CULTURE PRODUCT OF MICROORGANISM BELONGING TO THE GENUS WICKERHAMOMYCES

Abstract

A culture product comprising a large quantity of ethyl benzoate and has a more complex and fresher fruity aroma than a chemically synthesized product.

The culture product is obtained by culturing a microorganism belonging to the genus Wickerhamomyces in a milk component-containing culture medium.

Claims

1. A culture product obtained by culturing a microorganism belonging to the genus Wickerhamomyces in a milk component-containing culture medium.

2. The culture product according to claim 1, wherein the milk component-containing culture medium is a whey-containing culture medium.

3. The culture product according to claim 1, wherein the microorganism belonging to the genus Wickerhamomyces is Wickerhamomyces pijperi.

4. The culture product according to claim 1, wherein the microorganism belonging to the genus Wickerhamomyces is at least one selected from the group consisting of Wickerhamomyces pijperi NBRC1290 and Wickerhamomyces pijperi NBRC1887.

5. The culture product according to claim 1, wherein the milk component-containing culture medium further comprises at least one selected from the group consisting of benzoic acid and a salt thereof.

6. A flavoring composition comprising the culture product according to claim 1.

7. A cosmetic product comprising the culture product according to claim 1.

8. A food or a drink comprising the culture product according to claim 1.

9. A pharmaceutical product comprising the culture product according to claim 1.

10. A method for imparting an aroma to a cosmetic product, a food or a drink, or a pharmaceutical product, the method comprising: adding the culture product according to claim 1 to the cosmetic product, food, drink, or pharmaceutical product.

11. A flavoring composition comprising the culture product according to claim 2.

12. A flavoring composition comprising the culture product according to claim 3.

13. A flavoring composition comprising the culture product according to claim 4.

14. A flavoring composition comprising the culture product according to claim 5.

15. A method for imparting an aroma to a cosmetic product, a food or a drink, or a pharmaceutical product, the method comprising: adding the culture product according to claim 2 to the cosmetic product, food, drink, or pharmaceutical product.

16. A method for imparting an aroma to a cosmetic product, a food or a drink, or a pharmaceutical product, the method comprising: adding the culture product according to claim 3 to the cosmetic product, food, drink, or pharmaceutical product.

17. A method for imparting an aroma to a cosmetic product, a food or a drink, or a pharmaceutical product, the method comprising: adding the culture product according to claim 4 to the cosmetic product, food, drink, or pharmaceutical product.

18. A method for imparting an aroma to a cosmetic product, a food or a drink, or a pharmaceutical product, the method comprising: adding the culture product according to claim 5 to the cosmetic product, food, drink, or pharmaceutical product.

Description

EXAMPLES

[0038] The present invention will now be described in detail by way of Examples.

Example 1

(1) Strains and Pre-Preculture

[0039] (i) Used Strains

[0040] Wickerhamomyces pijperi YIT8095 (NBRC1290), Wickerhamomyces pijperi YIT12779 (NBRC1887)

COMPARATIVE EXAMPLE

Kluyveromyces marxianus YIT12612 (NBRC0260)

[0041] (ii) Pre-Preculture

[0042] Pre-preculture: 20 μl of a strain cryopreserved in 20% glycerol was inoculated into 2 ml of a Yeast and Mold (YM) culture medium (1% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract) and incubated with shaking at 160 rpm for 24 hours at 30° C.

(2) Analysis of Aroma Components Produced By Bacteria

[0043] (i) Preculture

[0044] Preculture: 20 μl of the culture solution obtained from the pre-preculture was inoculated into 2 ml of a whey-containing culture medium supplemented with 1% glucose and incubated with shaking at 160 rpm for 24 hours at 30° C.

[0045] As the above described whey-containing culture medium, the supernatant of the culture product which was obtained by culturing Streptococcus thermophilus YIT2084 (FERM BP-10879) in a 3% skimmed milk powder-containing culture medium was used. The result of analysis of this supernatant showed that the supernatant contained lactose at an amount of 1.1%, galactose at an amount of 0.4%, and lactic acid at an amount of 0.4%, and had a pH of 4.0.

[0046] (ii) Culture

[0047] Culture: 50 μl of the preculture solution was inoculated into 5 ml of a whey-containing culture medium supplemented with 1% glucose and incubated with shaking at 160 rpm for 24 hours at 30° C.

[0048] (iii) Analysis of Aroma Components

[0049] After completion of incubation, a sample was collected and centrifuged at 8,000 rpm for 5 minutes and 2 ml of the supernatant was collected in a 20 ml vial. Then, an aroma component in the culture product was identified by Headspace gas chromatography mass spectrometry (HS-GC-MS) (Tables 1 and 2). Furthermore, the identified aroma component was quantified by HS-GC-Flame Ionization Detector (FID) (Tables 3 and 4). The result is shown in Table 5. Furthermore, sensory analysis of the odor of the culture solution was performed using the criteria described below. The result is shown in Table 5.

(Evaluation Criteria for Sensory Analysis)

[0050] 5: A complex and fresh fruity aroma is strongly detected. [0051] 4: A complex and fresh fruity aroma is detected. [0052] 3: A complex and fresh fruity aroma is slightly detected. [0053] 2: A complex and fresh fruity aroma is hardly detected. [0054] 1: No complex and fresh fruity aroma is detected.

TABLE-US-00001 TABLE 1 HS Conditions: Device MPS2-xt Syringe size 2.5 ml Agitator temperature 50° C. Agitator speed 250 rpm Vial equilibration time 15 min Syringe temperature 100° C. Injection volume 1000 μL Injection rate 200 μL/s

TABLE-US-00002 TABLE 2 GC-MS Conditions: System MS700D (JEOL) Column InertCap Pure WAX 30 m × 0.25 mm i.d. × 0.25 μm (GL Sciences) Oven temperature 40° C.-250° C. (10° C./min) Carrier gas He Flow rate 0.7 ml/min Injection temperature 250° C. Split ratio Splitless MS resolution 1000 Ionizing current 300 μA Ionizing voltage 70 eV Chamber temperature 250° C. Injection volume 1 μL

TABLE-US-00003 TABLE 3 HS Conditions: Device Agilent 7697A Loop size (ml) 5 Oven temperature (° C.) 50 Loop temperature (° C.) 110 Transfer line temperature (° C.) 115 Vial equilibration (min) 15 Injection time (min) 1

TABLE-US-00004 TABLE 4 GC Conditions: System Agilent 7890B Column InertCap Pure WAX 30 m × 0.25 mm i.d. × 0.25 μm (GL Sciences) Oven temperature 40° C. (5 min)-10° C./min-250° C. (3 min) Carrier gas He Flow rate 3 ml/min Injection temperature 250° C. Split ratio 20:1 Split flow rate 60 ml/min Vial equilibration (min) 5 Injection time (min) 1

TABLE-US-00005 TABLE 5 Kluyveromyces Compounds (ppm) NBRC1290 NBRC1887 marxianus NBRC0260 Alcohol Isoamyl alcohol 10.07 10.79 29.07 Ethyl ester Ethyl benzoate 1.14 1.24 0.00 Evaluation by 5 5 1 sensory analysis

[0055] A complex and fresh fruity aroma was strongly detected in the culture solutions of the NBRC1290 strain and the NBRC1887 strain. The results of analysis of the aroma components and comparison of the contents of the compounds demonstrated the culture solutions characteristically had a high content of ethyl benzoate. For comparison with these culture solutions, aroma components of the culture solution obtained by culturing a different yeast species (Kluyveromyces marxianus NBRC0260 strain) in a whey-containing culture medium were analyzed and evaluated. The result showed the culture solution of the NBRC0260 strain had a strong rose-like aroma but hardly any fruity aroma, and contained no ethyl benzoate.

[0056] The culture solutions of the NBRC1290 strain and the NBRC1887 strain and a chemically synthesized product of ethyl benzoate alone in an amount equal to that in the culture solutions were subjected to sensory analysis. A complex aroma which the chemically synthesized product fails in producing was detected in the culture solutions of the NBRC1290 strain and the NBRC1887 strain. Thus, the culture solutions had a better aroma than the chemically synthesized product.

(3) Difference in Yields of Aroma Components By Bacteria Depending on Culture Media

[0057] An experiment was performed using the NBRC1290 strain and the NBRC1887 strain.

[0058] (i) Preculture

[0059] Preculture: 20 μl aliquots of the culture solution obtained from the pre-preculture were inoculated into 2 ml of a whey-containing culture medium supplemented with 1% glucose, 2 ml of a YPD culture medium (1% yeast extract, 2% peptone, and 2% glucose), and 2 ml of a YM culture medium (1% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract) and incubated with shaking at 160 rpm for 24 hours at 30° C.

[0060] (ii) Culture

[0061] Culture: 5 ml aliquots of the culture solution obtained from the preculture were inoculated into 2 ml of the whey-containing culture medium supplemented with 1% glucose, 2 ml of the YPD culture medium, and 2 ml of the YM culture medium, and incubated with shaking at 160 rpm for 24 hours at 30° C.

[0062] (iii) Analysis of the Content of an Aroma Component

[0063] After completion of incubation, the content of an aroma component in the culture product was analyzed by HS-GC-FID (Tables 3 and 4). The result is shown in Table 6. Sensory analysis of the odor of the culture product was also performed using the same criteria as described in Example 1 (2). The result is shown in Table 6.

TABLE-US-00006 TABLE 6 NBRC1290 NBRC1887 Compounds (ppm) Whey YPD YM Whey YPD YM Alcohol Isoamyl alcohol 17.07 242.14 70.07 7.53 235.7 98.31 Ethyl ester Ethyl benzoate 2.69 0.10 0.09 2.78 0.00 0.11 Evaluation by sensory 5 2 2 5 1 2 analysis

[0064] When the NBRC1290 strain and the NBRC1887 strain were cultured in the whey-containing culture media, a complex and fresh fruity aroma was strongly detected in the resulting culture solutions. The culture solutions contained a large quantity of ethyl benzoate. In contrast, when the above described strains were cultured in the YM culture medium and the YPD culture medium, the resulting culture solutions contained ethyl benzoate in some cases, but the content was low and a complex and fresh fruity aroma was hardly detected.

(4) Difference In Yields of an Aroma Component By Bacteria Depending On Culture Conditions

[0065] An experiment was performed by using the NBRC1887 strain.

[0066] (i) Preculture

[0067] Preculture: 20 μl of the culture solution obtained from the pre-preculture was inoculated into 2 ml of a whey-containing culture medium supplemented with 1% glucose and incubated with shaking at 160 rpm for 24 hours at 30° C.

[0068] (ii) Culture

[0069] Culture: 1 ml aliquots of the preculture solution were inoculated into 100 ml of the whey-containing culture medium supplemented with 1% glucose and were incubated with shaking at 160 rpm under the culture conditions of the temperature of 15° C., 20° C., 25° C., and 30° C. Samples were collected after 0, 24, 32, 48, and 56 hours of incubation.

[0070] (iii) Analysis of the Content of ethyl benzoate

[0071] The content of ethyl benzoate in the culture products was analyzed by HS-GC-FID (Tables 3 and 4). The result is shown in Table 7. Furthermore, sensory analysis of the odor of the culture products was performed using the criteria described below. The result is shown in Table 7.

(Evaluation Criteria for Sensory Analysis)

[0072] a: A complex and fresh fruity aroma is strongly detected. [0073] b: A complex and fresh fruity aroma is detected. [0074] c: A complex and fresh fruity aroma is slightly detected but the complexity is low. [0075] d: The odor of other aroma components is strong. The character of the odor is changed and a sake-like odor is detected. [0076] e: No complex and fresh fruity aroma is detected.

TABLE-US-00007 TABLE 7 Ethyl benzoate (ppm)/Evaluation by sensory analysis 0 hr 24 hr 32 hr 48 hr 54 hr 15° C. 0/e 0.73/c 2.48/c 2.24/c 2.18/c 20° C. 0/e 3.04/a 2.54/b 2.06/d 1.90/d 25° C. 0/e 2.48/b 1.89/b 1.65/d 1.67/d 30° C. 0/e 2.23/b 1.72/b 1.26/d 1.51/d

[0077] For the incubation at a temperature of 15° C., ethyl benzoate was produced by microbial fermentation. However, the odor of other aroma components was weak, and thus the complexity of the odor of the culture product was low. In contrast, the culture product resulting from incubation at temperatures ranging from 20 to 30° C. had a complex and fresh fruity aroma, that is, the culture product had a good odor. In the case of a culture time of 48 hours or more, the yield of aroma components other than ethyl benzoate increased due to microbial fermentation. Consequently, the odor of the other aroma components became stronger so that the character of the odor was changed and a sake-like odor was detected. Therefore, the preferable culture time was from 24 to 32 hours.

(5) Difference in Contents of ethyl benzoate Resulting from Addition of Benzoic Acid or a Salt thereof to the Culture Medium

[0078] An experiment was performed by using the NBRC1887 strain.

[0079] (i) Preculture

[0080] Preculture: 20 μl of the culture solution obtained from the pre-preculture was inoculated into 2 ml of a whey-containing culture medium supplemented with 1% glucose and incubated with shaking at 160 rpm for 24 hours at 30° C.

[0081] (ii) Culture

[0082] Culture: sodium benzoate was added to 100 ml of the whey-containing culture medium supplemented with 1% glucose to a final concentration of 0.01%. In a similar manner, each of benzoic acid, phenylalanine, and para-hydroxymethylbenzene was added to 100 ml of the whey-containing culture medium supplemented with 1% glucose to obtain the culture medium containing 0.01% benzoic acid, the culture media containing 0.01%, 0.05%, or 0.1% phenylalanine, and the culture media containing 0.01% or 0.05% para-hydroxymethylbenzene. Furthermore, 100 ml of the whey-containing culture medium supplemented with 1% glucose which had no additive was prepared as a control. One-milliliter aliquots of the preculture solution were inoculated into each of the culture media and incubated with shaking at 160 rpm at 30° C. Samples were collected after 0, 24, 32, and 48 hours of incubation.

[0083] (iii) Analysis of the Content of ethyl benzoate

[0084] The content of ethyl benzoate in the culture products was analyzed by HS-GC-FID (Tables 3 and 4). The result is shown in Table 8.

TABLE-US-00008 TABLE 8 The content of ethyl benzoate in each culture solution (ppm) 0.01% 0.05% Para- Para- 0.01% 0.01% 0.01% 0.05% 0.1% hydroxy- hydroxy- Sodium Benzoic Phenyl- Phenyl- Phenyl- methyl- methyl- Control benzoate acid alanine alanine alanine benzene benzene  0 Hours 0 0 0 0 0 0 0 0 24 Hours 2.44 3.30 2.41 2.43 2.38 2.66 0.71 0.07 32 Hours 3.54 12.59 15.31 3.37 3.39 3.65 2.17 0.34 48 Hours 2.89 27.67 23.53 2.48 2.58 2.28 2.78 2.12

[0085] The content of ethyl benzoate in the culture products significantly increased when benzoic acid or sodium benzoate was added to the culture medium and the culture time was 32 to 48 hours. The content of ethyl benzoate in the culture products hardly increased when other substances were added to the culture medium.

Preparation Example 1

[0086] In reference to the composition shown in Table 9, to a mixture of (5) and (6), were added (1) to (4). The mixture was stirred thoroughly to prepare a toner. This toner had a complex and fresh fruity aroma. In this Example, the culture solution was used after filtration, wherein the culture solution had been obtained by inoculating 1 ml of the preculture solution resulting from preculture in Example 1 (4) into 100 ml of a whey-containing culture medium supplemented with 1% glucose and incubating the culture with shaking at 160 rpm for 24 hours at 20° C.

TABLE-US-00009 TABLE 9 Raw material Quantity used (%) (1) Ethanol 5.0 (2) 1,3-Butylene glycol 2.0 (3) Polyoxyethylene hydrogenated 0.05 castor oil (4) Methyl parahydroxybenzoate 0.1 (5) Culture solution 10.0 (6) Distilled water Balance to 100

Preparation Example 2

[0087] In reference to the composition shown in Table 10, to (10) were added (7) to (9), and the mixture was heated and then emulsified by adding (1) to (6) thereto at 80° C. The mixture was allowed to cool down to the room temperature to prepare a milky lotion. This milky lotion had a complex and fresh fruity aroma. The culture solution used was the same culture solution as that in Preparation Example 1.

TABLE-US-00010 TABLE 10 Raw material Quantity (%) (1) Stearic acid 2.0 (2) Liquid paraffin 5.0 (3) Squalane 2.0 (4) Sorbitan monostearate 0.05 (5) Polyoxyethylene (20) sorbitan 2.0 monostearate (6) Butyl parahydroxybenzoate 0.05 (7) Glycerin 2.0 (8) Methyl parahydroxybenzoate 0.1 (9) Culture solution 3.0 (10)  Distilled water Balance to 100%

Preparation Example 3

[0088] In reference to the composition shown in Table 11, to (12) were added (9) to (11), and the mixture was heated and then emulsified by adding (1) to (8) thereto at 80° C. The mixture was allowed to cool down to the room temperature to prepare a cream. This cream had a complex and fresh fruity aroma. The culture solution used was the same culture solution as that in Preparation Example 1.

TABLE-US-00011 TABLE 11 Raw material Quantity (%) (1) Liquid paraffin 23.0 (2) Petrolatum 7.0 (3) Cetanol 1.0 (4) Stearic acid 2.0 (5) Beeswax 2.0 (6) Sorbitan monostearate 3.5 (7) Polyoxyethylene (20) sorbitan monostearate 2.5 (8) Butyl parahydroxybenzoate 0.05 (9) 1,3-Butylene glycol 1.0 (10)  Methyl parahydroxybenzoate 0.1 (11)  Culture solution 3.0 (12)  Distilled water Balance to 100