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Insights into the Biogenic Amine Metabolic Landscape during Industrial Semi-dry Chinese Rice Wine Fermentation Xiaole Xia, Qingwen Zhang, Bin Zhang, Wuji Zhang, and Wu Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01523 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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Journal of Agricultural and Food Chemistry

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Insights into the Biogenic Amine Metabolic Landscape during Industrial Semi-dry

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Chinese Rice Wine Fermentation

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Xiaole Xia1*, Qingwen Zhang1, Bin Zhang2, Wuji Zhang1, and Wu Wang1

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Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, P. R. China.

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* Corresponding Author:

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E-mail: [email protected]

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Phone: 008651085326829

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

Nantong Baipu Chinese Rice Wine Co., Ltd., Nantong, Jiangsu 226500, P. R. China.

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ACS Paragon Plus Environment


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ABSTRACT

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Inspired by concerns about food safety, the metabolic landscape of biogenic amines

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(BAs) were elucidated during industrial semi-dry Chinese rice wine fermentation. The

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main fermentation process represented the largest contribution to BAs formation which

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corresponding to 69.1% (54.3 mg/L). Principal component analysis (PCA) revealed that

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total acid and ethanol were strongly correlated with BAs, indicating that BAs formation

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favored acidic and stressful conditions. Other than Putrescine (PUT), spermidine (SPD)

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and spermine (SPM), 5 BAs exhibited strong relationships with the precursor amino acids

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(R2 > 0.85). PUT was mainly decarboxylated from arginine (ARG) (89.6%) whereas SPD

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(100%) and SPM (83.1%) were obtained from ornithine (ORN). Interestingly, some SPD

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could convert back to PUT (24.3%). All 8 BAs showed good relationships with the LAB

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(R2 around 0.75). Moreover, among the five main LAB genera, Lactobacillus had a

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positive correlation with BAs formation.

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Keywords: Biogenic amines; Semi-dry Chinese rice wine; Industrial fermentation;

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Principal component analysis; Lactobacillus; Decarboxylation

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INTRODUCTION

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Biogenic amines (BAs) are present in varying concentrations in food and are mainly

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formed by amino acid decarboxylation.1 It is commonly accepted that high concentrations

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of BAs represent a health hazard because of their undesirable physiological effects,

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particularly when alcohol is present.2 At elevated levels (100 mg/L) these compounds,

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mainly cadaverine (CAD) and putrescine (PUT) also exert an impact on the organoleptic

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properties of fermented foods.b The European Union (EU) has established regulations for

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histamine (HIS) levels, and many wine importers in the EU require a BA analysis.3

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Currently, the public health, technological and economic significance of BAs support the

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hypothesis that their contents should become a wine quality index.4 Additionally, the

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presence of BAs in food may serve as an indicator of undesired microbial activity and for

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the evaluation of good manufacturing practices (GMP).5 In wine, HIS and tyramine (TYR)

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are considered the most toxic products and are particularly relevant for food safety; PUT

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and CAD are known to potentiate these effects. Moreover, these amines cannot be

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inactivated by the thermal treatments used in food processing and preparation.6 Various

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studies have shown that BA formation in wines is related to the grape variety, type of

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vinification, wine pH, malolactic fermentation, ageing and their interactions.7

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Chinese rice wine is a traditional fermented alcoholic beverage that is rich in amino

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acids, oligosaccharides and microelements.8 Commonly, Chinese rice wines are classified

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into four types based on their total sugar levels; some differences also exist in their

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production techniques.9 Semi-dry Chinese rice wine is a kind of CRW which sugar

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content is between 15.1 to 40.0 g/L. Wheat Qu, Chinese koji, steamed rice and water are

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mixed to initiate a so-called parallel fermentation, during which starch saccharification

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and alcoholic fermentation proceed in parallel rather than sequentially.10 Similar to other

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types of rice wine, the indigenous bacteria, together with the abundant precursor amino

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acids available during fermentation, have raised concerns about the accumulation of BAs

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in Chinese rice wine.11 Lu et al. analyzed 14 Chinese rice wines from four wine-making

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regions of China, and detected HIS in all samples, followed by spermine (SPM), CAD,

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TYR and spermidine (SPD). The mean level of BAs was 107 mg/L and ranged from 39.3

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to 241 mg/L.12 Zhong et al. analyzed 39 samples from different manufacturers and

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showed that the most prominent BA was serotonin, followed by PUT, TYR, CAD and

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HIS. The total BA contents varied, ranging from 29.3 to 260 mg/L, whereas that of

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semi-dry Chinese rice wine is 102 mg/L on average.13 This value is higher than those of

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wine, beer, and Turkish cereal wine and lower than that of Korean rice wine.14,15

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Some studies have quantified the levels of BAs during the production of Chinese rice

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wines.16 Zhang et al. found that the BA content in the seed starter varied over a large

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range (16.43–87.72 mg/L). Moreover, in the first 2 days, the BA content increased

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sharply and then more gradually, and the content reached a maximum value on day 6.17

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Compared to wine, few studies have addressed the BA metabolic landscape during

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fermentation, particularly its interactions with metabolites and microorganisms.18 It has

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been commonly accepted that BAs are mainly decarboxylated by a decarboxylase derived

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from lactic acid bacteria (LAB), particularly Lactobacillus19 However, in a correlation

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analysis of bacteria community succession and the changes in the BA levels during

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Chinese rice wine fermentation, Liu et al. found that Lactobacillus might not be the main

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BA producer.20 Currently, few reliable methods that can be used to detect limiting BA

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formation in fermented alcoholic beverages exist. Guo et al. reported that the knock out

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of some specific genes in Saccharomyces cerevisiae decreased BA formation by 25.5%.21

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García-Ruiz demonstrated that some LAB isolated from fermented foods can degrade

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BAs by producing amine oxidase enzymes.22 Callejón identified a multicopper oxidase

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from LAB that can degrade amines in wine.23

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Hence, the risk mitigation options, which are based on analyzing and controlling those

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factors and interactions, should be systematically considered and ranked in Chinese rice

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wine fermentation. The objective of this work, which represents a preliminary part of a

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wider research project aimed at the control of BA formation in Chinese rice wine, is to

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identify the BA metabolic landscape during industrial semi-dry Chinese rice wine

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fermentation. In particular, the interactions and relationships between BAs, metabolites

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and microorganisms are investigated and analyzed. Based on these results, potential

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protocols for limiting and reducing BAs and enhancing the quality of Chinese rice wine

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are discussed.

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MATERIALS AND METHODS

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Materials and standards

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BA standards (2-phenylamine (PHE), PUT, CAD, HIS, TYR, tryptamine (TRY), SPD

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and SPM) were purchased from Sigma-Aldrich (USA). Acetonitrile, acetone and dansyl

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chloride for high-performance liquid chromatography (HPLC) were purchased from

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Merck (Germany), and ultrapure water was obtained from a Milli-Q system (Millipore,

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USA). Other reagents used in this study were analytical grade and were purchased from

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local companies. Rice, Chinese koji and wheat Qu were prepared by Nantong Baipu

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Chinese Rice Wine Co., Ltd. (China).

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Production and sampling of semi-dry Chinese rice wine

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The industrial fermentation of Chinese rice wine was performed at Baipu Chinese Rice

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Wine (Nantong, China). As shown in Scheme 1, rice was processed by washing and

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water steeping at 25-30°C for 2 days, and then, the steeped rice was steamed, which was

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considered the beginning of fermentation. The seed starter is unique to Chinese rice wine

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fermentation; its preparation involves mixing steamed rice and 0.5-1% (w/w) Chinese

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koji which amylase activity is higher than 100U/g , which is cultured at approximately

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25°C for 48 h. This process is known as canning and saccharification. The seed starter

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was mixed with the same volume of steamed rice, 200% water and 5% wheat Qu which

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amylase activity is higher than 500U/g and then entered the so-called main fermentation

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process, which typically consisted of incubation at 28-30°C for 6-7 days with intermittent

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oxygen filling. Subsequently, post-fermentation occurred at room temperature for

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approximately 10 days. After fermentation, the fermentation mash was filtered by

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pressing, clarification and sterilization. Eleven samples were taken from each batch, as

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summarized in Scheme 1 and Table S1. Triplicate samples were collected, filtered, and

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stored at 4°C. The method of Zhang et al.17 was used for the preparation of liquid samples

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and the derivatization of all of the extracted solutions.

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Scheme 1 BA determination

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A 5-mL aliquot of each sample was vortexed with 15 mL of 0.1-M hydrochloric acid to

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obtain a homogenous mixture. After centrifugation (6,000g, 10 min, and 4°C), the

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aqueous layer was collected, diluted to 25 mL with 0.1-M hydrochloric acid and stored

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under refrigeration. For analysis, 2 mL of the extract was added to 0.2 mL of

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1,7-diaminoheptane (100 mg/L in 0.1-M hydrochloric acid) as an internal standard, and

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then, the mixture was subjected to derivatization with dansyl chloride and determination

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by reversed-phase HPLC (RP-HPLC) with ultraviolet (UV) detection, as described

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previously.24

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Detection of microorganisms and metabolites

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To count the LAB, the samples were serially diluted 10-fold with distilled water, and

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100 µL of each dilution was spread on a 1% (w/v) deMan-Rogosa-Sharpe agar (MRS

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agar, Oxoid, UK) plate. The agar plates were incubated at 37°C for 20 h under aerobic

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conditions. Yeast and Mould were cultured on 3M™ Yeast and Mould Count Plates (3M,

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MN, USA) in a 20°C incubator for 68 h under aerobic conditions. The numbers of viable

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LAB and yeast were determined by calculating the colony forming units (cfu)/mL. The

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total sugar, acid, alcohol and amino acid contents of the fermentation mash were

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measured according to the National Standard of the People’s Republic of China for

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Chinese rice wine.25

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Analysis of LAB using 16s rDNA

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Ten milliliters of the fermentation broth were centrifuged at 17,000 × g for 6 min at

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4°C to collect the microbial cells. The resulting pellet was used to extract the total DNA

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with the Promega Wizard Genomic DNA Purification Kit according to the

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manufacturer’s instructions. Polymerase chain reaction (PCR) was used to amplify the

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16S

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(5’-CCTACGGGNGGCWGCAG-3’)

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TCC-3’).26 The amplified PCR products were sequenced with Illumina MiSeq to generate

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millions of reads by Personalbio (Shanghai Personal Biotechnology Co., Shanghai,

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China). The operational taxonomical units (OTUs), which were defined according to a

rDNA

V3-V4

region

using and

the 805R

universal

primers

341F

(5’-GACTACHVGGGTATCTAA

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3% distance level, were phylogenetically classified, and a taxonomy file describing the

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complete taxonomic information of each sequence in the RDP database from domain to

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genus was created.

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Statistical analysis

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Three batches of each Chinese rice wine (obtained by 3 separate fermentation trials)

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were analyzed in triplicate. Pearson’s correlation test and t-test were conducted using

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Origins 7.5 (OriginLab, MA, USA). For univariate statistics, analysis of variance

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(ANOVA) was performed using SPSS statistical software version 12.0 (SPSS Inc.,

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Chicago, IL). When ANOVA revealed P < 0.01, the data were further analyzed using

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Duncan’s test for multiple comparisons. For multivariate analysis, principal component

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analysis (PCA) was carried out to identify any possible patterns and outliers between

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samples.27

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RESULTS

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BA analysis during industrial fermentation

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To date, few studies have analyzed the BAs formed during fermentation, and no studies

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on the BA metabolic landscape on the industrial scale have been reported. It has been

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commonly accepted that BAs are mainly produced in the steeping and canning periods. No

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biogenic amines were detected in rice, Chinese koji and wheat Qu which

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in accordance with previous work.17

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Figure 1

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A different result was found in our work. As shown in Figure 1, unlike previous reports,

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BA formation was mainly detected in two periods, canning and the beginning of the main

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fermentation period. 38.1 mg/L was generated in the first 1.5 days, and 45.7 mg/L was

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generated on day 5. The main fermentation stage had the largest contribution to total BA

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formation: 69.1% (54.3 mg/L); only 7.4 mg/L was produced during post-fermentation.

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The final BA concentration was 78.5 mg/L, lower than the previously reported value of 102

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mg/L. This difference may have occurred because we discarded the seriflux, which is not

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part of the traditional method.16 The trend of the changes in the total BA levels over time

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was similar to those of the LAB and ethanol levels but different from those of mould and

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yeast. The mould and yeast grew rapidly in the first 3 and 6 days and then decreased

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sharply to 104 cfu/mL. According to Xie et al., the changes in microorganism composition

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lead to different functions at different fermentation time points.28

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Figure 2A shows that the fluctuation trends of the formation ratio of BAs during

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fermentation in 8 BAs are similar. The highest BA formation ratio was 11.4 mg/L*d on

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day 5 and that the next highest was 6.3 mg/L*d in the first 1.5 days which give the major

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contribution. The formation ratios were -5.7 and -1.2 mg/L*d on days 3 and 4 when

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200% water and then 100% steamed rice were added to the vat. The ratios of BA

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formation decrease to less than 0.3 mg/L*d and the concentrations of all 8 BAs only

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exhibited small increases after day 7. Among the 8 BAs, the highest formation ratio was

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exhibited by PUT, followed by TYR and HIS, which displayed similar concentrations.

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Figure 2B shows that PUT was the most abundant BA at the end of fermentation (35.8

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mg/L and 45.54%). This finding differs from that in wine, in which TYR and HIS are the

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most toxic BAs. Here, the concentrations of these species were 25.0 and 6.14 mg/L, and the

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final proportions were 31.84% and 7.82%, respectively. Among the 8 BAs, the highest

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formation ratio was exhibited by PUT, followed by TYR and HIS, which displayed similar

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concentrations.

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Figure 2 Changes in microorganisms and metabolites and their correlations with BAs

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The above result revealed that the BA concentrations exhibited the same trend as the

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LAB and ethanol, similar to the results reported in other studies.28 However, the exact

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correlation between BAs formation and other metabolites and microorganisms remained

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unknown. Shen et al. reported a PCA of these factors during the ageing of Chinese rice

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wine, and separation was found to be common, similar to the result of their previous

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study.29

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Figure 3

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Figure 3 shows the PCA performed on 8 variables (ethanol, total acid, total amino acids,

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pH, LAB, BA, yeast and mould), which were significantly different in 11 samples from

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different fermentations. All of the data were auto-scaled to prevent variables with high

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intensities from being considered more important than those with low intensities before

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PCA. The first two PCs accounted for 88.2% of the total variance in the raw data (PC1

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69.7% and PC2 18.5%). The separation between samples of various fermentation statuses

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was quite satisfactory. Overall, the pattern recognition revealed that PC1 clearly

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classified the 11 samples into two groups: canning and fermentation. However, PC2

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accounted for minor separation and classified samples into four groups: steeping, canning,

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main fermentation and post-fermentation; these groups perfectly match the brewing status.

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Pereira et al. reported factors governing BA formation, including the presence of an

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amino acid decarboxylase, the amount of free amino acid substrates, and appropriate

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reaction conditions.30 As shown in Figure 3, total acid and ethanol had good, positive

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relationships with BA formation which similar with total amino acids and LAB. These

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findings confirmed that amino acid decarboxylation favors acidic and other stressful

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media, resulting in increased pH and membrane energization.31

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Figure 4 shows that the R2 values of all four factors exceeded 0.80, which is rather

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good. The coefficients were different, with the lowest obtained for ethanol and the

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highest for the total amino acids: 0.0006 and 0.022, respectively. As shown in Figures 4C

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and 4D, the initial BA concentration was rather high (around 20 mg/L), suggesting that

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formation initiates at the very beginning of the process. This finding is opposite to that in

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wine, in which BAs are produced at concentrations of less than 4 mg/L before malolactic

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fermentation.32 This result confirmed that the decarboxylate reaction favors acidic and

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stressful conditions and that the amino acids and LAB are direct contributors. Therefore,

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BA formation during industrial Chinese rice wine fermentation may be related to

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metabolic activity, which was directly related to the decarboxylase activity. Hence, its

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relationships with amino acids and LAB were further investigated in detail.

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Figure 4 Correlation between BAs and amino acids

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Chinese rice wine contains 7 different essential amino acids, and the total amino acid

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concentration is much higher than those of other liquors, which is considered

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advantageous.12 BAs are mainly produced via the decarboxylation of amino acids; each

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BA has one specific precursor amino acid, except for PUT, SPM and SPD, which have

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two precursors: arginine (ARG) and ornithine (ORN).33 Figure 5 shows that the levels of

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all BAs are strongly correlated with those of their precursor amino acids throughout the

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fermentation process. All R2 values were approximately 0.85, except for those relating

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SPD to ARG and ORN, which were 0.70 and 0.72, respectively. Additionally, the initial

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concentrations of the PUT, TYR and HIS were high, similar to the result described above.

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This finding confirmed that the formation of all 8 BAs mainly resulted from the

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decarboxylation of their precursor amino acids during fermentation.

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Figure 5 Multivariate linear stepwise regression was used to investigate the relationships of SPD, SPM, and PUT with their precursors, as shown in Table 1.

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Table 1

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Based on these results, we determined the metabolic networks for PUT, SPD and SPM

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with ORN and ARG, as shown in Figure 6. PUT is mainly formed via two principal

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pathways: (i) direct decarboxylation of ORN and (ii) decarboxylation from ARG via

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agmatine. The first is usually more efficient because it is a one-step reaction.34 During

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industrial Chinese rice wine fermentation, both pathways appeared to have similar

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decarboxylation efficiencies, and the coefficients relating PUT to ORN and ARG were

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0.05 and 0.07, respectively. Regarding the relationships of PUT with SPD and SPM, SPD

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was approximately two-fold higher than SPM during the fermentation process. The

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coefficient relating SPD to ARG was -0.001, indicating that that it could convert back to

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PUT. In conclusion, PUT was mainly decarboxylated from ARG (89.6%), whereas SPD

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(>100%) and SPM (83.1%) were decarboxylated from ORN, and the ARG levels were

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3.40-fold higher than those of ORN. Interestingly, some SPD could convert back to PUT

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(24.3%). It may because some polyamine oxidases involved in the spermidine

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degradation.35 Unlike wine, lower amounts of SPD were produced in many samples

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compared with the control, whereas SPM was drastically increased in most of the

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samples compared with the control.36

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Figure 6 Correlation between the BAs and LAB

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Table 2 shows that the concentrations of all 8 BAs exhibited good correlations with the

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LAB throughout the fermentation process. Most of the R2 values were approximately

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0.75, lower than those for amino acids; PHE was the highest, with an R2 value of 0.85,

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whereas the lowest was SPD, with an R2 value of 0.61. The initial concentrations of PUT,

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TYR and HIS were high which concentrations are 5.23 mg/L, 6.84 mg/L and 0.93 mg/L,

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but those of the other BAs were low. This difference could be explained by the

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observation that the LAB had different capacities for producing different amines, and this

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capacity was associated with the specific fermentation status. Additionally, other bacteria

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can produce BAs at the beginning of the process.

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Table 2

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As shown in Figure 7A, analyzing the LAB in detail revealed that Streptococcus,

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Lactococcus, Lactobacillus, Leuconostoc and Pediococcus are the five main genera

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present during Chinese rice wine fermentation. Except at the beginning, Streptococcus is

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the most abundant LAB and represents approximately 60% of the LAB, followed by

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Lactococcus and Lactobacillus, which correspond to approximately 30% and 5%,

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respectively. As shown in Figure 7B, PCA was performed on the five LAB genera and

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BAs, which differed significantly in the 11 samples from different fermentation statuses.

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The first two PCs accounted for 80.8% of the total variance in the raw data (PC1 45.4%

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and PC2 35.4%). The separation between samples of various fermentation statuses was

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quite satisfactory. Overall, the pattern recognition revealed that PC1 and PC2 clearly

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classified the 11 samples into three groups: beginning, high metabolic activity and low

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metabolic activity; these match the BA metabolic status. Furthermore, only Lactobacillus

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were found to have a positive relationship with the BAs which R value is 0.15 which is

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consistent with previous reports. And within microbial groups, in many cases the capacity

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to produce biogenic amines is a strain specific characteristic. Then, to assess the

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incidence of specific bacteria with BA-producing potential, high-throughput DNA

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sequencing of the metagenome should be employed, as in other fermented foods.

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Figure 7 DISCUSSION

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Concern regarding food safety has raised interest in analyzing BAs in Chinese rice

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wine in recent years. And BAs concentration of Chinese rice wine is approximately 2

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times of wine, it may be because CRW has a high level of amino acids and LAB which

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according with previous reports.12 Although many related studies have been published,

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the relationship between the vinification conditions and BA formation in wine is not well

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understood.20 Here, the whole landscape of BAs metabolism during industrial Chinese

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rice wine fermentation was determined. Unlike previous reports, the main fermentation

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process was found to be the major contributor to BAs formation, possibly because the

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steeping water was discarded, which is used in the traditional brewing process. Moreover,

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the proportion of seed starter was 50% of the final volume, providing enough leavening

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power for fermentation. In addition, the detailed description about the relation between

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BAs formation and various variables was analyzed. The formation of all 8 BAs was

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found to be strongly related to amino acids and LAB, which favored acidic and other

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stressful conditions. Specifically, the metabolic network relating PUT, SPM and SPD

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with their precursors, ARG and ORN, was established. Interestingly, PUT is mainly

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produced from ARG, whereas SPD and SPM are produced from ORN, and SPD can

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convert back to PUT. This may be related to their function, because the production of

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polyamines, such as PUT, could interfere with physiological functions in bacteria, such as

306

osmotic stress and oxidative stress responses, and bacterial cross-talk.37

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Although we confirmed that the LAB and amino acids are direct contributors to BA

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formation, the detailed mechanism by which BAs are formed remains unclear. The

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European Food Safety Authority (EFSA) Panel on Biological Hazards (BIOHAZ) (2011)

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concluded that the accumulation of BAs in fermented foods is a complex process that is

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affected by multiple factors and their interactions, the combinations of which are

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numerous, variable, and product specific.38 As shown in Figures 8A and 8B, although the

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concentration of amino acids and lactic acid is high, interestingly, the formation ratio

314

decreases after the main fermentation, indicating that the amino acid decarboxylase

315

activity also decreases after day 7. As shown in Figure 8B, the trend of the BA formation

316

ratio is similar to those of the acid, ethanol, LAB and amino acids. This finding is similar

317

that of Pessione et al., who showed that amino acid decarboxylation could occur in wine

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when the bacterial population is in the exponential growth stage under stressful

319

conditions.39

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Figure 8

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The exact relationships with environmental factors, specific LAB species and

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decarboxylase activity were investigated. O’Sullivan reported the dominant species with

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TYR- and HIS-producing potential using high-throughput DNA sequencing.40 PCR

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primers and DNA probes were designed to detect HIS decarboxylase in wine which

325

determines HIS synthesis. Additionally, some studies have demonstrated that acidic and

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stressful conditions induce decarboxylase expression.41 In Chinese rice wine, the initial

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concentration of BAs is approximately 15 mg/L, and the concentrations of both

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metabolites and microorganisms are rather low. These results suggest that studies should

329

be performed in a standard system using selected strains to eliminate variables from the

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natural system and obtain a better understanding of BAs formation. And the described

331

works above are ongoing in our lab and will published soon.

332

It is important to identify mechanisms to reduce the final BAs contents, especially the

333

toxic BAs, in Chinese rice wine. Selection and addition strains, which free or reduce BAs,

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are the mainstays of current research. The absence of decarboxylase activity should be a

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criterion for the selection of strains intended for fermentation to obtain a BA-free product.

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And we can use some exist BAs degradation strains, especially Lactobacillus casei which

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can degrade BAs in wine and cheese fermentation.42 For industrial Chinese rice wine

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fermentation, high-quality raw materials and optimal technological conditions also are

339

crucial factors to ensure proper performance and reduced BA accumulation. Discarding

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the seriflux and focusing on the main fermentation, which is the major step in BA

341

formation, may be good strategies, particularly for the control of the metabolic activity of

342

LAB via an optimized brewing method. The proteolytic capacities of the strains to be

343

used in these fermentations should also be studied, particularly to control the formation of

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toxic BA precursors.

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CONFLICT OF INTEREST

346 347 348

The authors have no conflicts of interest to declare. FUNDING This work was supported by grants from Key research and development program of

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Jiangsu Province (Grant No. BE 2016331); the National Natural Science Foundation of

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China (Grant No. 31301540); and the Priority Academic Program Development of

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Jiangsu Higher Education Institutions.

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ACKNOWLEDGMENT

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We thank Xinhua Jin and Meifang Xia from Nantong Baipu Chinese Rice Wine Co.,

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Ltd., for their assistance with the industrial fermentation.

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ASSOCIATED CONTENT

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Supporting Information Available

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Changes in the levels of each BA during fermentation (Figure S1). Correlation

358

between the BAs and lactic acid (Figure S2). This material is available free of charge via

359

the Internet at http://pubs.acs.org.

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478

V. Potential of wine-associated lactic acid bacteria to degrade biogenic amines. Int. J.

479

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480 481

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482

FIGURE CAPTIONS

483

Scheme 1. The fermentation and sampling processes of semi-dry Chinese rice wine. The

484

number in the scheme is the sampling points.

485

Figure 1. Changes in the microorganism, alcohol and BA levels during fermentation.

486

Figure 2. A) Changes in the formation ratio of BAs during fermentation. B) The

487

proportion of BAs at the end of fermentation. PHE - 2-phenylamine, PUT - putrescine,

488

CAD - cadaverine, HIS - histamine, TYR - tyramine, TRY - tryptamine, SPD -

489

spermidine, and SPM - spermine.

490

Figure 3. PCA of the BA concentration, microorganisms and metabolite concentrations

491

during the main fermentation process. AA - amino acids, BA - biogenic amines, LAB -

492

lactic acid bacteria.

493

Figure 4. Correlations between the total BAs and the following variables: A) total acid, B)

494

ethanol, C) LAB, and D) total amino acids. BA - biogenic amines.

495

Figure 5. Correlation between BAs and amino acids.

496

Figure 6. The metabolic network of PUT, SPD and SPM with ORN and ARG; PUT -

497

putrescine, SPD - spermidine, SPM - spermine, ornithine-ORN, and arginine - ARG.

498

Figure 7. A) Changes of the main LAB during fermentation. B) PCA of the BA

499

concentration and main LAB throughout the fermentation process. BA - biogenic amines,

500

str - Streptococcus, lat - Lactococcus, lac - Lactobacillus, leu - Leuconostoc, ped -

501

Pediococcus

502

Figure 8. A) Correlations between the formation ratio of BAs with amino acids and LAB

503

during fermentation. B) Changes in the formation ratios of microorganisms, alcohol and

504

BAs during the main fermentation process.

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Page 26 of 37

Table 1 Equations of SPD, SPM, and PUT with their precursors Number

BA with their precursors

Equation

R2

1

SPD, SPM, PUT with ORN and

y=8.20+0.05X1+0.07X2

0.83

ARG 2

PUT with ORN and ARG

y=8.22+0.028X1+0.073X2

0.81

3

SPM with ORN and ARG

y=0.003+0.005X1+0.0002X2

0.98

4

SPD with ORN and ARG

y=-0.022+0.017X1-0.001X2

0.97

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Table 2 Correlation between the BAs and lactic acid bacteria BA

Equation

R2

2-phenylamine

y=0.50x+0.54

0.85

Putrescine

y=4.01x+7.08

0.77

Cadaverine

y=0.48x+0.59

0.78

Histamine

y=0.642x+1.04

0.74

Tyramine

y=2.53x+6.49

0.74

Tryptamine

y=0.11x+0.12

0.63

Spermidine

y=0.11x+0.09

0.61

Spermine

y=0.065x+0.047

0.70

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FIGURES Scheme 1

28


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Figure 1

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Figure 2

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Figure 3

31



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Figure 4

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Figure 5

33



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Figure 6

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Figure 7

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Figure 8

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TOC Graphic

37


Insights into the Biogenic Amine Metabolic Landscape during

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