Accumulation of citrulline by microbial arginine metabolism during the

Subscriber access provided by MT ROYAL COLLEGE

Article

Accumulation of citrulline by microbial arginine metabolism during the alcoholic fermentation of soy sauce FANG FANG, Jiran Zhang, Jingwen Zhou, Zhaohui Zhou, Tieqiao Li, Liling Lu, Weizhu Zeng, Guocheng Du, and Jian Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06053 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

Journal of Agricultural and Food Chemistry

1

Accumulation of citrulline by microbial arginine metabolism during the

2

alcoholic fermentation of soy sauce

3 4

Fang Fanga, b*, Jiran Zhanga, b, Jingwen Zhoua, Zhaohui Zhouc, Tieqiao Lic, Liling Luc,

5

Weizhu Zenga, Guocheng Dua,d Jian Chena,e

6

a

7

Biotechnology, Jiangnan University, Wuxi 214122, China

8

b

9

214122, China

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi

10

c

Guangdong Pearl River Bridge Biotechnology Co. Ltd., Zhongshan 528415, China

11

d

The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of

12

Education, Jiangnan University, Wuxi 214122, China

13

e

14

University, Wuxi 214122, China

15

*Corresponding author

16

Fang Fang, E-mail: [email protected], Telephone: +86-510-85918307

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan

17

1

ACS Paragon Plus Environment


18

Abstract: Citrulline, the major precursor of ethyl carbamate in soy sauce, is an

19

intermediate catabolite of arginine produced by bacteria present in soy sauce moromi

20

mash. Pediococcus acidilactici is responsible for the formation of citrulline during the

21

lactic acid fermentation process of soy sauce. However, citrulline accumulation

22

during the alcoholic fermentation process and the corresponding bacteria involved

23

have not been identified. Salt-tolerant, arginine-utilizing bacteria were isolated from

24

moromi mash during the alcoholic fermentation process. Under normal cultivation

25

conditions, arginine utilization by these strains did not contribute to citrulline

26

accumulation. However, the conversion of arginine to citrulline by these bacteria

27

increased when cultivated during the alcoholic fermentation process. Additionally, the

28

ethanol-enhanced solubility of free fatty acids in moromi mash stimulated the

29

accumulation of citrulline. Staphylococcus exhibited the highest capability in the

30

conversion of arginine to citrulline.

31

Keywords: ethyl carbamate, soy sauce, arginine deiminase pathway, citrulline, fatty

32

acids, ethanol

33

2


Page 2 of 27

Page 3 of 27


34 35

INTRODUCTION Ethyl carbamate (EC), a by-product of the fermentation of group 2A carcinogenic

36

compounds,1, 2 is widely present in beverages, fermented foods, and soy sauce.3-9 EC

37

is formed from ethanol and EC precursors including urea, citrulline, hydrocyanic acid,

38

and carbamyl phosphate.7 Even though the EC level in soy sauce is not as high as that

39

in beverages, understanding the formation of EC and its precursors in soy sauce is

40

critical to reduce its formation and characterize the mechanism of EC formation in

41

similar fermentation systems. Soy sauce, a popular condiment in Asia, is estimated to

42

be the major source (compared with plum liqueur and whisky) of EC in Korea.10 Both

43

urea and citrulline are detected in soy sauce, with citrulline being the main EC

44

precursor.11, 12 Additionally, citrulline is an EC precursor in rice wine.13 Citrulline is

45

formed from the metabolism of arginine through the bacterial arginine deiminase

46

(ADI) pathway.14 The ADI pathway provides energy to several microorganisms under

47

anaerobic conditions15, and the end-product of the ADI pathway is ornithine16. A

48

small quantity of citrulline is secreted into the broth17-19. Interestingly, certain

49

environmental factors, including pH and NaCl concentration, alter the ADI pathway

50

favoring the production of citrulline over ornithine19-21.

51

Our previous study demonstrated that there are two citrulline accumulation

52

periods during soy sauce fermentation: the lactic acid fermentation process (during

53

days 1 through 20) and the alcoholic fermentation process (during days 21 through

54

69).12 There are important differences between the lactic acid and the alcoholic 3



55

fermentation processes. The fermentation temperature is approximately 15ºC during

56

the lactic acid fermentation process and 30°C during the alcoholic fermentation

57

process when sodium-tolerant yeast Zygosaccharomyces rouxii or Candida versatilis

58

are added.11, 22-25 More importantly, the bacterial communities responsible for these

59

two fermentation processes are different. The dominant microorganisms in koji and

60

the lactic acid fermentation process are lactic acid bacteria, mainly from the Weissella

61

and Pediococcus genera.26 Staphylococcus, Micrococcus, and Bacillus are the

62

dominant bacteria during the alcoholic fermentation process due to the inhibition of

63

Pediococcus by salty brine.27, 28 The partial inhibition of arginine catabolism in

64

Pediococcus acidilactici by high saline concentrations lead to more citrulline from

65

arginine than that converted to ornithine. This is the predominant pathway of

66

citrulline production during lactic acid fermentation in soy sauce.12 However, neither

67

citrulline-producing strains nor citrulline accumulation during the alcoholic

68

fermentation process was reported. Therefore, understanding the citrulline

69

accumulation mechanism and identifying the corresponding bacteria during this

70

period are necessary to determine efficient solutions for controlling the levels of EC

71

precursors and EC in soy sauce.

72 73

MATERIALS AND METHODS

74

Industrial soy sauce fermentation. Cooked defatted soybean were mixed with

75

roasted wheat, inoculated with 0.1% to 0.2% of Aspergillus oryzae, and incubated at 4


Page 4 of 27

Page 5 of 27


76

37oC for 2 d to make koji. The resulting koji was subsequently mixed with 20% (w/v)

77

brine at a ratio of 1:1.7 (w/v), resulting in moromi mash. The moromi mash was held

78

at 15 ± 1oC for 20 d. Zygosaccharomyces rouxii was added, and the temperature of

79

the mash was raised to 30oC. Soy sauce was made from pressed, filtered, and

80

pasteurized moromi mash that had fermented for 120 to140 d. More details about this

81

process have been reported previously.22, 28.

82

Isolation of microorganisms from moromi mash. Fresh samples of 2 kg

83

moromi mash (fermentation day 0, 9, 21, 28, and 35) from different fermentation

84

batches were collected from soy sauce fermentation containers at Guangdong Pearl

85

River Bridge Biotechnology Co. Ltd. (Guangdong Province, China). The moromi

86

mash in the 60-m3 fermentation container was aerated with filtered air for 30 min

87

before sampling. The samples were serially diluted and plated onto MRS agar (Oxoid

88

Ltd., Basingstoke, England) on the collection day with the addition of 5% (w/v) NaCl

89

(final concentration) for cultivating lactic acid bacteria or nutrient agar containing 5%

90

(w/v) NaCl for cultivating other bacteria. All plates were incubated at 30°C for 36 h.

91

Screening of microorganisms capable of metabolizing arginine. To investigate

92

bacterial arginine utilization, a single colony of each bacterial strain isolated from

93

moromi mash was sub-cultured in a modified medium (the medium for detection of

94

biogenic amines production)29 supplemented with 10 g L-1 arginine and 5% (w/v)

95

NaCl (pH 5.5) referred to as the arginine consumption medium. Moromi isolates were

96

incubated in the arginine consumption medium at 30°C for 48 h. Strains able to 5



97

metabolize arginine were identified by the appearance of a purple halo, indicating the

98

pH change by ammonia that was produced from the degradation of arginine.

99

Growth conditions for evaluating citrulline accumulation. Fifty one days are

100

required for an adequate amount of citrulline to accumulate during the alcoholic

101

fermentation stage of soy sauce. A relatively high concentration of cell suspension is

102

used for the fast evaluation of citrulline production by isolated bacteria. A cell

103

suspension (5 mL, 109 CFU mL-1) of the isolated strain was pipetted into 50 mL of the

104

arginine consumption medium containing 18% (w/v) NaCl and 0 to 2% (v/v) ethanol

105

or pasteurized raw soy sauce collected during different fermentation stages and

106

supplemented with 10 g L–1 arginine (pH 5.5) and cultured at 30°C for 5 d. Ethanol

107

and a sterilized fatty acid mixture (50% oleic acid, 40% linoleic acid, and 10% stearic

108

acid) were added to the medium to assess their effects on citrulline accumulation.

109

Determination of amino acids. Arginine, citrulline, and ornithine in the samples

110

were quantiﬁed by HPLC.12 For HPLC analysis, samples were centrifuged to remove

111

solid materials and microbial cells, and the supernatants were diluted with 5% (m/v)

112

trichloroacetic acid. Concentrations of arginine, citrulline, and ornithine in the broth

113

were measured, and the values (moles) of citrulline production (∆Cit), arginine

114

consumption (∆Arg), and ornithine production (∆Orn) were calculated. To precisely

115

calculate citrulline produced from arginine from the ADI pathway (see below),15 the

116

amount of arginine (∆Arg) consumed in the ADI pathway was calculated as the sum

117

of citrulline production (∆Cit) and ornithine production (∆Cit). The conversion ratio 6


Page 6 of 27

Page 7 of 27


118

of arginine to citrulline was calculated by the following equation,

119

∆Cit/∆Arg = ∆Cit/(∆Cit + ∆Orn)

120 ADI

121

L-arginine + H2O L-citrulline + NH3 (1)

122

L-citrulline + Pi L-ornithine + carbamyl-P (2)

123

ATP + CO2 + NH3(3) Carbamyl-P +ADP

124 125

OTC

CK

Quantitation of free fatty acids. Free fatty acids were quantified using a free fatty acid quantification kit (Biovision, San Francisco, CA, USA).

126

DNA amplification. The genomic DNA of bacteria isolated from soy sauce

127

moromi was extracted using the E. Z. N. A. genomic DNA isolation kit (Omega

128

Bio-Tek, Winooski, VT, USA). The 16S rRNA gene sequences of the isolated strains

129

were amplified using primers 27F and 1492R 30 and analyzed as previously

130

reported12.

131 132

RESULTS

133

Accumulation of citrulline during alcoholic fermentation. In a previous study,

134

we reported a two-stage (0–7 d and 18–69 d) citrulline (the major EC precursor in soy

135

sauce) accumulation during soy sauce fermentation (Fig. 1) and demonstrated that P.

136

acidilactici was the main contributing bacterial strain in the first stage (0–7 d), which

137

corresponds to the lactic acid fermentation stage.12 In the second stage (18–69 d,

138

which corresponds to the alcoholic fermentation stage), citrulline in soy sauce moromi 7



139

increased by 4.3 mM (from 1.56 to 2.32 g L–1).12 Possible EC formation from these

140

quantities of citrulline has been previously reported.12 EC content in fermented foods

141

ranges from 20 to 800 µg/L or µg/kg,7 which is much less than citrulline (1–3 g/L) in

142

soy sauce. Therefore, a slight increase of citrulline (0.76 g/L) significantly affects EC

143

formation. In the second stage, a slight increase in ornithine concentration (2.3 mM)

144

was also detected (Fig. 1), confirming that the arginine-to-citrulline conversion ratio

145

during alcoholic fermentation was 65.2%. P. acidilactici is likely not the predominant

146

bacteria contributing to citrulline accumulation during this period, because its salt

147

tolerance in moromi mash is weaker than that of other bacteria (e.g., Tetragenococcus,

148

Bacillus, and Staphylococcus). Therefore, halotolerant bacteria with the ability to

149

utilize arginine from moromi during this period were isolated and evaluated for

150

citrulline production to investigate citrulline accumulation during the alcoholic

151

fermentation process of soy sauce.

152

Isolation and characterization of arginine-utilizing bacteria during the

153

alcoholic fermentation process. Bacteria able to metabolize arginine were isolated

154

during the alcoholic fermentation process of soy sauce using high throughput

155

screening. Among the 85 isolated bacterial strains, Staphylococcus (S. caprae and

156

Staphylococcus sp., 72%) was the dominant genus. Other strains identified included

157

Tetragenococcus halophilus (19%) and Bacillus amyloliquefaciens (9%). For each

158

species, the strain with the strongest capability of utilizing arginine was selected:

159

Tetragenococcus halophilus MRS1, Bacillus amyloliquefaciens B2, Staphylococcus 8


Page 8 of 27

Page 9 of 27


160

caprae ZJY10, and Staphylococcus sp. JY09. All these strains were able to convert

161

arginine to ornithine in the presence of 18% (w/v) NaCl with no or little citrulline

162

produced, except for S. caprae ZJY10, which produced 1.52 g L–1 citrulline and

163

exhibited the strongest capacity in converting arginine to citrulline (Table 1).

164

However, the conversion ratio of arginine to citrulline by S. caprae ZJY10 was 15.6%,

165

which was lower than the average conversion ratio (65.2%) observed during the

166

alcoholic fermentation process of soy sauce. Therefore, ethanol concentration and

167

other factors related to alcohol fermentation may affect the bacterial utilization of

168

arginine. The effect of ethanol concentration on the conversion ratio of arginine to

169

citrulline by arginine-utilizing bacteria was subsequently investigated.

170

Characterization of factors that affect the conversion of arginine to citrulline.

171

T. halophilus MRS1, B. amyloliquefaciens B2, S. caprae ZJY10, and S. sp JY09

172

strains isolated during the alcoholic fermentation process of soy sauce were

173

individually added to moromi mash samples obtained during different fermentation

174

periods to investigate the effect of moromi mash on the conversion ratio of arginine to

175

citrulline. Figure 2 shows that the production of citrulline by T. halophilus MRS1 and

176

B. amyloliquefaciens B2 was detected when they were cultivated in soy sauce samples

177

collected on days 21 through 35. However, there was no citrulline accumulation when

178

cultivated from soy sauce samples collected during the lactic acid fermentation

179

process (days 0 through 9). For S. caprae ZJY10 and S. sp. JY09, the conversion ratio

180

of arginine to citrulline increased dramatically when cultivated in soy sauce samples 9



181

collected during the alcoholic fermentation process (days 21 through 35).

182

Among the isolated strains, staphylococci exhibited the highest

183

arginine-to-citrulline conversion ratio. The conversion ratios of arginine to citrulline

184

in soy sauce moromi on day 35 were 78.2% and 68.0% for S. caprae ZJY10 and S. sp.

185

JY09, respectively (Fig. 2), which were higher than the average conversion ratio

186

(65.2%) of arginine to citrulline during the alcoholic fermentation process. These

187

results revealed an increase in the conversion of arginine to citrulline by

188

environmental factors, which were specific to alcoholic fermentation. We detected

189

slight changes in pH (decreased from 5.6 to 5.5) and NaCl concentration (decreased

190

from 18.1% to 16.7%) in moromi mash from days 21 through 35. However, the most

191

significant change during this period was ethanol concentration, which increased from

192

0.3% to 1.7% (Table 2).

193

Effect of ethanol on the conversion of arginine to citrulline. To investigate the

194

stimulation of ethanol on the conversion of arginine to citrulline, ethanol was added to

195

the soy sauce moromi sample cultivated for 21 days. Interestingly, we observed a

196

higher conversion ratio for all strains cultivated in the ethanol-containing moromi

197

sample than in the control sample (Fig. 3). The arginine-to-citrulline conversion ratios

198

significantly increased in all strains, which demonstrated the stimulation of citrulline

199

accumulation by ethanol. To confirm the independent effect of ethanol on bacterial

200

citrulline accumulation, we investigated the conversion of arginine to citrulline by

201

isolates cultivated in arginine consumption medium supplemented with ethanol. The 10


Page 10 of 27

Page 11 of 27


202

conversion of arginine to citrulline by isolated strains was not affected by the

203

presence of ethanol (Fig. 4). These results revealed that, in the presence of another

204

compound, ethanol may stimulate the conversion of arginine to citrulline.

205

Surfactants may enhance the formation of citrulline via the microbial ADI

206

pathway. We detected the presence of free fatty acids (FFAs) in soy sauce moromi

207

mash. The concentration of FFAs in moromi mash during the alcoholic fermentation

208

process (days 21 through 35) ranged between 5.7 and 12.3 mmol L–1. FFAs are not

209

soluble in acidic aqueous solutions; however, their solubility increases with the

210

addition of ethanol.31 In this study, FFAs were added to the arginine consumption

211

medium at a final concentration of 2.5 g L–1 to ensure that the FFA content was higher

212

than the soluble FFA content. As shown in Fig. 5, the conversion ratios of arginine to

213

citrulline for all isolates increased dramatically, including those (B. amyloliquefaciens

214

B2 and T. halophilus MRS1) do not accumulate citrulline in the presence of ethanol

215

(Fig. 4), when they were cultivated in the arginine consumption medium

216

supplemented with ethanol and 2.5 g L–1 FFAs. In the presence of FFAs, the

217

conversion ratio of arginine to citrulline increased with increasing ethanol

218

concentration. Two Staphylococcus strains exhibited a strong capacity to produce

219

citrulline in the presence of FFAs and 2% ethanol (Fig. 5), with 84% and 74%

220

conversion ratios for S. caprae ZJY10 and S. sp. JY09, respectively, which were

221

higher than the average conversion ratio (65.2%) of arginine to citrulline during the

222

alcoholic fermentation process. These results suggested that ethanol and FFAs 11



223

enhanced citrulline production by these strains.

224 225

DISCUSSION

226

The presence of EC precursors is the major cause of EC formation in beverages

227

and fermented foods. Citrulline, the main EC precursor in soy sauce, is produced by

228

bacteria through the ADI pathway. In this study, we identified microorganisms

229

responsible for citrulline accumulation during the alcoholic fermentation process of

230

soy sauce. In this process, FFAs in soy sauce moromi mash increased the conversion

231

ratio of arginine to citrulline by arginine-utilizing and salt-tolerant bacteria.

232

Lactic acid bacteria (LAB), especially Lactobacillus strains, are capable of

233

utilizing arginine through the ADI pathway. L. brevis and L. buchneri isolated from

234

wine and beer synthesize the highest levels of citrulline among all tested LAB.32 In

235

soy sauce moromi mash, the growth of most microorganisms was inhibited by 18%

236

(w/v) NaCl and low temperatures during the brine fermentation process. The bacterial

237

population decreases from 109–1010 CFU (g koji)–1 to 105–106 CFU (g moromi mash)–

238

1 27

239

mesophilic aerobic bacteria (Bacillus sp, and Staphylococcus sp.) and halophilic LAB

240

(T. halophilus MRS1; Table 1). Strains such as P. acidilactici and Weissella able to

241

accumulate citrulline in koji strains12 were not present in isolates obtained from the

242

moromi mash during the alcoholic fermentation period. This result suggested that

243

citrulline-producing strains from koji were not the dominant strains during alcoholic

. In this study, microorganisms able to metabolize arginine were halophilic and

12


Page 12 of 27

Page 13 of 27


244

fermentation and were not responsible for the accumulation of citrulline during this

245

period.

246

Unlike the accumulation of urea (an EC precursor) in rice wine by yeast nitrogen

247

metabolism33, the accumulation of citrulline in soy sauce is affected by environmental

248

factors. LAB strains including L. sakei, L. plantarum, Oenococcus oeni, Pediococcus

249

pentosaceus, and Leuconostoc possess the arc gene and are able to utilize arginine.34,

250

35

251

strains of food origin.36, 37 Previous studies have reported that environmental factors

252

such as osmotic pressure and pH affect citrulline accumulation in LAB through the

253

ADI pathway. High NaCl concentrations improve the conversion ratio of arginine to

254

citrulline in both P. acidilactici, a koji isolate, and L. fermentum IMDO 130101, a

255

sourdough LAB strain.12, 19 However, the conversion ratio was not affected by

256

temperature19. Moreover, citrulline accumulation was observed when L. fermentum

257

was cultivated at the optimal pH condition for growth, while ornithine was the main

258

product of the ADI pathway at sub-optimal pH conditions.20 The mechanism of

259

citrulline accumulation by Staphylococcus and Bacillus has not been elucidated.

260

Staphylococcus does not accumulate citrulline under normal conditions. 36 During the

261

alcoholic fermentation process of soy sauce, pH and NaCl concentration slightly

262

decreased. Therefore, neither pH nor NaCl was responsible for the increased

263

conversion of arginine to citrulline. During the wine making process, production of

264

citrulline by LAB is stimulated at pH 3.0 and in the presence of ethanol (10%–12%).32

ADI activity and its corresponding gene are present in Staphylococcus and Bacillus

13



265

In this study, ethanol affected citrulline accumulation in LAB and other bacteria.

266

Therefore, the increased conversion ratio of arginine to citrulline is more likely to be

267

affected by ethanol during the alcoholic fermentation process of soy sauce.

268

In our study, the addition of ethanol to the arginine utilization medium did not

269

increase the conversion of arginine to citrulline by soy sauce isolates (Fig. 4). Arena

270

demonstrated that ethanol concentration does not affect the conversion ratio of

271

arginine to citrulline by Lactobacillus hilgardii.38 Therefore, the effect of ethanol on

272

the conversion ratio of arginine to citrulline may be dependent on other factors,

273

especially detergents. It has been reported that the presence of surfactants has a

274

significant effect on citrulline formation. Streptococcus faecails is capable of

275

converting arginine to citrulline in the presence of cetyl trimethyl ammonium bromide

276

(CTAB).39 Presumably, the addition of surfactants increases the accumulation of

277

citrulline by enhancing cell membrane permeability and inhibiting ornithine

278

transcarbamylase activity.40 In soy sauce moromi mash, FFAs hydrolyzed from

279

soybean lipid are natural surfactants. The FFA concentration in soy sauce was 2.5 g

280

L-1, and FFAs consisted mainly of oleic acid (40%–50%), linoleic acid (30%–40%),

281

stearic acid (6%), and palmitic acid (2%).41 However, the solubility of FFAs is very

282

low under acidic conditions (~ pH 5.0 in soy sauce). Oleic acid, linoleic acid, and

283

stearic acid are practically insoluble in water, and the solubility of palmitic acid is

284

only 8.26 mg L–1 at 30°C. FFAs, however, are soluble in ethanol, which increases the

285

concentration of surfactants, which may increase cell membrane permeability and 14


Page 14 of 27

Page 15 of 27


286

enhance the release of citrulline to the extracellular environment during the alcoholic

287

fermentation process (Fig. 5). Moreover, the successful reduction of citrulline during

288

the lactic acid fermentation process significantly decreased both citrulline and EC

289

levels in soy sauce, confirming the effect of ethanol and other factors on citrulline

290

accumulation by soy sauce microorganisms.42

291 292

Funding

293

This study was financially supported by the National Key R&D Program of China

294

(2017YFC1600403), the National Natural Science Foundation of China (Grant No.

295

31371821), and the Self-determined Key Program of Jiangnan University (Grant No.

296

JUSRP51734B).

297 298

ACKNOWLEDGEMENTS

299

We are grateful to Professor Nam Sun Wang for the invaluable assistance received

300

during the preparation of the manuscript.

301 302

REFERENCES

303 304 305 306 307 308 309 310

(1) Beland, F. A.; Benson, R. W.; Mellick, P. W.; Kovatch, R. M.; Roberts, D. W.; Fang, J. L.; Doerge, D. R., Effect of ethanol on the tumorigenicity of urethane (ethyl carbamate) in B6C3F1 mice. Food Chem. Toxicol. 2005, 43, 1-19. (2) Riachi, L.; Santos, A.; Moreira, R.; De Maria, C., A review of ethyl carbamate and polycyclic aromatic hydrocarbon contamination risk in cachaça and other Brazilian sugarcane spirits. Food Chem. 2014, 149, 159-169. (3) Chen, D.; Ren, Y.; Zhong, Q.; Shao, Y.; Zhao, Y.; Wu, Y., Ethyl carbamate in alcoholic beverages from China: Levels, dietary intake, and risk assessment. Food 15



311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352

Control 2017, 72, 283-288. (4) Hasnip, S.; Crews, C.; Potter, N.; Christy, J.; Chan, D.; Bondu, T.; Matthews, W.; Walters, B.; Patel, K., Survey of ethyl carbamate in fermented foods sold in the United Kingdom in 2004. J. Agri. Food Chem. 2007, 55, 2755-2759. (5) Jiao, Z.; Dong, Y.; Chen, Q., Ethyl carbamate in fermented beverages: presence, analytical chemistry, formation mechanism, and mitigation proposals. Compr. Rev. Food Sci. Food Saf. 2014, 13, 611-626. (6) Ryu, D.; Choi, B.; Kim, E.; Park, S.; Paeng, H.; Kim, C.-i.; Lee, J.-y.; Yoon, H. J.; Koh, E., Determination of ethyl carbamate in alcoholic beverages and fermented foods sold in Korea. Toxicol. Res. 2015, 31, 289-297. (7) Weber, J. V.; Sharypov, V. I., Ethyl carbamate in foods and beverages-a review. In Climate Change, Intercropping, Pest Control and Beneficial Microorganisms, Lichtfouse, E., Ed. Springer Netherlands: 2009; Vol. 2, pp 429-452. (8) Wu, P.; Pan, X.; Wang, L.; Shen, X.; Yang, D., A survey of ethyl carbamate in fermented foods and beverages from Zhejiang, China. Food Control 2012, 23, 286-288. (9) Zhao, X.; Du, G.; Zou, H.; Fu, J.; Zhou, J.; Chen, J., Progress in preventing the accumulation of ethyl carbamate in alcoholic beverages. Trends Food Sci. Technol. 2013, 32, 97-107. (10) Choi, B.; Ryu, D.; Kim, C. I.; Lee, J. Y.; Choi, A.; Koh, E., Probabilistic dietary exposure to ethyl carbamate from fermented foods and alcoholic beverages in the Korean population. Food Add. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2017. doi: 10.1080/19440049.2017.1364433 (11) Matsudo, T.; Aoki, T.; Abe, K.; Fukuta, N.; Higuchi, T.; Sasaki, M.; Uchida, K., Determination of ethyl carbamate in soy sauce and its possible precursor. J. Agri. Food Chem. 1993, 41, 352-356. (12) Zhang, J.; Fang, F.; Chen, J.; Du, G., The arginine deiminase pathway of koji bacteria is involved in ethyl carbamate precursor production in soy sauce. FEMS Microbiol. Lett. 2014, 358, 91-97. (13) Wang, P.; Sun, J.; Li, X.; Wu, D.; Li, T.; Lu, J.; Chen, J.; Xie, G., Contribution of citrulline to the formation of ethyl carbamate during Chinese rice wine production. Food Add. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 2014, 31, 587-592. (14) Liu, S.-Q.; Pilone, G., A review : Arginine metabolism in wine lactic acid bacteria and its practical significance. J. Appl. Microbiol. 1998, 84, 315-327. (15) Sürken, M.; Keller, C.; Röhker, C.; Ehlers, S.; Bange, F.-C., Anaerobic arginine metabolism of Mycobacterium tuberculosis is mediated by arginine deiminase (arcA), but is not essential for chronic persistence in an aerogenic mouse model of infection. Int. J. Med. Microbiol. 2008, 298, 657-661. (16) Liu, S.-Q.; Pritchard, G.; Hardman, M.; Pilone, G., Citrulline production and ethyl carbamate (urethane) precursor formation from arginine degradation by wine lactic acid bacteria Leuconostoc oenos and Lactobacillus buchneri. Am. J. Enol. 16


Page 16 of 27

Page 17 of 27


353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

Viticult. 1994, 45, 235-242. (17) Arena, M.; Manca de Nadra, M., Comparative survey in Lactobacillus plantarum of the growth and metabolism of arginine and citrulline in different media. J. Agri. Food Chem. 2002, 50, 6497-6500. (18) Rollan, G.; Lorca, G.; de Valdez, G. F., Arginine catabolism and acid tolerance response in Lactobacillus reuteri isolated from sourdough. Food Microbiol. 2003, 20, 313-319. (19) Vrancken, G.; Rimaux, T.; Wouters, D.; Leroy, F.; De Vuyst, L., The arginine deiminase pathway of Lactobacillus fermentum IMDO 130101 responds to growth under stress conditions of both temperature and salt. Food Microbiol. 2009, 26, 720-727. (20) Vrancken, G.; Rimaux, T.; Weckx, S.; De Vuyst, L.; Leroy, F., Environmental pH determines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 130101. Int. J. Food Microbiol. 2009, 135, 216-222. (21) Mira de Orduña, R.; Liu, S. Q.; Patchett, M. L.; Pilone, G. J., Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria. FEMS Microbiol. Lett. 2000, 183, 31-35. (22) Luh, B., Industrial production of soy sauce. J. Ind. Microbiol. 1995, 14, 467-471. (23) Van Der Sluis, C.; Tramper, J.; Wijffels, R. H., Enhancing and accelerating flavour formation by salt-tolerant yeasts in Japanese soy-sauce processes. Trends Food Sci. Technol. 2001, 12, 322-327. (24) Chou, C. C.; Ling, M. Y., Biochemical changes in soy sauce prepared with extruded and traditional raw materials. Food Res. Int. 1998, 31, 487-492. (25) Kobayashi, M.; Hayashi, S., Supplementation of NaCl to starter culture of the soy yeast Zygosaccharomyces rouxii. J. Ferment. Bioeng. 1998, 85, 642-644. (26) Sulaiman, J.; Gan, H. M.; Yin, W. F.; Chan, K. G., Microbial succession and the functional potential during the fermentation of Chinese soy sauce brine. Front Microbiol. 2014, 5, 556. (27) Wei, Q.; Wang, H.; Chen, Z.; Lv, Z.; Xie, Y.; Lu, F., Profiling of dynamic changes in the microbial community during the soy sauce fermentation process. Appl. Microbiol. Biotechnol. 2013, 97, 9111-9119. (28) Tanaka, Y.; Watanabe, J.; Mogi, Y., Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis. Food Microbiol. 2012, 31, 100-106. (29) Chang, M.; Chang, H. C., Development of a screening method for biogenic amine producing Bacillus spp. Int. J. Food Microbiol. 2012, 153, 269-274. (30) Chen, L.; Zhang, H.; Liu, G.; Sha, W., First report on the bacterial diversity in the distal gut of dholes (Cuon alpinus) by using 16S rRNA gene sequences analysis. J. Appl. Genet. 2016, 57, 275-283. (31) Nagai, Y.; Kawano, S.; Motoda, K.; Tomida, M.; Tatebe, C.; Sato, K.; Akiyama, H., Solubility testing of sucrose esters of fatty acids in international food additive 17



395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

specifications. Biol. Pharm. Bull. 2017, 40, 284-289. (32) Araque, I.; Reguant, C.; Rozes, N.; Bordons, A., Influence of wine-like conditions on arginine utilization by lactic acid bacteria. Int. Microbiol. 2011, 14, 225-233. (33) Zhao, X.; Zou, H.; Fu, J.; Chen, J.; Zhou, J.; Du, G., Nitrogen regulation involved in the accumulation of urea in Saccharomyces cerevisiae. Yeast 2013, 30, 437-447. (34) Araque, I.; Gil, J.; Carrete, R.; Bordons, A.; Reguant, C., Detection of arc genes related with the ethyl carbamate precursors in wine lactic acid bacteria. J. Agri. Food Chem. 2009, 57, 1841-1847. (35) Rimaux, T.; Rivière, A.; Hebert, E. M.; Mozzi, F.; Weckx, S.; De Vuyst, L.; Leroy, F., A putative transport protein is involved in citrulline excretion and re-uptake during arginine deiminase pathway activity by Lactobacillus sakei. Res. Microbiol. 2013, 164, 216-225. (36) Sánchez Mainar, M.; Matheuse, F.; De Vuyst, L.; Leroy, F., Effects of glucose and oxygen on arginine metabolism by coagulase-negative staphylococci. Food Microbiol. 2017, 65, 170-178. (37) Maghnouj, A.; de Sousa Cabral, T. F.; Stalon, V.; Vander Wauven, C., The arcABDC gene cluster, encoding the arginine deiminase pathway of Bacillus licheniformis, and its activation by the arginine repressor ArgR. J. Bacteriol. 1998, 180, 6468-6475. (38) Arena, M.; Manca de Nadra, M., Influence of ethanol and low pH on arginine and citrulline metabolism in lactic acid bacteria from wine. Res. Microbiol. 2005, 156, 858-864. (39) Cottenceau, G.; Dherbomez, M.; Lubochinsky, B.; Lettellier, F., Immobilization and treatment of Streptococcus faecalis for the continuous conversion of arginine into citrulline. Enzyme Microb. Technol. 1990, 12, 355-360. (40) Kakimoto, T.; Shibatani, T.; Nishimura, N.; Chibata, I., Enzymatic production of L-citrulline by Pseudomonas putida. Appl. Microbiol. 1971, 22, 992-999. (41) Wan, S.; Wu, Y.; Wang, C.; Wang, C.; Hou, L., The development of soy sauce from organic soy bean. Agri. Sci. 2013, 4, 116-121. (42) Zhang, J.; Du, G.; Chen, J.; Fang, F., Characterization of a Bacillus amyloliquefaciens strain for reduction of citrulline accumulation during soy sauce fermentation. Biotechnol. Lett. 2016, 38, 1723-1731.

428

18


Page 18 of 27

Page 19 of 27


429 430

431 432 433 434

Table 1 Comparison of the ability to accumulate citrulline by arginine-utilizing bacteria Strains ∆Cit (g L-1) ∆Orn (g L-1) ∆Cit/∆Arg (mol mol-1) Tetragenococcus halophilus 0.11±0.01 3.17±0.13 2.6% MRS1 Bacillus amyloliquefaciens B2

0.17±0.01

7.92±0.08

0.2%

Staphylococcus caprae ZJY10

1.52±0.05

6.21±0.14

15.6%

Staphylococcus sp. JY09

0.05±0.00

6.97±0.20

0.05%

Strains were cultivated in media containing 18% NaCl (w/v) and 10 g L–1 arginine. ∆Cit, production of citrulline; ∆Orn, production of ornithine; ∆Cit/∆Arg, conversion ratio of arginine to citrulline.

19



435 436

437 438 439

Table 2 Variations in pH and concentrations of NaCl and ethanol during alcoholic fermentation process Fermentation (d) 21 35 P value pH

5.6±0.1

5.5±0.1

0.42

NaCl (w/v)

18.1±0.2

16.7±0.3

0.04

Ethanol (v/v)

0.3±0.1

1.7±0.1

0.01*

P values (t test) were calculated using software SPSS 19.0. *, P values < 0.05 represent significant difference.

20


Page 20 of 27

Page 21 of 27


440

Figure captions

441

Figure 1. Ornithine concentrations during soy sauce fermentation.

442

Solid squares, citrulline; open circles, ornithine. The values represent mean ± SD of

443

three independent experiments.

444 445

Figure 2. Conversion ratio of arginine to citrulline by arginine-utilizing

446

microorganisms isolated from moromi mash during alcoholic fermentation.

447

The values represent mean ± SD of three independent experiments.

448 449

Figure 3. Effect of ethanol on the conversion ratio of arginine to citrulline on day 21

450

soy sauce moromi mash.

451

Day 21, moromi mash of soy sauce fermented for 21 days. The values represent mean

452

± SD of three independent experiments.

453 454

Figure 4. Conversion ratio of arginine to citrulline of isolated strains cultivated in

455

arginine-consumption medium supplemented with ethanol.

456


457 458

Figure 5. Effect of ethanol and FFAs on the conversion ratio of arginine to

459

citrulline in arginine consumption medium

460


21



Figure 1

22


Page 22 of 27

Page 23 of 27


Figure 2

23



Figure 3

24


Page 24 of 27

Page 25 of 27


Figure 4

25



Figure 5

26


Page 26 of 27

Page 27 of 27


Table of contents (TOC)

27


Accumulation of citrulline by microbial arginine metabolism during the

Recommend Documents