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Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in simulated media based on wine components
Accepted Manuscript Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in simulated media based on wine components Ruiling Lv, Thunthacha Chantapakul, Mingming Zou, Man Li, Jianwei Zhou, Tian Ding, Xingqian Ye, Donghong Liu PII:
S0956-7135(18)30041-0
DOI:
10.1016/j.foodcont.2018.01.029
Reference:
JFCO 5960
To appear in:
Food Control
Received Date: 19 November 2017 Revised Date:
28 January 2018
Accepted Date: 29 January 2018
Please cite this article as: Lv R., Chantapakul T., Zou M., Li M., Zhou J., Ding T., Ye X. & Liu D., Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in simulated media based on wine components, Food Control (2018), doi: 10.1016/j.foodcont.2018.01.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in
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simulated media based on wine components
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Ruiling Lv a, Thunthacha Chantapakul a, Mingming Zou a, Man Li a, Jianwei Zhou a, Tian
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Ding a, c, Xingqian Ye a, c, Donghong Liu a, b, c, *
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a
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Food Technology and Equipment, Zhejiang University, Hangzhou 310058, Zhejiang, China
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Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, Zhejiang, China
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c
Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food
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Technology and Equipment, Hangzhou 310058, Zhejiang, China
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Department of Food Science and Nutrition, National Engineering Laboratory of Intelligent
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* Corresponding author. College of Biosystems Engineering and Food Science, Zhejiang
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University, 866 Yuhangtang Rd., Hangzhou 310058, China.
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E-mail address: [email protected] (D. Liu). [email protected] (R. Lv).
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[email protected] (T. Chantapakul).
[email protected] (M. Zou).
[email protected] (M. Li)
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[email protected] (J. Zhou).
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[email protected] (T. Ding).
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[email protected] (X. Ye) 1
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ABSTRACT Chinese rice wine, a traditional wine widely consumed in China, is typically sterilized in
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light of its sublethal alcohol content and nutritional richness. Nevertheless, several foodborne
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pathogens have been detected in final products, of which Bacillus cereus is one of the most
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heat-resistant. To study the effects of the wine matrix on bacterial inactivation, simulated
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media were established based on the main characteristics of the wine, including glucose
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content (23.48 ± 0.86 g/L), alcohol content (17.0 ± 0.5%), pH (4.14 ± 0.06), and protein
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content. In Chinese rice wine, log counts declined to 5.79 after 1 min heat treatment at
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60.0 °C, 4.99 at 70.0 °C, and 4.37 at 80.0 °C. The loss of viability was smaller in simulated
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media with protein and glucose than in sterile saline, implying that glucose and especially
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protein were protective. The inactivation kinetics of B. cereus vegetative cells were then
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assessed in these media, and modeled. Remarkably, thermal survival curves in Chinese rice
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wine and simulated media were linear and well-modeled by first-order kinetics with R2 values
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0.9501-0.9992. The data demonstrated that media with acidic pH were the most adverse
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environment against B. cereus, with D-value 0.61 ± 0.02 min at 60.0 °C. Similar values were
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obtained in media with 23.48 g/L glucose at 80.0 °C. Transmission electron micrographs
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showed heat-induced injuries in B. cereus through “coagulation” of the cytoplasmic content in
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all the media. Furthermore, modifications on the cell wall in acidic pH and alcohol media,
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leakage of cellular content observed in Chinese rice wine and glucose media. Conclusively,
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the results might help optimize the sterilization parameters to ensure the safety and quality of
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Chinese rice wine.
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Key words:
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Chinese rice wine
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thermal inactivation
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heat resistance
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D-value
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first-order kinetics
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morphological damage
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1. Introduction Chinese rice wine is one of the oldest wines in the world, with a history of nearly 5,000
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years (Wu, et al., 2015; Yu, Zhao, Li, Tian, & Ma, 2015). The typical wine is fermented from
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glutinous rice with wheat Qu and yeast. The wine is bright red in color, and has fascinating
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sweet flavor, unique aroma, low alcohol content, as well as high medicinal (Han & Xu, 2011),
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health (Lv, Huang, Zhang, Rao, & Ni, 2012), and nutritional value (Lu, et al., 2007). Thus, it
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is considered the national banquet wine in China (Lambert, 2003; Liu, et al., 2015), with
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annual consumption of more than 2 million tons (Zhang, et al., 2012). The manufacture of
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Chinese rice wine basically involves five steps namely, soaking rice for 5–10 days, steaming,
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simultaneous saccharification and fermentation, sterilization, and lastly, storing and ageing
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(Xu, et al., 2015; Xu, et al., 2016). Accordingly, the complex winemaking process has
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attracted attention as a potential food safety issue (Li, Wang, Wu, & Lu, 2014). For example,
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Chinese rice wine was reported to contain various bacteria (Kim, Park, Lee, Hwang, & Rhee,
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2014; Kim, et al., 2015; Li, et al., 2014; Liu, et al., 2015), including Escherichia coli and
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other foodborne pathogens such as Bacillus cereus, Campylobacter jejuni, Salmonella spp.,
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Staphylococcus aureus, and others. Of these, B. cereus was the most heat-resistant since it
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could produce spores (Kim, Park, et al., 2014; Kim, et al., 2015).
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B. cereus is an endospore-forming, facultatively anaerobic, Gram-positive foodborne
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pathogen and spoilage microorganism (Cronin & Wilkinson, 2008). It can isolate from low
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acid, chilled foods (Carlin, et al., 2000; Silva & Gibbs, 2010), and can cause foodborne
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diseases outbreaks such as diarrhea and emetic. Furthermore, B. cereus withstands prolonged 4
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cereus is considered a suitable indicator of microbiological safety (Yang, et al., 2017), and
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adequate management processes are needed to eliminate B. cereus and ensure product safety
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(Wang, Devlieghere, Geeraerd, & Uyttendaele, 2017).
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Thermal processing is the most widely used process to ensure microbiological safety in
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the food industry (Rawson, et al., 2011). But application of excessive heat to achieve lethality
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against specific foodborne pathogens also degrades the quality and sensory attributes of
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products (Li, et al., 2014; Wu, et al., 2014). Thus, thermal intensity and duration of exposure
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should be minimized as much as possible to retain product quality without compromising
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microbiological safety.
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Microbial heat resistance is mostly determined by duration of exposure and temperature,
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although environmental factors such as the pH, water activity (aw) may also have significant
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impact (Esteban, Huertas, Fernandez, & Palop, 2013). In particular, proteins and sugars had
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been reported to be protective (Black, Huppertz, Fitzgerald, & Kelly, 2007; Gao, Ju, & Wu,
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2007), along with high fat content or low aw values (Hereu, Bover-Cid, Garriga, & Aymerich,
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2012; Hereu, Dalgaard, Garriga, Aymerich, & Bover-Cid, 2012). As Chinese rice wine was
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rich in protein and contained different concentrations of sugar, it was typically sterilized after
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fermentation by boiling at 85-95 °C and immediately bottled (Wu, et al., 2012). Dry and
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Chinese rice wine were boiled at higher temperatures (90-95 °C) in which sugar content is
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lower than 15.0 and between 15.1 to 40.0 g/L respectively, while semisweet and sweet
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Chinese rice wine with sugar higher than 40.0 g/L were boiled at 85-90 °C to avoid
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melanoidin formation from sugars and amino acids (Wu, et al., 2012; Xia, Zhang, Zhang,
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Zhang, & Wang, 2016). Unfortunately, there had been little studies on the effects of wine
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components on sterilization, and the mechanism of thermal inactivation remained unclear. Due to the significance of B. cereus to food safety, and the gaps in knowledge of bacterial
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inactivation, we investigated the thermal inactivation kinetics of B. cereus in Chinese red
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wine. In particular, we studied components in Chinese rice wine that might promote the
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inactivation of B. cereus, modeled the thermal inactivation kinetics with a view to optimize
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sterilization parameters, and investigated the mechanism of inactivation using simulated
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media. The long-term goal was to help promote the development of traditional food in China.
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2. Materials and methods
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2.1 Sample collection and processing
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A sample of Chinese rice wine was provided by Zhejiang Guyuelongshan Shaoxing
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Wine Co., Ltd (Shaoxing, Zhejiang, China). The wine used in the study was fermented from
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glutinous rice by “wheat Qu” (made from raw wheat, inoculated with molds; bacteria and
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yeasts) under 30 °C for about 25 days, and thermally processed for 20 min at 85 °C before
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being bottled. It was then aged for 8 years.
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2.2 Analysis of the characteristics of Chinese rice wine
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Glucose content was determined at 30 °C and 1 mL/min on a Prominence LC-20A
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high-performance liquid chromatography system (Kyoto, Japan) fitted with a Platisil 5 µm
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NH2 exclusion column (250 mm × 4.6 mm), using 85 % acetonitrile as mobile phase, samples
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were analyzed in triplicate. Alcohol content was measured on an alcohol meter (74290: 0-40,
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Shanghai chemical laboratory equipment Co., Ltd., Shanghai, China) according to Chinese 6
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National Standard GB 13662-2008. The pH was tested on a PHS-550 pH meter (Lu Heng
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Biotechnology Co., Ltd., Hangzhou, Zhejiang, China).
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2.3 Preparation of simulated media Characteristics and composition of each media were listed in Table 1, and the
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preparations of simulated media were as followed. Aqueous solution containing glucose as
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glucose simulated media was prepared based on average values of glucose content. A
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simulated media of alcohol was alcohol-water solution having the same alcohol content as
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Chinese rice wine and a simulated media with the same pH was prepared using lactic
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acid/sodium lactate buffer. A media with the same protein content was prepared by dialyzing
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Chinese rice wine for 3 days at molecular weight cutoff 5,000 D to remove small molecules
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such as ethanol, sugar, acid, etc. And 0.85% sterile saline also prepared and sterilized as a
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control without ethanol, glucose, protein at neutral pH.
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2.4 Test suspensions
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B. cereus ATCC 11778-3 was obtained from Hope Bio-Technology Co., Ltd., Qingdao,
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Shandong, China, and routinely grown for 12 h until stationary phase at 37 °C and 150 rpm
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with population about 7.6 log CFU/mL (Abhyankar, et al., 2013), using standard nutrient
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broth (Hope Bio-Technology Co., Ltd., Qingdao, Shandong, China) with pH 7.2 ± 0.2, which
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contains 0.5 % NaCl, peptone, and beef extract powder. Cells were harvested by
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centrifugation for 10 min at 2,320 ×g and 4 °C (TGL-20M centrifuge; Kaida Scientific
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Instruments Co., Ltd., Changsha, Hunan, China), washed twice with 0.85% sterile saline.
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Ultimately, the suspension used in this work were free from spores determined by observation
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in a microscope (UPH 203i Phase Microscope, Aopu Photoelectric Technology Co., Ltd.,
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Chongqing, China)
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2.5 Sterilization by heat 0.5 mL bacterial suspensions were transferred into sterile glass tubes with a diameter of
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15 mm in which Chinese rice wine or simulated media (4.5 mL) were pre-heated by full
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immersion in a water bath (DKS-12, Jiaxing Zhongxin Medical Instruments Co., Ltd., Jiaxing,
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Zhejiang, China) at 60.0 °C, 70.0 °C, and 80.0 °C. Heat treatments were applied to each
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sample and each temperature with a control without treatment, samples were then
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immediately cooled in ice bath.
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2.6 Enumeration of surviving cells
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The viability of B. cereus was evaluated immediately after sterilization by plate counting.
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Briefly, 0.1 mL aliquots from selected dilutions were plated on tryptic soy agar (Hope
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Bio-Technology Co., Ltd., Qingdao, Shandong, China). The plates were then incubated for 48
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h at 37 °C, the number of colonies formed was counted and marked after 24 h, and then
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checked if there were some more colonies by heat injured cells at 48 h. Plates with 30-300
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CFUs were considered most suitable for analysis, and each sample was tested in triplicate.
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2.7 Modeling the kinetics of Bacillus cereus inactivation
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The thermal inactivation of B. cereus plotted as log microbial numbers vs. time, was best
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modeled by first-order kinetics (Evelyn & Silva, 2015). From these models, decimal reduction
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time (D-values or DT) to reduce microbial population by 90 %, was calculated from the
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reciprocal of the slope as
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log
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where N0 was the initial B. cereus concentration in the sample (CFU/mL), and N was the 8
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concentration of viable B. cereus after exposure to heat for time t (min).
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2.8 Transmission electron microscopy Cells were collected by centrifugation at 2,320 ×g for 10 min at 4 °C, washed twice with
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sterile normal saline, fixed with 2.5 % glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd,
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Shanghai, China) for more than 4 h, and washed another three times with 0.1 M phosphate
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buffer (Hangzhou Ke Yi Biotechnology Co., Ltd., Hangzhou, Zhejiang, China) pH 7.0 for 15
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min each. Samples were then post-fixed with 1 % OsO4 for 1-2 h, and again washed three
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times with 0.1 M phosphate buffer for 15 min. Subsequently, specimens were dehydrated over
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a graded ethanol series (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, and 100 %), transferred to
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absolute acetone, embedded in Spurr’s resin, incubated for 4 h at room temperature, and
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polymerized for 24 h in an oven at 65 °C. Finally, the specimens were sectioned in Leica EM
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UC7 ultramicrotome (Leica Biosystems, Nussloch, Germany), stained with uranyl acetate and
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alkaline lead citrate for 5-10 min, and visualized on Hitachi Science & Technology H-7650
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Transmission Electron Microscope (Hitachi, Ltd., Tokyo, Japan).
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2.9 Statistical analysis
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All experiments were conducted in triplicate for each treatment, from which the
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mean ± standard deviation was obtained and analyzed. Data were processed in Origin
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Software Version 9.2 (OriginLab Corp., MA, USA). Correlation analysis was performed by
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ANOVA in SPSS Statistics 20 (IBM Co., Chicago, USA).
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3. Results and discussions
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3.1 Characteristics of Chinese rice wine 9
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0.86 g/L and the alcohol percentage was 17.0 ± 0.5%, simulated media for glucose, alcohol,
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and pH were prepared accordingly. The characteristics of this wine were close to the average
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and typical Chinese rice wine (Shen, Wu, Wei, Liu, & Tang, 2017; Z. Z. Wu, et al., 2016).
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3.2 Inactivation kinetics of B. cereus
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After incubation about 12 h, the colonies of B. cereus were visible and there was no
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increase more 12 h later, so the inactivation results were based on the 12 h amounts. The
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number of viable B. cereus cells significantly dropped with duration of exposure to heat (Fig.
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1a-f). Notably, the media affected B. cereus inactivation, although inactivation generally
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followed the same trends in all media (Petruzzi, et al., 2017). The loss of viability was smaller
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in simulated media with protein and glucose than in Chinese rice wine or simulated media
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with pH 4.14 at the same temperature, meanwhile, the inactivation in sterile saline was
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between those, implying that glucose and especially protein were protective, in line with
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previous studies (Bover-Cid, et al., 2015). In contrast, inactivation was clearly accelerated in
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simulated media with alcohol and, especially, in simulated media with pH 4.14, indicating
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enhanced sterilizing effects. Survival was lowest in the latter where 6.05 log CFU/mL B.
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cereus remained viable after 1 min at 60.0 °C, which coincided with Black et al. (Black, et al.,
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2007). Similar reductions were observed in simulated media with glucose or protein at
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70.0 °C but only 4.59 log CFU/mL remained viable after 1 min in simulated media with pH
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4.14 at the same temperature.
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3.3 Modeling the thermal inactivation of B. cereus
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The thermal survival of B. cereus in Chinese rice wine and simulated media was linear 10
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temperatures (Fig. 1). The slopes were at the 0.05 level and significantly differently different
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from zero. D-values in different media and at different temperatures were listed in Table 2,
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and ranged from 0.28 to 1.13 min. Remarkably, the D-value at 60.0 °C in simulated media
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with pH 4.14 (0.61 ± 0.02 min) was comparable to that at 80.0 °C in simulated glucose media.
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Previous studies almost focused on the heat resistance about the bacteria or spores in single
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simulated media, and there was no study relating to the inactivation in Chinese rice wine and
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its components. This data indicated that components of Chinese rice wine affected B. cereus
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inactivation, among which more pronounced in simulated media of alcohol and pH than in
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simulated media of glucose and protein. Besides, the significant difference among the
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D-values in each media at different temperature showed that the media did affect the
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inactivation of B. cereus and the variety with increasing temperature differed a lot among
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media. However, the D-values in protein media decreased rapidly with increasing temperature,
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possibly due to protein denaturation (Atuonwu, Ray, & Stapley, 2017) and loss of protective
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properties. The D-values also reinforced the notion that alcohol and pH enhanced sterilization.
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We noted that models of the thermal inactivation of B. cereus had traditionally been based
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on thermodynamic arguments, which assumed that only a single critical site needed to be
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inactivated. To vegetative cells, thermal process seemed to affect the cells as an integral part
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of an entire complex system rather than one specific target. Accordingly, such arguments
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might turn on significant information on where and how bacterial cells were injured indeed
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(Lambert, 2003).
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3.4 Transmission electron microscopy
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changes in B. cereus. Untreated cells were found to be rod-shaped and intact (Fig. 2a), and
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different kinds of injuries were observed after heating in Chinese rice wine and various
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simulated media, in general, there were some “coagulation” of the cytoplasmic content and
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modifications on the cell wall (Li, et al., 2017). In Chinese rice wine (Fig. 2b) or glucose
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media (Fig. 2c), heating damaged the cell wall and other structures, and some leakage of
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cellular content was observed. However, the damage to organelles was less pronounced in the
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latter than in the former, likely because of the protective effects and tonicity of glucose. In
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simulated alcohol media, only minor changes in structure were observed, but cellular contents
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were heavily damaged (Fig. 2d) and the cell wall was significantly thinner. Remarkably, cells
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in simulated protein media appeared to be undergoing plasmolysis, but also appeared to have
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similar structure as untreated cells, with seemingly normal cell morphology and negligible
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intracellular damage (Fig. 2e). In contrast, cells heated at pH 4.14 exhibited the most
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pronounced changes in morphology, highlighting the effects of pH on sterilization. These
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cells were severely damaged, with cellular contents aggregated around cell walls that had
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become significantly thinner. In general, protein coagulation followed thermal process in all
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condition, which was one of major lethal events to the vegetative cells (Earnshaw, Appleyard,
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& Hurst, 1995).
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4. Conclusions
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In summary, this research indicated that components of Chinese rice wine affected
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sterilization. In particular, protein and glucose provided slightly different degrees of thermal 12
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sublethal treatments, especially by heat, were potentially significant food safety hazards, the
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data suggested that sterilization parameters should be customized according to the
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composition of the wine, and according to appropriate mathematical models of survival.
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Unfortunately, the underlying mechanisms of heat inactivation in various environments
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remain unclear, without considering the spores is still a limitation. Also, in the near future,
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thermal sterilization might be replaced or combined with other non-thermal technologies,
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including high hydrostatic pressure, ultrasound, and others. So, it is necessary to regard the
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inactivation mechanisms of the spores induced by thermal and non-thermal technologies on
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further studies. In any case, the Chinese traditional food industry should continue to innovate
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and keep pace with manufacturing advances.
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Formatting of Funding Sources
This study was supported by the National Key Research and Development Program of China [grant number 2016YFD0400301].
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Conflict of interest None
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Liu, S. P., Mao, J., Liu, Y. Y., Meng, X. Y., Ji, Z. W., Zhou, Z. L., & Ai-lati, A. (2015). Bacterial succession and the dynamics of volatile compounds during the fermentation of Chinese rice wine from Shaoxing region. World Journal of Microbiology & Biotechnology, 31(12), 1907-1921.
Lu, Y. M., Lu, X., Chen, X. H., Jiang, M., Li, C., & Dong, M. S. (2007). A survey of biogenic amines in Chinese rice wines. Food Chemistry, 100(4), 1424-1428.
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filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing. Journal of General and Applied Microbiology, 58(1), 33-42. Maria José Valera, F. S., Albert Mas, María Jesús Torija. (2017). Effect of chitosan and SO2 on viability of Acetobacter strains in wine. International Journal of Food Microbiology, 246 1–4. Petruzzi, L., Campaniello, D., Speranza, B., Corbo, M. R., Sinigaglia, M., & Bevilacqua, A. (2017).
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Thermal Treatments for Fruit and Vegetable Juices and Beverages: A Literature Overview. Comprehensive Reviews in Food Science and Food Safety, 16(4), 668-691. Rawson, A., Patras, A., Tiwari, B. K., Noci, F., Koutchma, T., & Brunton, N. (2011). Effect of thermal and non thermal processing technologies on the bioactive content of exotic fruits and their
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Silva, F. V. M., & Gibbs, P. A. (2010). Non-proteolytic Clostridium botulinum spores in low-acid cold-distributed foods and design of pasteurization processes. Trends in Food Science & Technology, 21(2), 95-105. Wang, X., Devlieghere, F., Geeraerd, A., & Uyttendaele, M. (2017). Thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth versus agar surface. International Journal of Food Microbiology, 243, 70-77. Wu, H. M., Chen, L., Pan, G. S., Tu, C. Y., Zhou, X. P., & Mo, L. Y. (2012). Study on the changing 15
ACCEPTED MANUSCRIPT chromatography/mass spectrometry. European Food Research and Technology, 235(5), 779-782. Wu, P. G., Cai, C. G., Shen, X. H., Wang, L. Y., Zhang, J., Tan, Y., Jiang, W., & Pan, X. D. (2014). Formation of ethyl carbamate and changes during fermentation and storage of yellow rice wine. Food Chemistry, 152, 108-112. Wu, Z. Z., Long, J., Xu, E. B., Wang, F., Xu, X. M., Jin, Z. Y., & Jiao, A. Q. (2016). A Feasibility Study
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Xu, E. B., Wu, Z. Z., Wang, F., Li, H. Y., Xu, X. M., Jin, Z. Y., & Jiao, A. Q. (2015). Impact of
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High-Shear Extrusion Combined With Enzymatic Hydrolysis on Rice Properties and Chinese Rice Wine Fermentation. Food and Bioprocess Technology, 8(3), 589-604. Xu, E. B., Wu, Z. Z., Wang, F., Long, J., Xu, X. M., Jin, Z. Y., & Jiao, A. Q. (2016). Effect of 'wheat Qu' addition on the formation of ethyl carbamate in Chinese rice wine with enzymatic extrusion liquefaction pretreatment. Journal of the Institute of Brewing, 122(1), 55-62. Yang, Y., Yu, X. F., Zhan, L., Chen, J. C., Zhang, Y. Y., Zhang, J. Y., Chen, H. H., Zhang, Z., Zhang, Y. J., Lu, Y. Y., & Mei, L. L. (2017). Multilocus sequence type profiles of Bacillus cereus isolates
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from infant formula in China. Food Microbiology, 62, 46-50.
Yu, H. Y., Zhao, J., Li, F. H., Tian, H. X., & Ma, X. (2015). Characterization of Chinese rice wine taste attributes using liquid chromatographic analysis, sensory evaluation, and an electronic tongue. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 997, 129-135.
Zhang, B., Kong, L. Q., Cao, Y., Xie, G. F., Guan, Z. B., & Lu, J. (2012). Metaproteomic
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concentration of ethyl carbamate in yellow rice wine during production and storage by gas
characterisation of a Shaoxing rice wine "wheat Qu" extract. Food Chemistry, 134(1), 387-391.
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Figures
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Fig. 1. Survival of B. cereus at 60-80 °C in a) Chinese rice wine, simulated media of b)
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Glucose, c) Alcohol, d) pH, e) Protein and f) Sterile saline. Data were mean ± standard
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deviation, and were fit to first-order kinetic models, R2 of first-order kinetic models also
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showed in the Figure. 19
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(a)
(b)
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(c)
(d)
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(e)
(f)
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Fig. 2. Transmission electron micrographs of (a) untreated B. cereus, and B. cereus heated for
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1 min at 80 °C in (b) Chinese rice wine and simulated media of (c) Glucose, (d) Alcohol, (e)
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Protein, and (f) pH 4.14.
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Tables
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Table 1.
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Characteristics and composition of each media (“+” means with that component, while “-” means without that component) Glucose
+
Acid
+
-
7.0
-
+
7.0
-
-
-
Acid
-
-
-
7.0
+
-
-
7.0
-
+
Alcohol
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Glucose
Protein
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Control (Sterile saline)
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Protein
+
pH
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pH
Chinese rice wine
Alcohol
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Table 2.
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D-values (min) for B. cereus heated at 60.0-80.0 °C in Chinese rice wine and simulated media based on wine characteristics pH
60.0
0.56 ± 0.05d, e, *
0.82 ± 0.04g
0.53 ± 0.06d, e, f
70.0
0.40 ± 0.10a, b, c
0.72 ± 0.04f, g
0.37 ± 0.04a, b
80.0
0.31 ± 0.08a
0.55 ± 0.02c, d, e
0.36 ± 0.03a, b
Protein
Sterile saline
1.13 ± 0.01h
0.76 ± 0.08f, g
0.32 ± 0.06a, b
0.66 ± 0.05e, f
0.54 ± 0.02c, d, e
0.28 ± 0.02a
0.47 ± 0.06b, c, d
0.37 ± 0.03a, b
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Alcohol
0.61 ± 0.02d, e, f
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*Values are the mean of triplicate measurements ± standard deviation; The different letters (a-h) indicate significant differences (P < 0.05).
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Chinese rice wine
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Temp (°C)
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S0956-7135(18)30041-0
DOI:
10.1016/j.foodcont.2018.01.029
Reference:
JFCO 5960
To appear in:
Food Control
Received Date: 19 November 2017 Revised Date:
28 January 2018
Accepted Date: 29 January 2018
Please cite this article as: Lv R., Chantapakul T., Zou M., Li M., Zhou J., Ding T., Ye X. & Liu D., Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in simulated media based on wine components, Food Control (2018), doi: 10.1016/j.foodcont.2018.01.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Thermal inactivation kinetics of Bacillus cereus in Chinese rice wine and in
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simulated media based on wine components
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Ruiling Lv a, Thunthacha Chantapakul a, Mingming Zou a, Man Li a, Jianwei Zhou a, Tian
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Ding a, c, Xingqian Ye a, c, Donghong Liu a, b, c, *
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a
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Food Technology and Equipment, Zhejiang University, Hangzhou 310058, Zhejiang, China
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b
Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, Zhejiang, China
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c
Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food
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Technology and Equipment, Hangzhou 310058, Zhejiang, China
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Department of Food Science and Nutrition, National Engineering Laboratory of Intelligent
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* Corresponding author. College of Biosystems Engineering and Food Science, Zhejiang
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University, 866 Yuhangtang Rd., Hangzhou 310058, China.
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E-mail address: [email protected] (D. Liu). [email protected] (R. Lv).
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[email protected] (T. Chantapakul).
[email protected] (M. Zou).
[email protected] (M. Li)
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[email protected] (J. Zhou).
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[email protected] (T. Ding).
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[email protected] (X. Ye) 1
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ABSTRACT Chinese rice wine, a traditional wine widely consumed in China, is typically sterilized in
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light of its sublethal alcohol content and nutritional richness. Nevertheless, several foodborne
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pathogens have been detected in final products, of which Bacillus cereus is one of the most
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heat-resistant. To study the effects of the wine matrix on bacterial inactivation, simulated
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media were established based on the main characteristics of the wine, including glucose
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content (23.48 ± 0.86 g/L), alcohol content (17.0 ± 0.5%), pH (4.14 ± 0.06), and protein
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content. In Chinese rice wine, log counts declined to 5.79 after 1 min heat treatment at
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60.0 °C, 4.99 at 70.0 °C, and 4.37 at 80.0 °C. The loss of viability was smaller in simulated
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media with protein and glucose than in sterile saline, implying that glucose and especially
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protein were protective. The inactivation kinetics of B. cereus vegetative cells were then
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assessed in these media, and modeled. Remarkably, thermal survival curves in Chinese rice
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wine and simulated media were linear and well-modeled by first-order kinetics with R2 values
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0.9501-0.9992. The data demonstrated that media with acidic pH were the most adverse
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environment against B. cereus, with D-value 0.61 ± 0.02 min at 60.0 °C. Similar values were
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obtained in media with 23.48 g/L glucose at 80.0 °C. Transmission electron micrographs
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showed heat-induced injuries in B. cereus through “coagulation” of the cytoplasmic content in
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all the media. Furthermore, modifications on the cell wall in acidic pH and alcohol media,
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leakage of cellular content observed in Chinese rice wine and glucose media. Conclusively,
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the results might help optimize the sterilization parameters to ensure the safety and quality of
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Chinese rice wine.
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Key words:
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Chinese rice wine
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thermal inactivation
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heat resistance
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D-value
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first-order kinetics
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morphological damage
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1. Introduction Chinese rice wine is one of the oldest wines in the world, with a history of nearly 5,000
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years (Wu, et al., 2015; Yu, Zhao, Li, Tian, & Ma, 2015). The typical wine is fermented from
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glutinous rice with wheat Qu and yeast. The wine is bright red in color, and has fascinating
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sweet flavor, unique aroma, low alcohol content, as well as high medicinal (Han & Xu, 2011),
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health (Lv, Huang, Zhang, Rao, & Ni, 2012), and nutritional value (Lu, et al., 2007). Thus, it
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is considered the national banquet wine in China (Lambert, 2003; Liu, et al., 2015), with
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annual consumption of more than 2 million tons (Zhang, et al., 2012). The manufacture of
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Chinese rice wine basically involves five steps namely, soaking rice for 5–10 days, steaming,
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simultaneous saccharification and fermentation, sterilization, and lastly, storing and ageing
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(Xu, et al., 2015; Xu, et al., 2016). Accordingly, the complex winemaking process has
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attracted attention as a potential food safety issue (Li, Wang, Wu, & Lu, 2014). For example,
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Chinese rice wine was reported to contain various bacteria (Kim, Park, Lee, Hwang, & Rhee,
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2014; Kim, et al., 2015; Li, et al., 2014; Liu, et al., 2015), including Escherichia coli and
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other foodborne pathogens such as Bacillus cereus, Campylobacter jejuni, Salmonella spp.,
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Staphylococcus aureus, and others. Of these, B. cereus was the most heat-resistant since it
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could produce spores (Kim, Park, et al., 2014; Kim, et al., 2015).
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B. cereus is an endospore-forming, facultatively anaerobic, Gram-positive foodborne
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pathogen and spoilage microorganism (Cronin & Wilkinson, 2008). It can isolate from low
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acid, chilled foods (Carlin, et al., 2000; Silva & Gibbs, 2010), and can cause foodborne
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diseases outbreaks such as diarrhea and emetic. Furthermore, B. cereus withstands prolonged 4
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cereus is considered a suitable indicator of microbiological safety (Yang, et al., 2017), and
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adequate management processes are needed to eliminate B. cereus and ensure product safety
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(Wang, Devlieghere, Geeraerd, & Uyttendaele, 2017).
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Thermal processing is the most widely used process to ensure microbiological safety in
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the food industry (Rawson, et al., 2011). But application of excessive heat to achieve lethality
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against specific foodborne pathogens also degrades the quality and sensory attributes of
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products (Li, et al., 2014; Wu, et al., 2014). Thus, thermal intensity and duration of exposure
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should be minimized as much as possible to retain product quality without compromising
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microbiological safety.
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Microbial heat resistance is mostly determined by duration of exposure and temperature,
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although environmental factors such as the pH, water activity (aw) may also have significant
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impact (Esteban, Huertas, Fernandez, & Palop, 2013). In particular, proteins and sugars had
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been reported to be protective (Black, Huppertz, Fitzgerald, & Kelly, 2007; Gao, Ju, & Wu,
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2007), along with high fat content or low aw values (Hereu, Bover-Cid, Garriga, & Aymerich,
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2012; Hereu, Dalgaard, Garriga, Aymerich, & Bover-Cid, 2012). As Chinese rice wine was
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rich in protein and contained different concentrations of sugar, it was typically sterilized after
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fermentation by boiling at 85-95 °C and immediately bottled (Wu, et al., 2012). Dry and
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Chinese rice wine were boiled at higher temperatures (90-95 °C) in which sugar content is
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lower than 15.0 and between 15.1 to 40.0 g/L respectively, while semisweet and sweet
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Chinese rice wine with sugar higher than 40.0 g/L were boiled at 85-90 °C to avoid
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melanoidin formation from sugars and amino acids (Wu, et al., 2012; Xia, Zhang, Zhang,
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Zhang, & Wang, 2016). Unfortunately, there had been little studies on the effects of wine
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components on sterilization, and the mechanism of thermal inactivation remained unclear. Due to the significance of B. cereus to food safety, and the gaps in knowledge of bacterial
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inactivation, we investigated the thermal inactivation kinetics of B. cereus in Chinese red
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wine. In particular, we studied components in Chinese rice wine that might promote the
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inactivation of B. cereus, modeled the thermal inactivation kinetics with a view to optimize
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sterilization parameters, and investigated the mechanism of inactivation using simulated
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media. The long-term goal was to help promote the development of traditional food in China.
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2. Materials and methods
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2.1 Sample collection and processing
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A sample of Chinese rice wine was provided by Zhejiang Guyuelongshan Shaoxing
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Wine Co., Ltd (Shaoxing, Zhejiang, China). The wine used in the study was fermented from
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glutinous rice by “wheat Qu” (made from raw wheat, inoculated with molds; bacteria and
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yeasts) under 30 °C for about 25 days, and thermally processed for 20 min at 85 °C before
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being bottled. It was then aged for 8 years.
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2.2 Analysis of the characteristics of Chinese rice wine
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Glucose content was determined at 30 °C and 1 mL/min on a Prominence LC-20A
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high-performance liquid chromatography system (Kyoto, Japan) fitted with a Platisil 5 µm
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NH2 exclusion column (250 mm × 4.6 mm), using 85 % acetonitrile as mobile phase, samples
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were analyzed in triplicate. Alcohol content was measured on an alcohol meter (74290: 0-40,
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Shanghai chemical laboratory equipment Co., Ltd., Shanghai, China) according to Chinese 6
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National Standard GB 13662-2008. The pH was tested on a PHS-550 pH meter (Lu Heng
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Biotechnology Co., Ltd., Hangzhou, Zhejiang, China).
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2.3 Preparation of simulated media Characteristics and composition of each media were listed in Table 1, and the
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preparations of simulated media were as followed. Aqueous solution containing glucose as
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glucose simulated media was prepared based on average values of glucose content. A
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simulated media of alcohol was alcohol-water solution having the same alcohol content as
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Chinese rice wine and a simulated media with the same pH was prepared using lactic
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acid/sodium lactate buffer. A media with the same protein content was prepared by dialyzing
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Chinese rice wine for 3 days at molecular weight cutoff 5,000 D to remove small molecules
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such as ethanol, sugar, acid, etc. And 0.85% sterile saline also prepared and sterilized as a
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control without ethanol, glucose, protein at neutral pH.
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2.4 Test suspensions
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B. cereus ATCC 11778-3 was obtained from Hope Bio-Technology Co., Ltd., Qingdao,
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Shandong, China, and routinely grown for 12 h until stationary phase at 37 °C and 150 rpm
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with population about 7.6 log CFU/mL (Abhyankar, et al., 2013), using standard nutrient
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broth (Hope Bio-Technology Co., Ltd., Qingdao, Shandong, China) with pH 7.2 ± 0.2, which
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contains 0.5 % NaCl, peptone, and beef extract powder. Cells were harvested by
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centrifugation for 10 min at 2,320 ×g and 4 °C (TGL-20M centrifuge; Kaida Scientific
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Instruments Co., Ltd., Changsha, Hunan, China), washed twice with 0.85% sterile saline.
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Ultimately, the suspension used in this work were free from spores determined by observation
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in a microscope (UPH 203i Phase Microscope, Aopu Photoelectric Technology Co., Ltd.,
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Chongqing, China)
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2.5 Sterilization by heat 0.5 mL bacterial suspensions were transferred into sterile glass tubes with a diameter of
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15 mm in which Chinese rice wine or simulated media (4.5 mL) were pre-heated by full
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immersion in a water bath (DKS-12, Jiaxing Zhongxin Medical Instruments Co., Ltd., Jiaxing,
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Zhejiang, China) at 60.0 °C, 70.0 °C, and 80.0 °C. Heat treatments were applied to each
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sample and each temperature with a control without treatment, samples were then
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immediately cooled in ice bath.
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2.6 Enumeration of surviving cells
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The viability of B. cereus was evaluated immediately after sterilization by plate counting.
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Briefly, 0.1 mL aliquots from selected dilutions were plated on tryptic soy agar (Hope
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Bio-Technology Co., Ltd., Qingdao, Shandong, China). The plates were then incubated for 48
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h at 37 °C, the number of colonies formed was counted and marked after 24 h, and then
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checked if there were some more colonies by heat injured cells at 48 h. Plates with 30-300
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CFUs were considered most suitable for analysis, and each sample was tested in triplicate.
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2.7 Modeling the kinetics of Bacillus cereus inactivation
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The thermal inactivation of B. cereus plotted as log microbial numbers vs. time, was best
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modeled by first-order kinetics (Evelyn & Silva, 2015). From these models, decimal reduction
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time (D-values or DT) to reduce microbial population by 90 %, was calculated from the
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reciprocal of the slope as
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log
=−
(1)
where N0 was the initial B. cereus concentration in the sample (CFU/mL), and N was the 8
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concentration of viable B. cereus after exposure to heat for time t (min).
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2.8 Transmission electron microscopy Cells were collected by centrifugation at 2,320 ×g for 10 min at 4 °C, washed twice with
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sterile normal saline, fixed with 2.5 % glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd,
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Shanghai, China) for more than 4 h, and washed another three times with 0.1 M phosphate
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buffer (Hangzhou Ke Yi Biotechnology Co., Ltd., Hangzhou, Zhejiang, China) pH 7.0 for 15
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min each. Samples were then post-fixed with 1 % OsO4 for 1-2 h, and again washed three
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times with 0.1 M phosphate buffer for 15 min. Subsequently, specimens were dehydrated over
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a graded ethanol series (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, and 100 %), transferred to
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absolute acetone, embedded in Spurr’s resin, incubated for 4 h at room temperature, and
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polymerized for 24 h in an oven at 65 °C. Finally, the specimens were sectioned in Leica EM
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UC7 ultramicrotome (Leica Biosystems, Nussloch, Germany), stained with uranyl acetate and
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alkaline lead citrate for 5-10 min, and visualized on Hitachi Science & Technology H-7650
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Transmission Electron Microscope (Hitachi, Ltd., Tokyo, Japan).
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2.9 Statistical analysis
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All experiments were conducted in triplicate for each treatment, from which the
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mean ± standard deviation was obtained and analyzed. Data were processed in Origin
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Software Version 9.2 (OriginLab Corp., MA, USA). Correlation analysis was performed by
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ANOVA in SPSS Statistics 20 (IBM Co., Chicago, USA).
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3. Results and discussions
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3.1 Characteristics of Chinese rice wine 9
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0.86 g/L and the alcohol percentage was 17.0 ± 0.5%, simulated media for glucose, alcohol,
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and pH were prepared accordingly. The characteristics of this wine were close to the average
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and typical Chinese rice wine (Shen, Wu, Wei, Liu, & Tang, 2017; Z. Z. Wu, et al., 2016).
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3.2 Inactivation kinetics of B. cereus
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After incubation about 12 h, the colonies of B. cereus were visible and there was no
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increase more 12 h later, so the inactivation results were based on the 12 h amounts. The
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number of viable B. cereus cells significantly dropped with duration of exposure to heat (Fig.
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1a-f). Notably, the media affected B. cereus inactivation, although inactivation generally
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followed the same trends in all media (Petruzzi, et al., 2017). The loss of viability was smaller
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in simulated media with protein and glucose than in Chinese rice wine or simulated media
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with pH 4.14 at the same temperature, meanwhile, the inactivation in sterile saline was
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between those, implying that glucose and especially protein were protective, in line with
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previous studies (Bover-Cid, et al., 2015). In contrast, inactivation was clearly accelerated in
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simulated media with alcohol and, especially, in simulated media with pH 4.14, indicating
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enhanced sterilizing effects. Survival was lowest in the latter where 6.05 log CFU/mL B.
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cereus remained viable after 1 min at 60.0 °C, which coincided with Black et al. (Black, et al.,
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2007). Similar reductions were observed in simulated media with glucose or protein at
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70.0 °C but only 4.59 log CFU/mL remained viable after 1 min in simulated media with pH
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4.14 at the same temperature.
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3.3 Modeling the thermal inactivation of B. cereus
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The thermal survival of B. cereus in Chinese rice wine and simulated media was linear 10
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temperatures (Fig. 1). The slopes were at the 0.05 level and significantly differently different
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from zero. D-values in different media and at different temperatures were listed in Table 2,
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and ranged from 0.28 to 1.13 min. Remarkably, the D-value at 60.0 °C in simulated media
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with pH 4.14 (0.61 ± 0.02 min) was comparable to that at 80.0 °C in simulated glucose media.
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Previous studies almost focused on the heat resistance about the bacteria or spores in single
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simulated media, and there was no study relating to the inactivation in Chinese rice wine and
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its components. This data indicated that components of Chinese rice wine affected B. cereus
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inactivation, among which more pronounced in simulated media of alcohol and pH than in
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simulated media of glucose and protein. Besides, the significant difference among the
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D-values in each media at different temperature showed that the media did affect the
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inactivation of B. cereus and the variety with increasing temperature differed a lot among
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media. However, the D-values in protein media decreased rapidly with increasing temperature,
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possibly due to protein denaturation (Atuonwu, Ray, & Stapley, 2017) and loss of protective
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properties. The D-values also reinforced the notion that alcohol and pH enhanced sterilization.
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We noted that models of the thermal inactivation of B. cereus had traditionally been based
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on thermodynamic arguments, which assumed that only a single critical site needed to be
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inactivated. To vegetative cells, thermal process seemed to affect the cells as an integral part
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of an entire complex system rather than one specific target. Accordingly, such arguments
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might turn on significant information on where and how bacterial cells were injured indeed
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(Lambert, 2003).
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3.4 Transmission electron microscopy
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changes in B. cereus. Untreated cells were found to be rod-shaped and intact (Fig. 2a), and
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different kinds of injuries were observed after heating in Chinese rice wine and various
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simulated media, in general, there were some “coagulation” of the cytoplasmic content and
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modifications on the cell wall (Li, et al., 2017). In Chinese rice wine (Fig. 2b) or glucose
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media (Fig. 2c), heating damaged the cell wall and other structures, and some leakage of
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cellular content was observed. However, the damage to organelles was less pronounced in the
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latter than in the former, likely because of the protective effects and tonicity of glucose. In
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simulated alcohol media, only minor changes in structure were observed, but cellular contents
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were heavily damaged (Fig. 2d) and the cell wall was significantly thinner. Remarkably, cells
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in simulated protein media appeared to be undergoing plasmolysis, but also appeared to have
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similar structure as untreated cells, with seemingly normal cell morphology and negligible
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intracellular damage (Fig. 2e). In contrast, cells heated at pH 4.14 exhibited the most
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pronounced changes in morphology, highlighting the effects of pH on sterilization. These
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cells were severely damaged, with cellular contents aggregated around cell walls that had
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become significantly thinner. In general, protein coagulation followed thermal process in all
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condition, which was one of major lethal events to the vegetative cells (Earnshaw, Appleyard,
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& Hurst, 1995).
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4. Conclusions
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In summary, this research indicated that components of Chinese rice wine affected
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sterilization. In particular, protein and glucose provided slightly different degrees of thermal 12
ACCEPTED MANUSCRIPT protection to B. cereus, while alcohol and acidic pH enhanced the lethality of heat. As
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sublethal treatments, especially by heat, were potentially significant food safety hazards, the
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data suggested that sterilization parameters should be customized according to the
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composition of the wine, and according to appropriate mathematical models of survival.
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Unfortunately, the underlying mechanisms of heat inactivation in various environments
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remain unclear, without considering the spores is still a limitation. Also, in the near future,
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thermal sterilization might be replaced or combined with other non-thermal technologies,
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including high hydrostatic pressure, ultrasound, and others. So, it is necessary to regard the
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inactivation mechanisms of the spores induced by thermal and non-thermal technologies on
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further studies. In any case, the Chinese traditional food industry should continue to innovate
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and keep pace with manufacturing advances.
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Formatting of Funding Sources
This study was supported by the National Key Research and Development Program of China [grant number 2016YFD0400301].
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Conflict of interest None
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Figures
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Fig. 1. Survival of B. cereus at 60-80 °C in a) Chinese rice wine, simulated media of b)
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Glucose, c) Alcohol, d) pH, e) Protein and f) Sterile saline. Data were mean ± standard
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deviation, and were fit to first-order kinetic models, R2 of first-order kinetic models also
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showed in the Figure. 19
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(a)
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(c)
(d)
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(e)
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Fig. 2. Transmission electron micrographs of (a) untreated B. cereus, and B. cereus heated for
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1 min at 80 °C in (b) Chinese rice wine and simulated media of (c) Glucose, (d) Alcohol, (e)
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Protein, and (f) pH 4.14.
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Tables
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Table 1.
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Characteristics and composition of each media (“+” means with that component, while “-” means without that component) Glucose
+
Acid
+
-
7.0
-
+
7.0
-
-
-
Acid
-
-
-
7.0
+
-
-
7.0
-
+
Alcohol
-
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Glucose
Protein
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Control (Sterile saline)
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Protein
+
pH
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pH
Chinese rice wine
Alcohol
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Table 2.
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D-values (min) for B. cereus heated at 60.0-80.0 °C in Chinese rice wine and simulated media based on wine characteristics pH
60.0
0.56 ± 0.05d, e, *
0.82 ± 0.04g
0.53 ± 0.06d, e, f
70.0
0.40 ± 0.10a, b, c
0.72 ± 0.04f, g
0.37 ± 0.04a, b
80.0
0.31 ± 0.08a
0.55 ± 0.02c, d, e
0.36 ± 0.03a, b
Protein
Sterile saline
1.13 ± 0.01h
0.76 ± 0.08f, g
0.32 ± 0.06a, b
0.66 ± 0.05e, f
0.54 ± 0.02c, d, e
0.28 ± 0.02a
0.47 ± 0.06b, c, d
0.37 ± 0.03a, b
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Alcohol
0.61 ± 0.02d, e, f
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*Values are the mean of triplicate measurements ± standard deviation; The different letters (a-h) indicate significant differences (P < 0.05).
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Chinese rice wine
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Temp (°C)
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