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Waste Management xxx (2012) xxx–xxx

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Waste Management

journal homepage: www.elsevier .com/locate /wasman

Review

Potential of chicken by-products as sources of useful biological resources

Adeseye Lasekan a, Fatimah Abu Bakar a,b,⇑, Dzulkifly Hashim a,b

a Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysiab Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

a r t i c l e i n f o

Article history:Received 5 April 2012Accepted 3 August 2012Available online xxxx

Keywords:Chicken by-productsProtein hydrolysatesProteasesPolyunsaturated fatty acidsBioresourceKeratin

0956-053X/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.wasman.2012.08.001

⇑ Corresponding author at: Faculty of Food SciencPutra Malaysia, 43400 UPM Serdang, Selangor, Malays+60 389423552.

E-mail address: [email protected] (F. Abu

Please cite this article in press as: Lasekan, A., ehttp://dx.doi.org/10.1016/j.wasman.2012.08.001

a b s t r a c t

By-products from different animal sources are currently being utilised for beneficial purposes. Chickenprocessing plants all over the world generate large amount of solid by-products in form of heads, legs,bones, viscera and feather. These wastes are often processed into livestock feed, fertilizers and pet foodsor totally discarded. Inappropriate disposal of these wastes causes environmental pollution, diseases andloss of useful biological resources like protein, enzymes and lipids. Utilisation methods that make use ofthese biological components for producing value added products rather than the direct use of the actualwaste material might be another viable option for dealing with these wastes. This line of thought has con-sequently led to researches on these wastes as sources of protein hydrolysates, enzymes and polyunsat-urated fatty acids. Due to the multi-applications of protein hydrolysates in various branches of scienceand industry, and the large body of literature reporting the conversion of animal wastes to hydrolysates,a large section of this review was devoted to this subject. Thus, this review reports the known functionaland bioactive properties of hydrolysates derived from chicken by-products as well their utilisation assource of peptone in microbiological media. Methods of producing these hydrolysates including theirmicrobiological safety are discussed. Based on the few references available in the literature, the potentialof some chicken by-product as sources of proteases and polyunsaturated fatty acids are pointed out alongwith some other future applications.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction 1995; Korniłłowicz-Kowalska and Bohacz, 2011). In addition,

The production and consumption of poultry products have beenon the increase globally. Statistics from poultry industry watchersshowed that United States, China and Brazil maintain their lead asthe biggest producers of poultry meat. Each is expected to produceup to 19,852, 18,102 and 12,200 thousand metric tons of poultrymeat, respectively, in 2011 (USDA, 2011a). However, China andBrazil top the list in the production of chicken meat, each havinga forecast of 13,000 and 11,750 thousand metric tons, respectively,in 2011 (USDA, 2011b). With this large production, thousands oftons of organic by-products in the form of viscera, feet, head,bones, blood and feathers are generated (Zhu et al., 2010). The vis-cera constitute about 30% of these wastes while feather could beup to 10% (Jamdar and Harikumar, 2005; Grazziotin et al., 2007).

In the past, it was a common practice to convert animal wastesinto livestock feed and organic fertilizers. As a result, processessuch as rendering, composting, chemical, microbial and thermaltreatment of poultry and other animal wastes were developedand widely researched (Salminen and Rintala, 2002; Cai et al.,

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e and Technology, Universitiia. Tel.: +60 192775012; fax:

Bakar).

t al. Potential of chicken by-pro

waste management methods such as dumping, landfilling andincineration are also practiced in different parts of the world(Salminen and Rintala, 2002). However, after the outbreak of theBovine Spongiform Encephalopathy (BSE) in the United Kingdom,United States and some countries in Europe coupled with the linkestablished between consumption of BSE infected meat and thedevelopment of Creutzfeldt-Jakob disease, there has been a sharpdecline in the conversion of animal by-products to feed and certainlegislations in some countries disallow indiscriminate dumping orlandfilling of animal wastes. Composting and anaerobic digestionwhich are considered as better options for converting biologicalwastes to value added products still possess certain drawbackswhich are hindering their upscale. Hence, researches on alternativemethods of processing animal wastes to useful products are stillongoing in different parts of the world. One such alternative in-volves development of techniques for recovering biological com-pounds from animal wastes which are later use for somedownstream processing or application. Since animal wastes arecomposed of biodegradable C, N, H, O and S compounds, thesecompounds can be recovered and utilised in different industrieswhile also helping to reduce volume of the starting waste product.

Thus, a large number of studies on chicken and other animal by-products have focused on extraction, isolation and utilisation of thebiomolecules that are trapped inside these products. Specifically,

ducts as sources of useful biological resources. Waste Management (2012),

http://dx.doi.org/10.1016/j.wasman.2012.08.001

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2 A. Lasekan et al. / Waste Management xxx (2012) xxx–xxx

different chicken by-products are known to contain appreciableamount of biomolecules like proteins, enzymes and lipids (Ocker-man and Hansen, 2000; Raju et al., 1997). These biomoleculescan be recovered and processed into products which can be usefulin microbiology, medicine, pharmaceuticals, human nutrition andcosmetics (Bueno-Solano et al., 2009). To this end, some attemptshave been made to test the suitability of different chicken by-prod-ucts for the production of specific value added products such asprotein hydrolysates, enzymes and polyunsaturated fatty acids(Kim et al., 2001; Grazziotin et al., 2007; Surówka and Fik, 1994;Rathina Raj and Mahendrakar, 2010; Patil and Nag, 2011). How-ever, there is so far a lack of publications addressing in a compre-hensive manner the subject matter of recovery and utilisation oforganic biomolecules from chicken by-products.

Thus, this review discussed the utilisation of poultry by-prod-ucts as sources of protein hydrolysates, enzymes and polyunsatu-rated fatty acids. Due to the diverse applications of proteinhydrolysates in different fields and industry coupled with the siz-able number of literature references available on utilisation ofchicken by-products as substrates for producing protein hydroly-sates, a large section of this review covers the functional and bio-active properties of these hydrolysates, their usage as sources ofpeptone, methods of production as well as their microbiologicalsafety. Based on the few literature references available, methodsof deriving industrial proteases and polyunsaturated fatty acids(PUFAs) from chicken wastes were reviewed along with some fu-ture applications.

2. Animal by-products: processing methods and utilisation

The Commission of the European Communities Regulation (EC)No. 1096/2009 defines animal by-products as, whole body of ananimal, parts from the body of an animal or products derived fromanimals which are not meant for human consumption (EuropeanCommission, 2009). These by-products are grouped into three cat-egories based on their level of risk for transmitting pathogens andtoxic substances. The third category which comprises animal by-products such as feather, skin, hides, blood, heads, feet, horn andhoof obtained from healthy animals are permitted to be used forcertain beneficial purposes (Table 1) while those in category 1are expected to be buried or incinerated at designated sites andby approved agency (Barrena et al., 2009). Animal by-productsclassified as category 2 are also considered as high risk productsbut can be used as feedstock for composting, biogas generationand energy production after pretreatment at high temperatureand pressure. These regulations have consequently led to somemodification of the conventional methods of processing and util-isation of these wastes while also creating an opportunity to searchfor novel waste processing and utilisation methods.

Table 1Methods of utilising chicken by-products and their corresponding categories accord-ing to the EU legislation.

Uses of chicken by-products EU classification of the by-productsa

Livestock feed Category 3Pet food Category 3Aqua feed Category 3Cosmetic products Category 3Compost Category 2 and 3Production of biogas Category 2 and 3Production of thermal and electrical

energyCategory 1, 2 and 3

Production of biofuel Category 1, 2 and 3

a The current European Union document on the utilisation of animal by-productsshould be consulted for guidelines on handling and processing of these by-products.

Please cite this article in press as: Lasekan, A., et al. Potential of chicken by-prohttp://dx.doi.org/10.1016/j.wasman.2012.08.001

One of the most common methods of converting solid wastes tovalue added products prior to these regulations is via the renderingprocess. This process uses heat treatment such as temperature ofabout 133 �C, a pressure of 3 bars and cooking time of about20 min to separate the fat and the proteinaceous portion of animalwastes (Salminen and Rintala, 2002). The fat can then be used asraw material for producing cooking oil, soap, detergents and cos-metics while the protein residues are dried and grounded into feedmeal such as meat and bone meal (MBM), feather meal and meatmeal for livestock (Shareefdeen et al., 2005). However, since theoutbreak of Bovine Spongiform Encephalopathy (BSE) the use ofanimal by-products for livestock feeding has come under strict reg-ulation such that, only the low risk materials (category 3) are per-mitted even in pet foods and as organic fertilizers (Cascarosa et al.,2012). The feeding of ruminant animals with meals from animalby-products has been banned in the EU and in some other coun-tries outside the EU. In addition, this ban was a welcome develop-ment for environmentalists who were concern about theenvironmental impacts of the odorous volatiles such as hydrogensulphide, ammonia, ketones and aldehydes released from theseanimal wastes prior and during the rendering process (Shareefdeenet al., 2005; Gwyther et al., 2011).

Apart from rendering, farmers have long used animal wastesand carcasses for improving soil fertility. But improved knowledgein agriculture and animal husbandry has revealed the demerits ofdirect application of animal wastes to farmland. These demerits in-clude the proliferation in the soil and transmission of pathogenicmicroorganisms to animals that graze on the land in addition tocontamination of water bodies via leaching and emission of green-house gases in some cases. Hence, animal wastes are usuallypassed through the process of composting before application as or-ganic fertilizers. Composting is an aerobic digestion process whichconverts organic waste materials (category 3 and excluding mor-talities) into a soil like stable form which can be mixed with soilor used directly as medium for plant growth (Barrena et al.,2009). It is essentially a decomposition process involving thermo-philic and mesophillic microorganisms. The heat generated by theaction of the thermophiles inactivates the pathogens thus makingit safe for application to soil. Although, it is widely accepted thatanimal by-products are viable compost raw material, very few lit-erature references are available regarding their use since moststudies made use of animal manure instead (Barrena et al.,2009). However, with respect to the process, there are some con-cerns about recontamination by opportunistic and pathogenicorganisms and also about ammonia emission. In addition, as notedby the work of Barrena et al. (2009) the need to maintain a temper-ature within the range of 60–70 �C throughout the reactor makesthis process to be prone to complication such as moisture losswhich is due to the high airflow necessary to maintain a uniformtemperature throughout the reactor. Hence, the process needsclose monitoring, control of the initial material and process param-eters such as temperature, aeration, moisture and C/N ratio, in or-der to guarantee safety and good quality compost.

Furthermore, organic wastes rich in protein and fat can be usedto generate biogas (methane) via anaerobic digestion, which canserve as an alternative to fossil fuel. Volatile fatty acids (VVFs) de-rived from the fermentation of polypeptides, amino acids and longchain fatty acids undergo a series of reaction involving acetogenicand methanogenic microorganisms to produce methane and car-bon dioxide. Salminen and Rintala (2002) gave an excellent reviewon the process of anaerobic digestion with emphasis on poultryslaughterhouse wastes. Palatsi et al. (2011) on the other hand re-ports the limiting effect of lipid biodegradability on the process ki-netic and the need for microbial enrichment for successfulanaerobic treatment. More so, some authors have pointed out thedifficulty of anaerobic digestion of slaughterhouse wastes. These



A. Lasekan et al. / Waste Management xxx (2012) xxx–xxx 3

wastes are rich in protein whose breakdown leads to generation ofammonia during anaerobic treatment which in turn inhibits themethanogenic microorganisms thus reducing the yield of biogas(Alvarez and Lidén, 2008; Resch et al., 2011). From the economicpoint of view, it has also being posited that the pretreatment thatis required before these wastes are used for biogas productionmakes it an expensive venture. In spite of these, this process isadvantageous because the remaining solid fraction can be madeinto compost via aerobic digestion.

The field of composite engineering and polymer science hasopened up another way of utilising animal by-products. Natural fi-bres that can be added to polymeric materials to create compositeshaving improved mechanical and thermal properties are currentlybeen sought (Faruk et al., 2012). These composites which can beused in the packaging, textile, medical and automobile industriesoffer the advantages of biodegradability and biocompatibility inaddition to other improved properties (Pandey et al., 2010).Although cellulose has been used extensively as natural fibres inboth organic and petroleum-based polymeric media, some of itsdisadvantages which includes its minimal association with hydro-phobic polymer, formation of aggregates and high affinity forwater has been reported thus giving room for novel natural fibres(Barone and Schmidt, 2005; Kalia et al., 2011). Increased knowl-edge about the chemical properties of keratin (the protein compo-nent of chicken feather, a poultry waste) which includes itsthermal resistance, hydrophobic nature, crystalline structure, highaspect ratio and durability makes it a viable candidate for use asreinforcement material in a polymeric matrix (Cheng et al.,2009a,b). In line with these properties, a study was carried out totest the reinforcement ability of chicken feather fibre (CFF) inpoly(lactic acid) PLA polymer medium and the result shows thatthe composite has improved mechanical and thermo-mechanicalproperties but lower load bearing ability (Cheng et al., 2009a,b).Another study revealed the challenge of getting chicken feather fi-bre-high density polyethylene composite with the best propertieswhich only occurs at temperature above 200 �C with some adverseeffect when this step is prolonged (Barone et al., 2005). The poten-tial of this poultry waste for the production of eco-friendly productwas however demonstrated in a recent work where chicken feath-er fibre was used in the production of thermoplastics with the aidof different plasticizers (Ullah and Wu, 2012). Researchers are stillon-going in this field to create better composites from differentanimal by-products that are durable and can withstand someindustrial thermal treatments.

Other waste management methods that are currently experi-encing increased popularity in some countries are advanced heattreatment methods known as gasification and pyrolysis. In thesemethods, waste biomass are burned in limited air or in an inertenvironment to generate heat and fuel products (synthesis gasand bio oil) used for producing electricity and eco-friendly auto-mobile fuel (Bulushev and Ross, 2011; Voets et al., 2011). However,researches in this area have mostly concentrated on plant material,poultry litter, animal wastes and treated municipal sludge fromhuman wastes (Mante and Agblevor, 2012; Shinogi and Kanri,2003; Yaman, 2004). Based on some recent studies, utilisation ofanimal by-products as substrates for gasification and pyrolysisseems to be receiving some attention (Cascarosa et al., 2012; Mar-culescu and Stan, 2011; Wisniewski et al., 2010). DudyDski et al.(2012) gave a detailed report about the utilisation of chicken feath-er for producing syngas using a fixed bed gasifier which has sincebeen used for energy production in an industrial plant. Theresearchers noted the economic viability of this waste disposalmethod for the poultry processing plant, the cost effectiveness ofthe gasification method used and the ease of control of the amountof pollutant generated.


3. Protein hydrolysates, proteases and polyunsaturated fattyacids from chicken and other animal by-products

It is evident that different branches of science are using theirexpertise to solve waste management issues arising from animalby-products. However, a paradigm shift might be necessary in orderto achieve a better utilisation of animal by-products using someprinciples in the field of chemistry, biochemistry and microbiology.For so many years, utilisation of these wastes has largely revolvedaround the raw material level which involved feeding of animalswith the pretreated wastes (in powdered form) or using them di-rectly as fertilisers. Usage of these wastes in their raw or pretreatedform has not been totally effective as had been discussed earlier.Consequently, a shift to the molecular level of utilisation, wherebythe intrinsic organic chemical compounds of the waste materialsare recovered and used for some beneficial purposes have been sug-gested as a better means of handling these wastes (Rulkens et al.,1998). The major advantages of dealing with these wastes at themolecular level include involvement of processes that are mostlymild, environmental friendly and which generate little effluentsand waste residues compared to the conventional methods. In addi-tion, biomolecules recovered from these wastes can find applica-tions outside the agricultural sector. Against this background, thissection will explore the recovery of biomolecules such as proteinhydrolysates, proteases and polyunsaturated fatty acids from differ-ent animal by-products but with emphasis on chicken by-products.This is done in order to show the little attention that chicken by-products has received in terms of recovery of useful biomoleculesfrom them as compared to by-products from other animals. Moreso, this section will provide the opportunity to compare the func-tionality of the bioresources obtained from chicken by-productswith those of other animals so that more attention can be focusedon the utilisation of chicken by-products for beneficial purposes.

Thus, apart from the traditional usage as animal feeds and as or-ganic fertilizers, by-products from animal processing are widelyconverted to protein hydrolysates (Gbogouri et al., 2004). Thismethod of utilisation is different from other conventional methodsbecause the chemical components of the by-products (protein) arefirst recovered and then utilised rather than direct use of the by-products. In the production of protein hydrolysates (Fig. 1), theproteins are first extracted from the organic material using water,alkali or acid extraction depending on the pH at which the proteinis soluble. The soluble protein can then be recovered from the clar-ified solution through precipitation and dried in order to obtain theprotein isolate. Partial enzymatic, chemical and chemical-enzy-matic hydrolysis of this isolate will produce a hydrolysate contain-ing a mixture of peptides of varying length and free amino acids(Wu et al., 2003; Neklyudov et al., 2000). Partially hydrolysed pro-tein products have been shown to possess improved functionalproperties like solubility, fat absorption, foaming stability andemulsifying properties (Klompong et al., 2007). Bioactive peptidesare also liberated during controlled hydrolysis of protein. Antioxi-dant, antimicrobial and antihypertensive properties (ACE inhibi-tors) of peptides from different hydrolysates have been studiedextensively (Hsu et al., 2009; Pihlanto-Leppälä et al., 2000). Innutrition, patients that cannot digest intact protein are fed withhydrolysates from food sources, while in microbiology they areused as carbon and nitrogen source in growth media (Clemente,2001; Ghorbel et al., 2005). Peptides and amino acids have alsobeen recognised as important flavour precursors (van Boekel,2006). As a result, the flavour industry is making use of hydroly-sates which provide a cheap and abundant source of these precur-sors to create interesting flavours in model system.

For many years, protein hydrolysates from edible food sourcesare largely utilised for their nutritional, functional and bioactive



Fig. 1. Process diagram for the production of protein hydrolysate.


properties (Chen et al., 1998; Periago et al., 1998). However, theincreasing global population and food insecurity in some parts ofthe world makes it necessary to look for alternative sources ofhydrolysates apart from edible food products (Plascencia-Jatomeaet al., 2002). More so, effort at reducing environmental pollutionis another element driving the search for alternative sources ofprotein hydrolysates.

Animal by-products have been recognised as rich sources ofprotein even though they are often discarded due to aesthetic rea-sons (Rivera et al., 2000). Since most of the protein fractions foundin animal wastes can be easily extracted, they have been usedextensively in the production of hydrolysates that can be used asfood ingredients.

Enzymatic hydrolysis has been used mainly for converting thesewastes to hydrolysates with a high degree of hydrolysis (DH) andyield (Ovissipour et al., 2012; Šlizyte et al., 2005). The degree ofhydrolysis (DH) is a term used to describe the extent of enzymatic


peptide cleavage of a protein substrate and is calculated as the per-centage ratio between the number of peptide bonds cleaved andthe total number of peptide bonds in the substrate under studyusing protocols such as TNBS, pH stat and OPA (Rutherfurd,2010). Factors such as the time of hydrolysis, pH of the reactionmedium, temperature, enzyme concentration and the nature ofthe substrate have been shown to affect the extent of enzymatichydrolysis and different combinations of the values of these factorsexist in the literature that give the optimum DH for differenthydrolysates as shown in Table 2. Most researches on the utilisa-tion of animal by-products as sources of hydrolysates have focusedon the fish processing by-products (Harnedy and Fitzgerald, 2012)and this might be due to the ease of isolation and hydrolysis of thefish proteins (Table 2). Fish is an important source of protein forhuman nutrition hence it is widely consumed globally. Largeamount of proteinaceous wastes in form of heads, scales, bonesand viscera are generated during fish processing and they have




been used for the production of protein concentrate and hydroly-sate. Functional properties such as solubility, emulsion capacity,emulsion stability and fat absorption of different fish wasteshydrolysates have been investigated. From these studies, it hasbeen shown that hydrolysates with high degree of hydrolysis showa higher solubility while limited hydrolysis favours emulsioncapacity, emulsion stability and fat absorption (Gbogouri et al.,2004). The solubility of protein hydrolysates is commonly mea-sured in terms of its percentage soluble nitrogen in water after stir-ring and centrifugation. Nitrogen solubility values between 75%and 100% have been reported for different fish protein hydrolysatesat pH range of 2–11 (Gbogouri et al., 2004; Kristinsson and Rasco,2000). The increased solubility has been attributed to disruption ofthe hydrophobic interaction and the resultant exposure of moreionisable groups which can interact with the water molecules. Inthe work of Gbogouri et al., 2004 using salmon by-products proteinhydrolysates having degree of hydrolysis from 11.5% to 17.3%,emulsifying capacity, emulsion stability and oil holding capacitywere found to increase up to a maximum followed by a declineacross the degree of hydrolysis values that were examined. Highantioxidant activity which is reflected by a high DPPH-radical scav-enging activity of about 87% was reported for the enzymatic hydro-lysate of Sardinelle by-products protein with a low degree ofhydrolysis of about 6% (Bougatef et al., 2010). Antihypertensivepeptides were also isolated from the hydrolysate of skate skin pro-duced after 12 h hydrolysis with a-chymotrypsin (Lee et al., 2011).The ability of protein hydrolysate to bind iron was demonstratedwith the aid of shrimp processing by-product (SPB) protein hydrol-ysates with the result showing a steady increase in the iron bindingcapacity for up to 6 h of hydrolysis with alcalase (DH 8%) followedby a decline (Huang et al., 2011) while a high antihypertensiveactivity was reported for hydrolysate obtained from clam mantle(Sun et al., 2011).

Among the meat by-products, the blood has been used exten-sively for producing hydrolysates with functional and bioactiveproperties. Antioxidant, antihypertensive and antitoxigenic prop-erties of the peptidic fraction of porcine haemoglobin and bovineblood plasma hydrolysates have been demonstrated (Hyun andShin, 2000; Park and Hyun, 2002; Chang et al., 2007). The skin ofdifferent animals is also converted to value added protein hydrol-ysates. Fish, bovine and porcine skins are rich in collagen whichcan be denatured and extracted leading to the formation of gela-tine which in turn can be hydrolysed by enzymes to produce

Table 2Optimal conditions for the enzymatic hydrolysis of different animal by-products.

Substrate Enzyme X1 (%)

Tuna waste Umamizyme 1.5Salmon by-products Alcalase 2.4.L 5.2Viscera waste protein of Catla (Catla catla) Alcalase 1.5Shrimp waste Alcalase 1.84Cuttlefish viscera Protamex 0.1

Flavourzyme 1.5Alcalase 0.1

Sardine viscera Protamex 0.1Flavourzyme 1.5Alcalase 0.1

Fish soluble concentrate Flavourzyme 5.0Kojizyme 5.0

Porcine blood meal Alcalase 10Herring by-products (head, gonad) Alcalase 0.5Chicken blood meal Prozyme-6 10Sheep viscera mass Protease P ‘‘Amano’’ 6 1.0Bovine haemoglobin Pepsin –Calf skin Alkaline proteinase containing

subtilisin DY4.0

X1: enzyme concentration, X2: pH, X3: temperature, X4: time, Y: degree of hydrolysis.


hydrolysate. This was shown by Vasileva-Tonkova et al. (2007)who studied the potential of calf skin hydrolysate as peptone forbacterial growth and the result revealed that all the test microor-ganisms grew well in 1% solution of the hydrolysate without theaddition of dextrose and NaCl. The functional and bioactive proper-ties of hydrolysates from collagen wastes of these animals havealso been studied extensively, most especially its antihypertensiveproperty. This is due to the fact that, collagen is rich in prolinewhich plays a key role in the antihypertensive reaction (Giménezet al., 2009; Gómez-Guillén et al., 2011).

The poultry by-products have also been receiving some atten-tion with respect to their utilisation as sources of functional andphysiological food ingredients. This might be due to the global in-crease in production and consumption of poultry products leadingto generation of large quantities of wastes. The blood, viscera, skin,bone, head, feet, mechanically deboned meat and feather are themajor by-products generated during chicken processing with eachhaving varying amount of protein (Table 3). The blood representsabout 2–6% of the live bird weight (Salminen and Rintala, 2002;Piazza et al., 2011). After slaughter, the blood is usually collectedseparately and often treated with chemicals to prevent coagula-tion. When filtered and dried, it becomes a highly concentratedprotein source known as blood meal which shows a high contentof lysine, arginine, methionine and cystine (Márquez et al., 2005;Huang and Liu, 2010) and is commonly used as animal feed supple-ment and fertilizer. The feather on the other hand constitute about5–7% of the live bird weight (Grazziotin et al., 2008). A wholefeather is almost a pure protein material with a crude protein con-tent of over 90% (Taskin and Kurbanoglu, 2011). Feather proteinknown as keratin is rich in glycine, serine and proline but deficientin some essential amino acids like histidine, methionine and lysine(Sangali and Brandelli, 2000; Ouled Haddar et al., 2009). However,keratin is highly resistant to proteolysis hence it is commonly sub-jected to thermochemical treatment to produce feather meal hav-ing an inferior protein quality. Chicken leg bone is a by-product ofdeboned chicken meat which is produced in large quantities insome parts of the world. It is made up of adhering meat, cartilageand connective tissues, thus making it a good source of protein.Chicken bone protein is essentially collagen, which account forup to 90% protein content in bone. Therefore, the presence ofhydroxylproline is one distinguishing feature of the amino acidprofile of this waste (Cheng et al., 2009a,b). The head, feet, skin(which are consumed in some parts of the world) and viscera have

X2 X3 (�C) X4 (min) Y (%) References

7.0 45 240 45 Guerard et al. (2002)8.0 57 120 17.2 Gbogouri et al. (2004)8.5 50 135 50 Bhaskar and Mahendrakar (2008)8.3 59 84 33 Dey and Dora (2011)8.0 50 1440 3.2 Kechaou et al. (2009)8.0 50 1440 6.88.0 50 1440 7.08.0 50 1440 3.1 Kechaou et al., 20098.0 50 1440 1.98.0 50 1440 3.36.0 45 360 62 Nilsang et al. (2005)6.0 50 360 686.24 54.2 180 28.89 Pérez-Gálvez et al. (2011)8.0 50 60 (18.3, 10.1) Sathivel et al. (2003)7.0 50 300 96 Huang and Liu (2010)7.0 43 45 34 Bhaskar et al. (2007)4.5 23 3–18.5 Daoud et al. (2005)8.0–8.3 50–55 300–360 65 Vasileva-Tonkova et al. (2007)



Table 3Protein content of different chicken by-products.

Chicken by-product Protein (%) References

Feather and feather meal 85–99 Sangali and Brandelli (2000) and Grazziotin et al. (2007)Blood meal 60–80 Huang and Liu (2010) and Sant’Anna et al. (2010)Heads and feet 16 Okanovic et al. (2009)Bone 23–24 Cheng et al. (2008a,b) and Zhang et al. (2010)Viscera 11–12 Jamdar and Harikumar (2008a,b) and Rivera et al. (2000)Chicken intestine 53–60 Jamdar and Harikumar (2005) and Muthukumar and Kandeepan (2009)Offal (heads, feet, viscera) 12–15 Cai et al. (1995) and Russell et al. (1992)


varying protein and lipid composition. The viscera have higher li-pid content while the head, skin and feet are rich in both collage-nous and keratinous proteins. Consequently, chicken by-productscan be converted to hydrolysates having functional, bioactive andnutritional benefits (Fig. 2). However, while information regardingthe bioactive properties of hydrolysates produced from these by-products is common, less is known about their functional proper-ties, as shown in Table 4.

Based on the limited reports available in the literature, kerati-nous hydrolysates usually show a stronger antioxidant activitywhile collagenous hydrolysates often display a better antihyper-tensive potential. According to Fakhfakh et al. (2011) the hydroly-sate obtained after the fermentation of chicken feather with thebacterium Bacillus pumilus A1 has a high antioxidant activity. Com-plete solubilisation of the feather was achieved after 5 days of fer-mentation in an alkaline medium using 50 g/l of substrate at 45 �Cand the highest DPPH radical scavenging activity of 0.3 mg/ml wasrecorded after 48 h. In addition, when the enzymatic hydrolysatesof meat meal (collagen waste) was compared with that of keratinwaste of chicken feather meal (also containing a mixture of hornand hoof from cow and buffalo), it was observed that the ACEinhibitory activities as shown by the IC50 of different fractions ofthe hydrolysate range from 1.1 to 3.5 in feather meal and 0.6 to2.8 in meat meal with the fraction having low molecular weightpeptides displaying the highest activity. However, the keratinwastes show stronger antioxidant activities as shown by DPPH rad-ical scavenging activities compared with the collagen wasteswhose DPPH activities was weak (Ohba et al., 2003). Also, it wasshown that hydrolysates derived from feather meal or raw feathersusing chemical method showed lower ACE inhibitory activity com-pared to other substrates like fish scale, bone, fish meal and porkcrackling (Karamac et al., 2005). The low ACE inhibitory activitycould result from the lower concentration of proline and hydrox-

Fig. 2. Overview of poultry by-products and their usag


ylproline in keratin waste compared with collagen waste (Ohbaet al., 2003; Byun and Kim, 2002). This probably account for thebetter ACE inhibitory activity observed in alcalase hydrolysedchicken leg bone, a collagen waste, as reported by Cheng et al.(2009a,b). Following the fractionation of this hydrolysate, the mostpotent antihypertensive fraction with IC50 value of 0.066, wasfound to contain 10 peptides with most having proline, lysineand alanine as part of their amino acid residues. However, in an-other study, the hydrolysate of chicken bone extract produced aftera 6 h hydrolysis with pepsin showed a strong ACE inhibitory activ-ity with IC50 value of 0.22 (lg/mL). The synthetic form of a peptidefraction isolated from this hydrolysate having the YYRA (Tyr-Tyr-Arg-Ala) amino acid sequence was found to exhibit relativelystrong antihypertensive activity (32.7 lg/mL) compared with othersynthetic ACE inhibitory peptides that are known to exist in otherfood sources (Nakade et al., 2008). When this fraction was orallyadministered to spontaneously hypertensive rats (SHRs), a signifi-cant decrease in their systolic blood pressure (SBP) was observed3 h after administration and this continues for the next 3 h. Theauthors suggested that peptides without proline can also have po-tent ACE inhibitory activity in vivo.

Enzyme type, degree of hydrolysis (DH) and the molecularweight of peptides are factors that influence the ACE inhibitoryactivity of hydrolysates. Alcalase hydrolysed substrates and lowmolecular weight peptides generally produced better ACE inhibi-tory activity (Cheng et al., 2009a,b; Huang and Liu, 2010). Alcalasehas been used extensively in most studies because of its broadspecificity and its ability to produce a high DH hydrolysate withina relatively short time frame (Benjakul and Morrissey, 1997). Thehydrolysis of chicken blood meal using enzyme alcalase corrobo-rates what has been found in previous studies that low molecularweights peptides gives a higher ACE inhibitory activity (Huang andLiu, 2010). The study revealed that prolonged hydrolysis time of

e as sources of hydrolysates, enzymes and lipids.



Table 4Bioactive and functional properties of hydrolysates from different chicken by-products.

By-product hydrolysates Functional and bioactive properties References

Chicken leg bone ACE inhibitor Cheng et al. (2009a,b), Saiga et al. (2008) and Nakade et al. (2008)Viscera Growth media and emulsifier Jamdar and Harikumar (2008a,b)

Antioxidant and ACE inhibitor Jamdar et al. (2012)Acid hydrolysed feather ACE inhibitor Karamac et al. (2005)Feather meal raw feather Antioxidant Ohba et al. (2003) and Fakhfakh et al. (2011)Chicken blood ACE inhibitor Huang and Liu (2010)


about 5 h and high enzyme concentration of 10% gives a hydroly-sate with a potential antihypertensive property having IC50 valueof 0.34 mg peptide/ml. When this hydrolysate was fractionatedinto three parts based on their molecular weights, it was shownthat the low molecular weight fraction (<3000 Da) exhibited thestrongest ACE inhibitory activity. In like manner, the hydrolysateof collagen extracted from chicken legs was found to have strongantihypertensive potential with the low molecular weight fraction(<3000 Da) having the strongest activity (Saiga et al., 2008). A sig-nificant reduction in the systolic blood pressure of orally adminis-tered SHRs was observed after 6 h. The most potentantihypertensive peptide from this fraction was an octapeptidecontaining glycine, proline, hydroxyproline, leucine and alanine.Chicken viscera hydrolysate obtained via autolytic hydrolysis atpH 2.8 and temperature 55 �C produced low emulsifying propertyand high solubility due to extensive hydrolysis carried out for 6 h(Jamdar and Harikumar, 2008a,b). The hydrolysate however, iscomparable with peptone as an excellent bacteriological media(Jamdar and Harikumar, 2008a,b). The study on autolytic hydroly-sate of chicken viscera was taken a step further by fractionating thepeptide fractions of the hydrolysates into three different molecularweight ranges viz >10 kDa, 3–10 kDa and <3 kDa (Jamdar et al.,2010). Different antioxidant assay procedures such as radical-scav-enging activity assays (DPPH, Hydroxyl, ABTS and Superoxide Rad-ical-Scavenging Activity), reducing power (RP), total antioxidantactivity (TAA) and antioxidant activity index (AAI) were used toevaluate the antioxidant potential of the chicken viscera hydroly-sate and its fractions. The data presented by the authors showedthat the viscera hydrolysate and its lowest molecular weight frac-tion exhibited the strongest radical scavenging activity with littleor no significant difference. However, the data obtained for theDPPH radical scavenging activity (43.4 ± 3.52%, 45.8 ± 3.88%) forthe first and second fraction respectively, seem to suggest thatthe size of the peptides does not significantly influence the DPPHactivity, which does not agree with other studies. Results fromother assays on the other hand corroborated previous works thatthe highest molecular weight fraction has stronger total antioxi-dant activity and reducing power. High ACE inhibitory activity ofIC50 0.65 mg/ml was however observed in the 3–10 kDa fractionand the peptide composition was noted to have stronger influenceon the antihypertensive potential compared to the molecularweights of fractions (Jamdar et al., 2012).

Apart from their functional and bioactive properties, proteinhydrolysates have also found uses in microbiology. Some microor-ganisms that are routinely used in biotechnology and those thatproduce useful industrial products such as antibiotics, organicacids and enzymes are in high demand. For large scale cultivationof these microorganisms and the economic feasibility of the pro-cess, suitable growth media for cultivating the microorganismsneed to be found (Brandelli and Riffel, 2005). Media that satisfythe requirements of optimum growth and high enzyme activityare chosen for large scale production. One important constituentof commercial growth media is peptone which provides the carbonand nitrogen source for the microorganism. Commercial peptone isa protein hydrolysate obtained from different plant and animal


sources, and this constituent is largely responsible for the high costof most commercial growth media (Kurbanoglu and Kurbanoglu,2002). Thus, some studies have been carried out to test the poten-tial of different by-products as alternative and cheap sources ofpeptone for different microorganisms (Vasileva-Tonkova et al.,2007; Safari et al., 2009). Taskin and Kurbanoglu (2011) preparedpeptone from chicken feather and compared the growth of threebacteria in this medium with other commercial media containingtryptone peptone, fish peptone and protease peptone. They foundthat, biomass yield for the bacterium Bacillus subtilis was highestin the chicken feather peptone compared with the others. This isan important finding because B. subtilis is an important microor-ganism used for producing different enzymes like alpha amylaseand proteases (Gupta et al., 2002; Najafi et al., 2005). In anotherstudy, it was demonstrated that acid hydrolysate of ram horncan be utilised as peptone for the growth of bacteria, fungi andyeast at concentration not more than 4% ram horn peptone (Kurb-anoglu and Kurbanoglu, 2002). Based on these few references it canbe seen that more researches are still needed to test the potentialof other chicken by-products as sources of peptone. A combinationof the collagenous and keratinous by-products of chicken might of-fer a peptone source that can support the growth of diversemicroorganisms.

Although keratinous hydrolysates from poultry can function assource of C and N in culture media, other studies have shown thatthe presence of unhydrolysed keratin in culture media can also in-duce the extracellular production of keratinases (keratin degradingenzymes) by certain microorganisms (Cheng et al., 2009a,b; Guptaand Ramnani, 2006; Cai et al., 2008 ; Casarin et al., 2008). The en-zymes can find uses in the detergent, cosmetic, medical and leatherindustry while the peptides and amino acids from the degradedfeather can be used for some downstream processes such as pep-tone or in cosmetic formulation. Consequently, different speciesof Bacillus and Streptomyces bacteria isolated from decomposingfeather and soil have been tested for their ability to produce kera-tinases in media containing keratin substrate (Mazotto et al., 2011;Cheng et al., 1995). The molecular structure of the keratin sub-strate used in the culture medium also influences the productionof keratinases as demonstrated by the work of Mazotto et al.(2011) where feather meal substrate gave a better recovery ofthe enzyme compared with substrate containing whole featherwhen cultured with three strains of Bacillus spp. The partial dena-turation of feather keratin during the production of feather mealmight be responsible for the better utilisation of the substrate assource of carbon and nitrogen by the microorganism. Conditionssuch as pH and temperature are on the hand dependent on the nat-ure of the keratinase producing microorganism with most havingoptimum enzyme production at alkaline pH and mesophilic tem-peratures (Brandelli and Riffel, 2005; Syed et al., 2009; Brandelliet al., 2010).

By following the trends in the recovery of useful biomoleculesfrom animal by-products, two important products that can be de-rived from the intestine or the viscera are proteases and lipids. Pro-teases are an example of enzymes whose application hastranscended the food industry and are now widely used in other




industries (Gupta et al., 2002). The demand for proteases has con-tinued to soar because they have found application in detergents,waste processing, pharmaceutical, leather and silk industries (Gup-ta et al., 2002; Nascimento and Martins, 2004). The need to reducethe reliance on chemicals in these industries because of environ-mental and safety consideration is one factor that is driving thisdemand for proteases (Choudhary et al., 2004). Up till now, mostof the commercial proteases were obtained from microbial sourcesespecially from the Bacillus species of bacteria (Kumar and Takagi,1999). Strains from Bacillus sp. are known to produce extracellularproteases even at the extreme of temperature and pH thus makingthem useful in industrial processes that take place at these condi-tions (Kumar and Takagi, 1999; Han and Damodaran, 1997; Anwarand Saleemuddin, 1998). As a result, the needs to achieve full util-isation of organic waste products from animal origin and also meetthe demand for proteases have initiated attempts aimed at recov-ering proteases from these wastes. Many reports are available onthe recovery, characterisation and utilisation of proteases from fishprocessing by-products (Espósito et al., 2009; Murado et al., 2009;Shahidi and Janak Kamil, 2001; Haard, 1998; Souissi et al., 2008).Protease recovery from chicken by-products on the other handhas only been seen in recent times. It has been shown that poultryviscera are rich in proteolytic enzymes (Rathina Raj and Mahendra-kar, 2010). These enzymes which are mostly acidic proteases havebeen utilised in vivo for the recovery of soluble protein which canbe incorporated into poultry or fish feed formulation. However, asshown by the work of Rathina Raj and Mahendrakar (2010) preser-vation methods such as acid ensiling and fermentation can affectthe activity of the proteases. For instance, a 66% and 57% reductionin the activity of acidic protease was observed in the extract of acidensiled and fermented chicken viscera respectively while reduc-tion of the activity of neutral and alkaline protease were between42% and 47% respectively. Thus, more investigations are still re-quire in this area in order to develop a method that can achievethe dual objective of maintaining the stability of the raw material(viscera) and the activity of the proteases. Chicken intestine hasalso been shown to be a good source of aminopeptidases (Damleet al., 2010) which are enzymes that are used in the food industryto remove bitterness from hydrolysates and also improve theirfunctional properties. An aminopeptidase known as aminopepti-dase N (APN) was successfully isolated and purified from chickenintestine. However, this enzyme only showed a better debitteringefficiency in the presence of other aminopeptidases (Mane et al.,2010). In another study, proteases in the chicken intestine wereused in situ for the hydrolysis of tannery fleshing, a waste productof leather processing (Raju et al., 1997). Sarangi et al. (2011) puri-fies alkaline protease from the same source using aqueous twophase system (ATPS).

Lipids are important nutrients required for the normal function-ing of the human body. The building blocks of all lipids are the fattyacids which can be classified as essential and non-essential fattyacids. Long chain n � 3 polyunsaturated fatty acids (PUFAs) includ-ing eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) aresome of the essential fatty acid in high demand because of theirimportant health benefits (Patil and Nag, 2011; Khoddami et al.,2009). Their roles in the reduction of risk of cardiovascular disease,hypertension, autoimmune and inflammatory diseases have beenestablished (Kim and Mendis, 2006). Also, they are involved inthe development of brain and nervous tissue in infants, and visualfunction (Innis, 2004; Uauy et al., 2001).

Different varieties of food that are rich in essential fatty acidsincluding PUFA, most especially fish, have been used to meet thebody’s requirement of these nutrients. However, the increasing hu-man population and awareness of the health benefit of PUFA aremaking researchers look for other sources of the nutrient. Fishproducts and their wastes have been used extensively in the re-


search for alternative sources of PUFA. This is due to the fact that,characterisation of oils obtained from different species shows high-er concentration of polyunsaturated fatty acids than monosaturat-ed or saturated fatty acids (Zuta et al., 2003; Sahena et al., 2009).More so, different authors have confirmed that different parts offish wastes viz liver, head, muscle, skin and intestine produce dif-ferent yield of PUFA (Khoddami et al., 2009; Sahena et al., 2010;Nazeer et al., 2009).

Methods of extraction include hexane extraction, vacuum distil-lation, urea complexation and conventional crystallization all ofwhich either requires high temperature leading to loss of valuablecompounds or use of toxic solvent (Sahena et al., 2010). Supercrit-ical carbon dioxide (SC-CO2) extraction and fractionation method iscurrently being tested by different groups as a safer, cheaper andnon-destructive method for the extraction of PUFA (Corrêa et al.,2008; Dunford et al., 1997). Although the viscera of some animalsincluding poultry are known to be rich in PUFA (Saha et al., 1990),few attempts have been made at extracting the oil from poultry by-products. Recently, Patil and Nag (2011) successfully recoveredPUFA from free fatty acids obtained by the alkaline hydrolysis ofchicken viscera. A two stage process involving crystallization withacetone at low temperature followed by urea fractionation wasused for the production of PUFA concentrate with a yield of about10%.

4. Hydrolysis methods for producing chicken by-productshydrolysates

Due to the multi-application of protein hydrolysate, this sectionwill further explore the production methods that are available forproducing hydrolysates for specific function. Although differenthydrolysis methods can be used to achieve the objective of peptidecleavage, the choice of method to use however can depend on thenature of the substrate, expected properties or application of thehydrolysates, the efficiency of the process and availability of mate-rials or equipment required for the process.

The functional and physiological activity of a protein hydroly-sate is usually influenced by the amino acid composition of thehydrolysate either in free form or bound to peptide and this isdetermined in part by the method and conditions of the hydrolysisprocess (Kristinsson and Rasco, 2000). More so, the molecularweight of the resulting peptides which influence the functionalproperties of the hydrolysate is largely determined by how wellthe reaction can be controlled and the type of enzyme that cataly-ses the reaction (Rossi et al., 2009).

Consequently, an appropriate method of hydrolysis has to bedecided upon in the course of producing a hydrolysate with a par-ticular functional or bioactive property. In general, chemical, ther-mal, microbial and enzymatic methods or a combination of theseare commonly used for the hydrolysis of organic materials. How-ever, the enzymatic method is a method of choice for producingfunctional and bioactive hydrolysate (Rossi et al., 2009). Theadvantages of this method lie in the ease of control of the hydroly-sis reaction, the reproducibility of the method, the specificity of theenzymes involved in the reaction and the nutritional quality of theresulting hydrolysate (Tarté, 2009).

Different investigators have used enzymatic method for thehydrolysis of poultry by-products. The efficacy of most commercialenzymes like alcalase and pepsin to hydrolyse chicken leg boneand head respectively has been reported (Cheng et al., 2009a,b;Surówka and Fik, 1994). Rossi et al. (2009) optimised the enzy-matic hydrolysis conditions of mechanically deboned poultry meatusing alcalase and flavourzyme. It was observed that the hydroly-sate derived from alcalase produced the highest nitrogen recoveryof 89% compared to that of flavourzyme which was around 67% and




this might be due to the broad specificity of the former. Further-more, in order to improve the palatability of pet food producedfrom low ash poultry meal, enzymatic hydrolysis of the meal wascarried out with enzymes used singly or in combination (Nchienziaet al., 2010). It was observed that hydrolysate derived fromsequential hydrolysis with alcalase and flavourzyme gave a goodyield of hydrolysable material containing low molecular weightpeptides and free amino acids. Most protein hydrolysates producedby alcalase and other endoproteases usually have bitter tastewhich has been attributed to the hydrophobic peptide fractionsgenerated by these enzymes (Seo et al., 2008). Therefore, introduc-ing flavourzyme which is an exoprotease will help cleave most ofthe hydrophobic amino acids from the peptide fractions thusimproving their palatability.

Feather protein (keratin) on the other hand is a recalcitrant bio-molecule which is not easily hydrolysed by most commercial en-zymes. Its resistance to proteolytic degradation stem from thedisulphide bonds, hydrogen bonds, salt bridges and the extensivecross linking of its structure (Sangali and Brandelli, 2000). Conse-quently, thermal and thermochemical methods are largely usedto modify the structure chicken feather keratin prior to its use asfeather meal (Moritz and Latshaw, 2001). These treatments usuallyinvolve heating feathers at high temperature and pressure alone orin an acid or alkaline solution in order to cleave the disulphidebonds and disrupt the hydrophobic interaction thus making theprotein more soluble and digestible (Wang and Parsons, 1997).These methods are however disadvantageous because they pro-mote destruction of certain heat labile amino acids and the racemi-zation of amino acids (Papadopoulos, 1989). Thus, keratinhydrolysates produced via these methods have low protein qualityand their use as animal feed is very limited due to poor amino aciddigestibility (Papadopoulos, 1989). The use of chemical pretreat-ment followed by enzymatic hydrolysis of feathers has been sug-gested by different authors as better method of producingfeather protein concentrate or feather keratin hydrolysate (Dalev,1990; Mokrejs et al., 2011). Pretreatment of a feather substratewith an alkaline solution (a reducing agent) under a mild temper-ature setting of about 70–80 �C can provide the redox required forthe cleavage of disulphide bonds leading to a more open proteinstructure which will improve the accessibility of the enzyme tothe active sites within the structure (Dalev, 1990; Coward-Kellyet al., 2006; Mokrejs et al., 2011). The combination of low temper-ature regime and short incubation time during the alkaline pre-treatment stage might help prevent loss and racemization ofamino acid in addition to preventing excessive peptide cleavage.Thus, this might be a cost effective, rapid and safe method of pro-ducing keratin hydrolysate.

In spite of the resistance of feather to proteolysis however, ithas been shown that this keratinous material does not accumulatein nature which indicates the existence of keratin degrading micro-organisms. Utilisation of these microbes for keratin degradationmight serve as cheap and environmental friendly means of obtain-ing biological products like peptides, amino acids and proteaseswhich can be used for different applications. Result from severalresearches show that some Bacillus species of bacteria, actinomy-cetes and some fungi can effectively degrade keratinous substrates(Fakhfakh et al., 2011; Korniłłowicz-Kowalska and Bohacz, 2011;Williams and Shih, 1989; Lin et al., 1995, 1999; Nagal and Jain,2010) which they use as sources of C, N, S and energy. An excellentreview by Korniłłowicz-Kowalska and Bohacz (2011) pointed outthat the Bacillus licheniformis strains were prominent among thekeratinolytic bacteria while among the actinomycetes and fungi,the genus Streptomyces and Chrysosporium respectively, were fre-quently observed. Although the mechanism of microbial degrada-tion of keratin substrate is still a subject of some debates, anumber of studies have reported that the synergistic relationship


between some intracellular reductases and the extracellular prote-ases of the microorganisms is responsible for their ability to de-grade keratin (Böckle and Müller, 1997; Ramnani et al., 2005).These authors explained that the intracellular reductases act asbiological reducing agents that cleave the disulphide bonds ofthe keratin thereby opening the protein structure to the extracellu-lar proteases which cleaves the peptide bonds of the protein. Thisexplanation was substantiated by Ramnani and Gupta (2007) whofound that some conventional proteases could degrade feather inthe presence of chemical or biological reducing agents. However,they noted that pepsin and trypsin can only degrade feather afterthe surface hydrophobic residues on the keratin structure havebeen cleaved by other proteases thus releasing the lysine and argi-nine active sites buried within the protein. This probably explainedwhat was observed when the nutritive value of feathers treatedunder aerobic condition was compared with those treated underanaerobic condition. It was shown that the product from the anaer-obic condition named feather lysate have improved amino aciddigestibility when fed to broiler chicks and consequently gave im-proved growth response compared with aerobically treated feather(Williams et al., 1991). Also, Fakhfakh et al. (2011) demonstratedthat B. pumilus A1 isolated from slaughter house polluted watercompletely solubilised chicken feather during fermentation for2 days at 45 �C in an alkaline medium. It was also reported thatthe hydrolysate possesses good antioxidant potential and can beused as functional food ingredient. Recently, it was established thatfeather hydrolysate obtained via the action of a mixed culture ofthermoactinomycetes can be used for soil enrichment since it im-proves the activities of soil microbes and consequently plantgrowth (Gousterova et al., 2012). In addition to its soil enrichmentpotential, analysis of the hydrolysate showed that it contains somepeptides which exhibit antifungal activity against four plant path-ogenic fungi.

Compared to other chicken by-products, viz., head, feet, legbone and feather, the viscera has a high content of endogenouspeptidases (Jamdar and Harikumar, 2005; Raju et al., 1997). Theseenzymes play key role in the digestion of food in live birds while atpost mortem they are involved in reaction that leads to rigor mor-tis and flavour development (Toldrá and Flores, 2000). Some con-siderations have been given for the utilisation of endogenousenzymes for the recovery of proteins as soluble peptides and aminoacids from poultry viscera (Jamdar and Harikumar, 2005; Sarangiet al., 2011). Cathepsin B, D, H and L, aminopeptidases and alkalineproteases are group of enzymes that have been isolated from dif-ferent parts of poultry viscera (Jamdar and Harikumar, 2005; Dam-le et al., 2010). These enzymes can be used to hydrolyse in vivoprotein in poultry viscera in a process known as autolysis. Cathep-sin D was found to predominate in the autolytic breakdown ofchicken intestine, with optimum activity at pH 2–2.5 and 60 �C(Jamdar and Harikumar, 2008a,b). The authors claimed that, prod-ucts derived via this method will have acceptable microbial qualitysince protein degradation occurs at acidic pH. However, the appli-cation of the hydrolysate derived from autolytic method can belimited. This hydrolysate can supplement animal feed and nitrogensources in growth media because these uses do not require exten-sive manipulation and control of the hydrolysis condition. Produc-tion of hydrolysates with improved functional and bioactiveproperties on the other hand involves careful control of hydrolysisconditions like temperature, time, pH and enzyme type. Hence,exogenous enzymes used singly or in combination are more suit-able for these purposes. In this instance, the hydrolysis conditionis amenable to manipulation hence, hydrolysates with desiredfunctional and bioactive properties are produced (Ohba et al.,2003; Cheng et al., 2008a,b). Thus, autolysis can be viewed as a costeffective way of recovering soluble proteins from chicken process-ing by-products.




Until recently, hydrolysis of biomass waste into peptides andamino acids has been achieved with the aid of chemicals, heatand enzymes, in combination or separately. Chemical treatment,although rapid, is often associated with loss of desired productslike some amino acids coupled with generation of effluents whichpollute the environment (Sereewatthanawut et al., 2008; Papado-poulos, 1989). Enzymatic treatment on the other hand can beexpensive and usually requires long processing times (Sere-ewatthanawut et al., 2008). Therefore, the development of a newenvironment-friendly method to overcome the drawback of chem-ical hydrolysis and the frequently long enzymatic hydrolysis isnecessary (Uddin et al., 2010). A new hydrolysis technology mak-ing use of subcritical water has been developed to overcome thesechallenges. Water is an abundant, non-toxic and cheap natural re-source. In its pure state, water exists as a polar liquid having a den-sity of 1000 kg m�3, a dielectric constant of 79.73 and an ionicproduct of 10�14. However, at subcritical temperatures and pres-sures, it shows low dielectric constant, viscosity and surface ten-sion. The ionisation constant of water on the other handincreases to a maximum value at subcritical region thus, releasingionic products which are of three orders of magnitude higher thanthat obtained under normal condition. As a result, subcritical wateris rich in both hydrogen and hydroxide ions which can take part inacid–base catalysed reaction (Galkin and Lunin, 2005; Shaw et al.,1991). This technology has been applied for the hydrolysis of dif-ferent biomass types and their wastes in recent years (Kanget al., 2001; Rogalinski et al., 2005; Tavakoli and Yoshida, 2006;Yoshida et al., 1999). Production of amino acids by hydrolysingchicken intestine in subcritical water has been carried out (Zhuet al., 2010). Amino acid yield of 11.49% was obtained at optimumtemperature 533 K, reaction time 28 min and H2SO4 concentrationof 0.02%. Cheng et al. (2008a,b) applied subcritical water for thehydrolysis of fish waste, chicken waste, feather and hair in orderto obtain amino acids. Reaction temperature, pressure and timewere noted as factors that affect the yield of amino acid with thehighest yield occurring at temperature between 200 and 290 �C,a pressure range of 6–16 MPa and a reaction time range of 5–20 min. In this study, short reaction time, mild reaction tempera-ture and pressure are important in obtaining a good yield of aminoacid and reducing the cost of the process. Thus, subcritical waterprovides an environmental friendly, simple and efficient meansof recovering value added products from waste biomass.

In general, depending on the effectiveness of the hydrolysismethod, little or no residues are left after hydrolysis of the by-products at the laboratory scale. Data from researches that lookat the volume of solid wastes generated during the production ofhydrolysate especially from a large scale process are rare or cur-rently unavailable to the best of our knowledge. Hence, it is advis-able that the amount of solid residues generated after a particularhydrolysis process should be adopted as a routine analysis in thelaboratory, pilot and industrial plants. However, since any residuesthat are left will be unhydrolysed components containing someamount of protein, they can be added to compost or growth mediaas source of carbon and nitrogen.

In addition, information about the actual financial implicationof these methods is rarely reported. Although the use of readilyavailable animal by-products rather than edible food products forthe production of protein hydrolysates offers some advantageswith regard to the cost raw material, it is pertinent to ascertainhow the cost of enzymes, microbes, chemicals and equipment forsubcritical extraction will influence the industrial feasibility ofthese methods. Furthermore, the nature of the desired end productmight be a factor that will contribute to the cost. For instance,hydrolysates requiring extensive peptides cleavage where veryshort peptides and free amino acids are required will likely involvea more costly process (for instance, addition high dose of enzyme


or high energy requirement), than those requiring minimal hydro-lysis. Thus, the economic feasibility of each method of hydrolysiscan be determined if the production scale, substrate for hydrolysisand specific properties of end products are standardised as this willgive room for unbiased comparison.

5. Microbiological safety of chicken by-product hydrolysates

Microbiological safety and storage stability of hydrolysates de-rived from waste sources have been the subject of some researches(Sangtherapitiku et al., 2005; Bueno-Solano et al., 2009; Rossi et al.,2009). These researches are carried out in order to address somepotential issues that might form the basis for consumer rejectionof products obtained from these by-products. High microbial loadand rapid deterioration of the waste material will affect consumerconfidence regarding the safety of the resultant products fromthese sources. However, Surówka and Fik (1994) observed the ab-sence of microbial pathogen in dried chicken head hydrolysate.This result was corroborated by Sangtherapitiku et al. (2005)who confirmed the absence of Salmonella and Escherichia coli in dif-ferent types of spent hen hydrolysate.

The microbial safety of the finished product, for instance pro-tein hydrolysate, depends on the microbial load of the startingmaterial. High initial microbial load of the viscera, bone and headwill increase the risk of microbial contamination and low shelf lifeof the finished product. Hence, traditional methods like heat ster-ilization, acid stabilization, chilling and fermentation are widelyused to enhance the shelf life of animal by-products, although withsome drawbacks (Giri et al., 2000; Kherrati et al., 1998). A novelmethod which can ensure prolonged shelf life and microbial safetyof poultry by-products was recently developed. This method madeuse of gamma radiation to decontaminate and improve storage sta-bility of poultry viscera (Jamdar and Harikumar, 2008a,b). Expo-sure of poultry viscera to radiation dose of 20 KGy extends itsshelf life to 62 days. A lower radiation dose of 5 KGy ensures stor-age stability for 5–10 days at 26 and 4 �C, respectively. Radiationdose between 5 and 10 KGy is just enough to reduce total viablecount of bacteria, yeast and mold by 4 and 5log10 units while coli-form was eliminated completely (Jamdar and Harikumar, 2008a,b).These research efforts showed that, value-added products that arefree from pathogens can be produced from chicken processing by-products. In general, since few literature references are currentlyavailable on the microbial quality of protein hydrolysates, we arerecommending that microbial analysis should be added as part ofthe routine analyses carried out on protein hydrolysates especiallythose derived from animal by-products in order to improve theiracceptance and guarantee their safety.

6. Future application of poultry by-products

There is still a wide area of research to be explored in the util-isation of chicken by-products for beneficial uses. Among the by-products, the viscera seem to be the most useful because all the va-lue-added products can be derived from it (Fig. 1). The functionaland bioactive properties of viscera hydrolysate obtained by con-trolled hydrolysis needs to be ascertained in order to clarify theirpotential uses as food ingredients. The presence of different kindsof protein such as keratin and collagen in these wastes makes themuseful substrates for specific function. To ensure reproducibility,enzyme type and optimum hydrolysis conditions for producinghydrolysates with specific functional property will have to beidentified.

In addition, different authors have confirmed the potential ofhydrolysates for the creation of thermal process flavouring in modelsystems (Hwang et al., 1997; Guo et al., 2010). Thermal flavouring




is a term used to describe the food flavouring generated by heatingprecursor materials, usually a reducing sugar and amino acid orpeptide, under a carefully controlled condition (Guo et al., 2010).The resultant product from this reaction can be added to food toimprove its sensory properties. Thus, hydrolysates from chickenviscera, heads and leg bone can easily be obtained for the genera-tion of process flavouring if method of oil removal is in place andappropriate enzyme is used. Also, the potential of hydrolysatesgenerated from feather for producing thermal flavouring can beattempted. The protein in feather, keratin, is known to contain highamount of cysteine (sulphur-containing amino acid) which canassist in the production of meat-like flavouring when heated withreducing sugar.

Lastly, the production of hydrolysates usually involves the gen-eration of two fractions namely, the soluble and the insoluble frac-tion or sludge. In most studies, only the soluble fraction is used forfurther analysis while the insoluble fraction is discarded. As a re-sult, information relating to the volume, properties, compositionand potential utilisation of the insoluble components are uncom-mon. Therefore, it will be worthwhile to characterise and suggestways of utilising the insoluble residues from poultry by-productshydrolysates. Based on some reports of hydrolysates from fishsources, it has been shown that protein, amino acids and sometrace elements tends to partition between the soluble and theinsoluble fractions (Liaset et al., 2003; Liaset and Espe, 2008). Gen-erally, insoluble fractions from these hydrolysates are low in ash,high in insoluble proteins, hydrophobic peptides or amino acids,have a high fat content and are mostly used in aquaculture feeding(Yin et al., 2011; Liaset et al., 2003; Liaset and Espe, 2008).

Thus, there are ample evidences to believe that residues fromchicken bone hydrolysates will contain trace elements which canbe added to soils or for the mineral enrichment of a feed formula-tion. More so, protein and oil rich residues from other chicken by-products hydrolysates can find uses in the conventional methodslike composting and anaerobic digestion, and can also be consid-ered for aquaculture feeding.

7. Conclusion

This review discussed the utilisation of animal by-products asprotein hydrolysates having functional and bioactive propertieswith emphasis on the chicken by-products. There exists moreinformation regarding the bioactive properties of hydrolysatesfrom these sources compared to functional properties. Differentmethods of hydrolysis of the by-product as well as their microbio-logical safety are dealt with. These by-products can also be con-verted to peptone for use in growth media or used directly assubstrates for culturing microorganisms that can produce usefulbiotechnological products. A short note was given on the potentialof poultry viscera or intestine as source of polyunsaturated fattyacid and proteases, although not much has been done in this re-gard, along with some future application of poultry by-products.

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