STUDY OF HYDROLYZED PROTEIN COMPOSITION, FREE AMINO ACID, AND TAURINE CONTENT IN DIFFERENT MUSCLES OF GALICIAN BLONDE BEEF

jmf_218 769..784

STUDY OF HYDROLYZED PROTEIN COMPOSITION, FREEAMINO ACID, AND TAURINE CONTENT IN DIFFERENT

MUSCLES OF GALICIAN BLONDE BEEF

DANIEL FRANCO1, LAURA GONZÁLEZ, ESPERANZA BISPO,PATRICIA RODRÍGUEZ, JOSÉ IGNACIO GARABAL and TERESA MORENO

Instituto Gallego de la Calidad Alimentaria (INGACAL), Centro de InvestigacionesAgrarias de Mabegondo, Apdo 10 15080 A Coruña, Spain

Accepted for Publication February 5, 2009

ABSTRACT

The aim of this study was to identify and quantify hydrolyzed and freeamino acids including taurine in different muscles of the Blonde Galicianbreed. The main essential amino acids present in each of the muscles werelysine and leucine, with aspartic acid and glutamic being the most importantin the non-essential fraction. The chemical score and the index of essentialamino acids were calculated as correlation parameters of the biological valueof the proteins, with the best values being obtained for the muscles longisimusdorsi and semitendinosus. An analysis was also carried out of the amino acidin the free fraction. Taurine was found to be the main free amino acid in allmuscles, with the exception of the cardiac muscle, where histidine was themain one present. The muscles that presented higher amounts of taurine werethe masseter muscle followed by semimenbranous, and biceps femori, wheretaurine represented 40, 35, and 31%, respectively, of the total content of thefree fraction.

PRACTICAL APPLICATIONS

This study provides practical and useful information on the nutritionalquality of muscles, which are widely consumed in Spain. Several of thesemuscles are highly appreciated by the consumers in restaurants such as, eye ofround, sirloin, and masseter muscle. These results will be relevant for the cattleindustry, because this data add information to the fatty acid profile descriptionof samples studied in the same breed. It will be also useful for nutritionists indescribing tables of the food composition.

1 Corresponding author. TEL: 00 34 988548277; FAX: 00 34 9885482276; EMAIL: [email protected]

Journal of Muscle Foods 21 (2010) 769–784.© 2010, INGACALJournal of Muscle Foods © 2010, Wiley Periodicals, Inc.

769

INTRODUCTION

The quality of a product is the set of attributes that characterize it, andwhich determines if it is acceptable or not to the consumers. Taking intoaccount different criteria, such as the hygienic quality, the nutritive value,and the organoleptic characteristics, the term “quality of the meat” has dif-ferent interpretations for the consumer (Ouali 1991). The nutritive qualitycan be defined as the one that assures the presence of nutritive components(proteins, essential amino acids, essential fatty acids, minerals, vitamins, andmicroelements) and their sufficient bio-availability. Nowadays, consumerswant to know more about the nutritive quality of food, and in the case ofmeat, this depends on the nitrogenous components, basically protein of highbiological value, free amino acids (FAA), dipeptides and other nonproteinnitrogenous compounds (NPN). These compounds are responsible for thetaste and flavor in food (Kato et al. 1989). In fact, it has been establishedthat oxidative muscles are tastier than glycolytic ones (Valin et al. 1982) andthis is in part because of the FAA and dipeptides content (Cornet andBousset 1990).

The biological value of a protein basically depends on its composition ofessential amino acids. However, knowledge of the amino acid profile is notenough to quantify the nutritional quality of the protein because there are otherfactors that have an influence, such as the age and the physiological conditionof humans. There are some indexes, based on chemical, microbiological, andbiological methods that establish a better relationship between the composi-tion of the proteins and their nutritional quality, such as the chemical score, thedigestibility of the protein, and the protein efficiency rate. The first patternused as a standard protein, was the egg protein, but its use has been heavilycriticized because its composition of amino acids is not constant, and thecontent of some amino acids are excessive. Therefore, most of the proteinsfrom different foods turn out to be in deficit when they are compared with theegg pattern protein. Later, in 1985, the World Health Organization (WHO)proposed a pattern protein, based on experimental studies (WHO 1985) thatevaluate the amount of nitrogen necessary to produce a balance of equilibratednitrogen. Currently, the studies are focused on the search of methods thatevaluate amino acids metabolism. In fact, in the last review of the NationalAcademy of Sciences of the U.S.A. (Institute of Medicine 2002), the amountestimated of amino acids for human consumption was based on studies thatmeasured the metabolic utilization of different amino acids. This organizationproposes a new protein pattern for children and adults to evaluate food proteinqualities. Furthermore, some non-constituent amino acids of the protein, suchas taurine, play an important biological role because accumulation of taurineis necessary to regulate the function of the eyes, heart, brain and central

770 D. FRANCO ET AL.

nervous system (Sturman 1993; Stapleton et al. 1997; Schuller-Levis and Park2003). Taurine required by the human body is synthesized from the dietarysulphur amino acids methionine and cysteine; a baby’s metabolism is not ableto do this. Taurine is also highly recommended for heart diseases like conges-tive heart failure, which occurs when the cardiac muscle cannot pump theblood efficiently enough (Azuma et al. 1985).

The aim of this study is to characterize the amino acidic profile ofdifferent muscles, as well as to quantify the taurine content in veal from theBlonde Galician breed, in order to evaluate the meat of this breed from anutritive point of view, and as there is a lack of pre-existing information. Theknowledge obtained from this data complements the information, relative tothe fatty acid profile and content in conjugated linoleic acid, present in thesame breed, in identical conditions of handling that have been presented inprevious works (Varela et al. 2004; Moreno et al. 2006).

MATERIALS AND METHODS

Animals and Treatment of the Samples

Three male veal calves of the Blonde Galician breed from the experimen-tal herd of the Agricultural Research Center of Mabegondo were used for thisstudy. Animals were born in October and were together in pasture for 4months. Then, the animals spent 4 months indoor before being slaughtered 8months old. The concentrate rations consisted of hay silage and concentrate“ad libitum.” Animals were conventionally slaughtered at a commercial abat-toir 4 km from the Research Center, with a live weight of 361.10 � 12.70 kg.Carcasses were weighed and chilled at 4C in a cold chamber immediately afterslaughtering for 24 h. At this point, the muscles were extracted from the lefthalf of each carcass. Samples were taken immediately to the laboratory underrefrigerated conditions. Samples were extracted from the middle of the fol-lowing muscles: semitendinosus (ST), biceps femoris (BF), semimembranosus(SM), longisimus dorsi (LD), Masseter (MS) from which visual fat and con-nective tissue were eliminated. From cardiac muscle (CM) a sample of the wallof left ventricle was extracted, free of fat and connective tissue. All sampleswere ground and freeze dried in a freeze dryer (Dura-Stop FTS-SYSTEM,New York, NY) and afterward they were vacuum packed and frozen at -20Cfor posterior analysis.

Amino Acids and Protein Extraction

FAA Extraction. The extraction of FAA was carried out according to themethodology of Konosu and Yamaguchi (1982). In brief, 5 g of dry meat were

771GALICAN BLONDE BEEF MUSCLES

homogenized with 7% trichloroacetic acid (TCA) with a solid liquid ratio of1:4 in a Turrax (Heidolph DIAX 9000, Schwabach, Germany). Afterward, thesolution was centrifuged at 4,000 g for 20 min at 4C. The precipitate wasextracted once again with TCA under the same conditions described above andafterward the two supernatant layers were removed and placed in a knownvolume. An equivalent volume of diethyl ether was added to eliminate theTCA from the aqueous phase by liquid–liquid extraction. The extractionprocess was repeated twice and the aqueous phase was taken to dryness in arotator evaporator (Buchi R-114, Flawil, Switzerland) at 40C. The driedextract was dissolved in 10 mL of distilled water and frozen at -20C untilposterior analysis.

Protein Hydrolysis. The hydrolysis of the protein was carried out on100 mg of precipitated protein (dry basis) with 5 mL of HCL (6 N) in anampoule glass sealed for 24 h at 110C. After the hydrolysis, the solution wasdiluted with 200 mL of distilled water; it was filtered through 0.45 mm (FilterLaboratory, Barcelona, Spain). A corresponding aliquot was frozen at -20Cand stored until its posterior analysis. Tryptophan determination was notpossible, because acidic hydrolysis transform it into ammonium.

Analytical Methods

Chemical Composition and Glycogen. The chemical composition ofsamples was determined by near infrared reflectance spectroscopy (NIRS). TheNIRS equipment was calibrated against reference methods from Official Ref-erence Laboratory of Galicia according to described by Moreno et al. 2007.

Amount of glycogen was expressed as a concentration of glucose, mul-tiplying the content of glucose by 0.9 constant. The determination of theglucose was carried out in duplicate according to the method of Dubois et al.1956, on the extract obtained during the extraction of the amino acids.d-glucose (Panreac, Barcelona, Spain) was used as a standard and calibrationwas made on spectrophotometer (Perkin Elmer Lambda EZ 210 UV/VIS,Norwalk, CT) at 750 nm.

FAA and Constituent Amino Acid from Protein

Chemicals and Chromatographic Instrumentation. AccQ.Fluorreagent kit (AQC, borate buffer) and AccQ.Tag Eluent A concentrate wereacquired from Waters (Milford, MA, U.S.A.). Acetonitrile (MeCN), disodiumethylenediaminetetraacetic acid, phosphoric acid, sodium acetate trihydrate,and sodium azide were from Baker (Phillipsburg, PA, U.S.A.); triethylamine


was purchased from Aldrich (Milwaukee, WI, U.S.A.). Amino acid standardswere from Sigma (St. Louis, MO, U.S.A.) and taurine from Panreac (Barce-lona, Spain).

High performance liquid chromatography systems used were a Waterssystem consisting of two pumps (Model 515), a 2,475 scanning fluorescencedetector and a system equipped with a column heater and 717 Autosampler.Waters Millennium Chromatography Manager (v 4.0) was used to controlsystem operation and results management.

Derivatization of Standards and Samples andChromatographic Analysis

To yield a total volume of 100 mL, 10 mL of sample was buffered to pH8.8 (AccQ.Flour borate buffer). Derivatization was initiated by the addition of20 mL of AccQ-Fluor reagent (3 mg/ml in MeCN). Reaction of the AQC withall primary and secondary amines was rapid and excess reagent was hydro-lyzed within 1 min. Completion of hydrolysis of any tyrosine phenol modifi-cation was accelerated by heating for 10 min at 55°C.

Separations were carried out using a Water AccQ-Tag column(3.9 ¥ 150 mm with a 4 mm of particle size) with a flow-rate of 1.0 mL/min andperformed at 37C. The gradient profile and composition of the mobile phase wasadapted from methodology developed by Van wandelen and Cohen (1997) andWaters AccQ-Tag (1993). Detection was accomplished by fluorescence withexcitation at 250 nm and emission at 395 nm. Amino acids were identified byretention time using an amino acid standard to which taurine was added.

Protein quality. Chemical score of amino acids

Once the amount of amino acids in the different muscles were deter-mined, the chemical score of the essential amino acids (SQEAA) was calcu-lated in relation to the reference on pattern protein proposed by the Institute ofMedicine 2002 applying the following equation:

SQEAAg EAA in tested protein

g EAA in pattern protein= ×100

The essential amino acids index (IEAA) was also calculated applying thefollowing equation described by Hepher 1988.

IEAA = ∗ × ∗ × ∗ × ×100 100 100 100a

a

b

b

c

c

j

jp p p p

n ......


Where: a, b, c, . . . . j = percentage of EAA in tested protein.ap, bp, cp, . . . . jp = percentage of EAA in tested protein or in pattern

protein.n = number of amino acids used.

Statistical Analysis

For the statistical analysis of the results, data were analyzed using theSPSS 15.0 for Windows (SPSS, Chicago, IL) software package. One wayanalysis of variance was used to analyze the effect of type muscle on proxi-mate composition, hydrolyzed protein composition and FAA content. The leastsquares mean were separated using Duncan’s t-test. All statistical test of LSMwere performed for a significance level <0.05.

RESULTS AND DISCUSSION

Proximate Composition

There were no significant differences in approximate composition foreach muscles sample under study, except for ash and NPN compounds(Table 1). Moisture values for all muscles were around 76%. Regarding theprotein content, in general, the values found were similar for muscles; onlythe MS muscle’s protein content did not reach 20%, probably because of thedifficulty in eliminating all the connective tissue during the homogenizationstage of sample. The intramuscular fat content was not greater than 3% in anyof the muscles, the highest value being that of the MS (2.92%). The glycogencontent does not reach more than 0.3% in any of the muscles. With regard tothe NPN, we found significant differences in LD compared with the othermuscles, where it reached an average value of 0.25% (0.08 versus 0.25%;P < 0.05). The proximate composition found for each of the muscles wassimilar to that reported by Pearson and Young (1989). However, this authorreported higher content of carbohydrates, ashes, and NPN fraction than valuesshowed in our study (Table 1); probably because of the early age at which ouranimals were slaughtered. Pearson and Young (1989) indicate that the valuesof these compounds increase with the age of the animals, basically because ofthe decrease in moisture content. Brea et al. (1998) also confirm the hypothesismentioned above, when they worked on modeling the growth of the BlondeGalician veal breed. Pearson and Young (1989) reported values for NPNfraction in the range of 1.5–1.8%; whereas, we only found, as a best value,0.3% in the MS muscle. This is due to the fact that this fraction is not formedexclusively of FAA and ammonium: it includes other compounds like, nucle-otides, nucleosides, basis nitrogenous, amines . . . , etc. Regarding the FAA


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content, our results are in agreement with Shewan (1974), who found a valueof 0.35% FAA for muscles of mammals.

Amino acid Profile of Protein

The results related to the amino acid content of the precipitated proteinappear in Table 2. As we can see, in general, amino acid composition (g/100 gprotein) in different muscles showed a similar pattern. In fact, there were nosignificant differences in any amino acid between muscles. This result wasexpected and was reported by Schweigert (1987) who found that amino acidcomposition of the protein remains constant in different commercial cuts.Nevertheless, we found a higher value for isoleucine in the CM and for leucine,in the LD, in relation to the other muscles studied. Glutamic and aspartic acidwere the major amino acids found in the non-essential fraction of all the musclesstudied, and they reached the highest values in the LD (18.59 and 11.46%,respectively). In the essential fraction, the major amino acids were lysine andleucine, and again we found the highest values in the LD (18.63 and 9.49%,respectively). In this fraction, methionine in the MS showed the lowest contentin relation to the other muscles. Similar values were reported by (Paul andSouthgate 1978; Gorska et al. 1988; Elgasim and Alkanhal 1992) when study-ing veal samples. In other red meats; such as lamb, goat, and camel (Elgasim andAlkanhal 1992) and in white meats such as hen (Paul and Southgate 1978;Elgasim and Alkanhal 1992) and ostrich (Sales and Hayes 1996), glutamic andaspartic acid in the non-essential fraction and lysine and leucine in the essentialfraction, were also the major amino acids. Within the sulphur amino acids(methionine and cysteine), the highest values were detected in the LD (Table 2).In general, our values were similar to the ones found by Elgasim and Alkanhal(1992); although these authors found slightly higher values for all amino acids,especially for histidine where they obtained quantities 4.5 times higher. Thepoor values for histidine in our samples explains why the essential amino acid(ESAA)/non-essential amino acid (NEAA) ratio was, in the best case, 0.79 forSM; whereas, these authors obtained a ratio of 0.86.

Nutritional Quality of Protein from Different Muscles

The nutritional quality for each type of muscle was evaluated. The chemi-cal score for each of the essential amino acids with respect to the patternprotein, as proposed by the Institute of Medicine (2002) for humans (childrenof more than 1 year old and adults) was calculated (Table 3). The profile ofother pattern proteins (egg protein and reference protein Food and AgricultureOrganization/WHO 1985) is also shown for comparative purposes. The analy-sis of the chemical score (SQ) allows the order of the restrictive amino acidsto be determined. We can observe that the SQ of the isoleucine was highest in


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all muscles, reaching a maximum value of 397 in the CM, in comparison withother amino acids. Histidine was the only limiting amino acid in the rest of themuscles, with the exception of the MS. The different muscles were not limitedin sulphur and aromatic amino acids in relation to the reference protein.However, with regard to sulphur amino acids (Met + Cys) and aromatic aminoacids (Phe + Tyr), we can observe that they were not limiting amino acids.After leucine, they were the least abundant, respectively. The IEAA index wasthe highest for LD, followed by ST and BF, whereas the lowest value wasobtained for the MS.

FAA Profile and Taurine Content

The fraction of FAA found in fresh muscle is shown in Table 4. Highvariability for each of the amino acids inside each muscle was found andsignificant differences was detected in histidine, alanine (P < 0.05), asparticand glutamic acid, taurine and in the total amino acidic amount (P < 0.01).With the exception of LD, we found a range of values between 215 and 278 mgFAA in 100 g of fresh meat. Taurine was the most abundant FAA in themajority of muscles with the exception of the CM, where histidine was themost abundant. The MS muscle was the most important in terms of taurinecontent with a mean value of 111 mg of taurine in 100 g of fresh meat,followed by SM and the pair BF and ST with similar a value. LD and CM hadsimilar values close to 18 mg taurine/100 g fresh meat. (Purchas et al. 2004)obtained similar values of taurine to the ones shown in Table 4 for the CM,three times higher for the MS and lower for the ST (38.6 mg against

TABLE 3.PROTEIN QUALITY INDEX OF DIFFERENT MUSCLES (SQ AND IAAE)

Egg Pattern1983

FAO/WHO1985

N.A.2002

ST(SQ)

BF(SQ)

SM(SQ)

LD(SQ)

MS(SQ)

CM(SQ)

Histidine 2.6 2.0 1.8 96.1 96.1 87.7 82.2 108 96.6Isoleucine 5.5 4.0 2.5 232 292 264 224 259 397Leucine 9.1 7.0 5.5 164 136 119 172 118 101Lysine 6.9 5.5 5.1 206 194 178 208 170 179Met + Cys 3.9 3.5 2.5 182 180 152 200 127 168Phe + Tyr 10.4 6.0 4.7 198 197 171 228 174 202Threonine 5.2 4.0 2.7 194 177 174 190 154 181Valine 6.7 5.0 3.2 191 195 172 207 168 200IAEE 178 176 158 181 154 174

Pattern proteins are expresses in (g/100 g protein). Values of SQ and IEAA are referred only respect toN.A. 2002 protein pattern.ST, semitendinosus; BF, biceps femori; SM, seminebranous; LD, longisimus dorsi; MS, masseter; CM,cardiac muscle; SQ, chemical score; IEAA, index of essential amino acids.


TAB

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4.FR

EE

AM

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AC

IDS

(mg

FRE

EA

MIN

OA

CID

S/10

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FRE

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)

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pone

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e

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MS

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71�

1.67

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1.21

5.55

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292.

16�

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6.63

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173.

32�

0.19

n.s.

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cine

6.00

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90�

0.59

2.40

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55�

0.37

3.35

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161.

79�

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46�

2.46

4.35

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324.

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0.71

1.64

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115.

95�

1.88

3.53

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2.43

1.55

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0.08

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59.81 mg). These differences can be attributed to the different breed and age ofslaughter (Watanabe et al. 2004), type of feed (Koga et al. 1985) and gender(Hollo et al. 2003).

Obviously, the methodological aspects relative to the treatment andextraction of the samples might also be an influence, because of the differ-ences in the methodology used by different researchers. For example, insidethe muscle chewer, if we take a major proportion of the masseter it mighthave an influence on the final content of taurine (Purchas et al. 2004). Thesame situation occurs with the CM, because this author found values in therange of 25.4–85.1 mg taurine/100 g fresh heart. The values found by us inthe round piece (ST) were slightly higher than those reported by Spitze et al.2003. This great variability of the concentrations of taurine among similarsamples reveals the great effect of variables, such as diet, handling, breedand environmental conditions on the final content of taurine. The levels oftaurine are directly related to the different oxidative pattern of the porcinemuscles (Cornet and Bousset 1990; Aristoy and Toldra 1998), the highestvalues of taurine being in the oxidative muscles. In this study, we did notanalyze the different types of muscular fibers, but according to the classifi-cation of Talmant et al. (1986) for bovine, the more oxidative muscles in ourstudy (major proportion of red fibers type I) in decreasing order are:MS > CM > BF > ST> LD> SM, where the MS is the most oxidative and theSM is the most glycolic. Cornet and Bousset (1990), working with pigs,reported levels of taurine seven times greater in the MS than to those in thecorresponding LD. We have an identical situation because the value oftaurine in the MS was seven times higher to that found in the LD. The muscleSM had a different behavior, because it was the second muscle in taurineamount. A similar situation occurs with the CM. Because of its function as atypically aerobic muscle, it should have a high proportion of taurine but itonly had an amount of 18.53 mg taurine/100 g fresh muscle. However, thistaurine content was in agreement with Purchas et al. 2004, because thisauthor found values of 22.3 mg taurine /100 g CM in beef.

If we exclude taurine, the most abundant FAA were histidine, alanine andglutamic acid, the sum of them representing values around 50% of the total,and 77% in the case of CM. Ma et al. (1961) studied at a qualitative level, thepresence of FAA in different muscles of veal (ST, LD); alanine, and glutamicacid were the most abundant FAA; Parrish et al. (1969) also obtained thehighest values for glutamic acid and alanine in the muscle LD, but the quan-tities they found were four times lower than the ones obtained by us. Feidtet al. (1996) found that valine was the major FAA followed by alanine andglutamic acid, but the values that these authors quantified were about 50 timeslower than the ones found by us. Even smaller amounts were reported byMullen et al. (2000) and Watanabe et al. 2004 in LD samples, from different


breeds of veal. However, Mullen et al. 2000 found histidine was the main FAAfollowed by alanine, while for Watanabe et al. 2004 it was alanine.

CONCLUSIONS

From the results obtained on the quality of the proteins of the differentmuscles of the Blonde Galician breed, we can conclude that they possess ahigh nutritional value. There are no limiting amino acids with the exception ofhistidine, and the protein biological value of each of the muscles is very high.A better evaluation of the protein quality of the abovementioned muscleswould be obtained if we considered the protein digestibility. This would allowus to calculate the protein digestibility corrected amino acid score which isobtained by multiplying the value of the SQ by the value of the digestibility.Taurine is the most important FAA in all muscles, with a maximum of111.21 mg per 100 g of fresh muscle. Although there is not a recommendeddaily intake of taurine, from the physiological requirement point of view, in atypically omnivorous diet, an average amount of 58 mg taurine/day wasreported as necessary by Rana and Sanders (1986). According to this data,100 g of the muscles ST, BF, and SM from the Blonde Galician would con-tribute to the abovementioned amount, while the MS doubles this level of dailytaurine ingestion.

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Documents

STUDY OF HYDROLYZED PROTEIN COMPOSITION, FREE AMINO ACID, AND TAURINE CONTENT IN DIFFERENT MUSCLES OF GALICIAN BLONDE BEEF