Waste Management 95 (2019) 399–415

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

Beyond plucking: Feathers bioprocessing into valuable protein

hydrolysates
Kelly Callegaro a, Adriano Brandelli b, Daniel Joner Daroit a,⇑
a
Programa de Pós-Graduação em Ambiente e Tecnologias Sustentáveis, Universidade Federal da Fronteira Sul (UFFS), Campus Cerro Largo, Av. Jacob Reinaldo Haupenthal
1580, 97900-000 Cerro Largo, RS, Brazil
b
Laboratório de Bioquímica e Microbiologia Aplicada, Instituto de Ciência e Tecnologia de Alimentos (ICTA), Universidade Federal do Rio Grande do Sul (UFRGS), 91501-970
Porto Alegre, RS, Brazil

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

Article history: The livestock production and subsequent processing of meat results in huge quantities of solid waste such
Received 4 April 2019 as viscera, bones, skin and keratin-rich materials, including feathers, hair, wool, claws and hooves. In par-
Revised 21 June 2019 ticular, the continuous growth of poultry industry generates massive amounts of feathers as major waste
Accepted 22 June 2019
material. The conversion of such by-products into materials with increased value has been studied.
Available online 25 June 2019
Hydrothermal, chemical or biological approaches have been investigated to achive effective conversion
of highly recalcitrant proteins that are abundant in animal waste, but increasing interest is devoted to
Keywords:
the development of biotechnological methods. The processing of feathers and other by-products into pro-
Bioconversion
Feather hydrolysates
tein hydrolysates may have industrial and commercial significance. Therefore, this review comprehen-
Hydrolysis sively addresses the postulated applications of hydrolysates obtained from keratinous biomasses.
Meat industry Examples on the utilization of feather hydrolysates as organic soil fertilizers, feed ingredients, cosmetic for-
Poultry waste mulations and biofuel production are described in the literature. Microbial feather hydrolysis can generate
bioactive peptides as well. The use of protein-rich waste from meat industry to produce hydrolysates with
biological activities constitutes a point of utmost interest for development of functional ingredients with
elevated value.
Ó 2019 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
2. Keratin structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
3. Poultry feathers: Waste or raw material? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
4. Microbial degradation of feathers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
5. Hydrolysates from feathers based keratin rich material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
6. Applications of keratin hydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
6.1. Nitrogen-rich fertilizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
6.2. Animal feed ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
6.3. Organic nitrogen source in culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
6.4. Biofuels production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
6.5. Hair care and cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
7. Biological activities of protein hydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
7.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
7.2. Protein hydrolysates derived from keratinous materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
8. Concluding remarks and future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412

⇑ Corresponding author.
E-mail address: daniel.daroit@uffs.edu.br (D.J. Daroit).

https://doi.org/10.1016/j.wasman.2019.06.040
0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
400
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
1. Introduction
Processing of livestock for human consumption generates enor-
mous amounts of organic wastes and by-products. Viscera, skins,
meat trimmings, bones, blood and epidermal appendages are
among these major residual biomasses, which need to be properly
managed (Lemes et al., 2016a). Collagen, elastin and keratins are
the prevalent fibrous proteins found in by-products of animal pro-
cessing for meat production (Ferraro et al., 2016). Among these
proteins, the particular recalcitrance of keratins further compli-
cates the management and recycling of keratin-rich by-products
such as feathers, hairs, wool, claws, hooves and beaks (Daroit and
Brandelli, 2014).
The conventional methods used for the disposal of animal by-
products, besides being cost-intensive, ultimately lead to the loss
of a potential income source. This perception prompts the meat
industry to search for alternative technologies to reclaim these
materials (Martínez-Alvarez et al., 2015; Mora et al., 2014). Innova-
tive approaches for the valorization of residual biomass form the
basis of the biorefinery concept, focusing on diminished wastage
and recycling as integral elements of agro-industrial sustainability
(Mirabella et al., 2014).
Microbial, biochemical and (physico)chemical processes are
versatile options available for biomass valorization. Specifically,
biotechnology encompasses an assembly of useful and
environmental-friendly processes to be used for the conversion
of residual biomasses. From this approach, multiple valuable prod-
ucts, such as biofuels, microbial enzymes, feed, fertilizers, chemi-
cals, cosmetics and bioactive molecules could be obtained (Toldrá
et al., 2016).
Previously published review articles already dealt with the uti-
lization of by-products from meat industry (Brandelli et al., 2015;
Di Bernardini et al., 2011; Ferraro et al., 2016; Jayathilakan et al.,
2012; Lafarga and Hayes, 2014; Lemes et al., 2016a; Martínez-
Alvarez et al., 2015; Mora et al., 2014; Sinkiewicz et al., 2018;
Toldrá et al., 2016). However, the efforts towards research and
development continue to expand the range of promising applica-
tions considering the environmental and economical significance
of this subject. Additionally, substantial knowledge is recently
accumulated regarding the strategies for recycling keratin-rich
materials. In this sense, this review focuses on biotechnological
approaches intended for the utilization of keratinous materials
derived from the meat industry, particularly chicken feathers, as
abundant and renewable resources to obtain protein hydrolysates.
These keratin-derived bioproducts present potential relevance for
human nutrition and health, as for biofuels production and animal
and plant production.
2. Keratin structure
Keratins are among the most important structural proteins
found in vertebrates, constituting the intermediate filaments of
eukaryotic cell cytoskeleton and composing the epidermis and epi-
dermal appendages such as feathers, nails and hair. The biological
specialties of these fibrous proteins are the result of their chemical
composition and spatial arrangement of polypeptide chains. Ker-
atins are heterogeneous proteins due to variations in the type of
secondary structures and the amino acids composition and/or
predominance. Considering the secondary structure, a-helices
and b-sheets are the dominant motifs in a-keratins and
b-keratins, respectively (Daroit and Brandelli, 2014; Ferraro et al.,
K. Callegaro et al. / Waste Management 95 (2019) 399–415
2016). Different covalent bonds (disulphide bridges) and non-
covalent bonds (hydrogen bonds and hydrophobic interactions)
confer to keratins their typical insolubility and resistance
(Sinkiewicz et al., 2018; Wang et al., 2016; Holkar et al., 2018).
A high cysteine content in the primary sequence of keratins, pre-
dominant at the protein N- and C-terminal regions, differentiate
these proteins from other fibrous proteins such as collagen and
elastin (Korniłłowicz-Kowalska and Bohacz, 2011). Its compact con-
formation and high stability are determined, to a major extent, by
disulfide bridges formed among cysteine residues (yielding cystine)
which occur both within and between keratin polypeptides
(Brandelli et al., 2010). Hydrogen bonds and hydrophobic interac-
tions also contribute to keratin stability. The latter are located at
the central portions of keratin sequence due to the abundance of
hydrophobic amino acids (Ferraro et al., 2016; Wang et al., 2016).
The a-keratins present superior rigidity and resistance when
compared to b-keratins, since the former possess a higher cystine
content and thus increased number of disulfide bridges, than the
latter (Daroit and Brandelli, 2014). In addition, post-translational
modifications, such as phosphorylation and glycosilation, result
in keratin filaments with different bioaccessibilities for degrada-
tion (Lange et al., 2016). These keratin properties are essential for
epidermal appendages to fulfill their biological functions. How-
ever, considering that these materials are abundantly generated
from animal processing for meat production, their recalcitrance
represents a potential obstacle for the adequate management of
keratin-rich wastes and by-products (Daroit et al., 2011).
3. Poultry feathers: Waste or raw material?
Feathers possess a high protein content. Nearly 90% of the
feathers mass, on a dry basis, are represented by keratins (Daroit
and Brandelli, 2014). Among keratins, a-keratins account for 41–
67%, b-keratin for 33–38% and amourphous keratins are present
in lower amounts (Lange et al., 2016). Such composition confers
to feathers its hydrophobic character and a high resistance to
diverse proteolytic microorganisms and enzymes, chemicals and
mechanical stresses (Brandelli et al., 2010).
Worldwide, around 5 million tons of feathers are generated by
poultry processing on an annual basis, making feathers the most
common keratin-rich by-product (Korniłłowicz-Kowalska and
Bohacz, 2011; Tesfaye et al., 2017). The United States, China and
Brazil are the global leaders regarding chicken meat production,
with Brazil as the top exporting country. In 2018, around 5.7 billion
chickens were slaughtered in inspected Brazilian abbatoirs, totaling
13.51 million tons of carcasses (IBGE, 2019). From these data and
considering that feathers account for 5–10% of the poultry weight,
around 680 thousand tons of feathers were generated by the Brazil-
ian poultry industry in 2018. To corroborate with this scenario, a
single slaughterhouse, processing 165 thousand poultry per day,
produces a daily stream of 18.5 tons of feathers (IPEA, 2012).
Management of these protein-rich materials encompasses enor-
mous challenges for the poultry industry. Landfilling is among the
traditional strategies for the disposal of feathers. However, such
practice demands increased areas, resulting in environmental lia-
bilities. Additionally, the uncontrolled anaerobic degradation of
these protein-rich materials in landfills tends to release ammonia
and hydrogen sulfide. Incineration is a management alternative,
almost instanstaneously reducing enormous feathers amounts.
Nevertheless, this is an expensive process with potential negative
 K. Callegaro et al. / Waste Management 95 (2019) 399–415
impacts on health and environment due to emission of toxic gases
(Salminen and Rintala, 2002; Sharma and Gupta, 2016). Hydrother-
mal treatments are also common methods for feathers reclama-
tion. After this energy-intensive process and milling, the
resulting feather meal has limited uses as ingredient in animal
feed. Nonetheless, feather meal is a low added-value product with
diminished digestibility and nutritional value (Lasekan et al., 2013;
Li et al., 2012; Onifade et al., 1998).
In view of the environmental risks, high costs and low prof-
itability of these traditional strategies, novel methods have been
considered to successfully manage keratinous materials (Verma
et al., 2017). Keratins and feathers are effectively converted into
composites, absorbents, films for food and packaging, textiles and
electronic devices. Such versatility of feathers benefit the automo-
tive, construction, plastics, among other industries (Reddy, 2015;
Tesfaye et al., 2017). Beyond these applications, keratinous materi-
als are cycled in nature through the action of microbial agents
(Brandelli et al., 2010). Therefore, increasing attention is devoted
to the development of biotechnological methods for feathers pro-
cessing, aiming further valorization of this abundant and low-
cost protein-rich biomass (Lasekan et al., 2013).
4. Microbial degradation of feathers
Microorganisms are the protagonists when it comes to decom-
position processes in the environment and this is true for the
degradation of keratinous materials as well. Keratinolytic microor-
ganisms are able to utilize keratins as carbon, nitrogen, sulfur and
energy sources for growth and maintenance (Brandelli, 2008;
Gupta and Ramnani, 2006). The most studied keratinolytic
microorganisms are bacteria belonging to the Bacillus, Chryseobac-
terium, Serratia and Stenotrophomonas genera, actinomycetes (for
instance, Streptomyces spp.) and some fungi (such as Chrysosporium
spp., Aspergillus spp.). Particularly, representatives of the Bacillus
genus, such as B. licheniformis, B. subtilis, B. pumilus and B. cereus
are among the most effective feather-degrading microorganisms
(Verma et al., 2017). Typically, the feather degradation process per-
formed by keratinolytic fungi occurs at slower rates than that
achieved by bacteria (Sivakumar and Raveendran, 2015).
Considering the complex structure of feathers and their pro-
teinaceous composition, microbial degradation occurs through
mechanisms involving proteolytic and sulfitolytic systems. Sulfi-
tolysis, indicating the breakdown of disulfide bridges in the kerati-
nous substrate, is achieved by disulfide reductases, release of
Fig. 1. Multiple enzymes involved in the putative mechanism for complete degradation of keratin. Please refer to the text for detailed explanations.
401
sulfite/thiosulfate and cell-associated redox systems, as usually
demonstrated for keratin-degrading bacteria, actinomycetes and
fungi. Proteolysis, the cleavage of peptide bonds in keratins, is per-
formed by diverse microbial proteases, with the particular rele-
vance of substrate-specific keratinases. In this sense, major
endopeptidases with keratinolytic activity can be involved with
initial degradation, but a combination of other endopeptidases,
exopeptidases and oligopeptidases seems to be helpful to exten-
sive keratin hydrolysis (Brandelli et al., 2010).
However, there is increasing evidence that a complex mixture
of enzymes is needed to achieve an efficient degradation of the
complex structure of keratins (Fig. 1). Despite the recognized role
of proteolytic enzymes, the participation of lytic polysaccharide
monooxygenases produced by fungi is hypothesized for keratin
decomposition, as suggested based on evidence with Onygena corv-
ina (Lange et al., 2016). The attack to glycosides at the N- and C-
terminal regions would cause a disorganization of keratin struc-
ture, providing easy access to proteolytic enzymes (Lange et al.,
2016). The potential roles of bacterial c-glutamyl transpeptidase
in keratinolysis has been suggested as well, as reported for Bacillus
subtilis CH-1 (Liu et al., 2014). This enzyme cleaves the bonds
between e-amine of lysine and c-glutamyl of glutamine, an isopep-
tide bond found in many keratinous materials. In addition, a puta-
tive cystathionine c-lyase produced by some bacteria possibly acts
on keratin hydrolysis through the breakdown of carbon-sulfur
bonds (He et al., 2018).
The understanding of microbial functionalities is crucial in
adapting ecological phenomena, such as organic matter decompo-
sition, for biotechnology (Daroit and Brandelli, 2014). Within this
perspective, microbial conversion of feathers and other keratin-
rich materials result into valuable products, such as microbial bio-
mass and enzymes, which are extensively reviewed (Daroit and
Brandelli, 2014; Brandelli et al., 2010, 2015; Gupta et al., 2013a,
2013b; Gupta and Ramnani, 2006; Verma et al., 2017). Recent
interest is dedicated to feather processing into protein hydroly-
sates, with diverse industrial and commercial applications
(Sinkiewicz et al., 2018), which are presented and discussed below.
5. Hydrolysates from feathers based keratin rich material
Protein hydrolysates are complex mixtures of peptides and
amino acids resulting from the cleavage of peptide bonds within
a polypeptide chain. Generally, the choice of a hydrolysis strategy
402 K. Callegaro et al. / Waste Management 95 (2019) 399–415
depends on the features of the protein substrate, the availability of
appropriate hydrolysis agents, the quality of the final product and
the desired applications of the hydrolysate (Hou et al., 2017;
Stiborova et al., 2016).
Hydrolysis of keratinous substrates could be performed through
chemical, enzymatic or microbial methods. Chemical processes
employ alkali, strong acids or other reagents (such as reducing
agents), frequently under high temperatures and pressure
(Bouhamed and Kechaou, 2017; Coward-Kelly et al., 2006; Lee
et al., 2016; Sinkiewicz et al., 2017; Stiborova et al., 2016). Chem-
ical hydrolysis is usually faster and simpler than enzymatic/micro-
bial processes, with high hydrolysis yields. However, chemical
hydrolysis occurs in a nonspecific manner, leads to the potential
destruction of amino acids and products with a high ash content
(Adler et al., 2018; Sinkiewicz et al., 2018; Taskin and
Kurbanoglu, 2011). Additionally, some chemicals (thiols, sulfites
and peroxides) present increased toxicity, severe hydrolysis condi-
tions (high temperature and/or pressure) demand high-energy
inputs and the treatment of process streams might be problematic.
Novel approaches have been investigated for the hydrolysis of
keratin-rich materials, aiming to avoid or minimize the negative
aspects of alkaline/acid hydrolyses. Among these are alternative
hydrothermal treatments, such as the utilization of superheated
water and steam flash explosion and the use of ultrasound or
microwaves to assist chemical hydrolysis (Bhavsar et al., 2017;
Holkar et al., 2016; Lee et al., 2016; Zhang et al., 2014a).
From the biotechnological perspective, protein hydrolysates are
obtained using the proteolytic potential of microorganisms, or
microbial proteases in the form of crude or partially purified
enzyme preparations (Lasekan et al., 2013; Samaranayaka and Li-
Chan, 2011). Purified keratinases are less frequently used
(Gegeckas et al., 2015). The main advantages of enzymatic hydrol-
ysis are the moderate process conditions, specificity of action,
preservation of amino acids and easiness of both controlling the
degree of hydrolysis and inactivating enzymes after the process.
Additional advantages reside on the utilization of immobilized
enzymes, allowing its recovery and reuse (Garcia et al., 2011;
Hou et al., 2017; Rai and Mukherjee, 2015).
Nevertheless, commercial availability of specific proteases (ker-
atinases) for the hydrolysis of keratin substrates is still limited
(Gupta et al., 2013a, 2013b). In addition, from the complexity of
keratinous materials, hydrolysis processes on an industrial scale
might require other enzymes, such as biocatalysts attacking the
disulfide bridges and the glycosyl moiety in keratins (Lange et al.,
2016). Although enzymatic hydrolysis is a usually rapid (per-
formed within hours), a main disadvantage of such approach
resides on the high costs of enzymes when compared to chemicals
used for alkaline/acid hydrolyses (Hou et al., 2017). Processes
employing sequential (thermo-)chemical-enzymatic hydrolyses,
are also explored. In these cases, the initial (thermo-)chemical
hydrolysis allows increased access to the subsequent action of pro-
teolytic enzymes towards the partially destabilized keratinous
substrate (Cheong et al., 2018; Dalev, 1990; Kim et al., 2002;
Łaba and Szczekala, 2013; Mokrejs et al., 2011).
The majority of hydrolysates from keratin-rich materials is pre-
pared through (thermo-)chemical methods or microbial bioconver-
sion (Brandelli et al., 2015). In microorganism-mediated hydrolytic
processes, protein hydrolysis depends on microbial growth and
secretion of extracellular proteases and other enzymes. This results
in extended hydrolysis periods when compared to chemical and
enzymatic methods, and the microorganisms consume part of the
released amino acids and peptides to sustain their growth (Gupta
et al., 2013a, Stiborova et al., 2016; Veselá and Friedrich, 2009).
Moreover, distinct efficacies are usually observable from the intrin-
sic variability of biological systems (Hou et al., 2017). Nevertheless,
the microbial bioconversion of feathers stands out as a promising
management strategy in terms of both environmental constraints
and financial costs. Within this perspective, bioprocessing consists
of an ecologically safe technology, demanding low energy inputs
and moderate operational conditions (Brandelli et al., 2010;
Vasileva-Tonkova et al., 2009). In these aspects, the biodegradation
of keratinous materials using microorganisms is more attractive as
compared to (thermo-)chemical processes.
6. Applications of keratin hydrolysates
6.1. Nitrogen-rich fertilizers
Nitrogen (N) is the main limiting nutrient for plant growth.
Although feathers contain nearly 15% (w/w) of N, its recalcitrance
leads to a slow degradation and N-mineralization in soils, hinder-
ing its direct application as fertilizers (Thuriès et al., 2001). Chem-
ical, enzymatic or microbial processing of feathers result into more
available N sources for plant uptake, thus presenting potential
agricultural uses (Choi and Nelson, 1996; Paul et al., 2014;
Tronina and Bubel, 2008).
Different methods have been used to produce keratin-derived
hydrolysates intended for use as fertilizers and those involving
microbial fermentations are widely investigated. Feather hydroly-
sates are usually produced through submerged cultivations with
mesophilic bacteria (5–20 g feathers/L, 30–40 °C, 24–96 h). Despite
the microorganism used, application of hydrolysates often
enhances plant vegetative parameters. This is also observed for
hydrolysates obtained through thermo-chemical methods, which
can be dependable on the process conditions (Table 1).
Particularly, promising results of feather hydrolysates were
reported for wheat, ryegrass, Bengal gram, rice, beans and vegeta-
bles. Increased seed germination, root lenght, above ground and root
weight, as well as enhanced productivity, are usually observed with
the application of hydrolysates obtained from keratin-rich materi-
als. In addition, higher macronutrient (such as nitrogen, phosphorus,
calcium and potassium) and micronutrient (such as iron and man-
ganese) contents of plant biomass are reported (Table 1).
Usually, besides inorganic N, feather hydrolysates contain
organic nitrogen, mainly in the form of peptides and amino acids
and some minerals (P, K, Ca, Fe, Mn, Zn, Cu), which typically reflect
feather’s composition (Bhavsar et al., 2016; Gurav and Jadav, 2013;
Kshetri et al., 2019; Nurdiawati et al., 2018; Tronina and Bubel,
2008). The low carbon-to-nitrogen ratio (C/N) of the hydrolysates
suggests the rapid mineralization of organic N by the soil micro-
biota, releasing mineral N capable of being uptaken by the plants.
However, other factors, such as the physical and chemical features
of hydrolysates and soils might affect the N mineralization rates
(Nurdiawati et al., 2019a).
Peptides and amino acids within hydrolysates might be directly
uptaken by the plant root and leaves, being translocated to other
plant tissues and potentially acting as growth stimulants (Colla
et al., 2015). Tryptophan, released from keratin hydrolysis and/or
produced through feathers microbial processing (Maciel et al.,
2017; Paul et al., 2014), is a fundamental amino acid for the syn-
thesis of indoleacetic acid (IAA), a hormone possessing important
functions on plant growth. Besides plant-produced IAA, diverse
keratinolytic bacteria are demonstrated to produce IAA using
feathers as substrates during submerged cultivations (Bhange
et al., 2016; Kshetri et al., 2019; Nafady et al., 2018), which poten-
tially contribute to the effects of feather hydrolysates on plant
growth promotion and represents an advantage over thermo-
chemical hydrolysates.
Application of feather hydrolysates can enhance populations
and activities of beneficial soil microorganisms, such as ammonify-
ing, nitrifying and nitrogen-fixing bacteria and inorganic
 K. Callegaro et al. / Waste Management 95 (2019) 399–415 403

Table 1
Hydrolysis of keratinous materials and potential applications of hydrolysates as fertilizers.a

Keratinous Hydrolysate production Application as fertilizers Reference

material
Method Agent (conditions) Use Results
Whole Microbial (SmF) Paenibacillus woosongensis TKB2 (7.5 g/L Soil Higher germination and roots mass (fresh and Paul et al. (2013)
feathers feathers, 30 °C, pH 8.5, 48 h) dry) and increased number of root nodules of
Bengal gram in comparison to unfertilized
controls
Thermoactinomyces sp. RM4 (20 g/L feathers, Soil Higher germination and roots mass (fresh and Verma et al. (2016)
60 °C, pH 10.0, 96 h) dry) of gram in comparison to unfertilized
controls
Amycolatopsis sp. MBRL 40 (5 g/L feathers, Soil Rice growth and dry mass (root and shoot) Tamreihao et al.
30 °C, 120 h) similar to urea (2017)
Stenotrophomonas maltophilia DHHJ (26.7 g/L Foliar Higher growth of Chinese cabbage in Cao et al. (2012)
feathers, 40 °C, pH 7.8, 72 h) comparison to unfertilized controls
Bacillus polymyxa B20 (10 g/L feathers, 30 °C, Soil Fresh mass and leaf chlorophyll content of Kucinska et al.
pH 7.2, 144 h) tomato, cucumber and white cabbage plants (2014)
similar to reference fertilization
Chryseobacterium sp. RBT (10 g/L feathers, Soil/ Earlier flowering of banana plants, earlier Gurav and Jadav
37 °C, pH 7.5, 30 h) Foliar harvest, higher banana fruit yields in (2013)
comparison to unfertilized controls
Microbial (SSF) Streptomyces sampsonii GS 1322 (500 g Soil Higher germination and growth of wheat in Jain et al. (2016)
feathers, 60% moisture, pH 8.0, 24 days) comparison to unfertilized controls
Hydrothermal Saturated steam (feathers or poultry litter: Soil Similar leaf area, dry weight and chlorophyll Nurdiawati et al.
water = 1:3, 160–180 °C, 0.6–0.9 MPa, 30 min) content of patchouli; similar trends for (2019b)
increased chlorophyll contents, pod length,
yield per plant and weight of hundred seeds of
mung bean when using feather and poultry
litter hydrolysates (5:1) with 50% of
recommended NPK when compared to 100%
NPK fertilization.
Thermo- Alkaline hydrolysis (feather:water = 1:5, 0.1 g – With or w/o phytotoxic effects on germination Nurdiawati et al.
chemical Ca(OH)2/g dry feather, 180 °C, 1 MPa, 30 min) index of Japanese mustard spinach seeds, (2018)
depending on production temperature and
hydrolysates dilution
Milled Microbial (SmF) Bacillus amyloliquefaciens 6B (5 g/L milled Soil Higher mung bean germination and growth in Bose et al. (2014)
feathers feathers, 37 °C, pH 7.2, 12 h) comparison to unfertilized controls, similar to
reference fertilization
Bacillus licheniformis ASU (10 g/L milled Soil Higher growth and dry weight (roots and Nafady et al.
feathers, 30 °C, pH 7.2, 120 h) shoot) and increased number and weight of (2018)
root nodules in faba bean when compared to
unfertilized controls; synergistic effects of
hydrolysate and inoculation with mycorrhizal
fungi on faba bean responses
Aspergillus niger (20 g/L milled feathers, 30 °C, Soil Higher growth and yield of cowpea in Adetunji et al.
pH 5.7, 168 h) comparison to unfertilized controls (2012)
Bacillus pumilis KHS-1 (5 g/L milled feathers, Soil Growth and dry weight (roots and shoot) of Kim et al. (2005)
40 °C, pH 6.0, 72 h) carrot and Chinese cabbage similar to reference
fertilization
Thermoactinomyces sp. 3H, 8H and M4 Soil Higher germination and growth of ryegrass in Gousterova et al.
(consortium) (7 g/L milled feathers, 55 °C, pH comparison to unfertilized controls (2011)
7.2, 72 h)
Enzymatic Bacillus licheniformis AS-S24-I keratinase Soil Germination, growth and fresh biomass of Rai and Mukherjee
immobilized in Fe3O4 magnetic nanoparticles Bengal gram similar or higher than unfertilized (2015)
(20 g/L milled feathers, 45 °C, pH 9.0, 48 h) controls
Thermo- Acid hydrolysis (30% H2SO4, 80 °C, 4 h) Soil After pH adjustment and addition of urea and Popko et al. (2015)
chemical K2SO4, fresh and dry mass of rapeseed plants
were similar to chemical fertilization (urea and
ammonium nitrate)
Wool waste Thermo- Alkaline hydrolysis (100 g/L wool waste, Soil Higher germination, growth and fresh biomass Nustorova et al.
chemical 0.15 M KOH-0.05 M NaOH, 120 °C, 20 min) of ryegrass in comparison to unfertilized (2006)
controls
Wool Chemical + Alkaline hydrolysis (20 g/L wool, 30 g/L KOH, Soil Higher germination and growth of wheat in Holkar et al. (2016)
ultrasound 2.4 g/L NaOH, 24 h) + acoustic cavitation comparison to unfertilized controls
(20 kHz, 40% of maximum power output of
750 W, 30 min)
Hydrothermal Superheated water (wool:liquor ratio
1, – Higher germination and root length of Bhavsar et al.
saturated steam at 170 °C, 7 bar, 60 min) Lepidium sativum in comparison to unfertilized (2016)
controls

Abbreviations: SmF: submerged cultivation; SSF: solid-state cultivation.

a
Detailed information on hydrolysates production (and also on upstream and downstream processing) and application schemes might be found in the cited references.

phosphate-solubilizers. In addition, hydrolysates were reported to and Mukherjee, 2015). Hence, protein hydrolysates might act
stimulate microorganisms that inhibit phytopathogens (Bose et al., directly on plant growth, but also indirectly through processes per-
2014; Jain et al., 2016; Nustorova et al., 2006; Paul et al., 2013; Rai formed by the soil microbiota (Colla et al., 2015).
404 K. Callegaro et al. / Waste Management 95 (2019) 399–415

6.2. Animal feed ingredients
Proteins are important and expensive ingredients in animal feed
formulations. Animal processing generates abundant and low value
protein-rich by-products that could be used as feed (Saarela et al.,
2017). In fact, the conversion of protein-rich by-products into ani-
mal feed is a biologically efficient strategy for nutrients recycling,
since the proteinaceous animal wastes are returned to animal pro-
duction in the meat supply chain (Freeman et al., 2009).
Hydrothermal processes conventionally used to produce com-
mercial feather meal (also known as hydrolyzed feather meal)
enhance the solubility of feather constituents. However, the recal-
citrance of keratins to the action of digestive tract enzymes (such
as pepsin and trypsin) negatively affects the nutritional value of
feather meal as feed ingredientes, especially for monogastric ani-
mals. In addition to the high production costs, hydrothermal treat-
ment causes the loss of thermolabile amino acids and the formation
of lanthionine and lysinoalanine, which diminishes bioavailability
and are potentially toxic (Brandelli, 2008; Fakhfakh et al., 2011).
In vivo and in vitro investigations, employing distinct hydrolysis
methods, indicate the potential of hydrolysates derived from ker-
atinous materials as alternatives to commercial feather meal and
as partial replacements to the typical protein sources used in ani-
mal feed, such as soybean proteins (Table 2).
However, in addition to its low digestibility, another particular
drawback is that feather keratins are deficient in amino acids such

Table 2
Hydrolysis of keratinous materials and potential applications of hydrolysates as animal feed.a

Keratinous material Hydrolysate production Application in animal feed Reference

Method Agent (conditions)
Whole feathers Microbial (SmF) Vibrio sp. kr2 (60 g/L feathers, 30 °C,
pH 6.0, 168 h)
Chryseobacterium sp. kr6 (50 g/L
feathers, 30 °C, 120 h)
Bacillus sp. kr16 (10 g/L feathers,
37 °C, 120 h)
Chryseobacterium sediminis RCM-SSR-
7 (50 g/L feathers, 30 °C, pH 7.5, 84 h)
Bacillus sp. MPTK6 (30 g/L feathers;
30 °C, pH 10, 48 h)
Bacillus subtilis AMR (10 g/L feathers,
26 °C, pH 8.0, 144 h)
Bacillus pumilus A1 (50 g/L feathers,
45 °C, pH 10.0, 48 h)
Chemical, with or w/o Alkaline hydrolysis (80 g/L feathers,
Enzymatic 1 M NaOH, 37 °C, 2–20 h), with or w/
o Enzyme INSTA-PROÒ (0.614 g/g
feather, 37 °C, 24 h)
Thermo-chemical Na2SO3 (75 g/L feathers, 2.1–3.6 g/L
+ Hydrothermal Na2SO3, 85 °C, 60 min) + Autoclaving
(133 °C, 2.4 bar, 60–90 min)
Hydrothermal Boiling (100 g/L feathers, 2 h soaking,
+ Enzymatic 10–20 min boiling) + Crude
keratinase from B. licheniformis ER-15
(480 U/g feather, 50 °C, pH 8.0, 8–
12 h)
Whole feathers (and Enzymatic + Lipase and protease (Allzyme FDÒ
blood) Hydrothermal (0.5 kg/ton substrate, 2.5 kg
Na2S2O5/ton substrate, 32 °C,
40 min) + autoclaving (110 °C,
200 kPa, 40 min)
Milled feathers Microbial (SmF) Bacillus pumilus A1 (50 g/L milled
feathers, 45 °C, pH 10.0, 48 h)
Bacillus licheniformis PWD-1 (milled
feathers:liquid bacterial culture = 1:4,
50 °C, 120 h)
Kocuria rosea LPB-3 (30 g/L milled
feathers, 40 °C, pH 7.5, 26 h)
Enzymatic Enzymatic preparation (INSTA-PROÒ)
(51.3 g/kg milled feathers, 60–
120 min)
Wool waste Microbial (SmF) Bacillus pumilus A1 (50 g/L wool,
45 °C, pH 10.0, 48 h)
Higher digestibility and biological Grazziotin et al.
value than feather meal (in vitro); (2006, 2008)
replacement of 20% soybean protein
in Wistar rat’s diets supplemented
with methionine
Amino acids enrichment, higher Maciel et al.
digestibility and biological value than (2017)
feather meal (in vitro)
Amino acids enrichment, higher Kshetri et al.
digestibility than feather meal (2019)
(in vitro)
Higher digestibility than whole Kumar et al.
feathers (in vitro) (2012)
Increment on nutritional quality of Mazotto et al.
cornmeal by blending with (2017)
hydrolysate (26%) through extrusion
Higher digestibility than whole Fakhfakh et al.
feathers (in vitro) (2011)
Increased solubility, digestibility and Kim et al. (2002)
amino acids digestibility (in vitro)
than raw feathers
Increased solubility, digestibility and Adler et al. (2018)
amino acid scores when compared to
commercial feather meal
In vitro digestibility, amino acids and Tiwary and Gupta
essential amino acids profiles were (2012)
similar (or higher) when compared to
literature data on microbial
hydrolysates (or chemical/
hydrothermal hydrolysates)
Higher apparent digestibility of Pacheco et al.
nutrients and gross energy, (2016)
metabolizability of gross energy and
digestible energy, when compared to
hydrothermal hydrolysates fed to
adult dogs
Higher growth (weight) of Wistar rats Fakhfakh et al.
through addition of 2.5% or 5.0% of (2012)
feather hydrolysate to standard diet
Replacement of up to 5% soybean Williams et al.
protein in broiler diets; (1991)
supplementation with amino acids
resulted in growth curves identical to
soybean protein
Higher true amino acids digestibility Bertsch and Coello
than feather meal on in vivo trials (2005)
with roosters
Reduction of up to 55% of fish meal by Mendoza et al.
the combination of feather (2001)
hydrolysate and soybean meal (2:1)
in diets of pacific-white shrimp
Higher digestibility than wool waste Fakhfakh et al.
(in vitro) (2013)

Abbreviation: SmF: submerged cultivation.

a
Detailed information on hydrolysates production (and also on upstream and downstream processing) and application schemes might be found in the cited references.
 K. Callegaro et al. / Waste Management 95 (2019) 399–415
as histidine, lysine, methionine and tryptophan (Grazziotin et al.,
2006; Kumar et al., 2012; Taskin and Kurbanoglu, 2011; Williams
et al., 1991). In this sense, microbial conversion is reported not
only to enhance the digestibility of feather proteins, but also to
enrich the hydrolysates with limiting amino acids due to microbial
synthesis (Saarela et al., 2017). Hence, microbial hydrolysis is pre-
ferred over thermo-chemical methods when considering the amino
acids deficiencies of keratins and the severe conditions generally
used in thermo-chemical hydrolytic processes.
Therefore, the nutritional quality of feathers might be
improved. In the case of amino acids deficiencies, this is easily cir-
cumvented by amino acids supplementation into feed (Maciel
et al., 2017). From the promising results reported, these hydroly-
sates present a remarkable potential for utilization as livestock
feed (Pan et al., 2016). However, legal restrictions and regulations,
such as prohibiting the intra-species recycling of animal by-
products, might apply (Saarela et al., 2017).
Rendered animal proteins are increasingly investigated as aqua-
culture feed, particularly as alternatives to the widely used fish-
meal (Naylor et al., 2009). Commercial feather meal was reported
to replace, without negative effects, 76% of fishmeal in diets for
European seabass (Dicentrarchus labrax) juveniles (Campos et al.,
2017). The growth and disease resistance of hybrid tilapia were
not affected by replacing up to 12% of cottonseed or soybean meals
with a commercial product constituted of rice bran mixed with a
so-called completely hydrolyzed feather meal. Replacement with
this keratin-containing product, obtained by autoclaving and acid
hydrolysis of feathers, also seemed to induce less stresses regard-
ing the immune status in the intestine and liver of this fish
(Zhang et al., 2014b).
Adler et al. (2018) indicated that chemical hydrolysis with
NaOH and/or Na2SO3, followed by hydrothermal treatment (auto-
claving), positively affected the solubility and in vitro protein
digestibility of feathers. However, Na2SO3 and autoclaving yielded
a more adequate amino acids composition aiming at a higher nutri-
tional value for Atlantic salmon diets. Of note, no effects on solubil-
ity or digestibility were observed when keratinases were added
(alone or in combination with chemicals/autoclaving) for feathers
hydrolysis (Adler et al., 2018). On the other hand, enzymatic
hydrolysates of milled feathers, blended with soybean meal (2:1),
were reported to reduce fishmeal by 55% in diets for Pacific-
white shrimp (Mendoza et al., 2001).
A mixture of feathers and blood was subjected to an enzymatic
treatment (protease and lipase) before steam hydrolysis and the
resulting meal was used to feed Beagle dogs. Increased coefficients
of total apparent digestibility of nutrients and gross energy (kcal/
kg of feed), metabolizability of gross energy and digestible energy
were observed, when compared to feed prepared only through
hydrothermal processing (Pacheco et al., 2016). These results indi-
cate that the enzymatic pretreatment improved feather meal qual-
ity as a protein source in diets for adult dogs. The utilization of
keratin hydrolysates in the pet food industry has been recently
focused and represent an interesting and expanding niche to be
explored (Martínez-Alvarez et al., 2015).
6.3. Organic nitrogen source in culture media
Substrates for the growth of microorganisms represent a major
cost for the production of microbial biomass and bioproducts.
Sources of organic nitrogen, peptides and amino acids, commonly
used as peptones, are the most expensive ingredients of culture
media, due to the relatively high value of the animal and plant
materials utilized to produce these components (Taskin et al.,
2012).
Protein hydrolysates from feathers and wool, produced through
thermo-chemical processes, are interesting sources of organic
405
nitrogen for culturing microorganisms, as substitutes to commer-
cial sources, such as casein, fish, meat and soy peptones (Taskin
and Kurbanoglu, 2011; Taskin et al., 2016). Furthermore, hydroly-
sis products are rich in important minerals, such as potassium, cal-
cium and sulfur, in part derived from the chemicals used for
hydrolysis, that are needed for microbial activities (Taskin et al.,
2011).
A comparative study on the growth of a model microorganism
(Escherichia coli) in culture media containing feather hydrolysates
(feather peptones) obtained by different methods was performed
by Stiborova et al. (2016). Those authors verified that the highest
biomass production occurred with hydrolysates obtained by alka-
line hydrolysis, due to a higher concentration of soluble peptides
in comparison to hydrolysates produced by enzymatic hydrolysis
or microbial conversion by Pseudomonas sp. P5. However, the
application of particular hydrolysis methods depends on the
intended use of the hydrolysate (Stiborova et al., 2016). Besides
microbial growth (biomass), thermo-chemical hydrolysates of
keratin-rich materials were successfully used to obtain microbial
pigments, enzymes, biopolymers and other bioproducts (Table 3).
Although there is a current predominance of thermo-chemical
approaches, microbial, enzymatic and combined methods have
also been investigated to obtain peptones from keratinous materi-
als (Table 3). Ramakrishnan et al. (2011) showed that a culture
medium formulated with basal salts (NaCl, 5.0 g/L; KH2PO4, 1.5 g/
L), agar (15 g/L) and feathers degraded by Streptomyces sp. IF 5
(10 g/L), presented the essential nutrients (such as C, N, S, Ca, Fe)
for the growth of bacteria and fungi, as evidenced by colony counts
comparable to that of a reference medium. A crude keratinase from
Streptomyces pactum DSM 40530 was utilized for the hydrolysis of
goose feathers pretreated by autoclaving with reducing agent. The
hydrolysate was successfully used as a source of carbon and nitro-
gen for the growth of and cyanophycin production by a recombi-
nant Escherichia coli strain (Altun et al., 2018). Such hydrolysates,
particularly those produced through microbial/enzymatic conver-
sion, deserve further efforts regarding their use as ingredients in
culture media. Increasing the yield of free aminoacids and small
peptides and, principally, the solubility and stability of hydroly-
sates towards heat, alkali, pH and salting-out, are examples of
major topics to be addressed aiming to at the production of pep-
tones from keratin-rich materials through biotechnological
approaches (Maciel et al., 2017; Orak et al., 2018).
6.4. Biofuels production
Production of biofuels from feathers, especially methane, has
been investigated. Methane is the energetic compound of biogas,
a gas mixture originating from the microbial degradation of
organic matter under anaerobic conditions. This complex process,
denominated anaerobic digestion, comprises multiple steps,
namely hydrolysis, acidogenesis, acetogenesis and methanogene-
sis, performed by the synergistic action of diverse groups of
microorganisms (Strong and Gapes, 2012).
The first step of anaerobic digestion, hydrolysis, involves the
disintegration of organic matter by hydrolytic enzymes secreted
by bacteria, yielding amino acids, fatty acids and simple sugars.
In the second step, acidogenesis, monomers from hydrolysis are
mainly converted into organic acids, alcohols, carbon dioxide and
hydrogen. Acetogenesis is the third step, where homoacetogenic
bacteria produce acetic acid from carbon dioxide and hydrogen.
The last step is methanogenesis, where methane is produced from
carbon dioxide and hydrogen by hydrogenotrophic methanogens,
or from acetic acid by acetotrophic methanogens (Patinvoh et al.,
2017).
Therefore, during anaerobic digestion of keratin-rich biomasses,
the hydrolysis of keratins originates amino acids and peptides that
406 K. Callegaro et al. / Waste Management 95 (2019) 399–415

Table 3
Hydrolysis of keratinous materials and potential applications of hydrolysates as ingredients in microbial culture media.a

Keratinous Hydrolysate production Application in culture media Reference

material
Method Agent (conditions)
Whole Microbial (SmF) Streptomyces sp. IF 5 (5 g/L feathers, 28 °C, 72 h) Only organic substrate for growth of Bacillus, Ramakrishnan et al.
feathers Staphylococus aureus, Streptomyces, Pseudomonas (2011)
aeruginosa, Proteus vulgaris, Aspergillus flavus
Enzymatic Partially purified keratinase from Pseudomonas sp. Only organic substrate for Escherichia coli growth Stiborova et al.
P5 (24.9–31.9 g/L feathers, 1–2 U keratinase/mg (2016)
feather, 50 °C, pH 7.5, 24–48 h)
Thermo-chemical Autoclaving with Na2SO3 (10 g/L feathers, 121 °C, Casamino acids substitute for growth and Altun et al. (2018)
+ Enzymatic 20 min, 10 mM Na2SO3) + lyophilized S. pactum cyanophycin biopolymer production by
DSM 40,530 crude keratinase (8,000 U keratinase/g recombinant Escherichia coli
feather/L/day, 50 °C, pH 8.0, 24 h)
Thermo-chemical Alkaline hydrolysis (24.9–31.9 g/L feathers, 6 g/L Only organic substrate for E. coli growth Stiborova et al.
KOH, 70 °C, 24 h) (2016)
Alkaline hydrolysis [150 mL of 2.5 N KOH, total 95 g Growth and pigment production by Monascus Orak et al. (2018)
feathers (four additions: 30 + 25 + 25 + 15 g), 6 h at purpureus
60 °C for each addition, then 4 h at 120 °C]
Microwave-assisted alkaline hydrolysis [60 g/L Nitrogen source for polyhydroxyalkanoates Benesova et al.
feathers, 0.5 M NaOH, microwave (600 W, total production by Cupriavidus necator on waste frying (2017)
10 min, on/off 1-min period)] oil
Milled Thermo-chemical Acid hydrolysis [three parallel hydrolysis (50 g Peptone substitute for microbial growth (Bacillus Taskin and
feathers milled feathers and 75 mL of 6 N H2SO4 or HCl or subtilis and E. coli) Kurbanoglu (2011)
H3PO4) at 70 °C for 24 h, then 130 °C for 4 h; after Peptone substitute for growth and carotenoids Taskin et al. (2011)
neutralization, the three hydrolysates were mixed] production by Rhodotorula glutinis MT-5
Substrate for biomass and exopolysaccharide Taskin et al. (2012)
production by Morchella esculenta
Acid hydrolysis [two parallel hydrolysis (50 g Peptone substitute for growth and glutathione Taskin (2013)
milled feathers and 100 mL of 6 N H2SO4 or H3PO4) production by Saccharomyces cerevisiae
at 80 °C for 24 h, then 120 °C for 5 h; hydrolysates Peptone substitute for growth and amylase and Baltaci et al. (2018)
were mixed and the solution was neutralized] lipase production by Bacillus licheniformis 016
Acid hydrolysis [two parallel hydrolysis (50 g Addition of feather hydrolysate to medium Ozdal and
milled feathers and 75 mL of 6 N H2SO4 or HCl) at enhanced the production of xanthan gum by Kurbanoglu (2018)
70 °C for 24 h, then 130 °C for 4 h; after Xanthomonas campestris MO-03
neutralization, the two hydrolysates were mixed]
Wool Thermo-chemical Alkaline hydrolysis [150 mL of 2.5 N KOH, total Peptone substitute for microbial growth (Aspergillus Taskin et al. (2016)
100 g wool (five additions of 20 g each), 6 h at 60 °C niger and E. coli)
for each addition, then 4 h at 120 °C]

Abbreviation: SmF: submerged cultivation.

a
Detailed information on hydrolysates production (and also on upstream and downstream processing) and application schemes might be found in the cited references.

could be fermented into organic acids, carbon dioxide, ammonia,
hydrogen and sulfur-containing molecules. Subsequently, part of
carbon dioxide, hydrogen and organic acids are converted into
methane (Xia et al., 2012).
Xia et al. (2012) demonstrated that feathers, added to anaerobic
digesters with swine manure or slaughterhouse sludge to repre-
sent 23–37% of total solids, were degraded during anaerobic diges-
tion, not affecting methane yields. However, the actual methane
yield from feathers and other keratinous materials is usually much
lower than the theoretical values (Costa et al., 2012), which might
be particularly related to feathers recalcitrance (Salminen et al.,
2003). In this sense, hydrolysis represents an initial rate-limiting
step for anaerobic digestion of recalcitrant biomasses, such as
keratin-rich wastes (Patinvoh et al., 2017). Hence, feather pretreat-
ments have been investigated in an attempt to accelerate and
increase methane production (Table 4).
Methane production from microbial feather hydrolysates was
assessed after 50 days of anaerobic digestion at thermophilic con-
ditions (55 °C), using the inoculum from a municipal solid waste
digester. Feather hydrolysates produced by Bacillus licheniformis
ATCC 53757 and a recombinant Bacillus megaterium ATCC 14945,
representing 38% of the volatile solids in the digesters, increased
methane production by 55% and 122% when compared to milled
feathers (Forgács et al., 2011). Similarly, hydrolysates obtained
after microbial pretreatment of feathers by Bacillus sp. C4 improved
methane production in comparison to untreated feathers, when
added as 33% of the volatile solids in digesters. Methane yields
were increased by 105–292% in relation to milled feathers,
depending on the feather hydrolysate tested and the source of
inoculum used for mesophilic (37 °C) anaerobic digestion
(Patinvoh et al., 2016).
Thermal, hydrothermal, chemical, enzymatic and combined
pretreatments (hydrothermal, then enzymatic) increased methane
yields by 5–51% as compared to untreated feathers in tests per-
formed with 62% of volatile solids from (un)treated feathers, using
digested sewage sludge as inoculum at mesophilic (35 °C) condi-
tions (Salminen et al., 2003). At thermophilic conditions (55 °C),
anaerobic digestion of a thermo-chemical feather hydrolysate, pro-
duced using calcium dihydroxyde, increased methane yields by
97–105% when compared to the untreated counterparts (Forgács
et al., 2014).
Forgács et al. (2013) reported that a feather hydrolysate,
obtained through enzymatic treatment of feathers with an alkaline
endopeptidase, increased the methane yield by 122% as compared
to untreated feathers, after 50 days of anaerobic digestion under
thermophilic conditions (55 °C). It was also noted that combined
pretreatments (hydrothermal, then enzymatic), although resulting
in higher feather degradation than the enzymatic pretreatments,
decreased methane yields (although still higher than untreated
feathers). Possibly, the higher content of amino acids and peptides
produced in the combined treatment allow a quicker release of
ammonia, inhibiting methanogenic bacteria (Forgács et al., 2013).
A decreased methane yield was also observed with thermo-
chemically (alkali) preteated feathers, as compared to methane
produced with untreated feathers, in anaerobic digesters using
granular sludge as inoculum and (un)treated feathers to represent
 K. Callegaro et al. / Waste Management 95 (2019) 399–415
Table 4
Hydrolysis of keratinous materials and potential applications of hydrolysates for biofuels production.a
Keratinous material Hydrolysate production
Method Agent (conditions)
Whole feathers Microbial (SSF) B. licheniformis KK1 (feather:
water = 1:2, 42 °C, pH 7.2, 120 h)
Microbial Fervidobacterium pennivorans DSM
(bioaugmentation) 9078
Thermo-chemical Alkaline hydrolysis [40 g/L feathers,
0.2 g lime (or NaOH)/g feathers, 90 °C,
1–1.27 bar, 120 min]
Milled feathers Microbial (SmF) Bacillus sp. C4 (50–200 g/L milled
feathers, 37 °C, pH 7.0, 24–192 h)
B. licheniformis ATCC 53,757 /
Recombinant B. megaterium ATCC
14,945 (40 g/L milled feathers, 37 °C,
pH 8.0, 24–192 h)
B. licheniformis KK1 (40 g/L milled
feathers, 43 °C, pH 8.0, 42–138 h)
Enzymatic Alkaline protease [15 g VS milled
feathers/L, 0.53–2.66 mL Savinase 16
LÒ/g VS feathers, 55 °C, pH 8.0, 0 h
(enzyme added to biodigester) or
2–24 h pretreatment]
Alkaline endopeptidase (100 g/L milled
feathers, 2–10 g/L enzyme, 55 °C,
2–24 h)
Chemical Alkaline hydrolysis (NaOH) (100 g/L
milled feathers, 2–10 g/L NaOH, 35 °C,
2–24 h)
Hydrothermal Autoclaving (15 g VS milled feathers/L,
pH 8.0, 120 °C, 10 min)
Autoclaving (120 °C, 5 min)
Hydrothermal Autoclaving (15 g VS milled feathers/L,
+ Enzymatic pH 8.0, 120 °C, 10 min) + Alkaline
protease [0.53–2.66 mL Savinase
16 LÒ/g VS feathers, 55 °C, pH 8.0, 0 h
(enzyme added to biodigester) or
2–24 h pretreatment]
Autoclaving (120 °C, 5 min) + Alkaline
endopeptidase (100 g/L milled feathers,
2–10 g/L enzyme, 55 °C, 24 h)
Thermo-chemical Wet air oxidation [10 g/L feathers
carbon, inert (nitrogen) or oxidative
(oxygen) atmosphere, 140 °C, 20 bar,
60 min]
Alkaline hydrolysis [100 g TS milled
feathers/L, 0.1–0.2 g lime/g TS feathers,
autoclaving (100–120 °C, 30–120 min)
Acid hydrolysis (50 g milled feathers
and 75 mL of 6 N H2SO4, 70 °C for 24 h,
then 130 °C for 4 h)
Abbreviations: SmF: submerged cultivation; SSF: solid-state cultivation; VS: volatile solids; TS: total solids; AB: anaerobic biodigestion.
a
Detailed information on hydrolysates production (and also on upstream and downstream processing) and application schemes might be found in the cited references.
407
Effect on biofuels production Reference
Increased methane yield (12%) when added Mézes and Tamás (2015)
(5%, m/m) to mesophilic anaerobic digesters
(38 °C; 30 days), in comparison to yield
without addition; higher concentration of
biodegraded feathers (>10%, m/m)
decreased methane yield
Addition of the bacterium (co-treatment) Costa et al. (2012)
diminished methane yield of feather waste
as compared to AB of feathers (65 °C,

70 days), though co-treatment increased
feather solubilization
Diminished methane yield, as compared to Costa et al. (2012)
AB of feathers at mesophilic conditions
(VShydrolysate:VSinoculum = 1.35, 37 °C,

30 days), although pretreatment increased
feathers solubilization
Increased methane yield (105–292%) in Patinvoh et al. (2016)
comparison to milled feathers during AB at
mesophilic conditions (VShydrolysate:
VSinoculum = 0.50, 37 °C, 55 days)
Increased methane yield (55% / 122%) in Forgács et al. (2011)
comparison to milled feathers during AB at
thermophilic conditions (VShydrolysate:
VSinoculum = 0.63, 55 °C, 50 days)
Biohydrogen production by Thermococcus Bálint et al. (2005)
litoralis
Increased methane yield (108–122%) in Forgács et al. (2013)
comparison to milled feathers during AB at
thermophilic conditions (VShydrolysate:
VSinoculum = 0.53, 55 °C, 50 days)
Increased methane yield (5–21%) in Salminen et al. (2003)
comparison to untreated feathers during AB
at mesophilic conditions (VShydrolysate:
VSinoculum = 1.67, 35 °C,
45 days)
Increased methane yield (13–32%) in Salminen et al. (2003)
comparison to untreated feathers during AB
at mesophilic conditions (VShydrolysate:
VSinoculum = 1.67, 35 °C,
45 days)
Increased methane yield (
11%) in Forgács et al. (2013)
comparison to untreated feathers during AB
at thermophilic conditions (VShydrolysate:
VSinoculum = 0.53, 55 °C, 50 days)
Increased methane yield (5–24%) in Salminen et al. (2003)
comparison to untreated feathers during AB
digestion at mesophilic conditions
(VShydrolysate:VSinoculum = 1.67, 35 °C,

45 days)
Increased methane yield (15–51%) in Forgács et al. (2013)
comparison to untreated feathers during AB
at thermophilic conditions (VShydrolysate:
VSinoculum = 0.53, 55 °C, 50 days)
Increased methane yield (37–51%) in Salminen et al. (2003)
comparison to untreated feathers during AB
at mesophilic conditions (VShydrolysate:
VSinoculum = 1.67, 35 °C,
45 days)
Increased methane yield (195–270%) during Strong and Gapes (2012)
AB at mesophilic conditions (36 °C, 70 days)
Increased methane yield (97–105%) in Forgács et al. (2014)
comparison to milled feathers during AB at
thermophilic conditions (55 °C, 50 days)
Nitrogen and mineral source for inoculum Serna-Cock et al. (2018)
preparation and ethanol production by
Saccharomyces cerevisiae in molasses; final
ethanol concentration and yield (after 24 h)
were 81% and 92% of controls (100%) using
urea and ammonium phosphate
408 K. Callegaro et al. / Waste Management 95 (2019) 399–415

58% of the volatile solids. Likewise, methane production was
diminished through anaerobic co-treatment (bioaugmentation) of
untreated feathers with the keratinolytic bacterium Fervidobac-
terium pennivorans DSM 9078 (Costa et al., 2012). Although the
thermo-chemical and bioaugmentation strategies increased feath-
ers solubilization, inhibitory concentrations of ammonia and other
soluble metabolites were reached in some of the anaerobic biodi-
gesters. Therefore, the limiting step for anaerobic digestion was
not the rate of feathers hydrolysis, but the conversion of soluble
organic matter to methane (Costa et al., 2012). Moreover, due to
the high concentration of disulfide bridges in keratins, degradation
under anaerobic conditions might also lead to H2S production.
Besides its toxicity, hydrogen sulfide is highly corrosive and repre-
sents an additional cost for biogas cleaning (Mézes and Tamás,
2015).
Therefore, the anaerobic digestion of keratin-rich materials
poses at least two related challenges for methane production. First,
the refractory character of these biomasses hinders bioaccessibil-
ity. However, solving this problem through hydrolytic pretreat-
ments convey to the second challenge, that is, the products of
hydrolysis and/or those originating from the anaerobic digestion
of hydrolysates might inhibit methane production. Independently
from the hydrolysis method, research results seem to indicate that
pretreatments represent the way forward to increase keratin appli-
cability for methane production. The inhibitory effects on anaero-
bic digestion process, particularly the direct effect of ammonia
originating from the rapid mineralization of the organic nitrogen
contained in feather hydrolysates, but also other mechanisms con-
trolling dominant methanogenic pathways at high ammonia/
ammonium concentrations that potentially affect methane yields
(Lü et al., 2013; Yang et al., 2018; Yin et al., 2018), can be avoided
by controlling the hydrolysates dilution. In this sense, such hydro-
lysates are indicated as nitrogen and nutrients sources in anerobic
co-digestion with nitrogen-poor organic wastes, as similarly pro-
posed for other biomasses (Shah et al., 2015).
In addition to methane, feather hydrolysates produced by Bacil-
lus licheniformis KK1 and supplemented with essential minerals,
were successfully utilized for biohydrogen production by Thermo-
coccus litoralis (Bálint et al., 2005). A feather hydrolysate obtained
through thermo-chemical treatment was used as a suitable source
of nitrogen and minerals for inoculum preparation and ethanol
production by Saccharomyces cerevisiae using molasses as carbon
source. The ethanol yield with hydrolysates (0.46 g ethanol/g
reducing sugars) was comparable to that using urea and ammo-
nium phosphate (0.50 g ethanol/g reducing sugars) (Serna-Cock
et al., 2018).
6.5. Hair care and cosmetics
Keratin peptides and hydrolysates also find applications in for-
mulations of pharmaceutical and cosmetic products (Table 5). A
peptide, synthesized based on the amino acids sequence of cuticu-
lar keratin type II from human hair, was investigated as a hair-
strengthening agent. This peptide displayed the capability of
recovering the mechanical and thermal properties of damaged
hair, with or without the aid of protein disulphide isomerase to
graft the peptide to hair through disulfide formation (Fernandes
and Cavaco-Paulo, 2012; Fernandes et al., 2012). Since applications
in the haircare and cosmetic industry might require specific pep-
tides to perform desired functions, enzymatic and/or microbial
hydrolysis of keratinous materials are preferred over chemical
ones.
A feather hydrolysate produced by Stenotrophomonas maltophil-
ia DHHJ was able to enter the hair cortex and form a transparent
film on the hair surface, promoting weight gain and improving
flexibility and strength of normal and damaged hair (Cao et al.,
2012). The < 1 kDa fraction of a feather hydrolysate produced by
Bacillus subtilis AMR was added (10%) to a mild shampoo and a
rinse-conditioner, resulting in enhanced hydration, softness and
brightness of hydrolysate-treated hairs (normal, colored,
bleached). Such effects were mainly observed after drying hair
with a straigthener (180 °C), which possibly collaborated to pep-
tide incorporation into the hair and the sealing of hair cuticles
(Villa et al., 2013).
A peptide preparation from enzymatic hydrolysis of wool ben-
efited skin hydration, elasticity and water retention capacity

Table 5
Hydrolysis of keratinous materials and potential applications of hydrolysates in hair care and cosmetic formulations.a

Keratinous Hydrolysate production Relevance Reference

material
Method Agent (conditions)
Whole feathers Microbial (SmF) Fervidobacterium islandicum AW-1 (8 g/L Ultrafiltration fractions (<1 kDa and 1–10 kDa) Yeo et al. (2018)
feathers, 70 °C, pH 7.0, 48 h) displayed anti-melanogenic activity in murine
melanoma cell line; diminished ultraviolet B
radiation-induced expression of matrix
metalloproteinases in human fibroblasts
Stenotrophomonas maltophilia DHHJ (26.7 g/L Hydrolysate incorporation into hair resulted in Cao et al. (2012)
feathers, 40 °C, pH 7.8, 72 h) increased weight, flexibility and strength, with
potential for hair protection and repairing
Bacillus subtilis AMR (10 g/L feathers, 28 °C, Ultrafiltration fraction (<1 kDa) increased Villa et al.
120 h) hydration, softness and brightness of hair after (2013)
hydrolysate application as mild shampoo and a
rinse-conditioner; better effects after hair
straightening (180 °C) which promoted
incorporation of keratin peptides to hair
Milled feathers Thermo-chemical Alkaline hydrolysis (milled feathers:0.3% Incorporation of feather hydrolysate to an ointment Mokrejš et al.
+ Enzymatic KOH = 1:50, 60 °C, 24 h) + Protease (5 g/100 g base increased skin hydration and diminished (2017)
feathers, 60 °C, 8 h) transepidermal water loss
Feather meal Enzymatic Pepsin + pancreatin (12.5 g/L feather meal, Ultrafiltration fraction (<3 kDa) exhibited enhanced Pongkai et al.
substrate:pepsin = 20:1, 37 °C, 120 min, then tyrosinase inhibition and anti-melanogenic activity (2017)
substrate:pancreatin = 1:20, 37 °C, 180 min) in murine melanoma cell line (however, some
cytotoxicity was observed)
Wool Enzymatic Not specified Keratin peptides (<1 kDa) improved hydration, Barba et al.
elasticity and water retention capacity of the skin (2008)

Abbreviations: SmF: submerged cultivation; SSF: solid-state cultivation.

a
Detailed information on hydrolysates production (and also on upstream and downstream processing) and application schemes might be found in the cited references.
 K. Callegaro et al. / Waste Management 95 (2019) 399–415
(Barba et al., 2008). The ultrafiltration fractions (<1 kDa and 1–
10 kDa) of a feather hydrolysate obtained from feathers bioconver-
sion by Fervidobacterium islandicum AW-1 diminished melanin for-
mation by a murine melanoma cell line (B16F10) (Yeo et al., 2018).
The probable mechanism for such outcome was the inhibition of
tyrosinase, an enzyme involved in controlling melanogenesis in
mammals. Thus, those authors indicated its application as poten-
tial skin-lightening agents. These fractions displayed minimal
cytotoxicity and inhibited ultraviolet B radiation-induced expres-
sion of matrix metalloproteinases-1 and -13 in human dermal
fibroblast cells, which is a major cause of skin ageing (Yeo et al.,
2018).
Pongkai et al. (2017) also reported that enzymatic hydrolysates
of feather meal inhibited a cell-free mushroom tyrosinase system.
Particularly, the <3 kDa hydrolysate fraction, obtained after ultra-
filtration of hydrolysates produced with pepsin-pancreatin treat-
ments, displayed better results. Considering the participation of
tyrosinase in melanin production, inhibitors of this enzyme pre-
sent potential applications in cosmetic products. When tested in
B16F10 cells, the <3 kDa fraction was demonstrated to inhibit
tyrosinase and melanin production, although the latter was not
correlated with tyrosinase inhibition.
7. Biological activities of protein hydrolysates
7.1. Overview
Traditionally, proteins are considered as sources of energy and
amino acids for nutrition, thus needed for body development and
maintenance of physiological functions. In recent decades, how-
ever, proteins received especial interest due to the biological activ-
ities presented by protein hydrolysates and the bioactive agents
within these hydrolysates, which usually are peptides (Ryan
et al., 2011; Sarmadi and Ismail, 2010). Bioactive peptides are com-
posed of specific amino acid sequences that contribute to regula-
tion and modulation of physiological functions, exerting
antioxidant, anti-hypertensive, antidiabetic, antimicrobial and
other activities. These peptides are inactive as part of the precursor
protein, becoming active only after their release through hydrolytic
cleavage (Korhonen and Pihlanto, 2003; McCarthy et al., 2013;
Möller et al., 2008; Samaranayaka and Li-Chan, 2011).
Although the structure-activity relationship of bioactive pep-
tides is not fully clarified, some similar features are identifiable.
For instance, peptides displaying some bioactivity contain 2–30
amino acids, a molecular mass below 6 kDa, usually presenting
hydrophobic and/or aromatic amino acid residues particularly
located at or near its C- or N-terminal regions. Protein hydrolysis
tends to increase the number of ionizable groups and to expose
hydrophobic groups, which might also contribute to the bioactive
potential of peptides (Sarmadi and Ismail, 2010; Zou et al., 2016).
7.2. Protein hydrolysates derived from keratinous materials
Bioactivities of hydrolysates and peptides obtained from
protein-rich wastes and by-products of animal origin have been
demonstrated, drawing increased attention to this topic (Di
Bernardini et al., 2011; Martínez-Alvarez et al., 2015; Toldrá
et al., 2016). Bones and skin (collagen-rich), ligaments (elastin-
rich) and blood (plasma proteins and hemoglobin), from bovine,
swine and fish are used as substrates to produce protein hydroly-
sates, especially through enzymatic hydrolysis, displaying various
bioactivities (Ferraro et al., 2016; He et al., 2013; Lafarga and
Hayes, 2014; Mora et al., 2014).
Utilization of keratin-rich materials as resources to obtain
bioactive hydrolysates and peptides is scarcely explored
409
(Brandelli et al., 2015; Sinkiewicz et al., 2018). However, prospect-
ing keratinous materials is promising (Jones et al., 2010). Consider-
ing that keratins are rich in hydrophobic amino acid residues,
which represent 50–60% of the polypeptide chain (Arai et al.,
1983; Silveira et al., 2009; Wang et al., 2016), such feature might
be relevant since such amino acids are usually related to the bioac-
tivities of protein hydrolysates and peptides (Lemes et al., 2016a).
In silico strategies, used to identify potential bioactive peptides
within the amino acid sequence of proteins (Lafarga and Hayes,
2014), indicate the presence of anti-hypertensive peptides within
the sequence of feather keratin (Choińska et al., 2011).
Hydrolysates of keratinous materials, particularly those
obtained through microbial bioconversion of feathers, are demon-
strated to possess antioxidant activities, generally displaying radi-
cal scavenging abilities, chelation of metal ions and reducing
power (Table 6). The relevance of such activity resides on its posi-
tive influence on biological systems and food products. For
instance, the deleterious action of free radicals and oxidative stress
are related to pathological conditions, such as hypertension,
atherosclerosis and cancer (Liu et al., 2016; Zou et al., 2016).
In this context, oxidative stress indicators were decreased in
Wistar rats receiving a diet containing feather hydrolysate pro-
duced by Bacillus pumilus A1, when compared to rats receiving a
standard diet. Specifically, diminished lipid peroxidation and a
decreased activity of enzymes related to antioxidant protection
(superoxide dismutase, glutathione peroxidase and catalase) were
observed in diverse internal organs of these animals (Fakhfakh
et al., 2012).
Oxidative reactions are also problematic for the food industry,
promoting undesirable alterations that affect both food quality
and safety (Sarmadi and Ismail, 2010). Furthermore, the potential
toxicity of some synthetic food antioxidants encourages the search
for natural ones (Dhaval et al., 2016). In this sense, Zeng et al.
(2013) indicated the ability of a bovine hair hydrolysate to inhibit
the oxidation of edible oils. Antioxidant keratin hydrolysates might
also inhibit lipid peroxidation by inactivating pro-oxidants and/or
acting on the cascade reactions that ultimately lead to peroxida-
tion (Fontoura et al., 2019). Feather hydrolysates produced through
acid hydrolysis displayed antioxidant activities, including the abil-
ity to inhibit b-carotene bleaching promoted by the action of lipid
(peroxyl) radicals in b-carotene/linoleic acid system. These hydro-
lysates showed good solubility and displayed some other interest-
ing functional properties for food applications, such as foaming,
emulsifying, water- and fat-binding capacitites (Bouhamed et al.,
2018).
Reports on the anti-hypertensive and antidiabetic potentials of
feather hydrolysates are scanty (Table 6). The former is usually
evaluated, in vitro, by the capability of hydrolysates to inhibit the
activity of angiotensin I-converting enzyme (ACE), which performs
a crucial role on controlling blood pressure and is the target of
drugs used for hypertension treatments (Martin and Deussen,
2019). The latter is commonly assessed in vitro by the inhibition
of dipeptidyl peptidase IV (DPP-IV), a ubiquitous enzyme that
cleaves and inactivates incretin hormones. Inhibition of DPP-IV is
among the therapeutic strategies to treat type 2 diabetes (Power
et al., 2014).
ACE inhibition was demonstrated for hydrolysates of milled
feathers obtained through enzymatic hydrolysis (Ohba et al.,
2003) and thermo-chemical processes (Karamać et al., 2005). Nev-
ertheless, anti-hypertensive (ACE-inhibitory) and antidiabetic
(DPP-IV-inhibitory) activities of feather hydrolysates produced by
microbial conversion were only recently demonstrated (Fontoura
et al., 2014). Callegaro et al. (2018) described, in vitro, the antioxi-
dant, antidiabetic and antihypertensive potentials of feather
hydrolysates obtained through feather bioconversion with three
keratinolytic bacterial strains.
 410
Table 6
Bioactivities of protein hydrolysates produced from keratin-rich materials.a

Keratinous Hydrolysate production Bioactive potential Reference

material
Method Agent (conditions) Activity Action mechanism EC50/IC50 (mg/mL)
Whole feathers Microbial (SmF) Kocuria rhizophila p3-3 (50 g/L feathers, 25 °C, AA Radical scavenging (ABTS, DPPH), Fe3+ – Łaba et al. (2018)
96 h) reduction
Fervidobacterium islandicum AW-1 (8 g/L AA Hydrolysate fractions (<1 kDa and 1–10 kDa) – Yeo et al. (2018)
feathers, 70 °C, pH 7.0, 48 h) exhibited radical scavenging (ABTS, DPPH)
Chryseobacterium sediminis RCM-SSR-7 (50 g/L AA Radical scavenging (DPPH) 0.102 Kshetri et al. (2019)
feathers, 30 °C, pH 7.5, 84 h)
Bacillus pumilus A1 (50 g/L feathers, 45 °C, pH AA Radical scavenging (DPPH), Fe3+ reduction DPPH: 0.3 Fakhfakh et al. (2011)
10.0, 48 h)
Bacillus sp. MPTK6 (30 g/L feathers; 30 °C, pH AA Radical scavenging (DPPH), Fe3+ reduction DPPH: 0.5 Kumar et al. (2012)
10, 48 h)
Chryseobacterium sp. kr6 (50 g/L feathers, 30 °C, AA Crude hydrolysate: Radical scavenging (ABTS, ABTS: 16.0–18.3 Fontoura et al. (2014)
pH 8.0, 48 h) DPPH)
Partially purified hydrolysate: Radical – Fontoura et al. (2019)
scavenging (ABTS, hydroxyl, peroxyl), Fe3+

K. Callegaro et al. / Waste Management 95 (2019) 399–415

reduction, inhibition of lipid peroxidation
Purified peptide (LPGPILSSFPQ): radical ABTS: 0.25
scavenging (ABTS, peroxyl)
AH ACE inhibition – Fontoura et al. (2014)
AD DPP-IV inhibition –
Bacillus sp. CL18; Bacillus sp. CL33A; Bacillus sp. AA Radical scavenging (ABTS, DPPH), Fe3+ Strain CL18: ABTS: 5.39; DPPH: Callegaro et al. (2018)
CL14 (10–40 g/L feathers, 30 °C, pH 7.0, 5– reduction, chelation of Fe2+ 15.12; Fe2+-chelation: 10.50
12 days) AH ACE inhibition Strain CL18: 1.61
AD DPP-IV inhibition Strain CL18: 1.52
Pseudomonas otitis H11 (10 g/L feathers, 40 °C, AA Radical scavenging (DPPH, superoxide) DPPH: 0.97 Huang et al. (2019)
pH 11.0, 24 h)
3+
Thermo-chemical Acid hydrolysis (HCl) (50 g/L feathers, 0.5 N AA Radical scavenging (DPPH, peroxyl), Fe and DPPH: 0.47–1.43 Bouhamed et al.
H2SO4, 90 °C, 100 min) Mo6+ reduction (2018)
Milled feathers Microbial (SmF) Bacillus pumilus A1 (50 g/L feathers, 45 °C, pH AA Chelation of Fe2+, DNA protection against – Fakhfakh et al. (2012)
10.0, 48 h) H2O2-induced oxidative damage; hydrolysate
added to Wistar rat diets decreased, in vivo,
lipid peroxidation and activity of antioxidant
enzymes in internal organs
Bacillus subtilis S1-4 (50 g/L milled feathers, AA Crude hydrolysate: Fe3+ reduction – Wan et al. (2016)
37 °C, 72 h) Purified peptide (SNLCRPCG): radical ABTS: 0.35; DPPH: 0.39; Fe2+-
scavenging (ABTS, DPPH), Fe3+ reduction, chelation: 1.85
chelation of Fe2+
Bacillus sp. P45 (43 g/L milled feathers, 30 °C, AA Radical scavenging (ABTS, DPPH), Fe3+ – Lemes et al. (2016b)
pH 7.0, 72 h) reduction
Enzymatic Protease from Bacillus subtilis (SabinaseÒ, 50 °C, AA Radical scavenging (DPPH) Crude hydrolysate and ultrafiltration Ohba et al. (2003)
pH 8.3, 1 h) fractions: < 10
AH ACE inhibition Crude hydrolysate: 1.8
Fraction > 10 kDa: 3.5
Fraction 1–10 kDa: 1.1
Fraction < 1 kDa: 2.1
Hydrothermal Moist heat (170–180 °C, 1 min) + Protease AA Scavenging of peroxyl radicals – Eremeev et al. (2009)
+ Enzymatic from Acremonium chrysogenum with Na2SO3
(feathers:water = 1:4–1:8, 5–15 U enzyme/g
substrate, 0.5% Na2SO3, 1–12 h, 55 °C, pH 10.0)
Thermo-chemical Acid hydrolysis [feathers:HCl solution AH ACE inhibition – Karamać et al. (2005)
(4.75 M) = 3:7, 110 °C, 12 h]
Wool waste Microbial (SmF) Bacillus pumilus A1 (50 g/L wool, 45 °C, pH 10.0, AA Radical scavenging (DPPH), Fe3+ reduction, DPPH: 0.14 Fakhfakh et al. (2013)
48 h) chelation of Fe2+
 K. Callegaro et al. / Waste Management 95 (2019) 399–415 411

As noted from Table 6, the vast majority of keratin-derived

Abbreviations: SmF: submerged cultivation; AA: antioxidant activity; AH: anti-hypertensive activity; AD: antidiabetic activity; ABTS: 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl;
bioactive hydrolysates and peptides is produced through microbial
Ohba et al. (2003)

processing. This is probably due to the selective cleavage per-

Zeng et al. (2013)

formed by microbial enzymes within the keratinous substrates,

contrasting with the broad hydrolysis of peptide bonds and the
destruction of some amino acids usually associated with chemical
methods. However, optimization of chemical hydrolysis, such as
the use of suitable reagents and moderate processing conditions,
might be potentially usefull to obtain bioactive hydrolysates
ABTS: 0.56; Superoxide radical: 1.52;

Crude hydrolysate and ultrafiltration

(Bouhamed et al., 2018). Alternatively, mild thermo-chemical pre-

treatments, intended to destabilize the structure of keratinous
materials without significant cleavage of peptide bonds before
Crude hydrolysate: 17.0

Fraction 1–10 kDa: 14.0

enzymatic or microbial hydrolysis, are interesting approaches for

Fraction > 10 kDa: 29.0
Hydroxyl radical: 1.19

the production of bioactive hydrolysates.

Post-hydrolysis processing is used to concentrate the bioactive
fractions: < 4.0

components comprised in the hydrolysates. Ohba et al. (2003)

Detailed information on hydrolysates production (and also on upstream and downstream processing) and application schemes might be found in the cited references.

observed that the content of anti-hypertensive and antioxidant

peptides was enriched after ultrafiltration of enzymatic feather
hydrolysates, especially in the 1–10 kDa fraction. Bioactive pep-
tides obtained through enzymatic hydrolysis of a mixture of horn
and hoof from cows and buffalo, were also concentrated in the
hydroxyl), inhibition of edible oil oxidation

1–10 kDa fraction (Ohba et al., 2003). Sequential ultrafiltration

was used to fractionate feather hydrolysates produced through
Radical scavenging (ABTS, superoxide,

feathers conversion by Fervidobacterium islandicum AW-1. In

antioxidant tests, scavenging of the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical was higher in the 1–10 kDa fraction,
Radical scavenging (DPPH)

whereas the scavenging of the 2,20 -azino-bis(3-ethylbenzothiazo

line-6-sulfonic acid) (ABTS) radical was similar for the < 1 kDa
ACE: angiotensin I-converting enzyme; DPP-IV: dipeptidyl peptidase IV; EC50/IC50: Half-maximal effective/inhibitory concentration.

and 1–10 kDa fractions (Yeo et al., 2018).

Purification strategies are essential aiming to identify the pep-
ACE inhibition

tides responsible for observed bioactivities. Zeng et al. (2013) used

two sequential steps of gel-permeation chromatography to purify
an antioxidant peptide (18.7 kDa) from a chemical hydrolysate of
bovine hair; however, no peptide identification was performed.
An antioxidant peptide of 848.8 Da (Ser-Asn-Leu-Cys-Arg-Pro-
Cys-Gly) was purified from feather hydrolysates produced through
AH
AA

AA

bioconversion with Bacillus subtilis S1-4 using sequential acid pre-

cipitation, cation-exchange chromatography and reversed-phase
Protease from Bacillus subtilis (SabinaseÒ, 60 °C,

fast performance liquid chromatography (Wan et al., 2016). More

recently, a new antioxidant peptide (Leu-Pro-Gly-Pro-Ile-Leu-Ser-
Alkaline hydrolysis (50 g/L bovine hair,

Ser-Phe-Pro-Gln) was identified from the hydrolysate obtained

with Chryseobacterium sp. kr6 growing on raw feathers (Fontoura
et al., 2019). Up to now, these are the only antioxidant peptides
0.125 M NaOH, 45 °C, 24 h)

identified from hydrolysates of feather protein.

Extracts from keratinous materials also display significant
bioactivities and functionalities (Rouse and Van Dyke, 2010). Ker-
atin extracts obtained from the solubilization of human hair pre-
sented antioxidant activity, protecting human dermal fibroblasts
pH 8.3, 1 h)

from the oxidative stress induced by hydrogen peroxide (Lai

et al., 2018). Nanoparticles prepared from feathers-extracted ker-
atin showed antioxidant potential and antibacterial activities
towards Staphylococcus aureus and Escherichia coli (Sundaram
et al., 2015). Extracts produced by chemical feather hydrolysis
were used to obtain spheric keratin microparticles with antioxi-
dant potential and anticancer activities towards tumor cell lines,
displaying important potentials for pharmaceutical and medical
Enzymatic
Chemical

uses (Sharma et al., 2017).

In addition, spheric keratin particles with low cytotoxicity were
synthesized from thermo-chemical hydrolysates of wool. These
biofunctional microspheres present potential for drug delivery,
Horns and hooves

wound healing, among other applications (Zhang et al., 2013).

Wool keratin extracts obtained by a sulphitolytic process were
Bovine hair

used to synthesize keratin nanoparticles through self-assembling

and desolvation techniques. Covalent binding of chlorin e6, a
strong photosensitizer used in photodynamic therapy for cancer
a

treatment, to wool keratin yielded nanoparticles that were able

412 K. Callegaro et al. / Waste Management 95 (2019) 399–415
to accumulate in the cytoplasm of tumor cells. Such accumulation
increased the antitumoral efficiency of chlorin e6 upon photoacti-
vation (Aluigi et al., 2016).
8. Concluding remarks and future work
Keratin-rich wastes represent a relevant management issue,
both from its environmental recalcitrance and voluminous produc-
tion. Hence, significant efforts are undertaken attempting to
expand its possible applicability. The production of protein hydro-
lysates from keratin-rich biomasses should be regarded as a
promising strategy within this scope. Various technological
approaches are available as options for keratin hydrolysis. How-
ever, there are wide opportunities for improvements regarding
optimization, process adaptation, as well as the proposition of
novel hydrolysis methods.
Keratin hydrolysates might be used in diverse areas, ranging
from biofuels to human health. Such flexibility, also considering
that most of the valuable knowledge about this topic has been
recently constructed and is expanding, indicates a large potential
for research and development. The potentials for agriculture, ani-
mal production and bioenergy, and the inherent relationship
among these areas, has key importance to put in perspective the
significance of keratin hydrolysates. Additionally, there is a
promising scenario for discovering and further unveil the bioactive
potential of molecules encrypted within keratin-rich biomasses.
Distinct hydrolysis systems generate hydrolysates with differ-
ent peptide and amino acid yields and compositions, which in turn
affect potential applications. When considering the use of hydroly-
sates as fertilizers, feed and for biofuels production, the main goal
of hydrolysis may simply be the breakdown of recalcitrant keratin-
rich biomasses to increase accessibility. However, if the interest
resides on bioactive properties, then hydrolysis should yield speci-
fic hydrolysates and peptides. Thus, enzymatic and microbial
methods are better suited.
From the outstanding bioactive potentials so far reported and
the proposed uses of keratin hydrolysates and peptides in human
health and feed formulation, information is still lacking regarding,
for instance, toxicity, stability during gastrointestinal digestion and
suitability for intestinal absorption. In addition, the identification
and characterization of specific peptide sequences might provide
information to synthesize bioactive peptides for designing drugs.
Therefore, keratinous wastes should in fact be better described
as largely available and low-cost raw materials demanding ade-
quate technological approaches for its recycling and valorization.
From this premise, the diverse production processes and applica-
tions of keratin hydrolysates share a common background, that
is, the benefits for human activities and well-being are clearly inte-
grated into a wider dimension where humans are both dependent
and part of the environment.
Acknowledgments
Authors thank the financial support of CNPq and CAPES (Brasília,
Brazil).
Declaration of Competing Interest
None to declare.
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