Bioresource Technology 100 (2009) 6665–6668

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Bioresource Technology

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Short Communication

Characterization of sucrose–glutamic acid Maillard products (SGMPs) degradingbacteria and their metabolites

Ram Chandra a,*, Ram Naresh Bharagava a, Vibhuti Rai b, Shio Kumar Singh c

a Environmental Microbiology Section, Indian Institute of Toxicology Research (CSIR), P.O. Box 80, M.G. Marg, Lucknow 226001, Uttar Pradesh, Indiab School of Studies in Life Sciences, Pt. Ravishankar Shukla University, Raipur 492010, Chhattisgarh, Indiac Pharmacokinetics and Metabolism Division, Central Drug Research Institute (CSIR), Chatter Manzil, Lucknow 226001, Uttar Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 May 2009Received in revised form 13 July 2009Accepted 14 July 2009Available online 8 August 2009

Keywords:Maillard productDegradationAlcaligenes faecalisBacillus cereusMetabolite characterization

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.07.029

* Corresponding author. Tel.: +91 522 2476051/2472228471.

E-mail addresses: rc_microitrc@yahoo.co.in, ramc(R. Chandra).

Two aerobic bacteria RNBS1 and RNBS3 capable to degrade and utilize sucrose–glutamic acid Maillardproducts (SGMPs) as carbon, nitrogen and energy source were isolated and characterized as Alcaligenesfaecalis (DQ659619) and Bacillus cereus (DQ659620) respectively by 16S rRNA gene sequence analysis.In present study, mixed bacterial culture was found more effective compared to axenic culture RNBS1and RNBS3 decolourizing 73.79%, 66.80% and 62.56% SGMPs, respectively. The SGMPs catabolizingenzyme was characterized as manganese dependent peroxidase (MnP) by SDS–PAGE yielding a singleband of 43 KDa. Further, the LC-MS–MS and other spectrophotometric analysis have revealed that mostof the SGMPs detected in control were diminished from bacteria treated samples. The disappearance ofSGMPs from bacteria treated samples could be related with the degradation of SGMPs.

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1. Introduction

Maillard reactions are common, non-enzymatic browning reac-tions taking place between sugars and amino acids during the ther-mal processing of food materials, sugarcane juice in sugarindustries and molasses in distilleries and produced a dark brownto black complex polymer known as melanoidins (Chandra et al.,2008), which is the major source of environmental pollution. Themolecular weight, structure and elemental composition of mela-noidins is strongly influenced by the ratio and type of reactantsas well as reaction conditions such as temperature, reaction time,pH and solvent used (Yaylayan and Kaminsky, 1998).

Large volume of dark colored distillery effluent released into theenvironment causes coloration and reduction of sunlight penetra-tion in aquatic environment as well as reduction in soil alkalinity,inhibition of seed germination and damage of vegetation (Chandraet al., 2008). Hence, it requires adequate pre-treatment prior to itsdischarge into the environment. Bacteria seem to be more effectivefor the bioremediation of environmental pollutants due to theirimmense environmental adaptability and biochemical versatility(Ghosh et al., 2002).

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Some workers have reported the microbial degradation ofmodel as well as molasses melanoidins (Miyata et al., 2000; Ghoshet al., 2002). But detail information regarding the melanoidinsdegrading enzymes and the nature of metabolic products of mela-noidins degradation has not reported so far. Hence, the objectivesof this study were to isolate and characterize the potential aerobicbacteria and enzymes capable to degrade SGMPs and metabolitesproduced during the bacterial degradation of SGMPs.

2. Methods

2.1. Preparation of sucrose–glutamic acid Maillard products

Sucrose–glutamic acid Maillard products (SGMPs) was preparedas reported earlier (Bharagava et al., 2009).

2.2. Isolation, screening and characterization of SGMPs degradingbacteria

The potential aerobic bacteria capable for SGMPs degradationwere isolated from distillery waste contaminated soil collectedfrom M/s Unnao distillery and brewery Ltd., Unnao (UP), India byenrichment technique and screened on the basis of growth andperoxidase activity on GPYM agar plates amended with SGMPssolution, 1.3% agar and 0.1% phenol red (w/v) (BDH Ltd.)(Bharagava et al., 2009). Further, the isolated bacteria were charac-terized following the standard procedures (Barrow and Feltham,

6666 R. Chandra et al. / Bioresource Technology 100 (2009) 6665–6668

1993) and identified by 16S rRNA gene sequence analysis (Bharag-ava et al., 2009).

2.3. Degradation and metabolization of SGMPs by axenic and mixedbacterial culture

The degradation experiments were performed in triplicate in250 ml Erlenmeyer flasks containing 100 ml of sterile SGMPssupplemented GPYM broth. The flasks were inoculated with 1%(v/v) over night grown culture and incubated at 37 �C undershaking flask condition (125 rpm) for six consecutive days. Thedegradation of SGMPs was monitored spectrophotometrically interms of bacterial growth, and decrease in color density (absor-bance) at 620 and 475 nm, respectively (Bharagava et al., 2009).

2.4. Assay and characterization of SGMPs degrading enzymes

Manganese dependent and independent peroxidase (MnP andMIP) activity was determined by the oxidation of phenol red(BDH Ltd.) and ABTS, respectively (Miyata et al., 2000; Bharagavaet al., 2009). The enzyme (MnP) was isolated and purified by coldacetone (�20 �C) and Sephadex G-100, respectively (Boer et al.,2006) and characterized by denaturing SDS–PAGE performed on10% polyacrylamide gel. Manganese peroxidase (Sigma–Aldrich,USA) and protein ladder (Bangalore Genei, India) was used as mar-ker standards to compare and estimate the molecular weight ofenzymes.

2.5. Metabolite characterization

The samples (treated and untreated) were centrifuged and ex-tracted thrice with equal volume of ethyl acetate (Shen et al.,2007). The ethyl acetate extract was vacuum dried and residuewas dissolved in HPLC grade methanol and used for metabolitescharacterization.

0.02

Fig. 1. Neighbor-joining tree showing the phylogenetic position of sucrose–glutamic acigene sequences. The GenBank accession number for each bacterium used in the analysi

The electrospray ionization-mass spectrum (ESI-MS) was re-corded with Micromass Quattro II triple quadruple mass spectrom-eter to know the ionization pattern of compounds. The Infrared (IR)spectra were recorded on a Pye Unicam SP3-200 instrument as thinKBr discs and values were expressed in cm�1 whereas samples forH1 NMR spectroscopy were exchanged thrice with D2O undernitrogen atmosphere, dissolved in 400 ll of D2O and transferredto a 5 mm NMR tube, scanned for 1H NMR at 25 �C with a Broker,advance DRX-200 NMR spectrometer and chemical shifts were re-corded in ppm (d) with TMS at 0.00 as internal standard. The LC-MS/MS analysis were performed using 2690 separation modulewith Quattro Ultima triple quadrupole mass spectrometry detector(Waters, UK) equipped with Atlantis� dC-18 column (2.1 � 50 mm,3 lm; Waters, USA).

3. Results and discussion

3.1. Bacteria isolation and identification

Two morphologically distinct SGMPs degrading bacteria RNBS1and RNBS3 were isolated and identified as Alcaligenes faecalis(DQ659619) and Bacillus cereus (DQ659620), respectively (Fig. 1).Bacterium RNBS1 was characterized as gram (�ve), small rods giv-ing positive tests for motility, catalase and oxidase activitywhereas RNBS3 was gram (+ve), long rods in chain giving positivetests for motility, catalase activity, casein, starch and gelatinhydrolysis.

3.2. Degradation and metabolization of SGMPs by axenic and mixedbacterial culture

In present study, mixed bacterial culture was found more effec-tive compared to axenic culture RNBS1 and RNBS3 decolourizing73.79%, 65.88% and 62.56% SGMPs, respectively (Fig. 2A). The deg-radation of SGMPs involved the production of extracellular H2O2

Alcaligenes faecalis st. cb-4 (FJ588233)

Alcaligenes faecalis st. MG-F5 (EU921230)

Alcaligenes sp. MH146 (FJ626617)

Alcaligenes sp. CC-ESB2 (DQ490983)

Uncultured bacterium (EU534884)

Uncultured Alcaligenes sp. clone HB-5 (EF032608)

Alcaligenes sp. F78 (EU443097)

Alcaligenes sp. IS-67(AY346140)

Alcaligenes sp. IS-J1 (EF599759)

Alcaligenes sp. XW3 (EU545399)

Uncultured bacterium gene (AB290346)

Alcaligenes faecalis st. CC2 (FJ226423)

Alcaligenes faecalis st. SP03 (EF427887)

Uncultured beta proteobacterium clone (DQ366010)

RNBS1 (DQ659619) Bacillus cereus st. BGSC (AY224382)

Bacillus cereus strain BGSC (AY224384)

Bacillus cereus st. BGSC (AY224385)

Bacillus cereus st. BGSC (AY224388)

Bacillus cereus st. CCM 2010 (DQ207729)

Bacillus cereus st. 2000031503 (AY138277)

Bacillus cereus st.2000031491 (AY138276)

Bacillus cereus st. G3317 (AY138278)

Bacillus cereus st. G9241 (AY425946)

Bacillus thuringiensis st. GS1 (FJ462697)

RNBS3 (DQ659620) Bacillus anthracis st. (AY138383)

d Maillard products degrading bacteria and their related species based on 16S rRNAs is shown in parenthesis after the species name.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 24 48 72 96 120 144Incubation time (hrs)

Abso

rban

ceS-1

S-3

S-1+3

CONTROL

S-1

S-3

S-1+3

0

0.5

1

1.5

2

2.5

3

3.5

0 24 48 72 96 120 144Incubation Time (hrs)

Enzy

me

Activ

ity (U

/ml)

MnP S-1

MnP S-3

MnP S-1+3

MIP S-1

MIP S-3

MIP S-1+3

KDa L M 1 3

68

44 43

C

B

A

Fig. 2. (A) Bacterial growth and degradation of sucrose–glutamic acid Maillardproducts (SGMPs) by axenic and mixed bacterial culture; (B) MnP and MIPproduction by bacteria during the degradation of sucrose–glutamic acid Maillardproducts. MnP: manganese dependent peroxidase; MIP: manganese independentperoxidase; (C) SDS–PAGE of bacterial MnP. Lane L: protein ladder (16–97.4 KDa);Lane M: marker protein (Horseradish peroxidase, 43 KDa); Lane1: BacteriumRNBS1; Lane 3: Bacterium RNBS3.

R. Chandra et al. / Bioresource Technology 100 (2009) 6665–6668 6667

and peroxidases, which catalyzes H2O2 dependent oxidation of Mn(II) to Mn (III) and Mn (III) catalyzes one-electron oxidation of phe-nolic and non-phenolic recalcitrant compounds by H2O2, promot-ing free radicals generation leading the degradation of SGMPs(Miyata et al., 2000). Bacteria RNBS1 and RNBS3 has shown opti-mum level of enzyme production (approx. 2 U/ml) at 96 h of incu-bation period (Fig. 2B) and denaturing SDS–PAGE of purified MnPenzyme has yielded a single band of 43 KDa (Fig. 2C), which waswithin the MnP family ranging between 37–46 KDa (Boer et al.,2006).

3.3. Characteristics of SGMPs and their metabolites

The electrospray ionization-mass spectra (ESI-MS) of controlSGMPs showed two major molecular ion peaks with molecularmass 382 (MH+) and 365. The fragmentation of molecular ion (m/e) 382 resulted four product ion peaks (m/e) 330, 314, 219 and203 with molecular mass 331, 314, 220 and 204 respectively.The ESI-MS of treated sample has shown only three major peaks(m/e) 197, 179 and 165 with molecular mass 198, 178 and 165,respectively. The fragmentation of these peaks produced severalion peaks (m/e) 113, 100, 86, 69 and 60 with molecular mass112, 100, 86, 69 and 60 respectively. Moreover, infrared (IR) spec-troscopy of control and degraded samples has shown stretchingfrequencies (t) at 3426, 2940, 1721, 1658, 1641, 1566 and1408 cm�1 for the presence of an alcoholic (–OH), –C–H stretching,ketonic (@C@O), aldehydic (–CHO), carboxylic (–COOH), carbon–carbon double bond (–C@C–) and an asymmetric –NO2 group,

respectively. Simultaneously, the 1H NMR spectrum of controland degraded samples has shown signals at d 4.88 ppm for meth-oxy group while signals at d 4.83 and d 2.20 ppm has indicatedthe multiplet of six protons responsible for two protons each forolefinic, enolic, alcoholic and aliphatic groups respectively.

The LC-MS–MS analysis of control SGMPs has shown three ma-jor molecular ion peaks at (m/z) 600, 605 and 621 with molecularmass 600, 605 and 621 respectively. Molecular ion with molecularmass (m/z) 600 was appeared due to formation of a diester productwith empirical formula C22H36N2O17. The fragmentation of thisdiester molecule resulted several ion peaks (m/e) 256, 165, 133,130, 127, 110, 101, 97 and 84 with molecular mass (m/e) 256,165, 134, 131, 126, 110, 100, 96 and 84 respectively. On the basisof IR and 1H NMR results, the detected SGMPs in control sampleswith molecular mass (m/z) 256, 165, 134, 131, 126, 110, 100, 96and 84 were characterized as palmitic acid (C16H32O2), 2-nitroace-tophenone (C8H7NO3), 2,20-bifuran (C8H6O2), methylindol (C9H9N),5-hydroxymethyl-2-furancarboxaldehyde (C6H6O3), 5-methyl-2-furancarboxaldehyde (C6H6O2), 2-methylhexane (C7H16), furan-2-carboxaldehyde (C5H4O2) and 2, 3-dihydro-5-methylfuran(C5H8O), respectively. The LC-MS–MS analysis of degraded sampleshas shown three major ion peaks (m/e) 433, 375 and 307 withmolecular mass 432, 374 and 306, respectively. The fragmentationof these high molecular weight compounds has resulted four prod-uct ion peaks (m/e) 165, 143, 83 and 81 with molecular mass 165,142, 84 and 81 respectively. On the basis of IR and 1H NMR results,the molecular ions with (m/e) 165, 142, 84 and 81 were character-ized as 2-nitroacetophenone (C8H7NO3), p-chloroanisol (C7H7ClO),2,3-dihydro-5-methylfuran (C5H8O) and N-methyl pyrrole(C5H7N) respectively. Compounds p-chloroanisol and N-methylpyrrole was produced as new metabolites during bacterial degra-dation of SGMPs. In this study, most of compounds detected incontrol samples were diminished from bacteria treated samplesindicating that disappearance of SGMPs from bacteria treated sam-ples could be related with color removal associated with the deg-radation of SGMPs.

4. Conclusion

This study revealed that mixed bacterial culture was moreeffective for SGMPs degradation compared to axenic culture RNBS1and RNBS3. It indicated that presence of each bacterium in culturemedium has cumulative enhancing effect on growth and degrada-tion of SGMPs rather than inhibition. The LC-MS–MS and otherspectrophotometric analysis has revealed that most of the SGMPsdetected in control samples were diminished from bacteria treatedsamples and the disappearance of SGMPs from treated samplescould be related with the degradation of SGMPs.

Acknowledgement

The financial assistance received by Mr. R.N. Bharagava, SRFfrom University Grants Commission and CSIR (NWP-19), New Del-hi is highly acknowledged.

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