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Article

Influence of LAB Fermentation on the Color Stability and Oxidative Changes in Dry-Cured Meat

by
Joanna Stadnik
1,
Paulina Kęska
1,*,
Patrycja Gazda
1,
Łukasz Siłka
1 and
Danuta Kołożyn-Krajewska
2
1
Department of Animal Food Technology, Faculty of Food Science and Biotechnology, University of Life Sciences in Lublin, Skromna 8, 20-704 Lublin, Poland
2
Department of Food Gastronomy and Food Hygiene, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences (WULS–SGGW), Nowoursynowska 159c, 02-776 Warsaw, Poland
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(22), 11736; https://doi.org/10.3390/app122211736
Submission received: 2 November 2022 / Revised: 12 November 2022 / Accepted: 16 November 2022 / Published: 18 November 2022
(This article belongs to the Special Issue Fermentation Technology in Food Production)
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Abstract

:
Consumption of food products with a high level of compounds that are products of fat or protein oxidation is associated with the onset of various diseases. Therefore, this study estimated the level of oxidation in a fermented long-maturing tenderloin inoculated with lactic acid bacteria strains. To estimate the level of fat and protein exposure to oxidative factors, thiobarbituric acid reactive substances (TBARS), oxidation–reduction potential (ORP), oxygenation index (Oxi) as well as surface hydrophobicity (HS) of protein, and the content of free sulfhydryl (SH) groups were used as indicators of oxidation status. To validate the results, changes in the color of the products were determined using instrumental methods. This study confirmed the relationship between fat oxidation (based on TBARS) and oxidation of myoglobin as a protein representative (based on Oxi). Indeed, statistical analysis showed that TBARS, Oxi and SH were correlated with each other and could be used as indicators of oxidation in fermented products. The findings of this study also showed the use of LAB as starter cultures for meat fermentation may have an impact on the level of oxidation; in particular, the BAUER strain showed a protective effect on proteins in the final stages of the production of dry-cured pork loins.

1. Introduction

For centuries, human nutrition has been based on meat. Meat is a source of protein, vitamins, minerals, and other bioactive compounds. Due to its chemical composition, such as high protein content, meat tissue is a potential medium for pathogens. Therefore, to extend the shelf life of meat, fermentation has long been used as a preservation method. Besides extending the shelf life, fermentation produces specific compounds that are responsible for the taste and smell of the fermented product [1]. Thus, fermentation is used as a biopreservation method although nowadays meat is fermented mainly to enhance taste and flavor [2]. There are no clearly defined starting and end stages of the fermentation process. The moment when lactic acid bacteria (indigenous or industrial) begin to grow and multiply on meat and when carbohydrates are converted into lactic acid are considered the starting stage of fermentation. The moment when microflora nutrients are exhausted is considered the end stage. In most industries, commercial and industrial micro-organisms are used as starter cultures in fermentation, such as lactic acid bacteria (LAB), especially Lactobacillus, whose fermentation properties result in a number of desired product changes and increase fermentation efficiency, thus leading to consistent product quality [3,4]. However, meat can also be fermented using environmental strains, but this process is difficult to control because of the way in which microflora develops and its final effects on the product are not certain. The primary advantage of using LAB in meat fermentation is their ability to break down carbohydrates into lactic acid, which lowers the pH of meat and acidifies it. Other studies have reported that the action of endogenous proteases is enhanced with the decrease in the pH of the meat matrix [5,6]. In addition, a low pH limits the growth and development of undesirable microflora in the meat, thereby increasing the microbiological purity of fermented meat products. The reduction in pH also suppresses the growth of spoilage bacteria, which are responsible for the poor microbiological quality of end products on the one hand and excessive proteolysis leading to texture defects on the other hand [2,7,8]. In addition, under low pH conditions, meat proteins undergo gelation, which improves the texture of the meat in dry-cured products [9]. Furthermore, fermentation enhances the organoleptic properties of the final product, giving it a slightly sour taste. During meat fermentation, complex processes shape the sensory perception of smell which consumers define as desirable. In addition, LAB reduces nitrate V to nitrite III in cured products, which is further broken down into nitric oxide, which in turn interacts with myoglobin and forms nitrosomyoglobin. Nitrosomyoglobin is responsible for the characteristic red color of the fermented meat products [10]. The enzymes derived from LAB also play a crucial role as they reduce the peroxide content and thus reduce the rancidity of fats and stabilize the color of products [11,12,13]. For instance, Lactobacillus plantarum AR113, Pediococcus pentosaceus AR243, and L. plantarum AR501 isolated from Chinese fermented foods showed high activity in scavenging α-diphenyl-β-picrylhydrazyl and hydrogen radicals and a strong inhibition of peroxidation [14].
The degree of advancement of the oxidation processes in meat products most often relates to changes in fat. The most popular is the thiobarbituric acid (TBA) reactive substance test (TBARS). Lipid oxidation proceeds through the free radical chain mechanism, yielding many end products, in particular aldehydes. One of the best-studied aldehydes is malondialdehyde (MDA), which reacts with the acid with TBA. Therefore, it is considered a marker of oxidative damage within the fat fraction [15]. Besides fats, proteins are also an important component of meat. During fermentation, aging, and storage of meat products, moderate protein degradation can effectively improve the taste and nutritional value of fermented meat, whereas excessive protein oxidation can adversely affect meat quality by changes in texture, color, and flavor. Protein oxidation is a free radical chain reaction, similar to lipid oxidation, in which the polypeptide backbone and amino acid side chains are susceptible to oxidative attack and the formation of protein carbonyls. Due to the progressive oxidation of proteins, the digestibility of proteins decreases, which reduces the nutritional value of meat products (e.g., by limiting the content of essential amino acids) and even causes product spoilage and deterioration, thus seriously affecting the safety of the product for consumption. The most studied modification of proteins as a result of oxidation in meat products is the formation of carbonyl compounds [16]. Secondly, the loss of thiol groups can be used as a marker of protein oxidation in meat products due to the high susceptibility of cysteine residues to oxidation [17]. Moreover, it is generally accepted that oxidation reactions lead to changes in the secondary and tertiary structures of proteins [18], and the hydrophobicity of the surface can be used to detect structural changes on the surface of proteins [19,20].
To maximize the quality and nutritional value of the fermented meat, Wang et al. [21] emphasized the need to optimize the rate of change of oxygen-sensitive food ingredients (such as fats and proteins) in order to prevent their excessive oxidation. Therefore, in this study, the influence of selected LAB strains on the color and oxidative changes of fermented meat products after 6, 9 and 12 months of aging was investigated.

2. Materials and Methods

2.1. Meat Product Preparation

Pork loins (m. Longissimus thoracis) with an average weight of 2.0 ± 0.2 kg were obtained from carcasses of Great White Polish fattening (hogs, live weight of approximately 120–130 kg). The animals were conventionally slaughtered after an electrical stunning in local abattoir (Lublin, Poland). Loins were excised at 24 h post mortem from half carcasses chilled at 4 °C. Then, all loins were dry-cured by a surface massage using a curing mix (99.97% sea salt, and 0.03% NaNO3) in a proportion of 2.8% per kg of loin. After salting, the loins were maintained at 4 °C for 24 h to allow for the penetration of the curing mixture. A part of the loins (n = 3) served as the control, whereas the remaining loins (n = 3 for each variant) were surface-inoculated with 0.2% (v/w) LAB strains to an initial level of 106–107 CFU/g of meat. The LAB used in meat fermentation were as follows: Lacticaseibacillus rhamnosus LOCK900 (LOCK), Bifidobacterium animalis subsp. lactis BB12 (BB), and Lactobacillus acidophilus Bauer Ł0938 (BAUER). The inoculum was prepared at the Department of Food Gastronomy and Food Hygiene (WULSSGGW, Warsaw, Poland) according to the procedure previously described by Wójciak et al. [22]. The prepared loins were kept in a disinfected laboratory aging chamber (Italfrost-De Rigo-GS, Pszczyna, Poland) with a relative humidity of 75% ± 0.3 at 16 ± 1 °C for 14 days. Then, whole pieces of loins were vacuum-packed in 80-µm polyamide/polyethylene bags (Wispak, Lublin, Poland) and aged at 4 ± 1 °C for 6, 9, and 12 months. All analyses were performed in triplicate.

2.2. pH and Water Activity (aw) of Products

The pH of the products was evaluated by homogenizing the ground samples with distilled water (1:10) for 1 min using a homogenizer (T25 Basic ULTRA-TURRAX, IKA Germany). The resulting suspension was allowed to stand for 15 min before measuring the pH using a CPC-501 digital pH meter (Elmetron, Poland) equipped with a pH electrode (ERH-111, Hydromet, Poland). Before measurements, the electrode was calibrated with standardized buffers at pH 4.0, 7.0, and 9.0.
The water activity (aw) of the products was measured in a ground meat sample at 20 °C using a LabMaster instrument (Novasina AG, Lachen, Switzerland), which provides temperature-controlled measurements.

2.3. Color Parameter

The parameters of brightness (L*), redness (a*), and yellowness (b*) in the L*a*b* system were measured by the reflection method using an X-Rite 8200 spherical spectrophotometer with a measuring hole of diameter 12.7 mm. Measurements of these parameters were carried out taking into account the gloss (SPIN) in the measuring range λ = 360 ÷ 740 nm using a standard light source D65 and a standard colorimetric observer with a field of view of 10°. Fresh 40-mm thick slices from three different locations of each batches were used to measure the color parameters.

2.4. Oxidation Status

2.4.1. Thiobarbituric Acid Reactive Substances (TBARS) Level

The TBARS index was determined using the method of Pikul et al. [23]. A mixture of 3 g of ground meat, 12 mL of 4% cold perchloric acid, and 200 µL of 0.01% ethanolic BHT solution (Sigma-Aldrich, St. Louis, MO, USA) was homogenized for 1 min using a laboratory homogenizer (T25 Basic Ultra-Turrax, IKA, Staufen, Germany) and separated on a filter paper. Then, 650 µL of the filtrate was mixed with 650 µL of 0.17% aqueous 2-thiobarbituric acid solution and incubated at 100 °C for 20 min. Absorbance was read at 532 nm at approx. 20 °C using a Nicolet Evolution 300 spectrophotometer (Thermo Electron Corp., Waltham, MA, USA). A mixture made of 650 µL of 4% cold perchloric acid and 650 µL of 0.17% aqueous solution of 2-thiobarbituric acid served as the reference sample. The results were expressed in milligrams of MDA (malondialdehyde) in 1 kg of the product, as shown in the following Equation:
TBARS [mg MDA/kg] = 5.5 absorbance value of the tested sample

2.4.2. Oxidation–Reduction Potential (ORP)

The oxidation–reduction potential was determined in an aqueous meat homogenate (10:1) by a combined platinum electrode, type ERPt-13, using a digital pH conductometer CPC-501 (Elmetron), following the method of Ahn and Nam [24].

2.4.3. Oxygenation Index (Oxi)

The Oxi was determined as the ratio of reflectance between 630 nm (maximum of the oxidized myoglobin) and 580 nm (maximum of the oxygenated myoglobin) measured using an X-Rite 8200 spherical spectrophotometer [25].

2.4.4. Surface Hydrophobicity

The hydrophobic surface (HS) of protein fraction solutions was determined following the method of Lieske and Konrad [26]. In this method, the surfactant Tween 80 (polyoxyethylene sorbate) was attached to meat slices using hydrophobic protein fragments, and then the complexing ability of the test dye (Bio-Rad) was quantified. The HS is expressed as the ratio of the designated nonpolar residues to the sum of polar and nonpolar residues [%]. To isolate proteins, the minced meat was homogenized thrice on ice with distilled water and centrifuged using a grinder with a disc having 3-mm holes. The supernatant was pooled after each centrifugation, and its protein content was determined using the Bradford method based on the BSA standard curve (0.019–5 mg mL−1). Protein extracts were adjusted to a concentration of 0.5% using distilled water, and their absorbance values with and without the addition of the surfactant were measured against 0.1 M phosphate buffer (pH 6.2) at λ = 595 nm using a Nicolet Evolution 300 spectrophotometer (Thermo Electron Corporation) after 10 min of incubation.

2.4.5. Free Sulfhydryl Group (SH) Contents

Meat protein samples were adjusted to 3 mg/mL in Tris–glycine–EDTA (TGE, pH 8.0), with 2.5% (w/v) sodium dodecyl sulfate (SDS). The protein solution (4 mL) was mixed with 0.04 mL of Ellman’s solution. The mixed solution was then shaken for 10 min at dark and centrifuged at 3000 rpm for 2 min. The supernatant was collected, and its absorbance was read at 412 nm using a spectrophotometer (UVmini-1240, Shimadzu, Japan) [27]. SDS–TGE buffer was used as the reference, reduced glutathione (GSH) was used as the calibration curve (0–1.0 mg/mL), and the absorbance value was converted to free SH groups [28].

2.5. Statistical Analysis

Each experiment was carried out three times, in three independent repetitions (n = 9), and data were statistically analyzed using two-way ANOVA and Tukey’s post hoc test (significance level of p < 0.05). The relationships between lipid and protein oxidation measurements and the Oxi were evaluated using Pearson’s correlation coefficients. For hierarchical cluster analysis (HCA), Ward’s method of linkage with squared Euclidean distance as a measure of similarity for multivariate analysis was used. All analysis were performed using the Statistica software, version 13.3 (Dell, Inc., Round Rock, TX, USA).

3. Results

The results of the evaluation of the physicochemical parameters (pH and aw) of the fermented meat products are presented in Table 1.
As shown in Table 1, the lowest pH value was observed in the LOCK test (5.607; p < 0.05), whereas the other research variants did not differ statistically significantly (p > 0.05) after 6 months of aging, reaching an average pH value of 5.666. With time, the pH value started fluctuating, i.e., it increased after 9 months and decreased in the 12th month of ripening of the pork loins. These changes were statistically significant only in the LAB samples. Among these, the biggest change was observed in the LOCK test, which increased the pH of the products by 0.361 units after 9 months. At the end of the research period (12 months), the influence of LAB on the pH value of the products was not observed, and there were no statistically significant differences (p > 0.05). The LOCK test was also characterized by the most dynamic changes in aw in the products, recording the largest increase in the analyzed period. In the 12th month of aging, the influence of LAB on aw was observed, where a statistically significantly (p > 0.05) higher aw was observed in LOCK and BB12 samples (average 0.831), whereas in BAUER samples, aw was significantly lower (0.810; p < 0.05) than that of samples without LAB.
The influence of LAB strains on the color of the fermented meat products is presented in Table 2. The influence of LAB strains on the brightness (L*) of the products was observed. The LAB samples were significantly brighter than the control samples (except for the BAUER test after 12 months of aging). In LOCK and BB samples, no effect of the storage time on the changes in the L* parameter was observed. However, samples inoculated with the Lactobacillus acidophilus Bauer strain L0938 (BAUER) were an exception in which significant darkening of the color was observed after 12 months of aging (decrease in the L* parameter by 5.26 units) compared with that at the beginning of the research period (6 months) (p < 0.05). Darkening was also observed in samples subjected to spontaneous fermentation (C) in the 9th month of aging (p < 0.05), but this change was not permanent as the color lightened to the initial level in the next research period.
The a* parameter determines the chromaticity ranging from red to green, with a higher a* value representing the higher redness of the meat. As presented in Table 2, a significant effect of using BB and BAUER strains on a* values was observed during the entire study period, whereas the LOCK test did not differ significantly from the control (C; p > 0.05). In C and LOCK samples, an increase in the redness (a*) was observed in the 9th month of ripening, followed by a decrease in a* in the 12th month. At the same time, in BB and BAUER tests, the value of a* decreased only in the 12th month, showing the higher stability of redness during production. It is also worth noting that in the 12th month, the highest loss of redness (decrease in a* by 5.49 units) in the general tone of color was observed in BB samples. Taking into account the distribution of values characterizing the b* parameter, a significantly higher b* value was observed in BB and BAUER tests compared with the control sample in the 6th month of aging (p < 0.05). These samples were found to be more yellow in the overall color tone. In addition, in these test variants, the b* parameter remained stable throughout the entire study period, whereas an increase in b* values was observed in C and LOCK samples in the 9th month (by 2.14 units and 3.49 units, respectively), and this trend was maintained until the 12th month of aging. The b* values after 9 and 12 months did not differ significantly (p > 0.05) between all variants. Color stability can also be determined using the Oxi calculated by the ratio 630/580 nm, which is correlated with the formation of metmyoglobin. The lower the value of Oxi, the more oxidized the myoglobin is in the sample. Taking these trends into account, the highest levels of metmyoglobin were observed in BAUER tests (p < 0.05) and the lowest in BB tests (p < 0.05) in the 6th and 9th months of aging. However, in the 12th month, the highest decrease in Oxi in BB tests was observed among all variants (p < 0.05), which corresponds to the decrease in the a* parameter, thus suggesting changes caused by myoglobin oxidative factors.
The TBARS are commonly used as indicators of the degree of lipid peroxidation. They are formed during the second stage of autoxidation, during which peroxides are oxidized to aldehydes and ketones. In this study, the influence of the LAB strain used on the TBARS value during cold storage was confirmed. The use of LAB strains was associated with an increase in the TBARS value, as shown in Table 3. Among the LAB variants, the highest TBARS value was observed in LOCK tests and the lowest in BB and BAUER tests (in the 9th month). Taking into account the time criterion, the TBARS value significantly (p < 0.05) decreased in all analyzed variants with an increase in the period of storage (p < 0.05), compared with the initial value. Another parameter related to the oxidative status in the meat matrix is the ORP index. It is used to assess the balance between the formation of reactive oxygen species and the degree of their neutralization. A lower ORP value indicates a higher ability to donate electrons and eliminate free radicals. Similar to TBARS, the ORP index decreased with an increase in the fermentation period, although from the 9th month, these changes were not statistically significant (p > 0.05) (except for the BAUER test, where a significant systematic decrease in the ORP index was observed). Taking into account the influence of LAB on the ORP index, in the first research period (6 months), BB samples showed the best (lowest) ORP index, although in the 9th and 12th months, the LOCK tests showed a more favorable ORP index compared with samples subjected to spontaneous fermentation.
Surface hydrophobicity is determined by the number of hydrophobic groups on the surface of the protein molecules. In the first research period (6 months), a decrease in surface hydrophobicity was observed in LAB samples compared with the control sample subjected to spontaneous fermentation. Further analysis of the data showed that surface hydrophobicity was stable in the next research period (9 months) and then decreased with an increase in the refrigerated storage time of the products, reaching the lowest value in the 12th month. The BAUER test is an exception, in which a systematic increase in surface hydrophobicity was observed, reaching the highest value after 12 months in all samples (Figure 1).
The free SH group content can be considered a characteristic of protein unfolding and/or denaturation. As shown in Figure 2, in 6-month-old meat products, the number of SH groups was similar in C, LOCK, and BB12 tests and was on average 0.178 mg/mL (p > 0.05). The lowest SH content (p > 0.05), amounting to 0.10 mg/mL, was observed in BAUER tests in the 6th month. After 9 months, a significant decrease in the SH content was observed in the control sample (C), as well as an increase in tests with the starter culture (p > 0.05), compared with the previous period. In the 9th month, the highest SH content was observed in LOCK and BB samples, averaging 0.23 mg/mL (p > 0.05), followed by BAUER (0.14 mg/mL) and C (0.12 mg/mL) samples (p > 0.05).
HCA was used to assess the direction of oxidative changes based on research methods (TBARS, ORP, OXi, and HS) and to determine the degree of oxidation of food ingredients. This method grouped the oxidation indices into relatively homogeneous effects, and the results of the analysis are shown in Figure 3. The results showed that the effects observed in the TBARS, OXi, and SH analysis showed similar trends, i.e., a separate group was created on the dendrogram. The BAUER test is an exception, in which the interaction of these parameters was different, i.e., SH showed effects more similar to ORP and HS than other oxidation indicators. For more representative results, interactions of the analyzed parameters with oxidation were shown in the 3D diagram. A strong correlation between the lower values of the surface hydrophobicity and the lower content of oxidized myoglobin was observed (illustrated by the higher value of OXi, which explains the strong negative correlation presented in Table 4) in C and LOCK samples, whereas in BAUER samples, these factors were positively correlated. Surprisingly, the higher the OXi, the higher the indices of parameters determining the degree of protein oxidation in BAUER samples (HS and SH), for which a strong positive correlation was observed (Table 4). However, it seems that protein oxidation was accompanied by a decrease in the TBARS content in BAUER tests, which may indicate a different intensity of oxidative processes in meat products conditioned by the presence of the LAB strain.

4. Discussion

Since LAB are considered natural antioxidants, several mechanisms are involved in their antioxidant effect, such as redox regulation, production of antioxidant metabolites, free radical scavenging, and metal ion chelation. For example, the cell-free LAB cell extract showed good Fe2+ and Cu+ chelating abilities. Therefore, LAB cells may contain related metal-chelating agents that determine their high antioxidant activity [29,30,31]. In addition, several studies have shown that the intracellular antioxidant activity of LAB is based on the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), nicotinamide adenine dinucleotide oxidase (NADH), NADH peroxidase, and GSH [31,32,33]. To illustrate this, the antioxidant activity of lactic acid bacteria P. pentosaceus, Lactobacillus curvatus, Lactobacillus brevis, and Lactobacillus fermentum isolated from dry Harbin sausage was evaluated. The in vitro results demonstrated that P. pentosaceus showed the highest resistance to H2O2, radical scavenging activity, reducing power, and inhibition of lipid peroxidation. Moreover, the SOD and GPx activities in P. pentosaceus were higher than in the other strains, and these results correlated with the lower rates of fat and protein oxidation in Harbin sausage [34].
Thus, antioxidant enzymes play an indispensable role in the high antioxidant activity of LAB. Therefore, LAB inoculation during the production of fermented meat products may influence the oxidation of meat during its long-term aging and storage.
However, the presence of LAB in meat products also affects their physical and chemical parameters (including pH), which is attributable to the specific characteristics of the ripening products. Based on the acidification of the meat tissue by LAB, pH values of, on average, 5.447 and 5.353 were observed in this study for the control sample and LAB variants, respectively. The lower pH value for the samples inoculated with the starter culture was due to the activity of micro-organisms capable of producing lactic acid, thus acidifying the meat matrix, as described by Libera et al. [35], Stadnik et al. [36], Kęska and Stadnik [37], and Wójciak et al. [38]. The effect of LAB on the water activity of meat products was also observed, where a higher value of water activity was recorded (an average value of 0.825) compared with that of the control samples (an average value of 0.811) during the storage of the test samples from 6 to 12 months. Water availability is correlated with water activity and is an important factor for the development of micro-organisms, including LAB, on dried meat products. However, aw can be a criterion for the microbiological safety of cured meat products. In general, the highest growth of micro-organisms is observed between aw values of 0.980 and 0.995, and the growth is inhibited at approximately 0.85 [39], which is in agreement with the present study. These findings are consistent with those of other studies using the same LAB strains.
Color stability during processing and storage is an important distinguishing feature of meat products. It influences the sensory attractiveness of these products and is the major factor encouraging the purchase by the customer. Discoloration of fermented meat products is caused by a variety of factors, such as dehydration, acidification, and oxidation. The critical factors affecting the color of the meat product are as follows: the content and chemical state of myoglobin (oxidation of myoglobin causes the accumulation of metmyoglobin and meat discoloration), reductase activity, and muscle structure (degradation and oxidation of meat proteins may relax muscle fibers and affect the scattering of light by the muscle while assessing the color, thus giving the impression of lightness in the meat). Changes in meat color coordinates (L*, a*, and b*) over time are closely related to changes in myoglobin due to oxidation. The increased metmyoglobin content is responsible for the browning of meat (associated with the loss of color stability), thus resulting in the loss of redness (a*) and changes in yellowness (b*). Tarladgis [40] reported that the iron–porphyrin coordination complex of the denatured globin molecule is responsible for the brownish gray color that occurs due to cold cuts. Modification of this structure leads to the degradation and the subsequent release of the heme molecule, which is one of the pro-oxidative factors. As a result of the chemical composition of the raw material used in this study, i.e., a high protein content with a lower fat content, the oxidative changes primarily affected proteins. Therefore, proteins may be responsible for the loss of color of meat products [41]. In the present study, the influence of LAB on color parameters, including brightness, was demonstrated, where samples subjected to spontaneous fermentation (C) were generally darker (p < 0.05) than other research variants. Otherwise, the brightness remained the same throughout the entire testing period, except for the BAUER test where a decrease in brightness was observed.
While evaluating the color stability caused by oxidation, the redness and yellowing parameters are more significant than brightness. Reduction in meat redness, which is correlated with the presence of an oxidized form of myoglobin and a brown shade of meat, was observed in the control and LOCK samples, whereas BB and BAUER samples showed a higher redness (except for the last study period, when a decrease in a* was observed). Moreover, BB and BAUER tests were characterized by a higher value of yellowness (b*), which indicates the higher influence of myoglobin oxidation on the color stability in these tests. As reported by Wang et al. [42], myoglobin oxidation is correlated with lipid oxidation, and one may exacerbate the other. In addition, oxidation of myoglobin (Fe2+) to metmyoglobin (Fe3+) produces pro-oxidants that may intensify further oxidation of lipids or other proteins in food [42]. The TBARS are the most commonly used indicators of the degree of oxidation in food products and are directly related to the fat content. In the present study, after 6 months of aging, a TBARS value of 2.446 mg/MDA was observed in the control sample, which is consistent with the reports of Libera et al. [35] in dry-cured necks (2.26 mg/MDA), although the same authors simultaneously reported a lower TBARS value in B. animalis ssp. lactis BB12 tests (1.52 mg MDA/kg (Libera et al. [35] as much as 1.666 mg MDA/kg [38]) than that achieved in the present study (2.476 mg MDA/kg) after 6 months. High TBARS values observed after 6 months of aging indicate a high content of oxidation products and may also be attributable to the reaction of MDA with amino acids, sugar, nitrates, and nitrites in the complex (Libera et al. [35]). Several authors reported a limiting effect of LAB strains on TBARS in the aging process of various meat products, but it was observed in this study, and even LOCK and BB tests showed slightly higher TBARS values than the control in the 6th month. Over time, TBARS values decreased in all variants, finally reaching (in the 12th month) the value of 1.191 mg MDA/kg in trial C and higher values, i.e., 1.166, 1.193, and 1.155 for LOCK, BB, and BAUER samples, respectively. Several authors reported a high increase in TBARS values between 4 and 6 months of aging [38,43] in fermented loins, necks, or sausages, followed by a decrease to a level similar to that recorded in the present study.
Generally, the oxidation of fats and proteins in food products follows a common mechanism as, in both cases, the mechanism is a chain reaction of initiation, transmission, and termination. These reactions are induced by active substances, such as reactive oxygen species (ROS) and reactive nitrogen species or indirectly by oxidative stress secondary products (such as already oxidized lipids or carbohydrate oxides). In particular, ROS can oxidize amino acid side chains and the protein backbone, leading to protein fragmentation or protein–protein crosslinking. In addition, oxidation processes, especially of proteins, can be catalyzed by metal ions (Fe2+, Cu2+, Mn2+), such as heme ions released during heme decay, in myoglobin [14,41].
Due to the oxidation of the protein molecule, its hydrophobicity increases, among others, as a result of changes in its secondary and tertiary structures and exposure to the hydrophobic groups on the protein surface [44]. Therefore, in the present study, surface hydrophobicity of proteins from the fermented pork loins during aging was determined to estimate oxidative changes. After 6 months of aging, the surface hydrophobicity of samples inoculated with LAB was significantly (p < 0.05; Figure 1) higher than that of samples subjected to spontaneous fermentation using environmental microflora. This is probably due to the relaxation of the protein structure by the action of oxidative factors. In the natural structure of proteins, hydrophilic amino acid residues are exposed to the water phase, whereas hydrophobic residues are usually found in molecules. The structure of proteins unfolds due to oxidation, revealing hydrophobic amino acids in peptide chains that were previously hidden in proteins, thereby increasing the surface hydrophobicity of proteins [44]. Indeed, surface hydrophobicity corresponds to the TBARS score, which was higher for LAB samples (Table 3), indicating the intensification of fat oxidative processes. With time, a decrease in the HS content was observed in all samples, except for BAUER samples. The reduction in surface hydrophobicity may also be due to the action of proteases. During the fermentation process, meat proteins undergo gradual time-dependent proteolysis, gradually breaking down long-chain fragments into small peptides, and thus may have a lower number of hydrophobic binding sites than larger peptides [45]. A previous study reported that LAB strains affect the degree of proteolysis during 360-day aging [46]. In addition, in the peptidomic approach, the highest influence of LAB on the meat proteome was observed in particular after 180 (BB12 and BAUER) and 270 (C and LOCK) days of aging in the range of sarcoplasm proteins [18]. These authors also emphasized the slight influence of the LOCK strain on changes in the myofibrillar protein fraction resulting from proteolysis compared with the control, regardless of the time criterion, whereas BAUER samples were the most different [18]. The decrease in HS values observed in this study may also be due to changes in free SH groups. SH groups are one of the most reactive functional groups in proteins, and SH groups of sulfur-containing amino acids (e.g., cysteines) are easily oxidized by hydroxyl radicals, thus forming intra- or intermolecular disulfide bonds [34,47]. Therefore, SH is another indicator of volatilization in muscle proteins [48]. In this study, free SH groups were probably oxidized to disulfide bonds, creating a relatively tight structure of the protein network. Due to the formation of protein networks, hydrophobic amino acids were probably buried in particles, which led to reduced hydrophobicity [49], as was observed in C, LOCK, and BB tests. A slightly different trend was observed in the BAUER test in which a systematic increase in the HS parameter was observed, reaching the highest surface hydrophobicity in all variants after 12 months (Figure 1) and a simultaneous increase in the SH value (Figure 2). Generally, in the first period of the analysis (in the 6th month), the effect of BAUER on the reduction in the SH content was observed (p > 0.05), whereas the remaining strains did not affect the SH content compared with the control (C; p < 0.05). The reduction in the SH content observed in BAUER samples was probably related to the oxidation of cysteine SH groups to form disulfide bonds [28]. In the present study, a decrease in the SH content was observed after spontaneous fermentation (C) in 9-month products, whereas LAB tests showed an increased SH content. This increase in the SH content in LAB-inoculated samples might be due to the stretching and unfolding of protein molecules, as a result of which new (previously unidentified) internal SH groups were exposed. Previously, most of the reactive/free SH groups in native proteins were masked from the attack by Ellman factors as they were located in hard-to-reach regions of the polypeptide chain [50]. As the conformational structure of the protein changed over time, the unmasking and activation of SH groups occurred, and they could be detected according to the Ellman reaction protocol. It should be emphasized that in 9-month-old products, the HS index for LAB tests was significantly lower (p < 0.05) than in C, which may indicate limited protein oxidation in these meat products.
This study confirmed the relationship between fat oxidation (based on TBARS) and oxidation of myoglobin—as a protein representative (based on Oxi)—as shown in Figure 3. Indeed, HCA showed that TBARS, Oxi, and SH were correlated among themselves, although the interaction of mechanisms in BB12 samples was not as interdependent as in other samples. However, the collinear relationship between fat oxidation (TBARS) and Oxi was strongly significant (p < 0.05) in the BAUER test (Table 4). Indeed, samples fermented by the L. acidophilus Bauer L0938 strain showed a lower (though not the lowest among the LAB variants) TBARS index and a higher Oxi (indicating a lower level of myoglobin oxidation) during aging. Moreover, a strong linear correlation between Oxi and indicators of other parameters reflecting the degree of protein oxidation was observed in BAUER samples (HS and SH). Comparing this finding with the observations described above, it can be concluded that the increase in oxidative changes within proteins (HS and SH) is accompanied by a decrease in the TBARS content in the BAUER test, which may indicate that the intensity of oxidative processes in meat products is dependent on the presence of this LAB strain.

Author Contributions

Conceptualization, J.S. and P.K.; methodology, P.K.; validation, P.K., P.G. and Ł.S.; formal analysis, P.K.; investigation, P.K., P.G. and Ł.S.; data curation, P.G. and Ł.S.; writing—original draft preparation, P.K.; writing—review and editing, J.S. and D.K.-K.; visualization, P.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 12 11736 g001 550
Figure 1. Surface hydrophobicity of the fermented meat products during storage [C-control sample; LOCK-samples inoculated with Lacticaseibacillus rhamnosus LOCK900; BAUER-samples inoculated with Lactobacillus acidophilus Bauer Ł0938; BB12-samples inoculated with Bifidobacterium animalis subsp. lactis BB12. (a–c) Within the same treatment, means followed by the same letter do not differ significantly (p < 0.05). (A–D) Within the same aging time, means followed by the same letter do not differ significantly (p < 0.05)].
Figure 1. Surface hydrophobicity of the fermented meat products during storage [C-control sample; LOCK-samples inoculated with Lacticaseibacillus rhamnosus LOCK900; BAUER-samples inoculated with Lactobacillus acidophilus Bauer Ł0938; BB12-samples inoculated with Bifidobacterium animalis subsp. lactis BB12. (a–c) Within the same treatment, means followed by the same letter do not differ significantly (p < 0.05). (A–D) Within the same aging time, means followed by the same letter do not differ significantly (p < 0.05)].
Applsci 12 11736 g001
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Figure 2. Free sulfhydryl group (SH) content in the fermented meat products during storage [C-control sample; LOCK-samples inoculated with Lacticaseibacillus rhamnosus LOCK900; BAUER-samples inoculated with Lactobacillus acidophilus Bauer Ł0938; BB12-samples inoculated with Bifidobacterium animalis subsp. lactis BB12. (a–c) Within the same treatment, means followed by the same letter do not differ significantly (p < 0.05). (A–B) Within the same aging time, means followed by the same letter do not differ significantly (p < 0.05)].
Figure 2. Free sulfhydryl group (SH) content in the fermented meat products during storage [C-control sample; LOCK-samples inoculated with Lacticaseibacillus rhamnosus LOCK900; BAUER-samples inoculated with Lactobacillus acidophilus Bauer Ł0938; BB12-samples inoculated with Bifidobacterium animalis subsp. lactis BB12. (a–c) Within the same treatment, means followed by the same letter do not differ significantly (p < 0.05). (A–B) Within the same aging time, means followed by the same letter do not differ significantly (p < 0.05)].
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Figure 3. Graphical representation of dependencies for oxidative indicators as a dendrogram (left) and 3D chart showing the interaction between TBARS (x), Oxi (y), and SH (z) (right).
Figure 3. Graphical representation of dependencies for oxidative indicators as a dendrogram (left) and 3D chart showing the interaction between TBARS (x), Oxi (y), and SH (z) (right).
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Table
Table 1. The pH and water activity (aw) of fermented meat products.
Table 1. The pH and water activity (aw) of fermented meat products.
ParameterTime [Month]Variants
CLOCKBBBAUER
pH65.658 ± 0.003 Ab5.607 ± 0.008 Bc5.657 ± 0.002 Ab5.681 ± 0.018 Aa
95.961 ± 0.015 Aa5.916 ± 0.016 Ab5.754 ± 0.004 Ba5.706 ± 0.037 Ba
125.923 ± 0.058 Aa5.968 ± 0.020 Aa5.743 ± 0.013 Ba5.643 ± 0.015 Ca
aw60.805 ± 0.004 Bb0.815 ± 0.003 Bb0.841 ± 0.003 Aa0.809 ± 0.003 Bb
90.814 ± 0.003 Ba0.834 ± 0.001 Aa0.834 ± 0.001 Ab0.819 ± 0.001 Ca
120.815 ± 0.002 Ba0.831 ± 0.001 Aa0.832 ± 0.001 Ab0.810 ± 0.001 Cb
C-control sample; LOCK-samples inoculated with Lacticaseibacillus rhamnosus LOCK900; BAUER–samples inoculated with Lactobacillus acidophilus Bauer Ł0938; BB12–samples inoculated with Bifidobacterium animalis subsp. lactis BB12. (a–c) Within the same treatment (column), means followed by the same letter do not differ significantly (p < 0.05). (A–C) Within the same aging time (row), means followed by the same letter do not differ significantly (p < 0.05).
Table
Table 2. The results of the evaluation of the color stability of fermented meat products during storage.
Table 2. The results of the evaluation of the color stability of fermented meat products during storage.
Color ParameterTime [Month]Variants
CLOCKBBBAUER
L*643.15 ± 1.19 Ba46.92 ± 1.16 Aa45.62 ± 1.81 ABa47.71 ± 3.40 Aa
938.75 ± 3.59 Bb45.35 ± 4.21 Aa44.56 ± 3.40 Aa48.19 ± 2.73 Aa
1243.82 ± 2.47 ABa46.10 ± 1.94 Aa46.32 ± 1.57 Aa42.45 ± 1.06 Bb
a*67.29 ± 0.27 Cb6.60 ± 0.76 Ca10.17 ± 0.92 Aa8.73 ± 0.98 Ba
99.27 ± 1.36 ABa8.26 ± 1.60 Ba10.70 ± 1.07 Aa8.68 ± 0.48 Ba
128.07 ± 1.02 Aab7.09 ± 0.81 ABa4.68 ± 0.69 Cb6.76 ± 0.37 Bb
b*66.96 ± 0.45 Bb5.28 ± 0.68 Cb8.95 ± 1.32 Aa9.43 ± 1.10 Aa
99.10 ± 1.46 Aa8.77 ± 1.68 Aa9.45 ± 1.31 Aa9.68 ± 0.81 Aa
128.43 ± 1.94 Aab9.17 ± 1.11 Aa10.69 ± 1.63 Aa10.16 ± 0.99 Aa
Oxi61.635 ± 0.02 Bb1.743 ± 0.10 Bab2.059 ± 0.13 Aa1.253 ± 0.043 Cb
91.919 ± 0.20 BCa1.943 ± 0.29 Bb2.141 ± 0.20 Aa1.608 ± 0.05 Ca
121.724 ± 0.10 Aa1.611 ± 0.07 Ba1.253 ± 0.04 Cb1.608 ± 0.05 Ba
C-control sample; LOCK-samples inoculated with Lacticaseibacillus rhamnosus LOCK900; BAUER-samples inoculated with Lactobacillus acidophilus Bauer Ł0938; BB12-samples inoculated with Bifidobacterium animalis subsp. lactis BB12. (a–b) Within the same treatment (column), means followed by the same letter do not differ significantly (p < 0.05). (A–C) Within the same aging time (row), means followed by the same letter do not differ significantly (p < 0.05).
Table
Table 3. Parameters determining the level of oxidation in fermented meat products.
Table 3. Parameters determining the level of oxidation in fermented meat products.
ParameterTime [Month]Variants
CLOCKBBBAUER
TBARS [mg MDA kg−1]62.446 ± 0.17 Ba2.990 ± 0.18 Aa2.476 ± 0.12 Ba2.915 ± 0.10 Aa
91.471 ± 0.31 Ab1.811 ± 0.24 Ab1.382 ± 0.32 Ab1.385 ± 0.26 Ab
121.191 ± 0.06 Ac1.166 ± 0.20 Ac1.193 ± 0.18 Ac1.155 ± 0.08 Ac
ORP [mV]6400.85 ± 4.03 Aa389.90 ± 4.24 Aa366.85 ± 2.47 Ba404.85 ± 0.78 Aa
9330.35 ± 2.19 Bb307.55 ± 2.19 Cb325.35 ± 2.19 Bb347.50 ± 5.94 Ab
12324.15 ± 3.18 Bb303.05 ± 0.49 Cb323.50 ± 7.78 ABb338.50 ± 3.54 Ac
C-control sample; LOCK-samples inoculated with Lacticaseibacillus rhamnosus LOCK900; BAUER-samples inoculated with Lactobacillus acidophilus Bauer Ł0938; BB12-samples inoculated with Bifidobacterium animalis subsp. lactis BB12. (a–c) Within the same treatment (column), means followed by the same letter do not differ significantly (p< 0.05). (A–C) Within the same aging time (row), means followed by the same letter do not differ significantly (p < 0.05).
Table
Table 4. Linear correlation coefficient for oxidation parameters (only statistically significant correlation (p < 0.05) is presented).
Table 4. Linear correlation coefficient for oxidation parameters (only statistically significant correlation (p < 0.05) is presented).
TBARSORPOxiHSSH
CTBARS
ORP 0.690
Oxi −0.787
HS −0.787
TG 0.690
TBARSORPOxiHSSH
LOCKTBARS 0.823
ORP
Oxi −0.920
HS0.823 −0.920
SH
TBARSORPOxiHSSH
BB12TBARS
ORP −0.777
Oxi
HS
SH −0.777
TBARSORPOxiHSSH
BAUERTBARS −0.823
ORP
Oxi−0.823 0.7390.739
HS 0.739 1.000
SH 0.7391.000
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MDPI and ACS Style

Stadnik, J.; Kęska, P.; Gazda, P.; Siłka, Ł.; Kołożyn-Krajewska, D. Influence of LAB Fermentation on the Color Stability and Oxidative Changes in Dry-Cured Meat. Appl. Sci. 2022, 12, 11736. https://doi.org/10.3390/app122211736

AMA Style

Stadnik J, Kęska P, Gazda P, Siłka Ł, Kołożyn-Krajewska D. Influence of LAB Fermentation on the Color Stability and Oxidative Changes in Dry-Cured Meat. Applied Sciences. 2022; 12(22):11736. https://doi.org/10.3390/app122211736

Chicago/Turabian Style

Stadnik, Joanna, Paulina Kęska, Patrycja Gazda, Łukasz Siłka, and Danuta Kołożyn-Krajewska. 2022. "Influence of LAB Fermentation on the Color Stability and Oxidative Changes in Dry-Cured Meat" Applied Sciences 12, no. 22: 11736. https://doi.org/10.3390/app122211736

APA Style

Stadnik, J., Kęska, P., Gazda, P., Siłka, Ł., & Kołożyn-Krajewska, D. (2022). Influence of LAB Fermentation on the Color Stability and Oxidative Changes in Dry-Cured Meat. Applied Sciences, 12(22), 11736. https://doi.org/10.3390/app122211736

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