Food Hydrocolloids 19 (2005) 923928 www.elsevier.

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Extraction of gelatin from sh skins by high pressure treatment

mez-Guille n*, B. Gime nez, P. Montero M.C. Go
o (CSIC), Ciudad Universitaria, 28040 Madrid, Spain Instituto del Fr Received 7 August 2004; revised 17 November 2004; accepted 7 December 2004

Abstract High pressure, at 250 and 400 MPa, for 10 or 20 min, was applied at either of two stages: during pre-treatment in acid at 10 8C to facilitate destabilisation of acid labile crosslinks, or during extraction in water at 45 8C to accelerate collagen hydrolysis. The resulting gelatins were evaluated in terms of yield of extraction, molecular weight distribution by SDS-PAGE, and viscoelastic properties of gelatin newly dissolved and after overnight cold maturation. Pressure level and time of treatment induced noticeable changes in molecular weight distribution and consequently affected viscoelastic properties. The use of high pressure to extract gelatin from sh skins is a useful alternative to the conventional procedure. Its utility lies basically in that the longest phase of the treatment can be drastically shortened, thus making it possible to produce a gelatin of high gelling quality in only a few minutes. q 2005 Elsevier Ltd. All rights reserved.
Keywords: Fish gelatin; Extraction; High pressure; Viscoelastic properties

1. Introduction Collagen, from which gelatin is derived, is the major structural protein found in skin and bones of animals. Collagen molecules, composed of three a-chains intertwined in the so-called collagen triple helix, adopt a 3D structure which is an ideal geometry for interchain hydrogen bonding (Johnston-Banks, 1990; Te Nijenhuis, 1997). In fact, the hydroxyproline content, through its OH group, has been reported to be largely responsible for the triple helix stabilisation (Burjandze, 1979; Ledward, 1986). There are also very short terminal regions, called telopeptides, which do not form triple helical structure, in which intra- and intermolecular covalent crosslinks, mainly involving lysine and hydroxylysine residues, are located. Because of the acid lability of cross-linking in sh skin collagen, mild acid treatment is normally enough to produce adequate swelling and cleavage of non-covalent intra- and as, Turnay, & inter-molecular bonds (Montero, Border Lizarbe, 1990; Norland, 1990; Stainsby, 1987). Subsequent thermal treatment above 40 8 C cleaves hydrogen
* Corresponding author. Tel.: C34 31 5492300; fax: C34 91 5493627. mez-Guille n). E-mail address: cgomez@if.csic.es (M.C. Go 0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.12.011

and a number of covalent bonds; this destabilises the triple helix by means of a helix-to-coil transition, leading to conversion into soluble gelatin (Djabourov, Lechaire, & Gaill, 1993). High molecular weight polymers may arise in the resulting gelatin through the possible persistence of crosslinks depending on the nature and degree of solubilisation. High pressure above 150 MPa has been shown to induce protein denaturation by disturbing the balance of the noncovalent interactions that stabilise the native conformation of many proteins (Masson, 1992). However, collagen has been reported to be scarcely affected by high pressure, given that hydrogen bonds are chiey pressure-insensitive (Gekko & Koga, 1983; Heremans, 1995). At relatively low pressure they could appear to be even somewhat strengthened, due to an associated small reduction in volume (Cheftel & Culioli, 1997). No signicant effects by high pressure treatment around 300400 MPa were found on intramuscular collagen solubility from beef (Suzuki, Watanabe, Ikeuchi, Saito, & ndez-Mart n, Takahasi, 1993) or pacic blue whiting (Ferna rez-Mateos, & Montero, 1998). However, changes in Pe bovine collagen solubility have been shown to vary with applied pressure; 200 MPa increased solubility, whereas 400 MPa reduced it (Kwiatkowska, Jankowska, & Korzeniowski, 2001).

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Regarding high pressure utilisation in gelatin, there are a few works focused on aspects related to induced ndez-D az, & Go mez-Guille n, gelation (Montero, Ferna 2002; Walkenstrom & Hermansson, 1997). However, no published articles are available in relation to gelatin extraction assisted by high pressure. mez-Guille n and Montero (2001) reported a proGo cedure for extracting gelatin with high gelling capacity from sh skins; the procedure was essentially based on a mild acid pre-treatment for collagen swelling followed by extraction in water at moderate temperature (45 8C). The entire process takes around 24 h. The point of applying high pressure in the sh gelatin extraction process is mainly to cut down the treatment times and/or to improve the quality of the gelatin produced. In conventional gelatin manufacturing procedure, high pressure can be applied at two stages: either during pre-treatment in acid at 10 8C to facilitate destabilisation of acid labile crosslinks, or during extraction in water at 45 8C to accelerate collagen hydrolysis. The aim of this work was to analyse the yield, molecular weight distribution and viscoelastic properties of gelatin extracted from sh skins using high pressure (250 or 400 MPa) either during mild acid pre-treatment at 10 8C or during extraction in water at 45 8C.

2.2. Electrophoretic analysis (SDS-PAGE) Dry gelatin was dissolved in distilled water at 60 8C and then 3-fold-concentrated loading buffer containing bmercaptoethanol was added until a nal concentration of 2 mg/ml of gelatine is reached, as described previously by mez-Guille n et al. (2002). Protein samples were heatGo denatured 5 min at 90 8C and analysed by SDS-PAGE according to Laemmli (1970) using 3% stacking gels and 5% resolving gels in a Mini Protean II unit (Bio-Rad Laboratories, Hercules, CA) at 25 mA/gel. The loading volume was 15 ml in all lines. Protein bands were stained with Coomassie brilliant Blue R250. Type I and type III collagens from fetal calf skin (Sigma Aldrich) were used as markers of a-chain, b- and g-component mobilities. 2.3. Viscoelastic properties Dynamic studies were performed on a Bohlin CRS-10 controlled stress rheometer rotary viscometer (Bohlin Instruments Ltd, Gloucestershire, UK) using a cone-plate geometry (cone angle 48, gapZ0.15 mm), according to mez-Guille n et al. (2002). Solutions were prepared by Go mechanical stirring for 1520 min of pre-weighed (6.67% w/v) dry gelatin in water at 40 8C. Also the samples were cooled in a refrigerator at 7 8C (maturation temperature) for 1618 h overnight to analyse the effect of maturation time on gelatin. Temperature ramps were performed at a scan rate of 1 8C/min, frequency 1 Hz, and oscillating applied stress of 3.0 Pa. The phase angle (8) was represented as a function of temperature. Values of elastic modulus (G 0 ) and viscosity modulus (G 00 ) were taken at 6 8C. The ramps were implemented from 40 to 6 8C and back to 40 8C for newly dissolved gelatin, to study gelatin gelation and subsequent melting. Gelatin matured overnight (1618 h) at 67 8C was also subjected to temperature ramps from 6 to 40 8C and then from 40 to 7 8C, to evaluate the effects of maturation time on the gelatin gel. Measurements were done at least in triplicate and results were expressed as mean values and standard deviation.

2. Materials and methods 2.1. Gelatin extraction Skins from Dover sole (Solea vulgaris) were collected and stored frozen at K20 8C until use. Cleaning of thawed sh skins and gelatin extraction procedure was carried out mez-Guille n & Montero, 2001). as previously described (Go Briey, the method consist of a mild acid (50 mM acetic acid) swelling step for 3 h and subsequent overnight (1618 h) gelatin extraction in distilled water at moderate temperature (45 8C). Extracted gelatin was air-dried until moisture was less than 15%. This procedure was considered as conventional treatment and the resulting gelatin was called control gelatin, in order to compare with the gelatins obtained by means of high pressure. For the pressure-assisted extractions, sh skins were frozen, thawed and cleaned as mentioned above. Pretreatment in 50 mM acetic acid at 10 8C was performed during 10 min at 250 MPa, followed by extraction in water at 45 8C during 1618 8C at atmospheric pressure (S250). Samples extracted in water at 45 8C by high pressure were all preceded by a pre-treatment (at atmospheric pressure) in 50 mM acetic acid at 10 8C during 3 h. In substitution of a conventional overnight extraction during 1618 h, high pressure was applied at 250 MPa for 10 min (E250-10 0 ) or 20 min (E250-20 0 ), and at 400 MPa for 10 min (E400-10 0 ) or by two pulses of 5 min each (E400-2!5 0 ).

3. Results and discussion The utility of the high pressure treatment was tested at two stages: during acid swelling to facilitate the conversion of the insoluble collagen into a form suitable for subsequent extraction; and during the actual gelatin extraction step in water at 45 8C. As a control for comparison of the gelatins from different extraction procedures, gelatin was extracted conventionally following the procedure described by mez-Guille n (2000), with a mild acid preMontero and Go treatment followed by overnight extraction at 45 8C.

 mez-Guille n et al. / Food Hydrocolloids 19 (2005) 923928 M.C. Go Table 1 Yield of extraction (expressed as g dry gelatin per 100 g of cleaned skins) Samples Control S250 E250-10 0 E250-20 0 E400-10 0 E400-2!5 0 Yield of extraction (%) 21.3 22.8 10.2 4.7 3.4 5.7

925

3.1. Yield of extraction Pressure-assisted acid swelling was performed at 250 MPa/10 min at 10 8C (S250), followed by an overnight extraction in water. These conditions were assayed with 50 mM acetic acid as in the control treatment, and also with 10 mM acetic acid. Preliminary results showed that no signicant amount of gelatin could be extracted at the low acetic acid concentration, even with 20 min pressurising. All the preliminary collagen swelling (pre-treatment) assays in the extracting step were therefore performed with 50 mM acetic acid. As Table 1 shows, the yield (expressed as grams of dry gelatin per 100 g of clean skin) was slightly higher when pressurisation was used for collagen pre-solubilisation (S250), than in conventional gelatin extraction (control). On the contrary, the yield was considerably lower in all cases when pressurisation was applied in the extraction step. The general impression was that increasing the pressure or prolonging pressurisation tended to reduce the yield. As regards the pressure level, more gelatin was extracted with 250 MPa than with 400 MPa. Prolonging of treatment with 250 MPa from 10 to 20 min also caused a reduction in the extraction yield. In the case of pressurising at 400 MPa, when samples were pressurised in two consecutive 5-min cycles, the amount of gelatin extracted was only 2% greater than when samples were pressurised continuously for 10 min in the same conditions. 3.2. Molecular weight distribution The electrophoretic (SDS-PAGE) proles of the various gelatin preparations are shown in Fig. 1. Samples extracted with high pressure at 400 MPa (E400-10 0 and E400-2!5 0 ) were characterised by a typical gelatin/collagen type I prole, with a notable amount of a-chains (a1/a2 ratio around 2), a considerable presence of b-components (achain dimers), and particularly of higher molecular weight polymers including g -components ( a -chain trimers) mez-Guille n et al., 2002). There were noticeably more (Go of these high molecular weight peptides than with the conventional extraction procedure; the reason may be either that high pressure favours more polymer extraction, or else that there is some aggregation during hydrolysis. Both in the swelling step (S250) and in the water extraction step (E25010 0 ), pressurisation at 250 MPa for 10 min led to gelatin

Fig. 1. Electrophoretic analysis of gelatin preparations (SDS-PAGE) in the presence of 2-b-mercaptoethanol: (a) control, (b) S250, (c) E250-10 0 , (d) E400-10 0 , (e) E400-2!5 0 and (f) E250-20 0 .

preparations in which peptides of MW%100 kDa predominated and dimers and trimers were practically absent. The electrophoretic proles and the higher yield of these pressure-assisted gelatin preparations indicate a higher degree of collagen solubilisation or hydrolysis. When pressurisation at 250 MPa was prolonged to 20 min (E25020 0 ) during extraction in water, the prole contained appreciably more high molecular weight polymers, which suggests a degree of pressure-induced aggregation. 3.3. Viscoelastic properties of newly dissolved gelatins Dry gelatins were dissolved at 40 8C. Fig. 2 shows changes in the phase angle (d) of the newly dissolved gelatin preparations (concentration 6.67% w/v) upon cooling to 6 8C and subsequent heating. As the cooling ramp shows, the transition curve from solution to gel was rapid and similar in all samples, the only variation being in the gelling temperatures. All samples presented low phase angles at 6 8C (d!58), indicating good gelling gelatins. The same was true during subsequent heating; the studied gelatins differed in melting temperature but all presented a sharp melting transition. The onset of gelling and melting occurred at slightly higher temperatures than in the control in gelatins extracted under 400 MPa pressure, but at considerably
70

Phase angle ()

60 50 40 30 20 10 0

60 50 40 30

Control S250 E250-10 E250-20 E400-10 E400-2*5

26

22

18

14

10

6 6

10

14

18

22

26

Temperature (C)

Temperature (C)

Fig. 2. Changes in phase angle d, monitored upon cooling from 40 to 6 8C and subsequent heating from 6 to 40 8C, of newly dissolved gelatins at 6.67% w/v.

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mez-Guille n et al. / Food Hydrocolloids 19 (2005) 923928 M.C. Go

Table 2 Viscoelastic properties of newly dissolved gelatines Samples Cooling ramp G 6 8C (Pa) Control S250 E250-10 0 E250-20 0 E400-10 0 E400-2!5 0 2520G63 868G22 731G18 2720G68 2700G70 1630G41
0

Heating ramp G
00
6 8C

(Pa)

g.t. (8C) 13 9 8 12 13 13

G 0 40 8C (Pa) 0.64G0.02 0.67G0.02 0.66G0.01 0.35G0.01 0.54G0.03 0.47G0.02

G 00 40 8C (Pa) 0.41G0.01 0.31G0.01 0.35G0.02 0.42G0.03 0.42G0.02 0.45G0.02

m.t. (Pa) 22 18 17 21 23 23

54.6G1.4 35.6G0.9 45.9G1.1 69.4G1.7 63.6G1.6 35.7G0.9

g.t., gelation temperature; m.t., melting temperature.

lower temperatures than the control in gelatins extracted under 250 MPa pressure for 10 min (S250 and E250). However, transition temperatures were higher when the pressurisation time was prolonged (E250-20). As reported by Johnston-Banks (1990), the increase in the gelling/melting temperatures is directly related to the increase in the average molecular weight of the studied gelatins. Table 2 shows gelling and melting temperatures and elastic modulus (G 0 ) and viscous modulus (G 00 ) values at 6 and 40 8C. The samples pressurised at 250 MPa for 10 min, both during swelling (S250) and during extraction in water (E250-10 0 ), presented signicantly lower G 0 and G 00 values as measured at 6 8C than the other gelatins. In samples pressurised at 400 MPa in two 5-min cycles, viscoelastic properties were again lower than with continuous pressurisation for 10 min. On the other hand, values of G 0 in 40 8C samples were low upon subsequent heating, indicating that the gels were all perfectly thermo-reversible and melted completely. 3.4. Viscoelastic properties of overnight cold matured gels The transition curves of the matured gelatin gels upon melting and subsequent cold renaturation (Fig. 3) again revealed differences among the samples, predominantly in the thermal transition temperatures rather than in the slope. Both melting and gelling temperatures of S250 and E250-10 0 gels were again notably lower than in the others. Also, phase angle values at 6 8C were higher in these samples, indicating poorer overall gel forming capacity as a result of cold maturation. In sample pressurised at 400 MPa for 10 min (E400-10 0 ), the cooling phase angle transition indicated the onset of collagen renaturation at around 20 8C, which was considerably higher than with the conventional treatment. As shown in Table 3, values of G 0 measured after overnight maturation at refrigeration temperature (around 6 8C) in E250-20 0 and E400-10 0 gelatin gels were signicantly higher than in the control gelatin. Again, all gels were completely melted at 40 8C. Upon subsequent cold renaturation, the E400-10 0 sample attained the highest values of G 0 and re-gelling temperature, which was in fact higher than in the corresponding newly dissolved gelatin. There is

clearly a relationship between the effect of pressure and the molecular weight of gelatin fractions. According to Stainsby (1987), the greater presence of a-chain dimers and trimers in these gelatins should make them more susceptible to renaturation to the full native collagen form. In addition, the presence of a signicant amount of higher molecular weight components acts also in favour of their slightly higher melting/gelling temperatures (Johnston-Banks, 1990). On the other hand, Sims, Bailey, and Field (1997) indicated that the generation of short triple helices by many interactions of free chains, rather than rapid completion of a longer triple helix, will form a more stable gel. In this sense, the way of triple helix formation upon cooling in E400-10 0 and E4002!5 0 samples having similar molecular weight prole, may explain the difference in their renaturation ability. High pressure-assisted extraction at 400 MPa for 10 min produced considerably less amount of gelatin than conventional extraction over 1618 h. However, the quality of the gelatin extracted in that short time was high, comparable to that of conventionally extracted gelatin as reported by mez-Guille n (2000). The molecular weight Montero and Go distribution of the gelatin extracted by high pressure was characterised by a high presence of a-chain, together with b- and g-components (dimers and trimers of a-chain, respectively), as well as a certain amount of greater polymers. All of them are key factors for high viscoelastic properties, gel strength and melting/gelling temperatures. After maturing overnight at 67 8C, the modulus of elasticity (G 0 ) of the gel from pressure-processed gelatin was greater than that of a conventional gelatin, and
80

Phase angle ()

70 60 50 40 30 20 10 0

Control S250 E250-10 E250-20 E400-10 E400-2*5

10

14

18

22

26

30

30

26

22

18

14

10

Temperature (C)

Temperature (C)

Fig. 3. Changes in phase angle d, monitored upon heating from 6 to 40 8C and subsequent cooling from 40 to 6 8C, of gelatin gels (6.67% w/v, 168 h maturation at 67 8C).

 mez-Guille n et al. / Food Hydrocolloids 19 (2005) 923928 M.C. Go Table 3 Viscoelastic properties of overnight matured gelatin gels Samples Heating ramp G 6 8C (Pa) Control S250 E250-10 0 E250-20 0 E400-10 0 E400-2!5 0 5240G131 566G14 882G22 8030G201 6160G154 5370G134
0

927

Cooling ramp G
00
6 8C

(Pa)

G 40 8C (Pa) 0.72G0.02 0.66G0.02 0.52G0.01 0.48G0.02 0.53G0.03 0.44G0.02

00
40 8C

(Pa)

m.t. (8C) 24 20 20 23 24 24

G 0 6 8C (Pa) 2870G72 312G7.8 602G15.5 3460G86 4720G118 2680G67

G 0 6 8C (Pa) 61.6G1.5 24.9G0.6 45.6G1.1 94.3G2.3 122.6G3.1 54.9G1.4

g.t. (Pa) 14 8 9 13 17 15

361G9.0 104G2.6 169G4.2 413G10.3 318G7.9 303G7.6

0.50G0.02 0.30G0.01 0.33G0.01 0.28G0.02 0.67G0.03 0.60G0.02

g.t., gelation temperature; m.t., melting temperature.

the melting temperature was slightly higher. This gelatin also presented greater renaturation ability upon subsequent cooling, but only with continuous extraction (E400-10 0 ); when extraction was carried out at 400 MPa in two 5-min cycles (E400-2!5 0 ) there was no improvement in renaturation ability even although the molecular weight prole was similar to that of gelatin E400-10 0 , from which we deduce that it is not only the amount but also the conguration of these fragments that matters. Pressurising at 250 MPa for 20 min produced a gelatin with less amount of dimers and trimers than with 400 MPa, however, viscoelastic properties were similar, and the value of G 0 after overnight maturation was even higher. With pressurising at 250 MPa, time was a key factor in the quality of the extracted gelatin: with 10 min pressurisation in these conditions, the peptides extracted were largely of relatively low molecular weight and there were insufcient a-chains for proper annealing of collagen molecules upon cooling. Although the yield was higher, this gelatin exhibited considerably poorer gel-forming ability than any of the others, including the control. In light of these results, we conclude that the use of high pressure to extract gelatin from sh skins is a useful alternative to the conventional procedure, essentially in that it drastically reduces the duration of the longest part of the process, so that a high-quality gelatin can be produced in a matter of minutes. Successive extractions at atmospheric pressure and increasing temperatures would improve the yield from original raw material. Further studies would be needed to optimise pressure/time/temperature settings so as to increase the yield in the rst extraction and in successive extractions from the same original material.

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Acknowledgements This research was nanced by the Comunidad de Madrid n under project 07G/0052/00, and by the Spanish Comisio a under project Interministerial de Ciencia y Tecnolog AGL2000-1497.

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