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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

 Slaughterhouse blood is an inevitable part of the meat production food chain and represents a rich source of protein. The physicochemical characteristics and utilization of animal blood in various food and industrial applications has been well explored. However, in recent years much attention has been paid to the generation of peptides with biological activities from food by-products including blood. This review examines the angiotensin-converting enzyme inhibitory, antioxidant, antimicrobial, and other bioactive peptides derived from various slaughterhouse animal blood sources. Furthermore, the effect of enzyme choice, degree of hydrolysis, and peptide sequence or size on the potency of these bioactivity is discussed.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

In the production of food for human consumption, the generation of by-products and waste is an integral part of the production chain. In meat production, the nature and quantity of these materials varies at each stage of production. The products from animals slaughtered for meat may be divided into 4 categories, the first being meat (high-value product), the second is inedible components which can be used for industrial purposes (for example, hides, bones, hooves, and blood), the third is low-value components (offal and meat meal), and, last, items of no useful purpose (for example wool slip, digestive tract content, effluents) which are disposed of as waste (Fallows and Verner Wheelock 1982).

Blood is an inevitable by-product of the meat industry representing up to 4% of the live animal weight or 6% to 7% of the lean meat content of the carcass (Wismer-Pedersen 1988). Blood contains a number of compounds, which have potential commercial value and represents a valuable source of protein (Piot and others 1986). Tons of blood are collected in abattoirs each year, that is either processed into blood meal and sold as low-value animal food and fertilizer or discarded as effluent (Anderson and Yu 2003; Yu and others 2010). Animal blood produced in slaughterhouses represents a problematic by-product of the meat industry due to the high volumes generated and its very high pollutant load when discarded directly into the environment (Del Hoyo and others 2008).

The use of blood and its derivatives in various industries has been explored to some extent. For example, plasma proteins are used as emulsifiers and whole blood is used in some traditional products such as black pudding. It is estimated that the food industry utilizes about 30% of the blood produced from slaughter (Gatnau and others 2001). However, issues surrounding the biological safety of blood collected from slaughtered animals (for example, the transmission of spongiform encephalopathies) have been raised (Hsieh and Ofori 2011). Religious constraints as well as negative consumer perception of blood for direct consumption have also contributed to its limited use in food applications. This has resulted in a search for alternative ways to use slaughterhouse blood, including the extraction of bioactive peptides.

Ockerman and Hansen (2000) suggested that human usage of animal blood may increase as worldwide protein deficiency increases and, therefore, sources of animal protein, such as blood, should be investigated now. The meat industry has been facing the task of better utilization of all slaughter products including blood (Gorbatov 1988). Finding new applications for blood components represents an important challenge for scientists (Silva and Silvestre 2003) and it is necessary to develop procedures and applications that will permit the utilization of animal blood on a larger scale, both to eliminate a sizable pollution hazard and prevent the loss of a potentially valuable material (Hyun and Shin 1998). As proteins constitute one of the main components of blood (Ockerman and Hansen 2000), the possibility of recovering proteins has been given considerable attention (Del Hoyo and others 2008). More recently, the recovery and extraction of bioactive compounds from blood, after suitable collection, has been seen as an opportunity to add economic value and generate new applications for slaughterhouse blood.

General Physicochemical Characteristics of Blood

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

Blood is a red fluid, which is made up of water, cells, enzymes, proteins, and other organic and inorganic substances that can be separated into 2 fractions, the cellular fraction and plasma. The cellular fraction corresponds to 30% to 40% of blood wet weight and is dispersed within the liquid fraction, which is known as the plasma (which comprises up to 60%). The most important cellular elements are red corpuscles (erythrocytes), white corpuscles (leukocytes), and platelets. The granular white blood cells or leukocytes exist as 3 types based on their differently staining granules: neutrophil, eosinophil, and basophil. Hemoglobin is the major protein constituent of red blood cells, having a molecular weight as a tetramer of 68 kDa and consisting of 4 separate polypeptides known as globins, of which the α- and β-chains are arranged in a spherical structure (Wismer-Pedersen 1988). An iron-containing heme group is located within each globin which can bind 1 molecule of oxygen, making a hemoglobin tetramer molecule capable of transporting 4 molecules of oxygen altogether.

Generally, 2 methods can be used to separate whole blood into fractions. These are either by centrifugation resulting in red cells and plasma or by allowing blood to clot in order to separate the red cells from the serum. Blood has a natural tendency to clot but the addition of an anticoagulant such as sodium citrate will prevent clotting. This happens when the citrate ions chelate calcium ions in the blood by forming calcium citrate complexes, thus disrupting the blood clotting mechanism. Other anticoagulants commonly used to prevent coagulation are heparin and ethylenediaminetetraacetic acid (EDTA).

Plasma, the part of the blood remaining after removal of the cells from unclotted blood, contains 6% to 8% proteins, consisting primarily of albumin, globulins, and fibrinogen. These, as well as more than 100 smaller proteins, have been well characterized. Some blood proteins and their molecular weights (based on human blood) are listed in Table 1.

Table 1. Proteins from human blood and some properties
 Molecular weight  
Protein(kDa)StructurepI
  1. Sources: Hsieh and Ofori (2011), Saguer and others (2012), Wismer-Pedersen (1988), Betgovargez and others (2005), Frost and others (2000), and Chung (1984).

Albumin66Globular protein∼4.8
Fibrinogen341Glycoprotein consisting of 2 identical subunits. Each one is composed of 3 nonidentical polypeptide chains (Aα, Bβ, and γ), held together by 29 disulfide bonds.∼5.5
Immunoglobulin M800 to 950Polymers where multiple immunoglobulins are covalently linked together with disulfide bonds 
Immunoglobulin G150Symmetrical Y-shaped structure composed of 2 heavy chains and 2 light chains, held together by both disulfide bonds and noncovalent interactions∼7 to 9.5
Hemoglobin68Four separate polypeptides known as globins, of which α- and β-chains are arranged in a spherical structure∼6.8
Prothrombin72Glycoprotein∼6.6
Transferrin80Iron binding monomeric glycoprotein∼6.3 to 6.4

Serum is qualitatively different from plasma in that the bulk of the fibrinogen has been removed by conversion into a fibrin clot together with the platelets which have either been physically bound in the fibrin matrix, or activated to form aggregates, or both. Varying amounts of other proteins are removed into the fibrin clot either by specific or nonspecific interactions (Lundblad 2005).

Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

Blood is maintained relatively constant in composition due to the activity of the excretory systems and its composition of hormones, enzymes, and other biologically active substances. Some differences do exist among different animal species. For example, while the erythrocytes of the majority of farm animals such as sheep, cattle, and pigs are round, erythrocytes have an oval shape in deer and camel blood (Gorbatov 1988). The number of erythrocytes also varies with cattle and pigs having 6 to 8 million/mL3, sheep 6 to 11 million/mL3, and goats 14 to 18 million/mL3. Table 2 summarizes the main constituents of blood from different animal species.

Table 2. Blood constituents from commonly farmed animals
 Content of blood (%)Levels of protein constituents in whole blood (%)
Animal speciesPlasmaCellular elementsaAlbuminα-globulinsβ-globulinsγ-globulinsFibrinogenHemoglobin
  1. 1

    Gorbatov (1988), 2White and Cook (1974), 3Youatt (1965).

  2. a

    Cellular elements represent red blood cells, white blood cells, platelets.

  3. N/A, data not available.

Cattle167.4532.553.610.510.530.630.610.3
Pigs156.4943.513.830.590.490.950.6514.2
Sheep172.0023.003.830.190.500.830.469.3
Deer2,350.5049.503.800.290.470.70N/A10.2

The properties of erythrocytes (weight and coalescence) affect their precipitation and separation from the cellular elements. Blood from pigs separates comparatively quickly in contrast to cattle and sheep blood (Gorbatov 1988). The plasma content of animal blood also varies, with sheep blood reported to have the highest plasma content (Table 2). Globulins in plasma can be divided into alpha 1, alpha 2, beta, and gamma globulins. The most significant group of gamma globulins is the immunoglobulins (IgGs).

Species effects, geographical altitude, and maturity of the animal can contribute to the differences reported for blood hematological parameters. Looking at deer blood as an example, we can see that in different species of deer (fallow, red, Colorado mule), hemoglobin levels can range from 13.2 to 18.9 g/dL (Table 3). Free-ranging red deer had a lower number of red blood cells (8.8 ± 0.9 × 1012/L) and white blood cells (2.2 ± 0.6 × 109/L) when compared to the other species of deer.

Table 3. Hematological parameters in different species of deer
ParameterFallow deer (Dama dama)Red deer (Cervus elaphus)Colorado mule deer (Odocoileus hemionus)
  1. 1(Agar and Godwin 1992),

  2. 2(Anderson and others 1970),

  3. 3(Padilla and others 2000)—Red deer at high altitudes,

  4. 4(Rosef and others 2010)—immobilized free ranging red deer.

  5. N/A, data not available.

Packed cell volume (%)47.1 ± 0.9145.6 ± 2.81146.7 ± 0.62
  35 ± 3.004 
Hemoglobin (g/dL)17.2 ± 0.3118.9 ± 0.97116.4 ± 0.32
  13.16 ± 1.164 
Red blood cell (number × 106/μL)9.91 ± 0.1019.67 ± 0.5119.2 ± 0.22
  8.8 ± 0.94 
Mean cell volume (f/L)47.5 ± 0.63147.0 ± 0.46152.8 ± 0.92
  39.7 ± 4.14 
Mean cell hemoglobin17.4 ± 0.2119.6 ± 0.411N/A
Mean cell hemoglobin concentration (g/dL)36.6 ± 0.8141.8 ± 1.12135.1 ± 0.22
  38.13 ± 8.54 
Leukocytes (109/L)N/A4.8 ± 1.3833.0 ± 0.12
  2.2 ± 0.64 
NeutrophilN/A40.07 ± 8.01340.6 ± 1.22
  42.27 ± 22.274 
Band Neutrophil (%)N/A0.44 ± 1.1531.1 ± 0.22
Lymphocyte (%)N/A48.13 ± 7.83343.4 ± 1.12
  49.09 ± 13.644 
Monocyte (%)N/A5.13 ± 2.7036.2 ± 0.452
Eosinophil (%)N/A2.94 ± 2.5738.3 ± 0.612
Basophil (%)N/A3.12 ± 2.2830.4 ± 0.072

Slaughterhouse Animal Blood Collection and Separation

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

Blood from slaughterhouse animals can be collected by 2 methods. The first, more primitive method, is open draining where blood from the animal is drained into buckets or trays. This method is more susceptible to contamination and blood collected this way is unlikely to be suitable for food applications. Blood, which is to be used for food applications has to come with a guarantee that it is sourced from veterinary-approved disease-free animals. In alive and healthy animals, blood is “sterile” (Dàvila Ribot 2007), in the sense that it can be consumed. However, contamination can occur during the blood collection process. Because of its high nutritional value, blood is particularly susceptible to bacterial growth.

The 2nd method is via a closed draining system, where blood from the slaughterhouse animal is not exposed to air and is drained directly from the body of the animal; for example, using a hollow knife connected to vacuum piping. However, this method can be more costly and slows down any slaughtering line speed (Dàvila Ribot 2007).

Recoverable blood could constitute up to 7% (carcass weight) of most animals (Wismer-Pedersen 1988). With the right collection procedures, this blood component can be used as raw material for products for human consumption as well as for technical products. Rigorous control of the temperature of the raw blood and plasma is necessary in order to ensure an end product that meets the exceptionally stringent requirements associated with food-quality ingredients. Many companies provide specialized equipment and systems for separating blood into high-grade plasma and hemoglobin, as well as specialized equipment for turning blood into feed protein products. Edible proteins must be processed with the utmost care if they are to be sold as a high-value item. Such processing must be configured to ensure gentle treatment that minimizes any shear effects that might rupture the red blood cells and contaminate plasma.

According to the FAOSTAT (2012) website, approximately 304 million cattle, 959 million sheep and goats, and 1374 million pigs were processed worldwide for their meat in 2010. Assuming a recovery of 15 L of blood from each head of cattle and 2 to 3 L per pig (Fallows and Verner Wheelock 1982), this would amount to huge volumes of blood (for example, cattle—4.56 billion L of blood), which represents a substantial resource and an interesting future opportunity for development. Blood from slaughterhouse animals is a better source when compared to donor animals in terms of volumes being generated. Utilizing slaughterhouse blood in novel ways, such as the production of bioactive peptides (Figure 1), may help in reducing blood discharge and pollution of the environment.

image

Figure 1. Animal blood collection and separation into usable fractions.

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Animal Blood in Human Food Applications

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

Blood is a good source of nutrients suitable for human use. Anecdotal evidence of tribes drinking animal blood (for example, the Maasai in Africa) suggests the potential use of blood for sustenance. As mentioned previously, blood collected at abattoirs can be a valuable by-product if appropriately collected under hygienic conditions. The use of blood or blood components in food products to utilize the benefits from its nutritional value and functional properties is described in Table 4.

Table 4. Examples of animal blood use in food applications
Blood fractionFunctionFood productReference
Whole animal bloodProtein sourceSpanish blood sausageSantos and others 2003
Bovine hemoglobinIron fortificationCookiesWalter and others 1993
Bovine globin and plasmaFat replacerHam pâtéViana and others 2005
Animal plasmaFat replacerBologna (fermented) sausageCofrades and others 2000
Porcine transglutaminase, fibrinogen, thrombinBinderRestructured meat productsTseng and others 2006
Porcine plasmaProtease inhibitorSurimiVisessanguan and others 2000
Chicken plasmaProtease inhibitorSurimiRawdkuen and others 2004
Bovine plasmaEgg white replacerCakesLee and others 1991
   Myhara and Kruger 1998
Processed bovine plasmaEmulsifier, StabilizerMinced meatsFurlán and others 2010

Several societies have utilized whole blood, as a source of protein, in products such as blood sausages, black puddings, or blood tofu. Several studies have also looked at using blood or its derivatives in other functional forms in food applications (Table 4). For example, Walter and others (1993) incorporated 6% bovine hemoglobin in cookies in a successful attempt to fortify the diets of schoolchildren with iron. The iron status of the children improved and the acceptability of the cookies was good (cookies had a chocolate flavor added to them and were indistinguishable from regular chocolate cookies).

Lee and others (1991) reported that nearly equivalent cake quality (firmness, cohesiveness, and color) was achieved by replacing egg whites with bovine plasma (in a 1 to 1.1 ratio). However, conflicting sensory results were reported, depending on the source of plasma protein and the method used to obtain it. For example, while Lee and others (1993) reported a 100% likeness for their plasma cakes, the majority of a sensory panel (64%) ascribed a strong objectionable flavor toward the cakes which had their egg whites replaced with bovine plasma in another study (Myhara and Kruger 1998).

Plasma proteins from pigs and chickens have also been tested for their use as protease inhibitors in the making of surimi, a form of fish gel (Visessanguan and others 2000). Also, a combination of porcine transglutaminase, fibrinogen, and thrombin has been used as a binder in restructured meat products (Tseng and others 2006).

Several companies have commercialized food-grade proteins from animal blood. ImmunoLin®, produced by Proliant Inc., Boone, Iowa U.S.A., is a serum concentrate from bovine plasma advertised as containing immunoglobulins, transferrin, mitogenic growth factors, and immune-regulating cytokine, which can be added to bars and drinks with the intention to boost the immune system. Fibrimex® is a combination of thrombin and fibrinogen concentrate precipitated from bovine plasma, manufactured by Sonac B.V, The Netherlands. The product can be used for the bonding of muscle tissue. Some controversy has arisen recently over the use of binding agents such as this, which have been labeled as “meat glue.” Sonac (a part of Vion Ingredients in The Netherlands) also produces Harimix P, P+, and C proteins, which are hemoglobin powders derived from bovine, porcine, and hemoglobin liquid from bovine and porcine blood. These products can be used as a natural meat colorant. Licán, a company operating in Brazil, Chile, and Paraguay, produces Prolican 70 (spray-dried bovine plasma) for emulsifying, gelling, and binding, Prietin (spray-dried porcine whole blood) for blood sausages, and Myored (red pigments from blood) for use as a natural colorant. Similarly, the Veos Group in Belgium markets a range of products under the Vepro® brand utilizing plasma and hemoglobin from animal blood for various food applications.

Animal Blood in Other Applications

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

Apart from the food applications mentioned above, animal blood has also been used in other industrial applications (Table 5). For example, whole blood from cow, sheep, pig, or chicken has been added to pet food as a cheap protein source, while, on the other side of the scale, compounds with higher value medical applications such as thrombin (EC 3.4.21.5) and plasmin have been recovered from animal blood.

Table 5. A summary of medical and industrial applications of animal blood
Blood sourceSectorFunction/useReference
Cow, sheep, pig, or chicken whole bloodPet foodProtein sourcePatent US4089978
Spray-dried bovine and porcine plasmaAnimal feedEnhancement of growth rate and feed intakePierce and others 2005
Porcine plasmaAnimal feedProtein source for weaned pigletsProglobulin® produced by Sonac (Vion Ingredients Group)
HemoglobinAquatic feedFood source for carnivorous fish or shrimpHemoglobin in aqua feed produced by Sonac (Vion Ingredients Group)
HemoglobinPet foodCattle, poultry, piglet, fish, and pet foodActipro® produced by the Veos Group
PlasmaAnimal feed  
Globin   
Bovine plasma albuminLaboratoryImmunohematologyTanaka and others 2001
  Testing for Rh factor in humansJayathilakan and others 2011
Porcine plasmin enzymeMedicalTo digest fibrin in blood clots in heart attack patientsJayathilakan and others 2011
Bovine thrombinMedicalPromotes blood coagulationJayathilakan and others 2011
  Treatment of wounds 
  Hold skin grafts in place 
Bovine plasmaMicrobiologyMedium for the growth of probiotic bacteria (Lactobacillus sp.)Hyun and Shin 1998
Bovine plasma powderFeed supplementFor nonruminant animalsBovogen Biologicals Pty Ltd (Australia)
Bovine hemoglobin powderPharmaceuticalUse as a raw material for pharmaceutical porphyrin derivative production.Bovogen Biologicals Pty Ltd (Australia)
Bovine fibrinogenMicrobiologyReagent in Baird Parker microbiological media for Staphylococcus aureus identificationBovogen Biologicals Pty Ltd (Australia)
 MedicalReagent for routine blood clotting in serology laboratories 
  Bioengineering scaffolding applications 
Bovine prothrombinMedicalUse as a precursor to thrombin production and purificationBovogen Biologicals Pty Ltd (Australia)
  Active ingredient in topical surgical hemostatic applications 
Abattoir derived: Defibrinated, lysed,Research Lampire Biological Laboratories (USA)
 laked blood   
Whole blood   
Red blood cells from bovine, calves,   
 porcine animals   

Pierce and others (2005) conducted experiments to evaluate the effects of dietary spray-dried porcine plasma (SDPP) and spray-dried bovine plasma (SDBP) and their various fractions on the performance of pigs weaned at approximately 14 or 21 d of age. In addition, the efficacy of various levels of the immunoglobulin G (IgG)-rich fraction of SDPP and SDBP were evaluated. The results indicated that both porcine and bovine plasma were beneficial to young pig performance during the 1st week after weaning and that the IgG fraction of plasma was the component responsible for the enhancement in growth rate and feed intake.

Bioactive Peptides from Food By-products

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

For the purpose of this review, bioactive food compounds are defined as naturally occurring nonessential constituents in or derived from plant, animal, or marine sources, which have the ability to modulate biochemical, physiological, and metabolic processes in the human body while exerting beneficial effects beyond basic nutritional functions (Kris-Etherton and others 2004).

Bioactive compounds include a large array of compounds such as peptides, oligosaccharides, fatty acids, enzymes, water-soluble minerals, and biopolymers. These compounds can be present naturally in food sources or be produced in vivo (gastrointestinal digestion), by industrial enzymatic digestion (food processing activities) or by microbial fermentation (Hernández-Ledesma and others 2011).

Bioactive peptides are short sequences of approximately 2 to 20 amino acids in length that exert physiological benefits. Their bioactivities depend on their amino acid composition and sequence. These peptides can be “encrypted” or inactive within the sequence of the parent proteins and may be released by proteolytic hydrolysis using commercially available enzymes, proteolytic microorganisms, or fermentation methods (Davies and others 2005; Vercruysse and others 2005). After digestion, bioactive peptides can be absorbed from the intestine and enter the blood stream directly, which facilitates their bioavailability in vivo enabling a physiological effect at the target site (Erdmann and others 2008).

A wide range of activities from bioactive peptides derived from food sources has been described, including antimicrobial properties, blood pressure-lowering (ACE-inhibitory) effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption/bioavailability, cyto- or immunomodulatory effects, and opioid activities. Some peptides can be multifunctional and are able to exert more than one effect (Lantz and others 1991; Korhonen and Pihlanto 2003).

Through the control and improvement of physiological functions, bioactive peptides have the potential to provide new therapeutic applications for the prevention or treatment of chronic diseases. Thus, by adding bioactive peptides as components of functional foods or nutraceuticals, bioactive peptides may improve commercial returns (McConnell and others 2001; Erdmann and others 2008). The use of multifunctional peptides from natural sources for therapeutic purposes (for example, those that have anti-inflammatory and antimicrobial activities) may have wider economic benefits, as they would be more appealing to consumers.

Some researchers have highlighted that the ability of bioactive peptides to exert a physiological effect in vivo is dependent on the bioavailability of the peptide. This factor is dependent on the resistance of the bioactive peptide to hydrolysis by peptidases present in both the intestinal tract and blood stream serum, and the ability of the peptides generated to be absorbed across barriers such as the intestinal epithelium. Consequently, in the course of identifying bioactive peptides for the development of food-based nutraceutical products, this fact should be taken into account (Ryan and others 2011). Foltz and others (2010) caution that the stimulatory or inhibitory effects on target proteins in vitro has often been used as the justification to test these compounds directly in vivo. Unfortunately, this research approach has an inherent flaw as the in vitro method tends to neglect the poor absorption, distribution, metabolism, and excretion (ADME) properties of peptides, which may result in low peptide bioavailability. Because peptides usually undergo extensive hydrolysis in the gastrointestinal tract by peptidases present in the stomach, most of them do not actually arrive or reach the absorption stage in the duodenum and jejunum. Therefore, a valid research approach should consider including the demonstration of peptide stability and evaluate its ADME properties before further establishing bioactivity in vivo (Foltz and others 2010).

Bioactives Isolated from Animal Blood

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

An emerging new area, focused on obtaining bioactive peptides from blood fractions, has been developing intensively over the past decade (Wanasundara and others 2002b; Nedjar-Arroume and others 2008; Wei and Chiang 2009; Parés and others 2011; Toldrá and others 2012). Results from several studies, using predominantly bovine and porcine blood, have indicated that bioactive peptides from blood sources have angiotensin I-converting (ACE)-inhibitory activity, antioxidant activity, antimicrobial properties, mineral-binding ability, and opioid activity. These will be described in detail in the following sections.

Angiotensin I-converting enzyme inhibitory peptides

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase (EC 3.4.15.1) that converts an inactive form of the decapeptide, angiotensin I, to a potent vasoconstrictor, octapeptide angiotensin II (Li and others 2004). ACE also inactivates bradykinin, which has a depressor or vasodilatational (widening of blood vessels) action. Through these actions, ACE elevates blood pressure (Figure 2). Therefore, by inhibiting the catalytic action of ACE, the elevation of blood pressure can be suppressed (Arihara and Ohata 2008) and an overall antihypertensive effect can be achieved (Shalaby and others 2006).

image

Figure 2. Blood pressure regulation by angiotensin I-converting enzyme (ACE).

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Various ACE-inhibitory drugs such as captopril, enalapril, alacepril, and lisinopril have been used in the treatment of high blood pressure. However, these synthetic inhibitors have certain side effects, such as cough, taste disturbances, and skin rashes. Serious side effects, such as proteinuria and blood dyscrasias, have been reported, especially when captopril is given in high dosage or to patients with renal failure (The Lancet Editorial 1980). Several peptides derived from food proteins (for example from milk, meat, seafood, and plant hydrolysates) have been shown to exert ACE-inhibitory activity and are considered to be milder and safer than synthetic drugs (Yu and others 2006). Furthermore, the activities reported for natural peptides with ACE-inhibitory activity usually have other bioactivities (multifunctional properties) and are easily absorbed (Lantz and others 1991; Korhonen and Pihlanto 2003; Yu and others 2006; Adje and others 2011b).

Effect of enzymes on the generation of hydrolysates with ACE-inhibitory activity

Although the activities of bioactive peptides in the sequences of the parent proteins are latent, they can be released by proteolytic enzymes. In this aspect, meat (and also blood) proteins have possible bioactivities beyond a nutritional source of amino acids alone (Arihara and Ohata 2008).

Proteolytic enzymes hydrolyze the peptide linkage between amino acids of proteins, yielding a mixture of peptides of different molecular size and free amino acids. As the ability of peptidases to hydrolyze proteins is highly variable, the selection of suitable enzymes for production of hydrolysates having defined physicochemical and nutritional characteristics is essential (Clemente 2000).

The choice of substrate and protease employed and the degree to which the protein is hydrolyzed can greatly affect the physicochemical properties of the resulting hydrolysates. Enzyme substrate specificity is also important to hydrolysate functionality because it strongly influences its molecular size and the hydrophobic/hydrophilic balance; the broader the protease specificity, generally the smaller are the peptides produced and the more complex the peptide profile becomes. For the economic production of bioactive peptides, high productivity in peptide production and a good availability of protein source and proteolytic enzyme is required. High productivity will be achieved by producing more active peptide fractions at higher yields (Hyun and Shin 2000).

Researchers have used a number of proteases to hydrolyze animal blood in the initial stages of attempting to obtain peptides with ACE-inhibitory activity. Alcalase (EC 3.4.21.62), Neutrase, pepsin (EC 3.4.23.1), papain (EC 3.4.22.2), trypsin (EC 3.4.21.4), and Flavourzyme have been utilized to determine which enzyme is best able to generate crude hydrolysates with the highest ACE inhibitory activities (Table 6).

Table 6. Enzymes used to generate animal blood-derived hydrolysates with ACE- inhibitory activity
Protein sourceSubstrate Enzyme-to-   
(substrate)concentrationProteasesubstrate ratioTemperaturepHReference
  1. LAPU, leucine amino peptidase units; N/A, information not available.

Bovine hemoglobinN/APepsin1% (w/v)23 °C5.5Adje and others 2011b
Bovine whole plasma0.09 g/mL in 50 mM PBS (pH 7.4) Protein concentration was then adjusted to 40 mg protein/mL with distilled waterAlcalaseNeutrasePepsinPapainTrypsin0.05% (w/w)55 °C7.5Hyun and Shin 2000
   1.0% (w/w)45 °C6.2 
   0.25% (w/w)37 °C2.0 
   2.0%(w/w)25 °C6.2 
   -   
Bovine albumin0.09 g/mL in 50 mM PBSAlcalase0.0005% (w/w)55 °C7.5Hyun and Shin 2000
  (pH 7.4) ProteinTrypsin0.0025% (w/w)37 °C7.5 
  concentration was then adjusted to 40 mg protein/mL with distilled water     
Bovine globulins0.09 g/mL in 50 mM PBSAlcalase0.01% (w/w)55 °C7.5Hyun and Shin 2000
  (pH 7.4) ProteinTrypsin1.0% (w/w)37 °C7.5 
  concentration was then adjusted to 40 mg protein/mL with distilled water     
Defibrinated bovine plasmaN/AFlavourzyme110 LAPU/g protein50 °C7.0Wanasundara and others 2002b
Porcine hemoglobinN/AAlcalase124g / 20kg50 °C9.0Mito and others 1996
Porcine red blood cell10 g/L in 0.1 mol/L PBSAlcalase1:5055 °C7.5Wei and Chiang 2009
 corpuscles (pH 7.4)Alcalase + Flavourzyme 55 °C7 
Plasma Trypsin 38 °C7 
Defibrinated plasma Trypsin + Chymotrypsin + Thermolysin 35 °C7.5 
Porcine globin30 mg/mL in 0.067 MPepsin1% (w/w)37 °C2.0Yu and others 2006
  PBS bufferTrypsin1% (w/w)37 °C7.5 
  Papain1% (w/w)37 °C7.5 
Porcine globin18%Pepsin8% pepsin35 °C followed by Ren and others 2011
    55 °C followed by3 
   3:1 (w/w)37 °C  
Cervine plasmaN/ATrypsin1:100 (w/w)37 °C8.0Liu and others 2010b

Alcalase (an endoprotease from Bacillus licheniformis) was chosen by several researchers as it has a low cost and high yield. For example, Alcalase was selected for the enzymatic digestion of bovine plasma proteins because it exhibited the highest proteolytic activity compared to trypsin, neutrase, pepsin, and papain (Hyun and Shin 2000). Neutrase, a metalloprotease containing Bacillus subtilis neutral proteases, was not able to hydrolyze the bovine whole plasma proteins efficiently. The protease pepsin, which is of animal origin, was able to hydrolyze the plasma proteins moderately, but papain (which is from the papaya—of plant origin) had a low degree of hydrolysis. Bovine plasma was not able to be hydrolyzed by trypsin. The authors believed that this was due to the presence of several trypsin inhibitors in whole bovine plasma (Hyun and Shin 2000). However, trypsin was able to hydrolyze isolated bovine albumin at a higher level, in comparison to isolated bovine globulin.

It has been documented that Alcalase is limited by its hydrolysis specificity and depending on the substrate; the highest degree of hydrolysis (DH) that can be reached is 20% to 25% (Adler-Nissen 1986; Pommer 1995). Wanasundara and others (2002a) preferred Flavourzyme to Alcalase. When the 2 enzymes were compared for their ability to hydrolyze defibrinated bovine plasma (DBP) proteins, Alcalase gave a maximum of 24.8 ± 1.5 DH% at the highest enzyme concentration (0.5 AU/g protein) and prolonged hydrolysis (24 h) while Flavourzyme was more efficient in hydrolyzing DBP and gave 48.3 ± 2.3 DH% at 24 h of hydrolysis when 100 LAPU/g protein was used. Flavourzyme is a fungal protease complex produced by Aspergillus orizae containing endo- and exopeptidases which give it broader specificity; thus, high DH values can be achieved. An endo-and exopeptidase complex can hydrolyze the peptide bonds in a protein molecule more completely than an endopeptidase such as Alcalase (Chang and others 2007). Bovine plasma proteins in their native state were not susceptible to hydrolysis by Alcalase or Flavourzyme but pretreatment heat denaturation (for example 90 °C for 20 min) prior to hydrolysis was found to be effective in improving hydrolysis values. The heat-denaturation of plasma proteins causes the molecules to unfold and makes them more accessible to proteases for hydrolytic reaction than in their native state (Wanasundara and others 2002a).

Yu and others (2006) performed the hydrolysis of globin obtained from porcine hemoglobin individually with 3 proteases at the same temperature (37 °C): pepsin (at pH 2.0), trypsin (pH 7.5), and papain (pH 7.5). The highest ACE-inhibitory activity was found using the pepsin hydrolysate (IC50 of 1.19 mg/mL) while trypsin, which cleaves peptide bonds that are C-terminal to Arg and Lys, had the lowest ACE-inhibitory activity of the 3 proteases used. Liu and others (2010b) were able to use trypsin for the hydrolysis of cervine plasma (pH 8.0, 37 °C). Pepsin was used by Ren and others (2011) for porcine globin hydrolysis (pH 3, 35 to 55 °C) and also by Adje and others (2011b) as the enzyme of choice for the hydrolysis of bovine hemoglobin at pH 5.5, 23 °C, in the presence of 30% ethanol which, according to the authors, would result in structured, hydrophobic, and positively charged peptides.

The enzyme-to-substrate (E/S) ratio could also be an important factor in obtaining hydrolysates with higher ACE-inhibitory activity. The ACE-inhibitory activity of porcine blood hydrolysates increased as the E/S ratio increased. An E/S ratio of 1:5 produced hydrolysates with the highest ACE-inhibitory activity when compared to hydrolysates from E/S ratios of 1:10 and 1:100 (Wei and Chiang 2009).

Effect of the Degree of Hydrolysis on the Generation of Hydrolysates with ACE-Inhibitory Activity

The degree of hydrolysis is a measure of the extent of hydrolytic degradation of proteins. It is the most used indicator for comparison among different proteolytic processes, although different methods such as pH-stat, osmometry, soluble nitrogen content, trinitrobenzene-sulfonic acid (TNBS), and o-phthalaldehyde (OPA) can be used to monitor the degree of hydrolysis (Nielsen and others 2001).

Different degrees of hydrolysis were obtained depending on the enzyme-to-substrate ratio and hydrolysis time employed when Flavourzyme was used to hydrolyze defibrinated bovine plasma. A 43% DH with the highest inhibiting ACE activity (78.93% or IC50 of 1.08 mg/mL) was achieved using an enzyme concentration of 110 leucine amino peptidase units/g protein and 15.5 h of hydrolysis time (Table 7). Unhydrolyzed defibrinated bovine plasma had a negligible amount of ACE-inhibitory activity (Wanasundara and others 2002b).

Table 7. Degree of hydrolysis and ACE inhibitory activity of blood-derived hydrolysates
   Peptide yield/ACE- 
  Hydrolysisdegree ofinhibitory 
Protein sourceProteasetime (h)hydrolysis (%)activityReference
  1. a

    Peptide yield from proteolytic reaction with 0.5% (w/w) protease.

  2. b

    IC50 (mg/mL).

  3. c

    % ACE inhibitory activity.

  4. N/A, data not available; ND, not detectable.

Bovine hemoglobinPepsin 1%N/AAdje and others 2011b
Bovine whole plasmaAlcalase258.0a2.53bHyun and Shin 2000
 Neutrase116.5a10.50b 
 Pepsin437.6a17.19b 
 Papain210.1a23.47b 
 Trypsin0.2a 
Bovine albuminAlcalase469.2a0.56bHyun and Shin 2000
 Trypsin258.3a0.89b 
Bovine globulinsAlcalase432.4a7.11bHyun and Shin 2000
 Trypsin816.2a8.14b 
Defibrinated bovine plasmaFlavourzymeUnhydrolyzed3.20 ± 1.45cWanasundara and others 2002b
  N/A1361.50 ± 1.57c 
  N/A2673.07 ± 1.86c 
  N/A3875.72 ± 0.82c 
  15.54378.93 ± 2.04c 
Porcine hemoglobinAlcalase215N/AMito and others 1996
Porcine globinPepsin12N/A1.19bYu and others 2006
 Trypsin12N/A8.79b 
 Papain12N/A2.21b 
Porcine red blood corpusclesTrypsin10 2.80bWei and Chiang 2009
 Trypsin + Chymotrypsin + Thermolysin6 0.58b 
 Alcalase2 1.60b 
 Alcalase + Flavourzyme10 1.24b 
Porcine plasmaTrypsin10 4.10bWei and Chiang 2009
 Trypsin + Chymotrypsin + Thermolysin2 0.80b 
 Alcalase6 1.24b 
 Alcalase + Flavourzyme2 1.37b 
Porcine defibrinated blood plasmaTrypsinND NDWei and Chiang 2009
 Trypsin + Chymotrypsin + Thermolysin2 1.06b 
 Alcalase24 2.20b 
 Alcalase + FlavourzymeND ND 
Porcine globinPepsin7N/A4.37bRen and others 2011

A hydrolysate of bovine albumin using Alcalase generated the most active ACE-inhibitory activity (IC50 of 0.56 mg/mL) at a hydrolysis time of 4 h using 0.5% (w/w) enzyme (Hyun and Shin 2000) with a peptide yield of 69.2%. The major plasma proteins were degraded due to hydrolysis; peptides of less than 1.04 kDa were dominant in the product when a high degree of hydrolysis was employed (Wanasundara and others 2002a).

Thus, the results from the above research demonstrate the importance and potential of obtaining an active ACE inhibitor from crude hydrolysates of blood by optimizing the hydrolysis processing conditions.

Effect of peptides on ACE-inhibitory activity. In bovine hemoglobin, peptides α 99 to 105 and α 100 to 105 both demonstrated ACE-inhibitory activity (Table 8). These 2 peptides are different by only 1 amino acid at the N-terminal end (Adje and others 2011b). However, the ACE-inhibitory activity of α 99 to 105 was much stronger than that of α 100 to 105. One possible explanation for this lies in the presence of a lysine residue at position 99, which contributes to an increase in the peptide's hydrophobicity, an important characteristic that contributes to ACE-inhibitory activity (Adje and others 2011b). ACE prefers to have substrates or competitive inhibitors that contain hydrophobic amino acid residues such as proline, phenylalanine, and tyrosine at 3 positions from the C-terminal end (Cheung and others 1980). The relationships between the structure and activity level of various ACE-inhibitory peptides indicate that binding to ACE is strongly influenced by the C-terminal tripeptide sequence of the substrate. A study on the peptic hydrolysis of bovine hemoglobin proposed that a short reaction time (about 20 min) would yield hydrolysates with a more hydrophobic nature, whereas an extensive reaction (about 16 h) was suggested to prepare hydrolysates with a more hydrophilic nature (Su and others 2006).

Table 8. Peptides with ACE inhibitory activity from blood-derived sources
SourcePeptide size (Da)Amino acid sequenceLevel of activity (IC50)Reference
  1. N/A, data not available.

Bovine albumin<1000N/A0.09 mg/mLHyun and Shin 2000
Defibrinated bovine156HThe fraction which containedWanasundara and others
plasma269H-(L or I) all the peptides listed to the 2002b
    left had an ACE-inhibitory activity of 63.18 ± 2.73% 
 279(L or I)- F (*LF is conserved within bovine serum albumin)  
 336GYP  
 416HPY (conserved within bovine serum albumin)  
 366SPY  
 416YPH  
 428HPGH  
Bovine hemoglobin4430.1α 67 to 106366 ± 16.10 μMAdje and others 2011b
  TKAVEHLDDLPGALSELSDLHAHKLRVDPVNFKLLSHSLL  
 3651.1α 73 to 105518.29 ± 37.3 μM 
  LDDLPGALSELSDLHAHKLRVDPVNFKLLSHSL  
 797α 99 to 10542.51 ± 3.96 μM 
  KLLSHSL  
 669α 100 to 1051095.5 ± 65.5 μM 
  LLSHSL  
Porcine hemoglobin1555α 34 to 464.92 μMYu and others 2006
  LGFPTTKTYFPHF  
 N/Aβ 34 to 396.02 μM 
  VVYPWT  
Porcine hemoglobinN/Aβ 130 to 1365.8 μMMito and others 1996
  FQKVVA  
 N/Aβ 130 to 1377.4 μM 
 N/AFQKVVAG  
 N/AFQKVVAK2.1 μM 
 N/Aβ 64 to 691.9 μM 
  GKKVLQ  
Porcine hemoglobinN/AWVPSV0.368 mg/mLRen and others 2011
 N/AYTVF0.226 mg/mL 
 N/AVVYPW0.254 mg/mL 
Porcine plasmaN/AGVHVV6 μMPark and others 1996
Porcine plasmaN/ALVL0.6 μg/mLHazato and Kase 1986
Cervine plasmaN/AVYNEGLPAP3.1 μMLiu and others 2010b

The lowest ACE-inhibitory activity was reported from a bovine albumin-derived peptide with a size of <1000 Da. ACE-inhibitory activity increased with decreasing MW cut-off and total peptides when bovine albumin hydrolysates were further processed and purified. This indicates that the molecular weights of active peptides are usually lower than 1000 Da (Hyun and Shin 2000). The ACE-inhibitory peptides identified in the study by Wanasundara and others (2002b) are very short and presumed to have originated from serum albumin and globulins, as this particular plasma protein product (defibrinated bovine plasma) is devoid of thrombin and fibrinogen.

There was little change in the ACE-inhibitory activity of the peptides after in vitro incubation with gastrointestinal proteases (pepsin, chymotrypsin, and trypsin), suggesting that these peptides might then be resistant to digestion in the gastrointestinal tract. Yu and others (2006) tested the stability of their 2 purified peptides obtained from the hydrolysis of porcine globin using pepsin. Shorter peptides are less likely to be cleaved by gastric proteases (Wanasundara and others 2002b). This is interesting as the antihypertensive effect of ACE inhibitory peptides is strongly influenced by their bioavailability, which is predominantly determined by the resistance to peptidase degradation and intestinal absorption.

Antioxidant peptides from animal blood sources

Methods for assessing antioxidant activity of a compound fall into 2 broad categories reflecting the focus on activity in foods or bioactivity in humans (Antolovich and others 2002), while antioxidant assays can be divided into in vitro and in vivo types. The antioxidant activity of protein hydrolysates or peptides is usually tested by in vitro assays (Di Bernardini and others 2011). On the basis of the chemical reactions involved, major antioxidant capacity assays can be roughly divided into 2 categories: (1) hydrogen atom transfer (HAT) reaction-based assays and (2) single electron transfer (ET) reaction-based assays (Huang and others 2005). There is no standardized method to study the antioxidant activity of a substance, and for this reason it is recommended to study the antioxidant activity with various oxidation conditions and different antioxidant methods (Frankel and Meyer 2000; Antolovich and others 2002; Sanchez-Moreno 2002). Antolovich and others (2002) also caution that the term “activity” as applied to antioxidants needs clarification as it can have a variety of meanings, and relevant aspects include: mechanistic intervention, for example, free radical scavenger, catalytic decomposition, and pro-oxidant suppression; rate of scavenging, for example, near-diffusion or controlled; medium or substrate selectivity (for example, aqueous, surface, or lipid phase); concentration effectiveness (moles of free radicals scavenged per mole of antioxidant); and synergistic effect for other antioxidants.

Effect of enzymes on the antioxidant activity of hydrolysates from animal blood sources

Different enzymes were utilized by researchers to generate hydrolysates with antioxidant activity (Table 9) in a similar fashion to that previously described for ACE-inhibitory peptides. To hydrolyze porcine hemoglobin, Chang and others (2007) used Alcalase and a combination of Alcalase followed by Flavourzyme, while Sun and others (2011c) used Flavourzyme, papain, A.S. 1398, Alcalase, pepsin, and trypsin to screen for hydrolysates with the highest antioxidant activity. For porcine plasma, Wang and others (2008) and Liu and others (2010a) used Alcalase as their protease of choice for hydrolysis, although at different enzyme-to-substrate ratios. Xu and others (2009) meanwhile used pepsin and papain in their hydrolysis of porcine plasma. The hydrolysis temperature for Alcalase was 50 to 55 °C, for Flavourzyme 40 to 45 °C, papain and pepsin 37 °C, and trypsin and A.S. 1398 45 °C. Optimal pH range or values reportedly used by researchers for each enzyme were pH 7.5 to 8.0 for Alcalase, pH 6.5 to 7.5 for Flavourzyme, pH 6.5 to 8.0 for papain, pH 2.0 for pepsin, pH 7.0 for A.S. 1398, and pH 7.5 for trypsin.

Table 9. Enzymes used to generate crude hydrolysates from animal blood sources with antioxidant activity
Protein source  Enzyme-to-   
(substrate)Substrate concentrationEnzymesubstrate ratioTemperaturepHReference
Porcine0.05 g/mL driedAlcalase2%50 °CpH 8.0Chang and others 2007
 hemoglobin hemoglobin in 0.1 NFlavourzyme1%40 °CpH 7.5 
  NaOH     
  Alcalase followed by Flavourzyme2%50 °CpH 7.5 
   1%   
Porcine hemoglobin1 mg/mLPepsin1.6% (w/w)40.4 °CpH 1.6Sun and others 2011a
Porcine hemoglobin0.05 g/mL dried hemoglobin in distilled waterFlavourzyme0.5% (w/w)45 °CpH 6.5Sun and others 2011c
  Papain0.3% (w/w)37 °CpH 6.5 
  A.S. 13980.2% (w/w)45 °CpH 7.0 
  Alcalase0.2% (w/w)55 °CpH 8.0 
  Pepsin1.6% (w/w)37 °CpH 2.0 
  Trypsin0.2% (w/w)45 °CpH 7.5 
Porcine plasma albumin and globulin0.08 g/mL of each powder in 50 mM PBS (pH 7.5)Alcalase0.1% (w/w)55 °CpH 7.5Wang and others 2008
Porcine plasma40 mg protein/mLAlcalase2:100 (w/w)55 °CpH 8.0Liu and others 2010a
Porcine plasma0.2 g/L in distilled waterPepsin25:1 (w/w)37 °CpH 2.0Xu and others 2009
  Papain20:1 (w/w)37 °CpH 8.0 

The effects of pepsin-assisted hydrolysis conditions on the antioxidant activity of porcine hemoglobin hydrolysate were investigated and the effects of temperature, pH, and enzyme-to-substrate ratio on the antioxidant activity were evaluated and compared using response surface methodology (Sun and others 2011a). Temperature and pH were found to be the major factors affecting the antioxidant activity of porcine hemoglobin hydrolysates, while enzyme-to-substrate ratio influenced antioxidant activity to a lesser extent. Properties of hydrolysates are largely dependent on the primary structure of the peptide residuals as a consequence of the type of hydrolyzing enzymes, coupled with hydrolysis conditions, degree of hydrolysis and substrate pretreatment. Therefore, a careful choice of process parameters is of utmost importance for successful hydrolysis.

Effect of the degree of hydrolysis on the antioxidant activity of hydrolysates from animal blood sources

Although Alcalase hydrolysis of porcine hemoglobin produced a higher degree of hydrolysis (about 17%) in comparison to pepsin (DH 7.7%), the DPPH scavenging activity of hydrolysates using pepsin was higher (67% compared to 27%) (Sun and others 2011c). The authors reported that there was no correlation between extent of hydrolysis and antioxidant activity (P > 0.05) and suggested that since pepsin preferably digests peptide bonds by cleaving after the N-terminal of aromatic amino acids, such as phenylalanine (F), tryptophan (W), and tyrosine (Y), the phenyl groups of the residues at peptide ends were likely to be scavenging the free radical to prevent DNA damage. This result indicates that the antioxidant activity of the hydrolysates is inherent to the characteristic amino acid sequences of peptides depending on protease specificities.

The DH of porcine albumin and globulin hydrolysates using Alcalase increased with increasing hydrolysis time and reached the highest values at 24 h of hydrolysis. However, the highest reducing power values of both hydrolysates were not obtained at the highest DH value but at 12 h for albumin, and 16 h for globulin (Wang and others 2008). Once again the lack of a direct relationship between antioxidant activity and DH suggested that the specific composition (such as type of peptides and ratio of different free amino acids) is an important factor regarding antioxidant abilities.

Conversely, other researchers found that fractions with a higher degree of hydrolysis, produced peptides with a higher antioxidant activity (Wanasundara and others 2003; Liu and others 2010a). Porcine plasma hydrolyzed using Alcalase with a degree of hydrolysis of 17.6% had higher reducing power, iron chelating ability, and DPPH radical-scavenging activity compared to less hydrolyzed fractions (Liu and others 2010a), while defibrinated bovine plasma had the highest iron-chelating and thiobarbituric acid-reactive substances (TBARS) activity at 43% degree of hydrolysis. The ability of porcine plasma hydrolysates to chemically inhibit lipid oxidation as demonstrated in the study was attributed, in part, to protein structural changes. Nonhydrolyzed plasma proteins, possibly because of their compact structure, had low antioxidant activity when measured. Likewise, Chang and others (2007) found that Alcalase hydrolysates of porcine hemoglobin had higher DPPH radical-scavenging activities than that of the native hemoglobin (Table 10).

Table 10. Hydrolysis and antioxidant activities of animal blood hydrolysates
      DPPH radical-   
  HydrolysisDegree of Iron-chelatingscavenging Superoxide radical- 
SourceProteasetimehydrolysisReducing powerability (%)activity(%)TBARSscavenging activityReference
  1. *Estimated values from figures available in corresponding reference.

  2. ND, not detectable.

  3. Note: Please refer to corresponding reference for protein concentration of hydrolysates used in the antioxidant activity tests.

Porcine hemoglobinNo treatment0%0.38 ± 0.06%74.87 ± 5.2321.53 ± 1.98  Chang and others 2007
 Alcalase4 h7.6%0.21 ± 0.00%34.50 ± 0.9651.57 ± 4.19   
 Flavourzyme6 h∼18.7%*0.08 ± 0.00%33.96 ± 4.13−2.81 ± 0.44   
 Alcalase +4 h +13.4%0.23 ± 0.01%63.54 ± 3.6941.94 ± 1.89   
  Flavourzyme6 h       
Porcine hemoglobinNo treatment0%  21.2 ± 1.61  Sun and others 2011c
 Flavourzyme1 h∼3%*  ∼4*   
 Papain1 h∼5.5%*  ∼18*   
 A.S. 13981 h∼4%*  ∼17*   
 Alcalase1 h∼17%*  ∼27*   
 Pepsin1 h7.7%  67.0 ± 1.84   
 Trypsin1 h∼2%*  ∼24*   
          
Porcine albuminAlcalase12 h∼65%*0.57* @ 700 nm    Wang and others 2008
Porcine globulinAlcalase16 h∼10%*0.65* @ 700 nm    Wang and others 2008
Porcine red blood corpusclesTrypsin24   20.2 ± 1.0  Wei and Chiang 2009
 Trypsin + Chymotrypsin + Thermolysin10   64.9 ± 3.9   
 Alcalase10   62.3 ± 1.8   
Porcine plasmaTrypsin24   11.5 ± 2.3  Wei and Chiang 2009
 Trypsin + Chymotrypsin + Thermolysin24   24.4 ± 2.4   
 Alcalase24   30.8 ± 3.2   
Porcine defibrinated blood plasmaTrypsinND   ND  Wei and Chiang 2009
 Trypsin + Chymotrypsin + Thermolysin24   22.4 ± 1.9   
 Alcalase24   23.4 ± 1.4   
Porcine blood plasmaNo treatment0%456.4 ± 20.3 μM 24.47 ± 0.10 20.04 ± 0.06Liu and others 2009
 Alcalase5 h17.6%1405.6 ± 7.6 μM 76.79 ± 0.02 63.99 ± 0.04 
Porcine bloodNo treatment0%456.4 ± 1.4μM5.62 ± 0.3021.43 ± 0.092.05 ± 0.01 mg/L Liu and others 2010a
plasmaAlcalase0.5 h6.2%713.1 ± 8.8μM6.80 ± 0.6231.16 ± 0.931.53 ± 0.01 mg/L  
  2 h12.7%1303.4 ± 8.2μM9.67 ± 0.3045.14 ± 1.511.48 ± 0.01 mg/L  
  5 h17.6%1407.9 ± 9.9 μM12.03 ± 0.5276.53 ± 1.511.37 ± 0.01 mg/L  
PorcinePepsin5 h  150% of EDTA48.4  Xu and others 2009
plasmaPapain16 h  61% of EDTA43.1   
Bovine plasma (defibrinated)No treatment 0% 60.0 27.6 Wanasundara and others 2003
 Flavourzyme 13% 95.0 28.8  
   26% 90.0 40.0  
   43% 100 45.0  

Effect of peptides on the antioxidant activity. Not much work has been done on sequencing peptides with antioxidant activity thus far. However, peptides of <3000 Da from porcine blood hydrolysates appear to have higher antioxidant activity than larger peptides in terms of reducing power, DPPH radical-, hydroxyl radical-, and superoxide radical-scavenging activities (Table 11). The antioxidant activity of porcine plasma hydrolysates, for example, was lowest for the peptide fraction above 10,000 Da. It has been suggested that the antioxidant ability of biopeptides in vitro depends on peptide size, amino acid composition of the peptide, and presence of free amino acids within the hydrolysates (Ryan and others 2011).

Table 11. Effect of peptide on antioxidant activity from animal blood
   DPPHHydroxylSuperoxide 
   radical-radical-radical- 
 PeptideReducingscavengingscavengingscavenging 
Sourcesize (Da)power (μM)activity (%)activity (%)activity (%)Reference
  1. *Estimated values from figures available in corresponding reference.

Porcine plasma<30001206.4 ± 31.570.07 ± 1.86  Liu and others 2010a
 3000 to 6000866.6 ± 12.550.38 ± 2.06   
 6000 to 10000664.9 ± 22.441.30 ± 1.23   
 >10000557.3 ± 16.331.11 ± 1.07   
Porcine hemoglobin<3000 83.4 ± 3.855.8 ± 2.227.8 ± 1.6Sun and others 2011b
 3000 to 5000 66.7 ± 3.131.6 ± 2.818.1 ± 1.3 
 5000 to 10000 54.2 ± 2.022.3 ± 5.714.8 ± 0.8 
 >10000 69.9 ± 1.738.0 ± 4.611.9 ± 1.1 
Porcine albumin<3000 ∼36*33.8 ± 0.660.9 ± 0.9Wang and others 2008
 3000 to 6000 ∼30*   
 6000 to 10000 ∼26*   
 10000 to 30000 ∼21*   
 >30000 ∼22.5*   
Porcine globulin<3000 ∼26*53.1 ± 0.472.5 ± 0.7Wang and others 2008
 3000 to 6000 ∼20*   
 6000 to 10000 ∼16*   
 10000 to 30000 ∼12*   
 >30000 ∼29*   

A recently published study used a nonenzymatic technique to produce porcine hemoglobin peptides at high temperatures (120 to 180 °C) and low pressures (4 MPa) under a nitrogen stream. Peptides presenting an average size of 3.2 kDa were obtained, with a yield of 84% with respect to the initial hemoglobin hydrolyzed. The main fraction of these peptides (40%) was composed of molecules smaller than 1 kDa, which possessed some good antioxidant properties (Álvarez and others 2012).

Antimicrobial peptides from animal blood sources

Antimicrobial peptides are usually used to inhibit the growth of pathogenic bacteria. The agar diffusion assay (or inhibition zone assay) method is a common method used to test the antimicrobial activity of hydrolysates and peptides. The antimicrobial effect of a hydrolysate or a peptide increases in accordance with the diameter of the zone of inhibition formed. Peptidic solutions can be placed in wells made in the agar or on sterilized filter paper discs placed on agar previously inoculated with test bacteria. An accurate way to study the antimicrobial activity of hydrolysates and/or peptides is the determination of their minimum inhibitory concentration (MIC). The MIC value defines the lowest concentration of an antimicrobial that inhibits 100% of the growth of a microorganism, and it is usually determined by liquid growth inhibition in a 96-well plate spectrophotometric method (Fogaça and others 1999) or in a multi-tube system.

Antimicrobial peptides from animal blood have been studied quite extensively. A comprehensive review by Yu and others (2010) focused on antimicrobial peptides, including defensins and cathelicidins, found in the blood of animals relevant to the Australasian meat (cattle, sheep, pigs, goats, and deer) and poultry (chicken, turkey, and ostrich) industries. Livestock animals such as cattle, pigs, goats, and sheep are more recently evolutionarily related and are thought to be more likely to contain similar antimicrobial defense systems (Tomasinsig and Zanetti 2005).

The mechanism of antimicrobial peptide activity is usually expressed by the disintegration of cell membrane, whereby the lipid bilayer of the cell membrane is the principal target. Interaction between the antimicrobial peptide and the cell membrane is an important requirement for antimicrobial activity. The majority of antimicrobial peptides that contain α-helical structures are cationic and amphipathic, while others are also hydrophobic α-helical peptides. The cationic properties of the peptide enable binding with the anionic phospholipid-rich membrane, which initiates cell lysis (Kitts and Weiler 2003). The determination of hydrolysis conditions in order to obtain peptides with desired molecular weights, which are able to adopt a α-helical structure in contact with the bacterial membrane, is now of considerable interest. The micro-environment of the lipid bilayer has also been shown to have a stabilizing effect on the α-helical structure of peptides (Adje and others 2011a) and is currently under investigation.

Effect of enzymes and hydrolysis on the antimicrobial activity of hydrolysates from animal blood sources

The use of enzyme hydrolysis to obtain antimicrobial peptides from animal blood is a relatively recent development starting with Froidevaux and others (2001). An antimicrobial peptide α1 to 23, active toward Micrococcus luteus A270, was isolated after enzymatic hydrolysis of bovine hemoglobin using pepsin. The initial identification of an antibacterial peptide sequence in bovine hemoglobin (α33 to 61) was actually first purified from the gut of a tick, Boophilus microplus, leading to the assumption that the proteolytic degradation of hemoglobin had taken place in the gut to provide a defense against microorganisms for the tick (Fogaça and others 1999).

Other methods used by researchers to obtain antimicrobial peptides from animal blood include crude extraction by lysing of cells or chemical treatment followed by separation of the peptide of interest using ion-exchange chromatography separation (bovine hemoglobin), or gel filtration chromatography (porcine neutrophils) (Hu and others 2010; Wessely-Szponder and others 2010). For antimicrobial peptides from sheep neutrophils, sonication of ovine white blood cells followed by gel permeation chromatography was utilized (Anderson and Yu 2003). Although some groups have reported that it is necessary to add neutrophil elastase to their crude cell extract to cleave the proregion and release the active peptides (Shamova and others 1999), the extraction process used by Anderson and Yu (2003) and Wessely-Szponder and others (2010) did not involve the addition of this enzyme. The latter authors reasoned that the cleavage was probably carried out by the neutrophil elastase naturally present in the crude extract.

The kinetics of the α1 to 23 peptide, which was the first antibacterial peptide to be isolated from bovine hemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 °C in a homogeneous-phase system (Choisnard and others 2002). The kinetics of peptide appearance was investigated in acetate buffer alone and in urea as a hemoglobin-denaturing agent. Two different hydrolysis mechanisms, a “one-by-one” for native hemoglobin hydrolysis and a “zipper” for denatured hemoglobin hydrolysis were proposed. Regardless of the hemoglobin state, native or denatured, and the hydrolytic mechanism, one-by-one or zipper, the antibacterial α1 to 23 peptide was found to be a transient peptide. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native hemoglobin with the yields of α1 to 23 peptide being 55% and 25%, respectively (Choisnard and others 2002).

The hydrolysis of bovine hemoglobin using pepsin was further utilized by Daoud and others (2005) to isolate another antimicrobial peptide at a low degree of hydrolysis (3%). The α107 to 136 peptide was the second antimicrobial peptide obtained by in vitro proteolysis of bovine hemoglobin. This peptide is active against 4 bacterial species: Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis.

The isolation and characterization of transient antibacterial peptides appearing in the course of bovine hemoglobin peptic digestion at a low degree of hydrolysis was further reported by the same group (Nedjar-Arroume and others 2006). A study of the time-dependent nature of peptic hydrolysis of bovine hemoglobin found that the released peptides detected during the early stage of hydrolysis (0 to 10 min) were all derived from the N- and C-terminal regions of bovine hemoglobin α- and β-chains: from 1 to 46 and 99 to 141 regions of α-chain; from 1 to 40 and 105 to 145 regions of β-chain. These regions possess relatively higher hydrophobicity. In the later stage of hydrolysis, pepsin hydrolyzed the middle part of the α-chain from N- to C-terminal, while little enzymatic cleavage occurred in the center region of β-chain due mainly to the high hydrophilic nature. It was concluded that the terminal positions of these regions and their relatively higher hydrophobicity may play important roles in their greater susceptibility to pepsin hydrolysis, because pepsin preferentially cleaves exposed and hydrophobic peptide bonds (Su and others 2006).

Approaches to facilitate the production of only intermediate peptides after peptic hydrolysis of bovine hemoglobin with alcohols (40% methanol, 30% ethanol, 20% propanol, or 10% butanol), used to induce structural change of hemoglobin and to realize a limited hydrolysis, is a recent development and has shown that new antimicrobial peptides can be obtained this way (Adje and others 2011a).

Effect of peptides on antimicrobial activity

Several studies have been carried out isolating and sequencing antimicrobial peptides from bovine, porcine, ovine, caprine, and cervine blood (Table 12). The hydrophobicity of the peptide has been shown to be related to its antimicrobial activity, as higher hydrophobicity is useful in the binding of lipopolysaccharides on the outer cell membrane of the bacteria. Lopes and others (2005) have shown both the important role of tyrosine (Y) in the interaction with membranes, and the 2 positively charged amino acids, arginine and lysine, which interact with negatively charged membrane phospholipids. Recent results from Catiau and others (2011a) showed that the peptide β114 to 145 and its peptic derivatives containing the sequence RYH exhibited antibacterial activity. RYH was then chemically synthesized because hydrolysis did not result in this sequence. They concluded that RYH was necessary for the antibacterial activity and that the RYH is the minimal antimicrobial peptidic sequence required. The sequence of chemically synthesized peptide RYH contains both tyrosine and 2 positively charged amino acids, arginine and histidine. This may explain the highest antibacterial activity measured for this peptide in addition to the highest release of carboxyfluorescein from the liposome.

Table 12. Effect of peptide on antimicrobial activity
     
 PeptideAmino acid  
Sourcesize (Da)sequence/Peptide nameAntimicrobial activityReference
  1. 1Inhibition zone of peptides.

  2. 2MIC.

Bovine hemoglobin1992.401VNFKLLSHSLLVTLASHLE. coli O11110.3 ± 0.1 mm1Hu and others 2010
   S. aureus NCTC41639.5 ± 0.1 mm1 
   C. albicans 3135A7.6 ± 0.2 mm1 
Bovine hemoglobin2236α 1 to 23M. luteus A270671 μM2Froidevaux and others 2001
  VLSAADKGNVKAAWGKVGGHAAE   
Bovine hemoglobin α 67 to 106E. coli35.27 μM2Adje and others 2011b
   L. innocua35.27 μM2 
   M. luteus A27039.2 μM2 
   S. aureus39.2 μM2 
Bovine hemoglobin3152α 107 to 136E. coli76 μM2Daoud and others 2005, Nedjar-Arroume and others 2006
  VTLASHLPSDFTPAVHASLDKL. innocua38 μM2 
  FLANVSTVLTSKYRM. luteus A27076 μM2 
   S. aureus76 μM2 
Bovine hemoglobin654α 137 to 141E. coli9 μM2Catiau and others 2011b
  TSKYRS. enteritidis4.6 μM2 
   S. aureus1 μM2 
   L. innocua1 μM2 
   M. luteus A2709 μM2 
Bovine hemoglobin768β 140 to 145S. enteritidis18 μM2Catiau and others 2011a
   E. coli45 μM2 
   L. innocua45 μM2 
   M. luteus A27018 μM2 
Bovine hemoglobin2196β 126 to 145S. enteritidis35 μM2Nedjar-Arroume and others 2006
  QADFQKVVAGVANALAHRYHE. coli71 μM2 
   L. innocua71 μM2 
   M. luteus A27071 μM2 
Porcine neutrophils2154.5Protegrin 1E. coli10 μg/mL2Wessely-Szponder and others 2010
 1955.6Protegrin 2   
 2055.5Protegrin 3   
Ovine neutrophils Peptide Pa/OaBac5E. coli4 μg/mL2Anderson and Yu 2003
   S. aureus10 μg/mL2 
   C. albicans10 μg/mL2 
Caprine leukocytes3375ChBac3.4E. coli ML35p2.3 ± 0.5 μM2Shamova and others 2009
  RFRLPFRRPPIRIH PPPFYPPFRRFLE. coli ATCC 259223.8 ± 1.5 μM2 
   E. coli M156.0 ± 2.0 μM2 
   P. aeruginosa6.3 ± 2.9 μM2 
   ATCC 27853  
   L. monocytogenes EGD2.8 ± 2.2 μM2 
   S. aureus 710A8.0 μM2 
   MRSA ATCC 3359110.0 ± 3.7μM2 
   C. albicans 820>16 μM2 
Caprine leukocytes5160.2ChBac5E. coli ML35p1.6 ± 0.5 μM2Shamova and others 2009
   E. coli ATCC 259223.1 ± 1.5 μM2Shamova and others 1999
   E. coli M154.7 ± 2.0 μM2 
   P. aeruginosa8.2 ± 2.9 μM2 
   ATCC 27853  
   L. monocytogenes EGD2.8 ± 2.2 μM2 
   S. aureus 710A>16 μM2 
   MRSA ATCC 33591>16 μM2 
   C. albicans 820>16 μM2 
Cervine neutrophils4460Peptide P15E. coli0.3 μg/mL2Treffers and others 2005
   S. aureus1 μg/mL2 
   C. albicans3 μg/mL2 

Meanwhile, KYR was the minimal sequence exhibiting an antibacterial activity from another study on the α-chain of bovine hemoglobin (Catiau and others 2011b). Comparing the KYR sequence with the sequence RYH obtained from the β-chain of hemoglobin, both sequences contain tyrosine and 2 positively charged basic amino acids, including arginine. The authors concluded that tyrosine (Y), arginine (R), and one positively charged basic amino acid such as lysine (K) or histidine (H) are required for antibacterial activity (Catiau and others 2011a). However, other peptides from bovine hemoglobin, which do not contain tyrosine or arginine (Froidevaux and others 2001; Hu and others 2010) still demonstrate antimicrobial activity, suggesting another mechanism of action.

Both Wessely-Szponder and others (2010) and Anderson and Yu (2008) suggested that antibacterial peptides from animal blood neutrophils could be significantly more active in combination. It is therefore recommended that better therapeutic results could be obtained if isolated peptides were used synergistically.

Other Blood Bioactivity Reported from Animal Blood Sources

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

Opioid peptides

Opioid peptides are short sequences of amino acids that bind to opioid receptors in the brain. Hemorphins are a class of naturally occurring, endogenous opioid peptides, which are found in the bloodstream; they are derived from the β-chain of hemoglobin. Opioid peptides have an affinity for an opioid receptor and have an effect on the nerve system (Pihlanto and Korhonen 2003). They also influence gastrointestinal functions. Typical examples of opioid peptides are endorphins, enkephalin, and prodynorphin (Arihara 2006). The isolation of 2 opioid peptides (hemorphins) from a bovine hemoglobin source through peptic hydrolysis was first reported by Piot and others (1992). On the basis of their study, they surmised that hemorphins are present in an inactive state within the intact β-chain of the globin protein sequence (Piot and others 1992). Hemorphins derived from hemoglobin have also been reported to have inhibitory actions on ACE activity (Lantz and others 1991). In Table 13, examples are given of peptides (YPWT and YPWTQ) which demonstrated an opioid activity of IC50 45.2 μM and 46.3 μM, respectively, obtained from the β-chain of bovine hemoglobin (Zhao and others 1997).

Table 13. Opioid, mineral-binding, and antigenotoxic activity from animal blood
Peptide/ Enzyme   
hydrolysatesSourcetreatmentAmino acid sequenceActivityReference
Opioid peptideBovine hemoglobinPeptic hydrolysisβ 34 to 37 and β 34 to 38 (YPWT)45.2 μM (IC50)Zhao and others 1997
   β 35 to 38 and β 35 to 39 (YPWTQ)46.3 μM (IC50) 
Opioid peptideBovine hemoglobinPeptic hydrolysisβ 31 to 4029.1 μM (IC50)Piot and others 1992
   (LVV-hemorphin-7) LVVYPWTQRF  
   β 32 to 4034.3 μM (IC50) 
   (VV-hemorphin-7)  
   VVYPWTQRF  
Calcium-binding peptidePorcine plasmaFlavourzyme with a 50:1 substrate-to-enzyme ratio (w/w) at 50 °C for 8 hVSGVEDVNCa binding 0.025 mM at a peptide concentration of 0.011 mMLee and Song 2009a
Iron-binding peptidePorcine plasmaFlavourzyme with a 50:1 substrate-to-enzyme ratio (w/w) at 50 °C for 8 hDLGEQYFKGFraction F222 had Fe binding of 0.056 mM at a peptide concentration of 0.021 mMLee and Song 2009b
Hydrolysates with antigenotoxic activity  -Mean tail length, μM:Park and Hyun 2002
 Bovine plasmaAlcalase 36.6 ± 11.5 
  Neutrase 60.9 ± 17.0 
  Pepsin 39.1 ± 9.1 
  Trypsin 70.4 ± 20.6 
      
 BovineAlcalase 26.2 ± 7.1 
  albuminPepsin 27.8 ± 6.4 
  Trypsin 35.8 ± 9.3 
 BovineAlcalse 50.8 ± 14.1 
  globulinPepsin 43.2 ± 11.0 
  Trypsin 51.4 ± 12.9 

The effect of the composition of the solvent on the peptic hydrolysis of bovine hemoglobin was studied to improve the preparation of 2 opioid peptides. Peptic hydrolysis was performed for 24 h at 23 °C in 0.1 M sodium acetate buffer, pH 4.5. The kinetics of appearance of hemorphins was investigated in the presence of either 20% (v/v) ethanol, a stabilizing solvent of hemoglobin, or urea, a denaturing agent. Ethanol improved the yield of VV-hemorphin-7, whereas urea improved the yield of the 2 hemorphins. Because the amounts of hemorphins observed in urea were greater than in ethanol, the denatured state of hemoglobin is more favorable to obtaining LVV-hemorphin-7 and W-hemorphin-7. The peptide bonds that on hydrolysis give rise to hemorphins would be more accessible to pepsin. LVV-hemorphin-7 and VV-hemorphin-7 are produced not only at pH 2, during the peptic hydrolysis of bovine hemoglobin, as reported by several authors, but also at pH 4.5, at which pepsin is far less active. The peptic hydrolysis of bovine hemoglobin in the denatured state or in the native state in the presence of 20% (v/v) ethanol permits a significant increase in the production of these hemorphins (Lignot and others 1999).

Mineral-binding peptides

Mineral-binding peptides, for example, caseino-phosphopeptides (CPP), which have been generated from milk proteins, function as carriers for minerals, including calcium. CPP has also demonstrated anticarcinogenic activity (Saïd and Dominique 2011). Two mineral-binding peptides obtained from porcine plasma after hydrolysis with Flavourzyme have been reported (Lee and Song 2009a). A calcium-binding peptide had the sequence VSGVEDVN, while an iron-binding peptide had the amino acid sequence of DLGEQYFKG (Table 13). The level of binding ability of the 2 peptides was relatively similar.

Antigenotoxic peptides

Hydrolysates from bovine plasma, globulin and albumin were tested for the antigenotoxicity potential (the ability to prevent damage to DNA) by measuring the reduction of DNA damage using the Comet assay (Park and Hyun 2002). Of the 4 enzymes used (Alcalase, Neutrase, pepsin, and trypsin), pepsin was the most effective protease for producing active peptides; and the peptic hydrolysate from bovine blood albumin was able to demonstrate the best antigenotoxic effect (Table 13). Antigenotoxic activities of the peptic hydrolysate of whole plasma and albumin increased when the treated concentrations were increased. The mechanism of action responsible for the antigenotoxicity activity of the peptides was not a direct chemical inactivation of the carcinogen MNNG, but a biological effect resulting from interaction with cells and changing the physiology or metabolism of detoxification. The aromatic amino acid residues, phenylalanine (F), tyrosine (Y), and tryptophan (W), were also thought to contribute to the antigenotoxic effect (Park and Hyun 2002).

Bacterial growth-stimulating peptide

A peptide with a bacterial growth-stimulating activity was isolated from a bovine hemoglobin hydrolysate by reversed-phase high-performance liquid chromatography. Native bovine hemo-globin was produced by adding erythrocytes to water and 4 M hydrochloric acid, and 300 L 5% denatured hemoglobin at pH 2 was obtained. For peptic digestion, 80 L of this hemoglobin solution was heated to 40 °C in a reactor and pepsin was added. Hydrolysis lasted for 8 h at pH 2.0, which was maintained by a pH-stat. The primary structure and molecular mass of the bioactive peptide, determined by amino acid analysis and fast-atom bombardment mass spectrometry, was identical to that of fragment 48 to 52 (STADA) of the β-chain of bovine hemoglobin. Microbiological tests in solid media demonstrated that this peptide exhibited growth-stimulating activity on Gram-negative bacteria. In the test group comprising enteric bacterial strains that colonize in an environment where hemoglobin is readily available, 7 of 10 strains of bacteria were stimulated by the peptide (Zhao and others 1996).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References

This review presents detailed coverage of the literature available on bioactive peptides from animal blood, and it explores the possibilities of utilizing animal blood from the slaughterhouse industry for deriving such peptides, besides the conventional uses in food and other industrial applications. Blood from slaughterhouse animals is a readily available protein source. The utilization of animal blood from the slaughterhouse as described in this review offers economic, nutritional, and environmental benefits. As such, there is the need for more effort to be directed toward using this valuable by-product more fully to ensure that maximum benefits are derived. Research on the bioactive functions of peptides obtained from blood fractions should move toward targeting novel activities such as immunomodulatory activities.

It is well known that religious restrictions on the use of blood do exist. For example, both Islamic sharia'a and Jewish shechita are very clear on the prohibition of blood consumption, which may be a hurdle denying the use of whole blood in food for some cultures. However, the transformation brought about by the hydrolysis process may provide a way toward the acceptability of blood-derived products. Religious opinions from scholars are yet to be presented.

The extent of hydrolysis of blood, as determined by degree of hydrolysis (DH), and the potency of bioactive peptides can be influenced by the choice of enzyme, the hydrolysis conditions including enzyme-to-substrate ratio, temperature, pH, and hydrolysis duration. Peptide size and amino acid sequence also contribute to differences in bioactivity. These critical control points must be studied and understood to derive the best activity or peptide. However, as most studies have only looked at the bioactivity of blood-derived peptides in vitro, further experiments are required to evaluate the effectiveness of these activities in vivo. This review of research conducted to date gives a strong indication that slaughterhouse blood is a good source of protein with the potential to provide many bioactive compounds.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. General Physicochemical Characteristics of Blood
  5. Comparison of the Physicochemical Characteristics of Slaughterhouse Animal Blood
  6. Slaughterhouse Animal Blood Collection and Separation
  7. Animal Blood in Human Food Applications
  8. Animal Blood in Other Applications
  9. Bioactive Peptides from Food By-products
  10. Bioactives Isolated from Animal Blood
  11. Other Blood Bioactivity Reported from Animal Blood Sources
  12. Conclusions
  13. Acknowledgment
  14. References
  • Adje EY, Balti R, Kouach M, Dhulster P, Guillochon D, Nedjar-Arroume N. 2011a. Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin. Intl J Biol Macromol 49:14353.
  • Adje EY, Balti R, Kouach M, Guillochon D, Nedjar-Arroume N. 2011b. α 67–106 of bovine hemoglobin: a new family of antimicrobial and angiotensin I-converting enzyme inhibitory peptides. Eur Food Res Technol 232:63746.
  • Adler-Nissen J. 1986. Enzymic hydrolysis of food proteins. New York, N.Y.: Elsevier Applied Science Publishers. p 427.
  • Agar N, Godwin I. 1992. Studies on the blood of fallow deer (Dama dama) and red deer (Cervus elaphus): haematology, red cell enzymes, metabolic intermediates and glycolytic rates. Comp Biochem and Physio Part B: Comparative Biochem 103:90911.
  • Álvarez C, Rendueles M, Díaz M. 2012. Production of porcine hemoglobin peptides at moderate temperature and medium pressure under a nitrogen stream. Functional and antioxidant properties. J Agric Food Chem 60:563643.
  • Anderson AE, Medin E, Bowden DC. 1970. Erythrocytes and leukocytes in a Colorado mule deer population. J Wildlife Manag 34:389406.
  • Anderson RC, Yu PL. 2003. Isolation and characterisation of proline/arginine-rich cathelicidin peptides from ovine neutrophils. Biochem Biophys Res Comm 312:113946.
  • Anderson R, Yu PL. 2008. Pilot-scale extraction and antimicrobial activity of crude extract from ovine neutrophils. Process Biochem 43:8826.
  • Antolovich M, Prenzler PD, Patsalides E, McDonald S, Robards K. 2002. Methods for testing antioxidant activity. The Analyst 127:18398.
  • Arihara K. 2006. Functional properties of bioactive peptides derived from meat proteins. In: Nollet L, Toldrá F, editors. Advanced technologies for meat processing. Boca Raton, Fla.: CRC Press. p 24573.
  • Arihara K, Ohata M. 2008. Bioactive compounds in meat. In: Toldrá F, editor. Meat biotechnology. New York, N.Y.: Springer New York. p 23149.
  • Betgovargez E, Knudson V, Simonian MH. 2005. Characterization of proteins in the human serum proteome. J Biomol Technol 16:30610.
  • Catiau L, Traisnel J, Chihib N-E, Le Flem G, Blanpain A, Melnyk O, Guillochon D, Nedjar-Arroume N. 2011a. RYH: a minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity. Peptides 32:14638.
  • Catiau L, Traisnel J, Delval-Dubois V, Chihib N-E, Guillochon D, Nedjar-Arroume N. 2011b. Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain: KYR. Peptides 32:6338.
  • Chang CY, Wu KC, Chiang SH. 2007. Antioxidant properties and protein compositions of porcine haemoglobin hydrolysates. Food Chem 100:153743.
  • Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW. 1980. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence. J Biol Chem 255:4017.
  • Choisnard L, Froidevaux R, Nedjar-Arroume N, Lignot B, Vercaigne-Marko D, Krier Fo, Dhulster P, Guillochon D. 2002. Kinetic study of the appearance of an anti-bacterial peptide in the course of bovine haemoglobin peptic hydrolysis. Biotech App Biochem 36:18794.
  • Chung MCM. 1984. Structure and function of transferrin. Biochem Edu 12:14654.
  • Clemente A. 2000. Enzymatic protein hydrolysates in human nutrition. Trends Food Sci Technol 11:25462.
  • Cofrades S, Guerra Ma, Carballo J, Fernández-Martín F, Colmenero FJ. 2000. Plasma protein and soy fiber content effect on Bologna sausage properties as influenced by fat level. J Food Sci 65:2817.
  • Daoud R, Dubois V, Bors-Dodita L, Nedjar-Arroume N, Krier F, Chihib NE, Mary P, Kouach M, Briand G, Guillochon D. 2005. New antibacterial peptide derived from bovine hemoglobin. Peptides 26:7139.
  • Davies C, McConnell M, Slobbe L, Haggarty N, Buchan G. 2005. Murine antigen-presenting cells are multifunctional in vitro biosensors for detecting the immunoactive potential of bovine milk products. J Nutri 135:26516.
  • Dàvila Ribot E. 2007. Advances in animal blood processing: development of a biopreservation system and insights on the functional properties of plasma. [Doctoral Thesis]. Girona, Spain: Universitat de Girona. p 184. Available from: Tesis Doctorals en Xarxa.
  • Del Hoyo P, Rendueles M, Díaz M. 2008. Effect of processing on functional properties of animal blood plasma. Meat Sci 78:5228.
  • Di Bernardini R, Harnedy P, Bolton D, Kerry J, O'Neill E, Mullen AM, Hayes M. 2011. Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products. Food Chem 124:1296307.
  • Erdmann K, Cheung BWY, Schröder H. 2008. The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J Nutri Biochem 19:64354.
  • Fallows SJ, Verner Wheelock J. 1982. By-products from the U.K. food system 2. The meat industry. Conserv Recycl 5:17382.
  • FAOSTAT. Food and Agriculture Organization of the United Nations. Available from: http://faostat.fao.org. Accessed 2012 Nov 22.
  • Fogaça AC, da Silva PI, Miranda MTM, Bianchi AG, Miranda A, Ribolla PEM, Daffre S. 1999. Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. J Biol Chem 274:253304.
  • Foltz M, van der Pijl PC, Duchateau GSMJE. 2010. Current in vitro testing of bioactive peptides is not valuable. J Nutri 140:1178.
  • Frankel EN, Meyer AS. 2000. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J Sci Food Agric 80:192541.
  • Froidevaux R, Krier F, Nedjar-Arroume N, Vercaigne-Marko D, Kosciarz E, Ruckebusch C, Dhulster P, Guillochon D. 2001. Antibacterial activity of a pepsin-derived bovine hemoglobin fragment. FEBS Lett 491:15963.
  • Frost C, Naudé R, Oelofsen W, Muramoto K, Naganuma T, Ogawa T. 2000. Purification and characterization of ostrich prothrombin. Intl J Biochem Cell Bio 32:11519.
  • Furlán LTR, Padilla AP, Campderrós ME. 2010. Functional and physical properties of bovine plasma proteins as a function of processing and pH, application in a food formulation. Adv J Food Sci Tech 2:25667.
  • Gatnau R, Polo J, Robert E. 2001. Plasma protein antimicrobial substitution at negligible risk. Cahiers Options Méditerranéennes 54:14150.
  • Gorbatov VM. 1988. Collection and utilization of blood and blood proteins for edible purposes in the USSR. In: Pearson AM, Dutson TR, editors. Edible meat by-products advances in meat research. London and New York, N.Y.: Elsevier Applied Science. p 167–96.
  • Hazato T, Kase R. 1986. Isolation of angiotensin-converting enzyme inhibitor from porcine plasma. Biochem Biophys Res Comm 139:525.
  • Hernández-Ledesma B, del Mar Contreras M, Recio I. 2011. Antihypertensive peptides: production, bioavailability and incorporation into foods. Adv Colloid Intl Sci 165:2335.
  • Hsieh YH, Ofori J. 2011. Food-grade proteins from animal by-products. In: Nollet LML, Toldra F, editors. Handbook of analysis of edible animal by-products. Boca Raton, Fla.: CRC Press. p 1335.
  • Hu J, Xu M, Hang B, Wang L, Wang Q, Chen J, Song T, Fu D, Wang Z, Wang S, Liu X. 2010. Isolation and characterization of an antimicrobial peptide from bovine hemoglobin α-subunit. World J Micro Biotech 27:76771.
  • Huang D, Ou B, Prior RL. 2005. The chemistry behind antioxidant capacity assays. J Agric Food Chem 53:184156.
  • Hyun CK, Shin HK. 1998. Utilization of bovine blood plasma obtained from a slaughterhouse for economic production of probiotics. J Ferment Bioengr 86:347.
  • Hyun CK, Shin HK. 2000. Utilization of bovine blood plasma proteins for the production of angiotensin I converting enzyme inhibitory peptides. Process Biochem 36:6571.
  • Jayathilakan K, Sultana K, Radhakrishna K, Bawa AS. 2011. Utilization of byproducts and waste materials from meat, poultry and fish processing industries: a review. J Food Sci Technol 49:27893.
  • Kitts DD, Weiler K. 2003. Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Curr Pharm Des 9:130923.
  • Korhonen H, Pihlanto A. 2003. Food-derived bioactive peptides-opportunities for designing future foods. Curr Pharm Des 9:1297308.
  • Kris-Etherton PM, Lefevre M, Beecher GR, Gross MD, Keen CL, Etherton TD. 2004. Bioactive compounds in nutrition and health research methodologies for establishing biological function: the antioxidant and anti-inflammatory effects of flavonoids on atherosclerosis. Annu Rev Nutri 24:51138.
  • Lantz I, Glamsta EL, Talback L, Nyberg F. 1991. Hemorphins derived from hemoglobin have an inhibitory action on angiotensin converting enzyme activity. FEBS Lett 287:3941.
  • Lee CC, Johnson LA, Love JA, Johnson S. 1991. Effects of processing and usage level on performance of bovine plasma as an egg white substitute in cakes. Cereal Chem 68:1004.
  • Lee CC, Love JA, Johnson LA. 1993. Sensory and physical properties of cakes with bovine plasma products substituted for egg. Cereal Chem 70:1818.
  • Lee SH, Song KB. 2009a. Purification of a calcium-binding peptide from hydrolysates of porcine blood plasma protein. J Korean Soc App Bio Chem 52:2904.
  • Lee SH, Song KB. 2009b. Purification of an iron-binding nona-peptide from hydrolysates of porcine blood plasma protein. Process Biochem 44:37881.
  • Li GH, Le GW, Shi YH, Shrestha S. 2004. Angiotensin I–converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutr Res 24:46986.
  • Lignot B, Froidevaux R, Nedjar-Arroume N, Guillochon D. 1999. Solvent effect on kinetics of appearance of haemorphins in the course of peptic hydrolysis of bovine haemoglobin. Biotech App Biochem 29(Pt 1):2530.
  • Liu Q, Kong B, Jiang L, Cui X, Liu J. 2009. Free radical scavenging activity of porcine plasma protein hydrolysates determined by electron spin resonance spectrometer. LWT—Food Sci Technol 42:95662.
  • Liu Q, Kong B, Xiong YL, Xia X. 2010a. Antioxidant activity and functional properties of porcine plasma protein hydrolysate as influenced by the degree of hydrolysis. Food Chem 118:40310.
  • Liu X, Song C, Chen R, Jiang X, Jin Y, Zou H. 2010b. Identification of angiotensin I-converting enzyme inhibitors in peptides mixture of hydrolyzed red deer plasma with proteomic approach. Chinese J Chem 28:166572.
  • Lopes SCDN, Fedorov A, Castanho MARB. 2005. Lipidic membranes are potential “catalysts” in the ligand activity of the multifunctional pentapeptide neokyotorphin. Chembiochem 6:697702.
  • Lundblad RL. 2005. Considerations for the use of blood plasma and serum for proteomic analysis. Internet J Gen Prot 1:2. Available from: http://www.ispub.com/journal/the-internet-journal-of-genomics-and-proteomics/volume-1-number-2/considerations-for-the-use-of-blood-plasma-and-serum-for-proteomic-analysis.html#sthash.SqY5H3gK.dpbs. Accessed 2012 June 15,.
  • McConnell MA, Buchan G, Borissenko MV, Brooks HJL. 2001. A comparison of IgG and IgG1 activity in an early milk concentrate from non-immunised cows and a milk from hyperimmunised animals. Food Res Intl 34:25561.
  • Mito K, Fujii M, Kuwahara M, Matsumura N, Shimizu T, Sugano S, Karaki H. 1996. Antihypertensive effect of angiotensin I-converting enzyme inhibitory peptides derived from hemoglobin. Eur J Pharmacol 304:938.
  • Myhara RM, Kruger G. 1998. The performance of decolorized bovine plasma protein as a replacement for egg white in high ratio white cakes. Food Qual Pref 9:1358.
  • Nedjar-Arroume N, Dubois-Delval V, Adje EY, Traisnel J, Krier F, Mary P, Kouach M, Briand G, Guillochon D. 2008. Bovine hemoglobin: an attractive source of antibacterial peptides. Peptides 29:96977.
  • Nedjar-Arroume N, Dubois-Delval V, Miloudi K, Daoud R, Krier F, Kouach M, Briand G, Guillochon D. 2006. Isolation and characterization of four antibacterial peptides from bovine hemoglobin. Peptides 27:20829.
  • Nielsen PM, Petersen D, Dambmann C. 2001. Improved method for determining food protein degree of hydrolysis. J Food Sci 66:64246.
  • Ockerman HW, Hansen CL. 2000. Animal by-product processing and utilization. Boca Raton, Fla.: CRC Press.
  • Padilla S, Bouda J, Quiroz-Rocha G, Davalos J, Sanchez A. 2000. Biochemical and haematological values in venous blood of captive red deer (Cervus elaphus) at high altitude. Acta Veterinaria Brno 69:32731.
  • Parés D, Saguer E, Carretero C. 2011. Blood by-products as ingredients in processed meat. In: Kerry JP, Kerry JF, editors. Processed meats: improving safety, nutrition and quality. Cambridge, UK: Woodhead Publishing Ltd. p 21842.
  • Park E, Won M, Lee H-H, Bin Song K. 1996. Angiotensin converting enzyme inhibitory pentapeptide isolated from supernatant of pig plasma treated by trichloroacetic acid. Biotech Technol 10:47980.
  • Park KJ, Hyun CK. 2002. Antigenotoxic effects of the peptides derived from bovine blood plasma proteins. Enzyme Microb Technol 30:6338.
  • Pierce JL, Cromwell GL, Lindemann MD, Russell LE, Weaver EM. 2005. Effects of spray-dried animal plasma and immunoglobulins on performance of early weaned pigs. J Anim Sci 83:287685.
  • Pihlanto A, Korhonen H. 2003. Bioactive peptides and proteins. In: Taylor SL, editor. Advanced food nutrition research. San Diego, U.S.A.: Elsevier Inc. p 175276.
  • Piot JM, Guillochon D, Thomas D. 1986. Preparation of decolorized peptides from slaughter-house blood. MIRCEN J App Micro Biotech 2:35964.
  • Piot JM, Zhao Q, Guillochon D, Ricart G, Thomas D. 1992. Isolation and characterization of two opioid peptides from a bovine hemoglobin peptic hydrolysate. Biochem Biophys Res Comm 189:10110.
  • Pommer K. 1995. New proteolytic enzymes for the production of savory ingredients. Cereal Foods World 40:7458.
  • Rawdkuen S, Benjakul S, Visessanguan W, Lanier TC. 2004. Chicken plasma protein: Proteinase inhibitory activity and its effect on surimi gel properties. Food Res Intl 37:15665.
  • Ren Y, Wan DG, Lu XM, Chen L, Zhang TE, Guo JL. 2011. Isolation and characterization of angiotensin I-converting enzyme inhibitor peptides derived from porcine hemoglobin. Scientific Res Essays 6:62629.
  • Rosef O, Nystøyl HL, Solenes T, Arnemo JM. 2010. Haematological and serum biochemical reference values in free-ranging red deer (Cervus elaphus atlanticus). Rangifer 24:7985.
  • Ryan JT, Ross RP, Bolton D, Fitzgerald GF, Stanton C. 2011. Bioactive peptides from muscle sources: Meat and fish. Nutrients 3:76591.
  • Saguer E, Alvarez P, Ismail AA. 2012. Heat-induced denaturation/aggregation of porcine plasma and its fractions studied by FTIR spectroscopy. Food Hydrocoll 27:20819.
  • Saïd B, Dominique B. 2011. Mineral-binding peptides from food. In: Hettiarachchy NS, Sato K, Marshall MR, Kannan A, editors. Bioactive food proteins and peptides. Applications in human health. Boca Raton, Fla.: CRC Press. p 11730.
  • Sanchez-Moreno C. 2002. Review: methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci Technol Intl 8:12137.
  • Santos EM, González-Fernández C, Jaime I, Rovira J. 2003. Physicochemical and sensory characterisation of Morcilla de Burgos, a traditional Spanish blood sausage. Meat Sci 65:8938
  • Shalaby SM, Zakora M, Otte J. 2006. Performance of two commonly used angiotensin-converting enzyme inhibition assays using FA-PGG and HHL as substrates. J Dairy Res 73:17886.
  • Shamova O, Brogden KA, Zhao C, Nguyen T, Kokryakov VN, Lehrer RI. 1999. Purification and properties of proline-rich antimicrobial peptides from sheep and goat leukocytes. Infect Immun 67:410611.
  • Shamova O, Orlov D, Stegemann C, Czihal P, Hoffmann R, Brogden K, Kolodkin N, Sakuta G, Tossi A, Sahl HG, Kokryakov V, Lehrer R. 2009. ChBac3.4: a novel proline-rich antimicrobial peptide from goat leukocytes. Intl J Peptide Res Therapeut 15:10719.
  • Silva VDM, Silvestre MPC. 2003. Functional properties of bovine blood plasma intended for use as a functional ingredient in human food. LWT—Food Sci Technol 36:70918.
  • Su R-X, Qi W, He Z-M. 2006. Time-dependent nature in peptic hydrolysis of native bovine hemoglobin. Eur Food Res Technol 225:63747.
  • Sun Q, Luo Y, Shen H, Hu X. 2011a. Effects of pH, temperature and enzyme to substrate ratio on the antioxidant activity of porcine hemoglobin hydrolysates prepared with pepsin. J Food Biochem 35:4461.
  • Sun Q, Luo Y, Shen H, Li X, Yao L. 2011b. Purification and characterisation of a novel antioxidant peptide from porcine haemoglobin hydrolysate. Intl J Food Sci Technol 47:14854.
  • Sun Q, Shen H, Luo Y. 2011c. Antioxidant activity of hydrolysates and peptide fractions derived from porcine hemoglobin. J Food Sci Technol 48:5360.
  • Tanaka K, Sawatani E, Shigueoka EM, Dias GA, Nakao HC, Arashiro F. 2001. Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology. Brazilian J Medical Bio Res 34:97783.
  • The Lancet Editorial. 1980. Captopril: benefits and risks in severe hypertension. The Lancet 316:12930.
  • Toldrá F, Aristoy MC, Mora L, Reig M. 2012. Innovations in value-addition of edible meat by-products. Meat Sci 92:2906.
  • Tomasinsig L, Zanetti M. 2005. The cathelicidins-structure, function and evolution. Curr Protein Pept Sci 6:2334.
  • Treffers C, Chen L, Anderson RC, Yu PL. 2005. Isolation and characterisation of antimicrobial peptides from deer neutrophils. Intl J Antimicrob Agents 26:1659.
  • Tseng T, Tsai C, Yang J, Chen M. 2006. Porcine blood plasma transgluataminase combined with thrombin and fibrinogen as a binder in restructured meat. Asian Austra J Anim Sci 19:10544.
  • Vercruysse L, Van Camp J, Smagghe G. 2005. ACE inhibitory peptides derived from enzymatic hydrolysates of animal muscle protein: a review. J Agric Food Chem 53:810615.
  • Viana FR, Silva VDM, Delvivo FM, Bizzotto CS, Silvestre MPC. 2005. Quality of ham pâté containing bovine globin and plasma as fat replacers. Meat Sci 70:15360.
  • Visessanguan W, Benjakul S, An H. 2000. Porcine plasma proteins as a surimi protease inhibitor: effects on actomyosin gelation. J Food Sci 65:60711.
  • Walter T, Hertrampf E, Pizarro F, Olivares M, Llaguno S, Letelier A, Vega V, Stekel A. 1993. Effect of bovine-hemoglobin-fortified cookies on iron status of schoolchildren: a nationwide program in Chile. Am J Clin Nutri 57:1904.
  • Wanasundara JPD, Pegg RB, Shand PJ. 2003. Value added applications for plasma proteins from the beef processing industry. Canadian Meat Science Assn. CMSA News September 2003. p 1015. Available from: http://cmsa-ascv.ca/documents/2003September-03Wanasundarapgs10-15.pdf. Accessed 2012 July 13.
  • Wanasundara PKJPD, Amarowicz R, Pegg RB, Shand PJ. 2002a. Preparation and characterization of hydrolyzed proteins from defibrinated bovine plasma. J Food Sci 67:62330.
  • Wanasundara PKJPD, Ross ARS, Amarowicz R, Ambrose SJ, Pegg RB, Shand PJ. 2002b. Peptides with angiotensin I-converting enzyme (ACE) inhibitory activity from defibrinated, hydrolyzed bovine plasma. J Agric Food Chem 50:69818.
  • Wang JZ, Zhang H, Zhang M, Yao WT, Mao XY, Ren FZ. 2008. Antioxidant activity of hydrolysates and peptide fractions of porcine plasma albumin and globulin. J Food Biochem 32:693707.
  • Wei JT, Chiang BH. 2009. Bioactive peptide production by hydrolysis of porcine blood proteins in a continuous enzymatic membrane reactor. J Sci Food Agric 89:3728.
  • Wessely-Szponder J, Majer-Dziedzic B, Smolira A. 2010. Analysis of antimicrobial peptides from porcine neutrophils. J Microbiol Methods 83:812.
  • White M, Cook RS. 1974. Blood characteristics of free-ranging white-tailed deer in southern Texas. J Wildlife Dis 10:1824.
  • Wismer-Pedersen J. 1988. Use of haemoglobin in foods—a review. Meat Sci 24:3145.
  • Xu X, Cao R, He L, Yang N. 2009. Antioxidant activity of hydrolysates derived from porcine plasma. J Sci Food Agric 89:1897903.
  • Youatt WG. 1965. Composition of milk and blood in nursing white-tailed does and blood composition of their fawns. J Wildlife Manage 29:7984.
  • Yu PL, van der Linden DS, Sugiarto H, Anderson RC. 2010. Antimicrobial peptides isolated from the blood of farm animals. Anim Prod Sci 50:6609.
  • Yu Y, Hu J, Miyaguchi Y, Bai X, Du Y, Lin B. 2006. Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin. Peptides 27:29506.
  • Zhao Q, Garreau I, Sannier F, Piot JM. 1997. Opioid peptides derived from hemoglobin: hemorphins. Biopolymers 43:7598.
  • Zhao QY, Piot JM, Gautier V, Cottenceau G. 1996. Isolation and characterization of a bacterial growth-stimulating peptide from a peptic bovine hemoglobin hydrolysate. App Micro Biotech 45:77884.