any one of claims 1 to 4 or a composition according to any one of claims 5 to 8 to milk or a dairy product, and fermenting the mixture at a temperature of 22°C to 43°C until a pH of 4.6 or less than 4.6 is achieved.

10. A fermented dairy product containing the bacteria described in any one of claims 1 to 4.

11. The fermented dairy product according to claim 10, wherein the fermented dairy product is obtained by the method described in claim 9.

12. The fermented dairy product according to claim 10 or 11, wherein the fermented dairy product maintains a pH greater than 3.8 when stored at 25°C for at least 28 days.

13. The aforementioned bacteria, at least 10 7 A fermented dairy product according to any one of claims 10 to 12, present at a concentration of CFU / g.

14. A food, feed, or pharmaceutical product comprising the bacteria described in any one of claims 1 to 4 or the composition described in any one of claims 5 to 8.

15. A food, feed, or pharmaceutical product according to claim 14, obtainable by the method described in claim 9.

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