Food Research International 73 (2015) 3–12

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Food Research International

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

Microbial enzymes for bioconversion of poultry waste into

added-value products
Adriano Brandelli a,⁎, Luisa Sala b, Susana Juliano Kalil b
a
Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, Brazil
b
Laboratório de Microbiologia, Escola de Química e Alimentos, Universidade Federal do Rio Grande, 96201-900 Rio Grande, Brazil

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

Article history: The continuous growth of poultry industry results in an increased amount of waste provided from both produc-
Received 1 November 2014 tion facilities and processing plants. The processing of poultry meat results in massive quantities of solid waste as
Received in revised form 19 January 2015 feathers, viscera, bones, and dead on arrival. The use of enzymes for bioconversion of such byproducts into ma-
Accepted 24 January 2015
terials with increased value is an interesting strategy. Enzymes can be useful to convert poultry waste into feed
Available online 31 January 2015
and fertilizers. The hydrolysis of animal byproducts can also generate bioactive peptides, which are important
Keywords:
molecules that may exert physiological effects in vivo, as antioxidant, angiotensin-I converting enzyme (ACE)-in-
Meat industry hibitory and dipeptidyl peptidase-IV (DPP-IV)-inhibitory activities. Thus, poultry byproduct hydrolysates show
Chicken feathers potential for use as functional ingredients. This review presents an overview on useful microbial enzymes for bio-
Byproduct meal conversion of poultry waste, such as proteases, lipases, combined enzyme preparations and, especially,
Keratinase keratinases for bioconversion of feathers. A discussion about production, purification and properties of
keratinases is presented. Main areas for further studies are large scale production and purification of keratinases,
and development of effective processes for production of bioactive molecules from poultry waste.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction clogging of soil by fat accumulation, and presence of pathogens in

Poultry industry is an important and diverse component of the food
sector. Poultry products, including eggs, chicken and turkey meat repre-
sents an important protein source in the diets of most people. Broiler
production greatly increased from the 1980s, due to the suggested nu-
tritional benefits of chicken meat compared with other meats and
through an extraordinary increase in consumption. This increase has
been also accredited to the poultry industry, which supply processed
products that are easier for the customer to prepare. This industry is
constantly growing and the largest producers worldwide, namely USA,
Brazil and China, account for more than 40 million tons per year produc-
tion (USDA, 2014). The European Union annual production is approxi-
mately 11 million tons (AVEC, 2014).
The increased production of poultry industry results in a huge
amount of waste that needs to be managed. Three primary waste prod-
ucts are dissolved air flotation sludge, compost from dead birds and
hatchery waste, and litter and manure from production facilities. The
overabundance of litter and manure resulted in excessive waste appli-
cation on cropland or imprudent stockpiling and dumping of waste
nearby waterways (Salminen & Rintala, 2002; Simpson, 1991). Al-
though a controlled utilization of such waste as soil fertilizer is feasible,
some concerns are due to the high levels of oxygen demand, physical
⁎ Corresponding author. Tel.: +55 51 3308 6249; fax: +55 51 3308 7048.
E-mail address: abrand@ufrgs.br (A. Brandelli).
dead, hatchery and litter compost.
The major waste materials generated in poultry processing plants
are feathers, soft meat, blood, deboning residue, and also dead on arriv-
al. These materials are currently converted into meat and bone meal,
feather meal, blood meal and fats/oils by rendering process (Lasekan,
Bakar, & Hashim, 2013; Salminen & Rintala, 2002). Although these
meals are a good source of protein, their utilization can be limited due
to nutritional restrictions associated with losses of essential amino
acids (Onifade, Al-Sane, Al-Musallam, & Al-Zarban, 1998), and with
high calcium, phosphorous and lysine content of the meat and bone
meal (Batal & Dale, 2011).
Feathers are an abundant waste of poultry industry since they
account for approximately 8% of the adult chicken weight and are con-
stituted by about 90% protein (Onifade et al., 1998). The major insoluble
structural protein of feathers is keratin, which is recognized for its recal-
citrance. Keratin-rich wastes are of difficult degradation since the
protein chains are tightly packed and strongly stabilized by several hy-
drogen bonds and hydrophobic interactions. In addition, cross-linking
of polypeptide chains by several disulfide bonds provides mechanical
resistance and impairs degradation by conventional proteolytic en-
zymes (Brandelli, 2008; Kornillowicz-Kowalska & Bohacz, 2011). How-
ever, some microbial enzymes can hydrolyze insoluble feather keratins,
allowing their conversion into feedstuffs, fertilizers, and films (Gupta &
Ramnani, 2006; Onifade et al., 1998). In addition, applications of these
enzymes for pharmaceutical and cosmetic purposes have been also

http://dx.doi.org/10.1016/j.foodres.2015.01.015
0963-9969/© 2015 Elsevier Ltd. All rights reserved.
4 A. Brandelli et al. / Food Research International 73 (2015) 3–12
described (Brandelli, 2008). Proteolytic enzymes can be also useful to
increase nutritional value of poultry byproducts like mechanically
deboned meat (Rossi, Flores, Venzke, & Ayub, 2009) and feather meals
(Odetallah, Wang, Garlich, & Shih, 2003; Pedersen, Yu, Plumstead, &
Dalsgaard, 2012). Thus, enzymatic catalysis can be an interesting alter-
native to convert byproducts generated during poultry processing into
added-value materials.
This article presents a discussion on useful enzymes that can be use-
ful in the management of byproducts of poultry industry. Considering
the relevance of protein-rich waste, emphasis is devoted to proteolytic
enzymes used in bioconversion of these waste materials, in particular
on the production, purification and applications of keratinases in bio-
conversion of keratin waste.
2. Byproducts of poultry industry
Poultry industry generates a large amount of byproducts, mostly in
the form of heads, legs, bones, viscera, skin and feathers, in addition to
whole carcasses in the form of dead on arrival. These materials repre-
sents a massive amount of solid waste that should be properly managed
to avoid environmental damage and loss of important raw materials
for feed industry and as biological resources (Jayathilakan, Sultana,
Radhakrishna, & Bawa, 2012; Lasekan et al., 2013).
Animal wastes generated in the meat industry contain considerable
amounts of insoluble and hard-to-degrade structural proteins like colla-
gen, elastin and keratin, which are major constituents of bones, organs
and hard tissues. These byproducts are often rich sources of protein,
which can be extracted and hydrolyzed to be used as feed or functional
ingredients. The protein amount of different poultry byproducts and
bioactive properties of the byproduct hydrolysates are presented in
Table 1.
2.1. Collagen-rich waste
The extracellular matrix that occupies the major part of the animal
tissue volume is composed by a diversity of proteins and polysaccha-
rides (mostly linked to proteins as proteoglycans) that maintain and
protect the cells and tissues (Fig. 1). Among these proteins, collagens
are found in all animals and are the most abundant proteins, reaching
25% of total protein mass of skin and bones (Mayne & Brewton, 1993;
Van der Rest & Garrone, 1991). Byproducts that are rich in collagen
can be heat-denatured and extracted leading to the formation of gelatin.
Also, hydrolysates from collagen-rich waste have been investigated be-
cause of its antihypertensive characteristics, which appears to be associ-
ated with the elevated proline content (Gómez-Guillen, Giménez,
López-Caballero, & Montero, 2011). Chicken leg bone is derived from
deboning chicken meat, and is basically constituted by collagen, thus
containing hydroxyproline as a peculiar feature. Both chicken bone
and blood have been used for producing hydrolysates with potential
bioactive properties (Cheng et al., 2009; Huang & Liu, 2010), as shown
Table 1
Protein-rich byproducts of poultry industry as source of bioactive and functional
hydrolysates.a
Poultry byproduct Protein (%) Functional and bioactive properties
Feathers and feather meal 85–99 Antioxidant, ACE inhibitor and DPP-IV
inhibitor b
Blood and blood meal 60–80 ACE inhibitor c
Bone 23–24 ACE inhibitor a
Viscera 11–12 Antioxidant, ACE inhibitor, emulsifier a
Chicken skin 17–20 ACE inhibitord
Offal (heads, feet, viscera) 12–15 Antioxidant e
a
Essentially compiled from Lasekan et al. (2013).
b
Fontoura et al. (2014).
c
Huang & Liu (2010).
d
Saiga et al. (2008).
e
Manhiani, Northcutt, Han, Bridges, & Dawson (2013).
in Table 1. Bioactive peptides are examples of important molecules
found in animal byproduct hydrolysates that can exert a physiological
effect in vivo, showing potential for use as functional ingredients in an-
imal feeds.
2.2. Feathers
Feathers, accounting to about 8% of live weight, are a major waste of
poultry processing plants. The large amount of waste feathers generated
each year by commercial poultry plants creates a serious environmental
problem. Disposal strategies like burning in incineration plants and
landfilling are restricted or banned in many countries. Feathers are
mostly composed by keratin protein and this byproduct has been fre-
quently converted into feather meal, a low-grade feed ingredient. Feath-
er waste has been also converted into fertilizers for agricultural purpose,
as bedding material, and for decorative purpose (Jayathilakan et al.,
2012). The development of suitable processes to convert feather waste
into digestible proteins and amino acids has been the main goal, and
biotechnological approaches based on microbial enzymes have been ex-
tensively investigated in the last decade (Gupta, Sharma, & Beg, 2013;
Kornillowicz-Kowalska & Bohacz, 2011). Feather hydrolysates could
be converted into methane gas and fuel pellets for heating (Dudynski,
Kwiatkowski, & Bajer, 2012; Ichida et al., 2001), and also utilized in
the production of biohydrogen (Bálint et al., 2005). The development
of an effective conversion of feather waste into fuels may address the in-
creasing interest for energy conservation and recycling.
2.3. Miscellaneous byproducts
Heads, gizzards and blood have been typically used for meal produc-
tion, whereas feet, skin, intestines and glands can be also a source of
poultry fat (Sams, 2001). Keratin-rich materials like nails and beaks
can be degraded by keratinolytic microorganisms (Riffel & Brandelli,
2006), and are often used in combination with viscera or blood for pro-
duction of animal feed (Sams, 2001). Blood is generally treated with
chemicals to prevent coagulation and dried to become a concentrated
protein source known as blood meal, which shows a high content of
basic (Lys and Arg) and sulfur (Cys and Met) amino acids (Márquez,
Bracho, Archile, Rangel, & Benítez, 2005).
Byproducts of hatchery include egg shells, unhatched eggs, infertile
eggs, and discarded chicken, which are utilized in animal feed. These
meals present high calcium content, limiting its use up to 5% into feed
(Jayathilakan et al., 2012).
2.4. Litter and manure
In addition to plant processing byproducts, waste from the production
phase is mainly found as poultry litter and manure. These byproducts
have been typically used as recycled feed and surface covering of agricul-
tural lands (Shih, 1993; Simpson, 1991). However, poultry manure and
slaughterhouse waste can be converted in methane as energy source by
some anaerobic organisms (Salminen & Rintala, 2002). Thermophilic
anaerobic digestion has been used to degrade chicken manure and pro-
duce biogas at high rate. The major byproduct of anaerobic digestion is
biogas, which is a combustible fuel gas and can be utilized for generating
electricity, heating and drying. The digester effluent is a liquid, and the
aquacultural use of this byproduct has been demonstrated (Shih, 1993).
Pathogenic microorganisms such as fecal coliforms are destroyed in the
thermophilic digester. Also, fungi are reduced by nearly 100% and oocysts
of the pathogenic protozoan Eimeria tenella lost both their infectivity
in vivo and sporulability in vitro (Shih, 1993).
3. Useful enzymes for bioconversion of poultry waste
Advances in microbial enzyme technology offer considerable oppor-
tunity for development of low-energy consuming technologies for
 A. Brandelli et al. / Food Research International 73 (2015) 3–12
Fig. 1. Schematic representation of the structure of extracellular matrix proteins in animal tissues. The extracellular matrix is composed by collagen fibrils, located outside the cells with
elastin, water molecules and diverse proteoglycans such as hyaluronan, chondroitin sulfate, dermatan sulfate, keratan sulfate and heparan sulfate.
bioconversion of poultry waste into added-value products (Darah,
Nur-Diyana, Nurul-Husna, Jain, & Lim, 2013; Onifade et al., 1998).
Enzymatic processing may be useful to recycle protein-rich waste gen-
erated by poultry industry into valuable products, thus protecting the
environment by reducing wastage (Daroit, Correa, & Brandelli, 2009;
Moreira-Gasparin et al., 2009). The valuable enzymes for bioconversion
of poultry waste are usually proteases with capacity to act on compact
substrates. The application of keratinolytic enzymes for valorization of
feather waste is discussed in the next section. Subsequently, some ex-
amples on the use of other enzymes, such as proteases, lipases, and
combined enzyme preparations are presented.
3.1. Bioconversion of feathers by keratinases
The recycling of feathers is a topic of interest because of its potential
as an inexpensive and alternative protein feedstuff. However, feather
protein has limited utilization due to its poor digestibility and low biolog-
ical value. The conventional hydrothermal degradation of feathers con-
verts this material in a more digestible feather meal, which sustains
losses of essential amino acids and contains non-nutritive amino acids
such as lanthionine and lysinoalanine. Thus, biotechnological approaches
using microorganisms and their keratinolytic enzymes have been used to
upgrade nutritional value of poultry feathers as feed supplements
(Brandelli, 2008; Gupta & Ramnani, 2006; Onifade et al., 1998).
Keratinases have the capacity to act on compact substrates, such as
keratin-rich waste, better than other comparable proteases (Brandelli,
2008). These enzymes are gaining importance due to their numerous
applications associated with hydrolysis of keratinous substrates, mainly
byproducts of agro-industrial processes (Anitha & Palanivelu, 2013).
Keratinases can convert the keratin present in feather waste, producing
protein hydrolysates that can be used as supplement in animal feed
(Onifade et al., 1998) and production of nitrogen fertilizers (Ichida et al.,
2001). These enzymes can be used in degradation of prions (Langeveld
et al., 2003; Yoshioka et al., 2007) and in the production of keratin pep-
tides with antioxidant (Fakhfakh et al., 2012), angiotensin-I converting
enzyme (ACE)-inhibitory and dipeptidyl peptidase-IV (DPP-IV)-inhibito-
ry activities (Fontoura et al., 2014). A summary of these potential applica-
tions of enzymatic feather hydrolysates is presented in Fig. 2.
3.1.1. Feathers conversion to animal feed
Feather degradation mediated by keratinase provides a viable alter-
native to alkali hydrolysis and steam pressure-cooking of feathers
resulting in products of better nutritional properties to be used as animal
feed (Brandelli, Daroit, & Riffel, 2010). Thus, several keratinolytic prote-
ases have been investigated with the aim to produce hydrolyzed feather
keratin for feed formulation (Brandelli et al., 2010; Gupta & Ramnani,
2006). Bacillus spp. seems to represent a major source of keratinolytic en-
zymes for feather processing. The original use of a keratinase enzyme as
feed additive was reported by Lee, Ferket, and Shih (1991). The
5
keratinase from Bacillus licheniformis increased the total amino acid di-
gestibility of raw feather from 30 to 66% and commercial feather meal
from 77 to 99%. The daily weight gains of 3-week-old broilers were
66 g for soybean diet, 50 g for feather meal, and 56 g for keratinase treat-
ment. The commercial product Versazyme®, based on the subtilisin-like
keratinase from B. licheniformis, has been successfully tested as a feed ad-
ditive (Odetallah, Wang, Garlich, & Shih, 2005).
Chicken feather hydrolysates obtained with a microbial keratinase
showed improved nutritional characteristics as compared with feather
meal and feather keratin. Increased in vitro digestibility and nutritional
parameters, such as protein efficiency ratio (PER), biological value (BV)
and protein digestibility-corrected amino acid score (PDCAAS) were ob-
served for the enzymatic hydrolysates (Grazziotin, Pimentel, de Jong, &
Brandelli, 2006). Kim and Patterson (2000) compared enzymatic and
sodium hydroxide treatments for processing feathers from dead hens.
Although the alkaline treatment was more rapid for separating feathers
from the carcass, feather-digesting enzyme improved the nutritional
quality of feathers.
The complete solubilization of whole feathers was achieved by
keratinases from Bacillus pumilus A1 after 6-h incubation at tempera-
tures ranging from 45 °C to 60 °C (Fakhfakh-Zouari, Haddar, Hmidet,
Frikha, & Nasri, 2010). The crude enzyme of B. pumilus A1 was able to
degrade chicken feathers keratin in the absence of a reducing agent.
This result allows the use of the crude keratinase of B. pumilus A1 in
the conversion of waste feathers into protein hydrolysates, which can
be used as an ingredient in animal feed.
The content of amino acids of chicken feathers degraded by crude
keratinases from Microsporum fulvum IBRL SD3 after 10 days of incuba-
tion was analyzed in terms of protein that contained essential amino
acids, like threonine, valine, methionine, isoleucine, leucine, lysine, his-
tidine, and tyrosine (Darah et al., 2013). This result demonstrate that
hydrolyzed feather protein represents a valuable source of protein
that may be employed in animal diets, especially for fish and prawns.
3.1.2. Feathers conversion to fertilizers
The hydrolysates obtained from the enzymatic conversion of
poultry feathers could be also useful for preparation of nitrogen fertil-
izers or soil amendments (Kornillowicz-Kowalska & Bohacz, 2011;
Vasileva-Tonkova, Gousterova, & Neshev, 2009). The capability of
microbial keratinases to accelerate the composting of dead chicken or
discarded feathers could be a feasible and eco-friendly method of
recycling these organic wastes into nitrogen-rich fertilizers (Ichida
et al., 2001). As an example, feather digests obtained with Bacillus
pumilus showed comparable results to a reference fertilizer in a 27-day
plant growth assay using carrots and Chinese cabbage (Kim, Choi, &
Suh, 2005). Keratin degradation by the fungal keratinase of Paecilomyces
marquandii resulted in products that are potentially useful for foliar fer-
tilization. A higher amount of amino acids was observed in the keratin
hydrolysates as compared to that obtained by microbial degradation,
6 A. Brandelli et al. / Food Research International 73 (2015) 3–12
Fig. 2. Bioconversion of feather waste from poultry industry by microbial keratinases. After hydrolysis by keratinolytic enzymes, the crude hydrolysates can be used in feed formulation and
as soil fertilizers. Alternatively, hydrolysates can be fractionated to obtain peptide fractions presenting antioxidant, ACE-inhibitory or DPP-IV-inhibitory activities.
indicating the advantage of using enzyme preparations instead of micro-
organisms for keratin hydrolysis because the microbial cells consumed
part of the solubilized products during growth (Veselá & Friedrich,
2009).
3.1.3. Feathers conversion to bioactive peptides
Antioxidant, antimicrobial, and antihypertensive peptides
obtained by controlled enzymatic hydrolysis of different proteins
have been broadly described (Ryan, Ross, Bolton, Fitzgerald, &
Stanton, 2011; Chakrabarti, Jahandideh, & Wu, 2014; Correa,
Daroit, Fontoura, Meira, Segalin, & Brandelli, 2014). However,
biological activities of keratin hydrolysates are largely unknown.
In fact, only hydrolysates produced by microbial conversion of chicken
feathers were reported to possess antioxidant activity (Fakhrah, Ktari,
Haddar, Mnif, Dahmen, & Nasri, 2011), and feather hydrolysates
obtained through acid hydrolysis showed angiotesin I-converting enzyme
inhibitory activity (Karamac, Flaczyk, Wanasundara, & Amarowicz, 2005).
More recently, Fontoura et al. (2014) used the keratinolytic bacterium
Chryseobacterium sp. kr6 for production of bioactive hydrolysates from
raw feathers. This bacterium produces an unusual metalloprotease that
belongs to the M14 family of peptidases and is the first enzyme of this
family associated with keratinolytic activity (Riffel et al., 2007). The hy-
drolysates obtained from raw chicken feathers displayed antioxidant,
ACE- and DPP-IV-inhibitory activities (Fontoura et al., 2014), suggesting
that feather hydrolysates constitute an interesting source of bioactive
peptides.
3.2. Enzymatic valorization of poultry byproducts
Poultry byproduct meals and poultry litter have been treated with
complex enzymatic preparations, aiming to improve the nutritional
features of such materials as animal feed. The enzyme complex Nutrase
Xyla from Bacillus subtilis was used as a supplement in rabbit diets
containing poultry litter. Results showed a better nutrient digestibility
by animals fed enzyme-supplemented diets with up to 30% poultry
litter inclusion levels (Ogunsipe, 2014). This enzyme complex contains
active enzymes such as cellulose, xylanase, glucanase, amylase,
pectinase and protease, and it was conceived to work in the breakdown
of polysaccharides. Thus, the enzyme would help to improve metaboliz-
able energy, growth rate, feed conversion rate, increase digesta passage
rate, decreased viscosity of the intestinal tract and reduce sticky
dropping.
Fermented broiler's litter was applied as supplement of high-fiber
diet along with 0, 1, 5, 10, 20 or 25 IU/kg of fungal enzyme mixture (cel-
lulose, amylase and pectinase) fed to broiler chicks for 28 days. Results
of the trials show a positive correlation between enzyme supplementa-
tion and body weight, feed efficiency, carcass weight, edible meat and
the meat:bone ratio (Onilude, 1999). The effects of the level of inclusion
of poultry byproduct and enzyme-prebiotic supplementation on the
diet digestibility and performance of broilers was investigated by
Kirkpinar, Açikgöz, Buzkurt, and Ayhan (2004). Body weights, feed in-
take and feed conversion efficiency were not affected by poultry
byproduct; however, enzyme-prebiotic had a significant positive effect
on feed conversion efficiency at 0 to 6 weeks. Protein efficiency ratio
(PER) values were increased by poultry byproduct at the rate of 40 g
per kg of feed and addition of enzyme-prebiotic.
The incorporation of poultry offal meal at 10% level was effective as
substitute of soybean meal in diets of Japanese quails in 15-week trials,
while supplementation with the enzyme Allzyme SSF (phytase, prote-
ase, pentosanase, glucanase, cellulose, amylase and pectinase) was ad-
vantageous only in diets containing 5% offal meal (Mutucumarana,
Samarasinghe, Ranjith, Wijeratne, & Wickramanayake, 2010). The use
of high-quality meals from poultry byproducts is also a viable alterna-
tive for diets in aquaculture. Enzyme-digested poultry byproduct meal
was tested as an effective replacement for fish meal in feeding trials
with red drum (Kureshy, Davis, & Arnold, 2000).
The improvement of nutritional value of feather protein via plastein
reaction using proteolytic enzymes has been also investigated. Both
alkaline proteinase B7-2 and trypsin were used to incorporate lysine
into feather keratin hydrolysates by plastein reaction between the
ε-NH2 of lysine and mostly γ-COOH groups of glutamate residues. The
increase of lysine content after enzyme modification indicates that
the nutritive value of feather keratin was increased (Dalev, Ivanov, &
Liubomirova, 1997).
 A. Brandelli et al. / Food Research International 73 (2015) 3–12
Mechanically deboned chicken meat (MDCM) is a byproduct of
poultry industry that corresponds to about 20% of fresh-cut chicken
carcass and is obtained by crushing tissues after meat removal. MDCM
is mostly obtained from neck and back parts of chicken and turkey
carcasses and is increasingly used in processed meats, such as meat
emulsions and chicken nuggets (Negrão et al., 2005). MDCM hydro-
lyzed with the commercial enzyme Alcalase was used to feed formula-
tions tested in the rat model. The MDCM diet resulted in a good net
protein utilization and true digestibility value reaching 96% (Rossi
et al., 2009). Although the amino acid composition of the hydrolysate
was balanced, methionine and cysteine were limiting amino acids.
More recently, MDCM hydrolysates were prepared with a combination
of Protamex and bromelain. The addition of these hydrolysates into
imitation fish paste improves gel characteristics and improved ACE-
inhibitory and free radical scavenging activities in vitro (Jin et al., 2014).
3.3. Enzymes for processing poultry fat
Animal fat has the advantage of wide availability and low cost, being
a byproduct of meat processing. Chicken fat is a waste product of poul-
try processing industry, which has been used to produce biodiesel
(Arnaud, Relkin, Pina, & Collignan, 2004). However, the composition
of poultry fat includes both essential and non-essential fatty acids.
Some essential fatty acids, such as the ω-3 and ω-6 polyunsaturated
fatty acids (PUFAs) and monounsaturated fatty acids (MUFAs) are
very relevant due to their significant health benefits. MUFAs are
known to reduce cholesterol levels, whereas PUFAs are important for
adequate development of nervous tissue, eye health and skin mainte-
nance, and have been associated with reduction of risk of cardiovascular
disease, hypertension, and inflammatory diseases (Gogus & Smith,
2010; Nordestgaard & Varbo, 2014). Although fish tissues and fish
byproducts are recognized as a major source of PUFAs, some other ani-
mal sources, including poultry, are known to be rich in MUFAs and
PUFAs (Patil & Nag, 2011; Saha, Bandyopadhyaya, & Dutta, 1990). Tri-
glyceride fractions enriched with MUFAs were extracted from chicken
fat by supercritical CO2. These tryglicerides were used to produce struc-
tured lipids by acidolysis reactions catalyzed by immobilized lipases of
Candida rugosa and Geotrichum candidum (Lee & Foglia, 2000). In addi-
tion, PUFAs could be successful recovered from free fatty acids obtained
by alkaline hydrolysis of chicken viscera (Patil & Nag, 2011).
Although enzymatic processing has not been detailed for extraction
and modification of fatty acids from poultry waste, the use of lipases
seems feasible and has been applied in other systems (Sharma, Chisti,
& Banerjee, 2001). Depending on the reaction conditions, lipases cata-
lyze hydrolysis, esterification, or exchange location of fatty acids in es-
ters (interestefication). Hydrolytic reaction of lipases can be used to
help removal of fat and obtaining fatty acids from meat and fish prod-
ucts. Saturated and monounsaturated fatty acids are often easily hydro-
lysed by lipolytic enzymes. However, the presence of cis- double bonds
in fatty acids results in bending of the chains, which may cause a steric
hindrance effect on lipases. This effect is noticeable for ω-3 PUFAs like
eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA;
22:6) due to the presence of 5 and 6 double bonds, respectively. Thus,
selective lipases for EPA and DHA allows separation and concentration
of these fatty acids from others in the remaining portion, providing
the possibility of producing ω-3 concentrates with dominance of either
EPA or DHA (Kapoor & Patil, 2011).
Through interesterification reactions, lipases are useful to modify
the properties of lipids by altering the location of fatty acid chains in
the glyceride and replacing one or more of the fatty acids with different
ones. Thus, a relatively inexpensive and less desirable lipid can be mod-
ified to a higher value fat (Sharma et al., 2001). Skin fat may be extracted
and react with different lipids, acids or alcohols through interesterifica-
tion reactions. Chicken skin is rich in oleic and palmitic acids, the former
may be maintained or increased, while the last may be decreased by in-
teresterification, becoming this source more applicable nutritionally or
7
technologically. The enzyme Lipozyme RM IM (1,3 specific lipase from
Rhizomucor miehei) was used to obtain different structured lipids rich
in oleic and linoleic acids from chicken skin and a mixture of branched
fatty acids derivative of lanolin (Feddern, Soares, & Xu, 2013).
4. Keratinases: production, purification and properties
4.1. Production of keratinases
Keratinolytic enzymes are produced by bacteria (Bach, Daroit,
Correa, & Brandelli, 2011; Rai & Mukherjee, 2011; Silva, Macedo, &
Termignoni, 2014), actinomycetes (Habbeche et al., 2014) and fungi
(Darah et al., 2013; Lopes et al., 2011). The microorganisms used in
keratinase production have been isolated from diverse environments,
including decomposing feathers (Nagal & Jain, 2010), penguin feathers
(Pereira, Lopes, Petry, Medina, & Brandelli, 2014), poultry waste
digestor (Williams, Richter, Mackenzie, & Shih, 1990), fish intestine
(Daroit et al., 2009), and slaughterhouse polluted water (Fakhfakh-
Zouari et al., 2010), among others.
The production of keratinase is usually performed on relatively
inexpensive growth media. Usually, the components of growth media
are salts and keratinous substrates as carbon and nitrogen sources,
which makes the enzyme obtainment highly attractive from the
economic point of view (Daroit & Brandelli, 2014). The production of
microbial keratinases is usually reported to be induced by keratins
(Brandelli et al., 2010), although other growth substrates, such as soy
flour (Wang & Shih, 1999) and gelatin (Casarin, Cladera-Olivera, &
Brandelli, 2008), might act as inducers of keratinase production.
Furthermore, the constitutive production of keratinases has also been
reported (Son, Park, Kim, & Lee, 2008). Lopes et al. (2011) studied differ-
ent keratinous substrates (human hair, pig hair, chicken feather meal,
raw chicken feathers, and bovine horn) for keratinase production by
Aspergillus niger, and found that chicken feather meal provided the
highest keratinolytic activity. Media containing chicken feather meal
have been described as the best keratin source concerning keratinase
production by different Bacillus strains (Daroit, Correa, & Brandelli,
2011; Fakhfakh-Zouari et al., 2010; Silva et al., 2014). Otherwise, raw
chicken feathers have been used as substrate to keratinase production
by Chryseobacterium sp. (Casarin et al., 2008).
The microorganisms used by keratinase production are usually
mesophilic and termophilic (Nagal & Jain, 2010; Zaghloul, Embaby,
& Elmahdy, 2011). In order to provide reduction of energy costs in
the application of enzymes in industrial processes, psychrotolerant
bacteria have been investigated. Strains of Lysobacter, Arthrobacter
and Chryseobacterium, isolated from Antarctic area, have been re-
ported as feather-degrading microorganisms (Pereira et al., 2014),
where Lysobacter sp. A03 was capable to degrade feather meal al-
most completely in 7 days of cultivation at 20 °C.
Another important factor that must be considered is the fact that
keratinases are generally secreted into the culture medium (Daroit &
Brandelli, 2014; Gupta & Ramnani, 2006). This facilitates the recovery
of these enzymes, since cell lysis is not necessary and therefore, the
costs of enzyme production are reduced.
Regarding the keratinase production scale, abundant reports on
shake flasks production are available, but few studies describe the
scale up to large-scale fermenters (Wang & Shih, 1999; Zaghloul et al.,
2011). The complete solubilization of feathers in a 14-L laboratory
scale fermentor was achieved after 24 h of incubation by recombinant
Bacillus subtilis. In this process, two parameters were evaluated, the aer-
ation (0.7 and 1.0 vvm) and agitation speed (500 and 700 rpm). The
keratinase produced under both combination sets were quite similar.
However, the combination of 0.7 vvm and 700 rpm accelerated the
rate of feather biodegradation and resulted a 1.92-fold enhanced level
of released soluble proteins after 24 h incubation. The feather hydroly-
sate can be used as feed additive since it was very rich in free amino
acids (Zaghloul et al., 2011).
8 A. Brandelli et al. / Food Research International 73 (2015) 3–12

Wang and Shih (1999) evaluated the scale up of keratinase produc-
tion by B. licheniformis PWD-1 and recombinant B. subtilis FD-29 from
shake flasks to 15-L and 150-L fermentors. After optimizing fermenta-
tion conditions in 15-L and 150-L fermentor, B. licheniformis PWD-1
maintained a higher enzyme yield and a much better productivity
when compared with B. subtilis FDB-29, being the keratinase production
approximately 40% higher. Three low-cost substrates (raw chicken
feathers, commercial chicken feather meal, and soy flour) were used
to compare their relative expenditures for the production of keratinase
by both Bacillus strains, and the most cost-effective production was
achieved using raw feathers or commercial feather meal. In this sense,
aiming industrial application of this enzyme, additional research should
be conducted to provide better understanding about the behavior of
keratinases production at large scale.
The co-production of enzymes, like alkaline proteases and thermo-
stable α-amylase, using chicken feathers may represent a cost-effective
process. Co-production of these enzymes by Bacillus licheniformis NH1
was achieved when chicken feathers were used as nitrogen and carbon
sources in the cultivation medium. The chicken feathers were completely
degraded after 24 h of incubation at 37 °C. The maximum activities were
obtained after 30 h incubation for protease (4000 U mL−1) and 48 h in-
cubation for α-amylase (10 U mL−1) (Hmidet et al., 2010).
4.2. Purification of keratinases
Generally, keratinases are purified for the determination of bio-
chemical properties, being obtained in high purification factors and
low recoveries. To obtain keratinase in high purity the use of various pu-
rification techniques is often required. The initial steps of keratinase pu-
rification protocols usually employed ammonium sulfate precipitation
following by dialysis, acetone precipitation following by dialysis, ultra-
filtration, and/or aqueous two-phase system. Moreover, the final tech-
niques typically include multiple chromatographic steps including ion-
exchange and gel filtration columns (Table 2). The advantages and dis-
advantages of these techniques are summarized in Fig. 3.
Keratinase from Bacillus megaterium was purified using fractionated
ammonium sulfate precipitation followed by ion-exchange chromatog-
raphy using a Q-Sepharose matrix. The first step of ammonium sulfate
precipitation was 0–30% and the second was 30–60%. The ammonium
sulfate precipitation did not promote enzyme purification (purification
factors lower than one unit). On contrast, the ion-exchange chromatog-
raphy was very efficient and the keratinase was purified 655-fold with a
recovery of 12.7% and specific activity of 544.7 U mg− 1 (Agrahari &
Wadhwa, 2012). Q-Sepharose is a strong anionic exchanger, which sug-
gest the keratinase was negatively charged at pH 8.0.
Correa et al. (2010) proposed a purification protocol for the crude
extract of Bacillus sp. P7, which involves ammonium sulfate precipita-
tion (60% saturation), gel filtration on Sephadex G-200, following by
two steps of ion-exchange chromatography using SP-Sepharose and
DEAE-Sepharose. The keratinase P7 was purified 29.8-fold with enzy-
matic recovery of 27% and specific activity of 12,966 U mg−1.
The purification protocol of keratinase from Chryseobacterium sp.
strain kr6 was based on ammonium sulfate precipitation (50% satura-
tion), followed by gel permeation on Sephadex G-100 and anion-
exchange chromatography on Q-Sepharose Fast Flow. The keratinase
kr6 was purified 40.2-fold with enzymatic recovery of 7.1 (Silveira
et al., 2010). For purification of β-keratinase from Brevibacillus sp. AS-
S10-II, 80% acetone precipitation following by Sephacryl S-200 gel filtra-
tion was used, where the keratinase was purified 18-fold and the enzy-
matic recovery was 58.5% (Rai & Mukherjee, 2011).
When large volume of purified enzyme is desired, non-
chromatographic techniques such as aqueous two-phase system
(ATPS) can be applied. An advantage of ATPS is that initial assays are
generally performed in small quantities, around 10 g, for optimization
of purification process. After the protocol is established, the ATPS can
be easily scaled-up. In addition, the components of ATPS are very inex-
pensive when compared, for example, with chromatography tech-
niques. ATPS would be interesting to obtain keratinases for industrial
applications, as an efficient and less expensive purification protocol, ca-
pable of scaling-up.
ATPS was applied in the purification of keratinases produced by Ba-
cillus sp. P45 (Sala et al., 2014) and Serratia marcescens P3 (Bach et al.,
2012). Two steps of ATPS promoted the purification of keratinase P45
in 5.6 fold with 90% of enzymatic recovery, while keratinase P3 was pu-
rified by one step ATPS and the purification factor and recovery were
around 2.7 and 69%, respectively. These ATPS were formed by polyeth-
ylene glycol (PEG) and salts. The top-phase was rich in PEG while the
bottom phase was rich in salts. In both cases, different keratinases pro-
vided the same behavior in ATPS, partition to the top phase. However,
the presence of PEG can interfere in some analytical determination,

Table 2
Purification factor (PF), enzymatic recovery (REC) and specific activity (SA) of keratinases purified by different protocols.

Source Purification protocol a PF REC SAb Reference

(%)

Actinomadura keratinilytica Cpt29 ASP, heat treatment, and GFC 48.1 11.6 70,000.0 Habbeche et al. (2014)
Aspergillus oryzae ASP, GFC, and IEC 11.4 33.3 303.6 Farag and Hassan (2004)
Aspergillus fumigatus TKF1 ASP and GFC 2.0 1.9 77.0 Paul et al. (2014)
Aspergillus parasiticus Acetone precipitation, dialysis, ASP, and 2.19 2.47 106.2 Anitha and Palanivelu (2013)
IEC
Bacillus megaterium ASP and IEC 655 12.7 544.7 Agrahari and Wadhwa (2012)
Bacillus pumilus KS12 UF and thermal precipitation 3.2 89.1 35,885.6Rajput and Gupta (2013)
Bacillus sp. P45 Two steps ATPS and UF 6.1 56 2582.7 Sala et al. (2014)
Bacillus sp. P7 ASP, GFC, and two steps IEC 29.8 27 12,966.6Correa, Daroit, and Brandelli (2010)
Bacillus subtilis MTCC (9102) ASP, IEC, and GFC 45.9 27.6 4181.8 Balaji et al. (2008)
Bacillus subtilis NRC 3 ASP, IEC, and GFC 31 20 5233.0 Tork, Shahein, El-Hakim, Abdel-Aty, and Aly (2013)
Bacillus thuringiensis serovar israelensis H14 ASP, GFC, and IEC 11.7 – 595.0 Poopathi, Thirugnanasambantham, Mani, Lakshmi, and
(IPS-82) Ragul (2014)
Brevibacillus sp. strain AS-S10-II Acetone precipitation and GFC 18 58.5 21,6000.0 Rai and Mukherjee (2011)
Chryseobacterium sp. strain kr6 ASP, GFC, and IEC 40.2 7.1 21,466.0 Silveira, Casarin, Gemelli, and Brandelli (2010)
Cunninghamella echinulata Acetone precipitation, and affinity 13.7 39.7 35.3 More, Sridhar, Prakash, Vishwakarma, and Umashankar
chromatography (2013)
Meiothermus sp. I40 ASP, hydroxyapatite IEC, and GFC 30.2 45.0 35,364.8 Kuo et al. (2012)
Purpureocillium lilacinum LPS # 876 ASP, GFC, two steps IEC, and GFC 19.9 1.3 1432.7 Cavello, Hours, Rojas, and Cavalitto (2013)
Serratia marcescens P3 ATPS 2.7 69 179.0 Bach et al. (2012)
Streptomyces sp. strain AB1 ASP, heat treatment, GFC, and IEC 86 24 67,000.0 Jaouadi et al. (2010)
a
Abbreviations: ASP, ammonium sulfate precipitation; GFC, gel filtration chromatography; IEC, ion-exchange chromatography; ATPS, aqueous two-phase system; UF, ultrafiltration.
b
The specific activity is expressed in enzyme units per mg protein (U mg−1).
 A. Brandelli et al. / Food Research International 73 (2015) 3–12
Fig. 3. Advantages and disadvantages of main purification techniques for keratinases.
like electrophoresis and protein determination by Lowry's method. Sala
et al. (2014) proposed the application of ultrafiltration in diafiltration
mode to remove PEG from keratinase P45, which results in a purifica-
tion factor and enzymatic recovery of 6.1 and 56.3%, respectively, with
specific activity of 2582.7 U.mg−1.
The initial purification techniques like acetone or ammonium sulfate
precipitation and ATPS promote high enzymatic recovery around 76.1–
91.0% (Rai & Mukherjee, 2011; Sala et al., 2014; Silveira et al., 2010),
whereas chromatographic techniques, such as ion-exchange in fixed
bed and gel filtration, provide high purification factor. The choice of
the most adequate protocol for keratinase purification will depend on
the purpose. For example, industrial applications often require higher
volumes of purified enzyme and therefore ultrafiltration, ammonium
sulfate or acetone precipitation, ATPS and ion exchange chromatogra-
phy in expanded bed can be applied. On the other hand, if the aim is
to investigate biochemical properties of the keratinase, ion-exchange
chromatography in fixed bed and gel filtration protocols can be used
for purification.
In recent years, some protein purification techniques have been im-
proved, such as ATPS and ultrafiltration. The use of ATPS with modifica-
tion by using substitute polymers or copolymers can be more effective
for separation, purification and increasing recovery of bioproducts
(Goja, Yang, Cui, & Li, 2013). In addition, a new ATPS based on the use
of ionic liquids and salts has been proposed for enzyme purification
(Dreyer & Kragl, 2008). The biggest disadvantage of ultrafiltration is
the reduction of the permeate flux through the membrane over time,
due the concentration polarization and fouling (Vardanega et al.,
2013). These effects cannot be completely eliminated, but the use of
force fields to membrane systems as magnetic (Vardanega et al., 2013)
and turbulence promoter (Sen, Roy, Das, Sadhu, & Bhattacharjee, 2010)
can minimize them. In the current literature, the aforementioned tech-
niques have not been reported for keratinase purification. Further,
there is a lacuna about purification of keratinase by ion-exchange chro-
matography in expanded bed mode. In this technique, higher volumes
of bioproducts are obtained when compared with fixed bed.
4.3. Properties of keratinases
The biochemical features of microbial keratinases are dependent on
the producer microorganism. The characteristics of some microbial
keratinases are summarized in Table 3. Microbial keratinases are
predominantly extracellular, however intracellular enzymes have
been described (El-Naghy, El-Ktatny, Fadl-Allah, & Nazeer, 1998).
Most keratinases are neutral or alkaline proteases, with the optimum
9
pH ranging 6.0–13.0. The optimum temperature of several microbial
keratinases is about 45–65 °C. Regarding the catalytic type, keratinases
are predominantly serine proteases, such as the typical keratinases pro-
duced by Bacillus spp. and Streptomyces spp., although the description of
keratinolytic metalloproteases has been increased (Brandelli, 2008;
Brandelli et al., 2010).
Keratinases and proteases produced by Aeromonas hydrophila K12,
Chryseobacterium indologenes A22 and Serratia marcescens P3 degraded
feather meal producing high amounts of soluble proteins and forming
thiol groups (Bach et al., 2011). The proteases of strains K12, A22 and
P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; while the optimal
temperature was in the range 45–55 °C. The proteolytic activity of strain
K12 was stimulated by organic solvents and by the detergent SDS, sug-
gesting its potential application for detergent formulations and peptide
synthesis. Strains A22, K12 and P3 have great potential for use in bio-
technological processes involving hydrolysis of keratinous byproducts.
The purified keratinase produced by Aspergillus oryzae hydrolyzed
different substrates, showing its highest proteolytic activity on bovine
serum albumin and casein followed by keratin, chicken feathers, colla-
gen, duck feathers and sheep wool (Farag & Hassan, 2004). Further-
more, both crude and purified keratinases from Bacillus sp. P45 were
able to catalyze the hydrolysis of various substrates, where the prefera-
bly by keratinase P45 was casein, following bovine serum albumin,
feather meal and heat-treated chicken feather (Sala et al., 2014). The hy-
drolysis of soluble substrates such as casein and albumin is typically re-
ported as more effective than the hydrolysis of insoluble substrates,
such as keratin present in feathers (Daroit, Correa, Segalin, & Brandelli,
2010; Han, Luo, Gu, & Yu, 2012; Sala et al., 2014).
From an industrial point of view, it is interesting that keratinase can
maintain its activity in a wide pH range, avoiding the need for a pH con-
trol system, in addition to presenting thermal stability. Keratinase pro-
duced by Bacillus subtilis S14 was stable at pH 8.0 for more than 200 h
at 4 °C and 25 °C. At 37 °C and 50 °C, the activity was reduced by 25%
and 58% after 12 h, remaining at this level for at least 216 h, respectively
(Silva et al., 2014). In the presence of ionic and non-ionic surfactants (1%
SDS, Tween 80, or Triton X-100), keratinase maintained more than 50%
of its initial activity, suggesting that it can be applied as an additive in
detergent formulation. Keratinase was active in the pH range 5.5–10.5,
and the optimum pH was 8.0. Keratinase S14 presents potential applica-
tion in biotechnological processes, like bovine horn degradation, which
require thermostability at 50 °C for long periods (Silva et al., 2014).
The purified β-keratinase produced by Brevibacillus sp. showed opti-
mum activity at 45 °C and pH 12.5–13.0. The β-keratinase retained 97%
of its original activity post-heating at 60 °C for 15 min. The biochemical
10 A. Brandelli et al. / Food Research International 73 (2015) 3–12

Table 3
Biochemical features of crude and purified keratinases produced by microorganisms.

Source of keratinase pHb T (°C)b Substrate MW (kDa)c Potential applications d References

Actinomadura keratinilytica Cpt29a 10.0 65 Keratin azure 29 DF, KH, dehairing Habbeche et al. (2014)
Aeromonas hydrophila K12 8.0 55 Azocasein – DF, KH, peptides synthesis Bach et al. (2011)
Aspergillus fumigatus TKF1a 6.0 50 Keratin 24.3 AF Paul et al. (2014)
Aspergillus parasiticusa 7.0 50 Azocasein 36 KH, AF, leather processing Anitha and Palanivelu (2013)
Bacillus subtilis S14 8.0 50 Azokeratin – DF, horn degradation Silva et al. (2014)
Bacillus pumilus A1 9.0 55–60 Keratin – AF, organic fertilizer Fakhfakh-Zouari et al. (2010)
8.5 55–60 Casein –
Brevibacillus sp.a 12.5–13.0 45 Keratin 83.2 Leather industry Rai and Mukherjee (2011)
Chryseobacterium indologenes A22 7.5 45 Azocasein – KH Bach et al. (2011)
Chryseobacterium sp. kr6a 8.5 50 Azokeratin 64 KH Riffel et al. (2007)
Serratia marcescens P3 6.0 45–50 Azocasein – KH Bach et al. (2011)
a
Purified enzyme.
b
Optimum values for pH and T are presented.
c
Molecular mass is only available for purified enzymes.
d
Abbreviations: DF, detergent formulations; KH, hydrolysis of keratinous byproducts; AF, feather degradation for use in animal feed.

features of β-keratinase like thermo-stability, high specificity activity,
high temperature optima, stability against surfactants, and activity at
high alkaline pH even in the presence of EDTA advocated the impor-
tance of this protease in laundry detergent formulations, and other bio-
technological processes. The keratinase was able to hydrolyze the
protein substrates, in order of preference, as chicken-feather keratin, ca-
sein, bovine serum albumin, bovine serum fibrinogen, hemoglobin, bo-
vine serum globulin. However, gelatin, collagen, and human hair (α-
keratin) could not be hydrolyzed by this keratinase (Rai & Mukherjee,
2011).
Some purified keratinases are not stable at high temperatures, for
example the purified keratinase from Aspergillus parasiticus retained
only 32% activity after one-hour incubation at 50 °C and pH 7.0
(Anitha & Palanivelu, 2013).
The increase of keratinase thermal stability can be achieved by addi-
tion of salts, such as CaCl2. Fakhfakh-Zouari et al. (2010) tested the effect
of CaCl2 on crude keratinase of Bacillus pumilus at 60 °C. In the presence
of 5 mM CaCl2, the keratinase had a half-life value of 43 min, while in the
absence of Ca2+ the half-life was 20 min. Similarly, the thermal stability
of the keratinase from Chryseobacterium sp. kr6 was significantly im-
proved, and the half-life of the enzyme increased 9.8 fold at 60 °C in
the presence of Ca2+ (Silveira et al., 2010).
There is a lacuna in the literature regarding the stabilization of
keratinases after purification. Some researchers relate about the addi-
tion of salts like Ca2+ ions (Fakhfakh-Zouari et al., 2010), or immobiliza-
tion of this enzyme (Cavello, Contreras-Esquivel, & Cavalitto, 2014;
Konwarh, Karak, Rai, & Mukherjee, 2009), as a form of stabilization. En-
zyme immobilization frequently improves the stability of the enzyme
toward temperature and pH, increasing its potential for use in several
applications. The immobilization may reduce the autolysis of
keratinases, thus enhancing the storage stability (Farag & Hassan,
2004; Wang, Swaisgood, & Shih, 2003). However, the use of stabilizing
agents, as for example polymers or ionic liquids, is not found in the cur-
rent literature.
Finally, the exact mechanism and mode of action of keratinases on
native keratin degradation is still not fully understood (Fang, Zhang,
Liu, Du, & Chen, 2013). It is recognized that the reduction of disulfide
linkages is required for efficient hydrolysis of native keratinolytic sub-
strates. In this sense, the keratinolysis needs the synergistic action of
the proteases with any of the various redox mechanisms, such as reduc-
tases, sulfite production, reducing agents, or redox potential of cells
(Rahayu, Syah, & Thenawidjaja Suhartono, 2012; Yamamura, Morita,
Hasan, Yokoyama, & Tamiya, 2002). The reducing agents often used
are β-mercaptoethanol and dithiothreitol (DTT). Yamamura et al.
(2002) purified and characterized a disulfide reductase-like protein
from Stenotrophomonas sp. and show its role to degrade keratin with
the cooperation of protease D-1. The proposed mechanism for extracel-
lular enzymatic keratin degradation by bacteria (or possibly even fungi)
comprises two steps: (i) initially, the disulfide bonds in keratin are
attacked by disulfide reductase-like enzymes, resulting in the increase
of free thiol groups and weakening protein structure (Nagal & Jain,
2010; Yamamura et al., 2002); and (ii) keratinase cleaves the peptide
bonds of β-pleated sheet and releases different amino acids and pep-
tides (Gupta & Singh, 2014; Nagal & Jain, 2010).
5. Conclusions
Poultry industry generates huge amounts of waste, which should be
properly managed due to environmental concerns, but also to generate
added valued products. The advances in enzyme technology indicate
that biocatalysis may be a very interesting biotechnological tool to con-
vert byproducts of poultry industry into useful products. Enzymes have
been used to improve nutritional features of poultry byproduct meals,
enhancing the quality of feed formulations. A significant research has
been devoted to keratinolytic enzymes, which can degrade resilient
proteins like keratins and allow the bioconversion of keratin-rich
waste. The hydrolysates may present improved nutritional characteris-
tics and can be used as feed additives or soil fertilizers. The interest on
enzymatic degradation of keratin-rich wastes also resulted in advances
on the chemistry and structure of proteolytic enzymes and about the
molecular mechanisms of keratin degradation. Several keratinases
have been purified and characterized, but large scale purification proto-
cols need to be established to allow the effective utilization of these en-
zymes at industrial level. The recent association of antioxidant, ACE-
inhibitory and DPP-IV-inhibitory activities with feather hydrolysates
suggests that poultry waste can be a relevant source of bioactive pep-
tides. Therefore, future research is necessary to establish experimental
conditions to reach higher yields of these bioactive molecules.
Acknowledgments
AB and SJK are research awardees of CNPq. LS is a PhD fellowship
from CAPES.
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