FURTHER EFFECTS OF ANIMAL AGE ON THE ALKALI PROCESS GELATIN MANUFACTURED FROM BOVINE HIDE

CGB Cole1 and JJ Roberts2.

1Leiner Davis Gelatin South Africa. PO Box 5019, West Krugersdorp. 1742. South Africa.

2Department of Food Science, University of Pretoria, Pretoria 0002. South Africa.

Published in the Proceedings of the Centinary Conference of the International Union of Leather Technologists and Chemists. London. 1997.

Introduction.

The first study of the effect of animal age in gelatine production was on Type A gelatine by Reich et al (1962a). In Cole and McGill (1988), a study of enzymic conditioning of hide showed that the gelatine extractability of hide was a function of animal age, however, the ages of the animals was based on calves, little horn growth and long horn growth. The primary subject for this study was the uncharted field of the origins of gelatine colour and the first hypothesis was that gelatine colour was a function of the alkaline conditioning process, because, tannery waste hide regularly gave gelatine of pale colour whereas gelatine from other sources was of variable and often dark colour. From our study of 1988 it was evident that no two hides could be relied upon to give the same performance, so we turned to ANPI to help us with the supply firstly of two different lots of hide from the same animal and then later the whole hide from animals of known age. We found that with hide from a single animal the random variances of gelatine extractability and yield disappeared and by conducting several experiments on one hide we were able to establish, once and for all, that variations in the alkali conditioning process had no effect on gelatine colour. We then fell back on animal age as the source of gelatine colour and found excellent correlations. Based on collagen studies by Sell and Monnier (1989), Tanaka et al (1988) and many others, this study was extended by the examination of the florescence and electrophoretic mobility of the gelatines produced, in order to gain further understanding of the effects of senescence at the molecular level.

Materials and Methods.

The methods used for the laboratory production and testing of gelatine were described by Cole and Roberts (1996a). Briefly, a salted hide was cut into ca. 10 cm x 10 cm pieces which were randomised in a tumbler and divided into lots of equal weight. The hide was then washed free of salt, immersed in a solution of lime and sodium sulphide for a specified time and at a controlled temperature of 22°C. Thereafter the hide was washed well, acidulated in sulphurous acid and again washed to an extraction pH of about 3.0. The gelatin was then extracted by warming the hide in water, in a water-bath at controlled temperatures starting at 45°C for the first extraction. The proportion of the total amount of gelatine available, that was extracted at 45°C was a measure of the extractability of hide or the effectiveness of the pretreatment (conditioning).

The methods used for measuring the fluorescence intensity of gelatine were described in Cole and Roberts (1996a) and the electrophoresis methodology was described in Cole and Roberts (1996b).

Results and Discussion.

The results of the determinations of colours of the gelatines from the hides of animals of various ages, are shown in Table 1.

Table 1. The colour of gelatines from the hides of bovines of various ages.
 
Animal Age (months) 10 18 40 58 78 144 152
45 deg. C Extract average colour in DGI units. 3.2 5.2 6.3 6.1 6.9 9.7 9.1
Average Overall colour in DGI units. 4.0 6.3 5.8 7.6 7.6 15.1 15.1

The linear correlation coefficients for animal age and gelatine colour were:

        Animal age and 45°C extract colour. r = 0.95

        Animal age and overall colour. r = 0.97

and for both correlations the probability of there being no correlation was less than 0.001. (5 degrees of freedom).

This study was based on the finding that, as far as gelatine manufacture was concerned, one hide was a uniform raw material which did not introduce a significant random variable into a series of experiments. In gelatine manufacture, it has been found that even after a standard conditioning process, the biggest variable encountered in practice was variation in the extractability at 45°C, (Cole and McGill, 1988). The next most significant variance was the yield from one hide to another. In a series of experiments, the standard deviation of yield on an anhydrous basis was shown to be 11.1% (Cole, 1995) due mainly to variances in hair content, fat content and lime and acid soluble constituents. Initial trials conducted using a mask and hide of the same weight from the same animal gave yields and extractabilities with very low variances and the results were considered satisfactory.

A series of experiments using a Brahman hide from an 18 month old animal in which the liming time was the only variable (Table 2), illustrates the methodology used and shows the reproducibility of the yield from one experiment to the next. It can also be seen that the 45°C extractability of 35% was the limit above which yield losses were observed. These were physical losses of hide during washing. Similar results were obtained with many series of experiments and was the basis on which it was accepted that the hide from a single animal was a homogeneous (invariant) raw material. It could also be noted that Table 2 illustrated the commonly accepted improvement in extractability resulting from alkali conditioning, that is applied for gelatine manufacture based on cow hide raw material.

Table 2. The effect of varying the liming time on the gelatine quality obtained from the salted hide of an 18 month old Brahman.
 
Conditioning time (weeks) 1 2 3 4 5 6
Gelatine Yield (% on RM1) 29.7 29.7 30.5 29.1 28.7 26.8
45 deg. C Extractability2 (%) 3.3 11.3 21.2 22.5 35.0 39.5
50 deg. C Extractability (%) 8.1 14.2 24.3 28.4 31.0 32.3
55 deg. C Extractability (%) 11.5 19.2 27.1 27.7 27.8 23.3
45 deg. C Gel. Bloom3 (g)  285 332 317 310 324 327
50 deg. C Gel. Bloom (g) 332 330 309 324 298 308
55 deg. C Gel. Bloom (g)  331 302 291 299 275 277
45 deg. C Gel. Viscosity4 (ms) 30.5 36.7 42.8 46.3 51.5 48.0
50 deg. C Gel. Viscosity (ms) 33.6 37.3 43.5 48.0 49.5 55.6
55 deg. C Del. Viscosity (ms) 33.2 41.2 39.5 39.4 50.1 49.7
45 deg. C Gel. Colour5 (DGI units) 5.6 5.2 5.2 5.6 6.0 5.2
Overall Colour (DGI units) 6.5 6.0 6.2 - 6.4 -

1. Yield based on raw material mass.
2. Extractability = % of total gelatine recovered .
3. Bloom gel strength by BS757:1975
4. Viscosity of solution by BS757:1975
5. Solution colour by comparison to the DGI standard.
 

Figure 1.The extractability of hide from bovines of various ages as a function of conditioning time.


The data shown in Figure 1 was extracted from the many trials similar to that shown in Table 2. The salient features of Figure 1 were the very high extractability of hide from a 10 month old animal hide, the continuously increasing extractability of hide from an 18 month old bovine with conditioning time and the very low extract-ability of hide from the 13 year old animal.

From Figure 1. it was concluded that as bovines aged they developed collagen cross-links that were more and more resistant to the liming process and it could be seen that even at 58 months of age there was evidence that liming for more than 4 weeks could not significantly increase the extractability of older animal hide.

Fluorescence.

Based on the studies of Tanaka et al (1988) and Sell and Monnier (1989) it appeared possible that the alkali stable cross-link deduced from Figure 1. could be due to the increased formation of the pentosidine cross-link with animal age, and as fluorescent pentosidine was a product of the Maillard reaction it could also be associated with the colour of gelatine. Hence, the fluorescence spectra of the gelatines were determined. Typical spectra are shown in Figures 2 to 5. It can be seen that the 335/385 pentosidine fluorescence of pale, colour 4.4, gelatine was much less than that of dark, colour 16, gelatine.

In Figures 2 and 3 the first peaks at 360 nm are attributed to Rayleigh scatter (Munck and de Francisco, 1989). Figure 2 shows the fluorescence emission spectrum (FES) of Type B Gelatine from a 10 month-old animal (i.e. alkali treated collagen). It was found to be limited to an excitation wavelength peak at 335 nm (Figure 4) with emission maxima at 385 and 410 nm. Figure 3 shows the FES of gelatine from a 12 year old animal. The marked increase in fluorescence intensity of the gelatine from the older animal is evident. Figure 4 shows the excitation scan for Type B gelatine and confirms the presence of a single fluorescence maximum at 335 nm (for alkali process gelatines).

Table 3. A comparison of bovine age, and the colour of the derived gelatine and its 335/385 nm pentosidine fluorescence.
 
FIRST EXTRACTION at 45 deg. C
Animal Age (months) 10 18 40 58 78 144 156
Gelatine colour (DGI* units) 3.6 5.6 6.6 5.6 8.4 8.9 10.0
Gelatine 335/385 nm fluorescence intensity. 38 104 102 104 141 133 187
SECOND EXTRACTION at 50 deg. C
Animal age (months) 10 18 40 58 78 144 156
Gelatine colour (DGI* units) 4.4 5.6 6.0 6.8 7.6 12.3 9.4
Gelatine 335/385 nm fluorescence intensity. 48 133 138 126 165 168 223
*Davis Gelatine Industries.
 

Type A gelatine was made from calf (less than 6 months of age) skin using the acid conditioning process (Reich et al, 1962b) usually used for the manufacture of gelatine from pigskin. This gelatine exhibited 335/385 nm fluorescence and it also had a 295 nm fluorescence excitation maximum as shown in Figure 5, which would be due to the pyridinoline cross-link which Eyre et al (1984) had showed to be alkali labile. Hence it followed that initially, in young animals, collagen was stabilised by the pyridinoline cross-link but with age this alkali labile cross-link was superseded in importance by the pentosidine

Figure 2. Fluorescence emission spectrum of pale
Colour 4.4, Type B gelatine from 10 month old
calf skin (YSA/2). Excitation 335 nm.


Figure 3. Fluorescence emission spectrum of dark,
Colour 16, Type B gelatine from a 12 year old Inguni
cow hide. Excitation 335 nm.
Figure 4. Fluorescence excitation spectrum of Type B gelatine.
Emission at 395 nm.


Figure 5. Excitation spectrum of Type A calfskin
gelatine. Emission at 385 nm.


cross-link which was stable to alkali as it persisted in Type B gelatine. A detailed correlation of the 335/385 pentosidine fluorescence intensity and gelatine colour or animal age is given in Table 3.

The linear correlation between animal age and the fluorescence of the gelatine extracted at 45°C.
r = 0.895

The linear correlation between animal age and the fluorescence of the gelatine extracted at 50°C.
r = 0.771

Both these correlations are significant at the 97.5% level of probability.

The reasons for the somewhat poor linear correlations between animal age and 335/385 nm fluorescence intensity will become apparent later.

Electrophoresis.

The electrophoresis of some of the gelatines produced from the hide of animals of known age was undertaken in order to gain an insight into the effect of animal age on the molecular structure of the gelatines. The electrophoretic densitograms of gelatin from a 10 month old animal labelled YSA and that from 144 month old animal labelled ST24 are shown in Figure 6. It can be seen that the YSA/1 gelatine (extracted at 45°C) and YSA/2 gelatine (extracted at 50°C), consisted very largely of collagen and chains and together constituted 62 % of the available gelatine. On the other hand only the ST24/1 gelatine (extracted at 45°C) consisted of largely and chains and this was only 9.6 % of the available gelatine.

From Figure 6 it was concluded that, after alkaline conditioning, the first gelatine to go into solution was thermally denatured collagen alpha and beta chains which dissolved from the polymer as a result of the hydrolysis of the alkali labile (pyridinoline) cross-links. As the animal aged, however, there was a build up of alkali stable cross-links which required, as a result of pentosidine formation, thermal hydrolysis of the collagen protein for solubilization of the collagen as gelatin. Hence, gelatin from older animals consisted largely of peptides of indeterminate molecular weight, as shown by the -/3 traces, whereas gelatin from young animals consisted largely of linear collagen subunits that had been thermally denatured but not hydrolysed. It should be stated that as a result of denaturation it would be impossible for the gelatine to behave like acid soluble collagen which could renature and reform collagen fibres from solution. Furthermore, from Figure 6, the similarity of the -/1 densitograms would suggest that at 45°C the temperature was too low to cause much thermal hydrolysis of the collagen chains, hence at this temperature and at pH 3, virtually the only product available was denatured alpha and beta chains (thus accounting for the low 45°C gelatine extractability of old-animal hide even after extended periods of alkaline conditioning).

Conclusions.

This study gave a further insight into what was occurring at the molecular level during the alkaline conversion of collagen to gelatine:

1. The fluorescence data showed that alkaline conditioning destroyed the pyridinoline cross-links of collagen and the electrophoresis data showed that with young animals the gelatine consisted mainly of collagen , and chains hence it appears that after alkaline conditioning young animal collagen is free to undergo thermal denaturation and simply disperse into solution.

There have been reports (Uchiyama et al, 1991), that the amount of pyridinoline cross-link is invariant with animal age, whereas others have contended that the pyridinoline cross-link changes (Eyre et al, 1984) and stabilises with age thus accounting for the toughening of meat and the increasing difficulty of gelatine extraction with animal age.
 

Figure 6. Electrophoretic densitograms of gelatines from 10 month old animal (YSA) and 144 month old
animal (ST24) extracted at 45 deg. C (-/1), 50 deg. C (-/2) or 60 deg. C (-/3)


2. This study also showed that there was increase in gelatine colour, reduced extractability, increased fluorescence and increased polydispersity of gelatine with animal age. All these phenomena are compatible with the continuing formation of the non-enzymic pentosidine cross-link in collagen with senescence. The cross-link is evidently stable to alkali, acid, and heat processes used in Type B gelatin manufacture, as the cross-link fluorescence persists in the gelatine. Hence it followed that even if the pyridinoline cross-link was constant with age, its influence was replaced by that of pentosidine, with the result that there was in practice little difference in the polydispersity of Types A and B gelatine. (Cole and Roberts 1996b). That is, in the presence of collagen cross-links, gelatine is largely a product of thermal hydrolysis of protein peptide bonds.

This study suggests, that the continuing formation of the non-enzymic pentosidine cross-link is in fact responsible for the change in collagen leading to reduced gelatine extractability with age and therefore possibly also to the toughening of meat. The probability is that the colour of gelatine was attributable also to the Maillard pentosidine reaction between serum glucose and the -amino group of lysine and the side chain of arginine. The lower coefficient of correlation between animal age and gelatine fluorescence could be due to the low amount of cross-links extractable at 45°C as confirmed by the electrophoresis data, or the fact that pentosidine is colourless and therefore the coloured product was a further reaction product of the Maillard complex. To that should be added the probability that there could well be more than one age related reaction causing gelatine colour.
 

Finally. from the practical point of view, this study shows that animal age can have a significant effect on products made from hide or collagen, hence, when uniformity of product is a consideration then animal age should be of major concern to the manufacturer. Put another way, there is no such thing as "hide", because at the molecular level there will be marked differences in the cross-linking structure from animal to animal and for the researcher it is even more important to take full cognisance of this fact. Furthermore, collagen fluorescence could well have applications in all industries using collagen as a raw material, as the technique is basically simple and does not need difficult preparation of the product for analysis.

ACKNOWLEDGEMENTS.

The authors wish to thank the Animal Nutrition and Production Institute (Irene, RSA) for the supply of hides from animals of known biological age.

Our thanks also go to Leiner Davis Gelatin SA for permission to present this study.
 

References.

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Cole C.G.B. Occurrence, measurement and origins of gelatine colour as determined by fluorescence and electrophoresis. 1995. Ph.D Thesis, University of Pretoria, Pretoria 0002, South Africa.

Cole C.G.B. and Roberts J.J. Gelatine fluorescence and its relationship to animal age and gelatine colour. SA Journal of Food Science and Technology. 1996a. 8(4):145-149.

Cole C.G.B and Roberts J.J. Changes in the molecular composition of gelatin due to the manufacturing process and animal age, as shown by electrophoresis. J. Soc. Leather Technol. Chem. 1996b. 80:136-141.

Eyre R.E., Paz M.A., Gallup P.M. Cross-linking in collagen and elastin. Annual Review of Biochemistry. 1984. 53:717-784.

Munck L, de Francisco A. Fluorescence analysis in foods. New York: Longman Scientific & Technical and John Wiley & Sons Inc. 1989:37.

Reich G., Walther S., Stather F. The influence of the age of cattle and pigskin on the yield and the quality of the gelatine obtained after the acid conditioning process. Deutsche Lederinstitut. Frieberg/SA. 1962a. 18:24-30.

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Tanaka S., Avigad G., Eikenberry E.F., Brodsky B. (1988). Isolation and partial characterisation of collagen chains dimerised by sugar-derived cross-links. J. Biol. Chem. 1988. 263:17650- 17657.

Uchiyama A, Ohishi T, Takahashi M, Kushida K, Inoue T, Jugie M, Horiuchi K. Fluorophores from ageing human articular cartilage. Journal of Biochemistry (Tokyo). 1991. 110:714-718.