# Patent

## Title
Bacterium of the genus bifidobacterium and fermented foods using the same

## Abstract

The present invention provides a bacterium belonging to the genus Bifidobacterium, which can exhibit a high viability in the storage under agitation and can produce a fermented food containing a large number of viable bacterial count. The present invention further provides a fermented food, which contains the bacterium belonging to the genus Bifidobacterium and exhibits various physiological effects such as inhibitory action against harmful intestinal bacteria and intestinal function controlling effects. The present invention further provides a bacterium belonging to the genus Bifidobacterium having a rate of viability of 30% or more, when the bacterium is stored in the aerobic condition under agitation at 10° C. for 14 days after culturing in a medium containing milk as a main ingredient until obtaining a viable count of 1×108 cfu/ml or more, and fermented food using the same.

## Background
TECHNICAL FIELD
The present invention relates to bacteria belonging to the genus Bifidobacterium having a high viability even after storing under agitation, and fermented foods containing the same.
BACKGROUND ART
Bacteria belonging to the genus Bifidobacterium are obligate anaerobes and are weak in an aerobic condition. In fermentation products, it is difficult to handle in view of proliferation during the production and viability under storing. In order to obtain physiological effects (intestinal function controlling effect or the like) of the bacteria belonging to the genus Bifidobacterium, they must reach the intestine in a viable form as much as possible. It is thought to be an important factor in the production of the fermented foods of this type to increase in the viability of bacteria in the fermented foods and an arriving rate of bacteria to the intestine after meal.
As a result of improvement in bacterial strains by various methods, strains improved in proliferation and viability have recently been prepared. In addition, an improvement in viability is performed also in fermented foods by an improvement of production process and addition of various agents for improving the viability, such as N-acetyl glucosamine, pantothenic acid, pantethine, pantetheine, peptides and lactulose.
However, in the production site of the fermented foods, it is difficult to pack all fermented products of Bifidobacterium bacteria on the same day from the viewpoint of production cost and working ability, and at present, semimanufactured products products admixed with other additives or the like are stored in the aerobic storage tank for several days. In order to stably supply a product with a superior viability satisfying a quality standard for maintaining high viable count even under such aerobic circumstance, further improvement in the viability is required.
Under such circumstance, viable bacteria living in severe conditions such as aerobic condition or addition of acid did not always exhibit increased viability. As a result, it was extremely difficult to obtain a bacterial strain having more superior viability as compared with known bacteria. Further, even by the improvement of the production process and the addition of various viability improving agents, it was difficult to maintain the viability of bacteria in the fermented foods which were aerobically stored in the production site, at a sufficiently high level even after shipment of the products.
Consequently, an object of the present invention is to provide a bacterium belonging to the genus Bifidobacterium having a high viable count and good viability, even after storing for desired term until packing into a vessel and shipping to the market after fermentation of the bacterium, and fermented foods produced by using the same.
DISCLOSURE OF THE INVENTION
Taking such actual circumstances into consideration, the present inventors studied extensively and paid attention on the fact that in the storage of fermented milk before shipment, an agitation treatment was commonly given in a tank in order to maintain uniformity of the fermented foods. During the production of fermented foods, it was thought that Bifidobacterium bacteria were forced to be exposed to severe environment for the bacteria. The present inventors found that the viability of bacterium in the fermented product was significantly improved, when the bacterium belonging to the genus Bifidobacterium having a nature exhibiting a high viable count after aerobic storage with agitation was selected, and the fermented product, produced by fermenting using the bacterium, was stored with agitation. The present inventors also found that viability of bacteria under the storing condition was significantly improved, when fermented foods were produced by using said fermented product, and thereby completed the present invention.
Namely, an object of the present invention is to provide a bacterium belonging to the genus Bifidobacterium having a rate of viability of 30% or more, when the bacterium is stored in the aerobic condition under agitation at 10° C. for 14 days after culturing in a medium containing milk as a main ingredient until obtaining a viable count of 1×108 cfu/ml or more.
Another object of the present invention is to provide a fermented food containing a fermented product fermented with the bacterium belonging to the genus Bifidobacterium. 
Further object of the present invention is to provide a fermented food containing bacteria belonging to the genus Bifidobacterium in a fermented food containing a fermented product fermented with one or more bacteria belonging to the genus Bifidobacterium, wherein a viable count of the bacteria belonging to the genus Bifidobacterium in said food immediately after production is 1×108 cfu/ml or more, and a rate of viability of any one of the bacteria belonging to said genus Bifidobacterium is 30% or more, when the food is stored in the aerobic condition under agitation at 10° C. for 14 days.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows viable counts of various strains, when fermented milk products containing each strain are stored under agitation at 10° C. in Example 2.
FIG. 2 shows viable counts of various strains, when fermented milk products containing each strain are stored under agitation at 10° C. for 1 week, packed and stored anaerobically at standing.



BEST MODE FOR CARRYING OUT THE INVENTION
Bacteria belonging to the genus Bifidobacterium of the present invention are the bacteria having a rate of viability of 30% or more, when the bacteria are stored in the aerobic condition under agitation at 10° C. for 14 days after culturing in a medium containing milk as a main ingredient until obtaining a viable count of 1×108 cfu/ml or more. The medium containing milk as a main ingredient herein is the medium containing milk as a main nutrient source and capable of growth of the bacteria belonging to the genus Bifidobacterium. Examples of the milk include cow's milk (whole milk) and skim milk as a processed product thereof.
Culture condition for culturing the bacteria belonging to the genus Bifidobacterium in the medium is not particularly limited, and may be suitably set depending on the growth condition of the bacteria belonging to the genus Bifidobacterium used. Culture is performed generally at 30 to 40° C., preferably at 33 to 37° C., preferably under anaerobic condition. Among the bacteria belonging to the genus Bifidobacterium, some strains are difficult to grow by a nutrient source of milk alone. In such case, essential nutrient source and growth promoting substance for Bifidobacterium bacteria such as various sugars, yeast extract, peptide and the like may be added. Further, it can also be simultaneously cultured with lactic acid bacteria such as Lactococcus lactis which can promote growth of Bifidobacterium bacteria by cocultivating therewith.
Among media used for culturing Bifidobacterium bacteria, a medium which can be generally used include a liquid medium containing 20% by weight (hereinafter simply designated as %) of skim milk and 0.03% of Meast. Almost all Bifidobacterium bacteria can be grown in said medium, and the bacteria of the present invention are able to exhibit a viability of 30% or more in the above storage condition under agitation after culturing up to a viable count of 1×109 cfu/ml or more.
As the Bifidobacterium bacteria of the present invention, the strain exhibiting a rate of viability of 30% or more after culturing up to a viable count of 1×109 cfu/ml or more in said medium is particularly preferable.
Generally, in the production of food and drink products containing Bifidobacterium bacteria such as fermented milk, since it is required to obtain a large viable count with small amount, the product is designed for maintaining a viable count of 1×107 cfu/ml or more in the product after storage. The strain, as like the present invention, having a viable count of 1×108 cfu/ml or more and a high rate of viability after storage with agitation has high tolerability (rate of viability) for agitation in the food and drink product with a high viable count, and at the same time, when the product is stored by standing after storage with agitation, i.e. after the product is manufactured into a commercial form, the rate of viability in the storage state is significantly high. Especially, strains having a high rate of viability after culturing up to a viable count of 1×109 cfu/ml or more are superior in the above effect.
Further, in the present invention, the storage with agitation means that cultured products, in which Bifidobacterium bacteria are cultured in the medium containing milk as a main ingredient, or fermented foods appropriately prepared by mixing with syrup or the like are stored under the condition with continued agitation by using stirring bar or mixing propeller in the aerobic condition.
Confirmation of the rate of viability of Bifidobacterium bacteria is performed by using a method, in which, specifically, 300 ml of cultured product or fermented food is charged into 300 ml flask with a stirring bar, cotton plugged, and stored under stirring at about 120 rpm by using magnetic stirrer in an incubator at 10° C.
Rate of viability indicates an extent to which the viable bacterial cells exist after storage, and viable count can be measured by the conventional method. For example, viable count can be obtained by spreading a properly diluted fermented product (food) containing viable cells on TYL agar plate medium, culturing anaerobically at 37° C. for 72 hours, and counting the number of colonies on the medium.
In the present invention, “rate of viability” means a rate of the viable count after storage under stirring (at 10° C. for 14 days) to the viable count of the cultured liquid, which is cultured up to a viable count of 1×108 cfu/ml or more in the medium containing milk as a main ingredient, or the viable count before storage in the fermented food immediately after production.
The Bifidobacterium bacteria having the rate of viability of 30% or more in such storage under agitation can not only give a high rate of viability, even after the storage under agitation in the tank, when a fermented food is produced by using said strain, but also maintain the high viability even after manufacturing and storing a commercial product. Consequently, Bifidobacterium bacteria of the present invention are quite useful.
In the present invention, species of the bacterium belonging to the genus Bifidobacterium is not particularly limited. For example, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium animalis, Bifidobacterium pseudocatenulatum and Bifidobacterium adolescentis can be suitably used. Among them, Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium longum are preferable, since viability improving effects thereof after production of fermented foods are high, and safeties thereof are sufficiently confirmed, and Bifidobacterium breve is particularly preferable.
Such strains having a high rate of viability after storing under agitation can be obtained, for example, by culturing parent strain of Bifidobacterium bacteria, storing the thus obtained fermented product with agitation for about 2 weeks, and selecting a highly resistant strain from viable cells.
The present inventors have selected two strains which were recognized to have a high rate of viability after storing under agitation. Strains Bifidobacterium breve YIT 10001 (FERM BP-8205) and Bifidobacterium breve YIT 10002 (FERMBP-8206) were deposited in International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Chuo No. 6, 1-1, Higashi-1-chome, Tsukuba, Ibaraki, 305-8566 Japan) on Aug. 14, 2001.
Bifidobacterium breve YIT 10001 and Bifidobacterium breve YIT 10002 have following taxonomical properties.










TABLE 1







YIT 10001
YIT 10002


















Gram stain
positive
positive


Morphology
Rods of various
Rods of various



shapes
shapes


Catalase
−
−


Gas production from glucose
−
−


Growth at


20° C.
−
−


30° C.
±
±


37° C.
+
+


40° C.
+
+


45° C.
−
−


in 0.2% bile
±
±


Hydrolysis of arginine
−
−


Benzidine test
−
−


Nitrate reduction
−
−



















TABLE 2







YIT 10001
YIT 10002




















Glycerol
−
−



Erythritol
−
−



D-Arabinose
±
±



L-Arabinose
−
−



Ribose
+
+



D-Xylose
−
−



L-Xylose
−
−



Adonitol
−
−



β-Methyl-xyloside
−
−



Galactose
+
+



D-Glucose
+
+



D-Fructose
+
+



D-Mannose
+
+



L-Sorbose
−
−



Rhamnose
−
−



Dulcitol
−
−



Inositol
−
−



Mannitol
−
−



Sorbitol
−
−



α-Methyl-D-mannoside
−
−



α-Methyl-D-glucoside
+
+



N-Acetyl glucosamine
−
−



Amygdaline
−
+



Arbutine
+
+



Esculin
+
+



Salicin
+
+



Cellobiose
+
+



Maltose
+
+



Lactose
+
+



Melibiose
+
+



Saccharose
+
+



Treharose
−
−



Inulin
−
−



Melezitose
+
+



D-Raffinose
+
+



Amidon
−
±



Glycogen
−
−



Xylitol
−
−



β-Gentiobiose
−
+



D-Turanose
+
+



D-Lyxose
−
−



D-Tagatose
−
−



D-Fucose
−
−



L-Fucose
W
W



D-Arabitol
−
−



L-Arabitol
−
−



Gluconate
−
−



2-ceto-gluconate
−
−



5-ceto-gluconate
−
−







+: positive,



W: weakly positive,



±: false positive,



−: negative






The strain having a high rate of viability after storage under agitation can also be obtained by treating with ultraviolet ray or mutagenic agents such as nitrosoguanidine (NTG) and ethyl methansulfonate (EMS).
The fermented food of the present invention contains at least one fermented product which is fermented by the above exemplified bacteria belonging to the genus Bifidobacterium. When the fermented food immediately after production (which contains Bifidobacterium bacteria having a viable count of 1×108 cfu/ml or more) is stored under agitation at 10° C. for 14 days in the aerobic condition, a rate of viability of the Bifidobacterium bacteria is 30% or more. Examples of the fermented food include fermented milk, fermented soybean milk, fermented fruit juice and fermented vegetable milk, and the fermented milk and fermented soybean milk are particularly preferable due to significantly improved viabilities thereof after storage of the product.
The fermented food preferably contains the Bifidobacterium bacteria having a viable count of 1×109 cfu/ml or more in the fermented product after cultivation, and contains 1×108 cfu/ml or more immediately after manufacturing the product by adding other components. When the bacteria are grown to such extent, a particularly superior improved effect in viability can be obtained as compared with use of other strains (e.g. parent strain).
In the production of the fermented food, microorganisms other than the Bifidobacterium bacteria of the present invention may be used in combination. Examples of such microorganisms include: bacteria belonging to the genus Lactobacillus such as Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus gallinarum, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus kefir, Lactobacillus paracasei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. bulgaricul, Lactobacillus helveticus, Lactobacillus zeae and Lactobacillus salivalius; bacteria belonging to the genus Streptococcus such as Streptococcus thermophilus; bacteria belonging to genus Lactococcus such as Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis; bacteria belonging to the genus Bacillus such as Bacillus subtilis; and yeast belonging to the genus Saccharomyces, Torulaspora and Candida such as Saccharomyces cerevisiae, Torulaspora delbrueckii and Candida kefyr. 
Among these, bacteria belonging to the genus Lactobacillus, the genus Streptococcus and the genus Lactococcus are preferable, because a fermented milk product, produced by combining one or more bacteria selected therefrom, has a superior acceptability and is easy to drink continually. These bacteria are also preferable due to improved effect for viability.
The fermented product of the present invention can be produced by the conventional methods. For example, in order to produce a fermented milk, firstly a bacterium belonging to the genus Bifidobacterium is inoculated alone or in combination with other microorganism in a sterilized milk medium, and cultured to obtain a fermented milk base after homogenization. A separately prepared syrup solution is added thereto and mixed, and homogenized by using a homogenizer, and then flavor is added to prepare a final product.
The thus obtained fermented milk of the present invention can be processed to any type of product such as plane type, soft type, fruit flavor type, solid type and liquid type.
Food materials used in the conventional fermented food such as various sugars, emulsifier, thickener, sweetener, acidifier and fruit juice can optionally be admixed. Specific examples thereof include: sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose and xylose; sugar alcohol such as sorbitol, xylitol, erythritol, lactitol, palatinit, reduced glutinous starch syrup and reduced glutinous maltose syrup; emulsifier such as sucrose esters of fatty acid, glycerin esters of fatty acid and lecithin; thickener (stabilizer) such as carrageenan, xanthan gum, guar gum, pectin and locust bean gum; acidifier such as citric acid, lactic acid and malic acid; and fruit juice such as lemon juice, orange juice and berry juice. Vitamins such as vitamin A, vitamin B, vitamin C, vitamin D and vitamin E and minerals such as calcium, iron, manganese and zinc can also be added.
EXAMPLES
The present invention will be explained in more detail by Examples, but the present invention is not limited to these Examples.
Example 1
A parent strain, a strain of Bifidobacterium breve (YIT 4065: FERM BP-6223, deposited in International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Chuo No. 6, 1-1, Higashi-1-chome, Tsukuba, Ibaraki, 305-8566 Japan) on Feb. 2, 1996) (cephalothin-streptomycin resistant strain) and Lactococcus lactis were inoculated into a medium containing 20% skim milk, and mix-cultured at 37° C. for 24 hours to prepare a bacterial solution. To this bacterial solution, a bacterial solution obtained by culturing Streptococcus thermophilus using 20% skim milk at 37° C. for 20 hours was added (mixing ratio=1:2). A syrup solution containing reduced glutinous maltose syrup was added to a final concentration of 5% to prepare a fermented milk product. The fermented milk product was poured into a glass bottle and was agitated using stirrer under aerobic environment with cotton plugged during storage period. Under this condition, the bottle was stored at 10° C. for 2 weeks. Viable bacteria were smeared on the medium, and 2,000 strains of single colony were isolated.
The fermented milk product was prepared with the isolated strains and the parent strain of the strain of Bifidobacterium A by the same way as described above. Into a 300 ml flask with stirrer, 300 ml of the mixture was poured, cotton plugged and stored with agitation at 120 rpm using stirrer. Viabilities of bacteria during the storage were compared, and two strains which showed superior viabilities than the strain A were obtained. Viability after 2 weeks of storage under agitation is shown in Table 3. Two strains (YIT 10001 and YIT 10002) exhibited 30% or more of viabilities after 2 weeks of storage under agitation. Viable counts were performed using the TYL medium having a composition shown in Table 4.











TABLE 3








Rate of viability
Initial viable



Strain
(%)
count (cfu/ml)




















Strain A
11
 5.6 × 108



YIT 10001
31
5.22 × 108



YIT 10002
33
5.54 × 108

















TABLE 4





Composition of TYL medium


















Trypticase peptone (BBL)
1.0 (%)



Yeast extract (Difco)
0.5



Lab lemco powder (Oxoid)
0.3



Potassium dihydrogen phosphate
0.3



Dipotassium hydrogen phosphate
0.48



Ammonium sulfate
0.3



Magnesium sulfate
0.02



L-cysteine
0.05



Lactose
1.0



Agar
1.2










Example 2
Viability Test
Bifidobacterium breve YIT 10001, YIT 10002 and strain A as the bacteria belonging to the genus Bifidobacterium were used to prepare fermented milk products.
Namely, 2 lit. of 20% milk medium was charged into a 2.5 L jar-fermenter, strains of the bacteria belonging to the genus Bifidobacterium hereinabove and Lactococcus lactis YIT 2027 were inoculated as seed starters in 2% and 0.01%, respectively, and cultured at 37° C. for 24 hours to prepare a bacterial solution by homogenizing at 15 Mpa. Streptococcus thermophilus was cultured in 20% skim milk at 37° C. for 20 hours to prepare a bacterial solution by homogenizing at 15 Mpa. These bacterial solutions were mixed at a ratio of 1:2, and a syrup solution containing reduced glutinous maltose syrup was added to the final concentration of 5% to prepare a fermented milk product (viable counts were: strain A 5.65×108 cfu/ml, YIT 10001 5.34×108 cfu/ml, and YIT 10002 5.45×108 cfu/ml).
Viable counts of these samples were measured by the same way as in Example 1 after storage under agitation at 10° C., or after agitating for 1 week and packing into a test tube, and then anaerobically stored by replacing the air with nitrogen gas. Results are shown in FIG. 1 and FIG. 2.
Viabilities of YIT 10001 and YIT 10002 stored for 14 days exceeded 30% and were improved as compared with the strain A. Viabilities of both strains after stored under agitation and maintained by standing storage were also improved significantly as compared with the strain A.
Example 3
In a 2.5 lit. jar-fermenter, 2 lit. of 24% milk medium was charged. Seed starters of Bifidobacterium breve YIT 10001 and Lactococcus lactis YIT 2027 were inoculated, in 2% and 0.01%, respectively, and cultured at 37° C. for 24 hours to prepare a bacterial solution by homogenizing at 15 Mpa. Seed starters of Lactobacillus casei and Lactobacillus acidophilus were inoculated each in 0.5% in 20% skim milk, cultured at 37° C. for 24 hours to prepare a bacterial solution by homogenizing at 15 Mpa. These bacterial solutions were mixed at a ratio of 2:3, and a syrup solution containing palatinose was added to a final concentration of 10% to prepare a fermented milk product (Initial viable count of YIT 10001: 5.76×108 cfu/ml).
In the fermented milk product, YIT 10001 exhibited a rate of viability of 30% or more after the storage at 10° C. for 2 weeks under agitation in the same way as in Example 1. Taste was also good.
INDUSTRIAL APPLICABILITY
Bacteria belonging to the genus Bifidobacterium of the present invention exhibits a high rate of viability even after storage under agitation, and a fermented food containing a large amount of viable microorganisms can be produced. Consequently, the fermented food of the present invention contains a high viable count, and exhibits a high physiological effect such as suppressive action against harmful intestinal bacteria and intestinal function controlling effects possessed by the bacteria belonging to the genus Bifidobacterium.

## Claims

1. A bacterium belonging to the genus Bifidobacterium having a rate of viability of 30% or more, when the bacterium is stored in the aerobic condition under agitation at 10° C. for 14 days after culturing in a medium containing milk as a main ingredient until obtaining a viable count of 1×108 cfu/ml or more, wherein the bacterium is Bifidobacterium breve YIT 10001.

2. A fermented food comprising a fermented product fermented by a bacterium belonging to the genus Bifidobacterium according to claim 1.

3. The fermented food according to claim 2 comprising fermented milk.

4. The fermented food according to claim 2, further containing a sweetener.

5. A fermented food comprising bacteria belonging to the genus Bifidobacterium in a fermented food comprising a fermented product fermented with one or more bacteria belonging to the genus Bifidobacterium according to claim 1, wherein a viable count of the bacteria belonging to the genus Bifidobacterium in said food immediately after production is 1×108 cfu/ml or more, and a rate of viability of any one of the bacteria belonging to said genus Bifidobacterium is 30% or more, when the food is stored in the aerobic condition under agitation at 10° C. for 14 days, and wherein the bacteria belonging to the genus Bifidobacterium are Bifidobacterium breve YIT 10001.

6. A method for producing a bacterium belonging to the genus Bifidobacterium having a rate of viability of 30% or more, when the bacterium is stored in aerobic conditions, the method comprising:
a) inoculating Bifidobacterium and Lactococcus lactis into a medium comprising 20% by weight of skim milk,
b) culturing the mixture of a) at 30 to 40° C. for 24 hours under anaerobic conditions,
c) adding to the culture of b) a bacterial solution, wherein the bacterial solution is a solution of (a) Streptococcus thermophilus or (b) Lactobacillus casei and Lactobacillus acidophilus, 
d) adding a syrup solution comprising a reduced glutinous maltose syrup to a final concentration of 5% to prepare a fermented milk product,
e) storing the fermented milk product under agitation and aerobic conditions at 10° C. for 14 days, and
f) smearing a viable bacteria on a medium and isolating the Bifidobacterium having a rate of viability of 30% or more,
wherein the bacterium belonging to the genus Bifidobacterium is Bifidobacterium breve YIT 10001.

7. The method for producing a bacterium belonging to the genus Bifidobacterium of claim 6, wherein the bacterial solution of c) is obtained by culturing Streptococcus thermophilus using 20% skim milk at 37° C. for 20 hours, and wherein a mixing ratio of the culture and the bacterial solution is 1:2.

8. The method for producing a bacterium belonging to the genus Bifidobacterium of claim 6, wherein the bacterial solution of c) is obtained by culturing Lactobacillus casei and Lactobacillus acidophilus using 20% skim milk at 37° C. for 20 hours, and wherein a mixing ratio of the culture and the bacterial solution is 2:3.