Deesanam et al. 59 Effect of Fermentation Temperatures on Quality of Naem Made from Raw Materials Treated with Plasma N. Deesanam1, N. Chomsri2, C. Dechthummarong3, and V. Thonglek3 1Dept. of Food Science and Technology, Faculty of Sciences and Agricultural Technology, Rajamangala University of Technology Lanna, Chiang Mai, Thailand 2Agricultural Technology Research Institute, Rajamangala University of Technology Lanna, Chiang Mai, Thailand 3Dept. of Electrical Engineering, Faculty of Engineering, Rajamangala University of Technology Lanna, Chiang Mai, Thailand Abstract—The objectives of this study were to investigate the effect of fermentation temperatures on quality of Thai traditional fermented sausage (Naem) made from ingredients treated with plasma. The non-thermal plasma at 15 kV was applied on raw materials used in Naem production. Fermentation temperatures at 30 and 35oC were carried out. Microbiological and chemical analyses revealed the impact of fermentation temperatures on Naem quality. The products made from ingredients with or without plasma treatment were not significantly different in organoleptic quality (p>0.05). Keywords—Non-thermal plasma, naem, fermentation temperature I. INTRODUCTION Naem is a fermented ground pork product made from minced pork, boiled pork rinds, cooked rice, garlic, salt, sugar, pepper, chili and sodium nitrite, generally packed in banana leaves or plastic sheets and allowed to ferment for approximately 3-4 days at 30-35oC [1]. Many factors affect Naem quality including fermentation temperature. Prevalence of unwanted bacteria such as Salmonella, Staphylococcus and Listeria in Naem product has been reported [2]. Prevention of pathogenic bacterial contamination in raw materials must be considered as the Thai fermented pork is usually consumed as a raw dish. Plasma processing generates energetic, reactive gases to (a) inactivate microorganisms in food as well as food processing environment [3]. This technology utilizes electricity and matter producing free radicals, discharges and various gases, e.g. ozone, oxygen, hydrogen, nitrogen, helium [4]. Microbial inactivation using plasma for food and food products is highly promising [5]. The interaction of charged particles with microorganism contaminants was proposed to explain the inactivation mechanism (Fig. 1). Many studies demonstrated that plasma has inactivation effects on bacteria through bacterial cell wall/ membrane or extracellular polymeric substance disruption or via action on intracellular components, metabolic activity or virulence factors, e.g. prevention of (b) biofilm formation leading to cell death [6]. Application of plasma in food processing has been widely Fig. 1. (a) Plasma interaction with microbial contaminants and (b) studied. For example, the corona discharge plasma jet could strategy for plasma application [5, 6]. reduce 2 log cfu/g of total aerobic bacteria contaminated on dried squid shreds [7]. In addition, no significant changes on appearance, color, flavor, taste and texture of the products were not detected. Since Thai fermented ground pork is II. METHODOLOGY usually consumed as a raw dish, it is possible that unwanted microorganisms existing in the fermented pork might be a A. Naem Preparation source of pathogen transfer. Thus, the objective of this study These Naem processing steps is shown in Fig. 2. The was to evaluate the effect of plasma application for Naem major ingredients of Naem were as follows (%): raw ground production. lean pork (45.5); cooked shredded pork rind (35); chopped garlic (8); cooked rice (8); monosodium glutamate 0.3); sugar (1) and nitrite salt (2.2). The 15 kV, 2.45 kHz, 50 mA and borosilicate glass material of dielectric barrier discharge Corresponding author: Napaporn Deesanam (DBD) plasma was applied on raw ground pork, cooked e-mail address: [email protected] shredded pork skin, chopped garlic and cooked rice for 10 min 60 International Journal of Plasma Environmental Science & Technology, Vol. 12, No. 2, JANUARY 2019 (Fig. 2). The size of DBD treatment chamber was 60 cm width, bag. Serial dilutions were prepared using 0.85% saline in order 45 cm length and 20 cm height (Fig. 3). Gap distance between to obtain colonies in a countable range, i.e. 30-300 colonies [8]. glass and raw materials was about 0.5 inch. Thickness of pork One ml of the appropriate dilutions was pour-plated onto samples was about 0.3±0.1 cm. Then all constituents were sterile plate count agar (Merck KGaA, Darmstadt, Germany) mixed, divided into portions, (40 g/ portion) and wrapped each and MRS agar (Merck KGaA, Darmstadt, Germany) plates for portion in the polypropylene plastic bag tightly. The total microbial and lactic acid bacteria counts, respectively [9]. fermentation was carried out at 30 and 35oC for 72 hours. The The plates were then incubated at 35oC for 48 h. Colony- Naem control was fermented at ambient temperature (30+3oC) forming units (cfu) were used to quantify results in as it was homemade or small scale enterprise produced in microbiological plating. Colonies were counted and counts Thailand. were expressed as cfu per gram. Concentrations of cfu were expressed using logarithmic notation, where the value shown B. Microbiological Analysis was the base 10 logarithm of the concentration. The mean The Naem sample was removed from the packaging viable count was calculated from three plates. aseptically. The sample of 25 g was used for microbiological analysis. It was mixed with 225 ml of 0.85% saline and C. pH and Lactic Acid Measurements homogenized for 2 min at normal speed in a sterile stomacher pH was measured by introducing a pH meter (Model C831, Belgium) into a mixture of sausage and distilled water (1:9). Total acidity (TA) was determined by diluting each 5 g of sample in 50 ml distilled water and then titrating to pH 8.2 using 0.1 N NaOH [8]. Titratable acidity was expressed as lactic acid. D. Sensory Evaluation The packaging was removed and the fermented Naem products were sliced about 1 cm thickness and cooked on the fried-pan. The 50-untrained panelists participated in the hedonic tests. Each sample was labeled, at random, with a three digit code number and presented on plate at the ambient temperature in a randomized order. The panelists were required to use water to cleanse the palate and neutralize the flavors between samples. The panelists were asked to assign scores for appearance, color, firmness, flavor, taste, sour and overall acceptability on a structured nine-point hedonic scale (9 = like extremely; 8 = like very much; 7 = like moderately; 6 = like slightly; 5 = neither like nor dislike; 4 = dislike slightly; 3 = dislike moderately; 2 = dislike very much; 1 = dislike extremely). Fig. 2. Flowchart of Naem making Analysis of variance (ANOVA) was used to compare mean differences of the samples. Significant differences between treatments were analyzed by Duncan’s new multiple range test (DNMRT) at a 0.95 significance level. All data were presented as mean value with their standard deviation indicated (mean ± SD). III. RESULTS A. Microorganism in Naem (a) The total microbial and lactic acid bacteria counts during fermentation were in the ranges of 2-6 and 5-7 log cfu/g in all samples, respectively (Fig.4a). Fermentation temperatures significantly influenced the total microbial population of total plate count in Naem during fermentation. There was a remarkably low count of the total microbial count (3.55 log cfu/g) in Naem fermentation at the temperature of 30oC for 36 hours. This might be explained by the microflora which could grow well at 30oC was overgrown by lactic acid bacteria. After a 12-hour lag phase of lactic acid bacteria, the cells grew (b) exponentially up to 24 hours, then remained in stationary phase until the entire course of fermentation. Fig. 3. Schmatics of DBD treatment (a) and its wave form (b) E. Statistical Analysis. Deesanam et al. 61 (a) (a) (b) (b) Fig. 5. Changes of total acidity (a) and pH (b) during fermentation. Fig. 4. Changes of total plate count (a) and lactic acid bacteria (b) during fermentation. C. Sensory Evaluation of Naem All sample Naem were lactic acid bacteria rage 5-7 log Plasma treatment and fermentation temperatures did not cfu/g. During fermentation at 0-72 h of 30oC ripen temperature significantly influenced hedonic scores of Naem products was more slightly growth rate of lactic acid bacteria than (p>0.05). The panelists considered the scores of appearance, control. color, firmness, flavor, taste, sour and overall among all Ripen temperature at 35oC at 36-60 h was decreased samples at high scale value of 7.3-7.6 indicating desirable slightly of lactic acid bacteria compare with control (Fig. 4b). quality of Naem products. B. Lactic Acid Content and pH during Naem Fermentation IV. DISCUSSION The pH and lactic acid content of the mixture immediately before wrapping was in between 4-5 and 0.4-1.4%, Many researchers reported that non-thermal plasma could respectively. (Fig. 5). As fermentation time increased, all inhibit gram positive bacteria, i.e. Enterococcus faecalis and Naem samples exhibited rapid decrease in pH during the first Staphylococcus aureus better than gram negative, i.e. 48 h (Fig. 5b), which continued to decrease reaching a final Pseudomonas aeruginosa but the most resistant plasma was value of 4.15 within 72 hours. Similarly, the average TA Candida albicans fungus [11, 12]. values continuously increased with the fermentation time in all The fermented meat product may be contaminated with batches of Naem (Fig. 5a). However, the drop of pH observed pathogenic bacteria, e.g. Staphylococcus aureus and resulted between 48-72 hours showed significant differences among in food poisoning, therefore, decrease their population after the Naem treatments. The capability of surface penetration fermentation is necessary [8]. Although the total microbial possibly affected the physicochemical and microbial population at the beginning of fermentation did not differ properties of raw material using for making resulting in the among samples, harmful bacteria might be destroyed.