Elsevier

Food Hydrocolloids

Volume 67, June 2017, Pages 229-242
Food Hydrocolloids

Interactions in casein micelle – Pea protein system (part I): Heat-induced denaturation and aggregation

https://doi.org/10.1016/j.foodhyd.2015.12.015Get rights and content

Highlights

  • Thermostability of legumin and vicilin increased in presence of casein micelles.

  • In admixture, denatured legumin yielded mainly disulfide-bonded aggregates.

  • Break-up of legumin subunits resulted in soluble and insoluble covalent aggregates.

  • In admixture, denatured vicilin resulted in soluble and non-covalent aggregates.

  • Protein change in heated mixtures resulted from unfolded pea proteins interactions.

Abstract

The aim of this work was to investigate the heat-induced interactions between pea proteins (vicilin 7S or legumin 11S enriched-fractions) in admixture with suspended casein micelles (SCM), at weight protein ratio of 1:1 and pH 7.1. The single-protein samples and mixtures thereof were prepared at concentrations of 18 and 36 mgprotein/gsample, respectively, then heated from 40 to 85 °C and incubated for 0–60 min. As compared to single-protein samples, differential scanning calorimetry (DSC) data indicated that the denaturation temperature of pea proteins increased of about 4 °C in the presence of SCM. Heat-induced change in protein composition of the soluble (SP) and micellar (MP) phases from centrifuged SCM – pea protein mixture was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry. Likewise SP was analyzed by size-exclusion chromatography (SEC-HPLC). While pea legumin 11S and vicilin 7S oligomers were markedly sedimentable in MP from their respective unheated mixture, thermal denaturation and protein aggregation (≥75 °C) resulted in increasing levels of dissolved pea proteins in SP. Heating of the SCM – legumin mixture (85 °C, 15–60 min incubation) resulted in the dissociation of the legumin subunits Lαβ into acidic Lα and basic Lβ polypeptides, yielding in comparable amounts soluble and insoluble disulfide-bonded aggregates, respectively. In contrast in the SCM – vicilin mixture, the heat-denatured vicilin polypeptides in a temperature range of 70–80 °C produced in majority soluble and non-covalent aggregates. Though the heat-induced interactions between pea proteins were altered in the presence of micelles, caseins would not be involved into pea proteins aggregation.

Introduction

Proteins are widely encountered in most of tailored food for both nutritional value and functional properties. Representing around 80% of total milk protein, casein micelles (CM) are widely used as ingredients in various dairy processes (Cayot & Lorient, 1998). Nevertheless, the rapid expansion of intensive livestock production on account of population growth and worldwide demand for animal-based food led to increasing environmental pressure, regarding water and land utilization, energy costs, pollutant emissions, climate change, therefore threatening human and animal health (FAO, 2008). Change in dietary based on plant protein sources was suggested to reduce the environmental impact due to the food production (FAO, 2013). In this regard, legume proteins could embody such change in food consumption patterns, owing to their diversity, low production cost, nitrogen fixation ability, and also valuable nutritional properties (Boye, Zare, & Pletch, 2010). However, both deficiency in certain essential (sulfur-containing) amino acids and reduced acceptability of western consumers towards non-conventional plant-based foodstuffs may discourage the choice of sustainable plant protein sources. Hence, a promising strategy may be based on the formulation of novel milk – plant protein blended products, displaying desirable textural properties and also complementary amino-acid composition (Beliciu & Moraru, 2011). From this perspective, processing challenges could originate from the little knowledge on the interaction between milk and plant proteins. To date, conflicting reports on this issue were attributable to the different processing history of each protein source, which affected thereafter their functional properties; scarce relevant studies investigated the rheological properties of casein micelles – soy protein systems, in the presence or absence of whey proteins (Beliciu and Moraru, 2011, Beliciu and Moraru, 2013, Roesch and Corredig, 2006). So as to develop in the present study food applications using traditional European crop rather than soybean, dry pea seeds (Pisum sativum L.) may represent an alternative plant protein source, by virtue of high protein content (20–25 wt%) and food texturation abilities (Shand, Ya, Pietrasik, & Wanasundara, 2007).

Numerous studies have focused on the gelation properties of milk proteins and plant ones, however in isolation. Heat-treatment of milk proteins is a prerequisite to enable enhanced firmness, homogeneity and reduced syneresis of acid gels during yoghurt manufacture (Guyomarc’h et al., 2003a, Guyomarc’h et al., 2003b). During heating of milk above 70 °C, whey proteins (WP) denatured and interacted with κ-casein via sulfhydryl-disulfide bond (S/S–S) exchange reactions (Singh & Fox, 1986). Depending on initial pH of milk, heating procedure and WP-to-CM weight ratio, the production of soluble and micelle-bound thermal co-aggregates affected further acid–gelation properties of CM and gelled network structuration.

Pea proteins are mainly globulins (≈75% of total protein composition), made up with heterogeneous subunits (Tzitzikas, Vincken, de Groot, Gruppen, & Visser, 2006). Legumin 11S is composed of six subunits Lαβ of molecular weight (Mw) of about 60 kDa, consisting of disulfide-bonded acidic Lα (≈40 kDa) and basic Lβ (20 kDa) polypeptides, whereas vicilin and convicilin 7S are constituted of three subunits, of Mw mainly of around 50 and 70 kDa, respectively (Marcone, Kakuda, & Yada, 1998a,b). Heat-induced pea protein gels are increasingly documented (O'Kane et al., 2005, Shand et al., 2007, Sun and Arntfield, 2010). In addition to protein extraction procedure, the effects of temperature, pH and salt composition were shown to affect the formation of soluble protein aggregates that could rearrange into gelled network. Alternatively, acid-induced gelation of pea proteins was carried out (Mession, Chihi, Sok, & Saurel, 2015). In this regard, the level of soluble pea protein aggregation (85 °C, pH 7.2, low ionic strength I) was observed to further influence the acid-gel strength; the disulfide bonded-legumin thermal aggregates displayed a decreasing solubility that would impair the acid–gelation properties, by comparison with quite soluble and non-covalent vicilin thermal aggregates.

Combining the functional properties of the two protein ingredients would enable original textures of the “mixed” gelled products. Thus it was of primary interest to investigate the heating step of milk – pea protein mixtures, since the resulting thermal aggregates may affect thereafter the acid gelation properties of the blended system. Better knowledge regarding the heat-induced interactions between pea proteins and CM was required, in respect of the reported role of the WP thermal aggregates on the texture of acid gels made with pre-heated milks (Roesch & Corredig, 2006). Therefore the present study was an attempt to elucidate the heat-induced protein interaction between globular pea proteins, namely legumin 11S (Leg) and vicilin 7S (Vic)-enriched fractions, in admixture with a WP-depleted casein micelle suspension (SCM), at pH close to neutrality. As in standard cow milk, total protein concentration of the SCM – pea protein mixtures was set at 36 mg/gmixture, while applying protein weight ratio of 1:1, respectively. Protein denaturation and aggregation phenomena were compared to those in the corresponding single-protein samples. The acid–gelation properties of the different SCM – pea protein mixtures were considered in a following work (Part II).

Section snippets

Materials

As described previously, the legumin and vicilin-enriched pea protein fractions were laboratory-prepared from defatted pea flour (Mession et al., 2015). These were available as freeze-dried samples. Microfiltrated half-skim milk was purchased in a local supermarket (Carrefour brand name, batch n° 7190, EMB 69013C, Arnas, France).

All other reagents and chemicals purchased from Sigma–Aldrich (St Louis, USA) were on analytical grade.

Chemical assays

Total moisture and ash contents were evaluated according to AOAC

Extraction of CM

The initial skim milk contained 0.6 wt% total nitrogen, of which ≈92% was PN, 5.1 wt% lactose and 0.5 wt% salts. After UF/re-UF procedure, protein, lactose and salt recoveries (in weight) in the retentate 2 were of about 66%, 12% and 59%, respectively, whereas these were found in both permeates at levels of 8.4%, 88% and 22% of initial amount, respectively. On a basis of 30 mmol/l total calcium concentration in the starting skim milk, it was found that around 10% was removed in permeates, in

Conclusion

The heat-induced protein interaction between pea globulins in with the presence of casein micelles (at protein weight ratio 1:1) was investigated at pH 7.1. Pea legumin 11S, and to a lower extent vicilin 7S oligomers, were of decreasing solubility in their respective unheated mixtures, possibly attributable to charge screening in the presence of exchangeable calcium cations arising from the micelles. In the mixtures, the denaturation temperature for each fraction was higher (+4 °C) than that

Notes

The authors declare no competing financial interest.

Acknowledgments

The authors would like to thank the GIP AgroSup Tech Est and the Conseils Régionaux de Bourgogne et de Franche-Comté for technical and financial supports. The Ingredia Group (Arras – France) is thanked for its contribution to the present project. All other moral support of this study and more generally people who are aware of the need to develop alternative food models are as well gratefully acknowledged.

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