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Physicochemical Characteristics and Protein Degradation During Fermentation of Plaa-Som, a Traditional Fermented Fish Product of North-Eastern Thailand

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Physicochemical Characteristics and Protein Degradation During Fermentation of Plaa-Som, a Traditional Fermented Fish Product of North-Eastern Thailand Indian Journal of Traditional Knowledge Vol. 14 (2), April 2015, pp. 220-225 Physicochemical characteristics and protein degradation during fermentation of Plaa-som, A traditional fermented fish product of North-Eastern Thailand Karnjana Chadong1, Sirinda Yunchalard2 & Weera Piyatheerawong 2, 3* 1Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand; 2Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand; 3Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand E-mail: [email protected] Received 16 October 2014, revised 06 February 2015 The involvement of lactic acid fermentation in physicochemical characteristics and fish protein degradation of Plaa-som, a Thai fermented fish produced from common silver barb (Barbonymus gonionotus), was investigated. pH of Plaa-som gradually decreased to 5.16 after 120 hrs of fermentation. In contrast, titratable acidity of Plaa-som rapidly increased and reached a peak of 0.73% (w/w) after 48 hrs then it slightly declined. Concentration of lactic acid was enhanced to 2.13% (w/w) after 120 hrs. Small amounts of other organic acids such as acetic, citric, succinic, and formic acids were also detected. Peptide content demonstrated an increasing trend while protein concentration illustrated a decreasing trend. Protein degradation was also observed using electrophoresis. Actin and tropomyosin were degraded after 96 and 120 hrs, respectively. Simultaneously, small proteins of 18 and 21 kDa were detected between 24 and 48 hrs and eventually degraded after 72 hrs, indicating complete degradation. Therefore, the degraded products, such as small proteins and peptides, could lead to development of health-promoting areas for the functional peptides obtained from Plaa-som consumption. Keywords: Physicochemical characteristics, Protein degradation, Plaa-som, Spontaneous fermentation, Fermented fish, Barbonymus gonionotus IPC Int. Cl.8: A61B 5/00, C07K, A23J, C12N 1/00, C12P 21/00, A21, A23, A47J27/00, A47J 37/00, A23L 1/325, A22C 25/00 Silver barb or java barb is shared the same wrapped plastic bag until a sour taste is developed by scientific name of Barbonymus gonionotus Bleeker1. indigenous microorganisms4. Lactic acid bacteria Generally, this freshwater fish, locally named (LAB) are mainly found to be the predominant Plaa-ta-pien, is distributed and consumed among bacterial species when the fermentation has Southeast Asian Nations especially the countries of progressed6,7,8. These LAB utilise carbohydrate Mekong basin such as Kingdom of Cambodia, Lao available in the recipe and produce organic acids, People's Democratic Republic, Kingdom of Thailand, mainly lactic acid, as a part of their metabolites4,8,9. and Socialist Republic of Vietnam1. It is widely used Thus, pH and acidity of the products are usually as either fish fillet in boiled soup (Tom-yam) or related to the quality of Plaa-som, and the dominant fermented fish (Plaa-som, Som-fug and Som-plaa)2-4. groups of LAB during spontaneous fermentation have Plaa-som and other types of fermented fish have been investigated7. Exploitation of these LAB as a gained popularity due to their high nutritional values starter culture could result in the development of from fish protein as well as their unique aroma and innovative production for accelerated fermentation, flavour characteristics2,5. This fermented product is improved flavour development and safety in consumed mainly in North-Eastern Thailand4. fermented food10,11. Physical, and chemical changes Production of Plaa-som is achieved by the during the fermentation of fish have been widely combination of whole fresh, cleaned, gutted and studied5,12,13. Protein degradation is usually detected scaled fish with salt, cooked rice or sticky rice, and during fermentation. Formation of the resulting garlic. The fish is then left to ferment in a tightly products, such as small proteins, peptides and amino acids, could also affect both texture and flavour —————— 14,15,16 *Corresponding author development . Since fish muscle protein contains CHADONG et al.: PHYSICOCHEMICAL AND PROTEIN DEGRADATION DURING FERMENTATION OF PLAA-SOM 221 a high concentration of myofibrillar protein and low scaled, slit along both sides of the trunk, and concentration of stroma protein, these proteins are thoroughly washed with clean tap water for several readily salt and water soluble17. During fermentation, times (Fig. 1B). Subsequently, the prepared fish was proteolysis is caused by endogenous enzymes in the soaked in 20% (w/v) brine solution for approximately fish muscle as well as from microorganisms16,18,19. 2 hrs (Fig. 1C). It was then rinsed once, and the However, little is known regarding the process of excessive brine was drained. Subsequently, these muscle protein degradation during Plaa-som freshly prepared fish was mixed with other fermentation. This study aims to examine the ingredients (Fig. 1D). In general, Plaa-som is involvement of spontaneous fermentation on some prepared by mixing the prepared fish with 5% (w/w) physicochemical characteristics and degradation of freshly crushed garlic, 10% (w/w) steamed jasmine fish protein during Plaa-som production. rice and 0.5% (w/w) sucrose4. After all of the ingredients were mixed with the freshly prepared fish, Methodology the fish was packed into plastic bags and pressed to Chemicals expel most of the air before closing with rubber bands Ammonium persulfate, 30% (w/v) Acrylamide/Bis- (Fig. 1E). Then, the fish was placed in plastic boxes acrylamide solution, Coomassie Brilliant Blue covered with a screw cap lid and left to ferment at R-250, β-mercaptoethanol, sodium dodecyl sulphate, 30ºC. Fermentation was performed for 120 hrs, and N, N, N’, N’- tetramethylenethyl-enediamine, Folin the samples were obtained at the following steps with Ciocalteu’s phenol reagent, bovine serum albumin three replicates, fresh fish from the market, the fish and tyrosine were purchased from Sigma-Aldrich Co. freshly mixing with all ingredients (the fermented fish LLC (St. Louis, Missouri, USA). Other chemicals at 0 hr) and the fermented fish at 24-120 hrs. were analytical grade. Determination of pH and titratable acidity Preparation of Plaa-som Plaa-som samples of 5.0 gm were collected from Thai silver barb (Barbonymus gonionotus) for the each sampling point, homogenised with 45 ml of size of 300 - 400 gm was purchased from the local CO2-free distilled water, and filtered through gauze. market (Khon Kaen, Thailand). Freshness of fish The filtrate was directly determined using a pH meter. samples was maintained by rapid transportation on ice Titratable acidity (TA) in the form of lactic acid to the laboratory within 30 min, and the samples were equivalents was determined using titration. The immediately prepared (Fig. 1A). The fish was gutted, filtrate was titrated against 25 mM NaOH with phenolphthalein solution as an indicator20. Determination of organic acid concentration Concentration of organic acids was determined using high-performance liquid chromatography (HPLC). Plaa-som samples of 5.0 gm were collected from each sampling point, homogenised with 25 ml of CO2-free sterile distilled water and centrifuged at 3,000 × g for 15 min. Protein in the supernatant was precipitated with 0.5 M perchloric acid at a ratio of 1:1. The mixture was left at room temperature for 5 min, and then centrifuged at 10,000 × g for 10 min to remove all of the precipitates. To eliminate fat, petroleum ether was added to the supernatant at a ratio of 1:1. 2.0 ml of the lower phase was collected and adjusted to 10 ml with the addition of 0.1% (v/v) phosphoric acid. The mixture was then collected and filtered through filter paper (Whatman No.1) and a Fig. 1—Preparation of Plaa-som (A) fresh fish; (B) the prepared membrane filter (Chrom tech. Inc., Minnesota, USA). fish; (C) the prepared fish soaking in 20% (w/v) brine solution; (D) the prepared fish mixing with ingredients; (E) the prepared Prior to HPLC analysis, 20 µl of filtrate was fish with ingredients packing into plastic bags injected into a Shimadzu HPLC system, including a 222 INDIAN J TRADIT KNOWLE, VOL 14, NO. 2, APRIL 2015 LC-10 AD pump, SPD-10A detector, VertiSep OA performed using SPSS statistical software, version 8 µm HPLC column, 7.8 × 300 mm column, and a 19.0 (SPSS Inc., Chicago, Illinois, USA). The results computer with a data analysis software program (LC were expressed as the means ± standard deviation. 45 Solution). The sample was analysed in an isocratic mode at 0.3 ml/min using 1.0 mM sulfuric acid as the Results and discussion mobile phase. The column temperature was steadily Physicochemical characteristics of Plaa-som maintained at 40°C. Lactic, acetic, citric, succinic, 4 Initial pH and TA of the prepared fish were 6.73 and formic acids were used as external standards . and 0.14% (w/w), respectively. During incubation, Determination of protein and peptide contents pH of Plaa-som gradually decreased from 6.36 to Plaa-som samples of 5.0 gm were collected from 5.16 after 120 hrs of fermentation. This pH profile each sampling point and homogenised for 5 min in is consistent with the advised characteristics of Plaa-som according to Thai community products 10 ml of sterile distilled water using a blender at a 21 constant grinding speed of 1,000 rpm. The mixture standard . Generally, pH is regarded as an indicating was subsequently filtered through gauze and filter factor to ensure the safety of the fermented 3,4 Plaa-som paper (Whatman No. 4). To remove particulate debris, products . In addition, TA of was extensively increased and reached a peak of 0.73% the filtrate was then centrifuged at 10,000 × g for (w/w) after 48 hrs then it slightly declined (Fig. 2). 15 min at 4°C. Amount of the supernatant was The high level of TA was mainly associated with adjusted to 10 ml by the addition of 25 mM sodium the major organic acid such as lactic acid, which phosphate buffer (pH 7.0).
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