Selection of Taste Markers Related to Lactic Acid Bacteria Microflora

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Selection of Taste Markers Related to Lactic Acid Bacteria Microflora Metabolism for Chinese Traditional Paocai: A GC-MS-based Metabolomics Approach Nan Zhao, Chuchu Zhang, Qin Yang, Zhuang Guo, Bo Yang, Wenwei Lu, Dongyao Li, Fengwei Tian, Xiaoming Liu, Hao Zhang, and Wei Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05332 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on February 29, 2016

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Selection of Taste Markers Related to Lactic Acid Bacteria Microflora

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Metabolism for Chinese Traditional Paocai: A GC-MS-based Metabolomics

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Approach

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Nan Zhao, † Chuchu Zhang, † Qin Yang, † Zhuang Guo#, Bo Yang, † Wenwei Lu, †

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Dongyao Li, † Fengwei Tian, †, § Xiaoming Liu, † Hao Zhang †, ‡, §, Wei Chen †, §*

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†

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and Technology, Jiangnan University, Wuxi 214122, P.R. China

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‡

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Technology & Business University, Beijing 100048, P.R. China

State Key Laboratory of Food Science and Technology, School of Food Science

Beijing Innovation Centre of Food Nutrition and Human Health, Beijing

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§UK-China

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#

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Chemical Engineering and Food Science, Hu Bei University of Arts and Science,

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Xiangyang 441053, China

Joint Centre on Probiotic Bacteria

Northwest Hubei Research Institute of Traditional Fermented Food, College of

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* Corresponding author: Wei Chen.

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Address: School of Food Science and Technology, Jiangnan University, Wuxi

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214122, P.R. China. Tel: 86-510-85912155; Fax: 86-510-85912155;

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E-mail addresses: [email protected] (W. Chen).

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ABSTRACT

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Traditional paocai brine (PB) is continuously propagated by back slopping and

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contains numerous lactic acid bacteria (LAB) strains. Although PB is important

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for the quality of paocai (Chinese sauerkraut), the taste features, taste-related

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compounds of PB-paocai and the effects of LAB communities from PB on the

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taste compounds remain unclear. An electronic tongue was used to evaluate the

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taste features of 13 PB-paocai samples. Umami, saltiness, bitterness, sweetness

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and aftertaste astringency were the main taste features of PB-paocai. Fourteen

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compounds were identified as discriminant taste markers for PB-paocai via

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GC-MS-based multi-marker profiling. A LAB co-culture (Lactobacillus plantarum,

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Lactobacillus buchneri and Pediococcus ethanoliduran) from PB could

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significantly increase glutamic acid (umami), sucrose (sweetness), glycine

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(sweetness), lactic acid (sourness) and γ-amino-butyric acid in PB-paocai, which

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would endow it with important flavor features. Such features could then facilitate

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starter screening and fermentation optimization to produce paocai-related foods

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with better nutritional and sensory qualities.

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KEYWORDS: paocai brine; paocai; lactic acid bacteria; metabolomics; taste;

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GC-MS.

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Introduction

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Paocai, one of the typical representatives of Chinese traditional fermented

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vegetables, is widely consumed in Asia.1 It has been recorded that the Chinese

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made paocai as early as 1000 B.C., and the traditional methods for its

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fermentation continue to be used to the present day. Vegetables are pretreated

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and immersed in paocai brine (PB) and then left at an ambient temperature

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(20-25 °C) for 6-10 days in pickle jars. PB which is continuously propagated by

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back slopping contains stable lactic acid bacteria (LAB) microflora and abundant

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flavor and nutritional metabolites that are traditionally used continuously for a

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long time, even up to a century for some old brands. In the previous studies,

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several species of LAB isolated from traditional paocai brine2-5. Xiong et al.

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isolated 180 LAB strains from paocai brine, and Lactobacillus plantarum NCU116

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showed excellent fermentation capability included higher cell growth and more

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rapid acidity producing in sauerkraut broth2. Luo et al. screen two LAB strains

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with outstanding inhibitory activity against Salmonella using a modeling

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method4. Although several species of LAB isolated from traditional paocai brine,

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the relation between taste of paocai and these LAB was paid less attention. The

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taste of traditional paocai, to a large extent, depends not only on the bacteria

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present on the vegetables, but also on the LAB communities in the PB. Although

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PB is critical to producing high-quality paocai, regardless of house or industry

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scales, less attention is paid to the PB. Thus, the main feature of PB-paocai and

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the effects that LAB communities from PB have on its taste compounds remain

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unclear.

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Metabolomics is an approach used to detect and quantify the metabolites in

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biological samples.6,

7

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clinically screen for biomarker metabolites8, 9. This strategy has also been used to

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identify marker taste compounds in food systems.10, 11 Although targeted flavor

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metabolites have been used to evaluate the taste quality of fermented vegetable

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elements such as sugar and organic acid in normal salt brine (SB)-paocai,1 these

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compounds might be limited to a synthetic and global representation of the

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flavors and nutritional chemicals in PB-paocai. Hence, an untargeted

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metabolomics analysis is used in the present study to compensate for the

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limitations of the aforementioned targeted methods when evaluating the taste

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compounds of more complicated paocai systems to establish the relationship

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between LAB and these taste features.

Numerous researchers have used metabolomics to

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The major objective of this research was to select the marker compounds in

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situ via the metabolomics method and reveal the effects of LAB communities,

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isolated from PB, on these markers in the process of paocai fermentation in vitro.

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MATERIALS AND METHODS Reagents. Pyridine, ribitol, methoxyamine hydrochloride and N-Methyl-N(trimethylsilyl)-trifluoroacetamide

(MSTFA)

were

4


purchased

from


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Sigma-Aldrich (Milwaukee, WI). The 14 standards of the discriminant marker

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compounds—lactic acid, butanedioic acid, fructose, glucose, sucrose, mannitol,

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valine, leucine, isoleucine, proline, glycine, glutamic acid, phenylalanine and

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GABA—were purchased from Sigma-Aldrich (Milwaukee, WI).

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Paocai preparation. Thirteen PB samples were collected from Sichuan

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province, and their basic information is listed in Table S1. L. plantarum

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SCYAPCZN11-1, L. buchneri SCYAPCZN11-4 and P. ethanoliduran SCYAPCZN11-8

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was isolated from PB11. PB, SB and SB plus mixed LAB (LABSB) were used to

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make paocai with same batch of radish. L. plantarum SCYAPCZN11-1, L. buchneri

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SCYAPCZN11-4 and P. ethanoliduran SCYAPCZN11-8 were inoculated in SB to

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make LABSB. The paocai samples fermented with different brines were divided

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into two groups. The first was to identify the taste features of paocai and screen

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for the marker taste compounds responsible (PB paocai vs SB paocai). The

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second was to reveal the effects of LAB on those compounds (SB paocai vs LABSB

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paocai). All of the brines contained 5-6% salt before fermentation. The SB and

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LABSB were 5% salt solution without or with LAB, respectively. The LAB 1

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inoculum in the SB was prepared according to Xiong, et al.

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were washed, dried, cut into small pieces and then put into 1 L pickle jars with

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these brines (800 ml). The jars were then sealed with water to exclude air and

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maintained at 25°C for 7 days. To identify the taste features of paocai and screen

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for marker taste compounds, the PB- and SB-paocai were sampled at 168 h. To

5


Radishes (250 g)

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reveal the effects of LAB on these marker compounds, the SB- and LABSB-paocai

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were sampled at 0, 12, 24, 36, 48, 60, 72, 96, 120, 144 and 168 h for further

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analysis.

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Electronic tongue analysis. The electronic tongue system (taste sensing

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system SA-402B, Intelligent Sensor Technology, Inc., Kanagawa, Japan) used

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comprised

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electrodes, an auto-sampler, an electronic unit for data acquisition and a

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personal computer with an advanced chemometric software package.12,

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Sample solutions were prepared by paocai juice with distilled water (20% v/v)

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and centrifuged for 10 min at 3000 g before analysis. paocai juice with boiling

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water (1% w/v) for 5 min and centrifuged for 10 min at 3000 rpm before

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analysis. Nine taste-indices—sourness, saltiness, bitterness, sweetness, umami,

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afterteaste-astringent, afterteaste-bitterness, richness and astringent—were

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evaluated, and all of the measurements followed Kobayashi, et al. 14

reference

electrodes, multichannel

lipid/polymer

membrane

13

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Extraction and derivatization for GC-MS analysis. For GC-MS analysis, an

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aliquot of 50 μL of paocai juice supernatant was transferred into a tube. Ribitol

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(15 μL of a 0.20 mg/mL solution) was added as an internal standard, and the

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samples were dried in a Speed-vac RC 1022 (Thermo Electron, San Jose, CA) for

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further derivation. The residues were redissolved and derivatized for 90 min at

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37°C in methoxyamine hydrochloride in pyridine (30 μL, 20 mg/mL), then

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incubated with N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) at 40°C

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for 30 min. An aliquot (10 μL) of all of the extracts was mixed as the quality

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control (QC) sample preparation. All of the samples (20 μL) were diluted with

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hexane (80 μL) and then transferred to GC vials for analysis.

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GC-MS Analysis. Samples (1 μL) were injected into the GC column in split

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mode (1:10) and the metabolites were analyzed via a GC (GC-2010 plus,

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Shimadzu, Japan) fitted with a quadrupole MS (GCMS-QP2010 Ultra, Shimadzu,

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Japan) using an Rtx-5MS capillary column (30 m, 0.25 mm ID, 0.25 μm thickness).

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The injection temperature was 240°C and the transfer line and ion source were

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220°C. Helium was used as the carrier gas at a constant linear velocity of 35.0

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cm/sec. The oven was increased at a rate of 5°C/min from 70°C to 230°C. Then,

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the temperature was increased to 320°C at a rate of 90°C/min and finally held

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for 5 min. The scan range was from 33 to 600 m/z.

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Spectral Data Processing. The chromatograms and mass spectra were

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evaluated using the XCMS online. For the feature detection, the XCMS

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matchedFilter was used with the following parameters: FWHM = 3, step = 0.25,

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mzdiff = 0.5, S./N. ratio cutoff = 10. The feature alignment was performed using

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the default parameters in XCMS with bw = 5 and mzwid = 0.25. The retention

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time correction was performed using peakgroups in XCMS with extra = 1 and

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missing = 1. To correct the MS response shift during the run, the spectra were

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normalized manually by adjusting the peak intensity against the ribitol internal

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standard.

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Multivariate Analysis. Principal component analysis (PCA) and redundancy

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analysis (RDA) were performed in MATLAB. Partial least squares discriminant

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analysis (PLS-DA) was performed in SIMCA-P 12.0 (Umetrics AB, Umea, Sweden).

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Data were Pareto-scaled to reduce the effect of noise in the chromatograms.

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For validating models, a 7-fold validation was applied to the PLS-DA model, and

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the reliabilities of the models were further rigorously validated by a permutation

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test (n = 100). Student’s t-tests were conducted in SPSS 16.0 (SPSS Inc., Chicago,

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IL, USA) to determine the significance. P values of less than 0.05 were considered

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to be significant.

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Metabolite Identification. The NIST 2011 standard mass spectral database

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was used to identify the metabolites. A potential metabolite was identified based

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on retention time and mass-spectral similarity (more than 80%) match.

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Authentic standards were used to confirm all selected discriminant taste

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markers.

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Microbial Isolation and Identification. The LAB isolation strategy followed

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Xiong, et al. 15 Subsequently, to identify the LAB isolates, the 16S rDNA gene was

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sequenced. The universal bacterial 16S rDNA primers (27F and 1492R) were

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used.16, 17 Amplification was conducted under the following conditions: initial

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denaturation at 95 °C for 3 min; 30 cycles of denaturation at 95 °C for 30 s,

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annealing at 55 °C for 30 s and extension at 72 °C for 1 min; and a final extension

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at 72 °C for 10 min.

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RESULTS AND DISCUSSION

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3.1 Effects of PB on Taste of Paocai

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To investigate the taste features of PB-paocai, an electronic tongue was used to

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evaluate nine taste indices of PB- and SB-paocai. The results were then combined

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with a multivariate analysis to identify the taste features of PB-paocai. According

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to PCA, paocai fermented by different brines (PB vs SB) was generally separated

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(Figure 1A). The results suggest that PB-paocai has different taste features from

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those of

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information about the taste index that contributed to the data differentiation. A

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Monte Carlo permutation test showed that the ordination model constrained by

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SB- or PB-paocai was significant (P = 0.048), with 40.2% of the variance

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explained by the canonical axis. Six taste-indices (umami, saltiness, sourness,

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sweetness, bitterness and aftertaste astringency) were identified as key variables

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well fit by the scores on the canonical axis (Figure 1B). As the RDA ordination

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plot revealed, five taste indices (umami, saltiness, sweetness, aftertaste A and

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bitterness) were located on the right side of the plot (Figure 1B), whereas

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sourness was on the left side of the plot. These results suggest that PB affects the

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tastes of paocai including umami, saltiness, bitterness and aftertaste A, whereas

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sourness was the major characteristic of SB-paocai.

SB-paocai. Subsequently, RDA was used to provide detailed

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3.2 Identification of Significant Taste Compounds for PB-Paocai through

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Untargeted Metabolomics.

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To obtain detailed information on the compounds responsible for the taste

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features of PB-paocai, a metabolomics approach was used. XCMS online was used

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to extract more than 5,000 ions from each sample. After preprocessing, the

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multivariate data matrix was subjected to multivariate analysis. PCA was used to

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investigate the compound differences between the two types of paocai (PB vs SB).

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A PCA score plot, derived from the 21 paocai samples (Figure S2), showed that

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the first two principal components clearly separated PB- from SB-paocai. The

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different brines used to ferment paocai strongly influenced the data separation.

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Subsequently, a PLS-DA model was constructed to explore the detailed

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information

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discrimination. The PLS-DA score plot demonstrated that the SB- and PB-paocai

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were clearly distinguishable, with the model parameters R2Y = 0.953, R2X =

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0.535 and Q2 = 0.917 (Figure 2A). The permutation result (n = 100, intercept of

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Q2 = -0.237) validated the stability and reliability of this PLS-DA model.

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Following the score plot, the S-plot was constructed to screen the markers that

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contribute to the differentiation (Figure 2B). According to the S-plot, 18

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compounds with P-values less than 0.05 and VIP (variable importance in the

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projection) values larger than 1.0 were selected as potential candidates for

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discriminant markers.18 The potential markers are summarized in Table 1. Most

regarding

marker

compounds

contributing

10


to

the

data


210

of these compounds contributed to the discrimination between SB- and

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PB-paocai in our study, and thus were considered as important taste substances

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in foods.11, 19, 20

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The concentrations of glucose, fructose and sucrose in PB-paocai were 3-, 5- and

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6-times greater than those in SB-paocai, respectively, which could affect the

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difference in sweetness between PB- and SB-paocai. Meanwhile, higher

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concentrations of lactic and butanedioic acids were also determined in PB-paocai,

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compared with SB-paocai. Although the organic acids, especially lactic acid in

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PB-paocai, were 2- and 2.9-times more than in SB-paocai, the sourness was not a

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taste feature of PB-paocai. This indicated that the high level of sugars in

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PB-paocai could also mask the sourness resulting from the organic acids

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present.21 Moreover, some amino acids were selected as discriminant markers in

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the PB-paocai. The glutamic acid content, which provides the umami taste in

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fermented foods,22 was 5.8-times higher in the PB-paocai. In addition, more

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proline,

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considered to be compounds contributing to sweetness and bitterness—were

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observed in the PB-paocai.23,

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physiological functions in animals and humans,25, 26 was identified in both SB-

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and PB-paocai, but its concentration was 17-times greater in PB-paocai than in

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SB-paocai.

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Previous studies have largely focused on SB-paocai in vitro with major interest in

glycine,

valine,

leucine,

24

isoleucine

and

phenylalanine—widely

Interestingly, GABA, which has numerous

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sugars and organic acids. Hence, there has been little information on PB-paocai

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and unbiased approaches for evaluating the traditional PB-paocai taste

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compounds in situ. We conducted global metabolomics to screen for the more

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targeted taste compounds related to the traditional PB-paocai taste. In general,

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our results indicate that PB affects the taste compounds in paocai, and 14

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compounds were identified as being responsible for the unique taste

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characteristics and nutritional quality of PB-paocai.

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3.3 Effects of LAB Communities Isolated from PB on the Marker Taste

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Compounds

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In previous studies, LAB strains have been considered key in improving the taste

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and nutritional quality of fermented vegetables.15, 27, 28 Different species of LAB

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inhabit PB,15,

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According to the results above, 14 compounds were identified as the marker

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taste compounds responsible for the taste of PB-paocai, but the effects of the LAB

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strains in the PB on the taste markers remain unclear.

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To investigate the LAB communities in the PB, the total microbial counts of LAB

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were determined by a culture-dependent method. The number of presumptive

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LAB isolates ranged from 1.1×105 to 2.6×107 CFU/mL (Table S1). Subsequently,

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according to the 16S rDNA gene sequences, three dominant LAB species were

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isolated from the PB samples, including Lactobacillus plantarum, Lactobacillus

29, 30

and they can be used as starters in paocai fermentation.

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buchneri and Pediococcus ethanoliduran. L. plantarum and P. ethanoliduran were

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isolated from all of the samples, and L. buchneri was not detected in the PB1 and

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PB3 samples. Our result is consistent with previous research, but Leuconostoc

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was not determined in all of the PB samples, perhaps due to the low pH of PB and

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the decreased acid-tolerance of Leuconostoc.1 We concluded that there were

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abundant LAB microflora in the PB, and L. plantarum, P. ethanoliduran and L.

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buchneri were the dominant LAB species in the PB.

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To investigate the effects of these indigenous LAB strains in the PB (L. plantarum,

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P. ethanoliduran and L. buchneri) on the taste compounds, we inoculated those

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three species to SB, and then monitored the changes in marker taste compounds

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during the fermentation. According to the PCA score plots based on the datasets

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for the marker compounds (Figure 3), the changes in the marker taste

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compounds in SB- and LABSB-paocai indicated that the indigenous LAB strains

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used as starters affected the accumulation of marker compounds during the

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process of paocai fermentation. SB-paocai performed separation from

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LABSB-paocai after 24 h. These results suggest that the LAB inoculum

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significantly modified the taste compounds in paocai fermentation. The inoculum

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improved the final level of those marker taste compounds for umami, sweetness

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and sourness, which included sucrose, lactic acid, glycine and glutamic acid

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(Figure 4). It is worth noting that the concentration of GABA was also

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upregulated by the LAB inoculation. The concentrations of mannitol, leucine,

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isoleucine, phenylalanine and valine, however, were significantly lower in

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LABSB-paocai than those in the SB-paocai (Figure 4), which mainly contribute to

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bitterness. As Figure 5B shows, during the entire fermentation process, the

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concentration of lactic acid in SB-paocai was significantly lower than that in

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LABSB-paocai (p < 0.05). Moreover, sucrose was at a similar level in SB- and

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LABSB-paocai before 36 h, but then dramatically decreased in SB-paocai (Figure

279

5A). These results indicate that sucrose might be metabolized mainly by L.

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mesenteroides in the early stage of paocai fermentation, which is a main LAB in

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the spontaneous fermentation process.1 The glycine content was similar at the

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early fermentation stage in both SB- and LABSB-paocai, and it increased more

283

rapidly in LABSB-paocai than in SB-paocai after 60 h (Figure 5C). Additionally,

284

glutamic acid, which contributes to umami, increased up to 60 h then declined,

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finally stabilizing after 120 h in both types of paocai. However, the level of

286

glutamic acid in LABSB-paocai was higher than that in SB-paocai during the

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major fermentation process. Interestingly, the level of GABA increased

288

dramatically after 60 h. These results indicate that the glutamic acid might

289

gradually be converted to GABA by the inoculum via glutamic decarboxylase

290

(GDH).31 Most of the marker taste compounds were upregulated through

291

inoculating indigenous LAB into SB, and the trends of those markers in LABSB

292

were generally in line with those in PB-paocai, which could indicate that the

293

major differences between PB- and SB-paocai stem from the LAB species in the

14



294

PB.

295

According to the metabolites found in the present study and Kyoto Encyclopedia

296

of Genes and Genomes (KEGG) LAB pathways (KEGG pathway map00250,

297

map00260, map00010, map00020, map00330, map00620, map00051), we

298

constructed a paocai fermentation metabolic pathway related to the marker taste

299

compounds’ metabolism (Figure 6). In our studies, inoculating these indigenous

300

LAB strains for paocai fermentation upregulated most of the concentrations of

301

the taste compounds, which are screened from PB-paocai. Thus, our results

302

indicate that these indigenous LAB strains in the PB contribute significantly to

303

the taste of PB-paocai. According to the pathways of the three LAB species, the

304

LAB inoculated has the metabolic pathways to produce some of the unique

305

metabolites in PB-paocai. When inoculating these LAB strains into SB, the

306

inoculum rapidly occupied the ecology niche and became the dominant strains in

307

paocai. Thus, the taste compounds of PB-paocai were significantly improved in

308

LABSB-paocai. For instance, the level of glutamic acid and GABA in LABSB- and

309

PB-paocai were higher than those in SB-paocai at the end of fermentation

310

because the L. plantarum and L. buchneri inoculated in our study have the gdh

311

and gad genes, which are responsible for generating glutamic acid and GABA.31, 32

312

We also found that the concentrations of leucine, isoleucine, phenylalanine and

313

valine were significantly lower in LABSB-paocai than in SB-paocai. L.

314

mesenteroides, which mainly grows in the early stage of paocai fermentation and

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can be inhibited by rapid acid producing strains such as L. plantarum,1, 15 has the

316

complete pathway for valine, leucine, phenylalanine and isoleucine production,

317

which could explain the higher levels of these four amino acids in SB-paocai than

318

in LABSB-paocai. It is worth noting that while the LAB used in the current study

319

have incomplete pathways for amino acid production from carbohydrates, the

320

LAB inoculum improved the levels of these compounds. This indicates that the

321

metabolic complementation among the different genera that appear in the

322

process of fermentation may play an important role in the catabolism of

323

specific-amino acids and possible flavor-formation pathways.33, 34

324

In summary, this investigation addresses the taste differences between

325

traditional PB-paocai and regular SB-paocai by screening for the marker taste

326

compounds responsible for these taste features by metabolomics analysis. More

327

importantly, we reveal that the indigenous LAB strains isolated from the PB play

328

important roles in improving most of these taste features. These results may

329

facilitate screening for better starters and optimization of the paocai

330

fermentation process to achieve better quality features. The detailed interaction

331

between this pathway and the microorganisms involved in real-time

332

fermentation information requires further investigation with improved

333

strategies.

334

ABBREVIATION USED

335

PB, paocai brine; LAB, lactic acid bacteria; SB, salt brine; LABSB, salt brine plus

16



336

mixed LAB.

337 338

ACKNOWLEDGMENTS

339

We would like to thank all people for providing PB samples.

340 341

SUPPORTING INFORMATION DESCRIPTION

342

Table S1 Main characteristics of analyzed paocai brine samples

343

Figure S2. Principal component analysis (PCA) scores plot of the taste feature of

344

Paocai fermented by different brine base on the compounds profiling data.

345 346

FUNDING SOURCES

347

This work was supported by the National Natural Science Foundation of China

348

(No. 31470161, 31125021), the key projects in the national science & technology

349

pillar program during the twelfth five-year plan period (No. 2013BAD18B01,

350

2013BAD18B02, 2012BAD28B08), the 111 Project B07029, the Program for

351

Changjiang Scholars and Innovative Research Team in University (IRT1249), and

352

Industry Development Program in Collaborative Innovation Center for Food

353

Safety and Quality Control, Jiangsu Province.

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FIGURE CAPTIONS

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Figure 1 (A) Principal component analysis (PCA) scores plot of the taste feature

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of paocai fermented by different brine; triangle ( ), PB paocai; open triangle ( ),

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SB paocai. (B) Biplot of the redundancy analysis (RDA). Constrained explanatory

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variables (PB Paocai/SB Paocai) are indicated by triangles. Upper left shows p

475

value of Monte Carlo Permutation Test.

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Figure 2 Results of the multiple pattern recognition of compounds in the SB and

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PB paocai (A) PLS-DA score plot (R2Y = 0.953, R2X = 0.535, and Q2 = 0.917);

479

triangle ( ), PB paocai; open triangle ( ), SB paocai. (B) PLS-DA S-plot. Each

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triangle in the S-plot represents an ion. The ions far away from the origin

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represent potential discriminant compounds.

482 483

Figure 3 Score plots of the principal component analysis (PCA) performed on the

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GC-MS of marker taste compounds during Paocai fermentation. The directions of

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the curved arrows indicate the routes of data points on the score plots during

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Paocai fermentation; triangle ( ), LABSB paocai; open triangle ( ), SB paocai.

487 488

Figure 4 Mean levels of marker taste compounds in SB and SB with LABs paocai

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sampled at 0 and 168 h. Y-axis is peak area normalized by the internal standard.

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Values are mean ± SD values with three replicates. *p < 0.05, **p < 0.01, ***p

Selection of Taste Markers Related to Lactic Acid Bacteria Microflora

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