Chapter 5 Mineral Composition of Blood Sausages – A Two-Case Study

It is well known that a balanced diet is essential in maintaining a good health; hence, the nutritional value of foods is an important aspect of food quality [1]. In this context, more and more people are becoming very concerned about the chemistry of what they eat. Conse‐ quently, food industry is interested in maintaining a high standard of quality of their manu‐ factured products which could meet the demands of an increasingly sophisticated consumer. Therefore, an important issue of food industry is the determination of food com‐ position and the establishment of analytical controls [2].


Introduction 1.Relevance of the assessment of mineral content in food
It is well known that a balanced diet is essential in maintaining a good health; hence, the nutritional value of foods is an important aspect of food quality [1].In this context, more and more people are becoming very concerned about the chemistry of what they eat.Consequently, food industry is interested in maintaining a high standard of quality of their manufactured products which could meet the demands of an increasingly sophisticated consumer.Therefore, an important issue of food industry is the determination of food composition and the establishment of analytical controls [2].Food scientists and food industry have long since been paying great attention to minerals in food, which has been mainly devoted to its essential role in human nutrition, i.e., physiological functions, humans' nutritional requirements, and mineral implication on safeness issues, i.e., mineral toxicity.There are more than 60 minerals in the human body, but only a few are considered to be essential, namely, iron, calcium, zinc, magnesium, phosphorus, sodium, potassium, manganese, selenium, copper.These minerals are absolutely essential to a host of vital processes, from bone and tooth formation, to the functioning of neurological, circulatory, renal and digestive systems, and some of them are necessary for regulation of enzyme systems [2,3].
Minerals deficiencies in human are common world-wide and there are evidences which suggest that deficiencies may play a main negative role in children's development, pregnancy and elderly health [3].In this context, Ca, K, Mg and Fe are the most commonly under-consumed minerals in humans' diet [4].Fe deficiency is the most common and widespread nutritional disorder in the world affecting both developing and industrialized nations [5].Insufficient intakes of Fe cause anemia, fatigue, poor growth, rickets and impaired cognitive performance in humans [3].On the other hand, the concentration of non-desired minerals in food can be increased by the persistent release of hazardous pollutants to the environment mainly derived from human industrial activity.This contamination of food supply can result in an increase of exposure of consumers to toxic metals such as lead, cadmium, arsenic and mercury, to levels higher than the tolerable daily intake [6].
The assessment of the mineral content in food is not only interesting from the nutritional and toxicological points of view.Since a few decades ago, instrumental analytical techniques based on atomic absorption or emission spectrometry applied to the determination of the mineral content coupled to multivariate statistical analysis have been proved to produce suitable methods to characterise food products, discriminate between food quality categories and control food authenticity, i.e., determination of the geographical origin of food, discrimination between cultivation methods (e.g.organic vs convenience crops), varieties of fruits and vegetables, or food processing practices [7][8][9][10].
The analysis of minerals in foods is challenging due to the wide range of concentrations present, which may vary from ppb to percent levels.The situation is further complicated by naturally occurring seasonal and varietal differences in concentrations within the same food [11].Official methods by de AOAC offers many single element methods based on colorimetric techniques: UV/Visible spectrophotometry, and flame and graphite furnace atomic absorption spectrophotometry.However, although no AOAC food methods currently employ Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES), it is a well-established multi-element technique that no requires the use of dangerous solvents from the environmental point of view [11].Its high specificity, multi-element detection capability and good detection limits result in the use of this technique in a large variety of applications.Detection limits typically range from parts per million (ppm) to parts per billion (ppb), although depending on the element and instrument, it can sometimes achieve even less than ppb detection [12].ICP-AES provides higher reproducibility and quantitative linear range compared to conventional AES, and reduces molecular interferences due to a higher temperature (7000-8000 K) in the excitation source (plasma).On the other hand, ICP-AES is more expensive than conventional AES, and in complex samples, emission patters can be of difficult interpretation [13].

Blood sausages, making process and chemical composition
Meat products are generally made from various raw materials (from different origins and suppliers), which are combined at the formulation stage in obedience to criteria of composition, technological factors, sensory characteristics, legal regulations and also economic efficiency and profit [14].
Among meat and meat products, muscle foods are the most commonly consumed.However several edible meat by-products and their derivatives are also importantly consumed in a Food Industry number of countries, where meat by-products are usually linked to traditional or ethnic foods.Meat by-products are traditionally sold to the lower income market however, by different reasons -one of them could be the increase in tourism -their consumption seems to be increasing and some of the by-products are becoming delicacies in niche markets.Advantageously, meat by-products consumption contributes to increase the edible portion of slaughter animals, Furthermore, meat edible by-products constitute an excellent source of nutrients like essentials amino acids, minerals and vitamins [15,16].Due to the great variety and specificity of edible meat by-products and their peculiar consumption patterns and their relative low economic value, there is relatively scarce information on their making process and chemical composition.
In some areas of the world, and to different degrees, blood is utilized as an edible meat byproduct.For example, for several ethnic groups of Africa and India, blood is the primary source of animal protein, where it holds ritualistic importance.However, in some cultures (Islamic and Jews), blood consumption is seen as a taboo [17,18].In Europe and Asia, animal blood has been traditionally used in making a variety of foods such as blood sausages, blood pudding, biscuits and bread, as well as blood soups and crackers [19,20].
From the nutritional point of view, blood is a good source of dietary protein, lysine and iron [19,21].The high iron content of blood (approximately between 400-500 mg of iron per liter), coupled with the high absorption of heme iron compared to non-heme iron, is particularly useful for food based strategies designed to combat iron deficiency anemia.Furthermore, the environmental concern associated with blood disposal at slaughterhouses, together with blood nutritive value, has fostered research and industrial efforts to recover blood or blood components, to be used into a wide range of food products or as dietary supplements [22].For example, blood or blood proteins (plasma or cellular fractions) are being used in meat products, primarily to increase protein levels and enhance water binding and emulsifying capacity.
Blood sausages are very popular traditional meat products in many parts of the world such as Europe, Latin America or Asia [23][24][25][26].In Europe, blood sausages are normally called morcilla and morcella in Spain and Portugal, black pudding in Great Britain, blutwurst and Thuringer blood sausage in Germany, blodpϕlse in Denmark, boudin noir in France, bloed worst in Belgium, blood-tongue sausage and black pudding in Austria, caltabosi cu singe in Hungary, vaerevorst in Estonia, kaszanka in Poland, biroldo in Italy.In Latin American countries, blood sausages are also produced and are named as relleno, prieta, moronga, mocillón in Mexico, Colombia, Peru or Argentina, and Morcela in Brazil; these sausages from Latin America show characteristics similar to those from Europe, especially to those of Iberian Peninsula [25].In this sense, blood sausages from Latin America can be included into the group of creole meat products, which means that they were originated from the adaptation of former Iberian meat products (brought to America by immigrants) to local condition and circumstances, thus, involving an innovation process at that time.
Nowadays, blood sausages are currently receiving worldwide increasing attention because they have become gourmet products in several countries, thus leading to an increase in their production and potential markets [27].Furthermore, increasing consumer demand for eth-nic specialties has renewed interest in such products, leading to a consequent need to assure safety and longer shelf-lives in an expanding market.Moreover, the Governmental Institutions, e.g., European Union, are getting more involved in the protection of high-quality traditional foods from specific regions or areas, which reflects a policy of supporting the inhabitants of rural areas and promoting regional products [23,27].
Blood sausages are basically made with blood, fat and a variety of vegetable origin food; Moreover the use of meat, pork skin or offal (e.g., liver, intestine) is common, mainly in German blood sausages [28][29][30][31].The vegetable-origin food used is enormously diverse so that, apart from spices and condiments, blood sausages can contain as main ingredients onion, leeks, cereals (rice, oat, flour, bread, etc.), sugar, fruits (apple, plum, etc.), nuts, etc. [32][33][34].Other ingredients such as eggs, cream, milk are used in some types of blood sausages in France [32].Moreover, as any meat product, blood sausages are added with common salt.The NaCl used in blood sausages from Mediterranean Europe tend to be between 1.2 and 1.5 % [35][36][37], and in blood sausages from Germany [38] and USA [16] tend to be higher, close to 2%.NaCl has a direct effect on the flavour and also increases the shelf-life, decreasing the water activity.Finally, several additives such as curing agents (nitrate and nitrite salts), pH modifiers (such as lactate or acetate salts) or emulsifiers can be also used [30,39].
The making process of blood sausages differs as a result of type, region and manufacturer.However, a common flow chart of the process of most of the blood sausages is depicted in Table 1 [21].The initial mixture of blood sausages is complex by the number of ingredients used and pre-treatments to which those ingredients have been undergone.For example, meat can be cured previously to the mixture preparation, or pork rind can be cooked and emulsified.Similarly, several ingredients, such as fat, onion or rice, can be cooked before the mixture is prepared.Once prepared, the initial mixture is normally stuffed into natural or artificial casings and the sausage is cooked in hot water until a temperature of 65-75 ºC is reached in the inner part of it [31], and then the sausage is chilled before refrigeration storage.Some varieties of blood sausages are dried and/or smoked after cooking.Once cooked and chilled, most of blood sausages present a dark-red to black colour and a rather firm and sliceable texture [30] due to the formation of a gel structure from the interaction of collagen, starch, blood proteins, etc.; nonetheless, some blood sausages are soft and spreadable.In general, meat and meat products are generally recognized as good sources of high biological-value proteins, group B vitamins, minerals as well as some other bioactive compounds [15].The composition of meat products depends on their formulation.Thus, the chemical composition of blood sausages is diverse and would depend on the ingredients and manufacturing process used.As a matter of reference, Table 2 shows the proximate composition of several blood sausages from Europe and Latin America.Moisture is expressed as percentage of fresh weight, and values of protein, fat, available carbohydrate, fibre and ash are expressed as percentage of dry matter.The literature sources for the data are the following (see Moisture content of blood sausages would depend inversely on the fat content and directly on the amount of moisture evaporated during an eventual drying/smoking stage.As can be seen in Table 2, the ranges of fat, available carbohydrate and protein in dry matter vary from 22.6 to 69.1, 0 to 51.1 and 7 to 58.9, respectively.There are great variations in dry matter composition between sausage types, which can be attributable to differences in the quanti-ties of the main ingredients used, i.e., pork fat, cereals, vegetables, meat or blood.Thus, the presence and levels of fibre are the result of the use of vegetables, namely onion, leek, fruits, etc.Finally, ash content is related to the amount of common salt used in the making process.
Regarding to the mineral content of blood sausages, the Fe content is the most reported in literature.Fe content of blood sausages is high due to the use of blood, and amounts reported vary from 6 to 16 mg per 100 g [16, 36,42,45].

Aim of the study
In spite of their popularity and increasing interest, literature on the composition and quality of blood sausages is to our knowledge scarce.The knowledge of the chemical composition of blood sausages presents potential usefulness regarding nutritional, product characterization and quality control aspects.Among the chemical composition, the mineral content of blood sausages seems to be a key point in those aspects.Therefore, the main aim of the present study is to describe and determine, as case studies, the manufacturing process and the chemical composition with particular interest on the mineral content, of two typical blood sausages produced in two different parts of the world: a typical blood sausage with white onion (Allium cepa), from the region of Leon (north-western Spain), known as Morcilla de Leon; and typical blood sausage with white cabbage (Brassica Oleracea var.capitata), from the region of Tumbes (north-western Peru), known as Relleno de Tumbes.

Making process of the blood sausages
In order to collect information about the making process of the blood sausage Morcilla de Leon, four interviews were conducted with the correspondent production managers at the four main local companies producing this sausage in Leon city.The two-member interview panel asked a set of questions regarding general company characteristics, raw materials used, making process and storage conditions.Moreover, collecting data on the making process of Relleno de Tumbes was carried out by standardized open-ended interviews conducted with 15 homemade manufacturers at the region of Tumbes (Tumbes city and small villages at Zarumilla province).The questions asked were to know information on the raw materials used and the making process followed.In both cases, the interviews were followed by the observation of the sausage making process.

Chemical analysis
A total of 8 samples of Morcilla de Leon were manufactured by local producers (city of Leon, north-western Spain) and were purchased from local markets.The sample weights were approximately 250 g.Once taken, the sausages were transported under refrigeration (<4 °C) to the laboratory of Department of Food Hygiene and Technology (University of Leon).On the other hand, a total of 12 samples of Relleno de Tumbes were obtained from Food Industry small local producers and retail stores in Tumbes City (north-western Peru) and small villages around the city.For each sausage sampled, a 300 g sample was packaged individually in a bag and transported in refrigerated containers to the laboratory in Tumbes.Subsequently, samples were frozen at -40° C and were transported to the laboratory at University of Leon where upon arrival at laboratory the samples were kept frozen at -40 °C until the analysis was performed.
Determinations of moisture, fat, protein and ash contents in the sausage samples were performed in duplicate according to methods recommended by the AOAC International [46] -Official Methods nos.950.46, 991.36, 981.10 and 920.153, respectively.Total dietary fibre was analysed following the AOAC 991.43 standard method [46], using the K-ACHDF 11/06 enzymatic kit (Megazyme, Wicklow, Ireland).Finally, the percentage of available carbohydrates was calculated by difference (100 -the percentage of the rest of components).
The analysis of mineral composition of sausages was performed by ICP-AES on wet digested samples.Duplicate aliquots of approximately 1 g (±0.01) of the previously homogenised samples were digested with 10 ml of concentrated HNO 3 in tightly closed screw cap glass tubes for 18 h at room temperature, and then for a further 4 h at 90 °C.For the analysis of sodium, potassium, sulphur and phosphorus, 1 ml of the mineralized solution was added with 8 ml of deionized water and 1 ml of scandium solution as internal standard.In order to determine the levels of calcium, copper, iron, magnesium, manganese and zinc, 3 ml of the digested solution was added with 6 ml of deionized water and 1 ml of Sc solution.

Statistical analysis
The software STATISTICA for Windows [47] was used for the statistical treatment of data.Furthermore, a principal component (PC) analysis, unrotated method, using the mineral composition as expressed as non-fat dry matter, was also performed.

Making process
The Morcilla de Leon (Figure 1), typically produced in the region of Leon (north-western Spain), is made from a mixture of chopped onion (used at amounts between 65 and 75 % of total weight), animal fat (lard and/or tallow; 10-20 %), blood (normally from pigs, 10-20 %), rice or breadcrumbs (2-10 %), salt (1-1.5 %), dry powdered paprika (1-2 %; including hot and sweet paprika), garlic and a mixture of spices (usually up to 1 g/kg) composed of several of the following: oregano, cumin, anis, cinnamon or pepper.Normally, onion and rice are precooked with the lard or tallow for 1-2 hours (until the onion becomes soft and tender).At the end of cooking, the condiments, spices and blood (liquid) are added and the mix is stirred from some minutes.Nevertheless, one manufacturer did not precook the onion and fat, and thus all ingredients (raw) were cold-mixed.The mixture, (hot if it was precooked or cold if not precooked) is stuffed in natural pork or beef casings of around 45 mm of diameter, tied or clipped forming 20-cm pieces.After the stuffing of the mix, the sausages are cooked in hot water at 80-90 °C for 20-45 min.After this step, sausages are drained hung at room temperature for a few hours and then chill-stored.This product is usually stored without packaging, and the shelf-life is around 12 days at refrigeration temperatures.Relleno de Tumbes (Figure 2) is a typical blood sausage from Northern Peru, which consists of a mixture of blood (approximately 30%), pork lard fat (10%), chopped cabbage (40%), chopped red and Chinese onion (5%), chopped fresh paprika (2%; including sweet and hot paprika local varieties), common salt (1.5%) and a number of herbs and spices at low quantities (spearmint, coriander, garlic, cumin, pepper) and a in-situ-prepared annatto oil extract; furthermore, the addition of glutamate is common.The amounts indicated above are roughly estimated because the manufacturers did not use scales and the interviewers did not carry a scale in order to weight the ingredients used in the making process.The blood (liquid or coagulated, sometimes precooked and shredded) is manually mixed with the lard, chopped vegetables and salt.Then, the mix is manually stuffed into natural pork casings (large intestine).The blood sausages are cooked in boiling water for approximately half an hour.After cooking, the blood sausages are cooled and then drained hung.

Proximate composition
The proximate composition of Morcilla de Leon and Relleno de Tumbes are shown in Tables 3 and 4, respectively.Moisture is expressed as percentage of fresh weight, and values of protein, fat, available carbohydrate, fibre and ash as percentage of fresh and dry matter weights.Moisture content variability would mainly depend on fat content and the degree of drying loss during cooling and storage.Furthermore, the presence and variability of protein, fat, available carbohydrates and total dietary fibre would be respectively explained mainly by the amounts of blood, lard or tallow, rice or breadcrumbs, onion or cabbage (plus other vegetal condiments and species) used in the formulation.In fresh weight basis, both types of blood sausages have a similar percentage of moisture.However, Morcilla de Leon shows lower amount of protein and higher of fat, fibre and available carbohydrates, than Relleno de Tumbes, both in fresh and dry weight basis.This is explained by a higher amount of blood and lower of vegetables and fat, being used in the Relleno de Tumbes making process, with respect to those being used for Morcilla de Leon.

Mineral composition
The mineral contents of Morcilla de Leon and Relleno de Tumbes are shown in Tables 5 and  6, respectively.Values (expressed as mg/100 g) are given in all fresh, dry and nonfat dry weight basis.Na is the mineral with the highest concentration, and the mean value seems slightly lower in Morcilla de León than in Relleno de Tumbes, where Na concentration shows a great variability between samples (high standard deviation).In average, Relleno de Tumbes contained higher amounts (approximately twice as much) of Ca and S macroelements and of Fe, Zn and Cu microelements.
The mineral content of blood sausages is the result of the sum of the contributions from all the ingredients used.In order to better ascertain the eventual contribution of ingredients to the mineral content of blood sausages, the mineral composition of the main ingredients used in Morcilla de Leon and/or Relleno de Tumbes is shown Table 7 [16,41,[48][49][50].From Table 7 and taking into account the quantities of the ingredients used in the making processes of the Food Industry blood sausages, it can be notice that blood appears to be the main source of Fe and Cu to blood sausages.On the other hand, onion and specially cabbage would be the main sources of K, Ca and Mn.Furthermore, S, P, Mg and Zn are importantly provided by both blood and vegetables, with the the high S content of cabbage being remarkable.Finally, lard seems not to be a good source of minerals and common salt, added at amounts of 1-2% to the sausage mixture, is the major source of Na in sausages (not shown in tables).In this context, the higher content of Fe and Cu in Relleno de Tumbes can be associated to the higher quantity of blood used.Similarly, the high content of Ca and S in cabbage together with the high quantity used in Relleno de Tumbes would account for the higher levels of those minerals with respect to Morcilla de Leon.From the nutritional point of view, comparing the mineral content of blood sausages (fresh weight basis) with that of pork meat or muscle meat products, such as frankfurters or chorizos [16-51], the blood sausages had considerably higher levels of Ca (more than three times), and Mn, Fe and Cu (more than ten times).On the contrary, amounts of K, P, S and Zn are slightly lower in blood sausages (up to 60% lower than those in meat).The levels of Mg were roughly comparable in meat and blood sausages, and those of Na depends on the quantities of common salt added.Having into account the Reference Labelling Values (RLVs) reported by the Scientific Committee of Food from the European Union [52], which are the following: K (2000 mg), Ca (1000 mg), P (700 mg), Na (600 mg), Mg (375 mg), Fe (14 mg), Zn (10 mg), Mn (2 mg) and Cu (1 mg); interestingly, a portion of 100 g of blood sausage equals or exceeds the recommended daily intake of Fe and contributes with 10-15% the recommended daily intake of Mn and Cu.Thus, the high iron content of blood, coupled with the high absorption of heme iron compared to non-heme iron, is particularly useful for food based strategies designed to combat iron deficiency anemia a major global malnutrition problem.Results of principal component analysis are shown in Figures 3 and 4. Principal component analysis was carried out with the mineral content expressed as mg per 100 g of nonfat dry matter for all the blood sausages analysed in this study.The first principal component (factor 1) accounted for a variance of 42.77% and the second of 19.89%.Figure 3 shows that samples from each type of blood sausage are located in two defined sets of results, which corroborate the differences in mineral contents found between both blood sausages.Figure 4 shows the projection of the variables (mineral contents) on the plane formed by the two principal components.The minerals with higher influence (factor loadings higher than 0.8) on factor 1 are Mn, Zn, and Ca.The mineral with higher influence on factor 2 was K (factor loading > 0.8).

Fresh weight
Moreover, in Figure 4 it can be seen that the most correlated mineral contents, as indicated by the highest proximity of points in the plain, were S with Ca and Mn with Zn.The first relation could be explained by the significant contribution of cabbage and onion to the S and Ca content in the sausage mixture.However, the second relation is difficult to explain from the contribution of ingredients.Other remarkable correlation is that of Fe with Cu, with blood being the main source of both of them.This correlation could be not as strong as expected due to the feasible migration of Fe ions to ingredients and sausage mixture from the surfaces of cast iron equipment, i.e., pans, knives, etc. [53], which are frequently present at Food Industry small homemade sausage producing facilities in small villages.This reason could be responsible for part of the distance between the Fe and Cu points.

Conclusion
The mineral content of two traditional blood sausages from different parts of the world: Morcilla de Leon and Relleno de Tumbes, as well as the proximate composition and general guidelines of the making process have been described in this study, which thus contribute to the chemical characterisation, diffusion and protection of these two traditional meat products.
The variety and quantities of ingredients used for blood sausage production have a significant relevance on their mineral content.Blood provides important quantities of Fe, Cu and Mn to the blood sausages from the nutritional point of view.The content of Fe of 100 g of Morcilla de Leon practically equals the daily requirements for adults and that of Relleno de Tumbes exceeds those requirements.
Food Industry

Figure 3 .
Figure 3. Principal component score plot (two first principal components or factors), considering mineral composition on non-fat dry matter basis, and showing samples according to sausage type: L, Morcilla de Leon; T, Relleno de Tumbes

Figure 4 .
Figure 4. Projection of the normalised factor coordinates of variables (mineral contents) in the 1 x 2 factor plane obtained by the principal component analysis

Table 1 .
General flow chart of blood sausage making process

Table 3 .
Proximate composition of the Spanish blood sausage Morcilla de Leon (n = 8).

Table 7 .
Essential mineral content (mg/100 g) of the main ingredients used in Morcilla de Leon and/or Relleno de Tumbes