The Effects of Grape Seed Flour on the Quality of Turkish Dry Fermented Sausage (Sucuk) during Ripening and Refrigerated Storage

The beef, beef subcutaneous fat and sheep’s tail fat pieces (~5 cm³ in size), spices, garlic, salt, sugar, GSF, starter cultures (Staphylococcus carnosus and Lactobacillus sakei; Bactoferm^TM; Chr. Hansen, Denmark) and sodium nitrite were mixed and minced in a grinder (Alveo, Turkey). Sausage formulations are shown in the Table 1. Each of resulting batches of sausage batter was rested for 12 h at 4℃ and stuffed into collagen casings (Naturin Darm, Germany) of 40 mm diameter. Each sample was washed under running water, and then a 10% potassium sorbate solution was sprayed on it. Samples were ripened for 14 d. Ripening temperature was started from 24℃ and then first 7 d decreased 1℃ for every day. For equilibration, the relative humidity was adjusted to 60±3% in the first 6 h of the ripening period and was then increased to 90±3% and decreased every day by 1 unit. At the end of the ripening period, sausages packed in a polyethylene bag by applying vacuum and stored at 4℃ for 80 d.

Ten grams of ground sausage was homogenized in 100 mL distilled water and the pH was measured using a pH meter (Orion 3-star, USA) equipped with temperature probe as outlined by Ockerman (1985).

Moisture, fat and protein were determined according to AOAC (2000). Protein was determined as crude protein using the Kjeldahl method. Fat was determined as crude fat using the Soxhelet extraction.

Ripening yield was determined as follows:

Yield (%) = W₁ × 100 / W₀

where W₀ is the weight before ripening, and W₁ is the weight on the 7th or 14th d of ripening period.

Diameter reduction was calculated as follows:

$DiameterReduction\left(\%\right)={\displaystyle \frac{\left(d_0-d_1\right)\times 100}{d_0^{}}}$

where d₀ is the diameter of patties before ripening and d₁ is the diameter on the 7th or 14th d of ripening period.

TBA (mg malonaldehyde/kg sausage) was determined according to the distillation method as outlined by Tarladgis et al. (1960). FFA was determined according to Egan et al. (1981). A 10 g ground sample was mixed with 25 mL of chloroform in the presence of anhydrous sodium sulphate for 5 min, and then filtered with filter paper. The free fatty acids in the filtrate were titrated with 0.1 N NaOH. Free fatty acid content was expressed as g oleic acid/100 g fat.

Free fatty acid (%) = (S × N × F) × 28.2 / W

where S is the volume of titration (mL), N is the normality of the sodium hydroxide solution, F is the factor of the sodium hydroxide solution and W is the fat weight (g) in the sample.

The colour values of the sausages were measured by using a portable colorimeter (Minolta CR-400, Japan). The instrument was standardised against a white standardisation plate before each measurement. The colour was measured according to CIELAB systems as L (lightness), a (redness) and b (yellowness) values. Eight measurements were performed for each sausage.

A 20 g sample was aseptically taken from each sausage and homogenised in 180 mL of sterile salt solution (0.85% NaCl; Merck). The count of total aerobic mesophilic bacteria was determined on Plate Count Agar (PCA; Merck) incubated at 37℃ for 48 h (APHA, 1992), while Lactic acid bacteria (LAB) was determined on De Man Rogosa Sharpe Agar (MRS; OXOID) incubated at 30℃ for 72 h. (APHA, 1992) and moulds-yeasts on Dichloran Rose Bengal Chloramphenicol Agar (DRBCA; Merck) incubated at 25℃ for 5 d (APHA, 1992). Enterobacteriaceae were cultured on Violet Red Bile Glucose Agar (VRBGA; Merck) incubated at 30℃ for 24 h (Harrigan, 1998).

Sensory analyzes were performed at the end of the ripening period. The sausages were sliced to ~2 mm thickness and coded with geometric shapes. They were served in a random order. Water and sliced bread pieces were served after each sample to remove traces of the previous sample from mouth. Eleven trained panel (academic staff of Adiyaman University, Food Process, Food and Beverage, and Cookery Departments) who were selected and trained according to Yetim and Kesmen (2009) assessed the sensory properties using a hedonic scale for the general appearance, cross-sectional surface appearance, outer surface color, cross-sectional surface colour, brittleness, odour, flavour, and overall acceptability. The values in the scale indicated the following range of reactions: 1: dislike extremely to 9: like extremely.

Two replicates were performed for this study. The data were subjected to analysis of variance (ANOVA), and the results were expressed as mean±standard deviation (SD). When there were differences among the samples, the differences were compared using Duncan’s multiple-range tests; a probability value of p<0.05 was considered significant.

The effects of GSF on the pH values of the sausages were not found to be significant (p>0.05, Table 2). Moreover, the effects of ripening and storage periods on the pH values of the sausages were found to be significant (p<0.01). As shown in Table 2, pH values decreased during the ripening period. However, the storage period increased pH values. LAB of fresh meat and starter cultures produces lactic acid which reduces the pH of dry fermented sausages (Bover-Cid et al., 2001). The increased pH in the storage period might result from basic components such as amines. Generally, such basic components are produced from the degradation of amino acids by proteolytic enzymes. Moreover, such increases in pH could result from decreasing lactic acid formation and increasing degradation of lactic acid. The pH values of the samples were found to be lower than the pH of typical sucuk. This result might be due to the use of an acidifying starter culture.

The effects of GSF on the moisture values of the sausages were found to be significant (p<0.01, Table 3). The addition of GSF decreased the moisture values of the sausages on the 7th d of the ripening period. Although the addition of GSF decreased the moisture values of the sausage on 0 and 14th d, these decreases were not found to be significant. Moreover, the effects of the ripening period on the moisture values were found to be significant (p<0.01). The moisture values of sausages were decreased during ripening period (Table 3). Gökalp et al. (1999) reported that moisture loss in sucuk increased with decreasing pH values to the isoelectric points of meat proteins. The decrease in the pH and relative humidity of the medium increased the moisture loss of the sucuk (Kurt, 2006). The addition of GSF did not cause a significant difference in fat and protein values of sausage (Table 3). However, the fat and protein values increased significantly (p<0.01) during the ripening period. Moisture loss in the sausages increased protein and fat values.

Although the addition of GSF decreased FFA values of the sausages, such differences were not found to be significant (p>0.05, Table 4). However, FFA values of the sausages increased significantly (p<0.01) during ripening and storage periods. The level of free fatty acids in dry fermented sausages depends on the hydrolytic activity of the lipases, the microbial metabolic processes, and the oxidative reactions (Soriano et al., 2006). Such lipases are mainly of endogenous origin. Lipase activities increase with decreasing pH value in dry cured meat products (Chizzolini et al., 1998; Toldrá, 1998; Vestergaard et al., 2000). Toldrá (1998) reported that phospholipid hydrolysis generated significant free fatty acids in dry cured meat products. Moreover, Toldrá (2006) reported that long processes with mild ripening conditions allowed for relatively higher enzyme activity.

The effects of GSF on the TBA values were found to be significant during ripening (p<0.05) and storage (p<0.01) periods (Table 4). 1.5% and 3% of the GSF decreased TBA values at the 14th day of the ripening period. Moreover, the addition of GSF decreased TBA values during the storage period. Such effects of GSF might result from its phenolic content which affects the oxidative stability of meat products (Lutterodt et al., 2011). Lutterodt et al. (2011) reported that the most abundant phenolic compound in concord and ruby red grape seed flours was catechin. Özvural and Vural (2011) reported that TBARS values in frankfurters decreased with increasing grape seed flour content.

The effects of the ripening and storage periods increased TBA values significantly (p<0.01). Sausage can be easily oxidized during ripening and refrigerated storage periods because of its fat content and environment (Gökalp et al., 1999). Increasing oxidation can increase secondary products, such as malonaldehyde. The effects of the ripening period on TBA values were in accordance with the results of Kurt (2006), who reported that TBA values increased with increasing ripening period of sucuk.

The effects of GSF on the diameter of sausages were found to be significant (p<0.01, Table 5). The addition of GSF decreased the diameter reduction of sausages during the ripening period. In particular, the rate of decrease on the 7th d of the ripening period was greater than that on the 14th d. The addition of GSF did not cause significant difference in ripening yield values (p>0.05, Table 5). The changes in the diameter reductions might be related to the fibre content of the GSF.

The effects of GSF on the L values of sausages were found to be significant (p<0.01) during the storage period (Table 6). Although the addition of GSF decreased L values during the ripening period (Table 6), its effect was found to be significant (p<0.05) on the 80th day of the storage period. Moreover, L values decreased during the ripening period and then increased significantly (p<0.01) with the storage period. Such decreases might be associated with moisture loss, in particular drying of the surface of the sausages. Bozkurt and Bayram (2006) reported that the L values of sucuk decreased from 38 to 28 during ripening period. Vacuum packaging and equilibration of the moisture content of the sausages might have affected L values during the storage period.

The effects of GSF on the a (redness) values of the sausages were found to be significant (p<0.01, Table 6). The addition of GSF decreased the a values of the sausages during the ripening and storage periods. The differences in the a values might be due to the colour pigment contents of GSF. The colour black grape seed flour was darker than the beef. This difference in the colour might be affected the colour values of beef patties. Özvural and Vural (2011) reported that grape seed, the by-product of the production of red wine, decreased the colour values of sausages significantly.

Although the ripening and storage periods decreased a values of the sausages significantly (p<0.01), the effects in control group were not found to be significant during storage period (Table 6). Moreover, GSF decreased the a values of the sausages more than the control group during the ripening period. Decreasing a values might be related to the moisture loss and natural colour pigments of the grape seed flour. Similarly, Kargozari et al. (2014) reported that the ripening period decreased a and b values of Turkish dry fermented sausage. Decreasing a values during the ripening period might have resulted from denaturation of colour pigments due to lactic acid formation. Pérez-Alvarez et al. (1999) reported that a decline in red values is related to partial or complete denaturation of myoglobin, nitrosomyoglobin and oxymyoglobin.

The effects of GSF on the b values of the sausages were found to be significant (p<0.01, Table 6). The addition of GSF decreased b values of the sausages during the ripening and storage periods. Such effect was the highest on the 14th d of the ripening period. The colour components of grape seed, such as polyphenols might have affected the b values of the sausages.

The b values of the sausages decreased significantly (p<0.01) during the ripening period. A GSF level of 3% decreased the b values of the sausages less than other levels of GSF during ripening period. Drying of the sausages during the ripening period could have contributed to the decreased in b values. Pérez-Alvarez et al. (1999) reported that a decline in yellowness values of Spanish-type drycured sausage may have been related to a decrease in oxymyoglobin due to oxygen consumption by microorganisms. Casaburi et al. (2007) and Lorenzo and Franco (2012) have agreed about the decreasing in L, a and b values during ripening period of sausages.

The effects of GSF on the counts of Enterobacteriaceae, moulds and yeasts were not found to be significant (p>0.05) on the 0th d of ripening period. Moreover, Enterobacteriaceae, moulds and yeasts were not detected on the 7th and 14th d. The decreasing pH values of the sausages might have prevented the growth of Enterobacteriaceae. However, the application of potassium sorbate on the sausages casings’ might have prevented the growth of moulds and yeasts. Özdemir et al. (1996) reported that the counts of moulds and yeasts in sucuk may decrease below the detection limit until 7th d of ripening period.

The effects of GSF on the count of total aerobic mesophilic bacteria were found to be significant (p<0.05) during the storage period (Table 7). As shown in Table 7, 1.5% and 3% levels of GSF decreased the count of total aerobic mesophilic bacteria during the storage period. Such effect might be due to the flavonoid contents of GSF. Anastasiadi et al. (2009) reported that flavonoids and flavonoid derivatives in the grape seed play an important role in its antimicrobial activity.

The effects of the ripening and storage periods on the count of total aerobic mesophilic bacteria were found to be significant (p<0.01, Table 7). The count of total aerobic mesophilic bacteria increased on the first day of the ripening period. The results of some studies (Ayhan et al., 1999; Gönülalan et al., 2004; Özdemir et al., 1996) have agreed with this result. The addition of starter cultures and ripening conditions might be responsible for the increased count of total bacteria on the first day of the ripening period. However, the storage period decreased the count of such bacteria. Decline in the count of total aerobic mesophilic bacteria might be associated with the vacuum packaging and refrigerated storage condition.

The effects of GSF on the count of Lactic acid bacteria (LAB) were found to be significant (p<0.05) during the storage period (Table 7). The addition of 1.5% and 3% GSF decreased the count of LAB on the 40th and 80th d of the storage period. Such an effect might be associated with the flavonoid content of GSF. Perumalla and Hettiarachchy (2011) reported that grape seed extract has an important antimicrobial activity. Metabolic activities of starter culture such as LAB are required for the desirable changes that determine the particular characteristics of dry fermented sausages (Erkkilä, 2001). Therefore, such antimicrobial activity of GSF is important during storage instead of ripening period.

LAB play the most important role in producing the required quality of sausages. Their count increased in the first days and then significantly (p<0.01) decreased during the last days of the ripening period (Table 7). Moreover, the count of LAB decreased during the storage period significantly (p<0.01). The addition of starter cultures and their adaptation to the meat fermentation environment might have increased the count of LAB in the first days of the ripening period. Increasing moisture loss and decreasing pH values in the last days of the ripening period could account for the decrease in LAB during that period. Some researchers (Aksu and Kaya, 2004; Gönülalan et al., 2004; Özdemir et al., 1996) reported that the count of LAB in the sausages increased during the first days of the ripening period and decreased slightly during the last days of the ripening period. Moisture loss and decreasing in pH values might have caused decrease in LAB count during the last days of the ripening period. However, low moisture, pH and refrigerated conditions could also have affected the count of LAB.

The effects of GSF on the sensory scores of the sausages were found to be significant (p<0.05) with the exception of brittleness scores (Table 8). The addition of grape seed flour at 1.5% did not cause a statistically significant difference in the sensory properties, with the exception of odour. Odour scores decreased with the addition of GSF significantly. The addition of GSF at the 3% level caused decreasing scores of the sensory parameters, as shown in Table 8. The decreasing sensory scores might be related to the components of GSF such as tannins. Lesschaeve and Noble (2005) reported that many phenolic compounds are characterized by bitterness and astringency.