ORIGINAL ARTICLE

Fermentative recovery of lipids and proteins from freshwaterfish head waste with reference to antimicrobialand antioxidant properties of protein hydrolysate

Ruthu & Pushpa S. Murthy & Amit Kumar Rai &N. Bhaskar

Revised: 26 April 2012 /Accepted: 1 May 2012# Association of Food Scientists & Technologists (India) 2012

Abstract Effectiveness of fermentation using lactic acidbacteria (LAB) for recovering lipids and proteins simulta-neously from freshwater fish head (FWH) was evaluated.Three different proteolytic LAB (Pediococcus acidilacticiNCIM5368, Enterococcus faecium NCIM5335 andPediococcus acidilactici FD3) isolated from fish processingwastes were employed in the fermentation process. Thefermentation conditions involved 10 % (w/w) glucose, 2 %(w/w) NaCl and 10 % (v/w) LAB cultures at 37 °C. Theprocess resulted in 38.4 % of degree of hydrolysis (in caseproteins) and a recovery of 63.6 % of the oil present in thematerial. The fermentation process did not affect the fattyacid profile of lipids. The hydrolyzed protein rich fermen-tation liquor exhibited DPPH radical scavenging activity(EC50 – 5.1 mg protein) as well as antagonistic propertiestowards several bacterial and fungal pathogens. The mini-mum inhibitory concentrations (MIC) of fermentated liquor(with E. faecium NCIM5335 as starter) were 10 and 12 mg/ml for Listeria monocytogenes and Salmonella itridicus,respectively. A higher MIC (60 and 96 mg/ml forAspergillus ochraceus and Penicillium chrysogenum, re-spectively) was observed in case of fungal pathogens.Both the oil and protein hydrolysate rich liquor from fish

head can be used as biofunctional ingredients in both humanfood as well as livestock feed formulations.

Keywords Fish head . Lactic acid bacteria . Lipids .

Protein . Antioxidant . Antimicrobial

Introduction

Fish heads (FH) are the major edible by-products generatedfrom fresh water fish processing industries, which are rich inlipids and protein. The fish production from freshwaterbodies through aquaculture currently stands in close to 33million metric tonnes, accounting for >22 % of world fishproduction (FAO 2010). Considering the fact that freshwater FH are a rich source of protein and polyunsaturatedlipids (Swapna et al. 2010) and constitute about 15–25 %(depending on the species) of the fresh water fish biomass, itcan be estimated that freshwater fish processing waste in theform of head alone amounts to >4.5 MMT. Hydrolysisprocesses have been developed to convert under-utilizedfish and fish byproducts into a marketable and acceptableform, which can be widely used in food rather than asanimal feed or fertilizer. Lactic acid fermentation (LAF)being an eco-friendly method results in biological silagewherein lactic acid bacteria (LAB) generate acid in situ forpreservation of waste or for recovery of biomolecules fromby-product. In addition to acid, LAB also produce proteasesfor the hydrolysis of protein (Jini et al. 2011) and some ofthe lactobacilli produce antimicrobial compounds, whichincrease the preservation effect. The process of LAF hasbeen employed to recover various biomolecules such asprotein, lipids, carotenoids and chitin from among host ofbiomolecules (Amit et al. 2011). LAF has been studied forthe simultaneous recovery of lipids and protein from fish

Ruthu :A. K. Rai :N. Bhaskar (*)Department of Meat, Fish & Poultry Technology, Central FoodTechnological Research Institute (CFTRI), CSIR,Mysore 570 020, Karnataka, Indiae-mail: bhaskar@cftri.res.in

N. Bhaskare-mail: bhasg3@yahoo.co.in

P. S. MurthyDepartment of Plantation Products Spices and Flavor Technology,CSIR-Central Food Technological Research Institute,Mysore 570 020, Karnataka, India

J Food Sci TechnolDOI 10.1007/s13197-012-0730-z

visceral waste (Amit et al. 2011) but that can only be usedfor aquaculture and animal feed.

The lipids from these edible wastes are rich in polyunsat-urated fatty acids that can be used for human consumption.Economic value of fish oil had a record increase in the pastfew years due lopsided demand–supply issues; and, isexpected to continue to increase in the near future (Turchiniet al. 2009). In order to offset the lesser supply and higherdemand, there is a great urgency to find and implementsustainable alternatives to fish oil. Lipids recovered from fishhead can be better alternative to fish oil and till some extentreduce the demand of fish oil production. Protein hydrolysatefrom fish muscle (Rajaram and Nazeer 2010) and visceralwaste (Amit et al. 2011) have been studied for antioxidantand antibacterial activity. Natural antioxidant such as ascorbicacid and α-tocopherol are preferred over synthetic antioxidantdue to their safety regard to health. In order to develop naturaland safer antioxidant having nutritional value synergistic ef-fect of amino acids, peptides and protein hydrolysate has beenattracting research attention considerably. Similarly, proteinhydrolysate after LAF can be used as a probiotic; and, use ofprobiotic as food additives is preferred over that of antibioticsas they do not exhibit any of the undesirable effects associatedwith the use of antibiotics viz, toxicity, allergy, bacterial drugresistance (Ajitha et al. 2004). In our present study, weassessed the effectiveness of LAF for the simultaneous recov-ery of lipids and protein from fresh water FH. The LAB usedfor the fermentation of fish head possesses good proteolyticproperties and produces bacteriocin having a broad antibacte-rial spectrum (Amit et al. 2009; Jini et al. 2011). Further, thefatty acid profile of recovered fish oil along with the antibac-terial and antioxidant activities of the hydrolyzed protein richliquor portion was also analyzed, in order to explore itspossible potential as a high value ingredient in human food.

Materials and methods

Materials Fresh water fish heads (FWFH) were obtained bydressing of freshly harvested Indian major carps (Rohu andCatla) procured from the local market and transported tolaboratory under iced conditions. DPPH (2, 2′-diphenyl-1-picrylhydrazyl), was purchased from Sigma-AldrichChemie (Steinheim, Germany). LAB isolates namelyEnterococcus faecium NCIM5335, Pediococcus acidilacticiFD3, Pediococcus acidilactici NCIM5368 and all the patho-gens were obtained from the institute culture collection. Allmicrobiological media were procured from Hi-Media (Hi-Media, Mumbai, India). All the solvents and chemicals wereof analytical grade.

Preparation of silage FWFH were crushed into small piecesusing bowl chopper (TC11, Scharfen 58413, West

Germany) followed by homogenization in a blender(Stephan Mill; Stephan UM, Germany) for 5 min. Thehomogenized mass was mixed and cooked under steam for20 min. After cooling, 10 % (w/w) dextrose and 2 % (w/w)salt were added and mixed thoroughly. The LAB cultures E.faecium NCIM5335, P. acidilactici FD3, P. acidilacticiNCIM5368 were grown in 100 ml of MRS-Broth (Hi-Media, India) for 24 h at 37 °C in a shaking incubator(Technico Ltd., India) set at 100 rpm. The cells were har-vested by centrifuging (C31 Cooling centrifuge, Remi-India, India) at 3000×g for 10 min, washed twice withsterile physiological saline and resuspended in physiologicalsaline (100 ml). The silage mix was placed in airtightcontainer and incubated for 72 h at 37 °C. After 24 h offermentation the content was centrifuged at 5000×g for20 min and upper oil layer was collected. The lower FWHfermented protein rich hydrolysate (FHPH) was analyzedfor degree of hydrolysis (DH) and antioxidant activities.

Yield, quality and fatty acid composition of lipids recoveredon fermentation Oil yield after fermentation of cooked ho-mogenized FWFH was calculated as % of total lipid contentin FWFH (Amit et al. 2011). Total lipid content in theFWFH as determined by the method of Bligh and Dyer(1959) was considered as 100 %; and, the yield of lipidson fermentation was computed as -

Yield %ð Þ ¼ Oil recovered; g� Total fat content; g½ �� 100

Acid and peroxide value of the recovered oil was esti-mated as per AOAC (2000) to ascertain the quality of oil.Fatty acid composition of recovered lipids was determinedby preparing the fatty acid methyl esters (FAME) of therecovered oil. Briefly, oil samples were transmethylatedusing 2 M methanolic sodium hydroxide followed by 2 Mmethanolic hydrochloric acid to obtain FAME. FAME wereanalysed by using a gas chromatography (GC) system(Shimadzu GC 2014; M/s Shimadzu, Kyoto, Japan) fittedwith a flame ionization detector (FID) for identifying theindividual fatty acids. FAME dissolved in hexane wereanalyzed using a fused silica capillary column (30 m×0.32 mm×0.25 μm) (Omegawax™ 320; M/s Supelco,Bellefonte, USA) with a split ratio of 1:30). The temper-atures (°C) of injector, column and detector were set at 250,200 and 260, respectively.

Degree of protein hydrolysis

DH of the FHPH resulted after lactic acid fermentation ofFH was estimated as per the methodology described by

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Amit et al. (2011) and was computed as - DH (%)0[10 %TCA soluble N2 in the sample ÷ Total N2 in the sample]×100.

Antioxidant activities of fermented head proteinhydrolysate

Preparation of sample for in-vitro antioxidant and antibac-terial assays The FHPH obtained was filtered using muslincloth to obtain filtered fermented liquor (FFL). FFL wasfurther centrifuged at 5000×g for 20 min to obtain asediment-free supernatant. This supernatant was referred toas centrifuged FFL (CFFL). CFFL was lyophilized and thelyophilized sample was used for antioxidant and antimicro-bial properties. The in-vitro antioxidant assays includedtotal antioxidant activity (TAO) and DPPH radical scaveng-ing activity.

Total antioxidant activity TAO was carried out as per themethod described by Amit et al. (2011). TAO of CFFL wasdetermined by mixing sample with 3 ml of reagent solution[3.0 ml; 0.6 M sulfuric acid: 28 mM sodium phosphate:4 mM ammonium molybdate (1:1:1 v/v/v)]. Reaction mix-ture was incubated in a water bath at 95 °C for 90 min,cooled to room temperature and the absorbance was mea-sured at 695 nm. TAO was expressed as of μg of ascorbicacid equivalents (AAE) per mg of protein in fermentedhydrolysed sample.

Diphenyl picrylhydrazyl (DPPH) radical scavengingactivity The DPPH radical scavenging capacity of CFFLwas determined by the method previously described inAmit et al. (2011). Briefly, 100 μl of sample and made upto 2 ml with distilled water followed by addition of 2.0 ml of0.16 mM DPPH solution (in methanol). The mixture wasvortexed and kept at room temperature for 30 min in thedark. Sample blank was prepared by replacing DPPH withmethanol. The absorbance of all the sample solutions wasmeasured at 517 nm. Methanol along with DPPH served asthe control. The scavenging activity (%) was calculated byusing the formula:

Scavenging activity %ð Þ¼ 1� Asample � Asample blank

� �=Acontrol

� �� �� 100

Antibacterial and antifungal susceptibility assay

Microorganisms Bacterial strains such as Bacillus cereusF4810, Bacillus subtilis MTCC441, Staphylococcus aureus

FRZ722, Streptococcus pyogenes MTCC1923, Listeriamonocytogenes Scott-A, Escherichia coli MTCC118,Pseudomonas aeruginosa MTCC741, and Yersinia enter-ocolitica MTCC859, Salmonella typhi FB231, S. paratyphiFB254, S. itriditicus FB256, Micrococcus luteus and fungalstrains Aspergillus flavus MTCC277, A. niger MTCC218,A. ochraceus MTCC4643, A. oryzae MTCC262, Fusariumoxysporum MTCC1755 and Penicillium chrysogenumMTCC616 were obtained from Microbial Type CultureCollection (MTCC), Chandigarh, India and culture collec-tion center maintained at CFTRI, India.

Preparation of spore suspension All the fungal cultureswere grown on potato dextrose agar (PDA) slants at 25 °Cuntil the spores ramified (7–10 days). Spores were sus-pended by adding Tween 20 solution (0.1 %v/v) in distilledwater and inoculated viable spores (106) per mL.

The antibacterial activity was carried out using agar welldiffusion method (Denton and Kerr 1998). The 18–24 h oldbacterial strains were grown on nutrient broth and incubatedat 37 °C under shaking condition. The culture broth of0.1 mL containing 108–109 cfu/mL was spread on nutrientagar plate (15 cm) by spread plate method. Wells were bored(8 mm diameter) in the agar plates and filled with proteinhydrolysate and were incubated at 37 ±1 °C for 24 h and48 h. Tetracycline (10 μl/ml) was used as positive control.The zone of growth inhibition was expressed in mm.

The antifungal properties of protein hydrolysate werestudied against various fungi (Singh et al. 2004). The knownconcentrations of the CFFL were added onto the plate alongwith the PDA before plating and the content was evenlymixed. Point inoculation of the respective fungi was carriedout. The PDA plate without CFFL was considered as con-trol. The plates were incubated at 27 ±1 °C temperature for5–7 days. Nystatin (1 μl) was used as the positive antifungalagent. At the end of the incubation period, the susceptibilityof the test organisms was determined by measuring themean growth values and were converted into percentage ofmycelial growth inhibition (MGI) in relation to the controltreatment, by using formula MGI (dc-dt)/dc X100, where dcand dt represent mycelial growth diameter in control andtreated petriplate, respectively. All the experiments werecarried out in triplicates.

Determination of Minimum Inhibitory Concentration (MIC)for bacteria and fungi The MIC of FHPH against selectedbacterial strain were carried out according to Thongson et al.(2004). Different concentrations 5, 10, 15, 20, 40, 60, 80,100 μl (protein concentration 7–8 mg/ml) of the CFFL and100 μL of the bacterial suspension (105 CFU/ml) wereadded aseptically to 10 ml of nutrient broth separately andincubated for 24 h at 37 °C. Growth was observed bothvisually and by measuring the optical density (OD) at

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600 nm. MIC was defined as the lowest concentration whichresulted in a reduction of >90 % in the observed absorbance.Likewise, the MIC of the fungal strains was carried outusing different concentration (5 to 100 μl) of CFFL (proteinconcentration 7–8 mg/ml) on A. ochraceus and Penicilliumsp using potato dextrose broth. These were incubated at27 °C for 4–5 days and observed for the growth inhibition.Triplicate sets of tubes were maintained for each concentra-tion of test sample.

Scanning electron microscopy (SEM) To determine the ef-ficacy of protein hydrolysate on the morphology of the cells,SEM studies were performed on L. monocytogenes and S.itriditicus treated with MIC of protein hydrolysate. Controlswere prepared without protein hydrolysate. The bacterialsamples were washed gently with 50 mM phosphate buffersolution (pH 7.3), fixed with 2.5 g/100 ml glutaraldehyde.The specimen was dehydrated using sequential exposure perethanol concentrations ranging from 30–100 %. After dehy-dration, the samples were spread on a double sided conduct-ing adhesive tape pasted on a metallic stub, subjected togold covering, and observed under a scanning electronmicroscope (LEO 435VP, UK) at 20 kV attached to Videocopy processor (Mitsubishi, Japan) . Photographs were tak-en using 35 mm camera (Ricoh, Japan) that was connectedto monitor optically through fibre optics.

Three-day-old fungal cultures of Penicillium sp. and A.ochraceus on PDA treated with sterile filter paper discs(10 cm diameter) soaked in protein hydrolysate placed onthe inner surface of the Petri dish lid. Then, the dishes weresealed with parafilm and incubated upside-down at 25 °C,and negative control without protein hydrolysate was usedfor SEM to study the mode of action of the protein. Thesegments measuring 5–10 mm were cut at periphery of thecolony from the cultures growing on the PDA plates andwere placed in vials containing 3 % glutaraldehyde in phos-phate buffer saline (PBS) (pH 7.4) at 4 °C and furtherprocessed as mentioned for bacterial cells.

Statistical analysis All the experiments were comducted intriplicates, wherever required. The data was subjected toanalysis of variance to determine the significant differencebetween different treatments and mean separation was ac-complished by Duncan’s multiple range test (Statistica1999).

Results and discussion

Recovery of lipids and protein from fresh water fishhead Fresh water fish head (Rohu and Catla) had a lipidand protein content of 26.47 % and 55.23 % (both on dry

weight basis), respectively. Oil recovery after fermentationof FWH using different LAB cultures is presented inTable 1. The total lipid content in FWH was 15–16 %(wwb). Oil recovery showed variations among differentLAB cultures used. E. faecium NCIM5335 gave highestoil recovery of (63.6 %), followed by P. acidilacticiNCIM5368 (58.3 %), the least being in P. acidilactici FD3(39.2 %). Hence, it can be said that fermentation using LABcan be an effective approach for oil recovery from freshwater fish head.

The oil recovered on fermentation using different LABcultures was assessed for its quality which was determinedwith respect to acid value and peroxide value. The acidvalue (mg NaOH g-1 sample) in the oil recovered is pre-sented in Table 1. Acid value of the recovered oil was in therange of 2.2–2.9 (mg NaOH g−1 sample) with the maximumin case of P. acidilactici FD3 (2.9) and P. acidilacticiNCIM5368 (2.8). Acid value of oil recovered from fishhead is lower compared to visceral waste, as viscera are richin lipases (Nayak et al. 2004) and lipolytic bacteria. Butlipase produced by LAB can increase the acid value of theoil. Due to the action of lipases, the lipids present in the fishviscera are hydrolyzed thus releasing the fatty acids, whichcontribute to acid value. Peroxide value indicates the degreeof oxidation of lipids which is presented in Table 1. Theperoxide value (meqv oxygen kg−1 sample) in the oil in-creased significantly from an initial value of 11.23 meqvoxygen kg−1 sample in fresh and was found to be maximumin case of P. acidilactici NCIM5368 and P. acidilactici FD3(42 and 48.4 meqv oxygen kg−1 sample), respectively.Peroxide value can be reduced by the addition of syntheticantioxidant such as TBHQ to the homogenized head beforefermentation. Changes in peroxide value, an indicator oflipid oxidation, during fermentation of fish viscera has beenreported by Ahmed and Mahendrakar (1996) where slightincrease was observed in peroxide value which later reduceddue to breakdown of oxidized products. Further they alsosuggested the use of antioxidants to prevent oxidation oflipids and development of rancidity during storage of fer-mented fish viscera. The fermentation may also result information of peptides possessing antioxidant activity, whichcan prevent lipid oxidation (Amit et al. 2011).

Fatty acid composition of recovered oil from hydrolysedfish head waste is presented in Table 2. There was nosignificant difference in the fatty acid profile of the oilrecovered on fermented homogenized head using differentLAB cultures when compared to the fresh oil. Unsaturatedfatty acids dominated the fatty acid profile of the recoveredoil. There was no significant difference in the amount ofsaturated fatty acids like palmitic acid (C16:0) stearic acid(C18:0) as well as unsaturated fatty acids such as eicosa-pentaenoic acid (EPA) (C20: 5), palmitoleic acid (C16:1),oleic (C18:1) and linoleic (C18:3) (Table 2). This

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observation correlated with Gbogouri et al. (2006) whereinno difference in fatty acid profile of oil recovered fromsalmon heads by solvent extraction and enzymatic hydroly-sis was noticed. Similarly, in case of fermentation of freshwater viscera using LAB, there was no change in fatty acid

composition in the recovered oil as reported by Amit et al.(2011).

Antioxidant activities of fermented protein hydrolysate Theprotein hydrolysates obtained from wastes/by-products ofanimal and fish processing industry exhibit antioxidant ac-tivity (Amit et al. 2009; Bijinu et al. 2011; Sachindra andBhaskar 2008). TAO and DPPH radical scavenging activityof CFFL obtained from FHPH by different LAB cultures ispresented in Table 1. DPPH has been used extensively as afree radical to evaluate reducing substance and is a usefulreagent for investigating the free radical scavenging activi-ties of protein hydrolysates. DPPH radical scavenging ac-tivity expressed in EC50 which was found to be higher incase of E. faecium NCIM5335 (5.1 mg protein), least beingin P. acidilactici NCIM5368 (7.2 mg protein). The methodis based on the reduction of methanolic DPPH solution inpresence of a hydrogen donating antioxidant due to forma-tion of a non-radical form of DPPH-H by the reaction andthis modification is visually noticeable as a discolorationfrom purple to yellow (Shon et al. 2003)

Total antioxidant activity (TAO) was expressed as mg ofascorbic acid equivalents per microgram of protein in CFFL(AAE/mg). E. faecium NCIM5335 showed highest TAO of(13.4 mg of AAE/mg of protein), followed by P. acidilacticiFD3 showing (11.3 AAE/mg protein) and P. acidilacticiNCIM5368 (11.1 AAE/mg protein). The TAO exhibitedby the CFFL could possibly be due to the peptides andamino acids resulting from the hydrolysis of fish wasteproteins. In this phosphomolybdenum method, molybde-num VI (Mo6+) is reduced to form a green phosphate/Mo5+

complex (Amit et al. 2009).

Antibacterial activity of FHPH The in- vitro antibacterialsusceptibility of the FHPH obtained on fermentation ofhomogenized fish head using different LAB showed an-tibacterial activity against bacterial pathogens (Table 3).The FHPH extract was found to be effective againstGram- positive bacteria such as S. aureus, L. monocyto-genes, B. cereus, B. subtilis, M. luteus and Gram- nega-tive pathogens such as E. coli, S. itriditicus, S. paratyphi,Y. enterocolitica and S. typhi. The inhibition zone usingFHPH obtained using different LAB ranged from 13.5–29.75 mm and maximum inhibition was obtained with L.monocytogenes (29.75±0.2) and S. itriditicus (28.25±1.25). The antibacterial activity exhibited by the liquorportion rich in hydrolysed portion could possibly bebecause of presence of antimicrobial peptides (bacterio-cin) produced during fermentation using LAB and pep-tides formed on hydrolysis of protein present in head.Kawai et al. (2007) have reported antagonistic propertyof bovine lactoferrin hydrolysate against mastitis patho-gens. Similarly, hydrolysates of food proteins by

Table 1 Summary of lactic acid fermentation for simultaneous recoveryof lipids and protein from fresh water fish head (n03)

Attributes E faeciumNCIM5335

P acidilacticiNCIM5368

P acidilacticiFD3

Lipid

Oil Recovery (%) 63.6±3.12a 58.3±3.87a 39.2±2.80b

Peroxide value 37.5±3.62a 42.0±2.71 a,b 48.4±3.63 b

Acid value 7.8±0.84a 8.2±1.14 a 6.9±0.40 a

Protein

DH (%) 38.4±2.18a 29.0±2.15b 36.0±4.18 a

TAO (mg AAE/mg) 13.4±1.17 a 11.1±0.83b 11.3±2.78b,c

DPPH (EC50) 5.1±0.42a 7.2±0.50b 6.7±0.92c

Peroxide value - meqv oxygen kg−1 sample, Acid value - mg NaOH/gof sample

DH: Degree of hydrolysis, TAO – Total antioxidant activity; DPPH :DPPH radical scavenging activity: EC50 : effective concentration ofprotein (mg) for 50 % radical scavenging activity. AAE: Ascorbic acidequivalent. Values within the same row with the same superscripts arenot singnificantly different (p>0.05) (n03)

Table 2 Fatty acid composition of oil recovered on fermentation offresh water fish head using different lactic acid bacteria (n03)

Head Fresh NCIM5335 FD3 NCIM5368

C14:0 3.7 3.8 3.8 3.7

C16:0 29.6 28.7 29.8 29.3

C18:0 4.5 4.6 4.3 4.3

C20:0 1.1 1.3 1.1 1.1

ΣSFA 36.4 39.8 40.4 38.5

C16:1 13.1 12.6 13.3 13.3

C18:1n-9 19.7 19.7 18.5 18.7

C18:1n-7 2.5 3.9 3.6 3.5

ΣMUFA 35.3 36.2 35.5 35.5

C18:2n-6 4.1 4.1 4.0 4.2

C18:3n-3 5.7 5.5 5.6 5.7

C20:4n-6 2.4 2.2 2.0 2.1

C20:5n-3 2.8 2.6 2.6 2.7

C22:5n-3 1.6 1.4 1.3 1.3

C22:6n-3 2.9 2.5 2.9 3.2

ΣPUFA 19.1 18.3 17.2 19.1

SFA Saturated fatty acid, MUFA Mono unsaturated fatty acid, PUFAPoly unsaturated fatty acid. NCIM5335 - Enterococcus faeciumNCIM5335, FD3 - Pediococcus acidilactici FD3, NCIM5368 - Ped-iococcus acidilactici NCIM5368; Fatty acid concentrations above0.5 % are only considered for the above

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gastrointestinal proteases have also been shown to beantibacterial as well as immunostimulatory in nature(Gediminas et al. 2006). Bactriocins are known to killthe pathogenic bacteria either by forming pores in thecytoplasmic membrane or by acting on the energizedmembrane vesicles to disrupt the proton motive force(PMF), inhibit uptake of amino acids, and cause releaseof accumulated amino acids (Abee et al. 1995). In caseof P. acidilactici FD3 FHPH, maximum inhibition wasobserved with S. aureus (29.75±0.25 mm) and minimuminhibition in case of B. subtilis with 16±1.5 mm. S.itriditicus (28.7.0±0.5 mm) showed good inhibition withFHPH of P. acidilactici NCIM5368 followed by E. coliand Y. enterocolitica. Minimum inhibition was found incase of B. subtilis and S. typhi when treated with E.faecium NCIM5335, P. acidilactici FD3 whereas P. acid-ilactici NCIM5368 exhibited minimum inhibition with S.aureus and B. cereus. This may perhaps be due to theresistance that may result probably from alteration ofbacterial membrane composition, destruction of the bac-teriocin by proteases or altered receptors (Martinez andDe Martinis 2005).

Determination of Minimum Inhibitory Concentration (MIC)of FWPH in bacteria The FHPH obtained on fermentationwith E. faecium NCIM5335 had better antibacterial spectrumand effective antibacterial activity against pathogen like L.monocytogenes (29.75±0.2) and S. itriditicus (28.0±1.25)and hence considered for further studies to find the MIC ofextracted protein hydrolysate on fermentation with E. faeciumNCIM5335.

MIC of FHPH obtained on fermentation with E. faeciumwas calculated for L. monocytogenes and S. itridicus whichwere 10 and 12 mg/ml, respectively. The antibacterial activityincreased with increasing concentration of the FHPH andindicates that antibacterial activity is dose dependent. Thepeptides have ability to depolarize the cytoplasmic membraneand are usually effective against Gram-positive microorgan-isms and are well supported by L. moncytogenes. LAB bac-teriocins have varied mechanisms to exert an antimicrobialeffect, but generally target the cell envelope. The initial elec-trostatic attraction between the target cell membrane and thebacteriocin peptide is thought to be the driving force forsubsequent events (Deegan et al. 2006). Anionic cell surfacepolymers like teichoic acid and lipoteichoic acid may beimportant in the initial interaction of cationic bacteriocins ofGram-positive bacteria (Jack et al. 1995). The exact mode ofaction of antimicrobial peptides is widely believed that thepeptides interact with and disrupt the cytoplasmic membrane,leading to the dissolution of the proton motive force andleakage of essential molecules, resulting in cell death.Bacteriocins may possess a bactericidal or bacteriostatic modeof action on sensitive cells, this distinction being greatlyinfluenced by several factors such as bacteriocin dose anddegree of purification, physiological state of the indicator cellsand experimental conditions (Cintas et al. 2001).

Scanning electron microscopy studies on bacterial cells TheFHPH effect observed under microscope revealed stronginhibition on cell growth. The cells of the bacteria appearedshriveled with degenerative size compared to untreatedsample (Fig. 1). The inhibition caused may be due to somepeptides. Moreover, bacteriocins are part of antimicrobialpeptides and are widely believed that the carnage mecha-nism of these peptides on bacteria involves an interactionwith the cytoplasmic membrane. Both groups act on sensi-tive cells by targeting either the inner membrane by poreformation or an intracellular target using enzymatic activitysuch as DNAse or RNAse (Abee 1995). Animal-derivedantimicrobial peptides exhibit inhibitory effect against widerange of microorganisms than those produced by bacteria, asthey are not affected by antibiotic- resistance mechanismslike other antibiotics (Rydlo et al. 2006).

Antifungal activity of FHPH Antifungal susceptibility wasobserved in all the three protein hydrolysate extracted

Table 3 Antimicrobial activity (zone of inhibition in mm) of fer-mented fresh water fish head protein hydrolysate

Organisms NCIM5335 FD3 NCIM5368

Antibacterial properties (zone of inhibition in mm)

E. coli MTCC118 28.0±2.75a 29.5±2.00 a 26.2±2.25 a

B. cereus F4810 18.7±0.25 a 18.7±0.25 a 14.0±0.50b

B. subtilis MTCC441 17.2±1.25 a 16.0±1.50 a 16.0±2.00 a

Y. enterocoliticaMTCC859

27.2 ±1.25 a 27.5±1.00 a 26.7±0.75 a

L. monocytogenesScott A

29.7±0.25 a 20.7±0.75b 13.5±1.00c

S. aureus FRZ722 28.2±0.92a 29.7±0.25a 13.5±0.50b

S. typhi FB231 17.5±0.50 a 15.0±0.50b 17.5±0.50 a

S. paratyphi FB247 25.5±0.50 a 23.2±0.25b 25.0±1.00 a

S. itriditicus FB257 28.2±0.50 a 28.7±1.00 a 27.0±1.50 a

M. leuteus 25.5±0.50 a 19.7±3.25b 20.5±3.00b

Antifungal activity (% of inhibition)

A. niger MTCC218 NI NI NI

A. ochraceusMTCC4643

28.0±1.00 a 18.0±0.50b 15.2±1.00c

A. flavus MTCC277 NI NI NI

A oryzae MTCC262 29.0±1.00 a 19.5±1.50b 16.20±1.00c

P. chrysogenumMTCC616

55.5±2.00 a 39.4±1.00b 40.0±2.00b

F. oxysporumMTCC1755

21.6±2.00 a 22.8±2.00 a 20.5±1.00 a

NCIM5335 - Enterococcus faecium NCIM5335, FD3 - Pediococcusacidilactici FD3, NCIM5368 - Pediococcus acidilactici NCIM5368;NI: No inhibition

Values within the same row with the same superscripts are not singni-ficantly different (p>0.05) (n03)

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Fig. 1 Scanning electronmicroscopy of bacterial cellstreated with proteinhydrolysates. (E faeciumNCIM5335). (a healthy cells ofL. monocytogenes, b treatedcells and c healthy cells of S.itriditicus, d treated cells)

Fig. 2 Scanning electronmicroscopy of hyphae treatedwith protein hydrolysates. (ahealthy hyphae of A.ochraceus,b treated organisms with PH ofE faecium NCIM5335, chealthy Penicillium and dtreated)

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(Table 3). The FHPH extract fermentation with E. faeciumNCIM5335 was found to be fairly efficient compared to P.acidilactici FD3 and P. acidilactici NCIM5368. The FHPHof NCIM5335 was effective against Penicillium comparedto A. oryzae and A. ochareus. The delay in sporulationexpressed as % of inhibition by FHPH of NCIM5335against Penicillium, A. ochraceus and A. oryzae were55 %, 28 %, and 29 %, respectively (Table 3), whereas A.niger and A. flavus were found to be resistant. Atanossova etal. (2003) reported to have characterized an antimicrobialproteinaceous compound from Lactobacillus paracasei subsp. paracasei M3, with broad antibacterial activity and withfungistatic effects against 4 of the 26 tested yeast species. Incontrast with the vast literature on bacteriocins, only fewreports exist on antifungal peptides of LAB (Roy et al.1996) and our results is one of the few to report the same.

Minimum inhibitory concentration of FHPH in fungi Theminimum inhibitory concentration of the protein hydroly-sate against A. ochraceus was 60 mg/ml and 96 mg/ml forPenicillium. Likewise our results also suggest that theNCIM5335 extracts exhibited antifungal activity against A.ochraceus and Penicillium. Delayed growth and sporulationin case of Penicillium was observed. Different mechanismsof action have been proposed for peptides such as alterationof enzymatic activity, inhibition of spore germination andinactivation of anionic carriers (Martinez and De Martinis2006). The FHPH did not exhibit antifungal activity againstA. niger and A. flavus which could be due to influence of theproteolytic enzymes produced by these fungi in the hydro-lysate (Batish et al. 1989). Possible explanations for theseobservations include synergistic effects between this peptideand host derived antifungal factors, such as endogenousantimicrobial proteins/peptides and reactive oxygen inter-mediates which has brought about inhibitory effect in caseof fish epidermis (Claire Hellio et al. 2002).

Scanning electron microscopy studies of fungal cells TheSEM pictures of A. ochraceus and Penicillium sp exhibitedalteration of the hyphal morphology when treated withNCIM5368 samples. The mycelium of untreated (control)A. ochraceus was illustrious with free and linearly shapedhyphae with anomalous branching (Fig. 2). But in case ofFHPH treated fungi, notching of the terminal hyphae, shriv-eled hyphae, severely collapsed, cell surface depressionsand squashed mycelium were observed (Fig. 2). This maybe due to interferences of cell wall synthesis, which causemorphological changes and inhibit growth. Such modifica-tions may be related to effect of peptides as enzymaticreactions regulate cell wall synthesis. This could be due tothe loss of integrity of the cell wall and as a result, plasmamembrane permeability might be affected, which explainchanges in morphology with orientation to size and shape of

the internal organelles. Also the antifungal activity involv-ing cytoplasmic granulation, cytoplasmic membrane ruptureand inactivation of intracellular and extracellular enzymescould have taken place. These biological events could takeplace separately or concomitantly, culminating in mycelialgermination inhibition. However, it is likely that a carefulreevaluation of other ‘bacteriocins’ might reveal furtherantifungal activities.

Conclusions

The study collates information regarding oil rich in PUFAand peptidic hydrolysates with antioxidant and antimicrobi-al properties isolated from FWFH. Antioxidant and antimi-crobial proteins isolated from fish head sources may be usedas functional ingredient in food formulations to impart hu-man health benefits and/or improve the shelf life of foods.Furthermore, fermented protein hydrolysate exhibited anti-microbial effects on various types of bacteria and finds itsapplication in food preservation, food supplements andpharmaceutical industry.

Acknowledgements Authors thank Department of Biotechnology(DBT), Govt. of India for partial funding of this work through Grant##BT/PR 9474/AAQ/03/345/2007. Authors place on record theirthanks to Director, CFTRI for encouragement and permission to pub-lish the work.

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