Novel lactic acid bacterium, dairy product using the strain, and method for producing the same

Abstract

To provide a novel lactic acid bacterium strain that is usable as a starter useful for production of dairy products, especially natural cheese, allows reduction of the production time in cheese production, and enables the produced cheese to retain a large number of live bacteria.SOLUTION: A lactic acid bacterium being Lactococcus cremoris strain 210 (Accession No.: NITE P-04049) or Lactococcus cremoris strain 215 (Accession No.: NITE P-04050).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to starter lactic acid bacteria for producing dairy products, which are fermentation microorganisms necessary for producing dairy products such as natural cheese. [Background technology]

[0002] Consumption of natural cheese in Japan has been increasing in recent years, and domestic cheese production has also increased accordingly. Natural cheeses have a wide variety of flavors, textures, and textures depending on the manufacturing process. Factors that affect taste include milk quality, milk processing methods, cheese starters (bacteria such as lactic acid bacteria, filamentous fungi, and yeast), enzymes, and aging conditions. In Europe, over a long history, these factors have been modified in a complex manner to produce cheeses that suit the tastes of people in each country and region, leading to the diversity of cheeses we see today. In recent years, with the spread of natural cheeses mentioned above, natural cheeses with a variety of flavors and textures have begun to appear on the market in Japan as well, but the variety is still limited compared to Europe. One of the important factors in determining the flavor of natural cheese is the cheese starter, such as lactic acid bacteria, used during production. The starter (main starter), which contributes to lactic acid fermentation during the cheese-making process, produces lactic acid early in the process, lowering the pH of the raw milk and inhibiting the growth of spoilage bacteria while promoting the coagulation of milk proteins by rennet. On the other hand, the auxiliary starter used in conjunction with the main starter does not have any particular effect on the early stages of cheese-making, but by breaking down proteins and lipids during the cheese's ripening period, it accelerates ripening and creates a unique flavor, aroma, and texture. Both main starters and auxiliary starters are commercially available in powder form, made by freeze-drying high concentrations of live bacteria, for convenience and hygiene reasons.

[0003] Currently, most cheese starters used in natural cheese production in Japan are imported from overseas. The lactic acid bacteria used in imported commercial cheese starters were isolated and identified from strains that had been passaged by traditional cheese makers. Cheese starters containing these bacteria alone or in combination have stable lactic acid production and are capable of producing high-quality cheese, so they are widely distributed worldwide. However, because there are only a limited number of global starter manufacturers, there are very few options for modifying starters to characterize cheese. Furthermore, while the lactic acid production ability and rate of lactic acid bacteria used in commercially available cheese starters are stable, this makes it difficult to shorten the production time in cheese production. Furthermore, the lactic acid bacteria that make up commercially available cheese starters are 1 x 10 9 The bacterial concentration increases to about cfu / g, but after about one month of aging, the bacterial concentration rapidly decreases. Cheese is a food source that provides lactic acid bacteria, but there was a problem in that the number of live lactic acid bacteria in cheese rapidly decreases as the aging period increases. Although lactic acid bacteria useful for natural cheese production have been reported (for example, Patent Documents 1 to 3, etc.), there has not yet been a report that can solve the above problems. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2004-507265 [Patent Document 2] Japanese Patent Application Publication No. 2017-221231 [Patent Document 3] Japanese Patent Application Publication No. 2019-047831 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel lactic acid bacteria strain that can be used as a starter useful in the production of dairy products, particularly natural cheese, that can shorten the cheese production time, and that has high survival rate in the produced cheese. [Means for solving the problem]

[0006] The present inventors conducted extensive research to solve the above problems and selected lactic acid bacteria with high milk fermentation ability from the lactic acid bacteria library held by the National Agriculture and Food Research Organization. After examining the selected lactic acid bacteria in detail for their characteristics related to cheese production, they found that the cheese production time could be shortened and that the produced cheese contained a large number of viable bacteria, leading to the completion of the present invention.

[0007] Specifically, the present invention provides the following: 1. A lactic acid bacterium selected from Lactococcus cremoris strain 210 (accession number: NITE P-04049) and Lactococcus cremoris strain 215 (accession number: NITE P-04050). 2. The lactic acid bacterium according to 1, which is used for producing dairy products. 3. The lactic acid bacterium according to 1, which is used as a cheese starter bacterium. 4. A dairy product containing the lactic acid bacteria according to any one of 1. to 3. 5. Cheese containing the lactic acid bacteria according to any one of 1. to 3. 6. A method for producing a dairy product, characterized by using the lactic acid bacterium according to any one of 1. to 3. 7. A method for producing cheese, characterized by using the lactic acid bacterium according to any one of 1. to 3. [Effects of the Invention]

[0008] The novel lactic acid bacteria of the present invention have excellent lactic acid production ability, and when used as a starter in the production of dairy products, particularly cheese, it is possible to shorten the time required for the curd formation process. Furthermore, the cheese obtained by the cheese production has the advantage of having a large number of viable bacteria, making it possible to develop new foods that contain lactic acid bacteria. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a graph showing the pH change every hour from the start of fermentation at a growth temperature of 30°C for seven species of lactic acid bacteria in Test 1 to confirm the properties of the novel lactic acid bacteria strain in Example 4. [Figure 2] 1 is a graph showing the pH change every hour from the start of fermentation at a growth temperature of 40°C for seven species of lactic acid bacteria in Test 1 to confirm the properties of the novel lactic acid bacteria strain in Example 4. [Figure 3] 1 is a graph showing the salt sensitivity of seven types of lactic acid bacteria in Confirmation Test 2 of the properties of the novel lactic acid bacteria strain in Example 5. [Figure 4] 1 is a graph showing the low-temperature growth of seven species of lactic acid bacteria in Test 2 for confirming the properties of novel lactic acid bacteria strains in Example 5. [Figure 5] 1 is a graph showing the high-temperature growth of seven species of lactic acid bacteria in Test 2 to confirm the properties of novel lactic acid bacteria strains in Example 5. [Figure 6] 1 is a graph showing the time (minutes) required for the three types of lactic acid bacteria to reach pH 5.0 in Test 1 to confirm the lactic acid production ability of the novel lactic acid bacteria strain in Example 6. [Figure 7] 1 is a graph showing the time (minutes) required for the pH to reach 5.6 in six test plots in Test 2 to confirm the lactic acid production ability of the novel lactic acid bacteria strain of Example 7. [Figure 8] 1 is a graph showing the viable cell count in cheese for each ripening period (0, 1, 30, 60, 90, 180, 360 days) for Gouda cheese produced using three test plots as a starter in a confirmation test of the viable cell count in cheese produced with the novel lactic acid bacteria strain of Example 8. [Figure 9] 1 is a graph showing the time (minutes) required for the pH to reach 5.6 in four test plots in Test 3 to confirm the lactic acid producing ability of the novel lactic acid bacteria strain of Example 10. [Figure 10] 1 is a graph showing the changes in moisture content in Gouda cheese obtained in Test 3 for Confirmation of the Lactic Acid Production Ability of the Novel Lactic Acid Bacteria Strain in Example 10, on the 1st, 30th, 60th, 90th, and 180th days after ripening. [Figure 11]1 is a graph showing the changes in fat content in Gouda cheese obtained in Test 3 for confirming the lactic acid production ability of the novel lactic acid bacteria strain in Example 10, on the 1st, 30th, 60th, 90th, and 180th days after ripening. [Figure 12] 1 is a graph showing the changes in pH value in Gouda cheese obtained in Test 3 for confirming the lactic acid production ability of the novel lactic acid bacteria strain in Example 10, on the 1st, 30th, 60th, 90th, and 180th days after ripening. [Figure 13] 1 is a graph showing the change in viable cell count in Gouda cheese obtained in Test 3 for confirming the lactic acid production ability of the novel lactic acid bacteria strain in Example 10, on the 1st, 30th, 60th, 90th, and 180th days after ripening. [Figure 14] 1 is a graph showing the change in the amount of free amino acids (μmol / g) in Gouda cheese obtained in Test 3 for confirming the lactic acid production ability of the novel lactic acid bacteria strain in Example 10 after 90 days of ripening. [Figure 15] 1 is a graph showing the change in the amount of free glutamic acid (μmol / g) in Gouda cheese obtained in Test 3 for confirming the lactic acid production ability of the novel lactic acid bacteria strain of Example 10 after 90 days of aging. [Figure 16] 1 is a graph showing the glutamic acid concentration (μmol / g) in Gouda cheese obtained in Test 3 for confirming the lactic acid production ability of the novel lactic acid bacteria strain of Example 10, on the 1st, 30th, 60th, 90th, and 180th days after ripening.

[0010] The present invention relates to Lactococcus cremoris strain 210 (accession number: NITE P-04049) and Lactococcus cremoris strain 215 (accession number: NITE P-04050), which were selected from the lactic acid bacteria library held by the National Agriculture and Food Research Organization. The present invention will be described in detail below.

[0011] <New lactic acid bacteria strain> The novel lactic acid bacteria of the present invention, Lactococcus cremoris strain 210 (accession number: NITE P-04049) and Lactococcus cremoris strain 215 (accession number: NITE P-04050), were selected from the lactic acid bacteria library held by the National Agriculture and Food Research Organization. Lactococcus cremoris strain 210 was isolated from pickled radish, and Lactococcus cremoris strain 215 was isolated from pickled mizuna. These lactic acid bacteria strains have been deposited at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation, an independent administrative institution, and their accession numbers are NITE P-04049 and NITE P-04050, respectively (deposit date: December 20, 2023). As will be described later, the two novel lactic acid bacteria strains of the present invention have the excellent characteristics of being highly capable of producing lactic acid during cheese production and remaining viable in cheese for an extremely long period of time even during the ripening process.

[0012] <Cheese production> Cheese production is divided into the curd forming process and the ripening process. The curd formation process separates the raw milk components into solids and water (whey) using acidification, enzyme reactions, heating, etc., and then solidifies the cheese by removing the whey. Lactic acid bacteria used in starters are essential biological ingredients for cheese production, as they are involved in the acidification of raw milk and part of the enzyme reactions. Their basic role is to lower the pH of the raw milk by breaking down lactose to produce lactic acid, preventing the growth of harmful microorganisms, and to promote the action of rennet, a milk-clotting enzyme, to promote curdling and syneresis from the curd. The time required for curd formation, which is the first step in cheese production, is greatly affected by the starter's ability to produce lactic acid. Therefore, if a starter with poor lactic acid production ability is used, syneresis will not progress and the time required for curd formation will be longer. The ripening process involves storing the formed curd at a constant temperature and humidity. During this process, the enzymes originally contained in the raw milk, rennet, and the extracellular and intracellular enzymes released into the curd by the autolysis of the starter lactic acid bacteria promote the decomposition of casein and milk fat, and the production of aromatic components, creating the texture and flavor specific to each cheese type. 9 Lactic acid bacteria, which grow to levels exceeding cfu / g, are the main source of enzymes, and a sufficient concentration of bacteria is required. The viable bacterial populations of the main starter bacteria, supplemental starter bacteria, and non-starter bacteria accidentally introduced from the environment that form the viable flora of ripened cheese depend on the characteristics of the strains that make them up. For example, the typical populations of Lactococcus lactis and Lactococcus cremoris, which form the main starter bacteria, reach their peak during the curd-forming process, gradually decrease after about one month of ripening, and reach approximately 1 / 1,000 after three months. As the main starter bacteria decrease, the source of enzymes that function in ripening shifts to supplemental starter bacteria and non-starter bacteria. In particular, in cheese production without supplemental starters or mold starters, the main starter bacteria contribute significantly to the ripening process, and the population and enzyme activity of the main starter bacteria directly affect the final quality of the cheese. Natural cheese is known as a food that allows you to consume live lactic acid bacteria, but hard and semi-hard cheeses require at least 3 to 4 months of aging before they reach the quality specific to each cheese variety and are ready to eat. Therefore, the number of bacteria derived from the main starter in cheese at the optimum time for consumption is 1 x 10 6 Many are below the cfu / g level.

[0013] The two novel lactic acid bacteria strains of the present invention are lactic acid bacteria selected for their properties suitable for use as main starters. When used as main starters, the two novel lactic acid bacteria strains of the present invention exhibit excellent lactic acid production ability in the curd molding step during cheese production, thereby exhibiting the significantly excellent effect of shortening the time required for cheese production. Furthermore, the two novel lactic acid bacteria strains of the present invention have the same level of lactic acid production ability as the standard strain of Lactococcus cremoris at the optimum growth temperature, and can grow at temperatures higher and lower than the upper growth temperature limit of the standard strain of Lactococcus cremoris, maintaining their high lactic acid production ability. It can also grow at a salt concentration of 5% sodium chloride, where ordinary Lactococcus cremoris cannot grow. The two novel strains of lactic acid bacteria of the present invention can grow at 10°C, which is often used as a cheese ripening temperature, and at a salt concentration of 2% sodium chloride, which is approximately the same as the salt concentration in cheese. The viable cell count of cheese produced using these two strains of lactic acid bacteria as the main starter was 1 x 10 in the cheese-forming process, which is similar to that of cheese produced using existing or commercially available starters. 9 cfu / g and increased to 1 x 10 9 It has the excellent effect of maintaining extremely high bacterial counts exceeding cfu / g. In cheese production, either one of the two strains of novel lactic acid bacteria of the present invention may be used, or the two strains may be used in combination.

[0014] <Cheese manufacturing method> The present invention relates to a method for producing dairy products using two novel strains of lactic acid bacteria, and in particular to a method for producing cheese. For example, a method for producing cheese using one or both of the two novel lactic acid bacteria strains will be briefly described. The origin of the milk used as the raw material is not limited, and milk such as cow's milk, goat's milk, sheep's milk, buffalo milk, and donkey's milk can be used, with cow's milk or goat's milk being preferred. Cheese is produced by adding one or both of the two novel strains of lactic acid bacteria as a main starter to raw milk for cheese production, which produces lactic acid to create a sour taste and flavor substances, and then adding a milk-clotting enzyme (rennet) to cause curdling and form curds. Next, the whey in the curds is removed by cutting and cooking, and the curds are then pressed into molds to remove the whey and form the cheese. After molding, salt is added to impart a salty taste and shelf life, and the water activity is adjusted as needed. The cheese can then be produced in a manner similar to known cheese-making methods, including aging, except for fresh cheese.

[0015] <Cheese> The cheese produced using the two novel lactic acid bacteria strains of the present invention is not particularly limited. Cheese is broadly classified into natural cheese and processed cheese. The two novel lactic acid bacteria strains of the present invention are suitable for the production of natural cheese, and because they have excellent lactic acid-producing ability in milk fermentation, they are also suitable for the production of fresh cheeses such as cottage cheese and mozzarella cheese. Furthermore, because they have the characteristic of achieving and maintaining a high viable cell count in cheese curds, they are also suitable for the production of aged cheeses such as Gouda cheese, Cheddar cheese, and Camembert cheese.

[0016] <Fermented dairy products> Fermented dairy products produced using the two novel lactic acid bacteria strains of the present invention are not particularly limited. Products produced by fermenting milk and other ingredients include fermented milk, dairy lactic acid bacteria beverages, and lactic acid bacteria beverages. The two novel lactic acid bacteria strains of the present invention are suitable for producing these products because of their excellent lactic acid-producing ability during milk fermentation. Furthermore, they can impart a flavor different from that of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, which have been widely used in the production of these products. For example, in milk fermentation using the two novel lactic acid bacteria strains of the present invention, the lactic acid bacteria rapidly produce lactic acid from the start of fermentation, lowering the pH to around 5. However, once the pH reaches 4.4 to 4.5, it does not drop below that level, preventing the fermented milk from becoming excessively sour (see Figure 1). A method for producing a fermented dairy product using one or both of the two novel strains of lactic acid bacteria will now be briefly described. The origin of the milk used as the raw material is not limited, and milk such as cow's milk, goat's milk, sheep's milk, buffalo milk, and donkey's milk can be used, with cow's milk or goat's milk being preferred. The production method involves adding one or both of the two novel strains of lactic acid bacteria to pasteurized raw milk and fermenting it for approximately 8 to 12 hours at a temperature suitable for lactic acid bacteria growth. To enhance palatability, sweeteners, fruit juice, gelatin, etc. may be added. Furthermore, homogenization using a blender or other device allows the production of drinkable or frozen fermented milk.

[0017] As described above, the two novel lactic acid bacteria strains of the present invention, Lactococcus cremoris strain 210, isolated from pickled radish, and Lactococcus cremoris strain 215, isolated from pickled mizuna, are highly salt-tolerant and can grow at a salt concentration of 5% sodium chloride. They can also grow at a low temperature of 10°C. For these reasons, the two novel lactic acid bacteria strains of the present invention are also useful as starters for fermented plant foods such as pickles.

[0018] In the present invention, dairy products refer to products that use milk as a raw material. Specific examples include yogurt, cheese, cream, butter, butter oil, concentrated whey, ice cream, concentrated milk, concentrated skim milk, unsweetened condensed milk, unsweetened condensed skim milk, sweetened condensed milk, sweetened condensed skim milk, whole milk powder, skim milk powder, cream powder, whey powder, protein-concentrated whey powder, buttermilk powder, sweetened milk powder, modified milk powder, fermented milk, lactic acid bacteria beverages, and milk beverages. Among these, cheese is preferred as the dairy product of the present invention. Examples of raw milk for dairy products include cow's milk, goat's milk, sheep's milk, buffalo milk, donkey's milk, etc. [Example]

[0019] The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0020] Example 1: Identification of a new lactic acid bacteria strain Lactococcus cremoris and Lactococcus lactis, both Lactococcus species, are typical starter species for dairy processing, but their roles in production are different, making it important to identify the bacterial species when considering industrial use. Both species produce lactic acid and contribute to lowering the pH of raw milk. However, Lactococcus cremoris often dies early in the ripening process and becomes a source of enzymes involved in the ripening process, whereas Lactococcus lactis often tolerates pH, temperature, and salt concentration, and remains active for a long period in products such as cheese, which require a long ripening period, before dying and becoming a source of enzymes. Based on the above circumstances, two novel strains of lactic acid bacteria of the present invention were identified. The names of the microbial strains used for species identification, whose genome information is publicly available, are listed below. 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains ATCC 19435T: Lactococcus lactis subsp. lactis type strain ATCC19435 T CCUG 32210T: Lactococcus lactis subsp. hordoniae type strain CCUG32210 T ATCC 19257T: Lactococcus cremoris subsp. cremoris type strain ATCC19257 T DSMZ 21502T: Lactococcus cremoris subsp. tractae type strain DSMZ21502 T ATCC stands for American Biobank, CCUG for Swedish Biobank, and DSMZ for German Biobank. The results are shown below.

[0021] [Table 1]

[0022] Strains 210 and 215 could not be clearly distinguished from one another by 16S rRNA gene sequencing, a commonly used method for identifying bacterial species. Furthermore, phenotypic classification identifies them as Lactococcus lactis. However, as shown in Table 1, the average nucleotide identity (ANI) method, which compares the homology of the entire genome, and digital DNA-DNA hybridization (dDDH) methods identified them as Lactococcus cremoris. The ANI method described above was performed by comparing the entire genome using assembled sequences. Similarity was calculated for aligned regions, and strains with a homology of 95% or higher (equivalent to a DDH homology of 70%) were considered to be of the same species. ANI is an index of species identification that uses the average similarity between DNA regions that show homology between two genomes. Here, orthoani (https: / / github.com / althonos / orthoani) was used to calculate the ANI between each genome sequence. dDDH is a genome sequence-based calculation method that shows a high correlation with DDH, a test method that indirectly calculates homology between base sequences. Here, DDH estimates were calculated on GGDC (https: / / ggdc.dsmz.de / ggdc.php#).

[0023] Example 2: Antimicrobial susceptibility of novel lactic acid bacteria strains To confirm whether the two novel lactic acid bacteria strains of the present invention are suitable for food manufacturing applications, a test to confirm their susceptibility to antimicrobial substances was carried out. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains CH1 strain: Lactococcus lactis CH1 strain (a milk fermentation starter bacterium manufactured by Christian Hansen) Strain 527: Lactococcus lactis strain 527 (a milk fermentation starter bacterium distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) (2) Test method Antimicrobial susceptibility testing was performed using the disk diffusion method. Each test organism was pre-cultured overnight in 5 mL of MRS medium (OXOID), washed once with 0.85% sodium chloride solution, and then suspended in an equal volume of 0.85% sodium chloride solution. 100 μL of the suspension was poured onto a plate of MRS agar medium, and SensiDiscs (Becton, Dickinson, and Company) containing the antimicrobials listed in Table 2 were placed on the plate using a dispenser (Becton, Dickinson, and Company). The plate was then incubated at 30°C for 24 hours. After incubation, susceptibility was determined by the presence or absence of an inhibition zone around each disk.

[0024] (3) Results The antimicrobial susceptibilities of the four strains of lactic acid bacteria tested are summarized in Table 2 below. In Table 2, "+" means susceptible, and "-" means not susceptible. [Table 2]

[0025] As shown in Table 2, strains 210 and 215 had antimicrobial susceptibility equivalent to that of the commercially available strains, confirming their applicability for food manufacturing purposes. Example 3: Sugar utilization by novel lactic acid bacteria strains To clarify the assimilation characteristics of the two novel lactic acid bacteria strains of the present invention, their sugar assimilation ability was tested. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains CH1 strain: Lactococcus lactis CH1 strain (a milk fermentation starter bacterium manufactured by Christian Hansen) Strain 527: Lactococcus lactis strain 527 (a milk fermentation starter bacterium distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) (2) Test method The sugar utilization test was performed using API50CHL Medium and API50CH (BioMérieux, Metropole de Lyon, France). Each test bacterium was anaerobically cultured overnight in 5 mL of MRS medium (OXOID), washed twice with 0.85% sodium chloride solution, and then suspended in 2 mL of suspension medium. An amount of suspension that achieved a McFarland turbidity of 2 was added to API50CHL Medium and stirred, then dispensed into each tube of an API50CH plate, and mineral oil was then overlaid on the cup portion. After 48 hours of culture at 30°C under aerobic conditions, the plates were evaluated, and the resulting biochemical profiles were analyzed using the APIWEB TM The data was entered into the database and the test bacteria were identified.

[0026] (3) Results The sugar utilization ability of the four strains of lactic acid bacteria tested is summarized in Table 3 below. The meanings of the symbols in Table 3 are summarized in Table 4. [Table 3] [Table 4]

[0027] As shown in Table 3, in the sugar utilization test, both strains 210 and 215 showed similar sugar utilization to the commercially available strains CH1 and 527 of Lactococcus lactis. TM In this study, strains 210 and 215 were identified as Lactococcus lactis with a 99.6% probability. This indicates that strains 210 and 215 differ from the common Lactococcus cremoris and have sugar assimilation properties similar to Lactococcus lactis. Furthermore, in this study, strains 210 and 215 were also able to assimilate D-mannitol, which differs from the commercially available strains CH1 and 527.

[0028] <Example 4: Test 1 to confirm the properties of the new lactic acid bacteria strain> To demonstrate the properties of the two novel lactic acid bacteria strains of the present invention, skim milk was fermented at 30°C, the optimum temperature for growth of Lactococcus cremoris, and at 40°C, the upper limit for growth of Lactococcus cremoris, and their lactic acid production ability was confirmed. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains H61 strain: Lactococcus cremoris H61 strain (a milk fermentation starter bacteria distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) Strain 527: Lactococcus lactis strain 527 (a milk fermentation starter bacterium distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) Strain 19257: Lactococcus cremoris type strain ATCC19257 T Strain 19435: Lactococcus lactis type strain ATCC19435 T CH1 strain: Lactococcus lactis CH1 strain (a milk fermentation starter bacterium manufactured by Christian Hansen) The strain name ATCC (American Type Culture Collection) stands for American biological resource bank. (2) Test method Each test bacterium was pre-cultured overnight in GM17 medium (M17 medium (Difco) supplemented with 1% glucose), washed once with 0.85% sodium chloride solution, and then suspended in an equal volume of 0.85% sodium chloride solution to prepare a test bacterium solution. The 10% skim milk test medium was sterilized by heating at 110°C for 5 minutes on the day before the test. Immediately before the test, the sterilized skim milk was stored at the test temperature for at least 1 hour to stabilize the temperature, and each test bacterium was added at 1 x 10 7 The culture medium was inoculated at a concentration of cfu / mL and immediately placed at the test temperature. The pH of the skim milk culture medium was measured every hour for 24 hours using a pH monitor (YUSB-01, manufactured by Yamagata Toa DDK). The pH changes at 30°C are shown in Figure 1, and the pH changes at 40°C are shown in Figure 2.

[0029] As shown in Figure 1, at the optimum growth temperature of 30°C for Lactococcus cremoris and Lactis, the pH of Cremolis strains 19257 and H61 and the Christian Hansen cheese starter strain CH1 decreased rapidly in the early stages of cultivation. The pH of strains 210 and 215 decreased at a similar rate to that of strain 527 and more rapidly than that of Lactococcus lactis type strain 19435. Furthermore, the final pH decreased to a similar level as that of strain 19257 and the dairy fermentation starter strains 527, H61, and CH1. As shown in Figure 2, at 40°C, which is above the upper growth limit of Lactococcus cremoris, strains 210 and 215 decreased in pH faster and reached a lower pH than Lactococcus lactis strains 527 and 19435 used for comparison. Strain CH1 decreased in pH quickly in the early stages of cultivation, but did not reach a pH of 5. Furthermore, strains 19257 and H61 of Lactococcus cremoris could not grow, and no decrease in pH due to lactic acid production was observed. The results in Figure 1 confirm that the novel lactic acid bacteria strains 210 and 215 of the present invention have lactic acid production capabilities at the optimum growth temperature of Lactococcus cremoris, 30°C, equivalent to those of the type strain of Lactococcus cremoris and strains 527, H61, and CH1, which have a proven track record as milk fermentation starters. Furthermore, the results in Figure 2 confirmed that the novel lactic acid bacteria strains 210 and 215 of the present invention have superior lactic acid production ability to all the comparative strains used in the test at 40°C, the upper growth limit for Lactococcus cremoris.

[0030] <Example 5: Confirmation test 2 of the properties of the new lactic acid bacteria strain> To demonstrate the properties of the two novel lactic acid bacteria strains of the present invention, salt sensitivity and low / high temperature growth were confirmed. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains H61 strain: Lactococcus cremoris H61 strain (a milk fermentation starter bacteria distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) Strain 527: Lactococcus lactis strain 527 (a milk fermentation starter bacterium distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) CH1 strain: Lactococcus lactis CH1 strain (a milk fermentation starter bacterium manufactured by Christian Hansen) (2) Test method Each test bacterium was inoculated into GM17 medium (M17 medium (Difco) with 1% glucose added) on the day before the test and cultured overnight to prepare the test bacterium solution. In the 10°C and 40°C culture tests, the culture solution was kept at the test temperature for more than 1 hour before the start of the test to stabilize the temperature. Each test bacterium was inoculated into GM17 medium (M17 medium (Difco) with 1% glucose added) on the day before the test and cultured overnight to prepare the test bacterium solution. 7 The bacteria were inoculated at a concentration of cfu / mL, immediately placed at the test temperature, and the absorbance (OD = 620 nm) was measured after 8, 24, and 48 hours. For salt concentration sensitivity tests, GM17 medium containing 5% sodium chloride was used, and the bacteria were cultured at 30°C for the specified time, after which the absorbance (OD = 620 nm) was measured. Salt sensitivity is shown in Figure 3, low-temperature growth in Figure 4, and high-temperature growth in Figure 5.

[0031] As shown in Figure 3, the salt tolerance of the novel lactic acid bacteria strains 210 and 215 of the present invention was higher than that of the type strain 19435 of Lactococcus lactis. T The fermentation activity of the H61 and 19257 strains of the Cremoris species was comparable to that of the 527 milk fermentation starter strain, but higher than that of the CH1 strain of Christian Hansen. T The strain failed to grow. As shown in FIG. 4, it was confirmed that the low-temperature growth ability of the novel lactic acid bacteria strains 210 and 215 of the present invention was higher than that of all the comparative strains used in the test. As shown in Figure 5, it was confirmed that the high-temperature growth ability of the novel lactic acid bacteria strains 210 and 215 of the present invention was as high as that of the lactis species milk fermentation starter strain 527 for 8 hours from the start of cultivation, and that the absorbance achieved after 24 and 48 hours of cultivation was higher than that of all the comparative strains used in the test.

[0032] <Example 6: Test 1 to confirm the lactic acid production ability of novel lactic acid bacteria strains> To demonstrate the properties of the two novel lactic acid bacteria strains of the present invention, the time required to reach a specific pH value was compared with that of commercially available milk fermentation starter bacteria to confirm their lactic acid production ability. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains CH1 strain: Lactococcus lactis CH1 strain (a milk fermentation starter bacterium manufactured by Christian Hansen) (2) Test method In order to confirm the lactic acid producing ability of the two novel lactic acid bacteria strains of the present invention in the process of producing fresh cheese, the time required for them to reach a specific pH was determined in the process of producing string cheese. In the case of fresh cheeses such as mozzarella and string cheese, after curd formation, the pH is lowered by lactic acid fermentation and heating is used to encourage the excretion of whey from the curd, and the curd is finally stretched and shaped in hot water. The target pH for stretching and shaping the curd is pH 5.0 to 5.4. Therefore, the target pH for this test was set at 5.0. The time (minutes) required to reach pH 5.0 is shown in FIG. Different letters on the bar graph in Figure 6 indicate significant differences at the 5% level in the Tukey-Kramer multiple comparison test.

[0033] As shown in Figure 6, it was confirmed that the novel lactic acid bacteria strains 210 and 215 of the present invention showed a significantly faster decrease in pH during production than the CH1 strain, which has a proven track record as a milk fermentation starter, i.e., they had a higher lactic acid production ability.

[0034] <Example 7: Confirmation test 2 of the lactic acid production ability of the novel lactic acid bacteria strain> To confirm the lactic acid production ability of the two novel lactic acid bacteria strains of the present invention in the hard and semi-hard cheese manufacturing process, the time required to reach a specific pH was determined in the Gouda cheese manufacturing process. In the curd-forming process for hard and semi-hard cheeses such as Gouda, the pH is lowered by lactic acid fermentation and heating is used to encourage whey to be released from the curd, which is then molded and pressed to complete the process. The target pH for the whey-removal process is about 5.6, after which the cheese moves on to the cooling process, which inhibits the vigorous growth of lactic acid bacteria, or the salting process. Therefore, the target pH for this test was set at 5.6. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain Strain 217: Lactococcus cremoris strain 215 Strain 527: Lactococcus lactis strain 527 (a milk fermentation starter bacterium distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) CHN11: CHN11 (Christian Hansen, a mixed starter containing several types of lactic acid bacteria) OUT strain: Lactobacillus paracasei OUT0010 strain (Accession number: NITE P-03014) (2) Test method All cheeses except those made with the commercial cheese starter CHN-11 were produced at the dairy processing laboratory at the National Agriculture and Food Research Organization's Ikenodai Plant. Gouda cheese was produced using four batches of strain 527 as the starter, eight batches using strains 527 and OUT together, four batches using strains 210 and OUT together, and four batches using strains 215 and OUT together. The pH of the cheese curds during the production process was measured and recorded over time by measuring the pH of the whey drained from the cheese curds. For each batch, the average value was calculated from the time the starter was added to the raw milk until the pH reached 5.6. Gouda cheese made with the commercial cheese starter CHN-11 was produced three times at a cheese factory in Hokkaido. Based on the production records, the average time from the time the starter was added to the raw milk until the curds reached pH 5.6 during cooling was calculated. FIG. 7 shows the time (minutes) required for the pH to reach 5.6 after the starter was added to the raw milk. Values marked with different letters on the bar graph in Figure 7 indicate significant differences at the 5% level in the multiple comparison test using the Tukey-Kramer method.

[0035] As shown in Figure 7, it was confirmed that the novel lactic acid bacteria strains 210 and 215 of the present invention showed a significantly faster pH decrease during production in the actual Gouda cheese manufacturing process than the strain 527, which has a proven track record as a milk fermentation starter, and the commercially available starter CHN11, i.e., they have a higher lactic acid production ability. From these results, it was revealed that the novel lactic acid bacteria of the present invention, when used as a starter bacterium in cheese production, have excellent lactic acid production ability in the curd molding process and can shorten the time required for cheese production. Furthermore, from the data in Figure 7 for the 527 strain alone and the combination of the 527 strain and the OUT strain, and the CHN11 strain alone and the combination of the CHN11 strain and the OUT strain, it is confirmed that the time (minutes) to reach pH 5.6 does not change significantly, and it is also clear that the lactic acid production ability does not change significantly even when the OUT strain is used in combination.

[0036] <Example 8: Confirmation test of viable cell count in cheese produced using a novel lactic acid bacteria strain> (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains Strain 527: Lactococcus lactis strain 527 (a milk fermentation starter bacterium distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) OUT strain: Lactobacillus paracasei OUT0010 strain (Accession number: NITE P-03014) (2) Test method The Gouda cheese used to measure the viable count of starter lactic acid bacteria was produced at the dairy processing laboratory at the Ikenodai Plant of the National Agriculture and Food Research Organization (NARO). The cheese was vacuum-packed in nylon polyfilm and aged in a ripening chamber set at 10°C. To measure the viable count, 1 g of shredded cheese curd was sampled in 50 mL of sterilized 2% sodium citrate solution, suspended in a blender, and serially diluted to prepare the sample. The viable counts of strains 210, 215, and 527 were measured on M17 agar plates. The viable count of strain OUT0010 was measured on MRS agar plates. Figure 8 shows the viable cell counts in Gouda cheese produced using three types of lactic acid bacteria as starter bacteria at each ripening period (0, 1, 30, 60, 90, 180, and 360 days).

[0037] As shown in Figure 8, the novel lactic acid bacteria strains 210 and 215 of the present invention were found to have higher viable cell counts in cheese throughout the entire ripening period than the strain 527, which has a proven track record as a milk fermentation starter. 5 Even in 180-day aged cheese, where the number of cfu / g decreased to about 1 × 10 8 A viable bacterial count exceeding cfu / g was confirmed.

[0038] Example 9: Tasting evaluation test of fermented milk products produced using novel lactic acid bacteria strains Drinkable yogurt was produced using strain 210, a novel lactic acid bacterium of the present invention, and a tasting evaluation was carried out. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain (2) Method for producing and evaluating drinkable yogurt As a production starter, 100 mL of fresh fermented milk was prepared using strain 210. To 8 L of sterilized raw milk, 1% (80 mL) of fresh fermented milk from 210 strains of bacteria was added as a starter, and 5% (400 g) of sucrose based on the weight of the raw milk was added and stirred well, then the mixture was left to ferment for 10 hours in a 30°C incubator. After fermentation was complete, the mixture was cooled in a 10°C refrigerator for approximately 12 hours, homogenized in a blender, filled into containers, and stored refrigerated until evaluation. This was used as the drink yogurt for evaluation. The tasting evaluation was carried out at three different locations on the 8th and 9th days after production, where evaluators (31 people in total, consisting of staff and students from the research project participating institutions) tasted and evaluated the drink yogurt. After tasting the drink yogurt, the evaluators verbalized what they felt was different from the commercially available products they regularly consume. The responses collected were compiled as follows to form the evaluation test results. (3) Results Of the responses given regarding features that were perceived as different from commercially available products, the number of respondents and percentages (%) of those mentioned by five or more people are shown in Table 5. [Table 5]

[0039] As shown in Table 5, 61% of the evaluators rated the drink yogurt sampled as "easier to drink" than the commercially available product. This is thought to be due to the preference for its mild acidity and moderate sweetness, and the smooth drinking experience. The sugar content of typical commercially available drink yogurts is around 5-8%, so the amount of sucrose added to the drink yogurt sampled was less than that of commercially available products. However, approximately 40% of the evaluators answered that it was "sweet." This suggests that the balance with the mild acidity allows the natural sweetness of milk (the sweetness of lactose) to be brought out. It is assumed that this is what the evaluator felt. In this evaluation test, no one gave negative answers to the food, such as "unpleasant taste, aroma, or texture." These results demonstrate that the novel lactic acid bacterium strain 210 of the present invention is suitable as a starter for fermented milk and various yogurts. Drinkable yogurt produced using strain 210 as a starter was found to be as palatable as or better than commercially available products, and was an easy-to-drink product in which the sweetness of the raw milk itself could be tasted.

[0040] <Example 10: Confirmation test 3 of the lactic acid production ability of the novel lactic acid bacteria strain> In Example 7, the results of Gouda cheese production were shown using the novel lactic acid bacteria strains 210 and 215 of the present invention in combination with the OUT strain. However, the novel lactic acid bacteria strains 210 and 215 of the present invention were used alone as starters to confirm the time required for a specific pH to be reached in the Gouda cheese production process. The pH reached in this test was set to 5.6. In addition, the changes in moisture content, fat content, and pH of the obtained Gouda cheese during the ripening period were confirmed. (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain 215 strains: Lactococcus cremoris 215 strains Strain 527: Lactococcus lactis strain 527 (a milk fermentation starter bacterium distributed by the Japan Dairy Technology Association, a public interest incorporated foundation) CH1 strain: Lactococcus lactis CH1 strain (a milk fermentation starter bacterium manufactured by Christian Hansen) (2) Test method Cheese production was performed at the dairy processing laboratory at the National Agriculture and Food Research Organization's Ikenodai Plant. Baby Gouda cheese was produced using 16 L of raw milk and strains 210, 215, 527, and CH1 as starters, with three replicates performed on different production days. Six baby Gouda cheese balls were produced per test plot. One ball was used for test sample preparation on days 1, 30, 60, 90, and 180 of ripening, and then shredded with a knife. The pH of the cheese curds during the production process was measured and recorded over time as the whey drained from the cheese curds. The average value was calculated for each test plot from the time the starter was added to the raw milk until the pH reached 5.6. FIG. 9 shows the time (minutes) required for the pH to reach 5.6 after the starter was added to the raw milk. Values marked with different letters on the bar graph in Figure 9 indicate significant differences at the 5% level in the multiple comparison test using the Tukey-Kramer method. The resulting Gouda cheese was aged at 10°C. Samples were prepared as described above on days 1, 30, 60, 90, and 180 after aging, and the moisture, fat, pH, viable bacterial count, and free amino acid content of the cheese were measured. The production test was performed three times, and the average values are shown in Figures 10 to 14. Furthermore, the amount of free glutamic acid (μmol / g) in the cheese after 90 days of aging is shown in Figure 15, and the glutamic acid concentration (μmol / g) in the cheese after 1, 30, 60, 90, and 180 days of aging is shown in Figure 16.

[0041] As shown in Figure 9, the novel lactic acid bacteria strains 210 and 215 of the present invention decreased the pH significantly faster during production in the actual Gouda cheese manufacturing process than the commercially available starter strain CH1, which has a proven track record as a milk fermentation starter. Furthermore, although there was no significant difference with the results of strain 527, they reached a pH of 5.6 within 400 minutes on average, suggesting their extremely high lactic acid production capacity. From these results, it was revealed that the novel lactic acid bacteria of the present invention, when used as a starter bacterium in cheese production, have excellent lactic acid production ability in the curd molding process and can shorten the time required for cheese production. As shown in Figure 10, the moisture content in Gouda cheese produced using the novel lactic acid bacteria strains 210 and 215 of the present invention tended to be higher throughout the entire maturation period than that of strains 527 and CH1, which have a proven track record as dairy fermentation starters. As shown in Figure 11, the fat content in Gouda cheese produced using the novel lactic acid bacteria strains 210 and 215 of the present invention tended to be lower than that of strains 527 and CH1, which have a proven track record as dairy fermentation starters, after 60 days of aging. As shown in Figure 12, it was confirmed that the pH value in Gouda cheese produced using the novel lactic acid bacteria strains 210 and 215 of the present invention tended to be equal to or lower than that of strains 527 and CH1, which have a proven track record as milk fermentation starters, after 60 days of ripening. The pH of cheese produced using strains 210 and 215 was stable from the early stages of ripening, suggesting little variation in quality. As shown in Figure 13, it was confirmed that the viable cell count in Gouda cheese produced using the novel lactic acid bacteria strains 210 and 215 of the present invention was higher throughout the entire ripening period after 60 days of ripening than the 527 and CH1 strains, which have a proven track record as milk fermentation starters. Generally, natural cheese is a food that allows lactic acid bacteria to be ingested, but a problem has been that the viable cell count of lactic acid bacteria contained in cheese rapidly decreases as the ripening period increases. However, it was suggested that this problem could be solved by using the novel lactic acid bacteria strains 210 and 215 of the present invention as starters. As shown in Figure 14, the free amino acid content of Gouda cheese produced using the novel lactic acid bacteria strains 210 and 215 of the present invention and aged for 90 days, the optimum aging period, showed characteristically high levels of certain components depending on the test group. Cheeses from strains 210 and 215 had high contents of glutamic acid (Glu), which provides umami, and sarcosine (Sar), which provides sweetness, suggesting the possibility of differentiating them from the reference cheese in terms of taste. Furthermore, cheeses from strains 210 and 215 had high contents of branched-chain amino acids (Val, Leu, Ile), which have been shown to maintain muscle function, and imidazole dipeptides (Ans, Car), which have been shown to have anti-fatigue effects, suggesting the possibility of differentiating them from the reference cheese in terms of functionality. Furthermore, because common Cremoris species do not exhibit arginine deiminase (ADI) activity, arginine (Arg) is detected as a metabolic product, but arginine (Arg) was hardly detected in cheese from strains 210 and 215. Instead, citrulline and ornithine, which are converted from arginine (Arg) by ADI, were detected in large amounts. This suggests that strains 210 and 215 are lactic acid bacteria with metabolic enzymes different from those of common Cremoris species. As shown in Figure 15, the amount of free glutamic acid in Gouda cheese produced using the novel lactic acid bacteria strains 210 and 215 of the present invention and aged for 90 days, the optimum period for aging, was found to be significantly higher than that of strains 527 and CH1, which have proven records as dairy fermentation starters. Values marked with different letters on the bar graph indicate significant differences at the 5% level in a multiple comparison test using the Tukey-Kramer method. Similarly, as shown in Figure 16, it was confirmed that the free glutamic acid concentration in Gouda cheese produced using the novel lactic acid bacteria strains 210 and 215 of the present invention was higher throughout the entire maturation period than that of the strains 527 and CH1, which have a proven track record as dairy fermentation starters.

[0042] <Example 11: Verification test 1 of fermented butter produced by a new lactic acid bacteria strain> (1) Lactic acid bacteria tested 210 strain: Lactococcus cremoris 210 strain Strain 217: Lactococcus cremoris strain 215 Ll23 strain: Lactococcus lactis Ll-23 strain (Danisuco) (2) Test method Butter was produced using commercially available fresh cream (Yotsuba Pure Fresh Cream NH47, milk fat content 47%). Fat globules and buttermilk were prepared from the fresh cream using a food processor (TESCOM) equipped with a butter preparation unit. After removing the buttermilk, the fat globules were washed twice with cold water, and the resulting fat globules were kneaded using a reverse sheeter (Nippon Kneader Co., Ltd.). The weight of the obtained butter was measured, and freeze-dried strains of each lactic acid bacteria were added so that the number of bacteria per weight was the same, followed by further kneading. These were filled into cups and cultured at 20°C for 2 days. A control was prepared in the same manner without the addition of lactic acid bacteria. Each fermented butter was prepared three times, and the number of lactic acid bacteria, pH value, water content and fat content (general components) in each fermented butter, and aroma components were analyzed using the following analytical methods. The average values of the lactic acid bacteria count and pH value in the fermented butter are shown in Table 6, the average values of the water content and fat content in the fermented butter in Table 7, and the average values of the aroma components in the fermented butter in Table 8. Each obtained value was subjected to a multiple comparison test using the Tukey-Kramer method. Values marked with different letters in Tables 6 to 8 indicate that there is a significant difference at the 5% level between the test intervals.

[0043] (3) Analysis method of aromatic components Each fermented butter was added with 3-octanol as an internal standard to prepare an analytical sample, which was then subjected to GC-MS analysis using solid-phase microextraction (SPME), and the relative area ratio of the peak area of each aroma component to that of the internal standard was calculated. The values in Tables 8 and 9 show the average ± standard deviation of three preparations of each fermented butter. <Solid Phase Microextraction (SPME)> SPME type: 50 / 30μm DVB / CAR / PDMS (Merck) Sample incubation: 50°C, 30 min Adsorption: 50°C, 30 minutes <gc-ms> ·GC equipment, analysis conditions Equipment: GC-2010 (Shimadzu Corporation) Injection: Splitless Inlet: 250℃ Sampling time: 300 seconds Gas: Helium Column: HP-INNOWax (60 m x 0.25 mm, 0.25 μm) Column flow rate: 1.0 mL / min Column temperature program: 40°C, 5 min hold → Heating rate 4°C / min → 220°C, 10 min hold (total 60 min) ·MS equipment, analysis conditions Equipment: GCMS-QP2010 (Shimadzu Corporation) Interface: 250℃ Ion source: 230℃ Measurement mode: Scan (40.00 to 350.00 m / z) Energy: 70 eV

[0044] [Table 6]

[0045] [Table 7]

[0046] [Table 8]

[0047] As shown in Tables 6 and 7, no significant differences were observed in the moisture and fat contents of the fermented butters produced. Furthermore, no differences were observed in the number of lactic acid bacteria in the fermented butters. On the other hand, the pH value of the fermented butter prepared using strain 210 was significantly lower than that prepared using the commercially available strain L123, and tended to be lower in the fermented butter prepared using strain 215. These findings demonstrate that the novel lactic acid bacteria strains 210 and 215 of the present invention have higher acid-producing ability than the commercially available strain L123, even in butter, which is a W / O emulsion with a low water content, and are therefore suitable for preparing fermented butter that suppresses the growth of unwanted bacteria due to its low pH. Furthermore, as shown in Table 8, when the proportions of diacetyl and acetoin, which are the main aroma components of fermented butter, were compared, no significant difference was observed for diacetyl except between the control and test sections, but the proportion of acetoin was significantly higher in strain 215 than in strain 210, and tended to be lower in strain 210 than in strain L123, a commercially available strain, while tending to be higher in strain 215. Since the intensity of these aromas affects palatability, it was suggested that by using the two novel lactic acid bacteria strains of the present invention appropriately, it may be possible to manufacture products tailored to customer needs.

Claims

1. A lactic acid bacterium selected from Lactococcus cremoris strain 210 (accession number: NITE P-04049) and Lactococcus cremoris strain 215 (accession number: NITE P-04050).

2. The lactic acid bacterium according to claim 1, which is used for producing dairy products.

3. The lactic acid bacterium according to claim 1, which is used as a cheese starter bacterium.

4. A dairy product containing the lactic acid bacteria according to any one of claims 1 to 3.

5. A cheese containing the lactic acid bacteria according to any one of claims 1 to 3.

6. A method for producing a dairy product, characterized by using the lactic acid bacterium according to any one of claims 1 to 3.

7. A method for producing cheese, characterized by using the lactic acid bacterium according to any one of claims 1 to 3.

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