Incorporation of Trigonella Foenum-Graecum Seed Powder in Nitrite-Free Meat Emulsion Systems with Olive Oil: Effects on Color Stability

Abstract

The replacement of nitrites and starch from Trigonella seed powder in the percentage of myoglobin and metamyoglobin as well as in the color factors (L*, a*, b*) in meat emulsions with olive oil was evaluated. The meat emulsions were prepared on the basis of complete replacement of sodium nitrite (NaNO2) and starch with Trigonella seed powder, where the fat was removed by the Soxhlet method. Thus, two samples emerged, namely, the first sample that was the control and contained 3% starch and sodium nitrite (Starch + NaNO2) in the amount of 150 ppm and the second sample containing Trigonella at 3% (Dtfg) where the fat was removed by the Soxhlet method. The Dtfg sample had a higher percentage of oxymyoglobin (P < 0.05) throughout the maintenance period and lower percentages of metamyoglobin (P < 0.05) up to the fifth day of maintenance compared to the Starch + NaNO2 sample. The factors L* (brightness) and a* (red color) decreased more strongly in the Starch + NaNO2 sample compared to the Starch + NaNO2 sample.

1. Introduction

The genus Trigonella belongs to the family of cumin (fabaceae or leguminosae), which probably got its name from the triangular shape of the leaves of its flower [1]. More specifically, in the work of Hutchinson [2], it is stated that the genus Trigonella is a member of the subfamily Trifoliae, together with the genera of alfalfa (Medicago), clover (Trifoliae), honeysuckle (Melilotus), and the genus Factorovekya, as the genus Trigonella is subdivided into many known species, of which the species Foenum-Graecum is the best known probably due to its actions in many areas of human activity. Historically, the species Foenum-Graecum appeared around 1500 BC in ancient Egypt from writings available—which happens to be an area of ​​great interest for the medicinal applications of plant species—and mainly, the seeds of the plant were used for therapeutic and embalming purposes [3]. However, the Latin name of this species, Foenum-Graecum, given by the scientists of the time, was attributed to the wider region of ancient Greece. Thus, in the work of Miller [4], it is mentioned that the well-known Greek physician Dioscouridis and “father” of pharmacology from Cilicia, from 65 AD, had included in his dissertation entitled “Materia Medica” the use of the plant for making ointments. The plant trigonella is also mentioned in the medical practice of Hippocrates [3].

It is important to note that technological development and the increase in per capita income from the second half of the twentieth century onward led to the abuse of meat and cold cuts. Specifically, for our country, the per capita consumption of meat products ranges between 8 and 10 kg [5]. The excessive consumption of meat and meat products, in addition to the adverse effects it can have on the body due to chemical additives, leads to a number of negative health effects due to the fat contained in it as well as cold cuts. In particular, Article 91 of the Food and Beverage Code stipulates that the maximum percentage of fat that can be contained in heat-treated meat sausages must not exceed 30% of the product as it stands (with the exception of mortadella, which can reach up to and 35%). The fat contained in these cold cuts, which mostly comes from the back of pigs (lard), plays a very important role in production from both physicochemical and microbiological and organoleptic points of view. However, its high content of saturated fatty acids makes it dangerous for the development of cardiovascular disease and obesity. In this light, scientific research has focused on two directions: one is to reduce the percentage of fat as it is in meat products, and the other is to change the profile of fatty acids by adding fat rich in monounsaturated and polyunsaturated fatty acids. Thus, the addition of olive oil to meat products over the last two decades has been a very interesting scientific and industrial challenge with many approaches in the international literature [6, 7, 8, 9].

In recent years, the effects of various food additives on the human body have been studied. In particular, many studies have been conducted, which deal with the negative effects of various chemical additives on the body and the need to find new ingredients of natural origin, which can replace the previous ones leading to the idea of functional food, i.e., the removal from the conceptual characterization of food as food intake and calories necessary for survival, but the consumption of foods that in addition to calories have potentially positive effects on human health in the long and short terms. Moving in this direction, research on the negative effects of chemical additives on the production of meat cold cuts has led to the discovery of harmful compounds created by the intake of nitrites and nitrates reduced to nitrites, which are added to cold cuts. Their association with free amino acids in the body leads to the formation of some very harmful substances with carcinogenic activity known as nitrosamines [10]. Thus, in recent years, a large field of research has been developed around the addition of plant extracts that can replace nitrite in these products [11, 12, 13]. Polyphenols are one of the most widespread and numerous groups of bioactive components with a very wide distribution in the plant kingdom and great diversity among different species of plant tissues. They are the products of the secondary metabolism of plants, which means that they are not a primary growth factor for the physiology of the young plant, but they play an important role in the subsequent metabolic and physiological activity of the plant organism. Polyphenols can be found in plant tissues in the form of phenolic acids, free and glycosylated flavonoids, and anthocyanins that are a subgroup of flavonoids [3].

The seeds of the plant Trigonella contain about 1–3% polyphenols [14], which are found in many forms, most notably glycosylated flavonoids [15, 16]. The plant has been known since ancient times for its beneficial properties both in human health and in its applications in increasing food preservation [3]. Regarding the phenolic components found in the seeds of the Trigonella plant, vanillic acid, 3-coumaric acid, genetic acid, and caffeic acid show larger amounts. Their amounts are, respectively, 0.585, 0.478, 0.358, and 0.210 mg/g seed [17, 18, 19]. However, other types of phenolics have been identified in the seed of the plant that are not mentioned as they are not, quantitatively, a significant percentage. In addition, the total phenolic content of the seeds ranges from 10 to 78 mg GAE/g, and the variation depends on the extraction and collection technique of the total phenolic components.

However, the seed of the Trigonella plant also contains a significant percentage of flavonoids, which, however, are mostly in glycosylated forms. In particular, the main aglycones contained in the seeds are apigenin with its glycosides accounting for about 40% of the total amount of flavonoids, camphorol with glycosides constituting about 15% of the total amount of flavonoids, and luteolin with glycosides constituting about 15% of the total amount of flavonoids. However, some of these glycosides have been identified for the first time in this plant, such as vicenin and its derivatives, which are xylose glycosides of apigenin, orientine, which is a glycoside of luteolin, and vitexin, which is a glycoside of apigenin [15, 16, 17, 18, 19, 20].

One of the ingredients added to meat emulsion products is olive oil, which is usually added to replace pork fat, in order to develop a healthy fatty acid profile, in the presence of monounsaturated fatty acids derived from olive oil. In meat mass, the application methods that have been proposed at research and industrial levels include the direct addition of olive oil in liquid form during processing in the cutter [21] and the pre-emulsification of olive oil [6]. The olive oil added during the production of cooked sausages must comply with the specifications of European Regulation 2568/912013, which sets the limits of the composition of fatty acids to qualify an oil as olive oil and, in particular, must contain 70–80% monounsaturated fatty acids (oleic acid), 6–16% omega-6 fatty acids, 0.3–1.3% omega-3 fatty acids, and 8–10% saturated fatty acids. In addition, in terms of organoleptic characteristics, olive oil used in the production of cooked sausages should have a fruity taste and smell as well as not show unacceptable organoleptic characteristics of taste and smell that refer to an oxidized product. The color of olive oil, which is influenced by factors such as storage conditions, place of production, and export system [22], is a result of chlorophylls where they are green in color as well as carotenoids (β-carotene and lutein) where they show yellow coloration. In meat products and especially in cooked sausages, olive oils rich in carotene and not in chlorophyll are preferable, as the development of green colors in the final products is considered unacceptable [23, 24].

The scope of this study was to evaluate the color preservation effect of T. foenum-graecum seed powder in meat emulsion systems and the potential replacement of nitrites with the specific seed powder.

2. Materials and methods

2.1 Materials

Fresh minced pork ham (M. biceps femoris, M. semitendinosus, M. semimembranosus) was purchased from a local market at 48-h postmortem. T. foenum-graecum seeds were purchased from a local market, and then, it was grounded using a laboratory grinder (Analytische Mühle, IKA). Sodium chloride (Kallas klassiko), corn starch (Bioygeia), and virgin olive oil (Altis Klassiko, ELAIS) were purchased from local markets. Sodium nitrite was obtained from CG Chemicalien (CG Chemicalien, Belgium).

2.2 Μeat emulsion preparation

The meat emulsions were prepared on the basis of complete replacement of sodium nitrite and starch with Trigonella seed powder, where the fat was removed by the Soxhlet method. Thus, two samples were manufactured, namely, the first control sample containing 3% starch and 150 ppm sodium nitrite, which is the upper limit according to European Regulation 1129/2011 on food additives. The second sample contained 3% deffated T. foenum-graecum seed powder, where the fat was removed by the Soxhlet method without the presence of sodium nitrite. The recipes of meat emulsions are presented in Table 1.

Ingredient	Quantity (g)
	Formulation 1—Control	Formulation 2
Beef minced meat	274.3	274.3
Water-Ice	125.7	125.7
Olive oil	75	75
Sodium chloride	10	10
Dtfg	—	15
Starch	15	—
Sodium nitrite (NaNO2)	0.075
Sum	500	500

Table 1.

Meat emulsion formulation.

2.3 Estimation of levels of oxymyoglobin Mb (Fe²⁺) and metamyoglobin MetMb (Fe³⁺) of fresh meat emulsions

The estimation of the levels of oxymyoglobin Mb (Fe²⁺) and metamyoglobin MetMb (Fe³⁺) in fresh meat emulsions was based on the method of Ning et al. [21] and Carlez et al. [22] with some modifications. Specifically, 2 g of the sample was homogenized with 20 ml of Na/K phosphate buffer at a concentration of 0.04 mol/L in a 50-ml Falcon flask in an Ultraturrax homogenizer for 20 s at 10,800 rpm. The samples were then centrifuged at 7000 rpm for 5 min after a total of 1 h in an ice basin, with the addition of aluminum foil externally to prevent oxidation. Immediately after filtration with whatman #1 filter type, the samples were supplemented with 25 ml of the same buffer. The absorbance of the samples at the following wavelengths was then measured: 525, 545, 565, and 572 nm, in a spectrophotometer (UV-1800, Shimadzu Co., Kyoto, Japan). The percentages of oxymyoglobin and metamyoglobin are calculated from the following equations [25]:

E1

E2

where factors R₁, R₂, and R₃ are the absorption ratios and are calculated as follows:

E3

2.4 Deffating

The defatting of T. foenum-graecum seeds was carried out based on the method of lipid extraction [26]. In this method, for the defatting of raw seeds, a Soxhlet apparatus was used utilizing diethylether as solvent. More specifically, a certain amount of T. foenum-graecum seed powder was weighed and placed in a cellulose extraction cartridge. The cartridge was plugged with cotton wool and then placed in the Soxhlet chamber, which was fitted to a pretared distillation flask containing dielthylether and two to three glass regulators. After extraction for 2 h in 50–60°C, the cartridge allowed to cool, and then, the cartridges were placed in an oven at 105°C for 12 h, following cooling in a desiccator and weighing. Placing in the oven for specific time, cooling in a desiccator and weighing was repeated until the difference between two consecutive weights was smaller than 2 mg.

2.5 Determination of color parameters

Instrumental color was conducted by taking a direct reading of raw meat emulsions with a colorimeter (Konica Minolta CR-400, USA). Standard observer was 2° (Closely matches CIE 1931 Standard Observer [(x2λ, yλ, zλ)]); 8-mm diameter circular aperture and d/0 (D65,Diffuse illumination/0 < ° viewing angle) illuminate were used. The CIE-L*, a*, and b* parameters were evaluated according to the methodology proposed by the American Meat Science Association (AMSA 2012). The calibration of colorimeter performed using a white plate, L* = 97.58, a* = 0.03, and b* = 1.08. For the color analysis of raw meat emulsions, 10 g of each sample was placed on a 9-cm diameter petri dish at 1-cm thickness. The average of nine readings was reported. All tests were performed at room temperature on the second day after manufacture and then 5 and 7 days after manufacturing.

2.6 Statistical analysis

All statistical analyses were performed with SPSS (Version 23, IBM, USA). The homogeneity of variances was tested with the Levene test in the case of non-normal data with SPSS. The normality of the residuals was tested using UNIVARIATE of SPSS, and consideration was given to the Shapiro–Wilk test for normality. Once it was determined that the assumptions of the analysis of variance (ANOVA) were met for these data, the GLM procedure of SAS with a fixed effect of treatment and a random effect of replication was used for the statistical determination of all variables. Tukey’s test (P < 0.05) was used to determine the differences between the treatment means. Least squares means were separated using a single-degree-of-freedom estimate statement to determine the difference between meaningful comparisons, which included Starch + NaNO₂ versus Dtfg (3% inclusion). Differences were considered statistically different at P < 0.05. This experiment was completed in its entirety three times.

3. Results and discussion

3.1 Estimation of levels of oxymyoglobin Mb (Fe²⁺) and metamyoglobin MetMb (Fe³⁺) of raw meat emulsions and determination of color parameters

In Figures 1–4, it is observed that in the Starch + NaNO₂ sample, the oxymyoglobin levels are lower than in the Dtfg sample throughout the shelf life of the meat emulsions. In addition, in the Starch + NaNO₂ sample, lower metamyoglobin values were observed after the fifth day of maintenance. These results could be correlated with the analysis of color parameters, as shown in Figure 5, where of particular interest is the sharp reduction of the factor L* (brightness) and a* (red color) in the Starch + NaNO₂ sample, in contrast to the Dtfg sample.

Figure 1.

Effect of deffated Trigonella foenum-graecum seed powder in oxymyoglobin content of meat emulsions with olive oil.

Figure 2.

Effect of deffated Trigonella foenum-graecum seed powder in metmyoglobin content of meat emulsions with olive oil.

Figure 3.

Effect of deffated Trigonella foenum-graecum seed powder in L* (lightness) factor of meat emulsions with olive oil.

Figure 4.

Effect of deffated Trigonella foenum-graecum seed powder in a* (redness) factor of meat emulsions with olive oil.

Figure 5.

Effect of deffated Trigonella foenum-graecum seed powder in b* (yellowness) factor of meat emulsions with olive oil.

These findings are particularly important; as in the present literature, there is difficulty in implementing strategies for the total replacement of nitrites with different plant materials. However, in some studies, an increase in color stability has been observed with the use of plant materials in meat products low in nitrite [12]. The stabilization of the color of meat products with the use of Trigonella plant powder is probably due to its high content of antioxidants, such as phenolic compounds and some steroids [27], as similar effects are observed during the incorporation of essential oils, with high content of antioxidants, in meat products with low nitrate content [26, 28]. These indications may be an important tool for the use of the Trigonella plant in the development of strategies for the reduction and replacement of nitrites in meat products, especially given that the samples of the present work were kept for 7 days without packaging in an environment with high redox potential without undergoing heat treatment.

4. Conclusion

The replacement of nitrates and starch from Trigonella seed powder in beef emulsions with olive oil, the stability of myoglobin to oxidation, and certain color factors were evaluated. Thus, the percentage of oxymyoglobin in the meat emulsion containing Trigonella was higher than in the meat emulsion containing nitrites and starch. In addition, the percentage of metamyoglobin in the meat emulsion containing Trigonella was lower until the fifth day of preservation than the meat emulsion containing nitrites and starch. Finally, the sample containing nitrites and starch underwent a sharp decrease in the values of L* (brightness) and a* (red color) during maintenance compared to the meat emulsion containing Trigonella, which constitutes its highest color stability. These observations lead to the hypothesis that T. foenum-graecum seed powder could be an efficient candidate for nitrite replacement from a color stability point of view in meat emulsion systems with high olive oil content.