METHOD OF PREPARATION OF BIFIDOBACTERIA-CONTAINING FERMENTED MILK

Abstract: Lactic acid bacteria or one or more strains selected from the group consisting of Lactobacillus casei C-2203 (FERM P-9106), Lactobacillus casei C-2204 (FERM P-­ 9107), Lactobacillus casei C-2205 (FERM P-9108) and Lactobacillus casei C-2254 (FERM P-9109; FERM BP-1723) and Bifidobacterium longum are cultivated in mixture or are mixed after being separately cultivated, and thus a bifidobacteria-containing lactic acid bacteria-fermented milk with an elevated survival rate of Bifidobacterium longum is obtained.

Description: FIELD OF THE INVENTION The present invention relates to a method of preparation of bifidobacteria-containing fermented milk in which the survival rate of Bifidobacterium longum has been elevated. In general, bifidobacteria have a significant relation to health for all of sucklings, infants and adults, and recently, it has even been said that the absence of bifidus bacteria in intestines is abnormal. Strains of Lactobacillus casei for use in the present invention have a function of extremely elevating the survival rate of Bifidobacterium longum as compared with common lactic acid bacteria, and the Bifidobacterium longum can well survive in the fermented milk products of the present invnetion during cold storage. Accordingly, as it is expected that a noticeable amount of living bacteria of Bifidobacterium longum could reach intestines, when the milk products are actually drunk or eaten, the products are considered to have significantly advantageous merits in the fields of healthy food industries and milk product industries. BACKGROUND OF THE INVENTION Hitherto, there are numerous medicines and foods prepared by utiliizng bifidobacteria, and in particular, many of milk products such as yogurt, fermented milk, powdery yogurt, etc. are bifidobacteria-containing products. For the preparation of such bifidobacteria-containing products, a method of adding a concentrated powder of bifidobacteria to a yogurt is often utilized. As the kind of the strains of the bacteria for use in the said method, there are recommended Bifidobacterium breve for products for sucklings and Bifidobacterium longum for products for infants and adults ( Kagaku-to-Seibutsu , Vol. 24, No. 1, pages 8-9). Further, it has also been tried to elevate the survival rate of bifidus bacteria by incubation of bifidus bacteria ( Bifidobacterium longum ) together with lactic acid bacteria ( Streptococcus thermophilus ) ( Japanese Journal of Dairy and Food Science , Vol. 33, No. 6, 1984, A203-A211). The present inventors cultivated known Lactobacillus casei , which is good in taste as lactic acid bacteria in mixture with Bifidobacterium longum , which is recommended for infants and adults, or alternatively blended the separately cultivated bacteria, so as to obtain various bifidobacteria-containing fermented milks, and then stored the thus-prepared milks for 7 days at 10°C to examine the survival rate of the bifidus bacteria in the respective milks. As a result, it was found that the survival rate of Bifidobacterium longum was only from 0.33 to 0.87 % or so in all the examined samples. In view of the fact that the general cold distribution period may be one week or so, such low survival rate of from 0.33 to 0.87 % means that the bifidobacteria could hardly reach intestines when the bacteira-containing products are actually drunk or eaten. SUMMARY OF THE INVENTION Having a conception that any other strains of Lactobacillus casei as derived from various sources than hitherto known strains of Lactobacillus casei may have a function of elevating the survival rate of Bifidobacterium longum , the present inventors earnestly carried out the selection and isolation of the intended strains, and as a reuslt, have found that four strains of Lactobacillus casei as newly isolated by the present invention are effective for elevating the survival rate of Bifidobacterium longum . The strains as newly isolated by the present invention include the following, which were deposited in Fermentation Research Institute, Agency of Industrial Science and Technology, Japan with the respective numbers parenthesized. Lactobacillus casei C-2203 (FERM P-9106), Lactobacillus casei C-2204 (FERM P-9107), Lactobacillus casei C-2205 (FERM P-9108) and Lactobacillus casei C-2254 (FERM P-9109; FERM BP-1723) "The present invention brings about a method of prepring bifidobacteria containing fermented milk, which comprises cultivating lactic acid bacteria in admixture with Bifidobacterium longum, characterized in that the lactic acid bacteria belong to one or several of the Lactobacillus casei strains: C-2203 (FERM P-9106), C-2204 (FERM P-9107, C-2205 (FERM P-9108) and C-2254 (FERM P-9109; FERM BP-1723). Lactic acid bacteria of one or more strains selected from the group consisting of the said strains are cultivated in mixture with Bifidobacterium longum , or alternatively, the separately cultivated bacteria or the processed materials obtained therefrom are blended, whereby the survival rate of the said bacteria of Bifidobacterium longum can be elevated up to about from 8.4 to 17.7 % or so under storage at 10°C for 7 days. DETAILED DESCRIPTION OF THE INVENTION Bacteriological properties of C-2203, C-2204, C-2205 and C-2254 are mentioned below, which are recognized to correspond to the properties of Lactobacillus casei . Regarding the surface appearance of BL agar medium, after grown thereon, C-2203, C-2204 and C-2254 showed a rough form, while C-2205 showed an intermediate state of a rough form and a smooth form. Bacteriological Properties of Four Strains of C-2203, C-2204, C-2205 and C-2254: Gram Staining : Positive, Catalase : Negative, Gas Production : Negative, Growth at 15°C : Positive, Growth at 45°C : Positive, Optical Activity of Lactic Acid : L(+), Assimilation of Carbohydrates: (+) : Rhamnose, Glucose, Mannose, Fructose, Galactose, Sucrose, Maltose, Cellobiose, Lactose, Trehalose, Melezitose, Ribose, Mannitol, Sorbitol, Amygdalin, Esculin, Salicin. (-) : Arabinose, Xylose, Melibiose, Raffinose, Starch. Cell morphology when cultivated on BL agar at 37°C for 48 to 72 hours: Size : 0.5 to 1.1 µm. Form : Usually bacilli, but often coccobacilli. Characteristics of colonies when cultivated on BL agar (by Nissui Pharamaceutical Co., Japan) at 37°C for 48 to 72 hours: Form : Circle. Projection : Cone. Circumferential edge: Smooth. Size : 0.5 to 2 mm Color : Dark brown in the center, while light brown to gray in the circumferential edge. Surface : Rough form (C-2203, C-2204, C-2254), Intermediate of rough form and smooth form (C-2205). One or more strains among the above-mentioned four strains of Lactobacillus casei are inoculated in a milk medium and incubated at 37°C for 16 hours or so to prepare a starter. As the Bifidobacterium longum for the present invention, anyone of known strains or newly isolated strains can be used. For example, there may be mentioned Bifidobacterium longum ATCC 15707, Bifidobacterium longum CZ-1907, etc. The strain of Bifidobacterium longum is incubated in a medium where the said strain can grow, for example, a reconstituted skim milk medium to which an yeast extract was added, at 37°C for 18 hours or so to prepare a starter. According to the present invention, the starter of the above-mentioned lactic acid bacteria and the starter of Bifidobacterium longum are cultivated in mixture or separately. When these are cultivated in mixture, the former starter is added to a milk medium in a proportion of 5 to 1 % and the latter starter 10 to 1 %, and cultivated therein at 37°C for 15 hours, to obtain a bifidobacteria-containing fermented milk. When these are cultivated separately, the starter of the lactic acid bacteria is added to a milk medium in a proportion of from 5 to 1 % and cultivated therein at 37°C for 18 hours to obtain a lactic acid bacteria-fermented milk. On the other hand, the starter of Bifidobacterium longum is added to the other milk medium in a proportion of from 10 to 1 % and cultivated therein at 37°C for 18 hours to obtain a cultivated material of Bifidobacterium longum . Next, a cultivated material (the lactic acid bacteria-fermented milk or the cells separated therefrom) prepared by the use of at least one strain of the four strains of Lactobacillus casei and a cultivated material of Bifidobacterium longum or a processed material thereof are blended. Alternatively, a processed material of the above-mentioned lactic acid cultivated material and a cultivated material of Bifidobacterium longum or a processed material thereof are blended. The cultivated material may be the cultivated liquid medium itself of the bacteria or the cells separated from the cultivated liquid medium, and the processed material includes a concentrated mateial, a spray-dried material, a frozen material or a vacuum freeze-dried material, or the like to be obtained from the cultivated material. These may be blended in any state, but it is preferred that the number of the living bacteria of Bifidobacterium longum is adjusted to be from 10⁸ to 10⁹/ml or so in the lactic acid bacteria-fermented milk. In the practice of the present invention, any other lactic acid bacteria may optionally added to the fermented milk, if desired. As the milk medium for the present invention, any conventional milk material medium which is generally used for preparation of fermented milk may be employed, e.g., including a cow milk, a skim milk, a reconstituted skim milk, etc. In accordance with the present invention as herein illustrated in detail, the strains of C-2203, C-2204, C-2205 and C-2254 are effective for extremely elevating the survival ratio of Bifidobacterium longum in a bifidobacteria-containing fermented milk during storage. The following examples are intended to illustrate the present invention. EXPERIMENTAL EXAMPLE 1 Test of Survival Rate of Bifidobacterium longum by Blend Cultivation Method: A 10 % reconstituted skim milk medium was autoclaved at 121°C for 15 minutes, and then 1 % of a starter of the strain of Lactobacillus casei (shown in Table 1 below) which had previously been grown in the same medium and 10 % of a starter of the strain of Bifidobacterium longum (shown in Table 1 below) which had been grown in a 15 % reconstituted skim milk medium (containing 0.4 % of yeast extract and autoclaved at 121°C for 15 minutes) were inoculated in the said medium in mixture and cultivated for 15 hours at 37°C. This was stirred and kept at 10°C. Measurement: Immediately after the preparation and after stored for 7 days at 10°C, the cultivated material was sampled, and the pH value and the number of the living bacteria in the respective samples were measured (by a modified Mitsuoka Method). (Almost no difference was admitted in the number of L . casei between immediately after the preparation and after stored for 7 days.) Strains Tested: Results: The results obtained are shown in Table 1 below. EXPERIMENTAL EXAMPLE 2 Test of Survival Rate of Bifidobacterium longum by Strain of Lactobacillus casei Method: A 10 % reconstituted skim milk medium was autoclaved at 121°C for 15 minutes, and then 3 % of a starter of the strain of Lactobacillus casei (shown in Table 2 below) which had previously been grown in the same medium was added thereto and cultivated for 15 hours at 37°C. On the other hand, 0.4 % of yeast extract was added to a 15 % reconstituted skim milk and autoclaved at 121°C for 15 min., and then 10 % of a starter of the strain of Bifidobacterium longum (shown in Table 2 below) which had previously been grown in the same medium was added thereto and cultivated for 15 hours at 37°C. 10 parts of the thus cultivated material were added to 90 parts of the lactic acid bacteria-fermented milk described above, and then well blended and stored at 10°C. Measurement: Immediately after the preparation and after stored for 7 days at 10°C, the cultivated material was sampled and the pH value and the number of the living bacteria in the respective samples were measured (by a modified Mitsuoka Method). (Almost no difference was admitted in the number of L . casei between immediately after the preparation and after stored for 7 days.) Strains Tested: Results: The results obtained are shown in Table 2 below. EXAMPLE 1 A 10 % reconstituted skim milk was pasturized under heating at 95°C for 30 minutes to prepare a milk medium, and 1 % of a starter of Lactobacillus casei C-2203 as cultivated in the same medium was inoculated in the said milk medium and cultivated for 18 hours at 37°C. On the other hand, 0.4 % of yeast extract was added to a 15 % a reconstituted skim milk and pasteurized under heating at 95°C for 30 minutes to prepare a milk medium, and 10 % of a starter of Bifidobacterium longum ATCC 15707 as cultiavted in the same medium was inoculated in the said milk medium and cultivated for 18 hours at 37°C. 10 kg of the bifidobacteria-fermented milk and 70 kg of the lactic acid bacteria-fermented milk previously described were blended and 20 kg of a sterilized water was added thereto, and the resulting mixture was homogenized with a homogenizer to obtain 100 kg of a fermented milk product with a non-fat milk solid content of 8 %. The product had a mild and favorable taste. Immediately after the preparation, this had a pH value of 4.1 and contained 2.6 x 10⁸ cells/ml of B . longum and 7.8 x 10⁸ cells/ml of L . casei . After stored for 7 days at 10°C, the number of the living B . longum was 1.0 x 10⁷ cells/ml. EXAMPLE 2 A 10 % reconstituted skim milk was pasteurized under heating at 95°C for 30 minutes to prepare a milk medium, and a starter of Lactobacillus casei C-2204 and a starter of Lactobacillus casei C-2254, which has been cultivated separately in the same medium, were inoculated in the said milk medium each in an amount of 0.5 % and cultivated for 18 hours at 37°C. On the other hand, 0.4 % of yeast extract was added to a 15 % reconstituted skim milk and pasteurized under heating at 95°C for 30 minutes to prepare a milk medium, and 10 % of a starter of Bifidobacterium longum CZ-1970 as cultivated in the same medium was inoculated in the said milk medium and cultivated for 18 hours at 37°C. 10 kg of the bifidobacteria-fermented milk and 70 kg of the lactic acid bacteria-fermented milk previously described were blended and 20 kg of a sterilized water was added thereto, and the resulting mixture was homogenized with a homogenizer to obtain 100 kg of a fermented milk product with a non-fat milk solid content of 8 %. The product had a mild and favorable taste. Immediately after the preparation, this had a pH value of 4.2 and contained 2.7 x 10⁸ cells/ml of B . longum and 7.8 x 10⁸ cells/ml of L . casei . After stored for 7 days at 10°C, the number of the living B . longum was 1.2 x 10⁷ cells/ml. EXAMPLE 3 A 10 % reconstituted skim milk was pasteurized under heating at 95°C for 30 minutes to prepare a milk medium, and 1 % of a starter of Lactobacillus casei C-2203 as cultivated in the same medium and 10 % of a starter of Bifidobacterium longum ATCC 15707 as cultivated in a milk medium (prepared by adding 0.4 % of yeast extract to a 15 % reconstituted skim milk and pasteurized under heat at 95°C for 30 minutes) were inoculated therein in mixture and cultivated for 15 hours at 37°C. A syrup prepared by dissolving 8 kg of sugar in 7 kg of water followed by pasteurization of the resulting solution at 100°C for 5 minutes and cooling was blended with 85 kg of the fermented milk containing Lactobacillus casei and bifidobacteria and homogenized to obtain 100 kg of a fermented milk product with a non-fat milk solid content of 8 %. The product had a mild and favorable taste. Immediately after the preparation, this had a pH of 4.4 and contained 2.2 x 10⁹ cells/ml of B . longum and 5.0 x 10⁸ cells/ml of L . casei . After stored for 7 days at 10°C, the number of the living B . longum was 3.9 x 10⁸ cells/ml. EXAMPLE 4 A 10 % reconstituted skim milk was pasteurized under heating at 95°C for 30 minutes to prepare a milk mdeium, and 1 % of a starter of Lactobacillus casei C-2254 as cultivated in the same medium was inoculated in the said milk medium and cultivated for 18 hours at 37°C. On the other hand, a liquid medium containing 5 % of protease-treated whey powder, 0.4 % of yeast extract and 1 % of calcium carbonate was pasteurized under heating at 95°C for 60 minutes, and after 0.2 % of ascorbic acid was added thereto, 5 % of a starter of Bifidobacterium longum CZ-1907 as cultivated in the same medium was inoculated and cultivated for 18 hours at 37°C. The cultivated cells were harvested by using a Scharples-type centriguge, washed and suspended in a 15 % reconstituted skim milk, pasteurized (95°C, 30 min), to which 1 % sodium glutamate had been added. The obtained cell suspension was lyophilized under the conditions of freezing temperature at -30°C, drying shelf temperature at 25°C and degree of vacuum at 0.01 Torr to obtain a bifidobacteria powder 1 kg of bifidobacteria powder and 85 kg of the lactic acid bacteria-fermented milk previously described were blended and 14 kg of sterilized water was added thereto, and the resulting mixture was homogenized with a homogenizer to obtain 100 kg of a fermented milk product with a non-fat milk solid content of 8 %. The product had a mild and favorable taste. Immediately after the preparation, this had a pH value of 4.4 and contained 3.1 x 10⁸ cells/ml of B . longum and 5.2 x 10⁸ cell/ml of L . casei . After stored for 7 days at 10°C, the number of the living B . longum was 2.7 x 10⁷ cells/ml. EXAMPLE 5 0.4 % of yeast extract was added to a 15 % reconstituted skim milk and pasteurized under heating at 95°C for 30 minutes to prepare a milk medium, and 10 % of a starter of Bifidobacterium longum CZ-1907 as cultivated in the same medium was inoculated in the said milk medium and cultivated for 18 hours at 37°C. On the other hand, a liquid medium containing 5 % of protease-treated whey powder, 0.4 % of yeast extract and 1 % of calcium carbonate was pasteurized under heating at 95°C for 60 minutes, and 1 % of a starter of Lactobacillus casei C-2205 as cultivated in the same medium was inoculated thereto and cultivated for 18 hours at 37°C. The cultivated cells were harvested by using a Sharples-type centrifuge, washed and suspended in a pasteurized 15 % reconstituted skim milk to which 1 % sodium glutamate had been added. The obtained cell suspension was liophilized under the conditions of freezing temperature at -30°C, drying shelf temperature at 25°C, and degree of vacuum at 0.01 Torr to obtain a lactic acid bacteria powder. 1 kg of the lactic acid bacteria powder and 85 kg of the bifidobacteria-fermented milk previously described were blended and 14 kg of sterilized water was added thereto, and the resulting mixture was homogenized with a homogenizer to obtain 100 kg of a fermented milk product with a non-fat milk solid content of 8 %. The product had a mild and favorable taste. Immediately after the preparation, this had a pH value of 4.4 and contained 4.5 x 10⁸ cells/ml of B . longum and 3.9 x 10⁸ cells/ml of L . casei . After stored for 7 days at 10°C, the number of the living B . longum was 5.7 x 10⁷ cells/ml. EXAMPLE 6 A liquid medium containing 5 % of the protease-treated whey powder, 0.4 % of yeast extract and 1 % of calcium carbonate was pasteurized under heating at 95°C for 60 minutes, and after 0.2 % of ascorbic acid was added thereto, 2 % of a starter of Bifidobacterium longum CZ-1907 as cultivated in the same medium was inoculated and cultivated for 18 hours at 37°C. The cultivated cells were harvested by using a Sharples-type centrifuge, washed, suspended in a pasteurized 15 % reconstituted skim milk, and frozen with dry ice. On the other hand, a liquid medium containing 5 % of protease-treated whey powder, 0.4 % of yeast extract and 1 % of calcium carbonate was pasteurized under heating at 95°C for 60 minutes, and 1 % of a starter of Lactobacillus casei C-2205 as cultivated in the same medium was inoculated thereto, and cultivated for 18 hours at 37°C. The cultivated cells were harvested by using a Sharples-type centrifuge, washed, suspended in a pasteurized 15 % reconstituted skim milk and frozen with dry ice. 1 kg of the former frozen material and 1 kg of the later frozen material were blended with 98 kg of milk containing 1 % of fat and 8.5 % of non-fat milk solid content, and homogenized with a homogenizer to obtain 100 kg of a bifidobacteria-containing milk. This contained 3.7 x 10⁸ cells/ml of B . longum and 4.6 x 10⁸ cells/ml of L . casei . After stored for 7 days at 10°C, the number of the living B . longum was 1.1 x 10⁸ cells/ml.

Claims: 1. A method of preparing bifidobacteria containing fermented milk, which comprises cultivating lactic acid bacteria in admixture with Bifidobacterium longum, characterized in that the lactic acid bacteria belongs to one or several of the Lactobacillus casei strains : C-2203 (FERM P-9106, C-2204 (FERM P-9107), C-2205 (FERM p-9108) and C-2254 (FERM P-9109 ; FERM BP-1723). 2. A method according to claim 1, characterized in that a material prepared by cultivating one or several of said strains is blended with a material obtained by cultivating Bifidobacterium longum or by processing the same. 3. A method according to claim 1, characterized in that a processed material of a material prepared by cultivating one or several of said strains is blended with a material obtained by cultivating Bifidobacterium longum or by processing the same. 4. A method according to any of claims 1 to 3, characterized in that the pH of the mixture, immediately after the preparation, is 4.1 to 4.5. 5. A method according to any of claims 1 to 4, characterized in that the mixture contains, immediately after the preparation, per ml, 2.6 x 10⁸ to 2.6 x 10⁹ living Bifidobacterium longum cells and living Lactobacillus casei cells of one or several of said strains. 6. Fermented milk product, which contains living lactobacillus and Bifidobacterium cells, characterized in that the Lactobacilli belong to one or several strains C-2203 (FERM P-9106), C-2204 (FERM P-9107), C-2205 (FERM-P-9108) and C-2254 (FERM P-9109 ; FERM BP-1723), and that the Bifidobacterium is Bifidobacterium longum. 7. Fermented milk product according to claim 6, characterized in that it contains, after 7 days storage at 10°C, 10⁷ to 4.6 x 10⁸ living Bifidobacterium longum cells per ml. 8. Fermented milk product according to claims 6 or 7, characterized in that its pH is 4.1 to 4.5 immediately after its preparation and after 7 days storage at 10°C.

Inventors: FUTAMI, AKIRA, HAYAKAWA, KUNIHIKO, SAITO, YOSHIO, TAKANO, TOSHIAKI
Applicants: THE CALPIS FOOD INDUSTRY CO., LTD.
Application year: 1988
Main classification: A23C9
IPC codes: A23C9, C12R1, C12N1