Food

Chemistry
Food Chemistry 86 (2004) 325–332
www.elsevier.com/locate/foodchem

Fourier transform infrared (FTIR) spectroscopic study

of acid soluble collagen and gelatin from skins and bones
of young and adult Nile perch (Lates niloticus)
a,b
J.H. Muyonga , C.G.B. Cole c, K.G. Duodu b,*

a
Department of Food Science and Technology, Makerere University, P.O. Box 7062, Kampala, Uganda
b
Department of Food Science, University of Pretoria, Pretoria 0002, South Africa
c
Davis Gelatine (South Africa), P.O. Box 5019 West Krugersdorp, 1742, South Africa
Received 25 April 2003; received in revised form 8 September 2003; accepted 8 September 2003

Abstract

Fourier transform infrared (FTIR) spectroscopy was conducted on type A gelatins derived from skins and bones of young and
adult Nile perch (Lates niloticus) by a sequential extraction process. Spectra for gelatins were compared to each other and to that of
acid soluble collagen from young Nile perch skins, in order to elucidate changes in protein secondary structure during collagen to
gelatin transformation. The first gelatin extracts showed diminished amide III bands while the last gelatin extracts showed distinct
amide III bands and their amide I bands consisted of a higher percent area of a component around 1690 cm
1 . The differences
suggested that the collagen to gelatin transition leads to loss of molecular order. The later gelatin extracts exhibited higher molecular
order than earlier gelatin extracts, probably because the former contained surviving crosslinks or/and because renaturation of the
low molecular weight gelatin fractions (later gelatin extracts) led to formation of more protein–protein linkages.
Ó 2003 Published by Elsevier Ltd.

Keywords: Nile perch; Gelatin; Collagen; FTIR; Protein structure

1. Introduction Fibrillogenesis (self assembly) of collagen has been

found to be associated with broadening and a slight shift
Fourier transform infrared (FTIR) spectroscopy has to lower wave number of the amide A peak (Milch,
been used to study changes in the secondary structure of 1964), increase in intensity and slight shift to lower wave
collagen and gelatin. It has been used to study collagen number of amide III peak (Jakobsen et al., 1983), band
crosslinking (Paschalis et al., 2001), denaturation (Friess broadening and shift of amide I peak to lower wave
& Lee, 1996), thermal self assembly (Jakobsen, Brown, number (Jakobsen et al., 1983; George & Veis, 1991;
Hutson, Fink, & Veis, 1983; George & Veis, 1991) as Prystupa & Donald, 1996) and shift of amide II peak to
well as gelatin melting (Prystupa & Donald, 1996). The lower wave number (Jakobsen et al., 1983; George &
spectral changes which are indicative of changes in Veis, 1991). Shift of amide I, II and III peaks to lower
collagen secondary structure have been shown to include wave numbers, increase in intensity of amide III and
changes in the amide A (Milch, 1964), amide I (1636– broadening of amide I are therefore associated with in-
1661 cm
1 ), amide II (1549–1558 cm
1 ) (Renugopala- creased intermolecular interactions (by hydrogen
krishnan et al., 1989) and the amide III (1200–1300 bonding) in collagen.
cm
1 ) regions (Friess & Lee, 1996). Denaturation of collagen, on the other hand, has
been found to lead to reduction in the intensity of amide
*
Corresponding author. Tel.: +27-12-420-3202/420-4299; fax: +27- A, I, II and III peaks (Friess & Lee (1996), narrowing
12-4202839. of amide I band (Prystupa & Donald, 1996), increase
E-mail address: [email protected] (K.G. Duodu). in amide I component found around 1630 cm
1 and

0308-8146/$ - see front matter Ó 2003 Published by Elsevier Ltd.

doi:10.1016/j.foodchem.2003.09.038
326 J.H. Muyonga et al. / Food Chemistry 86 (2004) 325–332

reduction in the intensity of amide I component, found determined and compared to those of acid-soluble col-
around 1660 cm
1 (George & Veis, 1991; Payne & Veis, lagen from the same species, in an effort to elucidate
1988; Renugopalakrishnan et al., 1989). changes in secondary structure that occur during the
Prystupa & Donald (1996) studied gelatin melting conversion of collagen to gelatin. The first and last
and found it to be associated with reduction in the 1678 gelatin extracts from skins and bones of young and adult
cm
1 peak and 1660/1690 cm
1 peak intensity ratio and Nile perch were studied.
increase in amide I components occurring around 1613,
1629 and 1645 cm
1 . These authors assigned the bands
occurring at 1645–1657 cm
1 to random coils and the 2. Materials and methods
1660 cm
1 band to triple helix, with contribution from a-
helix and b-turns. The amide I component, at 1690 2.1. Preparation of acid soluble collagen
cm
1 , has been attributed to helices of aggregated col-
lagen-like peptides (Doyle, Bendit, & Blout, 1975; Pry- Acid-soluble collagen was prepared from skins of
stupa & Donald, 1996). According to Doyle et al. (1975) young Nile perch (skin thickness < 0:4 mm), as de-
this peak vanishes with hydration of collagen or gelatin. scribed by G omez-Guillen & Montero (2001). Briefly the
As animals age, the extent of crosslinking of their method involved washing of the skins with chilled (5
collagen increases and the type of crosslinks change °C) water for a period of 10 min. During this time, the
(Sims & Bailey, 1992; Bailey, Paul, & Knott, 1998; Sims, skins were pressed intermittently by hand. The skins
Avery, & Bailey, 2000; Hickman et al., 2000). According were then washed with 0.8 M NaCl for 3 periods, of 10
to Bailey et al. (1998) collagen from skins of immature min each, followed by rinsing in running water, after
animals mainly contain the intermediate crosslinks de- each wash, with NaCl. Collagen was then extracted us-
hydroxylysinonorleucine (deHLNL), whereas collagen ing 0.5 M acetic acid solution (1:20 w/v). The extraction
from bones of immature animals contain hydroxylysin- was conducted for 16 h, during which the skins were
oketonorleucine (HLKNL). These intermediate divalent stirred intermittently. The viscous collagenous material
crosslinks are, respectively, converted to the more stable was separated from the insoluble components by sieving
trivalent histidinohydroxylysinonorleucine (HHL) and through cheesecloth and collagen was precipitated
pyridolines (PYR) during maturation. It has been shown using 0.9 M NaCl, washed with distilled water and
that differences in the quantities of the two types of freeze-dried.
crosslinks are manifested in the amide I region of the An attempt was made to extract collagen from bones
FTIR spectra of collagenous tissue (Paschalis et al., using 0.5 M acetic acid, but no collagen could be pre-
2001). There is a positive correlation between the ratio cipitated from the acetic acid liquor, after 5 days holding
of the components (1660/1690 cm
1 ) and the relative at room temperature.
abundance of PYR and HHL crosslinks.
Age-related increase in stability of collagen, through 2.2. Preparation of gelatins
increase in the amount and stability of crosslinks, affects
the stability of collagen to denaturation processes, e.g. The gelatins used in this study were derived from
heat. Collagens with more extensive crosslinks, e.g. Nile perch skins and bones by the acid process. Gel-
those from mature bovine hide, require a more severe atin was extracted from young (skin thickness < 0:4
process to break the crosslinks and allow collagen de- mm and skeleton length < 40 cm) and adult (skin
naturation and solubilisation into gelatin (Reich, Wal- thickness > 1:5 mm and skeleton length > 95 cm) fish.
ther, & Stather, 1962). During such severe processes, Briefly, extraction of skin gelatin involved acidulation
more peptide bonds are broken but some intermolecular with concentrated sulphuric acid to a pH of 2.5–3.0
crosslinks survive. The triple helices of collagen from and maintaining this pH range throughout the swell-
young animals are mainly held together by hydrogen ing period (16 h) by adding more acid solution until
bonds and Van der Waals forces. In such collagens, heat the skins were adequately swollen. The skins were
treatment mainly leads to breaking of hydrogen bonds then transferred to beakers, covered with warm (60
and the triple helical structure is more likely to decom- °C) water and gelatin extracted in water baths at 50,
pose, mainly to intact alpha chains. It is not clear, 60 and 70 °C, in a sequential process. In the case of
however, whether the secondary structures of gelatins young fish skins, extraction was conducted at only 50
derived from young and old animals differ. Nile perch and 60 °C because, after the 60 °C extraction, the
(Lates niloticus) is a warm water fish species, with po- residue left was very small and would give very small
tential for giving gelatin with gelling properties more amounts of gelatin at 70 °C. The gelatin extracts (light
similar to mammalian gelatins than cold water fish liquors) were filtered through compressed cotton wool.
species. The light liquor concentrations were determined by
In this study, the FTIR spectra of gelatins derived evaporating duplicate 10 ml portions to a stable
from young and adult Nile perch skins and bones were weight (48 h at 105 °C) and the concentration was
 J.H. Muyonga et al. / Food Chemistry 86 (2004) 325–332 327

used in calculation of % gelatin extractability as

ASC – Young Nile perch skins acid-soluble collagen, YS – Gelatin extracted from young fish skins, AS – Gelatin extracted from adult fish skins, YB – Gelatin extracted from young fish bones,

Numbers in brackets represent extraction temperature (°C) for the gelatin, Sh – Peak appearing as shoulder, – No common name for the spectral region, DM – Diminished peak, HB – Hydrogen
follows:

Halliday, and Mantsch (1995)

Amount of gelatin extracted at a given temp
 100%
sum of gelatin extracted at all temp

Jackson, Choo, Watson,

Abe and Krimm (1972)
Abe and Krimm (1972)

Abe and Krimm (1972)

Abe and Krimm (1972)
Jackson et al. (1995)
Jackson et al. (1995)
Jackson et al. (1995)
Jackson et al. (1995)
Jackson et al. (1995)
Sai and Babu (2001)
¼ %gelatin extractability at a given temp:
The light liquors were then passed through a column
of activated carbon (GRC 22, BHT water treatment,

Reference
Chloorkop, South Africa) at a rate of 5 bed volumes
per hour. The pH of all the light liquors was adjusted to
5.0 using 5% ammonia solution and the gelatin extract
was dried in a cross-flow air drier at 42 °C, until brittle.
The brittle sheets were broken into small pieces and

NH bend coupled with CN stretch

milled using a domestic coffee grinder to pass through a

NH stretch, coupled with HB

1 mm mesh sieve.

CH2 asymmetrical Stretch

C@O stretch/HB coupled

Bones used for gelatin extraction were cleaned, by

CH2 symmetrical Stretch

CH2 wagging of proline

scraping with a knife, to reduce the flesh contamination.
They were then decreased by tumbling in warm (35 °C)

Skeletal stretch
Skeletal stretch
water and demineralised using 3% HCl at room tem-

CAO stretch
Assignment
perature (20–25 °C) for a period of 9–12 days, with the

with COO-

CH2 bend

NH bend
liquor changed after every three days, until the bones
did not have any hard cores. The demineralised bones
were then treated in the same way as the acidulated
skins. The extractability and Bloom of the gelatins are AB(70)
presented in Table 1. 3478

1652

1540
1450

1236
1127
1076
874
671
Sh
YB(70)

2.3. Fourier transform infrared spectroscopy

3310

1656

1544
1451
1335
1243

1082

701
Sh

FTIR spectra were obtained from discs containing 2

mg sample in approximately 100 mg potassium bromide
AB(50)
3456

1644

1457
1402

1107
1006
DM
DM
DM

DM

(KBr). All spectra were obtained using a Bruker infrared

866
FTIR spectra peak position and assignments for Nile perch skin and bone gelatins

spectrophotometer (Bruker Instruments, Billerica, MA)

from 4000 to 500 cm
1 at data acquisition rate of 2 cm
1
YB(50)
3421
2924
2853
2355
1647

1558

1122
DM

per point. Background was subtracted using the Opus 870

software (Bruker Instruments, Billerica, MA). Triplicate 670
samples of collagen and gelatins were analysed and
AS(70)
3404
2923

1653

1541
1451
1335
1240
1082

spectra for the triplicate runs averaged. Fourier self

670

deconvolution was conducted on the average spectra for

the amide I band, using a resolution enhancement factor
YS(60)
3411

1654

1542
1452
DM

DM
DM

669

AB – Gelatin extracted from adult fish bones.

AS(50)

Table 1
3648
2924
2853
2356
1650

1541
1457

1011
DM

867
660
Peak wave number cm
1

Source, extractability and Bloom of gelatins used

Source Extraction Extractability Bloom
YS(50)

temperature (°C) (%) (g)

3623
2923
2853
2355
1648

1458

1234
1026
DM

DM

863
670

Fish skin gelatins

Adult fish 50 70.0 240
Young fish 50 86.5 217
Adult fish 70 10.6 134
ASC
3434
2924
2853
2355
1650

1542
1457

1235

871
670

Young fish 60 12.9 0

Fish bone gelatins
Amide III
Amide A

Amide II

Adult fish 50 33.0 84

Amide I
Region

bonding.

Young fish 50 33.3 156

Table 2

Adult fish 70 9.6 155

–
–
–

–
–

–
–
–
–

Young fish 70 22.6 0

328 J.H. Muyonga et al. / Food Chemistry 86 (2004) 325–332

of 1.8 and full height band width of 13 cm
1 . The self high temperature-extracted gelatins may be higher than
deconvolution provided information on the number and that in low temperature-extracted gelatins.
location of components. Curve fitting was then per- The gelatins extracted at higher temperature exhib-
formed using peakfit software (SPSS Inc., Chicago, IL, ited a much broader amide A than was observed for the
USA). low temperature-extracted gelatins and for acid-soluble
collagen. The amide A band in the high temperature-
extracted gelatins was in fact merged with the CH2
3. Results and discussion stretching band expected to occur at around 2930 cm
1 .
According to Kemp (1987), amide A tends to merge
3.1. Frequencies with the CH2 stretch peak when carboxylic acid groups
exist in stable dimeric (intermolecular) associations. It
The frequencies at which major peaks occurred for seems, therefore, that there are more associated com-
acid soluble collagen and the different gelatins and col- ponents in the high temperature-extracted gelatins. The
lagens are summarised in Table 2. high temperature extracted gelatins consist mainly of
low molecular weight peptides and, according to Led-
3.2. Spectra for skin gelatins ward (1986), gelling of low molecular weight gelatin
fractions entails more protein-protein linkages than
Nile perch gelatins, extracted at 50 °C, exhibited high gelling of high molecular weight gelatins. During drying,
Bloom (>200 g) and therefore have potential for therefore, it seems that the low molecular weight, high
substituting mammalian gelatins in gelling applications. temperature extracted gelatin fractions renatured slow-
Gelatins derived from young fish skins at 50 °C exhib- ly, forming a network with more protein-protein link-
ited spectra very similar to those for gelatins derived ages than the high molecular weight low temperature
from adult fish skins at the same temperature (Fig. 1), extracts.
but quite different from those extracted at higher tem- It is also possible that the high temperature-extracted
perature (70 °C for the adult and 60 °C for the young gelatins contain some covalent intermolecular bonds
fish skins) and from those of acid-soluble collagen. (surviving crosslinks) since they are derived from the
Compared to the spectra for acid-soluble collagen, the most crosslinked collagen, after the less crosslinked
low-temperature extracted gelatins showed lower inten- collagen is extracted during earlier (low temperature)
sity amide I and II bands and the amide III band was extractions. The stable intermolecular crosslinks may
almost non-existent. These changes are indicative of not break during extraction of gelatin. Instead, solu-
greater disorder (Friess & Lee, 1996) in gelatin and are bilisation may be achieved by cleavage of peptide bonds.
associated with loss of triple helix state. This is consis- As a result, the high temperature-extracted gelatin may
tent with changes expected as a result of denaturation of contain a significant amount of intermolecular cross-
collagen to gelatin. The gelatin extracted at the higher links. This may produce FTIR spectra showing a higher
temperatures, however, exhibited distinct amide III degree of molecular order. Paschalis et al. (2001) iso-
peaks. It seems therefore, that the extent of order in the lated stable (PYR and HHL) crosslinks from bovine
Amide III
Amide II
Amide I
Amide A

1
Absorbance

3500 3000 2500 2000 1500 1000

Wavenumber cm-1 5

Fig. 1. FTIR spectra for young Nile perch skin acid-soluble collagen (1), adult Nile perch skin gelatin extracted at 50 °C (2), young Nile perch skin
gelatin extracted at 50 °C (3), young Nile perch skin gelatin extracted at 60 °C (4) and adult Nile perch skin gelatin extracted at 70 °C (5).
 J.H. Muyonga et al. / Food Chemistry 86 (2004) 325–332 329

Amide III
Amide A

Amide II
Amide I
1 2
Absorbance

3500 3000 2500 2000 1500 1000

Wavenumber cm-1

Fig. 2. FTIR spectra for young Nile perch skin acid-soluble collagen (1), gelatin from young (2) and adult (3) Nile perch bones extracted at 50 °C and
from young (4) and adult (5) Nile perch bones extracted at 70 °C.

bone gelatin, supporting the assertion that intermolec- HHL and PYR but these were in concentrations less than
ular crosslinks may survive the process of gelatin 10% of those reported for bovine collagen.
extraction. Differences in the amide III region of the bone gela-
tins compared to acid-soluble collagen and skin gelatins
3.3. Spectra for bone gelatins are worthy of note, since the intensity of the amide III
band has been associated with the triple helical struc-
The spectra exhibited by bone gelatins differed from ture. The high temperature (70 °C)-extracted bone gel-
those exhibited by acid-soluble collagen and skin gelatins atins were found to exhibit low intensity peaks at
(Fig. 2). The amide I peaks in the bone gelatins were at around 1240 cm
1 . These peaks were not observed in the
lower frequencies than those of acid-soluble collagen. low temperature (50 °C)-extracted gelatins. It seems,
There were also differences in the amide III region. The 50 similar to the case of skin gelatins, that the 70 °C-ex-
°C-extracted Nile perch bone gelatins basically did not tracted bone gelatins had more intermolecular associa-
show absorption peaks in this region while the 70 °C- tions than the 50 °C-extracted gelatins.
extracted gelatins showed peaks. Nile perch bone gelatins
also exhibited sizeable peaks between 1000 and 1100 3.4. Amide I band components for Nile perch skin and
cm
1 . Absorption in this region is attributed to CAO bone gelatin
vibration due to carbohydrates (Jackson et al., 1995).
Carbohydrates in collagen are associated with glycation The amide I band, between 1600 and 1700 cm
1 , is
of collagen (Bailey et al., 1998) and carbohydrates are the most useful for infrared spectroscopic analysis of the
required in the formation of pentosidine crosslinks (Kent, secondary structure of proteins (Surewicz & Mantsch,
Light, & Bailey, 1985). It seems that Nile perch bone 1988). Deconvolution of the amide I band showed the
gelatins are more likely to contain pentosidine crosslinks band to consist of four components. The component
than Nile perch skin gelatins and acid-soluble collagen. peaks, their location and % areas are shown in Fig. 3
Cole (1995) reported presence of pentosidine crosslinks in and Table 3.
bovine hide collagen but studies on fish skin collagen, In agreement with Byler & Susi (1986), it is clear from
with hydrothermal isometric tension, show that they do Table 3 that protein segments with similar structures do
not contain substantial amounts of stable crosslinks, such not necessarily show band components with the same
as pentosidine crosslinks, even at advanced age (Cohen- frequencies. Overall, the variation in frequencies for
Solal, Le Lous, Allain, & Meunier, 1981). Hickman et al. particular band components in this investigation was
(2000) reported different types of crosslinks in fish swim not very different from that reported by Byler & Susi
bladder collagen. The stable crosslinks reported included (1986). They reported a variation of approximately 15
330 J.H. Muyonga et al. / Food Chemistry 86 (2004) 325–332

Fig. 3. Amide I band for Nile perch gelatins and collagens with fitted band components ASC – Young Nile perch skin acid soluble collagen, AB –
Gelatin extracted from adult fish bones, YB – Gelatin extracted from young fish bones, AS – Gelatin extracted from adult fish skins, YS – Gelatin
extracted from young fish skins. Numbers in brackets represent extraction temperature for the gelatin.

cm
1 for frequencies, attributable to b-structures of secondary structure estimates obtained by X-ray data
various components. and from infrared analysis (Byler & Susi, 1986; Surewicz
Quantitative band-fitting analysis of amide I areas, as & Mantsch, 1988).
applied in this investigation, has proved useful in One major observation was the consistently higher %
studying the nature and the extent of protein confor- area contributed by the 1690 cm
1 component for the
mational changes (Surewicz & Mantsch, 1988). Using higher temperature-extracted gelatins. In addition, the
this method, good correlations have been found for component around 1690 cm
1 occurred at higher wave
 J.H. Muyonga et al. / Food Chemistry 86 (2004) 325–332 331

Table 3
Location and percent area contribution of amide I components for Nile perch skin and bone gelatin and skin acid-soluble collagen
Material Component peak location (cm
1 ) and percent area (in brackets) contribution of total band
1 2 3 4
Young fish skin acid soluble collagen 1637 (69.0) 1652 (1.8) 1672 (16.7) 1696 (12.6)
Adult fish skin gelatin 50 °C 1634 (49.6) 1652 (10.9) 1674 (30.4) 1699 (9.2)
Adult fish skin gelatin 70 °C 1631 (18.8) 1658 (50.8) 1674 (2.1) 1690 (28.3)
Young fish skin gelatin 50 °C 1633 (45.1) 1652 (18.7) 1674 (25.2) 1697 (10.9)
Young fish skin gelatin 60 °C 1633 (32.9) 1657 (23.8) 1675 (8.4) 1694 (35.0)
Adult fish bone gelatin 50 °C 1632 (44.8) 1652 (19.0) 1673 (28.8) 1695 (7.4)
Adult fish Bone gelatin 70 °C 1631 (45.4) 1657 (24.4) 1673 (7.1) 1690 (23.1)
Young fish bone gelatin 50 °C 1633 (49.2) 1651 (15.0) 1674 (26.0) 1699 (9.8)
Young fish bone gelatin 70 °C 1631 (31.5) 1658 (31.8) 1672 (1.8) 1688 (34.9)
Figures derived from average spectra for triplicate determinations.
Fit quality (r2 ) between original and fitted spectra P 0.9998.

numbers in the higher temperature-extracted gelatins and decrease in molecular order. The extent of these
than in their low temperature-extracted counterparts, changes, in the case of Nile perch, seems to be affected
while the 1650 cm
1 component occurred at lower wave by the order (in a sequential extraction process) of gel-
numbers for the low temperature-extracted gelatins than atin extraction and the collagenous tissue from which
their high temperature-extracted counterparts. An am- gelatin is extracted. The secondary structure of gelatin
ide I component at around 1690 cm
1 has been reported obtained from the same raw material by sequential ex-
for gelatin (Payne & Veis, 1988; Prystupa & Donald, tractions may vary, with later extraction (higher tem-
1996; Paschalis et al., 2001) and collagen-like peptides perature) containing more intermolecular associations
(Doyle et al., 1975) and has been attributed to inter- in the dry state. The early extractions are obtained from
molecular associations. The bands around 1630, 1650 the least crosslinked collagen. Due to the relatively
and 1675 cm
1 have been assigned to imide residues milder extraction temperature, peptide hydrolysis is not
(and partly to b-sheet), random coils and b-turns, re- expected to be extensive and higher molecular weight
spectively, (Prystupa & Donald, 1996), while the helical gelatin fractions are produced. During drying, these
state is reported to show at 1660 cm
1 (Payne & Veis, form some protein-protein linkages but these are not
1988; George & Veis, 1991). The 70 °C-extracted gela- likely to be many. On the other hand, later extracts are
tins, however, had their component peaks showing at obtained from the more crosslinked collagen and con-
1657–1658 cm
1 . The corresponding peaks were found tain more low molecular weight fractions. These are
at 1651–1652 cm
1 for 50-°C extracted gelatins. These likely to form more protein–protein linkages which are
differences may be suggestive of differences in the sec- manifested as higher molecular order.
ondary structure of these gelatins. As earlier proposed, it
seems that the 70 °C-extracted gelatins contain a higher
degree of molecular order than the 50 °C-extracted
Acknowledgements
gelatins, probably due to protein–protein linkages
formed during drying of these low molecular weight
Thanks go to Prof JRN Taylor, Department of Food
gelatins. Based on their high content of the 1650 and
Science, University of Pretoria and Prof. Danita De
1675 cm
1 components, the 50 °C-extracted gelatins
Vaal, Department of Chemistry, University of Pretoria
seem to be made up, predominantly of random coils and
for their advice and support. Dr. Klaus Wellner, Insti-
b-turns.
tute of Food Research, Norwich, UK is gratefully ac-
The differences between bone and skin gelatins
knowledged for critical reading of the manuscript.
extracted at the same temperature may be due to
Author Muyonga acknowledges financial support from
structural differences between bone and skin colla-
Makerere University staff development committee. This
gens from the same species. Sims et al. (1992) reported
material is based upon work supported by the National
that the two types of tissue have different types of
Research Foundation (South Africa) under Grant
crosslinks.
number 1478.

4. Conclusions References

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