Use of Psychrotolerant Lactic Acid Bacteria (Lactobacillus spp. and

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Use of psychrotolerant lactic acid bacteria (Lactobacillus spp. and Leuconostoc spp.) isolated from Chinese traditional Paocai for the quality improvement of Paocai products Aiping Liu, Xiaoyan Li, Biao Pu, Xiaolin Ao, Kang Zhou, Li He, Shujuan Chen, and Shuliang Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00050 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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Journal of Agricultural and Food Chemistry

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Use of psychrotolerant lactic acid bacteria (Lactobacillus spp. and

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Leuconostoc spp.) isolated from Chinese traditional Paocai for the

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quality improvement of Paocai products

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Aiping Liu, Xiaoyan Li, Biao Pu, Xiaolin Ao, Kang Zhou, Li He, Shujuan Chen,

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Shuliang Liu*

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College of Food Science, Sichuan Agricultural University, Ya’an, Sichuan 625014, People’s

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Republic of China

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Corresponding Author: Shuliang Liu

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E-mail address: [email protected]

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Tel./Fax:+86-835-2882311

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ACS Paragon Plus Environment


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Abstract

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In order to improve the quality of Chinese traditional Paocai, 2 psychrotolerant

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lactic acid bacteria (LAB) strains were isolated from Paocai, and the quality of

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Chinese Paocai product using these 2 strains as starter cultures was compared to a

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control sample fermented with aged brine at 10 °C. The results suggested that the

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physicochemical and sensory features of Paocai fermented with psychrotolerant LAB

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were more suitable for industrial applications. The nitrite content of Paocai fermented

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with psychrotolerant LAB was 1 mg/kg, which was significantly lower than that of

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the control Paocai (P < 0.05). Low-temperature fermentation with the starter cultures

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of psychrotolerant LAB could effectively prevent overacidity and overripening of the

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Paocai products. Additionally, Paocai fermented with psychrotolerant LAB harbored a

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relatively simple microbial flora as revealed by polymerase chain reaction-denaturing

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gradient gel electrophoresis. This study provides a basis for improving the quality of

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Chinese traditional Paocai and the large-scale production of low-temperature Chinese

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traditional Paocai products.

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Keywords: Chinese traditional Paocai; lactic acid bacteria; PCR-DGGE; starter

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culture.

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1 Introduction

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Chinese traditional Paocai, believed to have originated in the Zhou Dynasty

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3,000 years ago,1 is a well-known side-dish in China. Unlike kimchi, which uses

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direct salting to withdraw juice from cabbage, the widely consumed Chinese

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traditional Paocai2 is a brine-salted and lactic-acid-fermented vegetable product.3 In

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traditional methods, Chinese Paocai is processed through spontaneous fermentation

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without sterilization of the raw materials. This results in the proliferation of various

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microorganisms, impeding the production of uniform and high-quality Paocai.4-6 In

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response to this, Lactobacillus inoculants have been increasingly used as starter

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cultures in the production of fermented vegetables in an attempt to improve quality.

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7-10

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Generally, mesophilic lactic acid bacteria (LAB) are widely applied in Chinese

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traditional Paocai production.3,11 However, the greatest harvest of vegetables to be

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salt-fermented during the production of Paocai occurs in the winter months. In this

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period, the fermentation rate of mesophilic LAB is slow, thus slightly inhibiting the

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growth of microbial contaminants. Consequently, high concentrations of NaCl are

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needed to prevent vegetable spoilage, but this requirement increases production costs

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because the finished products must subsequently be desalinated. In addition, organic

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acids and flavor compounds may be added to fulfill the quality requirements of

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Chinese Paocai products. These approaches not only influence the quality of Chinese

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Paocai but also limit industrial production. Since vegetable salinization occurs in

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winter, abundant psychrotolerant LAB may be isolated and then applied to the 3



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production process in order to address the above-mentioned issues.

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Recently, more and more studies have been focused on the application of

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psychrotolerant LAB in fermented vegetables.12-16 However, to the best of our

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knowledge, little detailed information exists on Chinese traditional Paocai prepared

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using psychrotolerant LAB. The present study was designed to standardize the

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fermentation and product quality of Paocai using 2 psychrotolerant LAB strains. The

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2 strains, namely, 3m-1 (Lactobacillus plantarum, L. plantarum) and 8m-9

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(Leuconostoc mesenteroides, L. mesenteroides), were isolated from Paocai products in

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Sichuan by the serial plate dilution method at 10 °C. Afterward, the Paocai was

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fermented with the starter cultures of these 2 psychrotolerant LAB, and the resulting

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product was compared to a control sample fermented with aged brine at 10 °C. The

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physicochemical characteristics, sensory features, microflora composition, and

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volatile flavor compounds of the products were investigated. The detailed profiling

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presented in this work will serve an important role in industrial applications.

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2 Materials and Methods

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2.1 Materials

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Cabbages (Brassica pekinensis) were purchased from a local market in Ya’an,

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Sichuan, China and used as the raw materials for fermentation. The outer leaves and

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inedible parts were removed. The aged brine used for the isolation of psychrotolerant

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LAB included 10 portions of locally ripened traditional Chinese Paocai products

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(spontaneously fermented) obtained from Chengdu, Meishan and Yaan cities in the

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Sichuan province. The aged brine was 3 to 5 years old, and contained 3% to 10% 4


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NaCl (W/V). The total aerobic and LAB counts were between 6.5 and 7.5 lg CFU/mL,

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respectively, and the yeast count was between 6.0 and 7.0 lg CFU/mL. The aged brine

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used for Paocai (2#) preparation comprised a mixture of 4 equal amounts of the above

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aged brine collected from Yaan city.

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2.2 Isolation and characterization of psychrotolerant LAB

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Briefly, the aged brine was inoculated into De Man Ragosa Sharpe (MRS) broth

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(pH 6.2-6.4) and kept at 10 °C for 96 h. The appropriate dilutions were prepared from

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the culture. Dilutions were plated by using MRS agar (MRS broth containing 1.2%

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agar) containing 1.5% CaCO3 via the pour plate method. Representative colonies with

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a dissolved calcium circle were randomly picked and purified. Gram-positive,

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catalase-negative isolates were considered as presumptive LAB isolates, and further

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screened by investigating growth status at 9, 8, 7, 6, 5, 4, 3 and 2 °C for 96 h,

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respectively. Those isolates with high tolerance to low temperatures were selected. In

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order to investigate whether the final isolates belonged to psychrotolerant LAB, the

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isolates at a density of 108 CFU/mL were inoculated into MRS broth (0.3%, V/V ),

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and growth at 2, 5, 8, 10, 15, 20, 25, 30, 34 and 37 °C was monitored for 48 h.

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Phenotypic

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gelatin liquefaction, litmus milk tests; acid production from aescinate, cellobiose,

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maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, melibiose,

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mannose, galactose, rhamnose, fructose, and xylose; growth at 15 °C; and tolerance to

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NaCl (2%, 4%, 6%, 8%, 10%, 12%, 14% and 16%, W/V), with reference to Ren et al.

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characteristics were assessed using several methods, including oxidase,

The isolates were sent to Sangon Biotech (Shanghai, China) for genus and species 5



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identification by 16S rRNA gene sequence analysis. The nitrite degradation

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characteristics of the final isolates (strains 8m-9 and 3m-1) were also studied

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according to the method of Oh et al.18 The original concentration of nitrite used for

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degradation was 125 µg/mL, and the nitrite content was examined every 12 h using

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hydrochloride naphthodiamide.19

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2.3 Preparation of starter cultures

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Strains 8m-9 and 3m-1 were activated on MRS agar for 2 d at 30 °C. The

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colonies of each strain were inoculated into MRS broth and the cultures were

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incubated at 30 °C for 24 h. The cell pellets were harvested and washed twice with

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0.9% saline. Afterward, the pellets were resuspended at a density of 1010 CFU/mL in

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0.9% saline.

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2.4 Paocai preparation

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Paocai was prepared using 2 different recipes. Paocai (1#) was processed as

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follows. Cabbages (1 kg) were washed with cooled boiled water, dried, cut into strips

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(2-4 cm × 5-10 cm) and placed in a 3 L ceramic jar. An equal amount of cooled boiled

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water containing 5% NaCl, 2% sucrose, 16% seasoning liquid, 0.3% CaCl2, and 1%

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Chinese liquor (50%, V/V, Langjiu, Sichuan Gulin langjiu Distillery Co., Ltd.,

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Sichuan, China) was added. Then, 0.3% (V/V) of mixed starter cultures (3m-1:8m-9 =

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2:1, V/V) at a density of 1010 CFU/mL were added. The jar was covered with a lid and

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water-sealed. The preparation of Paocai (2#), used as a control sample, was similar to

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that of Paocai (1#) except that the cell suspension and cooled boiled water were

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replaced with double diluted aged brine (diluted in cooled boiled water). All jars were 6


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kept at 10 °C. The seasoning liquid was prepared as follows: 12 g of Chinese Wang

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Shouyi thirteen spice powder (W/V, mainly contains pepper, dried ginger, cinnamon,

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star anise, garlic, fennel, cardamom, Pericarpium Citri Reticulatae [Chenpi in

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Chinese], Angelicae Dahuricae Radix [Baizhi in Chinese], Radix Aucklandiae

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[Muxiang in Chinese], and Syzygium aromaticum [Dingxiang in Chinese]; purchased

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from Zhumadian Wang Shouyi Shi San Xiang Multi-flavored Spice Group Co., Ltd,

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Henan, China) were wrapped in a cloth bag and heated in 100 mL of water at 80 °C

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for 3 h, and the collected supernatant constituted the seasoning liquid.

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2.5 Sampling and analysis of physicochemical characteristics

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The pickled cabbages and brine from Paocai (1#) and (2#) were aseptically

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collected at the same time points on day 0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15 and 20 in order

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to monitor the physicochemical characteristics. The jars were shaken prior to

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sampling. Each analysis was performed with homogenized cabbages and brine in

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triplicate, and the average result was reported.

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2.5.1 pH, nitrite and reducing sugar contents

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The pH was determined using a pH meter (Cyberscan PC 510, Eutech, Shanghai,

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China). The concentration of nitrite per kg was determined using hydrochloride

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naphthodiamide19. The reducing sugar was determined using a direct titration method

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with alkaline copper tartrate, as described by Hao et al.20

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2.5.2 Titratable acidity

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A 200 g of homogenized sample was mixed with an equal amount of cooled

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boiled water for 20 min, and the mixture was filtered using Whatman filter paper. 7



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Titratable acidity was determined by titrating the filtrate with 0.1 M NaOH using

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phenolphthalein (1% in 57% ethanol) as an indicator.

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2.5.3 Salinity

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The concentration of sodium chloride was determined as follows. First, 2 mL of

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the filtrate from the homogenized sample was mixed with 50 mL deionized water and

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1 mL potassium chromate (0.26 mol/L). The resulting mixture was titrated with

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standardized 0.1 mol/L silver nitrate until an orange color developed, and 50 mL of

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deionized water was used as a control. The concentration of sodium chloride was

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calculated as

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X=

( v1 − v2 ) × c × 0.0585 × 100 v m× 3 100

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where X is the concentration of sodium chloride (g/100 g), v1 is the volume (mL)

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of the silver nitrate solution used to titrate the filtrate, v2 is the volume (mL) of the

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silver nitrate solution used to titrate 50 mL of deionized water, v3 is the volume (mL)

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of the filtrate, c is the concentration of standardized solution of silver nitrate, m is the

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weight (g) of the sample, and 0.0585 represents the weight of sodium chloride that is

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equal to 1 mL of the standardized silver nitrate solution.

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2.5.4 Lactic acid and acetic acid contents

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Mashed samples from Paocai (1#), collected on day 1, 2, 3, 5 and 9, and Paocai

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(2#), collected on day 1, 2, 3, 5 and 11, were diluted to 50 mL in volumetric flasks for

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measurement. After soaking for 1 h, the solution was passed through filter paper and

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centrifuged at 10,000 × g for 5 min. The resulting supernatant was further

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membrane-filtered (0.45 µm) and injected into a Shimadzu HPLC (LC-10A2010C HT, 8


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Japan) equipped with UV detector, which was set at 210 nm. A Carbomix H-NP

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column with 8% crosslinking (7.8 × 300 mm, 10 µm, Sepax Technologies, Inc.,

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Newark, DE) was used. The mobile phase was 2.5 mM H2SO4 at a flow rate of 0.6

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mL/min. The column temperature was 58 °C. The calibration curve was established

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and the concentration versus peak area was calculated according to the least-squares

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method.

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2.6 Sensory analysis

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Sensory analysis was performed on the day when the ripened Paocai product was

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obtained. Panelists were trained to be familiar with the attributes of Paocai products,

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and several sensory characteristics, including appearance, color, odor, taste and

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texture were evaluated by 10 trained panelists on either a 10-point (appearance, color

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and odor) or 30-point (taste and texture) scale. On both scales, 1 represented the

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lowest score, while either 10 or 30 represented the highest score. The products were

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coded with random numbers and presented to the panelists on a tray. The serving

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orders were randomized, and water was provided between samples to cleanse the

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palate.20-23

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2.7 Microbial analysis

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2.7.1 Enumeration of microorganisms

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Samples (0, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, and 20 d) from Paocai (1#) and (2#)

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were removed aseptically, and 25 g of the sample was mixed with 225 mL of 0.9%

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NaCl. Further serial decimal dilutions were prepared from this mixture. The following

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incubation conditions were used: plate count agar for 2 d at 37 °C for total aerobes, 9



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MRS agar for 2 d at 37 °C (anaerobic) for LAB, potato dextrose agar for 5 d at 28 °C

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for mold-yeasts, and violet red bile agar for 2 d at 37 °C for total coliforms. All

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analyses were performed in triplicate and the mean values were calculated.

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2.7.2 Microflora composition

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Samples from Paocai (1#), collected on day 1, 2, 3, 5 and 9, and Paocai (2#),

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collected on day 1, 2, 3, 5 and 11 were analyzed through polymerase chain

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reaction-denaturing gradient gel electrophoresis (PCR-DGGE) according to the

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method presented by Liu et al.24 The 16S and 18S rDNA were ampliﬁed in 2 rounds

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of PCR, using primers A (F338-GC, 5'-CGCCCGCCGCGCGCGGCGGGCGGGGC

208 209 210

GGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3',

and

R518,

5'-GTATTACCGCGGCTGCTGG-3') and primers B (F1427-GC, 5'-CGCCCGCCG CGCGCGGCGGGCGGGGCGGGGGCACGGGGGGTCTGTGATGCCCTTAG

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ATGTTCTGGG-3',

and

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respectively.

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2.8 Volatile flavor compounds

R1616,

5'-GGTGTGTACAAAGGGCAGGG-3'),

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The samples were pretreated for solid-phase microextraction (SPME) analysis as

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reported by Zheng et al.25 In brief, ground raw materials (5 g) and samples (5 g) from

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Paocai (1#), collected on day 5 and 9, and Paocai (2#), collected on day 5 and 11,

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respectively, were placed in a 40-mL vial sealed with a PTFE/silicone septum

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(Supelco). Each vial was stored at 40 °C to equilibrate for 30 min in a water bath.

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Divinylbenzene/carboxen/polydimethylsiloxane

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exposed to adsorb the analytes for 30 min. Afterward, the fiber was withdrawn from

(DVB/CAR/PDMS)

10


fiber

was

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the needle, and introduced to a heated gas chromatograph injector for desorption and

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analysis. The fiber was held for 5 min in the injection port to desorb volatile

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compounds.

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Gas chromatography-mass spectrometry (GC-MS; Agilent-7890A-5975C;

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Agilent, USA) with an elastic capillary vessel column (HP-5MS, 30 m × 0.25 mm i.d.,

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0.25 µm film thickness) was utilized to analyze the volatile flavor compounds.

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Thermal desorption of the compounds from the fiber coating took place in the GC

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injector at 250 °C in splitless mode. Helium was used as the carrier gas at a flow

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velocity of 1 mL/min. The oven temperature gradient was programmed as follows:

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30 °C held for 2 min, then raised to 155 °C at a rate of 2 °C /min; raised to 180 °C at a

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rate of 5 °C/min; raised to 250 °C at a rate of 10 °C/min; and then held for 3 min. The

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mass spectra was obtained with a source temperature of 230 °C and a 70 eV

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ionization potential. The mass scan range was 30-400 AMU. Compounds were

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identified using the National Institute of Standards and Technology (NIST) library and

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profile

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determined by area normalization.

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2.9 Statistical analysis

analysis

(similarity

index>80).

The relative content of each peak was

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Each Paocai product was prepared and characterized with three independent

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replications. Mean and standard deviations were calculated and subjected to one-way

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ANOVA using SPSS 18.0. Statistical significance was determined at the 5% level (P
0.05)

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in both Paocai products. By contrast, the increase in the titratable acidity in Paocai (1#)

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was slower than that in Paocai (2#) (P < 0.05). Considering the pH and sensory

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evaluation of Paocai, well-ripened products from Paocai (1#) and (2#) were obtained

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on day 9 and 11, respectively. The suitable pH of Paocai (1#) stabilized after the

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ripening period, which contributed to an extended shelf life.8 The pH of Paocai (2#)

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declined from 3.63 to 3.51 after ripening but changed little thereafter. Initially, the

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salinity of both Paocai products increased. However, the salinity of Paocai (1#)

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reached a maximum of approximately 2.7% on day 5 and remained steady (P > 0.05),

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while the salinity of Paocai (2#) did not vary (P > 0.05) until day 15. The salinity of

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Paocai (1#) was much lower than that of Paocai (2#).

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In the initial stage of fermentation, nitrate was transformed into nitrite as a result 13



the

growth

of

nitrate-reducing

bacteria.

As

fermentation

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of

proceeded,

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nitrate-reducing bacteria were inhibited and nitrite was decomposed by LAB.

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Therefore, a decreasing trend in nitrite content was observed after the initial increase.

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The greatest nitrite content in Paocai (1#) was observed prior to that of Paocai (2#),

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and Paocai (1#) exhibited a smaller chromatographic peak possibly because the starter

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cultures, especially 3m-1, encoded a better degradation capacity for nitrite. It is worth

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noting that the nitrite content in ripened Paocai (1#) was close to the detection limit of

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the method employed (1 mg/kg), and lower than most of previous reports.11,

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Additionally, the nitrite content of ripened Paocai (2#) (2.5 mg/kg) also satisfied the

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local standard for Paocai (20 mg/kg).

28

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Before ripening, the contents of lactic acid and acetic acid were investigated. As

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shown in Figure 3, the lactic acid content in Paocai (2#) increased, while the lactic acid

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content in Paocai (1#) did not vary (P > 0.05) after day 5. The acetic acid content of

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both Paocai products increased over the course of the entire study. Although the

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increase in lactic acid and acetic acid contents was more rapid (P < 0.05) in Paocai (1#)

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than in Paocai (2#) from day 2 to day 5, the final concentrations of lactic acid and acetic

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acid in Paocai (2#) were much higher. As a consequence, Paocai (2#) was overripe.

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These changes were consistent with the pH and titratable acidity values, thus it was

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determined that low-temperature fermentation with the starter cultures of

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psychrotolerant LAB could effectively prevent overacidity and overripening of Paocai

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products.

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3.3 Sensory analysis 14


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The sensory evaluation of both Paocai products was shown in Figure 4. As

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depicted, there were no significant differences in appearance, color or texture of the 2

311

products (P>0.0.5). Conversely, the odor and taste of Paocai (1#) was better than that

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of Paocai (2#) (P < 0.0.5), and this finding was consistent with both the

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physicochemical characteristics as well as the analysis of volatile flavor compounds.

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Therefore, fermentation with the psychrotolerant LAB starter cultures could

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standardize and improve the product quality.

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3.4 Microbial analysis

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3.4.1 Microbial counts

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The changes in LAB, total aerobes and yeast counts in both Paocai products

319

during fermentation were shown in Figure 5. As LAB comprised the predominant

320

microbes and were able to propagate on PCA plates, the changes in LAB and total

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aerobes in both Paocai products showed a similar tendency. During the first 2 days,

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the psychrotolerant LAB inoculated in Paocai (1#) adapted rapidly to the

323

low-temperature environment. Thus, the CFU value of LAB increased exponentially

324

and reached a maximum. As a result, yeast growth was inhibited. However, the LAB

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count in Paocai (2#) was less (106 CFU/g) and comprised multiple species. Yeast

326

exhibited a certain resistance to cold, thus explaining their increase. After 2 d, LAB

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and total aerobe counts decreased slowly in both Paocai products (P > 0.05), while

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yeast counts increased due to the weakened inhibition from the LAB. After 4 d, LAB

329

inhibited by the low-temperature continued to grow, and there was a slow increase in

330

LAB and total aerobe counts. During the later period of the fermentation process, 15



331

LAB and total aerobe counts decreased sharply and then began to stabilize as a result

332

of the accumulation of metabolites and nutrient consumption.

333

Additionally, in order to assess the microbiological quality of the products,

334

counts of total coliforms in both Paocai products were investigated (Table 2). Mold

335

counts were always found to be lower than 10 CFU/g (data not shown). The number

336

of LAB in Paocai (1#) was greater than that in Paocai (2#) during the entire

337

fermentation process, whereas the number of yeast exhibited the opposite tendency. In

338

summary, low temperatures can reduce microbial contamination.

339

3.4.2 Microflora composition

340

A secondary PCR was performed to obtain adequate PCR products for a

341

successful DGGE analysis. Figure 6(A) indicated the DGGE profile of bacterial

342

diversity at different periods during fermentation. As depicted in Figure 6, certain

343

differences were observed in the bacterial diversity of both Paocai products. The

344

evident bands on the DGGE gel were reclaimed, amplified through PCR, and then

345

purified. The resulting products were ligated to a T-vector and transformed into E.

346

coli DH5a. Single colonies were collected and sent for sequencing. Table S1(A) listed

347

the types of bacteria identified based on the detectable DGGE bands. During

348

fermentation, the predominant LAB in both Paocai products were L. mesenteroides

349

and L. plantarum. The predominant LAB in aged brine were consistent with that

350

found by Xiong et al.29 Interestingly, Staphylococcus was observed in both Paocai

351

products, but the counts decreased over time in Paocai (1#) and were stagnant in

352

Paocai (2#). Figure 6(B) and (C) illustrated the similarity indices of the DGGE 16


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profiles of bacteria in Paocai (1#) and (2#). The bacterial populations differed

354

considerably during fermentation.

355

The DGGE profile of fungal diversity at different periods during fermentation

356

was shown in Figure 7. Table S1(B) listed the types of fungi identified based on the

357

detectable bands on DGGE. The main fungal flora detected was yeast, however,

358

reports on the molecular diversity of yeast in pickled cabbages were few. Compared

359

with the 14 types of yeast in our research, Chang et al.30 identified 8 types of yeast in

360

kimchi, but only Kluyveromyces marxianus was found in both studies. Figure 7(B)

361

and (C) illustrated the similarity indices of the DGGE profiles of fungi. The overall

362

similarity coefficient in the DGGE profile of fungi was higher than that of bacteria,

363

indicating that there was less variation in fungal genera. This observation was

364

consistent with the work of Chang et al.30

365

3.5 Volatile flavor compounds

366

The number of volatile flavor compounds and their relative percentage

367

composition in both Paocai products were shown in Table 3. Both Paocai products

368

were rich in volatile flavor compounds, such as esters, acids, alcohols and aldehydes,

369

which was similar to the findings of Wu et al.31 The main categories of volatile flavor

370

compounds included olefins, esters, alcohols and sulfides, and their relative contents

371

accounted for more than 90%. As fermentation proceeded, the total number of volatile

372

compounds increased due to chemical and enzymatic reactions or microbial

373

metabolites,32 and the total number and content of compounds in Paocai (1#) was

374

slightly more than that in Paocai (2#). 17



375

Sulfur compounds played an important role in determining the flavor quality of

376

kimchi products.33,

34

377

cabbage, radish, red pepper, gar garlic, ginger, and green onion.35 Ha et al.36 revealed

378

that the main flavor compound in kimchi fermented at 5 °C was dimethyl trisulfide,

379

which was also one of the sulfides identified in both Paocai products in our research.

380

This indicated that dimethyl trisulfide might also exert a certain influence on the

381

flavor of Paocai in the present study. The relative contents of esters and alcohols in

382

Paocai (2#) were lower than that of Paocai (1#), which might be associated with the

383

structure of the microflora.

The sulfur compounds in kimchi originated from Chinese

384

In summary, 2 psychrotolerant LAB strains were isolated and used for Paocai

385

production. Additionally, a detailed comparative study between Paocai fermented with

386

the starter cultures of these 2 psychrotolerant LAB and Paocai fermented with aged

387

brine was conducted. The results of this study could be helpful to the industrial

388

production of high-quality Chinese traditional Paocai. However, in order to provide a

389

reliable basis for the industrial production, future work should focus on the quality

390

control of various batches of products, quality comparisons among more products in

391

the market and whether they are typical of standard processed material.

392 393 394 395 396 18


Page 18 of 36

Page 19 of 36


397

Abbreviation Used

398

GC-MS, gas chromatography-mass spectrometry; LAB, lactic acid bacteria; MRS, De

399

Man Ragosa Sharpe; PCR-DGGE, polymerase chain reaction-denaturing gradient gel

400

electrophoresis.

401 402

Supporting Information Description

403

Table S1(A) Type of bacterial communities corresponding to the bands in Figure

404

6(A).

405

Table S1(B) Type of fungal communities corresponding to the bands in Figure 7(A).

406 407

Funding Support

408

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

409

(No. 31171726) and the Science & Technology Foundation of Sichuan Province

410

(14NZ0012, 2013NZ0055).

411 412 413 414 415 416 417 418 19



419

References

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highly active dextransucrase from lactate fermented vegetables. Int J Food Microbiol,

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32. Zhao, D.; Tang, J.; Ding, X. Analysis of volatile components during potherb 23



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mustard (Brassica juncea, Coss.) pickle fermentation using SPME–GC-MS.

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33. Cha, Y.; Kim, H.; Cadwallader, K. Aroma-active compounds in kimchi during

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fermentation. J Agr Food Chem, 1998, 46, 1944-1953.

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34. Hawer, W.S. A study on the analysis of volatile flavor of Kimchee. Anal Sci

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Technol, 1994, 7, 125-132.

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elephant (Great-headed) garlic, chive and Chinese chive. Uniquely high ally to methyl

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rations in some garlic samples. J Agric Food Chem, 1992, 40, 2418-2430.

517

36. Ha, J.H.; Seog, H.M.; Nam, Y.J.; Shin, D.W. Changes in the taste and flavour

518

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519

511-517.

520 521 522 523 524 525 526 527 528 24


Page 24 of 36

Page 25 of 36


529

Figure captions

530

Figure 1. The OD600nm value of strains 3m-1 and 8m-9 broth after incubation at

531

different temperatures for 48 h.

532

Figure 2. Changes in pH, titratable acidity, salinity, and nitrite and reducing sugar

533

contents of both Paocai products during fermentation.

534

(A) Paocai (1#). (B) Paocai (2#).

535

Figure 3. Changes in lactic acid (A) and acetic acid (B) contents in both Paocai

536

products during fermentation.

537

Figure 4. Sensory evaluation of Paocai products.

538

Figure 5. Changes in total aerobes, LAB, and yeast counts in both Paocai products

539

throughout fermentation.

540

(A) Paocai (1#). (B) Paocai (2#).

541

Figure 6. DGGE profile of PCR-amplified 16S rDNA and cluster analysis of DGGE

542

profile.

543

(A) DGGE profile of PCR-amplified 16S rDNA from Paocai (1#) and (2#). (B)

544

Cluster analysis of DGGE profile for Paocai (1#). (C) Cluster analysis of DGGE

545

profile for Paocai (2#).

546

Figure 7. DGGE profile of PCR-amplified 18S rDNA and cluster analysis of DGGE

547

profile.

548

(A) DGGE profile of PCR-amplified 18S rDNA from Paocai (1#) and (2#). (B)

549

Cluster analysis of DGGE profile for Paocai (1#). (C) Cluster analysis of DGGE

550

profile for Paocai (2#). 25



Page 26 of 36

Tables Table 1 The properties of strain 3m-1 and 8m-9 Strain 3m-1

Result

Strain 8m-9

Result

Catalase test

-

Catalase test

-

Oxidase

-

Oxidase

-

Grow at

Gelatin liquefaction

-

Litmus milk

-

Grow at

15 °C

+

14% NaCl

+

Decarboxylase (Lysine, Ornithine, and arginine)

10% NaCl Decarboxylase (Lysine,

-

Ornithine, and arginine)

+

-

Acid produced from

+

Acid produced from

Aescinate

+

Fructose

+

Cellobiose

+

Galactose

+

Maltose

+

Lactose

+

Mannitol

+

Maltose

+

Salicin

+

Mannose

+

Sorbitol

+

Xylose

+

Sucrose

+

Sucrose

+

Raffinose

+

Inulin

+

Lactose

+

Melibiose

+

Mannose

+

Galactose

-

Rhamnose

+

Symbols: “＋”, positive; “-”, negative.

26


Page 27 of 36


Table 2 The maximum probable number of total coliforms (CFU/100 g) Time (d)

1

2

3

4

5

7

9

11

13

15

20

Paocai (1#)

610

172

Use of Psychrotolerant Lactic Acid Bacteria (Lactobacillus spp. and

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