M.A. IN CULTURAL ASTRONOMY AND ASTROLOGY
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M.A. IN CULTURAL ASTRONOMY AND ASTROLOGY

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M.A. IN CULTURAL ASTRONOMY AND ASTROLOGY BATH SPA UNIVERSITY, BATH. SUBMITTED: 1 October, 2006 VENUS IN MYTHOLOGY, ASTROLOGY AND POETRY: What does the portrayal of Venus reflect about the experience of love in Western culture?
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Process Biochemistry 38 (2003) 1747 / 1759
www.else v ier.com/locate/procbio
Chemical composition and theoretical nutritional e v aluation of the produced fractions from enzymic hydrolysis of salmon frames with Protamex TM Bjørn Liaset * ,K˚areJulshamn,MaritEspe Institute of Nutrition, PO Box 185 Sentrum, N-5804 Bergen, Norway Recei v ed 4 July 2002; recei v ed in re v ised form 11 August 2002; accepted 30 August 2002
Abstract An amount of 200 kg fresh salmon frames were enzymic hydrolysed with the commercial protease mixture Protamex TM , which is known to produce non-bitter hydrolysates. After the enzymic procedure the frames were separated by centrifugation into fi v e fractions: an aqueous fraction rich in peptides, an insoluble fraction, an emulsion fraction, salmon oil and a bone fraction. Approximately 48% of total crude protein present in the salmon frames were found in the aqueous fraction, in which the lipid content was reduced to B / 0.1% in dry samples after ultramembrane filtration (UF fraction). The UF fraction was low in tryptophan, leucine and phenylalanine / tyrosine, but high in taurine. Nearly 19% of total crude protein present in the salmon frames were found in the insoluble fraction. This fraction was high in most of the indispensable amino acids. Approximately 77% of total lipids present in the salmon frames were isolated as salmon oil, which was high in both eicosapentaenic acid (EPA) and docosahexaenic acid (DHA). The bone fraction contained 62% of total ash present in the salmon frames and was high in the minerals Ca, P and Mg and also in the trace elements Cu, Fe, I, Mn, Se and Zn. All of the produced fractions were low in the undesirable substances As, Cd, Hg and Pb. For future studies the UF fraction and salmon oil might be interesting as health promoting agents, the insoluble fraction as dietary protein supplement and the bone fraction as dietary mineral supplement. # 2002 Else v ier Science Ltd. All rights reserved. Keywords: Salmon frames; Enzyme; Enzymic hydrolysis; Protamex TM ; Fish protein hydrolysate; Salmon oil; Fish bone; Chemical composition; Nutritional e v aluation
1. Introduction such as fish meal and fish silage [1] . From an ecological point of v iew, raw materials such as fresh salmon frames The salmon farming industry has grown enormously deser v e to be included in more high-v alued products. both in Norway and world-wide during the recent years. The salmon frames consist of three main fractions, Much of the salmon are sold to the customer as gutted, proteins, lipids and bones that each might be utilised for whole salmon, but significant amounts also are sold as different applications. From a nutritional point of v iew, fillets. In a typical automated filleting line, the fillets the salmon muscle protein has a well-balanced amino count for approximately 59 / 63% of the total wet weight acid composition, high in most of the indispensable in a salmon with body weight of 5 / 6 kg. Other products amino acids [2] . The salmon oil is rich in highly from the filleting line are salmon frame (9 / 15%), head polyunsaturated fatty acids, such as eicosapentaenic (10 / 12%) and trimmings (1 / 2%). In Norway today, acid (EPA) and docosahexaenic acid (DHA) [3] . The high-quality raw materials from se v eralspeciesmostlybmoinneeraflrsa.ctRioecnentmliyg,hftollbeianggotohdedsiuspcopl v ieerryooffdbieot v airnye are utilised in the production of low-v aluedproductsspongiformencephalopoatwhy(BSE)andtransmissible spongiform encephalopathies (TSE), there is an ongoing * Corr Tel.: / 47-55-905 discussion about the usage of protein hydrolysates and 299.espondingauthor.-114;fax: / 47-55-905-bone meal from farmed terrestrial animals [4 / 6] . These E-mail address: bjorn.liaset@nutr.fiskeridir.no (B. Liaset). incidences should make the utilisation of bones and 0032-9592/03/$ - see front matter # 2002 Else v ier Science Ltd. All rights reserved. doi:10.1016/S0032-9592(02)00251-0
1748 B. Liaset et al. / Process Biochemistry 38 (2003) 1747 / 1759 peptides of marine origin interesting in the substitution 2. Material and methods of the abo v e mentioned resources. Ob v iously, this demands proper treatment of the fresh salmon frames. Through enzymic treatment, the salmon frames can be 2.1. Raw material separated into an aqueous fraction rich in soluble led fish protein hydrolysate, FPH), an Frames without hea eshl illeted Atlantic innistroolugbelne(ncitarlogenfraction,anemulsionfraction,sal-salmon( Salmo salar ,Ld.s)fwroermetfrakenydifrectlyfromthe mon oil and a bone fraction rich in protein and minerals oduc ( Fig. 1 ).Theenzymichydrolysingprocessishighlypwreek,ttihoenflrionzeenanfdrafmroezsenwearte u / s2e0d 9 / i2n 8 tCh.eWenitzhyinmica controllable, and depending on the hydrolysing condi- olf Olsen Skarholm As-tions, the products are reproducible and well defined. In hydrolysing process. R en AS ( køy, Norway) kindly supplied the salmon frames. a pre v ious work, we focused on the nitrogen reco v ery in the aqueous fraction from the hydrolysis of salmon frames with Protamex TM at different hydrolysing condi-tions [7] . Fish protein hydrolysates can be v ery bitter, 2.2. Enzyme but this bitterness can be a v oided by controlling the degree of hydrolysis and minimise the amount of bitter Protamex TM (E.C. 3.4.21.62/ 3.4.24.28) is a Bacillus tasting peptides produced during the enzymic treatment protease complex from No v ozymes A/S (Bags v aerd, [8,9] . To produce fish protein hydrolysates of high Denmark), de v eloped for the hydrolysis of food proteins palatability, care must be taken in the choice of enzyme and fulfils the purity demands for food-grade enzymes, used. Therefore, the commercial protease mixture Pro- set by the Joint FAO/WHO Expert Committee on Food tamex TM , known to produce non-bitter hydrolysates, has Additi v es (JEFTA) and the Food Chemicals Codex been chosen in the present experiment. (FCC). The optimal working conditions for Protamex TM The aim of this study was to document the chemical are reported to be at pH 5.5 / 7.5 at a temperature of 35 / compositionofthefractionsproducedafterenzymic6A0ns 8 oCn.UPnrotamex TM h 1 a v Ietsadedacetcil v aarteidonatcetim v ipteyratoufre1.is5 hydrolysis of fresh salmon frames with Protamex TM and its (AU) g . to pinpoint possible utilisation of these, as a bottom-line reported to be 85 8 C for 10 min [10] . knowledge for further studies.
Fig. 1. Illustration of the raw material and produced fractions from the hydrolysis of salmon frames with Protamex TM .
B. Liaset et al. / Process Biochemistry 38 (2003) 1747 / 1759
Fig. 2. Flow scheme of the hydrolysing process of salmon frames with Protamex TM .
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2.3. Enzymic hydrolysing process the ultramembrane filtered fraction (UF fraction) was spray-dried (Niro Atomizer, Denmark, P-6.3 tower, The procedure in the present study ( Fig. 2 ) was T in / 200 8 C, T out / 84 8 C). performed at the pilot plant of No v ozymes A/S (Bags-v aerd, Denmark). The enzymic hydrolysis was per-2.4. Ch ical analyses formed at optimal conditions for Protamex TM with em regard to pH (neutral, / 6.5) and temperature (55 9 / 2 8 C), as described pre v iously [7] . The ratio of salmon Dry matter in the produced fractions was determined framestowaterwas1.14.Anenzymetosubstrateratio(grNa* v 6i.m2e5t)ricwaalslydetaeftremrinefdreeizne-adrnyiitnrgo.genCraundaelyzeprro(tePiEn of 11.1 AU kg 1 crude protein was used in the hydrolysis.After60minofenzymictreatment,the2d4et1e0rmiSnEeRdIEafSterIIt,heUmSeAt)h.odTootfalCoahmeinnoanadciSdtsrydwoerme temperature was ele v ated to 98 8 C, which was reached after 105 min. [ d 1 e 1 r ] i. v atTih v eespwheerneylisseoptahiroatceydanaotnea(PIWTaCte r)saPimcionoTaacgi d Large bones were retained in the hydrolysing tank, while small bones were remo v ed by filtering the hydro- Column (3.9 mm / 15cm)anddetecteedctboyanatultra v iolet lysate through a mesh ( Fig. 2 ).Thereafter,theinsolubledTeotteacltocrys(tWeianteerisn44th1eabsasomrppltieosnwdaestdetre)rmin2e5d4anftmer. fraction was remo v ed in a two-phase separator (West- oxidati c st falia,Germany,SC-35-36-177,15kW,7200rpm),(88%):onH 2 oOf 2 (y30e%in)e/(c v y/s v t)inteowyiitehld9:c1ystpeeircforacmiidc.aTcihde before the remaining mixture was separated in a three- sa le d analysis phaseseparator(Westfalia,Germany,SB-7-36-076,4desmcpribsedweraebofu v ret.heTrrtyrepattoepdhaasnthweaasmiannoalaysciedinthe kW, 8520 rpm) into salmon oil, emulsion fraction and samples after basic hydrolysis with Ba(OH) 2 for 20 h aqueous fraction. The aqueous fraction was concen- at 110 9 / 2 8 C. The samples were pH adjusted to 6.2, trated (NiroAtomizer, Denmark, Falling Film E v apora- separated on a HPLC (Shimadzu 6A/6B) equipped with tor, FF 100), filtered through an ultramembrane with a SUPELCOSIL TM LC-18 HPLC-column (4.6 mm / 15 nominal molecular weight limit of 100 000 (PCI cm) and detected in an UV-spectrophotometer (Shi-membrane systems, UK, FP100, 2.65 m 2 ) and finally madzu SPD 6A) at 280 nm. Free amino acids were
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B. Liaset et al. / Process Biochemistry 38 (2003) 1747 / 1759
extracted from dry samples with the addition of 1:2 ( v / v ) phosphate buffer pH 7, whirled for 1 min and centri-fuged at 2500 / g a v for 30 min. The supernatants were deproteinized with the addition of 1:1 ( v / v ) of 5% (w/ v ) sulfosalisylic acid. The samples were kept on ice for 30 min and centrifuged at 5000 / g a v for 15 min. The supernatants were mixed 4:1 ( v / v ) with internal standard (Norleucine). The free amino acid content was analyzed in a Biochrom 20 plus amino acid analyzer (Amersham Pharmacia Biotech, Sweden) equipped with a lithium column with post column ninhydrin deri v atization and colorimetric detection at 570 nm and 440 nm (proline and OH / proline). The amino acids were identified using an amino acid standard mixture (Sigma A-6282 and A-6407) and quantified by their response factor relati v e to the internal standard. The molecular weight distribution in the UF fraction was estimated through centrifugation in filter tubes with nominal molecule weight limits of 100, 50, 30, 10 and 5 kDa (Biomax PB polyethersulfone membrane, Millipore Corp., USA). The nitrogen amount in the filtrates relati v e to total amount of nitrogen in the samples was used to estimate the molecular weight distribution. The total lipid content was determined gra v imetrically as the sum of free and bound lipid. Free or loosely bound lipid was extracted with petroleum gasoline and dried at 103 9 / 1 8 C. The samples were thereafter hydro-lysed with HCl in a Tecator Soxtec hydrolysing unit to release the bound lipid, which was extracted with petroleum gasoline and dried at 103 9 / 1 8 C. Fatty acids were extracted from the samples with 2:1 chloroform: methanol ( v / v ). The samples were filtered, saponified and esterified in 12% BF 3 in methanol ( v / v ). Fatty acid composition of total lipids was analysed using methods described by Lie and Lambertsen [12] . Fatty acid methyl esters were separated using a Carlo Erba gas chromato-graph equipped with a 50 m CP-sil 88 (Chromopack, Middelburg, The Netherlands) fused silica capillary column (i.d. 0.32 mm). Lipid classes were determined as described by Bell et al [13] . Samples were analysed on an automated Camag HPTLC system (Switzerland) and separated on HPTLC silica gel 60 F plates (Merck). Total lipids were extracted as mentioned abo v e. An amount of 10 m g total lipid from each sample was analysed. The eluent for separation of phospholipids from neutral lipids (running at the sol v ent front) was methyl acetate:propanol:choloroform:methanol:0.25% KCl (w/ v ) (2.5:2.5:2.5:1.0:0.9, by v olume). To separate neutral lipids from cholesterol, the plates were further de v eloped in isohexane: diethylether: glacial acetic acid (8:2:0.1, by v olume). After separation, the HPTLC plates were bathed in a solution with 3% copper acetate and 8% phosphoric acid. The plates were charred at 160 8 C for 15 min and the lipid classes were quantified as amount of total lipids by reading optical densities at 546 nm in a Camaq TLC Scanner 3 (Switzerland). For
identification of lipidclasses, a mixture of standards (Sigma) was used. The ash concentration of the fractions was deter-mined gra v imetrically by burning all organic substances in a programmable furnace by increasing the tempera-ture gradually from ambient air temperature to 550 8 C and hold it at 550 8 C for 20 h. Prior to all element analyses, with the exception of iodine, the samples were wet digested in a Milestone microwa v e laboratory system (Milestone, Sorisole, Italy). To samples of approximately 0.2 g dry matter, 2 ml of nitric acid (65% (w/V) ultra pure quality) and 0.5 ml hydrogen peroxide (30% (w/V) analytical quality Merck) were added. The concentrations of Na, K, Mg, Ca, Mn, Fe, Cu and Zn were determined by flame atomic absorption spectrometry on a Perkin Elmer 3300 AAS (Norwalk, CT) [14 / 16] . The concentrations of P, As, Se, Cd and Pb were analysed by electrothermal atomic absorption spectrometry on a Zeeman atomic absorption spectro-meter (Perkin Elmer 4110 ZL, Norwalk, CT) equipped with a THGA graphite furnace and an AS 72 auto-sampler [15 / 17] . Matrix modifiers were used for all elements. Quantification was made by means of peak-height measurements and standard cur v e calibration, except for As and Se where peak area and standard addition procedure were used. Hollow cathode lamps were used for Mg, Ca, Mn, Fe, Cu and Zn, whereas Na and K were run in emission mode. EDLs were used for P, Se, Cd, Hg and Pb. The concentration of Hg was determined by flow injection-cold v apour atomic ab-sorption spectrometry (FI-CVAAS) [18] . Selenium in the bone fraction was analysed by inducti v ely coupled plasma-mass spectrometry (ICP-MS) on an Agilent 7500c ICPMS, due to the high amount of P present in this fraction. The analyses were carried out on the microwa v e digests as described abo v e. The selenium concentrations ( 82 Se) were calculated using external standard calibration and rhodium as internal standard. Iodine was determined by (ICP-MS) after the samples were extracted in tetramethylammonium hydroxide (TMAH) for 3 h at 90 8 C [19,20] .
3. Results and discussion 3.1. Yields The mass balance is shown in Table 1 . Approximately 48% of total crude protein present in the raw salmon frames were found in the aqueous fraction post enzymic treatment. This figure is in agreement with v alues pre v iously reported on nitrogen reco v ery in enzymic hydrolysis of salmon frames under similar hydrolysing conditions [7] . The insoluble fraction, the bone fraction and the emulsion fraction contained about 19, 11 and 3% of the total crude protein, respecti v ely. About 77%
B. Liaset et al. / Process Biochemistry 38 (2003) 1747 / 1759
Table 1 Mass balance from hydrolysis of 200 kg fresh salmon frames with Protamex TM Wet weight Dry weight Crude protein Salmon frames 200.0 90.3 32.4 Aqueous fraction a 245.0 18.7 15.5 Insoluble fraction 43.0 7.9 6.1 Salmon oil / 38.7 / Bone fraction 25.0 10.4 3.4 Emulsion fraction 8.8 1.7 0.9 Reco v ery (%) / 85.5 80.1 a Yield measured in supernatant before filtration.
of total lipids were isolated as salmon oil after hydro-lysis. The insoluble fraction, bone fraction, emulsion fraction and aqueous fraction contained approximately 3, 2, 2 and B / 1% of total lipids present in the raw salmon frames, respecti v ely. In addition to the hydro-lysing conditions per se, both the yield of crude protein in the aqueous fraction, and the yield of salmon oil is dependent on the separators used in the production. In the present study, the separators produced insoluble and emulsion fractions with relati v e large amounts of trapped aqueous fraction. More powerful separators might ha v e increased the dry matter content in the insoluble and emulsions fractions and thereby slightly increased the crude protein yield in the aqueous fraction. As can be seen from Table 1 , not all of the dry matter in the salmon frames was reco v ered after the hydrolysing process. Se v eral steps of the process could ha v e con-tributed to these losses. Filtering the hydrolysate through the mesh to remo v e bones ( Fig. 2 ) resulted in foaming and this foam was neither quantified nor chemically analysed. Furthermore, dead v olumes in the equipment such as the separators and pipes might ha v e trapped some of the hydrolysate. Finally, small aliquots in the beginning of each separation step were discarded. Taken together these losses might explain that less than 100% of crude protein, ash and lipid were reco v ered ( Table 1 ). The temperature during separation of oil from the aqueous fraction is critical for a successful separation and a temperature of 95 8 C is regarded as optimum for a highest possible separation. Ob v iously, such a high temperature may lead to unwanted reactions in the polyunsaturated fatty acids present in the salmon oil. In the present study, a separation temperature of approximately 90 8 C was chosen to achie v e an aqueous fraction with as low lipid content as possible to a v oid rancidity de v elopment in the protein hydrolysate, upon further storage. The bone fraction contained 62% of the total ash, while the aqueous, insoluble and emulsion fractions contained about 22, 3 and 1% of total ash present in the raw salmon frames ( Table 1 ). It has to be mentioned that this
Ash Total lipids 8.6 50.0 1.9 0.3 0.3 1.4 / 38.7 5.3 1.0 0.1 0.8 88.6 84.5
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study was performed in a pilot plant, using equipment not being optimised with respect to maximum reco v ery during the hydrolysing process. 3.2. Proximal chemical compositions The proximal chemical compositions in the different fractions produced are shown in Table 2 . Both the aqueous and UF fractions were high in crude protein, 829 and 855 g kg 1 dry weight, respecti v ely. The ultra membrane filtration reduced the lipid content in the aqueous fraction from 16 to less than 1 g lipid kg 1 dry sample. This is of rele v ance if a lipid-free salmon hydrolysate is of importance for the producer. The ultra membrane filtration slightly increased the ash content in the UF filtrate (122 g kg 1 dry weight) relati v e to the aqueous fraction (103 g kg 1 dry weight), as a cut off-limit of 100 000 Dalton will result in an increased concentration of minerals. The insoluble fraction was high in crude protein (774 g kg 1 dry weight) probably consisting of larger peptides or proteins that had been hydrolysed only to a smaller degree by the Protamex TM proteases. This might ha v e been caused either due to a lack of amino acid sequences recognised by the protease in these peptides or due to inaccessibility to the cutting sites caused by strong molecular interactions in these regions. The insoluble fraction also contained a sig-nificant portion of lipid (180 g kg 1 dry sample), perhaps caused by production of complexes of lipids with the larger peptides and proteins. Furthermore, the relati v e high lipid content might ha v e been due to the abo v e mentioned separation technique applied in the present study (i.e. a more powerful separator might ha v e reduced the lipid content). The emulsion fraction was high in both crude protein (516 g kg 1 dry sample) and lipid (490 g kg 1 dry sample). Both the insoluble and the emulsion fractions were low in total ash content, 37.0 and 33.0 g kg 1 dry sample, respecti v ely. The bone fraction contained mainly ash (514 g kg 1 dry weight) and crude protein (330 g kg 1 dry weight), but also a significant amount of lipid (96 g kg 1 dry weight). No
1752 B. Liaset et al. / Process Biochemistry 38 (2003) 1747 / 1759 Table 2 Proximal chemical compositions and total amino acids in the fractions obtained from the hydrolysis of salmon frames with Protamex TM Component Bone fraction Insoluble fraction Emulsion fraction Aqueous fraction UF fraction Dry matter (g kg 1 ) 415 9 / 1 184.3 9 / 0.8 194.3 9 / 0.1 76.4 9 / 0.4 n.a. Proximal chemical composition (g kg 1 dry sample) Protein 330 9 / 0.2 774 9 / 1 516 9 / 1 829 9 / 1 855.3 9 / 0.8 a Lipids 96 9 / 1 180.0 9 / 0.7 490 9 / 20 16 9 / 1 n.d. Ash 514.1 9 / 0.5 37.0 9 / 0.1 33.0 9 / 0.1 102.5 9 / 0.7 122 9 / 2 Amino acids (mmol g 1 protein) Arg 0.39 9 / 0.02 0.39 9 / 0.01 0.39 9 / 0.01 0.39 9 / 0.01 0.37 9 / 0.01 His 0.12 9 / 0.01 0.17 9 / 0.01 0.22 9 / 0.02 0.25 9 / 0.01 0.29 9 / 0.01 Ile 0.14 9 / 0.01 0.36 9 / 0.01 0.33 9 / 0.01 0.22 9 / 0.01 0.25 9 / 0.01 Leu 0.26 9 / 0.01 0.63 9 / 0.01 0.59 9 / 0.01 0.44 9 / 0.01 0.45 9 / 0.01 Lys 0.24 9 / 0.01 0.55 9 / 0.01 0.53 9 / 0.01 0.47 9 / 0.01 0.46 9 / 0.03 Met 0.17 9 / 0.01 0.17 9 / 0.01 0.15 9 / 0.01 0.13 9 / 0.01 0.11 9 / 0.01 Phe 0.18 9 / 0.01 0.29 9 / 0.01 0.26 9 / 0.01 0.18 9 / 0.01 0.18 9 / 0.01 Thr 0.27 9 / 0.02 0.44 9 / 0.01 0.42 9 / 0.01 0.34 9 / 0.01 0.34 9 / 0.02 Trp 0.01 9 / 0.01 0.07 9 / 0.01 0.06 9 / 0.01 0.02 9 / 0.01 0.03 9 / 0.01 Val 0.22 9 / 0.01 0.46 9 / 0.01 0.43 9 / 0.01 0.30 9 / 0.01 0.31 9 / 0.01 Total indispensable AA (IAA) 2.00 3.53 3.38 2.74 2.79 Ala 0.81 9 / 0.05 0.71 9 / 0.01 0.75 9 / 0.01 0.88 9 / 0.01 0.91 9 / 0.01 Asn / Asp 0.44 9 / 0.02 0.74 9 / 0.01 0.73 9 / 0.01 0.61 9 / 0.02 0.61 9 / 0.01 Cysteine (total) 0.03 9 / 0.01 0.11 9 / 0.01 0.09 9 / 0.01 0.05 9 / 0.01 0.05 9 / 0.01 Gln / Glu 0.66 9 / 0.03 0.83 9 / 0.01 0.79 9 / 0.01 0.86 9 / 0.02 0.84 9 / 0.01 Gly 2.6 9 / 0.2 0.84 9 / 0.01 0.98 9 / 0.01 1.45 9 / 0.01 1.26 9 / 0.02 OH / Pro 0.50 9 / 0.03 0.06 9 / 0.01 0.09 9 / 0.01 0.21 9 / 0.01 0.17 9 / 0.01 Pro 0.81 9 / 0.05 0.37 9 / 0.01 0.40 9 / 0.01 0.50 9 / 0.01 0.45 9 / 0.01 Ser 0.43 9 / 0.03 0.41 9 / 0.01 0.43 9 / 0.01 0.40 9 / 0.01 0.40 9 / 0.01 Tyr 0.11 9 / 0.01 0.23 9 / 0.01 0.20 9 / 0.01 0.13 9 / 0.01 0.14 9 / 0.01 Total dispensable AA (DAA) 6.39 4.30 4.46 5.09 4.83 Ratio IAA:DAA 0.31 0.82 0.76 0.54 0.58 Tau n.d. 0.02 9 / 0.01 0.03 9 / 0.01 0.05 9 / 0.01 0.06 9 / 0.01 Total AA including Tau 8.39 7.84 7.87 7.88 7.68 Abbre v iation: not detected, n.d. Data are gi v en as mean 9 / de v iation from the mean (duplicate analyses). a Detection limit set to 1.0 g lipid kg 1 sample. de-fatting of the bone fraction was done in the present aspargine, glutamine and v aline seemed to be slightly experiment, it was only washed with hot water before increased by the ultra membrane filtration ( Table 3 ). drying, which probably explains the relati v e high lipid The aqueous and UF fractions both were profoundly content found. increased in total amount of glycine and OH / proline, relati v e to the insoluble and emulsion fractions ( Table 3.3. Amino acid compositions 2 ). This indicates that some of the collagen, possibly from the extra-cellular matrix and other soft connecti v e Total amino acids are gi v en in Table 2 and free amino tissues were solubilised during the hydrolysing process. acids together with the estimated molecular weight of The total amino acid compositions of both the insoluble the UF fraction are shown in Table 3 . The ultra and emulsion fractions were quite similar ( Table 2 ) and membrane filtration ( Fig. 2 ) neither seemed to influence relati v e high in the amino acids isoleucine, leucine, on the total amino acid composition ( Table 2 ) nor the v aline, phenylalanine, cysteine and methionine, amino free amino acid content ( Table 3 ), as only minor acids that are expected to be found in a hydrophobic differences, with a few exceptions, were obser v ed en v ironment [21] . The cysteine content was highest in between the aqueous and UF fractions. Howe v er, the the insoluble fraction ( Table 2 ), probably due to its ammonia content seemed to be reduced by more than ability to participate in disulphide bridges, making one third through the filtration process ( Table 3 ). There cysteine rich peptides less a v ailable for hydrolysis by is no ob v ious reason for this, if not the ammonia the proteases used in the present study. It is reasonable specifically interacted either with retained particles or to belie v e the peptides in the emulsion fraction to be of a with the membrane material. The amounts of free smaller molecular weight as compared to the peptides in
B. Liaset et al. / Process Biochemistry 38 (2003) 1747 / 1759 1753 Table 3 Free amino acids and nitrogen molecular weight distribution in the fractions produced from the hydrolysis of salmon frames with Protamex TM Amino acid ( m mol g 1 protein) Insoluble fraction Emulsion fraction Aqueous fraction UF fraction b -Alanine 7.8 9 / 0.2 10.5 9 / 0.3 21.8 9 / 0.5 23.3 9 / 0.1 b -Aminoisobutyric acid 1.0 9 / 0.1 1.0 9 / 0.1 1.9 9 / 0.1 1.9 9 / 0.1 g -Amino-butyric acid 0.3 9 / 0.1 0.3 9 / 0.1 0.8 9 / 0.1 0.8 9 / 0.1 Alanine 14.0 9 / 0.3 18.7 9 / 0.1 39.8 9 / 0.1 42.2 9 / 0.1 Ammonia 26.9 9 / 0.6 37.6 9 / 0.1 72.1 9 / 0.2 45.0 9 / 0.1 Anserine ( b -alanyl-1-methyl-histidine) 44.2 9 / 0.6 56.0 9 / 0.1 115.0 9 / 1.0 116 9 / 2.0 Arginine 2.5 9 / 0.1 3.4 9 / 0.1 6.8 9 / 0.1 6.3 9 / 0.1 Aspargine 0.8 9 / 0.1 1.3 9 / 0.1 2.7 9 / 0.2 3.8 9 / 0.1 Aspartate 1.9 9 / 0.1 2.4 9 / 0.1 5.5 9 / 0.1 6.0 9 / 0.1 Cystine 0.4 9 / 0.1 0.5 9 / 0.1 1.1 9 / 0.1 1.1 9 / 0.2 Cystathionine 1.5 9 / 0.1 1.9 9 / 0.1 4.2 9 / 0.1 4.3 9 / 0.1 Glutamate 8.8 9 / 0.2 11.6 9 / 0.1 25.5 9 / 0.4 27.7 9 / 0.1 Glutamine 1.0 9 / 0.1 1.0 9 / 0.1 1.9 9 / 0.1 2.0 9 / 0.1 Glycine 7.0 9 / 0.1 9.4 9 / 0.1 20.2 9 / 0.1 22.2 9 / 0.1 Histidine 2.9 9 / 0.1 3.7 9 / 0.1 7.8 9 / 0.1 7.9 9 / 0.1 Isoleucine 3.5 9 / 0.2 4.8 9 / 0.3 9.8 9 / 0.1 9.8 9 / 0.1 Leucine 13.2 9 / 0.4 17.5 9 / 0.4 36.6 9 / 0.2 36.8 9 / 0.1 Lysine 6.8 9 / 0.1 8.9 9 / 0.1 18.3 9 / 0.1 18.5 9 / 0.1 1-Methyl histidine 8.5 9 / 0.2 11.0 9 / 0.2 22.6 9 / 0.1 22.9 9 / 0.1 3-Methyl histidine n.d. n.d. n.d. n.d. Methionine 4.2 9 / 0.2 4.6 9 / 0.1 11.6 9 / 0.1 12.0 9 / 0.1 Ornithine 0.4 9 / 0.1 0.5 9 / 0.1 1.0 9 / 0.1 0.9 9 / 0.1 OH / proline 0.6 9 / 0.1 0.7 9 / 0.1 1.8 9 / 0.1 1.8 9 / 0.1 Phenylalanine 5.5 9 / 0.3 7.4 9 / 0.2 15.5 9 / 0.1 15.5 9 / 0.2 Phosphoetanolamine 0.7 9 / 0.1 0.8 9 / 0.1 1.9 9 / 0.1 1.8 9 / 0.1 Phosphoserine 0.3 9 / 0.1 0.3 9 / 0.1 0.9 9 / 0.1 0.9 9 / 0.1 Proline 1.7 9 / 0.1 2.6 9 / 0.1 4.8 9 / 0.1 5.1 9 / 0.2 Sarcosine ( N -methyl glycine) 3.5 9 / 0.1 4.5 9 / 0.6 9.4 9 / 0.1 10.3 9 / 0.1 Serine 3.6 9 / 0.1 4.9 9 / 0.1 10.7 9 / 0.1 11.0 9 / 0.1 Taurine 15.5 9 / 0.2 20.6 9 / 0.1 43.4 9 / 0.2 37.5 9 / 0.1 Threonine 3.3 9 / 0.1 4.9 9 / 0.1 9.5 9 / 0.1 10.1 9 / 0.1 Tryptophan 1.2 9 / 0.1 1.7 9 / 0.1 3.4 9 / 0.2 3.4 9 / 0.1 Tyrosine 2.5 9 / 0.1 3.4 9 / 0.1 6.8 9 / 0.1 6.7 9 / 0.1 Urea 2.6 9 / 0.1 2.5 9 / 0.1 7.0 9 / 0.2 7.4 9 / 0.1 Valine 3.7 9 / 0.1 5.0 9 / 0.1 10.6 9 / 0.1 13.0 9 / 0.1 Total free amino acids 202.3 265.9 552.7 535.9 Amount free to total amino acids (%) a 2.6 3.4 7.0 7.0 Molecular weight fraction B / 10 000 Da (%) n.a. n.a. n.a. 100 9 / 1 b Molecular weight fraction B / 5000 Da (%) n.a. n.a. n.a. 89 9 1 b / Abbre v iations: not analysed, n.a.; not detected, n.d. Data are gi v en as mean 9 / de v iation from the mean (duplicate analyses). a Total amino acids from Table 2 . b Mean 9 / S.E.M. ( n / 6). the insoluble fraction, as these peptides were not ( Table 3 ). The amount of free amino acids relati v e to segregated in the first separation ( Fig. 2 ). The amino total amino acids were lowest in the insoluble fraction acid composition of the bone fraction was low in all the (2.6%), slightly increased in the emulsion fraction (3.4%) amino acids, relati v e to the other fractions, with the and more than doubled in the aqueous and UF fractions exceptions of glycine, OH / proline and proline ( Table (both 7.0%) ( Table 3 ). The contents of free amino acids 2 ). This amino acid pattern is in accordance with a together with small peptides are reported to affect the typical collagen composition [22] , indicating that most palatability of the hydrolysate [8,9] . The taste of the of the protein in the bone fraction was collagen and produced UF fraction has not been tested in an expert connecti v e tissues. panel, but no profound bitter taste was experienced In the present study, all the nitrogen present in the UF when we tasted the UF fraction, oursel v es. Consistently, filtrate seemed to be smaller than 10 000 Da and 89 9 / 1% only a small reduction in feed intake has been obser v ed (mean 9 / S.E.M., n / 6) seemed to be less than 5000 Da with rats fed the UF fraction at 200 g crude protein
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