Food Microbiology 77 (2019) 166–172 Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Comparative evaluation of spoilage-related bacterial diversity and metabolite profiles in chilled beef stored under air and vacuum packaging T Ahmad Rois Mansura,d,1, Eun-Ji Songb,d,1, Yong-Sun Choa, Young-Do Namb,d, Yun-Sang Choic, ∗ ∗∗ Dae-Ok Kime, Dong-Ho Seob, , Tae Gyu Nama, a Food Analysis Center, Korea Food Research Institute, Wanju, 55365, Republic of Korea b Research Group of Healthcare, Korea Food Research Institute, Wanju, 55365, Republic of Korea c Food Processing Research Center, Korea Food Research Institute, Wanju, 55365, Republic of Korea d Department of Food Biotechnology, Korea University of Science and Technology, Daejeon, 34113, Republic of Korea e Department of Food Science and Biotechnology, Kyung Hee University, Yongin, 17104, Republic of Korea ARTICLE INFO ABSTRACT Keywords: Microbial spoilage is a complex event to which different bacterial populations and metabolites can contribute Beef spoilage depending on the storage conditions. This study explored the evolution of spoilage and related volatile organic Bacterial communities compounds (VOCs) in chilled beef under air and vacuum packaging (VP). The results suggested that different Bacterial metabolites storage conditions affected changes in bacterial communities and metabolites in beef and consequently affected Volatile compounds the odor properties of the stored beef, thereby leading to spoilage. Bacterial species belonging to Pseudomonadaceae (Pseudomonas spp.) and lactic acid bacteria (Lactobacillus sp.) dominated the bacterial com- munities in beef stored under air and VP, respectively, with several VOCs associated with off-odors of the stored beef and most likely produced by both bacteria. Our results suggested several microbial VOCs that could be used as potential spoilage indicators, including acetic acid, butanoic acid, and 2-butanone in VP-stored beef and 3- methylbutan-1-ol, ethyl acetate, acetoin, 2-butanone, and diacetyl in air-stored beef. These findings might provide valuable information regarding the quality monitoring of beef during storage. 1. Introduction unacceptable levels, with the spoilage potential of SSOs depending on their ability to produce metabolites (Doulgeraki et al., 2012). SSOs Raw meat is a perishable product and can be considered un- represent a fraction of microorganisms that are favored by storage acceptable for human consumption when it spoils due to chemical and conditions (e.g., temperature and atmosphere) and can dominate other biological changes that alter its sensorial properties. It is well estab- microorganisms, reach high populations, and produce several metabo- lished that, in addition to lipid oxidation and autolytic enzymatic re- lites, thereby leading to the sensory rejection of the product (Wang actions, meat spoilage is mainly the result of decomposition and the et al., 2016). Understanding the dynamic changes in spoilage-related formation of metabolites resulting from microbial growth and enzy- microbial communities and metabolites, as well as their impact on matic activity (Casaburi et al., 2015). Therefore, preservation followed sensory quality, is a key factor for the development of a novel technique by adequate quality evaluation techniques is necessary to ensure meat for meat-spoilage evaluation. quality and shelf-life. Various conventional evaluation techniques, including micro- Hurdle techniques of refrigeration and vacuum packaging (VP) have biological [e.g., total viable counts of aerobic microorganisms (TVC)], been widely applied for the preservation of primal cuts of red meats, chemical [e.g., total volatile basic nitrogen (TVB-N)], pH, and sensorial such as beef (Chen et al., 2012). Temperature and packaging system (e.g., flavor/odor and color) assays have been broadly applied for meat- represent the most important extrinsic factors that determine the de- quality evaluation (Coombs et al., 2017; Wang et al., 2016). The recent velopment of spoilage-related microbial communities in stored meats. development of next-generation sequencing technology in food studies Spoilage usually occurs when specific spoilage organisms (SSO) grow to has enabled researchers to study and understand microbial-community ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (D.-H. Seo), [email protected] (T.G. Nam). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.fm.2018.09.006 Received 23 April 2018; Received in revised form 15 August 2018; Accepted 5 September 2018 Available online 06 September 2018 0740-0020/ © 2018 Elsevier Ltd. All rights reserved. A.R. Mansur et al. Food Microbiology 77 (2019) 166–172 diversity and dynamics during storage, as well as identify core spoilage was determined using a five-point scale [1 = severe off-odor (spoiled), microorganisms (Cao et al., 2017). Recently, volatile organic com- 2 = strong off-odor (spoiled), 3 = moderate off-odor (satisfactory), pounds (VOCs) have attracted increasing attention because of their 4 = slight off-odor, and 5 = no off-odor)]. The odor score of fresh possible impact on meat quality (Casaburi et al., 2015). Headspace samples before storage was determined at 5, and the stored sample was solid-phase microextraction (HS-SPME) coupled with gas chromato- considered spoiled when the median of the scale given was < 3. graphy mass spectrometry (GC-MS) has been employed to determine VOC levels to evaluate beef spoilage (Argyri et al., 2015; Jääskeläinen et al., 2016). A reliable compound for spoilage assessment should show 2.4. Volatile organic compounds (VOC) analysis good correlation with microbial growth, sensory score, and remaining shelf-life (Parlapani et al., 2015); therefore, establishing a new com- A sample (2 g from 25 g of beef) was weighted and placed into a prehensive study regarding relationships between VOC production, SSO 20 mL glass vial and sealed with a polytetrafluoroethylene/silicone growth dynamics, and sensorial and chemical changes is required in septum (Supelco; Sigma-Aldrich, St. Louis, MO, USA). The sealed vial order to identify reliable VOCs for potential application as beef-spoilage containing the sample was heated at 40 °C for 15 min (equilibrium indicators. Additionally, the use of high-resolution MS, such as time-of- time) and extracted at headspace for 15 min using a carboxen/poly- flight MS (TOF/MS), has not previously been employed for VOC de- dimethylsiloxane (Supelco; Sigma-Aldrich). The fiber was then ther- termination for the afore-mentioned purpose. mally desorbed into the GS-injection system equipped with a TOF/MS This study investigated the evolution of the spoilage of raw beef cuts (LECO Corp., St. Joseph, MO, USA). GC-TOF/MS conditions were the stored under air and VP at 4 °C through determination of VOCs using same as those described in our previous study (Mansur et al., 2017). HS-SPME and GC-TOF/MS along with its correlation with sensory The raw chromatograms were processed using ChromaTOF software (odor) changes, as well as correlations between VOCs and microbial (v.4.22; LECO Corp.) and extracted with a signal-to-noise ratio of 20. community in order to obtain reliable microbial VOC metabolites po- The peaks were identified tentatively by comparing the obtained mass tentially useful for evaluating beef spoilage. Conventional techniques, spectra with those available in the Wiley and NIST databases and using including TVC, TVB-N, and pH analyses, were also employed for quality a minimum similarity match of 900. Peak area was calculated using the monitoring. Our findings provide valuable information regarding the unique mass of each detected volatile compound. All of the peak in- potential use of several microbial VOC metabolites to monitor the formation from different chromatograms was aligned using the quality of raw beef cuts during storage. Statistical Compare (SC) option in the ChromaTOF software in order to obtain an accurate comparison between the volatile profiles present in 2. Materials and methods the beef samples. Results of the SC process were exported as .csv files and used in statistical analyses. The relative amounts of detected VOCs 2.1. Sampling and storage of beef cuts were expressed in arbitrary units using 3,3-dimethyl-2-butanol as the internal standard. Fresh beef tenderloin cuts were purchased from a local slaughter- house (Gyeonggi, Korea), and beef samples were transferred to the la- boratory in an ice box within 2 h. In the laboratory, the beef samples 2.5. Microbial-community analysis used for experimental analyses were stored under air and VP at 4 °C for 11 and 21 days, respectively. For aerobic storage, samples were placed DNA in sample (2 g from 25 g of beef) was extracted using a QIAamp in permeable polyethylene bags (200 × 300 mm). For VP storage, DNA stool mini kit (QIAGEN, Hilden, Germany), and concentration and samples were packed and sealed in polyethylene/polyamide bags purity were determined using a NanoDrop ND-1000 (NanoDrop 3 2 (200 × 300 mm) with an O2-transmission rate of 40 cm /m /day at Technologies Inc., Wilmington, DE, USA). The V1-V2 region of 16S 85% relative humidity and 23 °C. Each analysis, storage condition, and rRNA genes was amplified by polymerase chain reaction using a range sampling time was performed using triplicate samples (25 g each). of universal primers (8F and 338R) with barcode sequences for multi- plexing reads of each sample and a recombinant Taq polymerase 2.2. Microbiological