International Food Research Journal 25(1): 83 - 91 (February 2018) Journal homepage: http://www.ifrj.upm.edu.my Evaluation of antioxidant and nutritional properties of sago (Metroxylon sagu Rottb.) and its utilization for direct lactic acid production using immobilized Enterococcus faecium DMF78 1*Duque, S.M.M., 2Castro, I.J.L. and 3Flores, D.M. 1Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna, Philippines 2Environment and Biotechnology Division, Industrial Technology Development Institute - Department of Science and Technology, Bicutan, Taguig City, Philippines 3Department of Food Science and Chemistry, College of Science and Mathematics, University of the Philippines Mindanao, Mintal, Davao City, Philippines Article history Abstract Received: 1 November 2016 Sago flour obtained using the Argao process (indigenous Philippine flour extraction process) Received in revised form: was prepared into different mesh sizes (60, 100 and 200 mesh). The sieved flour was divided 25 November 2016 into fine (F) and course (C) fractions and were tested for percent process recovery, proximate Accepted:26 November 2016 analysis, total starch, total phenolic content, and antioxidant activity. Percent process recovery ranged from 77–89%. Crude fat content was less than 1% (w/w) for all samples while crude fiber content were significantly higher in the coarse fractions. Starch content(db) of the fine fractions were 88.31±1.78% (F60), 92.87±1.49% (F100), and 96.62±1.03% (F200) while Keywords coarse fractions had a total starch content(db) of 74.65±1.20% (C60), 68.66±0.96% (C100), and 64.67±1.14% (C200). Total polyphenol content of fine flour fractions (2.83–6.17mg Sago flour, Argao process, GAE/g sample) was significantly lower than the course fractions (31.50–42.76mg GAE/g Immobilization, sample). Moreover, sago was used as substrate for lactic acid production. Agar, alginate and Lactic acid production κ-carrageenan were tested as immobilization matrices for E. faecium DMF78. Alginate (3% w/v) extruded drop wise on 0.2M CaCl2, and cured for 1hr was found to be the most efficient immobilizing matrix. The use of immobilized and free cells led to the production of 3.011g/L and 4.80g/L lactic acid after 24hrs, representing lactic acid productivity of 0.125 g.L-1h-1 and 0.200 g.L-1h-1 and lactic acid yield (conversion) of 25.42% and 39.76%, respectively. © All Rights Reserved Introduction color acceptability, the dominant off-white sago flour obtained by a natural process should not be judged Sago flour is a cheap and abundant plant resource as undesirable for it contains considerable amount of in Southeast Asia that is derived from the pith of polyphenols (i.e. catechins) which are considered as Metroxylon sagu, growing extensively in the peat source of antioxidant in food (Ding, 2006). However, swamps of Malaysia, Indonesia, Papua New Guinea, there are no available literatures showing the yield and can also be found wild in marshy areas in the and physicochemical properties of sago flour obtained southern part of the Philippines. Starch accumulates using an indigenous process. in the trunk of the sago palm with a confirmed 25- Aside from being a staple food, sago flour ton per hectare per year of starch productivity specifically its starch content has a wide variety of (Ishizaki, 1997). So far, it has the highest record of uses. To take full advantage of this product, there productivity among starchy crops in the world. It has is a rising global interest in producing high value been consumed for thousands of years in Southeast products from sago starch, such as high fructose Asia and the Pacific and has been an important source syrup (Limbaga, 2007), dextrins (Wong et al., 2007), of dietary carbohydrate in Papua New Guinea. ethanol (Lee et al., 1987; Bandaru et al., 2006) and The recent awareness of consumers toward healthy lactic acid (Toleco et al. 2016). Lactic acid is a food has spurred the search of non-conventional valuable product that can be produced chemically flours in relation to dietary fiber content and potent (Demirci and Pometto, 1995) or by fermentation of antioxidants. The quest for sources like sago, banana, carbohydrates (Vick Roy et al., 1983). In lactic acid and squash flours had generated attention. In terms of fermentation, the most common substrate used is *Corresponding author. Email:[email protected] 84 Duque, S.M.M., Castro, I.J.L. and Flores, D.M. /IFRJ 25(1) : 83 - 91 glucose, which is expensive. The production of lactic Carbohydrates (Cbd) = Total Starch + Non- acid is therefore costly due to its process and high starch polysaccharides cost of glucose. In an effort to lower down the cost Dietary Fibre = Cbd + Cfbr of production, studies are being conducted to find cheaper substrates and more cost-efficient methods where MC, CF, Cfbr, CA, CP and Cbd refer to of production. This study aims to characterize sago moisture content, crude fat, crude fiber, crude ash, flour and utilize sago starch for the direct lactic acid crude protein, and carbohydrates (by difference) production using immobilized E. faecium DMF78. respectively. Materials and Methods Total starch content Total starch determination was done using Chemicals and materials anthrone method as described by McCready et al. Alginic acid and carrageenan (commercial (1950). grade, type 1, predominantly κ-carrageenan) were purchased from Sigma Co-Aldrich.Co., St. Loius Total phenolic content MO, agar powder from Nacalai Tesque Inc., Japan Total phenolic content was measured according and ρ-phenylphenol from Wako Pure Chemical to Singleton and Rossi (1965) with modifications. Industries, Ltd. Analytical and HPLC grade reagents Extraction of phenolic compounds was done using were used. 1g and 3g course and fine sago flour samples in 50mL 80% aqueous methanol placed in an ultrasonic Sago flour processing bath (Elma Ultrasonic LC30 H; Melrose, Park, Sago was obtained from Argao, Cebu, USA) for 20 mins. Two mL aliquot of the extracts Philippines. The sago log was debarked and the pith was centrifuged for 5mins at 20,000 x g using CT was made into thin strips and sun-dried for 3–4 days. 15RT refrigerated centrifuge (Techcomp, Ltd., The process is a local custom and shall henceforth Shanghai, China). One mL of the centrifuged be called the “Argao process”. The dried strips were aliquot was added into 100mL volumetric flask, ground using a Waring™ Blender. The ground sago containing 9mL deionized water. One mL of Folin- flour was first sieved through 20 mesh screen and Ciocalteau’s phenol reagent was added to the mixture was successively passed thru 60, 100 and 200 mesh and vortexed. After 5mins, the sample was added screens to produce the desired flour samples. Two with 10mL 7% Na CO solution, diluted to volume fractions were made in each mesh size: fine (F) and 2 3 using deionized water, and incubated for 90 mins at coarse (C) fractions. By definition, the fine fractions room temperature. The absorbance against prepared were those that passed through the sieve while the reagent blank was determined at 750nm using UV- course fractions were retained above the sieve. Vis Spectrophotometer Shimadzu (1601) (Kyoto, Japan) with gallic acid as standard. Total phenolic Proximate composition analysis content of sago flour was expressed as mg gallic acid The different sago flour fractions were subjected equivalents (GAE)/g sample. to the following analyses: total moisture content (air- oven method, AOAC 925.10), crude ash/minerals DPPH radical scavenging activity (AOAC 923.03), crude fat (AOAC 920.39), and crude Free radical scavenging activity was determined fiber (AOAC 984.04). The sum of all components will according to Budzianowski and Budzianowska be deducted from 100 to determine the % nitrogen- (2006) with some modifications. free extract (NFE) of the sample. Absolute methanol (100mL) was added to the flour sample (10g for fine fractions and 2.5g for Dietary fiber quantification course fractions as samples). The mixture was shaken Dietary fiber was quantified by combining at 250rpm in a rotary shaker for 30mins, placed in an the values of carbohydrates and crude fiber of ultrasonic bath for 15mins, filtered, and subjected to sago. Carbohydrates is computed as the total non- vacuum rotary-evaporator at 55°C. The dried extract starch polysaccharides (i.e. polyphenols) and total was reconstituted with 25mL absolute methanol that starch. The following formulas were used for the served as the stock solution. computation: Sago flour extract (200µL) sample solutions in Proximate Composition (100%) = MC + CF + methanol was mixed with 2.8mL of 100µM DPPH Cfbr + CA + CP + Cbd [1,1-diphenyl-2-picryl-hydrazyl (α, α-diphenyl-β- picrylhydrazyl)] in absolute methanol solution. The Duque, S.M.M., Castro, I.J.L. and Flores, D.M. /IFRJ 25(1) : 83 - 91 85 absorbance was read after 30 mins at 517nm. The In using к-carrageenan as the immobilizing free radical DPPH scavenging (i.e. reduction) activity matrix, 5mL cell suspension was mixed with 50mL was calculated using the equation: sterile 2.5% к-carrageenan solution. The resulting suspension was poured onto a sterile petri dish and A - Ax Activity (% DPPH Reduction) = x 100% was allowed to solidify for 30mins. The resulting ( A ) gel was cut into cubes (5mm x 5mm x 5mm) using a where A is the absorbance of DPPH solution with sterile scalpel, allowed to harden by soaking in 0.3M absolute methanol (control), Ax is the absorbance potassium chloride (KCl) solution for 1hr at 4°C, of DPPH solution with tested sago flour fraction or washed with sterile distilled water 3x, and stored in Vitamin E (positive control) solution. The antiradical sterile distilled water at 4°C until use.