Yunping Yao,
Guozhong Zhao,
Yuanyuan Yan,
Chen Chen,
Cong Sun,
Xiaoqiang Zou*,
Qingzhe Jin and
Xingguo Wang*
State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu Province, P. R. China. E-mail: wxg1002@qq.com; xiaoqiangzou@163.com; Fax: +86-510-85876799; Tel: +86-510-85876799
First published on 23rd December 2015
Freeze drying and spray drying are conventional methods for converting milk into powder in the dairy industry. The purpose of this study was to investigate the effects of these drying methods on the physical, chemical and structural features of milk fat globules (MFGs). The globule sizes increased after freeze drying and spray drying, but the integrity of their microstructures was still maintained. Compared with that in untreated MFGs, the composition of the fatty acids underwent fewer changes under the two different drying conditions. The main phospholipids showed some differences between the freeze-dried milk and spray-dried milk, and the saturation of the fatty acids of phospholipids was increased after the drying treatments. A reduction in the cholesterol content was observed after the freeze-drying and spray-drying processes. The level of unsaturation decreased as the diameter of the globules increased. The results indicated that freeze drying had a significant effect on the liquid-ordered domains in the MFG membrane, which was formed by sphingolipids and cholesterol. However, spray drying influenced the liquid-disordered domains, which were formed by glycerophospholipids. Thus, the drying method appears to affect the physical, chemical and microstructure characteristics of MFGs, which may affect the stability of the globules in milk and the way the milk is digested.
Applications of freeze drying or spray drying to convert milk into powder without changing its nutritional and sensory characteristics to extend the shelf-life of milk have been documented.2–4 Freeze drying, which is time consuming and expensive, is an important drying process for milk to conserve its flavour, bioactivities and other properties.5 However, the spray-drying process only requires a very short time.6 Both the freeze-drying and spray-drying methods can enhance the apparent solubility of milk concentrate powders.
The influence of the drying methods on the surface properties and morphological characteristics of milk fat globules (MFGs) has been reported. However, previous studies emphasised the changes in size, shape and surface proteins of milk powders produced by spray drying.7–11 The choice of drying methods affects some physical and chemical characteristics of milk. Both freeze-dried and spray-dried milk powders have essentially the same flavour characteristics. However, freeze-dried milk powder frequently acquires a fruity flavour that is not apparent in spray-dried milk.12 Freeze-dried and spray-dried MFG membranes (MFGM) are morphologically different when examined under light microscopy. The freeze-dried MFGM are irregular flaky translucent sheets with sharp edges, whereas the spray-dried MFGM are spherical particles.13 Drying methods also increase the degree of protein denaturation and surface fat coverage.14 It had been proved that adverse effects are reduced when using lower temperature than is conventionally used in spray drying.15 The drying methods also can alter the thermodynamic state of the phospholipids in MFGM.13 To our knowledge, there are few studies of the composition and changes in the microstructure of freeze-dried and spray-dried MFGM. Moreover, the chemical fingerprint of individual MFGs of different sizes from freeze drying and spray drying are also poorly described.
In the present study, the microstructure and lipid composition of freeze-dried and spray-dried milk were measured and compared. The differences in composition of the MFGs according to their sizes obtained with these methods were also reported. Our research will provide insight into the influence of the different methods of drying on MFGs.
Fatty acid methyl esters were measured on an Agilent 7820 GC (Agilent Corp., USA) equipped with a flame ionisation detector and a capillary column TRACE TR-FAME (60 m × 0.25 mm × 0.25 μm, Thermo Fisher, USA). The oven temperature was programmed as follows: 60 °C for 3 min, then raised to 175 °C at 5 °C min−1 and held for 15 min, and finally raised to 220 °C at 2 °C min−1 and held for 10 min. The injector and detector temperatures were 230 °C and 250 °C, respectively.
Size parameters | Raw milk | Freeze-dried milk | Spray-dried milk |
---|---|---|---|
a D3,2, average size of surface area; D4,3, average size of volume. | |||
D3,2 (μm) | 3.31 ± 0.11 | 8.67 ± 0.23 | 6.95 ± 0.41 |
D4,3 (μm) | 3.70 ± 0.31 | 5.02 ± 0.34 | 4.50 ± 0.33 |
Zeta-potentials (mV) | −9.44 ± 0.66 | −9.72 ± 0.43 | −10.35 ± 0.72 |
Surface tension (mN m−1) | 41.207 ± 0.023 | 41.993 ± 0.030 | 35.414 ± 0.029 |
![]() | ||
Fig. 1 Particle size distribution of the milk fat globules at different drying methods determined by laser light scattering. |
The size distribution in RM was characterised by a main peak at 3.89 μm, which was similar to previous results.23 This corresponded to D3,2 and D4,3 values of 3.31 ± 0.11 and 3.70 ± 0.31 μm, respectively. The shapes of the size distributions changed markedly with the treatments of freeze drying and spray drying, with significant decreases in the main peak at 3.89–5.04 μm. Particle size distribution curves were polydispersed and multimodal in FDM samples, with 3 peaks at 3.89, 11.99 and 16.96 μm, whereas the samples of SDM had bimodal size distributions at 5.04 and 13.08 μm. High values of D3,2 and D4,3 were characteristic of FDM and SDM that exhibited peaks that corresponded to high size values in the particle size distribution curves. These changes were commonly attributed to the formation of a small amount of MFGs bigger than those of the control. The size increases after heat treatment were presumably due to coalescence of the globules and increasing protein denaturation.24 The coalescence might be related to altered protein–lipid interactions in the membrane. Freezing was found to be very important in affecting the particle size. Two explanations for the growth of globule size during the freezing process have been proposed: (1) the ice crystal formation repelling foreign material away from the interstitials causes the aggregation of globules; and (2) the osmotic de-swelling of the globules causes them to recombine into larger globules.25 The drying processes caused a range of structural and physicochemical modifications, which in turn influenced the reconstitution and absorption of milk proteins and caused the increase in MFG sizes.
The zeta potential was thought to be particularly suitable to estimate the degree of MFG surface coverage by plasma proteins as an indicator of the degree of damage.26 The apparent zeta potential calculated for RM of −9.44 ± 0.66 mV was not significantly different from that of −9.72 ± 0.43 mV for FDM. However, the zeta potential of SDM (−10.35 ± 0.72 mV) was significantly higher than that of RM and FDM. The increase of about 9.6% in the absolute value of the apparent zeta potential may be related to the adsorption of casein micelles and whey proteins to the MFGM.27 This means that possible damage to MFGs during the spray-drying process may result in variation of the interface of the fat globules. Particularly, the heat treatment process can alter the composition of the membrane that may be accompanied by changes in the surface charge of the fat globules.
Surface tension can also be used to evaluate the degree of damage of MFGM. The values of surface tension of RM, FDM and SDM were measured and can be compared in Table 1. The values obtained in RM and FDM were not significantly different, whereas the value in SDM was lower than that in RM, which indicated that some surface-active materials were lost from the MFGM. Phospholipids, which have a polar head group and two hydrophobic hydrocarbon tails, are the most abundant lipids of MFGM. During the spray-drying process, the high temperature and high shear stress may cause the phospholipids to become a kind of surfactant that reduces the surface tension.
The emission fluorescence of Rh-DOPE in the form of red rings at the periphery of the MFGs was still detected after both the cold- and heat-drying methods (Fig. 2B2 and C2), indicating that the MFGM still maintained their structural integrity after processing. Despite the fact that no significant changes or disruption was found in the MFGM of the FDM and SDM samples, some small damage was still present in the globules. As observed in Fig. 2B2, the surfaces of some MFGs after freeze drying became thicker than those of RM. A possible explanation may be that the amphiphilic phospholipids tend to accumulate on the surface and then the fat globules coalesce with each other during the freeze-drying process.
Fig. 2C2 shows that some MFGs aggregated rather than evenly dispersed throughout an aqueous phase. At the same time, the results also revealed that freeze-dried milk powder dissolved in water showed better stability than spray-dried milk powder. One reason may be that with the changes that occur in the physical and chemical properties of MFGM with rapid water removal during spray drying, the powder particles are likely to join together due to changes in their surface properties.
Fatty acid (%) | Raw milk | Freeze-dried milk | Spray-dried milk |
---|---|---|---|
a SFA: saturated fatty acids; MUFA: total monounsaturated fatty acids; PUFA: total polyunsaturated fatty acids. Values are means ± standard. Calculations were based on 5 samples with three replicate measurement per milk sample.b The contents of fatty acids in freeze-dried milk and spray-dried milk were compared with the control (raw milk) separately. Means with different superscript letters are significantly different (*P < 0.05, **P < 0.01). | |||
C4:0 | 1.00 ± 0.04 | 1.49 ± 0.03** | 1.44 ± 0.06** |
C8:0 | 1.00 ± 0.03 | 1.16 ± 0.04** | 1.09 ± 0.03* |
C10:0 | 2.86 ± 0.09 | 3.10 ± 0.08* | 3.00 ± 0.07 |
C11:0 | 0.08 ± 0.01 | 0.08 ± 0.01 | 0.10 ± 0.02 |
C12:0 | 3.88 ± 0.09 | 4.00 ± 0.08 | 3.99 ± 0.07 |
C13:0 | 0.14 ± 0.02 | 0.15 ± 0.02 | 0.15 ± 0.04 |
C14:0 | 12.89 ± 0.23 | 12.77 ± 0.14 | 12.78 ± 0.15 |
C14:1 | 1.44 ± 0.08 | 1.45 ± 0.05 | 1.46 ± 0.05 |
C15:0 | 1.45 ± 0.13 | 1.43 ± 0.03 | 1.44 ± 0.06 |
C15:1 | 0.02 ± 0.00 | 0.02 ± 0.00 | 0.02 ± 0.00 |
C16:0 | 35.65 ± 0.24 | 35.09 ± 0.16 | 35.11 ± 0.11 |
C16:1 | 1.95 ± 0.10 | 1.82 ± 0.04 | 1.81 ± 0.05 |
C17:0 | 0.79 ± 0.07 | 0.78 ± 0.04 | 0.79 ± 0.04 |
C17:1 | 0.30 ± 0.02 | 0.29 ± 0.03 | 0.30 ± 0.04 |
C18:0 | 11.71 ± 0.11 | 11.67 ± 0.09 | 11.60 ± 0.13 |
C18:1t | 0.42 ± 0.05 | 0.43 ± 0.03 | 0.46 ± 0.04 |
C18:1 | 23.17 ± 0.15 | 23.06 ± 0.19 | 23.24 ± 0.13 |
C18:2t | 0.27 ± 0.02 | 0.36 ± 0.02** | 0.28 ± 0.03 |
C18:3n-6 | 0.06 ± 0.01 | 0.05 ± 0.01 | 0.07 ± 0.01 |
C18:3n-3 | 0.30 ± 0.03 | 0.30 ± 0.03 | 0.33 ± 0.02 |
C20:0 | 0.22 ± 0.02 | 0.20 ± 0.02 | 0.21 ± 0.01 |
C21:0 | 0.04 ± 0.01 | 0.04 ± 0.01 | 0.05 ± 0.01 |
C20:3n-6 | 0.15 ± 0.01 | 0.14 ± 0.03 | 0.15 ± 0.02 |
C22:1 | 0.01 ± 0.00 | 0.01 ± 0.00 | 0.02 ± 0.00 |
C20:5n-3(EPA) | 0.14 ± 0.02 | 0.05 ± 0.01** | 0.06 ± 0.01** |
C23:0 | 0.05 ± 0.01 | 0.04 ± 0.01 | 0.05 ± 0.01 |
C22:2 | 0.01 ± 0.00 | 0.01 ± 0.00 | 0.00 ± 0.00 |
SFA | 71.75 ± 0.87 | 72.00 ± 0.76 | 71.80 ± 0.81 |
MUFA | 27.31 ± 0.40 | 27.08 ± 0.34 | 27.31 ± 0.31 |
PUFA | 0.94 ± 0.09 | 0.91 ± 0.10 | 0.89 ± 0.09 |
The compositional data reported in Table 3 provided evidence that the drying methods affected the phospholipids. The proportion of phospholipids in FDM and SDM were significantly changed. We observed decreases in SM (from 34% to 29%) and PS (from 12% to 7%) in FDM (P < 0.01). However, the changes in SDM were different from those in FDM. Decreases were observed in the proportion of PC (from 29% to 26%) and PS (from 12% to 9%) in SDM (P < 0.01). SM in SDM pointed to high stability, whereas the relative distribution of PE and PI were increased.
Phospholipids (%) | Raw milk | Freeze-dried milk | Spray-dried milk |
---|---|---|---|
a PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; SM, sphingomyelin. Values are means ± standard. Calculations were based on 5 samples with three replicate measurement per milk sample. The contents of phospholipids in freeze-dried milk and spray-dried milk were compared with the control (raw milk) separately. Means with different superscript letters are significantly different (*P < 0.05, **P < 0.01). | |||
PE | 18.09 ± 1.65 | 26.63 ± 2.21** | 22.92 ± 1.55** |
PI | 5.76 ± 0.46 | 6.04 ± 0.37* | 6.73 ± 0.42** |
PS | 12.29 ± 0.79 | 7.23 ± 0.51** | 9.77 ± 0.73** |
PC | 29.72 ± 1.42 | 30.62 ± 1.27** | 26.59 ± 1.72** |
SM | 34.15 ± 1.38 | 29.48 ± 1.66** | 33.99 ± 1.84 |
The changes in the compositions of phospholipids seemed to be attributable to the locations of the phospholipids in the membrane. Native MFGs are enveloped by tri-layer membranes, with the inner layer originating from the endoplasmic reticulum and the outer bilayer originating from regions of the apical plasma membrane of mammary epithelial cells. PC and SM are present predominantly in the outer leaflet. PE and PI reside mainly in the inner leaflet, whereas PS is located almost exclusively in the inner leaflet of the plasma membrane.29 Therefore, PC and SM located in the outer leaflet of the membrane are more susceptible to treatment. According to the recent structural model of MFGM, the liquid-disordered phase is composed of the glycerophospholipids (PE, PC, PI and PS), whereas the liquid-ordered phase domain is composed of SM.30 In light of our results, we suspect that the liquid-disordered phase is more influenced by high temperature, but the liquid-ordered phase is more sensitive to low temperature. Whether the reported decreases in the respective contents of SM and PC in FDM and SDM are valid will require further investigation.
Fatty acid | Raw milk (%) | Freeze-dried milk (%) | Spray-dried milk (%) |
---|---|---|---|
a SFA: saturated fatty acids; MUFA: total monounsaturated fatty acids; PUFA: total polyunsaturated fatty acids. Values are means ± standard. Calculations were based on 5 samples with three replicate measurement per milk sample.b The contents of fatty acids of phospholipids in freeze-dried milk and spray-dried milk were compared with the control (raw milk) separately. Means with different superscript letters are significantly different (*P < 0.05, **P < 0.01). | |||
C4:0 | 0.14 ± 0.02 | 0.47 ± 0.04** | 0.54 ± 0.04** |
C6:0 | 0.16 ± 0.01 | 0.22 ± 0.02** | 0.57 ± 0.05** |
C8:0 | 0.19 ± 0.04 | 0.18 ± 0.02 | 0.66 ± 0.04** |
C10:0 | 0.59 ± 0.06 | 1.84 ± 0.07** | 1.22 ± 0.04** |
C11:0 | 0.11 ± 0.02 | 0.37 ± 0.03** | 0.26 ± 0.02** |
C12:0 | 1.19 ± 0.04 | 1.60 ± 0.03** | 1.76 ± 0.11** |
C13:0 | 0.16 ± 0.02 | 0.46 ± 0.06 | 0.39 ± 0.04 |
C14:0 | 6.87 ± 0.08 | 3.51 ± 0.10** | 5.66 ± 0.02** |
C14:1 | 0.47 ± 0.02 | 0.38 ± 0.02** | 0.59 ± 0.11** |
C15:0 | 1.12 ± 0.04 | 0.53 ± 0.03** | 1.14 ± 0.04 |
C15:1 | 0.20 ± 0.01 | 0.00 ± 0.00** | 0.21 ± 0.01 |
C16:0 | 27.71 ± 0.12 | 31.78 ± 0.16** | 28.73 ± 0.13** |
C16:1 | 1.80 ± 0.04 | 1.10 ± 0.20** | 1.43 ± 0.05** |
C17:0 | 0.82 ± 0.03 | 0.51 ± 0.04** | 0.91 ± 0.06 |
C17:1 | 0.30 ± 0.03 | 0.19 ± 0.02** | 0.50 ± 0.06** |
C18:0 | 16.40 ± 0.19 | 21.02 ± 0.22** | 18.45 ± 0.12** |
C18:1 | 27.48 ± 0.23 | 16.52 ± 0.19** | 20.91 ± 0.59** |
C18:2 t | 0.38 ± 0.04 | 0.54 ± 0.04** | 4.62 ± 0.44** |
C18:2 | 9.81 ± 0.12 | 11.26 ± 0.24** | 8.84 ± 0.14** |
C18:3n-6 | 0.10 ± 0.02 | 0.02 ± 0.00** | 0.11 ± 0.01 |
C18:3n-3 | 0.64 ± 0.03 | 0.86 ± 0.09** | 0.62 ± 0.08 |
C20:0 | 0.61 ± 0.03 | 0.77 ± 0.07** | 0.48 ± 0.06** |
C21:0 | 0.15 ± 0.02 | 0.26 ± 0.03** | 0.13 ± 0.02 |
C20:3n-6 | 0.65 ± 0.04 | 1.32 ± 0.02** | 0.56 ± 0.04 |
C22:1 | 0.27 ± 0.03 | 1.80 ± 0.11** | 0.26 ± 0.03 |
C20:5n-3EPA | 0.64 ± 0.03 | 0.00 ± 0.00** | 0.00 ± 0.00** |
C23:0 | 0.98 ± 0.10 | 2.54 ± 0.06** | 0.48 ± 0.04** |
C22:2 | 0.09 ± 0.01 | 0.00 ± 0.00** | 0.00 ± 0.00** |
SFA | 57.18 ± 0.82 | 66.03 ± 0.98 | 61.36 ± 0.87 |
MUFA | 30.51 ± 0.36 | 19.98 ± 0.54 | 23.89 ± 0.78 |
PUFA | 12.31 ± 0.29 | 13.99 ± 0.39 | 14.75 ± 0.71 |
Regarding the SFA composition of the phospholipids, C16:0 and C18:0 were significantly increased, whereas the UFA of C16:1 and C18:1 were significantly decreased in FDM and SDM. According to the data reported in the literature, SM was found to contain high amounts of long-chain fatty acids, with C16:0 being the major fatty acid (≥25%). C16:0 and C18:1 were the main fatty acids in PC, and a high amount of UFA existed as PE (C18:1 ≥ 50%).31,32 Therefore, the significant differences of fatty acids of phospholipids were related to the changes in the phospholipids profile during the drying processing. The increased SFA in FDM and SDM might be due to the respective decreases of PC and SM. Drying treatments may lead the PC, SM and PE to convert to phosphatidic acids, which are easy to extract.
Squalene is a precursor of cholesterol, and it may be viewed as a more flexible structure in the membrane than in the rigid cholesterol molecule. The function of squalene in the membrane is not yet clear, but it appears to have some roles in stabilising the membrane structure. The content of squalene increased from 9.1 mg/100 g in RM to 28.3 mg/100 g in FDM and to 12.3 mg/100 g in SDM under the effects of drying. The reason for the increase of squalene in FDM and SDM was unclear. This observed increase illustrates that modification of the structure of the MFGM during the drying processes resulted in increasing solubility in the extraction solvents.
The Raman spectra of MFGs in RM, FDM and SDM were different according to the size of the globules. As shown in Fig. 4 and 5, where visual differences in the peak heights of the spectra were noted, the relevant literature was consulted for information about the nature of these peaks. The prominent peaks in the Raman spectra were found at 1654 cm−1 (ν(CC) cis double bond stretching of RHC
CHR), 1443 cm−1 (δ(C–H) scissoring of –CH2), 1269 cm−1 (δ(C–H) bending at the cis double bond in R–HC
CH–R), 1303 cm−1 (C–H twisting of the –CH2 group) and 1742 cm−1 (ν(C
O) stretching of RC
OOR).36
![]() | ||
Fig. 5 Qualitative evaluation of the unsaturation level and the liquid/crystal fat ratio of RM, FDM and SDM fat globules with different sizes. |
The peak at 860 cm−1, which is quite broad (from 810 to 897 cm−1) (Fig. 4), was due to the contributions from the complex mixture of the polar head groups from the phospholipids.37 The intensity of the band at 860 cm−1 was decreased after the freeze-drying and spray-drying processes. According to the model proposed by Gallier et al.,35 the two ratios I1654/I1742 and I1654/I1443 are indicative of the degree of unsaturation of the samples. The band at 1269 cm−1 is a last indicator of the unsaturation level. In Fig. 4, the relative intensity of the band at 1269 cm−1 was weak compared with that of the band at 1303 cm−1, which was characteristic of the low degree of unsaturation. The Raman spectra of MFGs in RM, FDM and SDM showed decreased levels of unsaturation as the globule size increased (Fig. 5). This could be explained by the content of the triglyceride core increasing with the increasing size of the MFGs. The changing trends in the degree of unsaturation were similar under the freeze-drying and spray-drying processes for the small and medium MFGs, whereas the degree of unsaturation demonstrated a decreasing trend for MFGs with larger diameters.
Three intense bands near 1010, 1160 and 1530 cm−1 corresponded to the aromatic compounds of carotenoids.38 This carotenoid band appeared very strong for the small, medium and large MFGs in RM. However, the FDM and SDM presented very weak bands. This suggests the concentration of carotenoids was decreased by both the high- and low-temperature processing.
The Raman spectra in the region around 2600–3200 cm−1 contain information about the mobility and structure of the hydrocarbon chains of lipids. The C–C mobility of the hydrocarbon chains indicated by the intensities of the bands at 1065, 1080 and 1130 cm−1 and the increased intensity of the band at 2850 cm−1 relative to the band at 2885 cm−1 are indicative of higher mobility of the hydrocarbon chains. The change in the intensities of these bands after the freeze-drying and spray-drying processes indicated that the fatty acid composition of globules of different sizes varied during the drying processes. Therefore, MFGs of different sizes may have different melting points and could be at different states at the same temperature.
This journal is © The Royal Society of Chemistry 2016 |