C.G.B. Cole* and J.J. Roberts. Department of Food Science. University of Pretoria. Pretoria. 0002. RSA.

Originally published in the Journal of Leather Technologists and Chemists.(1996) Vol. 80, 136-141.


Type A gelatine was made from the skin of less than six month old calves by an acid process. Type B gelatine was made from a 10 month old calf skin as well as from the hides of mature bovines of 58 and 144 months of age, by an alkaline process. On electrophoretic separation the Type A calf skin gelatine closely resembled pigskin gelatine with a large number of bands below 100 kD while Type B calfskin gelatine contained unusually large amounts of intact collagen - and - and -chains. From the older animals, the best (first extraction) Type B gelatines contained large amounts of intact collagen chains the amounts of which appeared to decrease somewhat with increasing animal age. Type B gelatines were also observed to contain a peptide with a molecular mass of 82 kD. The quantity of intact collagen chains in Type B gelatine decreased markedly at higher extraction temperatures. Gelatine colour was observed to become darker with animal age, however there was no strict relationship between gelatine colour and molecular composition. The implications of these observations on the molecular mechanisms of collagen conversion and gelatine colour are discussed.


Electrophoresis is recognized as a primary method for investigating proteins at the molecular level.1-4 Hodney et al5 in their study distinguished age related changes in collagen due to glycation, by the degree of silver staining after SDS-PAGE however this technique was not pursued. In this study electrophoresis was used as a tool to ascertain whether differences in molecular weight profiles existed between gelatines of different colour.

It was accepted that gelatine was a protein derived from collagen by the thermal hydrolysis of an infinite polymer. Chalepakis3 showed that for acid conditioned pigskin gelatine, the molecular weight profile was more disperse than for alkali process gelatines. This meant that the alkali process gelatine contained larger amounts of the -chain protein (the monomer from which collagen is built) as well as dimers called -chains and relatively little protein with intermediate molecular weights. Acid process gelatines contained relatively large amounts of proteins with molecular weights between >300 kDalton (kD) and 1 kD. Superimposed on this polydispersity were amounts of - and -chain and other peptides. Furthermore, Chalepakis3 showed by means of SDS-PAGE electrophoresis, that the -chain material from acid conditioned pigskin had a faster migration rate than -chains from alkaline conditioned material.

In Type A gelatine manufacture, the acid conditioning of pigskin is simply a process of equilibration of the collagen to an acid pH prior to heating for extraction and Reich6 points out that this is not a conditioning process in the terms of the classic alkaline conditioning process. More recently Müller and Heidemann7 showed that acid thermal hydrolysis of collagen (that occurs during acid extraction) is not random in that there are preferred points of collagen hydrolysis.

Hence, it can be proposed that in the acid process, denaturation of the collagen resulted from thermal hydrolysis with the production of gelatine consisting of peptides with a largely random distribution of molecular weights (or polydisperse protein). The presence of , ß and chain material in pigskin gelatine could be explained by the probable presence of acid soluble collagen units8 in the collagen of very young animals (pigs are normally slaughtered at about 6 months of age). However, in the alkaline process the action of alkali results in the hydrolysis of "reducible" or labile collagen cross-links. This frees some of the -chain subunits allowing them to dissolve (first extraction) as soon as the stabilizing hydrogen bonds are sufficiently weakened by temperature and/or the presence of hydrogen bond breakers in the environment. An additional increase in temperature (second and third extractions) would then be expected to cause peptide bond hydrolysis and the conversion of collagen into gelatine which should be seen as having a polydisperse molecular weight profile.

Based on the above reasoning it was hypothesised that the electrophoretograms of darker gelatine, from old animals, could be distinguished from the electrophoretograms of paler gelatines from young animals (Table 1). Old animal gelatine should exhibit greater amounts of "random" proteolysis.

The objectives of this study were to:

  1. Use acid soluble collagen as the reference standard for collagen component electrophoretic mobility.
  2. Compare Type A and Type B gelatines from calf skin.
  3. Compare Type A gelatines from calf skin and pigskin.
  4. To compare Type B gelatines derived from young, middle-aged and old bovine's hide after similar alkaline conditioning treatments.

Materials & Methods.

Measurements were conducted in duplicate and the averages were reported. Analytical Reagent grade chemicals were used unless otherwise indicated.

The salted hides of animals of known date of birth and date of slaughter were supplied by the Irene Animal Production Institute, Pretoria, RSA.

Table 1. Manufacturing and analytical details of the gelatines tested.









pH Time














ASC < 6 A - - - - - - - - - - -
CA/1 < 6 A 50 3.63 5 43.2 351 55.9 4.0 48 10.1 0.27 6.2
D147 U A U U U U 289 58.4 2.0 44 8.4 0.20 4.5
YSA/1 10 B 45 3.06 5 35.3 326 55.1 3.6 44 9.1 0.46 5.8
YSA/2 10 B 50 3.08 5 26.6 313 50.8 4.4 44 9.9 0.28 5.6
YSA/3 10 B 55 3.28 5 20.9 286 47.5 4.8 48 10.8 0.58 5.6
5Y4/1 58 B 45 2.99 5 20.0 311 35.7 5.6 19 10.0 2.32 5.9
5Y4/2 58 B 50 3.37 5 20.8 269 34.5 6.8 21 9.9 3.94 5.8
5Y4/3 58 B 60 3.61 5 28.5 226 33.2 8.4 20 11.5 2.41 5.6
ST24/1 144 B 45 2.73 5 9.6 322 32.5 8.9 21 8.4 3.64 5.9
ST24/2 144 B 50 3.26 5 13.1 315 31.7 12.3 6 8.1 3.16 5.3
ST24/3 144 B 60 3.47 5 26.4 230 23.2 13.3 21 10.9 1.56 5.2
1 Temperature of extraction in C. 2 Extractability % . 3 Gelatine Viscosity (in millistokes) at 6.67% and 60C.
- Data not available or not applicable. U = Unknown.

Gelatine production.

Whole hide was cut into approximately 100 x 100mm pieces. A quantity (5-7 kg) was taken and washed free of salt overnight using a stainless steel tumbler (13 RPM) fitted with a continuous supply of water (through one axle) and a perforated plate in the door for drainage. The washed hide was placed in 20 kg of conditioning liquor containing sodium sulphide (2g/L) and calcium hydroxide (40g/L) (commercial lime and sodium sulphide were used). The hide was conditioned for 4 weeks at 22°C ± 2°C. The conditioning liquor was stirred every second or third day in order to prevent localised reductions in pH. After stirring the temperature was recorded. After conditioning, the hide was washed overnight in the tumbler, which removed lime and the hair and other non-collagen materials released from the hide during alkali conditioning. The hide was then acidulated using 5 x 20 L lots of sulphurous acid solution (0.1M) (commercial) over 4 days followed by soaking in tap water for 1 day. The material was then converted to gelatine by sequential 5 hr extractions in water (at 45°C, 50°C, 55°C) and finally by boiling (93°C) for 7 hr. After each extraction the gelatine solution was separated from the solid residue using a colander. The volume of each separated gelatine solution was determined. An aliquot of the solution was then filtered through Whatman 541 paper after which the concentration was determined gravimetrically in duplicate by drying 10 ml of solution at 105°C for 40 to 48 hours. The weight of the dry film was multiplied by 11.4286 to obtain the concentration (% gelatine containing 12.5% moisture) of the original solution. From the volume and gelatine concentration, the total amount of gelatine extracted was calculated. The gelatine produced in each extraction was expressed as the % of the total amount of gelatine recovered from a particular sample of hide. This was a measure of the "extractability" of a hide.

The bulk of each gelatine extraction liquor (excluding the boil solutions) was filtered through paper pulp and then vacuum evaporated (to about 10% concentration) at 40 to 42°C using a rising film glass evaporator. This was followed by refiltration through paper pulp. The concentrated gelatine liquor was then treated with a 5% ammonia solution as well as a 5% hydrogen peroxide solution until the liquor exhibited an approximate 10 ppm excess of peroxide and a pH of 5.0 to 5.5 (using Merck indicator strips). The solution was then set in a refrigerator, cut into slices and dried overnight in a current of air to about 10% moisture content. The sheets of dry gelatine were ground (using a domestic coffee grinder) to a powder to facilitate analysis using British Standard 757:1975 methods for the determaiation of Bloom gel strength, viscosity, moisture and ash contents etc. Bloom gel strength is a measure of the gel forming ability of gelatine and is the most important attribute in many applications.

The colour and clarity of the gelatine were determined by in-house methods:

a) Gelatine solution (60 ml at 40°C) from the Bloom gel strength determination (6.67%) was diluted to 100 ml using 40°C distilled water and then compared, using 100 ml Nessler tubes, to 100 ml of a 4% solution of a standard gelatine (with an ascribed colour of 8.0). Solution was poured out of the darker Nessler tube until a match was obtained. The colour of the unknown gelatine was then calculated in accordance with Beer's Law. For example, if 60 ml of unknown matched 100 ml of standard (colour 8) then the colour of the unknown was 100x8/60 = 13.3. This method was used to determine the colour of all the gelatines produced. This included the colour of the gelatine in the boil liquor. Using the quantity of gelatine in each extraction and its colour the overall or composite colour of the gelatine from each lot of hide was calculated.

b) Gelatine solution clarity was determined by filling a Turbidimeter (ICM Turbidimeter. ICM. 163 S.W. Freeman, Hillsboro, OR 97123, USA) cuvette (20 ml) with molten Bloom gel strength sample (6.67% gelatine) and reading the turbidity in NTU. The meter was calibrated daily against a 40 NTU standard supplied by the instrument manufacturer.

Gelatines used in this series of experiments:

ASC - Acid soluble collagen control. (loc sit.)

CA/1 - Type A gelatine from calf (less than 6 months of age) skin,
using the method of Reich.6 Liquor processing was as described under "Gelatine Production" above.

D147 - Type A pigskin gelatine. Details of manufacturing process were not known.

YSA/1 to YSA/3 - Type B gelatine from 10 month-old calf skin after 2 weeks of alkaline conditioning at 22°C.

5Y4/1 to 5Y4/3 - Type B gelatine from a 5 year-old Chianina after 4 weeks of alkaline conditioning at 22°C.

ST24/1 to ST24/3 - Type B gelatine from a 12 year-old bovine after 4 weeks of alkaline conditioning at 22°C.

The detailed analytical and manufacturing data is shown in Table 1.

These gelatines were chosen to span the animal age range for Type B gelatine manufacture as well as the extraction temperatures of 45, 50and 55°C. This range of gelatines could be run simultaneously on one electrophoresis gel. As the apparatus allowed the electrophoresis of two gels at the same time each gelatine could be run in duplicate.

For the gelatine electrophoresis, electrophoretic or analytical grade chemicals were used as appropriate. The methodology of Chalepakis3 was implemented with the following modifications:

1. Layering Solution.

50 ml t-Butanol

50 ml distilled water

Mixed well in a sealed glass container and then allowed to separate into a butanol upper layer saturated with water and a water rich lower layer. The upper layer was used for "layering".

2. Stain Solution.- Made up fresh daily, one lot for each gel.

Trichloroacetic acid (100 g) was dissolved in distilled water (200 ml).

Coomassie Blue (1.25 g) dissolved in methanol (200 ml).

Immediately prior to use, the above were mixed in a 500 ml volumetric flask and then diluted with distilled water.

3. Marker dye.

Bromophenol blue (0.05 g) was dissolved in sample buffer (10 ml).

4. Sugar solution.

Sucrose. (40 g).

Sodium dodecylsulphate. (0.2 g).

Marker dye. (5 ml).

Diluted to 100 ml with distilled water.

BioRad (2200 Wright Av. Richmond, California 94804, USA) PROTEAN II Slab Cell apparatus was used and the layering recommendations in the manufacturer's manual were followed. Teflon Combs for 20 wells (1.5 mm thick gels) were used.

Soluble collagen standard.

The production of a soluble collagen standard was based on the method described by Na et al.8 As they worked at 4°C throughout the process, it was not possible to follow their method precisely. Furthermore, they appeared to have disregarded the warning of Johns9 that if steps are not taken to remove neutral salt soluble collagen prior to the extraction of acid soluble collagen, then both types were extracted by acid solution.

1. Reagents:

Acetic Acid (0.5M) - 29 ml glacial acetic acid per l.

Saturated Salt Solution - 300 g NaCl/L

Sodium phosphate (NaP) (0.05M) buffer pH7:

NaH2PO4 (0.05 M) in 1.0 M NaCl (pH 3.4) - 160 ml

Na2HPO4 (0.05 M) in 1.0 M NaCl (pH 8.4) - 800 ml

2. A fresh calf skin was obtained from an abattoir and immediately tumbled overnight with coarse salt (50% of its weight). The hide was then allowed to drain for 24 hr. The drained hide was cut into approx. 20 mm x 20 mm pieces which were stored in a sealed plastic bag in a 5°C refrigerator until required.

3. For extraction of collagen, hide (125 g) was taken and washed overnight in a continuous flow of water. The hide was then drained of excess water and placed in a 2 L flask together with 1 L acetic acid (0.5 M) and agitated by means of a magnetic stirrer for 8 hr followed by 16 hr standing at ambient temperature. The insoluble residue was separated from the very viscous liquid using 300 nylon mesh. The liquid was then centrifuged at 63 hz (approx 2500 g) for 30 min. The supernatant (475 ml) was recovered and the collagen was precipitated by adding 1/5th volume (95 ml) of saturated salt solution very slowly with stirring. The precipitated collagen was separated by centrifugation to yield single precipitated acid soluble collagen (SPASC).

4. An attempt was made to follow the purification process of Na8 by dissolving the SPASC in NaP (0.05 M) buffer with continuous stirring overnight. This was followed by centrifuging and adding NaCl to 3M (174 g/L). By this method it was found that there was no sediment to remove by centrifugation nor was there any precipitation of collagen from the solution.

5. The SPASC was purified by redissolving in 250 ml acetic acid (0.5M) and stirring for 30 min. The solution was vacuum filtered through Whatman GF/A filter paper in a Büchner funnel. The collagen was reprecipitated by slow addition of 1/5th volume of NaCl (30%). The precipitate was recovered by centrifugation for 30 min. The supernatant was discarded and the solid material was transferred to a glass bottle for storage at 2°C until required as acid soluble collagen (ASC).

Preparation of samples for electrophoresis.

Using the method of Chalepakis3 gelatine (0.1 g) was dispersed in 10 ml Sample Buffer. After soaking for 30 min the samples were placed in a 40°C waterbath. The solutions were homogenised or mixed by means of a vortex mixer.

ASC standard. ASC (2 g) was weighed into a test tube and dispersed in sufficient boric acid/Tris buffer (15 ml, pH 8.8) to give a pH of 6 on a Merck pH strip. Saturated NaCl solution (approx. 10 ml) was added slowly with agitation until a precipitate was formed. The collagen was recovered by centrifugation at 50 hz for 10 min. The recovered solid was dissolved in 15 ml sample buffer (pH 9.2).

For electrophoretic runs, 0.5 ml of sample in a test tube was mixed well with 0.5 ml of sugar solution by means of a vortex mixer. Fifteen µL was loaded into each well of the polyacrylamide gels (5 %) by means of a Hamilton syringe.


A waterbath fitted with a cooling coil, thermostatically controlled heater and a circulating pump was set to maintain a temperature of 14°C. Cooling water from the outlet of the circulating pump was connected to the inlet of the central cooling tank of the electrophoresis apparatus and the outlet from the tank was returned to the waterbath using rubber tubing. Electrophoresis buffer (2.5 l) for each run was made up in an Erlenmeyer flask and cooled overnight in the 14°C waterbath. At the start of each run 2.15 L of cold buffer was poured into the lower buffer chamber (outer shell) of the apparatus.

The cast gels (5 %) were released from the casting apparatus and installed onto the central cooling tank of the electrophoretic apparatus to form the upper buffer (cathode) chamber. Care was taken to moisten the gaskets with buffer before clipping the gels into place. The positions of the wells in the stacking gel were marked on the outer plate of the gel using a waterproof marking pen. The combs were carefully removed from the gels and the central tank was placed in the outer tank. The remainder of the cold buffer (350 ml) was used to substantially fill the upper buffer chamber formed by the gels and the top of the inner tank. Each well was loaded with 15 µL of sample using a Hamilton syringe. The cover was then fitted to the apparatus and the leads connected to the DC power pack.

The electrophoresis of two gels was started with the current adjusted to 50 ma until the marker dye had moved into the resolving gel. The current was then increased to 70 ma. When the marker dye reached the bottom of the gels, the power pack was switched off and disconnected.

Post-electrophoresis treatment.

The gels were removed from the electrophoresis apparatus. The glass plates were removed from the holders and the plates were separated. Each gel was then marked by removing the bottom right corner so that the order of the lanes could be identified. The gel (still attached to one plate) was placed in a polythene container and covered with fresh dye solution (500 ml). The container was rocked from time to time over 30 min. During this period the gel and support plate separated. Dying of the gel was allowed to continue overnight. The dye solution was sucked from the container using a vacuum pump (tap water system) and was replaced with destaining solution (500 ml). The glass plate was also removed at this stage. After 15 min, the gel was manipulated onto a clean glass plate and transferred to a clean container in which destaining was completed over 7 to 8 hr. A total of 1 L of destaining solution per gel was used in 250 ml lots.

The gels were scanned using a Hoefer Scientific Instruments (San Francisco, USA) GS 300 Transmittance/Reflectance Scanning Densitometer with gain set to minimum. The output was plotted using a Lloyd Scientific Graphic 1000 (Lloyd Scientific, Johannesburg, RSA) plotter set to a 0 to 100 mv range and with gain set to a low level. This was necessary in order to minimise noise and to keep the plot within the width of the recording paper.

Results & Discussion.

Acid soluble collagen standard preparation.

The reason for the failure of the phosphate buffer precipitation in the ASC purification process of Na8 was possibly because the procedure was carried out at ambient temperature - It was observed that acid soluble collagen was completely soluble in electrophoresis buffer at ambient temperature (15°C) whilst under refrigerated storage conditions, it precipitated.


Preliminary experiments had shown that it was necessary to ensure the insolubilisation of the gelatine during the PAGE gel dying process. The dye solution of Chalepakis3 was found to be unsatisfactory due to the limited solubility of the Coomassie blue and the difficulty of filtration and the variable results obtained with filtered dye solutions. Hence, it was necessary to develop our own dye solution. This contained a high level of trichloroacetic acid (TCA) to prevent solution of gelatine and also methanol which was an excellent solvent for the Coomassie blue. Reaction between the methanol and TCA was observed but this did not adversely affect the dying of the gels nor did the oil formed, stain the gels. The oil was soluble in methanol for cleaning purposes.

Figure 1. Electrophoretic densitograms of acid soluble collagen (ASC),
Pigskin gelatine 147 (PIGSKIN 147), and first extraction gelatines from calf
skin, Type A = CA/1 (CALF Ty A/1) and Type B = YSA/1 (CALF Ty B/1).
Collagen gamma, ß and alpha chains are designated.

Figure 2. Electrophoretic densitograms of Type B gelatines from 10
month old bovine's hide. Collagen - and -chains designated. YSA/1 first
extraction, YSA/2 second extraction and YSA/3 third extraction.

The densitometric traces of the electrophoretic separations are shown in Figures 1 to 4. Figure 1 shows a comparison of the traces of Type A pigskin and first extraction Type A calf skin gelatines (CA/1) as well as Type B calfskin gelatine (YSA/1). Acid soluble collagen was also included for reference purposes. The observations made were:

1. The trace produced by the ASC standard showed well formed bands from gamma, beta and alpha chain collagen with the 1 and 2 proteins being well separated.

2. There was a marked similarity between the traces from the pigskin and the calf skin Type A gelatines and in particular the large number of bands with migration rates greater than ASC -chain were very similar to those obtained by Chalepakis3. It was observed also that with Type A gelatine and with ASC, the -chains were doublets or two bands of very similar mobility. The general impression was of a large number of peaks (bands) superimposed on a mass of polydisperse protein.

3. The Type B first extract calf skin gelatine was characterised by a far lower number of bands particularly with migration greater than 60. The small amount of protein with a molecular mass less than the -chain appeared to be a characteristic of Type B gelatines. Furthermore, the quantity of -chain collagen was considerably greater than in the Type A gelatines and also the -chain peaks were observed to be single instead of the doublets of Type A gelatine.

4. As shown by Chalepakis3, the migration rate of Type A gelatin -chain was somewhat faster than that of Type B gelatin -chain. In addition, it was observed that the migration rate of -chains from ASC and pigskin gelatine were the same. From this it was concluded that Type B gelatin -chain protein had a higher molecular weight than that of pigskin or ASC.

Figure 3. Electrophoretic densitograms of Type B gelatines from 58
month old bovine's hide. Collagen - and -chains designated. 5Y4/1 first
extraction, 5Y4/2 second extraction and 5Y4/3 third extraction.

Figure 4. Electrophoretic densitograms of Type B gelatines from 144
month old bovine's hide. Collagen - and -chains designated. ST24/1 first
extraction, ST24/2 second extraction and ST24/3 third extraction.

Figures 2, 3 and 4 show the electrophoretograms of Type B gelatines from 10 month old (YSA), 58 month old (5Y4) and 144 month old animals(ST24).

Figure 2 showed that the second (50°C) extraction gelatine (YSA/2) contained far larger amounts of - and -chain peptides than did the first (45°C) extraction gelatine (YSA/1). This indicated that the alkaline conditioning period of 2 weeks had not been sufficient to liberate all the -chains at an extraction temperature of 45°C and that additional thermal hydrolysis was needed to complete the liberation of -chains. The large amount of -chain protein in the second extraction gelatine also indicated that this collagen component was markedly resistant to thermal hydrolysis. Figure 2 also showed that as anticipated, the third (55°C) extraction gelatine (YSA/3) was almost entirely the product of random thermal hydrolysis resulting in polydisperse protein containing a negligible amount of the intact collagen (-chain) protein. What was of additional interest, however, was that all the Type B gelatines showed a band immediately after the -chain (at migration approximately 60) which had not previously been noted for these gelatines. From Table 2 it was deduced that this peak corresponded to peptide with molecular weight of about 82 kDalton. It is evident that Müller and Heidemann7 were aware of the discrete low molecular weight species in Type A gelatine. Hence, their demonstration of preferred points of collagen hydrolysis at or very close to arginine (due to temperature at low pH). The observation of the 82 kDalton fragment of the -chain in all type B gelatines would lend further weight to the contention that collagen possesses weak points of preferred (thermal) hydrolysis.

Table 2. Data for molecular weight calculations from ASC mobility.
Chain Molecular Weight D. Log Mw Migration Distance mm. Linear correlation.
Gamma 300 000 5.477 6.5 A = 5.565

200 000 5.301 24 B = -0.0118

100 000 5.000 47 r = 0.997
Log Mw = 5.565 - 0.0118 x distance. (p > 0.975).

Figures 2, 3 and 4 showed a remarkable similarity in the first (-/1, 45°C) extraction gelatines with respect to the amounts of -chain protein however there appeared to be a drop in the amount of -chain protein with animal age. The most marked differences due to animal age were seen in the second (-/2, 50°C) extraction gelatines where the reduction and eventual disappearance of the collagen -chain with age was noteworthy. Figures 3 and 4 again showed that the third extraction (-/3, 55°C) gelatines were largely devoid of intact collagen peptides and that the protein was polydisperse. Finally, the drop in collagen extractability with animal age as shown in Table 1 was noteworthy. The 35% extractability, at 45°C, for 10 month old animal, dropping to 10% for 144 month old animal was a clear indication of the extent of the formation of alkali resistant cross-links with animal senescence. This drop in extractability with animal age was also noted by Reich et al10 in their study of the acid conditioning process.

From the point of view of gelatine colour, gelatine YSA/3 had a colour of 4.8 and yet exhibited virtually no - and -chain peaks while gelatine 5Y4/1 had a colour of 5.6 and a large -chain content similar to that of gelatine ST24/1 with a colour of 8.9. Hence, it was evident that there was no strict correlation between gelatine colour and molecular composition. However, it was noted that for the same raw material, the less the amount of -chain material the darker the colour of the gelatine. Also, as the age of the animal increased so the amount of the (first extraction gelatine) -chain material decreased and the colour increased.


This study confirmed the findings of Chalepakis3 with regard to Type A gelatine but we have been able to compare Type A and Type B gelatine from calf skin and highlight the differences at the molecular level. Furthermore, it was found that pale gelatines contained more of the intact collagen (- and -chains) subunits than did dark gelatines indicating that stable cross-links were associated with gelatine colour.

In particular:

1. From the polydisperse nature of Type A gelatine electrophoretograms it was confirmed that this gelatine was largely the product of collagen peptide bond thermal hydrolysis. In particular, the large number of discrete bands with molecular weight less than the 100 kD collagen -chain seems to be characteristic of this gelatine.

2. First extraction Type B calf skin gelatine was the product of alkaline hydrolysis of cross-links between collagen -chains which allowed the intact collagen subunits to go into solution. Rising temperature (second and third extraction) permitted disintegration of the originally ordered structure. All Type B gelatines were observed to contain a protein band with a molecular weight of 82 kD confirming the finding of Müller and Heidemann7 that collagen had preferred points of hydrolysis.

3. With senescence Type B gelatines were affected by the formation of more stable cross-links which were not susceptible to alkaline hydrolysis resulting in the need for increased thermal hydrolysis to denature the collagen. This is shown by reduced extractability at 45°C (35% for calf skin reducing to 10% for 12 year old animal) and consequentially the recovery of greater amounts of polydisperse gelatine from older animals. It was noted that reduced extractability was accompanied by darker gelatine colour however there was apparently no relationship between molecular composition and gelatine colour.


1 Hames, B.D & Rickwood, D. Gel electrophoresis of proteins: a practical approach. IRL Press Limited, Oxford and Washington DC. (1981).

2 Koepff, P. The use of electrophoresis in the gelatin manufacture. International working group for photographic gelatin reports 1970 - 1982. Ammann-Brass, H. and Pouradier, J. (Ed.) (1984).

3 Chalepakis, von G., Tanay, I., Heidemann. E. Das Leder 1985, 36(1), 2-10.

4 Tanaka, S., Avigad, G., Eikenberry, E.F., Brodsky, B. The Journal of Biological Chemistry, 1988, 263, 17650-17657.

5 Hodney, Z., Struzinsky, R., and Deyl, Z. Journal of Chromatography 1992, 578, 53-62.

6 Reich, G., Walther, S., Stather, F. Deutsche Lederinstitut Freiberg/SA. 1962, 18, 15-23.

7 Müller, H.-T., Heidemann, E. Das Leder, 1993, 44, 69-79.

8 Na, C.G., Phillips, L.J., Freire, E.I. Biochemistry, 1989, 28, 7153-7161.

9 Johns, P. In: The Science and Technology of Gelatin. Ward, A.G., Courts, A. (Ed.). Academic Press. London New York San Francisco. p. 41-42. (1977).

10 Reich, G., Walther, S., Stather, F. Deutsche Lederinstitut Freiberg/SA. 1962, 18, 24-30.


Submitted in partial fulfilment of the requirements for the degree of Ph.D (Food Science). University of Pretoria. Thanks to Leiner Davis Gelatin (South Africa) for their support.