Original papers
Free Amino Acids and Biogenic Amines Content during Ageing of Dry-cured Pork Loins Inoculated with Lactobacillus casei ŁOCK 0900 Probiotic Strain
2015 Volume 21 Issue 2 Pages 167-174
Details
2015 Volume 21 Issue 2 Pages 167-174
The objective of this study was to investigate the effect of inoculation with a probiotic strain Lactobacillus casei ŁOCK 0900 and ageing time on the formation of free amino acids (FAAs) and biogenic amines in dry-cured pork loins. The predominant FAAs were glutamic acid, threonine, alanine and leucine. The total FAAs content increased significantly (p < 0.05) during ageing. After the longest examined ageing time, FAAs content in the loins inoculated with a probiotic strain represented significant increase of about 25% in comparison with the control sample. Differences in FAAs concentration were determinant for taste attributes of examined loins. Histamine was not detected in any sample, irrespective of ageing time. Inoculation with a probiotic strain L. casei ŁOCK 0900 proved to be a protective measure against the accumulation of cadaverine, putrescine and tryptamine even though the loins inoculated with L. casei were characterized by higher availability of FAAs.
Proteolysis resulting in generation of large-sized peptides, which are in turn degraded to oligopeptides, and then to free amino acids (FAAs), is one of the most important biochemical changes occurring during ageing of dry-cured fermented meat products (Hughes et al., 2002; Lorenzo et al., 2008). In addition to their biological value, released FAAs directly contribute to the basic taste of dry fermented meat products, and indirectly contribute to the development of their typical aroma since they are precursors of many volatile compounds (Aro Aro et al., 2010). However, an excessive amount of FAAs seems to be responsible for the onset of unpleasant sour and bitter taste (Sforza et al., 2006). Thus, FAAs play a key role in the nutritional value, consumer acceptance and sensory attributes of dry-cured fermented meat products (Beriain et al., 2000; Virgili et al., 2007). It is also of particular interest to take into account the evolution of FAAs during ageing of dry-cured fermented meat products due to their role in biogenic amines formation. These low molecular weight organic bases are mainly generated via decarboxylation of the precursor amino acids by substrate-specific enzymes of microbial origin under suitable conditions (Latorre-Moratalla et al., 2010b; Lorenzo et al., 2007). In dry-cured meat products, apart from the growth of a wide variety of bacteria potentially harbouring decarboxylase activity, acidification, and proteolytic phenomena occurring during fermentation and ageing make the environment particularly favorable for biogenic amines accumulation (Latorre-Moratalla et al., 2010a; Latorre-Moratalla et al., 2007). Their presence is undesirable due to the toxicological effects derived from their vasoactive or psychoactive properties such as hypertension, headache, diarrhea, nausea, rash, and localised inflammation when ingested in excessive amounts. Biogenic amines constitute a potential health risk especially when coupled with potentiating factors such as alcohol, monoamine oxidase inhibitor drugs and gastrointestinal diseases (Bover-Cid and Holzapfel, 1999; Lorenzo et al., 2007). In addition, they are potential precursors in the formation of carcinogenic N-nitroso compounds (Karovičová and Kohajdová, 2005).
Although biogenic amines have been studied for more than thirty years, the interest in their study is more relevant since food safety and food quality requirements are higher (Latorre-Moratalla et al., 2010b) and the growing number of consumers are sensitive to them (Ammor and Mayo, 2007). Hygienic quality of the raw materials and processing plant play a key role in biogenic amines accumulation, since most contaminant bacteria, such as Enterobacteriaceae and Pseudomonas, possess aminogenic capability being indicative of improper hygienic conditions (Suzzi and Gardini, 2003). However, optimization of hygienic conditions may not be enough to avoid excessive biogenic amines accumulation and other measures must be applied. There is therefore interest in exploiting technological strategies to reduce aminogenesis during the manufacture of dry-cured fermented meat products, such as the use of probiotic starter cultures which are not able to decarboxylate amino acids into biogenic amines and are well adapted to the ecology of fermented meat products (Latorre-Moratalla et al., 2010b; Naila et al., 2010).
Probiotics, literally meaning “for life”, are cultures of microorganisms, mainly lactic acid bacteria or bifidobacteria, which introduced orally in the gastrointestinal tract in adequate amounts, contribute positively to the activity of intestinal microflora and, therefore, to the host health (Nowak et al., 2010). Their addition may improve safety and stability of dry-cured fermented meat products extending the shelf life and provides diversity resulting in new sensory properties as well as health benefits (Lücke, 2000). Meat products which are processed by fermenting without heating may be suitable carriers for probiotics into the human gastrointestinal tract (Kołożyn-Krajewska and Dolatowski, 2009). In preventing aminogenesis in dry-cured fermented meat products the inability of the culture to form biogenic amines but also its ability to grow well at the temperature intended for processing of the product and competitiveness in suppressing the growth of wild amine producing microflora should be taken into consideration (Karovičová and Kohajdová, 2005; Suzzi and Gardini, 2003). The aminogenic potential among lacid acid bacteria (LAB) is low, however some species frequently involved in meat products fermentation, such as Lactobacillus curvatus, are usually amino acid decarboxylase positive (Ammor and Mayo, 2007; Latorre-Moratalla et al., 2010a). Therefore, to reduce biogenic amine formation in dry-cured fermented meat products, the selection of fermentative bacteria should be performed to a strain level, when the negative amino acid decarboxylase ability is concerned (Bover-Cid and Holzapfel, 1999).
Over the past decade the idea of using probiotic bacteria as starter cultures in meat products has developed and numerous studies have been carried out (Papamanoli et al., 2003; Pennacchia et al., 2006; Pidcock et al., 2002; Stadnik and Dolatowski, 2012). Most of them focus on dry sausages, only a few authors considered the use of cured meat cuts as vehicles for probiotics. Results of our previous research (Stadnik et al., 2014) showed that inoculation of L. casei had no significant effects on total viable counts. Due to probiotic culture inoculation, the count of lactic acid bacteria (LAB) in a sample with L. casei was higher by approximately one logarithmic level than controls. Loins inoculated with L. casei exhibited significantly lower (p < 0.05) pH than the control batches subjected to spontaneous fermentation which confirmed the effectiveness of the lactobacilli for acidification of loins. The lowest pH value observed in the inoculated sample (5.64 ± 0.2) corresponds to the highest LAB count in this sample (8.21 ± 0.13 log CFU g−1). Water activity values of dry-aged pork loins inoculated with L. casei were significantly lower (p < 0.05) than those obtained for the control. As to our knowledge, there is no available information in the literature concerning the effects of probiotic starter cultures on the development of free amino acids and biogenic amines in dry-cured pork loins which have undergone inoculation with a probiotic strain.
In consequence of this lack of knowledge, the aim of this work was to investigate the effect of inoculation with a probiotic strain Lactobacillus casei ŁOCK 0900 and ageing time on the formation of FAAs and biogenic amines in dry-cured pork loins. This meat product may act as a model system due to its homogeneous shape and structure and the short ageing time required.
Loins preparation Five trimmed loins (M. longissimus thoracis) from left half-carcasses of Polish Large White purebred fatteners with an average weight of 2.4 ± 0.4 kg were excised at 24 h post mortem. At 48 h post mortem all loins were dry-cured using a surface massage with a curing mixture (98.8 – 99.0% sodium chloride, 0.5 – 0.6% sodium nitrite, 0.5 – 0.6% sodium nitrate) at a ratio of 2.8% of the loin. After completion of salting, the loins were kept at 4°C for a total of 4 days to allow the curing mixture to penetrate. After salting, two loins were regarded as control samples. The surface of the three remaining loins was inoculated with 0.2% (v/w) of Lactobacillus casei ŁOCK 0900 (Motyl et al., 2011) to achieve initial level of 106 – 107 CFU/g meat. This strain was derived from the Collection of Pure Cultures of Industrial Microorganisms, Institute of Fermentation Technology and Microbiology at the Technical University of Łodź and was deposited in the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław under the number B/00019. The strain was selected on the basis of results of in vitro studies (Cukrowska et al., 2009), which comprised determination of resistance to the acidity of gastric juice and to bile, adherence to epithelial cells, and antimicrobial activity. All these studies were carried out following the FAO/WHO recommendations (2001, 2002). This species rank among the typical microflora of human intestines and therefore can be safely used for the production of probiotics preparations. This strain showed strong antagonistic activity against gram-positive and gram-negative pathogens (Cukrowska et al., 2009). In previous work (Neffe and Kołozyn-Krajewska, 2010) the probiotic strain Lactobacillus casei ŁOCK 0900 showed their ability to grow in the processing conditions of dry-cured pork loins and achieved a count of 107 log CFU/g, enough to consider those products to be probiotic. L. casei lyophilizate was produced as described by Nebesny et al. (2007). Subsequently, the loins were hung at 16°C in a drying room with a relative humidity of between 80 and 90% for 7 days. After that, the loins were cold smoked for an hour and then aged at 4°C for 14, 21 and 28 days. Analyses were performed at established ageing deadlines. At each sampling time all of the five loins were evaluated by means of cutting and analyzing one piece of them. An inner region of the sample was taken for aw measurements, and then the entire section was minced (ø 3 mm) and used for FAAs and biogenic amines determination.
Three repeated independent experimental trials (replications) were conducted. In each trial, measurements were made in triplicate.
Determination of dry matter Dry matter was determined by air drying the sample at 103°C ± 2°C until constant weight according to AOAC method (2006).
Free amino acids concentration The contents of free amino acids were determined using an automatic amino acid analyser AAA 400 (Ingos Ltd. Czech Republik) equipped with an Ostion LG ANB ion-exchange column (36x0.37 cm) kept at 70°C. The sample volume injected was 100 µL. FAAs were separated by stepwise gradient elution using a Na+/K+-citrate buffer-system with pH of 2.6 (A), 3.0 (B), 4.25 (C), 7.9 (D) (Ingos Ltd). Post-column derivatization with ninhydrin reagent followed by a spectrophotometric measurement at 570 nm and 440 nm was used for determination of components. The operating parameters were: eluent and reagent flow rates of 0.30 and 0.20 mL min−1, respectively; reactor temperature of 120°C; and total run time of 95 min. Determination of FAAs was confirmed by comparing the retention times and peak areas of the particular amino acid standards (Sigma-Aldrich) with those of the components present in the samples. The following amino acids were monitored: aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), proline (Pro), glycine (Gly), alanine (Ala), valine (Val), cysteine (Cys), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine (Lys) and arginine (Arg). Free amino acids content was expressed as mg/kg of dry matter (DM). The concentration of amino acids associated with sweet, bitter, acid and aged tastes has been calculated. In the text, the term “sweet” taste amino acids correspond to the sum of threonine, serine, proline, glycine and alanine. Sum of valine, methionine, isoleucine, leucine, phenylethylalanine, histidine and arginine is associated with “bitter” taste. “Acid” taste amino acids are aspartic acid, glutamic acid and histidine. “Aged” taste has been calculated as a sum of aspartic acid, tyrosine and lysine (Ordonez et al., 1999).
Biogenic amines concentration The contents of biogenic amines were determined using an automatic amino acid analyser AAA 400 (Ingos Ltd) equipped with an Ostion LG ANB ion-exchange column (11.5x0.45 cm) kept at 76°C. The sample volume injected was 100 µL. Biogenic amines were separated by stepwise gradient elution using a Na+/K+-citrate buffer-system with pH of 6.0 (A), 5.7 (B), 5.45 (C) (Ingos Ltd). Post-column derivatization with ninhydrin reagent followed by a spectrophotometric measurement at 570 nm was used for determination of components. The operating parameters were: eluent and reagent flow rates of 0.50 and 0.33 mL min−1, respectively; reactor temperature of 96°C; and total run time of 97 min. Determination of biogenic amines (cadaverine, histamine, putrescine, spermidine, spermine, tryptamine) was confirmed by comparing the retention times and peak areas of the particular biogenic amine standards (Sigma-Aldrich) with those of the components present in the samples. Biogenic amines concentrations were reported as mg/kg of meat.
Statistical analysis Obtained results were statistically analysed using the STATGRAPHICS Centurion XV (StatPoint Technologies Inc. Warrenton, Virginia, USA). A mean and standard deviation was calculated for each test. Results were analyzed by ANOVA followed by post hoc Tukey's multiple comparison test or Student's t-test as indicated in the table legends. Differences between means were considered statistically significant if the p value was less than 0.05.
Free amino acids concentration The changes in the content of FAAs observed during ageing of dry-cured pork loins are given in Table 1. Cysteine was not found in this work which is in agreement with data reported by Hughes et al. (2002) for semi-dry fermented sausages with similar ageing time. Methionine, tyrosine and arginine were present in low concentrations. Quantitatively the most important free amino acids were Glu, Thr, Ala and Leu. This free amino acids profile is largely consistent with those reported by Lorenzo et al. (2008) for dry-cured lacón. After 14 days of ageing these amino acids represented 41 and 43% of the total concentration of amino acids in the control and inoculated sample, respectively. Beriain et al. (2000) also reported that glutamic acid, alanine and leucine summed up the higher percentage of the amino acids in the salchichon. In more aged loins (with 21-day ageing period) they accounted for 35 and 38% of total free amino acids. At the end of the longest ageing period their share increased to 49 and 47%.
| Amino acid | Day 14 | Day 21 | Day 28 | |||
|---|---|---|---|---|---|---|
| control sample | sample with probiotic | control sample | sample with probiotic | control sample | sample with probiotic | |
| Asp | 7.80 ± 1.69*A | 10.86 ± 1.16A | 16.73 ± 2.34*B | 23.31 ± 2.83B | 37.98 ± 2.60*C | 43.76 ± 5.14C |
| Thr | 24.75 ± 3.73*A | 33.78 ± 3.07A | 43.76 ± 3.81*B | 54.70 ± 5.99B | 59.29 ± 6.64*C | 65.87 ± 5.25C |
| Ser | 11.66 ± 3.88A | 10.96 ± 3.40A | 32.44 ± 5.93B | 34.81 ± 4.63B | 54.96 ± 4.55*C | 63.40 ± 7.77C |
| Glu | 37.64 ± 5.54A | 41.38 ± 5.09A | 34.45 ± 6.07*A | 50.30 ± 9.16A | 61.16 ± 7.55*B | 70.87 ± 8.11B |
| Pro | 9.14 ± 2.49*A | 12.46 ± 2.21A | 18.38 ± 5.54B | 20.87 ± 6.58B | 43.86 ± 4.75*C | 53.86 ± 4.13C |
| Gly | 16.89 ± 3.80A | 19.78 ± 2.87A | 29.54 ± 5.69B | 33.94 ± 5.18B | 56.27 ± 6.11*C | 72.78 ± 7.66C |
| Ala | 27.89 ± 5.64*A | 33.93 ± 5.00A | 41.16 ± 4.64B | 43.26 ± 6.18B | 56.19 ± 5.92*C | 68.27 ± 6.18C |
| Val | 19.89 ± 6.75AC | 24.47 ± 5.53A | 27.54 ± 4.64*B | 34.99 ± 5.00B | 22.91 ± 5.39*BC | 57.68 ± 8.28C |
| Cys | nd | nd | nd | nd | nd | nd |
| Met | 10.01 ± 5.09A | 14.57 ± 5.65A | 10.85 ± 4.77AC | 14.20 ± 6.41A | 19.00 ± 5.46B | 16.22 ± 4.86A |
| Ile | 13.98 ± 4.31A | 17.75 ± 4.02A | 26.53 ± 5.74*B | 32.87 ± 6.32B | 44.09 ± 6.14*C | 51.37 ± 4.67C |
| Leu | 26.11 ± 5.85*A | 35.36 ± 4.89A | 38.02 ± 5.57*B | 47.55 ± 6.70B | 53.89 ± 6.13*C | 68.46 ± 5.21C |
| Tyr | 9.49 ± 4.34A | 10.71 ± 4.30A | 15.61 ± 4.58B | 11.44 ± 4.63A | 23.19 ± 4.88*C | 34.24 ± 5.48B |
| Phe | 14.98 ± 3.95*A | 19.97 ± 3.72A | 35.99 ± 4.65*B | 41.56 ± 5.12B | 42.72 ± 2.77*C | 59.49 ± 4.39C |
| His | 20.80 ± 3.40A | 20.22 ± 4.09A | 26.33 ± 5.44B | 23.79 ± 3.46A | 41.54 ± 4.01*C | 49.08 ± 4.05B |
| Lys | 19.92 ± 4.23A | 22.91 ± 4.46A | 35.42 ± 3.08B | 37.70 ± 3.83B | 62.55 ± 4.43*C | 78.43 ± 3.64C |
| Arg | 9.73 ± 3.14*A | 5.58 ± 1.89A | 16.90 ± 4.02B | 16.06 ± 2.47B | 34.58 ± 5.11C | 34.15 ± 3.62C |
| Total | 280.68 ± 43.42*A | 334.69 ± 40.46A | 449.65 ± 54.00*B | 521.36 ± 63.90B | 714.18 ± 68.90*C | 887.93 ± 64.14C |
nd-not detected
A–C Means followed by different letters differ significantly (p < 0.05) within the same sample during ageing (Tukey's test)
The average total free amino acids content increased significantly (p < 0.05) from 280.68 ± 43.42 and 334.69 ± 40.46 mg/100 g DM after 14 days of ageing to 714.18 ± 68.90 and 887.93 ± 64.14 mg/100 g DM at the end of the longest ageing period for the control and L. casei inoculated sample respectively. These final contents were lower than those reported by Garrido et al. (2012) and Lorenzo et al. (2008) for dry-cured lacón, which could be attributed to the shorter ageing period in the present study. Beriain et al. (2000) and Roseiro et al. (2008) also observed an increase in the total free amino acids content during ageing of dry cured sausages. In the present study, the increase in the total free amino acids content observed during ageing was consistent with the gradual increase in the content of individual free amino acids. The free amino acids that underwent the greatest increases were Lys, Gly, Ser and lipophilic ones, with except of methionine. Similar findings have been reported by Hughes et al. (2002). This free amino acid profile also basically coincides with those reported by different authors for dry-cured hams (Virgili et al., 2007). Aspartic acid, glutamic acid, tyrosine, histidine and arginine underwent moderate increase. Methionine in both samples and valine in the control sample suffered the least increase. Among the amino acids, lysine, tyrosine and histidine are the main precursors for biogenic amines (Stadnik and Dolatowski, 2010). These amino acids exhibited increases of 3.1- and 3.4- for lysine, 2.4- and 3.2 for tyrosine and 2.0 and 2.4-fold for histidine for the control and inoculated sample respectively after 28 days of ageing.
After 14 days of ageing the amino acids that underwent the greatest rise as a result of inoculation of the surface by Lactobacillus casei ŁOCK 0900 were Thr and Leu, whereas Arg significantly decreased (p < 0.05). In relation to the inoculation effect after 21 days of ageing, this produced an increase in the contents of most of the FAAs, especially Glu, Thr and Leu. In contrast, the levels of Tyr, His, and Arg decreased in comparison with the control sample, but the differences were not statistically significant (p < 0.05). At 28 days of ageing, the amino acids that suffered the greatest increase which can be attributed to the L. casei inoculation were Val, Phe, Lys and Leu. Statistically insignificant (p < 0.05) decrease in Met and Arg concentration as a result of inoculation with a probiotic strain was observed. At the end of the longest ageing period, the total FAAs content in the loins inoculated with a probiotic strain represented increase of about 25% in comparison with the control sample. Our previous research (Stadnik and Dolatowski, 2013; Stadnik and Dolatowski, 2014) also showed the greatest increase in non protein nitrogen (NPN) and free α-amino acids content during ageing in an inoculated sample. In the light of these results, we found an increased proteolytic activity in the samples inoculated with L. casei ŁOCK 0900, that could be attributed to an intense bacterial peptidase activity, which is in agreement with previous findings of Hughes et al. (2002) who observed differences between the control and S. carnosus MC1 inoculated sausages in the concentration of free amino acids during ripening. Aro Aro et al. (2010) also reported that the differences in the content of free amino acids in fermented sausages could be attributed to the starter culture activity.
Conversion of peptides to free amino acids through muscle and microbial aminopeptidases removing single residues sequentially from the N-terminus is the last proteolytic step. This action is of utmost importance to the overall acceptance of dry-cured fermented meat products, since FAAs are the source of specific tastes and odours, determining the final flavour characteristics (Roseiro et al., 2008). Since taste properties of dry-cured pork loins may be more accurately described by free amino acids grouped on the basis of their common taste characteristics rather than by single amino acids (Roseiro et al., 2008; Sforza et al., 2006), the concentration of amino acids associated with sweet, bitter, acid and aged tastes has been calculated in both samples (Table 2). The ageing time significantly (p < 0.05) influenced the development of basic tastes, with the differences between samples being increased. At 14 days of ageing, the loins showed similar concentration of the amino acid groups related to bitter, acid and aged tastes. Concerning the sweet taste amino acid group, the prevalence in the sample inoculated with a probiotic strain is relevant and resulted mainly from threonine level (24.75 ± 3.73 mg/100 g DM in the control sample and 33.78 ± 3.07 mg/100 g DM in the inoculated sample). Differences in bitter and aged characteristics between samples aged for 21 days were not significant (p < 0.05). At the end of the longest ageing period sample inoculated with L. casei ŁOCK 0900 had higher concentration of the amino acids associated with basic tastes than its counterpart. Taste profiles based on calculation of relative quantities of FAAs with common taste properties needs to be confirmed by a sensory evaluation, which will be the subject of future studies.
| Tastes | Day 14 | Day 21 | Day 28 | |||
|---|---|---|---|---|---|---|
| control sample | sample with probiotic | control sample | sample with probiotic | control sample | sample with probiotic | |
| Sweet | 90.33 ± 8.31*A | 110.91 ± 8.04A | 165.28 ± 19.15*B | 187.58 ± 19.20B | 270.57 ± 23.18*C | 324.18 ± 22.88C |
| Bitter | 115.50 ± 30.14A | 137.92 ± 26.96A | 182.16 ± 29.85B | 211.02 ± 31.90B | 258.73 ± 31.65*C | 336.45 ± 30.49C |
| Acid | 66.24 ± 6.68A | 72.46 ± 7.69A | 77.51 ± 7.96*B | 97.40 ± 11.50B | 140.68 ± 11.04*C | 163.71 ± 9.87C |
| Aged | 37.21 ± 7.24A | 44.48 ± 8.82A | 67.76 ± 7.23B | 72.45 ± 9.74B | 123.72 ± 8.80*C | 156.43 ± 8.87C |
A–C Means followed by different letters differ significantly (p < 0.05) within the same sample during ageing (Tukey's test)
Biogenic amines concentration The amounts of biogenic amines corresponding to different ageing times found in dry-cured pork loins are reported in Table 3. Examined loins contained five biogenic amines, namely, cadaverine, putrescine, spermidine, spermine, tryptamine. Histamine was not detected in any sample, irrespective of the ageing time. The same phenomenon has been observed with chorizo dry sausages (González-Fernández et al., 2003) and dry-cured ham (Virgili et al., 2007). The absence of this biogenic amine in dry-cured loins could be attributed to the fact that bacteria with capacity to decarboxylate histidine are uncommon in meat (González-Fernández et al., 2003). Cadaverine was present only in the samples with 28-day ageing period, though in very low amounts (below 3 mg/kg). The total concentration of biogenic amines decreased insignificantly (p < 0.05) during the ageing period, reaching a concentration of 46.26 mg/kg and 36.36 mg/kg for the control and inoculated sample respectively after 28 days of ageing. Similar trend was observed by Lorenzo et al. (2007) during the manufacturing process of dry-cured lacón. No direct correlation between FAAs and formation of biogenic amines was found (Fig. 1). This agrees with previous studies of Bover-Cid et al. (1999) who reported that the release of free amino acids as amine precursors occurred later than the early amine production and found no positive correlation between proteolytic activity of Staphylococcus xylosus (used as a starter culture) and biogenic amines production. The biogenic amines content in the examined loins was relatively low, compared to levels usually reported for dry-fermented meat products made from the whole cuts (Hernández-Jover et al., 1997; Lorenzo et al., 2007; Virgili et al., 2007). Those contradictory results could be attributed to differences in the ageing time.
| Biogenic amines | Day 14 | Day 21 | Day 28 | |||
|---|---|---|---|---|---|---|
| control sample | sample with probiotic | control sample | sample with probiotic | control sample | sample with probiotic | |
| Cadaverine | nd | nd | nd | nd | 2.20 ± 0.38* | 1.40 ± 0.28 |
| Histamine | nd | nd | nd | nd | nd | nd |
| Putrescine | 0.11 ± 0.04A | 0.14 ± 0.04A | 0.18 ± 0.04*B | 0.13 ± 0.03A | 0.26 ± 0.05*C | 0.17 ± 0.02A |
| Spermidine | 3.20 ± 0.30*A | 3.52 ± 0.26A | 2.81 ± 0.25*BC | 3.14 ± 0.20B | 2.63 ± 0.22C | 2.51 ± 0.24C |
| Spermine | 15.24 ± 2.03*A | 12.17 ± 2.52A | 14.67 ± 2.35*A | 10.22 ± 0.92BC | 14.52 ± 1.88*A | 10.06 ± 0.75C |
| Tryptamine | 33.90 ± 4.47*A | 24.72 ± 2.95A | 28.88 ± 3.06BC | 25.29 ± 4.44A | 26.65 ± 3.84*C | 22.22 ± 2.66A |
| Total | 52.45 ± 5.64*A | 40.55 ± 4.88A | 46.54 ± 5.30*A | 38.78 ± 5.30A | 46.26 ± 5.81*A | 36.36 ± 2.94A |
nd-not detected
A–C Means followed by different letters differ significantly (p < 0.05) within the same sample during ageing (Tukey's test)
Correlation between total free amino acids and total biogenic amines content
As presented in Table 3, tryptamine was found to be the main biogenic amine in dry-cured pork loins, representing about 60% of the total biogenic amines content. Tryptamine values in examined loins were slightly lower than those reported by Lorenzo et al. (2007), although in other works this biogenic amine was not detected (Latorre-Moratalla et al., 2010a; Latorre-Moratalla et al., 2007). During ageing of dry-cured loins, a significant decrease (p < 0.05) in the tryptamine content was observed in the control sample. In the inoculated sample tryptamine did not modify its content in a statistically significant way. Spermine was found to be the second dominant amine in examined loins, decreasing in the inoculated sample from an initial level of 12.17 mg/kg to 10.06 mg/kg. In the control sample the decrease was not statistically significant (p < 0.05). These values are slightly higher than those reported by Lorenzo et al. (2007) in dry-cured lacón. Quantitatively, the third biogenic amine was spermidine, which showed an average value of 2.97 mg/kg. Content of spermidine showed significant decrease during ageing for both samples (p < 0.05). These data are in agreement with the hypothesis that physiological polyamines (spermine and spermidine) are of endogenous origin and their levels are not influenced by the curing process or microbial activity (Latorre-Moratalla et al., 2007; Stadnik and Dolatowski, 2010). According to Hernández-Jover et al. (1997) spermine level can even drop, as was confirmed in the present study. There are two possible pathways of its degradation in dry-cured meat products: this polyamine can either be used as a nitrogen source by some microorganisms or it can be oxidized by polyamine oxidases which may be produced by bacteria (Lu et al., 2010). Putrescine was the only amine that showed an increase during ageing. This is in accordance with the results of Komprda et al. (2004), who also found a significant increase in putrescine content during ripening of dry fermented sausages. As illustrated in Table 3, putrescine was found to be present in a low concentration of 0.1 mg/kg after 14 days of ageing, but after 28 days exhibited about a 3-fold increase in the control sample. In the sample inoculated with L. casei putrescine did not undergo significant variations during ageing (p < 0.05). The final average values of putrescine were much lower than found in other meat products (González-Fernández et al., 2003; Lorenzo et al., 2007; Suzzi and Gardini, 2003; Tosukhowong et al., 2011; Virgili et al., 2007).
Irrespective of ageing time, the control loins had significantly higher (p < 0.05) concentration of total biogenic amines than loins inoculated with L. casei. As far as individual amines are concerned, the application of probiotic starter culture had different effect depending on the amine. Inoculation of the surface by Lactobacillus casei ŁOCK 0900 significantly reduced (p < 0.05) the content of tryptamine after 14 days of ageing but not of putrescine. In relation to the inoculation effect after 21 days of ageing, this produced a significant (p < 0.05) decrease in the contents of putrescine (27.8%). L. casei failed to reduce significantly the accumulation of tryptamine. At 28 days of ageing, the biogenic amine that suffered the greatest decrease which can be attributed to the L. casei inoculation was cadaverine (36.4%). This agrees with recent studies by Tosukhowong et al. (2011) who showed that accumulation of cadaverine, putrescine and histamine in Nham (a Thai traditional fermented pork sausage) has been significantly reduced by the addition of Lactobacillus plantarum BCC 9546. The ability to form biosurfactants, compounds not-susceptible to proteolytic enzymes - pepsin and trypsin, which exhibit antagonist activity with respect to Pseudomonas aeruginosa, Pseudomonas fluorescens, Lactobacillus acidophilus, Enterobacter aerogenes, expressed by the examined strain Lactobacillus casei ŁOCK 0900 can increase their competitiveness in suppressing the growth of wild amine producing microflora (Motyl et al., 2011). Lactobacillus casei ŁOCK 0900 has also antagonistic activity against food-borne pathogenic bacteria: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella enteritidis, Salmonella typhimurium, Listeria monocytogenes, Listeria innocua. This may contribute to improving the products' health and safety by inhibiting microbial growth and associated reduction of the content of biogenic amines. It is also possible that biogenic amines already produced, has been degraded by the probiotic strain applied in this study. To date, the ability to degrade biogenic amines by food microorganisms has been reported in the many papers summarized by Alvarez and Moreno-Arribas (2014). Identification and biochemical characterization of enzymatic activities involved on biogenic amines reduction is needed to verify this assumption.
According to some authors cited by González-Fernández et al. (2003) and Lu et al. (2010), addition of decarboxylase-negative starter cultures to carry out controlled fermentation could be an advisable practice for reducing aminogenesis during fermentation of dry-cured meat products. However, other studies cited by the same authors, failed to demonstrate the efficiency of starter cultures to reduce or inhibit the accumulation of biogenic amines, which have been attributed to their low competitiveness or difficulty to adapt to meat fermentation. The effectiveness of starter cultures in reducing biogenic amines accumulation seems to be strongly linked to the microbial quality of the raw material, as reported by Bover-Cid et al. (2001). The authors showed that the efficiency of an amine-negative starter culture (Lactobacillus sakei CTC494) in the reduction of biogenic amine production during the ripening of fermented sausages was strongly dependent on the hygienic conditions of the raw material used.
The values of free amino acids content in the dry-cured pork loins showed that the loins undergo moderate protein degradation during its ageing, less than that undergone in hams and other dry-cured meat products made from whole meat pieces, and that inoculation with a probiotic strain L. casei ŁOCK 0900 causes an increase in the degree of proteolysis that could be attributed to an intense peptidase activity. Examined loins differed in free amino acids concentration, which could be determinant for defining the taste and flavour attributes. Inoculation with a probiotic strain L. casei ŁOCK 0900 proved to be a protective measure against the accumulation of cadaverine, putrescine, spermine and tryptamine even though loins inoculated with a probiotic strain were characterized by higher availability of free amino acids, potential precursors of biogenic amines.
Acknowledgments The research described here has been supported by Ministry of Science and Higher Education. Project No. NN 312 275435. The authors sincerely thank Professor Z. Libudzisz, Head of the Institute of Fermentation Technology and Microbiology, Technical University of Łódź, for kindly providing of the strain of Lactobacillus casei ŁOCK 0900 and Professor D. Kołożyn-Krajewska, Head of the Chair of Food Hygiene and Quality Management Warsaw University of Life Sciences for preparing the starter cultures of a probiotic strain.
