Annual Research Conference Food, Nutrition & Consumer Sciences




НазваниеAnnual Research Conference Food, Nutrition & Consumer Sciences
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DENATURATION AND AGGREGATION OF ALPHA-LACTALBUMIN




K. Lišková1, P. McStay1, N.O’Brien2, A. Kelly2, A. Brodkorb1

1 Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland

2 Department of Food and Nutritional Sciences, University College Cork, Ireland



ABSTRACT

In this study, commercial bovine α-lactalbumin (1 to 5%) was heated at 80°C for different periods of time. The structural changes caused by the heat treatment were studied by reversed-phase and gel-filtration chromatography, polyacrylamide gel electrophoresis and intrinsic tryptophan fluorescence. It was found that heat-treatment caused aggregation of α-lactalbumin, induced by β-lactoglobulin present in the commercial product. Denaturation of α-lactalbumin proceeded faster and aggregation was more extensive with increasing concentration of protein. Unfolding of the protein, but not aggregation, could be followed by intrinsic tryptophan fluorescence.

INTRODUCTION

The whey protein α-lactalbumin (α-La) consists of 123 amino acids, including eight cysteine residues linked by four disulphide bonds. In the native state, α-La binds calcium with a high affinity. This calcium-bound form, holo-α-La, is more stable towards denaturation.1 A high degree of homology (85%) exists between the primary structures of α-La from human and bovine milk. α-La is a major protein in human milk (34% of total protein) but conventional whey protein-based infant formulas contain only about 14% α-La. Therefore, there has been a trend to enrich infant formulas with α-La in order to bring them closer to human milk. However, methods used for purification of α-La and following processing of infant formula can irreversibly alter its three-dimensional structure and consequently affect biological properties.

Recently, a complex with anti-tumour activity was discovered, that consists of α-La and oleic acid. It was suggested that this complex – called HAMLET/BAMLET (Human/Bovine Alpha-lactalbumin Made LEthal to Tumor cells) - could be naturally formed in the stomach of infants.2 The altered structure of α-La in infant formula could have an effect on the potential formation of BAMLET. The aim of this study was to examine the changes in α-La solutions occurring during heat-treatment.


MATERIALS AND METHODS

Bovine holo-α-La (purchased from Davisco) was dissolved in distilled water at increasing concentrations from 1 to 5%. To ensure that α-La is present in holo-form, CaCl2 was added in molar ratio 1:1 with protein. Solutions were reconstituted overnight at 4°C. Denatured protein was removed by isolelectric precipitation at pH 4.6. Prior to heating, the pH of solutions was adjusted to 7. Solutions were then heated at 80°C for different times (5-180 min) and immediately cooled on ice. Using a combination of gel-permeation and reversed-phase chromatography, the quantities of native, non-native monomer and aggregated protein in the solutions were measured as a function of time. To characterize the nature of the interactions in aggregates, samples were analysed by native and SDS (reducing and non-reducing) PAGE. For measurement of intrinsic tryptophan fluorescence of samples, the excitation wavelength was 280 nm and emission was measured between 300 and 400 nm.


RESULTS AND DISCUSSION

It was found that aggregation of α-La occurred in all heated samples. It is known that pure α-La does not form aggregates under these conditions, which is explained by the lack of free thiol groups in the protein. The aggregation occurring in our samples can be explained by the low content of β-lactoglobulin (about 10%) in the commercial α-La product. The free thiol group of Cys121 in β-lactoglobulin can initiate sulphydryl-disulphide interchange reaction upon unfolding and result in covalently linked aggregates. The denaturation of α-La proceeded faster with increasing concentration of protein (from 1% to 5% protein solution) and aggregation was more extensive. This is because at higher concentrations, proteins are in closer vicinity and an interaction with another protein is more likely to occur. A comparison of two samples is shown in the table below.


Sample

Heating [min]

Native monomer [%]

Non-native monomer [%]

Aggregate [%]

1%

60

62

25

13

5%

60

7

32

61


Native PAGE confirmed increasing amount of aggregates with the time of heating. In non-reducing SDS PAGE, the non-covalent interactions were disrupted, resulting in appearance of small bands of monomeric α-La and β-lactoglobulin. However, larger proportion of aggregates remained stable, which indicates that these were linked by covalent bonds. Addition of mercaptoethanol to the sample buffer resulted in the break-up of the disulphide bridges and only monomeric bands of α-La, β-lactoglobulin and BSA were present in the gels.

Heating of α-La samples had a great effect on their fluorescence spectra. Unfolding of protein resulted in a red shift of the emission maximum from 325 nm for native holo-α-La to 345 nm for completely denatured α-La. The emission intensity increased by 100% for denatured protein. Although heating of more concentrated samples produced substantially more aggregates (see above), fluorescence spectra of samples denatured to the same degree were practically identical.


CONCLUSIONS

Heating of α-La resulted in unfolding and aggregation of the protein. With increasing concentration of protein, the unfolding proceeded faster and aggregation to a greater extent. Both non-covalent and covalent interactions were involved in aggregation, with higher proportion of covalently linked aggregates. Intrinsic fluorescence spectra were correlated with the content of native protein but did not reflect the extent of aggregation.


ACKNOWLEDGEMENTS

This project was funded by Dairy Levy Trust Fund. Kamila Lišková was funded by the Teagasc Walsh Fellowship Scheme.


REFERENCES

1 Permyakov, E. A., & Berliner, L. J. (2000). FEBS Lett, 473(3), 269-274.

2 Svensson, M., Hakansson, A., Mossberg, A. K., Linse, S., & Svanborg, C. (2000). PNAS, 97(8), 4221-4226.


RECOVERY OF NATURAL PHENOLIC ANTIOXIDANTS FROM APPLE

(Malus domestica BORKH. cv Bramley’s Seedling) WASTE


L. Massini, A. B. Martín-Diana, C. Barry-Ryan and D. Rico

School of Food Science and Environmental Health, Functional Ingredient Food Unit (FIFU)

Dublin Institute of Technology, Dublin, Ireland


ABSTRACT

The recovery of phenolics from Bramley’s Seedling apple waste was investigated as an opportunity to enhance the traditional Irish cooking apple market, while extracting natural antioxidants in view of future functional food applications. Antioxidant, antiallergenic and anticancer properties have already been reported for apple phenolics. Hand-peeled apple skins were oven-dried or freeze-dried, then powdered; oven-dried peel flours were also pre-treated by dipping in ascorbic acid or blanching with steam or water. Crude extracts of phenolics obtained with aqueous acetone or ethanol were investigated for total phenolic and flavonoid content using Folin-Ciocalteu and aluminium-chloride assays. The antioxidant properties were evaluated with FRAP and DPPH· antiradical activity assays. Freeze-drying preserved peel phenolics better than oven-drying. Acetone recovered higher amounts of total phenolics than ethanol. The phenolic content of peels was significantly correlated with the antioxidant capacity, indicating the potential role of Bramley’s Seedling apple peel waste as a source of natural antioxidants.


INTRODUCTION

Apple is among the fruits that can be competitively grown in Ireland, also for further processing. Due to changes in lifestyle, the traditional cooking apple market based on the local variety Bramley’s Seedling has narrowed over the years, contributing to its general oversupply at low farm gate prices. The apple peel waste was investigated for recovery of natural antioxidant phenolics in view of functional food applications, in order to enhance the Irish culinary apple market while lowering the cost of waste disposal. Apple peels are known to be valuable waste products because of their antioxidant activities and other promising inhibitory effects (e.g. anticancer and antiallergenic), depending on the apple variety under investigation1. The antioxidant capacity was already reported in literature to be higher in apples with skins than without, suggesting the idea that apple antioxidant properties might be mainly attributed to its phenolic compounds2, rather than to vitamin C. It is also widely accepted that apple phenolics are mainly located in the skin than in the flesh and research already showed that the skin is rich in polyphenols not found in pulp such as quercetin glycosides and cyanidin glycosides3. The present research focused on the evaluation of Bramley’s Seedling apple waste as a profitable source of natural phenolic antioxidants.


MATERIALS AND METHODS

Hand-peeled skins obtained from apples purchased from a local store were oven-dried at 60°C in a ventilated oven or freeze-dried, then powdered. Oven-dried peels were preserved from further oxidation by dipping for 3 min in 0.5% (w/v) ascorbic acid and 0.05% (w/v) sodium chloride; by water-blanching at 100°C for 10 s; by steam-blanching for 1 min, 30 s and 10 s, prior to drying. Crude fresh and dried peel extracts were obtained with aqueous acetone or aqueous ethanol (80%, v/v) according to the method previously described by Wolfe and Liu4. After removal of the solvent, the extracts were resuspended in distilled water and stored in dark at -20°C. Total phenolic and flavonoid content of fresh and dried peels were assessed using the Folin-Ciocalteu and aluminium-chloride assays. The antioxidant power of apple crude extracts was evaluated as Ferric Reducing Antioxidant Power (FRAP) and antiradical capacity against DPPH·, the values were expressed as ascorbic acid equivalent. The effect of apple organic acids (e.g. citric and malic) was also investigated on the DPPH· antiradical power of peel phenolics, using ascorbic acid as a reference standard, in order to define possible interactions affecting the antiradical mechanisms of the fruit.


RESULTS AND DISCUSSION

Acetone extracted the highest amount of phenolics from fresh and freeze-dried peels; the crude extracts thus obtained also showed the highest antioxidant activities (p<0.05). Freeze-drying preserved the phenolic content and antioxidant activities of fresh specimens better than oven-drying. Pre-treatments applied before oven-drying did not enhance the phenolic content and the antioxidant activities of peel flours extracted with ethanol when compared to untreated samples (p>0.05). The addition of citric and malic acid significantly lowered the antiradical power of extracts of apple peel phenolics, regardless of solvent and drying method applied, while it synergistically enhanced the scavenging activity of ascorbic acid solutions at concentrations higher than 1 ppm, probably due to a different interaction between organic acids and fruit antioxidants in terms of radical scavenging mechanism. The phenolic content of crude apple extracts and the antioxidant capacities were significantly correlated (p<0.05).


CONCLUSIONS

The phenolic content of cv Bramley’s Seedling apple peel was similar to data reported in literature for other apple varieties. The significant correlation between phenolic content and antioxidant capacity of crude peel extracts indicated the potential use of Bramley's Seedling apple peels waste as a profitable source of natural antioxidant phenolics. Further characterisation on the type of extractable phenolics from apple peel waste is needed, including the investigation of the antioxidant mechanisms.


ACKNOWLEDGEMENTS

The authors would like to acknowledge the financial support of the DIT Strand III Research Project (2007-2010).


REFERENCES

1 Schieber, A., Stintzing, F.C., and Carle, R. (2001). Trends in Food Science and Technology. 12: 401-413.

2 Eberhardt, M.V., Lee, C.Y., and Liu, R.H. (2000). Nature. 405: 903-904.

3 Escarpa, A., and González, M.C. (1998). Journal of Chromatography A. 823: 331-337.

4 Wolfe, K.L., and Liu, R.H. (2003). Journal of Agricultural and Food Chemistry. 51: 1676-1683.


Determining curd moisture content using an online NIR sensor in cheese vat


M.J. Mateo1, D.J. O’Callaghan1, C.D. Everard1, C.P. O’Donnell2, M. Castillo3, F.A. Payne3

1Food Processing & Functionality Department, Teagasc, Moorepark Research Centre, Fermoy, Co. Cork, Ireland, 2Biosystems Engineering, UCD School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Dublin, Ireland and 3Department of Biosystems and Agricultural Engineering, University of Kentucky, Lexington, USA


ABSTRACT

The objective of this study was to evaluate the influence of fat and gel firmness at cutting on syneresis and curd moisture prediction using light backscatter at 980 nm.

A full factorial experiment involving milk fat level and gel cutting firmness level was performed. The trials were carried out using reconstituted whole milk in an 11 L cheese vat, in which a light backscatter online sensor was installed. Samples of curd/whey mixture were taken from the vat during syneresis using an online sampler at 10 min intervals and curd was separated from whey and curd moisture was determined. Fat in milk, gel firmness at cutting and time elapsed after gel cutting had a significant (P < 0.001) effect on curd moisture content in cheese-making, which could be predicted using a linear model, involving the two experimental variables and time after gel cutting in conjunction with light backscatter ratio (R2 = 0.93).

These results contribute to understanding the effect of milk fat levels and gel firmness at cutting on curd moisture content and can improve the control of curd moisture content in cheese-making.


INTRODUCTION

It is believed that a more sophisticated control of syneresis could improve the control of curd moisture and decrease the production of out-of-specification cheese leading to more consistent quality1. Cheese manufacturing control would benefit from online sensor technology for monitoring milk coagulation and syneresis. In this work, a light backscatter sensor was used, there were two objectives: (i) evaluating the influence of fat in milk and gel firmness at cutting on syneresis and (ii) curd moisture prediction using an online sensor under the above conditions.

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