ANIMAL SCIENCE REPORTER Animal Science Reporter, Volume 10, Issue 4, October, 2016 RESEARCH PAPERS 123-131 ORGANOLEPTIC, PHYSICO-CHEMICAL, AND MICROBIAL QUALITIES OF FRESH WATER FISH

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Animal Science Reporter, Volume 10, Issue 4, October, 2016

RESEARCH PAPERS

123-131 ORGANOLEPTIC, PHYSICO-CHEMICAL, AND MICROBIALQUALITIES OF FRESH WATER FISH (CATLA CATLA) AT VARIOUSSTAGES OF SUPPLY CHAIN VENDED IN RETAIL FISH MARKET

Rajesh Kumar Sahu, V.V. Deshmukh, C.D. Bhong, P.V. Yeotikar, M.S. Vaidya

132-142 ELICITATION AND CHARACTERIZATION OF SKIN GELATIN OFMALABAR SOLE FISH AT DIFFERENT EXTRTACTIONTEMPERATURES

R.R. Sawant, S.Y. Metar, V.H. Nirmale, J.M. Koli, V.V. Vishwasrao, S.B. Satam

143-148 ELECTROPHORETIC CHARACTERIZATION OF CASEIN OFREFRIGERATED COW AND BUFFALO MILK PRESERVED WITHBANANA PSEUDOSTEM JUICE

P.R. Ray, P.K. Ghatak, S.K. Bag, S. Maji

149-160 EFFECT OF POLYHERBAL FEED ADDITIVES ON GROWTH ANDFEED CONVERSION EFFICIENCY IN PIGS

A.C. Gyani, Ravindra Kumar, S.K. Sinha, Vijay Kumar

Animal Science Reporter(International Journal of Animal Science Research)

Volume 10, Issue 4, Page 121-160 CONTENTS October-December 2016

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Author attribution: 1PhD Scholar, Dept. of Veterinary Public health & Epidemiology, College ofVeterinary Science, Rajendranagar, Hyderabad, India-500030, 2Professor, 3Asst. Professor, Dept. ofVeterinary Public Health, 4Asstt. Professor, Dept. of Veterinary Biochemistry, 5Associate Professor,Dept. of Animal Genetics and Breeding, College of Veterinary and Animal Sciences, MaharashtraAnimal and Fishery Sciences University (MAFSU), Parbhani, Maharashtra, India – 431402. It is a partof the MVSc thesis in Veterinary Public Health of the first author, submitted to MAFSU in 2015.1Corresponding author (E-mail: [email protected]). Received: 12 March 2016, Accepted: 25July 2016. pp. 123-131

ORGANOLEPTIC, PHYSICO-CHEMICAL, AND MICROBIAL QUALITIES OFFRESH WATER FISH (CATLA CATLA) AT VARIOUS STAGES OF SUPPLY

CHAIN VENDED IN RETAIL FISH MARKET

Rajesh Kumar Sahu1, V.V. Deshmukh2, C.D. Bhong3, P.V. Yeotikar4, M.S. Vaidya5

ABSTRACT

This study elucidates the organoleptic (freshness), physicochemical, and microbialqualities of catla fish (Cyprinus catla) at various points of the supply chain retailed atthe fish market. The study conducted on 18 samples from three different stages ofsupply chains, viz., harvest, transportation and retail shop, evaluated by EuropeanUnion Freshness grading method indicated that the freshness quality of catla of allthe samples (100%) was of extra good quality (E grade) at the point of harvest. Therewas quality degradation in 67% of the samples during transport, but were of goodquality (A grade), and 100% of the samples at the retail shop, but were of satisfactoryquality (B grade). The quality of none of the samples was unsatisfactory (C grade).The supply chain was of four hours duration. There was significant (P0.01) differencein pH value at various points of the supply chain. The pH value was the highest (6.73± 0.05) at the point of harvest, and declined significantly (P0.01) during transportation(6.47 ± 0.04), with further (P0.01) dip at the retail shop (6.20 ± 0.03). There wassignificant (P0.01) difference in Thiobarbituric acid (mg/kg MDA) value at variouspoints of the supply chain. It was the lowest (0.65 ± 0.06) at the point of harvest, andincreased significantly (P0.01) during transportation (1.33 ± 0.18), with further rise(P0.01) at the retail shop (2.54 ± 0.23). There was significant (P0.01) difference inTotal Viable Count (Log CFU/gm) value at various points of the supply chain. TheTotal Viable Count value was the lowest (6.18 ± 0.03) at the point of harvest, andincreased significantly (P0.01) during transportation (6.36 ± 0.04), with furtherescalation (P0.01) at the retail shop (6.57 ± 0.02). It is concluded that freshwater fish(Catla catla) was of extra good quality at the point of harvest, but deterioratedprogressively during transport under cold chain and at the retail shop.

KEY WORDS

Catla Fish, Freshness, pH, Thiobarbituric acid, Total Viable Count

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INTRODUCTIONCatla (Catla catla) is a major carp cultivatedin the reservoirs of India (Ayappan et al.,2013). Fish is a nutrient dense diet(Ashano and Ajayi, 2003), and is a highlyperishable commodity. Thus, ensuringthe quality of fish is of paramountimportance for better economic returns inretailing. However, maintenance of fishquality is a complex concept due toinvolvement of a gamut of factors thatinduce deteriorative changes duringharvest, sorting, handling, transportationand storage with considerable adverseimpact on consumer acceptability (Jannatet al., 2007; Pamuk et al., 2011; Kapute etal., 2012).

The fish received at the landing site isgraded first by the receiver/ vendor onthe basis of freshness (Organolepticpropereties), linked with appearance,odour, texture and taste perceptions(Rahman et al., 2012; Cheng et al., 2014;Humaid and Jamal, 2014). Freshness is themost important quality attribute, but notthe only one. Fish quality is moremeticulously adjudged by sensory andinstrumental methods, which includesassessment of physical changes,biochemical and chemical changes, andmicrobiological changes, since fishspoilage results from three basicmechanisms, viz., enzymatic autolysis,oxidation and microbial growth withaccelerated pace with the rise in storagetemperature (Huss, 1995; Gram and Huss,1996; Abbas et al., 2008; Ghaly et al., 2010;Humaid and Jamal, 2014).

A spectrum of intrinsic and extrinsicfactors, e.g., species of fish (Ukekpe et al.,

2014), geographical location (Joseph,1989), water quality (Meador andGoldstein, 2003), water temperature(Gram and Hush, 1996), air temperature(Gang, 2014), microbial contamination(Nonga et al., 2015), method of fishing(Huss, 1995), nature of handling (Kaputeet al., 2012), and holding time (Gang, 2014)etc. influence freshness and quality of fish.

There is a meager quality appraisalresearch on sweet water catla fish(Cyprinus catla), and virtually none inrespect of catla fish harvested fromYelderi dam reservoir of Maharashtra(Image-1). Hence, this study was undertaken to assess the freshness and qualityof Catla catla fish in terms ofphysicochemical properties and microbialqualities, harvested from Yelderi damreservoir of Maharashtra at various stagesof supply chain and vended in the localretail fish market.

Image-1. Catla fish harvested from Yelderidam reservoir of Maharashtra.

MATERIALS AND METHODS

Fish: Catla catla fish were collected duringMarch-May from the harvest at Yelderidam reservoir in Parbhani district,Maharashtra, India, used for carp culture

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on co-operative basis. The Yelderi Damreservoir is in the back water of PurnaRiver flowing through Jintur taluka ofParbhani district. The harvest is beingmade in early morning hours. The catchis being made from the pre-laid netswhich were put during night hours. Thefish harvest is placed in plastic bags of 40-50 kg capacity. The cold chain starts afterweighing by putting ice flakes at the topof the fishes kept in gunny bags fortransportation to Parbhani city.

Sample collection: The samples werecollected in sterile polythene bags as perthe method described by ICMSF (1998).The samples were transported in ice to thelaboratory. A total of 18 samples fromthree different stages of supply chains viz.,harvest, transportation and retail fishshop were collected for organoleptic,physico-chemical, and microbiologicalanalysis.

Freshness: Organoleptic characteristics(freshness) of the fish samples based ongeneral appearance, colour, odour andtexture (Rahman et al., 2012; Humaid andJamal, 2014) were evaluated as perEuropean Union Freshness gradingmethod (EC, 1996). The freshness wereevaluated under four categories, viz., E(extra good quality), A (good quality), B(satisfactory quality), C (not suitablequality).

Physico-Chemical analysis: pH of fishsample was determined as per methodsprescribed in FSSAI (2012). Thiobarbituricacid (TBA) value of fish flesh wasdetermined as per the extraction methoddescribed by Witte et al. (1970) with partialmodification. Ten grams of fish flesh was

blended with 25 ml of pre-cooled 20%TCA in 2M orthophosphoric acid for 2min. The content was filtered throughWhatman filter paper No. 1 to get extract.Three (3) ml of this extract was mixed with3ml of TBA reagent (0.005M) in test tubesand placed in a dark room for 16 hours.A blank sample was prepared by mixing1.5 ml of 20% TCA, 1.5 ml distilled waterand 3 ml of 0.005M TBA reagent.Absorbance was measured at fixed wavelength of 532 nm usingspectrophotometer.TBA value wascalculated as mg malonaldehyde per kgof sample by multiplying absorbancevalue with the factor 5.2.

Microbial analysis: For evaluating totalviable count (TVC), Standard pour platetechnique was followed. Dilution ofinoculums was standardized for furtheruse. A quantity of 0.1 ml inoculum from10-3 and 10-4 dilutions were used for pourplate technique to which molten platecount agar (45-50oC, Hi-mediaLaboratories, Mumbai) was poured andmixed thoroughly by rotating plates.Incubation was done at 37 0C for 24 hours.TVC were calculated by using standardformula as per method described byAOAC (1997).

Statistical analysis: The data wasanalyzed as per Snedecor and Cochran(1967).

RESULTS AND DISCUSSION

Freshness: The study on freshness of fish(Table-1) indicated that all the samples(n=6) were of extra good quality (E grade)at the point of harvest. During transport,only 2 samples (33%) maintained E grade,while rest 4 (67%) showed quality

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degradation, and were of good quality (Agrade). At retail shop, there was qualitydegradation in all the samples. Two (2)samples (33%) were of good quality (Agrade), while the quality of rest 4 samples(67%) were graded satisfactory (B grade).

An earlier study on Labeo calbasu and Labeorohita collected from the local outlets ofSambalpur, Orissa had indicated excellentquality with sensory scores of 9.75 and9.2 respectively evaluated on 10-pointHedonic scale (Sahu et al., 2009).

Table-1. Organoleptic (Freshness) quality grades of Catla catla at various stages ofsupply chain.Stage Replication / Freshness grade

I II III IV V VI Harvest E E E E E E Transportation A E A A E A Retail shop B B A B A B Note: Grading scale as per EU grading method: E (Extra Good Quality), A (Good quality), B

(Satisfactory).

Table-2. Physico-Chemical and Microbial qualities of Catla catla at various stagesof supply chain.Parameter Supply chain

Harvest Transportation Retail Shop pH 6.73a ± 0.05 6.47b ± 0.04 6.20c ± 0.03 TBA Value (mg/kg MDA) 0.65a ± 0.06 1.33b ± 0.18 2.54c ± 0.23 TVC (Log CFU/gm) 6.18a ± 0.03 6.36b± 0.04 6.57c ± 0.02

Note: (1) Figures are presented as Mean ± SEM. (2) Means with different superscripts in arow differed significantly (P 0.01).

Declining pattern of freshness of fish fromharvest to retail shop might be due to timefactor, and transport under ambientcondition. It takes about four hours toreach fish from harvest to retail shop. Thedeterioration in freshness quality could bedue to holding fish for a longer time under

high environmental temperature, sincehigh ambient temperature and longerholding time negatively affects fishfreshness (Gang, 2014). Earlier, Triqui andBouchriti (2003) had also reported E gradeof fish upon harvest with deterioration offreshness quality subsequently.

Physico-Chemical Properties: Theresults of physico-chemical properties,viz., pH and TBA value are presented inTable-2 (Figures: 1&2).

pH: There was significant (P 0.01)difference in pH value at variouspoints of the supply chain. The pHvalue (Table-2, Figure-1) was thehighest (6.73 ± 0.05) at the point ofharvest, and declined significantly(P 0.01) during transportation (6.47 ±

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0.04), with further (P 0.01) dip at theretail shop (6.20 ± 0.03).

pH has been adjudged as a good physicalcharacteristic and is a reliable indicator forfreshness evaluation in fish, as there isremarkable correlation between these twocharacteristics (Abbas et al., 2008).Dhanapal et al. (2012) had reported anaverage pH of 6.8 ± 0.02 of raw Tilapiafish samples. Obemeata and Christopher(2012) had reported pH of 6.8 ± 0.41 infresh Tilapia fish that declinedsubsequently during storage. The pH offreshwater Tilapia fish has been reportedas 6.11 ± 0.12 (Kapute et al., 2012) and6.87 in catla (Rahman et al., 2012).

The decrease in pH value a few hours afterharvest indicate that fish have beenstressed during harvest. The typical pHof live fish muscle is approximately 7.0that declines in struggling fish due todepletion and metabolization of glycogenreservoirs of stressed fish muscles (Abbaset al., 2008).

Increase in acidity (i.e., drop in pH) hasbeen observed in Silver Catfish duringincreased storage interval due to bacterial(Lactobacil lus sp, Proteus spp,Staphylococcus aureus , Staphylococcusepidermis, Bacillus sp) contaminationexceeding 5.0 × 105 ICMSF standard forsafe fish product with strong negativecorrelation between pH and Free FattyAcid (r = - 0.121, P 0.01) and betweenPeroxide Value and pH (r = -0.313,P0.05), with strong positive correlation

(r = 0.97, P 0.01) between PV and FFA(Oyelese et al., 2013).

Thiobarbituric acid value (TBA): Therewas significant (P0.01) difference in TBA(mg/kg MDA) value at various points ofthe supply chain. The TBA (mg/kg MDA)value (Table-2, Figure-2) was the lowest(0.65 ± 0.06) at the point of harvest, andincreased significantly (P0.01) duringtransportation (1.33 ± 0.18), with furtherenhancement (P0.01) at the retail shop(2.54 ± 0.23). This may be due to increasedoxidative changes in fish muscles due totime factor.

Fish is an extremely perishable foodbecoming inedible within twelve hours attropical temperatures and spoilage beginsas soon as the fish dies. Lipid oxidation isa major cause of spoilage of fish.Oxidation of fish lipids, which consist ofhigh level of poly saturated fatty acidcause rancidity. The rancidity of oils in fishdenotes fish spoilage. Rancidity refers tounpleasant taste and smell of fatty foodsthat have undergone decomposition,liberating butyric acid and other volatilelipids. The keeping quality of fish isjudged by assessing the thiobarbituric acid(TBA) value of fish, evaluated as MDA(Ukekpe et al., 2014).

Earlier, Dhanapal et al. (2012) hadreported TBA value of 0.77 ± 0.01 mg/kgMDA of raw Tilapia fish collected fromMuthukur reservoir in Andhra Pradesh.Storage of fish under ambient conditionenhances the TBA value. Similar

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observations were recorded in presentstudy.

Total viable counts (TVC): There wassignificant (P 0.01) difference in TVC(Log CFU/gm) value at various points ofthe supply chain. The TVC value (Table-2, Figure-3), was the lowest (6.18 ± 0.03)at the point of harvest, and increasedsignificantly (P 0.01) duringtransportation (6.36 ± 0.04), with furtherenhancement (P0.01) at the retail shop(6.57 ± 0.02) indicating increasedcontamination with the advancement ofthe supply chain.

Microbial quality with respect to specificspoilage bacteria is a reliable indicator offish spoilage and consequently the shelf-life of fish (Huss, 1995). It is important,because microbiological shelf-life isshorter than organoleptic shelf-life in thesame fish (Humaid and Jamal, 2014). Fishspoilage is caused by microbial enzymes,particularly proteolytic enzymes(Engvang and Nielsen, 2001).

Bacteria colonising the skin, gills andintestines are usually harmless for livefish, and begin to replicate rapidly afterits death (Shamsuzzaman et al., 2011). Thefish could be contaminated after beingcaught or during transportation to retailmarkets. After contamination andreplication of microorganisms, decayoccurs and the consumption of decayedfish can be dangerous (Mol and Tosun,2011; Alparslan et al., 2014).

Many workers (Jayasekaran andAyyappan, 2003; Surendraraj et al., 2009;Mandal et al., 2009; Khatun et al., 2011;Hasan et al., 2012; Iqbal and Saleemi,2013; Sujatha et al., 2013) havesuccessfully used TVC counts forassessment of microbial quality of fishat different stages of supply chain. Ourresults are similar to Jannat et al. (2007),who have reported TVC values of 6×107

cfu/gm at harvest in raw hilsha fish.

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CONCLUSION

It is concluded that freshwater fish (Catlacatla) was of extra good grade at the pointof harvest, but the quality deterioratedduring transport and at the retail shop.

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Author attribution:1Research Scholar, 3Asst. Professor, FRM Division, 4,5Asst. Professor, FPTMDivision, College of Fisheries, Shirgaon, Ratnagiri, Maharashtra, India-415629, 2Curator (Asst.Professor), 6Asst. Research Officer, Marine Biological Research Station, Zadgaon, Ratnagiri,Maharashtra, India-415612. 2Corresponding author (E-mail: [email protected]) Received: 19March 2016, Accepted: 20 August 2016. pp.132-142

ELICITATION AND CHARACTERIZATION OF SKIN GELATIN OFMALABAR SOLE FISH AT DIFFERENT EXTRTACTION TEMPERATURES

R.R. Sawant1, S.Y. Metar2, V.H. Nirmale3, J.M. Koli4, V.V. Vishwasrao5, S.B. Satam6

ABSTRACT

Gelatin, derived from thermal denaturation of collagen from skin, skeleton andmuscles of mammals, birds and fish, has myriads of industrial applications. Fish ispreferred over mammals due to universal acceptability. There have been many studieson gelatin yield and properties in many species of fish, but none in Malabar sole fish(Cynoglossus macrostomus), which is a low value fish, and thus a more economicalsource of gelatine. This study was undertaken on yield, proximate composition, andphysical properties (colour and clarity) of gelatine, extracted from the skin of Malabarsole fish at three thermal schedules (40 0C, 45 0C, 50 0C). The results revealed significant(P0.05) difference in gelatin yield (%) between the three temperature groups. Theyield (%) of gelatin at 45 0C (8.32±0.03) was significantly (P0.05) higher than at 40 0C(6.9±0.02), but did not differ (P0.05) from 50 0C temperature group (7.47±0.04). Thedifference between the latter two was non-significant (P 0.05). The proximatecomposition (%) of gelatin showed significant (P0.01) variation at various extractiontemperatures with respect to all the proximate principles (moisture, protein, fat, ash).The protein content was maximum (88.73±0.04), and fat (1.30±0.10) and ash (1.70±0.03)contents were minimum at 45 0C extraction temperature. There was significant(P0.05) variation in the colour of gelatin pertaining to lightness (L*), redness (a*),and yellowness (b*) at different extraction temperatures. The colour of the extractedgelatin was predominantly light in colour (L*), and the lightness value was significantly(P0.05) higher at 450C (91.38±0.03) than at 40 0C and at 50 0C. The transmittance (%)was significantly (P0.05) higher at 450C (78.45±0.02) than at 40 0C and at 50 0C. Thestudy tends to conclude that gelatin yield, proximate principles (protein, fat, andash), gelatin colour and gel clarity were better at 450C extraction temperature than at40 0C and at 50 0C.

KEY WORDS

Extraction temperature, Gelatin, Malabar sole fish, Proximate composition,Physical properties, Skin

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INTRODUCTION

Gelatin is a hydrolyzed derivative ofcollagen, obtained by thermaldenaturation, and is the main structuralprotein present in the extracellular spacein various connective tissues of skin,bones, muscles, tendons, cartilage, andligaments of mammals, birds, and fish,and in edible insects, and is widely usedin food industry as a food supplementwith anti-aging and anti-inflammatoryproperties, since it contains almostseventeen essential amino acids that arenormally missing in the daily diet, besidesmyriad industrial applications includingcosmetology and medicine (Wasswa et al.,2007; Mariod and Adam, 2013).

The gelatin yield of fish skin has beenreported to be higher than from the bones(Koli et al. (2011). Moreover, the gelatinderived from fish skin contains moreprotein and less fat and ash than that ofbone gelatin (Sanaei et al., 2013).

Mammalian gelatins (porcine andbovine), although immensely popularand widely used, are subject toconstraints and skepticism due tosocio-cultural inhibit ions andepidemiological concerns, while fishgelatin grasp universal acceptability(Karim and Bhat, 2009). Fish gelatin(especially from warm-water fish)reportedly possesses s imilar

characteristics to porcine gelatin andis considered as a good substitute tomammalian gelatin for use in foodproducts (Jakhar et al., 2012; Killekaret al., 2012). Fish gelatin is howeversignificantly (P 0.05) superior tomammalian (bovine and porcine)gelat in in organolept ic qualities ,particularly with respect to the odourcharacteristics (Ninan et al., 2011).

Fishes belonging to the familyCynoglossidae, popularly known as solefish is a major by-catch of the shrimptrawler, and is a good and acceptablesource of gelatine world wide. The catchof sole fish in India during the year 2012was estimated as 61,859 tons (CMFRI,2013). Malabar sole fish (Cynoglossusmacrostomus), which is a low value fish, isthe predominant fish among all thespecies of flatfishes landed along the westcoast of India, in spite of paucity oftargeted fisheries for the species. With theincrease in targeted fisheries for shrimps,this species is also being heavily fished.

The yield, proximate composition, andphysical properties of fish skin gelatinhave been studied in different fish speciesby different extraction methods, atdifferent extraction temperatures, and atdifferent time intervals etc. But, very littlework has been done on these aspects inMalabar sole fish (Figure-1).

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This s tudy was undertaken todetermine the gelat in y ie ld (%),proximate composition (Moisture,Protein, Fat, and Ash), and physicalproperties (colour and clarity ) ofgelatin obtained from the skins ofMalabar sole f ish (Cynoglossusmacrostomus) at different extractiontemperatures (40 0C, 45 0C, 50 0C) dueto paucity of research work in thisdirection.

Figure-1. Malabar sole fish (Cynoglossusmacrostomus).


Malabar sole fish for the experiment wascollected from Mirkarwada landingcentre, Maharashtra. The skin wasremoved manually. The skin waswashed with clean water, and stored at–20 0C until further use. Gelatin wasextracted following the proceduredescribed by Koli et al. (2011) as per flowchart (Figure-2).

Gelatin yield (%) was calculated as(weight of dry gelatin/ wet weight of raw

fish skin) x 100, based on three replicatesat three extraction temperatures (40 0C,45 0C, and 500C). Proximate compositionwas carried out as per AOAC (2005).

Colour measurement was made by usinga Hunter Lab Scan XE colorimeter(Hunter Association Laboratory, Inc.,VA, USA). The tristimulus L*a*b*measurement mode was used as it relatesto the human eye response to colour. TheL* variable represents lightness (L* =0 forblack, L* =100 for white). The a* scalerepresents the red/green (+a* intensityof red and -a* intensity of green). The b*scale represents the yellow/blue (+b*intensity of yellow and -b* intensity inblue). The samples were filled into clearPetri dishes and readings were taken.

Clarity was determined by measuringtransmittance (%T) at 620 nm inspectrophotometer (Thermospectronic,Cambridge, U.K) through 6.67% (w/v)gelatin solution which were heated at600C for 1 h (Avena-Bustillos et al., 2006).

The data were analyzed usingappropriate statistical methods(Snedecor and Cochran, 1967, Zar, 1999).The differences between the treatmentswere determined by ANOVA. Thedifferences between the means oftreatments (P 0.05/0.01) were furthersubjected to SNK test.

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Figure-2. Flow chart: Extraction protocol of fish skin gelatin at differenttemperatures.

Fish skin

Washing with clean water to remove any suspended particles from skin

Treatment with 0.2% NaOH for 40 min (Repeated three times after draining the alkali)

Repeated washings with clean water

Treatment with 0.2% H2SO4 for 40 min (Repeated three times after draining the acid)

Treatment with 1% citric acid for 40 min (Repeated three times after draining the acid)

Rinsed with clean water

Rinsed with distilled water

Extraction of gelatin liquid in distilled water overnight at 40 0C, 45 0C, and 50 0C

Filtration of gelatin liquid extract through Whatman No.1 filter paper with Buchner Funnel Drying of gelatin filtrate in oven at 60 0C for 16 h

The thin film of dried gelatin was powdered, weighed, and packed in zip packaging bags

Storage of gelatin at ambient temperature (25±2 0C)


Gelatin yield: The differences in gelatinyield were significant (P0.05) betweenthe three temperature groups (Table-1,Figure-3). The gelatin yield (%) at 45 0C(8.32±0.03) was significantly (P 0.05)higher than at 40 0C (6.9±0.02), but did notdiffer (P0.05) from 50 0C (7.47±0.04). Theresults indicated that the yield of gelatinwas the maximum at 45 0C extractiontemperature.

Table-1. Yield of extracted gelatin fromskin of Malabar sole fish at differenttemperatures.

Factor Extraction Temperature40 0C 45 0C 50 0C

Yield (%) 6.9±0.02a 8.32±0.03b 7.47±0.04b

Note: (1) The figures are presented as Mean± SEM, and are based on three replicates. (2)The means with different superscriptsa,b,c in arow differed significantly at P 0.05.

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Gelatin yield have been reported to varyamong the fish species mainly due todifferences in skin collagen content, aswell as the skin matrix (Silva et al.,2014). I t may also happen due toincomplete hydrolysis of the collagenduring extraction (Jamilah andHarvinder, 2002). Variations in the yieldhave also been reported due to diversityin extraction methods (Gomez-Guillenet al., 2002; Jamilah and Harvinder,2002; Muyonga et al., 2004).

Gudmundsson and Hafsteinsson (1997)had recorded 14% yield of gelatin of codfish. Jamilah and Harvinder (2002) hadreported that the yields of red tilapiagelatin and black tilapia gelatin were7.81% and 5.39% respectively. Killekar etal. (2012) had reported 13.88% gelatinyield from skin of black kingfish. Koli etal. (2011) had reported that the yields ofgelatin from tiger-toothed croaker(Otolithesruber) and pink pearch(Nemipterus japonicus) were 7.56% and

Table-2. Proximate composition (%) of Malabar sole fish skin gelatin extracted atdifferent temperatures.

5.57% respectively, while the yields frombones were 4.57% and 3.55%respectively. Wangtueai and Noomhorm(2009) had reported 9.51% yield of gelatinfrom lizard fish (Saurida spp.) scales.Kittiphattanabawon et al. (2010) hadreported that the yield of gelatin fromthe skins of brown banded bambooshark (BBS) and black tip shark (BTS)extracted using different conditionsranged from 19.06 to 22.81% and from21.17 to 24.76%, respectively. Ninan etal. (2011) had reported that the gelatinyield of rohu and common carp were12.93% and 12%, respectively.

Component Extraction Temperature 40 0C 45 0C 50 0C

Moisture 9.78±0.034a 8.26±0.307b 7.66±0.02b Protein 86.57±0.08a 88.73±0.04b 87.74±0.04c Fat 1.51±0.06a 1.30±0.10a 2.15±0.08b Ash 2.12±0.04a 1.70±0.03b 2.45±0.06c Note: (1) The figures are presented as Mean ± SEM, and are based on three replicates. (2) The

means with different superscriptsa,b,c in a row differed significantly (P 0.01).

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Proximate composition: The proximatecomposition (%) of gelatin showedsignificant (P0.01) variation at variousextraction temperatures with respect tomoisture, protein and fat (Table-2).

Moisture content (%) was the highest at40 0C (9.78±0.034). It was significantly(P0.01) higher than the moisture contentat 45 0C (8.26±0.307) and at 50 0C(7.66±0.02). The difference between thelatter two was non-significant (P0.05).Moisture content was minimum at 50 0C.

Protein content (%) was the highest at 450C (88.73±0.04). It was significantly(P0.01) higher than the protein contentat 40 0C (86.57±0.08) and at 50 0C(87.74±0.04). The difference between thelatter two was also significant (P0.01).The results indicated that protein yieldwas the highest at 45 0C extractiontemperature.

Fat content (%) was the highest at 50 0C(2.15±0.08). It was significantly (P0.01)higher than the fat content at 40 0C(1.51±0.06) and at 45 0C (1.30±0.10). Thedifference between the latter two was non-significant (P0.05). The results indicatedthat the lowest fat content was obtainedat 45 0C extraction temperature.

Ash content (%) was the highest at 50 0C(2.45±0.06). It was significantly (P0.01)higher than the ash content at 40 0C(2.12±0.04) and at 45 0C (1.70±0.03). Thedifference between the latter two was alsosignificant (P0.01). The results indicatedthat the lowest ash yield was obtained at45 0C extraction temperature.

Our results indicated that maximumprotein content, and minimum fat and ashcontents were obtained at 45 0C extractiontemperature.

Our results on moisture content of solefish skin gelatin ranged between 7.66% (500C) and 9.78% (40 0C), which were wellbelow the prescribed limit of moisturecontent for edible gelatin, i.e., 15% (GME,2012). It was higher than the reportedvalue of 6.04% in Black kingfish, extractedat 45 0C (Killekar et al., 2012), but wassimilar to tilapia fish (8.71%) and snapperfish (7.63%), as reported by Pranoto(2006).

The moisture content varied not only withthe extent of drying, but also with thehumidity during storage (Ockerman andHansen, 1988). However, gelatin with low(6-8%) moisture content is veryhygroscopic and is a hurdle indetermining the physiochemicalattributes with accuracy (Cole, 2000).

Our results on protein content of sole fishskin gelatin at different extractiontemperatures ranged between 86.57% (400C) and 88.73% (45 0C). Killekar et al.(2012) had reported that the proteincontent of Black kingfish skin gelatin was88.72% at 45 0C. The protein content ofadult Nile perch skin gelatin was 88.8%at 50 0C (Muyonga et al., 2004). Theprotein content of red tilapia gelatin wasreported to be 89.70% (See et al., 2010).The protein contents of skin gelatin ofbigeye snapper and brown eye snapperfishes were 87.9% and 88.6%, respectively(Jongiareonrak et al., 2006). The protein

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content of gelatin extracted from scales oflizardfish (Saurida spp.) was reported tobe 86.9% (Wangtueai and Noomhorm,2009). The protein contents of gelatinextracted from the skin of tiger toothedcroaker and pink perch were 86.45% and72.63%, respectively at an extractiontemperature of 450 C indicating variationbetween the species (Koli et al., 2011).

Our results on fat content of sole fish skingelatin at different extractiontemperatures ranged between 1.3% (450C) and 2.15% (50 0C). The fat content oftilapia skin gelatin has been reported tobe 0.25% (Alfaro et al., 2013). The fatcontent of Ghol fish skin gelatin has beenreported to be 0.27% (Jakhar et al., 2012).The fat content of skin gelatin in cobrafish (1.6%) and croaker fish (0.6%) werereported by Silva et al. (2014). The fatcontent of the skin of catfish, pangasiuscatfish, snakehead, and red tilapiaranged between 0.47% and 2.63%, whileit was much lower (0.18%) in coldwaterfish (See et al., 2010). Apart from speciesvariation, defattening treatments likedegreasing of skin and chemicaltreatment before extraction can reduce fatcontent of gelatin (See et al., 2010; Shyaniet al., 2014; Ninan et al., 2015).

Our results on ash content of sole fishskin gelatin at different extractiontemperatures ranged between 1.7% (450C) and 2.45% (50 0C). In fact, ashreflects the mineral status. Thesevalues were less than therecommended maximum limit of 2.6%(Jones, 1977) and the limit given foredible gelatin i.e. 2% (GME, 2012). Lowash content sugges ted that theextracted gelatin was of high quality,as the ash content for a high qualitygelat in should be lower than 2%(Ockerman and Hansen, 1988).

The ash content of skin gelatin of blackkingfish has been reported to be 2.24%at 45 0C extraction temperature (Killekaret al., 2012). The ash content of skingelatin of tilapia (1.64%) and snapper(1.17%) were reported by Pranoto (2006).The reported ash content of skin gelatinof croaker and pink perch were in therange of 0.30-1.88% (Koli et al., 2011).Ash content can be reduced by pre-treatment of raw skin with chemicals(Ninan et al., 2011).

Physical properties: The physicalproperties pertaining to gelatin colour andgel clarity is depicted in Table-3.

Table-3. Gelatin colour and Gel clarity of extracted gelatin from skin of Malabarsole fish.Gelatin colour & Gel clarity Extraction Temperature

40 0C 45 0C 50 0C Lightness (L*) 88.97±0.55a 91.38±0.03b 89.76±0.04c Redness (a*) 1.78±0.2a 1.87±0.03b 2.15±0.02b Yellowness (b*) 4.28±0.05a 2.98±0.05b 3.58±0.04c Transmittance (%) 63.92±0.06a 78.45±0.02b 69.38±0.03c Note: (1) The figures are presented as Mean ± SEM, and are based on three replicates. (2) The

means with different superscriptsa,b,c in a row differed significantly (P 0.05).

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Gelatin colour & Gel clarity: Gelatincolour with respect to Lightness (L*),Redness (a*) and Yellowness (b*), andgel clarity as Transmittance (%) showedsignificant (P0.05) difference at differentextraction temperatures (Table-3).

There was s ignif icant (P 0.05)variation in the colour of skin gelatinof sole fish pertaining to lightness(L*), redness (a*), and yellowness (b*)at different extraction temperatures.The colour of the extracted gelatin waspredominantly light in colour (L*), andthe lightness value was significantly(P 0.05) higher at 450C (91.38±0.03)than at 40 0C (88.97±0.55) and at 50 0C(89.76±0.04). The difference betweenthe latter two was significant (P 0.05).

The transmittance (%) was significantly(P0.05) higher at 450C (78.45±0.02) thanat 40 0C (63.92±0.06) and at 50 0C(69.38±0.03). The difference between thelatter two was significant (P0.05).

Colour and clarity of gelatin gel areimportant functional properties forcommercial application. Consumersusually like pale colour than darkcolours, because lack of colour isconsidered to be associated with purity(Cole and Roberts, 1997).

The turbidity and dark colour of gelatinis commonly caused by inorganic

compounds, protein and mucosubstancecontaminants, acquired or not removedduring its extraction (Eastoe and Leach,1977). When protein is treated for a longtime at high temperature, aggregation isactivated and turbidity is increased(Johnson and Zabik, 1981). Increase inmolecular weight of aggregates can alsoincrease turbidity (Montero, et al., 2002).

Jamilah and Harvinder, (2002) hadreported 92.35 L* and -0.47 a* in the skingelatin of Red tilapia, and 93.32 L*, -0.56a* in the skin gelatin of Black tilapia.

See et al. (2010) had reported 44.36 L*,0.56 a* and -3.65 b* in the skin gelatinof Catfish, 40.40 L*, 0.71 a* and 2.86b* in red tilapia, and 91.89 L*, 0.35 a*and 2.76 b* in Rohu.

Koli et al. (2011) had reported 75.41 L*,2.79 a*, and 19.25 b* for gelatin colour, and49.43%T for gel clarity in Tiger-toothedcroaker, 71.74 L*, 2.74 a*, and 22.07 b* forgelatin colour, and 44.30%T for gel clarityin pink pearch, and 65.44 L*, 1.65 a*, and22.50 b* for gelatin colour, and 40.50%Tfor gel clarity in Tiger-toothed croaker.Ninan et al. (2011) had reported 90.15 L*,0.41 a* and 1.82 b* in the skin gelatin ofcommon carp. Killekar, et al. (2012) hadreported 86.76 L*, 2.33 a*, and 5.16 b*, forgelatin colour, and 41%T for gel clarity inBlack kingfish skin gelatin.

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CONCLUSION

The study revealed that gelatin yield,proximate principles (protein, fat, ash),gelatin colour and gel turbidity at 450Cextraction temperature were better thanat 40 0Cand 50 0C in Malabar sole fish.

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Author attribution: 1Associate Professor, 2Professor, Department of Dairy Chemistry,3Associate Professor, Department of Dairy Engineering, West Bengal University of Animaland Fishery Sciences, Mohanpur, Nadia, West Bengal, India-741252. 4PhD Scholar, DairyChemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India-132001.1Corresponding author (E-mail: [email protected]). Received: 9 April 2016, Accepted:24 August 2016. pp. 143-148

ELECTROPHORETIC CHARACTERIZATION OF CASEIN OFREFRIGERATED COW AND BUFFALO MILK PRESERVED WITH BANANA

PSEUDOSTEM JUICE

P.R. Ray1, P.K. Ghatak2 , S.K. Bag3, S. Maji4

ABSTRACT

Cow milk, a highly nutritious food item, is amenable to quality degradation due tolipolysis and proteolysis, as a sequel to the growth of spoilage microorganisms,particularly under ambient condition in tropical countries, with consequential loss ofaesthetic appeal and product output. Prescription antibiotics used for milkpreservation have many undesirable side effects on the health of consumers, besidesbolstering development of antibiotic resistant strains of bacteria. This paper has triedto elucidate the effect of treatment of raw cow milk and buffalo milk stored underrefrigeration with Banana (Musa paridasiaca) Pseudostem Juice (BPJ), a naturalantimicrobial agent on the electrophoretic profile of whole casein of milk, since BPJcan checkmate proteolysis with consequential alteration of molecular mass, on whichno study has been done earlier. Fresh cow milk and buffalo milk, procured from localmilk producers were treated with 0.3% (v/v) BPJ, and were kept under refrigerationat 7+2 0C for 5 days and 4 days respectively, while BPJ untreated (control) samples ofcow and buffalo milks were kept at same temperature for 3 days and 2 daysrespectively. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) profile of control and BPJ treated cow milk samples were determined after 3days and 5 days respectively, whereas the same was done for buffalo milk samplesafter 2 days and 4 days respectively. The SDS-PAGE pattern revealed that the wholecaseins of BPJ treated cow and buffalo milk samples resolved in to two bands of lowmolecular weight components ranging between 29 kD and 43 kD. The SDS-PAGEpattern of isolated whole caseins of BPJ treated cow milk and buffalo milk did notexhibit any deviation from control cow and buffalo milk caseins. It is concluded thataddition of BPJ (0.3%, v/v) to cow milk and buffalo milk did not alter the molecularconfiguration of milk casein under refrigerated (7+2 0C) preservation for 5 days and4 days respectively due to its inhibitory effect on proteolysis.

KEY WORDS

Banana Pseudostem Juice, Electrophoresis, Milk Casein

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INTRODUCTION

Cow milk is a highly nutritious food item,containing a variety of nutrients such asfats, proteins, carbohydrates, minerals,vitamins, and water, but extremelyperishable, since it also confers anexcellent medium for the growth ofspoilage microorganisms, particularlyunder ambient condition in tropicalcountries, leading to loss of aestheticappeal and product yield, especiallycheese (Prescott, 1999; Anderson et al.,2011; Ramos et al., 2015).

Spoilage behavior is attributed toincreased lipolysis as well as proteolysis.Proteolysis, which is the focus of thisstudy is caused by higher production ofprotease due to microbial action causingoff-flavour of milk and milk products,besides reducing the alpha-, beta- andkappa- casein fractions with consequentalteration of their molecular masses, andleading to industrial imbroglio due todecrease in cheese output (Deeth et al.,2002; Santos et al., 2003; Zhanq et al., 2013;Ramos et al., 2015).

A common practice, adopted by ruraldairy farmers of West Bengal andBangladesh to preserve raw milk isaddition of Banana (Musa paridasiaca)Ps eudo stem Juice (BPJ ) to che ckspoilage during transportation under

am bien t con di t io n du e t o no n-availability of transport vehicles withfreezer facility. The authenticity ofthis practice has been scientificallyva lida te d, as B PJ ha s pr ov enantimicrobial properties, presumablydue to its high tannin content (Duke,1985; Scalbert, 1991; Chung et al. ,1998; Ray, 2008; Ray and Ghatak,2013; Ray et al., 2015).

Earlier studies (Ray et al., 2015) haveindicated no-change in the lipid profile ofraw cow milk and buffalo milk storedwith BPJ at ambient temperature (30 ±20C). However, there is no study onproteolytic changes and molecularcharacteristics of milk casein of the milkstored with BPJ as preservative, underrefrigeration, since rural households inWest Bengal and Bangladesh generallystore BPJ added milk in refrigerator forpreservation. However, an earlier workhas revealed that some protein fractionsin semi-hard cheese samples duringMucor induced ripening were degradedinto peptides of low molecular weights(Zhanq et al., 2013).

The present study was conducted toevaluate the effect of addition of BPJ(0.3%, v/v) on the shelf-life andelectrophoretic profile of whole caseinunder refrigerated (7+2 0C) storage.

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Collection of milk: Fresh cow and buffalomilk samples were procured from thelocal milk producers.

Extraction of Banana Pseudostem juice:Banana pseudostem juice (BPJ) wasextracted, following the proceduredescribed by Biswas (2004) and Bharti(2005) with slight modification.

Martaman variety of banana (Musaparidasiaca) plant psudostem was choppedin to small pieces, and put in to a handdriven mechanical juicer. The pseudostempieces were subjected to high pressure inthe juicer and the juice coming out of thepseudostem was collected, and thenfiltered by Whatman filter paper no. 40 toobtain a clean juice. Banana pseudostemjuice was added to milk at the rate of 0.3%(v/v) as per Ray (2008).

Preservation of milk: Bananapseudostem juice 0.3% (v/v) treated cowand buffalo milk samples were kept underrefrigeration at 7+2 0C for 5 days and 4days, respectively (Ray, 2008). Controlsamples of cow and buffalo milks werealso kept under same temperature for 3days and 2 days respectively. PAGEprofile of control and treated cow milksamples were determined after 3 days and5 days respectively, whereas the same wasdone for buffalo milk samples after 2 daysand 4 days respectively.

Preparation of Casein: Casein wasobtained by adding 10% acetic acid to milkadjusting the pH at 4.6. Milk sampleswere diluted with distilled water at 1:1ratio and warmed at 350C before additionof acid. Coagulum was filtered withWhatman no. 42 filter paper, washed withwater, alcohol, petroleum ether, and driedsubsequently. One (1) mg of each driedsample of casein was dissolved in 500µlsample buffer and applied in to the lanesof PAGE.

Determination of PAGE profile of milkcasein: Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) of cow and buffalo milk casein wascarried out according to the method ofLaemmli (1970). A GENEI make RODGELelectrophoretic apparatus equipped withpower pack for maintaining constantvoltage was used to determine theelectrophoretic pattern. Bangalore Geneimake standard medium range proteinmarkers of five different molecularweights (14.3 kD, 29 kD, 43 kD, 68 kD,97.4 kD) were used to compare theelectrophoretic profile of milk caseinsamples.

Stacking Gel was prepared by using TrisHCl (pH6.8) , 10% w/v SDS, acrylamide/BIS acryl amide ( 30%, 0.8%, w/v),Ammonium persulfate (10% w/v) anddistilled water. Separating Gel wasprepared by using acrylamide/BIS acryl

146


amide (30%, 0.8%, w/v), 1.5 M TRIS (pH8.8), 10% w/v SDS, 10% w/vAmmonium persulfate and distilledwater. Staining solution was prepared byusing amido black, glacial acetic acid,methanol and distilled water, whereasdestaining solution was prepared byusing methanol, acetic acid and distilledwater. Gel preparation was completedwithin 20-30 minutes.

20 µl of marker and 40 µl of each samplewere applied in to the lane bymicropipette. Gel tubes were fixedvertically between upper and lowerelectrode of the electrophoresis apparatusand subjected to constant electric supplyof 9 mA for 3 hours after which gel tubeswere taken out. The gel was stainedovernight in Commassie Brilliant Bluesolution and was destained by repeated

Figure-1. (I) Electrophoregram of milk protein: (A) standard marker. (B) untreated cow milk. (C)banana pseudostem juice treated cow milk. (D) untreated buffalo milk. (E) banana pseudostem juicetreated buffalo milk. (II) Bands (1 and 2) represent casein band of molecular weights between 29 kDand 43 kD.

A B C D E

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CONCLUSION

It is concluded that addition of BPJ (0.3%,v/v) did not alter the molecularconfiguration of milk casein of raw cowmilk and buffalo milk stored underrefrigeration (7+2 0C) for 5 days and 4 daysrespectively due to its proteolysisinhibitory effect.

ACKNOWLEDGEMENT

The authors are thankful to the ViceChancellor, West Bengal University ofAnimal and Fishery sciences, Kolkata forproviding necessary infrastructurefacilities to carry out the work.

REFERENCES

Anderson, M et al. 2011. The microbialcontent of unexpired pasteurized milkfrom selected supermarkets in adeveloping country. Asian Pacific Journalof Tropical Biomedicine, 1 (3), 205-211.

Bharti, B. 2005. Preservation of raw milkthrough Pseudostem Juice of Banana Tree.MSc Thesis, West Bengal University ofAnimal and Fishery Sciences, Kolkata.

Biswas, P. 2004. Effect of BananaPseudostem Juice on water quality andfish growth: A study on the validation ofindigenous technical knowledge. MScThesis, West Bengal University of Animaland Fishery Sciences, Kolkata.

rinsing in the destaining solution till theseparated components becameprominent. After de staining, gels wereanalyzed under Gel DocumentationSystem (BIORAD make).


The electrophoretic (SDS-PAGE) behaviorof cow and buffalo milk samples ispresented in Figure-1. It revealed theappearance of two low molecular weightcaseins in untreated cow milk as well asbuffalo milk, so also in 0.3% (v/v) bananapseudostem juice (BPJ) treated cow milkand buffalo milk ranging between 29 kDand 43 kD, as compared to five molecularweight components (97.4 kD, 68 kD, 43kD, 29 kD and 14.3 kD) in downwarddecreasing order, in the standard markersamples.

The SDS-PAGE pattern of isolated wholecaseins of BPJ treated cow milk andbuffalo milk did not exhibit any deviationfrom control cow and buffalo caseins. Nonavailability of data on the effect of bananapseudostem juice on the PAGE pattern ofcow and buffalo milk caseins made itdifficult to compare the results of thepresent finding. However, the report ofKumar and Mathur (1989) regardingnegligible changes in various nitrogenfractions during preservation of buffalomilk under lactoperoxidase system wasin accordance with the present finding.

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Chung, KT et al. 1998. Tannins andhuman health: a review. CriticalReviews in Food Science and Nutrition,38 (6), 421-464.

Deeth, HC et al. 2002. Spoilage patternsof skim and whole milks. Journal of DairyScience, 69 (2), 227-241.

Duke, J .A. 1985. Handbook ofMedicinal Herbs. CRC Press, Inc., BocaRaton, Fla, U.S.A.

Kumar, S.; Mathur, B.N. 1989. Incidenceof lactoperoxide system on milkconstituents during preservation ofbuffalo milk. Part I. Ion exchangechromatographic and electrophoreticbehaviour of casein. Indian Journal ofDairy Science. 42, 185-189.

Laemlli, U.K. 1970. Cleavage of structuralproteins during the assembly of the headof bacteriophage T4. Nature, 227, 680-685.

Prescott, L.M. 1999. Microbial nutrition,growth and control and microbialdiseases and their control. In:Microbiology (Prescott, L.M.; Harley,J.P.; Klein, D.A.; eds). WCB/McGrawHill, Boston, 107–110.

Ramos, TM et al. 2015. Effect of somaticcell count on bovine milk proteinfractions. Journal of Analytical andBioanalytical Techniques, 6 (5), 1000269.

Ray, P.R. 2008. Preservation of raw milkand Paneer using Banana Pseudostemjuice. PhD Thesis, West Bengal Universityof Animal and Fishery Sciences, Kolkata.

Ray, PR et al. 2015. Effect of addition ofbanana pseudostem juice on fatty acidprofile of cow and buffalo milk duringstorage. Indian Journal of AnimalHealth, 54 (1), 27-36.

Ray, P.R. ; Ghatak, P.K. 2013.Phytochemical characterization andantimicrobial property of banana (Musaparidasiaca) pseudostem juice.International Journal of AgriculturalScience and Veterinary Medicine, 1 (4),November 2013.

Santos, MV et al. 2003. Effect of somaticcell count on proteolysis and lipolysisin pasteurized fluid milk during shelf-life storage. Journal of Dairy Science, 86(8), 2491-2503.

Scalbert , A. 1991. Antimicrobialproperties of tannins. Phytochemistry,30, 3875-3883

Zhanq, N et al. 2013. A briefinvestigation on the proteolysis andtextural modification of a semi-hardcheese ripened by Mucor spp.Biotechnology, 12 (1), 54-60.

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Author attribution: 1MVSc Scholar, 2Asst. Professor (LPM), 3Whole time teacher, AnimalNutrition, Ranchi Veterinary College (Birsa Agricultural University), Kanke, Ranchi,Jharkhand, India-834006, 4Touring Veterinary Officer, Saphi, Nawada, Bihar, India-805125.3Corresponding author (E-mail: [email protected]). Received: 13 April 2016, Accepted:27 August 2016. pp. 149-160

EFFECT OF POLYHERBAL FEED ADDITIVES ON GROWTH AND FEEDCONVERSION EFFICIENCY IN PIGS

A.C. Gyani1, Ravindra Kumar2, S.K. Sinha3, Vijay Kumar4

ABSTRACT

The current quest world wide is for invention and promotion of alternative growthpromoters as a substitute to antibiotic growth promoters, which are believed to developdrug resistance due to overuse. We have tried in this paper to elucidate the effect ofsix herbal growth promoter feed additives of Ayurvet Pharmaceuticals on growthcharacteristics, feed consumption, and feed conversion efficiency in Tamworth xNative (Desi) grower pigs. A total of 3 months old 42 piglets with an average bodyweight of 14.6±0.6 kg were randomly allocated to 7 treatments with 7 replicates of 6pigs per treatment in a 2×2 factorial design. The treatment groups were T1 (control),T2: Bacteriostatic growth promoter-1 (BGP-1), T3: Bacteriostatic growth promoter-2(BGP-2), T4: Antistressor & Immunomodulator growth promoter (AIGP), T5: Livertonic & Growth promoter Feed mash (LTGP-Mash), T6: Liver tonic & Growth promoterFeed bolus (LTGP-Bolus), and T7: Liver tonic & Growth promoter Syrup (LTGP-Syrup). The duration of the experiment was 6 fortnights (90 days). The charactersstudied were body weights (kg), average daily gain (ADG) in body weight (g), bodymeasurements (cm), viz., Body length, Chest girth, and Height at withers, feedconsumption (kg), and feed conversion ratio, that defined growth as a function offeed intake (F/G) at fortnightly intervals from 1st to 6th fortnight. The results indicatedsignificant (P0.05) positive effect of LTGP-Mash (T5) on growth characteristics andfeed conversion efficiency compared to the control (T1), and attained the highestbody weight (58.72±0.03), ADG (502.22±1.41), body length (98.50±0.13), chest girth(85.17±0.36), and height at withers (55.83±0.17) with the lowest feed conversion ratio(3.23±0.03) in 6th fortnight. It is concluded that the polyherbal preparation (LTGP-Mash) containing liver tonic & growth promoter herbs (Andrographis paniculata,Achyranthus aspera, Terminalia belerica) administered as feed mash @ 200 g/quintal ofthe basal ration was the most effective herbal product for better growth and feedconversion efficiency in pigs.

KEY WORDS

Body weight, Body measurements, Feed conversion efficiency, Pig, Poly herbaltreatment

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INTRODUCTION

Overuse of antibiotic feed additivesused as growth promoters in foodanimals are dissuaded because ofap pr eh en ded d ev elo pm en t ofantibiotic resistant bacterial strains,and residual effect on the health ofconsumers (Chattopadhyay, 2014).

Phytogenics represent a new andexciting group of feed additives ,originating principally from herbs,spices or other plants. Herbal feedadditives are good alternatives for useas feed addit ives with immensepotential and minimum adverse effect(Hashemi et al, 2011).

The present investigation was plannedto assess the effect of six polyherbalpreparations of Ayurvet Pharmaceuticalon growth characteristics and feedconversion efficiency in Tamworth xNative (Desi) grower pigs (Image-1).

Image-1: Tamworth x Native (Desi) pigs.


Experimental animals: The presentin ve s t iga t io n wa s co n du ct e d o nforty-two Tamworth x Native (Desi)gr owe r p igs of a lmo st sam e a gegroup (three months) and 14-15kgbody weights (Average = 14.6±0.6kg) were randomly allocated to 7treatments with 7 replicates of 6pigs per treatment in a 2×2 factorialdesign procured from Instructionalp i g b r e e d i n g f a r m , R a n c h iVeterinary College (RVC), Kanke,Ranchi.

M a n a g e m e n t : T h e p i g l e t s w e r edewormed 15 days prior to the starto f t h e e x p e r im e n t . T h e a n im a lswere given basal rations, preparedb y m i x i n g d i f f e r e n t f e e di n g r e d i e n t s , p r o v i d e d b yInstructional pig farm, RVC.

T h e p i g l e t s w e r e f e d w e i g h e dq u a n t i t y o f f e e d a d l i b i tu m i nseparate pans. Water was providedad l ibitum .

Polyherbal preparations: Thepolyherbal preparations required for theresearch work was supplied by AyurvetPharmaceuticals. The experimentalanimals were randomly divided intoseven groups (T1, T2, T3, T4, T5, T6, andT7) having six piglets in each group forthe trial.

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Table-1. Feed composition and drug schedule of experimental animals.

Group Treatment Schedule Feeding Schedule T1

Control

T1 was kept as control group maintained on basal ration consisting of 61 parts maize, 14 parts GNC, 16 parts wheat bran, 7.5 parts fish meal, 1 part mineral mixture and 0.5 part common salt.

T2

Bacteriostatic growth promoter-1 (BGP-1)

T2 was given bacteriostatic natural growth promoter consisting of Allium sativum, Zingibero fficinale, Curcuma longa and Terminalia belerica. This group was maintained on basal ration along with 25 gm/quintal growth promoter for 0-60 days.

T3 Bacteriostatic growth promoter-2 (BGP-2)

T3 was given bacteriostatic natural growth promoter (Allium sativum, Terminalia belerica, Woodfordia fruticosa, Zingiber officinale) @ 100g/quintal for 0-60 days along with basal ration.

T4 Antistressor & Immu-nomodulator growth promoter (AIGP)

T4 was given herbal antistressor & immunomodulator (Withania somnifera, Ocimum sanctum, Phyllanthus emblica, Aspaaragus racemosus, Glycerrhiza glabra) @100 g/quintal along with basal ration for 0-60 days.

T5 Liver tonic & Growth promoter Feed mash (LTGP- Mash)

T5 was given liver tonic & growth promoter (Andrographis paniculata, Achyranthus aspera, Terminalia belerica) @ 200g/quintal for 0-60 days along with basal ration.

T6 Liver tonic & Growth promoter Feed bolus (LTGP- Bolus)

T6 was given bolus of herbal liver tonic (Emblica officinale, Terminalia arjuna, Phyllanthus niruri, Eclipta alba, Achyranthus aspera, Tinospora cordifolia, Terminalia chebula) @ 1/2 bolus/piglet/day.

T7 Liver tonic & Growth promoter Syrup (LTGP- Syrup)

T7 was given liquid herbal liver tonic (Emblica officinale, Terminalia arjuna, Phyllanthus niruri, Eclipta alba, Achyranthus aspera, Tinospora cordifolia, Terminalia chebula, Andrographis paniculata.) @ 10 ml/piglet/day along with basal ration.

Feed Composition and Drug Schedule :The feed composition and drug scheduleare presented in Table-1. The polyherbalpreparations were administered to each

treatment group by mixing with basalrations as per the manufacturer’s prescribeddose schedule. The experimentation periodwas of 90 days (6 fortnights) duration.

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Recording of observations: Duringexperimentation, fortnightly Bodyweights (kg), fortnightly average dailygains in body weight (g), fortnightlybody measurements (Length, Chestgirth, and Height at withers, cm),fortnightly feed consumption (kg),and fortnightly feed conversion ratios,that defined growth as a function offeed intake (F/G) of the piglets weremeasured. The feed residues werecollected separately and weighed onnext day in all the groups.

Statistical Analysis: The data wereanalyzed using Analysis of Variance(ANOVA) and the Critical Difference(CD) to determine any significantdifference among the treatment meansas per standard statistical procedures(Snedecor and Cochran, 1989).


T h e p e r f o r m a n ce o f p i g s wi t hr e s p e c t t o b o d y w e ig h t s a n dm e a s u r e m e n t s , g a i n i n bo d yweight, feed consumption and feedconversion rat io are presented inTables 2-9 and figures 1-7.

Table-2. Initial body measurements (cm) and body weight (kg) in different treatmentgroups.

Parameters Treatment Groups T1 T2 T3 T4 T5 T6 T7

Length 58.92±0.24 59.00±0.16 59.00±0.18 59.08±0.15 60.00±0.18 58.92±0.15 59.00±0.22 Girth 53.25±0.21 53.25±0.21 53.33±0.17 53.25±0.21 53.08±0.15 53.50±0.18 53.08±0.15 Height 41.25±0.11 41.25±0.11 41.25±0.11 41.33±0.11 41.25±0.11 41.25±0.11 41.25±0.11 Weight 14.87±0.04 14.93±0.04 15.00±0.04 14.78±0.04 14.97±0.04 14.90±0.04 14.92±0.04 Note: The figures are presented as Mean ± SEM. Means without superscripts in rows were

not significantly different (P 0.05).

Table-3. Fortnightly (ftn) body weights (kg) in different treatment groups.

ftn Treatment Groups

T1 T2 T3 T4 T5 T6 T7 1 20.00±0.10a 20.40±0.04b 20.52±0.04bc 20.68±0.03cd 20.83±0.03d 20.65±0.02c 20.47±0.10b 2 26.42±0.08a 26.87±0.05b 27.12±0.02c 27.53±0.02e 27.73±0.02f 27.38±0.02d 27.03±0.07c 3 33.53±0.09a 33.83±0.11b 34.20±0.03c 34.77±0.03d 35.30±0.16e 34.55±0.03d 34.05±0.06bc 4 41.67±0.11a 42.05±0.26b 42.23±0.06b 43.02±0.04d 43.45±0.06e 42.68±0.03c 42.07±0.04b 5 48.27±0.08a 48.98±0.19b 49.65±0.03c 50.60±0.07e 51.18±0.04f 50.25±0.03d 49.40±0.08c 6 55.35±0.11a 55.98±0.23b 56.78±0.05c 58.08±0.03e 58.72±0.03f 57.57±0.04d 56.48±0.15c

Note: The figures are presented as Mean ± SEM. Means with disparate superscripts in rowsdiffered significantly (P 0.05).

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Table-4. Average daily gain in body weight (g) in different fortnights (ftn) indifferent treatment groups.


Table-5. Body length (cm) in different fortnights (ftn) in different treatment groups.


Table-6. Chest girth (cm) in different fortnights (ftn) in different treatment groups.


ftn Treatment Groups T1 T2 T3 T4 T5 T6 T7

1 333.33 ±5.71a

360.00 ±1.72b

367.78 ±2.05ab

378.89 ±1.11d

388.89 ±1.41e

376.67 ±1.49cd

364.44 ±5.62b

2 427.78 ±3.18a

431.11 ±2.81ab

440.00 ±2.98c

456.67 ±1.49e

460.00 ±1.72e

448.89 ±1.41d

437.78 ±4.10bc

3 474.44 ±2.68ab

464.44 ±5.62a

472.22 ±1.11ab

482.22 ±1.41b

504.44 ±11.24c

477.78 ±2.22ab

467.78 ±2.68ab

4 542.22 ±1.41

547.78 ±19.12

535.56 ±2.22

550.00 ±1.49

543.33 ±12.74

542.22 ±1.41

534.44 ±4.01

5 440.00 ±1.72a

462.22 ±22.94a

494.44 ±3.62bc

505.56 ±2.05bc

515.56 ±1.41c

504.44 ±2.22bc

488.89 ±2.81b

6 472.22 ±3.18a

466.67 ±9.27a

475.56 ±2.81ab

498.89 ±2.68cd

502.22 ±1.41d

487.78 ±2.05bc

472.22 ±4.69a


1 64.42±0.24 64.58±0.18 64.92±0.18 64.92±0.15 65.00±0.18 64.42±0.15 64.83±0.18 2 69.75±0.24a 69.50±0.24a 70.08±0.24ab 70.42±0.24b 70.83±0.22 69.83±0.24ab 69.83±0.17ab 3 75.33±0.25a 75.50±0.15ab 75.75±0.21ab 76.08±0.15b 76.67±0.28 75.42±0.20a 75.75±0.11ab 4 81.92±0.44a 82.17±0.36a 82.67±0.21ab 83.08±0.15b 83.42±0.20 82.50±0.18ab 82.50±0.18ab 5 89.75±0.42a 90.00±0.43ab 90.67±0.28bc 91.58±0.15d 92.42±0.20 90.83±0.25cd 89.75±0.11a 6 94.80±0.38a 94.92±0.40a 95.92±0.24b 97.58±0.24c 98.50±0.13d 97.42±0.20c 95.25±0.28ab


T1 T2 T3 T4 T5 T6 T7 1 57.08±0.15a 57.25±0.11a 57.33±0.17ab 57.75±0.21bc 57.92±0.15c 57.50±0.18abc 57.25±0.11a 2 62.08±0.15a 62.25±0.11ab 62.33±0.17ab 62.75±0.11cd 63.00±0.13d 62.58±0.15bc 62.25±0.11a 3 67.25±0.11a 67.25±0.11a 67.42±0.15ab 67.83±0.11cd 68.08±0.08d 67.67±0.11bc 67.33±0.11a 4 71.67±0.21a 71.67±0.21a 72.25±0.17b 72.33±0.17b 72.42±0.20b 72.17±0.11ab 72.08±0.15ab 5 76.67±0.21a 77.00±0.13a 77.08±0.15a 78.00±0.18cd 78.25±0.28d 77.67±0.11bc 77.17±0.11ab 6 83.58±0.24a 83.67±0.21a 83.92±0.20ab 84.33±0.17c 85.17±0.36d 84.17±0.11b 83.83±0.17ab

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Table-7. Height at withers (cm) in different fortnights (ftn) in different treatment groups.


T1 T2 T3 T4 T5 T6 T7

1 43.42

±0.15

43.42

±0.15

43.42

±0.08

43.50

±0.13

43.25

±0.11

43.25

±0.11

43.33

±0.11

2 45.50

±0.18

45.50

±0.18

45.42

±0.08

45.67

±0.17

45.42

±0.15

45.42

±0.15

45.33

±0.11

3 47.50

±0.18

47.58

±0.24

47.33

±0.11

47.75

±0.28

47.83

±0.31

47.67

±0.21

47.50

±0.18

4 50.58

±0.15

50.67

±0.17

50.50

±0.18

50.83

±0.11

50.50

±0.21

50.58

±0.15

50.50

±0.18

5 53.17

±0.11a

53.17

±0.11a

53.42

±0.15ab

53.50

±0.13b

53.58

±0.08b

53.42

±0.08ab

53.42

±0.08ab

6 55.00

±0.13a

55.08

±0.15ab

55.25

±0.17abc

55.67

±0.11cd

55.83

±0.17d

55.50

±0.13bcd

55.17

±0.17ab


Table-8. Feed consumption (kg) in different fortnights (ftn) in different treatment groups.



1 13.18 ±0.05

13.10 ±0.04

13.17 ±0.03

13.15 ±0.04

13.18 ±0.03

13.15 ±0.04

13.17 ±0.03

2 17.43 ±0.10

17.35 ±0.08

17.37 ±0.08

17.48 ±0.08

17.37 ±0.04

17.43 ±0.07

17.38 ±0.09

3 20.08 ±0.05ab

20.05 ±0.02a

20.08 ±0.05ab

20.20 ±0.03c

20.18 ±0.03bc

20.20 ±0.03c

20.15 ±0.06abc

4 22.47 ±0.03a

22.45 ±0.04a

22.60 ±0.04b

22.68 ±0.03b

22.47 ±0.05a

22.57 ±0.03ab

22.62 ±0.06b

5 23.53

±0.03abc 23.47

±0.05ab 23.65 ±0.06c

23.60 ±0.05bc

23.40 ±0.06a

23.55 ±0.04bc

23.60 ±0.06bc

6 24.37

±0.05ab 24.32 ±0.07a

24.37 ±0.09ab

24.52 ±0.06b

24.32 ±0.03a

24.42 ±0.06ab

24.42± 0.07ab

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Table-9. Feed conversion ratio in different fortnights (ftn) in different treatment groups.


T1 T2 T3 T4 T5 T6 T7

1 2.64±0.04d 2.43±0.02c 2.39±0.01bc 2.32±0.0ab 2.26±0.01a 2.33±0.02ab 2.41±0.04c

2 2.72±0.03e 2.68±0.02de 2.63±0.02cd 2.55±0.01ab 2.52±0.00a 2.59±0.02bc 2.65±0.04cd

3 2.82±0.02bc 2.88±0.03c 2.84±0.01bc 2.80±0.01b 2.67±0.06a 2.82±0.01bc 2.87±0.02c

4 2.76±0.01 2.75±0.09 2.82±0.01 2.75±0.01 2.77±0.08 2.78±0.01 2.82±0.02

5 3.57±0.01c 3.44±0.21bc 3.19±0.02 a 3.11±0.01a 3.03±0.01a 3.11±0.02a 3.22±0.02ab

6 3.44±0.03d 3.48±0.06d 3.42±0.02cd 3.28±0.02ab 3.23±0.03a 3.34±0.01bc 3.45±0.03d


T T T T T T T

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Body weight: The differences in bodyweights between the treatment groupswere significant (P 0.05) from 1st to6 th fortnight. The sixth fortnightlybody weights (kg) of the animalssubjected to growth promotertreatments (T2-T7) were significantly(P 0.05) higher than the T1 (control)group. It was the highest (58.72±0.03)in T5 (LTGP-Mash) group, which wassignificantly (P 0.05) higher than theother five treatment groups (T2, T3,T4, T6, and T7).

Better performance of T5 group mightbe due to its hepatoprotective, andantibacterial properties. Moreover,LTGP-Mash proved better than LTGP-Bolus and LTGP-Syrup, probably dueto better integration with the feed. Ourfindings are in conformity with theobservations of Sharma et al. (2008)and Sahoo et al. (2012).

Daily gain in body weig ht: Th edifferences in average daily gain(ADG) in body weight between thetreatment groups were significant(P 0.05) from 1 st to 6 th fortnight.The sixth fortnightly ADG (g) of T5(LTGP-Mash) group (502.22±1.41)was significantly (P 0.05) higherthan the ADGs of T1, T2, T3, T6,and T7, but did not differ (P 0.05)from T4 (AIGP).

Better performance of T4 group might bedue to its anti-stress, immunomodulatory,and antibacterial activities, while that ofT5 group might be due to thehepatoprotective and antibacterialactivities. Moreover, LTGP-Mashproved better than LTGP-Bolus andLTGP-Syrup, probably due to betterintegration with the feed. Findings ofthe present study agree with the findingsof Puri et al. (1993), Yin et al. (2007),

158


Nielsen (2008), and Ankita and Handique(2010).

Body measurements:

Body length: The differences in bodylength between the treatment groupswere significant (P 0.05) from 2nd to 6th

fortnight. The sixth fortnightly bodylength (cm) of the animals subjected togrowth promoter treatments (T2-T7)were significantly (P 0.05) higher thanthe control. It was the highest in T5(LTGP-Mash) group (98.50±0.13), whichwas significantly (P 0.05) higher thanthe control (T1) and other five treatmentgroups (T2, T3, T4, T6, and T7). Betterperformance of T5 group might beattributed to its hepatoprotective, andantibacterial properties. Moreover,LTGP-Mash proved better than LTGP-Bolus and LTGP-Syrup, probably due tobetter integration with the feed. Ourresults are similar to the findings of Parket al. (2000) in weaned pigs.

Chest girth: The differences in chestgirth between the treatment groupswere significant (P 0.05) from 1st to 6th

fortnight. The sixth fortnightly chestgirth (cm) of the animals subjected togrowth promoter treatments (T2-T7)were significantly (P 0.05) higher thanthe control (83.58±0.24). It was thehighest in T5 (LTGP-Mash) group(85.17±0.36) which was significantly

(P 0.05) higher than the control (T1)and other five treatment groups (T2, T3,T4, T6, and T7). Better performance ofT5 group might be attributed to itshepatoprotective, and antibacterialproperties. Moreover, LTGP-Mashproved better than LTGP-Bolus andLTGP-Syrup, probably due to betterintegration with the feed.

Height at withers: The differences inheight at withers between the treatmentgroups were significant (P 0.05) on 5th

and 6th fortnights. The sixth fortnightlyheight at withers (cm) of the animalssubjected to growth promotertreatments (T2-T7) were significantly(P 0.05) higher than the control. It wasthe highest in T5 (LTGP-Mash) group(55.83±0.17) which was significantly(P 0.05) higher than the control (T1)and other five treatment groups (T2, T3,T4, T6, and T7). Better performance ofT5 group might be attributed to itshepatoprotective, and antibacterialproperties. Moreover, LTGP-Mashproved better than LTGP-Bolus andLTGP-Syrup, probably due to betterintegration with the feed. Our findingsare in agreement with Kumar (2003) andAnkita and Handique (2010).

Feed consumption: Significantdifferences in feed consumptionbetween the treatment groups were

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observed from 3rd to 6th fortnight. Thesixth fortnightly feed consumption of T5(LTGP-Mash) group was the lowest(24.32±0.03), and did not differ (P 0.05)from other treatment groups (T1, T2, T3,T6, and T7) except T4 (AIGP) group(24.52±0.06), which was the highestamong all, and differed significantly(P 0.05) from T5 group.

More efficient feed utilization of T5(LTGP) group might be attributed to itshepatoprotective, and antibacterialpropert ies, while the dismalperformance of T4 (AIGP) group couldbe due to the stress-free condition of theanimals that culminated in higher feedconsumption. Our findings are inagreement with the findings of Hortonet al. (1991), Janz et al. (2007), and Wanget al. (2007), but contrary to the findingsof Holden and McKean (2002).

Feed conversion ratio: The differencesbetween the feed conversion ratios(FCR) between the treatment groupswere significant (P 0.05) from 1st to 6th

fortnight. The value of sixth fortnightlyFCR of T5 (LTGP-Mash) group was thelowest (3.23±0.03), and significantly(P 0.05) lower than T1, T2, T3, T6, andT7 groups, but not (P 0.05) from T4(AIGP) group (3.28±0.02). Our findingsare in agreement with Cullen et al.(2005) and Nielsen (2008).

CONCLUSION

It is concluded that the polyherbalpreparation containing liver tonic &growth promoter herbs (Andrographispaniculata, Achyranthus aspera, Terminaliabelerica) administered as feed mash @200g/quintal for 0-60 days along with thebasal ration was the most effective herbalproduct for better growth and efficientfeed utilization in pigs.

ACKNOWLEDGEMENT

Authors are highly thankful to Dean, RVCfor providing all the facilities to carry outthis research work at Pig Farm.

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Documents

ANIMAL SCIENCE REPORTER Animal Science Reporter, Volume 10, Issue 4, October, 2016 RESEARCH PAPERS 123-131 ORGANOLEPTIC, PHYSICO-CHEMICAL, AND MICROBIAL QUALITIES OF FRESH WATER FISH