Changes in the volatile compounds of pork loin ( fresh and marinated ) with different irradiation and packaging during storage

Se ha utilizado la cromatografía de gases/espectrometría de masas, la extracción mediante purga y trampa para estudiar los compuestos volátiles de lomo de cerdo fresco y adobado, tratados con electrones acelerados (1 y 2 kGy) y almacenado en refrigeración (4 y 8 °C) bajo diferentes atmósferas (aire, vacío y atmósfera modificada). Se observaron diferencias importantes entre las muestras de lomo fresco y adobado pero, en general, solo pequeñas diferencias fueron observadas en algunos compuestos volátiles de ambos tipos de lomo debidas al efecto de la temperatura, tiempo de almacenamiento, tipo de atmósfera o dosis de radiación. Se ha concluido que la aplicación de electrones acelerados es una tecnología muy eficaz para ampliar la vida útil del lomo de cerdo fresco y adobado sin que se detecten cambios en el olor de los productos.


INTRODUCTION
Worldwide, the population of pigs for human consumption rises to 956 million.The pork production contributes over 39% of the global production of meat for human consumption, an equivalent of 15.3 kg of pork consumed per person per year (MAPA, 2006).In Spain, the annual quantity per capita goes up to 58 kg of pork.To meet this demand, 37.5 million hogs are sent to slaughter annually.They are often killed when they turn 6 months old and weigh 100 kg.In the EU, this figure rises to 240 million pigs annually sent to slaughterhouses (MAPA, 2006).
The food industry has made great efforts to improve the maintenance of sanitary conditions and prevent the contamination of food, although a number of pathological processes associated with food still remain.The level of contamination can be reduced by good hygiene practices, but some pathogens are impossible to eliminate, especially in raw foods with minimal processing.Irradiation is presented as a possible method of decontamination for this food group.The most common alterations in the microorganisms in meat are Gram negative psychrotrophs which, in turn, are very susceptible to radiation because they are practically eliminated by a dose of 1 kGy (Monk et al., 1995).Irradiation is also a very effective way to eliminate the pathogens present in foods, including L. monocytogenes (Patterson and Damoglou, 1993, Sommers et al., 2003, Zhu et al., 2005) and Salmonella spp (Grant and Patterson, 1991, 1992, ICMSF, 1996, Patterson 1988, Tarkowski et al., 1984, Thayer et al., 1990, Cabeza et al., 2009, Cabeza et al., 2007).
The quality of the meat may be affected, depending on dose, temperature, and atmosphere during treatment as well as storage conditions.As seen in various studies, the irradiation of meat can produce changes in its aroma, color and flavor, which can significantly affect consumer acceptance (Thayer, 1993, Ahn et al., 1998, Ahn, et al., 2000;Chouliara et al., 2006, Jo and Ahn, 2000, Samelis et al., 2005).In addition, these factors influence CHANGES IN THE VOLATILE COMPOUNDS OF PORK LOIN (FRESH AND MARINATED) WITH DIFFERENT IRRADIATION… Irradiation had a significant impact on pork in the number and profile of volatile compounds.Butane, propane, mercaptomethane, dimethyl sulfide, methyl thioacetate and dimethyl disulfide were produced by irradiation, and were not detected in non-irradiated pork.Kim et al., (2008) also showed that irradiated pork samples formed a greater number of volatile compounds and increased their contents.They were identified by SPME GC / MS.On the other hand Huang et al., (2010) studied the contribution of the flavor of triglycerides and phospholipids of pork and observed a difference in taste between two breeds of pigs.The volatile compounds were extracted using solid phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS).Once identified, they were grouped into classes of lipid-derived aldehydes, Maillard derived aldehydes, alkanes, ketones, alcohols, sulfur compounds containing nitrogen-containing compounds, and furans.
The aim of this study was to investigate the effect of electron-beam irradiation on the volatile compounds in raw and marinated pork loin with different packaging and storage times.

Samples and Sample treatment
A total of fifty-four slices of fresh (Garcia-Marquez et al., 2012a) and marinated (Garcia-Marquez et al., 2012b) pork loin were packaged into low gas permeability laminated plastic bags (diffusion coefficient of 35 cm 3 /24 h m 2 bar for O2 and 150 cm 3 /24 h m 2 bar for CO2) with a 5:1 (v/w) gas/product ratio.Three batches were made.An aerobically packaged batch was used as control and the remainder were packaged in either a vacuum or a carbon dioxide enriched atmosphere (CO2/O2/N2) (30/20/50) (v/v/v) by means of a thermo forming packaging machine, model TMM 37/28 (Vapta, Madrid, Spain).
Samples were treated in an industrial electron beam radiation source working at the energy of 10 MeV.The radiation doses employed were 1 and 2 kGy.The dose absorbed by the samples was verified considering the absorbance of cellulose triacetate dosimeters (ASTM, 2000) simultaneously irradiated.Following the irradiation treatment, they were stored in termostated chambers at 4 and 8 °C, oxidative chemical changes (Katusin-Razem et al., 1992).
One of the main defects of irradiated meat is this characteristic odor, which is produced by the oxidation of lipids in the presence of oxygen.In raw meat, odors can be developed or disappear during cooking (Luchsinger et al., 1996, Hashim et al.,1995and Ahn et al., 1998).Most of the chemical changes in irradiated meat are associated with free radical reactions (Ahn and Lee, 2004).The characteristic odor of the irradiation process is supposed to be the result of oxidation of the free acids.Changes in the chemical oxidation by E-beam radiation depend on the dose and the presence of oxygen has a significant effect on the development of odor and its intensity (Merritt et al., 1975).Free radicals formed by this process interact with most organic molecules such as proteins, lipids, etc (Kim et al., 2008, Ahn 2001, Patterson and Stevenson, 1995) and they are clearly different from the characteristics of the oxidation of lipids.
Fatty acids are important precursors of the flavor of pork, because they are the main source of carbonyl compounds by heating (Selke et al., 1977(Selke et al., , 1980)).Therefore, carbonyl compounds are important for the odor of irradiation and its intensity depends on the essence of oxygen during irradiation (Reineccius, 1979).
The most important substance in the changes in meat quality are lipids, the effect of the fat content of irradiated meat is limited in the development of lipid oxidation, color changes or the production of volatiles production (Jo et al., 1999).A considerable amount of researches had been devoted to the study of the volatile compounds of meat.Among these studies, Ahn et al. (2001) researched the effect of irradiation on the volatile compounds of pork during storage, with different packaging.The volatiles were analyzed using the dynamic headspace GC / mass spectrometry method.Studying the gas chromatograms of irradiated raw pork suggested that the odor is caused by radiolytic protein degradation and lipid oxidation.
I. GARCÍA-MÁRQUEZ, M. NARVÁEZ-RIVAS, E. GALLARDO, C.M. CABEZA AND M. LEÓN-CAMACHO used as analytical signal.The quantification of individual volatile compounds was carried out using isoamyl butyrate as internal standard, which was prepared in refined sunflower oil (14.3407 mg 100 g -1 of oil).An equal relative response factor for any species was assumed.Isoamyl butyrate was used as a reference to calculate the relative retention time, due to the fact that it appears in all samples with high intensity at a mean retention time of 29.52 min.A representative chromatogram report of the volatile compounds of pork loin and their corresponding peaks are shown in Figures 1A and 1B.The relative retention time, molecular ion and base peak of the corresponding peaks are included in Table 2.
The volatile compounds identified were considered as chemical descriptors.A data matrix, whose rows are the samples and whose columns are the variables, was built.Each element of this matrix xij corresponds to the content of volatile compounds j for the sample i. Statistical analyses based on non-parametric techniques were used, including the Kolmogorov-Smirnov-Lilliefors test, which was used to evaluate the normality of each variable included in the study.Since the data distribution was not normal, non-parametric tests were applied.The Kruskal-Wallis test was used to find out significant differences among the variables with three levels.This test is considered as an ANOVA test for one factor.The Mann-Whitney U test was used to determinate the differences between two levels of a same variable.This test is considered similar to a t-Student test for independent samples groups.The calculations were made using the statistical package CSS: STATISTICA from StafsoftTM (Tulsa, OK, USA).

Purge and trap GC-MS analysis
A total of thirty seven volatile compounds were tentatively identified in the volatile fraction from pork loin (fresh and marinated) for the first time using P&T-GC-MS.A tentative assignment of the chromatographic peaks was done by comparing the spectra with those from NIST (National Institute of Standards and Technology) and WILEY libraries and verified by standards purchased from Sigma-Aldrich and Fluka (S. Louis, MO).
The volatile components of the samples were separated using a high polarity column and the conditions of the purge and trap system and GC-MS were previously described (Narváez-Rivas et al., 2010).Under the conditions used in the purge step no degradation of the matrix sample was observed.Repeatability was checked by consecutive analysis of one sample for 12 times and the values expressed as relative standard deviation ranged between 15.3 and 28.7%. the latter as an example of temperature abuse during product storage and distribution.Table 1 shows the E-beam treatment applied and the identification code assigned to each one.

Volatile compound analysis
Extraction of volatile compounds.The volatile compounds were isolated from 1.5 g of minced sample by the dynamic headspace technique and adsorbed on a Tenax trap, using a Purge and Trap (P&T) Concentrator apparatus Tekmar velocity XPT (Thousand Oaks, CA, USA), based on the method described by Narváez-Rivas et al., (2010).The purge conditions were as follows: sample temperature, 45 °C; Tenax trap temperature, 35 °C; purge gas flow, 350 mL min −1 of nitrogen; purge time, 14 min.After the purge time, the volatile compounds were desorbed by heating in the Tenax trap at 225 °C for 1min, and sent through the transfer line (kept at 150 °C) into the chromatograph injector.
Gas chromatography/mass spectrometry (GC/MS) analysis.The GC-ion-trap-MS analyses were performed using a Varian 3800 gas chromatograph coupled to a Saturno 2000 ion trap mass spectrometer (Varian, Palo Alto, CA, USA).The system was equipped with a 1079 injector operating in full scan mode from 50 to 600 amu at 1 scan sec -1 for the purpose of identification .The column used was a Supelcowax-10 (SUPELCO, Bellefonte, PA, USA) fused silica capillary column (60 m long × 0.25 mm i.d.× 0.25 μm film thickness).The GC conditions included hydrogen as carrier gas at 1.6 mL min −1 in constant flow mode.The oven temperature was held at 40 °C for 14 min and then raised to 91 °C at 1 °C min −1 , and then to 201 °C at 10 °C min −1 , and then to 220 °C at 5 °C min −1 , where it was held for 20 min.Split injection mode was used with a ratio of 1:5.The injector temperature was kept at 250 °C.The MS operating conditions were the following: ion source and transfer line temperatures were 200 and 290 °C, respectively; the electron energy was 70 eV with a resolution of 1 and the emission current 250 μA; dwell time and inter-channel delay were 0.08 s and 0.02 s, respectively.For GC-ion trap-MS, Varian MS Workstation version 6.3 software was used for data acquisition and processing of the results.The aldehydes and ketones present in the volatile fraction of the fat samples were identified by computer matching of their mass spectra with those from NIST (National Institute of Standards and Technology) and Wiley libraries and verified by standards purchase from Sigma-Aldrich and Fluka (S. Louis, MO).Peak area was used as analytical signal.

Quantitative analysis and statistical treatment
Thirty-seven volatile compounds were identified.The peak areas of the volatile compounds were

Volatile compounds in fresh and marinated loin
Table 3 shows the median minimum and maximum values of the volatile compounds analyzed in the loin (as mg kg -1 of fat) corresponding to the fresh and marinated.In this table, it can be deduced several interesting observation.Firstly, it would be interesting stand out that there are five compounds which have been detected only in marinated samples, they are: 2-beta-pinene, 2-nitrobutane, 3-carene, dl-limonene, and 2-ethyl-1-hexanol.The rest of volatile compounds are presented in both, fresh and marinated loin samples.
As far as we are aware, studies about changes in each volatile compound with different irradiation and packaging during storage have not been volatile compounds presented z-values up to 2.5 (in absolute value), except dl-Limonene, 2-pentylfurane, decanal and 1-octanol.
According to results obtained for the volatile compounds, the different packaging systems of of 107 cfu g -1 (Cabeza et al., 2007).Accordingly, the shelf-life of both fresh and marinated loin stored under the selected conditions will be different according to the strength of the method of microbiota previously reported.So, this is the first time that this type of study has been done.The end of the shelf-life of samples was established when the microbial load reach the value inhibition.However, the statistical analysis showed that these differences did not affect to the integrity of most phospholipid classes.Normality of the variables in the comparison groups was studied by No effect of the irradiation doses (until 2 kGy) on changes in the individual volatile compounds in fresh loin was observed which is valuable result since E-beam may be applied as an useful tool to extend the shelf-life of fresh loin without alterations.Only significant differences (p < 0.05) between 0 and 2 kGy are observed for dimethyl disulfide in marinated loin.

CONCLUSIONS
A study of the effect of E-beam irradiation and packaging on the volatile compounds from fresh and marinated pork loin has been carried out.Some differences were found between samples, namely in terpenes which only were detected in marinated sample due to the seasoning, which included paprika, source of those volatiles.Minor differences were found between the three type of packaging (air, vacuum and carbon dioxide) and storage temperatures (2 and 8 °C).However, in the context of the objective of the present work, the result of most concern is that no effect of the irradiation doses was found on changes in the individual volatile compounds in both products, even when 2 kGy was applied.Thus, the E-beams may be a very useful tool to extend the shelf-life of fresh and marinated pork loin.Additionally, this technology reduces the number of pathogens to negligible levels.
In Table 5, the results obtained from Mann-Whitney U test to study the effect of temperature are presented for both kinds of samples (fresh and marinated).Two temperatures (4 and 8 °C) have been applied for the storage.Only significant differences (p < 0.05) are observed for 2-propanone, 3,5,5-trimethyl-1-hexene and octanal in fresh loin.There are not references about the effect of temperature in volatile compounds.In marinated loin, significant differences have been found in a large number of compound, 2-propanone, 2-butanone, 2-butanol (p < 0.05), ethyl ester acetic acid, 2-penthyl-furane and ethyl ester hexanoic acid (p < 0.01), possibly because of the additives used.
As well, the effect of packaging atmosphere (air, MAP and vacuum) has been study applying a Kruskal-Wallis test.The results are in Table 6 and it can be deduced that there are significant differences (p < 0.05) between MAP and vacuum for 2-(1-methylethoxy)-1-propanol in the case of fresh loin, being the mean value highest in vacuum.This fact has not explanation and no references about it have been found.On the other hand, the packaging atmosphere has a major effect in volatile compounds from marinated loin, for 2-beta-pinene there are significant differences (p < 0.05) between MAP and air and (p < 0.001) between MAP and vacuum.Also, there are significant differences (p < 0.01) between MAP and air for 3,5,5-trimethyl-1-hexene.
Finally, several irradiation doses have been used for both types of loin (0, 1 and 2 kGy).The effect of this was also studied using a Kruskal-Wallis test, whose data are presented in Table 7.
Figure1GC-ion-trap-MS chromatograms in full scan mode of total volatile compounds profile from pork loin: A, from 0.0 to 25.0 minutes; B, from 25.0 to 80.0 minutes.Peaks identification: see table 2.

Table 1 Analysed intramuscular fat from pork loin samples
Peaks identification: see table 2.

Table 3 Median, minimun and maximun values (mg kg -1 ) for the volatile compounds determined in the analyzed loin samples and Mann-Whitney U Test By variable. Type Marked tests are significant at p < 0.05000
a for p < 0.05; b for p < 0.01; c for p<0.001 and d for p < 0.0001.CHANGES IN THE VOLATILE COMPOUNDS OF PORK LOIN (FRESH AND MARINATED) WITH DIFFERENT IRRADIATION…

Table 4 Mann-Whitney U Test by variable time for both types of samples
* For p <0.05; ** for p < 0.01 and *** for p < 0.001.

Table 6 Significant differences within both types of samples (fresh and marinated) for the volatile compounds classes analyzed according to the different packaging atmosphere (air, MAP and vacuum)
. GARCÍA-MÁRQUEZ, M. NARVÁEZ-RIVAS, E. GALLARDO, C.M. CABEZA AND M. LEÓN-CAMACHO means of Kolmogorov -Smirnov -Lilliefors test.In light of the results of this test, non parametric test, such as Kruskal-Wallis and Mann-Whitney U test were used for all between-group comparisons. I

Table 7 Significant differences within both types of samples for the volatile compounds analyzed according to the different irradiation doses (0, 1 and 2 kGy)
ns, not significant;* p<0.01.Comparison between irradiation doses using Kruskal-Wallis Test.CHANGES IN THE VOLATILE COMPOUNDS OF PORK LOIN (FRESH AND MARINATED) WITH DIFFERENT IRRADIATION…