Changes in the quality of superchilled rabbit meat stored at different temperatures

    Changes in the quality of superchilled rabbit meat stored at different temperatures Yang Lan, Yongbiao Shang, Ying Song, Quan Dong PII: DOI: Reference:

S0309-1740(16)30034-1 doi: 10.1016/j.meatsci.2016.02.017 MESC 6899

To appear in:

Meat Science

Received date: Revised date: Accepted date:

27 September 2015 20 January 2016 8 February 2016

Please cite this article as: Lan, Y., Shang, Y., Song, Y. & Dong, Q., Changes in the quality of superchilled rabbit meat stored at different temperatures, Meat Science (2016), doi: 10.1016/j.meatsci.2016.02.017

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ACCEPTED MANUSCRIPT Changes in the quality of superchilled rabbit meat stored at different temperatures

Yang Lan, Yongbiao Shang, Ying Song, Quan Dong *

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College of Food Science, Southwest University, Chongqing 400715, P. R. China

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ABSTRACT: This work studied the effects of a superchilling process at two different temperatures on the shelf life and selected quality parameters of rabbit meat. As the storage time increased, the rates at which the total aerobic count, total volatile basic nitrogen, thiobarbituric acid-reactive substances and pH

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value increased were significantly lower in superchilled rabbit meat stored at -4℃ compared to those in rabbit meat stored at -2.5℃ and 4℃. SDS-PAGE analysis indicated that the decrease in storage

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temperature could significantly reduce the degree of protein degradation. The lightness, redness, shear force, the integrity of muscle microstructure and water holding capacity decreased with increasing storage

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time. Compared with the samples frozen at -18℃, superchilled rabbit meat show a marked reduction in

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microstructure deterioration. These results suggest that shelf life of good-quality rabbit meat was 20 d under superchilling at -2.5℃ and at least 36 d under superchilling at -4℃, compared with less than 6 d

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under traditional chilled storage.

Keywords: Superchilling; Partial freezing; Chilling; Shelf life; Microstructure; Protein electrophoresis

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1. Introduction

Rabbit meat is an important product from an economical and nutritious perspective. China is one of the largest producers and consumers throughout the world. Over the past decade, China’s production of rabbit meat has doubled, reaching 723,975 tons in 2013 and accounting for 41.6% of global production (FAOSTAT). Rabbit meat is one of the healthiest meats because of its nutritive and dietetic properties, such as low fat content, low allergenicity and cholesterol, high digestibility and unsaturated fatty acid content (Vergara, Berruga, & Linares, 2005; Dalle Zotte, & Szendro, 2011). The superchilling process was described as early as 1920 by Le Danois, and the terms “superchilling”, “partial freezing” and “deep chilling” are used to describe a process by which food products are stored between their initial freezing point (-0.5℃~ -2.8℃ for most food) and 1~2℃ below this temperature in most reports (Chang, Chang, Shiau, & Pan, 1998; Duun & Rustad, 2007; Magnussen et al., 2008). Compared with traditional chilling technology, superchilling can retain better food quality and prolong

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ACCEPTED MANUSCRIPT the shelf life of most stored food by at least 1.5~4 times (Kaale, Eikevik, Rustad, & Kolsaker, 2011); it can also reduce the use of freezing/thawing and thereby a lower energy cost was involved in superchilling in terms of transportation and retailing compared with freezing technology (Zhou, Xu, & Liu, 2010).

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Previous studies have investigated the effects of superchilling on the shelf life and quality parameters of aquatic products only at a special temperature (Gallart-Jornet et al., 2007; Kaale, Eikevik, Rustad, &

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Nordtvedt, 2014; Beaufort, Cardinal, Le-Bail, & Midelet-Bourdin, 2009; Fernandez, Aspe, & Roeckel, 2009; Olafsdottir et al., 2006). Sivertsvik, Rosnes, and Kleiberg (2003) reported that a 21-day sensory shelf life was for superchilled (-2℃) salmon in air, whereas chilled (4℃) fillets were spoiled after 7 d. A

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combination of superchilling with modified atmosphere packaging (MAP) or vacuum packaging can greatly extend the shelf life (Duun et al., 2007; Wang, Sveinsdottir, Magnusson, & Martinsdottir, 2008).

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The formation and growth of intra- and extracellular ice crystals during superchilling storage is significant. These crystals accelerate the extent of myofiber detachment and breakage (Bahuaud et al.,

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2008; Kaale et al., 2013). However, there are few published studies describing the effect of different

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superchilling temperatures on the shelf life and microstructure of livestock products, and to the best of our knowledge, no literature concerning rabbit meat has yet been published. The purpose of this study was to

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investigate the effects of different superchilling temperatures (-4℃ and -2.5℃) on the shelf life, sensory and nutritional quality parameters of rabbit meat, including total aerobic count, total volatile basic nitrogen, color, tenderness and protein structure. At the same time, we also observed the effect of the

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growth of ice crystals on the microstructure of rabbit muscles at different freezing temperatures.

2. Materials and methods 2.1 Materials Forty male Ira rabbits (2-2.3 kg/live rabbit) from the experimental farm of the Southwest University (Chongqing, China) were slaughtered by standard commercial procedures, and the head, viscera and skin were removed. The carcasses were immediately transferred to chilled insulated EPS boxes (approximately 6℃) and then transported to laboratory within 1 h after killing. Unstained protein ladder was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Coomassie Brilliant Blue R250 and glycine used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All chemicals and reagents were of analytical grade.

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ACCEPTED MANUSCRIPT 2.2 Sample preparation The carcasses were chilled at 4℃ for approximately 24 h until rigor mortis dissipated, and the hind legs were then dissected. Every leg was weighed, placed in a polypropylene tray and wrapped with a

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low-oxygen-permeable polyvinyl dichloride film (thickness of 0.25 mm and oxygen permeability of 60

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cm3/m2/24 h/atm; Shiny-day Group Co., ltd., Hainan, China). Samples were subsequently cooled in a

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refrigeration cabinet at a temperature of -30℃ until the core temperature was approximately -2℃ （about 80min）and then immediately transferred to a cold storage room at -2.5±0.3℃ or -4±0.4℃for temperature equalization and storage for up to 36 d (freezing point of Ira rabbit hind legs is between -1.8℃ and

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-2.1℃). The day of processing was denoted day 0. Air temperature was recorded every 1 min during superchilled storage by loggers with an internal sensor (L91-1+, Hangzhou Loggertech Co., Ltd.,

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Zhejiang, China). References were stored at 4±1℃ for up to 10 d or frozen at -18℃ for 36 d. Prior to analysis, samples stored at sub-zero temperatures were transferred to storage at 4℃ and thawed for 12 h.

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Superchilled samples were analyzed on days 4, 8, 12, 16, 20, 24, 28, 32 and 36, whereas chilled

analyzed on day 36.

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2.3 Microbiological analysis

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references stored at 4℃ were analyzed on days 0, 2, 4, 6, 8, 10, and frozen references stored at -18℃ were

The determination of the total aerobic count (TAC) was performed as described by Liu et al. (2014). Ten grams of minced rabbit meat from each treatment was weighed aseptically and homogenized with 90 mL

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of 0.85% sterile physiological saline for 1 min. Serial tenfold dilutions were performed by adding 1 mL homogenates to 9 mL of 0.85% sterile physiological saline. The pour plates of the appropriate dilutions were incubated at 36℃±1℃ for 48 h. All counts were expressed as log CFU/g. 2.4 pH value A sample of 3 g minced rabbit was mixed with 30 mL KCl (pH7.0 0.1 mol/L) and homogenized by a homogenizer (XHF-D, Ningbo Scientz Biotechnology Inc., Zhejiang, China) for 1 min at 6000 rpm. The pH of the mixtures was measured by a digital pH meter (PHS-3+, Century Fangzhou Science & Technology Co., Ltd., Chengdu, China). 2.5 Total volatile basic nitrogen Total volatile basic nitrogen (TVB-N) was measured by semi micro steam distillation, as described by Zhang et al. (2011) with slight modifications. A sample of 10 g minced rabbit was dispersed in 100 mL distilled water and stirred for 30 min, and the mixture was then filtered. The TVB-N value was

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ACCEPTED MANUSCRIPT determined according to the consumption of hydrochloric acid and calculated using the following equation: TVB-N(mg/100 g) = (Y1-Y2)×C×2800, where Y1 is the titration volume of hydrochloric acid in the sample, Y2 is the titration volume in the blank, and C is the concentration of hydrochloric acid (0.01

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mol/L). 2.6 Thiobarbituric acid-reactive substances

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Lipid oxidation was evaluated by thiobarbituric acid-reactive substances (TBARS) according to the procedure developed by Lo Fiego et al. (2004) with some modifications. A sample of 10 g minced rabbit was mixed with 20 mL of 20% trichloroacetic acid (TCA) and homogenized (XHF-D, Ningbo Scientz

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Biotechnology Inc., Zhejiang, China) at 6000 rpm for 1 min. After centrifugation (5500rpm for 15 min at 4℃), the supernatant was filtered through filter paper. Five milliliters of filtrate were combined with 5 mL

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of 0.02 mol/L 2-thiobarbituric acid solutions (TBA) and heated in a boiling water bath for 20 min together with a blank containing 5 mL of 20% TCA and 5 mL TBA reagent. After the resulting solution was cooled

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under running water for 10 min, the solution’s absorbance was measured at 532 nm with a

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spectrophotometer (Model 722, Jinghua Science & Technology Co., Ltd., Shanghai, China). The TBARS value was expressed as mg of malonaldehyde/kg of rabbit sample and calculated using the following

solution.

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equation: TBARS (mg/kg) = (A532+0.002)×2.587, where A532 is the absorbance value of the assay

2.7 Protein electrophoresis

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Soluble protein was extracted according to the method described by Duun & Rustad (2008), and the amount of soluble protein was determined by Biuret colorimetry as described by Zheng et al. (2013). The degradation of the salt soluble protein was analyzed by SDS-PAGE according to the method described by Laemmli (1970) with some modifications. All equipment for electrophoresis was purchased from Bio-Rad Laboratories (Richmond, CA, USA). One milliliter of 1 mg/mL protein sample was mixed with 1 mL sample buffer (0.1 g SDS, 0.1 mL β- mercaptoethanol, 0.002 g bromophenol blue, 2 g glycerol and 2 mL Tris-Hcl of 0.05 mol/L pH 8.0 were mixed and deionized water was added to a total volume of 10 mL). The mixture was heated in boiling water for 3 min and subsequently centrifuged at 1800 g for 10 min. The concentrations of the running gel and stacking gel were 12% and 4%, respectively, and 20μL of supernatant was loaded into each well on the gel, but only 5μL unstained protein ladder was loaded into one well. The stacking gel and running gel were run on a Bio-Rad Mini-Protein II system at constant currents of 15 mA and 30 mA, respectively, and the total run time was approximately 2.5 h. The gels were

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ACCEPTED MANUSCRIPT stained for protein with Coomassie Brilliant Blue R250 and scanned using a Gel Imaging System (G:BOX, Genesys, California, USA). 2.8 Drip loss

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The drip loss of the hind legs was estimated as described by Lauzon et al. (2009) and calculated by the following equation: drip loss (%) = (initial weight raw material - weight after thawing) / (initial weight

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raw material) × 100. 2.9 Cooking loss

Samples measuring 5×5×2 cm were cut from the hind legs, wrapped in a retort pouch, and subsequently

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immersed completely in a constant-temperature water bath at 70℃ for 30 min (Honikel, 1998). After cooking, samples were removed and cooled to room temperature under tap water for 20 min. The cooking

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loss was estimated as described by Kim et al. (2013) and calculated by the following equation: cooking loss (%) = (weight of uncooked sample - weight of cooked sample) / (weight of uncooked sample) × 100.

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2.10 Shear force

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Sample pretreatment (cooking) was conducted by the method described previously (2.9). The tenderness of the samples was evaluated by shear force (Boccard et al., 1981) and measured by a TA-XT2i texture

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analyzer (Stable Micro Systems, Surrey, UK) equipped with a load cell of 5 kg and a HDP/BSW probe. Test samples measuring 1×1×2 cm were cut from the cooked meat. The measurements were conducted using a cross-head speed of 100 mm/min (Combes, Lepetit, Darche, & Lebas, 2004), and the tests were

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performed at two different locations of each sample; at least three samples were measured to obtain an average shear value. The result was expressed in Newtons. 2.11 Color measurement

Color was measured according to the method developed by Liu et al. (2014) with slight modifications. Lightness (L*), redness (a*) and yellowness (b*) were determined with a colorimeter (UltraScan PRO, Hunter Associates Laboratory, Inc., Virginia, USA) using a D65 light source, a 10°angle observer and a measuring area with a diameter of 10 mm at room temperature. The instrument was calibrated with a white standard plate before measurements. The analyses were performed at three different positions of each sample, and at least three samples were measured to obtain an average value. 2.12 Microstructure Observation Muscle blocks with a cross section of 2×2 mm and a length of 5 mm along the fiber axis were cut from

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ACCEPTED MANUSCRIPT the center of hind legs. The specimens were fixed in Bouin solution (75 mL trinitrophenol，25 mL formaldehyde, and 5 mL glacial acetic acid) for 24 h at room temperature. The blocks were then dehydrated in gradients of ethanol (70%, 80% and 90% for 10 min, respectively; 95% and 100% for 15

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min twice, respectively) and were subsequently immersed in xylene for approximately 15 min and in paraffin for 2 h at 55℃. Subsequently, these tissues were embedded in paraffin using an embedding

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machine (LEICA EG1150H, Leica Biosystems, Germany). The embedded samples were cut transversally and longitudinally into 5 μm thick slices using a rotary microtome (LEICA RM2235, Leica Biosystems, Germany) and then stained with hematoxylin and eosin solutions. The microstructure of these specimens

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was then observed, and images were acquired using a light microscope (Olympus BX43, Olympus Co., Tokyo, Japan).

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2.12 Statistical Analysis

Microsoft Excel 2007 (Microsoft Inc, U.S.A.) was used for data processing and SPSS 16.0 (SPSS Inc.,

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U.S.A.) for statistical analysis. OriginPro 8.6（OriginLab Corp., U.S.A.）and Photoshop CS6 (Adobe

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Systems Inc., U.S.A.) were used for image processing. All analyses were run in triplicate (except microstructure analyses). All data were subjected to analysis of variance (ANOVA). Duncan's multiple

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range test was used to determine the difference between means (significance was defined at P < 0.05).

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3. Results and discussion

3.1 Changes in total aerobic counts The changes in the TAC of rabbit hind legs stored under chilled and superchilled conditions are shown in Fig. 1. The initial TAC was approximately 4.6 log CFU/g. Although the TAC of all samples stored at different temperatures increased with storage time, the chilled samples showed higher rates of increase than the superchilled samples. It is clear that the chilled samples had already exceeded the microbiological acceptable limit (106 log CFU/g) (Fernández-Segovia, Escriche, Fuentes, & Serra, 2007; Fernandez, Aspe, & Roeckel, 2009) by the 6th day, whereas samples stored at -2.5℃ reached a value of 5.67 log CFU/g after 20 d. Duun et al. (2007) also reported that the TAC of superchilled fillets stored at -2.2℃ was markedly lower than that of chilled ones over 6 weeks of storage. Despite the higher microbiological acceptable limit of 7 log CFU/g adopted by Hong, Luo, Zhu, & Shen (2012), the author also claimed that TAC may not be suitable for a separate evaluation of the shelf life and deterioration of

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ACCEPTED MANUSCRIPT meat. Moreover, compared with the samples stored at -2.5℃, the superchilled samples stored at a lower temperature (-4℃) exhibited lower plate count numbers (4.56-5.66 log CFU/g) over the entire storage period, which indicates that lower storage temperatures could significantly inhibit the growth of

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microorganisms. Based on the microbiological limit of 6 log CFU/g, the shelf life of rabbit meat was less than 6 d at 4℃ and less than 24 d at -2.5℃, whereas it was longer than 36 d at -4℃.

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3.2 Changes in pH

The changes in the pH values of rabbit hind legs stored under different conditions showed the same trend with that in TAC. The pH decreased initially and then increased (Fig. 2). The initial decrease in pH value

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may be attributed to the accumulation of inorganic phosphoric acid resulting from the depletion of muscle adenosine triphosphate (ATP) (Scherer et al., 2005) and the production of lactic acid resulting from the

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decomposition of glycogen in rabbit hind legs (Koziol, Maj, & Bieniek, 2015). Compared with the slowly increasing trend observed for both superchilled treatments, the pH of the chilled hind legs exhibited the

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most rapid and dramatic increase between 2 and 10 d, mainly due to the production of ammonia, amines

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and other basic substances from the degradation of proteins by meat spoilage microorganisms and endogenous enzymes (Muela et al., 2010; Rodriguez-Calleja, Garcia-Lopez, Santos, & Otero, 2005).

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After 12 d, the samples stored at -2.5℃ had continuous growth until day 36 at pH 6.24. However, in the samples stored at -4℃, a significant decrease appeared from 12d to 28d (P<0.05) and an uptrend was observed after 28 d. Wang et al. (2008) reported a downtrend in modified cod loins stored at -1℃ between

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4 to 11 d, and Leygonie, Britz, & Hoffman (2012b) observed a slight decrease in pH over 30 d of frozen storage ( -20℃). Firstly,

the ionic balance and increased the concentration of free hydrogen ions (H+)

were altered, resulting from the loss of minerals and small protein compounds as exudates (Leygonie, Britz, & Hoffman, 2012a). Secondly, the production of free fatty acids during storage may acidify the rabbit meat (Hulot & Ouhayoun, 1999). 3.3 Changes in TVB-N TVB-N is widely used as an index for assessing the deterioration and shelf life of meat products and is mainly composed of ammonia, amine and trimethylamine. The TVB-N increased for all samples with storage time and showed higher rates of increase with the increase in storage temperature (Fig. 3). The TVB-N of chilled hind legs showed the highest rate of increase from 8.6 mg/100 g (initial value) to 23.4 mg/100 g over 10 d of storage, whereas the superchilled samples showed a steady increase over 20 d, which indicates that storage temperature greatly affects TVB-N values. The increase in TVB-N in hind

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ACCEPTED MANUSCRIPT legs is primarily attributed to the protein degradation reactions initiated by meat spoilage bacteria and endogenous enzymes (Muela et al., 2010; Rodriguez-Calleja et al., 2005), including the deamination and decarboxylation of free amino acids, degradation of nucleotides, and oxidation of amines (Lu et al., 2009).

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The TVB-N of superchilled samples stored at -2.5℃ increased sharply after 20 d compared with that of samples stored at -4℃. This increase may have been due to the large accumulation of TAC, which

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increased largely after 16 d (Fig. 1). Liu et al. (2014) reported a similar phenomenon in which a rapid increase in TVBN was observed after 2 weeks, and the TVB-N of samples stored at -3℃ was distinctly lower than that of samples stored at -1℃. High TVB-N values are not desirable and are associated with an

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unpleasant smell in meat. If a TVB-N value of 15 mg/100 g in rabbit meat is proposed as an acceptability limit, then according to our results the acceptable shelf life was less than 6 d at 4℃, 20 d at -2.5℃ and

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longer than 36 d at -4℃. Mi et al. (2013) reported that mirror carp stored at -3℃ had TVB-N values below 15 mg/100 g over 30 d of storage but only a 6 d shelf life at 3℃.

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3.4 Lipid oxidation

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The extent of lipid oxidation of rabbit hind legs stored at different conditions was evaluated by TBARS. The TBARS steadily increased from 0.05 mg/kg (initial value) to 0.6, 1.037 and 0.773 mg/kg in the

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samples stored at 4℃, superchilled at -2.5℃, and superchilled at -4℃, respectively, over the entire storage period (Fig. 4). Similarly to the TAC and the TVB-N, compared with that of samples stored under chilled conditions, the TBARS value of samples stored under superchilled conditions showed a lower rate

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of increase, which indicates that low temperature can restrain lipid oxidation to some extent. Ganhão, Estévez, & Morcuende (2011) reported that lipid oxidation produces hydroperoxides; these compounds then reactant and degrade into secondary products, including aldehydes, ketones and other carbonyl compounds, which are associated with the development of rancid, pungent and other off-flavors. Hansen et al. (2004) reported that these unpleasant flavors can be detected by trained sensory panels when the TBARS value exceeds 0.5 mg/kg. Considering this level, lipid oxidation was not a major problem before 8 d of storage at 4℃, 16 d of storage at -2.5℃ and 24 d of storage at -4℃. Furthermore, the TBARS value of superchilled samples clearly exceeded that of samples chilled at -2.5℃ after 24 d and that of samples chilled at -4℃ after 32 d (Fig. 4). Leygonie et al. (2012a) argued that frozen storage is not necessarily sufficient to prevent oxidation from occurring. Hansen et al. (2004) and Xia, Kong, Liu, & Liu (2009) reported that freezing and thawing of muscle tissues will accelerate lipid oxidation, which

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ACCEPTED MANUSCRIPT Benjakul & Bauer (2001) attributed to the fact that ice crystals can injure cells and cause the subsequent release of pro-oxidants for lipid oxidation, particularly non-heme iron. 3.5 Protein degradation

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The SDS-PAGE patterns of salt-soluble protein extracted from rabbit hind legs showed protein degradation under different storage conditions (Fig. 5). The lowest degree of protein decomposition

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occurred in muscles stored at -4℃ (Fig. 5). Major protein components, including myosin heavy chain (MHC), actin, and troponin, did not show any significant change during storage at -2.5℃ or 4℃. However, the band intensity of tropomyosin in hind legs stored at -2.5℃ clearly decreased after 24 d of

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storage, and more severe degradation could be identified in samples maintained at 4℃. In samples stored at 4℃, the band intensities of paramyosin, tropomyosin, and myosin light chain 2 (MLC2) decreased

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sharply, and the band intensity of MLC1 nearly disappeared; moreover, even new low-molecular-weight (MW) protein was generated. These findings indicate that the lower superchilled temperatures caused

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reduced proteolysis degradation. Duun et al. (2008) also reported a lower degree of protein degradation in

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superchilled salmon fillets stored at -3.6℃ compared with that observed in fillets stored -1.4℃. Protein degradation may be mainly due to endogenous enzyme activity and the activity of microorganisms,

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although low temperatures can inhibit these reactions (Ueng & Chow, 1998). 3.6 Drip loss and cooking loss

Changes in drip loss and cooking loss throughout the entire storage are presented in Table 1. These

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changes are indicators of the water holding capacity (WHC), which is regarded as an essential meat quality parameter (Duun et al., 2007). A decrease in WHC can cause products to become unacceptable because of the loss of nutrients during storage, which is of great importance to consumers (Xia et al., 2009). Overall, the drip loss and cooking loss of all treatments increased gradually as storage time increased, although higher values could be observed at day 4 of superchilled storage. The high drip loss of samples at day 4 may be attributed to a low pH (Fig. 2) close to the isoelectric point of myosin (Olsson, Seppola, & Olsen, 2007). Storage time had a significant effect (P<0.05) on the drip loss of chilled samples but not on that of superchilled samples over 32 d of storage (P>0.05). In the present study, the drip loss of chilled hind legs was significantly lower than that of superchilled samples (Table 1), indicating that freezing could destroy muscular tissues by causing mechanical damage to cell membranes and thus a loss in WHC (Anese et al., 2012; Srinivasan, Xiong, Blanchard, & Tidwell, 1997). Moreover, compared with the

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ACCEPTED MANUSCRIPT samples stored at -4℃, the superchilled samples stored at -2.5℃ exhibited a higher drip loss over the entire storage period; this phenomenon may have been the result of more severe proteolysis at higher temperatures caused by the activity of bacterial enzymes and endogenous enzymes (Olsson, Ofstad,

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Lødemel, & Olsen, 2003; Olsson et al., 2007). A similar phenomenon was reported by Duun et al. (2008), who observed that the drip loss of superchilled samples stored at -1.4℃ was higher than that of samples

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stored at -3.6℃.

The cooking loss of samples stored at -4℃ was always higher than that of samples stored at -2.5℃, indicating that a lower freezing temperature can cause more severe mechanical damage to cell membranes

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and thus higher water loss during cooking (Duun et al., 2007). Moreover, a significant increase (P<0.05) in cooking loss was observed at the end of storage, which may have been caused mainly by myosin

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denaturation and proteolysis (Xia et al., 2009). 3.7 Changes in shear force

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The tenderness of samples was evaluated in terms of shear force (Boccard et al., 1981). In this study, the

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shear force tended to decrease significantly (P < 0.05) with storage time in all treatments (Fig. 6), in agreement with a previous study that evaluated rabbit meat tenderness during storage (Vergara et al.,

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2005). Compared with the shear force measured for chilled hind legs, lower values were observed for both sets of superchilled samples. The breakdown of connective tissues and muscle fibers may be the main reason why the shear force decreased sharply, which can be attributed to the activity of enzymes,

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including bacterial enzymes and endogenous enzymes that can decompose key proteins (troponin, connectin and desmin). Moreover, the loss of membrane strength caused by ice crystal formation can also reduce the force used to shear meat (Leygonie et al., 2012a). Throughout the entire storage period, the shear forces were almost higher in superchilled legs stored at -4℃ than in superchilled samples stored at -2.5℃, in accord with the study of Duun et al. (2008). This discrepancy may have been due to the higher activity of enzymes at -2.5℃ and more severe protein degradation. 3.8 Changes in color Color plays an important role in the appearance, presentation and acceptability of rabbit meat. Lightness (L*) and redness (a*) were affected markedly by storage temperature and storage time; however, yellowness (b*) was not affected by either factor (Table 2). A similar phenomenon was reported for pork by Kim et al. (2013). The L* and a* of fresh rabbit hind legs were 64.07 and 2.37, respectively. L*

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ACCEPTED MANUSCRIPT decreased to 56.89 and 56.41 in samples superchilled at -4℃ and -2.5℃ over 28 d of storage. However, L* rapidly decreased to 56.21 in chilled samples over only 10 d of storage. These results indicate that superchilling could significantly inhibit the decrease in L* and a*. As shown in Table 3, both L* and a*

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decreased as the storage time increased, and lower L* and a* values could be observed in the samples superchilled at -2.5℃ compared with those observed in the samples superchilled at -4℃. Duun et al.

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(2008) also obtained a higher L* in salmon fillets stored at -3.6℃ than in fillets stored at -1.4℃. Generally, L* and a* tend to decrease as a function of freezing treatment and storage time (Jeong et al., 2006; Moore & Young, 1991). Throughout the entire storage period, the activity of the metmyoglobin

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reducing enzyme, which can degrade metmyoglobin into deoxymyoglobin and then oxygenate it back to oxymyoglobin (Ben Abdallah, Marchello, & Ahmad, 1999), will decrease as meat is frozen, and as a

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result, the metmyoglobin will accumulate gradually on the surface of the meat (Ballin & Lametsch, 2008) . Moreover, in this study, a decrease in L* and a* was observed with a concomitant increase in

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TBARS formation, which suggests that the oxidation of lipids contributes to the accelerated oxidation of

3.9 Changes in microstructure

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myoglobin, in agreement with previous studies (Leygonie et al., 2012a; Xia et al., 2009)

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Transverse sections (Fig. 7 a, c-h) and longitudinal sections (Fig. 7 b, i-n) of rabbit skeletal muscles were observed by light microscopy, and differences among fresh, superchilled and chilled samples were distinct. The structure of fresh muscle cells was intact (a, b). It is clear that every myofiber was

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surrounded by endomysium, and plenty of oval or rectangular nucleuses could be observed under the endomysium. There was little connective tissue around the myofibrils; however, a certain amount of connective tissue could be observed among the muscle bundles. As shown in Fig. 7, compared with chilling (e, k), freezing can cause obvious damage to the structure of myofibrils, including the shrinking and breaking myofibrils. As the storage time increased and the storage temperature decreased, the size and the number of gaps inside of cells increased both in the transverse and longitudinal sections, as did the detachment of the myofibrils, and the largest number of gaps and severest breakages occurred in the muscles of samples frozen at -18℃ (h, n). These effects may have been consequences of myofibrillar protein denaturation and the formation and development of intracellular and extracellular ice crystals during superchilling and freezing (Anese et al., 2012; Bahuaud et al., 2008; Mi et al., 2013).

4. Conclusions

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ACCEPTED MANUSCRIPT The results of this study indicate that compared with the shelf life afforded by traditional chilled storage, the shelf life of good-quality rabbit meat was increased by at least 3 times and 5.5 times by superchilled storage at -2.5℃ and -4 ℃, respectively, which suggests that subtle differences in superchilling

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temperature will exert a significant effect on shelf life. As the storage time increased, the samples stored at -4℃ maintained the lowest rates of increase with respect to TAC, TVBN, TBA and pH value compared

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to those observed for samples stored at -2.5℃ and 4℃ throughout the storage period. The results of SDS-PAGE, WHC, color and shear force analyses revealed that compared to other storage conditions, superchilled storage at -4℃ can markedly inhibit the deterioration of rabbit meat’s sensory and

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nutritional quality. However, the results obtained by light microscopy show that compared to superchilling at -2.5℃, superchilling at -4℃ produced larger and numerous gaps and more severe

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myofibrils breakages caused by the growth of ice crystals, whereas the most severe damage was observed

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in muscles frozen at -18℃.

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Acknowledgments

This study was supported by the Special Fund for Agro-scientific Research in the Public Interest of China

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（Grant No. 201303144）. The authors would like to acknowledge the Key Laboratory of Aquatic Science

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Figure Caption

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Fig. 1 Effects of superchilled storage at -4℃（■）, superchilled storage at -2.5℃ (●) and chilled storage at 4℃ (▲) on the total aerobic count of rabbit hind legs. The error bars indicate the standard deviation obtained from three analyses. The different letters (a-h) indicate significant differences between treatment times for the same treatment (P<0.05). Fig. 2 Effects of superchilled storage at -4℃（■）, superchilled storage at -2.5℃ (●) and chilled storage at 4℃ (▲) on the pH value of rabbit hind legs. The error bars indicate the standard deviation obtained from three analyses. The different letters (a-f) indicate significant differences between treatment times for the same treatment (P<0.05). Fig. 3 Effects of superchilled storage at -4℃（■）, superchilled storage at -2.5℃ (●) and chilled storage at 4℃ (▲) on the TVB-N of rabbit hind legs. The error bars indicate the standard deviation obtained from three analyses. The different letters (a-h) indicate significant differences between treatment times for the same treatment (P<0.05). Fig. 4 Effects of superchilled storage at -4℃（■）, superchilled storage at -2.5℃ (●) and chilled storage at 4℃ (▲) on the TBARS of rabbit hind legs. The error bars indicate the standard deviation obtained from three analyses. The different letters (a-i) indicate significant differences between treatment times for the same treatment (P<0.05).

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Fig. 5 Effects of superchilling and chilling on the salt soluble protein SDS-PAGE pattern of rabbit hind legs. M: molecular weight of standard protein; MHC: myosin heavy chain; MLC: myosin light chain.

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Fig. 6 Effects of superchilled storage at -4℃（■）, superchilled storage at -2.5℃ (●) and chilled storage at 4℃ (▲) on the shear force of rabbit hind legs. The error bars indicate the standard deviation obtained from three analyses. The different letters (a-h) indicate significant differences between treatment times for the same treatment (P<0.05).

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Fig. 7 Microstructure of transverse (T) and longitudinal (L) sections in rabbit skeletal muscles. Arrows: myofiber-myofiber detachments; g: gaps left by intracellular ice crystals; s: myofiber shrinking; b: myofiber breakages.

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ACCEPTED MANUSCRIPT Table Table 1 Effects of superchilled storage at -4℃, superchilled storage at -2.5℃ and chilled storage at 4℃ on the drip loss and cooking loss of rabbit hind legs. -4℃

-2.5℃

4℃

0

9.95±0.18a

2

0.4±0.1a

4

0.52±0.1ab

6

0.76±0.06abc

8

0.99±0.15bc

10

1.08±0.17c

10.66±2.06a 1.73±0.59a

1.75±0.81ab

T

4℃

-4℃

9.95±0.18a

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time (d)

Cooking loss

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Drip loss

Storage

12.83±2.32a

-2.5℃ 9.95±0.18a

12.32±2.22a

11.53±1.33a

12.15±0.35a

10.82±3.27a

14.02±2.26a

12.6±1.55a

12.37±1.89a 1.16±0.13a

1.32±0.28a

15.78±0.17ab 19.89±1.63b

1.11±0.05a

1.39±0.18a

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1.6±0.63a

2.21±0.16ab

13.6±2.44a

13.16±2.59a

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1.68±0.03a

1.76±0.06ab

16.26±2.21ab

12.68±0.78a

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1.77±0.2a

2.33±0.49ab

21.18±5.64bc

15.74±2.45ab

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2±0.21a

3.36±0.84ab

18.52±1.2ab

18.05±1.64bc

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2.32±0.36a

3.22±0.4ab

20.95±0.42bc

17.8±1.15c

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2.37±0.06a

3.66±0.8b

23.53±0.15c

19.34±0.65c

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ACCEPTED MANUSCRIPT Table 2 Effects of superchilled storage at -4℃ and -2.5℃ and chilled storage at 4℃ on the lightness (L*), redness (a*) and yellowness (b*) of rabbit hind legs. -4℃

storage

-2.5℃

L*

a*

b*

L*

0

64.07±1.07

2.37±0.51

10.53±0.83

64.07±1.07

a* 2.37±0.51

b*

L*

a*

10.53±0.83

64.07±1.07

2.37±0.51

10.53±0.83

1.53±0.59

10.05±1.27

61.49±1

0.44±0.72

9.48±1.16

59.48±3.16

0.15±0.33

10.5±0.88

57.02±2.03

-0.53±0.82

10.77±0.83

56.21±1.19

-0.51±0.79

12.64±2.91

T

time

4℃

4

62.07±1.48

61.64±1.38

2.1±0.26

11.26±0.83

61.51±1.07

1.74±1.12

8

60.12±1.05

1.01±0.28

9.69±0.49

59.62±1.48

9.23±1.1

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1.32±0.7

59.52±1.03

-0.27±0.5

10.25±0.71

58.07±0.63

0.8±0.49

10.94±1.82

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58.54±1.55

-0.07±0.45

9.09±1.38

58.94±1.31

0.59±0.77

10.42±0.58

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57.75±2.18

-0.98±0.25

11.26±0.83

57.65±1.01

0.65±0.92

10.68±0.64

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58.59±1.28

-0.68±0.53

9.51±0.56

57.32±1.81

-0.66±0.61

10.3±1.16

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56.89±0.95

-0.99±0.36

9.76±0.98

56.41±2.45

-0.94±0.37

10.44±0.65

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55.08±1.32

-0.85±0.91

11.6±0.8

55.49±0.76

-1.22±0.17

11.67±1.45

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55.63±1.27

-1.15±0.36

11.47±0.8

53.49±1.5

-1.23±0.28

11.17±0.59

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Values are presented as means±SD from six determinations.

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9.31±1.22

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b*

ACCEPTED MANUSCRIPT Title Page

Author names and affiliations:

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1.Yang(given name) Lan(family name), [email protected]; 2.Yongbiao(given name) Shang(family name)，[email protected]; 3.Ying (given name) Song(family name)，[email protected]; 4.Quan(given name) Dong(family name)，[email protected].

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Title: Changes in the quality of superchilled rabbit meat stored at different temperatures

（College of Food Science, Southwest University）for all authors.

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Corresponding author: Quan Dong Tel:+86 13370708378 E-mail address: [email protected]

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Present Address: College of Food Science, Southwest University, Beibei, Chongqing 400715, P.R. China

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