Harnessing the Potential of Blood Proteins as Functional Ingredients: A Review of the State of the Art in Blood Processing ´ lvarez Garc´ıa Sarah A. Lynch, Anne Maria Mullen, Eileen E. O’Neill, and Carlos A
Abstract: Blood is generated in very large volumes as a by-product in slaughterhouses all around the world. On the one hand, blood generation presents a serious environmental issue because of its high pollutant capacity; however, on the other hand, blood has the potential to be collected and processed to generate high-added-value food ingredients based on its exceptional nutritive value and its excellent functional properties. In this paper, we review the current state of the art for blood processing, from collection to final recovery of protein isolates, the functional properties of blood, impact of processing on functional properties, and potential applications as food ingredients. Furthermore, future challenges are outlined for this underutilized and abundant product from the meat industry. Keywords: blood processing, food additives, functional properties, protein
Introduction: Legal Frame and Potential Use of Blood either because they are parts not normally eaten or sold commerBlood, in European legislation, is defined as offal; which is the “fresh meat other than that of the carcass, including viscera and blood.” Blood is subjected to the same regulations as carcass meat as outlined in EC Regulation nr 853/2004, which lays down specific hygiene rules for food of animal origin. However, blood that is not intended for human consumption, and has not being hygienically collected, but has been subjected to veterinary inspection, is then classified as an animal by-product (ABP). According to European legislation (Article 3 of Regulation [EC] 1069/2009), blood collected in slaughterhouses, not intended for human consumption, is defined as an ABP. Specifically, this regulation defines ABP thus: “entire bodies or parts of animals, products of animal origin, or other products obtained from animals that are not intended for human consumption.” This includes a multitude of different materials such as catering waste, used cooking oil, former foodstuffs, butchering and slaughterhouse wastes, blood, feathers, wool, hides and skins, fallen stock, pet animals, zoo and circus animals, hunt trophies, manure, and ova, embryos, and semen not intended for breeding purposes. This legislation classifies by-products into 3 categories, based on the risk for human health. Categories 1 and 2 are considered the highest risk, while category 3 is considered as low risk. Category 3 includes parts of animals that have been passed as fit for human consumption but which are not intended for consumption,
CRF3-2016-1880 Submitted 11/14/2016, Accepted 1/3/2017. Authors Lynch, ´ Mullen, and Alvarez are with Teagasc Food Research Centre, Food Quality and Sensory Science, Ashtown, Dublin 15, Ireland. Author O’Neill is with Dept. of Food and Nutritional Sciences, Univ. College Cork, Cork, Ireland. Direct inquiries to author Garc´ıa (E-mail:
[email protected]). C 2017 Institute of Food Technologists®
doi: 10.1111/1541-4337.12254
cially; blood obtained in slaughterhouses falls within this 3rd category. Federal Regulation (2015) in the U.S. (9 CFR 310.20) states how to collect and process the blood when intended for human consumption. In recent years, there has been growing global pressure on the food industry to try and minimize the environmental impact of its activities and to improve sustainability. This has led to increased interest in the more complete recovery and optimum utilization of by-products from the food industry (Otles and others 2015). European Directives are focusing on implanting the triple philosophy of reducing raw material, recycling, and reuse of by-products. In particular, the 2008/98/CE directive encourages waste prevention and promotes recovery and recycling; this document defined for the first time the difference between waste and by-product. Hence, a by-product is defined as “when substances or objects resulting from a production process not primarily aimed at producing such substances or objects are by-products and not waste.” According to this directive, a by-product differs from a waste because (i) it will be used after the main process; (ii) it can be used directly, with no ulterior transformation different from the regular industrial practices; (iii) it is produced as an integral part of an industrial process; and (iv) its ulterior use is legal, for instance, the substance or object meets all the requirements for the specific application relative to the products and the environment and health protection, and it will not produce negative impacts either on human health or the environment. Within this same directive, the European Union has proposed a 5-step waste management hierarchy based on waste reduction, reuse, recycling, recovery, and finally barring all other steps of disposal. Until very recently, the potential of food by-products to create new opportunities and markets has been underestimated (Otles and others 2015); blood is one of these
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Blood processing and food applications . . . underutilized materials and it has been identified as a key source of extractable high-value molecules for applications in sectors such as the food, biomedical, and pharmaceutical industries (Lafarga and others 2015, 2016; Mullen and others 2015). Animal blood produced in slaughterhouses represents the most problematic by-product of the meat industry because of the high volumes routinely generated and its very high polluting power. Liquid blood has a total nitrogen content of approximately 30 g/L, a chemical oxygen demand (COD) of approximately 400 g/L and a biological oxygen demand (BOD) of approximately 200 g/L (Moure Fern´andez 2000). These values exceed the legal limits established by European Directive 91/271/CEE (further modified by 98/15/CE directive) that quotes a maximum COD value of 125 mg for the treated waste. Even more, environmental legislation of the European Union (Commission Decision 2006/12/EC) encourages the recovery and use of by-products in order to conserve natural resources. A comprehensive review about European regulations regarding ABPs can be found in Leoci (2014). The volume and polluting potential of blood make it a prime material for recovery of value. By 2001, it was estimated that only 30% of total blood produced in slaughterhouses was used as food ingredient, mainly in black pudding and related products (Ofori and Hsieh 2012). According to these authors, the use of blood in the food industry has increased; however, there are no data to confirm this. As described below and highlighted in a number of papers (Tybor and others 1975; Ramos-Clamont and others 2003; ´ Selmane and others 2008; Alvarez and others 2009, 2012a; Furl´an and others 2010; Par´es and others 2014), blood has many functional and nutritive attributes. While some products will use whole blood, many applications require some degree of processing that ranges from separation of blood into fractions through isolation of individual proteins (Yang and Lin 1998c). Multiple new technologies have been developed in recent years aimed at recovering purified proteins from blood: liquid–liquid extraction (Selvakumar and others 2012), chemical precipitation (Imeson and others 1978; Moure and others 2003), salting-out (Par´es and others 2014), or chromatographic techniques (Howell and Lawrie 1983). Recent publications have highlighted potential applications for blood proteins, in particular in the food industry (Di Bernardini and others 2011; Ofori 2011; Par´es and others 2011; Tarte 2011; Jayathilakan and others 2012; Leoci 2014). Taking these factors, legal framework, new technologies, together with increased industry research interest in capitalizing on the inherent value of blood (Galanakis 2012), this approach can lead to enormous economic, societal, competitive, and environmental benefits, in terms of new jobs and opportunities in the global food market (Ofori and Hsieh 2012).
Table 1–Chemical composition of whole blood from different species of livestock. Adapted from Gorbatov (1988). Components Water Dry solids Hemoglobin Other proteins Sugar Cholesterol Fat Fatty acids
Components g/100 g Cattle 80.89 19.11 10.31 6.98 0.07 0.19 0.06 –
Pigs 79.06 20.94 14.22 4.26 0.07 0.04 0.11 0.05
Sheep 82.17 17.83 9.29 7.08 0.07 0.14 0.09 0.05
methionine and isoleucine, can be used as a source of high-quality proteins for both animal feed and human consumption (Tybor and others 1975; Imeson and others 1978; Duarte and others 1999; Prata and Sgarbieri 2008; Lee and Song 2009; Ofori and Hsieh 2011; Bah and others 2013; Par´es and others 2014).
Blood Collection
The method of blood collection varies according to its final use. Blood, which will be rendered or used for compost, is collected following less stringent hygienic requirements, often using an open-drain system. Blood collected in an open-drain system drains from the animal into a centralized vat. In this type of system, there are many opportunities for contaminating the blood (Par´es and others 2011; Bah and others 2013). When blood is intended for food, research, or pharmaceutical purposes, it is important to collect it in a more careful and hygienic way, using a closed-drain system. In this system, contamination is reduced (Par´es and others 2011), and blood is not exposed to air. At slaughter, it is collected directly into a hollow sticking knife that is connected to a flexible tube to direct the blood flow to a collection vessel. The blood is often collected in a batch-type manner from several animals and is then passed for use when the corresponding carcasses have been approved by veterinary inspection as fit for human consumption. The knives can also be fitted with disposable bags that hang from the end of the knife and collect the blood directly. While gravity is normally used to draw blood from the animals, vacuum systems may be employed to speed up the bleeding process (Knipe 1988). Blood can be drawn directly from the carcass to a refrigerated storage vessel. Drawbacks associated with some vacuum systems include larger capital investment required for pulsed vacuum pump equipment and a slowing of the slaughter line, which is particularly obvious in pig slaughter (Par´es and others 2011). One study from the 1980s, which compared closed drainage systems with or without pulsed vacuum pump, suggested that a vacuum system confers no special advantages (Gorbatov 1988). In some cases, they may collapse the blood vessels, thus hinder the flow of blood to the knife (Ockerman and Hansen 2000). Two types of Nutritive Value of Blood closed-drainage systems exist and are distinct for the knife used: Blood has excellent nutritive value, not only because of its flat-bladed or hollow-bladed knives. high protein content, but also because of the bioavailability of the nutrients (Par´es and others 2011). Chemical composition of (a) Flat-bladed knives consist of a metal cup fitted around the blood from different animal sources is shown in Table 1. It is base of a standard sticking knife attached to a flexible tube important to note, however, that blood is deficient in the essential that carries blood to a covered receiving container. The cup amino acids methionine and isoleucine (Par´es and others 2011) is placed against the animal and funnels the blood from the (Table 2). Blood is a rich source of iron, which is contained stick wound into the flexible tube. This method may still in the hemoglobin of red blood cells (RBCs), and this heme pose a risk of contamination, as the cup may come into iron has a high bioavailability as it is more easily absorbed than contact with contaminated hides or carcass. nonorganic iron from plants or the ferrous salts commonly used (b) Hollow-blade knives are available in several varieties: the in the fortification of foods (In and others 2002). It has been Rizzi knife, the Esktam knife, and the cannula knife. The well reported that blood protein, despite the lower amounts in Rizzi knife consists of a 1-inch-diam tube, 1 end of which 2 Comprehensive Reviews in Food Science and Food Safety r Vol. 00, 2017
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Blood processing and food applications . . . Table 2–Amino acid profiles of porcine whole blood and various blood proteins in comparison to myosin from muscle. Adapted from Gorbatov (1988). Amino acid content of blood proteins (g/100 g) Amino acid Phenylalanine Tryptophan Arginine Histidine Lysine Methionine Threonine Leucine Isoleucine Valine Aspartic acid Glutamic acid Cysteine Tyrosine
Whole blood
Fibrin
Hemoglobin
Serum globulin
Serum albumin
Myosin
10.7 1.5 – 8.8 9.7 2.4 4.8 13.2 0.9 8.7 – – – 1.4
4.6 3.5 6.7 2.3 9.0 2.6 7.9 7.1 5.0 3.9 11.9 13.8 1.5 6.0
9.6 2.0 3.5 8.5 10.6 1.2 6.0 14.9 0.0 11.0 10.0 7.4 0.9 2.9
4.7 2.8 5.8 2.1 6.3 1.0 7.4 9.5 2.0 9.7 9.0 12.5 2.3 6.7
6.6 0.7 5.9 4.0 12.8 0.8 5.8 12.3 2.6 5.9 10.9 16.5 5.9 5.1
3.2 0.8 7.0 1.7 10.3 3.4 3.8 15.6 – 2.6 8.5 21.0 14 2.2
is formed into a sharp point and the other end is connected to a flexible tube that carries the blood into the holding container. The Esktam knife consists of a hollow 2-sided knife with a rod or a 2nd knife set in the arched space at right angles of the butt of the hollow knife. This second serves to hold the stick wound open allowing free flow of blood; it is probably the best system for reducing the contamination of blood. The cannula system consists of a 0.5-inch hollow tube at 1 end, formed into a sharp cone shape with slots cut into the tip of the cone for blood to enter. Some companies have developed rotatory systems for blood collection based on hollow knifes, and such a technology permits collections up to 85% of total blood from 1000 pigs per hour, addressing all the regulations regarding the hygienic collection of blood and the traceability of the final product. One example of such rotatory systems can be seen at the Butina home page (http://www.butina.eu/products/blood_collection/). Hemolysis during bleeding and collection needs to be minimized. If hemoglobin is released, due to the disruption of the red cells membranes, the quality of the plasma may be compromised. It can be prevented by rinsing all equipment with an isotonic anticoagulant solution. This will create an osmotic pressure outside the RBCs, equal to the pressure inside the cells. If the osmotic pressure is not equalized, higher pressure inside the cells will cause them to lyse, releasing hemoglobin leading to lower quality plasma (Halliday 1973). In the event, 1 animal’s blood is contaminated, or if a carcass is condemned after inspection, then the blood from that carcass, and any other blood with which it is mixed, loses its edible status and the entire blood collection line, from the knife to the batch holding tank, must be cleaned and sanitized. Blood that has been contaminated from contact with the surface of the animal, or by other means, should not be collected for use in foods (Dill and Landmann 1988). With this regard, the European legislation is clear: “The different categories of ABPs must be kept separate from each other at all times, to avoid cross-contamination. They must also be kept separate from food for human consumption” (Regulation [EC] nr 1069/2009, Article 26 Implementing Regulation [EC] nr 142/2011). The carousel system, where blood from individual animals is held in containers mounted on a carousel, can maintain correlation with individual carcasses as the carcasses progress down the line on slaughter floor. If individual storage is not possible, it is a regular practice in small-/medium-size abattoirs that only the blood from a small number of animals is C 2017 Institute of Food Technologists®
mixed; hence, if any carcass is condemned the volume of blood that has to be rejected is not excessive.
Collection rates Approximately 5% to 9% of animal (live weight) is accounted for as blood, with the actual amount varying with species (beef 8%, pigs 5%, and sheep 8%). Up to 50% of the total blood of an animal is in circulation, 16% is present in the spleen, 20% is found in the liver and, 10 % in the skin. Bleeding after slaughter will yield 40% to 60% of the animal’s total blood with the rest remaining in capillaries and organs (Gorbatov 1988; Ockerman and Hansen 2000; Par´es and others 2011). Again, blood yields will vary with breed, degree of fatness, and stunning and bleeding method. Yield of edible blood is approximately 3.2% for beef and 3% for pigs based on live weight (Gorbatov 1988). Cattle bleeding will yield 12 to 15 L of blood with 11.4 to 14.5 kg collected within 60 to 120 s, while in pigs, bleeding yields 3 L with 2.7 kg collected in 30% to 40 s. The yield can be maximized when the time between stunning and sticking is reduced (Knipe 1988). Standard bleeding times used on the slaughter line are 6 min for bovine, 4 to 5 min for ovine, and 6 min for porcine animals (Ockerman 2000). Immediate blood processing After bleeding, blood will begin to clot within 3 to 10 min depending on the environmental temperature. This clotting process is a natural process, due to the action of the enzyme thrombin converting soluble fibrinogen to insoluble fibrin, in a 3dimensional network together with platelets and other proteins (Mosesson 2005). As blood is a rich organic medium, it is important to chill it as soon as possible to 2 to 4 °C to minimize microbial growth, which would lead to blood spoilage. However, it is important to note that blood refrigeration has been reported to lead an enrichment in psychrophilic bacteria, such as Pseudomonas species, which are able to promote protein degradation (Par´es and others 2011). Thus, rapid processing to stabilize the product is required. Several researchers have demonstrated that common downstream processes, such as spray drying, ultrafiltration, or low-pressure evaporation, do not reduce significantly the microbial load (Par´es and others 1998; Dailloux and others 2002). When serum is the final product, collected blood is allowed to clot at the same time that it is chilled, after which clots can be removed, for example, by means of centrifugation or decanting. Under controlled conditions, the red cells become trapped in the fibrin net and are removed with the clot, yielding a serum free of red cells. More frequently, liquid blood or plasma is required for further processing. In these cases, anticoagulants are added to
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Blood processing and food applications . . . Table 3–Key quality parameters of plasma processed for biotechnological purposes. Data proportioned by SeraLab S.L. on February 2015. Type Bovine serum Chicken serum Fetal calf serum Horse serum Pig serum
Protein g/dL
Osmolality mIsm/kg H2 O
Hemoglobin mg/dL
Endotoxin ng/mL
pH
Glucose mg/dL
7.0 to 9.0 3.0 to 6.5 3.5 to 4.5 6.0 to 9.0 6.0 to 8.0
270 to 340 270 to 340 260 to 340 270 to 330 260 to 350
