BIFIDOBACTERIUM-FERMENTED MILK AND PROCESS FOR PRODUCING THE SAME

Abstract: A process for producing bifidobacterium-fermented milk by mix-culturing Bifidobacterium·breve with Lactococcus· lactis·subspecies·lactis , which forms neither diacetyl nor acetoin, on a medium composed mainly of milk; the bifidobacterium-fermented milk thus obtained; and Lactococcus·lactis·subspecies·lactis strain YIT2027 (FERM BP-6224). By the above process, an extremely large viable count of the bifidobacteria can be achieved without resort to any growth promoter and the survivability of the bifidobacteria during the storage of the fermented milk can be highly improved. Because of being free from diacetyl or acetoin, the obtained fermented milk has a good flavor.

Description: The present invention relates to bifidobacterium-fermented milk and production process thereof. The bifidobacteria are known to be useful bacteria found in the human intestines and exhibit inhibitory action against the growth of pathogenic bacteria occurring in conjunction with lowering of the pH in the intestines caused by the production of an organic acid. The bifidobacteria, in general, have however the characteristics as will be described below, which have posed problems from the viewpoints of the production of bifidobacterium-fermented milk and maintenance of a viable count in the fermented milk. 1. Owing to their complex and severe nutritional requirements, they do not proliferate in a pure milk medium not containing a growth promoter such as yeast extract. In addition, the growth promoter is usually expensive and moreover, the addition of it impairs the flavor of the cultured product. 2. Because of being obligate anaerobe, they do not grow and tend to die out in the presence of oxygen. 3. Compared with the ordinary lactic acid bacteria for dairy use, they have markedly low acid resistance so that it is difficult to allow them to grow for a long time in a low pH range as in fermented milk. various researches have been made with a view to improving the survivability of the bifidobacteria. For example, Japanese Patent Application Laid-Open No. Sho 54-14585 describes that improvements in the proliferative property and viable count of the bifidobacteria and lactic acid bacteria are brought about even under aerobic conditions by mix-culturing of the bifidobacteria with slow-acid-producing lactic acid bacteria such as Lactobacillus·acidophilus or Lactobacillus·salivarius. Furthermore, Japanese Patent Application Laid-Open No. Sho 63-94938 describes that the survivability of the bifidobacteria is improved by mix-culturing of the bifidobacteria with Streptococcus·diacetylactis . The above literature says that Streptococcus·diacetylactis , different from Streptococcus·cremoris or Streptococcus·lactis which is analogous thereto, is characterized by the formation of diacetyl and acetoin from citric acid in the fermentation substrate by the decarbonation reaction. Carbon dioxide formed by the reaction increases the anaerobic degree of the fermented milk and is therefore effective for the protection of the bifidobacteria. The Streptococcus · diacetylactis is however accompanied with the problem that it is not suited for practical use, because the resulting diacetyl and acetoin impart bifidobacterium-fermented milk with unfavorable flavor. Accordingly, an object of the present invention is to provide bifidobacterium-fermented milk improved in the survivability of bifidobacteria without using a growth promoter and without forming diacetyl and acetoin; and a production process thereof. Disclosure of the Invention With the foregoing in view, the present inventors have repeated extensive investigations to overcome the above-described problem. As a result, it has been found that by mix-culturing a specific lactic acid bacterium, which has been even regarded in Japanese Patent Application Laid-Open No. Sho-63-94938 to have no effects for improving survivability of the bifidobacteria and has also been regarded in Japanese Patent Application Laid-Open No. Sho 54-14585 to be unusable because it kills the bifidobacteria in the initial stage of the incubation, with a specific bifidobacterium, a markedly higher viable count of bifidobacteria than that obtained by the ordinary incubating method can be achieved even on a pure milk medium free from a growth promoter; and that the survivability of the bifidobacteria during the storage of the fermented milk can be highly improved, leading to the completion of the present invention. The present invention therefore provides a process for producing bifidobacterium-fermented milk, which comprises mix-culturing Bifidobacterium·breve with Lactococcus· lactis·subspecies·lactis , which forms neither diacetyl nor acetoin, on a medium composed mainly of milk; the bifidobacterium-fermented milk thus obtained; and Lactococcus·lactis·subspecies·lactis YIT2027 (FERM BP-6224) which is a novel strain. Best Modes for Carrying Out the Invention Lactococcus·lactis·subspecies·lactis is conventionally used for the production of cheese and the like, but that used in the present invention features that it forms neither diacetyl nor acetoin. Specific examples include strain YIT2027 (FERM BP-6224) and strain YIT2008 (ATCC 19435), of which the strain YIT2027 is particularly preferred. It should be noted that " Lactococcus · lactis · subspecies · lactis " according to new taxonomy embraces " Streptococcus·lactis·subspecies · diacetylactis " according to the old taxonomy which is employed in the above-described Japanese Patent Application Laid-Open No. Sho 63-94938, however, the latter one is eminently different from the strain to be used in the present invention because it forms diacetyl and acetoin. The lactic acid bacterium to be used in the present invention corresponds to " Streptococcus·lactis·subspecies·lactis " according to the old taxonomy which has been regarded to have capacity of forming neither diacetyl nor acetoin and have no effects for improving the survivability of the bifidobacteria in the above literature. The lactic acid bacterium to be used in the present invention is not included in the "slow-acid-producing lactic acid bacteria" referred to in Japanese Patent Application Laid-Open No. Sho 54-14585, because when it is subjected to monocyte incubation under aerobic conditions, the product obtained by incubation for 48 hours has an acidity of at least 10. It corresponds to a group of the lactic acid bacteria which has been regarded to bring the bifidobacteria into death in the initial stage of the incubation and therefore be unusable. Biochemical properties of the strain YIT 2027 belonging to Lactococcus·lactis·subspecies·lactis which forms neither diacetyl nor acetoin are shown in Table 1 and Table 2. YIT2027 Lactococcus·lactis·subspecies·lactis Lactococcus·lactis·subspecies·lactis Gram staining Positive Positive Positive Morphology Spherical Spherical Spherical Catalase Gas production from glucose Proliferation 10°C 40°C 45°C 60°C for 30 min. 2% NaCl 4% NaCl 6.5% NaCl 0.1% methylene blue milk 40% bile Hydrolysis of arginine Hydrolysis of esculin Hydrolysis of starch Hydrolysis of hippurate Reduction of nitrate V-P test GC content (%) 38.6 (Tm method) 35.8 YIT2027 Lactococcus·lactis·subspecies·lactis Lactococcus·lactis· subspecies·lactis Sugar fermentation test Arabinose Xylose Rhamnose Ribose Mannose Fructose Galactose Sucrose - Maltose Cellobiose Lactose Trehalose Melibiose Raffinose Melezitose Mannitol Sorbitol Esculin Salicin Amygdalin Sorbose Inositol Inulin Starch α-Methyl-D-glucoside Glucose Dextrin Glycogen As is apparent from Table 1 and Table 2, the strain according to the present invention coincides with the known Lactococcus·lactis·subsp·lactis in many properties, however, is different in the hydrolysis of hippurate and sugar fermentation properties by xylose, sucrose and dextrin. The applicant of the present invention therefore identified it as a novel strain and deposited it as FERM BP-6224 to National Institute of Bioscience and Human Technology, Ministry of International Trade and Industry (address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-0046, Japan) under the Budapest Treaty (date of original deposit: February 10, 1997). Examples of the Bifidobacterium·breve to be used in the present invention include the strain YIT4065 [FERM BP-6223, deposited to National Institute of Bioscience and Human Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-0046, Japan), date of deposition: February 29, 1996] and strain YIT4014 (ATCC 15700), of which the strain YIT4065 is particularly preferred. In the present invention, no particular limitation is imposed on the medium to be used upon mix-culturing of the above-described bifidobacteria and lactic acid bacteria insofar as it is composed mainly of milk. For example, any milk (whole milk, skim milk and reconstituted milk from such powdery milk) having a milk solid content concentration of about 8 to 20 wt.% which is usually provided for the incubation of lactic acid bacteria can be used. It is not necessary to use a growth promoter such as yeast extract or peptone or a reducing agent such as L-ascorbic acid or L-cysteine, which is often added to a medium conventionally used for bifidobacteria or lactic acid bacteria, but it can be added when the influence of it on the flavor of the final product falls within a permissible range or when it does not disturb the properties of the final product. The inoculation ratio of the starter of the bifidobacteria to the starter of the lactic acid bacteria preferably falls within a range of 10000:1 to 1:1, with 100:1 to 10:1 being particularly preferred. The inoculation amount is preferably 0.5 to 10 wt.% (which will hereinafter be called "%", simply), with 1 to 2 % being particularly preferred. The incubation is effected at a temperature permitting the growth of both bifidobacteria and lactic acid bacteria, more specifically, 35 to 39°C, with 36 to 38°C being particularly preferred. The incubation is carried out until the bifidobacteria and lactic acid bacteria reach the steady proliferation state. More specifically, it is preferred to conduct incubation until pH reaches 4.2 to 4.6, particularly, 4.3 to 4.5. The product obtained by incubation may be provided as a food containing bifidobacteria and lactic acid bacteria without any further treatment or may be provided as a beverage after added with a sweetener, fruit juice, water, fragrance or the like as needed to adjust its concentration and flavor. In addition, it may be provided as a food or preparation in the form of powders or tablets containing bifidobacteria and lactic acid bacteria after drying. No limitation is imposed on the processing means of the incubation product or form of the processed product insofar it does not cause these bacteria to death. As described above, Bifidobacterium·breve is recognized to exhibit both proliferation promoting effects and survivability improving effects when incubated together with Lactococcus·subsp·lactis which forms neither diacetyl nor acetoin. Some slow-acid-producing lactic acid bacteria such as Lactobacillus·acidophilus and Lactobacillus·bulgaris are recognized to have proliferation promoting effects of bifidobacteria but they do not bring about marked survivability improving effects. The reason for the exhibition of both effects is presumed to owe to that when bifidobacteria are used in combination with Lactococcus·subsp·lactis , incubation can be conducted under the conditions advantageous for bifidobacteria by controlling the temperature in consideration of the difference in the optimum incubation temperature between Lactococcus·subsp·lactis and Bifidobacterium·breve and a symbiotic relation permitting improvements in nutritional requirement and oxygen resistance of Bifidobacterium·breve is established between them. - Examples - The present invention will hereinafter be described more specifically by examples. It should however be borne in mind that the present invention is not limited by them. Example 1 Confirmation of proliferation promoting effects and survivability improving effects for bifidobacteria brought about by mix-culturing with lactic acid bacteria On each of 10% reconstituted skim milk mediums, which had been sterilized by heating at 95°C for 30 minutes and then cooled to 37°C, used as a fermentation substrate, 1% of a starter for Bifidobacterium . breve strain YIT4065 (FERM BP-6223) was inoculated. To the resulting medium, Streptococcus . thermophilus , Lactobacillus . bulgaris or Lactococcus . lactis . subspecies . lactis strain YIT2027 (FERM BP-6224) was inoculated in an amount of 0.01%. The inoculated medium and a control medium inoculated with only the above-described bifidobacteria were incubated at 37°C until the pH became 4.3. The viable count of bifidobacteria after incubation was measured. The cell solution after incubation was filled in a glass-made container, followed by anaerobic storage at 10°C for 2 weeks. The viable count of bifidobacteria after storage was also confirmed. The results are shown in Table 3. Lactic acid bacteria Viable count of bifidobacteria (/ml) when incubation is completed after storage Not added 8.0 x 10 8 Streptococcus·thermophilus 6.3 x 10 7 7.0 x 10 5 Lactobacillus·bulgaris 1.5 x 10 7 1.0 x 10 5 Lactococcus·lactis·subspecies·lactis 2.0 x 10 9 3.4 x 10 8 As is apparent from Table 3, it has been recognized that compared with not only the control but also the Bifidobacterium . breve incubated with other lactic acid bacteria, the Bifidobacterium breve incubated with the strain YIT2027 exhibited a larger viable count of the bifidobacteria, indicating marked bifidobacterium proliferation promoting effects. Even after the storage of the cell solution, the viable count of bifidobacteria is maintained high at a low pH, indicating marked survivability improving effects. Example 2 Confirmation of bifidobacterium survivability in a product having a non-fat milk solid content of 8% (anaerobic storage) To a medium of 20% reconstituted skim milk, 1% of a starter of Bifidobacterium breve strain YIT4065 (FERM BP-6223) and 0.01% of a starter of Lactococcus·lactis·subspecies·lactis strain YIT2027 (FERM BP-6224) were inoculated, followed by incubation at 37°C until the pH reached 4.4. The incubated product was homogenized in a conventional manner. To the homogenized product, a cell solution obtained by incubating Streptococcus·thermophilus , sweetener, a syrup containing HM pectin as a stabilizer and flavor were added for the adjustment of flavor, whereby a fermented milk product having a non-fat milk solid content of 8% was obtained. As a comparative example, Bifidobacterium·breve strain YIT4065 was incubated on a conventionally-employed medium of 10% reconstituted skim milk containing 0.05% yeast extract and a fermented milk product having a nonfat milk content of 8% was obtained similarly. These products were each charged in a glass-made container, followed by anaerobic storage at 10°C for 2 weeks. The viable counts of bifidobacteria before and after storage were confirmed. The results are shown in Table 4. Incubation method Viable count of bifidobacteria (/ml) Stored for 0 day Stored for 7 days Stored for 14 days Conventional method (using yeast extract) 3.1 x 10 8 4.5 x 10 7 1.7 x 10 7 Method according to the present invention (using lactis ) 6.9 x 10 8 5.1 x 10 8 1.8 x 10 8 As is apparent from Table 4, it has been confirmed that by the application of the present invention to the production of fermented milk beverage, a high viable count of bifidobacteria can be achieved and even after storage, the viable count at least 10 times as much as that of the conventional method using a growth promoter can be maintained. Example 3 Confirmation of bifidobacteria survivability in a product having a nonfat milk solid content of 4% (aerobic storage) To a medium of 20% reconstituted skim milk, 1% of a starter of Bifidobacterium . breve strain YIT4065 (FERM BP-6223) and 0.01% of a starter of Lactococcus . lactis . subspecies . lactis strain YIT2027 (FERM BP-6224) were inoculated, followed by incubation at 37°C until the pH reached 4.4. The incubated product was homogenized in a conventional manner. To the homogenized product, a cell solution obtained by incubating Streptococcus . thermophilus , sweetener, a syrup containing HM pectin as a stabilizer and flavor were added for the adjustment of flavor, whereby a fermented milk product having a nonfat milk solid content of 4% was obtained. As a comparative example, Bifidobacterium . breve strain YIT4065 was incubated on a conventionally-employed medium of 10% reconstituted skim milk containing 0.05% yeast extract and a fermented milk product having a nonfat milk content of 4% was obtained similarly. These products were each charged in a polystyrene-made container, followed by storage at 10°C for 2 weeks. The viable counts of bifidobacteria before and after storage were confirmed. The results are shown in Table 5. Incubation method Viable count of bifidobacteria (/ml) Stored for 0 day Stored for 7 days Stored for 14 days Conventional method (using yeast extract) 1.5 x 10 8 1.1 x 10 7 9.2 x 10 5 Method according to the present invention (using lactis ) 4.7 x 10 8 1.0 x 10 8 2.3 x 10 7 As is apparent from Table 5, it has been recognized that markedly high survivability improving effects are brought about even for a product having a low nonfat milk solid content or a product exposed to aerobic storage conditions after production so that high general-purpose properties of the present invention are confirmed. Capability of Exploitation in Industry As described above, according to the present invention, a markedly high viable count of the bifidobacteria can be achieved even without the addition of a growth promoter and in addition, the survivability of bifidobacteria during the storage of the fermented milk can be improved substantially. Because of being free from diacetyl and acetoin, the bifidobacterium-fermented milk obtained according to the present invention has a good flavor.

Claims: A process for producing bifidobacterium-fermented milk, which comprises mix-culturing Bifidobacterium ·breve with Lactococcus ·lactis ·subspecies ·lactis YIT2027 (FERM BP-6224), which forms neither diacetyl nor acetoin, on a medium composed mainly of milk. A process for producing bifidobacterium-fermented milk according to claim 1, wherein Bifidobacterium ·breve is YIT4065 (FERM BP-6223). Bifidobacterium-fermented milk obtainable by a process as claimed in claim 1 or 2. Lactococcus ·lactis ·subspecies ·lactis YIT2027 (FERM BP-6224).

Inventors: AKAHOSHI RYOICHI, MATSUI AKIHISA, MITA KAORI, MIZOBUCHI TAKAHIRO, AKAHOSHI, RYOICHI, MATSUI, AKIHISA, MITA, KAORI, MIZOBUCHI,TAKAHIRO, AKAHOSHI, RYOICHI, KABUSHIKI KAISHA YAKULT HONSHA, MATSUI, AKIHISA, KABUSHIKI KAISHA YAKULT HONSHA, MITA, KAORI, KABUSHIKI KAISHA YAKULT HONSHA, MIZOBUCHI,TAKAHIRO, KABUSHIKI KAISHA YAKULT HONSHA
Applicants: YAKULT HONSHA KK, KABUSHIKI KAISHA YAKULT HONSHA
Application year: 1998
Main classification: A23C9
IPC codes: A23C9, C12N1, C12R1