Effect of feed pH and non-volatile dairy components on flavour concentration by pervaporation

Journal of Food Engineering 107 (2011) 60–70

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Effect of feed pH and non-volatile dairy components on flavour concentration by pervaporation Amy R. Overington a,b,⇑, Marie Wong b, John A. Harrison c a

Fonterra Research Centre, Private Bag 11029, Palmerston North, New Zealand Institute of Food, Nutrition and Human Health, Massey University, Private Bag 102904, Auckland, New Zealand c Institute of Natural Sciences, Massey University, Private Bag 102904, Auckland, New Zealand b

a r t i c l e

i n f o

Article history: Received 31 March 2011 Received in revised form 29 May 2011 Accepted 31 May 2011 Available online 7 June 2011 Keywords: Pervaporation Non-volatile Dairy Protein Fat Lactose

a b s t r a c t Pervaporation was used to concentrate acids, esters and ketones in model flavour mixtures. The characteristics of the feed mixture (pH and presence of dairy ingredients) were found to alter the pervaporation behaviour of the flavour compounds. This effect was partially due to a reduction in driving force (caused by a lower activity in the aqueous phase as evidenced by the mole fraction in the headspace above the feed), and partially due to the sorption and diffusion behaviour of the flavour compounds. Acids were concentrated most effectively at pH 3.5 or below, when they were in their undissociated forms; their enrichment factors were reduced by up to 84% when the pH was increased to 7. Milk fat reduced the pervaporation enrichment of flavour compounds by up to 95%, as the flavour compounds partitioned into the fat phase and hence did not pass through the membrane as easily. In the presence of either milk protein isolate or lactose, enrichment factors were reduced by 45–67% for short-chain esters and ketones, for which permeation through the membrane was limited by the sorption step. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Potent flavouring ingredients can be created by concentrating flavour compounds beyond the levels at which they are naturally found in foods. Pervaporation is one of several techniques by which flavour compounds may be concentrated (Karlsson and Trägårdh, 1997). In this technique, hydrophobic flavour compounds can be selectively permeated through a non-porous hydrophobic membrane. Permeants evaporate as they pass through the membrane because the downstream side of the membrane is kept under vacuum. To evaporate, permeant compounds must have some degree of volatility, which means that in a food system, the pervaporation permeate largely consists of water and flavour compounds (as well as other volatile compounds that do not contribute to flavour, for example hydrocarbons). Several papers review the principles of flavour pervaporation (Karlsson and Trägårdh, 1993, 1996; Baudot and Marin, 1997; Pereira et al., 2006). The permeation rate of each compound is a product of the membrane permeability for that compound (a function of the compound’s sorption in, and diffusion through, the membrane) and the driving force (which results from the activity difference across the membrane) (Baudot and Marin, 1997; Pereira et al., 2006). There⇑ Corresponding author at: Fonterra Research Centre, Private Bag 11029, Palmerston North, New Zealand. Tel.: +64 6 350 4649; fax: +64 6 356 1476. E-mail address: [email protected] (A.R. Overington). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.05.045

fore, any factor that affects a permeant compound’s feed side activity, or its solubility in the membrane material, or its diffusivity through the membrane, can be expected to influence the pervaporation performance. Non-volatile components typically present in dairy systems (milk fat, milk protein and lactose) may influence the ability of pervaporation to concentrate flavour compounds present in these systems. Although these non-volatile substances should not pass through pervaporation membranes (Baudot and Marin, 1996, 1997; Kattenberg and Willemsen, 2001; Aroujalian et al., 2006), they could nonetheless affect the pervaporation behaviour of volatile compounds by altering their feed side vapour pressures, hence altering the driving force for mass transfer across the membrane. In most cases, interactions between flavours and food components have been studied from a sensory perspective: if less flavour is released due to flavour compounds binding to or associating with non-volatile substances, consumers’ perception of these flavours will be reduced (McGorrin and Leland, 1996; Leland, 1997). The same principle applies to pervaporation: flavours that are associated with non-volatile feed components will not be available to pass into the membrane. In a flavour mixture containing water and fat, a certain proportion of each flavour compound will dissolve in the fat phase, depending on its hydrophobicity. This reduces the volatility of the flavour compound, because the proportion in the fat phase cannot volatilise easily (Hatchwell, 1996; de Roos, 1997; Leland,

A.R. Overington et al. / Journal of Food Engineering 107 (2011) 60–70

1997). In contrast, proteins reduce the volatility of certain flavour compounds by binding rather than acting as a solvent (Hatchwell, 1996). Various flavour compounds, including ketones and esters, can bind to milk proteins (Mills and Solms, 1984; Hansen and Booker, 1996; Guichard and Langourieux, 2000; Kühn et al., 2007). Carbohydrates can decrease the volatility of flavour compounds through intermolecular attractions, or increase their volatility by salting-out (Godshall, 1997). Lactose contains many hydroxyl groups and is thus able to bind certain flavour compounds through hydrogen bonding (Kellam, 1998). All of these flavour/non-volatile interactions could, potentially, alter the feed side behaviour of flavour compounds during pervaporation. Therefore, the objective of this study was to determine the impact of milk fat, milk protein and lactose on the effectiveness of pervaporation for concentrating flavour compounds, and to relate these findings to the impact of these non-volatile dairy components on the volatilities of flavour compounds in a model dairy mixture. 2. Materials and methods 2.1. Feed mixtures The feed used for pervaporation experiments was a mixture of flavour compounds typically found in dairy products, with or without non-volatile dairy ingredients added, made up to volume with distilled water. All feed mixtures contained the same nine flavour compounds at the concentrations listed in Table 1 (Sigma–Aldrich Co., St. Louis, MO, USA; P98% purity), plus additional dairy ingredients as follows:  Cream (Anchor brand, Fonterra Co-operative Group Ltd., Auckland, New Zealand), containing 40.0 g fat and 2.0 g protein per 100 g. Various ratios of cream to water were used to create mixtures containing 0.5%, 1%, 5%, 10%, 20% and 38% (w/v) fat. The 38% (w/v) fat mixture corresponded to 100% cream. Mixtures were stirred only, not homogenised. A creamy layer rose to the top of mixtures with 0.5% and 1% fat, but feed mixtures with higher fat levels remained homogeneous. Cream was chosen as a convenient fat source because it contains the right mix of fats, at the right fat globule size, to be directly applicable to dairy systems.  Four percent (w/v) milk protein isolate (Fonterra Co-operative Group Ltd., New Zealand), equivalent to 3.4% total protein in the feed solution. Milk protein isolate contains a blend of casein and whey proteins. This protein level was chosen because it is almost double the protein level in cream, while being comparable with the amount of protein in milk (Swaisgood, 1996).  Six percent (w/v) lactose (extra pure grade; Scharlau, Barcelona, Spain). This concentration of lactose was chosen because it is double the amount of lactose in cream, while being similar to the lactose level in milk (Swaisgood, 1996). Table 1 Flavour compounds and their concentrations in the feed solution. Compound

Molecular weight (g mol1)

Feed concentration (mg L1)

Acids Acetic acid Butanoic acid Hexanoic acid Octanoic acid

60 88 116 144

105 107 111 105

Esters Ethyl butanoate Ethyl hexanoate Ethyl octanoate

116 144 172

101 100 10.4

Ketones 2-Heptanone 2-Nonanone

114 142

9.8 9.8

61

 Distilled water only (no dairy ingredients added to the flavour mixture).  No dairy ingredients added, but feed pH adjusted (from an initial pH of 3.5) to 2.5, 4.8 or 7.0 by the addition of 1 mol L1 hydrochloric acid or potassium hydroxide. 2.2. Pervaporation of flavour compounds in the presence or absence of non-volatile dairy components Details of the pervaporation procedure have been published previously (Overington et al., 2008). Triplicate pervaporation runs were carried out with each of the feed mixtures listed above, using a 0.012 m2 hydrophobic polydimethylsiloxane membrane (active layer thickness 0.5 lm; supplied by Helmholtz-Zentrum Geesthacht – Centre for Materials and Coastal Research, Geesthacht, Germany). The feed temperature was 30 °C, and the feed flow rate was 1 L min1 for all runs. The permeate pressure was maintained at either 1.5 kPa or 2.0 kPa (absolute). Following runs with aqueous feed solutions, the feed side of the pervaporation unit (including the membrane) was rinsed with water, then distilled water was re-circulated through the feed side overnight, with the permeate side open to the atmosphere. When the feed solution contained fat or protein, the membrane module was bypassed, and the feed tank and feed lines were cleaned with a 1% solution of Reflux B620 alkaline detergent (Orica Chemnet, New Zealand). As this cleaning solution was beyond the pH limit of the membrane, the membrane module was cleaned separately by soaking in absolute ethanol. The feed side and module were then rinsed with distilled water overnight, as with aqueous feed solutions. The total flux through the membrane was determined by weighing the permeate collected over a period of four hours. The enrichment factor for each compound was calculated as the ratio between that compound’s concentration in the permeate and its concentration in the feed. 2.3. Partitioning of flavour compounds between fat and water Four feed solutions, containing the standard model flavour compounds (Table 1) and cream, were made as described in Section 2.1, with 5%, 10%, 20% and 38% (w/v) fat, respectively. After holding at room temperature for at least one hour, triplicate samples of each solution (approximately 45 mL) were added to centrifuge tubes. Samples were separated into fat and aqueous phases by centrifuging in a Heraeus Multifuge 1 S-R (Kendro, Germany) at 4700 rpm (4618g) for 1 h at 40 °C. Fat and aqueous phases were extracted and analysed separately, to determine the concentration of flavour compounds in each phase. This is a similar procedure to that followed by Hansen and Booker (1996). 2.4. Measurement of flavour compound mole fractions in the feed headspace in the presence of non-volatile dairy components The apparatus depicted in Fig. 1 was used to measure the mole fractions of flavour compounds in the headspace vapour phase above feed mixtures with and without dairy ingredients (described in Section 2.1). The feed container was filled with 50 mL of the feed mixture. This was first frozen with liquid nitrogen while the headspace above the feed mixture was evacuated. The inlet and outlet needle valves were then shut to isolate the feed container from the rest of the system, then the contents of the feed container were thawed by placing in a 20 °C water bath, stirring with a magnetic stir bar as soon as enough ice had melted to make this possible. Five to ten minutes were allowed for thermal equilibration after the feed solution had completely thawed, then the outlet needle valve was opened slightly to draw material from the headspace

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Pressure gauge Air inlet needle valve

Outlet needle valve

Vacuum pump

Feed container

Cold trap Water at 20°C

Liquid nitrogen

Safety trap Liquid nitrogen

Fig. 1. Schematic diagram of apparatus for measuring mole fractions in the headspace above the feed. Fig. 2. Effect of fat on total flux (mean ± standard error of three replicates), at a permeate pressure of 2.0 kPa.

of the feed container into the cold trap, using the vacuum pump. The air inlet valve was also opened slightly, so that the feed container remained at atmospheric pressure. Collection was continued until approximately 1 g of material had been collected in the cold trap. The measured concentrations of each compound in the cold trap enabled the mole fraction of each compound in the headspace vapour to be calculated.

The total flux did not differ significantly with the addition of 4% milk protein isolate or 6% lactose to the feed. The highest and lowest pH mixtures both had fluxes statistically similar to a feed mixture with the standard pH of 3.5, under the same conditions. However, when milk fat was included in the feed mixture, the total flux was found to decrease with increasing concentrations of fat (Fig. 2).

2.5. Analysis of flavour compounds by gas chromatography Flavour compound concentrations in all permeate samples, as well as retentate samples not containing dairy ingredients, were determined using gas chromatography (GC), with the same conditions as described previously (Overington et al., 2008). In the case of retentate samples that contained dairy ingredients, the GC parameters were the same as described previously (Overington et al., 2008), but the extraction procedure was modified to include solid phase extraction (SPE) prior to GC analysis. Samples (2.0 g) were mixed well with 6 g sodium sulphate, 100 lL internal standard (an aqueous mixture of 873 mg L1 propyl butanoate and 918 mg L1 heptanoic acid, both from Sigma–Aldrich), 0.3 mL of 5 m sulphuric acid, 5 mL heptane and 5 mL diethyl ether (all from Scharlau). A 3 mL portion of the extract (top layer) was passed through a GracePure aminopropyl SPE cartridge (conditioned with heptane); the eluate was known as ‘extract 1’ and analysed for esters and ketones, using propyl butanoate as the internal standard peak. A 2:1 (v/v) mixture of chloroform and isopropanol (3 mL; both from Scharlau) was passed through the SPE cartridge, and the eluate was discarded. The acids were then eluted off the SPE cartridge with a solution of 6% formic acid in 2:1 (v/v) heptane/diethyl ether; this eluate was known as ‘extract 2’ and analysed for acids, using heptanoic acid as the internal standard peak.

3. Results 3.1. Effect of feed components on pervaporation performance 3.1.1. Total flux The total flux is a measure of the permeation rate of all components through the membrane. Under baseline conditions (feed pH 3.5, no non-volatile components added, feed temperature of 30 °C, and permeate pressure of 2.0 kPa), the total flux was 410 ± 41 mg m2 s1. As the majority of the permeate consisted of water, the total flux can be considered to be negligibly different from the water flux.

3.1.2. Enrichment factors of flavour compounds The enrichment factor in pervaporation is the ratio of a component’s concentration in the permeate to its concentration in the feed; thus giving a measure of the degree to which each flavour compound can be concentrated. As shown in Fig. 3, the feed pH strongly influenced the pervaporation of acids, but had little or no effect on the other flavour compounds. Acidifying the feed solution to pH 2.5 (from an initial pH of 3.5) had little effect on the enrichment of any flavour compound. However, increasing the feed pH to 4.8 (close to the pKa of these acids; Table 2) reduced the enrichment factors of acetic, butanoic and hexanoic acids by 48%, 58% and 51%, respectively. When the feed pH was further increased to 7.0, the enrichment factors of these acids were reduced by 68%, 84% and 84%, respectively, compared with the initial pH of 3.5. Octanoic acid was affected by a lesser degree than the smaller acids: its enrichment factor was 20% lower at pH 4.8 and 50% lower at pH 7.0. Ethyl hexanoate, ethyl octanoate and 2-nonanone had slightly higher enrichment factors at pH 4.8 and 7.0 compared with pH 3.5, although the enrichment factors of these compounds at pH 2.5 were not significantly different from those at higher pH levels. Adding fat to the feed mixture resulted in large decreases in the enrichment factors of all compounds (Fig. 4), except octanoic acid for which the results were inconclusive. For example, with 0.5% fat in the feed mixture, the flavour compound enrichment factors were 13–85% lower than the corresponding enrichment factors with a fat-free feed; at the highest fat level tested (38%), the enrichment factors were reduced by 88–95%. At fat levels of 10% and higher, only 2-heptanone and ethyl butanoate could be concentrated using pervaporation; the enrichment factors of all the other compounds had decreased to less than one, meaning that the permeate was less concentrated than the feed. In contrast, both protein and lactose affected the enrichment factors of some flavour compounds but not others (Fig. 5). When 4% milk protein isolate was included in the feed, the enrichment factors of acids were reduced by up to 96%, ethyl butanoate by 45% and 2-heptanone by 36%, compared with their enrichment when the feed was an aqueous solution without any protein.

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2.0

10 1.5 pH 2.5

8

pH 3.5 (control) pH 4.8

6

1.0

pH 7.0

4 0.5

Enrichment factor (acids)

Enrichment factor (ketones and esters)

12

2

id

id

ac

ac

no ic ct a O

an

oi c

ac H ex

oi c an

tic Bu t

yl Et h

id

id ac

te oc

Ac e

ta

xa no he

yl Et h

yl Et h

no a

at

e at bu

N on a

ta no

no

on e 2-

an H ep t 2-

e

0.0 ne

0

Fig. 3. Effect of feed pH on enrichment factor (mean ± standard error) of each flavour compound, at a permeate pressure of 1.5 kPa.

Table 2 pKa values of acids used in the feed mixture, at 25 °C (James and Lord, 1992), and proportions of each acid in the undissociated form (calculated using the Henderson– Hasselbalch equation). Compound

pKa

Acetic acid Butanoic acid Hexanoic acid Octanoic acid

4.75 4.83 4.88 4.89

Proportion in undissociated form (%) pH 2.5

pH 3.5

pH 4.8

pH 7.0

99.4 99.5 99.6 99.6

94.7 95.5 96.0 96.1

47.1 51.7 54.6 55.2

0.6 0.7 0.8 0.8

Similarly, 6% lactose resulted in decreases of 67% and 57%, respectively for ethyl butanoate and 2-heptanone. However, only slight reductions in the enrichment factors of acetic acid, butanoic acid and hexanoic acid, and a slight increase in the enrichment factor of octanoic acid, were observed with lactose. The enrichment factors of 2-nonanone and ethyl octanoate were not affected by either protein or lactose. 3.2. Effect of fat, protein and lactose on driving forces of flavour compounds 3.2.1. Partitioning of flavour compounds between fat and aqueous phases Fig. 6 shows how the nine flavour compounds were distributed between the fat and aqueous phases, in mixtures with various levels of fat. The mass of each compound in the fat and aqueous phases, in a given volume of feed, was calculated from the measured concentrations of that compound in each phase. Some errors may have been introduced with the 5% fat results, as it was difficult to completely separate this small amount of fat from the aqueous phase before analysis. This is a contributing factor to the large standard errors at 5% fat. The fraction of each compound in the aqueous phase was then determined according to Eq. (1):

F i;water ¼

mi;water mi;total

ð1Þ

where Fi,water is the fraction of compound i in the aqueous phase of the feed, mi,water is the mass of compound i in the aqueous phase in a

given volume of feed, and mi,total is the mass of compound i added to a given volume of feed mixture. The more fat available in the feed, the lower the proportion of each compound in the aqueous phase. Fig. 6 shows that the majority of each acid was associated with the aqueous phase, in contrast to esters and ketones which were mainly associated with the fat phase. This result is in agreement with the relative water solubility of each flavour compound: solubilities of the compounds in this study ranged from 790 to 106 mg/L for acids, 70 to 4900 mg/L for esters and 380 to 4300 mg/L for ketones (Howard and Meylan, 1997).

3.2.2. Headspace mole fractions of flavour compounds in the presence of fat, protein or lactose Fig. 7 shows how the level of fat in the feed mixture affected the mole fractions of flavour compounds in the headspace vapour phase above the feed mixture. The headspace mole fractions of ketones and esters decreased as the fat level increased. However, the headspace mole fractions of all acids, except acetic acid, changed very little between 0% and 38% fat. For the esters and ketones, the headspace mole fraction was positively correlated to the fraction in the aqueous phase given by Fig. 6. Table 3 shows the correlation coefficients between the fraction in the aqueous phase (Fig. 6) and the headspace mole fraction (Fig. 7). The headspace mole fractions of esters and ketones were also positively correlated to the enrichment factor (Fig. 8). The headspace mole fraction of each compound depended on its concentration in the feed mixture, which is why ethyl butanoate and ethyl hexanoate (approximately 100 ppm each) had much higher headspace mole fractions than ethyl octanoate and the ketones (approximately 10 ppm each). Although the concentrations of acids were approximately ten times greater than the ketones, their headspace mole fractions were in the same range (Fig. 7), reflecting their lower volatility. Notwithstanding the concentration differences, headspace mole fractions decreased with increasing carbon chain length within each functional group, except that butanoic, hexanoic and octanoic acids all had similar mole fractions. Fig. 9 shows how the headspace mole fractions of flavour compounds were affected by the addition of 4% milk protein isolate or

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20

(a)

2-Heptanone 2-Nonanone

Enrichment factor

15

10

5

0 0

20

5

10

15 20 25 Fat level (% w/v)

30

35

40

3.0

(b)

Ethyl butanoate Ethyl hexanoate Ethyl octanoate

2.5

2.0

10

1.5

1.0

Ethyl octanoate enrichment factor

Enrichment factor

15

5 0.5

0 0

5

10

15

20 25 Fat level (% w/v)

30

35

0.0 40

0.7

Enrichment factor

(c) 0.6

Acetic acid Butanoic acid

0.5

Hexanoic acid Octanoic acid

0.4 0.3 0.2 0.1 0.0 0

5

10

15 20 25 Fat level (% w/v)

30

35

40

Fig. 4. Effect of fat on enrichment factors of (a) ketones, (b) esters and (c) acids, at a permeate pressure of 2.0 kPa. Data points are the mean (± standard error) of three replicates.

6% lactose to the feed mixture. The results were very similar for both protein and lactose. The added protein or lactose decreased the headspace mole fractions, and hence the driving forces, of esters, ketones and octanoic acid by up to 94%. However, the headspace mole fractions of the smaller acids were increased in the presence of protein or lactose.

4. Discussion Each alteration to the feed mixture (pH adjustment or addition of non-volatile dairy ingredients) influenced the pervaporation enrichment of certain flavour compounds. To determine the mechanisms by which feed alterations affected the pervaporation of fla-

A.R. Overington et al. / Journal of Food Engineering 107 (2011) 60–70

1.0 0.8

15

0.6

10

0.4

5

0.2

0

0.0

Enrichment factor (acids)

20

Aqueous feed solution (pH 3.5) 4% Milk protein isolate (pH 6.3) 6% Lactose (pH 3.9)

2-

H ep ta no 2ne N on Et an hy on lb e ut Et an hy oa lh te ex an Et hy oa lo te ct an oa te Ac et ic Bu ac id ta no i c H ac ex id an oi c O a ct ci an d oi c ac id

Enrichment factor (esters and ketones)

25

Fig. 5. Effect of protein and lactose on enrichment factors of flavour compounds, at a permeate pressure of 2.0 kPa. Data are means (± standard errors) of three replicates.

vour compounds, the headspace mole fractions of the flavour compounds were measured above each feed mixture. The pervaporation driving force can be approximated by the partial pressure difference across the membrane; the higher the headspace mole fraction of a volatile component above the feed, the higher its vapour pressure and the higher it’s driving force. By comparing the driving force data with the enrichment data, it can be seen whether the influence of each feed alteration was caused by its effect on the driving force or by other factors. These proposed mechanisms are described in the following sections, and are summarised in Fig. 10. 4.1. Influence of feed pH on pervaporation of flavour compounds The permeation of acids depended on the feed pH, because the pH determined the proportion of each acid in its dissociated and undissociated forms. The undissociated (uncharged) form is more permeable, because charged compounds should not pass through pervaporation membranes (Baudot and Marin, 1997; Lipnizki et al., 2004). Charged compounds will be less soluble than uncharged compounds in the non-polar polydimethylsiloxane membrane. At low pH, a greater proportion of each acid was in the undissociated form, as shown in Table 2. At pH 2.5–3.5, all acids were more than 94% undissociated, and their corresponding enrichment fac-

65

tors were therefore relatively high (Fig. 3). The proportion of each acid in the undissociated form was much lower when the feed pH was increased to 4.8 (close to the pKa values of these acids) or further increased to neutral pH. Therefore, the acids had lower enrichment factors at higher pH levels. Ikegami et al. (2005) confirmed that the affinity of succinic acid for a hydrophobic membrane material (silicalite) decreased with increasing pH, as the proportion in the undissociated form decreased. The current study extends this finding to different acids with a polydimethylsiloxane membrane. The reason for the positive effect of increased pH on 2-nonanone, ethyl hexanoate and ethyl octanoate enrichment is less obvious. A possible explanation is that with lower levels of acids entering the membrane at higher pH, there would have been less competition between permeants for sites in the membrane, enabling esters and ketones to be more highly enriched. Ethyl butanoate and 2-heptanone already had high enrichment factors, so the lack of competition would bring no further improvement in their enrichment. 4.2. Influence of milk fat on pervaporation of flavour compounds Of the non-volatile components tested (fat, protein and lactose), only fat had a significant effect on the total (water) flux. This reduction in the flux may have been caused by an increase in the feed viscosity with higher levels of fat (leading to a less turbulent feed flow), or by membrane fouling. The fat may form a boundary layer on the membrane, which would act as an additional resistance to mass transfer. Moreover, the added fat affected the permeation of flavour compounds to a greater extent than the permeation of water, as shown by the decreasing enrichment factors with increasing fat level (Fig. 4). These results confirm Baudot and Marin’s (1996) prediction that fat would reduce the pervaporation yield of hydrophobic compounds. By comparing the enrichment factors (Fig. 4) with the distribution of each compound between fat and water (Fig. 6) and with the driving forces (Fig. 7), the reasons for the effect of fat on pervaporation can be deduced. Esters and ketones will be discussed first, followed by acids. As the fat level was increased, relatively more of each ester and ketone became associated with the fat phase of the feed (as opposed to the water phase). Because flavour compound volatilities

Fig. 6. Fraction of each flavour compound in the aqueous phase (compared with the total feed), in feed mixtures with various amounts of fat. Data are means (± standard error) of three replicates.

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(a)

6

Mole fraction in vapour phase (x 10 )

7 2-Heptanone 2-Nonanone

6 5 4 3 2 1 0 0

10

20 Fat level (% w/v)

30

40

(b)

6

Mole fraction in vapour phase (x 10 )

100 Ethyl butanoate Ethyl hexanoate Ethyl octanoate

80

60

40

20

0 0

5

10

15 20 25 Fat level (% w/v)

30

35

40

(c)

6

Mole fraction in vapour phase (x 10 )

16 Acetic acid Butanoic acid Hexanoic acid

14 12

Octanoic acid 10 8 6 4 2 0 0

10

20 Fat level (% w/v)

30

40

Fig. 7. Mole fractions of (a) ketones, (b) esters and (c) acids in vapour phase above feed mixtures with various levels of fat (mean ± standard error of two replicates, measured at 20 °C).

tend to be much lower in fat than in water (Landy et al., 1996; Meynier et al., 2003), the portion dissolved in the fat phase should not contribute significantly to the driving force. A comparison of Fig. 6 with Fig. 7 shows that this was true for the esters and ketones in this study: at higher fat levels, when relatively less of each compound was in the aqueous phase and more in the fat phase of the feed, the headspace mole fractions were lower. A similar finding was reported by Meynier et al. (2003), who measured the volatility of five esters and aldehydes in water, skim milk, anhydrous milk fat and cream. They observed that air/cream partition coefficients were 94–99% lower than the air/water partition coefficients,

indicating that the flavour compounds were less volatile in cream than in water. The enrichment factors of esters and ketones decreased with added fat in a similar pattern to their driving forces, suggesting that the effect of fat on pervaporation of these compounds was a direct result of the decreased driving force. This is also shown by the linear relationship between enrichment factors and headspace mole fractions for esters and ketones in Fig. 8. In contrast to the esters and ketones, the acids were mainly associated with the aqueous phase of the feed rather than the fat phase (Fig. 6), and there was either a negative correlation (acetic

67

4

20

2

0

0

Et hy lH

ut an

6

Mole fraction in vapour phase (x 10 )

40

oa te lO ct an oa 2te H ep ta no ne 2N on an on Ac e et ic A Bu cid ta no ic H Ac ex id an oi c Ac O ct id an oi c Ac id

6

Et hy lb

acid; hexanoic acid) or no correlation (butanoic acid; octanoic acid) between the fraction in the aqueous phase and the driving force (Table 3). There was also no linear relationship between the headspace mole fraction and the enrichment factor of any of the acids with various levels of fat (R2 < 0.45; data not shown). The headspace mole fractions of all acids, except acetic acid, changed very little between 0% and 38% fat (Fig. 7c) (for one replicate, an anomalously high mole fraction was recorded for acetic acid at 38% fat, but its average mole fraction at 38% fat was within standard error limits of the other fat levels). This result agrees with Roberts and Acree (1996), who found that the volatility of butanoic acid was similar in both water and an oil/water matrix. These findings reveal that, although the added fat reduced the enrichment factors of acids in a similar pattern to esters and ke-

8

60

te

0.89 0.99 0.96 0.90 0.80 0.98 0.48 0.75 0.03

80

Et hy

Ethyl butanoate Ethyl hexanoate Ethyl octanoate 2-Heptanone 2-Nonanone Acetic acid Butanoic acid Hexanoic acid Octanoic acid

10 No lactose or protein 4% Milk protein isolate 6% Lactose

oa

Correlation coefficient

100

ex an

Compound

6

Table 3 Correlation coefficients between the fraction in the aqueous phase of the feed (Fi,water) and the mole fraction in the vapour phase above the feed.

Mole fraction in vapour phase (x 10 )

A.R. Overington et al. / Journal of Food Engineering 107 (2011) 60–70

Fig. 9. Effect of added protein and lactose on mole fraction of flavour compounds in vapour phase above feed (measured at 20 °C; mean ± standard error of two replicates).

tones, it did so via a different mechanism. The added cream increased the pH of the feed (Table 4), which caused a greater proportion of each acid to be in its less-permeable dissociated form, as discussed in Section 4.1. In summary, the added fat reduced the enrichment factors of esters and ketones by lowering their driving forces as a result of their partitioning into fat. In contrast, the added fat reduced the enrichment factors of acids by lowering their sorption in the membrane as a result of the increased pH.

Fig. 8. Relationship between enrichment factors and mole fractions of (a) ketones and (b) esters in the headspace vapour above the feed, for fat levels between 0% and 38%.

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Alteration to 6. mixture feed

Interaction with flavour compounds

Main effect on driving force or permeability (sorption × diffusion)

Resulting effect on pervaporation enrichment

Fat addition

Hydrophobic compounds partition into fat phase

Lower driving force (mole fraction in vapour phase) for esters and ketones

Decrease in enrichment for compounds that partition into fat

pH adjustment

pH increase leads dissociation of acids

Lower sorption for acids

Decrease in enrichment of acids

Protein addition

Certain compounds bind to protein or lactose

Lower sorption (and lower driving force for some compounds)

Decrease in enrichment of proteinor lactose-bound compounds that are sorptionlimited (i.e. short-chain esters and ketones)

to

Lactose addition

Fig. 10. Summary of mechanisms by which feed alterations affected enrichment of flavour compounds by pervaporation.

4.3. Influence of milk protein on pervaporation of flavour compounds Milk protein reduced the effectiveness of pervaporation for concentrating certain flavour compounds (Fig. 5). This result was expected, because any flavour molecules that bound to the protein would be unable to enter the membrane. Fig. 9 shows that most of the tested flavour compounds were bound by protein, resulting in lower mole fractions in the headspace above a protein-containing feed compared with a protein-free solution. Within each homologous series, the longer-chain compounds were bound by the protein to a greater extent, which is consistent with the findings of other researchers (Landy et al., 1995, 1996; Kühn et al., 2006; Nongonierma et al., 2006). The reason that milk protein isolate increased the headspace mole fractions of small, hydrophilic acids was probably a salting-out effect. Apart from protein, milk protein isolate also contains small amounts of minerals and sugars, both of which may affect the flavour compound volatilities. However, if the effect of protein on pervaporation was purely due to a reduced driving force as a result of flavour binding, flavour compound enrichment factors with and without protein would follow a similar pattern to the headspace mole fractions in Fig. 9. This was not the case; for esters and ketones, the added protein had a greater effect on the headspace mole fractions of larger compounds, but reduced the enrichment factors of only the smaller compounds. Similarly, the protein reduced the headspace mole fraction of only the largest acid, but reduced the enrichment factors of all acids, especially those with low molecular weights. Therefore, the protein did not only affect the driving forces, but also the permeabilities of the flavour compounds. Similar to fat-containing feeds, the result for acids can be attributed to pH differences; the feed pH was 6.3 with 4% milk protein isolate, compared with 3.5 for the standard aqueous feed solution. As explained earlier, the acids were mostly in the dissociated form at the higher pH, so their fluxes were lower. This result could

Table 4 pH values of mixtures containing flavour compounds and various amounts of cream. Fat level (%, w/v)

pH

0 5 10 20 38

3.5 4.8 5.5 6.1 6.1

potentially be reversed, or at least reduced, by manipulating the feed pH. However, too low a pH could lead to denaturation of the proteins as the isoelectric point is approached. Denaturation can either increase or decrease the flavour binding capacity of proteins (Matheis, 1998; Kühn et al., 2006). The different behaviour between large and small esters and ketones is best explained by the mass transfer mechanism of the individual flavour compounds. Mass transfer in pervaporation involves permeant molecules (flavour compounds) first being transported to the membrane by convection, and then sorbing into the membrane, followed by diffusion through the membrane. As the protein was only present on the feed side of the membrane, it could not affect the diffusion through the membrane. Also, the fact that the total flux was not influenced by protein shows that the convective transport to the membrane was not affected. Therefore, protein in the feed should affect the sorption step only. In an earlier paper (Overington et al., 2008), it was discussed how smaller, more hydrophilic compounds have a lower degree of sorption in hydrophobic membranes, but a higher rate of diffusion than larger compounds. Therefore, provided that the greatest mass transfer resistance is transport through the membrane (sorption and diffusion) rather than transport on the feed side, then smaller compounds are more likely to be sorption-limited, and larger compounds are more likely to be diffusion-limited. If diffusion through the membrane is the rate-limiting step, then the flux should not be affected by the degree to which the protein slows down transport and sorption on the feed side, provided that the rate of molecules sorbing in the membrane does not become less than the rate of diffusion. Hence, even though the protein bound larger compounds to a greater extent, this binding affected the enrichment of only the smallest esters and ketones, for which sorption was the rate-limiting factor for mass transfer. In contrast to the findings in the current study, Aroujalian et al. (2003) found that 10 g L1 soy protein did not significantly affect the flux or selectivity during pervaporation of an ethanol/water mixture. Soy proteins are known to bind alcohols (Chung and Villota, 1989), but it is unlikely that this level of protein bound enough of the 2% ethanol to see a significant effect. Assuming a molecular weight of at least 150,000 g mol1 for soy protein (Fukushima, 2004), Aroujalian et al.’s (2003) feed solution contained 7  105 mol L1 protein and 0.43 mol L1 ethanol. Soy protein has been found to contain 18–40 binding sites for n-butanol and n-hexanol per protein molecule (Chung and Villota, 1989); assuming it has a similar number of binding sites for ethanol, the protein

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in Aroujalian et al.’s (2003) feed solution would have bound less than 1% of the ethanol present. In contrast, Fig. 9 shows that 4% milk protein isolate bound the flavour compounds in the present study by up to 94%, as measured by the reduction in their mole fractions in the feed headspace. van Willige et al. (2000a,b) investigated the effect of milk proteins, skim milk and whole milk on the sorption of flavour compounds into linear low-density polyethylene. Sorption was decreased if the flavour compound bound to milk proteins, which is consistent with the findings in the current study. van Willige et al. (2000a,b) found that the effect depended on both the flavour compound and the type of milk protein. 4.4. Influence of lactose on pervaporation of flavour compounds Compared with an aqueous feed, Fig. 5 shows that the enrichment factors of the smaller compounds within each functional group were reduced in the presence of lactose. For esters and ketones, the effect of lactose on pervaporation followed the same trends as the effect of protein. Likewise, lactose decreased the headspace mole fractions of the same compounds that were affected by protein (Fig. 9). Lactose is known to bind certain flavour compounds (Kellam, 1998), but the mechanism of flavour binding by sugars is unclear (Solms and Guggenbuehl, 1990; Matheis, 1998; Reineccius, 2006). Lactose, like all sugars, contains hydroxyl groups that can hydrogen-bond with other compounds. However, this does not explain why it had a greater negative influence on the headspace mole fractions of long-chain compounds, which are more hydrophobic than smaller compounds. The most plausible explanation parallels a hypothesis for the same effect with sucrose: when the sugar is dissolved, the solution becomes more hydrophobic, and hence more favourable to hydrophobic flavour compounds and less favourable to short-chain, hydrophilic compounds (Reineccius, 2006). As the enrichment factors and driving forces for esters and ketones followed the same patterns with both protein and lactose, the results can be explained in the same way. Although Fig. 9 shows that lactose bound all of the esters and ketones tested to some extent, resulting in lower headspace mole fractions compared with an aqueous feed mixture, Fig. 5 shows that lactose reduced the enrichment factors of only the smaller compounds within these homologous series. This fits with the theory proposed in an earlier paper (Overington et al., 2008) that sorption into the membrane was the rate-limiting factor for mass transfer of these smaller compounds. However, it is less clear why lactose increased the headspace mole fractions of the three smallest acids, yet it decreased their enrichment factors. Lactose did not affect the feed pH to the same extent as protein (the feed solution with 6% lactose had a pH of 3.9, compared with 3.5 for the standard aqueous feed), but this pH difference may still have caused the enrichment of acids to decrease. Other researchers have found that sugars and sugar alcohols can have positive, negative or neutral effects on pervaporation. Aroujalian et al. (2006) studied the effects of glucose and xylose on pervaporation of a 2% ethanol/water solution. Both sugars caused the total flux to decrease, with the effect being greater at high permeate pressures. Glucose also lowered the ethanol selectivity, but xylose increased the selectivity under certain conditions. Ikegami et al. (1999) also found that glucose, lactose, myo-inositol and xylitol all caused the total flux of an ethanol/water solution to decrease. Glucose, lactose and myo-inositol caused a slight decrease in the ethanol flux and a larger decrease in the water flux, resulting in an increased separation factor. However, xylitol caused the ethanol flux to decrease more than the water flux, so that the selectivity was lowered. In contrast, lactose did not significantly affect the flux or selectivity of dairy flavour compounds methylthiobutanoate

69

(Baudot et al., 1996) or diacetyl (Rajagopalan et al., 1994). These results suggest that the effect of sugars on pervaporation may depend on the operating conditions and type of permeant as well as the type of sugar. Aroujalian et al. (2006) explained the flux decrease in terms of sugars increasing the vapour pressure of ethanol and decreasing the vapour pressure of water. Although a higher selectivity would normally be expected if this were the case, they explained that the addition of sugars raised the ethanol concentration inside the polydimethylsiloxane membrane, causing the selectivity to be lowered in some instances due to membrane plasticisation. Unlike Aroujalian et al. (2006) , Ikegami et al. (1999) assumed that sugars reduced water sorption into the silicalite membrane, rather than affecting the vapour pressure in the feed. 5. Conclusion This study has shown that the composition of the feed mixture has a significant effect on flavour compound pervaporation. Pervaporation became less effective for concentrating flavour compounds when the feed mixture contained fat, as a result of the hydrophobic flavour compounds partitioning into the fat phase. The addition of fat (in the form of cream) also caused the feed pH to increase, as did protein (in the form of milk protein isolate). This resulted in a decrease in the enrichment factors of acids. Esters and ketones were affected in a similar way by both protein and lactose, suggesting that both these non-volatile components interacted with flavours via a binding mechanism. However, binding of a flavour compound to protein or lactose (as evidenced by the reduction in that compound’s mole fraction in the headspace vapour above the feed, when protein or lactose were present in the feed) did not automatically mean that the flux or enrichment of that compound would be lowered. Added protein or lactose affected the sorption of permeants into the membrane, but not their diffusion through the membrane. Hence, these dairy components only reduced the fluxes and enrichment factors of flavour compounds for which sorption was the rate-limiting factor for mass transfer. Of the three dairy components tested (fat, protein and lactose), only fat caused the total (water) flux to decrease. Therefore, protein and lactose were not able to reduce the feed side mass transfer (convective transport to the membrane) under the conditions tested in this study, meaning that their effects on pervaporation performance were purely due to their interactions with the flavour compounds. Fat, however, reduced the mass transfer on the feed side as well as reducing the amounts of flavour compounds available for pervaporation. Although pervaporation has potential as a method for concentrating flavours in food process streams, this study has shown that its effectiveness can be reduced if non-volatile dairy components are present, or if the pH is too high (in the case of acidic flavour compounds). Acknowledgements This research was funded by Fonterra Co-operative Group Ltd. and the Foundation for Research, Science and Technology (New Zealand). We thank Lílian Ferreira for her useful guidance. Pervaporation membranes were kindly supplied by Helmholtz-Zentrum Geesthacht – Centre for Materials and Coastal Research (Germany). References Aroujalian, A., Belkacemi, K., Davids, S.J., Turcotte, G., Pouliot, Y., 2003. Effect of protein on flux and selectivity in pervaporation of ethanol from a dilute solution. Separation Science and Technology 38 (12–13), 3239–3247.

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