Changes in proteolysis during the dry-cured processing of refrigerated and frozen loin

Accepted Manuscript Changes in proteolysis during the dry-cured processing of refrigerated and frozen loin Adela Abellán, Eva Salazar, Javier Vázquez, José Ma Cayuela, Luis Tejada PII:

S0023-6438(18)30513-9

DOI:

10.1016/j.lwt.2018.06.002

Reference:

YFSTL 7186

To appear in:

LWT - Food Science and Technology

Received Date: 19 October 2017 Revised Date:

31 May 2018

Accepted Date: 2 June 2018

Please cite this article as: Abellán, A., Salazar, E., Vázquez, J., Cayuela, José.Ma., Tejada, L., Changes in proteolysis during the dry-cured processing of refrigerated and frozen loin, LWT - Food Science and Technology (2018), doi: 10.1016/j.lwt.2018.06.002. 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.

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Changes in proteolysis during the dry-cured processing of refrigerated and frozen loin

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Adela Abellán, Eva Salazar*, Javier Vázquez, José Mª Cayuela and Luis Tejada

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Departamento de Tecnología de la Alimentación y Nutrición, UCAM-Universidad Católica de

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Murcia, Campus de los Jerónimos, 30107 Guadalupe, (Murcia), Spain.

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*e-mail: [email protected]

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Abstract

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The influence of frozen storage of dry-cured loin before its processing has been evaluated in terms

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of proteolysis, pH, dry matter (DM), NaCl and sensorial acceptance. The frozen storage did not

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affect total nitrogen (TN), non-protein nitrogen (NPN), or proteolysis index (PI), showing values at

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day 50 of 1.14 and 1.11 g nitrogen/kg of DM; 0.093 and 0.091 g nitrogen/kg of DM and 8.2-8.2, in

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refrigerated (R) and pre-cure frozen (PF), respectively. Initially, PF loin values of DM were higher

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(2.91 g/kg of dry-cured loin) than R (2.71 g/kg of dry-cured loin). NaCl content of PF loin was

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higher than R throughout the processing (1.15 and 1.38 g/kg of DM at day 50, respectively). The

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total amino acid ((TFAA) concentration was higher in PF than in R, with the major differences at

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day 50 (27.5 and 19.9 g/kg, respectively). The concentration of all free amino acids (FAA) was

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affected by the freeze-thaw process. From day 30 onwards, the concentration of FAA increased in

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PF to such an extent that after 50 days significantly higher values were observed for all FAA except

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arginine, methionine and valine. There were no differences in consumer acceptance between R and

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PF dry-cured loin.

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Highlights

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Fax: +34 968278578

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- Nitrogen fractions were not affected by frozen storage in dry-cured loin 1

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- Frozen storage of the raw muscle caused a release of amino acids in dry-cured loin

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- Pre-cure freezing did not influence the sensory acceptance of dry-cured loin

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- Pre-cure freezing of loin provides technological and economic advantages

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Keywords free amino acids; proteolysis; dry-cured loin; pre-freezing

1. Introduction

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The proteolytic enzyme system in muscle is quite complex and is comprised of endo and exo-

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peptidases. Initially, proteolytic phenomena are due to the endo-peptidases (calpains I and II or

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cathepsins B, D, H and L), which hydrolyse the proteins in interior molecular areas and generate

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smaller fragments of polypeptide chains. Subsequently, these polypeptides are degraded to small

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peptides by tripeptidase and dipeptidase enzymes. Certain aminopeptidase enzymes, including

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alanyl, leucyl and tyrosyl, then act upon them releasing the terminal amino acids at the end of the

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polypeptide chains (Toldrá, 2006).

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Freezing meat and meat products may cause changes in their physical characteristics (moisture loss,

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changes in texture), chemical characteristics (lipolysis, oxidation, denaturation and protein

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aggregation), and sensory characteristics (Pérez-Palacios, Ruíz, Martín, Barat, & Antequera, 2011).

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However, scientific studies that describe the effect that freezing the meat has on the quality of cured

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products are very scarce. The majority of studies carried out have been on ham (Bañón, Cayuela,

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Granados, & Garrido, 1999; Flores, Soler, Aristoy, & Toldrá, 2006; Flores, Aristoy, Antequera,

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Barat, & Toldrá, 2009; Pérez-Palacios, Ruíz, Barat, Aristoy, & Antequera, 2010). Specifically, with

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respect to proteolysis, disparate results have been observed regarding the effect of prior frozen

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storage on non-protein nitrogen (NPN) during processing. For Iberian hams, no significant

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differences were observed between the concentration of NPN in refrigerated hams and those that

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had been frozen/thawed (Flores et al., 2006; Pérez-Palacios et al., 2010). However, Wang (2001)

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obtained NPN content during the maturation process of Taiwanese ham manufactured from

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refrigerated meat than that produced from frozen/thawed meat. On the other hand, Bañón et al.

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(1999) observed higher NPN values in prefrozen hams. This is due to the fact that the proteins are

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modified during freezing, and these modifications provide a more favourable environment for

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muscle protease activity.

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The concentration of free amino acids demonstrates the degree of proteolysis reached during the

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maturation of cured meat products (Flores et al., 2009; Toldrá, Aristoy, & Flores, 2000). There are

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no studies that evaluate the effect of freezing on the evolution of amino acids in dry-cured loin.

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Only a few studies have been carried out on dry-cured ham and they are not consistent with each

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other. In Iberian ham, Pérez-Palacios et al. (2010) observed a greater concentration of amino acids

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in pre-cure frozen dry-cured ham, than in that which was refrigerated. Flores et al. (2006) observed

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the same in dry-cured ham obtained from a commercial crossbreed genotype. However, Flores et al.

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(2009) did not observe differences in free amino acid content between fresh and thawed Iberian

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hams throughout the whole process. On the other hand, Wang (2001) noted higher amino acid

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content in refrigerated hams than in the prefrozen hams.

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Dry-cured loin is a different product from dry-cured ham. The anatomical piece, muscles that

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compose it and chemical composition are different. Dry-cured loin is obtained from the

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Longissimus thoracis et lumborum while dry-cured ham is obtained from the whole pork leg

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(composed by different muscles as Biceps femoris, Semitendinosus or Semimembranosus).

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Therefore, the piece is higher and muscles are different so, freezing affects it in a different way. On

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the other hand, processing is different too. Dry-cured loin muscle is seasoned with a commercial

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mixture of salt, dextrose and nitrate/nitrite and keep for 2 days at 4º C to allow the seasoning

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mixture to penetrate. After that, a commercial mixture (Loin marinade spices) of paprika and

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natural spices is added at 10 g/kg weight and the loins are keep for a further 4 days at 4 Cº (Salazar, 3

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Abellán, Cayuela, Poto and Tejada, 2016). Dry-cured ham production involves a salted stage for 9-

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15 days, depending on the piece weight (1 day/kg). In addition, processing stages and ripening

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conditions (relative humidity and temperature) are different. Dry-cured loin total processing time is

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50 days while in dry-cured ham is 18 months (at least) with highly different stages (green stage,

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salting, post-salting, drying and ripening stage; Pérez-Palacios et al., 2010).

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Thus far, no studies have been carried out on the effect that frozen storage has on proteolysis in dry-

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cured loin during processing. There has only been one study conducted, which found that freezing

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meat before its processing affected the oxidative stability of the meat proteins (Lorido, Ventanas,

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Akcan, & Estevez, 2016). The storage of loins in freezing temperatures before the manufacturing

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process could have a series of technological and economic advantages. For example, it would make

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it possible to process cuts with a more homogenous weight and, as such, produce loins with lower

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variability. In addition, the seasonality of the product and the consequent changes in market prices

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could be avoided. Another advantage would be that it would allow greater independence with

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regards to the pace of work in slaughterhouses and flexibility in production levels. This is

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particularly important when it comes to native pig breeds, which are less standardised and produced

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in lower numbers.

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Furthermore, the proteolytic activity of cured meat products is affected by dry extracts, pH and salt

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concentration (Toldrá, Cerveró, & Part, 1993), meaning it is necessary to evaluate how frozen

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storage influences these parameters. In addition, it is also important to assess the sensory acceptance

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of the dry-cured loins, as proteolysis significantly influences the organoleptic characteristics and

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quality of cured products (Sforza et al., 2006).

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The aim of this work was to evaluate the influence of the pre-cure freezing of loin on

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physicochemical parameters, sensory acceptance, proteolytic changes and the evolvement of amino

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acids during processing.

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2. Materials and Methods 2.1 Raw materials and dry-curing process

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12 loins were obtained from the Longissimus thoracis et lumborum (LTL) muscle of the Chato

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murciano pig breed. The pigs (females) were reared on a pig farm (intensive system) and fed a

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commercial diet from 45 - 50 days of age until their slaughter. Feed and water were provided ad

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libitum. The pigs were all periodically weighed from 100 days of age until the day of departure for

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slaughter. The growth rates were 420 - 510 g/day. The average live weight at the time of slaughter

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was 120 kg (at 9 months of age). Whole muscles were removed from the left side of the carcasses.

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The loins were frozen in an industrial freezer at - 40°C and stored for 12 months at - 20°C. They

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were then thawed and stored at 3°C for 4 days.

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Twelve months later, another 12 raw loins were obtained from pigs of the same genetic breed.

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These pigs were also fed the same diet as those used for the frozen (PF) loins. However, they were

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slaughtered 2 days before the PF loins were fully thawed. Both batches were processed into dry-

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cured loins following the traditional process at a meat-processing plant, as described by Salazar et

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al. (2016). Samples (60g) were taken on days 0, 7, 30 and 50 in the middle of the muscle. Samples

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were trimmed of adipose fat and connective tissue and ground in a commercial mincer (Moulinex

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A320R1, SEB group, Selongey, France), obtaining a homogenized sample of > 0.1 mm of particle

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size. Immediately, physicochemical analyses were conducted and the remaining sample was frozen

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before analysing nitrogen fractions and free amino acids. After sampling 7 and 30, samples were

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covered with paraffin, to allow normal ripening. On processing day 50, muscles where cut into two

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pieces, one of which was used to determine physicochemical, nitrogen fractions and free amino

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acids, and the other for sensory analysis. The samples were then minced and stored at - 80 °C until

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analysis. All samples were analysed in triplicate.

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2.2 Physicochemical determination

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Dry matter was determined after sample desiccation at 105°C in an oven (until it reached a constant

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weight). Dry matter content was calculated as follow: Dry matter (g/kg) = (Weight of dried

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sample/Weight of sample) × 100. The results were expressed in g/kg of dry-cured loin as mean

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values.

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To determine chloride content, 10 g of muscle was boiled for 1h in 100 mL ethanol (400 g/L), then

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deproteinised by adding 2 mL of a solution of ferrocyanide (150 g/L) and zinc acetate (300 g/L)

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The mixture was filtered and the chloride content was determined using the Volhard method

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(AOAC, 2000). NaCl values were expressed in g/kg of dry matter as mean values. The pH was

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measured by probing the loin directly with the glass electrode of a Beckman 3500 digital pH-meter

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(Beckman Coulter, Inc., Brea, United States)

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2.3 Determination of nitrogen fractions

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Total nitrogen (TN) content and non-protein nitrogen (NPN) was determined using the Kjeldahl

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method (AOAC, 2000), according to Stadnik and Dolatowski (2013). The results were expressed in

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g nitrogen/kg of dry matter as mean values. The proteolysis index (PI) was calculated as the

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percentage ratio between NPN and TN.

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2.4 Determination of amino acids

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Amino acid content was determined following the procedure described by Abellán et al. (2012).

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Aminoacidic nitrogen was extracted through peptide precipitation of the non-protein fraction with

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sulfosalicylic acid (100 g/L). Free amino acid extract was pre-column derivatized with ortho-

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phthaldialdehyde (OPA) according to Jones, Pablo, and Stein (1981). The OPA derivatives were

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analysed in a HPLC (Shimadzu LC-10AD, Kyoto, Japan) equipped with a fluorescence detector

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(RF-10A XL) and an eclipse XDB-C18 column (5 µm / 4.6 x 250 mm). The column temperature

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was kept at 30 ºC. The separation was achieved in 60 min using a gradient between two solvents:

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0.05 mol/L sodium acetate (solvent A) and methanol (solvent B). The flow rate was 1.5 mL / min

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and the solvent gradient was: initial 12 % B maintained for 0.01 min; then a linear change to 25 %

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B over 15 min maintained for 0.01 min, 50 min linear change to 100 % B and then 10 min liner

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change to 0 % B.

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Identification and quantification was based on retention times and peak area integration of the

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reference compounds (Sigma, St. Louis, MO).

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Solutions between 0.00175 g/L and 0.028 g/L were prepared of the follow L-amino acids: aspartic

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acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), threonine (Thr), glycine (Gly),

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arginine (Arg), alanine (Ala), tyrosine (Tyr), histidine (His), methionine (Met), tryptophan (Trp),

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valine (Val), phenylalanine (Phe), isoleucine (Ile), leucine (Leu) and lysine (Lys).

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The results were expressed in g /kg of dry matter as mean values.

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2.5 Consumer evaluation

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A hedonic test of the two batches of dry-cured loin was carried out following the procedure

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described by Salazar et al. (2013) and in line with UNE-ISO 6658:2005 (AENOR, 2008) and UNE

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4121:2003 (AENOR, 2006) standards, with a consumer panel made up of 50 tasters. All

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participants in the study took part voluntarily without having received any kind of previous training.

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The panel of consumers tasted, in two different sessions, three samples taken in combinations of

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two (two with similar pre-treatment -R or PF loin- and one with the other pre-treatment- R or PF

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loin), following an experimental design in which all were tasted by consumers. The samples were

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presented to each consumer on dishes marked with a random three-digit code, along with an

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evaluation form. The form included a section for the acceptability of the product's appearance,

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smell, texture and taste, which were evaluated by assigning a numerical value with a verbal hedonic

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scale between 1 (I dislike it very much) and 5 (I love it).

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The results are presented as averages of the scores awarded by the consumer panel for each

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attribute.

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2.6 Statistical analysis

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Statistical analysis was carried out using the SPSS version 21.0 software package (IBM

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Corporation, Armonk, NY, USA). Analysis of variance using a one-way ANOVA procedure and

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Tukey’s test were performed to determine the effect of treatment (refrigeration or freezing) and

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processing time on the amino acid content of dry-cured loin.

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3. Results and discussion

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3.1 Physicochemical parameters

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Table 1 shows the pH values, dry matter (DM) and NaCl during the dry-cured processing of PF and

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R loins. Frozen storage and processing time significantly affected the pH values of the dry-cured

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loin (P < 0.05 and P < 0.01, respectively). In raw meat (day 0), the values were higher in PF loins

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than in R loins, probably due to both pre-slaughter factors (including transport, housing, perhaps

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fighting.). These differences in pH were also observed in meat of different species after frozen

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storage (Ablikim et al., 2016; Farouk and Swan, 1998, Seong et al., 2017). However, Pérez-Palacios

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et al. (2010), found no significant differences in Iberian dry-cured ham.

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processing, the pH in both types of dry-cured loin was similar, due to the fact that in PF it decreased

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more than in R during the processing.

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Frozen storage significantly affected the DM content of the loins (P < 0.05), with higher values

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observed in PF raw meat than in R due to the water loss that occurs during thawing process

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After 50 days of

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(Jalang'O Saul, & Lawrie, 1987). However, the water loss was higher in PF during the early days of

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processing. Consequently, after day 7 there was no significant difference found between the DM

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content of each type of loin. This could be due to the higher water content of R loins before

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processing and because they have a lower water-holding capacity (WHC) than PF loins due to a

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lower pH and NaCl content. There are no references on the effect that the frozen storage of the raw

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muscle has on physicochemical parameters in dry-cured loin, only in dry-cured ham. Grau,

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Albarracín, Toldrá, Antequera, and Barat (2008) observed that the use of either fresh or thawed raw

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material made no difference to the moisture content, water activity or weight loss of Iberian hams at

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the end of the post-salting stage. Bañón et al. (1999) observed greater DM values in dry-cured hams

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that were frozen before curing.

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Processing time and frozen storage affected NaCl content significantly (P < 0.001, in both cases). In

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both R and PF, the greatest increase took place during the first 7 days of processing. This is due to

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the penetration and diffusion of salt through the muscle mass via osmosis phenomena (Gou,

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Guerrero, Gelabert, & Arnau, 1996). Consequently, this period is also when the greatest loss of

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moisture took place, showing a positive correlation between DM and NaCl (P < 0.05) due to the

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increase of booth. The increase in NaCl concentration was more intense in PF than in R, with

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significantly higher values (P < 0.05) observed on days 7, 30 and 50 of processing. Similar results

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were obtained in dry-cured hams (Bañón et al., 1999; Grau et al., 2008). The process of

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freezing/thawing the meat produces changes in the muscle structure, favouring the penetration of

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salt, due to the increase in free water content (Bañón et al., 1999; Wang, 2001).

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3.2 Parameters relating to proteolysis

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Table 1 shows the effect of freezing on total nitrogen (TN), non-protein nitrogen (NPN) content and

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on the proteolysis index (PI) of dry-cured loin during processing.

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NPN, PI, and total free amino acid (TFAA) values increased significantly (P < 0.001) during

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processing in R and PF, while TN decreased (P < 0.001). Frozen storage did not affect the TN, NPN

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content, or PI (P > 0.05), with very similar values for PF and R at the raw meat stage (day 0) and

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throughout processing according to Pérez-Palacios et al. (2010) in Iberian dry-cured ham. Flores et

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al. (2009) observed that thawed hams showed similar cathepsin activity values to fresh salted

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Iberian hams and that this activity was maintained after the post-salting stage. Wang (2001)

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obtained higher NPN content during the processing of Taiwanese ham prepared with refrigerated

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meat than that made with frozen/thawed meat. On the other hand, Bañón et al. (1999) obtained

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higher PI values in ham that underwent a preliminary freeze/thaw, as the proteins were more

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vulnerable to attacks by proteolytic enzymes. In this regard, Lawrie and Ledward (2006) affirm that

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ice crystals (produced by freezing intramuscular water) cause a denaturation of proteins, due to a

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double mechanism of ionic strength and translocation of intra-extracellular water. The denatured

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proteins are more sensitive to the proteolytic enzymes released in the sarcoplasm and extracellular

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spaces as a consequence of membrane perforations by ice crystals. The fact that levels of NPN were

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not influenced by these changes during the freezing of the loin may be due to the differences

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observed between R and PF in terms of pH, DM, and salt values. The higher values of DM and,

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above all, of salt observed in PF prevented a further increase in the NPN and PI values, although the

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freezing process created more favourable conditions for protease tissue. With regards to this, it has

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been observed that protease activity (calpain and cathepsins) decreases with an increase in salt

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content (Martín, Córdoba, Antequera, Timón, & Ventanas, 1998; Ruíz-Ramírez, Arnau, Serra, &

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Gou, 2006; Sárraga, Gil, Arnau, Monfort, & Cusso, 1989; Toldrá, Miralles, & Flores, 1992) and a

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reduction in water content (Toldrá et al., 1993; Toldrá, Flores, & Sanz, 1997). Proteases can be

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denatured by an increase in ionic strength caused by an increase in salt content, as observed in

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cathepsins (Toldrá et al., 1992)

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3.3 Evolution of free amino acids

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Table 2 shows the free amino acid values (mg/g dry extract) in the PF and R loins on days 0, 7, 30

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and 50 of processing. The relevant values from the ANOVA and the multiple comparison of

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individualised means via Tukey’s test are also shown.

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The total free amino acid content (TFAA) concentration was higher in PF than in R throughout

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processing, being statistically significant on days 0, 30 and 50. The greatest differences are found

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on day 50 of processing, as a result of a greater release of amino acids in the frozen-stored loin.

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Pérez-Palacios et al. (2010) obtained similar results in Iberian dry-cured ham, where the total amino

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acid and dipeptide content was higher in ham that had been frozen prior to curing than in

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refrigerated hams at the raw meat stage, as well as in the drying and the final stage. They were

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relatively similar during the intermediate phases. In commercial crossbreed genotype dry-cured

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ham, a higher concentration of free amino acids is also found in frozen/thawed ham than in

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refrigerated ham during the salting and post-salting stages of manufacturing. This is due to higher

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proteolytic enzyme and aminopeptidase activity in the frozen/thawed hams (Flores et al., 2006).

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Salt influences the activity of exopeptidases, thus, it has been observed that it activates arginine

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aminopeptidase and reduces the activity of alanylaminopeptidases, pyroglutamyl aminopeptidases

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(Flores et al., 1997; Toldrá et al., 2000) and dipeptidylpeptidase (Sentandreu and Toldrá, 2001). In

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our study, the concentration of total free amino acids was higher in PF loins, showing that structural

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changes in muscle during the freezing/thawing process had a greater influence on the enzymatic

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activity than the NaCl concentration.

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It is noteworthy that in Iberian dry-cured ham at the raw meat stage the concentration of TFAA was

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higher in the samples stored in the freezer than in those that were refrigerated (991.10 and 762.63

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mg/100 g dry muscle matter) (Pérez-Palacios et al., 2010), the same as the Chato murciano dry-

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cured loin. Therefore, a release of amino acids took place during the frozen storage of the different

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muscles, which means that aminopeptidase activity took place. With regard to this, Khan (1966)

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reported that free amino acid and peptide content increased during frozen storage, something which

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suggests residual cathespin activity.

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The concentration of all individual free amino acids was affected significantly by the freeze/thaw

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process (table 2). The evolution of the free amino acid content in the R and PF dry-cured loins was

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not the same during processing. At the raw meat stage (day 0), the concentration of His, Gly+Thr,

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Arg, Tyr, Ala, Trp, Phe, Ile, Leu and Lys was significantly higher in PF than R (P < 0.05). This is

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because they were released by proteolytic activity that takes place during frozen storage, as

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mentioned above. In Iberian dry-cured ham, it has also been observed that most amino acids were

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present in higher levels in PF than in R ham at the raw meat stage (Pérez-Palacios et al., 2010). On

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day 7, the concentration of most amino acids was the same in both PF and R dry-cured loins, while

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significant differences were maintained in His, Tyr, Ala, Leu, and Lys. It is worth noting that Met

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values were significantly higher in the R than in PF loins from day 7 to day 30 of processing (P <

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0.05). Finally, from day 30 onwards the concentration of amino acids increased significantly in PF

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dry-cured loin to such an extent that on day 50 significantly higher values were observed (P < 0.05)

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for all amino acids except Arg, Met and Val. Pérez-Palacios et al. (2010) also noted a higher content

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of most of the amino acids in the previously frozen hams at the end of the processing. As

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mentioned, this fact highlights increased aminopeptidase activity in the PF dry-cured loin at the end

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of processing. This is probably due to an increased release of these enzymes caused by the physical

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change brought about by ice crystals (Flores et al., 2006), along with the fact that after the freezing

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proteins are more vulnerable to their activity (Bañón et al., 1999).

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3.4 Consumer evaluation

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In order to evaluate the effect of proteolysis on sensory acceptance, a consumer evaluation of the R

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and PF dry-cured loin was carried out on days 30 and 50 of processing (table 3). There were no

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significant differences (P > 0.05) between the acceptance of the appearance, smell, texture, or

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flavour of R and PF in either of the two periods studied. The consumers gave high scores to both

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types of dry-cured loin. The differences observed in the concentration of amino acids in PF did not

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influence its sensory acceptance.

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The freezing of the raw material did not influence consumers’ opinions of commercial crossbreed

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genotype hams either, despite a more intense proteolysis being observed (Bañón et al., 1999). Other

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authors are also in agreement that the sensory quality of dry-cured ham is retained when the raw

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material is frozen. For Iberian dry-cured ham, the consumer panel noted significantly less hardness

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in thawed hams, while not detecting any differences in the smell, taste or acceptability of the ham

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(Motilva, Toldrá, Nadal & Flores, 1994).

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4. Conclusions

A year of frozen storage did not affect non-protein nitrogen (NPN) content or proteolysis index (PI).

290

Although structural changes in the muscle during the freezing/thawing process created favourable

291

conditions for protease tissue, this effect is counteracted by a higher salt content.

292

Pre-cure freezing causes a greater release of amino acids in the dry-cured loin during processing

293

(mainly in the final stage). This is probably due to more intense enzyme activity due to changes

294

produced in the muscle during freezing. The concentration of most of the individual amino acids

295

was higher in PF, mainly at the end of the processing. During the frozen storage, a release of amino

296

acids occurs. Despite changes being produced in the amino acid content, these did not influence the

297

acceptance of the product by the consumer.

298

Frozen storage of the LTL for twelve months, for its later use as a dry-cured loin, did not have a

299

negative effect on proteolysis during processing nor on the sensory acceptance of the final product.

300

Therefore, pre-cure freezing could be used in the manufacture of dry-cured loin, providing both

301

technological and economic advantages.

AC C

EP

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289

13

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Acknowledgements

303

The authors thank the UCAM-Universidad Católica de Murcia (Spain) for their financial support

304

for the project (PMAFI-21/2010).

305

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ACCEPTED MANUSCRIPT Table 1 Changes in physicochemical parameters and parameters related to proteolysis during drycured processing of refrigerated and frozen loins. The results are presented as means values.

pH

Dry matter1

NaCl2

Total Nitrogen3

Non Protein Nitrogen3

Proteolysis Index4

Refrigerated

5.63abc

2.71d

0.11a

1.34a

0.048a

3.6a

Pre-cure frozen

5.85d

2.91e

0.13a

1.28a

0.045a

3.5a

Refrigerated

5.66abc

3.09a

0.94b

1.31a

0.054ab

4.1ab

Pre-cure frozen

5.73cd

3.19a

1.17cd

1.28a

0.056ab

4.4ab

Refrigerated

5.65abc

3.51b

0.99b

Pre-cure frozen

5.70bcd

3.57b

1.27d

Refrigerated

5.59a

3.92c

1.15c

Pre-cure frozen

5.61ab

4.03c

SE6

0.03

Time Treatment

Processing time Treatment

Day 30

SC

Day 7

RI PT

Day 0

0.066b

5.5b

1.21ab

0.064b

5.3b

1.14b

0.093c

8.2c

1.38de

1.11b

0.091c

8.2c

0.06

0.05

0.01

0.001

0.3

0.0121

0.0001

0.0001

0.0012

0.0001

0.0001

0.0412

0.0312

0.0152

0.5732

0.7013

0.6162

Interaction 0.9801 0.7321 0.7912 0.9801 0.7303 g/ kg of dry-cured loin 2 g/kg of dry-matter 3 g nitrogen /kg of dry-matter 4 Expressed as percentage of dry-matter; Proteolysis Index: 100 × (non protein nitrogen/total nitrogen). 5 Significant at P < 0.05. 6 Standard Error of ANOVA a,b,c,d,e Values within a column with different superscripts differ significantly at p < 0.05(Tukey Test).

0.7910

Day 50

P-value5

AC C

1

EP

(n= 96)

TE D

Results of ANOVA

M AN U

1.21ab

ACCEPTED MANUSCRIPT

Table 2. Changes in the free amino acid content during dry-cured processing of refrigerated and frozen loins.

Refrigerated

0.05ab

0.40a

Pre-cure frozen

0.03a

0.43a

Refrigerated

0.05ab

0.28a

Pre-cure frozen

0.05ab

Refrigerated

Asn

Ser

His

Arg

Tyr

Ala

0.061ab

0.21a

1.14a

0.6a

1.25a

0.12ab

0.55a

0.61b

0.033a

0.27ab

1.44b

0.9b

1.60b

0.31de

1.023c

0.090b

0.18a

2.05bc

0.8ab

1.68b

0.09a

0.43a

0.31a

0.079ab

0.22a

0.87a

1.0bc

0.09b

0.69b

0.262c

0.41bc

1.93b

1.1bc

Pre-cure frozen

0.16c

0.91b

0.331d

0.44c

2.14bc

1.4c

Refrigerated

0.28d

1.58c

0.481e

0.75d

2.72c

Pre-cure frozen

0.35e

1.96d

0.601f

0.98e

3.82d

SE2

0.02

0.09

0.024

0.05

0.11

P-value1

Time

0.032

0.042

0.040

0.031

(n= 96)

Treatment

0.000

0.000

0.000

0.000

Interaction

0.008

0.023

0.000

0.081

Gly+Thr

Processing time Treatment Day 0

Met

Val

Phe

Ile

Leu

Lys

TFAA

0.610de

0.74b

0.37ab

0.58b

0.25b

0.42a

7.9a

0.73c

0.531cd

0.82b

0.50c

0.70c

0.58c

0.86b

10.7cd

0.45a

0.461

0.33

0.27

0.35

0.14

1.22

c

a

a

a

a

c

8.9ab

1.71b

0.19bc

1.00c

0.52ab

0.242b

0.44a

0.30a

0.43a

0.30b

1.02ab

9.7bc

1.28a

0.24cd

0.68ab

0.51ab

0.540cd

0.82b

0.41b

0.66bc

0.46bc

1.20c

10.8d

1.15a

0.37e

0.72b

0.45a

0.131a

1.21c

0.55c

0.70c

0.51bc

1.29cd

12.9e

2.1d

2.33c

0.65f

1.52d

0.58b

0.611de

2.05d

0.51c

1.05d

0.97d

1.68d

19.9f

3.2e

2.41c

0.76g

2.43e

0.92d

0.678e

2.12d

0.88d

1.61e

1.56e

3.24e

27.5g

0.1

0.08

0.03

0.09

0.04

0.025

0.06

0.03

0.03

0.04

0.08

0.4

Day 30

EP

TE D

Day 50

Results of ANOVA

M AN U

Day 7

Trp

RI PT

Glu

SC

Asp

0.000

0.042

0.001

0.000

0.000

0.000

0.036

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.065

0.000

0.000

0.001

0.000

0.000

0.000

0.000

0.000

0.000

0.000

AC C

0.006

Results (mean) are expressed as g/Kg of total solids. TFAA: Total free amino acid 1 Significant at P < 0.05. 2 Standard Error of ANOVA a,b,c,d,e Values within a column with different superscripts differ significantly at p < 0.05 (Tukey Test).

ACCEPTED MANUSCRIPT

Table 3 Changes in the acceptability of the sensory attributes1 during dry-cured processing of refrigerated and frozen loins. The results are presented as means values. Appearance

Odour

Refrigerated Pre-cure frozen

3.9a 4.1a

3.8a 3.9a

Refrigerated Pre-cure frozen

3.8a 3.8a

3.8a 3.6a

SE3 Time Treatment Interaction

0.2 0.60 0.44 0.55

0.2 0.85 0.48 0.49

Texture

Taste

Processing time Treatments

Day 50

2

P-value (n=48)

3.4a 3.6a

3.7a 3.8a

3.4a 3.4a

3.6a 3.0a

0.2 0.61 0.76 0.59

0.2 0.19 0.05 0.16

SC

Results of ANOVA

RI PT

Day 30

AC C

EP

TE D

M AN U

1 Each attribute was scored assigning a numerical value through a verbal hedonic scale between 1 (I dislike very much) and 5 (I like very much). Results are expressed as means. 2 Significant at P < 0.05. 3 Standard Error of ANOVA a Values within a column with different superscripts differ significantly at P < 0.05(Tukey Test)