214 International Conference on Food Security and Nutrition IPCBEE vol.67 (214) (214) IACSIT Pre, Singapore DOI: 1.7763/IPCBEE. 214. V67. 19 Biochemical Change of Salt-Fermented Tuna Vicera (Dayok) and It Effect on Hitamine Content During Fermentation Jeebel R. Bea 1 and Erlinda I. Dizon 2 1 Department of Fiherie and Marine Science, College of Fiherie and Marine Science, Southern Philippine Agribuine and Marine and Aquatic School of Technology, Malita, Davao del Sur, Philippine 812 2 Intitute of Food Science and Technology, College of Agriculture, Univerity of the Philippine Lo Bano, College, Laguna, Philippine - 431 Abtract. Dayok i a mixture of tuna vicera, alt and mall amount of pice (garlic and ginger) fermented at ambient room temperature for 7 day and ha not been ubjected for heat treatment. The effect of varying alt concentration (1%, 17.5% and 25%), fermentation temperature (3-35 o C and 4 o C) and fermentation period (3 and 7 day) on microbial, chemical and biochemical change of tuna vicera during fermentation were invetigated. Effect of the different fermentation condition on microbial, chemical and biochemical change on tuna vicera were monitored. The reult demontrated that the level of ph, lactic acid, amino nitrogen and TVB-N increaed during fermentation. The formation of hitamine during fermentation i affected by ph, lactic acid, amino nitrogen, TVB-N, total plate count and LAB count. An increae in ph with a correponding decreae in lactic acid, increae in amino nitrogen, TVB-N, total plate count and LAB count produce a correponding increae in hitamine. Furthermore, formation of hitamine during dayok fermentation wa found to be influenced by alt concentration and fermentation period and not by fermentation temperature. Keyword: tuna vicera, hitamine, fermentation 1. Introduction In recent year, concern about food afety, together with conumer demand for afe and healthier product have promoted tudie of compound with harmful effect on human health. Fih fermentation, an old preervation technique, ha been found to contain high content of hitamine uch a fih auce and fih pate [1]. Hitamine (or combroid) fih poioning i a foodborne chemical intoxication caued by eating poiled or bacterially contaminated fih [2]. Scombroid fih uch a tuna, mackerel, bonito and aury are often implicated in combroid poioning incident when not properly proceed and tored [3]. In the Philippine, tuna vicera are widely ued a a raw material in the manufacture of fermented fih pate. Dayok i a mixture of tuna vicera, alt and mall amount of pice (garlic and ginger) fermented at ambient room temperature for 7 day and ha not been ubjected for heat treatment. Little information i available on the ue of tuna vicera in fermented product uch a fih pate. With the increaing number of conumer being expoed to thi product, it eemed appropriate to evaluate more on the afety rik that dayok might poe to the conumer to hitamine poioning. Thi tudy wa undertaken to invetigate the proceing condition affecting hitamine formation in alt-fermented tuna vicera in order to find way to control thi hazard at level below the allowable limit. The effect of varying alt concentration (1%, 17.5% and 25%), fermentation temperature (3-35 o C and 4 o C) and fermentation period (3 and 7 day) on microbial, chemical and biochemical change of tuna vicera during fermentation were invetigated. Correponding author. Tel.: + 63 917 8418475. E-mail addre: jillbea@yahoo.com. 97
2. Methodology 2.1.Fermented Tuna Vicera Preparation Yellowfin tuna (Thunnu albacare) vicera, excluding the bile ac and heart, wa ued in the tudy. Freh tuna vicera were obtained from evicerated yellow fin tuna and placed in ice box with a fih to ice ratio of 1:2 (w/w) and were tranported to the laboratory. Upon arrival, the tuna vicera wa wahed, drained and mixed with alt at 1%, 17.5% and 25% concentration. The ample were placed in terilized ealed bottle and were allowed to ferment for 7 day at varying temperature (ambient temperature and 4 o C). Sample of alted tuna vicera obtained at, 3, and 7 day of fermentation were imultaneouly analyzed for microbial and chemical analye. 2.2. Microbiological and Phyical Analyi Total bacterial plate count wa counted on PCA plate while colonie on MRS agar + 1% CaCO 3 howing clearing around the colonie wa counted a LAB after the incubation period at 3 C for 48 hour. The bacterial count of the fermented product wa expreed a log 1 colony forming unit (CFU)/g. 2.3. Determination of ph, Salt Content, Total Titratable Acidity and Amino Nitrogen The ph value of the ample were meaured uing a ph pen (Hannah intrument model HI19617). Five gram (5g) of homogenized tuna vicera ample were added with 1 ml of ditilled water and ph value were meaured. The ph pen wa calibrated with ph 4. and ph 7. tandard buffer prior to it ue. The alt content wa determined by diluting ample (5 g) with 1 ml of ditilled water. Five (5) ml of the olution wa placed in a 125 ml Erlenmeyer flak then 1 ml of 2% potaium chromate olution wa added. The olution wa titrated with 1/5 N ilver nitrate olution to a light orange endpoint. Total titratable acidity (expreed a percent lactic acid) wa determined by diluting five (5) ml ample with 1 ml ditilled water. Twenty-five (25) ml of the olution wa placed in a 125 ml Erlenmeyer flak and titrated againt a tandardized.1 N NaOH to ph 8.2. Amino nitrogen content wa determined by the Formol titration method. The ample ued in determining the total titratable acidity which wa previouly neutralized with.1 N NaOH olution to ph 8.2 wa ued. The ample wa added with 1 ml of neutralized formaldehyde (38%, v/v). (Note that for protein-rich ample, the ph of the ample i expected to decreae upon addition of formaldehyde). The mixture wa titrated with tandard.1 N NaOH to ph 8.2. Amino nitrogen wa expreed a mg%. 2.4. Determination of Total Volatile Bae Nitrogen (TVB-N) and Hitamine Content The TVB-N content of the ample wa meaured by the Conway Microdiffuion method while hitamine wa analyzed following the AOAC Fluorometric method [4]. 2.5. Statitical Analyi Tet for ignificance for the analytical tudy were done uing analyi of variance (ANOVA). Value of p<.5 wa regarded a ignificant. Pearon correlation and multiple linear regreion wa carried out to determine the relationhip between ph, alt content, total bacterial count, lactic acid bacteria count, ph, amino nitrogen, TVB-N and hitamine in fermented tuna vicera. Statitical analyi wa performed uing the Statitical Analyi Sytem (SAS) program. 3. Reult and Dicuion Tuna vicera (dayok) traditional fermentation proce involve mixing tuna vicera with 25% alt, fermented at ambient temperature (3-35ºC) for 3 day, draining, chopping or mincing, addition of pice (ginger and garlic) and then packed in bottle without heating. In thi tudy, the formation of hitamine i aeed by monitoring the hitamine content in every proceing tage during the production of dayok in relation to the different fermentation parameter to hitamine content. 3.1. Influence of Salting Concentration on Hitamine Content during Fermentation After tuna vicera wa mixed with alt at varying concentration, the varying level of alt concentration ignificantly affect hitamine formation. The initial hitamine content of raw material (49.6 ppm) 98
ignificantly increaed upon addition of alt with value of 77.2, 83.6 and 11.5 ppm for 1%, 17.5% and 25% alt concentration, repectively. The hitamine content increae with increaing alt concentration. Thee value are higher than thoe reported by [1] on fih pate at 26.3 ppm. Thi could be attributed to the difference in fih pecie or part ued for proceing, fermentation condition aide from the high initial hitamine content of the raw material. However, no ignificant correlation exit between ph, alt content, lactic acid, amino nitrogen, TVB-N and fermentation temperature. During the alting proce, hitamine formation wa not influenced by thee factor. 3.2. Influence of Fermentation Time on Hitamine Content during Fermentation A hown in Table 1, hitamine formation on tuna vicera i ignificantly affected by alt concentration and temperature when fermented for three (3) day but only to ome extent. High level of hitamine were formed at 1% NaCl fermented at 4 o C (11.9 ppm) which i twice higher than the FDA regulatory limit of 5 ppm while lower value were obtained at higher alt concentration of 17.5% and 25% NaCl. However, no ignificant difference were oberved on tuna vicera at 17.5% NaCl fermented either at ambient temperature (3-35 o C) and 4 o C with hitamine value below the regulatory limit at 46.4 and 43.1 ppm, repectively. On the other hand, ample fermented at 1% and 25% NaCl at ambient temperature have higher hitamine content above 5 ppm at 63.6 and 84.8 ppm, repectively. The increae in hitamine content at 25% NaCl could be attributed to the very high alt concentration which affect the rate of alt penetration into the vicera caued by it aturation point creating low diffuion of alt into the ample while at 17.5% alt wa completely diolved in the olution, thu fater rate of penetration. The formation of high hitamine level at 1% NaCl indicate that hitamine forming microorganim are moderately halophilic or alt tolerant and hitidine decarboxylae activity i better at 4 o C which fall within the optimum growth temperature of enzyme at 37-4 o C. In addition, the fermentation temperature of 3-35 o C fall within the optimal growth temperature between 2 o C and 37 o C for mot bacteria containing hitidine decarboxylae enzyme [5] which alo explain the high hitamine value of tuna vicera at alt concentration of 1% and 25% fermented at ambient temperature. Table 1. Hitamine content of tuna vicera alt fermented for three day at varying alt concentration and fermentation temperature. Salt concentration (%) Fermentation temperature Hitamine content (ppm) ( o C) After alt addition After fermentation 1 3-35 61.6+1.77 63.6+15.5 4 92.8+16.23 11.9+24.83 17.5 3-35 88.+15.39 46.5+11.33 4 79.2+13.85 43.1+1.5 25 3-35 95.3+16.67 84.8+2.66 4 17.8+18.85 45.8+11.16 Reult are expreed a mean +SD (n=3) Initial hitamine content of tuna vicera prior to alting = 49.6ppm After even day of fermentation, only alt concentration ignificantly affect hitamine formation regardle of fermentation temperature (Fig. 1). Reult have the ame trend with that of tuna vicera fermented for only three day but value increae with prolonged fermentation time. Hitamine content of tuna vicera fermented at 1% NaCl increae from 82.7 ppm (3 day) to 112.2 ppm (7 day). Similar trend but ignificantly lower hitamine content compared to 1% NaCl wa oberved at 17.5% and 25% NaCl with 47.8 and 61.2 ppm, repectively. The reult how that tuna vicera fermented for 3 and 7 day at 1% alt concentration have hitamine value higher than the tandard limit for afety. The formation of hitamine in tuna vicera fermented for 7 day i trongly influenced by ph, lactic acid, amino nitrogen and TVB-N. A ph increae, there i a correponding decreae in lactic acid and hitamine content increae. It hould be noted that the ph of tuna vicera fermented for even day range from 5.23 to 6.3 thu, creating a favorable condition for hitamine production. The activity of amino acid decarboxylae 99
Hitamine (ppm) Hitamine (ppm) i mot active in the acidic olution at ph 3 to 6 [6]. Furthermore, bacteria are encouraged to produce thee enzyme a part of their defene mechanim againt the acidity [5]. A amino nitrogen and TVB-N increae, hitamine content increae. Thi indicate that there i an increae availability of free amino acid (hitidine) neceary for the formation of hitamine due to the degradation of protein and polypeptide by proteolytic enzyme. Amino nitrogen and TVB-N are ued a indicator of protein decompoition but TVB-N value are mot ueful a index for poilage in freh and fermented fih product [7]. Thu, a ignificant direct correlation between TVB-N and the formation of hitamine content implie that bacterial poilage contribute to hitamine formation. Furthermore, higher TVB-N and amino nitrogen value were oberved in tuna vicera fermented at 1% NaCl baed on the reult of the tudy. Bacillu a predominant bacteria found in fermented fih pate i capable of decarboxylating one or more amino acid [6]. Hitamine forming bacteria belonging to the genu of Bacillu iolated from fih include Bacillu coagulan, Bacillu megaterium and Bacillu pumilu [8]. A 12 1 8 6 4 2 1% NaCl 17.5% NaCl 25% NaCl 3 7 Fermentation Time (day) Fig. 1: Change in hitamine (ppm) level during dayok fermentation at varying level of (A) alt concentration and (B) fermentation temperature. 1 5 B n n 3-35 C 4C n n 3 7 Fermentation Time(day) n n 3.3. Influence of the Addition of Spice to Hitamine Content during Fermentation Hitamine value of tuna vicera fermented for three and even day added with pice namely, ginger and garlic, both at the rate of 2% (w/w) per pice. The trend in hitamine formation i imilar with that of fermented ample before pice were added. Hitamine value of ample at 1% NaCl continuouly increaed until even day from 76.6 to 9.2 ppm while thoe fermented at 17.5% and 25% NaCl decreaed at even day from 56.5 to 44.2 ppm and 95.9 to 61.9 ppm, repectively. Tuna vicera fermented at 1% and 25% NaCl have hitamine value higher than the tandard limit of afety even after the addition of pice. Hitamine value of tuna vicera are not ignificantly different before and after pice were added. Thi implie that addition of pice at 2% concentration doe not ignificantly lower hitamine content. 3.4. Microbiological Change of Tuna Vicera during Fermentation The total plate count (TPC) increaed while lactic acid bacteria count decreae during the fermentation period. Microbial count decreaed with increaing alt concentration. Only alt concentration affect the microbial count and LAB count in the fermentation of tuna vicera (p<.5). Reult further how that dayok produced from tuna vicera at 1% alt concentration had total plate count of 1 7 cfu/g higher than the recommended value for total plate count of 1 5 cfu/g (p<.5) and higher than thoe produced from ample at higher alt concentration with 1 4 and 1 5 cfu/g for 17.5% and 25% alt concentration, repectively. The preence of high viable count of aerobic microorganim indicate that the product i prone to poilage epecially on tuna vicera fermented at 1% NaCl. 1
Lactic acid bacteria (log CFU/g) Total plate count (log CFU/g) Tuna vicera fermented at 1% NaCl had ignificantly higher LAB count of 1 6 cfu/g compared to thoe fermented at 17.5% (1 4 cfu/g) and 25% NaCl (1 3 cfu/g). Thi indicate that LAB preent in the fermented product wa able to grow at 1% NaCl a reflected by it high viable count. Hitamine formation i alo ignificantly influenced by total plate count (Fig. 2). The final total plate count of fermented tuna vicera of 1 7 cfu/g in 1% NaCl i ignificantly higher compared to 17.5% and 25% NaCl. It ha been reported that hitamine production occur only after aerobic plate count reach to 7 log CFU/ml [9]. Thi obervation wa confirmed in thi tudy. 4. Concluion Hitamine formation during fermentation i affected by ph and total plate count. It i controlled by alt concentration (>17.5%) and length of fermentation (7 day). 8 7 6 5 4 3 y =.447x + 2.195 2 R 2 1 =.4829* 5 1 15 9 8 7 6 5 4 3 2 1 y =.4199x + 3.1432 R 2 =.6578* 5 1 15 Hitamine (ppm) Hitamine (ppm) Fig. 2: Relationhip of total plate count and LAB count to hitamine formation on tuna vicera fermented for 7 day. 5. Acknowledgement Thi reearch wa financially upported by the Southeat Aian Regional Center for Graduate Study and Reearch in Agriculture (SEARCA) and the Commiion on Higher Education (CHED) Higher Education Development Program, Philippine. 6. Reference [1] Y.H. Tai, C.Y. Lin, L.T. Chien, T.M. Lee, C.I. Wei, and D.F., Hwang, Hitamine Content of Fermented Fih Product in Taiwan and Iolation of Hitamine-Forming Bacteria, Food Chemitry, Vol. 98, 26, pp. 64-7. doi:1.116/j.foodchem.25.4.36 [2] L. Lehane and J. Olley, Hitamine Fih Poioning Reviited, International Journal of Food Microbiology, Vol. 58, 2, pp. 1-37. [3] S.L. Taylor, Hitamine Food Poioning: Toxicology and Clinical Apect, Critical Review in Toxicology, Vol. 17, 1986, pp. 91-117. doi:1.319/1484486923767 [4] AOAC, 199.Official Method of Analyi, 15 th ed. Aociation of Official Analytical Chemit, Wahington, DC, USA. [5] J, Karovicova, and Z, Kohajidova, Review: Biogenic Amine in Food. Chem. Pap. Vol. 59, 25, pp. 7-79. [6] P.C. Sanchez, Philippine Fermented Food. Principle and Technology, The Univerity of the Philippine Pre, Diliman, Quezon City, 28. [7] Y. Hu, X. Wenhui and X.Y. L. Xiaoyong, Change in biogenic amine in fermented ilver carp auage inoculated with mixed tarter culture, Food Chemitry. Vol. 14, 27, pp. 188-195. [8] M.Z. Zaman, A.S. Abdulamir, F.A. Bakar, J. Selamat and J. Bakar, A Review: Microbiological, Phyicochemical 11
and Health Impact of High Level of Biogenic Amine in Fih Sauce, American Journal of Applied Science, Vol. 6, No. 6, 29, pp. 1199-1211. doi:1.3844/ajap.29.1199.1211 [9] K, Bjorndottir. 29. Detection and control of hitamine producing bacteria in fih. PhD Food Science. 1 February 21 from http://www.lib.ncu.edu/thee/available/etd-32429-11524/unretricted/etd.pdf 12