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LWT - Food Science and Technology 53 (2013) 191e197

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LWT - Food Science and Technology

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Effect of commercial proteases on shelf-life extension of Iberian dry-curedsausage

M.J. Petrón a,*, J.M. Broncano a, J. Otte b, L. Martín a, M.L. Timón a

aDepartment of Animal Production and Food Science, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Ctra. de Cáceres s/n, 06007 Badajoz, SpainbDepartment of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

a r t i c l e i n f o

Article history:Received 20 July 2012Received in revised form7 February 2013Accepted 14 February 2013

Keywords:Proteolytic proteaseDry-cured sausageAntioxidant activityShelf-life

* Corresponding author. Tel.: þ34 924286200; fax:E-mail address: mjpetron@unex.es (M.J. Petrón).

0023-6438/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.lwt.2013.02.014

a b s t r a c t

Forty dry-cured sausages from Iberian pigs were manufactured using 3 different proteases (batch 1:without proteases, batch 2: neutral bacterial proteases, batch 3: fungal proteases and batch 4: fungal proteasesconcentrate). The effect of proteases on formation of low molecular weight (LMW) compounds withantioxidant activity to extend shelf-life of Iberian dry-cured sausages was evaluated. The use of proteasessignificantly increased the antioxidant activity (P < 0.001) and the amount of fraction rich in LMWcompounds (P < 0.001). Extracts from batch 4 showed a high antioxidant activity and with the highestamounts of LMW-rich fractions (P < 0.001). In addition, samples from batch 4 displayed a higher stabilityagainst lipid oxidation and degradation of red colour (P < 0.001) in comparison with the rest of samples.

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1. Introduction

Lipid oxidation is the major quality deteriorative process thatoccurs during the processing and storage of meat and meat prod-ucts (Sammet et al., 2006). It is known that pigment degradation isrelated to lipid oxidation phenomena and changes in colour are oneof the main determinants of the shelf life of meat products(Hernández, Ponce, Jaramillo, & Guerrero, 2008). In order to pre-vent foods from undergoing such deterioration it is important toinhibit lipid oxidation occurring during processing and storage.Packaging (vacuum or modified atmosphere) is one of the toolsused to minimize oxidative rancidity and extend the shelf-life ofmeat products (Parra et al., 2012). Antioxidants are also used topreserve meat products by retarding discolouration and deterio-ration as a result of oxidation (Decker, Warner, Richards, & Shahidi,2005). However, the meat industry is increasingly searching fornatural solutions to minimize oxidative rancidity rather than syn-thetic additives that have been found to exhibit various negativehealth effects in animals (Saito, Sakagami, & Fujisawa, 2003).

It is well known that LMW substances, which are naturallypresent in the skeletal muscle (Arihara, 2006), could remain inmeat after processing in products such as fermented sausages(Candogan, Wardlaw, & Acton, 2009; Lorenzo, García Fontán,Franco, & Carballo, 2008). Some of these compounds including

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carnosine and anserine (Casaburi et al., 2008; Mora, Sentandreu &Toldrá, 2008) which have been reported to be good free radicalscavengers (Aruoma, Laughton, & Halliwell, 1989).

In addition, compounds formed from protein hydrolysis occur-ring during processing and storage ofmeat products such as peptidesand free amino acids may also have antioxidant activity (Gebicki &Gebicki, 1993; Karel, Schaich, & Roy, 1975; Kikugawa, Kato, &Hayasaka, 1991; Kohen, Yamamoto, Cundy, & Ames, 1988). In thesesense, Broncano, Otte, Petrón, Parra, and Timón (2012) isolatedmanyof the compounds listed above from dry-cured sausage.

Studies on the final products of proteolysis have describedvarious low and medium weight peptides, oligopeptides and freeamino acids with antioxidant effect in protein extracts from fer-mented sausages (Sun et al., 2009; Va�stag, Popovi�c, Popovi�c,Petrovi�c, & Peri�cin, 2010). Commercial proteases have been usedin dry-cured sausages to inhibit lipid oxidation (Broncano, Timón,Parra, Andrés, & Petrón, 2011). Therefore, the aim of this study isto determine if the use of exogenous proteases increases theamount of bioactive peptides with antioxidant effect, and if thesecompounds are able to extend shelf-life of the sausages by pre-venting rancidity and discolouration during storage.

2. Material and methods

2.1. Samples

This study was carried out using forty Iberian dry-cured sau-sages which were manufactured in the pilot plant of the School of

M.J. Petrón et al. / LWT - Food Science and Technology 53 (2013) 191e197192

Agricultural Engineering of Badajoz. Four batches of sausages weremade: a control batch (batch 1, n ¼ 10), where no protease wasadded, batch 2 (n ¼ 10), with added validase BNPL (bacterial pro-tease from Bacillus subtilis var., 0.5 g kg�1), batch 3 (n ¼ 10), withadded validase FP II (fungal protease from Aspergillus oryzae,0.5 g kg�1) and batch 4 (n ¼ 10), with added validase FP Concen-trate (fungal protease concentrate from A. oryzae, 0.5 g kg�1).Commercial enzymes were purchased from Valley Research (ValleyResearch Iberica, Madrid, Spain).

The processing of the Iberian sausage “Chorizo” has beendescribed by Broncano et al. (2011). The sausages (n ¼ 40) weredivided into four pieces, each one assigned into one of the foursampling days. At 0 day, samples were taken at the end of pro-cessing (no storage). The rest of the samples were packaged undervacuum and were stored under illumination (600 lx) at 18 �C (tosimulate commercial conditions) and maintained up to 45 days(enough time to appreciate if this kind of “chorizo” develops lipidand colour oxidation).

All samples were minced vacuum packed and stored at �80 �Cfor further analysis. LMW (<3 kDa) compounds were extractedfrom samples at day 0. The extracts were then evaluated for RSA(DPPH radical scavenging activity), RP (reducing power) and ILAA(Inhibition of linoleic acid autoxidation), RP-HPLC (reversed-phasehigh performance liquid chromatography) and HILICeESI-MS/MS(hydrophilic interaction liquid chromatography).

Samples from days 0, 15, 30 and 45 were also evaluated forTBARs and instrumental colour.

2.2. Materials

Chemicals required for the assays included: Linoleic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, and malondialde-hyde (MDA) were purchased from Sigma Chemical Co. (St. Louis,MO, USA, Steinheim, Germany and St. Louis, MO, USA, respectively).Trichloroacetic acid (TCA) and iron (II) chloride 4-hydrate PRS werepurchased from Panreac Química SAU (Barcelona, España). Butyl-ated hydroxyanisole (BHA) was supplied from Acr�os Organics NewJersey, USA. Other chemicals and used reagents were analyticalgrade and commercially available.

A spectrophotometer from Thermo Fisher Scientific ModelBioMate 3 was used.

2.3. Methods

2.3.1. Extraction of low molecular weight (LMW) (<3 kDa)compounds from dry-cured sausage

LMW compounds were extracted following the methoddescribed by Broncano et al. (2011). Briefly, frozen dry-curedsausage samples were homogenised in perchloric acid (3%). Thehomogenate was centrifuged and the supernatant was collectedand ultrafiltered. The permeates were finally lyophilized, redis-solved in milliQ water [20 mg/ml] and stored at �20 �C untilanalysis.

2.3.2. Determination of DPPH radical scavenging activity (RSA)The RSA of sausage extracts (<3 kDa) was determined according

to the method described by Broncano et al. (2011). Ascorbic acid(vitamin C) was used as positive control at the same concentrationof sample [20 mg/ml]. RSA was calculated as follows:

RSAð%Þ ¼ ½ðAcontrol� AsampleÞ=Acontrol� � 100;

where Asample is the absorbance of the test sample and Acontrol isthe absorbance of distilled water.

2.3.3. Reducing power (RP)The RP was determined according to the method described by

Broncano et al. (2011). Vitamin C was used as positive control at thesame concentration of sample [20 mg/ml]. Absorbance wasmeasured at 700 nm.

2.3.4. Inhibition of linoleic acid autoxidation (ILAA)The capacity of ILAA in model system of the sausage extracts

was measured based on the method described by Broncano et al.(2011). Butylated hydroxyanisole (BHA) was used as positive con-trol at the same concentration of sample [20 mg/ml]. A negativecontrol (CTRL), consisting in distilled water was also assayed.Colour development indicating linoleic acid oxidation wasmeasured spectrophotometrically at 500 nm.

2.3.5. Fractionation of the extracts by RP-HPLCSausage extracts of samples (n ¼ 40) were separated by reversed-

phase high performance liquid chromatography (RP-HPLC) (HewlettPackard Series 1100) according to the method developed by Quiróset al. (2007) with some modifications. 100 ml of extracts were injec-ted, and the componentswere separated on an Inertsil ODS-3, C8, Ph3column(4.6�250mm,5mm,GLScience, Japan)usinga lineargradientof acetonitrile (10e40%, in 33 min) containing 0.08% trifluoroaceticacidataflowrateof1.0mlmin�1. Thecompoundsweredetectedat214and 280 nm and collected automatically (Fraction Collector, AgilentTechnologies Series 1200). Each peak was pooled from the extracts,neutralized by KOH and lyophilized. Samples were redissolved inmilliQ water [2.5 mg ml�1] and stored at�20 �C until analysis.

2.3.6. HILICeESI-MS/MS separation and identification of isolatedLMW compounds

Components of each peak were analysed by HILICeMS/MS usingan Agilent 1100 LCMSD Trap (Agilent Technologies A/S, Naerum,Denmark) based on the method developed by Andersen,Schlichtherle-Cerny, and Ardö (2008), with some modifications.10 mL of these isolated fractions were injected, and the componentswere separated on a column SeQuant� ZIC�-HILIC (150 � 2.1 mm,3.5 mM,100 Å) (PEEK HPLC, Merck KGaA, Darmstadt, Germany) using6.5 mM ammonium acetate in acetonitrile/water as the mobilephase. Buffer A contained 90% acetonitrile, and buffer B 40%. Agradient of 10%e90% B in 90 min was used, followed by 100% B for20min and reequilibration at the starting conditions with a flow rateof 0.05 ml min�1. Nitrogen was used as sheath gas. AutoMS spectrawere recorded using the standard range from 50 to 700 mz�1 at thenormal scan resolution and the target mass set to 205 mz�1.

The standard compounds: amino acids (Alanine, Valine, Leucine,Isoleucine, Methionine, Phenylalanine, Proline, Tyrosine, Trypto-phan, Lysine, Arginine, Histidine and Glutamic acid), L-carnosine,L-carnitine and taurine (SigmaeAldrich, St. Louis, MO, USA), tryp-tophan, creatine and creatinine (Merck, Darmstadt, Germany) weredissolved in acetonitrile:water (60:40 v/v) and analysed at a con-centration of 2 mg ml�1. Compounds were identified by compari-son with standards with respect to retention time or according toCID fragments as published in databases (http://www.massbank.jp/) and in scientific published literature.

2.3.7. Thiobarbituric acid-reactive substances (TBARs)TBA reactive substances were measured following the extraction

methoddescribedbyAndrés,Cava,Ventanas,Muriel, andRuiz (2004).

2.3.8. Instrumental colour measurementColour measurements were taken in sausage samples immedi-

ately after opening the package in accordance with the recom-mendation on colour determination of the American Meat ScienceAssociation (AMSA, 1991).

Table 2Inhibition capacity of lipid autoxidation (ILAA) measured in linoleic acid modelsystem for 4 days in presence of dry-cured sausage extracts (day 0).

Batch Day 1(Abs 500 nm)

Day 2(Abs 500 nm)

Day 3(Abs 500 nm)

Day 4(Abs 500 nm)

Negativecontrol

0.54 � 0.06d 0.82 � 0.06d 1.21 � 0.08e 1.71 � 0.11e

1 0.11 � 0.02bcy 0.44 � 0.04cz 0.73 � 0.09dz 1.00 � 0.09dz

2 0.11 � 0.02bcy 0.24 � 0.02by 0.44 � 0.03cy 0.62 � 0.04cy

3 0.13 � 0.03cy 0.26 � 0.11by 0.39 � 0.11cy 0.41 � 0.10bx

4 0.07 � 0.01abx 0.14 � 0.01ax 0.24 � 0.02bx 0.41 � 0.05bx

Positivecontrol

0.04 � 0.00a 0.07 � 0.01a 0.09 � 0.02a 0.11 � 0.01a

P Batch P < 0.001 P < 0.001 P < 0.001 P < 0.001

Results are expressed as means � standard deviations.aee Means with different letters in the same column indicate significant differencesbetween batches negative control, 1, 2, 3, 4 and positive control.xez Means with different letters in the same column indicate significant differencesbetween batches 1, 2, 3 and 4 (P < 0.05).Batch 1 (without proteases), batch 2 (validase BNPL), batch 3 (validase FP II) andbatch 4 (validase FP concentrate).Negative control: distilled water.Positive control: BHA.

M.J. Petrón et al. / LWT - Food Science and Technology 53 (2013) 191e197 193

The colour parameters were determined using aMinolta CR-300colorimeter reflectance spectrophotometer (Minolta Camera Co.,Osaka, Japan) (Illuminant D65/0� standard observer and 0.8 cmport/viewing area). a* (redness) and b* (yellowness) values wereused to calculate spectral colour (hue ¼ arctan [b*/a*]). The mea-surements were repeated on three randomly selected locations oneach sample and averaged for statistical analysis.

2.4. Statistical analysis

Means and standard deviations of the meanwere obtained fromthe analytical experiments. Results were analysed using an ANOVAtest with the GLM procedure of SPSS 15.0 (SPSS Institute Inc., Cary,NC). When statistically significant differences were found a Tukey’stest was performed. A Pearson’s correlation test was performed toevaluate any relationship between analysed parameters. The levelof significance was set to p < 0.05. Principal components analysiswas carried out using the variables studied.

3. Results and discussion

3.1. Antioxidant activities of dry-cured sausage extracts (<3 kDa)

The antioxidant activity of sausage extracts was measured onday 0 (no storage) using RSA, RP (Table 1) and ILAA (Table 2)methods. The antioxidant activity (RSA, RP and ILAA) in sausageextracts treated with proteases (batches 2, 3 and 4) was signifi-cantly higher (P < 0.001) than in sausage extracts from batch 1(control without added proteases), except on day 1 for ILAA test. Inthis sense, the use of proteases in our study could have increasedthe amount of peptides and free amino acids with antioxidant ac-tivities in our sausage extracts.

Comparing the antioxidant activity of the sausage extracts withadded proteases (batches 2, 3, 4), no clear differences were observedamong them by RSA and RP determinations. Nevertheless, ILAAanalysis showed that batch 4 (FP Concentrate) had the highest in-hibition autooxidation capacity, being statistically significant(p < 0.001) on days 2, 3 and 4. These differences may be due to boththe quantity and type of peptides and amino acids generated by theaction of the different proteases. Some authors suggest that theantioxidant activity of protein hydrolysates is influenced by the de-gree of hydrolysis, the size of non-protein nitrogen fractions and thetype of peptides and amino acids generated by proteolysis (Donget al., 2008; Kong & Xiong, 2006; Pihlanto, 2006).

3.2. Isolation of major compounds in extracts by RP-HPLC

RP-HPLC analysis showed the same nine peaks in all of thesausage samples (n ¼ 40, from batches 1, 2, 3 and 4) but the use of

Table 1Evaluation of the antioxidant capacity of dry-cured sausage extracts by RSA and RPmeasurements (day 0).

Batch RSA (%) RP (Abs 700 nm)

1 58.47 � 3.92c 0.25 � 0.02c

2 74.31 � 2.01b 0.33 � 0.04ab

3 70.65 � 3.90b 0.30 � 0.04b

4 71.79 � 2.81b 0.36 � 0.06a

Positive control 89.70 � 1.03a d

P Batch P < 0.001 P < 0.001

Results are expressed as means � standard deviations.aec Means with different letters in the same column indicate significant differences.Batch 1 (without proteases), batch 2 (validase BNPL), batch 3 (validase FP II) andbatch 4 (validase FP concentrate).Positive control: vitamin C.d: The antioxidant activity of vitamin C at [20 mg/ml] was too high for the spec-trophotometer measurement RSA: radical scavenging activity RP: reducing power.

proteases significantly affected the quantity of each one. The size(a.a.u) of the nine fractions is showed in Table 3. Fractions 1, 2, 3, 7, 8and 9 were the largest in all of the batches, the remaining fractions(4, 5 and 6) showing lower sizes. Fraction 4 of batch 1 (withoutadded enzyme) was significantly higher than fraction 4 of batcheswith added proteases (p < 0.001). In contrast, batch 1 containedlower amount of the fractions 3, 6, 7, 8 and 9 (p < 0.001). Regardingthe batches treated with proteases, batch 4 showed the highesta.a.u in fractions 6�9 (p < 0.001).

Differences between batches with respect to the summation ofthe nine isolated fractions (

PF.1e9) are shown in Table 3. Batch 1

(control) had lower a.a.u ofP

F.1e9 than batches treated withproteases. On the other hand, extracts of batch 4 had the highesta.a.u of

PF.1e9 (p < 0.001).

3.3. Identification of major compounds in isolated fractions

Major compounds isolated from the nine fractions were iden-tified by HILICeMS/MS (Table 4).

Fraction 1 was mainly consisted of small peptides (PheeGlyeGly, AspeMet, carnosine and anserine). Trace amounts of freeamino acids (Glu, His, Arg and lys) were also determined in fraction1. Many studies have demonstrated the presence of small peptidesand free amino acid in fermented sausage from proteolysis pro-cesses (Broncano et al., 2012; Hughes et al., 2002) and also thatthese compounds can be effective in inhibiting oxidation of fattyacids (Guo, Kouzuma, & Yonekura, 2009), chelating metal ions (Wu,Chen, & Shiau, 2003) and also showing radical scavenging activity(Rajapakse, Mendis, Jung, Je, & Kim, 2005). Carnosine and anserineare natural dipeptides characteristic of meat and fermented sau-sages (Casaburi et al., 2008; Mora et al., 2008), and have been re-ported to be good free radical scavengers (Aruoma et al., 1989).

Fraction 2 contained mainly creatinine which has already beenfound in dry-cured ham (Mora, Hernández-Cázares, Sentandreu, &Toldrá, 2010). To the best of our knowledge, there is no studydealing on the antioxidative effect of creatinine, further in-vestigations being necessary to assess its antioxidant activity.

Fractions 3, 4 and 5 did not contain compounds that could beidentified by HILICeMS/MS and only the compounds with masses137 and 245 could be described.

Fractions 6�9 mainly contained free amino acids (Met, Tyr, Pheand Trp, respectively). Many studies have shown the ability of theseamino acids to quench radicals (Gebicki & Gebicki, 1993; Karel et al.,

Table 3Arbitrary area units (a.a.u) of the fractions isolated by RP-HPLC from dry-cured sausage extracts (day 0).

Fractions Batches

1 2 3 4 Pbatch

F.1 22,070.5 � 1404.5a 18,755.9 � 1829.6b 19,523.1 � 4127.9ab 21,311.1 � 1775.3ab <0.030F.2 13,730.1 � 2891.3b 14,879.5 � 3307.5b 16,664.5 � 1599.0ab 19,145.4 � 1595.5a <0.001F.3 6636.0 � 667.80b 10,978.1 � 1327.8a 10,796.1 � 2028.1a 9988.5 � 1159.8a <0.001F.4 12,019.5 � 560.0a 5144.7 � 404.2b 3799.0 � 1140.0c 5481.9 � 1100.0b <0.001F.5 4576.7 � 331.5c 7913.7 � 782.4a 6372.5 � 1324.9b 5525.0 � 1084.7bc <0.001F.6 1046.4 � 68.4c 2757.7 � 274.7b 2614.5 � 454.9b 4211.5 � 415.2a <0.001F.7 5099.7 � 800.5c 16,575.3 � 1319.6b 16,094.2 � 2397.6b 24,563.0 � 2085.0a <0.001F.8 5222.7 � 685.7c 19,375.0 � 1668.2b 20,314.9 � 3126.3b 31,119.5 � 3458.2a <0.001F.9 4264.0 � 678.2c 16,230.5 � 1445.4b 15,482.8 � 2486.7b 27,103.8 � 2506.9a <0.001P

F.1eF.9 74,665.6 � 7985.2c 112,610.4 � 11,526.2b 111,661.6 � 16,356.5b 148,449.7 � 13,177.1a <0.001

Results are expressed as means � standard deviations.aec Means with different letters in the same row indicate significant differences.F.1, F.2, F.3, F.4 F.5, F.6, F.7, F.8 and F.9 are the fractions isolated by RP-HPLC from dry-cured sausage extracts.Batch 1 (without proteases), batch 2 (validase BNPL), batch 3 (validase FP II) and batch 4 (validase FP concentrate).

M.J. Petrón et al. / LWT - Food Science and Technology 53 (2013) 191e197194

1975; Kikugawa et al., 1991; Kohen et al., 1988). In a previous study,mainly free amino acids and dipeptides in addition to metabolitescharacteristic of meat were also found by HILICeMS/MS method inextracts from sausages produced without the addition of proteases(Broncano et al., 2012).

3.4. Correlations analysis of antioxidant activities of sausagesextracts and the isolated fractions

Table 5 shows the correlation analysis between the antioxidantactivity (RSA, RP and ILAA) of sausage extracts and the summitrysize of all the isolated fractions (

PF.1e9).

Results in Table 5 indicate a significant correlation (p < 0.001)between the parameters of antioxidant activity and the

PF.1e9 of

the sausage extracts. These correlations indicate that high values ofantioxidant activity were associated with high values of

PF.1e9.

Thus, Batch 1 (control without added protease), which showedthe lowest antioxidant ability (RSA, RP and ILAA (Tables 1 and 2))also presented lower amounts of

PF.1e9 (p < 0.001) (Table 3). On

the other hand, extract from batch 4 developed a good antioxidantactivity, especially for the ILAA, which could be related to their highvalues of

PF.1e9.

Therefore, it could be established that the high antioxidant ac-tivity of sausage extract of batch 4 could be related to the higher

Table 4Compounds identified by HILICeMS/MS in the isolated fractions of the dry-cured sausagmost abundant fragment ions from each compound are underlined.

Fraction Retention time (min) Mass [M þ H] Fragments

1 43.4 148.0 84.3; 102.1; 130.0

50.3 280.0 86.2; 104.1; 167.9; 220.9;46.7e49.8 156.0 110.1; 138.061.2 265.3 127.9; 248.7

227.0 110.0; 138.0; 156.0; 180.065.5 241.0 109.1; 150.1; 168.0; 170.93.0 175.1 60.5; 70.3; 112.2; 116.0;96.4 147.1 84.2; 130.0

2 11.7 114.0 86.2; 103.823.4 118.2 72.3

3 6.7e7.4 230.2 212.18.5-9.6 137.0 No fragments

4 8.5e9.5 137.0 No fragments5 3.2 245.1 74 4; 100 2; 116 0; 129 16 20.6 150.1 56.6; 104.1; 132.97 24.3 182.0 136.0; 164.98 13.5 166.0 120.19 15.8 205.0 188.0

a n.i.: not identify.

amounts of peptides (carnosine, anserine, PheeGlyeGly, AspeMet)and free amino acids (Met, Tyr, Phe and Trp) present in theirextracts.

3.5. Evaluation of the oxidative stability of dry-cured sausagesamples during storage

Malondialdehyde values of sausage measured during the stor-age period ranged from 0.4 to 4.3 mg kg�1, corresponding theminimumvalues at 0 day of storage (0.4e1.3 mgMDA kg�1) and themaximum at the end of the storage period, 45 days (2.4e4.3 mgMDA kg�1) (Table 6). Other authors have also determined similarvalues of MDA in dry-cured sausage packaged under vacuum(Summo, Caponio, & Pasqualone, 2006), or even higher levels(>5 mgMDA kg�1) (Rubio, Martínez, García, Rovira, & Jaime, 2008).

The evolution of the oxidative stability of sausages duringstorage showed an intense lipid oxidation during the first 15 days,despite that sausages were packaged under vacuum. The increasein MDA content during storage of vacuum packed sausages hasbeen already described in several studies (Rubio et al., 2008;Sammet et al., 2006; Summo et al., 2006). According to these au-thors, the increasing oxidation could be attributed both to the re-sidual oxygen in the inner part of the sausages and to thepermeability of the plastic film packaging (Fernández, Rozas,

e extracts (day 0). The major compounds in each fraction are given in bold, and the

Propose compound Reference

Glutamic acid (trace) Rogalewicz, Hoppilliard, and Ohanessian(2000). Standard

240.0 Phe-Gly-GlyHistidine (trace) Rogalewicz et al. (2000). StandardAspeMet

; 210.0 Carnosine http://www.massbank.jp0; 224.0 Anserine (trace)157.0 Arginine (trace) Rogalewicz et al. (2000). Standard

Lysine (trace) Rogalewicz et al. (2000). StandardCreatinine StandardValine Rogalewicz et al. (2000). Standardn.i.a

n.i.n.i.

; 215.0 n.i.Methionine Rogalewicz et al. (2000). StandardTyrosine Rogalewicz et al. (2000). StandardPhenylalanine Rogalewicz et al. (2000). StandardTryptophan Rogalewicz et al. (2000). Standard

Table 7Evolution of the colour parameters (a*, b* and Hue*) of the external surface of dry-cured sausage during storage period.

Day Batch a* b* Hue*

Day 0 1 3.6 � 1.0bx 13.4 � 2.5x 74.1 � 5.1a2 4.6 � 1.1bx 13.4 � 1.6x 70.5 � 5.5ay

3 4.1 � 1.2bx 13.9 � 2.6x 72.8 � 5.0 ay

4 8.3 � 1.5ax 14.7 � 2.8x 60.2 � 4.5bz

P Batch *** ns ***Day 15 1 2.5 � 1.2bxy 9.7 � 2.1by 76.2 � 6.4a

2 3.1 � 0.8by 9.2 � 1.9by 74.8 � 4.8axy

3 2.7 � 1.0by 9.6 � 1.8by 75.9 � 3.3axy

4 5.0 � 0.9ay 13.4 � 1.1ay 65.8 � 4.0by

P Batch *** *** ***Day 30 1 2.2 � 0.2by 10.4 � 1.1 by 79.1 � 4.2a

2 2.5 � 1.0by 10.2 � 1.3 by 78.1 � 4.0ax

3 2.0 � 0.8by 9.9 � 1.6 by 78.9 � 5.1ax

4 4.4 � 0.9ay 12.4 � 1.7 ay 72.3 � 5.0bx

P Batch *** ** *Day 45 1 2.2 � 1.1by 10.5 � 2.9y 78.7 � 5.1a

2 2.6 � 1.0by 10.4 � 1.0y 76.4 � 4.2abx

3 2.4 � 0.7by 10.5 � 1.4y 78.0 � 4.7axy

4 4.1 � 0.7ay 11.9 � 2.6y 72.1 � 3.6bx

P Batch *** ns *P day 1 ** * ns

2 *** *** **3 *** *** *4 *** ** ***

Results are expressed as means � standard deviations.aec Means with different letters at the same day indicate significant differencesbetween batches.xeyMeans with different letters in the same batch in differents days indicate sig-nificant differences between days.Batch 1 (without proteases), batch 2 (validase BNPL), batch 3 (validase FP II) andbatch 4 (validase FP concentrate).*p < 0.5; **p < 0.01; ***p < 0.001; ns: not significant.

Table 5Pearson’s correlation coefficients between the summatory of isolated fractions(P

F.1e9) and the antioxidants effects (RSA, RP and ILAA days 1e4) of dry-curedsausage extracts (day 0).

RSA RP ILAA

Day 1 Day 2 Day 3 Day 4P

F. 1�9 0.65*** 0.70*** �0.46* �0.82*** �0.88*** �0.81***

RSA: radical scavenging activity.RP: reducing power ILAA: inhibition of linoleic acid autoxidation.*p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

M.J. Petrón et al. / LWT - Food Science and Technology 53 (2013) 191e197 195

Romero, & Vázquez, 1997). In addition, our sausages were exposedto light, and thus the phenomenon of photo-oxidation could havealso induced the increase in MDA content (Bekboelet, 1990).

The levels of MDA remained constant between 15 and 45 days,probably due to the previous consumption of the residual oxygenduring storage. This stabilization of lipid oxidation observed be-tween 15 and 45 days could be responsible for the antioxidantactivity of compounds (<3 kDa) found in the sausages. In this sense,some studies described a decrease in TBARs values in meat and fishsamples when protein hydrolysates rich in peptides and free aminoacids were added (Dekkers, Raghavan, Kristinsson, & Marshall,2011; Samaranayaka & Li-Chan, 2008). Furthermore, other au-thors have observed a significant increase in proteolysis products(peptides and amino acids) during the storage period of fermentedsausages packed under vacuum (Dalmis & Soyer, 2008). It istherefore possible that the proteolytic activity of the sausages hasbeen maintained during the storage period, thus producing pep-tides and amino acids with ability to inhibit lipid oxidation.

Regarding the different batches of our study, sausages formu-lated with protease FP concentrate (batch 4) showed the lowestvalues of TBARs from 15 days of storage until the end the storageperiod (P < 0.001). These differences among the batches areprobably in relation to the highest capacity to inhibit the autoxi-dation of linoleic acid that the sausage extracts from batch 4exhibited (Table 2). Broncano et al. (2011) also observed that theuse of protease improved the oxidative stability of dry-cured sau-sages due to the antioxidant activity of compounds (<3 kDa) whichwere present in sausages.

Considering the results obtained and regarding the levels ofsausage oxidation in the batch 4, it can be stated that the use ofprotease FP concentrate can be an efficient strategy to increaseshelf-life of dry-cured sausages.

3.6. Evaluation of instrumental colour of dry-cured sausage duringstorage

Table 7 shows the values obtained for the a*, b* and Hue* pa-rameters of the external surface of the vacuum packaged sausage

Table 6Evolution of the oxidative stability of dry-cured sausage during storage measured byTBARs (mg MDA/kg of sausage).

Batch Day 0 Day 15 Day 30 Day 45

1 1.2 � 0.3az 4.1 � 0.3ax 3.3 � 0.3ay 4.0 � 0.4abx

2 1.3 � 0.1az 4.0 � 0.4ax 3.3 � 0.7ay 4.3 � 0.3ax

3 0.4 � 0.1cz 3.9 � 0.6ax 2.7 � 0.5by 3.6 � 0.6bx

4 0.7 � 0.1bz 2.6 � 0.4bx 1.9 � 0.2cy 2.4 � 0.5cx

P Batch/day P < 0.001 P < 0.001 P < 0.001 P < 0.001

Results are expressed as means � standard deviations.aec Means with different letters at the same day indicate significant differencesbetween batches.xezMeans with different letters in the same batch in different days indicate signif-icant differences between days.Batch 1 (without proteases), batch 2 (validase BNPL), batch 3 (validase FP II) andbatch 4 (validase FP concentrate).

during storage at constant temperatures (18 �C) and exposed tolight.

The intensity of red colour (a*) significantly decreased duringstorage, from values ranging from 3.6 to 8.3 on day 0e2.2e4.1 after45 days of storage. The decrease in red colour of sausages wasprobably due to the oxidation of pigments present in cured sau-sages (myoglobin, nitrosylmyoglobin and carotenoid from paprika).This phenomenon has been already referred by other authors(Hernández et al., 2008). The pigments are known to be oxidizedwhen samples are exposed to light, temperature and oxygen con-ditions and alsowhen there is a decrease in antioxidant compounds(Klieber & Bagnato, 1999).

In the same way as was described for MDA, an intense oxidationof the pigments occurred between days 0 and 15 of storage.Pigment oxidation could be ascribed to both the direct light inci-dence on the sausage surface or/and to the presence of residualoxygen in the pack (Fernández et al., 1997). Values of a* remainedconstant between day 15 and day 45 of storage.

The b* parameter decreased from 13.4 to 14.7 (Day 0) to 10.4e11.9 (Day 45) which is probably in relation to the lipid oxidationprocess that occurred especially during the first 15 days, similar tothe trend observed for the TBARs values. Yellowness degradationhas been described in several studies in samples with high TBARsvalues (Pérez, Sayas, Fernández, & Aranda, 1999; Rubio et al., 2008).

The value of Hue angle in fresh meat normally takes positivevalues near to 0�, matching shades of red. In this study Hue* valueincreases progressively during storage period in batches 2 and 4,which indicates that the intensity of red colour is reduced. How-ever, Hue* values were constant during the 45 days of storage forbatches 1 and 3.

Regarding the differences between batches throughout thestorage period, batch 4 showed the highest a* and b* values and thelower Hue* values (p < 0.001). Therefore, the batch where protease

-2,0 -1,0 0,0 1,0 2,0

Principal component 1 (48.8%)

-1,0

0,0

1,0

2,0

Prin

cip

al co

mp

on

en

t 2 (13.2%

)

Fig. 1. Principal components analysis of Iberian dry-cured sausage without proteases added (batch 1) and with them added (batch 2 with validase BNPL, batch 3 with validase FP IIand batch 4 with validase FP concentrate) using the following variables: Antioxidant activities of dry-cured sausage extracts (RSA, PR and ILAA days 1e4), a.a.u of isolated fractionsfrom dry-cured sausage extracts (F.1�9), TBARs values and colour parameters of dry-cured sausage during storage period. Batch 1 Batch 2 Batch 3 Batch 4

M.J. Petrón et al. / LWT - Food Science and Technology 53 (2013) 191e197196

FP concentrate was added, the typical colour of this kind of sausagewas maintained longer during storage.

3.7. Principal components analysis

A principal components analysis (PCA) has been carried out tostudy the relations among antioxidant analysis, quantification offractions, lipid oxidation and colour parameters during the storagetime. PCA data are displayed in Fig. 1, which shows the plot of theloadings for the first two partial least squares components. The firstcomponent was able to predict 49% of the variation whereas thesecond one explained 13%. A good association among sausages ofeach group was found. The samples from batch 1 (control withoutproteases) are separated from the batches treated with proteases(2e4), probably due to its shorter shelf-life and the lower amount ofLMW compounds with antioxidant activity present in their ex-tracts. Batch 4 (protease FP Concentrate) had the longer shelf-lifeand the highest amount of LMW with antioxidant activity, andthus their samples in the plot are situated in the opposite side ofbatch 1 and separated from the batches 2 (protease BNPL) and 3(protease FP II). It is also reflected in the PCA plot that the proteasesused in batches 2 and 3 had quite similar effects, which is sup-ported by the TBARs and colour results obtained from both batches.

4. Conclusions

The use of commercial proteases in Iberian dry-cured sausage isa good strategy for increasing the amount of antioxidant LMWcompounds in order to improve the shelf life of this product. Pro-tease FP Concentrate is the most suitable enzyme in order to ach-ieve these effects.

Acknowledgements

This study has been possible thanks to the project PDT07A005supported by the Junta de Extremadura and FEDER.

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