Indian Journal of Biotechnology Vol 8, July 2009, pp 304-310 Influence of Pediococcus acidilactici as a starter on the flavour of tempoyak (fermented durian) Neti Yuliana 1* and Virgilio V Garcia 2 1Jurusan Teknologi Hasil Pertanian (THP), Fakultas Pertanian, Universitas Lampung (UNILA). Jalan Sumantri Brojonegoro #1 Bandar Lampung, Indonesia 2 Institute of Food Science and Technology, University of the Philippine at Los Banos, Philippines Received 24 July 2008; revised 28 November 2008; accepted 10 February 2009 Pediococcus acidilactici UP02 was used as a starter for the fermentation of tempoyak ( Durio zibethinus Murr.), a traditional Indonesian fermented fruit. The flavour of tempoyak was compared to those in spontaneous fermentation (no starter added). The results showed that addition of P. acidilactici UP02 in tempoyak fermentation decreased the quantity of sulphur components, reduced the sugar content, and increased various non-sulphur compounds as well as, non-volatile acidity and contents of organic acids (lactic, malic, and acetic). Sensory evaluation showed that there was no significant difference among the samples for sourness and aroma. However, the P. acidilactici UP02-inoculated sample was more acceptable than the sample with no-starter treatment. On the basis of the results of GC-MS, the flavour components of tempoyak with P. acidilactici UP02 treatment were observed to have 48 compounds, mainly composed of dimethyl disulfide and diethyl disulfide followed by ethane,1-1-bis ethylthio. Keywords: Pediococcus acidilactici , tempoyak, malic acid, sulphur components Introduction final pH of 3.8 to 4.6 whereas, the acidity content of Tempoyak ( Durio zibethinus Murr.) is a fermented tempoyak from Indonesia is 2.55 and 2.56% (as lactic durian, which is a popular side dish and condiment in acid) 6-8. This high acidity of tempoyak is attributed to Malaysia and Indonesia, mainly in Sumatra and lactic acid bacteria (LAB) 4,9,10 . Several researchers Kalimantan islands 1,2 . This product is creamy, yellow- have isolated many species of LAB from tempoyak. white in colour, and has a distinctive aroma. It is Lactobacillus plantarum, L. brevis, L. mali and L. prepared by placing durian pulp in jars to which salt fermentum have been found in Malaysian (2-15%) is added and carefully mixed. The jars are tempoyak 10,11 , whereas L. plantarum, L. casei and L. then tightly closed and kept for 7 d to let spontaneous corynebacterium have been found in tempoyak from fermentation to take place 3,4 . During fermentation, the Indonesia 12 . A new species, Lactobacillus durianis , texture of the durian pulp changes from solid to a isolated from Malaysian tempoyak, has also been semisolid mass accompanied by a strong flavour; the reported 13 . Other LAB found in tempoyak are flavour is the result of a unique combination of Leuconostoc mesenteroides 12 , Pediococcus various sugars, organic acids as well as various acidilactici and Weissella mesenteroides 4. Historically volatile organic compounds. The volatile organic the species of Lactobacillus, Leuconostoc, compounds in tempoyak consist of sulphur-containing Pediococcus, and Streptococcus are the main species compounds; 3,5-dimethyl-1,2,4 trithiolane being the of LAB. highest, and non-sulphur compounds with butanoic There are numerous examples of controlled acid, 2-methyl ethyl ester being the highest 4. Three fermentation using LAB as a starter for the processing organic acids include acetic, lactic and malic; malic of foods, such as, green olive, fermented soymilk, and being the most abundant 5. These organic acids not minced mackerel production 14-16. The use of a starter only contribute to taste compounds, but also increase culture provides consistency and reliability of the acidity of tempoyak. The acidity of tempoyak performance. Reports on the LAB as starters for produced in Malaysia is 3.6% (as acetic acid) with a tempoyak are limited. Fermentation, using starters, may affect the flavour of tempoyak. LAB can vary ________________ *Fax: 62-0721-781498 metabolite reactions that produce various 17-19 E-mail: [email protected] compounds . These compounds are important as YULIANA & GARCIA: INFLUENCE OF P. ACIDILACTICI ON FLAVOUR OF TEMPOYAK 305 they impart characteristic taste and aroma to the final Evaluation of Individual Organic Acids product. The objective of this research was to evaluate A 20 µL of tempoyak sample was injected after the flavour in tempoyak using P. acidilactici UP02 as filtering through a 0.45 µm membrane filter in a a starter, which has homofermentative nature and its Seppack C-18 cartridge (Waters Associate, USA). origin from tempoyak has been reported in a previous HPLC conditions were as follows: Merck Polysphere study. OA HY column (6 mm × 300 mm); mobile phase 0.01 N H 2SO 4; flow rate 0.8 mL/min. The peak areas of Materials and Methods each organic acid in the samples were calculated from Preparation of Inoculum the integrator system of the HPLC data station. Pediococcus acidilactici UP02 was obtained from Organic acid standards used were lactic, malic, acetic, the Institute of Food Science and Technology, succinic and citric. University of the Philippines, Los Banos, Philippines. This isolate was cultured and sub-cultured Determination of Total Volatile Acid Content anaerobically in the MRS medium (Difco, USA). The To an approximately 10 g homogenized sample cultures were centrifuged for 15 min at 3000 rpm and contained in a distilling flask, 100 mL of distilled resuspended in saline solution (0.85% NaCl) to yield water was added. Distillation was conducted rapidly 10 8CFU/mL for a working starter. until 100 mL of distillate was collected. The distillate was titrated when still hot with standard 0.1 N NaOH, Preparation of Tempoyak using phenolphthalein as an indicator. The results The flesh of the durians was aseptically taken and have been expressed as mg malic acid/g sample (wet divided into two sets of four representative treatments basis). comprising both with and without starter, and the Determination of Total Non-volatile Acid Content number of fermentation days (0,4,8 and 12). Two A 25 mL of sample (15 g diluted to 150 mL) was replications were maintained in this experiment. Salt evaporated to dryness in an evaporating dish on a was added to each treatment at the rate of 3% (w/w), steam bath and dried for 30 min in an oven at 100 oC. based on the durian flesh weight, and each lot (200 g) The residue was dissolved in 25 mL of distilled water was packed in a plastic container. One lot was and titrated with 0.1N NaOH using phenolphthalein inoculated with P. acidilactici UP02 and the other lot as an indicator. The amount of non-volatile acids was was naturally fermented (without adding starter). The calculated in terms of mL malic acid/g sample (wet inoculation rate was 1%, based on the weight of basis). durian. The samples were then kept in a cabinet at room temperature (27±1 oC) for evaluation. Samples Evaluation of Volatile Flavour Compounds from each treatment were withdrawn at 0, 4, 8, and 12 A 75 g of sample was put into an Erlenmeyer flask, d fermentation for the analysis of total volatile acids, sealed with aluminum foil with a protective seal, and non-volatile acids and reducing sugars. Data were fitted with an solid phase microextraction (SPME) gas calculated as mean of two replications. tight syringe in such a way that the syringe would be just above the food sample. The flask was placed in a o Analysis of Tempoyak water bath at 50 C with the syringe still fitted. The Procedures described by AOAC 20 were used for the volatiles released from the sample during 30 min examination of total volatile and non-volatile acids, period at 50 oC were swept onto the SPME. These were whereas reducing sugars were determined by Nelson- then analyzed using an HP 5890A gas chromatograph Samogyi method 21 . Evaluation of flavour compounds connected to an HP 5970 mass spectrometer (Hewlett- was done using the solid phase micro extraction Packard). Gas chromatography-mass spectrometry (SPME) trapping method and the quantification of (GC-MS) was operated at 70 eV in the electron impact individual organic acids by HPLC 22 as well as sensory (EI) mode over the range 35-450 amu; column used analysis was done on the 8 d fermented samples. To was SPB-5; 30 m ×0.25 mm, film thickness = 0.25 µm, assess the development of flavour during tempoyak (Supelco, Sigma-Aldrich Co.). The carrier gas was fermentation, sensory evaluation on the aroma and helium at a flow rate of 1 mL/min. The collected sourness was performed by scoring test, although volatiles were thermally desorbed at 250 oC for 2 min those on the general acceptability were performed by after desorption. The oven was heated rapidly to 60oC the hedonic test. and maintained at this temperature for 2 min before the 306 INDIAN J BIOTECHNOL, JULY 2009 temperature was increased at 10 oC min -1 to 220 oC (10 as homofermentative 24 . However, the result of this min). The constituents of samples were identified by study showed that malic acid was higher than lactic matching their mass spectra with those recorded in the acid. The homofermentative LAB produced mainly computer library (NIST98 and Wiley library). lactic acid from sugar metabolism, but significant changes in product formation could occur with sugar Evaluation of Reducing Sugars Reducing sugars were determined by the Nelson- limitation and aeration. Co-metabolism of sugars and organic acids was common and often led to the Samogyi method. To 1 mL of the sample, 1 mL of a 18 freshly prepared mixture of 25 parts copper reagent A production of a different product . to 1 part copper reagent B was added.