Umami and related components in “chilled” pork for the Japanese market

    Umami and related components in “chilled” pork for the Japanese market T.M. Ngapo, L. Vachon PII: DOI: Reference:

S0309-1740(16)30142-5 doi: 10.1016/j.meatsci.2016.05.005 MESC 7002

To appear in:

Meat Science

Received date: Revised date: Accepted date:

29 February 2016 22 April 2016 5 May 2016

Please cite this article as: Ngapo, T.M. & Vachon, L., Umami and related components in “chilled” pork for the Japanese market, Meat Science (2016), doi: 10.1016/j.meatsci.2016.05.005

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Umami and related components in “chilled” pork for the Japanese market.

PT

T.M. Ngapo* and L. Vachon

Saint Hyacinthe Research and Development Centre, Agriculture and Agri-Food Canada, 3600

SC

RI

boul. Casavant Ouest, St-Hyacinthe, Québec, Canada, J2S 8E3

MA

Pork; chilled; ageing; umami; taste; export

NU

Keywords

*Corresponding author at: Saint Hyacinthe Research and Development Centre, Agriculture and

D

Agri-Food Canada, 3600 boul. Casavant Ouest, St-Hyacinthe, Québec, Canada, J2S 8E3

AC CE P

TE

Email address: [email protected] (T. M. Ngapo)

1

ACCEPTED MANUSCRIPT Abstract

The aim of this study was to evaluate umami-related components and their evolution in

PT

Canadian pork destined for the Japanese market. Export quality pork loins for Japan were subjectively selected on-line for marbling, colour and firmness; remaining loins were retained for

RI

the domestic market. At 48 h post-mortem, samples were aged 5 d at 4.0°C (fresh) or 13, 28, 43

SC

or 58 d at -1.7°C (chilled). Meat qualities differed only in pH (˂ 1 pH unit; P˂ 0.05). Generally, free amino acid concentrations increased and nucleotide concentrations decreased with longer

NU

ageing periods. The equivalent umami concentration (EUC) was highest in the pork aged 5 d at 4.0°C and at 43 d at -1.7°C (P˂ 0.05) which is estimated as the transportation time for Canadian

MA

chilled exports to Japan. A lack of differences in EUC between domestic and export pork and between fresh and 43 d chilled ageing demonstrates that Canadian chilled pork in Japan has the

AC CE P

TE

D

EUC of its fresh 5 d counterpart.

2

ACCEPTED MANUSCRIPT 1. Introduction “Chilled” meat exportation involves chilling meat within 48 h post-mortem (p.m.) to

PT

strictly controlled temperatures below 0°C without freezing and holding under these conditions for several weeks (Scrine, 1982; Bowater, 1996). This mode of storage is used for export to high

RI

value markets where these chilled products compete with fresh counterparts, such as New

SC

Zealand and Australian lamb in North American and European markets, or pork and beef from these latter markets in Japan. With respect to the Japanese pork market, the high value is a

NU

consequence of not only the mode of transport, but also the strict physical criteria set by importers reflecting market and/or consumer demand and which require production specifically

MA

for this market or on-line meat selection.

Regardless of very specific Japanese import criteria and trade conditions making for a challenging market to enter and maintain, there are few scientific reports of the sensory quality

D

of pork after chilled transportation and with respect to the import criteria. Generally, the

TE

literature on chilled exportation of meat focuses on microbiological quality and when included, sensory evaluation is used to detect off-flavours and determine shelf-life (for example,

AC CE P

McMullen & Stiles, 1994; Jeremiah, Gibson, & Argnosa, 1995). Hence a study was undertaken to compare the sensory quality of pork in the local Canadian market with that selected on-line for export to Japan and the storage conditions for the local market with chilled transportation (Ngapo, et al., 2012a; Ngapo, Riendeau, Laberge, & Fortin, 2012b). It was observed that irrespective of the selection process, the physico-chemical and sensory measures of the domestic and export pork quality were similar. However, the ageing processes resulted in significant differences in the pork in the two markets with the chilled transport (43 d, −1.7°C) resulting in stronger sweet taste and caramel flavour, a greater number of panellists detecting sensory notes and a greater number of sensory notes detected when compared to domestic market ageing (5 d, 3.1°C). In addition, consumers judged tenderness, juiciness, taste liking and overall acceptability significantly higher with the longer, cooler ageing. The higher taste liking scores for the chilled meat supported the taste differences observed by the trained panel and the lack of differences in the taste strength with ageing indicated that, while the two meats tasted different, neither was stronger in overall taste.

3

ACCEPTED MANUSCRIPT Nishimura, Rhue, Okitani and Kato (1988) suggested that the gradual breakdown of the myofibrillar protein structure during ageing not only results in tenderisation, but the generation of peptides and amino acids also change flavour. A number of studies report a positive effect on

PT

eating quality with increasing ageing under conditions similar to that for the domestic market, that is, aged at about 4°C for up to 10 d p.m (Agerhem & Tornberg, 1993; Bejerholm, 1991;

RI

Bryhni et al., 2003; Buchter & Zeuthen, 1971; Channon, Baud, Kerr, & Walker, 2003; Channon,

SC

Kerr, & Walker, 2004; Kepčija, 1972; Nishimura et al., 1988; Wood et al., 1996, 1999). Extrapolating from the studies on pork in domestic market conditions, it might be concluded that

NU

the longer ageing time in chilled pork likely results in a greater breakdown of the myofibrillar protein structure and thereby a greater concentration of peptides and amino acids than in the

MA

fresh pork. However, it is not only the ageing period, but also the temperatures at which ageing takes place that differ. Cooler temperatures often mean reduced enzyme activity and in chilled export may impact the rate of ageing potentially reducing or even negating any effect of

D

extended time under chilled storage.

TE

Increased Maillard reaction indicative of the sweet and caramel tastes observed by Ngapo et al. (2012a, b) could be explained by greater protein breakdown in the chilled than the fresh

AC CE P

pork. Furthermore and importantly for the Japanese market, the increased total sensory notes and higher consumer taste liking scores also observed may indicate increased umami content. In recent years Japanese importers have discussed umami levels of imported pork, and while not an obligation, are often undertaken at the exporters cost in a voluntary evaluation of meat quality upon arrival. Umami was identified in 1908 by a Professor at the Tokyo Imperial University, Kikunae Ikeda, but it was not until 1985 and after much scientific debate that umami was officially recognised as the fifth basic taste joining sweet, sour, salt and bitter (see Yamaguchi, 1998, for a general review of umami). Umami represents the taste of a mix of glutamate and nucleotides and is described as pleasant, brothy or meaty. This taste induces salivation and a furriness sensation on the tongue, stimulating the throat, the roof and the back of the mouth. By itself, umami is not palatable and is pleasant only within a narrow concentration range, its fundamental effect being the ability to balance taste and round the total flavour of a dish. Many foods that may be consumed daily are rich in umami, the taste being common to foods that contain high levels of L-glutamate, inosine monophosphate (IMP) and guanosine monophosphate (GMP), including fermented and aged products, and dry-cured meats. Dry4

ACCEPTED MANUSCRIPT cured meats are most often pork products which have higher concentrations of protein breakdown products than their fresh meat counterparts due in part to the extended ageing process (Alfaia et al., 2004). Chilled meats are neither dry-cured nor fermented, but like these meat

PT

products, undergo an extended ageing process.

The aim of this study was to evaluate umami-related components and their evolution in

RI

Canadian pork destined for the Japanese market with particular reference to the long, slow

SC

ageing process in transport and the export selection criteria imposed by Japanese importers.

NU

2. Materials and Methods

MA

2.1. Chemicals, reagents and water

All chemicals and reagents were analytical grade or higher quality and water was

AC CE P

2.2. Pork and collection

TE

D

deionised.

Pork loins were acquired from a commercial slaughter-line. The pigs from which the loins were obtained had been slaughtered at about 110 kg live weight and the dressed carcasses hung at 2ºC. At 24 h post-mortem (p.m.), one loin (boneless, short cut) from each of 80 pigs was collected on-line. Abattoir staff sorted the loins achieving 40 pairs each of export and domestic quality. The selection of export quality pork for the Japanese market was achieved subjectively by visual estimation of marbling score (2-4 on the NPPC scale; NPPC, 1999) and colour (3-4 using the Japanese Pork Colour Standards; Nakai, Saito, Ikeda, Ando, & Komatsu, 1975) along the loin surface, and firmness by touch at the ends of the loins. Loins not meeting these selection criteria were retained for the Canadian domestic market. After sorting, loins were individually vacuum packaged, placed in boxes of four with cardboard dividers separating the loins, and removed to refrigeration at 0.5ºC. At 48 h p.m. the loins were transported under refrigeration to the research laboratory.

2.3. Portioning and ageing/chilling 5

ACCEPTED MANUSCRIPT

Working at 5°C, a 2.5 cm slice was removed from the centre of each loin for drip loss, colour and pH measures and for determination of the sex of the animal from which the meat was

PT

obtained. Three 7.5 cm sections were then taken from either side of the centre slice, individually vacuum packaged and one randomly selected section held in a walk-in refrigerator at 4.0°C

RI

(±0.3°C) monitored by four HOBO RH/temp dataloggers (H08-003-02, Onset Computer

SC

Corporation, Bourne, MA, USA). At 5 d ageing (that is, at 7 d p.m.) these loin sections were portioned. The remaining five vacuum packaged sections of loin were stacked in the same boxes

NU

as used commercially in the transport of loins to Japan and held at a mean core meat temperature of −1.7°C (±0.1°C) monitored by two Class ‘A’ platinum resistance temperature detectors

MA

(PT100 RTDs) with hermetically sealed sensor tips connected to OM-CP-QUADRTD 4-channel temperature data-loggers (Omega Engineering, Inc., Stamford, CT, USA). Core temperature was monitored in loins used only for this purpose. The temperature of the air circulating in the chiller

D

as well as that inside the boxes holding the loin sections were also monitored using four and two

TE

PT100 RTDs, respectively, mounted in open-end stainless steel housing (average ± 1 s.d of −1.84°C ± 0.17°C and −1.75°C ± 0.13°C, respectively). The refrigeration system had a daily

AC CE P

thawing cycle. All RTDs had perfluoroalkoxy alkanes (PFA) insulated leads. During the 58 d chilled storage period, readings were taken every 5 min. A randomly selected section from each loin was removed at 13, 28, 43 and 58 d ageing (that is, at 15, 30, 45 and 60 d p.m.) and portioned.

At completion of the 0, 5, 13, 28, 43 and 58 d ageing periods (that is, at 2, 7, 15, 30, 45 or 60 d p.m.), the loin sections were portioned at 5°C. The m. longissimus thoracis et lumborum was isolated from surrounding muscles, when present. Each section was cut in half along the fibre direction giving two 7.5 cm long pieces which were then cut into 3 portions of about equal size (a total of six portions per section of loin). The portions were individually vacuumpackaged, randomly assigned to an analysis with one spare and stored at -40ºC until required.

2.4. Drip loss, colour and pH

Drip loss, colour and pH were measured during portioning. The drip loss was measured in triplicate by the EZ-DripLoss method of Christensen (2003) with modifications. At 48 h p.m., 6

ACCEPTED MANUSCRIPT two cores (25 mm, parallel to the fibre direction) were taken from the same sites of each slice, individually placed in EZ-DripLoss containers, sealed and stored for 48 h at 4ºC. The meat sample was then removed and the drip loss calculated by difference and expressed as the weight

PT

percent of the original wet weight of the pork sample. The slice of loin was then bloomed 45 min at room temperature and CIE L*a*b* colour parameters were measured as the mean of

RI

readings on the same three sites of the M. longssimus thoracis et lumborum in each slice using a

SC

Minolta Chroma Meter (D65 light source with 0° viewing angle; Minolta Co., Ltd., Osaka, Japan) calibrated with a white tile. The pH was measured as the mean of readings taken using a

NU

glass probe with temperature compensation at the same two sites on each slice of loin.

MA

2.5. Proximate analyses

Protein, moisture and intramuscular fat (IMF) contents were measured according to

The loin slice in its packaging was partially thawed in a circulating

TE

International, 2015).

D

AOAC official methods of analyses 992.15, 985.14 and 2008.6, respectively (AOAC

waterbath (25°C, 30 min), then removed from packaging and the m. longissimus thoracis et

AC CE P

lumborum isolated. A homogenous sample was achieved by mincing at low speed (2 x 15 s) in a GE Deluxe Chopper 169121R (Bentonville, AR, USA). Protein content was measured and the remaining minced meat was vacuum packaged, frozen and stored at -40ºC. When required for analyses of moisture and IMF contents, the frozen minced meat was thawed at room temperature and mixed well.

Protein content was measured in triplicate (0.2 g pork per replicate) by combustion using a Leco FP428 Nitrogen and Protein Determinator (Leco Corporation, St Joseph, MI, USA). At least ten blanks (empty tin capsules) and six EDTA standards (0.2 g each) were used each sampling day. A conversion factor of 6.25 was used to calculate protein content which was expressed as weight percent of wet weight of the pork sample. Moisture and IMF contents were analysed in duplicate by rapid microwave drying and NMR using a CEM SMART System5 Moisture Analyser and SMART Trac Fat Analyzer Model 907955 (CEM Corporation, Matthews, NC, USA). Accurately weighed pork (3 g) was thinly spread onto the rough side of a glass fibre pad, covered with a second pad and dried to a constant weight (85% power). Moisture content was calculated by difference and expressed as the weight percent of the wet 7

ACCEPTED MANUSCRIPT weight of the pork sample. The pads with dried sample were then placed in a tube and fat content recorded.

PT

2.6. Sex determination and balanced loin selection

Briefly, extraction of DNA from pork loins was achieved using an

SC

Ngapo et al. (2012).

RI

Sex of the animals from which the loins were obtained was determined according to

Agencourt® RNAdvance Tissue kit (Beckman Coulter, MA, USA) for fibrous tissue by plate

NU

format without optional DNase treatment. Sample (10 mg) was homogenised in a Tissue Lyser small bead mill (Qiagen, ON, Canada; 25 min, 5 mm metal beads). Plates were incubated at

MA

37°C for 45 min. The DNA was extracted from homogenized lysates (400 µl) and eluted in RNAse free water (50 µl; Gibco, ON, Canada). No-sample extraction and positive extraction controls (female and male animals) were also included.

D

The PCR amplifications were carried out in Eppendorf Mastercycler® Gradient

TE

thermocycler using a HotStarTaq® Plus PCR kit. The reaction mixture (20 µl) contained the sex determination primers SRYB-5 and SRYB-3 (80 nM each; Pomp, Good, Geisert, Corbin, &

AC CE P

Conley, 1995) and PCR validation primers P1-5EZ and P2-3EZ (160 nM each; Aasen & Medrano, 1990). An initial incubation step of 5 min at 95°C was followed by 35 cycles of incubation at 94°C for 1 min, 55°C for 45 sec and 72°C for 1 min. Fragments of 163 bp, present only in male samples, were generated by the SRYB PCR products. Fragments of 445 bp or 447 bp were produced from female or male genomic DNA, respectively (corresponding to ZFX or ZFY genes, respectively) served as amplification controls. Upon determination of the sex of the animals from which the loins were obtained, a subset of 40 loins was selected from the 80 loins collected at the abattoir. The selection was balanced for domestic and export quality and for male and female animals. Data for these 40 loins only is reported here.

2.7. Sarcomere length

Sarcomere length (SL) was measured by the method of Cross, West and Dutson (19801981). Vacuum packaged loin portions were partially thawed in cold tap water and a sample (2.5 8

ACCEPTED MANUSCRIPT g) cut from the core of each portion. The samples were homogenised (Polytron PT-MR 3100 with a PT 3012/2T dispersing aggregate; Kinematica AG, Littau, Switzerland; 25 s, 16,500 rpm) in a 0.2 M sucrose solution (25 ml). A drop of homogenate was transferred to a slide and with

PT

cover slip and examined by phase microscopy (400x). Images were captured with Infinity Analyze (Version 6.3.0, 2006-2013, Lumenera Corporation, Ottawa, Canada) and analysed with

RI

Image-Pro Premier (Version 9.1.28, 2012-2014, Media Cybernetics, Inc., Rockville, MD, USA)

SC

calibrated with a stage micrometer.

The mean SL of each muscle was calculated from the average of 25 myofibrils, each

NU

from which the length of 10 sarcomeres was measured.

MA

2.8 Particle size

Particle size analyses were based on the method of Karumendu, van de Ven, Kerr, Lanza,

D

& Hopkins (2009) with modification. Vacuum packaged loin portions were partially thawed at

TE

room temperature. Duplicate samples (2.0 g) were cut from the core of each loin portion and homogenised at 11,000 rpm in ice cold buffer (30 ml) using a Polytron homogeniser (PT-MR

AC CE P

3100 with a PT 3012/2T dispersing aggregate; Kinematica AG, Littau, Switzerland). The buffer comprised 0.1 M KCl, 1 mM EDTA, 25 mM potassium phosphate (7 mM KH2PO4 and 18 mM K2HPO4, pH 7.0 at 4˚C), and 1 mM sodium azide. The samples were homogenised twice for 30 s with a 30 s break on ice. After homogenisation, the myofibril suspensions were filtered (polyethylene 1.0 mm2 mesh) to remove connective tissue, facilitated by washing with 20 ml of buffer.

The filtrate was centrifuged (1,000g, 10 min, 2°C), the supernatant decanted and

discarded, and the pellets re-suspended in 40 ml of buffer. Particle size was analysed using a laser diffraction particle size analyser (Mastersizer 2000E, Malvern Instruments Ltd., Malvern, United Kingdom connected to a Hydro 2000MU large volume manual wet sample dispersion unit) with a pump speed of 1500 rpm. Myofibril suspension (6-15 ml) was added to stirring water (800 ml) in which the head of the dispersion unit was immersed to achieve a light obscuration of 10-11%. The suspension was allowed to stabilise 1 min after which time the particle size data was obtained, including mean and median particle size, the standard deviation of the mean and quantile distributions.

9

ACCEPTED MANUSCRIPT 2.9 Oligopeptides

Extraction of TCA-soluble oligopeptides was undertaken according to the extraction

PT

method of Hughes et al. (2002) for free amino acids. Vacuum packaged loin portions were partially thawed at room temperature and duplicate samples (5 g) cut from the core of each

RI

portion. The samples were homogenised (IKA T25 DS1 Digital Ultra-turrax with S25N-18G

SC

dispersing tool, IKA®-Werke GmbH & Co. KG, Staufen, Germany) for 1 min (twice for 30 s with a 30 s break) on ice at 13,500 rpm in 2% TCA (20 ml). The homogenate was centrifuged

NU

(10,000g, 20 min, 4˚C) and the supernatant retained.

The 2% TCA-soluble oligopeptides were determined by spectrophotometric assay using a

MA

bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich, Saint Louis, MO, USA). In this assay, BCA reagent (2 ml; 50 parts of a solution containing BCA, sodium carbonate, sodium tartrate and sodium bicarbonate in 0.1 M NaOH and 1 part 4% copper (II) sulphate pentahydrate)

D

was mixed with protein sample (that is, the supernatant), standard or blank (0.1 ml). The

TE

mixtures were gently vortexed and incubated at 37˚C for 30 min. Absorbance was measured at 562 nm. Bovine serum albumin (BSA) was used as the standard and the oligopeptides expressed

AC CE P

as the mg BSA equivalent.

2.10 Free Amino Acids

Extraction of free amino acids (FAA) was undertaken according to Hughes et al. (2002). Vacuum packaged loin portions were partially thawed at room temperature and duplicate samples (5 g) cut from the core of each portion. The samples were homogenised (IKA T25 DS1 Digital Ultra-turrax with S25N-18G dispersing tool, IKA®-Werke GmbH & Co. KG, Staufen, Germany) for 1 min (twice for 30 s with a 30 s break) on ice at 13,500 rpm in 2% TCA (20 ml). The homogenate was centrifuged (10,000g, 20 min, 4˚C) and the supernatant filtered using syringe driven filter units (Millex-AP 25 mm filter unit, glass fibre filter; Millipore, Japan). The FAA were determined by RP-HPLC according to the Waters AccQ·Tag method (Waters Corporation, Milford, MA, USA) with modification. This method involved pre-column derivatization and conversion of the amino acids to stable fluorescent derivatives by reaction with AccQ·Fluor reagent (6-aminoquinolyl-N-hydrosuccinimidyl carbamate) (Cohen & Michaud, 1993). The HPLC system (Waters Corporation, Massachussetts, USA) comprised a 10

ACCEPTED MANUSCRIPT 600E Multisolvent Delivery System, a 717 Autosampler, a column heater and a 474 Scanning Fluorescence Detector. Separation was achieved by an AccQ·Tag 4 μm column (3.6 x 150 mm; Waters Corporation, Ireland) maintained at 37ºC. Elution was by means of a gradient of filtered

PT

(0.45 μm nylon membrane filters, Millipore Corporation, Ireland) solvents A (Waters AccQ.Tag eluent A), B (acetonitrile) and C (20% methanol) at a flow rate of 1 ml/min. The gradient was as

RI

follows: initial eluent 100% A; 99% A and 1% B at 0.5 min; 91% A and 9% B at 18 min; 87% A

SC

and 1% B at 19 min; 79% A and 21% B at 29.5 min; 60% B and 40% C at 33 min; 100% A at 36 min and held under these conditions for 11 min. Supernatant (20 ul) was injected in duplicate

NU

and the eluent monitored at an excitation wavelength of 250 nm and an emission wavelength of 395 nm. Individual amino acids were identified by comparison of their retention times with

MA

those of calibration standards and by spiking the samples with individual standards. Peak areas were processed using Empower Pro (Empower 2 software, 2005, Waters Corporation, Milford, MA, USA). Quantification of peak areas was based on norleucine as an internal standard and

D

calibration curves of external standards of amino acids. The concentrations of individual amino

AC CE P

2.11 Nucleotides

TE

acids were expressed as μg/g.

Extraction and analyses of nucleotides were undertaken according to Ryder (1985) with modifications. Vacuum packaged loin portions were partially thawed at room temperature and a sample (3 g) cut from the core of each portion. The samples were homogenised (IKA T25 DS1 Digital Ultra-turrax with S25N-18G dispersing tool, IKA®-Werke GmbH & Co. KG, Staufen, Germany) for 1 min (twice for 30 s with a 30 s break) at 14,000 rpm in 0.6 M perchloric acid (15 ml). The homogenate was centrifuged (3,000g, 10 min, 4˚C) and the supernatant retained. The precipitate was resuspended in 0.6 M perchloric acid (15 ml) and the homogenisation and centrifugation repeated. The supernatants were combined and made up to 37.5 ml with 0.6 M perchloric acid. An aliquot (10 ml) was transferred to a 50 ml conical tube, neutralised to pH 6.5-6.8 with 1 M KOH and made up to 20 ml with water. Neutralized supernatant (about 2 ml) was stored at -80˚C until analyses. Just prior to analyses, the supernatant was thawed at room temperature and filtered using Acrodisc LC syringe driven filter units (0.45 μm, 13 mm filter unit, PVDF filter; Waters Limited, Mississauga, Canada). 11

ACCEPTED MANUSCRIPT Nucleotides were determined using an RP-HPLC system (Waters Corporation, Massachussetts, USA) comprised of a 600E Multisolvent Delivery System, a 717 Autosampler, a column heater and an Hewlett Packard G1314A Variable Wavelength Detector (Series 1100,

PT

Hewlett Packard, Japan). Separation was achieved by an Atlantis T3 5 μm column (4.6 x 250 mm; Waters Corporation, Ireland) maintained at 30ºC. The mobile phase of 40 mM KH2PO4–60

RI

mM K2HPO4 without pH adjustment was used at a flowrate of 2 ml/min. Buffer solutions were Corporation, Ireland).

SC

prepared daily and were filtered through a 0.45 μm nylon membrane filter (HAWP, Millipore Thawed supernatant (10 ul) was injected in duplicate and the eluent

NU

monitored at 250 nm. Individual nucleotides were identified by comparison of their retention times with those of calibration standards and by spiking the samples with individual standards.

MA

Peak areas were processed using Empower Pro (Empower 2 software, 2005, Waters Corporation, Milford, MA, USA). Quantification was based on calibration curves of external standards of

D

nucleotides and the concentration of the nucleotides expressed as μg/g.

TE

2.12. Equivalent umami concentration

AC CE P

The equivalent umami concentration (EUC, g MSG/100 g) was estimated according to Chiang, Yen and Mau (2007). The EUC is the concentration of MSG equivalent to the umami intensity given by the mixture of MSG and the 5’-nucleotide and is represented by the following addition equation (Yamaguchi, Yoshikawa, Ikeda, & Ninomiya, 1971): Y = Σaibi + 1218 (Σaibi) (Σajbj)

where Y is the EUC of the mixture in terms of g MSG/100 g; ai is the concentration (g/100 g) of each umami amino acid (Asp or Glu); aj is the concentration (g/100 g) of each umami 5’nucleotide (5’-IMP, 5’-GMP, 5’-XMP or 5’-AMP); bi is the relative umami concentration (RUC) for each umami amino acid to MSG (Glu, 1 and Asp, 0.077); bj is the RUC for each umami 5’nucleotide to 5’-IMP (5’-IMP, 1; 5’-GMP, 2.3; 5’-XMP, 0.61 and 5’-AMP, 0.18) and 1218 is a synergistic constant based on the concentration of g/100 g used.

2.11. Statistical analyses

12

ACCEPTED MANUSCRIPT The effects of ageing and meat type and their interactions on protein breakdown variables were tested by repeated analysis of variance (ANOVA) using the MIXED procedure of SAS (2007). Significant interactions were investigated by ANOVA for each level of an effect in the

PT

interaction term. Significant differences were determined using the LSEMANS statement and the Bonferroni option of the MIXED procedure. For each of the 12 combined treatments of

RI

ageing (6 ageing periods) x meat type (2 types), 20 repetitions (animals) were achieved. The

SC

replicates of laboratory measures correspond to replicate measurements on the same animal with

NU

the mean of these replicates being used in the statistical analyses.

MA

3. Results

3.1. Physico-chemical characteristics of the loins

D

Results of physico-chemical analyses are presented in Table 1. Sarcomere length

TE

measures at day 0 ageing were all ≥1.58 μm indicating that none of the pork was cold shortened (Dransfield & Lockyer, 1985). Sarcomere length was also measured on samples aged 5 d at

AC CE P

4.0°C and 43 d at -1.7°C. No differences were observed at the three ageing times and so sarcomere length was not measured in the pork samples stored 13, 28 and 58 d at -1.7°C (P > 0.05). A significant difference in pork characteristics was only observed for pH measured at 48 hours which was an average of almost one unit higher in the export than domestic pork (P = 0.032). No difference in IMF was observed with meat type (P = 0.288). No influence of sex of the animal from which the loins were obtained on the physico-chemical characteristics was observed (P > 0.05). Noting that marbling is one of the selection criteria for the Japanese market, the IMF contents of all 80 loins collected from the abattoir were measured.

No

significant differences in the mean values were observed for meat type (1.97% in domestic, n=40, and 2.26% in export pork, n=40; P = 0.23) or sex (2.30% in castrates, n=37, and 1.96% in females, n=43; P = 0.14).

3.2 Particle size and oligopeptide concentration

13

ACCEPTED MANUSCRIPT The Sauter (D[3,2]) and De Brouckere (D[4,3]) mean diameters were all significantly smaller with ageing than the control (P ˂ 0.001), but no differences among the aged samples were observed (P > 0.05; Table 2). Nor was an influence of meat type observed (P > 0.05).

PT

Plotting the mean particle size against the 10%, 20%, 50%, 80% and 90% quantiles illustrates the evolution in the chilled meat particle size attaining a plateau at 28 d ageing (Figure 1).

RI

Also presented in Table 2 are the oligopeptide concentrations which were all greater with

SC

ageing than the control (P ˂ 0.001). The oligopeptide concentrations of the samples aged 5 d at 4.0°C and 43 d chilled were not significantly different (P > 0.05) and were lower than the 13, 28

NU

and 58 d chilled samples (P ˂ 0.001). An effect of meat type was also observed, with the export meat having a higher concentration of oligopeptides than the domestic meat type (P = 0.025). A

MA

meat type by ageing interaction shows that this was in fact the tendency and that it was only the amplitude that differed, for all but the 58 d chilled ageing (Table 3).

TE

D

3.3 Free amino acids

All of the free amino acids measured in this study increased with increasing ageing at

AC CE P

chilled temperatures (P ˂ 0.001; Table 4). Generally the concentrations of the free amino acids in samples aged 5 d at 4.0°C were greater than the concentrations at 0 d (no ageing), and were not significantly different from that at 13 d chilled ageing (P > 0.05). Notable exceptions in samples aged 5 d at 4.0°C were of glutamic acid which was intermediate between 13 and 28 d chilled ageing and significantly different from both, methionine which was lower than that of 13 d chilled ageing and alanine which was not significantly different to that of 28 d chilled ageing. The total of the free amino acids increased with increasing chilled ageing time (P ˂ 0.001) and that of the samples aged 5 d at 4.0°C was between 13 and 28 d chilled ageing. The concentrations of aspartic acid, serine, alanine, valine, isoleucine and phenylalanine were all greater in the export than domestic quality pork samples (P ˂ 0.05), but no difference in the total free amino acids was observed with meat type (P = 0.061). Interactions between meat type and ageing period were observed for concentrations of aspartic acid and proline (Table 3). While the differences were only significant for 13 and 58 d chilled ageing (P = 0.030 and P = 0.004, respectively), a general pattern of greater aspartic acid concentration in the Japanese than domestic type meat was evident with only the amplitude 14

ACCEPTED MANUSCRIPT differing. In proline, the only significant difference between the two meat types was observed at 5 d ageing, again being higher in the Japanese than domestic meat (P = 0.037).

PT

3.4 Nucleotides

RI

The concentrations of CMP, UMP, IMP and GMP decreased with increasing ageing time,

SC

whereas XMP increased with ageing time and no pattern of influence of ageing time on AMP was apparent (Table 5). The total nucleotide concentration decreased with ageing time, largely a

NU

consequence of IMP concentration which was from four to ten-fold the combined concentrations of the other nucleotides. Both the CMP and AMP concentrations were significantly different

MA

with meat type (P = 0.030 and P = 0.004, respectively), the former higher in the export meat and the latter higher in the domestic.

Interactions between meat type and ageing period were observed for concentrations of

D

CMP and UMP. In the control samples, that is, the samples that were not aged, the concentration

TE

of CMP was greater in the Japanese than the domestic meat (P = 0.010). However, in the aged samples there were no differences in CMP concentration for meat type (P > 0.05). The only

AC CE P

difference in UMP concentration was a significantly lower concentration in the Japanese than the domestic meat at 13 d chilled ageing (P = 0.015).

3.5 Umami

The equivalent umami concentration (EUC) was highest in the meat samples stored at 4.0°C (P ˂ 0.001; Table 6). Only after 43 d chilled ageing was the EUC not significantly different than that of the sample aged 5 d at 4.0°C (P > 0.05). However, at 58 d chilled ageing, the mean EUC was again significantly lower than that of the sample aged 5 d at 4.0°C. No effect of meat type or interactions between meat type and ageing period were observed (P > 0.05).

4. Discussion

Pork for the Japanese market is subjectively sorted on-line based on marbling and colour scores along the outer surface of the loin surface which is often covered in a thin layer of fat, as 15

ACCEPTED MANUSCRIPT well as firmness to the touch generally estimated at the loin ends. Marbling score is a visual estimate of IMF and is approximated to be of the same range in % as the score itself. The marbling score sought in pork for export to Japan is 2–4 on the NPPC scale (NPPC, 1999) and

PT

therefore corresponds to about 2–4% IMF. In the current study, the chemical measures showed that the average IMF of the export pork fell within the sought after range, albeit, at the lower end

RI

at 2.12%. However, a relatively high standard deviation (0.48%) demonstrates that there was

SC

much variation. Indeed, the IMF content ranged from 1.4 to 3.0% with 10 of the 20 export quality loins having less than 2% IMF. Similar findings were observed for the domestic quality

NU

pork which ranged from 1.2 to 3.1% with 12 of the 20 loins having IMF contents less than 2%. Furthermore, the IMF contents for the two meat types were not significantly different (P˂ 0.05).

MA

In fact, the laboratory analyses showed that the domestic and export meat qualities only differed significantly in pH (P˂ 0.05) and this difference was less than one unit. In an earlier study, it was also observed that there were no differences in IMF content with meat quality, and the only

D

significant difference of the traits measured here was that the export quality meat was darker

TE

than domestic quality, with both being in the normal range (CIE L* 51 vs 55, respectively; P˂ 0.05) (Ngapo et al., 2012a).

The NPPC standards were produced with the intent that

AC CE P

estimates be made on a cut surface of the muscle which is not an option for the commercial export of entire loins. It was suggested that the large range in IMF after sorting and the lack of differences between the meat types likely reflects the crude means employed to estimate the marbling score (Ngapo et al., 2012a) and this explanation is equally relevant to the lack of colour differences in the present study.

So, although the aim of the online sorting process is to achieve a meat satisfying the requirements of the Japanese importer, the physico-chemical analyses suggest that the quality of the domestic and export pork is actually very similar. With the only meat quality difference being a relatively small, albeit significant difference in pH, it is therefore surprising that differences in the concentrations of oligopeptides, six amino acids and two nucleotides were observed with meat type. Ageing by meat type interactions showed that for the nucleotides CMP and UMP and the free amino acid proline, the meat type effect was specific to one or two ageing times only.

However, the concentrations of oligopeptides and six other free amino acids

(aspartic acid, serine, alanine, valine, isoleucine and phenylalanine) were generally significantly higher in the export than domestic quality meat. 16

ACCEPTED MANUSCRIPT In contrast to the limited impact of meat type, ageing time influenced almost all of the measures related to protein breakdown in this study. The particle size was significantly smaller and the oligopeptide concentration greater with ageing beyond 2 d p.m. than without, although

PT

no differences or trends among the aged meat were evident for either of these measures. In addition, the FAA generally increased with ageing time, while most of the nucleotides decreased.

RI

These trends in the breakdown products of fresh meat with ageing times have been observed by

SC

other workers (for example, Nishimura et al., 1988; Koutsidis et al., 2008). Almost all of the amino acid concentrations were greater in ageing conditions simulating chilled export (43 d at -

NU

1.7°C) than domestic ageing conditions (5 d at 4.0°C). Indeed, pork aged under conditions mimicking those of the domestic market had FAA and nucleotide concentrations that were most

MA

often intermediate between the 0 d ageing control and that at 13 d chilled ageing, or not different from the latter.

At 58 d of chilled ageing, by far the largest increase in FAA relative to the concentration

D

prior to ageing was in the aspartic acid at 13 times the control concentration, followed by

TE

methionine at seven times. Precursors of volatile sulfur containing compounds are formed from sulfur containing amino acids, particularly, methionine and cysteine, which breakdown during

AC CE P

the cooking of meat (Davídek, Velíšek, & Pokorný, 1990). While the cysteine only increased by four times its original concentration, Koutsidis et al. (2008) explained that the reaction between cysteine and ribose when meat is cooked results in potent sulfur containing compounds which are reported to be crucial in cooked meat aroma (Mottram, 1998). Finally, leucine, isoleucine and serine increased by a factor of five and valine and phenylalanine a factor of four during the 58 d ageing period at -1.7°C. These amino acids are important in flavour formation, providing Strecker aldehydes and other aroma compounds (Koutsidis et al., 2008). After 5 d ageing at 4.0°C, all the FAA except alanine, ornithine, histidine and glycine had increased in concentration compared to the 0 d ageing control. However, the relative increases compared to the control concentrations were smaller than not only after 58 d chilled ageing, but also than after 43 d chilled ageing, the latter of which simulates time and temperature conditions for pork exports from Canada to Japan. In particular, methionine was only a factor of four times greater than the control pork and cysteine, a factor of two, whereas in the 48 d chilled aged pork, the concentrations were six and four times greater than the control pork, respectively. In other words, at 43 and 58 d chilled ageing the methionine and cysteine concentrations were about 2-3 17

ACCEPTED MANUSCRIPT times those of the pork aged for 5 d at 4.0°C. In addition, leucine, isoleucine, phenylalanine, valine and serine were from 2 to 5 times greater in the pork aged 43 and 58 d chilled than 5 d at 4.0°C. The first four of these FAA were classed as contributors to bitter taste and serine to sweet

PT

taste in the study by Chiang et al. (2007) who divided amino acids into several classes on the basis of taste characteristics described by Komata (1969). These same classes are presented in

RI

Table 7 with the exception of arginine which was not measured in the current study. On both

SC

weight per dry weight (for comparison to Chiang et al., 2007) and moles per wet weight of meat (for comparison to sensory evaluation by Ngapo et al., 2012a) bases, all classes of taste

NU

characteristics increased with ageing time. Noting that most of the FAA increased with ageing time and that these classes are merely sums of groups of FAA, this result is hardly surprising.

MA

Increases in the sweet and total characteristics with ageing appear to support the observed increases in sweet and overall notes by the trained sensory panel (Ngapo et al., 2012). However, bitter notes were not observed by trained panel, yet the bitter class was the most concentrated.

D

While the concentrations of these contributors can readily be summed, the relative impacts of

TE

each class on taste are unknown and comparisons across classes cannot therefore be made. Furthermore, the class concentrations observed are low when considering that Li et al. (2002)

AC CE P

used concentrations of 10 mMol/l of serine and leucine as levels that do not elicit response by human taste receptors suggesting that not only the differences with ageing, but also the actual levels of these FAA as well as the sweet and bitter classes are likely insignificant. While the MSG-like taste characteristic was estimated by Komata (1969) as the sum of glutamic and aspartic acids, a more complex equation was derived by Yamaguchi et al. (1971) from sensory evaluation studies to estimate the equivalent umami concentration (EUC). This equation incorporates the same two amino acids, as well as four nucleotides. Noting that most of the nucleotides in the current study decreased with increasing ageing period, the resulting values and trends of the EUC differ from the simply calculated concentrations of MSG-like taste characteristic FAA, a finding also observed by Chiang et al. (2007). In particular, the relatively high concentration of IMP compared to the other nucleotides (from four to ten-fold the combined concentrations of the four other nucleotides) combined with the relative umami constant of 1 mean that the EUC was very much influenced by the IMP concentration. Indeed, the EUC was highest in the pork aged 5 d at 4.0°C and was not significantly different from the pork aged 43 d at -1.7°C. Iida et al. (2016) found a similar pattern of umami intensity peaking at about 4 and 40 18

ACCEPTED MANUSCRIPT d in dry aged beef. A lack of differences in the EUC between the domestic and export ageing conditions and meat type is good news for the exporters and importers alike. However, the levels of EUC are very low (Mau, 2005). Noting that umami is not a taste in itself but impacts

PT

on other flavours upon attaining a threshold concentration, at these low levels one must question if the threshold has been achieved. This is particularly so in the taste dynamics of a food as

RI

complex as meat. Even if the umami threshold has been met, there may not be sufficient impact

SC

on the sensory quality of the pork that it would be perceived in its effect on other flavours. Indeed, other compounds may be of greater importance in this type of low temperature chilled

NU

meat ageing.

Model Maillard reaction systems have not only identified the FAA cysteine as a potent

MA

precursor of meat flavour volatiles, but also ribose and ribose 5-phosphate (Mottram & Whitfield, 1995). While these latter compounds were not measured in the current study, both compounds result from IMP degradation. Mottram and Nobrega (2002) demonstrated that both

D

ribose and ribose 5-phosphate were far more reactive than IMP and likely have a major effect on

TE

flavour formation. Therefore, while the decreasing IMP concentration with ageing corresponds to a reduced equivalent umami concentration, IMP degradation products might result in

AC CE P

increased flavour formation. Sensory and consumer studies showed that pork ageing for 43 d at 1.7°C was more flavoursome or of a better taste, respectively, than pork aged for 5 d at 3°C (Ngapo et al., 2012a, 2012b). The studies also showed that more flavour was observed when grilled than roasting or shabu shabu. Grilling subjects the surface of the pork to temperatures conducive to Maillard reactions, much more so than roasting and the Maillard reaction would not occur using the shabu shabu method which involves immersing the pork in boiling water. Therefore the findings of the sensory evaluation support the findings in the current study suggesting that the global improvement in taste is a result, at least in part, of increased FAA concentrations and not umami concentrations. In particular, the trained sensory panel observed increases in the sweeter notes which are indicative of Maillard reaction compounds.

5. Conclusions

Although pork for the Japanese market is subjectively sorted on-line based on marbling and colour scores along the loin surface, and firmness to the touch at the loin ends, laboratory 19

ACCEPTED MANUSCRIPT analyses showed that the domestic and export meat qualities only differed significantly in pH and this difference was less than one unit. While the quality of the meats may be very similar, the different ageing that the meat undergoes when destined for the local Canadian market versus the

contribute to taste.

PT

Japanese export market result in different levels of protein breakdown products, of which many Indeed, oligopeptides and free amino acids were found in greater

RI

concentrations in the pork aged 5 d at 4°C or up to 58 d at -1.7°C than control pork, and in

SC

particular, the free amino acids generally increased with longer ageing periods. The equivalent umami concentration (EUC) was highest in the pork aged under conditions simulating fresh pork

NU

in the domestic environment (5 d at 4.0°C) and was not significantly different from that aged under conditions simulating chilled exports (43 d at -1.7°C). A lack of differences in the EUC

MA

between the domestic and export ageing conditions and meat type demonstrates that Canadian pork supplied chilled to Japan has the equivalent umami content of the fresh (5 d aged at 4°C)

D

counterpart.

TE

Acknowledgements

AC CE P

The authors would like to acknowledge the in-kind support provided by Olymel S.E.C./L.P, and sincere thanks are extended to P.-P. Martin and the Vallée Jonction team for meat collection and valuable technical advice. Thanks are also expressed to Mr Claude Laberge of Experts-Conseils STATEX for the statistical analyses. Finally, the authors would like to acknowledge the technical assistance of D. Leblanc, A.-R. Rivière, A. Martinez-Lopez and J. Chamarande.

20

ACCEPTED MANUSCRIPT References Aasen, E., & Medrano, J. F. (1990). Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats. Bio/Technology, 8, 1279–1281.

PT

Agerhem, H., & Tornberg, E. (1993). Eating quality of pig meat. Meat Focus International, 2, 159–161.

RI

Alfaia, C.M., Castro, M.F., Reis, V.A., Prates, J.M., de Almeida, I.T., Correia, A.D., & Dias,

SC

M.A. (2004). Cahnges in the profile of free amino acids and biogenic amines during the extended short ripening of Portuguese dry-cured ham. Food Science and Technology

NU

International, 10, 297-304.

AOAC International (2015). Official methods of analysis. Last accessed 9 November 2015.

MA

http://www.eoma.aoac.org/

Bejerholm, C. (1991). The effect of ageing on the eating quality of normal pork loins. Proceedings 37th International Congress of Meat Science and Technology (pp48–51), 1–6

D

September 1991, Kulmbach, Germany.

TE

Bowater J. (1996). The preparation and transportation of meat products, with particular emphasis on chilled meat shipments. Paper presented at the Intermodal Conference, 1996. Last

AC CE P

accessed 20 April 2016. http://www.fjb.co.uk/wpcontent/themes/fjb/publication. Bryhni, E. A., Byrne, D. V., Rødbotten, M., Møller, S., Claudi-Magnussen, C., Karlsson, A., Agerhem, H., Johansson, M., & Martens, M. (2003). Consumer and sensory investigations in relation to physical/chemical aspects of cooked pork in Scandinavia. Meat Science, 65, 737–748.

Buchter, L., & Zeuthen, P. (1971). The effect of ageing on the organoleptic qualities of PSE and normal pork loins. Proceedings 2nd International Symposium on Condition and Meat Quality of Pigs (pp. 247–254). 22–24 March 1971, Zeist, The Netherlands. Channon, H. A., Baud, S. R., Kerr, M. G., & Walker, P. J. (2003). Effect of low voltage electrical stimulation of pig carcasses and ageing on sensory attributes of fresh pork. Meat Science, 65, 1315–1324. Channon, H. A., Kerr, M. G., & Walker, P. J. (2004). Effect of Duroc content, sex and ageing period on meat and eating quality attributes of pork loin. Meat Science, 66, 881–888. Chiang, P.-D., Yen, C.-T., & Mau, J.-L. (2007). Non-volatile taste components of various broth cubes. Food Chemistry, 101, 932-937. 21

ACCEPTED MANUSCRIPT Christensen, L. B. (2003). Drip loss sampling in porcine m. longissimus dorsi. Meat Science, 63, 469–477. Cohen, S. A., & Michaud, D. P. (1993). Synthesis of a fluorescent derivatizing agent, 6-

PT

aminoquinolyl-N-hydroxysuccinimdyl carbamate, and its application for the analysis of hydrolysate amino acids via high performance liquid chromatography. Analytical

RI

Biochemistry, 211, 279-287.

SC

Cross, H. R., West, R. L., & Dutson, T. R. (1980–1981). Comparison of methods for measuring sarcomere length in beef semitendinosus muscle. Meat Science, 5, 261–266.

NU

Davídek, J., Velíšek, J., & Pokorný, J. (1990). Developments in food science 21. Chemical changes during food processing. Prague: Avicenum, Czechoslovak Medical Press.

MA

Dransfield, E., & Lockyer, D. K. (1985). Cold-shortening toughness in excised pork. Meat Science, 13, 19–32.

Hughes, M. C., Kerry, J. P., Arendt, E. K., Kenneally, P. M., McSweeney, P. L. H. & O’Neill, E.

D

E. (2002) Characterization of proteolysis during the ripening of semi-dry fermented

TE

sausages. Meat Science, 62, 205-216. Iida, F., Miyazaki, Y., Tsuyuki, R., Kato, K., Egusa, A., Ogoshi, H., Nishimura, T. (2016).

AC CE P

Changes in taste compounds, breaking properties, and sensory attributes during dry ageing of beef from Japanese black cattle. Meat Science, 112, 46-51. Jeremiah, L. E., Gibson, L. L., & Argnosa, G. C. (1995). The influence of controlled atmosphere and vacuum packaging upon chilled pork keeping quality. Meat Science, 40, 79–92. Karumendu, L. U., van de Ven, R., Kerr, M. J., Lanza, M., & Hopkins, D. L. (2009). Particle size analysis of lamb meat: Effect of homogenisation speed, comparison with myofibrillar fragmentation index and its relationship with shear force. Meat Science, 82, 425-431. Kepčija, D. P. (1972). Investigations of some chemical and structural changes in beef and pork treated thermically at different stages of ageing. Acta Veterinaria, Yugoslavia, 22, 87–98. Komata, Y. (1969). The taste and constituents of foods. Nippon Shokuhin Kogyo Gakkashi, 3, 26.

Cited by Chiang, P.-D., Yen, C.-T., & Mau, J.-L. (2007). Non-volatile taste

components of various broth cubes. Food Chemistry, 101, 932-937. Koutsidis, G., Elmore, J. S., Oruna-Concha, M. J., Campo, M. M., Wood, J. D., & Mottram, D. S. (2008). Water-soluble precursors of beef flavour: Part II. Effect of post-mortem conditioning. Meat Science, 79, 270-277. 22

ACCEPTED MANUSCRIPT Li, X., Staszewski, L., Xu, H., Durick, K., Zoller, M., & Adler, E. (2002). Human receptors for sweet and umami taste. Proceedings of the National Academy of Sciences of the United States of America, 99, 4692-4696.

PT

Mau, J.-L. (2005). The umami taste of edible and medicinal mushrooms. International Journal of Medicinal Mushrooms, 7, 119-125.

RI

McMullen, L. M., & Stiles, M. E. (1994). Quality of fresh retail pork cuts stored in modified

SC

atmosphere under temperature conditions simulating export to distant markets. Meat Science, 38, 163–177.

NU

Mottram, D. S. (1998). Flavour formation in meat and meat products: a review. Food Chemistry, 62, 415-424.

MA

Mottram, D. S., & Nobrega, I. C. C. (2002). Formation of sulfur aroma compounds in reaction mixtures containing cysteine and three different forms of ribose. Journal of Agricultural and Food Chemistry, 50, 4080–4086.

D

Mottram, D. S., & Whitfield, F. B. (1995). Volatile compounds from the reaction of cysteine,

TE

ribose and phospholipid in low moisture systems. Journal of Agricultural and Food Chemistry, 50, 4080–4086.

AC CE P

Nakai, H., Saito, F., Ikeda, T., Ando, S., & Komatsu, A. (1975). Standard models of pork-colour. Bulletin of the National Institute of Animal Industry, 29, 69–74 (abstract). Ngapo, T. M., Riendeau, L., Laberge, C., Leblanc, D., & Fortin, J. (2012a). “Chilled” pork — Part I: Sensory and physico-chemical quality. Meat Science, 92, 330-337. Ngapo, T. M., Riendeau, L., Laberge, C., & Fortin, J. (2012b). “Chilled” Pork – Part II: Consumer perception of sensory quality. Meat Science, 92, 338-345. Nishimura, T., Rhue, M. R., Okitani, A., & Kato, H. (1988). Components contributing to the improvement of meat taste during storage. Agricultural and Biological Chemistry, 52, 2323–2330. NPPC (1999). Pork quality standards. Des Moines, Iowa, USA: National Pork Board. Pomp, D., Good, B. A., Geisert, R. D., Corbin, C. J., & Conley, A. J. (1995). Sex identification in mammals with polymerase chain reaction and its use to examine sex effects on diameter of day-10 or ‐11 pig embryos. Journal of Animal Science, 73, 1408–1415.

23

ACCEPTED MANUSCRIPT Ryder, J. M. (1985). Determination of adenosine triphosphate and its breakdown products in fish muscle by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry, 33, 678-680.

PT

SAS (2007). SAS users guide: Statistics. Version 9.1. Cary, USA: SAS Institute Inc. Scrine, G.R. (1982). Factors affecting the carriage of meat in containers. International Journal of

RI

Refrigeration - Revue Internationale du Froid, 5, 302-309.

SC

Wood, J. D., Brown, S. N., Nute, G. R., Whittington, F. M., Perry, A. M., Johnson, S. P., Enser, M. (1996). Effects of breed, feed level and conditioning time on the tenderness of pork.

NU

Meat Science, 44, 105–112.

Wood, J. D., Enser, M., Fisher, A. V., Nute, G. R., Richardson, R. I., & Sheard, P. R. (1999).

MA

Manipulating meat quality and composition. Proceedings of the Nutrition Society, 58, 363– 370.

Yamaguchi, S. (1998). Basic properties of umami and its effect on food flavour. Food Reviews

D

International, 14, 139-176.

TE

Yamaguchi, S., Yoshikawa, T., Ikeda, S., & Ninomiya, T. (1971). Measurement of the relative

846-849.

AC CE P

taste intensity of some L-α-amino acids and 5’-nucleotides. Journal of Food Science, 36,

24

ACCEPTED MANUSCRIPT 400 0 d (control) 5 d at 4.0°C

350

PT

13 d at -1.7°C 300

RI

28 d at -1.7°C 43 d at -1.7°C

SC NU

250

MA

200

TE

D

150

100

50

0 0

AC CE P

Particle size (um)

58 d at -1.7°C

20

40

60

80

100

Quantile (%) Fig 1. Mean particle size against quantiles for pork samples without ageing (0 d), aged at 4.0°C for 5 d, or aged at -1.7°C for 13, 28, 43 or 58 d.

25

ACCEPTED MANUSCRIPT Table 1. Physico-chemical characteristics of the 40 pork loins according to meat type and sex of the animal from which the meat was obtained (means with standard deviations in parentheses below;

Type

Sex of the animal

2.56 (1.88)

3.11 (1.59)

2.01 (2.03)

Drip loss48 (%)

4.16 (2.32)

4.88 (1.92)

3.44 (2.50)

4.26 (2.39)

4.07 (2.30)

0.052

0.799

pH

5.67 (0.13)

5.62 (0.11)

5.71 (0.12)

5.69 (0.10)

5.65 (0.15)

0.032

0.346

CIE L*

49.89 (2.45)

50.62 (1.68)

49.16 (2.90)

49.82 (2.52)

49.97 (2.45)

0.065

0.850

CIE a*

8.02 (1.09)

8.03 (1.27)

8.01 (0.91)

8.33 (1.03)

7.71 (1.08)

0.972

0.074

CIE b*

4.28 (0.94)

4.36 (0.89)

4.21 (1.00)

4.37 (0.88)

4.20 (1.00)

0.598

0.584

Protein (%)

24.19 (0.76)

24.22 (0.83)

24.16 (0.71)

24.02 (0.75)

24.36 (0.75)

0.795

0.154

2.04 (0.47)

1.96 (0.45)

2.12 (0.48)

2.08 (0.43)

2.00 (0.51)

0.288

0.577

73.22 (0.58)

73.27 (0.59)

73.17 (0.59)

73.26 (0.62)

73.18 (0.55)

0.580

0.662

Sarcomere length (day 0; μm)

1.83 (0.11)

1.84 (0.11)

1.81 (0.11)

1.84 (0.13)

1.82 (0.09)

0.367

0.660

Sarcomere length (day 7, 4°C; μm)

1.79 (0.08)

1.78 (0.08)

1.79 (0.09)

1.81 (0.07)

1.77 (0.09)

0.559

0.122

Sarcomere length (day 45, -2°C; μm)

1.80 (0.11)

1.83 (0.10)

1.77 (0.12)

1.78 (0.81)

1.81 (0.13)

0.090

0.457

40

20

20

20

20

Number of animals

NU

MA

D

TE

AC CE P

Castrate 2.76 (1.87)

2.35 (1.93)

0.070

0.500

SC

Drip loss24 (%)

Moisture (%)

Export

P-value sex

Female

Intramuscular fat (IMF; %)

Domestic

P-value type

RI

Overall

PT

significant differences, P˂ 0.05, shown in bold).

26

ACCEPTED MANUSCRIPT Table 2. The Sauter mean diameter (D[3,2]), De Brouckere mean diameter (D[4,3]) and oligopeptide concentration in the 40 pork loins according to ageing and meat type (means with

0

5 a

13 b

28 b

43 b

35.1 (13.5)

50.2 (20.3)

32.5 (7.2)

35.3 (12.5)

31.6 (9.0)

D [4, 3] (µm)

121.4 (39.6)

165.3a (57.2)

113.0b (22.0)

124.1b (38.8)

105.8b (28.0)

Oligopeptides (mg BSA equiv/g meat)

16.7 (4.8)

10.6c (3.9)

15.2b (4.3)

20.2a (4.1)

19.8a (2.8)

40

40

40

40

40

Number of animals

MA

A

28.6 (7.4)

35.3 (13.6)

34.9 (13.5)

˂ 0.001

0.872

111.1b (23.7)

109b (20.8)

121.3 (41.6)

121.4 (37.7)

˂ 0.001

0.987

16.0b (2.8)

18.6a (3.2)

16.2 (4.6)

17.2 (5.0)

˂ 0.001

0.025

40

40

20

20

AC CE P

TE

D

Means in a row without a common superscript are significantly different (p<0.05) for ageing time

27

P-value Type

32.4 (7.4)

b

Export

P-value Ageing

Domestic

NU

D [3, 2] (µm)

58

b

SC

Overall

Meat type

RI

Ageing time (5 d 4.0°C or 13-58 d at −1.7°C)A

PT

standard deviations in parentheses below; significant differences, P˂ 0.05, shown in bold).

ACCEPTED MANUSCRIPT Table 3. Mean oligopeptide, free amino acid and nucleotide concentrations according to meat type by ageing interactions in the 40 pork loins (significant differences, P˂ 0.05, shown in bold). Ageing time (5 d 4.0°C or 13-58 d at −1.7°C)A

(µmol/g meat)

0

5

13

28

58

P-value Ageing

PT

Free amino acid

43

15.1b (3.9)

18.9ab (3.5)

18.4ab (1.9)

15.2b (2.6)

19.5a (3.4)

˂ 0.001

Export

11.2c (3.7)

15.2bc (4.7)

21.4a (4.3)

21.1a (3.0)

16.6b (2.8)

17.6b (2.8)

˂ 0.001

0.291

0.933

0.060

0.002

0.069

0.075

P-value Meat Type

Aspartic acid (µmol/g meat)

NU

Domestic

9.9c (4.0)

SC

RI

Oligopeptide (mg BSA equiv/g meat)

0.017d (0.007)

0.058bc (0.025)

0.037c (0.024)

0.082b (0.037)

0.139a (0.068)

0.190a (0.081)

˂ 0.001

Export

0.021e (0.006)

0.062d (0.020)

0.054d (0.026)

0.096c (0.040)

0182b (0.083)

0.306a (0.148)

˂ 0.001

0.089

0.534

0.030

0.253

0.083

0.004

Proline (µmol/g meat)

D

0.245b (0.088)

0.232b (0.052)

0.249b (0.055)

0.281b (0.056)

0.301ab (0.092)

0.372a (0.103)

˂ 0.001

0.247c (0.046)

0.269c (0.059)

0.239c (0.057)

0.281c (0.066)

0.351b (0.102)

0.455a (178)

˂ 0.001

0.954

0.037

0.557

0.997

0.115

0.078

3.57a (1.64)

1.65b (0.54)

1.34b (0.31)

0.89c (0.31)

0.92c (0.21)

0.91c (0.25)

a

b

c

cd

cd

d

AC CE P

Domestic

TE

P-value Meat Type

Export

P-value Meat Type

MA

Domestic

CMP (µg/g)

Domestic

˂ 0.001

4.88 (1.32)

1.77 (0.48)

1.22 (0.44)

1.02 (0.32)

1.00 (0.34)

0.79 (0.29)

0.010

0.457

0.359

0.215

0.358

0.172

Domestic

9.80a (1.10)

8.80b (0.73)

8.69b (0.88)

7.10c (0.78)

6.03d (0.69)

5.13e (0.61)

˂ 0.001

Export

9.47a (1.18)

8.69a (0.85)

7.98b (0.94)

7.22c (0.98)

6.29d (0.85)

5.41e (0.73)

˂ 0.001

0.372

0.680

0.015

0.697

0.294

0.179

Export P-value Meat Type

˂ 0.001

UMP (µg/g)

P-value Meat Type A

Means in a row without a common superscript are significantly different (p<0.05) for ageing time

28

ACCEPTED MANUSCRIPT Table 4. Free amino acids in the 40 pork loins according to ageing and meat type (means with standard deviations in parentheses below; significant differences, P˂ 0.05, shown in bold). Ageing time (5 d 4.0°C or 13-58 d at −1.7°C)A Overall

0

5 e

13 d

28 d

43 c

58 b

a

Domestic

Export

P-value Ageing

P-value Type

0.087 (0.076)

0.120 (0.120)

˂ 0.001

0.004

0.104 (0.102)

0.019 (0.007)

0.060 (0.022)

0.045 (0.026)

0.089 (0.039)

0.160 (0.078)

0.248 (0.131)

Serine

0.849 (0.456)

0.324e (0.084)

0.631d (0.135)

0.575d (0.144)

0.882c (0.173)

1.157b (0.265)

1.5250a (0.388)

0.779 (0.408)

0.918 (0.491)

˂ 0.001

0.002

Glutamic acid

0.826 (0.385)

0.320f (0.085)

0.663e (0.118)

0.582d (0.121)

0.853c (0.103)

1.154b (0.178)

1.386a (0.221)

0.846 (0.383)

0.807 (0.389)

˂ 0.001

0.189

Glycine

1.205 (0.322)

0.966c (0.231)

1.111bc (0.240)

0.988c (0.194)

1.178b (0.211)

1.470a (0.302)

1.516a (0.251)

1.198 (0.332)

1.211 (0.312)

˂ 0.001

0.791

Histidine

1.148 (0.293)

0.883e (0.243)

1.092cd (0.221)

1.037d (0.231)

1.197bc (0.271)

1.239b (0.180)

1.438a (0.273)

1.142 (0.293)

1.153 (0.294)

˂ 0.001

0.810

Alanine

1.852 (0.380)

1.563c (0.249)

1.833b (0.344)

1.603c (0.237)

1.873b (0.292)

2.141a (0.362)

2.097a (0.369)

1.789 (0.386)

1.915 (0.365)

˂ 0.001

0.040

Proline

0.293 (0.107)

0.246d (0.070)

0.250d (0.058)

0.244cd (0.056)

0.281c (0.060)

0.326b (0.099)

0.414a (0.149)

0.280 (0.089)

0.307 (0.121)

˂ 0.001

0.126

Cysteine

0.520 (0.265)

0.193e (0.083)

0.393d (0.113)

0.380d (0.104)

0.566c (0.120)

0.736b (0.167)

0.854a (0.218)

0.529 (0.264)

0.512 (0.266)

˂ 0.001

0.566

Tyrosine

0.535 (0.288)

0.185e (0.039)

0.365d (0.076)

0.363d (0.089)

0.591c (0.107)

0.772b (0.166)

0.935a (0.211)

0.512 (0.275)

0.559 (0.301)

˂ 0.001

0.056

Valine

0.524 (0.279)

0.249e (0.047)

0.377d (0.072)

0.355d (0.074)

0.513c (0.090)

0.709b (0.173)

0.940a (0.291)

0.483 (0.238)

0.565 (0.311)

˂ 0.001

0.006

Methionine

0.371 (0.210)

0.098f (0.024)

0.236e (0.048)

0.265d (0.066)

0.421c (0.069)

0.545b (0.104)

0.664a (0.142)

0.356 (0.197)

0.387 (0.222)

˂ 0.001

0.067

Ornithine

0.043 (0.031)

0.029bc (0.025)

0.047ab (0.037)

0.029c (0.019)

0.037bc (0.018)

0.061a (0.035)

0.057a (0.031)

0.046 (0.034)

0.041 (0.027)

˂ 0.001

0.257

Lysine

0.478 (0.257)

0.183e (0.042)

0.345d (0.080)

0.322d (0.084)

0.490c (0.093)

0.682b (0.147)

0.844a (0.216)

0.456 (0.245)

0.499 (0.268)

˂ 0.001

0.077

Isoleucine

0.390 (0.215)

0.149e (0.038)

0.268d (0.060)

0.261d (0.068)

0.404c (0.071)

0.552b (0.117)

0.709a (0.175)

0.364 (0.200)

0.417 (0.226)

˂ 0.001

0.007

Leucine

0.718 (0.383)

0.277e (0.063)

0.517d (0.115)

0.475d (0.118)

0.743c (0.127)

1.016b (0.221)

1.277a (0.300)

0.685 (0.360)

0.750 (0.404)

˂ 0.001

0.060

Phenylalanine

0.358 (0.179)

0.139e (0.027)

0.255d (0.042)

0.261d (0.048)

0.377c (0.062)

0.494b (0.088)

0.624a (0.135)

0.342 (0.166)

0.374 (0.191)

˂ 0.001

0.035

Total FAA

10.22 (3.690)

5.82f (0.707)

8.45e (1.136)

7.78d (1.069)

10.49c (1.145)

13.21b (2.003)

15.53a (2.802)

9.894 (3.481)

10.535 (3.875)

˂ 0.001

0.061

Number of animals

40

40

40

40

40

40

40

20

20

SC

MA

D

TE

AC CE P

RI

Aspartic acid

NU

(µmol/g meat)

Meat type

PT

Free amino acid

A

Means in a row without a common superscript are significantly different (p<0.05) for ageing time

29

ACCEPTED MANUSCRIPT Table 5. Nucleotides measured in the 40 pork loins according to ageing and meat type (means with standard deviations in parentheses below; significant differences, P˂ 0.05, shown in bold).

Overall

0

5 a

13 a

28 b

43 a

Meat type

58 a

a

Domestic

Export

P-value Ageing

P-value Type

33.70 (6.60)

30.33 (7.50)

˂ 0.001

0.004

32.01 (7.25)

31.52 (7.70)

35.52 (9.27)

28.21 (5.58)

32.70 (5.49)

33.13 (7.13)

30.99 (5.86)

5’-CMP

1.67 (1.39)

4.23a (1.62)

1.71b (0.51)

1.28c (0.38)

0.96d (0.32)

0.96d (0.28)

0.85d (0.28)

1.55 (1.20)

1.78 (1.56)

˂ 0.001

0.030

5’GMP

10.85 (3.86)

14.29a (3.08)

12.87a (2.62)

12.36ab (3.37)

10.19b (2.80)

8.05c (2.69)

7.33c (2.90)

10.87 (3.73)

10.83 (3.99)

˂ 0.001

0.912

5’IMP

385.6 (150.2)

593.9a (49.0)

498.9b (55.5)

453.1c (46.9)

327.8d (47.3)

244.9e (39.0)

188.8f (32.6)

379.8 (150.28)

389.3 (150.64)

˂ 0.001

0.347

5’UMP

7.55 (1.75)

9.63a (1.14)

8.75b (0.79)

8.33c (0.97)

7.16d (0.88)

6.16e (0.77)

5.27f (0.68)

7.59 (1.84)

7.51 (1.66)

˂ 0.001

0.674

5’XMP

1.80 (2.25)

0.09c (0.41)

0.09c (0.35)

0.90bc (1.63)

1.91b (2.01)

3.53a (2.03)

4.26a (1.93)

1.74 (2.15)

1.85 (2.35)

˂ 0.001

0.668

Total 5’-nucleotides

438.5 (154.0)

653.7a (49.1)

557.8b (58.3)

504.2c (49.1)

380.8d (47.6)

296.7e (42.1)

237.5f (32.5)

435.3 (154.6)

441.6 (153.9)

˂ 0.001

0.535

40

40

40

40

40

40

20

20

A

40

SC

NU

MA

D

Number of animals

RI

5’-AMP

TE

(µg/g meat)

Ageing time (5 d 4.0°C or 13-58 d at −1.7°C)B

PT

NucleotideA

B

AC CE P

5’-AMP, 5’-adenosine monophosphate; 5’-CMP, 5’-cytosine monophosphate; 5’-GMP, 5’-guanosine monophosphate; 5’-IMP, 5’-inosine monophosphate; 5’-UMP, 5’-uridine monophosphate; 5’-XMP, 5’-xanthosine monophosphate. Means in a row without a common superscript are significantly different (p<0.05) for ageing time

30

ACCEPTED MANUSCRIPT Table 6. Equivalent umami concentration (EUC) of the 40 pork loins according to ageing and meat type (means with standard deviations in parentheses below; significant differences, P˂ 0.05, shown

0

5

13

28

43

EUC (g MSG/100 g dry weight)

2.02 (0.55)

1.37c (0.32)

2.42a (0.50)

1.94b (0.45)

2.11b (0.40)

2.18ab (0.43)

EUC (g MSG/100 g wet weight)

0.540 (0.146)

0.366c (0.085)

0.649a (0.135)

0.519b (0.118)

0.564b (0.106)

Number of animals

40

40

40

40

40

Export

2.10b (0.56)

2.04 (0.51)

1.99 (0.59)

˂ 0.001

0.628

0.582ab (0.113)

0.561b (0.146)

0.546 (0.136)

0.534 (0.156)

˂ 0.001

0.660

40

40

20

20

NU

MA

D TE 31

P-value Type

Domestic

Means in a row without a common superscript are significantly different (p<0.05) for ageing time

AC CE P

A

P-value Ageing

58

SC

Overall

Meat type

RI

Ageing time (5 d 4.0°C or 13-58 d at −1.7°C)A

PT

in bold).

ACCEPTED MANUSCRIPT Table 7. Contents of taste characteristic FAA of the 40 pork loins according to ageing and meat type (means with standard deviations in parentheses below; significant differences, P˂ 0.05, shown

Ageing time (5 d 4.0°C or 13-58 d at −1.7°C)A 5

13

28

43

Taste characteristic (mg/g dry weight)

Meat type

58

Domestic

Export

RI

0 B

PT

in bold).

P-value Ageing

P-value Type

0.19f (0.05)

0.39d (0.07)

0.34e (0.08)

0.51c (0.07)

0.71b (0.13)

0.89a (0.18)

0.51 (0.25)

0.50 (0.27)

˂ 0.001

0.749

Sweet

0.65f (0.09)

0.86d (0.14)

0.76e (0.11)

0.97c (0.15)

1.17b (0.20)

1.30a (0.25)

0.91 (0.27)

1.00 (0.28)

˂ 0.001

0.010

Bitter

0.92e (0.18)

1.34d (0.20)

1.28d (0.23)

1.71c (0.20)

2.10b (0.32)

2.60a (0.52)

1.60 (0.58)

1.72 (0.68)

˂ 0.001

0.083

Tasteless

0.23e (0.04)

0.44d (0.09)

0.42d (0.10)

0.67c (0.12)

0.90b (0.19)

1.09a (0.25)

0.60 (0.32)

0.65 (0.35)

˂ 0.001

0.073

Total

1.97e (0.28)

3.03d (0.45)

2.80d (0.46)

3.87c (0.46)

4.88b (0.78)

5.88a (1.14)

3.61 (1.38)

3.86 (1.55)

˂ 0.001

0.075

Bitter Tasteless Total

Number of animals A

NU

MA

D 0.72d (0.13)

AC CE P

Sweet

0.34f (0.09)

TE

Taste characteristicB (µmol/g meat) MSG-like

SC

MSG-like

0.63e (0.14)

0.94c (0.12)

1.31b (0.23)

1.63a (0.33)

0.93 (0.45)

0.93 (0.50)

˂ 0.001

0.875

1.89f (0.28)

2.47d (0.41)

2.18e (0.32)

2.76c (0.40)

3.30b (0.56)

3.62a (0.67)

2.57 (0.73)

2.83 (0.77)

˂ 0.001

0.005

1.70e (0.31)

2.51d (0.37)

2.39d (0.42)

3.23c (0.37)

4.01b (0.63)

4.99a (1.02)

3.02 (1.12)

3.26 (1.34)

˂ 0.001

0.047

0.37e (0.07)

0.71d (0.15)

0.69d (0.17)

1.08c (0.19)

1.45b (0.30)

1.78a (0.41)

0.97 (0.52)

1.06 (0.53)

˂ 0.001

0.058

4.29f (0.58)

6.41d (0.95)

5.88e (0.91)

8.01c (0.93)

10.08b (1.56)

12.02a (2.31)

7.49 (2.74)

8.08 (3.10)

˂ 0.001

0.035

40

40

40

40

40

40

20

20

Means in a row without a common superscript are significantly different (p<0.05) for ageing time

B

MSG-like, glutamic acid + aspartic acid; sweet, alanine + serine; bitter, histidine + isoleucine + leucine + phenylalanine + valine; tasteless, lysine + tyrosine

32

ACCEPTED MANUSCRIPT Highlights Canadian pork for the local and Japanese markets differed only in pH (˂ 1 unit)



Ageing conditions influenced the concentrations of protein breakdown products



Oligopeptides and amino acids generally increased with longer ageing periods



Nucleotides generally decreased with longer ageing periods



Umami did not differ in pork aged 5 d at 4.0°C and 43 d at -1.7°C (chilled export)

AC CE P

TE

D

MA

NU

SC

RI

PT



33