Materials and Methods
1.Mantı samples Wheat flour, minced beef meat, onion, black pepper, salt and water were used as the raw materials of mantı. Dough was prepared by mixing wheat flour (65%w/w of dough) and water (35%w/v of dough). The filling was prepared by mixing minced beef meat (23%w/w of mantı), kneaded onion (5%w/w of mantı), salt (2%w/w of mantı) and black pepper (0.5%w/w of mantı). The filling material was added to each square of dough material in an equal amount, averaging four measurements of 1.275 g. The heat treatment was performed in an oven with a product centre temperature of 60°C and treatment time of 4 min. The temperature was monitored by using a manual thermo logger. The product was left to cool at ambient temperature (∼20°C) and immediately transported under refrigeration (4°C) for MAP treatment.
2.MAP of the samples Samples of mantı were packaged under different gas combinations at the plant Antalya Altın Et Entegre Tesisleri, Turkey. Samples were placed in polypropylene (PP) trays (155x197x55 mm) with a water vapour transmission rate of 0.629 g/(m2 · day) and trays were sealed with a 25 µm thick polyolefin based film, an oxygen transmission rate of 24 cm3/m2 · day · bar and with an 18 g/(m2 · day) (38°C, % 100 RH) water vapour transmission rate. MAP was carried out using G. Mondini (Italy) packaging machine. Sealing temperature was 150°C and a 2/1 gas volume to mantı weight ratio in each pack was applied. Gas mixtures, volume based, were designed for MAP 1 (80% CO2 + 20% N2), MAP 2 (40% CO2 + 60% N2), MAP 3 (60% CO2 + 40% N2) and Control (atmospheric gas composition) treatments.
Samples from MAP 1, MAP 2 and MAP 3 were stored at 4°C, 50 – 63% RH for 126 days. Control samples were stored at 4°C, 50 – 63% RH until they reached the spoilage period, then mould mycelia inside the tray was observed. Analyses were performed on days 0, 1, 3, 5 and 7 and then at seven day intervals up to the day 126. Maximum storage day was determined by spoilage record, both visually and growth data of microorganisms.
3. Microbiological analysis
3.a) Analysis performed before MAP treatment Before and after heat treatment, the dough and filling of mantı samples were analysed separately for total aerobic mesophilic microorganisms (TAMB), Staphylococcus aureus, C. perfringens, Salmonella spp., Lactobacillus spp., yeast and mould, Coliform and Escherichia coli. Petri dishes were incubated at 30°C for 24 – 48 h for all aerobic mesophilic microorganisms; other analyses were performed as detailed below. Microbiological data were expressed in logarithm basis for the number of colonies (CFU/g).
3.b) Analyses performed after MAP treatment Lactobacillus spp., total anaerobic mesophilic microorganisms, S. aureus, yeast and mould, Coliform, E. coli, Salmonella spp., C. perfringens and total aerobic psychrophilic microorganisms were analysed during the storage period.
Maximum Recovery Diluent (Merck, Darmstadt-Germany) was used for homogenization and dilution, except in the Salmonella spp. analyses where buffered peptone water (Merck) was used (Anonymous, 2007).
Total aerobic psychrophilic microorganisms and total anaerobic mesophilic microorganisms were enumerated at Plate Count Agar (Merck). Anaerobic mesophilic counts were incubated at 25°C for 24 – 48 h by using Anaerocult A (Merck) in jars, and total aerobic psychrophilic microorganisms were incubated aerobically at 4°C for seven days (Anonymous, 2007).
MRS agar (de MAN, ROGOSA and SHARPE (Merck) was used for Lactobacillus spp. and were incubated anaerobically by using Anaerocult A (Merck) in jars at 25°C for two days (Anonymous, 2007). Morphologic and biochemical analyses were performed for identification. Baird Parker Agar (Merck) was used to estimate Staphylococcus spp. counts. After incubation at 35°C for 45 – 48 h, typical colonies were tested for detection of coagulase production. For this purpose, Staphylase test kits (Oxoid, Hampshire-UK) were used. Coagulase positive colonies were counted as S. aureus on the plates containing 20 – 200 colonies, coagulase (-) clear colonies were evaluated as coagulase negative Staphylococcus spp. (Anonymous, 2007).
Yeast and mould were counted using Potato Dextrose Agar (Merck) and incubated at 25°C for seven days (Anonymous, 2007). For identification of the mould species, media characteristics and cell morphology were investigated (Samson et al., 1981).
Enumerations of Coliform and E. coli were performed separately within a solid and broth medium. For this purpose, double layer Fluorocult Violet Red Bile (VRB) Agar (Merck) and Fluorocult Lauryl sulphate broth (LST) Broth (Merck) were used and incubated at 35°C for 16 – 18 h and 35°C for 24 – 48 h, respectively. After the incubation period, samples were exposed to a long wave (366 nm) UV light (Merck) in a darkened area and colonies or tubes that reflect fluorescence were counted as E. coli on plates and tubes by using the most probable number tables with biochemical tests. Gram stain, indole production, Voges-Proskauer reactive compounds, Methly red reactive compounds and citrate utility tests were evaluated to differentiate Coliform group bacteria (Anonymous, 2007).
Analyses of Salmonella spp. were carried out in four steps. Briefly, 25 grams of samples were weighed in Buffered Peptone Water (Merck) for non-selective pre enrichment step for 16 – 20 h at 35°C. Following the incubation, for selective enrichment step 0.1 mL and 10 mL were transferred to 10 mL of Rappaport Vassiliadis Broth (Merck) and 100 mL of Selenite Cystein Broth (Merck) and incubated at 42°C and 35°C for 24 h, respectively. After incubation a loopful were streaked on both Salmonella Shigella Agar (Merck) and XLT4 Agar (Merck), and incubated at 35°C for 24 – 48 h. Colonies isolated from each media were further analysed by biochemical reactions (lactose, gas from glucose, H2 S, urea, indole, citrate, lysine decarboxylase), where necessary API 10 E (Biomérieux, Marcy I'Etoile-France) was used to identify the bacteria (Anonymous, 2007).
4.Statistical analysis Each trial was repeated twice and triplicate samples were tested at each sampling time. Data were subjected to variance analysis in order to determine the effect of gas composition and storage time on each variable. The analysis was performed using analysis of variance (ANOVA) one-way analysis, and statistical package (SPSS 15.0, USA) was used to identify the different groups. The Duncan's post hoc test was applied; significance level was p < 0.05. Control group samples were not included in statistical analysis owing to the earlier spoilage record on day 21st.