EFFECT OF ROSEMARY EXTRACT AND GREEN TEA EXTRACT ON COLOUR STABILITY AND QUALITY OF FERMENTED SAUSAGE

Danica Savanović¹, Slavica Grujić¹, Radoslav Grujić², Jovo Savanović^3
1 Faculty of Technology, University of Banja Luka, Bosnia and Herzegovina
² Faculty of Technology Zvornik, University of East Sarajevo, Bosnia and Herzegovina
³ ZD.I. - PRODUKT d.o.o. Široki Brijeg, Branch Laktaši, Bosnia and Herzegovina

ABSTRACT

The aim of this study was to investigate antioxidant effect of natural rosemary extract and green tea extract on colour stability and quality of dry-fermented beef sausage "sucuk" type during ripening and storage period, comparing with effect of the other antioxidants. Samples were produced in industrial conditions. Different commercial additive blends with antioxidant property were added in seven sausage model samples separately to each, and sausages produced without antioxidants addition were used as control. Instrumental colour measurement, chemical composition, weight losses, pH and sensory quality were determined during 19 days of ripening, and 6 months of storage. Measured colour parameters (L*, a*, b*), investigated quality parameters and results of sensory analysis indicated that rosemary extract had statistically significant (p<0.05) positive impact on colour stability and high sensory quality of beef sausages produced for this research, and that green tea extract had positive impact on colour stability, but sausages had unacceptable sensory quality.

Key words: beef sausage, antioxidants, sensory quality, colour stability.

INTRODUCTION

Dry-fermented sausage "sucuk" type is a very popular meat product in Turkey and in most Middle Eastern Countries and Europe [7, 36, 51]. Manufacturing of the sucuk varies regionally, and different formulations exist. Ingredients used for its production usually are: beef, beef backfat, tail fat, salt, sugar, garlic, nitrite and/or nitrate and various spices [50]. Sucuk acceptance by the consumer is often based on colour, texture and flavour. The meat and spice combinations and the processing conditions have impact on the flavour quality. Spices and herbs are generally used in sucuk production for enhancing the flavour and/or colour attributes [5, 24].

Colour forming during production and colour stability are very important quality attributes of fermented meat products. Pigment responsible for the characteristic bright red colour of cured meat in fermented sausages is the nitrosylmyoglobin (MbFeIINO), in which an axial ligand nitric oxide (NO) is coordinated to central FeII of heme [28, 44]. The chemical reactions leading to the cured meat pigment forming are a complex series of processes involving microbially, enzymatically and/or chemically catalysed steps, which depend on pH, pigment concentration, redox potential, curing agent distribution, temperature and relative humidity [18]. Antioxidants act as catalysts in chemical reactions that take place during the meat curing. These substances accelerate the reduction of nitrite to nitric oxide and prevent oxidative changes of colour and taste of the product [44, 47].

The heme pigments in meat are the compounds that can be easily changed due to various physicochemical factors impact [33, 43] and they can be easily oxidized, as a consequence of their unsaturated character [12, 38, 45]. Oxidative discolouration of fermented sausages is characterized by conversion of MbFeIINO to nitrate and the brown pigment, metmyoglobin (MbFeIII). This process depends on partial oxygen pressure and myoglobin reducing systems in general and related is to the subsequent lipid oxidation [18, 49]. Processes occurring in fats (lipid oxidation) are additional factors with impact on the changes of colour, aroma, taste and nutritional value of the product. Products of lipid oxidation and free radicals that contribute to oxidation of myoglobin, and leading to forming of metmyoglobin and browning [12].

Therefore, it is desirable to control oxidation processes and improve the quality of food products by addition of inhibitory substances [20]. The rate of lipid oxidation can be effectively controlled or minimized, by natural or synthetic antioxidants addition [1, 17, 27, 31, 35]. Commonly used antioxidants in the meat products are: α-tocopherol, ascorbic acid, sodium ascorbate and ascorbyl palmitate [17, 34, 45]. Synthetic phenolic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl, octyl, and dodecyl gallates (PG, OG, DG), are easily available and largely used in different food products [13, 23]. The increasing consumer awareness and health consciousness, however, results in pressure to avoid synthetic additives use, which necessitates the use of natural additives to extend shelf life and/or improve product safety [3, 17, 48]. Sources of natural antioxidants usually are spices, herbs, teas, oils, seeds, cereals, cocoa shell, grains, fruits, vegetables [10, 19, 24, 31].

In the past culinary herbs were used as flavouring agent and naturally occurring antioxidant. Today, natural plant extracts are increasingly produced and sold primarily for their antioxidant properties, but scientific opinion on safety of their use are considering. The processing of extract can be optimised to enhance the antioxidative function and to reduce flavoring properties [14]. Amongst well known herbs, rosemary has antioxidative properties, and its effectiveness for achieving higher sensory scores and retarding lipid oxidation in various foods were reported [15, 17, 34, 35, 48]. According to theRegulation (EC) No 1333/2008 on food additives, exstracts of rosemary (E 392) was included in list of additives other than colours and sweeteners [42]. Rosemary (Rosmarinus officinalis) extracts contain antioxidant compounds, the most active being phenolic diterpenes such as carnosol, carnosic acid, rosmanol, epirsomanol, isorosmanol, methyl carnosate and other phenolic acids, such as rosmarinic acid [35]. This phenolic diterpenes act breaking free radical chain reactions by hydrogen atom donation. When added to lard, the antioxidant activity of isorosmanol is comparable with the activities of the synthetic butylated phenolic antioxidants hydroxianisole (BHA) and hydroxytoluene (BHT) [17]. The oxidation processes controlling with antioxidants such as rosemary should reduce the rate of myoglobin colour degradation.

Green tea (Camellia sinesis) contains several groups of polyphenols that include flavonds, caffeine, phenolic acid, theanine, flavor compounds and leucoanthocyanins, accounting for up 40% of dry leaf weight. Catechins are the main bioactive constituents of green tea leaves and account for 25–35% of their dry weight. Tea catechin can act as antioxidants by donation of a hydrogen atom, as an accepter of free radicals, interrupting chain oxidation reaction or by chelating metals. Addition of 1–2 % of green tea extract to ground beef meat was adequate in reduced meat lipid oxidation and stabilized the meat color through decreased metmyoglobin formation after refrigeration [15]. Bozkurt[5] reported that addition of green tea extract (Thymbra spicata oil) in Turkish dry-fermented sausage (sucuk) reduced TBARS, putrescine, histamine and tyramine formation significantly. This study pointed out that natural antioxidants were more effective than synthetic antioxidants (BHT), so they could be easily used in sucuk to enhance quality and provide safer products.

The aim of this study was to investigate antioxidant effect of natural rosemary extract and green tea extract on the colour stability and quality of dry-fermented beef sausage "sucuk" type, during ripening and storage periods, compared with effect of the other antioxidants, whose use is common in food industry (sodium ascorbate,  ascorbic acid, ascorbyl palmitate, tocopherols and butylated hydroxy-anisole) and with the sausage model sample, produced without antioxidants addition, used as control.

MATERIALS AND METHODS

Sausage formulation and processing
Dry-fermented beef sausage "sucuk" type was used in the experiment as a model-product. Samples were produced in industrial conditions, according to the producer’s specification. The following ingredients were used (quantity shown in descending order): beef, beef fat, salt, dextrose, starter culture, spices and preservative E 250.

Different commercial additive blends with antioxidant property and containing selected antioxidants (sodium ascorbate, rosemary extract, green tea extract, ascorbic acid, ascorbyl palmitate, tocopherols and butylatedhydroxyanisole) were added in seven sausage model samples in the recommended concentrations separately to each (Tab. 1). The eighth sausage model sample was produced without antioxidants addition, and used as a control in the research.

Frozen beef meat and beef fat, sliced into smaller pieces using the machine for frozen meat cutting (Farm Power, Type: SM 501, Slovakia), were used for the production of sausages model samples. Required amounts of meat and fat were weighed on an electronic platformer scale (Level, Type: Sigma 5ND12/600, Slovenia), and the required amount of additives and spices were weighed on an electronic scale (Level, Type: GAMMA PTE-N, Slovenia). Ingredients were chopped in cutter (Seydelmann, Type: DC 8 206 K, Germany).

Packing and storage of samples
Prepared sausage butter was filled into artificial collagen casings (43 mm diameter), on filling machine (Handtmann, Type: HF 622, Germany) and the sausages, approximately mass of 760 g, were closed on both sides with metal clips (Poly Clip, Type: FCA 3430-18, Germany). The sausages were fermented and ripened in climate chambers (Mauting, Czech Republic) for 19 days during which the temperature declined from 25 to 17°C and the relative humidity dropped from 90 to 77%. Technological parameters of the process during the ripening period are shown in Table 2. After ripening process the sausages, approximately mass of 530 g, were individually vacuum packaged, using packaging machines (Multivac, Type: R 230, Germany). Multilayer film, made of polyamide (PA) and polyethylene (PE), the structure of PA / PE / PA / PE, in which the thickness of the lower sheet was 200 µm and the upper film 100 µm, was used for sausages packaging. The samples were stored in vacuum packaging, at a temperature 4°C, under usual storage conditions as the same kind of products.

Sampling
Six dry-fermented beef sausages "sucuk" type from each batch were removed after 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 days of ripening and after 1, 2, 3, 4, 5, 6 months of storage. Instrumental colour measurement, chemical composition and pH values were analysed in sausages during the ripening and storage periods. Sausages weight losses were analysed during the ripening and sensory analysis were carried out immediately after ripening and during 6 month storage period, each month successively.

Instrumental colour measurement
Colour measurements were performed using Spectrophotometer CM-2600d (KONICA MINOLTA SENSING INC, Japan), with 8 mm port size, illuminant D65 and a 10° standard observer, in a room with fluorescent lighting and after standardization of the instrument with respect to the white calibration plate. Colour parameters, expressed as CIE L*, a* and b* values, were determined as indicators of lightness, redness and yellowness. Five measurements were taken on three cross sections of two sausages, for each treatment. The measurements were realised immediately after the sausage cutting. The mean of 30 measurements was recorded for each colour parameter.

Weight losses
Determination of the sausages weight loss were done by measuring the mass of each of 6 model sausages samples after 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 days of ripening. Differences in weight were expressed as a percentage relative to the initial mass, measured immediately after stuffing the sausage butter into casings. The mean of the six measurements was recorded.

Chemical composition
Moisture content (drying at 105°C to constant mass), fat content (according to the Soxhlet method) and protein content (according to the Kjeldahl method) were determined according to AOAC procedures [2]. Analysis of chemical composition was carried out in Laboratory for food analysis on the Faculty of Technology, University of Banja Luka, Bosnia and Herzegovina. All analyses were carried out with 6 replicates (6 sausages from each treatment).

pH measurement
pH values were measured using a pH meter (Testo 252, Lenzkirch, Germany) equipped with a stab glass electrode (Testo pH Electrodes, Typ 01-06, Germany) for the direct measurement of pH in meat and meat products. Before and during the values reading, calibration of the pH meter was realized using standard buffer solution (pH buffer calibration was 7.02 and 4.00 to 20°C). Measurements were carried out with 6 replicates (6 sausages from each treatment).

Sensory analysis
Sensory analysis was performed immediately after sausages production and repeatedly once in a month during the 6 months of storage, in the Laboratory for sensory analysis of foods on the Faculty of Technology in Banja Luka, Bosnia and Herzegovina. Sensory evaluation was carried out by 5 assessors, expert category [21] familiar with the nature of the product and with the aim of testing. The sausage casing was removed and the sausage was cut in slices (4 mm thick) on the machine for meat products cutting (Bosch, Type: MAS9101). Eight labelled different model samples were delivered to assessors in 3 sets (3 + 3 + 2 model of the sample), and 5 slices of each sample. Water, room temperature (20–23°C) and cubes of white bread were served to assessors for cleaning the mouth between samples evaluation. Given the fact that the model samples produced with additives addition with potential impact on the stability of the colour, the assessors were instructed to pay close attention to the colour of the product. For evaluation of sausages quality descriptive sensory analysis (scoring method) and consensus test were used. In initial preparation of sausages sensory evaluation by the scoring method [22, 40], coefficient of significance (Cs) was determined for each selected sensory attribute (sum of them are 20). Appropriate coefficient of significance (Cs) was multiplied with score given after sensory evaluation of each selected attribute (in scale from 5 for very good quality, to 1 for very bad, unacceptable quality) and after that, addition of all results for evaluated sensory attributes, gives corrected score expressed as percentage of maximum possible product quality, or 100%  for the best quality. For fermented sausages the most important sensory attributes were evaluated: outside appearance (Cs=2), cross section appearance (Cs=5), colour of cross section (Cs=3), the odour, aroma and taste (Cs=7); consistency Cs=3 [41].

Statistical analysis
Statistical analysis of the results was performed using the software package Minitab 14. The data obtained for chemical composition, pH and weight losses were statistically analysed using one-way analysis of variance (ANOVA). The data collected for instrumental colour measurement and sensory analysis were statistically analysed by a two-factor factorial arrangement. The factors were: (A) ripening / storage time (1, 3, 5, 7, 9, 11, 13, 15, 17, 19 days of ripening and 1, 2, 3, 4, 5, 6 months of storage) and (B) type of antioxidants used for producing sausages model samples.

Effect of rosemary extract on colour of fermented beef sausage "sucuk" type was compared with effect of the other antioxidants, after 19 days of ripening and after 6 months of storage, applying t-test. Also, effect of green tea extract on colour of fermented sausage "sucuk" type was compared with effect of the other antioxidants, after 19 days of ripening and after 6 months of storage, on the same way, applying t-test. The level of significance p<0.05 was used for all comparisons [26, 29].

RESULTS AND DISCUSSION

Changes of L*-, a*-, b*-values
The production of dry-fermented beef sausages consists in a skilled control and monitoring of processing parameters important for biochemical and microbiological transformations, as well as physical processes which take place in the bulk of the sausage mixture, during production and the post-manufacture maturation [39].

Selected ingredients and additives, depending on their concentrations, can exert a significant influence on the quality and sensory characteristics of finished sausages. Colour is one of the most important quality attributes of sucuk, since it affects overall quality and product acceptability on the market. The results of colour parameters, measured on the cross section of dry-fermented beef sausage "sucuk" type during ripening, expressed as CIE L*, a*, b* values, are given in Tables 3a,b,c.

The aim of this study was to investigate antioxidant effect of natural rosemary extract (sample 2), and natural green tea extract (sample 3) on the colour of dry-fermented beef sausage "sucuk" type, during ripening and storage periods comparing measured parameters with the effect of other antioxidants, whose use is common in food industry (sodium ascorbate, ascorbic acid, ascorbyl palmitate, tocopherols and butylated hydroxy-anisole) and with the sausage model sample, produced without antioxidants addition (sample 8), used as control.

Sausages colour stability during 19 days fermentation
Two-way ANOVA for colour parameters (L*, a*, b*) measured during 19 days fermentation, showed statistically significant difference for crossed mean values at the p<0.05 confidence level, and it was concluded that fermentation and 19 days storage time (FA) and type of antioxidants (FB) added into the sausage batter had impact on considered analysed values (p<0.05). In the case that difference is identified, a t-test was used to determine which of two sets of data are significantly different from each other.

Analysed lightness (L* value) on the cross section of all model samples decreased significantly during the ripening period (FA=46.20; p=0*; FB=6.61; p=0*) and the samples become darker.

To investigate between which of analyzed sausages had been statistically significant difference in L*-values at the end of fermentation (19. day), pairs of values measured for sample 2, produced with rosemary extract were compared with effect of the other added antioxidants and without it (sample code x=1, 3, 4, 5, 6, 7, 8) applying t-test statistics. The same analyse applying t-test statistics were repeated for values measured on sample 3 produced with green tea extract, which were compared with effect of the other added antioxidants and without it (sample code x=1, 2, 4, 5, 6, 7, 8). Results are shown in Table 3a.

Comparing measured values for sample 2 (produced with rosemary extract) after 19 days of ripening showed statistically significant difference in average values for lightness (L*-values) between samples 2 and 5, and between sample 2 and control sample 8 (Tab. 3a). Compared measured values for sample 3 (produced with green tea extract) after 19 days of ripening showed similar statistically significant difference in average value for lightness (L*-values) between samples 3 and 5; between samples 3 and 6; and between sample 3 and control sample 8 (Tab. 3a).

According to that, it could be concluded that, at the end of fermentation, there was no statistically significant difference in measured colour parameters for sausages produced with   rosemary extract and green tea extract and with sodium ascorbate or ascorbic acid, antioxidants whose use is common in food industry and that they had similar impact on colour quality and stability of sausage. The decrease in the L*-values during ripening, indicated formation of dark colour in the sucuk due to the browning reaction [6]. Similarly, Bozkurt [5] and Kayaardi and Gok [25] found that Hunter L-values of sucuk generally decrease during the 15 days of ripening.

The a* value could be indicator of changes in product during formation of desired red colour, important quality parameter for increase of the sausages acceptability and consumption. During 19 days of ripening, the average redness values (a*) statistically significant (p<0.05) changed, as result of ripening time and sausages fermentation process impact (FA) and (FB) antioxidants added into the sausage batter and without addition (FA=9.11; p=0*; FB=2.43; p=0.029*).

To investigate if there are statistically significant difference in a*-values between analyzed sausages at the end of fermentation (19. day), pairs of values measured for sample 2, produced with rosemary extract were compared with the effect of other added antioxidants and without it (sample code x=1, 3, 4, 5, 6, 7, 8) applying t-test statistics. There was no statistically significant difference in a*-values between analyzed sausages at the end on the ripening (19. day), and could be concluded that all analysed sausages had similar intensity of colour redness. Results of t-test for values measured on sample 3 produced with green tea extract, which were compared with the effect of other added antioxidants and without it (sample code x=1, 2, 4, 5, 6, 7, 8) showed statistically significant difference in average value for redness between samples 3 and 5; between samples 3 and 7, the difference identified and confirmed by descriptive sensory test. During development of ripening process, he average a*-value indicated on changes, they increased during the first 3 days of sausages ripening, but during further ripening period less change of a* values were recorded (Tab. 3b).

During the first days of ripening, nitrogenous compounds present in meat, combined with myoglobin, produce the desired red colour pigment, therefore a-values would be increased [24]. After this period, during the further ripening period, denaturation of formed pigment takes place, so a-values decreased [5, 6]. Kayaardi and Gok [25] found that a* values of sucuk increased during the first 5 days of the ripening period and then decreased during further ripening. Also, Muguerza et al. [32] observed that a* values of sausages increased during the ripening period. Similarly, Perez-Alvarez et al. [37] reported that measured a* values of Spanish type dry-cured sausage increased during the fermentation period, and then decreased during the ripening period. They reported that the formation of nitrosylmyoglobin and moisture loss could be the reasons for increasing a* values at the beginning of ripening, and the possible reason for decreasing a* values might be partial or total denaturation of nitrosylmyoglobin, as result of lactic acid impact during ripening.

Average values for colour parameter (b*), indicator for yellow shade in full colour, decreased during the ripening period and indicated on statistically significant (p<0.05) changes (FA=167.58; p=0*; FB=4.89; p=0*) as shown in Table 3c. With aim to investigate effect of rosemary extract on the colour of dry-fermented beef sausage "sucuk" type after ripening (sample 2), measured average b*-values were compared with the other. The results of t-test statistic analyse indicated on statistically significant (p<0.05) difference in the average b*-values between samples 2 and 5; and between sample 2 and control sample 8 (Tab. 3c), the same changes as of parameter L*-values. According to that, it could be concluded that, rosemary extract showed similar impact on colour quality of sausage at the end of fermentation, as the other antioxidants whose use is common in food industry. Results of t-test for colour parameters values, measured on sample 3 cross section, sausages produced with green tea extract, compared with the effect of other added antioxidants and without it (sample code x=1, 2, 4, 5, 6, 7, 8) showed the same statistically significant difference, in average value parameter b*, as impact of rosemary extract, between samples 3 and 5; between sample 3 and control sample 8, also between samples 3 and 1; samples 3 and 6; samples 3 and 7 (Tab. 3c).

The greatest decrease and change in the b* value, for analysed sausages were recorded after 3 days of ripening (Tab. 3c). These results were in agreement with Kayaardi and Gok [25]. The b* values of Spanish type dry-cured sausages, also decreased during the fermentation and ripening periods and authors Perez-Alvarez et al. [37]  explained the decreasing of b* values as result of microorganisms oxygen consumption and consequent decrease in oxymyoglobin, which contributes to the yellow colour shade. The decrease in the b* value indicated that colour of sucuk turned to lower intensity of yellow shade in sausage colour. This could be due to the browning reactions, as yield of melanoidins that have a brown colour [5].

Sausages colour stability during 6 month storage
After production and packing, slow changes and transformations in the products could result in deterioration of sausages and changes in colour shades. For that reason, application of antioxidants could be an effective method allowing preventing undesirable changes [39]. Colour stability of sausages were analysed during storage period, comparing changes in colour parameters (L*, a*, b*) measured on cross section of beef sausages produced with antioxidants, whose use is common in food industry (sodium ascorbate, ascorbic acid, ascorbyl palmitate, tocopherols and butylated hydroxy-anisole), with the sausage model sample, produced without antioxidants addition, used as control (sample 8) on one side, and with colour parameters measured on sausages produced with natural herb extracts as antioxidants, rosemary extract (sample 2),  and green tea extract (sample 3), separately.

Two-way ANOVA for colour parameters (L*, a*, b*) measured on fresh cross section of dry-fermented beef sausage "sucuk" type during 6 month storage, showed statistically significant difference for crossed mean values (p<0.05), and it was concluded that storage time (FA) and type of antioxidants added into the sausage batter and without it (FB) had impact on considered values. Ascorbic acid and ascorbates are commonly used as antioxidants with good results in the beef sausages production, and could be used as standard for control effect of other antioxidants, especially ascorbic acid. That was the reason for analyse effect of rosemary extract (sample 2), and green tea extract (sample 3) with these and other antioxidants, with special attention on relations between quality parameters measured on sausage samples 1 and 4. Statisitcala analyse was realised comparing colour parameters expressed as CIE L*, a*, b* values, measured after 6 months of storage on the cross section of sample 2 (produced with rosemary extract) with sample 1 (produced with sodium ascorbate); with sample 4 (produced with ascorbic acid); with sample 3 (produced with green tea extract) and with control sample 8; and sausages produced with some other antioxidants,  sample 5 (produced with ascorbyl palmitate); sample 6 (produced with tocopherols) and sample 7 (produced with butylated hydroxy-anisole). The same procedure was repeated for sample 3 (produced with green tea extract).

With aim to investigate effect of rosemary extract on the colour parameter L* measured on dry-fermented beef sausage "sucuk" type (sample 2) and to compare it with measured values on other sausages cross section at the end of storage period, a t-test was used for analyse which two sets of data were significantly different. Comparing average measured value for lightness (L*-values) showed statistically significant difference (p<0.05) between samples 2 and 1, as sample 1 was lighter; between samples 2 and 5, where sample 5 was relative darker; and between sample 2 and control sample 8, which had the lowest measured value (L*=45.74) of all eight samples (Tab. 4a) after 6 month storage.

Analyse of green tea extract (sample 3) effect on parameter L*-values showed statistically significant difference (p<0.05) between samples 3 and 1, between sample 3 and control sample 8, the same as for sample 2. Differences were identified also between samples 3 and 6.

Model samples produced with different antioxidants (samples 1–7) after 6 months of storage had higher L* values than values measured immediately after ripening (19. day), and samples during storage become slightly brighter (Tab. 4a). These changes were not observed in control sample 8, produced without addition of antioxidants.

Red colour is one of important quality attributes of sucuk, connected with impression related to the sausage acceptability. Two-way ANOVA for measured colour parameter a* during 6 months of storage showed statistically significant difference for crossed mean values (p<0.05), and it was concluded that storage time (FA) and type of antioxidants (FB) added into the sausage batter and without it, had impact on considered values (FA=132.33; p=0*; FB=7.75; p=0*).

Values for the colour parameter a* (redness), measured on the cross section of all sausages model samples, decreased during the storage period. With aim to investigate effect of rosemary extract on measured colour parameter a* (sample 2) and to compare it with values measured for the other sausages at the end of 6 month storage period, a t-test was used. Comparing average measured values for two sets of data for redness (a*-values) for sample 2 and each other samples (code 1, 3, 4, 5, 6, 7, 8) after 6 months storage showed statistically significant difference (p<0.05) between samples 2 and 1 which had relatively brighter shade of red colour, non-specific for the tested beef sausages; 2 and 4; 2 and 5; 2 and 6. All those samples had relative higher mean values for redness than sample 2 (a*=9.74). Analyse of green tea extract (sample 3) effect on mean values for parameter a*, showed statistically significant difference (p<0.05) between samples 3 and 1, between samples 3 and 6 (Tab. 4b).

Measured values for yellowness (b*) showed statistically significant difference for crossed mean values (p<0.05), and it was concluded that storage time (FA) and type of antioxidants added into the sausage batter and without it (FB) had impact on analysed values (FA=4.63; p=0.001*; FB=16.16; p=0*). Measured b* values on sausage samples 1, 2, 3, 4, 5 and 6, increased during first months of storage, after that had relative small decrease during 6 month of storage (Table 4c). During further storage period there was relatively small yellowness value changes, with trend of relative small increases and decreases, but after 6 months of storage the values were higher than measured immediately after ripening. For sample 7 and 8 b* values had a tendency to decrease during the storage period.

Results of t-test showed statistically significant difference (p<0.05) for colour parameter b*-values, measured after 6 months of storage, between samples 2, produced with rosemary extract and sample 7 produced with antioxidant butylatedhydroxyanisole; and between samples 2 and 8 (control produced without antioxidant addition). These results were confirmed with sausages colour sensory evaluation. The sample 1, had extremely bright red colour, and the other compared samples had a dark red colour with more or less greyish shade, after 6 months of storage. Analyse of green tea extract effect (sample 3) on mean values for parameter b*, showed statistically significant difference (p<0.05) between samples 3 and 7, and between samples 3 and control sample 8.

Results of different oxidation process are visible as change of colour shade or as some other deterioration indicators. Peroxides created by the oxidation process can impact on iron oxidation in porphyrin ring and lead to the appearance of unacceptable green and brown shades of colour [47]. The differences in the measured L*, a*, b* values on the cross sections of sausages model samples during 6 months of storage can be connected to the presence of various antioxidants and their activity in the analysed samples. Desirable attributes of the dry fermented beef sausage are the relatively higher values for colour parameter a* (redness), medium values for colour parameter L* (lightness) and a lower values for colour parameter b* (yellowness).

Weight loss
Weight losses of the dry-fermented beef sausage "sucuk" type are shown in Figure 1. As a result of drying all samples had significant weight lost during ripening process (F=1472.86; p<0.05) and at the end of ripening, weight losses amounted for more than 30% relative to the initial mass, measured immediately after sausage stuffing, which was in accordance with the expected values. Weight losses in fermented sausages depend on several factors, such as the temperature, relative humidity and air velocity in the ripening chamber, meat mixture, particle size, product formulation and casing material [32, 50].

Moisture, fat and protein content
Moisture, fat and protein content in model sausages during ripening are analysed. All samples were produced in the same conditions of industrial production and variations of the observed parameters between the samples were relatively small, as was expected. Moisture, fat and protein content of beef sausages had statistically significant changes during the ripening period (p<0.05). Moisture content significantly decreased (F=255.44; p<0.05) from initial values of 49.20–52.80% to 24.50–27.40% after 19 days of ripening (Fig. 2). During the ripening period, due to a decrease of the water content, the content of other ingredients proportionally increased. The fat content changed significantly (F=80.53; p<0.05) and ranged from 27.90 to 32.60% at the beginning of ripening, and from 44.00 to 49.80% at the end of the ripening period (Fig. 4). Protein content had statistically significant change (F=123.40; p<0.05), as expected, and increased from initial values of 11.20–13.10% to 20.00–23.10% (Fig. 6). Similar results were obtained by other authors [6, 25, 50].

The sausage samples were packed in vacuum packaging and stored in a refrigerator at a temperature of 4°C, during 6 months of storage. There was no any statistically significant changes (p>0.05) in the basic chemical composition as expected, moisture content (F=1.24; p=0.303), fat content F=0.56; p=0.758), and protein content F=0.92; p=0.488) in the sausages model samples during storage period, shown in Figures 3, 5, 7.

pH Value
Changes in the pH values of dry-fermented beef sausage "sucuk" type during ripening are shown in Figure 8. The ripening time had statistically significant effect on pH values (F=211.91; p<0.05). During the first 3 days of ripening, pH values decreased rapidly, possibly due to production of organic acids by bacteria [5, 6, 8]. The initial pH values ranged from 5.79 (sample 3) to 5.63 (sample 1). The pH measured on the third day of ripening were in the range from 4.67 (sample 6) to 4.85 (sample 3). During the further ripening period pH of samples increases slightly, and at the end of ripening reaches values between 4.92 (sample 5) and 5.20 (sample 3). These results were in agreement with reported in literature, where pH values of sucuk decreased sharply during the first 3 days of ripening, due to the production of acid and then its value increased during the further ripening period [8, 18]. The changes were explained with lactic acid production in the first 3 days (in the fermentation period) of ripening, but after that, lactic acid level became constant. The decline in the pH value during the first days of ripening, when fermentation occurs, is very important, due to the formation of desired quality and safety changes in sucuk, as are the inhibition of undesired bacteria growth, rate of conversion of colour, and formation of desired flavour, reducing the possibility of the growth and propagation of microorganisms whose presence is not desired in the product, such as Staphylococcus aureus, Salmonella, Clostridium botulinum, etc. [24, 47].

Measured pH values during 6 months storage are shown in Figure 9. The pH values were affected (F=2.57; p<0.05) by storage time. At the end of the storage time, pH of the samples ranged from 5.02 (sample 4) to 5.39 (sample 3). According to the Turkish Standard Institute [24] pH for high quality sucuk should be in the range of 4.7–5.4. The dry-fermented beef sausage "sucuk" type prepared with rosemary extract with green tea extract as a natural antioxidants, had values close to this range. Based on the obtained results it can be seen that the pH of all model samples were in the range of standard values.

Overall sensory quality
The overall sensory quality of dry-fermented beef sausage "sucuk" type were affected by the storage time (FA= 39.02, p<0.05) and addition of the antioxidants into the sausage batter (FB=6.19, p<0.05). Results of sensory analysis showed that all model samples had expected, uniform quality immediately after production (ripening). All evaluated sensory attributes had maximum scores, and all samples had 100% of maximally possible quality (Fig. 10).

The overall sensory quality of all sausage model samples decreased during six months of storage.  Sample 1, produced with sodium ascorbate, had stable quality during the first two months of storage (scored as 100% of maximally possible quality), but during further storage period its sensory attributes changed, especially the colour of cross section, odour, aroma and taste. After 6 months of storage, the colour of cross section was bright red, nonspecific for sucuk and odour, aroma and taste were slightly rancid, resulting in overall sensory quality sample 1 value 71.50% of maximally possible quality.

In sample 2, produced with rosemary extract, a significant changes have taken place during the first two months of storage, which led to a stabilization and a higher level of sensory quality in relation to changes in other samples, and after 2 months of storage sample 2 had expected quality (scored as 100% of maximally possible quality), what justifies the use of rosemary extracts as antioxidants in the production of fermented beef sausages. On the other hand, sensory quality of the sample 3, produced with green tea extract, was deteriorated during the first 3 months of storage. Green tea extract showed different impact on the overall sensory quality of the sausage during storage period, depending on the process flow that took place in the product during 6 months of storage. As the biggest defects of sample 3 sensory quality, unpleasant aroma and greyish shade in the colour of sausage cross section were identified. Comparing sensory quality of samples produced with natural plant antioxidants, sample 2 and sample 3, after 3 months of storage, the differences of cross section colour, odour, aroma and taste and consistency were observed. Sample 2 had desired red colour, specific for this product, but colour on the cross section of the sample 3 had bad greyish shade. Odour, aroma and taste of sample 2 were expressed, typical and pleasant, but sample 3 had very low quality with unpleasant aroma. The consistencies of both samples were similar after 3 months of storage. During further storage period sensory quality of these samples decreased. After 6 months of storage, the colour on cross section of both samples was dark red with a light greyish shade, but the sample 3 had noticeable and undesirable yellowish shade. Sample 3 had strong rancid, unacceptable aroma, and in sample 2 the panellists detected a slight rancid aroma. Consistency of sample 3 was softer than sample 2. Sample 2, produced with the addition of rosemary extract, after 6 months of storage was scored as 75.00% of the maximum possible quality and had an acceptable sensory quality. The overall quality of sample 3 was evaluated as worse, with 59.50% of the maximum possible quality.

Rosemary extracts contain antioxidant compounds, the most active being phenolic diterpenes that break free radical chain reactions by hydrogen atom donation [17]. Coronado et al. [9]showed that wieners sausages containing 0.03% rosemary extract appeared to have slower rates of oxidation than those without antioxidant. Martinez et al. [30] reported that addition of rosemary (1000 ppm) plus ascorbic acid (500 ppm) retarded discolouration effectively in sausages illuminated with the UV-filter, reaching a display life of 16 days, equal to that of sausages maintained in the dark. Georgantelis et al. [17] demonstrated positive effects of chitosan, added in combination with rosemary, on both retarding lipid oxidation and improving colour retention of beef burgers during frozen storage (-18°C) for 180 days. Rosemary extract was added at a concentration of 200 mg/kg, and chitosan was used at the level of 10 mg/kg, concluding that this combination could have for commercial use to improve preservation of beef burgers.

Comparing the effects of selected antioxidants on sensory quality of dry fermented beef sausages it was found that use of ascorbic acid provide stable, consistent sensory quality of dry fermented beef sausages during 6 months of storage in controlled conditions. Sample 4, produced with ascorbic acid, had the highest overall sensory quality after 4, 5 and 6 months of storage. Changes of evaluated sensory attributes during storage were less expressed after 6 months of storage. Outside appearance, cross section appearance and consistency of sample 4 were without greater mistakes, colour of cross section was dark red, specific for this product, odour, aroma and taste were pleasant and expressed. Ascorbic acid possesses antioxidant properties, although it may act as antioxidant or pro-oxidant depending on its concentration, the presence of metallic ions and the tocopherol content [30].  It is known that ascorbic acid is also added to meat products in order to produce nitric oxide from nitrite. Nitric oxide reacts with myoglobin to form nitrosylmyoglobin which is unstable in air and discolouration can be rapid. Also, ascorbate improves the stability of colour [16].

Lipid oxidation, leading to rancidity, is one of the major reasons of meat products’ quality deterioration. This irreversible change contributes to the development of unacceptable sensory characteristics. The products of fatty acid oxidation produce off-flavours and odours usually described as rancid. Oxidative processes are also associated with discolouration of meat products [17, 39]. The most undesirable discoloration of sausages during 6 months of storage were found in samples 5, produced with ascorbyl palmitate, sample 6, produced with ascorbyl palmitate and tocopherols and sample 7, produced with synthetic antioxidant butylated hydroxyanisole. After 6 months of storage cross section colour of sample 5 was pale and gray, and colour of sample 6 and sample 7 were pale and gray with yellowish hue. Odour, aroma and taste of these samples were unpleasant and rancid. Overall sensory quality of the sausages samples 5, 6, 7 after 6 months of storage were evaluated with 57.30, 54.60 and 53.90% of maximally possible quality, respectively. The results obtained during the sensory analysis indicated that the sample 5, 6, 7 did not have expected stability of the quality during storage period. The effect of tocopherols on lipid and colour stability was detected after refrigerated storage or cooking of meat [11]. Georgantelis et al.[17] have demonstrated the positive effect of α-tocopherol (60 mg/kg), added individually or in combination with other antioxidants, on lipid oxidation and colour stability of frozen (-18°C) beef burgers stored for 180 days.

Sample 8, produced without addition of antioxidants and used as a control, had the most acceptable sensory quality immediately after production, a very pleasant aroma and colour formed during the ripening period, but during storage the sensory quality of sample 8 decreased. However, during 3 months of storage these sensory attributes were rapidly changed as result of unwanted oxidation processes, and this sample had an unpleasant taste and aroma, colour of the cross section was darker than expected and greyish shade was noticeable. At the end of storage period, sample 8 had dry surface, expressed rancid aroma, dark red colour with grey shade, and it was scored as 62.50% of maximally possible quality.

One of the main causes of the quality deterioration of the sausage during storage is lipid oxidation [27, 46]. Oxidative processes are also associated with discolouration of meat products, as lipid oxidation results in the formation of pro-oxidants capable of reacting with oxymyoglobin, which lead to metmyoglobin formation. Appearance of meat products is one of the major determinants of their appeal to consumers and, consequently, sales of the product [17]. Overall sensory quality of sucuks depends on formation of desired aroma, colour and texture [24]. Lactic acid bacteria produce acids, aldehydes and ketones which give aroma to sucuk during the fermentation [4, 47]. Colouring compounds (nitrosylmyoglobin) are formed, gelatinization of proteins occurs, and these changes increase overall sensory quality of sucuks. Bozkurt and Erkmen [8] found that the overall sensory scores of sucuk decreased (p<0.05) during 36 days of storage at 50% RH and 30°C. It was observed that control sample (without antioxidant) was found to have the worst quality (p<0.05), and increasing the nitrate/nitrite concentration increased (p<0.05) the overall sensory quality of sucuks. Bozkurt [5] found that addition of green tea extract, T. spicata oil or BHT increased (p<0.05) the overall sensory quality of sucuk sausages.

L *, a *, b * values measured on the sausages cross section depend of the used ingredients and storage conditions[32, 46]. In our research, statistical analyse showed that at the end of fermentation and after 6 month storage, there was no statistically significant difference in colour parameters (L*, a*, b*) measured on fresh cross section of dry-fermented beef sausage "sucuk" type between sausages produced with rosemary extract (sample 2), and green tea extract (sample 3). It could be concluded that there was no statistically significant difference in colour parameter L*, when compared sample 2 and sample 3 with sample 4, evaluated as sausage with the best overall sensory quality and the best red colour of compared samples. We find significant difference for parameters L* and a*, when compared sample 2 and sample 3 with sample 1 (produced with sodium ascorbate), for which colour was described as relative lighter than expected for beef sausage. There was no statistically significant difference for mean b* values measured on sample 2 and sample 3 when compared with values measured for sample 4 or sample 1 after 6 month storage. It could be concluded that compared samples had similar yellow shade.

When connect analyse of measured colour parameters (L*, a*, b*) on fresh cross section of dry-fermented beef sausage "sucuk" type with results of sensory analysis, it could be concluded that rosemary extract had positive impact on colour stability and sensory quality of sausage, the same as ascorbic acid in sample 4, produced for this research, and that it could be used as antioxidant in dry-fermented beef sausage "sucuk" type production. It was concluded that green tea extract, used in our research, did not show acceptable antioxidant effect. Instrumentally measured colour parameters on cross section of sausages produced with  green tea extract showed acceptable stability, but sensory analyse indicated on some kind of changes in flavour and colour shade after 3 month storage, which could impact on acceptability of the product, especially after expected 6 month storage. 

CONCLUSION

Investigating antioxidant effect of natural rosemary extract and green tea extract, separately, on the colour stability and quality of dry-fermented beef sausage "sucuk" type, during ripening and storage periods, were realised comparing their quality parameters with the effect of the other antioxidants, whose use is common in food industry and with sausage model sample, produced without antioxidants addition, used as control.

• It was concluded that rosemary extract had positive impact on colour stability and sensory quality of dry-fermented beef sausages "sucuk" type, and that it could be used as antioxidant in sausages production, the same as ascorbic acid which had the best effect on sausages quality in realised research.
• Results of descriptive sensory analysis of beef sausages produced with green tea extract did not demonstrate expected quality level. Instrumentally measured colour parameters on cross section of sausages produced with green tea extract showed acceptable stability, but sensory analyse indicated on changes in flavour and colour shade after 3 month storage, which could impact on unacceptable overall product quality, connected with deterioration especially before expected 6 month storage expired. 
• Sausage produced with addition ascorbic acid as antioxidant, demonstrated the best colour stability and sensory quality of all sausages analysed in the research.   
• The overall sensory quality of sausage model samples produced with antioxidant sodium ascorbate, decreased during storage. After 6 months of storage, the colour of cross section was bright red, nonspecific for sucuk and odour, aroma and taste were slightly rancid, resulting in lower overall sensory quality score.
• Beef sausages produced without addition of antioxidants (used as a control), had the most acceptable sensory quality immediately after production, a very pleasant aroma and colour formed during the ripening period, but after 3 months of storage these sensory properties were rapidly changed, sausages had an unpleasant taste and aroma, colour of the cross section was darker than expected and greyish shade was noticeable. At the end of storage period, rancid aroma was expressed, and colour was dark red with grey shade
• The most undesirable discoloration and sensory quality of sausages during 6 months of storage were found in samples produced with added antioxidants ascorbyl palmitate (sausages had pale and gray colour), the sausages produced with ascorbyl palmitate and tocopherols, the same as sausages with added synthetic antioxidant butylated hydroxyanisole, which were pale and gray with yellowish hue. Odour, aroma and taste of these samples were unpleasant and rancid.

Acknowledgments

These results are part of the project No 06/0-020/961-103/09, which was financially supported by Ministry of Science and Technology, Republic of Srpska (Bosnia and Herzegovina).

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