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protein mixtures in humid air
ARTICLE IN PRESS
Journal of Crystal Growth 295 (2006) 231–240 www.elsevier.com/locate/jcrysgro
Crystallization of spray-dried lactose/protein mixtures in humid air A. Shawqi Barhama,, Md. Kamrul Haqueb, Yrjo¨ H. Roosb, B. Kieran Hodnetta a
Materials and Surface Science Institute, University of Limerick, Limerick, Ireland Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland
b
Received 30 November 2005; received in revised form 24 July 2006; accepted 9 August 2006 Communicated by M. Schieber
Abstract An in situ crystallization technique with X-ray diffraction analysis complemented by ex situ scanning electron microscopy and chromatographic analysis of the a/(a+b) solid-state anomeric ratios has been developed to study the crystallization of lactose/protein mixtures in humid air. This technique was used to determine changes in phase composition and morphology during crystallization. Following an induction period during which water is sorbed, crystallization is rapid and the predominant phase observed using the in situ method in spray-dried lactose/sodium-caseinate, albumin and gelatin is a-lactose monohydrate. However, in the case of spray-dried lactose/whey protein isolate (WPI) the predominant phase that appears is the a/b mixed phase with smaller amounts of a-lactose monohydrate. With pure lactose the a/b mixed phase appears as a transient shortly after the onset of crystallization and a-lactose monohydrate and b-lactose both appear as stable crystalline phases at longer times. Another transient phase with 2y ¼ 12.21, 20.71 and 21.81 was observed in spray-dried lactose/albumin. This phase decomposed as a-lactose monohydrate developed. Three phases seem to persist in the case of spray-dried lactose/gelatin, namely the phase with peaks at 2y ¼ 12.21, 20.71 and 21.81, a-lactose monohydrate and b-lactose for the duration of the in situ experiment. r 2006 Elsevier B.V. All rights reserved. PACS: 64.70.Dv; 60.70.Kb; 87.14.Ee Keywords: A1. Crystallization; A1. SEM; A1. X-ray diffraction; A1. Relative vapor pressure; B1. Proteins; B1. Lactose
1. Introduction Lactose–protein mixtures are commonly used in foods and can be prepared by a variety of methods including spray-drying and freeze-drying [1–5]. Following preparation, these mixtures are amorphous and form free flowing powders, which is a desired property for handling purposes [6,7]. The physical state of lactose–protein mixtures is important for the stabilization of food products during processing and storage. However, during processing and storage, amorphous lactose mixtures may undergo timedependent changes with increasing rate at increasing temperatures and water contents. These mixtures and pure lactose itself are very hygroscopic and readily sorb moisture from the ambient air [8]. Within 1 h of exposure Corresponding author. Tel.: +353 612324159; fax: +353 61213529.
E-mail address: [email protected] (A. Shawqi Barham). 0022-0248/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcrysgro.2006.08.006
to moist air lactose can sorb up to 10% by mass of water and the material becomes sticky, loosing its free flowing properties [4,5,9]. The glass transition temperature (Tg) of lactose becomes lower as the moisture content increases. Typical values for Tg of dry lactose have been reported of ca. 105 1C but this value decreases to ca. 5 1C when the moisture content is close to 10 wt% [4,5,10,11]. The material then coagulates, crystallizes and becomes very hard and difficult to grind into a fine powder [4,5]. Lactose, milk sugar, is a disaccharides consisting of two moieties of D-glucose and D-galactose joined by a b-1, 4-glycosidic linkage. There are two isomeric forms of lactose a- and b-lactose, which differ in the configuration of the terminal hydroxyl group of the glucose moiety [12]. However, several crystalline forms of lactose are known, such as, a-lactose monohydrate [13,14], b-lactose [15], hygroscopic anhydrous a-lactose [16], stable anhydrous a-lactose [17] and mixed compounds of the two anomers
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of a- and b-lactose. The mixed lactose compounds, namely, 5:3 [18,19], 4:1 [18], 3:2 [20] and 1:1 a:b phases [21], appear to be discrete crystalline phases rather than physical mixtures of crystals of pure a- and b-lactose [22]. In terms of crystallization from aqueous solution, the alactose monohydrate phase is the most extensively studied [23,24]. Most studies have been carried out in conditions where the a- and b-isomers are both present at ratios determined by the equilibrium mutarotation of the isomers [23–25]. This means a-lactose monohydrate has a propensity to form as a pure crystalline phase even in the presence of large amounts of the b-isomer [6,26]. Literature data seem to indicate that there is very little incorporation of the b-isomer into the a-lactose monohydrate phase [24–26]. However, the presence of the b-isomer does influence the crystalline habit of a-lactose monohydrate [12]. When the b-isomer is absent, such as when dimethyl sulfoxide (DMSO) is used as solvent (suppressing mutarotation), needle or platelet shaped particles of a-lactose monohydrate form. When the b-isomer is present, growth of the ð0 1¯ 1Þ facets of a-lactose monohydrate is inhibited and the well-known tomahawk shaped crystalline habits prevails [12]. Previous in situ X-ray diffraction (XRD) studies of the crystallization of spray and freeze-dried lactose have demonstrated the validity of this method for the observation, in particular, of phases which only appear at short times after the onset of crystallization, before the eventual formation of the final stable composition at longer times [27,28]. In particular, for both spray and freeze-dried lactose, the stable phase present at extended crystallization times are a-lactose monohydrate and b-lactose. However, a mixed isomer crystalline phase, previously reported as a a/ b mixed phase was observed shortly after the onset of crystallization but decomposed at longer times. A complementary SEM analysis showed that the crystalline material continuously evolved until platelet shaped particles eventually predominated at long crystallization times [27,28]. This paper describes a study of the crystallization of spray-dried lactose–protein mixtures (3:1 w/w) in moist air followed by in situ X-ray powder diffraction analysis, with complementary water sorption data, ex situ X-ray powder diffraction, scanning electron microscopy, and gas liquid chromatography. The proteins studied here were sodiumcaseinate, whey protein isolate (WPI), albumin, and gelatin. 2. Experimental procedure 2.1. Materials a-Lactose (Dairy Gold, Ireland), WPI (Protarmor 865) (Armor Proteines, 35460 Saint Brice En Cogles, France), sodium-caseinate (Dairy Gold, Ireland), egg albumin (Sanovo Egg Products Limited, UK) and gelatin (Croda Colloids Limited, UK). Molecular sieves (calcium, sodium
1 in pellets, nominal pore diameter 5 A˚), alumino-silicate, 16 phosphorus pentoxide anhydride (P2O5, 98%), potassium chloride (KCl, 99%) and sodium chloride (NaCl, 99.5%), sodium nitrite (NaNO2, 99.5%) were purchased from Sigma-Aldrich Ltd., Ireland. Dry pyridine (99.8%), dimethyl sulfoxide (DMSO, 99.7%), N-trimethylsilylimidazole (TMSIM, 98.0%) were purchased from SigmaAldrich, Ireland, and were used to derivatize lactose for GLC analysis. Aqueous solutions (15% w/w) of a-lactose, a-lactose/ WPI (3:1 w/w), a-lactose/Na-caseinate (3:1 w/w), a-lactose/ albumin (3:1 w/w) and a-lactose/gelatin (3:1) were prepared and spray-dried using a Niro Atomizer spray-drier (Copenhagen, Denmark) with an inlet temperature of 185–190 1C and outlet temperature of 80–85 1C. To enhance solubility, Na-caseinate solution was made slightly alkaline (pH 7.5) using sodium hydroxide (BDH Chemicals Ltd., Poole, England). The residual water content was removed by keeping samples in vacuum desiccators over P2O5 at room temperature (22–23 1C) for at least 5 days. The powdered samples were collected, immediately, packaged into glass containers, and desiccated over P2O5 [0% relative humidity (RH) at 25 1C]. Before experiments, residual moisture was removed by drying the lactose samples in a vacuum oven at 50 1C for 24 h [4,5].
2.2. Ex situ crystallization of spray-dried lactose and lactose/protein (3:1 w/w) mixtures Anhydrous spray-dried lactose and lactose/protein (3:1 w/w) mixtures in vials were stored in evacuated desiccators over saturated salt solutions at room temperature (22–23 1C) for 144 h. The salts used were Mg(NO3)2, NaNO2 and NaCl for 55%, 66% or 76% RH, respectively, giving a water activity (aw) of 0.01 % RH at equilibrium [29]. 2.3. In situ crystallization of spray-dried lactose and lactose/protein (3:1 w/w) mixtures In situ X-ray powder diffraction was performed using a Philips X’pert PRO MPD PW3040/60 X-ray diffractometer with nickel filtered copper Cu Ka radiation (l ¼ 1.542 A˚) as the X-ray source. The Cu Ka diffractometer anode was run under a tension of 40 kV and a current of 35 mA. Sample preparation consisted of grinding the sample to a powder (o45 mm) and loading it into a Macor sample holder cup (Anton Paar, Austria) approximately 17.5 mm in diameter by 2 mm deep and applying a smooth surface finish. The sample holder cup was loaded onto the rotating sample stage and placed inside a HTK1200 Oven Camera (Anton Paar, Austria). The temperature in the in situ sample chamber was independently controlled by a temperature control unit TCU1000 (Anton Paar Gmbh, Austria) which controls the wire heater of the sample and the inner wall of the chamber. Gas of a specified humidity was formed by
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passing dry nitrogen (N2) into 1 L cylindrical steel vessel filled with zeolite (molecular sieves, Sigma-Aldrich Ltd., Ireland), which was saturated with water. The temperature of the saturator (wet N2) was controlled using a Clifton (Nickel Electro LTD, England) water bath. The gas flow rate of the dry N2 was set at 100 mL min1 using a mass flow meter (Brooks, Instrument B.V., Holland). By variation of the temperature of the water bath (saturator) and for a set temperature of 25 1C in the in situ XRD chamber (HTK 1200), different RH values were generated. The temperature of the saturator and the in situ XRD chamber were 22 and 25 1C, respectively. The vapor pressure of water at 22 and 25 1C was 2.9 and 3.3 kPa, respectively, giving a RH of 88% [30]. The sample height inside the HTK1200 was accurately set using the X-ray beam (the sample was aligned when the maximum beam intensity at 2y ¼ 01 was halved by the sample). The sample was scanned over a range of 2y ¼ 5401 using a step size of 0.01671 2y and a scan speed of 0.03561 2y/s. The sample was rotated at 10 Hz during analysis. Scans were recorded every 17 min during in situ studies. 2.4. Electron microscopy The electron micrographs were obtained using a JEOL JSM-5600 electron microscope. A small amount of ungrounded material was scattered evenly onto the surface of an aluminum stub covered with a 12 mm diameter carbon tab. Excess material was removed using dry compressed air, and the sample was sputter coated with a thin conductive film of gold for approximately 2 min in an Edwards S150B sputter coater. The accelerating voltage used was 20 keV.
3. Results Water sorption data for spray-dried lactose are presented in Fig. 1, Trace E. These data, recorded at a RH value of 76%, clearly show the immediate uptake of moisture by lactose followed by its expulsion to an equilibrium value of ca. 4 wt%. The stoichiometric requirement for a-lactose monohydrate is 5 wt%. As crystallization proceeds there is a tendency to expel excess moisture. Table 1 summarizes the phases observed from pure spray-dried lactose and spray-dried lactose–protein mixtures treated in humid air at 55%, 66% or 76% RH for 144 h. The persistent crystalline phases, namely the phases present after prolonged exposure (144 h) to moist air (55%, 66% or 76% RH) observed with spray-dried lactose were a-lactose monohydrate and the anhydrous b-lactose. For spray-dried lactose-WPI (3:1 w/w) exposed to humid air at 55% RH, the predominant phase was the pure a/b mixed phase. At 66% RH, the predominant phases were mixtures of a/b mixed phase and a-lactose monohydrate for the same material. This situation is somewhat different for all lactose samples (3:1 w/w) mixed with gelatin, Na-caseinate or albumin at 55–76% RH. The predominant phase that appears was only the a-lactose monohydrate. Table 1 also presents the a/(a+b) solid-state anomeric ratios in the solid state for spray-dried lactose–protein mixtures (3:1 w/w). Values very close to 0.50 were recorded for all the anhydrous samples. For spray-dried lactose this value increased to about 0.65 after exposure to moist air. This ratio only changed slightly for spray-dried lactose/ WPI upon exposure to moist air ca. 0.53 at 55% RH. For
Spray-dried lactose Spray-dried lactose/Whey Protein Isolate(WPI)(3:1w/w) Spray-dried lactose/Na-Casinate(3:1w/w) Spray-dried lactose/Albumin(3:1w/w) Spray-dried lactose/Gelatin(3:1w/w)
20 Water Content (g/100g dry solids)
2.5. Gas liquid chromatography The instrument used was a Hewlett-Packard 4890A gas chromatograph equipped with a split injector and a flame ionization detector (FID) operating at 275 1C [31]. The column was a nonpolar capillary Rtx-5, 15 m 0.25 mm i.d. with 0.1 mm film thickness (Restek, UK). Oven temperature of the GC was 215 1C. Carrier gas was nitrogen at a flow rate of 1.0 mL min1 and 35 kPa. For routine GLC analysis, dissolution and derivatization must be rapid, and there must be negligible mutarotation. Therefore, we derivatized the dry samples with a mixture consisting of 19.5% DMSO, 22% N-trimethylsilylimidazole (TMSIM), and 58.5% pyridine by volume (mixture A) [31]. Approximately 1 mg of dry lactose–protein samples was dissolved in 1 mL of mixture A. The solutions were kept for 60 min at 60 1C before the derivatization was complete. The injection volume was 1 mL, and the run time used was 22 min. The lactose components eluted after 14 and 19 min for a-lactose and b-lactose, respectively.
233
16
12 (A) (B) (C) 8
(D)
4
(E)
0 0
5
10
15 Time (h)
20
25
Fig. 1. Water sorption for (A) spray-dried lactose/Na-caseinate (3:1 w/w), (B) spray-dried lactose/albumin (3:1 w/w), (C) spray-dried lactose/gelatin (3:1 w/w), (D) spray-dried lactose/whey protein isolate (WPI) (3:1 w/w) and (E) spray-dried lactose (76%RH) [4,5].
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Table 1 Summary of all phases observed following exposure to moist air for 144 h, glass transition temperatures and the solid-state anomeric composition of lactose–protein mixtures determined by GC analysisa Anhydrous
L–SD L3–WPI L3–CAS L3–GEL L3–ALB
RH 55%
RH 66%
RH 76%
XRD phases
Tg1 (1C)
a/(a+b)
XRD phases
a/(a+b)
XRD phases
a/(a+b)
XRD phases
a/(a+b)
Amorphous Amorphous Amorphous Amorphous Amorphous
105 111 103 113 108
0.45 0.47 0.48 0.47 0.50
a+b a/b a a a
0.63 0.53 0.85 0.98 0.94
a+b a+a/b a a a
0.66 0.79 0.86 0.63 0.88
a+b a a a a
0.65 0.93 0.94 0.88 0.96
a
Lx–protein ¼ lactose–protein at molar ratios of x:1. RH X% ¼ relative humidity at X%. Tg1 ¼ onset of glass transition (1C) [4]. a/(a+b) ¼ anomeric composition of lactose determined by GLC. a ¼ a-lactose monohydrate. b ¼ b-lactose anhydrous. a/b ¼ a/b mixed phase.
the lactose–protein samples the a/(a+b) solid-state anomeric ratio increased to close to 1.00 for those samples with the greatest propensity to form a-lactose monohydrate, consistent with a mechanism whereby the a:b ratio in the lactose–protein moisture mixtures was adjusting to its equilibrium value as more and more a-lactose monohydrate was crystallizing. Fig. 2 presents the in situ XRD patterns of spray-dried lactose exposed to water vapor for up to 420 min at 25 1C (RH ¼ 88%). The starting material contained small amounts of b-lactose (note the small peak at 2y ¼ 10.51), but these seemed to disappear and the sample became X-ray amorphous after 80 min. After 100 min, the features of b-lactose started to appear and were very strong at 110–125 min (note peaks at 2y ¼ 10.51 (1 1 0) and 211 (1 1 1)). At 110 min, peaks of other phases of lactose also started to appear including a-lactose monohydrate (2y ¼ 12.51 and 16.41), but the three main characteristic peaks of this phase (2y ¼ 19.11, 19.61 and 19.91) have not developed fully and the principal peak present in this pattern is at 20.11. There are additional small peaks at 2y ¼ 18.31 and 22.11. These features are characteristic of the a/b mixed phase [18]. The crystal structure of this phase has recently been determined [21]. It is made up of dimers of a-lactose and b-lactose. Earlier studied have labelled this phase of the 5:3 phase corresponding to its solid state a:b anomeric ratios [18]. Below this phase will be described as the a/b mixed phase [28]. The other principal peaks of this phase, namely at 12.41, 19.11 and 20.11 are all obscured by the principal peaks of a-lactose monohydrate. Fig. 2B presents the temporal development of the a-lactose monohydrate, b-lactose and the a/b mixed phase. Clearly, the a/b mixed phase is unstable and decomposes at intermediate crystallization times, whereas the a-lactose monohydrate and b-lactose phases persist for the duration of the experiment. Interestingly, for spray-dried lactose the a/(a+b) solid-state anomeric ratio shown in Table 1, always show values close to 0.5, corresponding to the crystalline composition of this material with approximately equal amount of a-lactose monohydrate and b-lactose. In another study of lactose–salt mixtures this phase was also observed. It formed at the onset of crystallization and disappeared as a-lactose monohydrate and b-lactose
formed [28]. In that study the formation of the a/b mixed phase was associated with restricted molecular mobility in the highly viscous lactose–protein–water mixtures, forming the mixed anomeric phase in preference to a-lactose monohydrate or b-lactose [28]. Fig. 3A presents the XRD patterns recorded using the in situ method for lactose Na-caseinate mixtures that were exposed to humid air for the times indicated. Following an induction period of 70 min during which strong moisture uptake occurred, peaks characteristic of a-lactose monohydrate started to appear and increased in intensity within about 100 min (see Fig. 3B) after which time no further changes in the XRD patterns was observed. When the same material was treated in humid air at 55%, 66% or 76% RH for 144 h and when the sample was ground prior to the ex situ XRD analysis the same diffraction pattern was recorded, namely that of a-lactose monohydrate. Moisture uptake data for the lactose Na-caseinate material are presented in Fig. 1, Trace A, and shows that uptake of water reduces to a maximum of ca. 20% and relaxed to an equilibrium value of ca. 12%, far in excess of the 5% stoichiometric requirement for a-lactose monohydrate. Of interest also is the data as the a/(a+b) solid-state anomeric ratios in Table 1. All the lactose–caseinate mixtures exhibited high ratios after exposure to moist air. The situation is somewhat different for samples of lactose mixed with WPI (see Fig. 4). The predominant phase that appears is the a/b mixed phase. Smaller amounts of a-lactose monohydrate also appear at the same time, and all these phases seem to persist for the duration of the in situ experiment (6 h). Each of the XRD patterns correspond to almost pure a/b mixed isomer phase, with just small peaks associated with a-lactose monohydrate. We selected the peaks at 2y ¼ 18.31 and 22.11 as the characteristic peaks of the a/b mixed phase, because the stronger peaks associated with this phase at 2y ¼ 19.11 and 20.11 are difficult to observe when a-lactose monohydrate is also present in the sample. At longer exposure times in the ex situ experiments (144 h), the a/b mixed phase persists at the lowest RH value used (note the a/(a+b) solid-state anomeric ratio for this sample (Table 1) was 0.53). aLactose monohydrate was the predominant phase observed at extended exposure times at RH values of 66% and 76%.
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235
β-lactose (10.5)°2θ α-lactose monohydrate, (16.4)°2θ α/β mixed phase, (18.3)°2θ α/β mixed phase, (22.1)°2θ
β-lactose
420 min
Intensity (a.u.)
α-lactose monohydrate
Area
250 min
220 min α/β mixed phase
α/β mixed phase 125 min 80 min 0 min
10 (A)
15 Pos. [°2Th.]
20
0
50
100
150
200
250
300
Time (min)
(B)
Fig. 2. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose and (B) crystallization profile for spray-dried lactose exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
α-lactose monohydrate, (16.4)°2θ
α-lactose monohydrate
Area
Intensity (a.u.)
340 min
221 min
102 min 85 min 68 min 0 min
10 (A)
15 20 Pos. [°2Th.]
0
25 (B)
50
100
150 200 Time (min)
250
300
350
Fig. 3. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/Na-caseinate (3:1 w/w) and (B) crystallization profile for spray-dried lactose/ Na-Caseinate (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
Moisture uptake was similar to the case of lactose Nacaseinate. This situation contrasts with that observed with pure spray-dried lactose [4,5]. In that case the a/b mixed phase, a-lactose monohydrate and b-lactose phases all appeared simultaneously, but the a/b mixed phase only
persisted for about 1 h, whereas the other two phases appeared to be stable once they had formed. The crystallization behavior of lactose albumin mixtures is presented in Fig. 5. Again a-lactose monohydrate is the predominant phase, but peaks at 2y ¼ 12.21, 20.71 and
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α/β mixed phase
α/β mixed phase
α-lactose monohydrate, (16.4)°2θ α/β mixed phase, (18.3)°2θ α/β mixed phase, (22.1)°2θ
α-lactose monohydrate
Area
Intensity (a.u.)
408 min
136 min
119 min 102 min 85 min 0 min
10 (A)
15 20 Pos. [°2Th.]
25
0
50
100
(B)
150 200 Time (min)
250
300
350
Fig. 4. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/whey protein isolate (WPI) (3:1 w/w) and (B) crystallization profile for spraydried lactose/whey protein isolate (WPI) (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
21.81 (see Fig. 5C) appeared shortly after the onset of crystallization and had disappeared after 200 min. The phase associated with these peaks has not been identified. a-Lactose monohydrate was the only phase observed in samples of spray-dried lactose–albumin mixtures for longer exposure times possible in ex situ conditions. Moisture uptake for this sample is shown in Fig. 1 (Trace B), and the a/(a+b) solid-state anomeric ratio was close to 1. The behavior of lactose–gelatin mixtures is presented in Fig. 6. Again a-lactose monohydrate is the predominant phase observed using the in situ method, but the peaks at 2y ¼ 12.21, 20.71 and 21.81 persist for the duration of the in situ experiments. These peaks were absent from the XRD patterns recorded ex situ following longer exposure times at 55%, 66% and 76% RH values. Moisture uptake by all the lactose–protein mixtures was stronger than for pure lactose. Typically the maximum values recorded for the former were in the range 18–20 wt% with equilibrium values of 8–12 wt%. The corresponding values for pure lactose were 12% and 4 wt%. Fig. 7 (A and B) presents the SEM micrographs of the anhydrous pure spray-dried lactose and the lactose/Nacaseinate mixtures. Pure spray-dried lactose exhibits wellseparated, solid spherical particles, while those with the added Na-caseinate were spherical but featured hollow spaces inside the particles. All the lactose–protein mixtures studied in this work showed similar features. These features are consistent with the bulk density measurements presented in Table 2, which show a greatly diminished bulk density for the lactose–protein mixtures. Fig. 7(C and D)
shows the SEM micrographs of the final crystalline product of the moisture exposed pure spray-dried lactose and the lactose/Na-caseinate mixtures from the in situ experiments, generally after 6 h exposure to moist air. A notable feature of the spray-dried pure lactose was the appearance of platelet shaped particles of which exhibit crystalline habit [27,28]. This feature was absent from all the spray-dried lactose–protein mixtures following crystallization, as shown in Fig. 7D for lactose/Na-caseinate. 4. Discussions This discussion will focus on three aspects of the crystallization of spray-dried lactose and lactose–protein mixtures, namely (i) a comparison of the phase compositions observed after long exposure to moist air, (ii) the role of transient phases in lactose crystallization and (iii) a comparison of the crystalline habits observed with pure lactose and lactose–protein mixtures. In this study the b-anhydrous form of the crystalline lactose was observed only in the sample of pure spray-dried lactose. This phase was not observed with any of the lactose–protein mixtures. In pure spray-dried lactose, alactose monohydrate also appeared as a stable phase, which contrasts sharply with the a/b mixed phase which appears to be unstable in humid air (Fig. 2A and B). The sorption of water was less for the pure lactose than for the lactose–protein mixtures [4,5]. The lower water content observed with pure lactose imply higher super-saturation levels in this sample by comparison with the mixtures and this factor may favor the formation of the b-anhydrous
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237
α-lactose monohydrate, (16.4)°2θ 2θ = 12.2°
α-lactose monohydrate
(21.8)°2θ
2θ = 20.7° 2θ = 21.8°
Intensity (a.u.)
476 min
Area
136 min
119 min
85 min 68 min 0 min
10 (A)
15 20 Pos. [°2Th.]
0
25
50
100
150
200
250
300
350
Time (min)
(B)
2θ = 20.7°
2θ = 12.2°
Intensity (a.u.)
2θ = 21.8°
10 (C)
15 20 Pos. [°2Th.]
25
Fig. 5. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/albumin (3:1 w/w), (B) crystallization profile for spray-dried lactose/albumin (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa) and (C) X-ray diffraction pattern of spray-dried lactose/albumin (3:1 w/w) at 85 min.
phase. Another equally plausible explanation is that the protein molecules present in the mixtures may inhibit nucleation or growth of the b-anhydrous phase. The in situ technique developed for this study does illustrate the role of transient phases, namely the phases that appear and disappear on the time scale of the in situ experiments during the crystallization of lactose and lactose–protein mixtures (Figs. 2–6). The first point of
interest to emerge from these studies is that the crystallization in the condition used is rapid. An induction period of intermediate duration, during which moisture is sorbed is observed prior to the onset of crystallization, but the crystallization event is itself rapid. The main transient phases observed in this work is the a/b mixed phase. The temporal behavior of this phase varied widely according to the nature of the lactose sample.
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α-lactose β-lactose 2θ = 12.2° monohydrate
β-lactose (10.5)°2θ α-lactose monohydrate, (16.4)°2θ (21.8)°2θ
2θ = 20.7° 2θ = 21.8°
Intensity (a.u.)
442 min
Area
136 min
102 min
85 min 68 min 0 min
10 (A)
15 20 Pos. [°2Th.]
0
25 (B)
50
100
150
200
250
300
350
Time (min)
Fig. 6. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/gelatin (3:1 w/w) and (B) crystallization profile for spray-dried lactose/gelatin (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
Fig. 7. SEM micrographs of (A) anhydrous spray-dried lactose, (B) anhydrous spray-dried lactose/Na-caseinate (3:1 w/w), (C) spray-dried lactose exposed to water vapor at time ¼ 6 h and (D) spray-dried lactose/Na-caseinate (3:1 w/w) exposed to water vapor at time ¼ 6 h.
ARTICLE IN PRESS A. Shawqi Barham et al. / Journal of Crystal Growth 295 (2006) 231–240 Table 2 Bulk density of anhydrous spray-dried lactose and lactose/protein mixtures (3:1% w/w) Bulk density (g/cm3)
Phase Spray-dried Spray-dried Spray-dried Spray-dried Spray-dried
lactose lactose/WPI lactose/Na-Casinate lactose/Gelatin lactose/Albumin
0.8 0.3 0.2 0.3 0.4
No transient phases were observed for the lactose Nacaseinate mixture (Fig. 3A and B). With pure spray-dried lactose the a/b mixed phase appeared for about 1 h following the onset of crystallization; thereafter it decomposed. With the lactose–WPI mixture this was almost the only feature to appear in the in situ X-ray patterns (Fig. 4A and B). It was again the main feature in the ex situ work following exposure of the sample to 55% RH for up to 144 h, but had decomposed at 66% and 76% after 144 h (see Table 1). This phase is associated with the rapidity of the crystallization event coupled with the viscous nature of the lactose–protein–water mixtures present at the onset of crystallization. High viscosities imply restricted molecular motion, so that the a- and b-isomers present in the mixtures become trapped into a mixed phase by the rapid crystallization [9,27,28]. This phase appears to be inherently unstable, at least during exposure to moisture, and probably via a mechanism of dissolution and re-crystallization, transforms into a-lactose monohydrate in the case of lactose WPI and into a-lactose monohydrate and blactose anhydrous in the case of pure spray-dried lactose [18,23]. A second transient phase was observed in this work for lactose–albumin and lactose–gelatin samples (Figs. 5 and 6). This phase exhibit powder diffraction peaks at 2y ¼ 12.21, 20.71 and 21.81 but it has not been reported previously in connection with lactose crystallization (Fig. 5C). It appears as a minor component for both samples. It appears and disappears very rapidly in lactose–albumin but persists in lactose–gelatin for the duration (6 h) of the in situ experiment (Fig. 6A and B). However, it is not present at 55%, 66% or 76% values after 144 h. A final point of discussion concerns the appearance in scanning electron microscopy of the lactose and lactose–protein mixtures before and after exposure to moisture (see Fig. 7A–D). The principal feature to emerge is the platelet nature of the lactose crystallites and the absence of any characteristic crystalline particle shapes with the lactose– protein mixtures (see Fig. 7C, D). Previous studies have been indicated that the a-lactose monohydrate exhibits a ‘‘tomahawk’’ particle habit when crystallized from an aqueous solution of the a- and b-isomers [6,8,11,18,23]. In the absence of the b-isomers in solution, platelet shaped particles is formed [18]. This observation has been explained on the basis that the b-isomer inhibits growth
239
of the ð0 1¯ 1Þ facet of a-lactose monohydrate crystals so favoring the formation of the tomahawk shaped crystals [18]. In this work platelet shaped crystals were observed with spray-dried lactose. Again, the highly viscous nature of the lactose water mixture at the point of the crystallization means that molecular motion is restricted and bisomer molecules may not be free to move and attach themselves to the ð0 1¯ 1Þ facet of the developing a-lactose monohydrate crystals, so allowing the development of platelet shapes [10,18]. The absence of clear crystallization features in the SEM images of the lactose–proteins mixtures, even after 144 h exposure to moist air may be associated with indiscriminate attachment of protein to all facets of the a-lactose monohydrate crystals. The large amounts of protein present (25%) may simply have the effect of covering the lactose crystals and hiding the underling morphology. Acknowledgments The authors acknowledge financial support from PRTL1 Cycle3 of the Higher Education Authority of Ireland. References [1] B.R. Bhandari, T. Howes, J. Food Eng. 40 (1999) 71. [2] P. Faldt, B. Bergenstahl, Lebensm.-Wiss. u.-Technol. 29 (1996) 438. [3] A. Gombas, I. Antal, P. Szabo-Revesz, S. Marton, I. Eros, Int. J. Pharm. 256 (2003) 25. [4] M.K. Haque, Y.H. Roos, J. Food Sci. 69 (2004) 23. [5] M.K. Haque, Y.H. Roos, J. Food Sci. 69 (2004) 384. [6] X.M. Zeng, G.P. Martin, C. Marriott, J. Pritchard, J. Pharm. Pharmacol. 52 (2000) 633. [7] R. Price, P.M. Young, J. Pharm. Sci. 93 (2004) 155. [8] M.F. Mazzobre, G. Soto, J.M. Aguilera, M.P. Buera, Food Res. Int. 34 (2001) 903. [9] M.F. Mazzobre, J.M. Aguilera, M.P. Buera, Carbohydr. Res. 338 (2003) 541. [10] K. Jouppila, J. Kansikas, Y.H. Roos, J. Dairy Sci. 80 (1997) 3152. [11] T.J. Buma, S. Henstra, Neth. Milk Dairy J. 25 (1971) 75. [12] T.D. Dincer, G.M. Parkinson, A.L. Rohl, M.I. Ogden, J. Crystal Growth 205 (1999) 368. [13] T.J. Buma, G.A. Wiegers, Neth. Milk Dairy J. 21 (1967) 208. [14] D.C. Fries, S.T. Rao, M. Sundaralingam, Acta Crystallogr. B 27 (1971) 994. [15] K. Hirotsu, A. Shimada, Bull. Chem. Soc. Japan 47 (8) (1974) 1872. [16] C. Plattea, J. Lefebvre, F. Affouard, P. Derollez, Acta Crystallogr. B 60 (2004) 453. [17] C. Platteau, J. Lefebvre, F. Affouard, J.F. Willart, P. Derollez, F. Mallet, Acta Crystallogr. B 61 (2005) 185. [18] T.D. Simpson, F.W. Parrish, M.L. Nelson, J. Food Sci. 47 (1982) 1948. [19] J.H. Bushill, W.B. Wright, C.H.F. Fuller, A.V. Bell, J. Sci. Food Agric. 16 (1965) 622. [20] N. Drapier-Beche, J. Fanni, M. Parmentier, J. Dairy Sci. 81 (1998) 2826. [21] J. Lefebvre, J.F. Willart, V. Caron, R. Lefort, F. Affouard, F. Danede, Acta Crystallogr. B 61 (2005) 455. [22] W.L. Earl, F.W. Parrish, Carbohydr. Res. 115 (1983) 23. [23] S.L. Raghavan, R.I. Ristic, D.B. Sheen, J.N. Sherwood, J. Phys. Chem. B 104 (2000) 12256. [24] S.L. Raghavan, R.I. Ristic, D.B. Sheen, J.N. Sherwood, J. Pharm. Sci. 90 (7) (2001) 823.
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A. Shawqi Barham et al. / Journal of Crystal Growth 295 (2006) 231–240 S. Garnier, S. Petit, G. Coquerel, J. Crystal Growth 234 (2002) 207. A. Olano, N. Corzo, I. Castro, Milchwissenschaft 38 (8) (1983) 471. A.S. Barham, B.K. Hodnett, Cryst. Growth Des. 5 (2005) 1965. A.S. Barham, A.M.E. Omar, Y.H. Roos, B.K. Hodnett, Cryst. Growth Des. 6 (2006) 1975.
[29] T.P. Labuza, A. Kaanane, J.Y. Chen, J. Food Sci. 50 (1985) 385. [30] D.R. Lide (Ed.), CRC Handbook of Chemistry and Physics, 73rd ed., CRC Press, Boca Raton, FL, 1992. [31] S.K. Dwivedi, A.G. Mitchell, J. Pharm. Sci. 78 (1989) 1055.
Journal of Crystal Growth 295 (2006) 231–240 www.elsevier.com/locate/jcrysgro
Crystallization of spray-dried lactose/protein mixtures in humid air A. Shawqi Barhama,, Md. Kamrul Haqueb, Yrjo¨ H. Roosb, B. Kieran Hodnetta a
Materials and Surface Science Institute, University of Limerick, Limerick, Ireland Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland
b
Received 30 November 2005; received in revised form 24 July 2006; accepted 9 August 2006 Communicated by M. Schieber
Abstract An in situ crystallization technique with X-ray diffraction analysis complemented by ex situ scanning electron microscopy and chromatographic analysis of the a/(a+b) solid-state anomeric ratios has been developed to study the crystallization of lactose/protein mixtures in humid air. This technique was used to determine changes in phase composition and morphology during crystallization. Following an induction period during which water is sorbed, crystallization is rapid and the predominant phase observed using the in situ method in spray-dried lactose/sodium-caseinate, albumin and gelatin is a-lactose monohydrate. However, in the case of spray-dried lactose/whey protein isolate (WPI) the predominant phase that appears is the a/b mixed phase with smaller amounts of a-lactose monohydrate. With pure lactose the a/b mixed phase appears as a transient shortly after the onset of crystallization and a-lactose monohydrate and b-lactose both appear as stable crystalline phases at longer times. Another transient phase with 2y ¼ 12.21, 20.71 and 21.81 was observed in spray-dried lactose/albumin. This phase decomposed as a-lactose monohydrate developed. Three phases seem to persist in the case of spray-dried lactose/gelatin, namely the phase with peaks at 2y ¼ 12.21, 20.71 and 21.81, a-lactose monohydrate and b-lactose for the duration of the in situ experiment. r 2006 Elsevier B.V. All rights reserved. PACS: 64.70.Dv; 60.70.Kb; 87.14.Ee Keywords: A1. Crystallization; A1. SEM; A1. X-ray diffraction; A1. Relative vapor pressure; B1. Proteins; B1. Lactose
1. Introduction Lactose–protein mixtures are commonly used in foods and can be prepared by a variety of methods including spray-drying and freeze-drying [1–5]. Following preparation, these mixtures are amorphous and form free flowing powders, which is a desired property for handling purposes [6,7]. The physical state of lactose–protein mixtures is important for the stabilization of food products during processing and storage. However, during processing and storage, amorphous lactose mixtures may undergo timedependent changes with increasing rate at increasing temperatures and water contents. These mixtures and pure lactose itself are very hygroscopic and readily sorb moisture from the ambient air [8]. Within 1 h of exposure Corresponding author. Tel.: +353 612324159; fax: +353 61213529.
E-mail address: [email protected] (A. Shawqi Barham). 0022-0248/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcrysgro.2006.08.006
to moist air lactose can sorb up to 10% by mass of water and the material becomes sticky, loosing its free flowing properties [4,5,9]. The glass transition temperature (Tg) of lactose becomes lower as the moisture content increases. Typical values for Tg of dry lactose have been reported of ca. 105 1C but this value decreases to ca. 5 1C when the moisture content is close to 10 wt% [4,5,10,11]. The material then coagulates, crystallizes and becomes very hard and difficult to grind into a fine powder [4,5]. Lactose, milk sugar, is a disaccharides consisting of two moieties of D-glucose and D-galactose joined by a b-1, 4-glycosidic linkage. There are two isomeric forms of lactose a- and b-lactose, which differ in the configuration of the terminal hydroxyl group of the glucose moiety [12]. However, several crystalline forms of lactose are known, such as, a-lactose monohydrate [13,14], b-lactose [15], hygroscopic anhydrous a-lactose [16], stable anhydrous a-lactose [17] and mixed compounds of the two anomers
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of a- and b-lactose. The mixed lactose compounds, namely, 5:3 [18,19], 4:1 [18], 3:2 [20] and 1:1 a:b phases [21], appear to be discrete crystalline phases rather than physical mixtures of crystals of pure a- and b-lactose [22]. In terms of crystallization from aqueous solution, the alactose monohydrate phase is the most extensively studied [23,24]. Most studies have been carried out in conditions where the a- and b-isomers are both present at ratios determined by the equilibrium mutarotation of the isomers [23–25]. This means a-lactose monohydrate has a propensity to form as a pure crystalline phase even in the presence of large amounts of the b-isomer [6,26]. Literature data seem to indicate that there is very little incorporation of the b-isomer into the a-lactose monohydrate phase [24–26]. However, the presence of the b-isomer does influence the crystalline habit of a-lactose monohydrate [12]. When the b-isomer is absent, such as when dimethyl sulfoxide (DMSO) is used as solvent (suppressing mutarotation), needle or platelet shaped particles of a-lactose monohydrate form. When the b-isomer is present, growth of the ð0 1¯ 1Þ facets of a-lactose monohydrate is inhibited and the well-known tomahawk shaped crystalline habits prevails [12]. Previous in situ X-ray diffraction (XRD) studies of the crystallization of spray and freeze-dried lactose have demonstrated the validity of this method for the observation, in particular, of phases which only appear at short times after the onset of crystallization, before the eventual formation of the final stable composition at longer times [27,28]. In particular, for both spray and freeze-dried lactose, the stable phase present at extended crystallization times are a-lactose monohydrate and b-lactose. However, a mixed isomer crystalline phase, previously reported as a a/ b mixed phase was observed shortly after the onset of crystallization but decomposed at longer times. A complementary SEM analysis showed that the crystalline material continuously evolved until platelet shaped particles eventually predominated at long crystallization times [27,28]. This paper describes a study of the crystallization of spray-dried lactose–protein mixtures (3:1 w/w) in moist air followed by in situ X-ray powder diffraction analysis, with complementary water sorption data, ex situ X-ray powder diffraction, scanning electron microscopy, and gas liquid chromatography. The proteins studied here were sodiumcaseinate, whey protein isolate (WPI), albumin, and gelatin. 2. Experimental procedure 2.1. Materials a-Lactose (Dairy Gold, Ireland), WPI (Protarmor 865) (Armor Proteines, 35460 Saint Brice En Cogles, France), sodium-caseinate (Dairy Gold, Ireland), egg albumin (Sanovo Egg Products Limited, UK) and gelatin (Croda Colloids Limited, UK). Molecular sieves (calcium, sodium
1 in pellets, nominal pore diameter 5 A˚), alumino-silicate, 16 phosphorus pentoxide anhydride (P2O5, 98%), potassium chloride (KCl, 99%) and sodium chloride (NaCl, 99.5%), sodium nitrite (NaNO2, 99.5%) were purchased from Sigma-Aldrich Ltd., Ireland. Dry pyridine (99.8%), dimethyl sulfoxide (DMSO, 99.7%), N-trimethylsilylimidazole (TMSIM, 98.0%) were purchased from SigmaAldrich, Ireland, and were used to derivatize lactose for GLC analysis. Aqueous solutions (15% w/w) of a-lactose, a-lactose/ WPI (3:1 w/w), a-lactose/Na-caseinate (3:1 w/w), a-lactose/ albumin (3:1 w/w) and a-lactose/gelatin (3:1) were prepared and spray-dried using a Niro Atomizer spray-drier (Copenhagen, Denmark) with an inlet temperature of 185–190 1C and outlet temperature of 80–85 1C. To enhance solubility, Na-caseinate solution was made slightly alkaline (pH 7.5) using sodium hydroxide (BDH Chemicals Ltd., Poole, England). The residual water content was removed by keeping samples in vacuum desiccators over P2O5 at room temperature (22–23 1C) for at least 5 days. The powdered samples were collected, immediately, packaged into glass containers, and desiccated over P2O5 [0% relative humidity (RH) at 25 1C]. Before experiments, residual moisture was removed by drying the lactose samples in a vacuum oven at 50 1C for 24 h [4,5].
2.2. Ex situ crystallization of spray-dried lactose and lactose/protein (3:1 w/w) mixtures Anhydrous spray-dried lactose and lactose/protein (3:1 w/w) mixtures in vials were stored in evacuated desiccators over saturated salt solutions at room temperature (22–23 1C) for 144 h. The salts used were Mg(NO3)2, NaNO2 and NaCl for 55%, 66% or 76% RH, respectively, giving a water activity (aw) of 0.01 % RH at equilibrium [29]. 2.3. In situ crystallization of spray-dried lactose and lactose/protein (3:1 w/w) mixtures In situ X-ray powder diffraction was performed using a Philips X’pert PRO MPD PW3040/60 X-ray diffractometer with nickel filtered copper Cu Ka radiation (l ¼ 1.542 A˚) as the X-ray source. The Cu Ka diffractometer anode was run under a tension of 40 kV and a current of 35 mA. Sample preparation consisted of grinding the sample to a powder (o45 mm) and loading it into a Macor sample holder cup (Anton Paar, Austria) approximately 17.5 mm in diameter by 2 mm deep and applying a smooth surface finish. The sample holder cup was loaded onto the rotating sample stage and placed inside a HTK1200 Oven Camera (Anton Paar, Austria). The temperature in the in situ sample chamber was independently controlled by a temperature control unit TCU1000 (Anton Paar Gmbh, Austria) which controls the wire heater of the sample and the inner wall of the chamber. Gas of a specified humidity was formed by
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passing dry nitrogen (N2) into 1 L cylindrical steel vessel filled with zeolite (molecular sieves, Sigma-Aldrich Ltd., Ireland), which was saturated with water. The temperature of the saturator (wet N2) was controlled using a Clifton (Nickel Electro LTD, England) water bath. The gas flow rate of the dry N2 was set at 100 mL min1 using a mass flow meter (Brooks, Instrument B.V., Holland). By variation of the temperature of the water bath (saturator) and for a set temperature of 25 1C in the in situ XRD chamber (HTK 1200), different RH values were generated. The temperature of the saturator and the in situ XRD chamber were 22 and 25 1C, respectively. The vapor pressure of water at 22 and 25 1C was 2.9 and 3.3 kPa, respectively, giving a RH of 88% [30]. The sample height inside the HTK1200 was accurately set using the X-ray beam (the sample was aligned when the maximum beam intensity at 2y ¼ 01 was halved by the sample). The sample was scanned over a range of 2y ¼ 5401 using a step size of 0.01671 2y and a scan speed of 0.03561 2y/s. The sample was rotated at 10 Hz during analysis. Scans were recorded every 17 min during in situ studies. 2.4. Electron microscopy The electron micrographs were obtained using a JEOL JSM-5600 electron microscope. A small amount of ungrounded material was scattered evenly onto the surface of an aluminum stub covered with a 12 mm diameter carbon tab. Excess material was removed using dry compressed air, and the sample was sputter coated with a thin conductive film of gold for approximately 2 min in an Edwards S150B sputter coater. The accelerating voltage used was 20 keV.
3. Results Water sorption data for spray-dried lactose are presented in Fig. 1, Trace E. These data, recorded at a RH value of 76%, clearly show the immediate uptake of moisture by lactose followed by its expulsion to an equilibrium value of ca. 4 wt%. The stoichiometric requirement for a-lactose monohydrate is 5 wt%. As crystallization proceeds there is a tendency to expel excess moisture. Table 1 summarizes the phases observed from pure spray-dried lactose and spray-dried lactose–protein mixtures treated in humid air at 55%, 66% or 76% RH for 144 h. The persistent crystalline phases, namely the phases present after prolonged exposure (144 h) to moist air (55%, 66% or 76% RH) observed with spray-dried lactose were a-lactose monohydrate and the anhydrous b-lactose. For spray-dried lactose-WPI (3:1 w/w) exposed to humid air at 55% RH, the predominant phase was the pure a/b mixed phase. At 66% RH, the predominant phases were mixtures of a/b mixed phase and a-lactose monohydrate for the same material. This situation is somewhat different for all lactose samples (3:1 w/w) mixed with gelatin, Na-caseinate or albumin at 55–76% RH. The predominant phase that appears was only the a-lactose monohydrate. Table 1 also presents the a/(a+b) solid-state anomeric ratios in the solid state for spray-dried lactose–protein mixtures (3:1 w/w). Values very close to 0.50 were recorded for all the anhydrous samples. For spray-dried lactose this value increased to about 0.65 after exposure to moist air. This ratio only changed slightly for spray-dried lactose/ WPI upon exposure to moist air ca. 0.53 at 55% RH. For
Spray-dried lactose Spray-dried lactose/Whey Protein Isolate(WPI)(3:1w/w) Spray-dried lactose/Na-Casinate(3:1w/w) Spray-dried lactose/Albumin(3:1w/w) Spray-dried lactose/Gelatin(3:1w/w)
20 Water Content (g/100g dry solids)
2.5. Gas liquid chromatography The instrument used was a Hewlett-Packard 4890A gas chromatograph equipped with a split injector and a flame ionization detector (FID) operating at 275 1C [31]. The column was a nonpolar capillary Rtx-5, 15 m 0.25 mm i.d. with 0.1 mm film thickness (Restek, UK). Oven temperature of the GC was 215 1C. Carrier gas was nitrogen at a flow rate of 1.0 mL min1 and 35 kPa. For routine GLC analysis, dissolution and derivatization must be rapid, and there must be negligible mutarotation. Therefore, we derivatized the dry samples with a mixture consisting of 19.5% DMSO, 22% N-trimethylsilylimidazole (TMSIM), and 58.5% pyridine by volume (mixture A) [31]. Approximately 1 mg of dry lactose–protein samples was dissolved in 1 mL of mixture A. The solutions were kept for 60 min at 60 1C before the derivatization was complete. The injection volume was 1 mL, and the run time used was 22 min. The lactose components eluted after 14 and 19 min for a-lactose and b-lactose, respectively.
233
16
12 (A) (B) (C) 8
(D)
4
(E)
0 0
5
10
15 Time (h)
20
25
Fig. 1. Water sorption for (A) spray-dried lactose/Na-caseinate (3:1 w/w), (B) spray-dried lactose/albumin (3:1 w/w), (C) spray-dried lactose/gelatin (3:1 w/w), (D) spray-dried lactose/whey protein isolate (WPI) (3:1 w/w) and (E) spray-dried lactose (76%RH) [4,5].
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Table 1 Summary of all phases observed following exposure to moist air for 144 h, glass transition temperatures and the solid-state anomeric composition of lactose–protein mixtures determined by GC analysisa Anhydrous
L–SD L3–WPI L3–CAS L3–GEL L3–ALB
RH 55%
RH 66%
RH 76%
XRD phases
Tg1 (1C)
a/(a+b)
XRD phases
a/(a+b)
XRD phases
a/(a+b)
XRD phases
a/(a+b)
Amorphous Amorphous Amorphous Amorphous Amorphous
105 111 103 113 108
0.45 0.47 0.48 0.47 0.50
a+b a/b a a a
0.63 0.53 0.85 0.98 0.94
a+b a+a/b a a a
0.66 0.79 0.86 0.63 0.88
a+b a a a a
0.65 0.93 0.94 0.88 0.96
a
Lx–protein ¼ lactose–protein at molar ratios of x:1. RH X% ¼ relative humidity at X%. Tg1 ¼ onset of glass transition (1C) [4]. a/(a+b) ¼ anomeric composition of lactose determined by GLC. a ¼ a-lactose monohydrate. b ¼ b-lactose anhydrous. a/b ¼ a/b mixed phase.
the lactose–protein samples the a/(a+b) solid-state anomeric ratio increased to close to 1.00 for those samples with the greatest propensity to form a-lactose monohydrate, consistent with a mechanism whereby the a:b ratio in the lactose–protein moisture mixtures was adjusting to its equilibrium value as more and more a-lactose monohydrate was crystallizing. Fig. 2 presents the in situ XRD patterns of spray-dried lactose exposed to water vapor for up to 420 min at 25 1C (RH ¼ 88%). The starting material contained small amounts of b-lactose (note the small peak at 2y ¼ 10.51), but these seemed to disappear and the sample became X-ray amorphous after 80 min. After 100 min, the features of b-lactose started to appear and were very strong at 110–125 min (note peaks at 2y ¼ 10.51 (1 1 0) and 211 (1 1 1)). At 110 min, peaks of other phases of lactose also started to appear including a-lactose monohydrate (2y ¼ 12.51 and 16.41), but the three main characteristic peaks of this phase (2y ¼ 19.11, 19.61 and 19.91) have not developed fully and the principal peak present in this pattern is at 20.11. There are additional small peaks at 2y ¼ 18.31 and 22.11. These features are characteristic of the a/b mixed phase [18]. The crystal structure of this phase has recently been determined [21]. It is made up of dimers of a-lactose and b-lactose. Earlier studied have labelled this phase of the 5:3 phase corresponding to its solid state a:b anomeric ratios [18]. Below this phase will be described as the a/b mixed phase [28]. The other principal peaks of this phase, namely at 12.41, 19.11 and 20.11 are all obscured by the principal peaks of a-lactose monohydrate. Fig. 2B presents the temporal development of the a-lactose monohydrate, b-lactose and the a/b mixed phase. Clearly, the a/b mixed phase is unstable and decomposes at intermediate crystallization times, whereas the a-lactose monohydrate and b-lactose phases persist for the duration of the experiment. Interestingly, for spray-dried lactose the a/(a+b) solid-state anomeric ratio shown in Table 1, always show values close to 0.5, corresponding to the crystalline composition of this material with approximately equal amount of a-lactose monohydrate and b-lactose. In another study of lactose–salt mixtures this phase was also observed. It formed at the onset of crystallization and disappeared as a-lactose monohydrate and b-lactose
formed [28]. In that study the formation of the a/b mixed phase was associated with restricted molecular mobility in the highly viscous lactose–protein–water mixtures, forming the mixed anomeric phase in preference to a-lactose monohydrate or b-lactose [28]. Fig. 3A presents the XRD patterns recorded using the in situ method for lactose Na-caseinate mixtures that were exposed to humid air for the times indicated. Following an induction period of 70 min during which strong moisture uptake occurred, peaks characteristic of a-lactose monohydrate started to appear and increased in intensity within about 100 min (see Fig. 3B) after which time no further changes in the XRD patterns was observed. When the same material was treated in humid air at 55%, 66% or 76% RH for 144 h and when the sample was ground prior to the ex situ XRD analysis the same diffraction pattern was recorded, namely that of a-lactose monohydrate. Moisture uptake data for the lactose Na-caseinate material are presented in Fig. 1, Trace A, and shows that uptake of water reduces to a maximum of ca. 20% and relaxed to an equilibrium value of ca. 12%, far in excess of the 5% stoichiometric requirement for a-lactose monohydrate. Of interest also is the data as the a/(a+b) solid-state anomeric ratios in Table 1. All the lactose–caseinate mixtures exhibited high ratios after exposure to moist air. The situation is somewhat different for samples of lactose mixed with WPI (see Fig. 4). The predominant phase that appears is the a/b mixed phase. Smaller amounts of a-lactose monohydrate also appear at the same time, and all these phases seem to persist for the duration of the in situ experiment (6 h). Each of the XRD patterns correspond to almost pure a/b mixed isomer phase, with just small peaks associated with a-lactose monohydrate. We selected the peaks at 2y ¼ 18.31 and 22.11 as the characteristic peaks of the a/b mixed phase, because the stronger peaks associated with this phase at 2y ¼ 19.11 and 20.11 are difficult to observe when a-lactose monohydrate is also present in the sample. At longer exposure times in the ex situ experiments (144 h), the a/b mixed phase persists at the lowest RH value used (note the a/(a+b) solid-state anomeric ratio for this sample (Table 1) was 0.53). aLactose monohydrate was the predominant phase observed at extended exposure times at RH values of 66% and 76%.
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235
β-lactose (10.5)°2θ α-lactose monohydrate, (16.4)°2θ α/β mixed phase, (18.3)°2θ α/β mixed phase, (22.1)°2θ
β-lactose
420 min
Intensity (a.u.)
α-lactose monohydrate
Area
250 min
220 min α/β mixed phase
α/β mixed phase 125 min 80 min 0 min
10 (A)
15 Pos. [°2Th.]
20
0
50
100
150
200
250
300
Time (min)
(B)
Fig. 2. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose and (B) crystallization profile for spray-dried lactose exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
α-lactose monohydrate, (16.4)°2θ
α-lactose monohydrate
Area
Intensity (a.u.)
340 min
221 min
102 min 85 min 68 min 0 min
10 (A)
15 20 Pos. [°2Th.]
0
25 (B)
50
100
150 200 Time (min)
250
300
350
Fig. 3. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/Na-caseinate (3:1 w/w) and (B) crystallization profile for spray-dried lactose/ Na-Caseinate (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
Moisture uptake was similar to the case of lactose Nacaseinate. This situation contrasts with that observed with pure spray-dried lactose [4,5]. In that case the a/b mixed phase, a-lactose monohydrate and b-lactose phases all appeared simultaneously, but the a/b mixed phase only
persisted for about 1 h, whereas the other two phases appeared to be stable once they had formed. The crystallization behavior of lactose albumin mixtures is presented in Fig. 5. Again a-lactose monohydrate is the predominant phase, but peaks at 2y ¼ 12.21, 20.71 and
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236
α/β mixed phase
α/β mixed phase
α-lactose monohydrate, (16.4)°2θ α/β mixed phase, (18.3)°2θ α/β mixed phase, (22.1)°2θ
α-lactose monohydrate
Area
Intensity (a.u.)
408 min
136 min
119 min 102 min 85 min 0 min
10 (A)
15 20 Pos. [°2Th.]
25
0
50
100
(B)
150 200 Time (min)
250
300
350
Fig. 4. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/whey protein isolate (WPI) (3:1 w/w) and (B) crystallization profile for spraydried lactose/whey protein isolate (WPI) (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
21.81 (see Fig. 5C) appeared shortly after the onset of crystallization and had disappeared after 200 min. The phase associated with these peaks has not been identified. a-Lactose monohydrate was the only phase observed in samples of spray-dried lactose–albumin mixtures for longer exposure times possible in ex situ conditions. Moisture uptake for this sample is shown in Fig. 1 (Trace B), and the a/(a+b) solid-state anomeric ratio was close to 1. The behavior of lactose–gelatin mixtures is presented in Fig. 6. Again a-lactose monohydrate is the predominant phase observed using the in situ method, but the peaks at 2y ¼ 12.21, 20.71 and 21.81 persist for the duration of the in situ experiments. These peaks were absent from the XRD patterns recorded ex situ following longer exposure times at 55%, 66% and 76% RH values. Moisture uptake by all the lactose–protein mixtures was stronger than for pure lactose. Typically the maximum values recorded for the former were in the range 18–20 wt% with equilibrium values of 8–12 wt%. The corresponding values for pure lactose were 12% and 4 wt%. Fig. 7 (A and B) presents the SEM micrographs of the anhydrous pure spray-dried lactose and the lactose/Nacaseinate mixtures. Pure spray-dried lactose exhibits wellseparated, solid spherical particles, while those with the added Na-caseinate were spherical but featured hollow spaces inside the particles. All the lactose–protein mixtures studied in this work showed similar features. These features are consistent with the bulk density measurements presented in Table 2, which show a greatly diminished bulk density for the lactose–protein mixtures. Fig. 7(C and D)
shows the SEM micrographs of the final crystalline product of the moisture exposed pure spray-dried lactose and the lactose/Na-caseinate mixtures from the in situ experiments, generally after 6 h exposure to moist air. A notable feature of the spray-dried pure lactose was the appearance of platelet shaped particles of which exhibit crystalline habit [27,28]. This feature was absent from all the spray-dried lactose–protein mixtures following crystallization, as shown in Fig. 7D for lactose/Na-caseinate. 4. Discussions This discussion will focus on three aspects of the crystallization of spray-dried lactose and lactose–protein mixtures, namely (i) a comparison of the phase compositions observed after long exposure to moist air, (ii) the role of transient phases in lactose crystallization and (iii) a comparison of the crystalline habits observed with pure lactose and lactose–protein mixtures. In this study the b-anhydrous form of the crystalline lactose was observed only in the sample of pure spray-dried lactose. This phase was not observed with any of the lactose–protein mixtures. In pure spray-dried lactose, alactose monohydrate also appeared as a stable phase, which contrasts sharply with the a/b mixed phase which appears to be unstable in humid air (Fig. 2A and B). The sorption of water was less for the pure lactose than for the lactose–protein mixtures [4,5]. The lower water content observed with pure lactose imply higher super-saturation levels in this sample by comparison with the mixtures and this factor may favor the formation of the b-anhydrous
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α-lactose monohydrate, (16.4)°2θ 2θ = 12.2°
α-lactose monohydrate
(21.8)°2θ
2θ = 20.7° 2θ = 21.8°
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Time (min)
(B)
2θ = 20.7°
2θ = 12.2°
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Fig. 5. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/albumin (3:1 w/w), (B) crystallization profile for spray-dried lactose/albumin (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa) and (C) X-ray diffraction pattern of spray-dried lactose/albumin (3:1 w/w) at 85 min.
phase. Another equally plausible explanation is that the protein molecules present in the mixtures may inhibit nucleation or growth of the b-anhydrous phase. The in situ technique developed for this study does illustrate the role of transient phases, namely the phases that appear and disappear on the time scale of the in situ experiments during the crystallization of lactose and lactose–protein mixtures (Figs. 2–6). The first point of
interest to emerge from these studies is that the crystallization in the condition used is rapid. An induction period of intermediate duration, during which moisture is sorbed is observed prior to the onset of crystallization, but the crystallization event is itself rapid. The main transient phases observed in this work is the a/b mixed phase. The temporal behavior of this phase varied widely according to the nature of the lactose sample.
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α-lactose β-lactose 2θ = 12.2° monohydrate
β-lactose (10.5)°2θ α-lactose monohydrate, (16.4)°2θ (21.8)°2θ
2θ = 20.7° 2θ = 21.8°
Intensity (a.u.)
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10 (A)
15 20 Pos. [°2Th.]
0
25 (B)
50
100
150
200
250
300
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Time (min)
Fig. 6. (A) Typical in situ X-ray diffraction patterns of spray-dried lactose/gelatin (3:1 w/w) and (B) crystallization profile for spray-dried lactose/gelatin (3:1 w/w) exposed to water vapor (T ¼ 25 1C; RH ¼ 88%; pH2O ¼ 3.3 kPa).
Fig. 7. SEM micrographs of (A) anhydrous spray-dried lactose, (B) anhydrous spray-dried lactose/Na-caseinate (3:1 w/w), (C) spray-dried lactose exposed to water vapor at time ¼ 6 h and (D) spray-dried lactose/Na-caseinate (3:1 w/w) exposed to water vapor at time ¼ 6 h.
ARTICLE IN PRESS A. Shawqi Barham et al. / Journal of Crystal Growth 295 (2006) 231–240 Table 2 Bulk density of anhydrous spray-dried lactose and lactose/protein mixtures (3:1% w/w) Bulk density (g/cm3)
Phase Spray-dried Spray-dried Spray-dried Spray-dried Spray-dried
lactose lactose/WPI lactose/Na-Casinate lactose/Gelatin lactose/Albumin
0.8 0.3 0.2 0.3 0.4
No transient phases were observed for the lactose Nacaseinate mixture (Fig. 3A and B). With pure spray-dried lactose the a/b mixed phase appeared for about 1 h following the onset of crystallization; thereafter it decomposed. With the lactose–WPI mixture this was almost the only feature to appear in the in situ X-ray patterns (Fig. 4A and B). It was again the main feature in the ex situ work following exposure of the sample to 55% RH for up to 144 h, but had decomposed at 66% and 76% after 144 h (see Table 1). This phase is associated with the rapidity of the crystallization event coupled with the viscous nature of the lactose–protein–water mixtures present at the onset of crystallization. High viscosities imply restricted molecular motion, so that the a- and b-isomers present in the mixtures become trapped into a mixed phase by the rapid crystallization [9,27,28]. This phase appears to be inherently unstable, at least during exposure to moisture, and probably via a mechanism of dissolution and re-crystallization, transforms into a-lactose monohydrate in the case of lactose WPI and into a-lactose monohydrate and blactose anhydrous in the case of pure spray-dried lactose [18,23]. A second transient phase was observed in this work for lactose–albumin and lactose–gelatin samples (Figs. 5 and 6). This phase exhibit powder diffraction peaks at 2y ¼ 12.21, 20.71 and 21.81 but it has not been reported previously in connection with lactose crystallization (Fig. 5C). It appears as a minor component for both samples. It appears and disappears very rapidly in lactose–albumin but persists in lactose–gelatin for the duration (6 h) of the in situ experiment (Fig. 6A and B). However, it is not present at 55%, 66% or 76% values after 144 h. A final point of discussion concerns the appearance in scanning electron microscopy of the lactose and lactose–protein mixtures before and after exposure to moisture (see Fig. 7A–D). The principal feature to emerge is the platelet nature of the lactose crystallites and the absence of any characteristic crystalline particle shapes with the lactose– protein mixtures (see Fig. 7C, D). Previous studies have been indicated that the a-lactose monohydrate exhibits a ‘‘tomahawk’’ particle habit when crystallized from an aqueous solution of the a- and b-isomers [6,8,11,18,23]. In the absence of the b-isomers in solution, platelet shaped particles is formed [18]. This observation has been explained on the basis that the b-isomer inhibits growth
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of the ð0 1¯ 1Þ facet of a-lactose monohydrate crystals so favoring the formation of the tomahawk shaped crystals [18]. In this work platelet shaped crystals were observed with spray-dried lactose. Again, the highly viscous nature of the lactose water mixture at the point of the crystallization means that molecular motion is restricted and bisomer molecules may not be free to move and attach themselves to the ð0 1¯ 1Þ facet of the developing a-lactose monohydrate crystals, so allowing the development of platelet shapes [10,18]. The absence of clear crystallization features in the SEM images of the lactose–proteins mixtures, even after 144 h exposure to moist air may be associated with indiscriminate attachment of protein to all facets of the a-lactose monohydrate crystals. The large amounts of protein present (25%) may simply have the effect of covering the lactose crystals and hiding the underling morphology. Acknowledgments The authors acknowledge financial support from PRTL1 Cycle3 of the Higher Education Authority of Ireland. References [1] B.R. Bhandari, T. Howes, J. Food Eng. 40 (1999) 71. [2] P. Faldt, B. Bergenstahl, Lebensm.-Wiss. u.-Technol. 29 (1996) 438. [3] A. Gombas, I. Antal, P. Szabo-Revesz, S. Marton, I. Eros, Int. J. Pharm. 256 (2003) 25. [4] M.K. Haque, Y.H. Roos, J. Food Sci. 69 (2004) 23. [5] M.K. Haque, Y.H. Roos, J. Food Sci. 69 (2004) 384. [6] X.M. Zeng, G.P. Martin, C. Marriott, J. Pritchard, J. Pharm. Pharmacol. 52 (2000) 633. [7] R. Price, P.M. Young, J. Pharm. Sci. 93 (2004) 155. [8] M.F. Mazzobre, G. Soto, J.M. Aguilera, M.P. Buera, Food Res. Int. 34 (2001) 903. [9] M.F. Mazzobre, J.M. Aguilera, M.P. Buera, Carbohydr. Res. 338 (2003) 541. [10] K. Jouppila, J. Kansikas, Y.H. Roos, J. Dairy Sci. 80 (1997) 3152. [11] T.J. Buma, S. Henstra, Neth. Milk Dairy J. 25 (1971) 75. [12] T.D. Dincer, G.M. Parkinson, A.L. Rohl, M.I. Ogden, J. Crystal Growth 205 (1999) 368. [13] T.J. Buma, G.A. Wiegers, Neth. Milk Dairy J. 21 (1967) 208. [14] D.C. Fries, S.T. Rao, M. Sundaralingam, Acta Crystallogr. B 27 (1971) 994. [15] K. Hirotsu, A. Shimada, Bull. Chem. Soc. Japan 47 (8) (1974) 1872. [16] C. Plattea, J. Lefebvre, F. Affouard, P. Derollez, Acta Crystallogr. B 60 (2004) 453. [17] C. Platteau, J. Lefebvre, F. Affouard, J.F. Willart, P. Derollez, F. Mallet, Acta Crystallogr. B 61 (2005) 185. [18] T.D. Simpson, F.W. Parrish, M.L. Nelson, J. Food Sci. 47 (1982) 1948. [19] J.H. Bushill, W.B. Wright, C.H.F. Fuller, A.V. Bell, J. Sci. Food Agric. 16 (1965) 622. [20] N. Drapier-Beche, J. Fanni, M. Parmentier, J. Dairy Sci. 81 (1998) 2826. [21] J. Lefebvre, J.F. Willart, V. Caron, R. Lefort, F. Affouard, F. Danede, Acta Crystallogr. B 61 (2005) 455. [22] W.L. Earl, F.W. Parrish, Carbohydr. Res. 115 (1983) 23. [23] S.L. Raghavan, R.I. Ristic, D.B. Sheen, J.N. Sherwood, J. Phys. Chem. B 104 (2000) 12256. [24] S.L. Raghavan, R.I. Ristic, D.B. Sheen, J.N. Sherwood, J. Pharm. Sci. 90 (7) (2001) 823.
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