EQUOL-PRODUCING LACTIC ACID BACTERIA-CONTAINING COMPOSITION

Abstract

An equol-producing lactic acid bacteria-containing composition comprises, as an essential component thereof, a lactic acid bacterial strain belonging to the genus Lactococcus having an ability to utilize at least one daidzein compound selected from the group consisting of daidzein glycosides, daidzein, and dihydrodaidzein to produce equol. Such a composition is effective for the prevention and alleviation of malaise inclusive of climacteric disturbance in middle-aged and elderly women for which no effective prophylactic method or alleviating means has heretofore been available.

Claims

1. An equol-producing lactic acid bacteria-containing composition comprising, as an essential component thereof, a lactic acid bacterial strain belonging to the genus Lactococcus having an ability to utilize at least one daidzein compound selected from the group consisting of daidzein glycosides, daidzein, and dihydrodaidzein to produce equol.

2. The composition according to claim 1, wherein said lactic acid bacterial strain belonging to the genus Lactococcus is Lactococcus garvieae.

3. The composition according to claim 2, wherein said lactic acid bacterial strain belonging to Lactococcus is Lactococcus 20-92 deposited under FERM BP-10036.

4. The composition according to claim 1 further comprising at least one member selected from the group consisting of daidzein compounds and daidzein compound-containing ingredients.

5. The composition according to claim 4, wherein the daidzein compound-containing ingredient is soybean flour or soy milk

6. The composition according to claim 4 which is in the form of a beverage or a milk product.

7. The composition according to claim 4 further comprising equol.

8. The composition according to claim 7 which is in the form of a fermentation product of soy milk.

9. A method of producing equil comprising the step of letting a lactic acid bacterial strain belonging to the genus Lactococcus having an ability to utilize a daidzein compound to produce equol act on at least one member selected from the group consisting of daidzein compounds and daidzein compound-containing ingredients.

10. The method according to claim 9, wherein said lactic acid bacterial strain belonging to the genus Lactococcus is Lactococcus garvieae.

11. The method according to claim 10 wherein said lactic acid bacterial strain belonging to the genus Lactococcus is Lactococcus 20-92 deposited under FERM BP-10036.

12. The method according to claim 9, wherein the daidzein compound-containing ingredient is soybean flour or soy milk.

13. A lactic acid bacterial strain belonging to the genus Lactococcus as deposited under FERM BP-10036.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG. 1 is a diagrammatic representation showing the relationship of incubation time to viable cell count as determined by the test protocol described in Test Example 1.

[0130] FIG. 2 is a diagrammatic representation showing the relationship of incubation time to equol-producing ability (score) as determined by the test protocol described in Test Example 1.

[0131] FIG. 3 is a diagrammatic representation showing the relationship of incubation time to equol output as determined by the test protocol described in Test Example 1.

[0132] FIG. 4 is a diagrammatic representation showing the time course of concentration of each of daidzein compounds and equol in the culture as monitored in accordance with the protocol described in Test Example 2.

[0133] FIG. 5 is a diagrammatic representation showing the relationship of storage period to equol-producing ability (score) as determined in accordance with the test protocol described in Test Example 3.

[0134] FIG. 6 is a diagrammatic representation showing the incubation time-dependent behaviors of the equol-producing strain (changes in growth performance, equol-producing ability, and equol output) as determined by the experiment described in Test Example 1-3.

[0135] FIG. 7 is a diagrammatic representation showing the relationship of incubation time to viable cell count as determined by the test protocol described in Test Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

[0136] The following examples of production of the equol-producing lactic acid bacteria-containing composition of the invention are intended to describe the present invention in further detail and should by no means be construed as defining the invention.

Example 1

(1) Production of a Fermented Soy Milk Beverage

[0137] The following ingredients were taken according to the formula and blended to prepare the composition of the invention in the form of a fermented soy milk beverage.

TABLE-US-00003 Fermentation culture of 100 mL water-soluble soy protein Vitamins & minerals q.s. Flavoring q.s. Water q.s Total 150 mL

[0138] The above fermentative culture of water-soluble soy protein was obtained by dissolving 13 g of water-soluble soy protein in 100 ml of water, adding 10.sup.8-10.sup.9 cells of Lactococcus 20-92 (FERM BP-10036), and carrying out fermentation at 37 C. for 24-48 hours. The water-soluble soy protein used contained about 1-2 mg, calculated as daidzein, of daidzein compounds in each one gram.

(2) Production of a Fermented Milk

[0139] The following ingredients were taken according to the formula and blended to prepare the composition of the invention in a fermented milk form.

TABLE-US-00004 Lactococcus 20-92 fermented milk 100 mL Vitamins & minerals q.s. Flavoring q.s. Water q.s. Total 150 mL

[0140] The Lactococcus 20-92 fermented milk was obtained by adding 10.sup.8-10.sup.9 cells of Lactococcus 20-92 (FERM BP-10036) to 1L of cow's milk (having a nonfat milk solids content of 8.5% or greater and a milk fat content of 3.8% or greater) and carrying out fermentation at 37 C. for 24-48 hours.

(3) Production of a Freeze-Dried Powder of Fermented Soy Milk

[0141] Using about 10.sup.9 cells of Lactococcus 20-92 (FERM BP-10036) and 100 g of soy milk (soy solids 10%, daidzein compound content 10-15 mg calculated as daidzein), lactic acid fermentation was carried out at 37 C. for 72-96 hours for the production of equol. This fermentation product was freeze-dried to prepare a powder. The equol content of the powder as determined by HPLC was 0.1-0.3 weight %.

[0142] The powder obtained above and various other ingredients were weighed out according to the following formula and blended to prepare the composition of the invention in the powder form (food form and pharmaceutical product form).

TABLE-US-00005 Freeze-dried powder of fermented soy milk 2.2 g (equol content 0.005 g) Excipient (corn starch) 17 g Vitamins & minerals q.s. Flavoring q.s. Total 20 g

(4) Production of a Powder

[0143] The following ingredients were weighed out according to the formula and blended to prepare the composition of the invention in a powder form (food form and pharmaceutical product form).

TABLE-US-00006 Freeze-dried powder of Lactococcus 20-92 4.1 g Excipient (lactose) 1.0 g Vitamins & minerals q.s. Flavoring q.s. Total 20 g

[0144] A freeze-dried powder of Lactococcus 20-92 was obtained by culturing Lactococcus 20-92 (FERM BP-10036) in a suitable liquid growth medium (MRS) (37 C., 24-48 hrs), harvesting and suspending grown cells in 10% skim milk, and lyophilizing the suspension. The cell content of the powder was 10.sup.9-10.sup.10 cells/g.

[0145] The above powder may be made into a daidzein-containing powder by blending it further with 4.1 g of semi-purified soy isoflavine powder.

[0146] Intake of the daidzein-containing powder thus obtained results in the urinary equol excretions of about 5 moles (about 1.2 mg) per day, indicating clearly that the amount of equol corresponding to the above excretions can be produced in vivo.

(5) Production of Granules

[0147] The following ingredients were weighed out according to the formula and blended to prepare the composition of the invention in a granular form (food form and pharmaceutical product form).

TABLE-US-00007 Semi-purified soy isoflavone powder 4.1 g Freeze-dried powder of Lactococcus 20-92 1.0 g Sucrose acid ester q.s. Vitamins & minerals q.s. Flavoring q.s. Total 20 g

[0148] The freeze-dried powder of Lactococcus 20-92 used was the same as the one used above in (1).

[0149] Intake of the above composition results in the concurrent delivery of daidzein and equol-producing bacteria to the large intestine, thus enabling production of equol in the large intestine.

[0150] Test Examples relating to the equol-producing lactic acid bacterial strain of the invention are presented below.

Test Example 1

Test for Growth Performance, Equol-Producing Ability (Activity), and Equol Output

(1) Test Protocol

[0151] Lactococcus 20-92 (10.sup.7-10.sup.9 cells/g) was incubated in 5 mL of BHI broth [a liquid medium for growth (basal medium)] anaerobically at 37 C. for 24 hours and the culture was diluted to 10.sup.2 and 10.sup.4 cells with the basal medium.

[0152] The culture obtained at completion of incubation and its dilutions prepared above were respectively taken, 0.2 mL each, and blended with 5 mL each of daidzein-supplemented basal medium (daidzein added to BHI broth at a final concentration of 10 g/mL), cow's milk and soy milk, respectively, and cultured anaerobically at 37 C. The incubation time was set to 8, 24, 48, 72, and 96 hours in the case of 10 g/mL daidzein-supplemented basal medium and soy milk, and 8, 24 and 48 hours in the case of cow's milk.

[0153] Before the start of incubation and at the end of each incubation period, 0.1 mL and 0.2 mL portions of the culture were sampled and respectively subjected to the counting of cells and assay of equol-producing ability (activity). Furthermore, for 10 g/mL daidzein-containing basal medium and soy milk, 0.5 mL of each culture was sampled before the start of incubation and at the end of each incubation period and the amount of equol produced in each sample was determined.

[0154] The number of bacteria was determined in the following manner. Each 0.1 mL sample was diluted with PBS(-) (product of Nissui Co.) to prepare 10.sup.4, 10.sup.5, 10.sup.6 and 10.sup.7-fold dilutions and 0.1 mL each of these dilutions were respectively coated on GAM agar medium and incubated aerobically at 37 C. for 24 hours. The colonies formed on the medium were counted for use as the number of bacteria.

[0155] The equol-producing ability (activity) was assayed as follows. Each 0.2 mL sample was blended with 5 mL of daidzein-supplemented basal medium (each in triplicate) and incubated anaerobically at 37 C. for 96 hours. At completion of incubation, 0.5 mL samples of the respective cultures were taken and respectively extracted twice with 5 mL portions of ethyl acetate and the daidzein, dihydrodaidzein (intermediate), and equol in the extract were quantitated by HPLC. Moreover, based on the total amount, the percentage of equol was calculated. The results were scored on the following 5-point scale and the average score of 3 samples was used as an index of equol-producing ability (activity). [0156] 4: Equol (90% or greater) [0157] 3: Equol produced, with daidzein diminishing to less than 50% (formation of intermediate) [0158] 2: Equol produced, residual daidzein (50% or greater) (formation of intermediate) [0159] 1: Intermediate formed, equol not produced [0160] 0: Neither intermediate nor equol produced, with daidzein not diminishing

[0161] The amount of equol produced was determined as follows. Each 0.5 mL sample was extracted twice with 5 mL portions of ethyl acetate and the amounts of daidzein, dihydrodaidzein (intermediate), and equol in the extract were quantitated by HPLC. Then, the respective concentrations were used to calculate the amount of equol produced.

(2) Test Results

(2-1) The Results of Counting of the Cells (Growth Performance) are Presented in FIG. 1.

[0162] In the diagrammatic representation, (1) represents the result obtained in the case where the daidzein-supplemented basal medium was used, (2) represents the result obtained in the case where soy milk was used, and (3) represents the result obtained when cow's milk was used. In each diagram, the horizontal axis represents incubation time (hr) and the vetical axis represents viable cell count (Log CFU/mL).

[0163] It can be seen from the respective diagrams, the growth performance of the strain of the invention is good and, regardless of the inoculum size used, the stationary phase of growth was invariably attained in 8 hours of incubation in all the daidzein-supplemented basal medium, soy milk and cow's milk. The viable cell count was found to be steady at 10.sup.9.1-9.4 CFU/mL in the daidzein-supplemented basal medium, 10.sup.8.5-8.7 CFU/mL in soy milk, and 10.sup.8.0-8.4 CFU/mL in cow's milk.

(2-2) The Equol-Producing Ability (Activity) Values Found are Presented in FIG. 2.

[0164] In FIG. 2, (1) represents the result obtained in the case where the daidzein-supplemented basal medium was used, (2) represents the result obtained in the case where soy milk was used, and (3) represents the result obtained in the case where cow's milk was used. In each diagram, the horizontal axis represents incubation time (hr) and the vetical axis represents activity score.

[0165] It is obvious from the results presented in FIG. 2 that the equol-producing ability (activity) tends to increase with time in any of the daidzein-supplemented basal medium, soy milk, and cow's milk. It could also be confirmed that even in the cases where cow's milk and soy milk were used, the equol-producing ability (activity) of the strain of the invention is sustained.

(2-3) Results of the Amount of Equol Produced Determination

[0166] The quantities of equol produced in the daidzein-supplemented basal medium and soy milk (about 80 g/mL calculated as daidzein) were as shown in FIG. 3.

[0167] Referring to FIG. 3, (1) represents the result obtained in the case where the daidzein-supplemented basal medium was used and (2) represents the result obtained in the case where soy milk was used. In each diagram, the horizontal axis represents incubation time (hr) and the vetical axis represents equol concentration (g/mL).

[0168] In both media, the production of equol began to be noticed at hour-48 following the start of incubation. In the case where soy milk was used, the amount of equol produced varied with inoculum size and particularly at the inoculation level of 4.00%, the production of equol was as large as 57.0 g/mL at hour-96 of incubation.

[0169] Although, in soy milk, not less than 90% of daidzein serving as the precursor of equol is present in the form of glycoside (in the form of glucose attached), the peak corresponding to the glycoside was no longer observed on the post-incubation chromatogram and this fact suggests that the strain of the invention decomposes the glycoside (-glucosidase activity) to give daidzein and further metabolizes this daidzein to equol.

Test Example 2

Equol Production Pathway in Lactococcus 20-92

(1) Test Protocol

[0170] Lactococcus 20-92 (10.sup.7 cells/mL) was aerobically cultured in 5 mL of BHI broth (a liquid medium for growth, basal medium) at 37 C. for 24 hours and 0.2 mL of the resulting culture was blended with 5 mL of daidzein-supplemented basal medium and the mixture was incubated anaerobically at 37 C. The incubation time was set to 8 hr, 24 hr, 30 hr, 36 hr, 48 hr, 51 hr, 54 hr, 60 hr, 84 hr, and 96 hr.

[0171] Before the start of incubation and at the end of each incubation period, 0.5 mL samples were taken and the concentrations of daidzein, dihydrodaidzein (intermediate), and equol in each sample were determined.

(2) Results

[0172] The results obtained are presented in FIG. 4.

[0173] FIG. 4 is a diagrammatic representation of the changes with time in the concentrations of daidzein (left), dihydrodaidzein (center), and equol (right). In each diagram, the horizontal axis represents incubation time (hr) and the vetical axis represents the concentration (g/mL) of the corresponding substance.

[0174] It will be apparent from the data presented in FIG. 4 that the concentration of daidzein began to decline at hour-48 of incubation, the intermediate compound dihydrodaidzein was formed during the period from hour-48 to hour-60, and the production of equol began at hour-48. It can also be confirmed that the metabolism of daidzein to equol had substantially gone to completion by hour-60.

[0175] While the above results indicated that the metabolism from daidzein to equol occurred via said intermediate compound dihydrodaidzein, the results also suggested that the formation of dihydrodaidzein and the metabolism thereof to equol took place in parallel.

Test Example 3

Low-Temperature Stability of Lactococcus 20-92 Strain-Containing Fermented Milks

(1) Test Protocol

[0176] Lactococcus 20-92 was cultured in 5 mL of a liquid medium for growth (basal medium) anaerobically at 37 C. for 24 hours, the resulting culture was used to inoculate 1L and 2L of cow's milk and 1L of commercial skim milk (10% solids), respectively, at the level of 4% and cultured aerobically under stationary conditions at 37 C. for 48 hours. The cultures were stored at 4 C.

[0177] In the case of cow's milk, the equol-producing ability (activity) was monitored on a weekly basis following completion of culture through week 4 of low-temperature storage at 4 C. Furthermore, two of the tubes were reserved and stored till day-42 and day-51, respectively, and the activity was determined in each case.

[0178] In the case of skim milk, the activity was determined at completion of culture and at week-1 and day-34 of low-temperature storage at 4 C.

[0179] The activity scores before storage and at the end of each storage period were generated by the above-described method comprising inoculating 5 mL of 10 g/mL daidzein-supplemented basal medium at the level of 4% (0.2 mL) in triplicate, culturing the microorganism anaerobically at 37 C. for 96 hours, and determining the concentrations of daidzein, dihydrodaidzein (intermediate), and equol for activity scoring.

(2) Results

[0180] The results are presented in FIG. 5. In FIG. 5, the horizontal axis represents storage period (days) and the vetical axis represents activity score.

[0181] It is apparent from this diagrammatic representation of results that, as far as cow' milk is concerned, the equol-producing ability (activity) is sustained to week-4 of low-temperature storage at 4 C. after completion of culture in both cases of 1L and 2L. Moreover, in the case of 2L of cow's milk, the activity was found to be sustained to day-51, that was the last day of monitoring of the storage stability at 4 C. In the case of 1L of commercial skim milk, too, the equol-producing ability (activity) was apparently sustained to day-34, the last day of monitoring of the low-temperature storage stability at 4 C. after completion of culture.

[0182] The foregoing results indicate that the fermented milk prepared by using Lactococcus 20-92 is capable of retaining the activity even under low-temperature storage conditions and, therefore, is also suitable for food distribution.

[0183] The relationship of the growth performance of Lactococcus 20-92 to its equol-producing ability (activity) and the amount of equol produced as deducible from the results obtained in the above Test Examples 1-3 can be diagrammatically represented as shown in FIG. 6.

[0184] Thus, although cultural conditions varied with different culture media, the equol-producing ability (activity) can be maintained in both the growth phase and the stationary phase. On the other hand, with regard of equol output, it appears that the enzyme begins to be expressed or activated to produce equol after a certain lag time in the stationary phase.

Test Example 4

[0185] Gastric Juice Tolerance Test of the Fermented Milk Prepared by using Lactococcus 20-92

(1) Test Protocol

[0186] Lactococcus 20-92 was anaerobically cultured in 5 mL of a liquid medium for anaerobic growth (BHI broth, basal medium) at 37 C. for 24 hours. The resulting culture (10.sup.9 cells/g) was used to inoculate 1L of cow's milk at the 4% level and incubated aerobically under stationary conditions at 37 C. for 48 hours. After completion of culture, the milk was stored at 4 C. and, regarding it as fermented milk, was subjected to the following test.

[0187] As artificial gastric juices, 0.045% pepsin-supplemented 50 mM glycine-HCl buffers (pH 2.5 and pH 3.0) were prepared. As control, 50 mM glycine-HCl buffer (pH 6.0) was prepared.

[0188] To 9 mL of each artificial gastric juice, 1 mL of the fermented milk stored at low temperature was added and the mixture was incubated (cultured) aerobically under stationary conditions in an incubator at 37 C.

[0189] The incubation time was set to 1 hr, 2 hr, and 3 hr, and 0.1 mL and 0.2 mL aliquots of each culture were respectively sampled before the start of incubation and at the end of each incubation period and subjected to the determination of viable cell count (in the case of 0.1 mL) and equol-producing ability (activity) (in the case of 0.2 mL).

[0190] The determination of viable cell count was carried out in accordance with the procedure described above in Test Example 1-(1), which comprises sampling 0.1 mL of each culture, diluting the sample 10.sup.4, 10.sup.5, 10.sup.6 and 10.sup.7-fold with Nissui's PBS(-), coating 0.1 mL of each dilution on GAM agar medium, incubating the inoculated medium aerobically at 37 C. for 24 hours, and counting the colonies formed on the GAM agar.

[0191] The determination of equol-producing ability (activity) was carried out in accordance with the procedure described above in Test Example 1-(1), which comprises inoculating 5 mL of daidzein-supplemented basal medium with 0.2 mL (4%) of the sample (in triplicate), incubating the inoculated medium anaerobically at 37 C. for 96 hours, and measuring the concentrations of daidzein, dihydrodaidzein (intermediate), and equol in the medium for activity scoring.

(2) Results

[0192] The results obtained are presented in FIG. 7-A (viable cell count) and B (equol concentration).

[0193] In FIG. 7-A, the horizontal axis represents incubation time (hr) and the vetical axis represents viable cell count (Log CFU/ml in milk).

[0194] In FIG. 7-B, the horizontal axis represents incubation time (hr) and the vetical axis represents equol activity (score).

[0195] The following can be deduced from the results presented in FIGS. 7-A and 7-B. Thus, in the buffer solution at pH 6.0, the viable cell count was sustained at the 10.sup.8 cells/mL level up to hour-3, and, here, the equol-producing ability (activity) was also sustained. In the artificial gastric juice at pH 3.0, the viable cell count was sustained up to hour-3 of incubation and, here, the activity was also sustained. On the other hand, in the artificial gastric juice at pH 2.5, a marked decrease in viable cell count began to take place at hour-2 of incubation and the activity also disappeared.

[0196] When studied in the same test system as above, the probiotics (microorganisms which find their way alive into the intestinal canal and exhibit physiological activity there) on the market are reportedly unchanged in viable cell count at pH 3.0 but decreased significantly at pH 2.5. This means that the tolerance to gastric juice at pH 3.0 allows these microorganisms to pass through the stomach alive. Therefore, the fermented milk prepared by using Lactococcus 20-92 is reasonably expected to deliver the organisms alive to the intestines to let them exhibit sustained activity in the lower part of the small intestine and in the large intestine.

Test Example 5

Bile Tolerance Test of Lactococcus 20-92

[0197] The tolerance to bile was determined with VITEK GPI Card (Nippon Biomrieux Co., Ltd) and evaluated by using the growth performances of the strain of the invention in 10% and 40% biles as indicators.

(1) Test Protocol

[0198] Lactococcus 20-92 (10.sup.8-9 cells) was smeared on 5% sheep blood-supplemented Trypticase-Soy agar and aerobically cultured at 37 C. for 24 hours. The colonies on the medium at completion of culture were hooked with a platinum loop and a homogeneous suspension thereof in 0.5% sterile saline was prepared. This suspension was placed in VITEK GPI Card and, after 15 hours' incubation at 35 C., the growth performance of the strain in the presence of bile was evaluated by using the dye (pH indicator). The bile was prepared by dissolving a predetermined amount of bile powder in sterile distilled water and placed in the Card in advance.

(2) Results

[0199] The results of the above test indicate that Lactococcus 20-92 grows in 10% and 40% biles, showing tolerance to 40% bile.

Test Example 6

Hemolysis Test of Lactococcus 20-92

(1) Test Protocol

[0200] Lactococcus 20-92 (10.sup.8-9 cells) was smeared on 5% sheep blood-supplemented Trypticase Soy agar and cultured anaerobically (N.sub.2:CO.sub.2:H.sub.2=8:1:1) at 37 C. for 24-48 hours. The portion around the colony formed on the medium after completion of culture was observed, and the hemolytic potential was evaluated according to the extent of decomposition of blood components (depigmentation or discoloration).

(2) Results

[0201] As the result of the above test, depigmentation (appearance of a transparent, colorless zone) was not observed around the colony, indicating that Lactococcus 20-92 does not cause -hemolysis and, in this respect, is a safe microorganism.

Test Example 7

Cell-Infiltrating Enzyme Activity Test of Lactococcus 20-92

[0202] Regarding the systemic invasion of ingested lactic acid bacteria, a depression in the defensive function of the mesenterium or impairment of the mesenterium itself may be considered as the factor on the host side. As the factor on the side of bacteria, their enzymatic activity (cell-infiltrating enzymes) decomposing the lipid-protein complex proteoglycans constituting the mesenterium can be mentioned.

[0203] This test was intended to investigate whether Lactococcus 20-92 has cell-infiltrating enzyme activities, namely collagenase (gelatinase), hyaluronidase and sialidase (neuraminidase) activities, or not and was carried out in the following manner.

(1) Test Protocol

[0204] Lactococcus 20-92 was smeared (smear size: 10.sup.8-9 cells) on blood-supplemented agar medium and cultured anaerobically (N.sub.2:CO.sub.2:H.sub.2=8:1:1) at 37 C. for 24-48 hours.

[0205] The colonies on the medium after culture were hooked up with a platinum loop and suspended in sterile distilled water to prepare a homogeneous suspension. Using this suspension, the presence or absence of collagenase (gelatinase) was investigated with Api (Nippon Biomrieux Co., Ltd.) using the degradation of gelatin as the indicator.

[0206] Moreover, the test as to whether Lactococcus 20-92 has hyaluronidase and sialidase (neuraminidase) activities or not was carried out by the procedure comprising incubating Lactococcus 20-92 in Tris-HCl buffer solution (pH 7.0) containing hyaluronic acid or sialic acid as the substrate (37 C., aerobic, 15 min. for sialidase activity and 24 hr for hyaluronidase activity) and measuring the degrees of decrease in the concentrations of the respective substrates.

(2) Results

[0207] Lactococcus 20-92 showed none of collagenase (gelatinase), hyaluronidase, and sialidase (neuraminidase) activities.

[0208] Thus, in view of the fact that the strain of the invention lacks cell-infiltrating enzymes which constitute a factor in infectivity, the strain was confirmed to be a highly safe microorganism from infectivity points of view as well.

Test Example 8

Vancomycin Resistance Test

[0209] The acquisition of resistance to antibiotics (mutation) by bacteria has been a matter of serious concern in recent years. It is not rare that patients infected with bacteria which have acquired such resistance to antibiotics succumb to death because they do not respond to the antibiotics. Particularly, the emergence of vancomycin-resistant bacteria (VRE) is a matter of serious concern in the field of clinical medicine today. Moreover, there is the apprehension that if the ingested organisms harboring the vancomycin resistance gene reach and settle in the intestines where they come into contact with virulent or infectious microorganisms (pathogenic bacteria), the vancomycin resistance gene may be transferred to the pathogenic bacteria, with the result that these bacteria also acquire vancomycin resistance. Therefore, it is necessary, at least, that microorganisms which are used as probiotics should not be vancomycin-resistant organisms.

[0210] This test was intended to investigate the susceptibility of Lactococcus 20-92 to vancomycin and was performed as follows.

(1) Test Protocol

[0211] The vancomycin susceptibility test was performed using Sensi-Disk (product of Nippon Becton-Dickinson Company, Ltd.). Lactococcus 20-92 was smeared (smear size: 10.sup.8-9 cells) on GAM agar medium, a disk containing 30 g of vancomycin was placed on the medium, and an aerobic culture was carried out at 37 C. for 24 hours. After the above incubation time, the diameter of the inhibition zone formed around the disk was measured and evaluated according to the evaluation table.

(2) Results

[0212] The diameter of the inhibition zone for Lactococcus 20-92 was 11.90.2 mm and the susceptibility evaluation according to the evaluation table was positive (susceptible: 10 mm). This result indicated that the strain of the invention is not a vancomycin-resistant strain and, therefore, is considered safe.

[0213] Presented in Example 2 below is an example of the production of equol from daidzein by using the lactic acid bacterial strain of the invention.

Example 2

Production of Equol

[0214] One mL of suspension containing 10.sup.7-10.sup.9 cells of Lactococcus 20-92 (FERM BP-10036) in GAM medium for culture of anerobic bacteria was prepared and this suspension was added to 100 g of soy milk (solids concentration: ca 2.2%). The mixture was incubated anaerobically at 37 C. for 72-96 hours and the equol produced in the culture was monitored by HPLC. The daidzein compound content of said soy milk was 95 g/mL calculated as daidzein.

[0215] The result indicated the formation of 10.76.3 g/mL (meanstandard deviation of 3 experiments) of equol in the above soy milk culture.

[0216] The above finding shows clearly that by exploiting the microorganism of the invention, equol can be produced from the daidzein compounds contained in food materials with good efficiency and at low cost.