Bioresource Technology 98 (2007) 388–394

Utilization of meat industry by products:Protein hydrolysate from sheep visceral mass

N. Bhaskar a,¤, V.K. Modi a, K. Govindaraju b, C. Radha b, R.G. Lalitha b

a Department of Meat, Fish and Poultry Technology, Central Food Technological Research Institute, Mysore 570 020, Indiab Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570 020, India

Received 28 October 2005; received in revised form 9 December 2005; accepted 14 December 2005Available online 2 February 2006

Abstract

Protein hydrolysate was prepared from pre-treated sheep visceral mass (including stomach, large and small intestines) by enzymatictreatment at 43§ 1 °C (at the in situ pH 7.1§ 0.2 of the visceral mass) using fungal protease. The enzyme readily solubilized the proteinsof the visceral mass as indicated by the degree of hydrolysis (34%) and nitrogen recovery (>64%). Hydrolysis with an enzyme level of 1%(w/w of total solids) at 43§ 1 °C with a pH around 7.0 for 45 min was found to be the optimum condition. The yield of protein hydroly-sate was about 6% (w/w). The amino acid composition of the protein hydrolysate that was very hygroscopic, was comparable to that ofcasein.© 2005 Elsevier Ltd. All rights reserved.

Keywords: Protein hydrolysate; Sheep viscera; Fungal protease; Amino acid

1. Introduction

Animals are mainly slaughtered for meat. Meat henceforms the major product while all other oVals become by-products. These by-products are sub divided into edible andnon-edible materials. By-products constitute nearly 60% to70% of the slaughtered carcass, of which nearly 40% formsedible and 20% inedible (Ranganayaki and Srinivasan,1999). Also, some by-products of sheep slaughter are con-sidered edible in most of the developing countries, apartfrom being used as casings for sausages in the developedcountries. On an average, proteins associated with the meatindustry by-products constitute more than one-eighth oftotal protein in the lean meat (Webster et al., 1982).Although use of these by-products in feeds/fertilizers afteremploying various technological processes—like rendering,dry reduction etc.—are technologically and economicallyviable, a growing market also exists for protein hydroly-

* Corresponding author. Tel.: +91 821 2514840; fax: +91 821 2517233.E-mail address: bhasg3@yahoo.co.in (N. Bhaskar).

0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2005.12.017

sates. These hydrolysates may be used as Xavor enhancers,functional ingredients or simply as nutritional additives tofoods of low protein quality.

Protein hydrolysates Wnd application mainly in nutri-tional management of individuals who cannot digest whole/intact protein. The most prevalent application of proteinhydrolysates has been for feeding infants with food hyper-sensitivity (Silvestre, 1997). Also, there are documentedreports on improvement in functionality of enzymaticallyhydrolysed proteins (Mahmood et al., 1992). Protein hydro-lysates rich in low molecular weight peptides, especially,di- and tri-peptides with as little as possible free amino acidshave been shown to have more dietary uses with high nutri-tional and therapeutic values (Vijayalakshmi et al., 1986).Most properties of protein hydrolysates depend on thehydrolysis conditions and the starting materials (Mahmoodet al., 1992; Silvestre, 1997).

Visceral mass (including stomach and intestines) formsan important by-product of the meat industry and consti-tute nearly 4% of the live weight of the slaughtered animal(Ranganayaki and Srinivasan, 1999). Although, vast

N. Bhaskar et al. / Bioresource Technology 98 (2007) 388–394 389

majority of work exists on the preparation of proteinhydrolysates using plant, meat and Wsh proteins as sub-strates, very little work appears to have been carried outusing fungal and bacterial proteases to solubilize the pro-tein of meat industry by-products including that of poultry.This study presents a method for preparation of proteinhydrolysates from the visceral mass of sheep in turn provid-ing a means for better utilization of slaughterhouse waste.The characteristics of protein hydrolysate prepared fromsheep visceral mass are also presented.

2. Methods

2.1. Materials

Sheep visceral mass (comprising stomach and intestines)was collected from the local slaughterhouse. Commerciallyavailable food grade fungal enzyme (protease P “Amano”6), having not less than 60,000 u/g proteolytic activity,was procured from M/s. Amano Pharmaceutical Co. Ltd.,Japan. The optimum pH and temperature requirementsof this enzyme were 8.0 and 43 °C, respectively. Sodiumdodecyl sulfate (SDS), 2,4,6-trinitrobenzene sulfonic acid(TNBS), L-leucine, tricine, and acrylamide were obtainedfrom Sigma Chemicals, USA. Standard protein markersfor molecular weight determination were obtained fromGenei (M/s. Bangalore-Genei, Bangalore, India). All otherreagents used were of Analar grade.

2.2. Methods

The visceral mass were thoroughly cleaned in runningwater to remove the intestinal contents, dipped in boilingwater for 5� and cut into small pieces. The diced visceralmass was then sterilized at 121 °C under pressure (15 lbs)for 15 min. The heat treated (sterilized) visceral mass(HTVM) was then cooled and minced in a Waring blender(Stephen mill, UM5 Universal, Hong Kong) followed bycentrifugation at 6000g for 30 min at 4 °C. After centrifuga-tion, the material separated into three phases with top layercontaining most of the fat, middle layer with water and pro-tein rich sediment in the bottom. Both the fat and waterlayer were discarded and only the protein rich sediment wascollected and designated as partially defatted visceral mass(PDVM). The PDVM had a pH of 7.1§0.2, almost same asthat of raw material (6.9§0.1). Initially, both HTVM andPDVM were used for hydrolysis experiments. The substratewas mixed with equal quantity of water (w/v) before theaddition of enzyme. Rate of hydrolysis was measured byviscosity measurements and by determining the absorbanceof TCA-soluble material at 280 nm (indicating the absor-bance by peptides and aminoacids) and 330 nm (indicatingthe scattering occurring due to fat or fat related substances)as a function of time. Preliminary analysis of rate of hydro-lysis and viscosity measurements during hydrolysis, using1% enzyme (w/w of total solids) revealed PDVM to be abetter substrate for protein hydrolysate preparation when

compared to HTVM. Hence, further experiments to stan-dardize the enzyme to substrate ratio, hydrolysis time andpreparation of protein hydrolysate were restricted toPDVM only.

2.3. Enzyme to substrate ratio

The defatted material was mixed with equal quantity ofwater (w/v) and was hydrolysed with three diVerent concen-trations of fungal protease viz., 0.5%, 1.0% and 1.5% (w/wof total solids) and hydrolysed for 90 min at 43§ 1 °C in athermostatically controlled reactor vessel, without alteringthe pH. The whole mixture was being constantly stirredduring hydrolysis. The reaction was stopped by heating themixture for 5 min at 85§1 °C. The hydrolysate was centri-fuged at 6700g, for 20 min at 15 °C, to collect the superna-tant. The supernatant was spray dried at 190–200 °C. Thewhite hygroscopic powder was used in all further studies.Total nitrogen content, nitrogen recovery and degree ofhydrolysis were determined for each of the conditions todetermine the optimum enzyme to substrate ratio.

2.4. Protein hydrolysate preparation

Protein hydrolysate from PDVM was prepared using 1%(w/w) enzyme to substrate ratio. The hydrolysis was done at43§ 1 °C, which was the optimum temperature for fungalprotease, for 45 min. The pH of the material was not alteredfor hydrolysate preparation. All viscosity measurementswere made using viscometer (Rheology International, UK)using a Windows based software. All spray drying stepsemployed the spray drier (Labplant SD05, LP Technolo-gies, UK). Proximate composition of the raw material andWnal product was estimated as per AOAC (1995) method.All protein measurements in the samples were carried outby Kjeldahl method using Kjeltec protein analyzer (FossTecator, Sweden).

The degree of hydrolysis (DH) was determined spectro-photometrically by the trinitrobenzene sulfonic acid (TNBS)method (Adler-Nissen, 1979). Amino acid composition wasdetermined using phenyl isothiocyanate (PITC) pre-columnderivatization (Bidlingmeyer et al., 1984) by employingWater’s PicoTag Column and Workstation. Sodiumdodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out by the method of Laemmli (1970) on12% gels of 0.75 mm thickness. The tristimulus Hunter colorparameters (L—lightness, a—redness and b—yellowness)were measured using the Hunter Color measurement system(Labscan XE, USA).

In vitro digestibility of the prepared protein hydrolysatewas determined by the method of Akeson and Stahman(1964). Pepsin followed by pancreatin digestion was carriedout using sample equivalent to 100 mg protein. Initiallythe sample was digested with 1.5% pepsin (w/w) in 0.1 NHCl at 37 °C for 3 h. Following neutralization with 0.2 NNaOH, the digestion was carried out using 4% pancreatin(w/w) at 37 °C for an additional 24 h. These enzymes were

390 N. Bhaskar et al. / Bioresource Technology 98 (2007) 388–394

inactivated using 10% tri-chloroacetic acid. The digest wereWltered and quantitatively made up to 100 ml. Digestibilitywas calculated based on the soluble and total nitrogen con-tent.

Considering contents of the nine essential amino acids(EAA), protein digestibility corrected amino acid score(PDCAAS) was calculated as per Sarwar and McDonough(1990) by determining the un-corrected amino acid scoreusing the reference protein requirement levels (mg pergram of crude protein)—for pre-school (2–5 years), school(10–12 years) children along with adults—prescribed byFAO/WHO (1985). The un-corrected amino acid score iscalculated by dividing the mg of EAA in 1 g of test proteinby milligram of amino acids in 1 g of the reference protein,which is the requirement for a particular group. Finally,multiplying the lowest of the un-corrected amino acidscores by the foods true digestibility value yields thePDCASS score.

The experiments were carried out in four batches of vis-cera. The means of all the parameters were examined forsigniWcance by analysis of variance (ANOVA) and in caseof signiWcance diVerence, mean separation was carried outby Duncan’s multiple range test using STATISTICA soft-ware (Statsoft, 1999).

3. Results and discussion

3.1. Pre-process conditions for hydrolysis

The material characteristics of the fresh visceral massfrom sheep are presented in Table 1. The raw materialappeared to carry a high bacterial load (7.1–9.0 logs)because of the intestinal contents. The heat treatment stephad the dual eVect of sterilizing the material and denaturingthe proteins which aided in accelerating hydrolysis. Anincrease 16.34% was observed in the fat content followingthe heat treatment of visceral mass. This may be due toloss of moisture during heat treatment. The fat contentdecreased by 62.80% when the HTVM was partially defat-ted by centrifugation. Defatting step resulted in an increasein the visceral mass protein (%) contents, for obvious rea-sons. In the initial experiments, both HTVM and PDVMwere hydrolysed using 1% (w/w of total solids) fungal pro-tease. Changes in the absorbance at 280 and 330 nm (Fig. 1)and viscosity measurements during hydrolysis revealed

PDVM to be better substrate than HTVM. As can be seenfrom the viscosity measurements (Fig. 2), partial removal offat also resulted in a sharp reduction in the viscosity (from15 to 4 mPas) in a span of 25 min, indicating the betterhydrolysis due to fat removal. Hence, PDVM was con-cluded to be the better form of the material for preparationof hydrolysate.

3.2. Standardization of enzyme to substrate ratio and hydrolysis time

In order to avoid the use of acid or alkali to adjustthe pH, the hydrolysis was carried out at the in situ pH ofthe material (7.01§0.2). The temperature for hydrolysis(43§1 °C) was Wxed as per the reported optimum tempera-ture for the protease used (based on the supplier’s datasheet). Thus, only time along with enzyme to substrate ratiowas standardized for the process. Both 1.0% and 1.5% pro-vided the same rate of hydrolysis reaching the peak absor-bance at 280 nm between 40 and 50 min (Fig. 3). The degreeof hydrolysis and nitrogen recovery at an enzyme concentra-

Fig. 1. Changes in the absorbance values during hydrolysis of partiallydefatted visceral mass (PDVM) and heat treated visceral mass (HTVM) at280 and 330 nm (n D 4).

0.0

0.5

1.0

1.5

2.0

0 20 40 60 80

A280-HTVM

A330-HTVM

A330-PDVM

A280-PDVM

Abs

orba

nce

at 2

80 o

r 33

0 nm

Time, min

Table 1Proximate composition (g 100 g¡1) of the sheep visceral mass at various stages of processing and hydrolysis (n D 4)

a As % of fresh viscera.b Nitrogen content.

Moisture Fat Protein Ash Yielda

% as raw material % Nitrogen recovered

Fresh viscera 85.53 § 1.65 5.20 § 0.33 9.45 § 0.95 0.90 § 0.04 – ¡Heat treated 83.10 § 1.06 6.05 § 0.45 10.68 § 0.63 1.11 § 0.09 84.20 § 1.39 95.20 § 1.64Partially defatted 76.40 § 0.85 2.25 § 0.19 20.38 § 1.05 1.44 § 0.14 40.30 § 0.79 86.90 § 1.94Protein hydrolysate 3.00 § 0.45 1.23 § 0.10 16.90 § 1.34b 0.93 § 0.05 5.78 § 0.26 64.60 § 2.30

N. Bhaskar et al. / Bioresource Technology 98 (2007) 388–394 391

tion of 1.0% and 1.5% was signiWcantly higher (P60.05)than that of enzyme at 0.5% level (Fig. 4). However, between1.0% and 1.5% level, there was no signiWcant diVerence(P > 0.05) as evidenced by degree of hydrolysis (34.7–34.9%)and nitrogen recovery (63.5–64.6%). Hence, an enzyme tosubstrate ratio of 1.0% (w/w of total solids) was used as the

Fig. 2. Changes in the viscosity of the visceral mass (with and without fatremoval) during hydrolysis (nD 4). Control indicates HTVM with noenzyme treatment. Experimental conditions—ASTM 2 spindle, 80 rpm.

0

10

20

30

40

0 10 20 30 40Time, min

Vis

cosi

ty, m

Pas

Control

HTVM

PDVM

Fig. 3. Changes in absorbance at 280 nm of the partially defatted visceralmass (PDVM) hydrolysed with diVerent level of enzyme concentrations(nD 4).

0.0

0.5

1.0

1.5

2.0

0 25 50 75 100

Time, Minutes

Abs

orba

nce

at 2

80 n

m

0.5% 1.0% 1.5%

optimum concentration with a hydrolysis time of 45 min.Haque-HaWz et al. (1994) recovered 81.25% protein frombovine lungs by papain hydrolysis. Kijowski et al. (1992)found that the recovery of nitrogenous compounds from thechicken bone residues was 23–24% and the hydrolysates hadhigh (80%) nitrogenous compounds solubility.

Comparison of the color characteristics of the proteinhydrolysates (Table 2) revealed that lightness index was notsigniWcantly diVerent between 0.5% and 1.0% enzyme con-centration. However, whiteness of the protein hydrolysateprepared with 0.5% was signiWcantly (P 6 0.05) superior tothe other two concentrations. In a work on Harp seal meathydrolysate, Shahidi et al. (1994) reported that higher DHvalues generally resulted in lighter colored protein hydroly-sates. However, in the present study higher DH resulted incomparatively low lightness. Similarly, protein hydrolysateprepared with an enzyme concentration of 1.5% had moreyellowness (P 6 0.05) compared to that of the other twoconcentrations with the least yellow among the three beingthe one prepared with 0.5% of enzyme.

3.3. Preparation of protein hydrolysate

Protein hydrolysate was prepared from PDVM usingthe optimized conditions—i.e., 1% enzyme, 43§ 1 °C and45 min of hydrolysis time. The hydrolysis was followed byinactivation at 85§2 °C for 5 min and centrifugation tocollect liquid protein hydrolysate in the supernatant. The

Fig. 4. Degree of hydrolysis and yield of protein hydrolysates as a functionof diVerent enzyme concentrations (n D 4). Similar superscripts on aparameter indicate no signiWcant diVerence (P > 0.05).

0

20

40

60

80

0.5% 1.0% 1.5%

Enzyme level, w/w of solids

DH

(%

) / N

itro

gen

reco

very

(%

)

DH Recovery

x

yy

a

b b

392 N. Bhaskar et al. / Bioresource Technology 98 (2007) 388–394

liquid protein hydrolysate was spray dried to obtain a whitepowder. The characteristics of the protein hydrolysate incomparison to the materials during the diVerent stages ofprocessing are detailed in Table 1. More than 64% of thenitrogen in the original material was recovered in the pro-tein hydrolysate. This clearly indicated that the proteaseeVectively degraded proteins associated with the visceralmass. The fat content in the protein hydrolysate wasconsiderably low (1.23%§ 0.10%). The beef hydrolysates,as reported by Maria et al. (1999) had 1.4% fat, whichwas very near with present Wndings. Further, the extent ofhydrolysis was supported by the disappearance of largeproteins in the electrophoretic proWle of the protein hydro-lysate (Fig. 5). The peptides in the protein hydrolysate hadan average molecular weight of >10 kDa indicating the use-fulness of this material as a source of some or several bioac-tive peptides. Sephton et al. (1996) prepared hydrolysatesfrom beef liver using the commercial proteolytic enzymeAlcalase 2YL at reaction conditions of 55 °C and pH 8.5.They found that the resultant hydrolysate contained lowmolecular weight peptides in the range 0.3–3 kDa. Theamino acid composition (Table 3) of the protein hydro-lysate clearly indicated the presence of several essentialamino acids like leucine (7.23%), lysine (6.93%), valine(4.72%), isoleucine (3.59%) and phenyl alanine (3.5%), thus,making it nutritionally beneWcial. Jeong-Bae and Kang

Lyung (1995) have also reported dark meat protein hydro-lysates to be rich in free amino acid contents.

DiVerences in protein digestibility of various foods arisefrom inherent diVerences in the nature of the food protein,presence of non-protein constituents or from processingconditions that alter the release of amino acids from pro-teins by enzymatic processes (Kies, 1981; Sarwar, 1987).The relatively lower digestibility (Table 3) compared to apure protein like casein could be due to matrix eVects, suchas fat and processing conditions.

The PDCAAS values calculated from analyzing aminoacid data is also presented in Table 3. PDCAAS accu-rately depicted protein quality because it rates proteinfoods relative to a given reference protein (Sarwar andMcDonough, 1990) and was based on the needs ofhumans rather than on the protein eYciency ratio (PER)criterion of a protein’s ability to support growth in youngrats. The PDCAAS values for the sheep visceral hydroly-sate were 0.67, 0.76 and 0.93, respectively for pre-school(2–5 years), school (10–12 years) children and adults. Thehighest PDCAAS score under these guidelines that anyprotein can achieve is 1.00. The PDCAAS scores for avariety of food proteins include 1.0 (casein, egg white, soyprotein isolate), 0.92 (beef protein), 0.68 (kidney beans)and 0.40 (whole wheat). Under the given set of experimen-tal conditions, the product seemed to have the desirable

Table 2InXuence of diVerent enzyme to substrate ratio on the color parameters of protein hydrolysates prepared from PDVM using fungal protease

PDVM, partially defatted visceral mass; L, lightness; a, redness; b, yellowness; w, whiteness index; a–n: values with similar superscript in the same columnare not signiWcantly diVerent (P 6 0.05).

1 % of solid weight.

Enzyme, conc.1 L a b w

0.50 82.21 § 0.54a ¡1.50 § 0.05c 9.66 § 0.04g 13.03 § 0.66l

1.00 82.54 § 0.16a ¡1.38 § 0.03d 10.30 § 0.04h 11.48 § 0.83m

1.50 78.94 § 0.11b ¡0.92 § 0.03e 12.28 § 0.17i 6.33 § 0.33n

Fig. 5. Electrophoretic pattern of visceral protein hydrolysate: (A—standard marker proteins; B1–B3—sheep visceral protein hydrolysate).

N. Bhaskar et al. / Bioresource Technology 98 (2007) 388–394 393

PDCAAS values and comparable to that of beef protein.The protein hydrolysate was white in color and corro-borated well by the color readings and lacked any percep-tible bitterness in a 5% solution. This indicated thepotential of this material as an ingredient in nutritionalformulations or in beverages where amino acid supple-mentation was required, provided the protein from theanimal sources was statutorily permitted for use in suchfood items.

4. Conclusions

It could be concluded that the slaughter house by-products, especially visceral mass from sheeps, held aconsiderable potential for preparation of protein hydroly-sates that was rich in some of the essential amino acids.Fungal protease could be a better enzyme for preparationof protein hydrolysates as evidenced by the considerablygood degree of hydrolysis and higher recovery of nitrogenin the Wnal product. As the protein hydrolysate containedlow molecular weight peptides, this hydrolysate couldcontain several bio-active peptides which should be inves-tigated. Relatively higher PDCAAS values and in vitrodigestibility could make the protein hydrolysate suitableas a nutritional supplement in various foods. As the pro-cess involved sterilization of the material, the proteinhydrolysate was safe for incorporation into several food

Table 3Amino acid composition and protein digestibility corrected amino acidscore (PDCAAS) values of sheep visceral protein hydrolysate

Standard PDCAAS values (mg g¡1crude protein) as per FAO/WHO(1985) for histidine, threonine, valine, cystine + methionine, isoleusine,leusine, tyrosine + phenylalanine and lysine are—19, 34, 35, 25, 28, 66, 63and 58 (for 2–5 year olds); 19, 28, 25, 22, 28, 44, 22 and 44 (10–12 yearolds) and 16, 09, 13, 17, 13, 19, 19 and 16 (adults), respectively.

a Mean § SD; n D 3.

Amino acids Content (mg g¡1

crude protein)Uncorrected aminoacid score

2–5 10–12 Adult

Essential amino acidsHistidine 20.0 1.05 1.05 1.25Threonine 36.9 1.09 1.32 4.10Valine 47.2 1.35 1.89 3.63Cystine + methionine 22.4 0.90 1.02 1.32Isoleusine 35.9 1.28 1.28 2.76Leusine 72.3 1.10 1.64 3.81Tyrosine + phenylalanine 60.0 0.95 2.73 3.16Lysine 69.3 1.19 1.58 4.33Tryptophan – – – –

Other amino acidsAspargine/aspartate 88.1 – – –Glutamine/glutamate 152.3Serine 43.5 – – –Glysine 108.9 – – –Alanine 73.8 – – –Proline/hydroxy proline 62.5 – – –In vitro digestibility (%)a 74.6 § 1.3 – – –PDCAAS 0.67 0.76 0.93

items, wherever permitted. Further, this process can beeasily integrated with the rendering process in the com-mercial set up.

Acknowledgements

The authors thank the Director, CFTRI for technicalhelp and encouragement during the work. Thanks also toDr. A.G. Appu Rao and Dr. D. Narasimha Rao for theirvaluable suggestions during the work.

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