Melanoidins as major colourant in sugarcane molasses based distillery effluent and its degradation

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 4648–4660

Review

Melanoidins as major colourant in sugarcane molasses baseddistillery effluent and its degradation

Ram Chandra a,*, Ram Naresh Bharagava a, Vibhuti Rai b

a Environmental Microbiology Section, Industrial Toxicology Research Centre, Post Box No. 80, M.G. Marg, Lucknow 226001, U.P., Indiab School of Studies in Life Sciences, Pt. Ravi Shankar Shukla University, Raipur 492010, C.G., India

Received 26 May 2007; received in revised form 19 September 2007; accepted 21 September 2007Available online 7 November 2007

Abstract

Melanoidins are natural condensation products of sugar and amino acids produced by non-enzymatic Maillard amino–carbonyl reac-tion taking place between the amino and carbonyl groups in organic substances. Melanoidins extensively exist in food products, drinksand wastewaters released from distilleries and fermentation industries. Melanoidins are very important from the nutritional, physiolog-ical and environmental aspects and due to their structural complexity, dark colour and offensive odor, these pose serious threat to soiland aquatic ecosystem that release of melanoidins cause increased load of recalcitrant organic material to natural water bodies. This thencauses the problems, like reduction of sunlight penetration, decreased photosynthetic activity and dissolved oxygen concentrationwhereas on land, it causes reduction in soil alkalinity and inhibition of seed germination. Further, due to the possibility of complexationreactions of introduced melanoidins with metal ions, they could influence the biogeochemical cycle of many constituents in naturalwaters. This review presents an overview to dramatic progress to understand the synthesis, chemical structure and degradation pathwayof melanoidins as well as microbial strategies for the degradation and decolourisation of melanoidins.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Melanoidins; Sugar cane molasses; Degradation; Chemical; Microorganism

1. Introduction

Melanoidins are dark brown to black coloured naturalcondensation products of sugars and amino acids producedby non-enzymatic browning reactions called Maillard reac-

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.09.057

Abbreviations: MRPs, maillard reaction products; MnP, manganese depeprotonic nuclear magnetic resonance; CP-MAS, crossed polarized-magneticelectrochemical diode; DAD, diode array detector; GC, gas chromatography;ionization; CE, capillary electrophoresis; MALDI-TOF, matrix assisted laser dspectrometry; NBT, nitroblue tetrazolium; ELISA, enzyme linked immunosorhigh performance liquid chromatography; CEC, cation exchange chromatograLMW, low molecular weight; Dp, degree of polymerization; IR, infrared; PMCOD, chemical oxygen demand; BOD, biological oxygen demand; MSW, mforming unit; MDE, melanoidin decolourizing enzyme; p-CMB, p-chloromercuSDS-PAGE, sodium dodecyl sulphate–polyacrilamide gel electrophoresis; DMsulfonate].

* Corresponding author. Tel.: +91 0522 220107/207/214118/227332; fax: +9E-mail addresses: [email protected], ramchandra_env@indiatimes.

tions (Plavsic et al., 2006). Naturally melanoidins are widelydistributed in food (Painter, 1998), drinks and widely dis-charged in huge amount by various agro-based industriesespecially from cane molasses based distilleries and fermen-tation industries as environmental pollutants (Kumar and

ndent peroxidases; MIP, manganese independent peroxidases; H1 NMR,angle spinning; HPLC, high performance liquid chromatography; ECD,FAB, fast atom bombardment; MS, mass spectrometry; ESI, electro sprayesorption/ionization-time-of-flight; LC–MS, liquid chromatography–massbent assay; FM-AA, furoylmethyl amino acids; RP-HPLC, reverse phase-phy, HA, humic acid; KDa, kilo Dalton; DNA, deoxy ribose nucleic acid;

DE, post-methanated distillery effluent; MWW, melanoidin wastewater;olasses spent wash; MDA, melanoidin degrading ability; CFU, colony

ribenzoic acid; N-BSI, N-bromosuccinylimide; HTL, heat treatment liquor;P, 2,6-dimethoxyphenol; ABTS, 2,20-azino-bis[3-ethyl-6-benzothiazoline

1 0522 228227/471.com (R. Chandra).

mailto:[email protected]

mailto:[email protected]

R. Chandra et al. / Bioresource Technology 99 (2008) 4648–4660 4649

Chandra, 2006; Gagosian and Lee, 1981). The structure ofmelanoidins is still not completely understood but it isassumed that it does not have a definite structure as its ele-mental composition (Ikan et al., 1990) and chemical struc-tures largely depend on the nature and molarconcentration of parent reacting compounds and reactionconditions as pH, temperature, heating time and solvent sys-tem used (Yaylayan and Kaminsky, 1998). Carbon isotoperatios support the stoichiometric ratios for the combinationof sugars with amino acids, which are based on the elementalcomposition data of melanoidins (Ikan et al., 1990).

Melanoidins have commercial, nutritional and toxico-logical significance as these have significant effect on thequality of food, since colour and flavours are importantfood attributes and key factor in consumer’s acceptance(Plavsic et al., 2006; Borrelli et al., 2003). Food and drinksas bakery products, coffee and beer having brown colouredmelanoidins exhibited antioxidant, antiallergenic, antimi-crobial and cytotoxic properties as in vitro studies haverevealed that Maillard reaction products (MRPs) may offersubstantial health promoting effects as they can act asreducing agents, metal chelators and radical scavengers.Besides, these health-promoting properties, in vitro studieshave also revealed some harmful effects of melanoidins asmutagenic, carcinogenic and cytotoxic effects (Silvanet al., 2006; Borrelli et al., 2003).

The wastewaters released from distilleries and fermenta-tion industries are the major source of soil and aquatic pol-lution due to presence of water-soluble recalcitrantcolouring compounds called melanoidins (Evershed et al.,1997), which are highly resistant to microbial attack, andconventional biological processes such as activated sludgetreatment process are insufficient to treat these melanoidinscontaining wastewater released from distilleries and fer-mentation industries. Hence, these wastewaters require pre-treatment before its safe disposal into the environment(Mohana et al., 2007; Kumar and Chandra, 2006). Degra-dation and decolourisation of these wastewaters by chemi-cal methods (Chandra and Singh, 1999), flocculationtreatment and physicochemical treatment such as ozona-tion (Kim et al., 1985) and activated carbon adsorptionhave been accomplished, but these methods are not eco-nomically feasible on large scale due to cost limitationwhereas biological decolourisation by using fungi such asCoriolus, Aspergillus, Phanerochaete and certain bacterialsp. as Bacillus, Alkaligenes and Lactobacillus (Kumar andChandra, 2006; Kumar et al., 1997; Ohmomo et al.,1987, 1985b; Aoshima et al., 1985) have been successfullyachieved and thus can be applied as a bioremediationtechniques.

However, the biological decolourisation of wastewaterscontaining melanoidins depends on pH, temperature, con-centration of nutrients, oxygen and inoculum size while theenzymatic system responsible for the degradation of mela-noidins consists mainly sugar oxidases and peroxidases assarbos oxidase, glucose oxidase, manganese dependentand independent peroxidases (MnP and MIP) (Watanabe

et al., 1982; Ohmomo et al., 1985a; Aoshima et al., 1985).Since, MnP and MIP showed melanoidin decolourizingactivity in presence of H2O2 and the decolourizing activityof both sugar oxidases and peroxidases were found opti-mum at a particular pH, temperature and substrate specific(Boer et al., 2006). Hence, this review contributes and sum-marizes the structure, chemistry, properties, and enzymesdegrading molasses melanoidins. The enzymatic degrada-tion of melanoidins by fungal and bacterial system at var-ious environmental conditions has been duly emphasized.

2. Chemistry of melanoidins

2.1. Melanoidin formation pathway

In general melanoidins are natural condensation prod-ucts of sugar and amino acids produced by Maillard reac-tions, which is one of the major reactions taking placeduring the thermal processing, household cooking, andstorage of foods (Painter, 1998). The mechanism proposedby Hodge (1953) for the synthesis of melanoidins fromamino–carbonyl reaction between sugar and amino acids/protein has been accepted as the most appropriate pathwayof melanoidins synthesis. Later, Hayashi and Namiki(1986) proposed a new pathway for browning reactionsinvolving the cleavage of sugar moiety of Schiff base at ini-tial stages of amino–carbonyl reaction before Amadorirearrangement followed by formation of C2 (glyoxal dial-kylamine) product(s) and free radicals.

One year later, Hayase et al. (1982)) have reported theformation of a C3 sugar fragment in early stages of brown-ing reaction between sugar and amines or amino acids,which was identified as methylglyoxal dialkylamine. Fayand Brevard (2004) had studied the initial steps of Maillardreaction and reported that the first stable intermediatecompound produced in the initial stages of Maillard reac-tion are called as Amadori compounds that are N-substi-tuted 1-amino-1-deoxyketoses representing an importantclass of Maillard intermediates, which are produced duringthe initial phases of Maillard reaction by Amadori rear-rangement of corresponding N-glycosyl amines.

This type of rearrangement was named after M. Amadoriwho was the first to demonstrate the condensation of D-glu-cose with an aromatic amine, which would yield two struc-turally different isomers, one N-substituted glycosyl-amine,which is more labile than the other towards hydrolysis.Hence, these intermediates of Maillard reaction are termedas Amadori compounds. It has been suggested that marinehumic and fulvic acids are formed by the condensation ofsugars with amino acids or proteins via Maillard reaction.Further, the results indicate that various heterocyclic moi-eties are the main building blocks of humic substancesrather than aromatic benzenoid structures (Ikan et al.,1992). Hayashi and Namiki (1986) have also observed thatthe course of C3 imine formation followed the pattern of C2

imine formation, and well correlated to decrease in theamount of glucosylamine and an increase in the formation

4650 R. Chandra et al. / Bioresource Technology 99 (2008) 4648–4660

of Amadori products. Reaction of Amadori products withn-butylamine rapidly produced C3 compound in a mannersimilar to that of glucose-n-butylamine system. Theseresults indicated the possibility of participation of Amadoriproducts in the formation of C3 compound. In spite oflarge research work done on the Maillard reaction, manyparts as mechanism of melanoidins formation at later finalstages of Maillard reaction are still obscure. However, theproposed mechanisms reviewed above present a clear pic-ture of melanoidins formation through Maillard amino–carbonyl reaction. Still, much is required to bring out theactual mechanisms involved in melanoidins formation.

2.2. Structure of melanoidin polymer

Melanoidins are high molecular weight amino–carbonylcompounds produced by non-enzymatic browning reac-tions called as Maillard reactions during the food process-ing and preservation. In general, the separation ofmelanoidins from food and other biological samples is verydifficult and therefore, most of the chemical and biologicalstudies about melanoidins have done on model melanoi-dins. Although the chemical structure of melanoidins isnot understood clearly, but some part of the chemicalstructure of model melanoidins have recently been eluci-dated by different spectral studies such as 1H NMR, CP-MAS NMR, etc. by various workers (Ikan et al., 1990,1992; Larter and Douglas, 1980). The chemical investiga-tions have revealed that natural and synthetic melanoidinsboth have similar elemental (CHON) compositions, spec-troscopic properties and electrophoretic mobilities at vari-ous pH values (Migo et al., 1997; Ikan et al., 1990, 1992).However, the nitrogen contents, acidities and electropho-retic behavior of the polymers all reflect functional groupdistributions inherited from the amino acids (Hedges,1978). Benzing et al. (1983) have studied xylose-glycine(N15) melanoidin by 15N cross polarized-magnetic anglespinning (CP-MAS) NMR and reported that the nitrogenin melanoidin polymers exists mainly in secondary amideform and some as pyrole and/or indole nitrogen and theyalso revealed that sterically hindered secondary amidebonds are very resistant to acid hydrolysis. According toHayase et al. (1984) the melanoidins structure seems tohave CH3–COR moiety and C-terminal structures origi-nated from glucose existing in melanoidins are suggestedas follows:

1. CH3–CO–R,2. CH3–C (H or OH)@C (H or OH)–CO–R00

3. R–CO–CO–R 0

4. R–CO–CH(CH3)–CO–R 0

5. R–CO–CH2–CO–R 0

6. R–CO–CH2–CH2–CO–R 0

7. CH3–CH(OH)–CO–R, etc.

However, Cammerer et al. (2002) have proposed thebasic structure of melanoidins formed from 3-deoxy-

hexosuloses and Amadori reaction products. Melanoidinshave net negative charge and therefore, different heavymetal ions (Cu2+, Cr3+, Fe3+, Zn2+

, Pb2+ etc.) form largecomplex molecules with melanoidins, amino acids, proteinsand sugars in acidic medium and get precipitated (Migoet al., 1997). According to Hayase et al. (1986) the satu-rated and aliphatic carbon atoms are supposed to comprisethe principal skeleton or backbone of melanoidins. More-over, the ozone treatment is supposed to lead the cleavageof C@C or C@N bonds and these unsaturated bonds havebeen suggested to be important for the structure of mela-noidins chromophore. Further, it was speculated that thenitrogen in melanoidins was mainly due to the conjugatedenamine linkage and partly to amine linkage.

In spite of these studies, the melanoidins chromophorehas not been yet identified. Hence, the chemical structureof the so-called melanoidin is still not clear but probablyit does not have a definite one and there exists various typesof melanoidins differing in structure depending on parentreactants and reaction conditions as pH, temperature andreaction time. Moreover, it further needs intensive investi-gations with more refined recent and advanced techniquesfor the elucidation of chromophore structure to deducethe main skeleton of melanoidin polymer.

3. Analysis of Maillard reaction products

3.1. Direct analysis of Maillard reaction products (MRPs)

Maillard reaction products (MRPs) could be analyzedby column chromatography employing an automaticamino analyzer and a post-column reaction with ninhy-drine (Silvan et al., 2006; Ames, 1998). Alternatively, thesecompounds may be also analyzed by high-performanceliquid chromatography (HPLC) with differential refrac-tometry detection (Shen et al., 2007; Ames et al., 1998).However, the sensitivity of this method does not allowthe detection of low concentration of MRPs and toimprove the sensitivity, HPLC methods involving derivati-sation have also been proposed. A recent method usinghigh-performance anion-exchange chromatography cou-pled with an electrochemical (ECD) and/or diode arraydetector (DAD) is regarded as a powerful technique forthe detection and monitoring of known MRPs. Althoughgas chromatography (GC) shows better separation effi-ciency as compared to HPLC. The MRPs need to be con-verted into volatile compounds prior to analysis and thenecessity to derivatize and the ability of GC to separatetautomeric forms of MRPs are the major drawbacks of thismethod. The detection of MRPs can also be achieved byfast atom bombardment (FAB) tandem mass spectrometry(MS) and the particular advantage of this method is itssimplicity as the samples are directly introduced into theion source. However, the simultaneous analysis of severalMRPs is difficult with this method and it has been almostcompletely replaced by electro spray ionization (ESI) since,this technique offers the advantage of a very soft ionization

Table 1Analytical techniques used for the analysis of Maillard reaction products(MRPs)

Maillard reaction products (MRPs) Analytical techniques

Amadori compounds (Directanalysis)

Column chromatographyHPLC differential refractometrydetectionHPLC involving derivatisationHPAEC coupled electrochimemicaland/or DADFAB–MSESI coupled HPLC and ECEC coupled MSMALDI-TOFNMRLC–MSNBTELISAImmunoblotting (lactosylatedproteins)

Indirect analysis (2-FM-AA) Ion-pair RP-HPLCCEC UV-detectionHPLC–MS

Unreactive lysine Colorimetric and fluorimetricmethods

Advanced Maillard productsGeneral AGEs

FASTHPLC-DAD

CML RP-HPLCRP-HPLC o-phthalaldehyde pre-column derivatisationGC–MSELISAImmunoblotting

Pyrraline Amino acid analysis with PADRP-HPLC

Cross linking products Lysinedimmers Arginine–lysine

LC–MS with ESILC–MS with ESIIon-exchange chromatographyFAB–MS

Other amino acid derivatesArgypirimidine HPLC-coupled GC–MSOMA ELISAPIO RP-HPLC/LC–ESI–TOF–MS/

NMRPyrazinones HPLC with UV and fluorescence

detectionLysine aminoreductone HPLC-DAD

Final stage MRP’sGeneral melanoidins HPLC, NMR, MS, UV, IR

spectrometryMALDI-TOF mass spectrometry

Pronyl-L-lysine GC–MS chemical ionization

Shen et al. (2007), Silvan et al. (2006), Jones et al. (1998), Ames et al.(1998).


and easy coupling to on-line separation techniques likeHPLC and capillary electrophoresis (CE) (Ames et al.,1998; Ames, 1998; Jones et al., 1998). The last technique(CE) is complementary to HPLC, with the advantage ofa higher resolving power for the first dimension of separa-tion and consumption of solvents and chemicals is alsolow. CE coupled to MS is an alternative and powerful ana-lytical tool allowing a rapid separation and identification ofMRPs. Various analytical techniques such as MALDI-TOF, electrospray and nuclear magnetic resonance(NMR) have been developed for the analysis of MRPs indairy products because of its usefulness as quality controlindicator. A selective liquid chromatography (LC–MS)procedure allowed the detection and quantification ofMRPs in milk samples after complete enzymatic hydrolysisand the main problems related to the direct quantificationof MRPs in different samples is the lack of a pure standardand the synthetic strategies proposed so far are rather time-consuming and do not yield a consistent rate of pureMRPs. A colorimetric method employed in foods for thedirect detection of MRPs is NBT based on the reducingability of fructosamines (glucose joined protein molecules)in alkaline solution, this procedure is fast, cheap and easilyautomated (Silvan et al., 2006).

3.2. Indirect analysis of MRPs

Peptide-bound MRPs (fructosyllysine, lactulosyl-lysineor maltulosyl-lysine) are mainly quantified by indirectmethods either based on the quantification of reactivelysine or involves an acid hydrolysis to give 2-furoylmethylamino acids (2-FM-AA). The later can be measureddirectly by ion-pair RP-HPLC or cation exchange chroma-tography (CEC) both using UV-detection. HPLC–MS is atechnique especially suitable for the identification of 2-FM-AA, which is a common indicator used to monitor theMaillard reaction during the heat treatment and storageof foods. 2-Furoylmethyl-lysine, also called as furosine isa well-known indicator used to express the extent of dam-age in processed foods or stored foods with a long shelf life.Furosine can also be determined by ion-pair RP-HPLC butthe main drawbacks of this technique are the time of anal-ysis and the fact that only part of MRPs (�30%) is con-verted into 2-FM-AA. The MRPs can also be estimatedindirectly by rapid colorimetric or fluorimetric methodsbased on the analysis of unreactive lysine, which can bemeasured through the reaction of a dye reacting with thefree –NH2 groups of lysine (Silvan et al., 2006).

4. Recent techniques available for the analysis of Maillard

reaction products (MRPs)

The qualitative and quantitative determination of theMaillard reaction products (MRPs) in foods or physiolog-ical samples is difficult since they are converted during theacid hydrolysis of proteins, thus making it impossible todetect them with routine amino acid analysis. This is

caused by the large amount of products formed duringthe Maillard reaction and the difficulties encountered intheir purification, identification and quantification of purecompounds. Yet several direct and indirect methods havebeen developed as mentioned in Table 1. (Shen et al.,2007; Silvan et al., 2006; Jones et al., 1998; Ames et al.,


1998), which can be of helpful to identify the reaction stepsinvolved in the formation of biologically active Maillardreaction products (MRPs).

5. Biological properties of Maillard reaction products

(MRPs)

The Maillard reaction products (MRPs) can cause bothdeterioration and enhancement of food quality. In past alot of scientific work was focused on the negative biologicaleffects of Maillard reaction products (MRPs) as theseinhibits the microbial growth by sequestering ammonia,amino acids and peptides, while the brown polymeric endproducts (Melanoidins) inhibit by cross-linking polypep-tide chains and sequestering essential multivalent metal cat-ions (Painter, 1998). The formation of anti-nutritional andtoxic MRPs has been reported frequently and in vitro stud-ies have revealed some harmful effects such as mutagenic,carcinogenic and cytotoxic effects of MRPs. Excessive gly-cation has also resulted the destruction of essential aminoacids, inactivation of enzymes, inhibition of regulatorymolecule binding, cross-linking of glycated extra-cellularmatrix, abnormalities of nucleic acid function, altered mac-romolecular recognition, endocytosis and increased immu-nogenicity whereas the formation of beneficial MRPsduring Maillard reaction having antioxidant, antialler-genic, antimicrobial and cytotoxic properties are amongstothers dominantly detected compounds and many studieshave focused the high antioxidant capacity of MRPs inmodel systems and foods such as beer, coffee and bakeryproducts (Silvan et al., 2006; Plavsic et al., 2006). It appearsthat especially low molecular weight MRPs exhibit antiox-idant effects in organism after they get absorbed in smallintestine. Both consumers and regulatory organizationsdemand high quality, healthy and safe food (ingredients)while food scientists attempt to develop new processes toobtain these. Most of the work focused on the biologicalproperties of MRPs has demonstrated the formation of alarge pool of compounds without knowing accurately,which one is responsible for a particular biological activityand the analysis of known indicators can help to under-stand at what stage of Maillard reaction the health-pro-moting compounds are produced and these indicatorsmay then be used to control the industrial production ofthese health-promoting MRPs, which are regarded aspromising new functional food ingredients (Borrelli et al.,2003; Silvan et al., 2006).

6. Behavior and reactions of melanoidins in environment

Melanoidins are found in natural waters, river waters,estuarine, coastal and open waters represent a key link inthe transformation of labile organic matter (polysaccha-rides, amino acids) into more recalcitrant humic materialin nature/environment and their similarities with HA makethem important as possible buffer compounds for metallicions (Ikan et al., 1992). The extent of complexation

depends on the amino acids used to make the melanoidinand the highest complexing capacity is obtained for mela-noidin prepared from glucose and the basic amino acidlysine (Painter, 1998).

However, the presence of calcium and magnesium ionsand other macro and micro constituents further influencesthe complexing properties of melanoidins towards metalions. Melanoidins prepared using condensation timeslonger than two days exhibit complexation propertiestowards copper ions that appear to depend on the basicityof the amino acid precursor and the molecular mass of theproduct (Plavsic et al., 2006). The adsorption at lower pHis more pronounced, but is weak at pH 6.0 and it is becauseof the additional binding sites for copper ions available asmore groups are dissociated at higher pH values. Thehigher pH value could cause the ligand configuration tochange and make more available binding sites. The highestcopper ion complexing capacity values were obtained formelanoidins obtained from Gluc-lys with molecular mass>10 kDa as lysine contains two amino groups while glu-tamic acid and valine contains only one (Plavsic et al.,2006). Basic amino acids (i.e., those containing more aminogroups than carboxylic groups) preferentially condensewith sugars to form nitrogen-rich polymers (Hedges,1978), which are good complexing agents for copper ions.

Metal cations can be classified as A or B-type cationsaccording to the number of electrons in the outer shell.Type A, or b (Hard sphere) cations have the inert gas type(d) electron configuration and are difficult to polarize (e.g.,Na+, K+, Ca2+, Mg2+) and they preferentially form com-plexes with fluoride ion and ligands having oxygen as adonor whereas Type B, or b (Soft sphere) cations are ofhigher polarizability (e.g., Hg2+, Cd2+, Au+, Cu+). TypeB cations coordinate preferentially with bases containingI, S, or N as the donor atom. Cu2+ ions forms very stableorganic complexes and are preferentially bound to ligandscontaining N, S or O as donor atoms (Morales et al., 2005;Painter, 1998).

Melanoidins behave as anionic hydrophilic polymers,which can form stable complexes with metal cations andreported that ketone or hydroxyl groups of pyranone orpyridone residues act as donor groups in melanoidins andparticipate in the chelation with metals as melanoidinshave net negative charge and therefore, different heavymetals (Cu2+, Cr3+, Fe3+, Zn2+, Pb2+, etc.) form largecomplex molecules with melanoidins, amino acids, proteinsand sugars in acidic medium and get precipitated (Migoet al., 1997). It is also possible that other metals, e.g., triva-lent metal ions like Fe3+, are bound to the melanoidin. Tri-valent metal ions form stronger complexes with HA thanmonovalent and divalent metal ions (Painter, 1998). Therewere no significant differences between the different mela-noidins from the Maillard model systems, but widely differ-ent behavior was observed in the ability to bind ironamong melanoidins isolated from commercial coffee, sweetwine and beer due to bilinear behavior means the presenceof at least two different types of binding sites (Morales

Table 2Pyrolysis product of nitrogenous polymer

% Area Compound

3.52 Acetic acid, methyl ester25.23 Acetic acid6.04 1-Hydroxy-2-propanone2.77 1-Methyl-1H-pyrrole2.69 2,3-Butanediol1.96 Pyridine0.43 4-Methyl-pyridine3.43 Methyl-pyrazine2.02 2-Furancarboxaldehyde0.96 1H-Pyrrole-2,4-dimethyl2.87 Cyclopent-2-en-l,4-dione0.76 1-(2-Furanyl)-ethanone3.88 2,6-Dimethyl-pyrazine0.88 2,3-Dimethyl-pyrazine1.72 5-Methyl-2-furancarboxaldehyde2.16 Trimethyl-pyrazine0.67 1-(1H-pyrrol-2-yl)-ethanonel.53 3-Hydroxy-2-methy1-4H–pyran-4-one (maltol)1.89 5-Methyl-1H-pyrrole-2-carbonaldehyde2.43 2,3-Dihydro-3,5-dihydroxy-2-methyl-4H-pyran-4-one

Yaylayan and Kaminsky (1998).


et al., 2005). Due to antioxidant and antimicrobial proper-ties, low molecular weight compounds bounded to mela-noidins exerts antioxidant and antimicrobial activitiesusually higher than those of the pure melanoidins to whomthey are linked. In case of antihypertensive activity, it hasbeen found that the main activity is related to the melanoi-din core (Henares and Morales, 2007; Silvan et al., 2006).However, Melanoidins obtained from His–Glu reactionmixture are elucidated as furan ring and nitrogen contain-ing brown compounds having peroxyl radical scavengingactivity, an indicator of highest antioxidative activity deter-mined by conjugated diene formation from peroxidation oflinoleic acid (Yilmaz and Toledo, 2005).

Brands et al. (2000) demonstrated that heated sugar–casein model melanoidins consisting variable sugars exhibitdifferent mutagenic activity. For example, ketose sugars(fructose and tagatose) showed a remarkably higher muta-genicity compared with their aldose isomers (glucose andgalactose) and generated active oxygen species resultingin DNA strand breaking and mutagenesis. Some otherMRPs were also reported to induce chromosome aberra-tions in Chinese hamster ovary cells and gene conversionin yeast. Mutagenicity and DNA strand breaking activityof melanoidins from a glucose–glycine model was demon-strated by Hiramoto et al. (1997) who reported that theLMW fractions act as lipid sink (Larter and Douglas,1980) and induced DNA damage, where the effectincreased with the concentration added. High concentra-tions (1%) seems to be cytotoxic for the cells, but also lowerconcentrations between 0.05% and 0.2% reduced cell prolif-eration and cell viability.

7. Degradation of melanoidins

Melanoidins, the complex bio-polymer of amino–carb-olyl compounds are very recalcitrant in nature and existsextensively not only in foods but also in wastewatersreleased from various agro-based industries as sugarcanemolasses based distillery and fermentation industries andkeeping in view the hazardous nature of melanoidins, itschemical and microbial degradation has been attemptedto reduce its pollution load and also to characterize itschemical structure so that better strategies could be madefor its degradation and decolourisation.

7.1. Chemical degradation

Many workers have studied the chemical degradationand decolourisation of model as well as natural melanoi-dins present in post methanated distillery effluent. Cam-merer et al. (2002) have studied the chemical degradationof model melanoidins formed from oligomeric and poly-meric carbohydrates with amino acids and they reportedthat with increasing the degree of polymerization (dp) ofcarbohydrates used as starting material in Maillard reac-tion, the release of monosaccharides was also increasedfrom about 3% (Glc) to 95% (Dex). However, the investiga-

tions on lactose melanoidins indicated that carbohydratespreferentially were released from the terminal end of sac-charides incorporated in melanoidins because galactose(Gal) is detected as main hydrolytic product from a lactosemelanoidins. Subsequent hydrolytic reactions performedon melanoidins, showed that the amount of intact mono-saccharides released was dependent on reaction conditionsused for melanoidins synthesis. For the release of Glc froma Glc/Gly model melanoidin formed under water-free reac-tion conditions, an explanation could be given by transgly-cosylation reactions taking place during the Maillardreaction. In contrast, in aqueous systems Glc mainlyundergoes retro-aldol reactions to form highly reactiveC2, C3 and C4 dicarbonyl compounds involved in Maillardreaction instead of Glc molecule. Using di- and oligosac-charides as carbonyl components in Maillard reaction, asignificantly higher amount of sugars than in monosaccha-ride model melanoidins can be released by acid hydrolysis.These results confirm that under given reaction conditionsthe glycosidic bonds in di- and oligosaccharides mostlyremain unchanged, which leads to the formation of mela-noidins skeleton with carbohydrate side chains whereasadditional carbohydrate side chains might be formed bytransglycosylation reactions. The glycosyl-cation necessaryfor this reaction can be formed more easily from Mal thanfrom Glc because Glc is a better leaving group than OH-group and with increasing reaction time and water contentthe stability of glycosidic bond in di- and oligosaccharidesis mainly responsible for the structure of melanoidinsformed. However, the pyrolytic degradation of nitrogenousand non-nitrogenous polymers produced in the initial andlater stages of Maillard reaction respectively produced anumber of pyrolysis products (Tables 2 and 3) (Yaylayan

Table 3Pyrolysis product of non-nitrogenous polymer

% Area Compound

B1 B2 Sucrose

2.18 2.36 1.71 Formic acid7.47 5.30 5.33 Acetic acid0.00 1.82 0.11 2-Methylfuran1.60 4.30 0.85 1-Hydroxy-2-propanone7.65 10.21 39.50 2-Furancarboxaldehyde0.48 0.73 0.78 2-Furanmethanol0.36 0.40 0.35 2(3H)-Furanone-5-methyl2.67 1.24 0.0 Cyclopent-2-en-1,4-dione0.60 0.70 0.57 1-(2-Furanyl)-ethanone0.00 1.49 0.45 1,3-Cyclopentanedione6.91 2.35 0.33 5-Methyl-2-furancarboxaldehyde0.00 0.13 0.0 2,2 0 -Bifuran0.00 0.25 0.0 2-Hydroxy-3-methyl 2-cyclo-penten-1-one2.46 1.46 0.0 2-Furancarboxylic acid0.00 0.84 1.43 3-Furancarboxylic acid, methyl ester0.00 0.26 0.0 2H-Pyran-2-one0.02 0.06 0.13 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol)8.64 4.32 2.07 2,3-Dihydro-3,5-dihydroxy-2-methyl-4H-pyran-

4-one1.48 0.86 0.0 3,5-Dihydroxy-2-methyl-4H-pyran-4-one

34.5 35.59 33.57 5-(Hydroxymethyl)-2-furancarboxylic acid0.07 0.00 0.0 [2,2 0-Bifuran]-3-carboxylic acid0.48 0.00 0.30 5-[(5-Methyl-2-furanymethyl)1-2-furan

carboxaldehyde

Yaylayan and Kaminsky (1998).


and Kaminsky, 1998) during the pyrolytic degradation ofnitrogenous and non-nitrogenous polymers.

Later, the chemical decolourisation of model melanoi-dins prepared from glucose-glycine system by hydrogenperoxide treatment studied by Hayase et al. (1984) whoreported about 64% and 97% decolourisation of melanoi-din [6.72% (v/v)] using hydrogen peroxide in neutral (pH7.0) and alkaline (pH 10.0) medium respectively and themean molecular weight of melanoidins after H2O2 treat-ment decreased from 5300 to 3500 and the major compo-nents in ether soluble fraction obtained from melanoidinsby oxidative degradation of alkaline H2O2 were identifiedas 2-methyl-2, 4-pentanediol, N,N-dimethylacetamide, phe-nol, acetic acid, oxalic acid, 2-furancarboxylic acid, furan-dicarboxylic acid and 5-(hydroxymethyl)-2-furancarboxylicacid.

Kim et al. (1985) had also studied the degradation anddecolourisation of non-dialyzable melanoidins preparedfrom glucose–glycine by ozone treatment and they reportedthat melanoidins were decolourised up to 84% and 97%after ozonolysis at �1 �C for 10 min and 90 min, respec-tively and the mean molecular weight of melanoidins afterozonolysis for 40 min, decreased from 7000 to 3000.

7.2. Microbial degradation

Microorganisms (bacteria/fungi/actinomycetes) due totheir inherent capacity to metabolize a variety of complexcompounds have been utilized since long back for biodeg-

radation of complex, toxic and recalcitrant compoundspresent in various industrial wastes for environmentalsafety. Microbial degradation and decolourisation ofindustrial wastes is an environment-friendly and cost com-petitive alternative to chemical decomposition process ofwastes minimization (Mohana et al., 2007; Pant and Adho-leya, 2007). Moreover, the utility of microbes in industrialwastes treatment process is largely depends on the enzy-matic setup, nutrient requirement of microbes as well asnature and chemical structure of recalcitrant compoundsand environmental conditions (Ohmomo et al., 1988b).The degradation of various complex, toxic and recalcitrantcompounds present in industrial wastes is measured interms of decrease in absorbance at a particular wavelengthcorresponding to the recalcitrant compounds, bacterialgrowth, increase in biomass, and reduction in colour inten-sity. However, the wastewaters released from distilleries isknown as spent wash, which is highly acidic in natureand has a variety of recalcitrant colouring compounds asmelanoidins, phenolics and metal sulfides which are mainlyresponsible for the dark colour of distillery effluent andgive a high inhibitory and antimicrobial activity to thiswastewater (Dahiya et al., 2001b), thus slowing down theanaerobic digestion process of distillery spent wash. ThepH of distillery spent wash increases from 4.5 to 8.5 duringthe anaerobic treatment process and finally it is called aspost methanated distillery effluent (PMDE). Many workershave isolated several aerobic and anaerobic bacterialstrains and studied the degradation and decolourisationof PMDE by these isolated bacterial strains in terms ofdecrease in absorbance at 475 nm, bacterial growth at620 nm, increase in biomass, and reduction in colour inten-sity (Mohana et al., 2007; Adikane et al., 2006; Kumar andChandra, 2006; Sirianuntapiboon et al., 2004; Benito et al.,1997). Kumar and Viswanathan (1991) had isolated somebacterial strains from sewage and acclimatized on increas-ing concentrations of distillery waste, which were able toreduce COD by 80% in 4–5 days without any aerationand the major products left after the degradation processwere biomass, carbon dioxide and volatile acids. Pseudo-

monas fluorescence decolourised melanoidin wastewater(MWW) up to 76% under non-sterile conditions and upto 90% in sterile samples (Dahiya et al., 2001a) and this dif-ference in decolourisation might be due to the fact thatmelanoidin stability varies with pH and temperature andat higher temperature during sterilization melanoidinsdecompose to low molecular weight compounds (Ohmomoet al., 1987). Marine cyanobacteria such as Oscillatoria bor-

yna have also been reported to degrade melanoidin due toproduction of H2O2, hydroxyl, per hydroxyl and activeoxygen radicals, resulting in the decolourisation of theeffluent (Kalavathi et al., 2001). Patel et al. (2001) havereported 96%, 81% and 26% decolorization of distilleryeffluent through bioflocculation by Oscillatoria sp., Lyn-gbya sp. and Synechocystis sp., respectively. Ghosh et al.(2004) have also isolated some bacterial strains capableof degrading recalcitrant compounds from anaerobically


digested spent wash from soil of effluent discharge sitewhich were Pseudomonas, Enterobacter, Stenotrophomonas,Aeromonas, Acinetobacter and Klebsiella all of which couldcarry out degradation of PMDE and maximum 44% CODreduction was achieved using these bacterial strains eithersingly or collectively. Sirianuntapiboon et al. (2004) usedan acetogenic bacterium to obtain a decolourisation yieldof 76.4% under optimal nutrient conditions.

In recent years, several basidiomycetes and ascomycetes

type fungi have been used for the decolourisation of natu-ral and synthetic melanoidins in connection with colourreduction of wastewaters from distilleries (Thakkar et al.,2006; Raghukumar et al., 2004; Raghukumar and Rivon-kar, 2001; Miranda et al., 1996) but higher fungi are noteasily adopted for aquatic habitats. Ohmomo et al.(1985a) used Coriolus versicolour Ps4a for MWW decolo-urisation and obtained 80% decolourisation in darknessunder optimum conditions. Later, they used autoclavedmycelium of Aspergillus oryzae Y-2-32 that adsorbed lowerweight fractions of melanoidin and degree of adsorptionwas influenced by the kind of sugars used for cultivation.Colour elimination from MSW using Aspergillus nigerwas studied by Miranda et al. (1996) and reported thatunder optimal nutrient concentration 83% of the total col-our removed was eliminated biologically and 17% byadsorption on the mycelium. Colour removal from distill-ery effluent using a marine fungus, Flavodon flavus has beenreported by Raghukumar and Rivonkar (2001), which wasmore effective in decolourizing raw MSW than was themolasses wastewater collected either after anaerobic treat-ment or after aerobic treatment. The larger molecularweight fractions of melanoidin were decolourised rapidly,while the small molecular weight fractions remained insolution and were metabolized slowly. The decolourisationwas less in sterilized spent wash than in non-sterile solu-tion. The decrease in colour removal efficiency in this studymight be due to the fact that the effluent taken for studywas alkaline (pH 8.5) and the melanoidins responsible forcolour were more soluble in the alkaline pH. In the acidicpH, the melanoidins might be precipitated and removedeasily. It was suggested that decolourisation by fungi takesplace due to the destruction of coloured molecules and par-tially because of sorption phenomena. Ohmomo et al.(1985b) concluded that microbial decolourisation of mela-noidins is due to two decomposition mechanisms; in thefirst the smaller molecular weight melanoidins are attackedand in the second the larger molecular weight melanoidinsare attacked. Under nutrient limiting conditions, fungalcells generally cannot remain active during a long-term cul-tivation. Therefore, the continuous-culture method is notpractical and the semi-batch or repeated-batch methodcan be an alternative for long-term cultivation.

Nevertheless, the feasibility of application of the processto full-scale would need further research in this continuousculture set-up, in order to minimize the added nutrientsand extend the biomass activity for a longer period. Anunderstanding of complete profile of the effluent and the

structures of recalcitrant colouring compounds would alsobe helpful in achieving the appropriate treatment solutions.

8. Effect of environmental factors on melanoidin

decolourisation

Environmental factors like pH, temperature, aerationand nutrients play vital role in microbial degradation pro-cess of industrial wastes as the activity of enzymes is greatlyinfluenced by these environmental factors. Several studieshave been made by various workers to understand the roleof various environmental factors in degradation of mela-noidins to achieve the maximum degradation and decolo-urisation by different microbes (Mohana et al., 2007;Kumar and Chandra, 2006; Sirianuntapiboon et al.,2004). However, Hayase et al. (1984) while studying thedegradation of melanoidins by hydrogen peroxide at differ-ent pH (3.0–13.0) found that melanoidin decolourisation inalkaline pH proceeds more rapidly than in acidic and neu-tral pH and it reached 94% at pH 10. Mohana et al. (2007)have reported the highest decolourisation (67%) at pH 7.0as the solubility of melanoidins depends on pH i.e. it is lesssoluble in acidic pH than in alkaline pH and pH more orless than pH 7.0 led to decrease in decolourisation activityas well as the growth of microbes. They also observed thatincrease in temperature from 20 to 37 �C was accompaniedwith increase in decolourisation from 35% to 67% and fur-ther increase in temperature above 40 �C adversely affectedthe decolourisation ability of microbes as melanoidin dec-olourizing ability of microbial cells was lost on exposureat high temperature (>40 �C) for long time. However, theisolated bacterial consortium exhibited maximum decolo-urisation in static conditions whereas aerated culturesalthough grew well and also brought about higher CODreduction (66%) but showed low decolourisation yield(Mohana et al., 2007).

Ohmomo et al. (1987) have studied the effect of variouscarbon and nitrogen sources on the MDA of Aspergillusfumigatus G-2-6 and reported that glucose was the best car-bon source allowing the maximum degradation of melanoi-dins and further increase in glucose concentration (>1%)resulted only increase in mycelial biomass but no furtherincrease in decolourisation yield. The effect of variousorganic and inorganic nitrogen sources studied on the deg-radation and decolourisation of melanoidins showed thatsupplementation of organic nitrogen source harmfullyaffected the degradation of melanoidins as addition oforganic nitrogen sources such as yeast extract, peptone,beef extract and tryptone brought down the level of decolo-urisation whereas inorganic nitrogen sources such assodium nitrate and ammonium nitrate reduced the decolo-urisation values. It has also been observed that decolouri-sation of melanoidins increases with increase in inoculumsize and maximum decolourisation was achieved at 15%(v/v) inoculum size (approx. 11 · 106 CFU/ml) and furtherincrease in inoculum size did not improve the decolourisa-tion of melanoidins (Mohana et al., 2007).


9. Degradation pathway of melanoidins

Many workers have studied the chemical and microbialdegradation and decolourisation of model as well as natu-ral melanoidins present in sugarcane molasses based distill-ery effluent. But no body has given the clear idea/clueabout the degradation mechanism of melanoidins. It ismainly due to the complexity of Maillard reactions anduncertainty about the definite structure of melanoidinspolymer because the elemental composition and chemicalstructure of melanoidins mainly depend on the natureand molar concentration of parent reactants (Ikan et al.,1990, 1992) and reaction conditions as pH, temperature,heating time and water content during the reaction.

10. Enzymes involved in the degradation of melanoidins

Although the enzymatic system related with the degra-dation of melanoidins is yet to be completely understoodand it seems greatly connected with fungal ligninolyticmechanisms. Several studies regarding the degradation ofmelanoidins, humic acids, and related compounds bymicroorganisms have also suggested the participation ofdifferent categories of enzymes as Watanabe et al. (1982)have reported the enzymatic degradation of melanoidinsby Coriolus sp. No. 20 having an intracellular enzyme,which required active oxygen molecules and sugars in reac-tion mixture. This enzyme was later identified as sorboseoxidase having molecular weight about 200,000 kDa. Thepurified enzyme aerobically decolourised the melanoidinpigment in presence of glucose, sorbose, galactose, xyloseand maltose. But the maximum melanoidin decolourizingactivity of enzyme was observed with sorbose as well asglucose.

Melanoidins were suggested to be decolourised by theactive oxygen (O�2 , H2O2) species produced by the reactionscatalyzed by enzymes probably oxidases because the reac-tions with pure enzyme was accompanied by the oxidationof glucose into gluconic acid. According to them, the straincould probably produce sugar oxidases other than sorboseoxidase because the crude preparation utilized arabinose,fructose and mannitol while sorbose oxidase did not utilizethese compounds and they finally concluded that almost allsugars contained in distillery wastewater might be oxidizedby Coriolus sp. to form a large amount of highly active oxy-gen species.

Aoshima et al. (1985) reported that the melanoidins dec-olourizing activity (MDA) of Coriolus versicolor Ps4a wasmainly due to intracellular enzymes that are induced bymelanoidins. The induced enzyme consisted of two compo-nents, namely a sugar dependant enzyme forming two-third part and other sugar independent part constitutingone-third part of the system. The sugar dependent enzymemight be the same as that of sorbose oxidase as reportedearlier by Watanabe et al. (1982) from Coriolus sp. No.20 or a same type of sugar oxidase and this was the firstreport about the existence of sugar independent enzymes

decolourizing melanoidins. Ohmomo et al. (1985a) purifiedthe melanoidins decolourizing enzyme (MDE) isolatedfrom C. versicolor Ps4a and reported that MDE of thisstrain was an intracellular enzyme consisted a major P-fraction and a minor E-fraction. The P-fraction has at leastfive enzymes, which were of two types and required sugaror no sugar for the decolourizing activity. They tookP-III and P-IV in the P-fraction as typical MDE for furtherstudies of their enzymatic properties. Enzyme P-III hadmolecular weight 48,400–50,000, optimum pH 5.5 andoptimum temperature 30–35 �C, but it was weeklyinhibited by p-chloromercuribenzoic acid (p-CMB),N-bromosuccinylimide (N-BSI), silver cation (Ag+), andO-phenanthroline. The decolourizing activity of P-III towardsvarious melanoidins was almost same as that of PIV.

The enzyme P-IV had molecular weight of 43,800–45,000, optimum pH 4.0–4.5 and optimum temperature30–35 �C. Unlike P-III, P-IV could decolourise the mela-noidins in absence of sugar or glucose and oxygen andwas inhibited weakly by Ag+, p-CMB and N-BSI. P-IV isthe enzyme that attacks the melanoidins directly in com-parison of P-III, which attacks the melanoidin polymerindirectly as in sub reaction of sugar oxidase. Furthermore,a multiplicative effect between P-III and P-IV for decolo-urisation activity was observed. But the catalytic mecha-nism of these two enzymatic reactions remained to beresolved.

Miyata et al. (1998) reported that melanoidin decolouri-sation by Coriolus hirsutus pellet was mainly due to theproduction of extra cellular hydrogen peroxide (H2O2)and peroxidases. The culture fluid contained two majorextra cellular peroxidases, one manganese independent per-oxidase (MIP) and other manganese dependent peroxidase(MnP) since both MIP and MnP showed melanoidin deco-lourizing activity in presence of H2O2. Hence, they con-cluded that melanoidin decolourisation by C. hirsutus

cultures involved the production of extra cellular H2O2

and peroxidases. Later, while investigating the decolourisa-tion of heat treatment liquor (HTL), a melanoidin contain-ing wastewater by the fungus Coriolus hirsutus theyreported that in pretreated HTL (i.e. before treatment withactivated sludge) the fungus produced a large amount ofmanganese independent peroxidase (MIP) and the additionof Mn(II) to pretreated HTL caused a further increase indecolourisation efficiency of fungus and a marked increasein manganese peroxidase (MnP) activity (Miyata et al.,2000). Further, they also reported that the peroxidaseactivity require hydrogen peroxide (H2O2), which was pro-duced during glucose oxidation, thus establishing thenecessity to add extra carbon source.

11. Physicochemical properties of melanoidins decolourizing

enzymes

Boer et al. (2006) have isolated and purified the melanoi-din decolourizing enzyme (MnP) from L. edodes andobserved two peaks for MnP as MnP1 and MnP2, which


was isolated by gel filtration method as shown in Fig. 1(Boer et al., 2006). They revealed that the purified enzymeyielded a single band after denaturing SDS–PAGE asshown in Fig. 2 (Boer et al., 2006). A molecular mass of46 kDa was estimated after SDS–PAGE, and this molecu-lar mass was confirmed by sephadex G-100 gel filtration.The carbohydrate content of MnP2 was estimated to be17.8% by using the phenol–sulfuric acid method withD-mannose as standard. The molecular weight of enzymeis well within the range of the MnP family, which is usuallybetween 37 and 46 kDa.

11.1. Temperature stability and pH optimum

The effect of pH on enzymatic activity and stability wasexamined at pH values ranging from 3.0 to 8.0 using DMPas substrate (Boer et al., 2006). The enzyme was completelystable in a wide pH range (4.0–6.0) and presented an opti-mum pH value of 4.5. The optimum temperature for theoxidation of substrate was 40 �C. The purified enzymewas stable at temperature up to 40 �C for several hoursand presented a half-life of 20 min at 50 �C, but at 4 �C,the enzyme was stable for several days. The optimum pH4.5 for the enzyme was also usual for this family. MnPpurified from L. edodes had relatively high thermostabilitywhen compared with other white-rot fungus MnP. It didnot lose activity at 40 �C for at least 24 h and retainedabout 80% of its original activity at 45 �C for 2 h and highthermo stability is a desirable feature of an enzyme for var-ious industrial applications (Boer et al., 2006).

Fig. 2. Denaturing SDS–PAGE of L. edodes MnP2 Silver staining. Lane1: Standard protein, 1: Bovine serum albumin (66 kDa), 2: Ovaalbumin(45 kDa), 3: Pepsin (34.7 kDa), 4: Trypsinigen (24 kDa), 5: b-lactoglobulin(18.4 kDa). Lane 2: L. edodes MnP2. Boer et al. (2006).

11.2. Effect of some chemicals on MnP activity

The effects of several chemicals on MnP activity studiedwere presented in Table 4 (Boer et al., 2006) and it wasfound that MnP was strongly inhibited by Hg2+, whileFe3+, Ca2+ and Ni2+ did not cause any alteration in theactivity. MnP was also resistant to presence of up to

Fig. 1. Elution profile of MnP activity on sephadex G-100. A280 nm (h),MnP activity (d) Boer et al. (2006).

5 mM H2O2, but it was inhibited in presence of 10.0 mMperoxide. The enzyme showed a high percentage of activityin reaction mixtures containing 10% (v/v) of differentorganic solvents. The susceptibility of MnP to inactivationby H2O2 in absence of substrate was also studied and it wasfound that the enzyme was very stable in presence of up to0.25 mM H2O2 but in presence of higher amounts of H2O2,the MnP activity rapidly decreased Fig. 3 (Boer et al.,2006).

In terms of industrial applications, the stability of MnPin presence of high concentrations of H2O2 is a very impor-tant property as MnP requires H2O2 for its activity butH2O2 could also inactivate MnP quickly when it wasapplied in high concentrations. When compared withreported MnPs from other white-rot fungi, the L. edodes

MnP purified was appeared to be more resistant towardsthe high H2O2 concentrations (Boer et al., 2006).

11.3. Substrate specificity

As study revealed that the enzyme was Mn2+ dependentfor its activity; i.e. the substrates DMP and ABTS were oxi-

Table 4Effect of some chemicals on MnP activity

Reagent (mM) Final concentration (%) Residual activity

None – 100CaCl2 10.0 105NCl2 10.0 97CuSO4 1.0 108CuSO4 10.0 86FeCl3 10.0 95HgCl2 1.0 21H2O2 0.1 97H2O2 0.5 93H2O2 1.0 92H2O2 5.0 75H2O2 10.0 17Acetone 10 92Isopropanol 10 107Ethanol 10 86Acetonitrile 10 71

Boer et al. (2006).

Fig. 3. Effect of H2O2 on the stability of L. edodes MnP. Control (d)H2O2 0.25 mM (s). 0.5 mM (j), 1.0 mM (h), 5.0 mM (m) Boer et al.(2006).


dized at a faster rate in presence of Mn2+ than in absenceof Mn2+. It appeared that MnP oxidized ABTS faster thanDMP. Like MnPs from other white-rot fungi, this MnPwas not able to oxidize veratryl alcohol, a non-phenolic lig-nin model compound. The Michaelis–Menten constants(KM) of MnP for H2O2 and Mn2+ were 20.8 and22.2 · 10�3 mM, respectively, which indicates high affinityof these enzymes for their substrates (Boer et al., 2006).

12. Proposed mechanisms of enzymatic degradation of

melanoidins

The structural complexity of melanoidins and very lessknowledge about the enzymatic system involved in its deg-radation/decolourisation has created a lot of problems toelucidate the mechanism involved in the degradation ofmelanoidins. Yet various workers have studied the enzy-matic degradation of melanoidins and built the concept

about the same as Hayase et al. (1984) suggested that thedegradation of melanoidins by active oxygen species suchas hydrogen peroxide (H2O2), which is secondarily pro-duced by the enzymatic oxidation of glucose into gluconicacid by glucose-oxidase enzyme. They proposed thathydrogen peroxide reacts with hydroxyl anion to give per-hydroxyl anion (HOO�), which has a strong nucleophilicactivity and per hydroxyl anion was considered to attackthe nucleophilically carbonyl groups of melanoidins.

Kim et al. (1985) proposed that the degradation of mel-anoidins and decrease in molecular weight are mainly dueto the cleavage of carbon-carbon double bonds by ozonol-ysis as ozone attacks organic compounds electrophilicallyand is known to exist as a resonance hybrid of four canon-ical forms. Ozone especially as an amphoteric ion makes anelectrophilic attack in an electron-rich p system such as acarbon–carbon double bonds and cause cleavage of bonds.Melanoidins have several carbon–carbon double bondsthat are cleaved by ozonolysis leading to its degradation/decolourisation.

Ohmomo et al. (1988a) proposed that the microbial deg-radation and decolourisation of melanoidins might be dueto two decomposition mechanisms that is an attack mainlyagainst smaller molecular weight components of melanoi-dins and other against the larger molecular weight compo-nents of melanoidins. Further, they proposed that thefacultative anaerobic bacteria use the former while Basidi-omycetes and Actinomycetes use the later, but they couldnot elucidate the detailed mechanism of enzymatic degra-dation and decolourisation of melanoidins and finally, theystated that a detailed mechanism of melanoidins degrada-tion and decolourisation by various enzymes as MIP andMnP is still to be explored.

Acknowledgements

The authors wish to thank Director, Industrial Toxicol-ogy Research Centre, Lucknow, India. The financial assis-tance received from the University Grants Commission,New Delhi as Senior Research Fellowship by Mr. RamNaresh Bharagava is duly acknowledged.

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Melanoidins as major colourant in sugarcane molasses based distillery effluent and its degradation