Effect of Inoculation with Probiotics and Ageing Time on Selected Functional Properties and Oxidation of Proteins in Dry-Cured Pork Loins

Abstract

The effect of inoculation with a probiotic strain Lactobacillus casei ŁOCK 0900 on changes in selected functional properties and protein oxidation of dry-cured pork loins during ageing was studied. The protein carbonyls content tended to increase significantly (p < 0.05) throughout ageing to reach a maximum level which was approximately two-fold the initial level in both samples. No statistically significant differences (p < 0.05) in carbonyl content were found between the control sample and sample inoculated with L. casei. The passage of ageing time was coupled with a statistically significant (p < 0.05) increase in the trichloroacetic acid soluble peptides concentration in examined loins. Inoculation with a probiotic strain did not have an influence on trichloroacetic acid soluble peptides concentration during the shortest ageing period. Results of measurements carried out for the control sample after 21 and 28 days of ageing were significantly lower (p < 0.05) than those obtained for the sample inoculated with L. casei ŁOCK 0900. Free α-amino acids content in both samples increased continuously with increasing ageing time (p < 0.05). Inoculation of loins with a probiotic strain Lactobacillus casei ŁOCK 0900 resulted in significantly higher peptides and free amino acids content. These differences might affect their organoleptic characteristics that need further investigations.

Keywords:

• Probiotics
• Dry-cured meat products
• Functional properties
• Oxidation
• Ageing

INTRODUCTION

The main role of diet is to provide nutrients to meet host physiological requirements, while giving the consumer a feeling of satisfaction and well-being. However, the increase in knowledge of nutrition has led to the development of foods which, beyond meeting nutrition needs, also controls and modulates various functions in the body. Therefore, the development of the market for the so-called functional foods or foods that provide additional health benefits beyond basic nutrition is on the rise.[1 ⁻ 4] Probiotics represent one of the largest functional food markets. Among the available probiotic products, most are some form of dairy, such as milk, ice cream, yogurt, cheese, and frozen desserts, despite the continuous growth of the non-dairy sector.[2]

The use of probiotics is an attractive approach for the development of health beneficial meat products.[4 ⁻ 13] Hammes and Hertel[8] suggested the possibility of developing starter cultures that exhibit specific probiotic properties and which also produce the required technological and sensory tasks in the meat matrix. Suitable probiotic cultures may provide two mechanisms of safety. Firstly, by inhibiting pathogenic bacteria in the meat product and, secondly, by inhibiting pathogens within the gastrointestinal tract.[12]

Meat products processed by fermentation without heating may be adequate for the carriage of probiotics into the human gastrointestinal tract.[4,13,14] Inoculation with lactic acid bacteria results in a rapid decrease in pH, which affects the firmness, colour, aroma, and flavour development of fermented meat products.[15] Their addition to meats may improve safety and stability of the product extending its shelf life and provides diversity resulting in new sensory properties as well as health benefits.[9,16,17]

Probiotics are cultures of living microorganisms-mainly intestinal strains of Lactobacillus and Bifidobacterium-which contribute positively to the activity of intestinal microflora and, therefore, to the host health when introduced orally in the gastrointestinal tract in adequate amounts.[2 ⁻ 5,7,10 ⁻ 12,18] The addition of probiotics to the intestine may re-establish the colonic and intestinal microbial balance. The colonisation of the gut by probiotic bacteria prevents the growth of harmful bacteria by competitive exclusion in the intestinal tract and by the production of antimicrobial compounds such as bacteriocins, organic acids, and hydrogen peroxide.[6,7,18,19] It has also been shown that probiotics positively affect immunoglobin production, antibody response, and other cellular immune responses that may contribute to good health and disease resistance.[18]

During the manufacture of dry-cured fermented meat products, different physicochemical changes take place, of which proteolysis is one the most important phenomena. Muscle proteins during ageing of fermented meat products undergo changes in solubility and are also degraded, particularly by the enzymes naturally present in the muscle.[20] Proteolysis is one of the main degradation mechanisms affecting proteins during ageing of fermented meat products. Proteolysis contributes to texture by breakdown of the muscle structure, to taste through the generation of small peptides and free amino acids and to aroma by further degradation of some free amino acids. There are several consecutive stages in proteolysis: breakdown of major myofibrillar proteins by the action of calpains and cathepsins, generation of polypeptides that act as substrates for peptidases to generate small peptides, and intense generation of free amino acids by the action of aminopeptidases.[21]

In recent years interest in probiotics in meat products has greatly increased and numerous studies have been carried out.[6,7,10 ⁻ 12] Efforts have been primarily directed to the study of the therapeutic utility of probiotic strains for their potential application to meat fermentation. But, until recently, there is no information regarding the effects of probiotics on the basic functional properties of proteins of dry-cured fermented meat products. Some researchers[22,23] have investigated the effect of high hydrostatic pressure on the oxidative stability of proteins of vacuum-packaged Iberian dry-cured ham. Nevertheless, the occurrence of protein oxidation in the muscles of dry-cured loins inoculated with probiotic has not been studied so far. The aim of this research was to investigate the effect of inoculation with a probiotic strain Lactobacillus casei ŁOCK 0900 on changes in selected functional properties and protein oxidation of dry-cured pork loins during ageing.

MATERIALS AND METHODS

Preparation of the Experimental Dry-Cured Loins

Fifteen loins (M. longissimus thoracis) with an average weight of 2.4 ± 0.4 kg were excised at 24 h post mortem from left half-carcasses of Polish Large White purebred fatteners chilled at 4°C. At 48 h post mortem all loins underwent salting using a surface massage with a mixture of 20 g of NaCl, 9.7 g of curing salt, and 0.3 g of KNO₃ per kg of loin. After completion of salting, the loins were kept at 4°C for a total of four days to allow the curing mixture to penetrate. Five of the loins were regarded as control samples. The remaining 10 loins were inoculated with 0.2% (v/w) of Lactobacillus casei ŁOCK 0900 (Patent No. P-382760) to achieve initial level of 10⁶–10⁷ CFU/g meat. The strain was derived from the Collection of Pure Cultures of Industrial Microorganisms, Institute of Fermentation Technology & 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. Selection of the strain was based on the results of in vitro studies, which comprised determination of resistance to the acidity of gastric juice and to bile, adherence to epithelial cells as well as antimicrobial activity. These studies were carried out according to FAO/WHO recommendations.[24,25] This species rank among the typical microflora of human intestines and therefore can be safely used for the production of fermented meat products and probiotics preparation. This strain showed strong antagonistic activity against gram-positive and gram-negative pathogens. Lactobacillus casei lyophilizate was produced according to the procedure previously described by Nebesny et al.[26] Subsequently, the loins were hung at 16°C in a drying room with a relative humidity of between 80 and 90% for seven days. After that, the loins were cold smoked for an hour and then aged at 4°C for 14, 21, and 28 days. Samples for analyses were taken at each established stage. Before analyses, the outer layer (1 cm) of the dry-cured loins was discarded and then the entire section was minced (ø 3 mm).

pH Measurement

For pH measurements 10 g of minced sample was homogenised with 100 ml of distilled water for 1 min using a homogenizator (T25 Basic ULTRA-TURRAX, IKA Germany). The resulting suspension was allowed to stand for 15 min before the pH was measured with a digital pH-meter CPC-501 (Elmetron, Poland) equipped with a pH electrode (ERH-111, Hydromet, Poland).

Determination of Trichloroacetic Acid (TCA) Soluble Peptides

TCA soluble peptides content was determined according to the method of Greene and Babbitt.[27] Minced sample (3 g) was added to 27 ml of cold 5% (w/v) TCA. The mixture was homogenised (IKA Ultra-Turrax T25 Basic, Germany) at 9500 rpm for 1 min and kept on ice for 1 h followed by centrifugation at 12,000 g for 15 min to collect the supernatant. The soluble peptides content in the supernatant was measured by the Biuret method[28] and expressed as micromoles of tyrosine released/g sample.

Determination of Free α-Amino Acids

Free α-amino acids were determined according to the method of Benjakul and Morrissey.[29] Minced sample (1 g) was homogenised (IKA Ultra-Turrax T25 Basic, Germany) with 29 ml of 1% sodium dodecyl sulfate (SDS) at 9500 rpm for 1 min. The mixture was incubated at 85°C for 15 min and then centrifuged at 13,500 g for 10 min at room temperature. The reaction mixture containing 125 μ l of supernatant and 2 ml of 0.2M phosphate buffer, pH 8.2 was mixed with 1 ml of 0.01% (w/v) 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution and incubated in dark at 50°C for 30 min. The reaction was terminated with 2 ml of 0.1M sodium sulfite. The absorbance was measured at 420 nm using a UV-VIS spectrophotometer (Nicolet Evolution 300 BB, Thermo Fisher Scientific, Waltham, USA). Free α-amino acids were calculated and expressed as micromoles of leucine/g sample.

Protein Oxidation

Protein oxidation was measured by estimation of carbonyl groups formed during incubation of purified myofibrils[30] with 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCL following the method described by Oliver et al.[31] Carbonyl concentration was expressed as nanomoles of DNPH fixed per milligram of protein using an absorption coefficient of 21.0 mM⁻¹ cm⁻¹ at 370 nm. Protein oxidation was expressed as nmol carbonyls/mg protein.

Statistical Analysis

Measurements were made in triplicate and the obtained results were statistically analysed using the Statgraphics v. 5 (Manugistics Inc., Rockville). A mean and standard error was calculated for each test. Two-way analysis of variance (ANOVA) was performed to analyse the effect of inoculation and time of ageing. Mean comparisons were run by Tukey's test (at the significance level p < 0.05).

RESULTS AND DISCUSSION

pH

The changes in pH values of the samples are shown in Fig. 1. Obtained results pointed out that inoculation with L. casei ŁOCK 0900 had a statistically significant (p < 0.05) effect on the pH of dry-cured pork loins. During the whole ageing period dry-cured loins inoculated with L. casei ŁOCK 0900 exhibited significantly lower (p < 0.05) pH than the control. The pH values found after ageing confirmed the effectiveness of the inoculation with lactobacilli for acidification of loins. Similar or greater reductions in pH have previously been observed in other meat products fermented by intestinal lactobacilli.[6,12] A decrease in pH was probably due to an increased concentration of lactic acid produced by lactobacilli. The pH of dry-cured pork loins generally increased with increasing ageing time to 5.4 and 6.0 at the end of the longest ageing period (28 days) for the inoculated and control sample respectively, but the differences were not statistically significant (p < 0.05).

Figure 1 Changes of pH in dry-cured fermented pork loins during ageing within the same ageing time; means followed by the common small letter do not differ significantly (p < 0.05). Means within a given treatment followed by the common capital letter do not differ significantly (p < 0.05). Error bars represent ± standard error.

Lactobacilli are the major producers of lactic acid responsible for the decrease in pH during manufacturing of dry-cured meat products.[32] Authors cited by Aro Aro et al.[1] contribute the reduction in pH to the reduction or elimination of undesirable bacteria, such as Enterobacteriaceae. Moreover, lactic acid is often suggested to be major contributor to the acid aroma and taste of fermented meat products and may play an important role in the development of its texture due to acid-induced changes in myosin[33] and collagen.[34]

TCA Soluble Peptides

Figure 2 presents the results of the determination of TCA soluble peptides concentration in dry-cured fermented pork loins inoculated with L. casei ŁOCK 0900 and control at various stages of ageing. The ageing period had a significant effect (p < 0.05) on TCA soluble peptides concentration in examined loins (Fig. 2). The passage of ageing time was coupled with an increase in the TCA soluble peptides concentration, with a mean value of 37 ± 1.77 for the loins inoculated with L. casei ŁOCK 0900 aged for 14 days, and 79 ± 2.99 for these samples with 28-day ageing period. As far as the control sample is concerned, the results were as follows: 33 ± 1.51 after 14 days of ageing and 61 ± 2.41 for the most aged samples. The behavior of this parameter corresponds to the course described by Visessanguan et al.[15,32] for fermented pork sausage.

Figure 2 Changes in TCA soluble peptides in dry-cured fermented pork loins during ageing within the same ageing time; means followed by the common small letter do not differ significantly (p < 0.05). Means within a given treatment followed by the common capital letter do not differ significantly (p < 0.05). Error bars represent ± standard error.

Inoculation with L. casei probiotic strain did not have an influence on TCA soluble peptides concentration during the shortest ageing period. Results of measurements carried out for the control sample after 21 and 28 days of ageing were significantly lower (p < 0.05) than those obtained for the sample inoculated wit L. casei ŁOCK 0900. The most noticeable differences were noted between samples aged for 28 days.

The content of peptides increases in all meats during post mortem ageing. The proteolytic enzyme system in muscle is quite complex and comprises endo- and exopeptidases. Main endopeptidases are calpains and cathepsins B, L, H, and D while main exopeptidases are tri-peptidylpeptidases I and II and di-peptidylpeptidases I, II, III, and IV as well as alanyl, arginyl, methionyl, leucyl, and pyroglutamyl aminopeptidases.[21] Calpains are probably the most extensively researched protease family with regard to meat science and it is widely accepted that they are the primary proteolytic system responsible for post mortem proteolysis. As far as cathepsins are concerned many research groups have discarded the contribution of cathepsins to meat proteolysis on the basis of a number of observations.[35]

Proteolytic reactions that occur during fermentation of dry-cured meat products have been studied extensively.[21,36 ⁻ 40] The continuous increase in TCA soluble peptides concentration during ageing of dry-cured loins is thought to reflect the activity of proteases, but the respective roles of indigenous and bacterial enzymes in protein degradation during ageing of fermented meat products have been a source of controversy.[40] Kato et al.[37] reported that since most bacteria grown in fermented meat products have faint or almost no proteolytic activity, the degradation of proteins is not greatly affected by the bacteria. However, lactic acid bacteria may form somewhat favourable conditions for the meat protein degradation by causing a decrease in pH, which results in increased activity of muscle proteases. As reviewed by Hierro et al.,[36] several species of Lactobacillus and Pediococcus used as starter cultures played no role in protein hydrolysis. Nevertheless, the intracellular peptidasic activities of lactic acid bacteria could contribute to the increased levels of free amino acids. Despite this, the lactic acid bacteria are considered to be weakly proteolytic compared with many other groups of bacteria (Bacillus, Proteus, Pseudomonas, coliforms) which may be present in fresh meat and other ingredients, but there is no direct evidence that these microorganisms contribute significantly to the flavor of fermented meat and it is doubtful whether their enzymes play any role at all in meat proteolysis. Molly et al.[38] concluded that initial protein degradation seemed to be originating from cathepsin D like muscle enzymes. In a later stage, bacterial enzymes become more important in further degradation of the protein fragments formed. Hierro et al.[36] found that the proteolytic activity occurring during at least the first 50 days of the ripening of dry fermented sausages is due to endogenous proteases. The present bacteria play a minor role, if any, in this phenomenon.

Free α-Amino Acids

The changes in the content of free α-amino acids observed in dry-cured fermented pork loins during ageing are given on Fig. 3. Free α-amino acids content in both samples increased continuously with increasing ageing time (p < 0.05). The behavior of this parameter corresponds to the course reported by Visessanguan et al.[15,32] for Nham and Hierro et al.[36] for dry fermented sausages. Loins inoculated with L. casei ŁOCK 0900 generally had higher increase in free α-amino acids content, compared with the control (p < 0.05). The total free α-amino acids content of the loins constituted 22–26 μmole leucine/g on 14 day in the control and inoculated sample, respectively. On day 21 an increase in the content of amino acids was observed and ranged between 37–42 μmole/g. A further increase up to the range of 44–53 μmole/g was observed after 28 days of ageing. The marked difference in the content of free amino acids among samples was noticeable at the end of the longest ageing period. In agreement with the results of this study, several other workers have shown that proteolysis during the ripening of fermented meat products is reflected by an increase in free amino acids concentration.[1,15,32,41]

Figure 3 Changes in free α-amino acids in dry-cured fermented pork loins during ageing within the same ageing time; means followed by the common small letter do not differ significantly (p < 0.05). Means within a given treatment followed by the common capital letter do not differ significantly (p < 0.05). Error bars represent ± standard error.

The pattern of proteolysis in fermented meat products is influenced by several variables such as product formulation, processing conditions, and starter culture.[41] During the ageing process, the pH of samples was at the acidic range (Fig. 1). These conditions might favour the hydrolysis of muscle proteins caused by cathepsins.[38] The length of the process is also important for the activity of the proteolytic enzymes. As longer is the process, a more pronounced action of the enzymes can be observed.[41] Thus, a more intense proteolysis can be expected when longer is the ageing time as was observed in the present study conditions.

The higher TCA soluble peptides and free α-amino acids content in dry-cured pork loins inoculated with L. casei ŁOCK 0900 suggested that the greater degradation took place. This might be associated with the formation of flavouring compounds or enhanced taste due to the several low molecular weight compounds, including peptides, amino acids, aldehydes, organic acids, and amines.[42] McCain et al.[43] observed a significant correlation between organoleptic determinations of dry-cured ham taste and the concentration of free amino acids and peptides. Compounds such as peptides and amino acids may also contribute to the taste by exceeding the taste properties of the pure compounds due to synergistic interactions.[44]

Protein Oxidation

Figure 4 showed the time course of protein oxidation in dry-cured fermented pork loins as measured by carbonyl groups accumulation. In the present study, no statistically significant differences (p < 0.05) in carbonyl content were found between the control sample and sample inoculated with L. casei ŁOCK 0900. The length of ageing time significantly (p < 0.05) affected the accumulation of carbonyls.

Figure 4 Changes in carbonyl content in dry-cured fermented pork loins during ageing within the same ageing time; means followed by the common small letter do not differ significantly (p < 0.05). Means within a given treatment followed by the common capital letter do not differ significantly (p < 0.05). Error bars represent ± standard error.

The initial level of protein oxidation was 5.0 and 5.2 nmole carbonyls/mg proteins in the inoculated and control sample, respectively. The content of protein carbonyls tended to increase throughout ageing to reach a maximum level which was approximately two-fold the initial level in both samples. The carbonyl content increase was most pronounced on the day 21.

These results of carbonyls (5.0–10.3 nmole carbonyls/mg proteins) were quite similar to previous data reported by Cava et al.[22] for sliced dry-cured Iberian ham (5.5–10.9 nmole carbonyls/mg proteins). However, the carbonyl compounds were much higher than that described by Wang et al.[45] (0.33–1.57 nmole carbonyls/mg proteins) for dry-cured Xuanwei ham.

Oxidative processes are one of the primary mechanisms of quality deterioration in meat and meat products. Protein oxidation results in modifications of functional properties of the proteins due to formation of protein carbonyls, hydroperoxides, and sulfoxides, and further to protein fragmentation, cross-linking and aggregation, and to decrease protein solubility. The oxidation of muscle proteins involves the loss of essential amino acids and decreases protein digestibility affecting its nutritional value. Proteolytic enzymes are inactivated by oxidation, and oxidative modification of proteins has been found to result in reduced tenderness and juiciness. Moreover, colour deterioration of meat products has been related to protein oxidation phenomenon.[46]

CONCLUSIONS

Ageing time showed a remarkable influence on the selected functional properties and oxidative status of dry-cured fermented pork loins through the modification of their TCA soluble peptides and free α-amino acids concentration and carbonyl content. Analysis proved that inoculation of loins with a probiotic strain Lactobacillus casei ŁOCK 0900 resulted in significantly higher peptides and free amino acids content. Further investigations are needed to explain how these changes in functional properties contribute to the organoleptic characteristics of dry-cured meat products.

ACKNOWLEDGMENTS

The research described was 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 strains of Lactobacillus casei ŁOCK 0900.