Effect of Starter Culture and Packaging on the Chemical, Microbiological and Sensory Quality of Turkish Soudjouck (Sucuk)

Abstract

Lactobacillus plantarum, Micrococcus aurantiacus, and Pediococcus pentosaceus in different combinations and amounts were used for the production of fermented soudjouck. The effects of starter cultures on total aerobic bacteria, and selected chemical properties of samples were determined. Additionally, the effects of starter culture and packaging on the sensory characteristics of final product were studied. It was found that nitrite and sugar content of samples decreased during ripening, while protein and fat content relatively increased with drying. Mesophilic aerobic bacteria counts increased in the first days of ripening and then decreased, similar trend was also observed for the counts of all three starter cultures. Sensory results showed that reducing amount of starter culture negatively affected and incorporation of glucano delta lactone (GDL) into formula along with starter culture had an adverse effect on the sensory properties. It was demonstrated that vacuum packaging improved the sensory properties; therefore, it should be preferred over normal packaging.

Keywords:

• Starter culture
• Fermented soudjouk
• Ripening

INTRODUCTION

Turkish style soudjouck (sucuk) is a most popular fermented meat product in Turkey. Since its production is dependent on natural conditions; ripening and drying steps take a lot of time, which adversely affects the productivity, and profitability for the producer. Generally, starter cultures are not used in the traditional production of Turkish style soudjouck, and the fermentation occurs because of microflora which was inoculated by chance. Therefore, the end product exhibits wide variations in terms of chemical, microbiological and sensory characteristics.[1] In addition, the standardization of properties and shelf life of the product is a very strong interest to the meat industry.[2] For these reasons starter cultures can be used in fermented meat products. Prerequisite of a high quality soudjouck production is the use of meat and animal fat with good hygienic quality and allow desired microorganisms to become dominant by providing necessary conditions in the beginning of production, so that microflora would grow easily and produce desired enzymes and other compounds during ripening period.[3,4]

Starter cultures in fermented meat products were firstly used by Jensen and Paddock[5] and since then many researchers have been concerned about the issue. A number of studies were carried out to determine the potential use of starter cultures in fermented meat products. For example, Johansson, Berdagué, Larsson, Tran, and Borch;[6] Garriga, Hugas, Gou, Aymerich, Arnau, and Monfort;[7] Olesen and Stahnke;[8] Erkkila, Suihko, Eerola, Petaja, and Mattila-Sandholm;[9] S⊘ndergaard and Stahnke;[10] Pinto, Ponsano, Franco, and Shimokomaki;[11] Larrouture-Thiveyrat, Pepin, Leroy-Sétrin, and Montel,[12] and Olesen, Meyer, and Stahnke[13] studied the effect of starter cultures on the aroma production or sensory characteristics of fermented meat products. Gonzalez and Diez;[14] Lahti, Johansson, Honkanen-Buzalski, Hill, and Nurmi;[15] Bredholt, Nesbakken, and Holck;[16] Dicks, Mellett, and Hoffman;[17] Deumier and Collignan;[18] Työppönen, Markkula, Petäjä, Suihko, and Mattila-Sandholm;[19] and Sameshima, Magome, Takeshita, Arihara, Itoh, and Kondo[20] investigated the antimicrobial effects of starter cultures to be added into meat products. Sunesen, Trihaas, and Stahnke[21] and Bruna, Ordóñez, Fernández, Herranz, and de la Hoz[22] explored the antioxidative effects of starter cultures in meat products. In addition, the potential use of starter cultures for the production of novel meat products was also studied.[20,23,24] Homofermentative lactic acid producing bacteria such as Lactobacillus spp., micrococcus spp. and yeast Debaromyces hansenii are among those which are commonly used as starter cultures in fermented meat products.

In this research, the use of P. pentosaceus, M. aurantiacus, and L. plantarum cultures in different combinations and amounts for the production of soudjouck was investigated. Additionally, the effects of those starter cultures on the counts of total aerobic bacteria, fecal streptococcus and coliform bacteria were determined as well as on certain chemical properties (moisture, protein, fat and nitrite, and sugar contents) during ripening period. Another objective was the determination of the effect of vacuum or ordinary packaging on the sensory characteristics of final products after one month of ripening.

MATERIALS AND METHODS

Soudjouck Preparation

Soudjouck samples were prepared according to the following formulation. Sample dough contained 8500 g of beef meat, 1500 g tallow, 100 g black pepper, 150 g red pepper, 30 g allspice, 250 g garlic, 250 g sucrose, and 250 g salt. Sodium nitrite ratio in the final product was estimated to be 0.02% after drying, for that reason 1.5 g of sodium nitrite was added to the soudjouck dough. All materials were provided by Pınar Meat Plant (İzmir).

Fresh meat and tallow in small pieces, other ingredients and starter culture dissolved in water were mixed and the dough was stored overnight in a refrigerator for a better distribution of ingredients into meat. Soudjouck mixture was then minced using a machine of 3 mm hole diameter and filled into natural casings obtained from intestine. Soudjouck samples were placed in a ripening chamber and relative humidity and temperature were controlled. Soudjoucks were ripened as follows: 1 day at 90% RH and 24 ± 1°C, and 1 day at 85% and 22 ± 1°C, and then soudjoucks were dried at 18 ± 1°C for 12 days until the water content decreased under 40%. Samples were inoculated with starter culture combinations as following.

1. Control sample: no starter culture.

2. M. aurantaicus+L. plantarum (50:50).

3. P. pentosaceus+M. aurantiacus+L. plantarum (20:50:30).

4. P. pentosaceus+M. aurantiacus+L. plantarum (25:35:40).

5. M. aurantiacus+L. plantarum (65:35).

6. P. pentosaceus+M. aurantiacus+L. plantarum (20:50:30). Same as sample 3 but the amount is 1/3 less.

7. M. aurantiacus+L. plantarum (50:50). Same as sample 2 but glucono-delta-lactone (GDL) (% 0,2) was also incorporated.

8. M. aurantiacus+L. plantarum (60:35) same as sample 5 but the amount is 1/2 less.

L. plantarum CH-1 and P. pentosaceus (Flore Carn P.) were obtained from Chr. Hansens Lab. A/S (Denmark). M. aurantiacus M. 56 was provided by Federal Meat Research Institute (Kulmbach –Germany). All starter cultures were pure and freeze dried. After the determination of microbial activity during pretrials, the mixtures of combinations were prepared. Two types of packaging were used in this study: vacuum and ordinary packaging. For vacuum packaging, two-layer plastic films made of polyethylene and polyamide having an oxygen permeability of <15 cm³/m² in 24 h at 22°C and 80% RH and water permeability of <1.5 g/m² in 24 h at 23°C and 85% RH. Ordinary packaging was carried out in bags of polyamide/polyethylene.

METHODS

Analytical Methods

Moisture, ash, protein, carbohydrate, and fat content of samples were determined according to AOAC[25] procedures. Residual nitrite level (mg NaNO₂/kg sample) was determined according to standards ISO/DIS 2918.[26] All analytical measurements were carried out before and after ripening period.

Microbiological Methods

Microbiological analyses of samples were carried out before and after inoculation of starter culture to the soudjouck mixture and 2, 4, 6, 8, 10, and 14 days of storage during ripening/drying process. Ten grams of sample were aseptically homogenized in 90 ml sterile 0.1% peptone water. Serial dilutions were aseptically prepared and plated onto various media. For the enumeration of aerobic-mesophilic bacteria, samples on plate count agar (PCA) plates (Biolab) were incubated at 25°C for 3 days.[27] MRS agar (Merck) was utilized for the identification and enumeration of L. plantarum.[28] Samples were plated by drop method and enumeration was performed after 72 incubation at 37°C. Mannitol Salt Agar (Merck) was used to enumerate M. aurantiacus.[29,30] After incubating samples for 48 h at 37°C, red-purple zone forming colonies were counted for M. aurantiacus. To determine the counts of P. pentosaceus of samples, the media prepared from glucose, peptone, yeast extract, gelatin, and agar was used, and pin-tip like white colonies were enumerated after incubation.[31]

Sensory Analysis

Sensory evaluation of samples were carried out after a month of ripening and drying period according to the hedonic test described by Cross, Moen, and Stanfield[32] for the evaluation of sensory quality of meat products was utilized. The sensory panel consisted of 8 experienced personnel of the meat plant. The panelists were familiarized with the soudjouck samples during pre-trials and were asked to evaluate the samples for appearance, sliceability and cross section, color, structure and flavor. The analysis was performed in Pınar Meat Company under white fluorescent lights with an intensity of approximately 350 lx in individual booths constructed according to the specifications of the International Standards Organisation (ISO DP 6658).[33,34] Two slices of each sample (approximately 4 mm thick) were served to the assessors. The grilled soudjoucks were heated to a core temperature of 72°C then cooled down to room temperature and served to panelists for flavor evaluation. Water at room temperature and unsalted crackers were also provided to clean the palate between samples. According the importance of a particular sensory attribute, a whole number was given to sample by the panel: 1–5 for appearance (1: very unpleasant 5: very pleasant); 1–10 for sliceability, color and structure (1: very unpleasant 10: very pleasant); 1–15 for flavor (1: very unpleasant 15: very pleasant) (total of 50). Results were expressed as the predominant score given by panelists.

Statistical Analysis

Results were statistically analyzed by Statistical Analysis System[35] software. The effects of storage period and treatments on microbiological and analytical results were investigated by using two-factor Analysis of Variance (ANOVA) and Duncan test. Sensory results were analyzed by one-way ANOVA.

RESULTS AND DISCUSSION

Chemical Composition of Samples

Chemical composition values of samples were presented in Table 1. It was determined that all samples were in accordance with Turkish Soudjouck Standard[36] in terms of chemical composition. It was found that the samples after ripening and drying period contained moisture 36–38, fat 37–39 and protein 14.9–16.7 kg/100 kg sample protein. The moisture content of samples decreased during ripening and drying period as expected; therefore, fat and protein contents were relatively increased. Carbohydrate content of samples inoculated with starter culture was also decreased, because microorganisms converted sugar to the lactic acid and utilized in their metabolism. Nitrite present in the sausage sample was bound to myoglobin, hence its content also diminished. Gökalp[37] reported that nitrite content of Turkish soudjouck decreased during ripening period which decreased the counts of proteolytic and lipolytic bacteria.

Table 1 Chemical composition of samples before and after ripening period

Sample	Moisture (%)	Fat (%)	Protein (%)	Carbohydrate (%)	Nitrite (ppm)
Control	52.50–37.65	32.00–39.50	12.95–15.75	1.34–0.99	38.0–10.5
2	53.10–37.20	31.85–38.65	13.30–15.35	1.33–0.81	36.0–7.50
3	53.15–37.70	34.15–39.70	12.90–14.90	1.28–0.79	37.5–8.00
4	52.85–36.45	33.25–39.95	12.60–16.70	1.20–0.45	35.0–7.50
5	51.85–37.75	30.95–38.95	13.10–15.65	1.42–0.85	36.5–10.0
6	52.85–37.65	32.30–38.65	12.45–16.65	1.20–0.40	30.0–8.50
7	52.90–38.30	31.70–39.15	13.00–15.60	1.25–0.87	17.5–9.50
8	54.00–37.05	31.45–37.65	13.35–16.20	1.33–0.90	38.5–11.50
First value represents before filling and the second for the final product.

Microbiological Results

Results for mesophilic aerobic bacteria counts in soudjouck samples were given in Table 2. It was found that there was a significant (p < 0.05) difference between the counts of mesophilic aerobic bacteria before filling into cases and freshly filled samples. On the other hand, the number of mesophilic-aerobic bacteria in the samples inoculated with different starter cultures initially increased and then they tended to decrease. For example, those of control samples increased regularly until reaching to maximum at the 6th day of storage then the number went slightly down towards to the end of storage. This trend is related to the ripening conditions of the samples as relative humidity was higher in the first five days and then it was reduced. Similar results were reported by Bozkurt and Erkmen[38] as they determined that aerobic bacteria in sausages increased during the first 8 days then they decreased. ANOVA results showed that starter culture addition significantly (p < 0.05) affected the growth of mesophilic aerobic bacteria during ripening period. Vural and Oztan[39] studied the effects of various starter commercial starter cultures on the mesophilic aerobic bacteria counts for Turkish soudjouck and determined significant differences in terms of the those bacteria among samples with different starter cultures. Coventry and Hicks[40] reported that L. plantarum and P. pentosaceus addition to dry fermented soudjouck formulations did not allow the growth of other microflora in considerable amount, which was an important aspect for the production of fermented soudjouck with high quality. They also added that P. pentosaceus was particularly effective in the inhibition of microflora other than starter culture.

Table 2 Change in the total mesophilic-aerobic count of sample during storage (log cfu/g)

	Before inoculation	After inoculation	Storage time (days)
Sample			2	4	6	8	10	14
Control	5.65	5.74	5.87	7.21	7.95	7.49	7.60	7.78
2	5.19	7.12	7.29	7.80	7.97	8.01	8.12	7.97
3	5.90	7.28	7.27	7.12	6.89	7.03	7.00	6.93
4	5.27	6.98	7.19	6.99	6.92	6.89	6.71	6.91
5	5.7	7.16	6.98	7.72	8.06	8.08	8.08	7.93
6	6.04	7.15	7.19	6.86	6.79	6.82	6.70	6.91
7	5.67	6.97	7.06	7.43	7.47	7.51	7.28	6.77
8	5.5	7.07	6.90	8.7	7.67	7.65	8.00	7.67

M. aurantiacus was added to all samples with different combination or amounts except for control, hence the growth of M. aurantiacus of samples with starter cultures were significantly higher than that of control. Figure 1 illustrates the change in M. aurantiacus count with respect to storage period. As it can be seen from the figure, the highest level of M. aurantiacus for the samples with starter culture was reached after 48 hr of ripening.

Figure 1 The effect of ripening period on the counts of M.aurantiacus.

The results of L. plantarum enumeration showed the similar trend observed for mesophilic-aerobic bacteria as they increased in the first days of ripening period then decreased toward to the end of storage. Pearson and Dutson[41] reported that lactobacilli were dominant microflora in fermented meat products. They added that most of those were L. plantarum and also a few L. casei and its strains were present. Lactic acid bacteria produce bacteriocin like compounds that inhibits the growth of pathogenic and spoilage bacteria.[17,18,15,42] Dicks et al.[17] reported that two bacteriocins (curvacin and Plantaricin) produced by L. plantarum and L. curvatus inhibited the growth of Listeria monocytogenes in ostrich meat salami.

Samples 2, 3, and 5 were inoculated with P. pentosaceus and it was determined that count of that bacterium increased during first two days of ripening at 24°C; after that ripening temperatures were dropped to lower than 20°C, which cause a decrease in the number of P. pentosaceus. for example P. pentosaceus count of sample 5 was 2.20 × 10⁶ cfu/g sample after inoculation and increased to 13.20 × 10⁶ cfu/g sample during first 2 days of ripening. However the counts were decreased back to 2.63 × 10⁶ cfu/g sample after 14^th day of ripening period.

Sensory Analysis

Sensory results of soudjouck samples ripened for a month after vacuum or ordinary packaging were illustrated in Table 3. Results indicated that vacuum packaging had a significant effect (p < 0.05) on the sensory characteristics of samples. All sensory attribute values were higher for vacuum packaged samples as compared to those of ordinary packaged samples.

Table 3 The effect of starter culture and packaging type on the sensory scores of sausage samples

Sample	Packaging	Appearance	Sliceability	Color	Structure	Flavor
Control	Ordinary	4.35	7.68	8.43	7.63	9.16
	Vacuum	4.76	9.06	9.31	9.03	14.00
2	Ordinary	4.41	8.34	8.58	8.37	12.02
	Vacuum	4.85	9.15	9.29	9.16	13.97
3	Ordinary	4.43	8.25	8.64	7.70	11.14
	Vacuum	4.78	9.18	9.36	9.03	12.86
4	Ordinary	4.34	9.02	8.32	8.15	11.08
	Vacuum	4.87	9.11	9.33	9.18	12.79
5	Ordinary	4.62	9.16	9.20	9.16	13.90
	Vacuum	4.93	9.56	9.36	9.35	14.03
6	Ordinary	3.88	5.51	8.34	5.89	11.69
	Vacuum	4.27	6.89	8.83	7.45	12.10
7	Ordinary	4.02	5.26	7.10	5.11	10.14
	Vacuum	4.23	6.58	8.31	7.32	12.47
8	Ordinary	4.00	5.17	6.52	5.11	9.77
	Vacuum	4.23	6.17	6.70	6.11	9.93

ANOVA results revealed that there was a significant effect (p < 0.05) of starter culture addition on the appearance of samples. It was observed that the sample 5 received the highest score for appearance for both vacuum and ordinary packaged samples. On the other hand, appearance score of samples 6, 7 and 8 were significantly lower than that of other samples. It was determined that starter culture addition significantly (p < 0.05) affected the sliceability and cross sectional view of samples. Similar to the appearance, the best results for sliceability was obtained from sample 5. On the hand sample 7, prepared with GDL addition was very difficult to slice and its cross section was not as smooth as other samples.

The color of sample was affected from starter culture addition. While the sample 5 received the best preference for the color, sample 8 received the lowest score for both ordinary and vacuum packaged samples. The color of samples inoculated with Pediococcus became partially darkened after ripening and drying period. This discoloration could be result of moisture loss or nitrosomyoglobin oxidation because of oxygen exposure.

When the texture of samples evaluated by the panel, highest score was given to samples with M. aurantaicus+L. plantarum. The sample 5 whose M. aurantiacus+ L. plantarum combination ratio was 65:35 was best in terms of texture of sample among all samples and the same starter culture combination with 50:50 ratio was rated second. It could be concluded that the use of combination of two starter cultures resulted in highly preferred texture. On the other hand, the sample which was prepared with the addition of GDL had a sandy texture and was not received a good preference from the panel members. It could be speculated that unpleasant texture of those samples occurred because, binding properties of the sausage did not properly progressed in GDL samples due to the fast pH drop resulting from gluconic acid release. ANOVA results showed that the texture of samples was significantly (p < 0.05) affected from starter cultures. In addition, different texture scores of sample 3 and 5 and samples 2 and 7 (both inoculated with same starter culture but in different amount) indicated that the amount of starter culture also played an important role for the textural properties; hence, higher amount of microorganisms yielded better sample in terms of textural results.

Statistical analysis of taste scores of samples revealed that there was a significant (p < 0.05) effect of starter cultures and sample 5 was found the best regarding to taste and it was significantly different when compared to that of other samples. However, when the taste scores of vacuum and ordinary packaged sample were separately analyzed after one month, it was observed that for ordinary packaging, control sample received significantly lower score than that of others while it received the second highest score for vacuum packaging.

Total sensory scores of samples are illustrated in Figure 2. Total scores were best correlated with texture scores (R² = 0.977), as similar results were obtained for the texture of samples, the samples (5 and 2) inoculated with M. auranticus and L. plantarum received the higher scores when compared to others Considering all sensory results, it could be summarized that M. aurantiacus and L. plantarum (65:35) added soudjouck sample received highest preference from panel; reducing the amount of starter culture addition negatively affected the sensory properties, incorporation of GDL into formula along with starter culture had an adverse effect on the taste, consistency and sliceability of samples. It could be concluded that the ratio of starter culture in combination is as important as the type and the concentration of cultures. Also, the amount of starter cultures should be high enough for them to become dominant microflora for the production of fermented soudjouck with desired quality. Additionally, it was demonstrated that vacuum packaging favorably affected the sensory properties, therefore, it should be preferred over normal packaging.

Figure 2 Total sensory scores of samples.