- Email: [email protected]
Protein recovery from slaughterhouse wastes
Bioresource Technology 70 (1999) 129±133
Protein recovery from slaughterhouse wastes mez-Ju nb, J. Figueroaa C. Go areza, R. Castellanosa, T. Ponce-Noyolaa,*, V. Caldero a
Departamento de Biotecnologõa y Bioingenierõa; CINVESTAV-IPN. Av. Instituto Polit ecnico Nacional 2508, Zacatenco 07300 DF, M exico b Departamento de Bioquõmica, CINVESTAV-IPN. Av. Instituto Polit ecnico Nacional 2508, Zacatenco 07300 DF, M exico Received 9 July 1998; received in revised form 17 February 1999; accepted 18 February 1999
Abstract Only 15% of the waste blood generated in the Mexican meat industry is used for animal feed. However, waste blood is a slaughterhouse by-product that has potential for both animal feed and human food, because of its high protein concentration and quality. The aim of this work was to recover protein from red cells of bovine blood. Red cells were hydrolyzed with papain and up to 32% of the heme group released by this process, was separated by ultra®ltration. Treating the retentate with sodium hypochlorite produced a white product, which was 75% protein, nearly 1% ash, and almost tasteless. Results indicated that the protein contained in the white fraction had a good amino acid pro®le for use as an ingredient in formulated foods for human consumption. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Decolorisation; Blood; Protein
1. Introduction Bovine blood is a by-product of the meat industry. In Mexico, approximately 15% of the waste blood is used for animal feed (Martõnez R., 1993. Personal communication). The remaining blood is disposed by municipal sewers and land®lls, causing severe environmental problems due to the associated high organic pollutant (biochemical oxygen demand, BOD) and microbial loads. For instance, the world meat industry generates a potential pollutant load of 1:7 106 ton/y of BOD due to blood only, which is equivalent to the organic wastewater pollution caused by 11 000 000 people (Dart, 1974). In Mexico, 6 118 000 cattle were slaughtered in 1995, (INEGI, 1996) and generated 91 770 000 l of blood. This blood represented a BOD load of 7:74 104 ton. Protein recovery from waste blood in Mexico could signi®cantly reduce this pollution problem. Although waste blood contains a high concentration of good quality protein, its use for animal feed and human consumption is limited due to consumer aesthetic concerns. In eect, its brown color gives dark hues to food and feed products formulated with blood. Several techniques exist for decoloration of bovine blood, such as treatments with acid acetone (Tybor et al., * Corresponding author. Tel.: 0052-747-7000; fax: 0052-747-7003; e-mail: [email protected]
1975), hydrogen peroxide (Oord et al., 1979), carboxymethyl-cellulose (Autio et al., 1984), and enzymatic hydrolysis (Hald-Christensen, 1979; Adler-Nissen, 1986; Dondero et al., 1990). Some of these treatments are not cost-eective, while others impart a bitter or salty taste to the recovered protein. The aim of this work was to recover a palatable protein from bovine waste blood, by using a partial hydrolysis treatment. 2. Mathods 2.1. Waste blood Bovine waste blood was sampled from the Tlalnepantla municipal slaughterhouse, Edo. de Mexico, Mexico. The blood was collected in 50 l clean, refrigerated plastic containers. Sodium citrate at a concentration of 3 g lÿ1 was added to prevent blood coagulation. Blood was immediately centrifuged in an Alfa-Laval centrifuge (Mod. YEB 1334A-60) at 9500 rpm to separate plasma and red cell fractions (RCF). The RCF were lysed with water using a ratio of 1:3 RCF:water (v/v). The solutions were centrifuged to further separate hemoglobin from the erythrocyte membranes. The hemoglobin fraction was dried in a spray dryer (Niro Atomizer model P.63), at 190°C inlet temperature and 90°C outlet temperature. Dry solids were stored in airtight containers at 5°C.
0960-8524/99/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 3 0 - 9
130
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133
2.2. Reagents and treatment All chemical analyses were conducted using analytical grade reagents from J.T. Baker, Merck and Sigma. Commercial grade sodium hypochlorite was used for decoloration (5% concentration active chlorine). Soluble protein was determined over the pH range 2.5 to 10.5 according to the procedure reported in AOAC (1980). The heme group concentration was determined as described by Hayakawa et al. (1986) at the same pH values as above. Chemical analyses (protein, ash) were performed following AOAC methods (1980). Total iron was determined by atomic absorption spectrophotometry according to the Ocial Mexican Method NOM117-SSA1-1994 (Diario O®cial de la FederacioÂn, 1994). Amino acids were determined in a Beckman-Spinco auto-analyser, model 118 CL following the procedure outlined in the Beckman manual. The decoloration process is depicted in Fig. 1. A solution containing 8% (w/v) protein was prepared from dry hemoglobin. The pH was adjusted to 2.5 with 0.1 N HCl, and the temperature was kept constant in a 47°C
bath water. Papain (Mixim Laboratories, Mexico, 0.445 enzymatic activity/g) was added to give a concentration of 3 Anson units (AU). Hydrolysis was performed for 2 h while pH was maintained at 2.5 with 0.1 N NaOH. Hydrolysis was stopped by increasing temperature to 70°C for 20 min. After this, the hydrolysate was diluted 1:3 (v/v) with distilled water and the low molecular weight peptide fraction (less than 10 kDa) was separated by ultra®ltration (Millipore Pellicon Standar 13 l/min No. Cat. XX42PEL60, 5 ft2 membrane, Millipore No. Cat. PTGC00005). The retentate was further diluted 1:1 (v/v) with distilled water. For decoloration NaOCl was added (0.3 g/100 g protein) and mixed at 4500 rpm in a lab agitator (pH 2.5; 50°C). This concentration of NaOCl is permitted for bleaching of ¯ours by the Food and Drug Administration (FDA). The decolorized protein fraction was recovered by centrifugation at 2800 rpm (Manuel Tamez mod. 305 SPQ), washed with hot distilled water and dried in a spray dryer (Niro Atomizer mod. P. 63, air inlet temperature 190° and outlet temperature 90°C). All chemical determinations were done in triplicate and results were analyzed for one way analysis of variance with the program SAS (1992). 3. Results and discussion
Fig. 1. Procedure to obtain a protein concentrate from blood.
The evolution of protein during hydrolysis at pH's 2.5, 5.5, 7.5 and 10.5 are shown in Fig. 2. Soluble protein concentrations were lower at the extreme pH's (2.5 and 10.5) than corresponding values at pH's 5.5 and 7.5. The highest level of hydrolysis and soluble protein yield were obtained at pH 7.5, consistent with the pH required for optimum activity of papain. Drepper et al. (1981) showed that a higher protein degradation, produces higher formation of bitter-taste peptides. This problem is more acute at high values of hydrolysis. The fact that the proportion of released heme increases when the pH decreases is shown in Fig. 3. Also, at pH values lower than 4.5, the hemoglobin dissociation increases. At the lowest pH values, degradation of the prosthetic group is very high (An®nsen et al., 1964). In this work, a compromise solution for the hydrolysis step was set at pH 2.5, 47°C, enzyme concentration of 3 AU, substrate concentration 8% and a total reaction time of 2 h. In this way, a HD 3.6 was obtained. By-products of the hydrolysed heme group were eliminated by UF. These conditions yielded a moderately hydrolysed protein, good elimination of the heme group, and minimal generation of bitter taste peptides. The overall mass recovery was 70% of decolored dry product with a 75% protein content. Guigoz and Solms (1976) found that peptides with molecular weights of 3000 Da were associated with the bitter taste. On the other hand, Adler-Nissen and Olsen (1979) reported that the peptides with molecular weights
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133
131
Fig. 2. Evolution of soluble protein during hydrolysis of red cells from bovine blood.
Fig. 3. Eect of the hydrolysis pH on the percentage conversion of heme into the soluble fraction.
Fig. 4. Absorption spectra of the heme group that was subjected to dierent treatments - - - - non-treated hemoglobin, . . . globin obtained according to Tybor et al. (1975) procedure, ±±± globin treated in this work (decolored with NaOCl).
132
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133
Table 1 Chemical analysis of non-treated hemoglobin and globin decolored with NaOCl Component (%)
Non-treated hemoglobin
Protein Ash Total Fe
83.60 0.11 3.22 0.25 0.191 0.00
a
Globin decolored with NaOCl a
75.87 0.21 0.98 0.03 0.009 0.00
Mean value and standard deviation.
of approximately 1000 Da were responsible for the bitter taste of protein concentrates. Aubes-Defau and Combes (1997) found bitter peptides in the fraction corresponding to 500±5000 Da after concentrating a hemoglobin hydrolysate through UF. Thus, in this work using an UF membrane with a molecular weight cut-o of 10 kDa produced 32% elimination of the heme group and all bitter peptides. The results of the decoloration step are presented in terms of absorbance of the heme group in Fig. 4. The absorbance peak at 395 nm of the decolored sample in this work decreases signi®cantly, as compared to the natural hemoglobin. Weismer-Pedersen (1987) reported that the peak absorbance at 395 nm is due to hematin, and the absorbance at 275 nm is eected by the monomeric heme associated with globin. Fisher and Dodebeck (1940) and Heikel (1958) hypothesized that the oxidant NaOCl presumably cleaves the protoporphyrinic ring into two fragments via oxidation of the two methylene groups, causing the disappearance of the peak absorbance due to hematin. The chemical analyses of the white concentrate are summarized in Table 1. The protein concentration is high (75%) and ash content is signi®cantly lower than that in the non-treated hemoglobin. This reduction could be due to the hot water washings performed for
Table 2 Amino acid pro®les of non-treated hemoglobin and Globin decolored with NaOCl Amino acid
Non-treated hemoglobin
globin decolored with NaOCl g/100 g protein
1973 FAO Provisional pattern of essential amino acids
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Phenylalanine
10.01 0.15 5.85 0.07 3.61 0.22 11.81 0.08 5.53 0.04 5.95 0.16 9.29 0.16 5.04 0.13 11.45 0.20 5.31 0.15 0.34 0.05 13.80 0.11 8.08 0.07
10.47 0.25 1.83 0.02 4.76 0.03 13.54 0.27 2.38 0.10 5.19 0.11 7.49 0.30 3.54 0.05 6.73 0.33 6.55 0.14 0.48 0.02 15.37 0.25 8.67 0.07
5.5
eliminating NaOCl carry-overs. Iron content reduction eected by the treatment was 94%. The amino acid pro®les in Table 2 show that the white protein concentrate recovered in this work has substantial concentrations of both essential amino acids lysine and leucine. Valine and phenylalanine contents are higher than those recommended by the FAO/WHO (1973). Histidine concentration, however, is much lower than that in the non-treated hemoglobin. Since histidine is bonded to the heme group, and the latter is subjected to the hydrolytic attack during the treatment, histidine is lost and consequently its amount decreases. Isoleucine is the limiting amino acid in the protein concentrate. 4. Conclusions A white protein concentrate was recovered from bovine waste blood with a treatment consisting of red cells hemolysis, enzymatic hydrolysis, ultra®ltration with a 10 kDa cut-o membrane, decolorisation with NaOCl and drying. The concentrate represented a very good recovery yield of mass (70%) and was high in protein content (75%), almost tasteless and showed an adequate concentration of essential amino acids. Contents of histidine, isoleucine and methionine were low. However, this shortcoming could be easily overcome with speci®c amino acid supplementation or balancing the formulation with other foods high in these important nutrients. Overall, the concentrate seems to be a promising ingredient for human consumption, particularly when used as an additive to improve the nutritional value of protein-de®cient foods. Acknowledgements The authors wish to thank Prof. Noemi Rinderknecht-Seijas (ESIQIE del IPN) for the critical reading of the manuscript. Funding for this research was provided by the Mexican Council of Science and Technology (CONACYT, Research Project No. F374-A9304) and Department of Biotechnology CINVESTAV. A graduate scholarship to one of the authors (CG-J) from CONACYT is also gratefully acknowledged, and Blanca R. Arizona, J. Vega E. and B. Altamirano M. for their technical assistance.
4.0
References
5.0 4.0 7.0 6.0
Adler-Nissen, J., Olsen, H.S., 1979. The in¯uence of peptide chain length on taste and functional properties of enzymatically modi®ed soy protein. ACS, Symp. Ser. 92, 125±146. Adler-Nissen, J., 1986. A review of food proteins hydrolysis speci®c areas. In: Enzymic hydrolysis of food proteins. pp. 97±99. Elsevier, New York.
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133 An®nsen, B.C., Anson, L.M., Edsall, T.J., Richards, M.F., 1964. In: Advances in Protein Chemistry. 19, p. 78. Academic Press, New York. AOAC, 1980. Ocial methods of analysis. 13th ed. Assoc. of Ocial Analytical Chemists. Washington DC. Aubes-Defau, I., Combes, D., 1997. Eect of dierent proteases on bitterness of hemoglobin hydrolysates. Appl. Biochem. Biotechnol. 67 (1/2), 127±138. Autio, K., Kiesvaara, M., Malkii, Y., Kanko, S., 1984. Chemical and functional properties of blood globin prepared by a new method. J. Food Sci. 49, 859±862. Dart, M.C., 1974. Treatment of wastewaters from the meat industry. Proc. Biochem. 9 (5), 11±14. Diario O®cial de la Federaci on, 1994. Norma O®cial Mexicana NOM117-SSA1-1994. Bienes y Servicios. Metodo de prueba para la determinaci on de ®erro en alimentos, agua potable y agua puri®cada por absorci on at omica. Dondero, C.M., Kaiser, L.S., Pascual, S.J., Coneo, G.M., 1990. Aprovechamiento de sub-productos c arnicos IV. Decoloraci on enzimatica de sangre de bovino. Alimentos 3 (15), 5±13. Drepper, G., Drepper, K., Ludwig-Bush, 1981. Neue eiweiûprodukte aus schlachttierblut. Fleischwirtsch 9 (61), 1393±1395. FAO/WHO, 1973. Energy and protein requirements. Report of a joint FAO/WHO Expert Group. FAO Nutrition Meetings Report. Rome, Italy. Fisher, H., Dodebeck, H., 1940. Uber die pentdyopent-reaktion. Z. Physiol. Chem. 263, 125.
133
Guigoz, Y., Solms, J., 1976. Bitter peptides, occurrence and structure. Chem. Senses ¯avour 2, 71±84. Hald-Christensen, V., 1979. Decolourization of slaughter blood by partial enzymatic hydrolysis. The Meat Hygienist, Aug./Sep. 22± 26. Hayakawa, S., Matsuura, Y., Nakamura, R., Sato, Y., 1986. Eect of heat treatment on preparation of colorless globin from bovine hemoglobin using soluble carboxymethyl-cellulose. J. Food Sci. 51, 786±796. Heikel, T., 1958. A paper electrophoretic and paper chromatographic study of pentdyopent. Scand J. Clin. and Lab. Investigation 10, 191. INEGI, 1996. Anuario Estadõstico del Sector Alimentario en Mexico. Instituto Nacional de Estadõstica, Geografõa e Inform atica, Mexico DF, Mexico. Oord, A.H., Vaden, A., Wesdorp, J., 1979. Decoloration enzymatique du cruor D'abattoirs. Procede et aplications du produit. RTVA 22 (193), 29±35. SAS, 1992. Statistical Analysis System, Ver. 6. SAS Institute, Cary, North Carolina. Tybor, P.T., Dill, C.W., Landmann, 1975. Functional properties of proteins isolated from bovine blood by a continuous pilot process. J. Food Sci. 40, 155±159. Weismer-Pedersen, J., 1987. Decoloration of blood by heme oxidation. Proceedings of the 33th International Congress of Meat Science and Technol I, p. 119.
Protein recovery from slaughterhouse wastes mez-Ju nb, J. Figueroaa C. Go areza, R. Castellanosa, T. Ponce-Noyolaa,*, V. Caldero a
Departamento de Biotecnologõa y Bioingenierõa; CINVESTAV-IPN. Av. Instituto Polit ecnico Nacional 2508, Zacatenco 07300 DF, M exico b Departamento de Bioquõmica, CINVESTAV-IPN. Av. Instituto Polit ecnico Nacional 2508, Zacatenco 07300 DF, M exico Received 9 July 1998; received in revised form 17 February 1999; accepted 18 February 1999
Abstract Only 15% of the waste blood generated in the Mexican meat industry is used for animal feed. However, waste blood is a slaughterhouse by-product that has potential for both animal feed and human food, because of its high protein concentration and quality. The aim of this work was to recover protein from red cells of bovine blood. Red cells were hydrolyzed with papain and up to 32% of the heme group released by this process, was separated by ultra®ltration. Treating the retentate with sodium hypochlorite produced a white product, which was 75% protein, nearly 1% ash, and almost tasteless. Results indicated that the protein contained in the white fraction had a good amino acid pro®le for use as an ingredient in formulated foods for human consumption. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Decolorisation; Blood; Protein
1. Introduction Bovine blood is a by-product of the meat industry. In Mexico, approximately 15% of the waste blood is used for animal feed (Martõnez R., 1993. Personal communication). The remaining blood is disposed by municipal sewers and land®lls, causing severe environmental problems due to the associated high organic pollutant (biochemical oxygen demand, BOD) and microbial loads. For instance, the world meat industry generates a potential pollutant load of 1:7 106 ton/y of BOD due to blood only, which is equivalent to the organic wastewater pollution caused by 11 000 000 people (Dart, 1974). In Mexico, 6 118 000 cattle were slaughtered in 1995, (INEGI, 1996) and generated 91 770 000 l of blood. This blood represented a BOD load of 7:74 104 ton. Protein recovery from waste blood in Mexico could signi®cantly reduce this pollution problem. Although waste blood contains a high concentration of good quality protein, its use for animal feed and human consumption is limited due to consumer aesthetic concerns. In eect, its brown color gives dark hues to food and feed products formulated with blood. Several techniques exist for decoloration of bovine blood, such as treatments with acid acetone (Tybor et al., * Corresponding author. Tel.: 0052-747-7000; fax: 0052-747-7003; e-mail: [email protected]
1975), hydrogen peroxide (Oord et al., 1979), carboxymethyl-cellulose (Autio et al., 1984), and enzymatic hydrolysis (Hald-Christensen, 1979; Adler-Nissen, 1986; Dondero et al., 1990). Some of these treatments are not cost-eective, while others impart a bitter or salty taste to the recovered protein. The aim of this work was to recover a palatable protein from bovine waste blood, by using a partial hydrolysis treatment. 2. Mathods 2.1. Waste blood Bovine waste blood was sampled from the Tlalnepantla municipal slaughterhouse, Edo. de Mexico, Mexico. The blood was collected in 50 l clean, refrigerated plastic containers. Sodium citrate at a concentration of 3 g lÿ1 was added to prevent blood coagulation. Blood was immediately centrifuged in an Alfa-Laval centrifuge (Mod. YEB 1334A-60) at 9500 rpm to separate plasma and red cell fractions (RCF). The RCF were lysed with water using a ratio of 1:3 RCF:water (v/v). The solutions were centrifuged to further separate hemoglobin from the erythrocyte membranes. The hemoglobin fraction was dried in a spray dryer (Niro Atomizer model P.63), at 190°C inlet temperature and 90°C outlet temperature. Dry solids were stored in airtight containers at 5°C.
0960-8524/99/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 3 0 - 9
130
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133
2.2. Reagents and treatment All chemical analyses were conducted using analytical grade reagents from J.T. Baker, Merck and Sigma. Commercial grade sodium hypochlorite was used for decoloration (5% concentration active chlorine). Soluble protein was determined over the pH range 2.5 to 10.5 according to the procedure reported in AOAC (1980). The heme group concentration was determined as described by Hayakawa et al. (1986) at the same pH values as above. Chemical analyses (protein, ash) were performed following AOAC methods (1980). Total iron was determined by atomic absorption spectrophotometry according to the Ocial Mexican Method NOM117-SSA1-1994 (Diario O®cial de la FederacioÂn, 1994). Amino acids were determined in a Beckman-Spinco auto-analyser, model 118 CL following the procedure outlined in the Beckman manual. The decoloration process is depicted in Fig. 1. A solution containing 8% (w/v) protein was prepared from dry hemoglobin. The pH was adjusted to 2.5 with 0.1 N HCl, and the temperature was kept constant in a 47°C
bath water. Papain (Mixim Laboratories, Mexico, 0.445 enzymatic activity/g) was added to give a concentration of 3 Anson units (AU). Hydrolysis was performed for 2 h while pH was maintained at 2.5 with 0.1 N NaOH. Hydrolysis was stopped by increasing temperature to 70°C for 20 min. After this, the hydrolysate was diluted 1:3 (v/v) with distilled water and the low molecular weight peptide fraction (less than 10 kDa) was separated by ultra®ltration (Millipore Pellicon Standar 13 l/min No. Cat. XX42PEL60, 5 ft2 membrane, Millipore No. Cat. PTGC00005). The retentate was further diluted 1:1 (v/v) with distilled water. For decoloration NaOCl was added (0.3 g/100 g protein) and mixed at 4500 rpm in a lab agitator (pH 2.5; 50°C). This concentration of NaOCl is permitted for bleaching of ¯ours by the Food and Drug Administration (FDA). The decolorized protein fraction was recovered by centrifugation at 2800 rpm (Manuel Tamez mod. 305 SPQ), washed with hot distilled water and dried in a spray dryer (Niro Atomizer mod. P. 63, air inlet temperature 190° and outlet temperature 90°C). All chemical determinations were done in triplicate and results were analyzed for one way analysis of variance with the program SAS (1992). 3. Results and discussion
Fig. 1. Procedure to obtain a protein concentrate from blood.
The evolution of protein during hydrolysis at pH's 2.5, 5.5, 7.5 and 10.5 are shown in Fig. 2. Soluble protein concentrations were lower at the extreme pH's (2.5 and 10.5) than corresponding values at pH's 5.5 and 7.5. The highest level of hydrolysis and soluble protein yield were obtained at pH 7.5, consistent with the pH required for optimum activity of papain. Drepper et al. (1981) showed that a higher protein degradation, produces higher formation of bitter-taste peptides. This problem is more acute at high values of hydrolysis. The fact that the proportion of released heme increases when the pH decreases is shown in Fig. 3. Also, at pH values lower than 4.5, the hemoglobin dissociation increases. At the lowest pH values, degradation of the prosthetic group is very high (An®nsen et al., 1964). In this work, a compromise solution for the hydrolysis step was set at pH 2.5, 47°C, enzyme concentration of 3 AU, substrate concentration 8% and a total reaction time of 2 h. In this way, a HD 3.6 was obtained. By-products of the hydrolysed heme group were eliminated by UF. These conditions yielded a moderately hydrolysed protein, good elimination of the heme group, and minimal generation of bitter taste peptides. The overall mass recovery was 70% of decolored dry product with a 75% protein content. Guigoz and Solms (1976) found that peptides with molecular weights of 3000 Da were associated with the bitter taste. On the other hand, Adler-Nissen and Olsen (1979) reported that the peptides with molecular weights
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133
131
Fig. 2. Evolution of soluble protein during hydrolysis of red cells from bovine blood.
Fig. 3. Eect of the hydrolysis pH on the percentage conversion of heme into the soluble fraction.
Fig. 4. Absorption spectra of the heme group that was subjected to dierent treatments - - - - non-treated hemoglobin, . . . globin obtained according to Tybor et al. (1975) procedure, ±±± globin treated in this work (decolored with NaOCl).
132
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133
Table 1 Chemical analysis of non-treated hemoglobin and globin decolored with NaOCl Component (%)
Non-treated hemoglobin
Protein Ash Total Fe
83.60 0.11 3.22 0.25 0.191 0.00
a
Globin decolored with NaOCl a
75.87 0.21 0.98 0.03 0.009 0.00
Mean value and standard deviation.
of approximately 1000 Da were responsible for the bitter taste of protein concentrates. Aubes-Defau and Combes (1997) found bitter peptides in the fraction corresponding to 500±5000 Da after concentrating a hemoglobin hydrolysate through UF. Thus, in this work using an UF membrane with a molecular weight cut-o of 10 kDa produced 32% elimination of the heme group and all bitter peptides. The results of the decoloration step are presented in terms of absorbance of the heme group in Fig. 4. The absorbance peak at 395 nm of the decolored sample in this work decreases signi®cantly, as compared to the natural hemoglobin. Weismer-Pedersen (1987) reported that the peak absorbance at 395 nm is due to hematin, and the absorbance at 275 nm is eected by the monomeric heme associated with globin. Fisher and Dodebeck (1940) and Heikel (1958) hypothesized that the oxidant NaOCl presumably cleaves the protoporphyrinic ring into two fragments via oxidation of the two methylene groups, causing the disappearance of the peak absorbance due to hematin. The chemical analyses of the white concentrate are summarized in Table 1. The protein concentration is high (75%) and ash content is signi®cantly lower than that in the non-treated hemoglobin. This reduction could be due to the hot water washings performed for
Table 2 Amino acid pro®les of non-treated hemoglobin and Globin decolored with NaOCl Amino acid
Non-treated hemoglobin
globin decolored with NaOCl g/100 g protein
1973 FAO Provisional pattern of essential amino acids
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Isoleucine Leucine Phenylalanine
10.01 0.15 5.85 0.07 3.61 0.22 11.81 0.08 5.53 0.04 5.95 0.16 9.29 0.16 5.04 0.13 11.45 0.20 5.31 0.15 0.34 0.05 13.80 0.11 8.08 0.07
10.47 0.25 1.83 0.02 4.76 0.03 13.54 0.27 2.38 0.10 5.19 0.11 7.49 0.30 3.54 0.05 6.73 0.33 6.55 0.14 0.48 0.02 15.37 0.25 8.67 0.07
5.5
eliminating NaOCl carry-overs. Iron content reduction eected by the treatment was 94%. The amino acid pro®les in Table 2 show that the white protein concentrate recovered in this work has substantial concentrations of both essential amino acids lysine and leucine. Valine and phenylalanine contents are higher than those recommended by the FAO/WHO (1973). Histidine concentration, however, is much lower than that in the non-treated hemoglobin. Since histidine is bonded to the heme group, and the latter is subjected to the hydrolytic attack during the treatment, histidine is lost and consequently its amount decreases. Isoleucine is the limiting amino acid in the protein concentrate. 4. Conclusions A white protein concentrate was recovered from bovine waste blood with a treatment consisting of red cells hemolysis, enzymatic hydrolysis, ultra®ltration with a 10 kDa cut-o membrane, decolorisation with NaOCl and drying. The concentrate represented a very good recovery yield of mass (70%) and was high in protein content (75%), almost tasteless and showed an adequate concentration of essential amino acids. Contents of histidine, isoleucine and methionine were low. However, this shortcoming could be easily overcome with speci®c amino acid supplementation or balancing the formulation with other foods high in these important nutrients. Overall, the concentrate seems to be a promising ingredient for human consumption, particularly when used as an additive to improve the nutritional value of protein-de®cient foods. Acknowledgements The authors wish to thank Prof. Noemi Rinderknecht-Seijas (ESIQIE del IPN) for the critical reading of the manuscript. Funding for this research was provided by the Mexican Council of Science and Technology (CONACYT, Research Project No. F374-A9304) and Department of Biotechnology CINVESTAV. A graduate scholarship to one of the authors (CG-J) from CONACYT is also gratefully acknowledged, and Blanca R. Arizona, J. Vega E. and B. Altamirano M. for their technical assistance.
4.0
References
5.0 4.0 7.0 6.0
Adler-Nissen, J., Olsen, H.S., 1979. The in¯uence of peptide chain length on taste and functional properties of enzymatically modi®ed soy protein. ACS, Symp. Ser. 92, 125±146. Adler-Nissen, J., 1986. A review of food proteins hydrolysis speci®c areas. In: Enzymic hydrolysis of food proteins. pp. 97±99. Elsevier, New York.
C. G omez-Ju arez et al. / Bioresource Technology 70 (1999) 129±133 An®nsen, B.C., Anson, L.M., Edsall, T.J., Richards, M.F., 1964. In: Advances in Protein Chemistry. 19, p. 78. Academic Press, New York. AOAC, 1980. Ocial methods of analysis. 13th ed. Assoc. of Ocial Analytical Chemists. Washington DC. Aubes-Defau, I., Combes, D., 1997. Eect of dierent proteases on bitterness of hemoglobin hydrolysates. Appl. Biochem. Biotechnol. 67 (1/2), 127±138. Autio, K., Kiesvaara, M., Malkii, Y., Kanko, S., 1984. Chemical and functional properties of blood globin prepared by a new method. J. Food Sci. 49, 859±862. Dart, M.C., 1974. Treatment of wastewaters from the meat industry. Proc. Biochem. 9 (5), 11±14. Diario O®cial de la Federaci on, 1994. Norma O®cial Mexicana NOM117-SSA1-1994. Bienes y Servicios. Metodo de prueba para la determinaci on de ®erro en alimentos, agua potable y agua puri®cada por absorci on at omica. Dondero, C.M., Kaiser, L.S., Pascual, S.J., Coneo, G.M., 1990. Aprovechamiento de sub-productos c arnicos IV. Decoloraci on enzimatica de sangre de bovino. Alimentos 3 (15), 5±13. Drepper, G., Drepper, K., Ludwig-Bush, 1981. Neue eiweiûprodukte aus schlachttierblut. Fleischwirtsch 9 (61), 1393±1395. FAO/WHO, 1973. Energy and protein requirements. Report of a joint FAO/WHO Expert Group. FAO Nutrition Meetings Report. Rome, Italy. Fisher, H., Dodebeck, H., 1940. Uber die pentdyopent-reaktion. Z. Physiol. Chem. 263, 125.
133
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