# Effect of droplet size on oxidation and release of volatiles in protein-stabilised food emulsions

Claude Genot, Michèle Viau, Angélique Villière, Isabelle Bronnec, Laurent Lethuaut, Chantal Brossard

► To cite this version:

Claude Genot, Michèle Viau, Angélique Villière, Isabelle Bronnec, Laurent Lethuaut, et al.. Effect of droplet size on oxidation and release of volatiles in protein-stabilised food emulsions. 3rd world congress on emulsions, Sep 2002, Lyon, France. pp.320. hal-01944581

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Submitted on 4 Feb 2022

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
GENOT Claude

VIAU Michèle

VILLIERE Angélique

BRONNEC Isabelle

LETHUAUT Laurent

BROSSARD Chantal

Adresse postale / mailing address

INRA - LEIMA, Nantes

Id.

Id.

Id.

Id.

Id.

INRA-LEIMA BP 71627 44316 Nantes cedex3 France

## RESUME

De nombreux aliments sont des émulsions huile-dans-l'eau stabilisées par des protéines dans lesquelles les lipides s'oxydent, détériorant l'arôme et réduisant la durée de vie des produits.

L'influence de la taille des gouttelettes d'huile sur l'oxydation a été déterminée sur des émulsions stabilisées par la sérum albumine bovine et présentant des diamètres moyens variant entre 0,5 et 27 µm. Diènes conjugués (DC) et volatils ont été mesurés respectivement par absorption UV et CPG après prélèvement SPME. Les altérations de la protéine et la formation de produits terminaux de l'oxydation ont été évalués par fluorescence frontale.

Les résultats montrent des effets de la quantité d'oxygène disponible et de la taille des gouttelettes. La taille influence à des degrés divers, la vitesse de formation des DC, des produits fluorescents et des volatils et les détériorations des protéines. Ces différences s'expliquent notamment par des effets antagonistes de l'aire interfaciale et de la protéine adsorbée à l'interface. Les cinétiques de formation du pentane et de l'hexanal diffèrent.

Les conséquences de ces observations sur la stabilité chimique et la flaveur des émulsions sont discutées notamment au regard de propriétés de partage des arômes

Mots clés

Oxydation, composés volatils, taille des gouttelettes, odeur, aliment.

## ABSTRACT

Many foods are oil-in-water emulsions stabilised by proteins in which the lipid phase is subject to oxidation, contributing to a decrease of product acceptability.

The influence of oil droplet size on oxidation was determined on emulsions made of sunflower oil and stabilised by bovine serum albumin (BSA). Emulsions with monomodal size droplet distribution (D[4,3] : 0.5 to 27 µm), were produced. Conjugated dienes (CD) were evaluated from UV spectra. Volatile compounds in the headspace were analysed by GC after sampling with solid phase microextraction (SPME) fibber. Front-surface fluorescence gave an insight of alterations of the protein and formation of oxidised lipid-amino acid reaction products (OLAARP).

Effects of available O₂ and oil droplet size were observed. The size influenced, to different extents, initial rates of CD formation, formation of OLAARP and volatiles and protein degradation, due to balanced pro-oxidant and antioxidant effects of interfacial area and protein adsorbed on the interface. Kinetics of pentane and hexanal release differed.

The likely consequences on the chemical stability and the flavour of emulsions are discussed as regard of the partition behaviour of volatile compounds.

Keywords

Lipid oxidation, volatiles, droplet size, odour, food emulsion.
# EFFECT OF DROPLET SIZE ON OXIDATION AND RELEASE OF VOLATILES IN PROTEIN-STABILISED FOOD EMULSIONS

## 1/ INTRODUCTION

Many foods can be described as oil-in-water emulsions stabilised by proteins. In these products, the lipid phase is often subject to oxidation phenomena that contribute to a decrease of product acceptability via the formation of various aroma compounds.

In these systems, oxidation is influenced by the way of physical and chemical properties of the droplet membrane (1). The total interfacial area, which is determined by the size distribution of the oil droplets, is often claimed to play an important role through its impact on the access of pro-oxidant factors present in the aqueous phase to the lipid phase. However, experimental evidences of the effective impact of this parameter in food-related model emulsions were scarce and contrasted (2-5). Recently, we published some work dedicated to the development of oxidation in emulsions made of sunflower oil and stabilised by bovine serum albumin (BSA) as a function of the oil droplet size (6-8).

This work is aimed to summarise these data and to give new results focusing on the influence of oil droplet size on the formation of volatile secondary products of oxidation.

## 2°/ MATERIAL AND METHODS

2.1 - Experimental design

The emulsions contained 30 % (v/v) sunflower oil stripped from tocopherols and 70 % of aqueous BSA solution (20 g/L) containing NaN₃ to avoid bacteria growth. They were prepared so that different average droplet sizes with nearly narrow and monomodal distributions were obtained. Samples were stored in rotated closed vessels kept in the dark at controlled temperature and analysed at time intervals selected with the purpose of evaluating the early stage of oxidation development as well as the latest stages. As lipid oxidation is a train of chain reactions, oxidation was evaluated with different methods making it possible to evaluate formation of primary products of oxidation (conjugated dienes : CD), development of selected volatile products (pentane and hexanal) and that of oxidised lipid/amino acid reaction products (OLAARP). The degradation of the protein was evaluated from front-surface measurements of fluorescence of the protein. Measurements were performed on 3 separate sets of experiments wherein the experimental conditions were specifically adapted.

**2.2 - Preparation and characterisation of emulsions**

Large oil droplet emulsions (LE) were obtained by homogenizing BSA solution and stripped oil with a rotor-stator homogenizer. To reduce the size of the oil droplets, LE was circulated through a one valve pressure-homogenizer set at 15 bars (medium droplet size emulsions : ME) or a two valves high-pressure homogenizer with first valve set at 200 — 300 bars (small droplet size emulsions : SE). Aliquots of emulsions were distributed in 22.4 mL headspace vials sealed with PTFE/silicon septa and aluminum crimp seals. Droplet size distribution of the emulsions was measured with laser granulometer just after preparation and during their storage.

**2.3 — Methods**

Set 1 Emulsions (n=3) with volume mean diameter (D[4,3]) equal to 0.5 ± 0.03 m (SE), 1.9 ± 0.1 m (ME) and 27 ± 6.9 m (LE) were prepared with pH 4.4 BSA solution (NaN₃, 0,4 g/L) distributed (3.2
mL) in the vials, kept at 47 °C and analysed every one or two hours during 24 h. Controls consisted in 3.2 mL oil. Conjugated dienes were evaluated from UV absorbance at 233 nm of the lipids solubilized in isopropanol (6). They were expressed in mmoles conjugated dienes per kilogram of oil (mmol CD.kg⁻¹ oil) using 27 000 M⁻¹.cm⁻¹ as the molar extinction coefficient. The initial rate of conjugated diene formation (vᵢ,CD) was calculated by linear regression in the linear part of the curve. Alteration of the protein was evaluated from the decrease of fluorescence intensity of its tryptophanyl residues (excitation : 290 nm ; emission : 333 nm) measured in front face mode (8; Rampon et al., this congress). Oxygen concentration in the headspace of samples was evaluated with GC-MS (7).

Set 2 SE (D[4,3] = 0.5 ± 0.03 m) and ME (2.0 ± 0.1 m) were prepared in triplicate with pH 4.4 BSA solution (NaN₃, 0.4 g/L), distributed (3.2 mL), kept at 47 °C and analysed every hour for 5 days and then twice a week until 15 days. OLAARP formation was evaluated from the intensity of the fluorescence measured at 420 nm (excitation 355 nm) in front-surface arrangement (8).

Set 3 SE (0.5 m), ME (2.3 m) and LE (16.0 m) prepared with pH 4.0 BSA solution (NaN₃, 2 g/L) were distributed (1.5 mL) in the vials, kept at 40 °C, analysed for CD and volatiles every two hours the first 16 hours then regularly until 15 days. Volatile compounds released in the headspace of the emulsions were sampled for 5 min at 40 °C with solid phase microextraction (SPME) polydimethylsiloxane coated (100 m thickness) fiber and analysed by gas chromatography on a DB 624 capillary column. They were identified by MS. The major peaks, pentane and hexanal were quantified from their surface area as detected by flamme ionisation detector.

## 3/ RESULTS

### 3.1 — Kinetics of primary products of lipid peroxidation (Set 1 of experiments)

CD formation and O₂ uptake in emulsions were closely related, the former becoming steady when the latter reached less than 20 % of the initial amount of O₂, that is less than 5 % O₂ in the headspace (Fig.1.). This agrees with previous results showing the influence of oxygen pressure on lipid oxidation (9). The maximum amount of CD in the lipids is also closely related to the ratio between total available oxygen and lipids. Namely, maximum CD amount in oil is one third in oil than in emulsions because oil control contained 3-fold more lipid than 30 % (v/v) emulsions. Experiments performed with the same volume of unemulsified oil than in emulsions confirmed this assumption (6). The kinetics of CD formation in emulsions prepared with stripped oil was characterised by a very fast development of primary products of oxidation with no lag phase whereas a lag phase was found in the oil. This evidences the great pro-oxidant effect of emulsification.
<figure><img src="image_2.png" /><figcaption>**Figure 2.** Influence of specific surface area on initial rate of CD formation in emulsions (47iC, pH 4.4).</figcaption></figure>

<figure><img src="image_3.png" /></figure>

Initial rates of CD formation were calculated and plotted as function of the interfacial area developed in the emulsions varying with oil droplet size as shown on Fig. 2. It was evidenced that, in these conditions, the smaller the size of the oil droplets, the greater the initial rate of CD formation. Mean values of rates of CD formation were 23.5 (±4.3), 16.7 (±2.1) and 12.3 (±0.7) mmol CD·kg⁻¹·h⁻¹ for SE, ME and LE, respectively with a significant effect of the emulsion type (p=0.0064).

Favored diffusion and access of oxygen and metal catalysts to the oil phase could explain the enhancement of lipid oxidation in emulsions when the interfacial area increased. However, initial rate of CD formation was not increased in the same proportions than interfacial area. This can be attributed to an antioxidant activity of the protein adsorbed at the interface that would lower the pro-oxidant effect of a wider interface.

## 3.2 - Protein modifications (Set 1 of experiments)

When irradiated by UV light, proteins that contain the aromatic amino acid, tryptophane (Trp), emit fluorescence. This fluorescence depends both on the amounts fluorescent residues and on their environment. The maximum fluorescence intensity for an excitation wavelength set at 290 nm was directly measured on the emulsions using the front-surface arrangement and plotted as function of emulsions ageing (Fig. 3). Fluorescence intensity of Trp in emulsions decreased during ageing for both
ME and SE. The decrease was more pronounced in SE during the first hours of the kinetics corresponding to the rapid phase for CD formation and O₂ uptake. In contrast, in ME, significant decrease of fluorescence occurs only after 2 hours. Data were compared with UV absorption spectra and with protein balance between the aqueous phase and the interface (not shown). This allowed to conclude to a probable quantitative loss of Trp residues of the protein during the first step of oxidation. Analysis of 4th derivative fluorescence spectra in SE and ME also demonstrated that Trp adsorbed at the interface but in hydrophilic environment were altered (7, 8).

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Influence of oil droplet size on fluorescence intensity of tryptophanyl residues of BSA during oxidation of emulsions. (excitation : 290 nm ; emission : 333 nm ; pH 4.4 ; 47°C ; n=3)</figcaption></figure>

When emulsions were stored for longer time (up to 15 days), a linear decrease of Trp fluorescence was observed which was attributed to both oxidation and aggregation of the protein (7).

### 3.3 - Formation of oxidised lipid/amino acid reaction products (Set 2 of experiments)

During ageing of emulsions, a broad peak of fluorescence with excitation and emission maxima set at around 355 and 420-450 nm developed.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Formation of OLAARP during oxidation of emulsions of different oil droplet size (excitation : 355 nm ; emission : 420 nm ; 47°C ; pH 4.7 ; n=3)</figcaption></figure>

This peak was assigned to the formation of fluorescent chromophores due to the reaction of oxidizing lipids or secondary products of oxidation (OLAARP) with selected side-chains of proteins. These reactions probably involve Lys residues (7) whereas His residues have also been proposed (10).

The maximum intensity of OLAARP fluorescence increased regularly during the ageing or emulsions (Fig. 4) with an apparent lag phase during the first hours (8). The fluorescence developed faster and higher in SE than in ME because more protein is directly in contact with the oxidized lipids in SE due to larger interface area.

### 3.4 - Formation of volatile compounds (Set 3 of experiments)

In previous experiments we had found no influence of droplet size on volatile formation (6). This was attributed to probable insufficient sensitivity of the method used to evaluate the volatiles (manual static headspace) and to a too short period for production of primary products of oxidation. Thus, in this set, emulsion volume was diminished to prolong the period of CD formation. The pH of the BSA solution and the temperature were also slightly decreased.
As expected, CD reached a higher level than previously observed (set 1) (result not shown). However, we did not observe any influence of droplet size on initial rate of CD formation. This result which apparently contradicts previous set of experiments could result from the different pH and ionic strength conditions. More than 20 volatile compounds were identified in the headspace of the oxidizing emulsions. Pentane and hexanal were the major compounds and chosen as indexes of lipid oxidation. Pentane showed an induction period during the first 8 hours of oxidation and then increased continuously even after 15 days of ageing (Fig. 5-A). In contrast, hexanal shot up from the beginning of the storage of the emulsions (Fig. 5.B), that evidences an early decomposition of hydroperoxydes. Hexanal increased dramatically until 12h of storage and then tended to level off.

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Release of pentane (A) and hexanal (B) during oxidation of emulsions (pH 4.0°; 40°C).</figcaption></figure>

Oil droplet size differentiates only SE from ME and LE. The effect is different according to the volatile: release of pentane in the headspace was enhanced when that of hexanal was slowered down during the first hours of ageing. Quantitative calibration with external standards is now needed to calculate the actual amounts of volatile compounds generated in the oil phase.

## 4/ DISCUSSION AND CONCLUSION

These results demonstrate first the limiting effect of the total available O₂ on the final CD level and the influence of O₂ concentration on rates of CD formation. Second, it is shown that the formation of volatile compounds and OLAARP and the decrease of Trp fluorescence continue even when O₂ pressure has become the limiting factor for formation of primary products. These latter phase of the chemical degradation of emulsions does not need the presence of oxygen inasmuch oxidation phenomena has been previously induced.

A decreased size of the oil droplets tends to enhance the chemical degradations of the emulsions at different levels. During the initial stage of oxidation, when O₂ pressure is not the limiting factor, droplet size may influence the rate of CD formation, but the influence is small and would be modulated by several factors as antioxidant properties of the protein at the interface and pH. Trp losses, as evaluated from fluorescence intensities, depend also on droplet size: early and acute Trp losses were observed only in emulsions with small droplet size and large proportions of proteins at the interface. During the latest stages of oxidation, when O₂ pressure is a limiting factor for CD formation, droplet size has a great influence on the formation of OLAARP and a smaller effect on Trp fluorescence intensity. Both phenomena are directly related to the quantity of protein adsorbed at the interface.
Finally kinetics of release of volatiles in the headspace of the ageing emulsions, were influenced, to a small extent, by oil droplet size. This observation integrate both kinetics of volatile formation in the oil phase and their transfer to the aqueous and then to the headspace. Interestingly, kinetics were also shown to differ according to the nature of the volatile (pentane and hexanal). This result will be completed with data concerning the other volatiles produced during oxidation, their quantification and their partition in the emulsions oil and aqueous phases, and then paired with measurement of threshold odour of the volatiles in emulsions. Indeed, flavour perception from food polyphasic systems depends on the distribution of aroma into the food and their practical ability to release vapours into the air phase. Proteins adsorbed at the interfaces or present in the aqueous phase could change the partition of volatiles, like aldehydes, through covalent binding (10). Also, in BSA stabilised oil-in-water emulsions, odour intensity of single aroma compounds is directly related to the aroma concentration in the aqueous phase (11). In fact, in oxidising emulsions, intensity and quality of perceived odour relates to the complex aroma composition in the continuous phase in contact with the air. Therefore, the implication of a large bunch of different flavour molecules with different kinetics of formation accounts for the great complexity of flavour perception of aromas in mixtures. To date, uncovering the links that relate the sensory characteristics of single odorants and the complex odor released by their mixes is a pending challenge (12).

5/ REFERENCES

(1) McClements J., E.A. Decker (2000) Lipid oxidation in oil-in-water emulsions: impact of molecular environment on chemical reactions in heterogeneous food systems, J. Food Sci. 65:1270-1282

(2) Shimada K., et al. (1996) Involvement of chelating action and viscosity in the antioxidative effect of xanthan in an oil/water emulsion, Biosci. Biotechnol. Biochem. 60:125-127

(3) Gohtani S. et al. (1999). Effect of droplet size on oxidation of docosahexaenoic acid in emulsion system, J. Dispersion Sci. Technol. 20: 1319-1325

(4) Jacobsen C. et al. (1999) Oxidation in fish-oil-enriched mayonnaise 1. Assessment of propyl gallate as an antioxidant by discriminant partial least squares regression analysis, Eur. Food Res. Technol. 210:13-30

(5) Jacobsen C. et al., (2000) Oxidation in fish-oil-enriched mayonnaise 3. Assessment of the influence of the emulsion structure on oxidation by discriminant partial least squares regression analysis, Eur. Food Res. Technol. 210:242-257

(6) Lethuaut L. et al. (2002) Effect of droplet size on lipid oxidation rates of oil-in-water emulsions stabilized by protein J. Amer. Oil Chem. Soc. 79(5), in press

(7) Rampon V. et al. (2001) Interface characterization and ageing of bovine serum albumin stabilized oil-in-water emulsion as revealed by front-surface fluorescence, J. Agric. Food Chem. 49:4046-4051

(8) Rampon V. et al. (2002) Modification of BSA and formation of fluorescent pigments in oxidizing emulsions as revealed by front-surface fluorescence, In: Recent Res. Dev. Agric. & Food Chem.: Food Emulsions & Dispersions. M. Anton & S.G. Pandala, eds Res. Signpost, Kerala, (Inde). (in press)

(9) Marcuse R., P.O. Fredriksson (1968) Fat oxidation at low oxygen pressure: I. Kinetic studies on the rate of fat oxidation in emulsions. J. Amer. Oil Chem. Soc. 45°:400-407

(10) Alaiz M., J. Giron (1994) Modification of histidine residues in bovine serum albumin by reaction with (E)-2-Octenal. J. Agric. Food Chem. 42:2094-2098

(11) Brossard C. et al.; (2002) Odour perception over liquid emulsions containing single aroma compounds: Effects of aroma concentration and oil volume fraction. J. Sensory Studies (in press).

(12) Wise P.V. et al. (2000). Quantification of odor quality. Chem. Senses, 25, 429-443.
We thank Région Pays de la Loire for the financial support (VANAM Program).