Accumulation of Citrulline by Microbial Arginine Metabolism during

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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

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Journal of Agricultural and Food Chemistry

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Accumulation of citrulline by microbial arginine metabolism during the

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alcoholic fermentation of soy sauce

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

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Weizhu Zenga, Guocheng Dua,d Jian Chena,e

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a

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Biotechnology, Jiangnan University, Wuxi 214122, China

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b

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214122, China

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

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

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c

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

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d

The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of

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Education, Jiangnan University, Wuxi 214122, China

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e

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University, Wuxi 214122, China

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*Corresponding author

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

National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan

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1

ACS Paragon Plus Environment


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

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

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

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

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during the alcoholic fermentation process and the corresponding bacteria involved

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

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moromi mash during the alcoholic fermentation process. Under normal cultivation

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

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

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

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

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accumulation of citrulline. Staphylococcus exhibited the highest capability in the

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conversion of arginine to citrulline.

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

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acids, ethanol

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

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

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

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

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

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

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

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

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

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

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formed from the metabolism of arginine through the bacterial arginine deiminase

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

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

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

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

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favoring the production of citrulline over ornithine19-21.

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Our previous study demonstrated that there are two citrulline accumulation

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

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

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



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fermentation processes. The fermentation temperature is approximately 15ºC during

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

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process when sodium-tolerant yeast Zygosaccharomyces rouxii or Candida versatilis

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

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

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

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

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

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

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Pediococcus acidilactici by high saline concentrations lead to more citrulline from

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

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

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citrulline-producing strains nor citrulline accumulation during the alcoholic

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fermentation process was reported. Therefore, understanding the citrulline

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accumulation mechanism and identifying the corresponding bacteria during this

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

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precursors and EC in soy sauce.

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MATERIALS AND METHODS

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Industrial soy sauce fermentation. Cooked defatted soybean were mixed with

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


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

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

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

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

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

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process have been reported previously.22, 28.

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

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

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batches were collected from soy sauce fermentation containers at Guangdong Pearl

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

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

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

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

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

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

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Screening of microorganisms capable of metabolizing arginine. To investigate

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

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

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

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

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



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

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

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Growth conditions for evaluating citrulline accumulation. Fifty one days are

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

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

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

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

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

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or pasteurized raw soy sauce collected during different fermentation stages and

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

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

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

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

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

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

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

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

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

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

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

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


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of arginine to citrulline was calculated by the following equation,

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∆Cit/∆Arg = ∆Cit/(∆Cit + ∆Orn)

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

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

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

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OTC

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

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

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

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

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

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reported12.

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RESULTS

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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citrulline production to investigate citrulline accumulation during the alcoholic

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fermentation process of soy sauce.

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Isolation and characterization of arginine-utilizing bacteria during the

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alcoholic fermentation process. Bacteria able to metabolize arginine were isolated

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during the alcoholic fermentation process of soy sauce using high throughput

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

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

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

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

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Tetragenococcus halophilus MRS1, Bacillus amyloliquefaciens B2, Staphylococcus 8


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

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

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

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

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

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

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

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

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

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citrulline by arginine-utilizing bacteria was subsequently investigated.

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

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

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strains isolated during the alcoholic fermentation process of soy sauce were

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individually added to moromi mash samples obtained during different fermentation

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

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

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

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

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cultivated from soy sauce samples collected during the lactic acid fermentation

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

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



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

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Among the isolated strains, staphylococci exhibited the highest

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

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

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

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

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

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

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

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

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

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0.3% to 1.7% (Table 2).

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

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

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

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

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

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

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

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

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


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

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

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

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Surfactants may enhance the formation of citrulline via the microbial ADI

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

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

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

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

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

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

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

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

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

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

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

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conversion ratio of arginine to citrulline increased with increasing ethanol

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concentration. Two Staphylococcus strains exhibited a strong capacity to produce

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

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

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

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



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enhanced citrulline production by these strains.

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DISCUSSION

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

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

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

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responsible for citrulline accumulation during the alcoholic fermentation process of

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

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

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

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

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

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

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

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

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1 27

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

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

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

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

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

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

12


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

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period.

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

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

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

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

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35

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

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

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

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

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

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

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

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

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citrulline accumulation by Staphylococcus and Bacillus has not been elucidated.

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

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

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

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

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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



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

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

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

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

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

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demonstrated that ethanol concentration does not affect the conversion ratio of

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

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

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

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significant effect on citrulline formation. Streptococcus faecails is capable of

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

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

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citrulline by enhancing cell membrane permeability and inhibiting ornithine

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

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

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

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

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

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

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

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


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

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

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the lactic acid fermentation process significantly decreased both citrulline and EC

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

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accumulation by soy sauce microorganisms.42

291 292

Funding

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

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

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

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JUSRP51734B).

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ACKNOWLEDGEMENTS

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

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during the preparation of the manuscript.

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REFERENCES

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428

18


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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


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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

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Figure 2

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Figure 3

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Figure 4

25



Figure 5

26


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Table of contents (TOC)

27


Accumulation of Citrulline by Microbial Arginine Metabolism during

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