JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 76, No. 1, 29-32. 1993 The Effect of pH and Calcium Ions on the Destabilization of Melanoidin VERONICA P. MIGO, 1 MASATOSHI MATSUMURA, 3. ERNESTO J. DEL ROSARIO, 2 A~rD HIROSHI KATAOKA 3 National Institutes of Biotechnology and Applied Microbiology, l Institute of Chemistry? University of the Philippines at Los Banos, College, Laguna, Philippines, and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan3 Received 16 February 1993/Accepted 16 April 1993 Synthetic melanoidin, prepared from a glucose-glycine system showed similar characteristics, UV spectra and molecular weight pattern with that of molasses-derived melanoidin. It is non-volatile, highly soluble in water, with an absorption maximum at 297 nm and a molecular weight of about 19 kDa. pH greatly affects the behavior and destabilization of melanoidin. It is precipitated at around pH 2.5 and showed an increase in mo- lecular size at higher pH by gel chromatography. Calcium had a destabilizing effect and flocculated melanoidin at pH > 12. The results of decolorization of dialyzed molasses slops with ferric salt and calcium oxide as dual coagulants were also presented. The brown pigments collectively known as "mela- noidins," which are produced by the nonenzymic brown- ing reaction between aldoses and amines (1), are present not only in foods but also in cane molasses wastes as well. Studies on the chemical and physicochemicai pro- perties of melanoidin have been conducted largely out of environmental considerations, specifically from the point of view of visible pollution. The empirical formula of the pigment isolated by dialysis (2) from cane molasses was Ct7_IsH26-27OIoN (3) which on catalytic hydrogena- tion yielded four amino acids, sorbitol and mannitol (4). These brown polymeric pigments can also be prepared from model systems. The melanoidins prepared in the present investigation were obtained from an equimolar reaction between glucose and glycine which was previously reported to have a repeating unit that closely approximates C6H602N(CH2)(COOH)0.5 (5). The chemical changes and the ambivalent nature of melanoidin as affected by heat, 02, pH, temperature and addition of transition metals have been described (6). Melanoidins resemble humic substances in their chemi- cal properties. These substances are acidic, polymeric and highly dispersed colloids which are negatively charged due to the dissociation of carboxylic and phenolic groups. The sign and magnitude of the primary negative charge on these colloidal particles are affected by the pH and the ionic environment (7), and in effect influence the effective- ness of the coagulation process. Effective coagulation occurs within a certain pH range. Metal coagulants like aluminum and iron salts are particularly effective in decolorizing molasses wastewater at low pH values (Soneda, N., unpub- lished thesis, University of Tsukuba, 1992). In some cases, however, added alkalinity is important to neutralize the acidity of the metal coagulant and maintain a neutral pH. In their previous study, the authors showed that ferric salts and calcium (as CaO) are effective in destabilizing molasses-derived melanoidin at high pH values (8). In the treatment of actual wastewater, however, complex equilibrium reactions are involved, so much so that it is rather difficult to determine the extent of effective coagula- tion and to explain the mechanism of the coagulation * Corresponding author. 29 process. In this regard, our present work deals mainly with simple model systems, namely, synthetic melanoidin and dialyzed molasses slops. In the previous paper, it was also inferred that calcium may play two possible roles: by acting as a coagulant in itself, and by reacting with fluoride which was found to be inhibitory to the decolorization of molas- ses wastewater at concentrations greater than 100 ppm. The present study aims to evaluate the role of calcium ions as an aid in the decolorization process and to inves- tigate the effect of pH on the behavior and destahilization of rnelanoidin particles. MATERIALS AND METHODS Preparation of synthetic melanoidin Synthetic melanoidin was prepared by dissolving 1 M glucose, 1 M glycine and 0.5 M Na2CO3 in 1 l of deionized water and autoclaving for 3 h at 121°C. Dialysis Fresh distillery slops from Godo Shusei, Co., Ltd. were dialyzed against deionized water for 3 d using a hollow fiber module (Kuraray model KL-2-30B, molecular weight cut-off of 10,000). A dialyzed sample was checked for the presence of ions and the analysis showed negative results. The absorbance and pH of fresh slops before and after dialysis were 33.55, 5.02 and 19.79, 5.31, respectively. Molecular weight determination 0.50 ml of a sample was eluted on a Biogel A-5 m (01.0 × 86.7 cm, BIORAD) gel filtration column with Tris buffer (50 mM Tris, 145 mM NaC1, 4raM EDTA, pH7.6) at a flow rate of 3.14 ml/h. The absorbance of eluted fractions was measured at 475 nm. Absorption spectra Absorption spectra were deter- mined from 200-800 nm using a Hitachi UV-VIS recording spectrophotometer, model U-2000. Effect of pH The pH of the sample was adjusted using HCI or NaOH and was measured using a TOA pH meter model HM-305. Effect of calcium on decolorization Five ml of a sam- ple was pipetted into a small vial, after which the desired amount of calcium (as CaO) was added. Flash mixing was done for 5 min followed by mild mixing for 10 min. The mixture was then centrifuged (3,400 rpm, 10 min) at 30°C