Mixed Starter Culture Regulates Biogenic Amines Formation via

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Food and Beverage Chemistry/Biochemistry

Mixed Starter Culture Regulate Biogenic Amines Formation via Decarboxylation and Transamination during Chinese Rice Wine Fermentation Xiaole Xia, Yi Luo, Qingwen Zhang, Yang Huang, and Bin Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01134 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Journal of Agricultural and Food Chemistry

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Mixed Starter Culture Regulate Biogenic Amines Formation

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via Decarboxylation and Transamination during Chinese

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Rice Wine Fermentation

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Xiaole Xia1, a, *, Yi Luo1, a, Qingwen Zhang1, Yang Huang1, Bin Zhang2

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

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a

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*

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

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

These authors contributed equally to this work. Corresponding Author:

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Xiaole Xia

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E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT 13

Utilize amine-negative starter based on understanding of nitrogen metabolism is a

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useful method for controlling biogenic amine (BA) in Chinese rice wine (CRW)

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fermentation. The contribution of brewing materials to protein degradation was

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analyzed, wheat Qu protein had no effect and yeast autolysis generated 10% amino

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nitrogen. Milling degree of rice was strongly correlated with BAs formation (R2 = 0.99).

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Subsequently, Lactobacillus plantarum and Staphylococcus xylosus were co-inoculated

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as amine-negative starter at an optimized ratio of 1:2. Co-inoculation induced a

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significant reduction in total BAs (43.7%, 44.5 mg L-1), putrescine (43.0%, 20.4 mg

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L-1), tyramine (42.8%, 14.3 mg L-1), and histamine (42.6%, 3.5 mg L-1) content.

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Notably, BAs degradation ability of Staphylococcus xylosus was stronger than the

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suppression effect of Lactobacillus plantarum, and higher lactic acid bacteria (LAB)

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amount has a positive correlation with lower BAs content. Overall, mixed strains

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exerted a synergistic effect in lowering BAs accumulation via decarboxylation and

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transamination.

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KEYWORDS:

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Staphylococcus xylosus, Lactobacillus plantarum, mixed starter

biogenic

amine,

Chinese

rice

wine,

nitrogen

metabolism,

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2


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INTRODUCTION 30

Chinese Rice Wine (CRW) is one of the most popular alcoholic beverages in China on

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account of its abundant amino acids, trace nutritional substance and low alcoholicity.1

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CRW brewed via a typical open semi-solid-state fermentation process. Recently,

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nitrogen metabolism hazards in CRW fermentation, including biogenic amine (BA) and

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ethyl carbamate, have raised significant public concerns. Ethyl carbamate is a

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carcinogenic compound resulting from the spontaneous reaction between urea and

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ethanol and its formation in brewing process of CRW has been widely investigated.2-3

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Among the nitrogenous compounds, BAs are synthesized through microbial

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decarboxylation of precursor amino acids.4 The most common amines, histamine (HIS),

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tyramine (TYR), tryptamine (TRY), phenethylamine (PHE) and cadaverine (CAD), are

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generated from histidine, tyrosine, tryptophan, phenylalanine and lysine, respectively.5

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Two independent pathways, via ornithine decarboxylase and agmatine deiminase, that

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generate putrescine (PUT) have been identified to date.6-7 And spermidine (SPD) and

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spermine (SPM) arise from putrescine that can mutually interconverted.8

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Some types of BAs are undesirable in fermented products since they trigger

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headaches, hypotension and digestive problems, especially in sensitive person.9 Among

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the BAs identified, HIS and TYR, displaying the greatest toxicity, have been widely

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studied. And malolactic fermentation was the main process to produce HIS and TYR in

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winemaking process. Additionally, polyamines such as PUT and CAD, with lower

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pharmacological activity, are reported to interact with amine oxidases to exacerbate the

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side effects of HIS and TYR.10 The main BAs determined in CRW are PUT, CAD, HIS,

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TYR, SPM and SPD.11 Héberger, Ket al. observed a significant association between

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free amino acids and BAs concentration in wine.12 The concentration of BAs in wine 3



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vary from trace amounts up to 130 mg L-1.13 HPLC with a pre-column derivatization

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was the most prevalent analytical technique for determining BAs. Recently, Xie and

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co-workers reported that the amino acid composition in CRW is highly associated with

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total protein content of raw material.14 Some aliphatic amines exist in wheat Qu

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(saccharifying agent) and glutinous rice(< 2.88 mg kg-1).15 In many cases, the capacity

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to produce BAs is related to nitrogen compounds and microbial decarboxylase

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activity.16 To our knowledge, no detailed studies have been conducted on protein

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metabolism and consequent effects on BAs formation during CRW fermentation

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process.

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At the beginning of CRW brewing, wheat Qu and Chinese koji (Aspergillus oryzae)

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are added to provide abundant microorganisms in the fermentation mash. Lactic acid

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bacteria (LAB) constitute the dominant BAs producers in CRW, similar to other

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fermented products. In 2011, Zaman et al. detected Lactobacillus rhamnosus as the

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main strain responsible for BAs formation in five wineries.17 Histamine is obtained by

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decarboxylation of free histidine through Lactobacillus hilgardii and Pediodoccus

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parvulus in wine.18 Production of tyramine was identified as a species-level trait of

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Lacobacillus brevis IOEB9809 in wine.19 And Enterococcus faecalis was responsible

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for putrescine biosynthesis.20 Several strategies have been established to control BAs

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accumulation in food based on the mechanisms underlying the formation of these

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compounds. Physical methods include cold treatment, hydrostatic pressure, irradiation

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to inhibit its microbial activity, and destroy producer strain cells.21 However, microbial

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growth is crucial to obtain fermented foods and these physical techniques may affect

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the flavor and quality of the final product. Another approach to control BAs formation

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is modulation of protein catabolism by utilizing specific starter culture that possessing

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low decarboxylase and significant amine oxidase activities.22-23 Moreover, disruption 4


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of autochthonous microbiota may aid in reducing the occurrence of BAs producer

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strains. Callejón et al. isolated Lactobacillus plantarum J16 and Pediococcus

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acidilactici CECT 5930 possessing laccases and multicopper oxidases responsible for

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amine degradation in wine.24 The use of Staphylococcus xylosus result in

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amine-degradation, this was accompanied by producing aldehyde, hydrogen peroxide,

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and ammonia via amine oxidase activity.25 Dehaut et al. demonstrated that inoculating

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fermented anchovies with Staphylococcus xylosus reduced the total BAs content by

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16%, especially in PUT, CAD, and TYR content.26 Mixed starter culture comprising

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Lactobacillus plantarum and Saccharomyces cerevisiae drastically reduced the

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accumulation of PUT and CAD level in fermented silver carp sausage, through their

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rapid growth and antimicrobial activity.27 Make use of Lactobacillus spp. and

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Staphylococcus spp. as starter culture in meat products has been widely investigated.

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However, little information is available on microbial interference to obtain CRW of low

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BA yield, especially with regard to effects on nitrogen metabolism and interactions

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between the starter and microbial community.

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The development of effective control strategies requires clarification of the BAs

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formation mechanisms. Therefore, identification of the protein sources and

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microorganisms involved in BAs production is necessary for enhancing food safety of

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fermented products. In this study, we evaluated the relationship between BAs formation

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and protein metabolic pathways, and the origin of nitrogen in raw materials employed

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for brewing CRW. Fermentation mash was co-inoculated with S. xylosus

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(CGMCC1.8382) and L. plantarum (ACBC271), which with a view to limiting

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decarboxylation and fortifying transamination, as well as inhibiting the growth of

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indigenous producer strains, and consequently, controlling BAs formation. The overall

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mechanism underlying BAs production and the effect of co-inoculation with the two 5



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strains on regulating BAs during fermentation were discussed.

MATERIALS AND METHODS 104

Brewing and Sampling of Chinese Rice Wine

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The brewing procedure of CRW was performed as described previously.28 Samples

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were collected from the top, center, bottom part of fermented mash for each

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fermentation stage in triplicate, and stored at 4 °C after filtration. The liquid samples

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were used for preparation of derivatization prior to HPLC.

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BAs determination

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Quantitative assay of BAs was performed via reversed-phase HPLC (RP-HPLC),

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eluted compounds were determined by a diode array spectrophotometer (Hitachi, Japan)

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and measuring absorbance at 254 nm.29 5 mL of sample was homogenized with 15 mL

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of hydrochloric acid (0.1 M). After centrifuging at 6000g (10 min, 4 °C), the

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supernatant was collected and diluted with hydrochloric acid (0.1 M) to 25 mL, stored

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at 4 °C for analysis. A 2 mL aliquot of the extract was supplemented with 0.2 mL of 1,

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

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mixture was derivatized with 2 mL of dansyl chloride at 45 °C for 1 h before analysis.

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The mobile phase consisted of A (acetonitrile) and B (water) at flow rate of 0.8 mL

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min-1. The gradient elution program as described by Annalisa Tassoni et al.,30 and with

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some modification as follows, 55% A at 5min, 65% A at 10min, 70% A at 20 min, 90%

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A at 27 min, 100% A at 30 min and 55% A at 35 min. Chromatography was performed

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at 30 °C and injection volume was 20 µL.

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Enumeration of microorganisms and detection of physico-chemical properties

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For microbiological enumeration, the number of viable LAB and yeast cells were

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determined as colony-forming units (CFU mL−1), samples were serially diluted 10-fold

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with sterile saline (0.85% NaCl) then spread 0.1 mL of each dilution on agar plates.

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LAB strains were routinely incubated on MRS (de Man-Rogosa–Sharp, Oxoid, Milan,

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Italy) medium at 37 °C for 24 h under anaerobic conditions. The pH of medium was

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adjusted to 5.8 with NaOH before sterilization for 15 min at 121 °C. Yeast was cultured

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on 3M count plates (MN, United States) at 25 °C for 68 h under aerobic conditions.

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Protein content was determined by the method described by Lowry et al.31 The

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analysis of starch was performed according to the method of Frei, M. et al.32

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Physico-chemical parameters (amino nitrogen, total sugar, acid, alcohol, and amino

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acid content) of fermentation mash were measured according to the National Standard

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of the People’s Republic of China of CRW.33

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Investigation of protein metabolism of wheat Qu, yeast autolysis and different

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levels of milled rice

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We investigated the contribution to protein metabolism of wheat Qu by determining

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protein and amino nitrogen content during fermentation. Fermentation was conducted

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with wheat Qu as control group, another fermentation group used enzyme (amylase,

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glucoamylase and protease, each 10 U g-1) to replace wheat Qu, and group 3 employed

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enzyme (amylase, glucoamylase and protease, each 10 U g-1) plus inactivated wheat Qu.

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Amylase (thermostable alpha-amylase, from Bacillus licheniformis), glucoamylase

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(1,4-alpha-D-glucan glucohydrolase, from Aspergillus niger) and protease (Acid

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protease, from Aspergillus oryzae) were purchased from Sigma Chemical Co. (St. 7



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Louis, MO, U.S.A.).

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To determine the effect of yeast autolysis on nitrogen metabolism, Chinese rice

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wine-like medium was inoculated with yeast cells. Saccharomyces cerevisiae (Angle,

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China) was inoculated in sodium lactate buffer (50 mmol L-1, pH 4.2, 13% v/v) and

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incubated at 30 °C for 7 days, the yeast cells were collected after centrifuging and

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washing three times with sterilized water. Rice was steamed after soaking, followed

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with saccharification by amylase (10 U g-1) at 85 °C for 0.5 h and with glucoamylase

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(10 U g-1) at 60 °C for 4 h. Then filtered and diluted to 13 ° Bx with water which pH

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value was adjusted to 4.2 ± 0.1 with lactic acid, and autoclaved at 121 °C for 15 min.

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The sterilized mixture as Chinese rice wine-like medium, which was inoculated with

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collected yeast cells and incubated at 15 °C for 21 days. Amino nitrogen content and

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yeast count were determined every 3 days.

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Brown rice was polished to different milling degrees of rice (100%, brown rice, A;

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91%, B; 87%, C; 82%, D), and subjected to CRW fermentation. The milling degree was

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calculated by using the following expression, which is an important indicator of rice

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quality.

Milling degree (%) =

weight of milled rice *100 weight of brown rice

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The physico-chemical parameters of different milled rice are shown in Table 1. Total

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sugar, ethanol, amino nitrogen and yeast count were evaluated during fermentation

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process.

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Table 1

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Identification of the ability of Lactobacillus plantarum (ACBC271) and

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Staphylococcus xylosus (CGMCC1.8382) to control BAs formation

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Low BAs-producing capability of Lactobacillus plantarum (ACBC271) was assessed

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in MRS broth, compared with Lactobacillus sp.30a (ATCC33222) which is able to

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produce HIS, PUT and CAD. MRS broth supplemented with 100 mg L-1 of each

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precursor amino acid (histidine, tyrosine, tryptophan, phenylalanine, lysine, arginine,

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and ornithine). The ability of BAs degradation by Staphylococcus xylosus

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(CGMCC1.8382) grown on Mannitol Salt Agar (MSA) broth, which supplemented

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with 100 mg L-1 of each amine (histamine, tyramine, tryptamine, phenethylamine,

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cadaverine, putrescine, spermidine and spermine). BAs concentration were examined

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to evaluate the control ability of the microorganism. After incubation at 37 °C for 48 h,

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samples were derivatized and analyzed by HPLC. The standard amino acids and BAs

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were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

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CRW fermentation with amine-negative starter culture

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Lactobacillus plantarum (ACBC271) and Staphylococcus xylosus (CGMCC1.8382)

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were prepared in MRS and MSA medium, respectively. Culture was conducted at 37 °C

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for 24 h prior to fermentation. Strains were inoculated into CRW fermentation with the

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initial cell density of 3.5 × 107 CFU mL-1. A non-inoculated sample of CRW

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fermentation was prepared as the control. Additionally, single-strain fermentation with

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L. plantarum (ACBC271) or S. xylosus (CGMCC1.8382) was conducted, respectively.

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Mixed strains were co-inoculated at ratios of 1:1and 1:2 to determine the relative

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amounts of strains for optimal fermentation. Samples of 10 mL were collected at

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different time and subjected to the analysis of BAs and other parameters. 9



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Data Analysis

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All experiments were performed in triplicate and presented as the mean with standard

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deviation (mean ± SD) for parallel samples. Pearson’s correlation and t tests were

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performed using Origins 8.0 (Origin Pro, MA, United States). A one-way analysis of

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variance (ANOVA) test was applied by SPSS statistical software version 12.0 (SPSS

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Inc., Chicago, IL, United States), which P value was < 0.01. Multiple linear regression

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using principal component analysis (PCA) was conducted to identify possible patterns

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and outliers between samples.

RESULTS 197

Contributions of wheat Qu and yeast autolysis to nitrogen metabolism

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BAs are mainly formed by decarboxylation of the corresponding precursor amino acids.

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While several reports have documented the effects of related microorganisms, few

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investigations have focused on the role of protein metabolism.34-35 Except rice, wheat

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Qu and yeast autolysis possess protein that may contribute to BAs formation in CRW

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fermentation. To determine the specific contributions of these proteins (other than rice

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protein) to amino nitrogen and BAs formation, the landscape of protein metabolism

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during fermentation requires clarification. Protein and amino nitrogen content were

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monitored in different wheat Qu containing fermentation samples (Figure 1), which

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showing a homologous trend in all three groups. The protein content (0.08 g L-1) in

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control group was lower than that in other groups, and amino nitrogen content (0.46 g

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L-1) was the highest at the end of fermentation. This finding may be explained that

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normal fermentation mash possesses abundant microorganisms with high protease

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activity, which convert protein into amino nitrogen through nitrogen metabolism. 10


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Interestingly, the amino nitrogen contents of Groups 2 and 3 were nearly equivalent

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with the concentration of 0.23 and 0.24 g L-1, respectively, indicating that proteins of

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wheat Qu have no influence on nitrogen metabolism during CRW fermentation. In the

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later stages of brewing, high protein content with a slow degradation rate was observed,

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suggesting that microbial activity is a crucial factor for nitrogen metabolism in CRW.

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Figure 1

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Yeast autolysis releases peptides and amino acids that affect BAs formation in

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sparkling wines.36-37 To investigate the contribution of yeast autolysis to nitrogen

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metabolism, Saccharomyces cerevisiae was inoculated into CRW-like medium.

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Changes and correlations of yeast and amino nitrogen patterns are presented in Figure 2.

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The amino nitrogen concentration increased to 0.047 g L-1 at day 15 and yeast count

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decreased progressively from 4.9 × 109 CFU mL−1 to 7 × 106 CFU mL−1. Furthermore,

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the amino nitrogen content (0.049 g L−1) from yeast autolysis accounted for 10% of

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total amino nitrogen, and a positive correlation (R2 = 0.94) existed between yeast and

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amino nitrogen.

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Figure 2

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Influence of rice protein on protein metabolism and BAs formation during CRW

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fermentation

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Rice is raw material of CRW fermentation, which provide protein substrate of

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microbial growth. The milling degree is an indicative of the overall protein content.

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We conducted fermentation using four levels of rice with different milling degree to

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investigate the metabolism of rice proteins. To better confirm that the influence of

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milling degree on BAs formation, all the data on parameters (ethanol, total sugar,

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milling rate, amino nitrogen, starch, protein, and yeast) and total BAs, were used to 11



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perform a principal component analysis (PCA) as shown in figure 3A, indicating that

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significantly different under the 4 levels of milling degree (100%, 91%, 87% and 82%)

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examined. As a result, two PCs accounted for 96.48% (PC1, 58.97%; PC2, 37.51%),

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brown rice and milled rice clearly separated into two distinct groups. Ethanol, amino

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nitrogen (AN) and yeast count appeared to be positively correlated with BAs. At the

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end of fermentation, the range of ethanol is 8.9-13.2% v/v, and corresponding total

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BAs concentration increased from 41.7 to 82.5 mg L-1. The correlation of ethanol with

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BA was presented in Figure 3B (R2 = 0.99), this suggests that a stressful environment

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containing high ethanol content may thus promote decarboxylation reactions to a

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certain extent, which might be due to amine formation as a protective mechanism of

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microorganisms against unfavorable growth conditions which is consistent with

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previous report.38

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Particularly, the group of fermentation with brown rice (group A) show a low

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ethanol (8.9% v/v) and low total sugar content (21 g L-1) at the end of fermentation.

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That might attribute to the rice protein was enveloped by rice bran. Therefore, PCA

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performed on 8 variables (ethanol, total sugar, BAs, milling degree, amino nitrogen,

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starch, protein, and yeast) except for group A, as shown in Figure 3C. This suggests

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that milling degree was positively correlated with BAs other than brown rice, and

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linear regression analysis revealed an R2 value of 0.99 (Figure 3D). The presence of

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BAs depend on the activities of at least two protein types, including (1) those involved

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in proteolysis into peptides, which further convert into precursor amino acids, (2)

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those enzymes responsible for decarboxylation of precursor amino acids.39 Therefore,

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considering the ethanol, amino nitrogen content and sugar utilization rates, the

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appropriate milling degree of 87-91% is a good choice for CRW fermentation. 12


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Figure 3

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Accumulation of BAs results from decarboxylation of specific precursor amino

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acids, which was strongly associated with protein metabolism of rice used for CRW. A

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close correlation between precursor amino acids and eight BAs during CRW

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fermentation was confirmed (R2 > 0.80), it is consistent with previous reports.11, 28

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Changes in amino acid content and BAs formation rate during CRW fermentation are

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shown in Figure 4. A significant increase of amino acids was observed at every stage.

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However, the high content of free amino acid did not generate homologous BAs

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formation rate. Especially at the end of fermentation, the amino acid concentration was

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3.2 g L-1 while the corresponding BAs formation rate of 0.55 mg L-1 d-1. This difference

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might attribute to a part of free amino acids is involved in catalytic actions,

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microorganism levels were in the stable or declining phase, which lead to weaker

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activity of BAs generating enzymes. Indicating that BAs formation during CRW

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fermentation is dominated by microorganism activity. Hence, the use of an

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amine-negative starter appears to present a promising means to diminish BAs

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concentrations in CRW.

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Figure 4

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Ability of L. plantarum (ACBC271) and S. xylosus (CGMCC1.8382) to control

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BAs

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Utilization of amine-negative starter to control the accumulation of BAs is broadly

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applied in fermented foods owing to high security and low interference with

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technological process. To verify the low BAs producing ability of L. plantarum

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(ACBC271) and degradation ability of S. xylosus (CGMCC1.8382) in medium. MRS

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broth was supplemented with seven precursor amino acids (each 100 mg L-1) for 13



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growth of L. plantarum. Low BAs producing activity was compared to BAs producer

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strain Lactobacillus sp.30a (ATCC33222). MSA with eight BAs (each 100 mg L-1) was

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used for evaluating degradation ability of S. xylosus (CGMCC1.8382). BAs content

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was detected after incubating for 48 h, as presented in Table 2. BAs formation during

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fermentation in L. plantarum (ACBC271) culture was less 58.91% than producer strain,

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and S. xylosus (CGMCC1.8382) showing a degradation percentage of 31.45% of total

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BAs. This result could be attributed to the lower decarboxylase activity of L. plantarum

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(ACBC271) and amine-oxidase of S. xylosus (CGMCC1.8382). Based on these

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findings, L. plantarum (ACBC271) and S. xylosus (CGMCC1.8382) were utilized as

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starter culture for further characterization.

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Table 2

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Influence of mixed starter on BAs accumulation during Chinese rice wine

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fermentation

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To our knowledge, no studies on optimizing starter culture to reduce BAs accumulation

297

during CRW fermentation has been conducted. In current investigation, fermentation

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mash was co-inoculated with L. plantarum (ACBC271) and S. xylosus

299

(CGMCC1.8382), and the ability to reduce BAs accumulation was analyzed as shown

300

in Figure 5. At the end of fermentation, control, single inoculation of L. plantarum

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(ACBC271) or S. xylosus (CGMCC1.8382) and co-inoculation samples contained total

302

BAs level of 79.1 mg L-1, 70.3 mg L-1, 61.7 mg L-1 and 58.2 mg L-1, respectively

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(Figure 5A). Compared with control group, reduction of total BAs was 11.1% in the L.

304

plantarum (ACBC271) and 22.0% in the S. xylosus (CGMCC1.8382) inoculation

305

groups. Indicating that the inhibitory effect of degradation is stronger than low

306

production efficiency. Moreover, an increased capability to diminish total BAs was 14


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observed, which with 25.5% reduction in mixed strains fermentation. Linear

308

regression analysis was performed on BA (excluding day 4) as shown in Figure 5B.

309

Overall, the slope of co-inoculation was the lowest at 0.96, suggesting that BAs

310

formation rate was the slowest. The two strains thus appear to exhibit a synergistic

311

suppressive effect on BAs accumulation. The BAs formation rate in co-inoculation

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group was lower than those of other groups at every stage (Figure 5C), which might

313

be attribute not only to the degradation activity of S. xylosus (CGMCC1.8382) and low

314

decarboxylation effect of L. plantarum (ACBC271) but also to the competition effect

315

with endogenous bacteria, particularly those with amino acid decarboxylase activity.

316

Our findings support the effective application of mixed starter culture with degradation

317

activity and low decarboxylase in reducing BAs accumulation during CRW

318

fermentation.

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Figure 5

320

L. plantarum (ACBC271) and S. xylosus (CGMCC1.8382) exhibited a synergistic

321

effect in reducing BAs formation. Since the suppressive effect of S. xylosus

322

(CGMCC1.8382) was stronger than that of L. plantarum (ACBC271), mixed culture

323

fermentation was conducted with ratio of 1:2 (L. plantarum (ACBC271) to S. xylosus

324

(CGMCC1.8382)). Compared with non-inoculated group, the BAs level was reduced to

325

25.5% and 43.0% at co-inoculation ratios of 1:1 and 1:2, respectively (Figure 6A).

326

These results indicated that the higher efficiency of BAs suppression at a co-inoculation

327

ratio of 1:2, which might due to amine oxidation of S. xylosus (CGMCC1.8382). As

328

shown in Figure 6B, piecewise linear regression analysis was performed, which divided

329

the whole process into main fermentation and post-fermentation stages. In main

330

fermentation stage, the slopes of these two groups were 2.03 (LP+SX, 1:1) and 1.44

331

(LP+SX, 1:2), respectively. At the post-fermentation stage, two groups displayed 15



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332

approximate slopes of 0.35 (LP+SX, 1:1) and 0.40 (LP+SX, 1:2), suggesting that

333

regulation on BAs was mainly happen in main fermentation stage.

334

Figure 6

DISCUSSION 335

The potential toxicity of BAs has been a subject of increasing research focus. The main

336

factor contributing to the presence of BAs is decarboxylation of precursor amino

337

acids.13 The influence of raw materials in fermentation mash on nitrogen metabolism

338

remains ambiguous. This work suggested that proteins of raw materials other than rice

339

had no significant influence on BAs formation, while microorganisms of wheat Qu are

340

an important contributory factor to nitrogen metabolism. Numerous studies to date have

341

focused on controlling BAs generation in fermented food. However, the utility of

342

physical methods is restricted, owing to demanding microbial growth conditions.

343

Capozzi et al. reported that BAs reduction is species- and strain-dependent, and

344

inoculation with amine-negative starter culture is an effective method to control BAs

345

generation.40 In this study, the fermentation mash was co-inoculated with L. plantarum

346

(ACBC271) and S. xylosus (CGMCC1.8382), with the aim to control BAs

347

accumulation during CRW brewing. Our findings indicated that the mixed strains

348

evoke low content of BAs through cooperative suppression of decarboxylation and

349

fortification of transamination activities. The S. xylosus (CGMCC1.8382) harbors

350

amine oxidase and L. plantarum (ACBC271) possesses low decarboxylase activity,

351

leading to low BAs yield. In addition, S. xylosus exhibited high antimicrobial activity

352

and competed with natural (endogenous) amine-producing microbiota of mash to

353

interfere with decarboxylation that metabolize precursor substrates into BAs.41

354

Generally, formation of BAs in CRW can be controlled by using appropriate raw 16


Page 17 of 32


355

materials, strict manufacturing environments and the inhibition of detrimental

356

microorganisms.

357

Although we confirmed prominent reduction of BAs using mixed starter culture,

358

the landscape of individual strains was not clear over all the fermentation stages. LAB

359

counts, ethanol, total acid content and their association with BAs production were

360

evaluated, as shown in Figure 7. The results showed similar changes of LAB, ethanol

361

and total acid levels. At the end of fermentation, the level of LAB in co-inoculation and

362

L. plantarum (ACBC271) inoculation groups were significantly higher than those of

363

control and S. xylosus (CGMCC1.8382) inoculation groups. All BAs showed similar

364

trends during fermentation, the concentration of individual BAs at the end of

365

fermentation were shown in Figure 7B. Compared with control group, PUT displayed

366

the highest content, followed by TYR and HIS, and the level of these three main BAs

367

was significantly decreased by > 42% in co-inoculation (LP+SX, 1:2) group. PCA was

368

performed on 7 variables (LAB, ethanol, total acid, BAs, PUT, TYR, HIS) in mixed

369

starter culture group (LP+SX, 1:2) as shown in Figure 7C. The first two PCs accounted

370

for 98.73% (93.90% PC1 and 4.83% PC2). LAB was negatively correlated with BAs,

371

i.e., higher LAB amount was associated with lower BAs content, similar to PUT, TYR,

372

and HIS. This suggests that the characteristic low decarboxylase activity of L.

373

plantarum (ACBC271) leads to diminished BAs production.

374

Figure 7

375

In natural and inoculated microbial communities within CRW fermentation system,

376

diverse microbial strains coexist and interact each other, which induce variations in

377

functions and specific microbial structures. Suppression of BAs was cooperatively

378

ascribed to amine oxidase activity of S. xylosus (CGMCC1.8382) and low amino acid

379

decarboxylase activity of L. plantarum (ACBC271), especially for HIS, PUT and TYR. 17



Page 18 of 32

380

BAs production ability is reported to be both species- and strain-dependent.42 Inhibition

381

of other strains and regulation of the relevant enzymes are complex dynamic processes.

382

Further elucidation of metabolic pathways is required in conjunction with species

383

identification to deep insight into the interactions among microbial community at the

384

molecular level, high-throughput DNA sequencing analyses are ongoing to explore the

385

microbial landscape during mixed strains fermentation. Additionally, Lactobacillus

386

plantarumcan produce diketone, diacetyl, 4-vinylphenol which are specific flavor

387

metabolites in wine.43 Staphylococcus xylosus also can promote polyphenol formation

388

in winemaking.44 Further research is underway to explore the species at molecular level

389

to reveal details of interactions and establish the effects of S. xylosus (CGMCC1.8382)

390

and L. plantarum (ACBC271) on flavor profiles.

391

Supporting Information

392

Scheme S1. The conversion pathways of amino acids and biogenic amines.

393

Scheme S2. The fermentation and sampling processes of Chinese rice wine.

394

Corresponding Author

395

Xiaole Xia,

396

E-mail: [email protected], Phone: 008651085326829

397

Funding

398

This work was supported by grant from National Key Research and Development Plan

399

(2017YFC1600401), The Fundamental Research Funds for the Central Universities

400

(JUSRP51734B), The Key Research and Development Program of Jiangsu Province

401

(BE 2016331), The 111 Project (No. 111-2-06).

18


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402

Notes

403

The authors declare no competing financial interest.

404

19



Page 20 of 32

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concentrations on biogenic amine contents in chorizo dry sausage. Food Microbiol. 2003, 20, 275-284. 24. Callejon, S.; Sendra, R.; Ferrer, S.; Pardo, I. Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine. Appl. Microbiol. Biotechnol. 2014, 98, 185-98. 25. Beck, H. C.; Hansen, A. M.; Lauritsen, F. R. Metabolite production and kinetics of branched-chain aldehyde oxidation in Staphylococcus xylosus. Enzyme & Microbial Technology 2002, 31, 94-101. 26. Dehaut, A.; Himber, C.; Mulak, V.; Grard, T.; Krzewinski, F.; Le Fur, B.; Duflos, G. Evolution of volatile compounds and biogenic amines throughout the shelf life of marinated and salted anchovies (Engraulis encrasicolus). J. Agric. Food. Chem. 2014, 62, 8014-22. 27. Nie, X.; Zhang, Q.; Lin, S. Biogenic amine accumulation in silver carp sausage inoculated with Lactobacillus plantarum plus Saccharomyces cerevisiae. Food Chem. 2014, 153, 432-6. 28. Xia, X.; Zhang, Q.; Zhang, B.; Zhang, W.; Wang, W. Insights into the Biogenic Amine Metabolic Landscape during Industrial Semidry Chinese Rice Wine Fermentation. J. Agric. Food. Chem. 2016, 64, 7385-7393. 29. Soufleros, E. H.; Bouloumpasi, E.; Zotou, A.; Loukou, Z. Determination of biogenic amines in Greek wines by HPLC and ultraviolet detection after dansylation and examination of factors affecting their presence and concentration. Food Chem. 2007, 101, 704-716. 30. Tassoni, A.; Tango, N.; Ferri, M. Comparison of biogenic amine and polyphenol profiles of grape berries and wines obtained following conventional, organic and biodynamic agricultural and oenological practices. Food Chem. 2013, 139, 405-13. 31. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265-275. 32. Frei, M.; Siddhuraju, P.; Becker, K. Studies on the in vitro starch digestibility and the glycemic index of six different indigenous rice cultivars from the Philippines. Food Chem. 2003, 83, 395-402. 33. Gereral Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China & Standardization Administration of the People's republic of China: China, 2008. GB 13662-2008. 34. Flasarova, R.; Pachlova, V.; Bunkova, L.; Mensikova, A.; Georgova, N.; Drab, V.; Bunka, F. Biogenic amine production by Lactococcus lactis subsp. cremoris strains in the model system of Dutch-type cheese. Food Chem. 2016, 194, 68-75. 35. Henríquez-Aedo, K.; Durán, D.; Garcia, A.; Hengst, M. B.; Aranda, M. Identification of biogenic amines-producing lactic acid bacteria isolated from spontaneous malolactic fermentation of chilean red wines. LWT - Food Sci and Technol. 2016, 68, 183-189. 36. Kemp, B.; Alexandre, H.; Robillard, B.; Marchal, R. Effect of production phase on bottle-fermented sparkling wine quality. J. Agric. Food. Chem. 2015, 63, 19-38. 37. Perpetuini, G.; Di Gianvito, P.; Arfelli, G.; Schirone, M.; Corsetti, A.; Tofalo, R.; Suzzi, G. Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation. Yeast 2016, 33, 303-12. 38. Mazzoli, R.; Lamberti, C.; Coisson, J. D.; Purrotti, M.; Arlorio, M.; Giuffrida, M. G.; Giunta, C.; Pessione, E. Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine. Amino Acids 2009, 36, 81-9. 39. Linares, D. M.; Martin, M. C.; Ladero, V.; Alvarez, M. A.; Fernandez, M. Biogenic amines in dairy products. Crit. Rev. Food Sci. Nutr. 2011, 51, 691-703. 40. Capozzi, V.; Russo, P.; Ladero, V.; Fernandez, M.; Fiocco, D.; Alvarez, M. A.; Grieco, F.; Spano, G. Biogenic Amines Degradation by Lactobacillus plantarum: Toward a Potential Application in Wine. Front Microbiol 2012, 3, 122. 41. Papamanoli, E.; Kotzekidou, P.; Tzanetakis, N.; Litopoulou-Tzanetaki, E. Characterization of Micrococcaceae isolated from dry fermented sausage. Food Microbiol. 2002, 19, 441-449. 42. Russo, P.; Spano, G.; Arena, M.; Capozzi, V.; Grieco, F.; Beneduce, L. Are consumers aware of the risks related to biogenic amines in food. Curr Res. Technol. Edu. Top. Appl. Microbiol. Microb. Biotechnol 2010, 1087-1095. 43. Pozo-Bayón, M. A.; G-Alegría, E.; Polo, M. C.; Tenorio, C.; Martín-Álvarez, P. J.; Banda, M. T. C. D. L.; Ruiz-Larrea, F.; Moreno-Arribas, M. V. Wine Volatile and Amino Acid Composition after Malolactic Fermentation: Effect of Oenococcus oeni and Lactobacillus plantarum Starter Cultures. J. Agric. Food. Chem. 2005, 53, 8729-35. 44. Papadopoulou, C.; Soulti, K.; Roussis, I. G. Potential antimicrobial activity of red and white wine phenolic extracts against strains of Staphylococcus aureus, Escherichia coli and Candida albicans. Food Technol. Biotechnol. 2005, 43, 41-46.

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Page 22 of 32

FIGURE CAPTIONS 518

Figure 1. Changes of protein (continuous line) and amino nitrogen (dotted line)

519

along fermentation with different starters. Control: routine wheat Qu, square; Group 2:

520

amylase, glucoamylase and protease (10 U g-1), circle; Group 3: glucoamylase,

521

amylase, protease (10 U g-1) and sterilized wheat Qu, triangle.

522 523

Figure 2. Changes (A) and correlation (B) of yeast and amino nitrogen in Chinese rice wine-like medium. Yeast (square) and amino nitrogen (triangle).

524

Figure 3. Principal Component Analysis (PCA) of BAs, ethanol, total sugar,

525

starch, amino acids, milling degree (the mass ratio of after milled rice and brown rice),

526

yeast in four milled rice fermentation samples (A), correlation of ethanol (% v/v) with

527

BA (B), PCA in three rice samples with different milling degree (C), correlation of

528

milling degree with BAs content (D).

529

Figure 4. Changes in amino acid (A) and BAs formation rate (B) during Chinese

530

rice wine fermentation. Amino acids: histidine (His), tyrosine (Tyr), phenylalanine

531

(Phe), lysine (Lys), ornithine (Orn), arginine (Arg). BAs: histamine (HIS), tyramine

532

(TYR), tryptamine (TRY), phenethylamine (PHE), cadaverine (CAD), spermidine

533

(SPD), spermine (SPM), putrescine (PUT). Samples were taken at 1.5, 3, 4, 5, 6, 7, 11,

534

16 and 21 days.

535

Figure 5. Changes in total BAs (A), linear regression (B), and BAs formation rate

536

(C) during Chinese rice wine fermentation. Strains were inoculated into CRW

537

fermentation after subculture. Control group, inverted triangle; single-strain

538

fermentation with Lactobacillus plantarum (ACBC271), square; single-strain

539

fermentation with Staphylococcus xylosus (CGMCC1.8382), circle; co-inoculation

540

with mixed strains of L. plantarum (ACBC271) and S. xylosus (CGMCC1.8382) at a 22


Page 23 of 32


541

ratio of 1:1, triangle. Samples were taken at 1.5, 3, 4, 5, 6, 7, 11, 16 and 21 days.

542

Figure 6. Changes in BAs (A) and piecewise linear regression (B) during CRW

543

fermentation in the presence of mixed starter culture at different ratios. Mixed strains

544

were co-inoculated at ratios of 1:1 and 1:2, respectively. LP+SX (1:1): L. plantarum

545

(ACBC271): S. xylosus (CGMCC1.8382) of 1:1, squares; LP+SX (1:2): L. plantarum

546

(ACBC271): S. xylosus (CGMCC1.8382) of 1:2, circle. Samples were taken at 1.5, 3, 4,

547

5, 6, 7, 11, 16 and 21 days.

548

Figure 7. Changes in total acid, ethanol and lactic acid bacteria during

549

fermentation with mixed starter culture(A), and content of eight BAs at the end of the

550

fermentation (B). Principal Component Analysis on 7 variables (LAB, ethanol, total

551

acid, BA, PUT, TYR, HIS), (C). Control, inverted triangle; LP-inoculated,

552

Lactobacillus plantarum (ACBC271), square; SX-inoculated, Staphylococcus xylosus

553

(CGMCC1.8382), circle; LP+SX-inoculated, Lactobacillus plantarum (ACBC271)

554

and Staphylococcus xylosus (CGMCC1.8382) at a ratio of 1:2, triangle. BAs: histamine

555

(HIS), tyramine (TYR), tryptamine (TRY), phenethylamine (PHE), cadaverine (CAD),

556

spermidine (SPD), spermine (SPM) and putrescine (PUT). Samples were taken at 1.5, 3,

557

4, 5, 6, 7, 11, 16 and 21 days.

23



Page 24 of 32

Table 1 Physicochemical properties of rice with different milling degree

Milling degree

Thousand grain

Starch

Protein

(%)

weight (g)

(g per 100 g)

(g per 100 g)

A

100

23.2

70.1

9.53

B

91

21.0

74.3

8.68

C

87

20.1

77.5

8.12

D

82

19.0

82.6

7.84

Number

Table 2 BAs concentration detection in medium of two starter strains BAs concentration (mg L-1)

L. plantarum (ACBC271)

S. xylosus (CGMCC1.8382)

Tryptamine

1.70 ± 0.14

81.68 ± 2.50

Phenethylamine

1.10 ± 0.06

88.36 ± 1.31

Putrescine

1.82 ± 0.06

80.40 ± 1.08

Cadaverine

2.52 ± 0.05

77.30 ± 1.49

Histamine

0.26 ± 0.13

53.94 ± 1.27

Tyramine

5.33 ± 0.19

65.08 ± 0.83

Spermine

0.57 ± 0.06

49.07 ± 2.03

Spermidine

0.75 ± 0.02

52.60 ± 3.65

Total BAs

14.05 ± 0.30

548.43 ± 4.26

24


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Figure 1

25



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Figure2

26


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Figure 3

27



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Figure 4

28


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Figure 5

29



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Figure 6

30


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Figure 7

31



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TOC graphic

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Mixed Starter Culture Regulates Biogenic Amines Formation via

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