From sticky mucus to Probing our Past: Aspects and problems of the Biotechnological use of Macromolecules

Food and Biopharma Processes imposing stresses on macromolecules:

• Thermal Processing• Irradiation• Freeze-thaw• Spray drying • Filtration • Extrusion • Lyophilisation

• Thermal Processing• Irradiation• Freeze-thaw• Spray drying • Filtration • Extrusion • Lyophilisation

Food and Biopharma Processes imposing stresses on macromolecules:

Courtesy of C. Faille, INRA, France

Stability in Response to Bioprocessing I:Thermal processing, D, z and F values

Steve Harding

Courtesy of C. Faille, INRA, France

EXOSPORIUM

PEPTIDOGLYCAN CORTEX

DEHYDRATED PROTOPLAST

COAT

DORMANT SPORE: Bacillus cereus

• Most bacteria (Salmonella, Listeria etc) can be destroyed by pasteurisation, but the spore forming ones such as Clostridium botulinium require severe heat treatment at temperatures >100oC.

• Other bacterial spores may not be pathogenic but can cause spoilage to the foods

• Thermal processing is the most common form of treatment – but can also destroy important components of the food from small vitamins to large proteins and polysaccharides

• The goal of Thermal Processing is to make the food safe for the consumer but minimising the disruption of the food components

So this presentation is about how the Processing Industry goes about doing this, the criteria used for safety (based on D, z and F values) and the consequences for the food components

Theories for the dehydration and heat resistance of spores

1. Lewis, Snell & Burr: Contraction of the peptidoglycan cortex is responsible.

2. Gould & Dring (Unilever, Bedford UK): high osmotic activity and expansion of the cortex is responsible, brought about by the presence of many unshielded acidic groups.

3. Ellar (Cambridge): dehydration of the protoplast occurs at an earlier stage by osmotis of the mother cell before the formationof the cortex & that the rigid cortex maintains the dehydrated state until the spore is ready to germinate.effectsof infection, such as release of endotoxin, do not occur.

So at the onset of germination Theory 1 predicts an initial large volume expansion, Theory 2 an initial contraction, Theory 3 no dramatic change. The relatively rapid method of Dynamic Light Scattering was used to test for this, and showed that the Ellar theory was the more likely.

1. Drop in Optical Density (Absorbance) with time shows the progress of germination of the spores:

2. Progress of diffusion coefficient coefficient (a measure of size) with time shows no dramatic change in volume

Kinetics of bacterial (and nutrient) destruction• Processing industry has to bring contamination down to a safe level based on agreed criteria but minimisingdamage to the nutrients

•Destruction of bacteria (and nutrients) follow a first order process

• Destruction rate dN/dt is proportional to N, the number of bacteria remaining

•Similar relations exist for molecular/macromolecular nutrients, except normally deal with number or weight concentrations, c (mol/ml) or C (g/ml)

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Effect of heat treatment of Polysaccharides

There have been only a few studies on this, and what data we have suggests that the damage depends very much on the type of polysaccharide. Consider 3 studies:

• Bradley and Mitchell (1988) – alginate, carboxymethylcellulose and κ-carrageenan

• Morris et al (1999) - low methoxy pectins

• Morris et al (2002) – high methoxy pectins

Effect of heat treatment of Polysaccharides

1. Study by Bradley and Mitchell (1988)

• used specially designed “slit viscometer” for measurement at high temperature (up to 100oC)

•Alginate, carboxymethylcellulose, κ-carrageenan

•Measured change of specific viscosity ηsp with time at different temps. T

•Use Mark-Houwinkrelation [η] = K’Ma to obtain molecular weight information

•Alginate much less stable

Effect of heat treatment of Polysaccharides

2. Study by Morris et al (1999)

• used specially designed analytical ultracentrifuge for measurement of sed. coeffs. and mol. wts. at temps up to 60oC –intrinsic viscosities measured too

•Low methoxy pectins

•No clear degradation - s, [η], M show little change with temp of measurement

No clear degradation - s, [η], M show little change with temp of measurement:

Effect of heat treatment of Polysaccharides

3. Study by Morris et al (2002)

•High methoxy pectins

•In contrast to the result for LM pectins, clear degradation - s, [η], M all decrease with temp of measurement, although conformation (from [η] = K’Ma and s = K”Mb

analyses) ~ unaltered

•Thermal stability of pectins seems to depend strongly on degree of esterification

[η] = K’Ma ; a~ 0.84

References• Bradley, T.D. and Mitchell, J.R. (1988) The determination of the kinetics

of polysaccharide thermal degradation using high temperature viscosity measurements, Carbohydrate Polymers, 9, 257-267

• Femenia, A., García-Pascua, P., Simala, S. and Rossellóa, C. (2003) Effects of heat treatment and dehydration on bioactive polysaccharide acemannan and cell wall polymers from Aloe barbadensis Miller, Carbohydrate Polymers, 51, 397-405.

• Harding, S.E. (1986) Applications of light scattering in microbiology, Biotechnology & Appl. Biochem. 8, 489-509.

• Morris, G.A., Butler, S.N.G., Foster, T.J., Jumel K. and Harding, S.E. (1999) Elevated temperature analytical ultracentrifugation of a low-methoxy polyuronide, Prog. Coll. Polym. Sci. 113, 205-208.

• Morris, G.A., Foster,T.J. and Harding, S.E. (2002) A hydrodynamic study of the depolymerisation of a high methoxy pectin at elevated temperatures. Carbohyd. Polym. 48, 361-367.

• Singh, R.P. and Heldman, D.R. (2009) Introduction to Food Engineering (4th Edition) Academic Press, London & New York.