Harnessing the Potential of Blood Proteins as

Functional Ingredients: A Review of the State

of the Art in Blood Processing
Sarah A. Lynch, Anne Maria Mullen, Eileen E. O’Neill, and Carlos Álvarez Garcı́a

Abstract: Blood is generated in very large volumes as a by-product in slaughterhouses all around the world. On the
one hand, blood generation presents a serious environmental issue because of its high pollutant capacity; however, on the
other hand, blood has the potential to be collected and processed to generate high-added-value food ingredients based
on its exceptional nutritive value and its excellent functional properties. In this paper, we review the current state of the
art for blood processing, from collection to final recovery of protein isolates, the functional properties of blood, impact
of processing on functional properties, and potential applications as food ingredients. Furthermore, future challenges are
outlined for this underutilized and abundant product from the meat industry.
Keywords: blood processing, food additives, functional properties, protein

Introduction: Legal Frame and Potential Use of Blood either because they are parts not normally eaten or sold commer-
Blood, in European legislation, is defined as offal; which is
the “fresh meat other than that of the carcass, including viscera
and blood.” Blood is subjected to the same regulations as car-
cass meat as outlined in EC Regulation nr 853/2004, which lays
down specific hygiene rules for food of animal origin. How-
ever, blood that is not intended for human consumption, and
has not being hygienically collected, but has been subjected to
veterinary inspection, is then classified as an animal by-product
(ABP). According to European legislation (Article 3 of Regu-
lation [EC] 1069/2009), blood collected in slaughterhouses, not
intended for human consumption, is defined as an ABP. Specif-
ically, this regulation defines ABP thus: “entire bodies or parts
of animals, products of animal origin, or other products obtained
from animals that are not intended for human consumption.” This
includes a multitude of different materials such as catering waste,
used cooking oil, former foodstuffs, butchering and slaughter-
house wastes, blood, feathers, wool, hides and skins, fallen stock,
pet animals, zoo and circus animals, hunt trophies, manure, and
ova, embryos, and semen not intended for breeding purposes.
This legislation classifies by-products into 3 categories, based on
the risk for human health. Categories 1 and 2 are considered the
highest risk, while category 3 is considered as low risk. Category
3 includes parts of animals that have been passed as fit for hu-
man consumption but which are not intended for consumption,
CRF3-2016-1880 Submitted 11/14/2016, Accepted 1/3/2017. Authors Lynch,
Mullen, and Álvarez are with Teagasc Food Research Centre, Food Quality and
Sensory Science, Ashtown, Dublin 15, Ireland. Author O’Neill is with Dept. of
Food and Nutritional Sciences, Univ. College Cork, Cork, Ireland. Direct inquiries to
author Garcı́a (E-mail: carlos.alvarez@teagasc.ie).
cially; blood obtained in slaughterhouses falls within this 3rd cate-
gory. Federal Regulation (2015) in the U.S. (9 CFR 310.20) states
how to collect and process the blood when intended for human
consumption.
In recent years, there has been growing global pressure on the
food industry to try and minimize the environmental impact of its
activities and to improve sustainability. This has led to increased
interest in the more complete recovery and optimum utilization of
by-products from the food industry (Otles and others 2015). Eu-
ropean Directives are focusing on implanting the triple philosophy
of reducing raw material, recycling, and reuse of by-products. In
particular, the 2008/98/CE directive encourages waste prevention
and promotes recovery and recycling; this document defined for
the first time the difference between waste and by-product. Hence,
a by-product is defined as “when substances or objects resulting
from a production process not primarily aimed at producing such
substances or objects are by-products and not waste.” According
to this directive, a by-product differs from a waste because (i) it
will be used after the main process; (ii) it can be used directly,
with no ulterior transformation different from the regular indus-
trial practices; (iii) it is produced as an integral part of an industrial
process; and (iv) its ulterior use is legal, for instance, the substance
or object meets all the requirements for the specific application
relative to the products and the environment and health protec-
tion, and it will not produce negative impacts either on human
health or the environment. Within this same directive, the Euro-
pean Union has proposed a 5-step waste management hierarchy
based on waste reduction, reuse, recycling, recovery, and finally
barring all other steps of disposal. Until very recently, the potential
of food by-products to create new opportunities and markets has
been underestimated (Otles and others 2015); blood is one of these


C 2017 Institute of Food Technologists®

330 Comprehensive Reviews in Food Science and Food Safety r Vol. 16, 2017 doi: 10.1111/1541-4337.12254
Blood processing and food applications . . .
underutilized materials and it has been identified as a key source of
extractable high-value molecules for applications in sectors such as
the food, biomedical, and pharmaceutical industries (Lafarga and
others 2015, 2016; Mullen and others 2015).
Animal blood produced in slaughterhouses represents the most
problematic by-product of the meat industry because of the high
volumes routinely generated and its very high polluting power.
Liquid blood has a total nitrogen content of approximately 30 g/L,
a chemical oxygen demand (COD) of approximately 400 g/L and
a biological oxygen demand (BOD) of approximately 200 g/L
(Moure Fernández 2000). These values exceed the legal limits es-
tablished by European Directive 91/271/CEE (further modified
by 98/15/CE directive) that quotes a maximum COD value of
125 mg for the treated waste. Even more, environmental legisla-
tion of the European Union (Commission Decision 2006/12/EC)
encourages the recovery and use of by-products in order to con-
serve natural resources. A comprehensive review about European
regulations regarding ABPs can be found in Leoci (2014). The
volume and polluting potential of blood make it a prime material
for recovery of value. By 2001, it was estimated that only 30% of
total blood produced in slaughterhouses was used as food ingre-
dient, mainly in black pudding and related products (Ofori and
Hsieh 2012). According to these authors, the use of blood in the
food industry has increased; however, there are no data to confirm
this.
As described below and highlighted in a number of pa-
pers (Tybor and others 1975; Ramos-Clamont and others 2003;
Selmane and others 2008; Álvarez and others 2009, 2012a; Furlán
and others 2010; Parés and others 2014), blood has many func-
tional and nutritive attributes. While some products will use whole
blood, many applications require some degree of processing that
ranges from separation of blood into fractions through isolation
of individual proteins (Yang and Lin 1998c). Multiple new tech-
nologies have been developed in recent years aimed at recovering
purified proteins from blood: liquid–liquid extraction (Selvakumar
and others 2012), chemical precipitation (Imeson and others 1978;
Moure and others 2003), salting-out (Parés and others 2014), or
chromatographic techniques (Howell and Lawrie 1983). Recent
publications have highlighted potential applications for blood pro-
teins, in particular in the food industry (Di Bernardini and others
2011; Ofori 2011; Parés and others 2011; Tarte 2011; Jayathilakan
and others 2012; Leoci 2014).
Taking these factors, legal framework, new technologies, to-
gether with increased industry research interest in capitalizing on
the inherent value of blood (Galanakis 2012), this approach can
lead to enormous economic, societal, competitive, and environ-
mental benefits, in terms of new jobs and opportunities in the
global food market (Ofori and Hsieh 2012).
Nutritive Value of Blood
Blood has excellent nutritive value, not only because of its
high protein content, but also because of the bioavailability of
the nutrients (Parés and others 2011). Chemical composition of
blood from different animal sources is shown in Table 1. It is
important to note, however, that blood is deficient in the essential
amino acids methionine and isoleucine (Parés and others 2011)
(Table 2). Blood is a rich source of iron, which is contained
in the hemoglobin of red blood cells (RBCs), and this heme
iron has a high bioavailability as it is more easily absorbed than
nonorganic iron from plants or the ferrous salts commonly used
in the fortification of foods (In and others 2002). It has been
well reported that blood protein, despite the lower amounts in

C 2017 Institute of Food Technologists® Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 331
Table 1–Chemical composition of whole blood from different species of
livestock. Adapted from Gorbatov (1988).
Components Components g/100 g
Cattle Pigs Sheep
Water 80.89 79.06 82.17
Dry solids 19.11 20.94 17.83
Hemoglobin 10.31 14.22 9.29
Other proteins 6.98 4.26 7.08
Sugar 0.07 0.07 0.07
Cholesterol 0.19 0.04 0.14
Fat 0.06 0.11 0.09
Fatty acids – 0.05 0.05
methionine and isoleucine, can be used as a source of high-quality
proteins for both animal feed and human consumption (Tybor and
others 1975; Imeson and others 1978; Duarte and others 1999;
Prata and Sgarbieri 2008; Lee and Song 2009; Ofori and Hsieh
2011; Bah and others 2013; Parés and others 2014).
Blood Collection
The method of blood collection varies according to its final
use. Blood, which will be rendered or used for compost, is col-
lected following less stringent hygienic requirements, often using
an open-drain system. Blood collected in an open-drain system
drains from the animal into a centralized vat. In this type of system,
there are many opportunities for contaminating the blood (Parés
and others 2011; Bah and others 2013). When blood is intended
for food, research, or pharmaceutical purposes, it is important to
collect it in a more careful and hygienic way, using a closed-drain
system. In this system, contamination is reduced (Parés and others
2011), and blood is not exposed to air. At slaughter, it is collected
directly into a hollow sticking knife that is connected to a flexible
tube to direct the blood flow to a collection vessel. The blood is
often collected in a batch-type manner from several animals and
is then passed for use when the corresponding carcasses have been
approved by veterinary inspection as fit for human consumption.
The knives can also be fitted with disposable bags that hang from
the end of the knife and collect the blood directly. While gravity
is normally used to draw blood from the animals, vacuum sys-
tems may be employed to speed up the bleeding process (Knipe
1988). Blood can be drawn directly from the carcass to a refrig-
erated storage vessel. Drawbacks associated with some vacuum
systems include larger capital investment required for pulsed vac-
uum pump equipment and a slowing of the slaughter line, which is
particularly obvious in pig slaughter (Parés and others 2011). One
study from the 1980s, which compared closed drainage systems
with or without pulsed vacuum pump, suggested that a vacuum
system confers no special advantages (Gorbatov 1988). In some
cases, they may collapse the blood vessels, thus hinder the flow of
blood to the knife (Ockerman and Hansen 2000). Two types of
closed-drainage systems exist and are distinct for the knife used:
flat-bladed or hollow-bladed knives.
(a) Flat-bladed knives consist of a metal cup fitted around the
base of a standard sticking knife attached to a flexible tube
that carries blood to a covered receiving container. The cup
is placed against the animal and funnels the blood from the
stick wound into the flexible tube. This method may still
pose a risk of contamination, as the cup may come into
contact with contaminated hides or carcass.
(b) Hollow-blade knives are available in several varieties: the
Rizzi knife, the Esktam knife, and the cannula knife. The
Rizzi knife consists of a 1-inch-diam tube, 1 end of which
Blood processing and food applications . . .

Table 2–Amino acid profiles of porcine whole blood and various blood proteins in comparison to myosin from muscle. Adapted from Gorbatov (1988).

Amino acid content of blood proteins (g/100 g)

Amino acid Whole blood Fibrin Hemoglobin Serum globulin Serum albumin Myosin
Phenylalanine 10.7 4.6 9.6 4.7 6.6 3.2
Tryptophan 1.5 3.5 2.0 2.8 0.7 0.8
Arginine – 6.7 3.5 5.8 5.9 7.0
Histidine 8.8 2.3 8.5 2.1 4.0 1.7
Lysine 9.7 9.0 10.6 6.3 12.8 10.3
Methionine 2.4 2.6 1.2 1.0 0.8 3.4
Threonine 4.8 7.9 6.0 7.4 5.8 3.8
Leucine 13.2 7.1 14.9 9.5 12.3 15.6
Isoleucine 0.9 5.0 0.0 2.0 2.6 –
Valine 8.7 3.9 11.0 9.7 5.9 2.6
Aspartic acid – 11.9 10.0 9.0 10.9 8.5
Glutamic acid – 13.8 7.4 12.5 16.5 21.0
Cysteine – 1.5 0.9 2.3 5.9 14
Tyrosine 1.4 6.0 2.9 6.7 5.1 2.2

is formed into a sharp point and the other end is connected
to a flexible tube that carries the blood into the holding
container. The Esktam knife consists of a hollow 2-sided
knife with a rod or a 2nd knife set in the arched space at
right angles of the butt of the hollow knife. This second
serves to hold the stick wound open allowing free flow
of blood; it is probably the best system for reducing the
contamination of blood. The cannula system consists of a
0.5-inch hollow tube at 1 end, formed into a sharp cone
shape with slots cut into the tip of the cone for blood to
enter.
Some companies have developed rotatory systems for blood
collection based on hollow knifes, and such a technology permits
collections up to 85% of total blood from 1000 pigs per hour,
addressing all the regulations regarding the hygienic collection
of blood and the traceability of the final product. One example
of such rotatory systems can be seen at the Butina home page
(http://www.butina.eu/products/blood_collection/).
Hemolysis during bleeding and collection needs to be mini-
mized. If hemoglobin is released, due to the disruption of the red
cells membranes, the quality of the plasma may be compromised.
It can be prevented by rinsing all equipment with an isotonic an-
ticoagulant solution. This will create an osmotic pressure outside
the RBCs, equal to the pressure inside the cells. If the osmotic
pressure is not equalized, higher pressure inside the cells will cause
them to lyse, releasing hemoglobin leading to lower quality plasma
(Halliday 1973).
In the event, 1 animal’s blood is contaminated, or if a carcass
is condemned after inspection, then the blood from that carcass,
and any other blood with which it is mixed, loses its edible status
and the entire blood collection line, from the knife to the batch
holding tank, must be cleaned and sanitized. Blood that has been
contaminated from contact with the surface of the animal, or by
other means, should not be collected for use in foods (Dill and
Landmann 1988). With this regard, the European legislation is
clear: “The different categories of ABPs must be kept separate
from each other at all times, to avoid cross-contamination. They
must also be kept separate from food for human consumption”
(Regulation [EC] nr 1069/2009, Article 26 Implementing Regu-
lation [EC] nr 142/2011). The carousel system, where blood from
individual animals is held in containers mounted on a carousel,
can maintain correlation with individual carcasses as the carcasses
progress down the line on slaughter floor. If individual storage
is not possible, it is a regular practice in small-/medium-size
abattoirs that only the blood from a small number of animals is
mixed; hence, if any carcass is condemned the volume of blood
that has to be rejected is not excessive.
Collection rates
Approximately 5% to 9% of animal (live weight) is accounted
for as blood, with the actual amount varying with species (beef
8%, pigs 5%, and sheep 8%). Up to 50% of the total blood of an
animal is in circulation, 16% is present in the spleen, 20% is found
in the liver and, 10 % in the skin. Bleeding after slaughter will yield
40% to 60% of the animal’s total blood with the rest remaining
in capillaries and organs (Gorbatov 1988; Ockerman and Hansen
2000; Parés and others 2011). Again, blood yields will vary with
breed, degree of fatness, and stunning and bleeding method. Yield
of edible blood is approximately 3.2% for beef and 3% for pigs
based on live weight (Gorbatov 1988). Cattle bleeding will yield
12 to 15 L of blood with 11.4 to 14.5 kg collected within 60 to
120 s, while in pigs, bleeding yields 3 L with 2.7 kg collected in
30% to 40 s. The yield can be maximized when the time between
stunning and sticking is reduced (Knipe 1988). Standard bleeding
times used on the slaughter line are 6 min for bovine, 4 to 5 min
for ovine, and 6 min for porcine animals (Ockerman 2000).
Immediate blood processing
After bleeding, blood will begin to clot within 3 to 10 min de-
pending on the environmental temperature. This clotting process
is a natural process, due to the action of the enzyme throm-
bin converting soluble fibrinogen to insoluble fibrin, in a 3-
dimensional network together with platelets and other proteins
(Mosesson 2005). As blood is a rich organic medium, it is im-
portant to chill it as soon as possible to 2 to 4 °C to minimize
microbial growth, which would lead to blood spoilage. However,
it is important to note that blood refrigeration has been reported
to lead an enrichment in psychrophilic bacteria, such as Pseu-
domonas species, which are able to promote protein degradation
(Parés and others 2011). Thus, rapid processing to stabilize the
product is required. Several researchers have demonstrated that
common downstream processes, such as spray drying, ultrafiltra-
tion, or low-pressure evaporation, do not reduce significantly the
microbial load (Parés and others 1998; Dailloux and others 2002).
When serum is the final product, collected blood is allowed to
clot at the same time that it is chilled, after which clots can be
removed, for example, by means of centrifugation or decanting.
Under controlled conditions, the red cells become trapped in the
fibrin net and are removed with the clot, yielding a serum free
of red cells. More frequently, liquid blood or plasma is required
for further processing. In these cases, anticoagulants are added to

332 Comprehensive Reviews in Food Science and Food Safety r Vol. 16, 2017 
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Blood processing and food applications . . .
Table 3–Key quality parameters of plasma processed for biotechnological purposes. Data proportioned by SeraLab S.L. on February 2015.
Protein Osmolality
Type g/dL mIsm/kg H2 O
Bovine serum 7.0 to 9.0 270 to 340
Chicken serum 3.0 to 6.5 270 to 340
Fetal calf serum 3.5 to 4.5 260 to 340
Horse serum 6.0 to 9.0 270 to 330
Pig serum 6.0 to 8.0 260 to 350
blood as close to the collection point as possible. Many hollow
knives are designed to facilitate this (Ockerman 2000) or, alter-
natively, anticoagulants are sprayed onto the blood in the open
drain collection system. Anticoagulants, such as sodium citrate or
ethylenediaminetetraacetic acid (EDTA), function by converting
the calcium present in blood into a nonionized form and inhibit-
ing its role in coagulation. Proteolytic enzymes have also been
used, in place of anticoagulants, to prevent clotting. Blood is then
subjected to centrifugal force to separate it into the plasma and
cellular fractions (Ockerman 2000).
While dried whole blood can be used, for example, to obtain
blood meal or can be directly used as a food ingredient (black
pudding), blood utilization by the food industry is minimal due to
the darkening of the products to which it is added (Mielnik and
Slinde 1983). Thus, whole blood often is separated into the plasma
and cellular fractions by centrifugation (Tybor and others 1973;
Tybor and others 1975) (see next section). While this can give a
plasma fraction with important functional proteins, many proteins
and iron are present in the cellular fraction that is more difficult
to use due to problems such as unpleasant flavors and unwanted
color (Caldironi and Ockerman 1982). Once plasma, serum, or
red cells are obtained, they need to be frozen or dried to preserve
quality and safety. Those not intended for human consumption
can be treated with chemical preservatives (Ockerman and Hansen
2000).
Blood centrifugation
For the best results, separation should be carried out as soon
as possible to minimize the hemolysis of RBCs. If high-quality
plasma is required with minimum color (color is due to the pres-
ence of hemoglobin released when hemolysis takes place), the
plasma is usually separated as soon as possible and prior to chilling.
In this case, the chilling of the separated fractions is carried out
directly after separation (Knipe 1988). This can be carried out
with either a continuous or noncontinuous process. For small vol-
umes (up to 3 to 4 L), discontinuous processes are more suitable
as they result in higher quality plasma in terms of low hemoglobin
content. Table 3 summarizes the specification that plasma has to
have to be considered of high quality for biotechnological, clinical,
and lab uses (according to SeraLab Industries, personal commu-
nication, West Sussex, U.K.). When plasma is employed for other
purposes such as pet food, or food ingredients, just the total pro-
tein content, hemoglobin content and the pH are considered as
quality parameters. From a microbiological point of view, blood
used in liquid form as an ingredient has to be heat-treated. While
the European legislation (EC No 2073/2005) on microbiological
criteria for foodstuffs does not mention specifically blood prod-
ucts, in general terms, the absence of salmonella bacteria in 25 g
is used to define the safety of these products. When not employed
for human consumption, regulation (EU) No 142/2011 imple-
menting Regulation (EC) nr 1069/2009 is applied; it requires the
absence of salmonella bacteria in 5 samples of 25 g each, and no
more than 300 enterobacteriacea per gram of sample.

C 2017 Institute of Food Technologists®
Hemoglobin Endotoxin Glucose
mg/dL ng/mL pH mg/dL
<50 <1 7.0 to 8.5 30 to 80
<80 <10 7.0 to 8.5 80 to 200
<20 <1 7.0 to 8.5 80 to 200
<50 <10 7.0 to 8.0 <140
<50 <5 7.0 to 8.5 35 to 85
Different combinations of speed, time, and temperature can be
used to obtain a good separation, and examples from the literature
are presented in Table 4. It has been reported that increasing time,
speed, and centrifugation volume increase the yield of plasma
obtained; however, the hemoglobin concentration in plasma is
increased as well and hence the plasma quality is reduced (Moure
Fernández 2000). On the other hand, when higher volumes have
to be processed, continuous centrifuges are the best option. It
is possible to separate 150 to 1000 L of blood per hour using a
plate-type separator; however, these will require periodic cleaning,
resulting in downtime for the equipment as well as the possibility of
introducing water into the blood (Knipe 1988). Several companies
(AlfaLaval, GEA Westfalia, and Eppendorf) have designed specific
blood centrifuges able to work in a continuous process. In this
type of equipment, the plate speed is approximately 10000 rpm
(8000 × g), though these values can vary. The capacity of such
centrifuges varies from 50 L/h to more than 4000 L/h and the
most recent models feature a CIP (clean-in-place) system, which
allows for reduction in the downtime of cleaning and maintenance.
Centrifugation of 100 kg of blood can yield 66 kg of plasma with
a protein content of 8% (5.3 kg of plasma protein) and 33 kg of
the cellular concentrate with a protein content of 38% protein
(12.5 kg of cellular protein) (Wismer-Pedersen 1988).
As it has been stated by several authors (Gorbatov 1988;
Ockerman and Hansen 2000), differences exist between the species
with regard to the ease of separation of the fractions and the qual-
ity of plasma obtained. Bovine blood appears to be less sensitive
to hemolysis and mechanical disruption of the cellular fraction
than porcine blood. This may be due, in part, to the composition
of the blood; porcine blood has a higher content of the cellular
fraction than ovine or bovine blood. Another possible factor is
the difference in the characteristics of the erythrocytes of blood
in the different species. In porcine blood, the diameter is largest
at 6.2 μm, while in ovine and bovine blood, it is 5.0 and 5.1
μm, respectively. In addition, the difference in the specific gravity
between the plasma and cellular fractions is greater in bovine than
in porcine blood, making centrifugal separation easier.
Fractionation of Plasma Proteins
Plasma proteins are highly functional and can be prepared as
crude or purified isolates. When isolating or fractionating pro-
teins, it is important that yield and functionality are maximized.
The conditions that proteins are subjected to during processing
will affect the functionality of the recovered protein (Tybor and
others 1973; Ockerman 2000). Numerous methods have been re-
ported for plasma fractionation and can be classified in a number
of ways: (i) differential solubility where a precipitant decreases the
attraction between protein surface and solvent, resulting in in-
creased protein–protein interactions and ultimately precipitation;
examples include alcohol fractionation (Cohn and others 1946),
polyethylene glycol (PEG) fractionation (Lee and others 1987), or
salting-out (Parés and others 2014); (ii) different interaction with
solid media or chromatographic methods (Ramos-Clamont and
Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 333
Blood processing and food applications . . .

Table 4–Examples of conditions used to separate plasma and cellular fractions from blood.

(Imeson and others (Del Hoyo and (Duarte and (Parés and others (Álvarez and (Lee and others (Silva and
1978) others 2007) others 1999) 2014) others 2012a) 1987) Silvestre 2003)
Time (min) 20 30 15 15 15 15 10
Speed (× g) 22000 20000 5000 2530 3000 1020 1000
Tempertaure (°C) 20 4-6 10 4 8 6 Room
temperature

others 2003; Moure and others 2003); and (iii) differential inter- difficulties with stirring and pumping (Ingham 1990). Usually,
action with physical parameters such as temperature or membrane buffers are employed in both protein and PEG stock solutions, to
filtration (Del Hoyo and others 2007; Del Hoyo and others 2008). control the pH and ionic strength. A disadvantage associated with
this method, however, is the effort required to remove PEG from
Differential solubility the supernatant fraction. Techniques such as ultrafiltration, gel
Ethyl alcohol fractionation. This method is the principal permeation, or electrodialysis can be employed to overcome this
method for albumin production for pharmaceutical use. It is based (Ingham 1990). By using PEG fractionation, 91% of fibrinogen,
on differential solubility of plasma proteins in ethanol/water mix- 88% of immunoglobulin, and 92% of serum albumin have been
tures (Cohn and others 1946). Organic solvents interact with apo- recovered. The parameters employed for precipitating fibrinogen
lar protein groups, displacing water from the protein surface. This were pH 5.5, PEG 9.06%, and NaCl 1.4 M. Subsequently, PEG
leads to protein aggregation and a decrease in solubility leading 12.6% and NaCl 0.35 M were added to the resultant supernatant
to precipitation of the proteins. The concentration of any solvent at pH 8.22 to precipitate immunoglobulins and to obtain albumin
required to precipitate a protein depends on the intrinsic proper- in the supernatant (Lee and others 1987).
ties of the target proteins (Moure Fernández 2000). Plasma is first Precipitation of plasma proteins using anionic polysaccharides.
diluted to 50% with distilled water giving a protein concentration It has been demonstrated that proteins in blood plasma, like those
of 30 to 35 mg/mL. To minimize the risk of denaturation due to in cheese whey, can be recovered by precipitating the protein from
ethanol the separations should be carried out at temperatures as solution using another polyelectrolyte such as a hydrocolloid. Pro-
close to 0 °C as possible. Phases can be separated by centrifuga- tein from cheese whey can be recovered using various materials,
tion with the precipitated target protein(s) in the sediment and the including sodium alginate, carboxymethyl cellulose (CMC), and
remaining proteins in the supernatant. This supernatant is used as polyacrylic acid (Hill and others 1982). Interaction of CMC and
feedstock for the subsequent fractionation(s). The main parame- whey proteins has been extensively reported, and it has been ob-
ters that drive the differential precipitation of plasma proteins are served that the amount of protein precipitated is closely dependent
pH, ionic strength, temperature, and protein concentration. The on the pH, ionic strength, as well as the degree of substitution of
fractions that are obtained are: fibrinogen (8% ethanol, pH 7.2); γ - the cellulose derivative. With optimum conditions, more than
globulins (19% ethanol, pH 5.5); α, β-globulins (40% ethanol, pH 90% of protein can be removed from solution, and the recovered
5.5), and albumin (40% ethanol, pH 4.5). After extraction, precip- protein has excellent functional properties (Imeson and others
itated proteins are easily redissolved in phosphate buffer at pH 7.2 1978).
to give an equivalent volume to the original plasma used (Álvarez Anionic polysaccharides have been used to extract other proteins
and others 2009). Although this method was developed mainly from solution besides whey protein. They have been successful in
for clinical applications, it can also be applied for processing blood the recovery of soy proteins, casein, yeast protein, and sunflower
collected in slaughterhouses into these fractions (Moure and oth- seed albumins. Sodium alginates and sodium pectate are the 2 main
ers 2003). The advantages (safety, cost of reagents [ethanol can be polysaccharides used to precipitate proteins, because they possess
reused], and ease of scale-up) and disadvantages (temperature con- the ability to form thermostable gels, a property that could be
trol, protein loss, and risk of handling flammable solvents) of this beneficial in the utilization of the recovered proteins in processed
method have been previously reviewed (Denizli 2011). By using meat products (Imeson and others 1978). When using anionic
this method, a complete protein precipitation can be achieved, polysaccharides for protein precipitation from plasma, sodium al-
yielding 4 protein-rich fractions and a protein-free supernatant ginate appears to be the most efficient and also the least affected by
rich in ethanol that can be recovered by distillation and reused increases in salt concentration. The higher charge per unit residue
in the next precipitation cycle. Ethyl alcohol fractionation can be for alginate, compared with pectate and CMC, may contribute to
coupled with chromatographic processing to obtain more pure the greater protein recovery yields achieved at high ratios (Imeson
fractions and to remove salts and low-molecular-weight organic and others 1978). A recent patent (Van Alstine and others 2014)
compounds (Moure and others 2003). refers to the use of polyacids, such as CMC, polyacrylic acid, or
Polyethylene glycol fractionation. PEG is a nontoxic, water- polyvinylsulfonic acid for plasma fractionation. According to the
soluble synthetic polymer that has been employed as a protein authors, this method can be employed in a selective manner to
precipitant. PEG holds several advantages over ethanol as a separate specific abundant proteins by the rigorous control of pH
precipitating agent as it results in less foaming, is less expensive, and salt composition.
is not flammable, and does not require strict temperature control Salting-out. Salting-out occurs with solutions of high ionic
to prevent denaturation (Lee and others 1987). In addition, com- strength, where the salt draws water molecules away from pro-
pared to ethanol and other precipitants, PEG requires less time to tein surfaces, thereby encouraging proteins to interact and coag-
exert its effects and its precipitation curve is relatively unaffected ulate. Since proteins differ noticeably in their solubility at high
by temperature changes. PEG with an average molecular weight ionic strength, salting-out can be used to separate proteins from
of 4000 to 6000 Da is the most commonly used; no significant a mixed solution such as plasma (Parés and others 2014). When
differences are observed when higher-molecular-weight PEGs using salting-out as a method of protein fractionation, the larger
are employed, but the viscosity is notably increased resulting in

334 Comprehensive Reviews in Food Science and Food Safety r Vol. 16, 2017 
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Blood processing and food applications . . .
the molecule, the more readily it is precipitated (Allen and others
1977). Ammonium sulfate is a commonly used salt that is diluted
to the concentration required to selectively precipitate the protein
of interest. The aggregated protein is then separated by centrifu-
gation, leaving the remaining proteins in the supernatant (Parés
and others 2014). Plasma proteins can be separated by salting-out
using a 100% saturated solution of food grade ammonium sulfate
in 10 mM Tris-HCl at pH 7.4, by adding it dropwise to fresh
plasma. This increases the ionic strength and progressively precip-
itates each fraction. Saturation percentages of ammonium sulfate
to precipitate the protein fractions are 20% for fibrinogen and 60%
for globulins. Serum can be obtained by removal of precipitated
fibrinogen after centrifugation. Finally, albumin is recovered in the
supernatant after other proteins present in the plasma have been
precipitated (Parés and others 2014).
Aqueous 2-phase extraction. An aqueous 2-phase system
(ATPS) is a gentle liquid/liquid partitioning system based on 2
types of phase-forming components. The 2 immiscible phases are
formed by mixing, for instance, a polymer (usually PEG) and a salt
(such as phosphate, sulfate, or citrate) or 2 polymers and water, and
they can be effectively used for the separation and purification of
proteins (Nath and others 2015). The partitioning between both
phases is dependent on the various physicochemical properties of
the proteins and on the properties of the 2-phase system (Asenjo
and Andrews 2011). Typically, the 1st stage of extraction yields a
lower phase, containing contaminants and cell debris, and an up-
per phase containing the product of interest, in this case protein.
The 2nd stage involves concentration of protein in the bottom
phase. This phase is further concentrated by ultrafiltration to yield
a protein concentrate (Rito-Palomares 2004). ATPS extraction
(using PEG and phosphate buffer) has been successfully employed
to isolate and recover 85% of bovine serum albumin (BSA), and an
overall protein recovery of 65% for hemoglobin subunits (α and
β) and Immunoglobulin (IgG) (Rito-Palomares and others 2000).
This shows its potential for the generic application as a primary
recovery and partial purification process of target protein solutes
from a complex biological suspension such as blood.
Chromatographic methods: ion-exchange chromatography
Protein separation and purification by differential interaction
of the soluble protein phase with a solid medium is certainly the
most common method used at laboratory scale. Affinity and ion-
exchange chromatography are the most important of these pro-
cesses (Burnouf 1995). Ion-exchange chromatography has been
selected as a method of plasma protein fractionation by many
research groups (Howell and Lawrie 1983; Tomono and others
1983; Goheen and Hilsenbeck 1998; Moure and others 2003),
because of its high binding capacity and the absence of denatu-
rating agents such as ethanol or solvents, thus leading to proteins
with properties of relevance even for pharmaceutical applications
(Moure and others 2004). Of the various modes of chromato-
graphic operations available, the most suitable for proteins has
been shown to be adsorption onto the whole column followed
by stepwise elution with buffered salt solution (Leaver and others
1987). Ionic-exchange resins may also be used to remove inor-
ganic salts from plasma, as a previous cleaning step for protein
applications in the food, pharmaceutical, or cosmetic industries
(Noordman and others 2002; Simon and others 2002; Benhabiles
and others 2012). In such cases, salts were removed in consecu-
tive steps by using strong anionic and cationic resins; and up to
a total 80% of total present salt can be removed. However, loss
of protein (approximately 14%) and a significant pH change may

C 2017 Institute of Food Technologists® Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 335
occur, which may negatively affected protein functionality (Del
Hoyo and others 2007).
The main drawback of chromatographic methods is that not all
chromatography media are suitable for large-scale use. However,
new composites based on agarose beads have been developed,
which enable scaling-up of the process. It can be employed either
for size-exclusion or ion-exchange chromatography. Such material
has the advantage to be easily scaled-up due to the fact that agarose
bead performance is not affected by a high-flow process or by high
internal pressure.
Membrane filtration
Membrane technology has become an important separation
technology in recent decades. Some of the main advantages asso-
ciated with this technology make it suitable for industrial and, in
particular, food applications; features are: no chemicals required,
relatively low use of energy, low processing costs, and easy to
scale-up (Muro and others 2013). Membrane separation tech-
niques have been used for human blood processing to recover
human serum albumin and immunoglobulins (Legallais and others
1994; Wan and others 2002; Mayani and others 2009). Membrane
filtration has been used for protein concentration and demineral-
ization of plasma, and also to reduce the salt content introduced
as anticoagulant. After plasma demineralization, it was reported
that approximately 80% of total ions can be removed with no
appreciable protein loss (<1%) when a 10 kDa molecular weight
cutoff (MWCO) membrane is used (Del Hoyo and others 2007,
2008). Ultrafiltration may also be used after ethanol precipita-
tion for protein concentration in the supernatant; however, it was
found that ethanol reduces the flux due to lower protein diffu-
sivity, and membrane-fouling is noticeably increased (Jaffrin and
others 1997).
Membrane dialysis is frequently used for desalting following
fractionation of plasma using salting-out procedures, but this is
only useful for lab or small-scale production. Parés and others
(2014) proposed to replace dialysis with tangential-flow filtration
(TFF), since membrane dialysis at industrial scale can take several
days and also requires large volumes of water and much sample
manipulation. TFF can overcome these issues with the added ad-
vantage that protein solutions can be concentrated and diafiltered.
A pilot-scale system was employed using a 30-kDa MWCO and
0.32 m2 active area modified-polyacetonitrile spiral module mem-
branes followed by a diafiltration process to complete salt removal.
After the process, 92% of total salt is removed with no loss of
protein.
Processing of Cellular Fraction of Blood
The 2nd fraction obtained from blood after centrifugation is the
cellular fraction or RBCs, with hemoglobin representing around
31% of total fraction weight. Based on its abundance of highly
bioavailable iron, protein content, and technofunctional proper-
ties, this fraction is interesting from a food ingredients perspective
(Toldrà and others 2004). Table 5 shows the chemical composition
of red cell blood from different animal sources.
Hemoglobin extraction
The 1st step in hemoglobin extraction is to disrupt the red cells,
allowing release of the protein to the reaction media. Three main
techniques are commonly employed: (i) osmotic shock; (ii) ul-
trasonic disruption; or (iii) enzymatic procedures. Osmotic shock
requires water and a disadvantage is the dilution of the final prod-
uct (Álvarez and others 2009). Ultrasonic waves are able to disrupt
Blood processing and food applications . . .
Table 5–Chemical composition of blood cellular fraction from different or adding ascorbic acid (Tybor and others 1973). The addition of
species. Amounts expressed as g/100 g. Adapted from Gorbatov (1988). acidified acetone will remove the heme group, and the globin pro-
Cattle Pigs Sheep tein precipitates allowing for easier recovery by filtration, before
Water 59.21 62.56 60.48
Dry solids 40.81 37.44 35.92
Hemoglobin 31.67 32.68 30.33
Other proteins 6.42 1.92 7.85
Sugar – – –
Cholesterol 0.34 0.05 0.24
Fatty acids – 0.01 – that it contained all of the essential amino acids and the levels of
the cell membrane; however, this can raise the temperature and
lead to intense shear forces that can alter the tertiary structure and
potentially have an impact on the functional properties of proteins
(Jambrak and others 2014). A continuous process has been recently
developed (Garcia and others 2015), where 91% of hemoglobin
was released using 48 Kw/L of acoustic power density (APD) and
short ultrasonic residence times (300 ms). Short residence times
will limit protein denaturation as there are no increases in tempera-
ture. Enzymes are often used to promote the formation of pores in
red cell membranes by cleaving the membrane phospholipids; such
enzymes, known as hemolysins, are generally toxins produced by
pathogenic organisms. However, these are more oriented toward
laboratory research (Moraveji and others 2014). When high-purity
hemoglobin is required, cell membranes have to be removed. For
food and pharmaceutical applications ultra-high-speed centrifu-
gation can be used since it does not involve the use of solvents
(Toldrà and others 2004) unlike other methods, which include
solvent extraction using chloroform, or by filtration (Chang and
others 2007).
Hemoglobin color and processing
Despite its potential for use in human nutrition, the dark brown-
ish color that hemoglobin imparts on food formulations greatly
restricts its use as a raw material or ingredient in the food indus-
try (Fontes and others 2004; Fontes and others 2010; Pereira and
others 2014). With a view to extending the use of hemoglobin,
various efforts have been focused on its discoloration. Most of
the techniques are based on transforming heme iron or ferrous
(Fe2+ ) ion into nonheme iron or ferric iron (Fe3+ ), as the later
has a bright yellow color that may be more suited to inclusion
in food formulations. The other approach to hemoglobin discol-
oration is removing the heme group, leaving behind the globin
protein. Hemoglobin contains a heme prosthetic group that has
an iron atom at its center, which is responsible for the color.
The heme group of hemoglobin is bound to the globin chains by
noncovalent chains, and an equilibrium exists between free and
bound heme; addition of acid will shift this equilibrium to favor
dissociation (Anson and Mirsky 1930). Traditionally, the prepara-
tion of a colorless globin has been based on the splitting of the
protein and pigment moieties of hemoglobin. Organic solvents
with acidified cold acetone are the most commonly used due to
their efficiency in removing heme; however ethanol, butatone, and
methylethylketone have also been used (Wismer-Pedersen 1988).
Acidified acetone discoloration. Acidified acetone has been
used to extract the heme pigment from the globin protein (Tybor
and others 1973 1975; Yang and Lin 1998). A brief overview of
the process is presented. The pH of a hemoglobin solution, with a
maximum protein concentration of 12%, is brought to less than 4
by the addition of ascorbic acid. The hemoglobin is converted to
choleglobin (separation of heme and protein is more easily carried
out in this form) by bubbling air through the mixture for 1 h
336 Comprehensive Reviews in Food Science and Food Safety r Vol. 16, 2017
being washed with an ether ethanol solution. The globin protein
is dissolved in water and the pH is readjusted to approximately
pH 7. Globin isolated using the acidified acetone method was
found to have a crude protein content of 88% to 92% (Tybor and
others 1975; Yang and Lin 1998). Analysis of the protein showed
lysine, leucine, threonine, valine, tryptophan, and phenylalanine
are indicative of good nutritional value (Tybor and others 1975).
A modification of this method has been reported: acidic ace-
tone solutions, containing 10% of hydroxide peroxide, are able
to produce insoluble discolored globin (Moure Fernández 2000).
In this process, a rich hemoglobin solution is added slowly to a
stirred acid acetone/H2 O2 solution, to a final proportion of 1:3
hemoglobin:acetone; the size of the drop and the speed of addi-
tion determines the size of the protein aggregates formed, which
is larger at higher addition ratios. In such conditions, globin is
extremely insoluble and can be easily recovered by filtration or
centrifugation. In order to minimize the denaturating effect of
organic solvents, which negatively affect the globin solubility and
other functional properties, a low temperature is applied during
the discoloration process. Lactose has also been shown to have a
protective effect on the functionality of plasma and globin pro-
teins. (Tybor and others 1973; Tybor and others 1975). Overall,
the recovery of globin using acidified acetone is problematic not
only because of the reduced functionality of the protein, but also
because of the risk of harmful residues (Yang and Lin 1998).
Heme sequestration. Recovery of globin from the cellular por-
tion of blood using adsorption has been proposed as an alternative
to the separation using organic solvents. Examples of adsorbing
agents that have been investigated include carboxymethylcellulose
(CMC), activated carbon, silicic acid, and manganese dioxide. For
example, a hemoglobin solution shifted to pH 2 is mixed with di-
luted CMC, after addition of CMC, the mix is centrifuged, with
the globin protein in the resulting supernatant and the precipitated
CMC-heme in the sediment. The pH of the protein solution is
readjusted to 3 and concentrated using ultrafiltration before spray-
drying (Autio and others 1984; Yang and Lin 1998). Isoelectric
focusing, carried out on blood globin recovered with the CMC
process, showed that the protein retained its ordered structure even
after spray-drying. One advantage to the use of CMC for heme
separation is that the CMC-heme precipitate may be used as a
source of heme iron. Others have employed alginates to obtain
colorless globin (Lee and others 1990). Optimal conditions for
globin separation were pH 2.25, 0.348% NaCl, and 0.107% al-
ginate; about 65% of total protein was recovered free of 97.6%
of heme. Other polymers of interest include chitin, chitosan, and
pectin. Some of these have been used for protein recovery from
different sources, such as surimi waste water (Wibowo and oth-
ers 2005) or whey (Li and others 2012; Souza and Garcia-Rojas
2015).
In order to avoid the use of organic solvents or extreme pH
conditions, a method for globin discoloration, which involves the
use of ion exchange resins, was developed by Yang and Lin (2000).
It was considered that Amberlite IR-120 (strongly acidic cation
exchange resin) was the best one in terms of color reduction.
However, this process is limited by the saturation of the resin and
the need of regeneration to retrieve its ion exchange capability.
Discoloration by hydrolysis. Several attempts aiming to sepa-
rate the heme group, assisted by enzymatic hydrolysis, have been

C 2017 Institute of Food Technologists®
Blood processing and food applications . . .
carried out. The method is based on cleaving the hemoglobin
molecule from the globin and subsequent and further separation
of the heme group by precipitation, chromatographic techniques,
centrifugation, or membrane filtration. It has been reported that
the use of single proteases (Alcalase, trypsin, or pepsin) was un-
successful for hemoglobin discoloration; only the combined use
of trypsin and pepsin was efficient for hemoglobin discoloration
(Toldrá 2002). However, the degree of bitterness in the final prod-
uct was extremely high and the functional properties were neg-
atively affected. The use of enzymatic hydrolysis assisted by high
hydrostatic pressures (HHPs) was able to produce a degree of dis-
coloration by using trypsin as the unique enzyme, but only after
24 h of hydrolysis (Toldrá and others 2011). When pepsin was
used, the discoloration was higher when compared to the control
after 30 min of hydrolysis. However, the protein loss was increased
as discoloration increased.
The use of subcritical water hydrolysis has been reported as a
tool to generate discolored hemoglobin (Álvarez and others 2012b,
2016). In this case, the hemoglobin hydrolysis is a result of high
pressure and high temperature. The released heme group was ox-
idized in the process, achieving discoloration comparable to that
following the HPP-assisted method. Protein recovery yield was
80% in form of small peptides. According to these authors, oxy-
gen injection was more effective than nitrogen for hemoglobin
discoloration.
An issue with discoloration is a loss of functional properties in
the resulting globin fraction. In addition, a bitter taste can be de-
veloped as a consequence of a higher exposure of the hydrophobic
amino acids present in the hemoglobin.
Color stabilization by means of carbon monoxide. The methods
previously discussed present various disadvantages, such as the use
of reagents that must be subsequently removed, thus decreasing
functional properties and reducing the nutritional value by
eliminating heme iron. A method that can overcome these is
based on saturation with CO, resulting in the conversion of
hemoglobin to carboxy hemoglobin that has a cherry red color
(Fontes and others 2004; Fontes and others 2010; Pereira and
others 2014). Such a method was applied to whole blood, which
was placed in a reactor where pure CO was bubbled through
a porous disc (1 L/min) with gentle stirring until complete
saturation. It was found that lower pH leads to shorter times
to completely saturate the hemoglobin. However, the red color
can be lost during storage due to an oxidation process. A recent
paper reported how blood, which was color-stabilized by this
method, can be applied in the formulation of mortadella (Fontes
and others 2015). The final product showed good protein
availability and what the authors considered excellent nutritional
quality.
Processing for Storage Stability
Protein preparations may need to be stored for an extended
period while retaining their original structural integrity and/or
activity. Depending on the nature of the protein, the method used
for stabilization and conditions of storage “shelf-life” can vary
from a few days to more than a year. When isolating proteins for
use in the food industry, it is best to prepare them as a dry product.
The dry state optimizes the microbial and flavor stability during
storage and, also, is most economical for transport and storage
of the product (Dill and Landmann 1988). Drying reduces the
moisture content from up to 90% to 5% to 10%. Any method
generally used for drying liquid foods may be used provided that

C 2017 Institute of Food Technologists® Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 337
it has minimal negative influence on characteristics such as flavor,
color, or functionality.
It is usually more economical to first concentrate the protein
in blood plasma before subjecting the fraction to the drying pro-
cess. For the most part, concentration has been achieved by the
evaporation process, but membrane filtration is a method that is
also used to concentrate proteins in solution. Membrane filtra-
tions will allow the removal of water and salts from the solution as
was previously discussed. Finally, concentrated or whole blood or
blood fractions can be dried by spray-drying, freeze-drying, flu-
idized bed-drying, ball-drying, or roller-drying (Parés and others
1998; Toldrà and others 2004; Polo and others 2009; Ishwarya and
others 2015).
Evaporation
There are 2 main methods for concentration by evaporation: the
falling film evaporator and the centriterm evaporator (Ockerman
and Hansen 2000). Falling film evaporator involves a vertical bat-
tery of steel plates enclosed in a steam jacket. Plasma flows down
the inner margin of the pipes as a thin film, which is spread out by
the steam evaporating from the liquid. The evaporator normally
operates under reduced pressure, this gives the plasma a boiling
point of around 36 °C, which is below the denaturation tempera-
ture of plasma proteins (Knipe 1988). Plasma is concentrated from
about 85% water content to between 25% and 27% (Ockerman
and Hansen 2000). Centriterm Evaporator creates a thin layer (less
than 0.1 mm) of the product on the surface of the evaporator and
it is spread out by centrifugation. The protein solution is driven
from the center of the cone to the edge by the rotation of the
cones at a speed of about 600 rpm. This gives a short drying
period (around 1 s), which will reduce the amount of protein
denaturation (Ockerman and Hansen 2000).
Spray-drying
During spray-drying, liquid blood is forced through a nozzle
under pressure, this pressure releases the liquid in very fine par-
ticles that dry as they are passed through a current of heated air;
common parameters for this process at pilot plant scale are: air
flow 64 m3 /h; compressor pressure 2 bar; inlet 180 °C; outlet
80 °C; and input flow 800 mL/h (Toldrà and others 2004). To
avoid denaturation, the dried blood should not be subjected to
high temperatures for longer than a few seconds. Spray-drying is
probably the most widely used method of drying. Spray-drying,
however, is not without its disadvantages, including the unfavor-
able alteration to the flavor and functionality of products (Knipe
1988). Spray-drying can be used for both plasma and cellular frac-
tions, but due to the possible color crossover, separate equipment
is used for each fraction. Advantages of spray drying include
(1) Easy to design to any capacity required.
(2) Operation can be done in a continuous way.
(3) It can be applied to heat-sensitive products, meaning that
proteins are not denaturized and they can preserve its orig-
inal properties (Patel and others 2009).
It has been reported that spray-drying is not suitable for reducing
the microbial content in blood; however, the microbial load of the
powder obtained can be decreased my means of other techniques
such as HHP (Toldrà and others 2004). A slight decrease in solu-
bility at the isoelectric point can be found in blood proteins, this
may be due to the occurrence of slight heat denaturation during
the spray-drying process (Etheridge and others 1981)
Blood processing and food applications . . .

Table 6–Functional properties of blood proteins based on type of purification process.

WHC(%water
Emulsifying(% of retention or g Foaming (Foam
Solubility oil emulsified or H2 O/g of vol. regarding
Protein Processing (%)(pH 6) EAI in m2 /g) protein) LGC (%) Tgel (°C) solution vol.)
Plasma Centrifugation (1, 5) 86 77% 56% 6-7 71.4 1
Ultrafiltrated plasma (2) 85 78% 65% – – <0.5
IEX demineralized (2) 55 90% 65% – – <0.5
Red cells Spray-drying (3) 92 30 m2 /g 45% 10 – 4
SD-HHP (3) 80 nd 46% 10 – 3.5
Fibrinogen Ethyl fractionation (4) 7 100% – 4 56.8 2
Globulins Ethyl fractionation (4) 100 77% to 81% – 2 44 to 57 1
Maillard modification (5) 10 100% – 10 69 to 72 nd
Albumin Ethyl fractionation (4) 80 30% 44% 4 67 2
Maillard modification (5) 50 40% – 12 82 Nd
Hemoglobin Centrifugation (4) 80 57% 3.27 g H2 O/g – 59 1
Maillard modification (5) 80 100% – 14 77 nd
Red globin Acid acetone (4) 65 92% 5.5 g H2 O/g 10 – 1
Yellow globin Acid acetone (6) 5 5 m2 /g 4 g H2 O/g 10 – <0.5
Hydrogen peroxide (6) 35 7 m2 /g 4 g H2 O/g 10 – <0.5
Acid acetone/H2O2 (6) 32 15% – – – 1
Enzymatic hydrolysis (6) 38 18 m2 /g No retention – – <0.5
CMC sequestration (7) 28 9 m2 /g 4 g H2 O/g 10 – <0.5
Ion exchange (8) 5 to 6 14 m2 /g – – – 1
Succinylated (8) 12 38 m2 /g – – – 1.2
Acetylated (8) 4 9 to 10 m2 /g – – – 0.5
Sources: (1) Furlán and others (2010); (2)Del Hoyo and others (2008); (3)Toldrà and others (2004); (4)Álvarez and others (2009): (5)Álvarez and others (2012a); (6) Yang and Lin (1998); (7) Autio and others
(1984); and (8)Yang and Lin (2000).

Freeze-drying
Freeze-drying is based on the sublimation of moisture in the
blood. The liquid blood is frozen to from ice crystals; then the
pressure is reduced leading to a sublimation of the ice crystals at
a lower temperature and thereby protecting the integrity of the
protein. The prerequisite for sublimation is that the vapor pres-
sure and the temperature are held below the triple point of water
(0.65 kPa and 0.01 °C) (Mumenthaler and Leuenberger 1991).
After ice sublimation, a porous structure is formed, which confers
to the product a very high hygroscopic ability. Particles obtained
generally have a high product quality, but the long drying times,
batch nature, low temperatures, high vacuum, and the resultant
high operational costs limit the usage of the freeze-drying tech-
nique in a processing environment (Lopez-Quiroga and others
2012). Usually, freeze-drying is applied only in very high added-
value blood products such as thrombin or plasmin enzyme (Jayathi-
lakan and others 2012). In order to reduce the time required for
freeze-drying, a new technology has been described: spray-freeze-
drying (SFD) (Ishwarya and others 2015). SFD is a 3-step process
that involves a liquid or solution being atomized into droplets,
solidified by contact with a cold fluid, and sublimated at low tem-
perature and pressure. SFD is thus a unique drying technique as it
is a combination of both spray-drying and freeze-drying (Leuen-
berger 2002). This has been demonstrated with BSA (Wang and
others 2006) and trypsinogen (Henczka and others 2006). While
this technique results in more stable particles, with a regular size
and employing a 60% less time than traditional freeze-drying; the
cost and the complexity of the process are currently prohibitive
unless used for very-high-added-value products.
Blood Proteins and Derivatives with Functional
Properties
Functionality
Research on the functionality of blood proteins has been
carried out since the 1970s, and their excellent technofunctional
properties have been shown to be due to the combined effects of
the 3 main protein types: serum albumin, globulins, and fibrino-
gen. Whole blood and cellular proteins are limited for use as food
ingredients due to the unpalatable flavor and color they impart to
final foods. The plasma fraction has more desirable characteristics
in that it has little color or flavor, making it easier to incorporate
into foods without altering the sensory properties of the end
product. For this reason, plasma has gained more attention and
use (Tarte 2011). The main functional properties and, hence, uses
of plasma proteins are as emulsifying and binding agents, gelation
under thermal treatment, and as solubility, foaming, or leavening
agents (Etheridge and others 1981; Tarte 2011; Parés and others
2014). Hence, the use of blood proteins as ingredients in the
formulation of processed foods is an obvious choice for adding
value to these products (Lawrie and Ledward 1988). Functional
properties affect the sensory characteristics of food, but they can
also influence its physical behavior during preparation, transfor-
mation, or storage (Ramos-Clamont and others 2003; Silva and
Silvestre 2003). The main desirable functional properties and im-
pact of processing conditions on blood proteins are summarized in
Table 6.
Many publications focus on assessing protein functionality at
a fundamental level, as, for example, testing of pure protein so-
lutions or very simple ones (salt buffer or water/oil mixtures).
While the definitive understanding of protein functionality must
be arrived at through inclusion in an accepted model system or in
the final product, these observations give us valuable insight into
downstream applications for the protein (Hettiarachchy and others
2012). Table 7 shows the potential application of blood proteins
based on the functionality displayed at a fundamental level. Fur-
ther information about meat and nonmeat usage of blood proteins
can be found in a bibliography by Ofori and Hsieh (2012). Once
assessed at a fundamental level, testing in a real food system is es-
sential as processing conditions and other ingredients can interact
to modify protein functionality (Foegeding and Davis 2011; Kudre
and others 2013; Arfat and others 2014; Heertje 2014; Intarasiri-
sawat and others 2014; Rocha and others 2014). This section will
mainly focus on technofunctional properties of isolated blood pro-
teins as few studies have been published on real food systems (Polo

338 Comprehensive Reviews in Food Science and Food Safety r Vol. 16, 2017 
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Blood processing and food applications . . .

Table 7–Potential application of blood proteins in several foodstuffs according to requirements.

Food Functional property required Optimal blood protein

Salad dressing Emulsifying, viscosity Fibrinogen, demineralized plasma,
conjugated globulins, conjugated
hemoglobin, red cells
Meat products Water absorption, gelation, fat absorption, Plasma, globulins, fibrinogen, conjugated
emulsifying hemoglobin
Cakes and pastries Foaming, color, fat absorption Red globin, fibrinogen, plasma, hemoglobin
Biscuits Solubility Plasma, globulins, albumin, hemoglobin
Yogurt Solubility, gelation, emulsifying Plasma, globulins, albumin, hemoglobin
Beverages Solubility Plasma, globulins, albumin, hemoglobin
Ice cream Emulsification, foaming Fibrinogen, demineralized plasma,
conjugated globulins
Simulated meats Fiber formation Fibrinogen,
Protein shakes Foaming, solubility Plasma, yellow globin
Gelatin, gels Gelation Globulins, fibrinogen, albumin
Soups and gravies Solubility, gelation, viscosity Fibrinogen, demineralized plasma,
conjugated globulins, conjugated
hemoglobin, red cells
Feeding formula Solubility Plasma, globulins, albumin, hemoglobin
Animal feed Nutritive, Plasma, red cells, albumin, hemoglobin,
globin.
Pet food Palatability Plasma, red cells, hemoglobin, globin.
Adapted from Hettiarachchy and others (2012).

and others 2005; Polo and others 2009; Hurtado and others 2011; Whole hemoglobin is soluble across a broad pH range. As the
Pereira and others 2014; Fontes and others 2015). preparation of hemoglobin does not involve thermal, chemical,
or aggressive processes that can alter its structure, its solubility is
Processing influence on functionality in general very good. Globin, however, which must be separated
Assuming the product meets all legislative and regulatory re- from the heme group, is more susceptible to denaturation than
quirements, plasma can be used directly as a food ingredient and hemoglobin, and hence, tends to be less soluble (Autio and others
only requires stabilization by using concentration or drying, for 1984). Solubility of plasma proteins is only mildly dependent on
example. While, as discussed above, drying can negatively impact pH, displaying a high solubility over the pH range characteristic
functionality, it has been reported that ultrafiltration and freeze- for processed meat products (pH from 4 to 7), making them ideal
drying can affect positively the functionality of plasma, in par- for inclusion in formulations (Tybor and others 1973; Parés and
ticular solubility, emulsification capacity, and foam stability (Del others 2011). Plasma proteins isolated by the Cohn method have a
Hoyo and others 2008; Furlán and others 2010; Parés and others solubility of >50% over a pH of range 3 to 8, with the exception
2014). Adding salt can also improve functional properties such of fibrinogen that has lower solubility (>20%). Albumin, the most
as gelation and foaming (Oshodi and Ojokan 1997). If plasma is abundant plasma protein, has an isoelectric point of 4.8 to 5.7
fractionated by ethanol precipitation, the proteins are reversibly depending on the attachment of fatty acids to the molecule (Fort
unfolded and the isolated fractions shown better functional prop- 2010), and it is very soluble in aqueous solutions and reasonably
erties than those reported for whole plasma (Álvarez and others soluble in salts solution. The solubility of porcine albumin is
2009). The functionality of freeze-dried serum and globin proteins negatively affected within the pH range of 4 to 6, which may
is improved by addition of lactose before freeze-drying; such effect be a result of polymerization that the protein exhibits in an acid
is more pronounced for serum proteins (Tybor and others 1973). medium, or because the isoelectric point of the protein. For
The influence of discoloring conditions on protein functionality globulins, the minimum solubility is seen at pH 4; beyond this
was described above. pH value, the overall solubility of plasma protein is between 80%
and 100%, with the maximum at pH 3 (Álvarez and others 2009).
Solubility
High solubility is desired for protein application in numerous
food formulations. Solubility is the ability of a protein to be dis- Gelling properties
solved and to form a homogeneous solution of the solute in the Protein gels are defined as structures in which polymer–polymer
solvent. As pH varies across food products, solubility of proteins and polymer–solvent interactions occur in an ordered manner re-
is an important prerequisite and indicator for protein functionality sulting in the immobilization of large amounts of liquid by a
(Etheridge and others 1981). Solubility is highly affected by pH small amount of protein. Cross-linking is essential for gel forma-
changes, and it is usually the most affected functional property tion; which together with the fluidity of the immobilized solvent
due to processing, hence impacting on other functionalities (Ty- gives gels their characteristic elasticity, flow behavior, and strength.
bor and others 1973). Solubility is highly dependent on a protein’s Important characteristics of a gel include water-holding capacity
3-dimensional structure and it can be nonreversibly modified by (WHC), the least gel concentration (LGC), as well as the rheo-
chaotropic reagents (ethanol, acetone, urea, strong acids, and al- logical behavior of formed gels. Testing a protein’s gelling quali-
kalis), high pressures, and extreme temperatures (Smeller 2002). ties provides useful information regarding texture and palatability
When protein solubility is plotted against pH, the graph has a U- (Kinsella 1979). Due to their good gelling properties, blood pro-
shape; the lower solubility corresponds to the isoelectric pH or iso- teins have been used extensively as binders in the meat industry;
electric point (pI), which is the pH value where protein molecules they can be used as an alternative to the traditionally used egg albu-
have no net charge, thus facilitating protein–protein interaction men (Hickson and others 1980); although plasma powder showed
causing the protein to aggregate, which means reduced solubility. a lower binding capacity.


C 2017 Institute of Food Technologists® Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 339
Blood processing and food applications . . .
Plasma proteins exhibit both excellent gelling and water-
binding capacity, properties that are used to improve the texture,
sliceability, and yield losses of processed meat products (Wismer-
Pedersen 1988; Parés and others 2014). For example, plasma
has been used as a phosphate substitutive in frankfurter-type
sausages (Hurtado and others 2011). The final product had
improved WHC, lower fat, and higher protein content compared
to controls. While hardness and chewiness were slightly higher,
they were still within an acceptable range.
Globin gels are softer than plasma gels; however, they have a
greater capacity to bind water (Autio and others 1985). WHC
of globin is reduced with the addition of NaCl, as salt enhances
the tendency of the protein to form random aggregates (Autio
and others 1984; Bah and others 2013). Bovine globin has bet-
ter gelling capacity than porcine globin prepared by the same
method; also, the WHC of bovine globin is higher than porcine
globin (Autio and others 1985). Globin recovered using CMC
was found to have superior gelling ability when compared to en-
zyme hydrolyzed globin, because peptides are less able to gener-
ate sufficient protein–protein interaction to form a 3-dimensional
structure (Yang and Lin 1998).
Emulsifying properties
An emulsion is defined as a 2-phase system, consisting of at least
1 immiscible liquid, intimately dispersed in another, in the form of
droplets. These are thermodynamically unstable and tend to col-
lapse. Proteins, as amphiphilic molecules, are used to stabilize food
emulsions by reducing interfacial tension (O’Riordan and others
1989). Emulsifying capacity is usually determined by measuring
the amount of emulsified oil per gram of protein employed. The
response of emulsifiers in foods is complex and is influenced by
many variables (Tybor and others 1973).
The globulins have high emulsion capacity, with γ -globulins
showing a better performance than α- and β-globulins, possibly
due to the increased hydrophobic regions available for interact-
ing with the oil phase of the emulsion. The emulsifying capacity
of fibrinogen reached 100% at all concentrations; this is note-
worthy considering the low solubility of this protein. This may
be due to the structure of the protein that contains 29 disul-
fide bonds (Allen and others 1977) capable of hydrophobic in-
teractions. Strong emulsifying properties are observed for whole
plasma, with fibrinogen and globulin appearing to have a greater
affect than albumins (Álvarez and others 2009). For some proteins,
especially albumin and globulins, there can be a reduction in emul-
sifying capacity with increases in concentration. This may be due
to an increase in protein–protein interactions, leading to an ag-
gregation process. Such protein–protein interactions are stronger
than hydrophobic or hydrophilic interactions, which are required
to stabilize the emulsion, thus less protein is available to form the
emulsion. While porcine albumin is very soluble, its emulsifying
capacity is low (emulsifies 25% to 30% of added oil) (Pearce and
Kinsella 1978). The low performance may be due to the globu-
lar structure of albumin, which prevents its hydrophobic residues,
buried in the center of the molecule, from interacting with the
oil phase (Álvarez and others 2009). However, addition of dried
serum albumin into processed meat products serves to stabilize
the fat content of the products. In some cases, the albumin and
fat are processed separately before addition to the product, in this
way, the fat is added in the form of a stabilized emulsion that can
be added at any stage in the processing and still remains stable
(Halliday 1973).
340 Comprehensive Reviews in Food Science and Food Safety r Vol. 16, 2017
For hemoglobin and its derivatives, red globin and yellow
globin, the emulsifying capacity increases with an increase in pro-
tein concentration. Globin has an emulsifying capacity equal to
or greater than hemoglobin for concentrations of 2 to 6 g/L, this
may be due to the structure of the globin. As the heme moiety,
which usually resides in the hydrophobic pockets of the protein,
has been removed, these hydrophobic residues may be available
for interaction with the oil phase of the emulsion. The reduction
of emulsifying capacity, when globin is compared to discolored
globin, is an indication of the effect of the discoloration pro-
cess had on the protein functionality and structure. The protein
is denatured and so its ability to bind to fat is lowered (Álvarez
and others 2009). However, a very good emulsifying ability was
reported for discolored globin prepared by ionic exchange chro-
matography, probably due to a low denaturation degree (Yang and
Lin 2000). Other authors reported that globin isolated by means
of acidified acetone or CMC performed better than sodium ca-
seinate (a common food industry emulsifier) in the absence of salt;
however, in the presence of salt, caseinate was superior (Silva and
Silvestre 2003). When globin is compared to ovalbumin or whole
hemoglobin, its emulsion capacity is remarkably higher; however,
in its isoelectric zone, globin is not a good emulsifier, because of
the aggregates formed (Nakamura and others 1984).
Foaming
Foam may be defined as a 2-phase system in which a distinct
gas bubble phase is surrounded by a continuous liquid lamellar
phase and is characterized by high viscosity. The foaming capacity
of a protein is the amount of foam formed per unit of solution
volume; it represents the interaction between the protein solution
and the air. To maintain the foam surface, agents are needed. The
stability of the foam is also an important factor, and is determined
by the length of time the foam maintains its volume. Foams are
inherently less stable than emulsions, because unlike emulsions
the volume occupied by the dispersed phase (air) is greater and
there is a higher interfacial surface (Penteado and others 1979).
Protein foams exhibit good stability close to pI. It is important to
note that foaming may be a desired (as in some food products as
meringues) or undesirable (excessive foaming during processing)
characteristics.
Blood proteins are able to create notable volumes of foam, but
these are not stable over time. Freeze-dried red cells produced 4
times the foam volume compared to the nonfreeze-dried (Toldrà
and others 2004), although the foam destabilizes rapidly. When
compared with egg albumen, plasma is able to generate the same
volume of foam; however, more stable foams are formed using egg
proteins (Tybor and others 1975). When compared with casein,
or whey and soya isolates, blood proteins are able to create more
stable foams and higher volumes (O’Riordan and others 1989).
Modification of functional properties
Food proteins do not necessarily possess the required func-
tional properties when they are in their native form. Structural
modifications, by physical, chemical, or enzymatic means, are fre-
quently used to improve functionality (Hettiarachchy and others
2012). Moreover, there is growing interest in modifying proteins
for industrial food applications. Most common modifications are
acylation, alkylation, phosphorylation, cross-linking, deamidation,
and glycosylation. Glycosylation is the preferred mechanism as it
occurs naturally under controlled conditions of temperature, time,
pH, and moisture, and it does not require addition of chemicals.
Such modifications have been widely studied on vegetable proteins

C 2017 Institute of Food Technologists®
Blood processing and food applications . . .
as those from faba bean, pea, or corn; and animal proteins such as
milk, fish, or egg proteins (Stanic-Vucinic and others 2013; Chen
and others 2014; de Oliveira and others 2014; Perusko and oth-
ers 2015). It has been reported that solubility can be remarkably
improved, and hence, thereby improve other properties such as
textural (gelling ability), foaming, and stability (Oliver and others
2006).
Regarding blood proteins, research in this field has been lim-
ited. It was reported that plasma conjugated with galactomannan
(Matsudomi and others 1995) displayed better solubility and en-
hanced emulsifying ability than native plasma, and emulsions
created 10 times more stable than those made using unmodi-
fied plasma. Dextran (high-molecular-weight carbohydrate) was
used to modify ethyl-fractionated plasma proteins and hemoglobin
(Álvarez and others 2012a); with results showing improved emul-
sion ability, better thermal stability, and higher temperature gela-
tion. However, glycated proteins had a remarkably lower solubility.
Oliver and others (2006) published an excellent review on protein
modification by the Maillard reaction in which it was reported
that only very specific degrees of glycation produced a better pro-
tein solubility influenced by variable (inherent) properties of the
native protein. Recently, it has been reported that the structural
changes in pure BSA after dextran conjugation protect BSA from
denaturation and aggregation during dry-heating (Xia and others
2015).
Chemical modification of globin was investigated by Yang and
Lin (2000) who reported that succinylated globin was 50% more
soluble than the control. On the other hand, acetylated globin
presented a lower solubility when compared with the control.
The succinylated proteins showed an improvement for emulsify-
ing activity and foam capacity at neutral pH. Emulsifying ability
of globin was improved after acetylation in the isoelectric zone
(pH ࣈ7), unlike in the acidic zone where it was 50% lower than
native protein (Nakamura and others 1984). The same authors
reported that globin digested with pepsin in the presence of 1.0%
CMC displayed improved emulsion ability and stability in the
acidic range.
Future Challenges for the Blood Protein Industry
Currently, major efforts are being carried out in food waste re-
covery from pulses, seeds, and the dairy industry (Galanakis 2012).
In spite of the high nutritional value and potential high added-
value opportunities for blood, it remains an underutilized prod-
uct. It has been estimated in 1999 that only 15% to 20% of blood
produced in slaughterhouses was destined for the food industry
(Gómez-Juárez and others 1999), while in 2001, 30% was used,
another 30% of blood was also used in the pet food sector (Gatnau
and others 2001). To the best of our knowledge, no real estima-
tion of blood usage has been reported recently however. While
blood can be used for higher end applications such as diagnostics
and medical sectors, it is likely that a high volume is currently
used for low added-value products. Of course, there are consumer
concerns that need to be taken into consideration with regard
to use of animal blood. According to Ofori and Hsieh (2012), 3
main factors are concerning the consumer: fraudulent usage, reli-
gious and ethical reasons, and health reasons. Strong legislation is
in place (European Directive 1333/2008 regarding food additives)
to prevent fraudulent use of blood and ensure clear labeling of
ingredients. This EU legislation also protects the consumer from
a health and safety perspective. Finally, clear guidelines exist to
instruct nonusage of blood for food purposes according to Halal
and Kosher dietary laws (Regenstein and others 2003).

C 2017 Institute of Food Technologists® Vol. 16, 2017 r Comprehensive Reviews in Food Science and Food Safety 341
More efficient and higher value use of blood proteins faces
several challenges that science and technology need to address.
These challenges somewhat match the processing steps as dis-
cussed in this review. (I) More efficient and easy-to-use tech-
nologies for hygienic blood collection are required. (II) Improved
(such as low hemoglobin content and high protein content) pro-
cesses for industrial-scale plasma production. (III) Greener, more
feasible, and more sustainable processes for protein separation and
purification need to be developed. Current processes are based on
the use of organic solvents, ethyl alcohol fractionation, or chro-
matographic techniques. (IV) Efficient and effective discoloring
of hemoglobin. (V) A better understanding of how novel pro-
cesses, such as ultrasound, pulse electric fields, cold atmospheric
plasma, or high pressures can affect protein functional proper-
ties is required (Saravacos and Kostaropoulos 2016). (VI) Build a
knowledge database on functionality in real food systems and how
the common food processing procedures affect their final perfor-
mance. (VII) Development of novel products, based on formu-
lations that include blood proteins, targeting consumer demands
for “healthy,” protein rich, and tasty products. (VIII) In depth
consumer studies can be applied to evaluate consumer perception
and acceptance of blood and blood derived products to develop a
suitable strategy to promote the excellent nutritive properties of
these components. (IX) Application of blood proteins in the de-
velopment of new biomaterials such as biofilms or food coatings,
and focusing on good rheological and emulsifying properties to be
imparted in carriers or encapsulants used for delivery of bioactive
compounds such as antioxidants or antimicrobials.
Conclusions
This review presents the current state of the art for blood col-
lection, processing, and downstream purification and modification
techniques to generate functional blood proteins for use in food ap-
plications. The influences of processing conditions on the protein
characteristics and functionalities are presented. Blood represents a
key resource for extracting additional value from the meat sector.
While there are some examples of it being utilized in an optimal
fashion, a concerted approach is lacking across the sector. Current
legal and political frameworks, along with new funding initiatives
focused on waste reduction, are helping to set an encouraging sce-
nario for more research groups and food companies to join efforts
and invest time and resources on improved blood processing. Con-
sidering the need for implementing more sustainable and higher
value practices, coupled with the global protein demand, many
opportunities can be exploited by the sector through focusing on
blood proteins.
Acknowledgments
This work forms part of the ReValueProtein Research Project
(Exploration of Irish meat processing streams for recovery of high
value protein based ingredients for food and nonfood uses, Grant
Award nr 11/F/043) supported by the Dept. of Agriculture, Food
and the Marine (DAFM) under the National Development Plan
2007-2013 funded by the Irish Government.
The authors declare no conflict of interest with the information
shared in this work.
Author contributions
Sarah Lynch drafted sections Nutritive Value, Blood Collec-
tion, Processing of Red Cell Fraction, and Processing for Stor-
age. Eileen O’Neill reviewed the drafted manuscript and aided in
Blood processing and food applications . . .

drafting all sections of the manuscript. Anne Maria Mullen re- Denizli A. 2011. Plasma fractionation: conventional and chromatographic
viewed the drafted manuscript, drafted Introduction, section Fu- methods for albumin purification. Hacettepe J Biol Chem 39(4):
315–41.
ture Challenges, and Conclusions. Carlos Álvarez reviewed drafted
Di Bernardini R, Harnedy P, Bolton D, Kerry J, O’Neill E, Mullen AM,
manuscript, drafted Introduction, sections Plasma Fractionation, Hayes M. 2011. Antioxidant and antimicrobial peptidic hydrolysates
Functional Properties, Future Challenges, and Conclusions. from muscle protein sources and by-products. Food Chem 124(4):1296–
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