Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a

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Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a Product of Rice Koji Cultivation, in Japanese Sake Brewing. Toshihiko Ito, Mahito Konno, Yoichiro Shimura, Seiei Watanabe, Hitoshi Takahashi, and Katsumi Hashizume J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01031 • Publication Date (Web): 15 May 2016 Downloaded from http://pubs.acs.org on May 21, 2016

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Journal of Agricultural and Food Chemistry

RESEARCH ARTICLE

TITLE: Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a Product of Rice Koji Cultivation, in Japanese Sake Brewing.

AUTHORS: Toshihiko Ito, # Mahito Konno, # Yoichiro Shimura, # Seiei Watanabe, § Hitoshi Takahashi, § and Katsumi Hashizume * , #

AFFILIATIONS:

#

Department of Biological Resource Sciences, Akita

Prefectural University, Nakano Shimoshinjyo, Akita 010-0195, Japan §

Akita Research Institute for Food & Brewing, 4-26 Sanuki, Araya-machi,

Akita 010-1623, Japan

1

ACS Paragon Plus Environment


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ABSTRACT

2

The formation of guaiacol, a potent phenolic off-odor compound in the Japanese

3

sake brewing process, was investigated.

4

and one contained guaiacol and 4-vinylguaiacol (4-VG) at extraordinary high

5

levels: 374 and 2433 µg/kg dry mass koji, respectively. All samples contained

6

ferulic and vanillic acids at concentrations of mg/kg dry mass koji. Guaiacol

7

forming microorganisms were isolated from four rice koji samples.

8

identified as Bacillus subtilis, B. amyloliquefaciens/subtilis, and Staphylococcus

9

gallinarum using 16S rRNA gene sequence.

Eight rice koji samples were analyzed,

They were

These spoilage bacteria convert

10

vanillic acid to guaiacol and ferulic acid to 4-VG.

However, they convert very

11

little ferulic acid or 4-VG to guaiacol. Nine strains of koji fungi tested produced

12

vanillic acid at the mg/kg dry mass koji level after cultivation. These results

13

indicated that spoilage bacteria form guaiacol from vanillic acid, which is a

14

product of koji cultivation in the sake brewing process.

15 16 17

KEYWORDS: guaiacol, vanillic acid, spoilage bacteria, koji, sake brewing

18

2


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INTRODUCTION

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Phenolic acids, represented by ferulic acid, contained in the plant cell wall are

21

bio-catalytically degraded by microorganisms which often produce volatile

22

phenols, for example; 4-vinyl guaiacol (4-VG) or guaiacol.1-2 In the

23

degradation process, bioconversion of vanillic acid to guaiacol has been found

24

in Rhodotorula yeast, Bacillus, Pseudomonas and Streptomyces bacteria.3-6

25

Guaiacol has been recognized as a potent phenolic off-odor compound of

26

Alicyclobacillus spoilage in apple or other fruit juices, and its formation has

27

been eagerly studied.7-9

28

preparations,10-11 and has been identified in wines that have been barrel-aged

29

or aged with toasted oak chips and staves 12-13 or wines made using grapes that

30

have been affected by bushfire smoke.14

31

specific marker for torrefied malts and its levels in beer increase with age.15

32

addition to guaiacol, 4-VG, 4-vinylphenol (4-VP), 4-ethylguaiacol (4-EG), and

33

4-ethylphenol (4-EP) are known as influencing phenolic off-odor constituents in

34

wines.16-17

35

Pof+ Saccharomyces cerevisiae, lactic acid and other bacteria in the wine

36

making process, while 4-EG and 4-EP are formed by Bretanomyces/Dekkera

37

sp. spoilage yeast.16

38

ferulic acid by Pof+ beer yeast and it is also formed during thermal processing in

39

beer production.18

40

wheat Qu, a moulded cereal used as a saccharifying enzyme source, rather

41

than from yeast.19

42

It is a representative constituent of smoke flavoring

In beer brewing, guaiacol is a In

4-VG and 4-VP are formed by various microorganisms, including

A previous report suggested that 4-VG is formed from

In Chinese rice wine production, 4-VG originates from

Japanese sake, a traditional alcoholic beverage, is made from polished

43

rice, rice koji, and water.

The polished rice is steamed after soaking and used

44

for moromi mash preparation or rice koji cultivation (Figure 1). Solid state

3



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fermentation by koji mold, Aspergillus oryze, produces a variety of enzymes

46

and metabolites required for alcoholic fermentation of the moromi mash.

47

Ferulic and p-coumaric acids are released from ingredient rice grains during the

48

fermentation process and they may be converted to 4-VG and 4-VP by

49

Staphylococcus and/or Bacillus species.20

50

reduced to 4-EG and 4-EP by Pichia spoilage yeast.20

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that guaiacol contributes to the 4-VG-like phenolic off-odor in sake, 21 however,

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its formation in the sake brewing process has never been studied.

53

maximum temperature reached in the sake brewing process is ca. 100 ℃ in

54

the steaming rice which is far lower than the temperature in the torrefied malts

55

making process (final roasting temperature reaches 225 ℃) 15 where guaiacol

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is produced by thermal reaction.

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sake brewing factories, suggesting spoilage microorganisms were involved in

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its production.

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microbiological factors affecting on guaiacol formation in the Japanese sake

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brewing process, focusing on rice koji cultivation process in which the so-called

61

open fermentation style is employed, in which contamination with spoilage

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microorganisms is possible. Volatile phenols and phenolic acids in rice koji

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were quantitated and guaiacol forming microorganisms in the rice koji were

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isolated and identified.

4-VG and 4-VP are sometimes Recently, we reported

The

Guaiacol was originally detected in particular

The aim of this study was to elucidate chemical and

65 66

MATERIALS AND METHODS

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Chemicals.

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2-Methoxyphenol (guaiacol) (98%) was purchased from Tokyo Chemical

69

Industry Co. (Tokyo, Japan).

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4-ethyl-2-methoxyphenol (4-ethylguaiacol) (95%), 3-methylphenol (m-cresol)

2-Methoxy -4-vinylphenol (4-vinylguaiacol),

4


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(98%), 4-methyphenol (p-cresol), 4-ethylphenol (95%), 2-6-dimethoxyphenol

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(98%) , 4-hydroxybenzoic acid (95%), 4-hydroxyphenylacetic acid (98%),

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4-hydroy-3-methoxybenzoic acid (vanillic acid) (95%), 4-hydroxycinnamic acid

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(p-coumaric acid) (98%), 4-hydroxy-3-methoxycinnamic acid (ferulic acid) (98%),

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3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid) (97%),

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4-hydroxy-3,5-dimethoxybenzoic acid (syringic acid), 4-n-butylphenol, ethanol

77

(95%), ethyl acetate (infinity pure grade) and methanol were obtained from Wako

78

Pure Chemical Industry Co. (Osaka, Japan).

79

pyridine were obtained from Sigma-Aldrich Japan Co. (Tokyo, Japan).

80

Acetonitrile (LC/MS grade) and BSTF+10 % TMCS were obtained from Thermo

81

Fisher Scientific Co. (Kanagawa, Japan）

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Materials. Eight rice koji samples from 4 sake brewing factories (2 samples

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from each) in the Akita prefecture were used.

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until use.

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guaiacol sake, as reported in our previous study.21 Strains of Aspergillus oryzae

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were kindly gifted by the National Research Institute of Brewing

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(Higashihiroshima, Japan)

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Analysis of Volatile Phenols in Rice Koji.

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mL of a 23.8 % (v/v) ethanol aqueous solution and stirred at 120 rpm for 3 h at

90

30 °C.

91

was diluted with an equal volume of distilled water. Volatile phenols in the

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diluted sample were analyzed by GC-MS after its preparation as an ethyl acetate

93

extract (Figure S1), according to our previously reported method for sake

94

samples.21

95

limits for guaiacol and 4-VG were 0.3 and 0.7 µg/L in the extract, and 1.6 and 3.7

96

µg/kg dry mass koji, respectively.

4-Vinylphenol solution and

Samples were stored at -20 °C

Factories C and D (Table 1) were selected because of their high

Rice koji (4 g) were mixed with 20

The solution was then filtered using filter paper. The filtrate (10 mL)

4-n-Butylphenol was used as an internal standard. The detection

The recovery rate for guaiacol spiked at 50

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µg/L was 104.5±10.1 % and 91.9 ±1.7 % for 4-VG spiked at 200µg/L.

98

moisture content of rice koji samples was measured using the loss of sample

99

weight after drying at 135 °C for 3 h.

The

100

Analysis of Phenolic Acid in Rice Koji. Phenolic acids in the rice koji were

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identified by GC-MS after solvent extraction, condensation, HPLC fractionation,

102

and trimethylsilylation.

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ethanol aqueous solution and stirred at 120 rpm for 90 min at 30 °C. The

104

extract was obtained through paper filtration. After ethanol removal by rotary

105

evaporation (60 °C, 120 hPa), the pH of the aqueous solution was adjusted to

106

1.4 with 1 M H2SO4 and then applied to a pre-conditioned Bond Elute C18 LRC

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500MG (Agilent Technologies, Santa Clara, CA, USA).

108

washed with water and trapped constituents were eluted with 2 mL of methanol.

109

The solution was concentrated to ca. 100 µL by rotary evaporation (60 °C, 40

110

hPa) and then fractionated by HPLC using a Capcell Pak C18 Type MG column

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（4.5 mm x 250 mm）(Shiseido, Tokyo, Japan).

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solvent B was 0.1 % phosphoric acid / water.

113

A: B=5: 95 to A: B=25: 75 over 30 min at a flow rate of 1.0 mL/min. The

114

absorbance at 280 nm was monitored. Each peak fraction was collected and

115

dried under vacuum and methylsilylated using a mixture of 100 µL of

116

BSTF+10 % TMCS and pyridine at 70 °C for 15 min.

117

analyzed using 7890A GC/5975C inert XL MSD (Agilent Technologies)

118

apparatus and a fused silica column (30 m x 0.25 mm i.d., coated with a 0.25 µm

119

film of DB-5MS; Agilent Technologies) using splitless injection mode.

120

column temperature was held at 80 °C for 8 min and then raised from 80 °C to

121

220 °C at a rate of 6 °C/min and held for 10 min at 220 °C.

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temperature was 220 °C, and the flow rate of the helium carrier gas was 1.5

Rice koji (4 g) was mixed with 20 mL of a 38 % (v/v)

The column was

Solvent A was acetonitrile, and

A linear gradient was used from

The sample (2 µL) was

6


The

The injector

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mL/min. The mass spectrometer was used with an ionization voltage of 70 eV

124

(EI) and an ion source temperature of 230 °C.

125

comparison with the retention time and mass spectrum of the trimethylsilylated

126

authentic phenolic acid and the NIST library.

127

conducted by HPLC without trimethylsilylation, applying 20 µL of the same

128

extract solution used for the volatile phenol analysis (Figure S2). HPLC

129

conditions were the same as the fractionation experiment. Quantitation was

130

performed using an external calibration method. Calibration curves were

131

constructed using 23.8% ethanol aqueous solutions of standard phenolic acids.

132

They showed good linearity (R2=0.9994 - 1.000) and detection limits for vanillic

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and ferulic acids were 0.01 and 0.02 mg/L, and 0.07 and 0.08 mg/kg of koji

134

sample, respectively.

135

acids at 0.667 mg/L in the extracted solution were 97.1 ± 7.0 % and 100.5 ±

136

9.3 %, respectively.

137

Isolation of Microorganisms able to Convert Vanillic Acid to Guaiacol.

138

YSG media (pH 3.722 and pH 6.1 without pH adjustment) were used in the

139

isolation. Ten grains of rice koji were suspended in a Tween 20 aqueous

140

solution (0.05 % (v/v)). After vigorous mixing, the liquid phase was diluted and

141

inoculated onto an agar (3%) plate. After incubation at 30 °C for 24-48 h,

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formed colonies were picked and inoculated into the liquid media (5 mL,

143

containing 10 mg/L vanillic acid) and cultivated at 30 °C for 24 h, with shaking at

144

120 rpm.

145

and HPLC. HPLC conditions were the same as for analysis of phenolic acid,

146

with one exception; a gradient condition was changed to A: B=40: 60 to A: B=60:

147

40 over 30 min. Guaiacol forming ability of the isolated microorganism was

148

confirmed repeatedly by plate and liquid cultivation tests.

Components were identified by

Quantitation of phenolic acids was

The percentage recovery of spiked vanillic and ferulic

Other phenolic acids tested also showed similar results.

Phenolic compounds in the liquid media were analyzed by smelling

7



149

Identification of Isolated Microorganism. Isolated microorganisms were

150

identified with 16S rRNA gene sequence using NCBI-BLAST

151

(https://blast.ncbi.nlm.nih.gov/); The gene was amplified by PCR using the 27F

152

and 1492R primer pair, the amplified fragment was sequenced using 27F, 519F,

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1114F, 518R, 806R, and 1492R primers,23-27 and then merged sequence was

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applied to the BLAST program.

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Bioconversion Ability of Phenolic Compounds from the Isolated

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Microorganism. An aliquot (0.2 mL) of pre-cultivated microorganism in YSG

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liquid medium (pH 6.1) was inoculated into 5 mL of liquid media (pH 6.1)

158

containing phenolic compounds at 10 mg/L.

159

shaking at 120 rpm.

160

After cultivation, the gas in the head space of the glass tube was inhaled to

161

check for guaiacol odor. The sample was centrifuged at 14,000 × g for 10 min,

162

and a 20 µL aliquot was applied to HPLC. Quantitation was performed using an

163

external calibration method.

164

23.8% ethanol aqueous solution of standard phenolic compounds except for

165

2,6-dimethoxy-4-vinylphenol.

166

0.06 and 0.03 mg/L, respectively.

167

(canolol) was confirmed by its GC-MS spectrum,27 and the concentration was

168

estimated by the molecular absorbance coefficient value.28 When ferulic or

169

vanillic acid were tested as substrates, guaiacol was analyzed using GC-MS

170

according a previously reported method.21

171

Effects of Ethanol Concentration and pH on Growth and Guaiacol

172

Formation in the Isolated Microorganism.

173

the liquid medium containing vanillic acid at 10 mg/L was adjusted by adding a

174

95 % aqueous ethanol solution and the pH was adjusted by the addition of lactic

It was cultured at 30 °C for 24 h,

Formed phenolic compounds were determined by HPLC.

Calibration curves were constructed using a

The detection limits for guaiacol and 4-VG were Formation of 2,6-dimethoxy-4-vinylphenol

The concentration of ethanol in

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acid. To test pH, ethanol was not added to the media.

Cultivation was

176

conducted under the same conditions as the test for bioconversion ability. Cell

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growth was monitored by absorbance at 660 nm and guaiacol formation was

178

analyzed by HPLC, as mentioned above.

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Analysis of Phenolic Acid in the Rice Koji Cultivation Process.

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of 60 % polished rice grain was soaked in water for 1 h, the water absorbance of

181

the soaked rice grains was adjusted to 30 %. The rice was then steamed for 50

182

min and cooled to room temperature, before it was put into a 300 mL Erlenmeyer

183

flask and autoclaved at 110 °C for 15 min.

184

oryzae (RIB128) spores was inoculated and mixed.

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for 22 h, the mix was transferred to two sterilized plastic plates (90 mm) and

186

cultivated at 35 °C for 19 h, and then 37 °C for 7 h covered with filter paper.

187

oryzae cultivation was initiated by the addition of one rice grain of the koji seed

188

which contained adequate spores for the strain comparison test.

Phenolic acid

189

was analyzed using the same conditions for the rice koji sample.

Production of

190

phenolic acid by A. oryzae RIB128 was also examined using a Czapek-Dox agar

191

plate (0.3 % NaNO3, 0.1 % K2HPO4, 0.05 % KCl, 0.05 % MgSO47H2O,

192

0.001 % FeSO47H2O, 0.01 % glucose, 0.5 % starch, 3.0 % agar, 20 mg/L

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phenolic acid) by cultivating at 30 °C for 3 days.

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was sampled and mixed with an equal weight of 70 % (v/v) aqueous ethanol

195

solution. After extraction at 30 °C for 90 min at 120 rpm, the supernatant

196

solution was analyzed by HPLC.

After 40 g

After cooling, 1 mg of Aspergillus After cultivation at 30 °C

A.

Agar medium and fungus body

197 198

RESULTS AND DISCUSSION

199

Quantitation of Phenolic Compound in Rice Koji. The concentrations of

200

volatile phenols in rice koji samples are shown in Table 1.

9


Guaiacol was only


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found in one sample (no. 7, factory D). It contained guaiacol and 4-VG at

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extraordinary high levels, 374 and 2433 µg/kg dry mass koji, respectively.

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There was little difference in the concentration of other volatile phenols among

204

the other samples. Sample no. 7 showed the highest water content. The

205

water-rich koji culture conditions may be profitable for bacterial growth. In the

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common sake brewing process, rice koji is used at 20 % of the total rice material,

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and ca. 2.15 L of jyunmai-type sake was brewed from 1 kg of rice (Figure 1) the

208

volatile phenols in the koji sample are diluted about ten times. Sample no. 7

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may produce guaiacol and 4-VG in the brewed out sake at the sensory

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acceptable level (guaiacol, 14.6 µg/L: 4-VG, 141 µg/L),21 if volatile phenols in the

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rice koji are transferred to sake with no decrease in concentration.

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value in sample no. 7 may be due to vigorous bacterial activity which often

213

produces organic acids. More than 45 years ago, Nakamura et al. reported that

214

rice koji used in sake brewing contained vanillic acid and several other phenolic

215

acids,29 howerver, no further studies on vanillic acid in rice koji for sake brewing

216

have been reported.

217

after HPLC fractionation and trimethylsilylation: p-hydroxybenzoic,

218

p-hydroxyphenylacetic, vanillic, and syringic acids were detected. The level of

219

syringic acid was far lower than the other phenolic acids. After identification,

220

phenolic acids were quantitated by HPLC without trimethylsilylation.

221

p-Hydroxybenzoic, p-hydroxyphenylacetic and vanillic acids were present at

222

more than 1.0 mg/kg dry mass in all samples (Table2). The amount of ferulic

223

acid was higher than 2.7 mg/kg dry mass in all samples which was comparable

224

to that of previous report.30

225

Isolation and Identification of Microorganisms Forming Guaiacol from

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Vanillic Acid in Rice Koji. An isolation trial using YSG medium (pH 3.7)22

The low pH

We analyzed phenolic acids in the sample no.1 by GC-MS

10


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suitable for detection of Alicyclobacillus sp. bacteria did not find any

228

microorganisms in the rice koji samples. A subsequent study using YSG

229

medium without pH adjustment (pH 6.1) produced many colonies after

230

incubation at 30 °C for 24-48 h. Each colony was tested for guaiacol forming

231

ability using liquid medium containing vanillic acid at 10 mg/L.

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microorganisms were isolated from sample no. 3, 6, 7, and 8 (Table 3).

233

Samples no. 6, 7 and 8 were from high guaiacol sake producing factories,

234

whereas sample no. 3 was not. Three of the microorganisms were identified as

235

Bacillus sp. and the other 2 microorganisms were Staphylococcus sp.

236

Kaneoke20 reported that S. gallinarum is a spoilage bacteria in the sake brewing

237

process that produces 4-VG from ferulic acid, however the Bacillus sp. bacteria

238

found in this study were different to those previously reported.20 It has been

239

reported that B. subtilis is able to form guaiacol from vanillic acid,6, 31 and it also

240

may decarboxylate p-coumaric acid and ferulic acid to form 4-VP and 4-VG.32

241

Thus, the isolated spoilage bacteria have an ability to convert various phenolic

242

compounds.

243

Bioconversion Ability of Isolated Bacteria. Three of the bacteria isolated

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were examined for their bioconversion ability of various phenolic compounds

245

using YSG liquid media (pH 6.1) containing phenolic compounds at 10 mg/L.

246

Ferulic, vanillic and p-coumaric acids were rapidly decarboxylated by the three

247

bacteria but only B. subtilis decarboxylated sinapic and syringic acids (Table 4).

248

In contrast, the three bacteria showed little ability for the bioconversion of 4-VG

249

to guaiacol. In both tests of ferulic acid and 4-VG addition, the characteristic

250

odor of guaiacol was detectable by smelling, however, it was not detected by

251

HPLC. Hence we analyzed it by GC-MS.

252

low. It was suggested that the bacterial conversion abilities were enough to

The

The determined level was extremely

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produce the level of guaiacol measured in rice koji sample no.7 from vanillic acid.

254

Since the observed guaiacol forming ability of the isolated bacteria was

255

comparable, high guaiacol level in samples no.7 might be due to the high levels

256

of both bacteria in the koji culture.

257

Effects of Ethanol Concentration and pH on the Growth and Guaiacol

258

Formation of Isolated Bacteria.

259

pH are important factors in protecting moromi mash from bacterial spoilage: the

260

effects of these two factors on growth and guaiacol formation in isolated bacteria

261

were examined (Figure 2-A, 2-B).

262

gallinarum were suppressed at 5 % (v/v) of ethanol, while Bacillus sp. were

263

suppressed at 10 % (v/v) ethanol. In contrast, low pH, less than 4, suppressed

264

growth and guaiacol formation in all bacteria. In the common sake brewing

265

process, the pH of the fermentation starter is adjusted to below 4.0 at the starting

266

point of cultivation by lactic acid to suppress spoilage bacteria. In the early

267

stage of the kimoto starter making process, a traditional fermentation starter

268

method prepared by mixing only steamed rice, rice koji, and water, the pH is

269

greater than 5 which may permit bacterial growth, and lead to higher guaiacol

270

production. Our results showed that Bacillus sp. produced higher levels of

271

guaiacol per absorbance (growth) than S. gallinarum, indicating that Bacillus sp.

272

might cause more serious spoilage effects when they grow.

273

Analysis of Phenolic Acid in the Rice Koji Cultivation Process.

274

isolated bacteria had low conversion rates for ferulic acid or 4-VG to guaiacol,

275

suggesting vanillic acid is required for high guaiacol production.

276

release ferulic and p-coumaric acids from steamed rice grains in the sake

277

brewing process,30,33 however the bioconversion ability from ferulic acid to

278

vanillic acid has never been reported.34,35 We analyzed phenolic acid

In sake brewing, ethanol concentration and

Growth and guaiacol formation of S.

12


The

A. oryzae

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production in the rice koji culture, using steamed rice and A. oryzae RIB128.

280

Phenolic acids, except ferulic acid, were not detected at the starting point of

281

cultivation, however, phenolic acids, including vanillic acid, were detected during

282

cultivation, until 41 h (Figure 3). It is suggested that the fungi produce the

283

phenolic acids from the degraded compounds of steamed rice.

284

may be converted to vanillic acid by A. oryzae RIB128 and it is possible that

285

other fungi could perform this conversion, including A. niger.36-39 We also

286

cultivated A. oryzae RIB128 on an agar plate containing ferulic acid at 20 mg/L.

287

Vanillic acid was detected on the second day, but the level was very low (< 0.1

288

mg/kg medium) and had disappeared by the third day (data not shown). The

289

difference in culture media might affect vanillic acid production in A. oryzae.

290

Previous analysis of phenolic acids in food rice, whose polishing rate was ca.

291

90 % reported that vanillic acid was found at low levels,40,41 however, we could

292

not detect it in steamed rice grains.

293

milling ratio (60 %) of used rice and the soaking and steaming procedures.

294

Differences in phenolic acid production among A. oryzae strains were also

295

examined (Table 5). All strains tested produced vanillic acid at more than 1.1

296

mg/ kg dry mass koji, indicating that vanillic acid, a precursor of guaiacol, is

297

constantly supplied in the common sake brewing process.

298

General Discussion.

299

at more than ca. 1 mg/kg dry mass koji in the common rice koji culture, and

300

spoilage bacteria, Bacillus sp. and S. gallinarum, present in the rice koji may

301

convert vanillic acid to guaiacol. The vanillic acid level in rice koji and the

302

bioconversion ability of the spoilage bacteria seems to be adequate to account

303

for the existence of guaiacol in sake at sensory significant levels when the

304

spoilage bacteria grow notably. Guiraud et al. reported that A. oryzae converts

Ferulic acid

The difference might be due to a high

This study revealed that A. oryzae produces vanillic acid

13



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305

vanillic acid to guaiacol during cultivation when vanillic acid is the sole carbon

306

source, however, we did not detect guaiacol in the six rice koji samples listed in

307

Table 1 in spite of the existence of vanillic acid at mg/kg concentrations.

308

observation supported the results of Suezawa et al. who reported that A. oryzae

309

in sake brewing does not form guaiacol from vanillic acid.

310

conditions of the two experiments differed and further studies are required to

311

clarify this point. We believe this is the first report on guaiacol formation in

312

Japanese sake brewing. The results of this study suggested that thorough

313

microbial control is important in rice koji culture to prevent the occurrence of

314

phenolic off-odors in sake.

The cultivation

315 316 317

ASSOCIATED CONTENT

318

Supporting Information

319

Chromatograms used for the identification and quantitation of the volatile

320

phenols (Figure S1) and phenolic acids (Figure S2).

(PDF)

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AUTHOR INFORMATION

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Corresponding Author

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*(K.H.) Fax: +81-18-872-1676; E-mail: [email protected]

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NOTE

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The authors declare no competing financial interest.

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460

FIGURE CAPTIONS Figure 1. a

The junmai-type sake brewing process.

+24 and +2 indicate increases in water.

Numerical values are common and

approximate weight proportions.

Figure 2.

Effects of ethanol concentration (A) and pH (B) on growth and

guaiacol formation of isolated bacteria.

–△-; Absorbance at 660 nm, -■-; Ratio

of conversion from vanillic acid to guaiacol (%).

Data are means ± the S.D. for

triplicated experiments.

Figure 3.

Concentration of phenolic acids in the rice koji cultivation process.

-----; temperature, -●-; vanillic acid, -○-; p-hydroxybenzoic acid, -◇-; p-hydroxyphenylacetic acid, -◆-; p-coumaric acid, -■-; ferulic acid. Data are means ± the S.D. for three measurements.

Table 1. Water and Volatile Phenol Contents of Rice Koji Samples a 4-EG, 3-MP, 4-VG, 4-EP, 2,6-DMP, and 4-VP are 4-ethylguaiacol, 3-methylphenol, 4-vinylguaiacol, 4-ethylphenol, 2,6-dimethoxyphenol, and 4-vinylphnol, respectively. b Data are means ± the S.D. for three measurements.

Table 2. Phenolic Acid Content of Rice Koji Samples [mg/kg dry mass koji] a Data are means ± the S.D. for three measurements.

Table 3. Isolated and Identified Guaiacol Forming Microorganisms from Rice Koji Samples

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Table 4. Phenolic Compound Converting Ability of Identified Microorganisms a Data are means ± the S.D. for triplicated experiments.

Table 5. Effects of Aspergillus oryzae on Phenolic Acid Levels in Rice Koji [mg/kg dry mass koji] a Data are means ± the S.D. for three measurements.

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Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a

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