Modified atmosphere packaging decreased Pseudomonas fragi cell metabolism and extracellular proteolytic activities on meat

Accepted Manuscript Modified atmosphere packaging decreased Pseudomonas fragi cell metabolism and extracellular proteolytic activities on meat

Guangyu Wang, Fang Ma, Leyin Zeng, Yun Bai, Huhu Wang, Xinglian Xu, Guanghong Zhou PII:

S0740-0020(17)31166-8

DOI:

10.1016/j.fm.2018.07.007

Reference:

YFMIC 3048

To appear in:

Food Microbiology

Received Date:

12 December 2017

Accepted Date:

16 July 2018

Please cite this article as: Guangyu Wang, Fang Ma, Leyin Zeng, Yun Bai, Huhu Wang, Xinglian Xu, Guanghong Zhou, Modified atmosphere packaging decreased Pseudomonas fragi cell metabolism and extracellular proteolytic activities on meat, Food Microbiology (2018), doi: 10.1016 /j.fm.2018.07.007

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Modified atmosphere packaging decreased Pseudomonas fragi cell

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metabolism and extracellular proteolytic activities on meat

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Guangyu Wanga, Fang Mab, Leyin Zengc, Yun Baic, Huhu Wang a, #, Xinglian Xua,

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Guanghong Zhoua

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a Key Laboratory of Meat Processing and Quality Control, Nanjing Agricultural

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University, Nanjing, Jiangsu, China

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b College of Veterinary Medicine, Nanjing Agriculture University, Nanjing, Jiangsu

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210095, P. R. China

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c Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality

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and Safety Control

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Running title: MAP inhibited P. fragi cell metabolism

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#Corresponding author: Huhu Wang

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Mailing address: Weigang No.1, Nanjing Agricultural University, Nanjing, Jiangsu,

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China. E-mail address: [email protected], Tel.: +86 25 84396457, Fax: +86 25

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84395730

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Abstract

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Modified atmosphere packaging (MAP) is considered an effective method for

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extending the shelf life of meat. The use of optimal mixture of gases (CO2 and N2) in

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food packaging containers has been proved to effectively inhibit the growth of

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microorganisms in poultry meat. In general, a minimum CO2 concentration range of

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20%-30% is required for the inhibitory effect. The aim of this study was to investigate

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the mechanism by which MAP (CO2/N2 30%/70%) inhibits Pseudomonas fragi, a

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dominant spoilage microorganism in aerobically stored chilled meat. The cell

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physiological changes were determined by measuring membrane integrity, membrane

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potential, ATP level, and extracellular proteolytic activity. The results showed that

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samples stored under MA retained cell membrane integrity, but lost significant

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membrane potential and ATP synthesis activity. Furthermore, the peptides issued from

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2 structural proteins (myosin and actin) were mainly identified in air samples, indicating

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that these fragments result from bacterial proteolytic activity while MAP inhibited this

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activity. Overall, the study found that cell metabolism and extracellular protease

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activity decreased under MAP conditions. This study showed that MAP is an effective

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food preservation strategy and revealed mechanisms by which MAP inhibits spoilage.

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Keywords: Modified atmosphere packing; Pseudomonas fragi; Spoilage inhibition;

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Cell viability

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1. Introduction

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Meat spoilage through physical damage or resulting from bacterial growth and

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metabolic activities is a major concern of the food industry. Apart from lipid oxidation

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and endogenous enzyme reactions, spoilage is mostly ascribable to undesired microbial

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activity (Casaburi et al., 2015). Despite the diversity of bacteria found in meat,

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Pseudomonas spp. are considered as main spoilers of raw meat that has been stored

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under aerobic refrigerated conditions. Pseudomonas fragi is the most frequently found

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species, with an incidence between 56.7% and 79.0% on spoiled meat (Mohareb et al.,

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2015; Nychas et al., 2008). P. fragi is recognized as one of the principal agents of meat

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spoilage. This psychrotrophic species is favored by the chill chain applied to fresh meat

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products, from slaughter houses to markets.

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Modified atmosphere packing (MAP) has been reported to prolong the shelf life

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of chilled meat (Al-Nehlawi et al., 2013; Meredith et al., 2014). Shelf life is largely

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affected by the number of bacterial contaminants. However, decreased quality of meat

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during storage depends not only on the quantities of bacteria, but also on the activities

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of bacterial metabolism. For example, proteases generate free amino acids which can

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be further metabolized by the bacteria, resulting in the off-flavors, off-odors, and slime

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associated with spoilage. The application of MAP can have an inhibiting effect on

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Pseudomonas species. Previous studies have demonstrated that gas mixture with 30%

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CO2 and 70% N2 used for MAP can prolong the shelf life of chilled chicken meat. The

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samples stored under such MAP had a lower count of Pseudomonas spp. than the air

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packed samples by 0.8-2.39 log CFU/g throughout the storage period (Zhang et al., 3

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2015). Although significant growth can still be observed in MAP samples, the activities

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of Pseudomonas spp. that lead to spoilage are lower in meat stored in MAP compared

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to that stored in air (Remenant et al., 2015). Therefore, MAP may affect bacterial

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growth and also metabolism.

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Recent investigations have focused on the effect of food preservation strategies,

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such as the use of essential oils, bacteriocin or organic acids, on bacterial viability and

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metabolism (Liu et al., 2015; Wang et al., 2015; Zhang et al., 2016). These strategies

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appear to share a common mechanism of action against spoilage bacteria. Specifically,

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they act on the cytoplasmic membrane, resulting in the release of intracellular

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substances and the dissipation of the proton motive force. Previous studies have

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demonstrated that MAP can affect bacterial surface properties and synthesis of

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Extracellular Polymeric Substances (Wang, Li, et al., 2017; Wang, Ma, et al., 2017).

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While MAP is known to inhibit spoilage activity of Pseudomonas spp., it is unknown

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how MAP affects Pseudomonas spp. viability and metabolism and what mechanisms

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underlie spoilage inhibition by MAP.

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The aim of the present study was to investigate the mechanisms by which MAP

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inhibits spoilage of meat by P. fragi. Changes in cell physiology were examined in the

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late exponential phase of bacterial growth after packing. Understanding how MAP

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inhibits spoilage potential of this species is key to develop the most effective packaging

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technologies that will safely extend the shelf life of fresh meat.

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2. Materials and methods

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2.1 Bacteria isolates and culture conditions 4

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P. fragi NMC25, a strain with strong spoilage potential, was previously isolated

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and identified from spoiled chilled chicken (Wang, Li, et al., 2017; Wang, Wang, et al.,

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2017). The isolate was streaked onto tryptone soy agar (TSA) from a frozen (−80 °C)

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glycerol stock, a single colony of bacteria was then transferred into tryptone soy broth

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(TSB) for 24 h at 28 °C. Stationary phase inoculum was prepared prior to use.

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For this work, the isolate was incubated in the following two atmospheres:

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modified atmosphere and air. The MAP used in this study was packaged by a SMART

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500 (ULMA Packaging, Barrio Garibai, Spain) under a modified atmosphere

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containing 70% N2 and 30% CO2 using Lid 1050 MAP films (Cryovac, Sealed Air Co.,

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Ltd, Shanghai, China). The film has an oxygen permeability of 6 cm3 m−2.day−1.atm−1

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at 0% RH/4 °C and a water vapor permeability of 0.1 g m−2.day−1 at 100% RH/4 °C.

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All samples were stored at 15 °C.

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2.2 RJA assay

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A meat-based raw-chicken juice agar (RJA) medium was prepared as described in

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previous publication (Wang, Wang, et al., 2017). RJA provides only the nutrients

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available in chicken as the energy source and was used to evaluate the spoilage potential

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of the bacteria. The isolate suspension was adjusted to a final concentration of 5 log

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CFU/mL. Four replicate 2-μL aliquots of the isolate were spotted onto RJA and

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incubated under MAP and air. The total viable counts (TVC) of the bacteria colony,

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decomposition zone diameter (DZD), and bacteria colony diameter (BCD) were

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measured for 7 days. At each sampling date, The DZD and BCD were measured with a

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precision caliper to the nearest 0.01 mm. The agar containing bacteria colony were 5

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transferred aseptically to a stomacher bag and homogenized with 0.9% NaCl solution.

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The mixture was transferred from the stomacher bag for additional serial-dilution steps

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according to the National food safety standard-Food microbiological examination:

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Aerobic plate count (GB 4789.2-2010). Aliquots of the appropriate dilutions (0.1 mL

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each) were spread on TSA in duplicate, and the agar plates were incubated aerobically

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at 28 °C for 24 h before counting.

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Differences in spoilage potential between the MAP and air group were determined

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using this preliminary step. Cell physiological changes were examined further in vitro

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and in meat.

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2.3 Inoculation of NMC25 in vitro and in meat

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Meat samples (10 g) were sliced from chilled chicken breasts and sterilized via

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irradiation at a dose of 6 KGy (Wang, Wang, et al., 2017). A stationary phase culture

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of P. fragi NMC25 was inoculated in TSB and on the surface of meat pieces at a

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concentration of 3 log CFU/mL (g). All the samples were packaged and stored as

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described above. Growth conditions were determined by a pre-experiment assessment.

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Briefly, sampling was performed 12 h apart. Meat samples were transferred aseptically

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to a stomacher bag and homogenized with 90 mL 0.9% NaCl solution. The viable

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counts of the mixture were performed by the standard method as described above.

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According to the pre-experiment assessment, late exponential phase cultures were

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harvested and used immediately for further characterization (Table 1). For the

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collection of strains grown on meat, the dispersed biomass on each meat sample was

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gently harvested using a polypropylene inoculating loop and suspended in 0.9% NaCl 6

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solution.

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2.4 Measurement of cell membrane integrity

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The cell membrane structural integrity was analyzed using the Live/Dead

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BacLight bacterial viability kit (Invitrogen, Carlsbad, CA, USA), according to the

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manufacturer’s instructions. One milliliter-sized late exponential phase cultures from

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TSB or meat were washed by 0.9% NaCl solution prior to staining. Ethyl alcohol -

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treated cells were used as dead control (75% v/v, 60 min). A 3-μL volume of an equal

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proportion of SYTO 9 and PI mixture was added to the sample and incubated in the

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dark for 15min at room temperature. After centrifugation and resuspension, the cells

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were examined under an Accuri C6 flow cytometer (BD, Franklin Lakes, NJ, USA).

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The flow cytometer was fitted with a 488-nm excitation laser. SYTO 9 fluorescence

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was measured through a 525-nm band-pass filter (FL1), and PI fluorescence was

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measured through a 620-nm band-pass filter (FL3).

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2.5 Measurement of the membrane potential

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The membrane potential was examined using a BacLight bacterial membrane

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potential kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s

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instructions. One milliliter late-exponential phase cultures from TSB and meat were

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diluted in sterilized PBS to 106- 107 CFU/mL. CCCP was used as a negative control as

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it eliminates the proton gradient. Before adding DiOC2(3) to samples, 1mM EDTA was

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added to facilitate dye uptake by bacteria cells. The samples were incubated for 30 min

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at 37 °C. Then the stained bacteria were analyzed on Accuri C6 flow cytometer.

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Fluorescence is collected in the green and red channels. The membrane potential was 7

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calculated as the ratio of red and green fluorescence intensity.

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2.6 Quantification of intracellular ATP

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Intracellular ATP levels were quantified using the BacTiter-Glo Microbial Cell

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Viability Assay Kit (Promega, Madison, WI, USA), according to the manufacturer’s

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instructions. Aliquots of 100 μL of bacteria were mixed with an equal volume of the

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BacTiter-Glo reagent into 96-well white plates and incubated for 5 min at room

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temperature. The emitted luminescence was detected by a TECAN infinite M200 plate

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reader and was expressed as relative luminescence units (RLU). The relative ATP

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concentration was calculated as the ratio of RLU to TVC.

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2.7 Bacterial extracellular proteolytic assay

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A gel-based liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)

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approach, using protein prefractionation by SDS-PAGE and in-gel digestion (IGD),

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followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS), was

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used for the identification of peptides degraded by the bacterial extracellular proteases.

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In order to collect the extracellular proteases from bacteria, rather than endogenous

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proteolytic enzymes (e.g. cathepsins) from meat tissues, the contaminated meat was

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shaken in a stomacher bag at a constant shaking speed of 120 rpm for 30s with 90 mL

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of sterilized PBS.

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2.7.1 SDS-PAGE and IGD

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The stationary phase cultures (1 mL TSB or 1 mL PBS mentioned above) were

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centrifuged at 10,000 g for 5 min. The supernatant was used as the microbial extract.

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Chicken sarcoplasmic and myofibrillar proteins were extracted as described by 8

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Mauriello et al. (2002). Briefly, meat sample was homogenized with phosphate buffer

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(0.02M, pH 6.5) and centrifuged at 13,000 g for 20 min at 4 °C. The supernatant was

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used as sarcoplasmic protein. The resulting pellet was then homogenized with

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phosphate buffer (0.03M, pH 7.4) containing 0.1% Triton X-100. After centrifugation

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at 13,000 g. for 20 min at 4 °C, the pellet was washed three times and resuspended in

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nine volumes of phosphate buffer (0.1M, pH 7.4) containing 0.7M KI. After

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homogenization, the suspension was centrifuged at 13,000 g for 20 min at 4 °C. The

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supernatant was used as myofibrillar protein. The protein extracts were filter sterilized

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(0.22 μm) and then adjusted to a concentration of 1 mg/mL. The microbial extracts (200

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μl) were incubated with sarcoplasmic and myofibrillar proteins (1 mL) for 1d at 15 °C.

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Next, SDS-PAGE was performed based on the method of Fadda et al. (1999).

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Before MS analysis, the proteins in 5 gel slices from every separated protein were

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digested as previously described (Piersma et al., 2013). Briefly, gel bands were

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destained with dd H2O and 50 mM NH4HCO3/50% acetonitrile, repeated wash-step

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until the color fades. Subsequently, gel bands were reduced (10 mM DTT) at 56 °C for

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60 min, alkylated (55 mM iodoacetamide) for 45 min protecting from light, wash (50

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mM NH4HCO3, followed by 50 mM NH4HCO3/50% acetonitrile and pure acetonitrile)

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and tryptic digested (trypsin) overnight at 37 °C. After digestion, peptides were

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extracted and sequentially rinsed with 50% acetonitrile, pure acetonitrile. Finally, the

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extracts were combined and desalted by Sep-Pak C18 cartridges (Waters, Milford, MA,

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USA). The desalted peptides were quantified by NanoDrop spectrophotometer before

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subjecting to NanoLC-MS/MS for further identification. 9

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2.7.2 NanoLC–MS/MS analysis and database searching

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Extracted peptides were identified using a nano liquid chromatography system

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(Ultimate 3000, Dionex, Sunnyvale, CA, USA) coupled with LTQ Orbitrap (Thermo,

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Bermen, Germany) mass spectrometry. For LC separation, an Acclaim PepMap 100

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capillary column (C18, 3 μm, 100 Å) (Dionex) with a 15 cm bed length was used with

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a flow rate of 300 nL/min. Two solvents, A (0.1% formic acid) and B (aqueous 90%

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acetonitrile in 0.1% formic acid), were used to elute the peptides from the nanocolumn.

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The gradient went from 5 to 40% B within 40 min and from 40 to 95% B within 5 min,

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with a total run time of 60 min. The mass spectrometer equipped with a

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nanoelectrospray ionization source was operated in the data-dependent mode so as to

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automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Full-scan

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MS spectra (from m/z 350 to 1800) were acquired in the Orbitrap with a resolution r =

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60000 at m/z 400, allowing the sequential isolation of the top 10 ions that were

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dependent upon signal intensities. The fragmentation on the linear ion trap was

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performed under collision-induced dissociation at 35% normalized collision energy.

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Protein identification was performed using the Proteome Discoverer software

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(version 1.4, Thermo Fisher, Waltham, MA, USA) against the Uniprot Gallus gallus

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database (2017. 02. 19 version, 29484 entries). Data matching was performed with a

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fragment ion mass tolerance of 0.02 Da and a parent ion tolerance of 10 ppm. Trypsin

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was used in tryptic peptides search. The mass value was monoisotopic. Oxidation (M)

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and carbamidomethyl (C) were chosen as variable and mixed modifications,

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respectively. Two missing cleavages were allowed. Protein mass was unrestricted. 10

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Database was set as Decoy. Peptide FDR was <0.01. Peptide identifications were

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accepted if they could be established at a PeptideProphet probability >99%

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2.8 Statistical Analysis

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All experiments were performed using 4 independent biological replicates. Tests

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for statistical significance were performed using the SPSS statistics program (Version

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22, USA). One-way analysis of variance (ANOVA) and Duncan's multiple range test

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were applied on data obtained from the RJA assay. A student's t-test was applied to the

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membrane potential and ATP concentration. In all cases, P < 0.05 was used to

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determine statistical significance.

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3 Results

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This study was conducted in two steps. In the first step, P. fragi NMC25,

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previously isolated from spoiled chicken, was cultured in RJA to monitor bacterial

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growth and decomposition zone under MAP and air throughout the storage period. In

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the second step, the biological responses of P. fragi under MAP were investigated by

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inoculating the isolate into either TSB or meat and characterizing cell viability and

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extracellular proteolytic activities.

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3.1 Preliminary characterization of spoilage potential under MAP

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The mean DZD and BCD on RJA were measured over time (Fig. 1a and c). In

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MAP group, no decomposition zone was observed, and the colony size did not

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significantly changed (P >0.05) over 5 days (Fig. 1a). After 7 days incubation, the BCD

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increased from 3.26 mm to 4.20 mm. However, in the air group, the decomposition

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zone and colony size significantly increased (P <0.05) over time. Notably, MAP 11

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inhibited the growth of NMC25, but when the two groups reached the same number of

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CFUs (e.g. after 3 days in air and 7 days in MAP), there was still no decomposition

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zone in the MAP group.

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3.2 Effect of MAP on cell viability

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Cell viability here was determined by the impermeability of the membrane to

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nucleic acid dyes, such as propidium iodide (PI), and the presence of metabolic activity,

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as indicated by the production of ATP or by maintenance of a membrane potential.

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To better understand the changes in membrane integrity between the two

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packaging treatments, the permeability of the bacterial membrane was characterized by

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staining with SYTO 9 and PI after incubation. The SYTO 9 green fluorescent stain

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labels all cells. In contrast, PI only penetrates bacteria with altered membrane

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permeability and its fluorescence enhanced upon binding nucleic acids. This causes a

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reduction in the SYTO 9 stain fluorescence signal when both dyes are present (Manteca

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et al., 2005). Air samples, whether in TSB or on meat, remained greater than 90% viable,

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whereas alcohol-treated cells were less than 0.1% viable (Fig. 2a, c and e). As measured

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by straining, the viability of the cells in MAP was similar to those cultured in air,

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indicating that MAP treatment did not affect the cell membrane integrity in either

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culture substrate (Fig. 2b and d). The percentage of viable cells in the MAP samples

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were a little bit higher than in the air samples.

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Another way to msesure cell viability is by straining cells with DiOC2(3), which

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emits a red fluorescence if it accumulates in cells with an intact electrical potential

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gradient (Spindler et al., 2011) but emits a green fluorescence to indicate membrane 12

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depolarization, as seen in samples treated with CCCP. In MAP samples, there was

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greater green fluorescence, resulting in a significantly decreased (P <0.05) fluorescent

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intensity ratio of red/green (Fig. 3a). This result suggested that MAP may lead to a

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decrease in membrane potential of NMC25 cells.

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The BacTiter-Glo microbial assay measures ATP as an indicator of metabolic

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activity (Fig. 3b). The cells in air samples had higher levels of ATP production when

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grown in either TSB or on meat (Fig. 3b, A). However, the MAP group displayed

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significantly lower (P <0.05) levels of ATP, suggesting that MAP inhibited ATP

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synthesis in the NMC25 cells. Amongst these three measures of cell viability, the overall patterns were similar

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whether the cells were grown in TSB or on meat.

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3.3 Proteolytic activity and GeLC-MS/MS assay

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The SDS-PAGE results are shown in Fig. 4. Comparison of the air samples, MAP

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samples and control revealed no significant differences in the sarcoplasmic protein

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bands. However, changes could be observed for the myofibrillar proteins incubated

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with air samples: three bands with molecular weights between 170 kDa to 100kDa, two

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bands between 70 kDa to 50kDa, one band of approximately 40 kDa, one band of

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approximately 30 kDa, and one band of approximately 15 kDa. In myofibrillar proteins

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incubated with MAP samples, the microbial extract showed no proteolytic activity,

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similar to the control. The overall proteolytic patterns in meat group were stronger than

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those in TSB. Peptides from eight bands (Fig. 4, a-h) can be matched against the Gallus gallus

283 13

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database. The Proteome Discoverer software can simulate the digestion of protein with

285

trypsin and get theoretical peptides with the carbon-terminal cleavage sites as Lys and

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Arg. The sequence of the peptides in each band obtained from LC-MS/MS were

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matched with these theoretical peptides. As a result, a total of 13 proteins were

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identified from the 8 excised band fractions, as list in Table 2. These 8 bands are

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fragments derived from 13 proteins, mainly myosin and actin, suggesting that both

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myosin and actin are degraded during P. fragi NMC25 spoilage. The myosin heavy

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chain was represented by 12 peptides that were present in several bands, while actin

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was identified by 8 common peptides in three digestion bands, this indicates that the

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bacterial proteases may work on specific cleavage sites of these two proteins (Table 3).

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4 Discussion

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In the present study, the physiological characteristics of P. fragi NMC25 were

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evaluated during growth under MAP and air both in vitro and in meat. While MAP has

297

been shown to be an effective method to prolong the shelf life and inhibit spoilage, the

298

available studies on MAP are based on viable cell counts or focus on the occurrence

299

and dynamics of some particular species (Argyri et al., 2015; Jaffres et al., 2009;

300

Parlapani et al., 2014). There is a complete lack of information on the mechanisms by

301

which MAP inhibits the spoilage bacteria. It is well known that growth of spoilage

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bacteria is not the only factor affecting the spoilage process, and that bacterial viabilities

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and metabolites (e.g. extracellular enzyme, pigment) are also very important.

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In the first assay in this study, at a later stage of incubation, MAP inhibited both

305

the degradation of chicken meat component and cell motility, despite the high bacterial 14

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counts. Bacterial motility and extracellular enzyme activity ensure that the bacteria can

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easily obtain nutrients from the food source, and when that food source is meat, these

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activities lead to spoilage. The spoilage inhibition by MAP may be related to changes

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in these physiological characteristics.

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From these results, the effects of MAP on cell viability and extracellular

311

proteolytic activity were assessed. Changes in membrane integrity showed that MAP

312

did not impair the membrane structure. Other antimicrobials used for food preservation

313

(e.g. essential oils, bacteriocin, organic acids, and electrolyzed water) generally

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increased cell membrane penetrability and caused leakage of the cytoplasm, thus

315

leading to cell death (Liu et al., 2017; Liu et al., 2015; Zeng et al., 2010). This suggests

316

that the modes of action of MAP and these antimicrobials are not the same and that the

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overall responses can be referred to as bacteriostatic and bactericidal, respectively.

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Membrane potential reflects active and passive transport mechanisms across the

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membrane that create an electrochemical gradient (Caron and Badley, 1995). Cells

320

typically maintain a transmembrane proton motive force, constituted of a membrane

321

potential and a pH gradient, that supports energy generation and transport of various

322

compounds into the cytoplasm (Spindler et al., 2011). The results suggested that MAP

323

may cause partial depolarization of the cytoplasmic membrane. Previous studies have

324

demonstrated that destruction of membrane integrity may lead to a loss of membrane

325

potential and that membrane depolarization is often associated with bacterial death

326

(Guo et al., 2017; Qian et al., 2012). However, in this work, MAP resulted in the partial

327

depolarization of the membrane yet the membrane remained intact. Notably, the 15

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lipophilic nature of CO2 allows it to pass through membranes and concentrate inside

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the cell, lowering intracellular pH (Hotchkiss et al., 2006). The dissipation of membrane

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potential may lead to a decrease in proton motive force, and thus interruption of

331

transmembrane transport and ATP synthesis. ATP is an indicator of cell metabolic

332

activity. We determined that ATP decreased significantly in MAP samples. It is known

333

that across the membrane, the electrochemical gradient of protons is essential for

334

bacteria to maintain the synthesis of ATP and the transportation across the membrane

335

(Xu et al., 2008). A dramatic loss of membrane potential reduces the ability of a cell to

336

synthesize ATP, and endogenous ATPases would destroy the remaining ATP. If ATP

337

synthesis were inhibited, then one would expect to observe a decrease in cell

338

metabolism, and thus a decrease in the production of extracellular enzymes. Here we

339

showed that a partial collapse of the membrane potential led to a reduction in the ATP

340

level, which is in agreement with previous studies (Glasser et al., 2014; Rao et al., 2008).

341

It is generally known that Pseudomonas spp. are obligate aerobes and that

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anaerobic conditions inhibit their aerobic respiration. Bacterial responses to MAP and

343

to antibiotics are different. Exposure to antibiotics likely stimulates cell respiration and

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consequently accelerates endogenous formation of reactive oxygen species (ROS),

345

resulting in cell death (Hassett and Imlay, 2007; Mieszkin et al., 2017). However, MAP

346

inhibits respiration and therefore does not directly induce cell death. In addition, the

347

CO2 in MAP can dissolved in the medium and food. Previous studies established that

348

CO2 has a direct effect on the membrane fluidity, enzymatic processes within cells, and

349

metabolic processes such as decarboxylation reactions and DNA replication (Dixon and 16

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Kell, 1989; Hong and Pyun, 2001; Sears and Eisenberg, 1961; Stretton and Goodman,

351

1998). Thus, the dissolution of CO2 may be a major contributor to the inhibition effect

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of MAP to P. fragi spoilage. MAP treatment decreased the bacterial membrane potential and caused a

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metabolic imbalance, possibly due to the inhibition of respiratory function.

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Deterioration of the sensory properties of meat results first from the degradation

356

of principal components, such as proteins and lipids. Bacterial proteases play important

357

roles in the hydrolysis of meat proteins, which results in the release of peptides and

358

amino acids (Cocconcelli and Fontana, 2010; Pisacane et al., 2015). The degraded

359

peptides identified in air samples were mainly fragments of myosin heavy chain and

360

actin, clearly showing that myosin and actin are substrates of extracellular bacterial

361

proteases. Myosin is the primary protein in the myofibril and contributes to meat

362

structure, while actin is the second most abundant protein in the myofibril and is the

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primary protein in the thin filament (Huff Lonergan et al., 2010). Degradation of

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myosin has the potential to influence protein solubility and fiber shear strength. Even

365

small changes can alter important meat sensory traits such as tenderness, juiciness, and

366

water-holding capacity. P. fragi has been reported to produce volatile metabolites

367

compounds (e.g. 3-methyl-1-butanol, benzaldehyde, 4-ethylbenzaldehyde, thiophene

368

and dimethylsulfone) in the headspace of contaminated meat samples (Ercolini et al.,

369

2010). These compounds were likely derived from the catabolism of amino acids in

370

meat. Thus, proteolysis may contribute to the meat spoilage caused by P. fragi NMC25.

371

As predicted in our first assay, MAP influenced the activities of bacterial extracellular 17

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enzymes. The corresponding spoilage characteristics, including juice loss and slime,

373

also disappeared (as determined through sensory analysis; data not shown). Moreover,

374

MAP inhibited ATP synthesis. Since protein synthesis consumes more ATP than any

375

other metabolic process (Russell and Cook, 1995), this inhibition of ATP synthesis may

376

be how MAP inhibited the proteolytic activity. In addition, several peptides appeared

377

repeatedly among the different myosin and actin fragments, indicating that the

378

proteolytic enzymes of bacteria may work on specific cleavages sites of the meat

379

proteins. Since spoilage can be a subjective judgment by the consumer, it is often

380

difficult to identify the spoilage indicators (Nychas et al., 2008). Therefore,

381

identification of these degradation products may be usable as objective meat spoilage

382

biomarkers. Further studies of these specific fragments will lead to a better

383

understanding of the bacterial proteolytic activities involved in meat spoilage.

384

5. Conclusion

385

The present study has produced new information on the mechanisms by which

386

MAP can inhibit spoilage of meat by P. fragi. MAP altered P. fragi NMC25 cell

387

physiology, influencing both growth as well as basal metabolism, in particular

388

extracellular proteolytic activities. Additionally, this study reported differences in

389

proteolytic changes in vitro and in meat, suggesting that such activities are more

390

reliably investigated in specific food matrices. As phenotypic changes are controlled by

391

changes in expression of genes related to metabolic pathways, further studies are

392

needed to gain an in-depth understanding at the genetic level by high throughput

393

techniques such as RNA-Seq analysis. 18

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Acknowledgments

395

This study was supported by the project funded by a grant from the Natural

396

Science Foundation of Jiangsu Province in China (BK20150678), Jiangsu Agriculture

397

Science and Technology Innovation Fund (JASTIF) and the China Agriculture

398

Research System (CARS-41) funded by the China Ministry of Agriculture.

399 400

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Table 1. Growth conditions and sampling time of P. fragi NMC25 in late exponential phase at 15 °C. Culture medium

524 525

Log CFU/ml or g (sampling time) Air

MAP

TSB

7.84±0.15 (36 h)

7.94±0.05 (48 h)

Meat

9.12±0.15 (36 h)

9.29±0.10 (84 h)

Values are expressed as the mean ± standard deviation (n =4).

25

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Table 2. Proteins identified from the band fractions using GeLC–MS/MS. Protein name No. of Peptide1 Myosin heavy chain 225 Myosin heavy chain 15 Myosin-binding protein C 25 M-protein 17 c Myosin heavy chain 6 Alpha-1,4 glucan phosphorylase 9 Myosin heavy chain 9 d Pyruvate kinase 17 Myosin heavy chain 6 e Beta-enolase 22 Calsequestrin-2 7 f Myosin heavy chain 54 Actin 8 Creatine kinase M-type 10 Actin 10 g Triosephosphate isomerase 13 h Myosin heavy chain 18 Actin 15 Myosin light chain 1 35 Myosin regulatory light chain 2 26 Troponin T variant TnTx7-e16 11 1The amount of peptides identified to the proteins in each band. 2 Protein ID according to UniProt database. Band a b

527 528 529

26

Accession no.2 P13538 P13538 P16419 Q02173 P13538 Q5ZME4 P13538 P00548 P13538 P07322 P19204 P13538 P68139 P00565 P68139 P00940 P13538 P68139 P02604 P02609 O57559

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Table 3. The potential spoilage biomarkers identified in the degraded fragments of air samples.

532 533

Accession no.2 Common peptide sequences1 Bands Origin proteins VIQYFATIAASGEK d, e, f Myosin heavy chain P13538 MQGTLEDQIISANPLLEAFGNAK a, d, e, f LASADIETYLLEK d, e, f, h LTGAVMHYGNLK a, d, f GQTVSQVHNSVGALAK d, e, f, h NKDPLNETVIGLYQK a, e, h GSSFQTVSALFR a, b, f, h TLEDQLSEIK a, b, c NLQQEIADLTEQIAEGGK a, b, c SELQASLEEAEASLEHEEGK a, b, c IAEKDEEIDQLKR a, b, c ANLLQAEVEELR a, b, c, e AVFPSIVGR f, g, h Actin P68139 DSYVGDEAQSK f, h IWHHTFYNELR f, g, h VAPEEHPTLLTEAPLNPK f, g, h SYELPDGQVITIGNER f, g, h KDLYANNVMSGGTTMYPGIAD f, h f, g, h R EITALAPSTMK f, g, h QEYDEAGPSIVHR 1The same sequences of peptides identified to corresponding proteins in different bands. 2 Protein ID according to UniProt database.

27

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Fig. 1. Decomposition zone diameter (DZD), bacteria colony diameter (BCD) (a, c) and growth pattern (b) changes of P. fragi NMC25 under modified atmosphere and air conditions in RJA at 15 °C. No decomposition zone was observed in MAP group. RJA, raw-chicken juice agar; A, air group; M, MAP group; a - f: BCD values with different lowercase letters in the same package condition are significantly different (P <0.05). A - G: DZD values with different uppercase letters in the same package condition are significantly different (P <0.05).

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Fig. 2. Flow cytometric dot plots of P. fragi NMC25. Cells were cultured in TSB (a, b) or on meat (c, d) under air (a, c) and MAP (b, d) conditions at 15 °C then strained with SYTO 9 and PI. e, Ethyl alcohol-treated cells as dead control. A, air group; M, MAP group. X-axis represents fluorescence intensity of SYTO 9, y-axis represents fluorescence intensity of PI. Numbers in each gate represent the percentage of events.

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Fig. 3. Membrane potential (a) and relative ATP concentration (b) changes of P. fragi NMC25 grown in TSB or on meat under air and MAP conditions at 15 °C. A, air group; M, MAP group; CCCP, negative control. Values with asterisks are significantly different (** P <0.01, *** P <0.001).

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FIG. 4. SDS-PAGE of sarcoplasmic (a) and myofibrillar (b) protein samples degraded by proteases of P. fragi NMC25 grown in TSB or on meat under air and MAP conditions at 15 °C. Lane M, molecular weight markers; lanes 1 and 4, control; lanes 2 and 5, air samples; lanes 3 and 6, MAP samples. The arrows indicate protein fragments.

ACCEPTED MANUSCRIPT Highlights: 

The action mechanism of MAP against P. fragi was studied



MAP exhibited a bacteriostatic effect to P. fragi



MAP retained cell membrane integrity



Bacteria membrane depolarization and ATP synthesis decrease were observed



MAP inhibited the bacterial proteolytic activity mainly on myosin and actin