Food Microbiology Changes in the Microbial Communities in Vacuum
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Food Microbiology 77 (2019) 26–37 Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Changes in the microbial communities in vacuum-packaged smoked bacon during storage T ∗ Xinfu Lia,b,d, Cong Lia,b,d, Hua Yea,b, Zhouping Wanga,b, Xiang Wud, Yanqing Hand, Baocai Xub,c,d, a State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China b School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China c School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China d State Key Laboratory of Meat Processing and Quality Control, Yurun Group, Nanjing, 211806, China ARTICLE INFO ABSTRACT Keywords: This study aimed to gain deeper insights into the microbiota composition and population dynamics, monitor the Microbial communities dominant bacterial populations and identify the specific spoilage microorganisms (SSOs) of vacuum-packed Smoked bacon bacon during refrigerated storage using both culture-independent and dependent methods. High-throughout High-throughput sequencing (HTS) sequencing (HTS) showed that the microbial composition changed greatly with the prolongation of storage time. The diversity of microbiota was abundant at the initial stage then experienced a continuous decrease. Lactic acid bacteria (LAB) mainly Leuconostoc and Lactobacillus dominated the microbial population after seven days of storage. A total of 26 isolates were identified from different growth media using traditional cultivation isolation and identification method. Leuconostoc mesenteroides and Leuconostoc carnosum were the most prevalent species since day 15, while Lactobacillus sakei and Lactobacillus curvatus were only found on day 45, suggesting that they could be responsible for the spoilage of bacon. Serratia, Rahnella, Fusobacterium and Lactococcus underwent a dramatic increase at some point in individual batchs which may be considered as potential contributors to the spoilage. 1. Introduction physicochemical attributes (such as flavour, colour, appearance, TVB- N, pH and fatty acid composition) and palatability (such as saltiness Food spoilage is a great economical problem which is caused by the and crispiness) (Person et al., 2005; Soladoye et al., 2015). TVB-N, pH- growth and metabolism of specific microorganisms (Huis in't Veld, value and colour changes have been suggested as chemical indicators of 1996). Packaged meat products of animal’ origin are susceptible to microbial spoilage in meat and meat products (Borch et al., 1996; natural contamination caused by various bacteria (Borch et al., 1996; Huang et al., 2014; Metaxopoulos et al., 2002). In recent years, mi- Casaburi et al., 2015; Chaillou et al., 2015; Korkeala and Björkroth, crobial spoilage has become a major concern for the bacon industry in 1997; Samelis et al., 2000a). The shelf-life of packed meat products China. It is necessary to investigate the dynamics of contaminating ranging from several days to several weeks. Bacon is a cured meat bacteria and predominant bacterium species during storage. product produced mainly from pork belly which is widely consumed Culture-dependent traditional microbial cultivation and character- around the world (Soladoye et al., 2015). The consumption of cooked ization methods have been extensively used to identify the micro- meat products especially smoked bacon is steadily increasing in China. organisms in meat and meat products (Barakat et al., 2000; Björkroth The higher moisture content, lower salt, near-neutral pH and avail- et al., 1998; Metaxopoulos et al., 2002; Samelis et al., 2000a). A great ability of nutrients in most modern bacon (Hughes, 1988; Sheard, 2010; diversity of bacteria can be found in different types of cooked meat Sheard et al., 2001) facilitated microbial growth, which led to the products include Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus spoilage. The spoilage can be considered the result of microbial activity alimentarius, Weissella viridescens, leuconostocs, Enterobacteriaceae, Bro- of a wide variety of microorganisms, indicated by off-flavors, off-odors, chothrix thermosphacta, yeasts and moulds (Borch et al., 1996; Cayré discoloration, slime, decrease in pH or unacceptable to the consumer et al., 2005; Korkeala and Björkroth, 1997; Samelis et al., 2000a, before the sell-by date (Borch et al., 1996; Gram et al., 2002; Nychas 2000b). However, only 0.1–3% bacteria can be cultivated which are et al., 2008). The bacon quality, including microbial quality, unable to represent the whole microbiota (Amann et al., 1995; Cocolin ∗ Corresponding author. School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China. E-mail address: [email protected] (B. Xu). https://doi.org/10.1016/j.fm.2018.08.007 Received 1 March 2018; Received in revised form 15 August 2018; Accepted 16 August 2018 Available online 17 August 2018 0740-0020/ © 2018 Elsevier Ltd. All rights reserved. X. Li et al. Food Microbiology 77 (2019) 26–37 et al., 2013). Now, one of the main challenges for microbiologists is to spoilage biota at different storage periods, five batches were prepared develop strategies to understand the microbial physiology and its at different times and numbered 1 to 5. Samples were quickly trans- control. Although the culture-dependent method has some limitations, ported to the laboratory with Drikold and then stored at 0–4 °C. The it is still an increasing interest due to its versatility (Alain and fresh smoked bacon (0 day) were analysed at vacuum-packaged and Querellou, 2009). then at 7, 15, 22, 30, and 45 days during storage. Samples of each batch Culture-independent methods are more advanced and reliable when were refrigerated and stored for testing in triplicate at each time point. analyzing microbial communities. Polymerase Chain Reaction (PCR) cloning and the sequencing of 16S ribosomal RNA gene (rRNA) are 2.2. Physicochemical analysis frequently applied as the standard methods for the cultivation-in- dependent survey (Klindworth et al., 2013). After decades of develop- 2.2.1. pH ment, fingerprinting molecular methods and PCR-denaturing gradient Sample (10 g) was harvested from the triplicates samples which gel electrophoresis (PCR-DGGE) have been frequently employed to were minced and mixed at each sampling point. They were then study the microbiota changes within the shelf-lives of meat and meat homogenised in 100 mL distilled water with KCl (0.01 mol/L) for 1 min products (Cocolin et al., 2013); however, small fragment size, low re- in a stomacher with filter net (Pro-media, SH-IIM; Elmex Ltd, Tokyo, solution power, background disturbance and difficulties in quantitative Japan; stomacher bags: PX0020, Elmex, Ltd., Japan). Impurities were analysis hindered the use of PCR-DGGE (Polka et al., 2015). In com- filtered by the filter net. These steps were conducted multiple times for parison, HTS offers a more advanced method which assists scientists’ all samples. The pH values were recorded using a pH meter (S210 assessment and understanding of microbial communities, and it pro- SevenCompact™ pH meter, Mettler Toledo). vides a comprehensive description of the complex interactions among the species (Polka et al., 2015). This is because it can generate far more 2.2.2. Instrumentally measured colour reads than traditional culture-independent methods and facilitate the Lightness (L*), redness (a*), and yellowness (b*) were used as in- discovery of more microbiota diversity as well as the detection of dicators for surface colour of samples. The indicators were measured by bacteria involved in high or low quantities of unknown origin (Ercolini, a Chroma meter CR-400 (Konica Minolta Sensing Inc, Osaka, Japan), 2013). HTS is more efficient for monitoring the evolution of microbiota with an 8 mm diameter measuring area, a D65 illuminant and 10° of meat and meat products within their shelf-lives. As evidence, 454 standard observer, expressed in CIE Lab colour space (two decimal pyrosequencing had been successfully applied in Zhenjiang Yao meat places). Five spots randomly selected for colour measurement were during refrigerated storage by Xiao et al. (2013). Although the entire measured on individual samples (L*, a*, b*). microbial diversity can be revealed by HTS, identification of dominant spoilage species cannot be obtained. Concerning the dominant species 2.2.3. Total volatile base nitrogen analysis (TVB-N) from spoilage microorganisms by more scientists, the purpose to further The TVB-N content in bacon was measured using a steam distillation study spoilage potential and exploit controlling methods species of method, in accordance with Chinese Standard GB 5009.228–2016. spoilage microorganisms. Compared to HTS, the traditional approach Samples (10 g) were ground individually by a processor (JYL-C012, provides scientists a better method for isolation and identification of the Jiuyang, China). Steam distillation was carried out using a Kjeldahl specific species inducing meat spoilage (Casaburi et al., 2011). The distillation unit (KDN-103F, Shanghai Xian Jian., China), and TVB-N specific species or strains can be obtained by culture-dependent results were calculated according to the consumption of hydrochloric methods (microbial cultivation) for the in-depth study of physiology, acid (0.01 mol/L). genetics and measures of industrial application (Alain and Querellou, 2009; Prakash et al., 2013). Culture-independent must be improved by 2.3. Culture-dependent methods accompanying developments