Microstructural evolution of whipped cream in whipping process observed by confocal laser scanning microscopy
It is useful to carefully observe the evolution of foam structures to elucidate the factors affecting cream during whipping. In this study, confocal laser scanning microscopy and a double dyeing technology were used to investigate the microstructural evolution of a rigid foam structure in whipped cream. The location of fat and proteins were determined according to the signals they produce at different characteristic wavelengths. Protein membranes on the surface of air bubbles were clearly observed. A simple yet comprehensive characterization of the whipping process was established according to the micrographs and supported by relevant theories. The formation of a rigid foam structure depends on foaming of the protein in the plasma phase and partial coalescence of fat globules. The formation of protein foam in the cream, creation of net structure, and system breakage and collapse phenomena occurring throughout the whole whipping evolution process was depicted and distinguished visually by different colors.
Introduction
Like all food products, whipped cream must be acceptable to the consumer both physically and organoleptically. High-quality whipped cream generally has fat content around 30–40% and is easy to whip into rigid foam with high overrun value (over 80–90%).[1] Accurately and comprehensively assessing the factors that affect the foam structure is necessary to improve the stability and whipping characteristics of whipped cream. It is useful to observe the structure evolution of whipped cream for this purpose – especially the locations of fat globules and proteins and the air bubble microstructures.
Factors at work during the whipping process and interfacial changes occurring in cream during whipping have been observed by previous researchers via light microscopy, scanning electron microscopy, and transmission electron microscopy, among other microscopy techniques.[2–7] The whipping process is often described as having three stages.[1,8,9] At the first stage, the overrun increases rapidly and large air bubbles are introduced. This initial foam is stabilized by protein. At the second stage, air bubbles gradually decrease in diameter and the extent of fat globule aggregation increases. In the third stage, a thin protein membrane on the surface of air bubbles is penetrated by fat globules and sparsely distributed fat crystals which settle in the plane of the air–plasma interface.[10] A partially coalesced framework of fat globules is built up at this point that gives stiffness to the whipped cream structure.
Previously published descriptions of the whipping process were dependent on electron microscopy observation and theoretical considerations, and lacked detailed analysis of the changes occurring throughout the whole process.[8,9,11] Large magnification is also typically used, and the micrographs used to analyze the aggregation and distribution of fat globules at the interface show only a bubble surface or connected area among several bubbles. Changes in the fat globules in the plasma phase at the primary stage are rarely investigated, and previous researchers have been unable to identify the location of proteins (especially in regards to the protein membrane on the surface of air bubbles).
We applied confocal laser scanning microscopy (CLSM) in this study, which has multiple channels available for detecting the fluorescence signals of a given sample. We also used a dual staining technique to make accurate distinctions between fat and proteins in whipped cream samples. Nile red (NR) has been extensively used for staining the fat phase in bulk palm oil systems and cream.[12,13] Fluorescein isothiocyanate (FITC) can also be applied to label proteins non-covalently.[14] Protein and fat produce signals at different wavelengths, which can be marked in different colors.
The purpose of this article was to provide micrographs detailing the complete whipping process with different magnifications and to provide morphological evidence supporting theoretical considerations of the whipping process. We gathered detailed data by measuring the overrun, firmness, and particle size distribution of whipped cream during the whole whipping process. The mechanisms at work behind changes in foam structure were analyzed to provide a simple, yet comprehensive characterization of the whipping process. Protein membranes on the surface of air bubbles were clearly observed in the micrographs, which supports our theoretical analysis that the surface consists of a protein membrane encompassing fat globules.
Materials and methods
Microstructure
Sample preparation: Commercially available UHT whipped cream (Anchor, New Zealand) with a composition of 35% milk fat was purchased from a local supermarket. Its ingredients include cream, milk solid (skimmed milk), emulsifiers (Glycerin Monostearate and Tween 80), and stabilizer (guar gum, xanthan gum and carrageenan). After storage at 4°C for 24 h, 200 g of whipped cream sample was poured into a beaker. Before whipping, 2.0 mL of 0.02% (w/t) NR (Solarbio, China) and FITC (Solarbio, China) were added to the samples to stain the fat and proteins, respectively.[12,15] Whipping was performed at 4°Cwith a kitchen mixer (AHM-P125A ACA, China) at two speed levels. Low speed (950 rpm) applied for 30 s allow for maximum air inclusion, then the mixer was switched to high speed (1100 rpm); the cream was whipped for varying durations (0, 15, 30, 60, 90, 120, 130, 160, 200, and 320 s) for comparison. After whipping, the stained samples were immediately placed on a slide for observation.
CLSM: A Leica inverted microscope (TCS SP2 CLSM, Leica Microsystems, Heidelberg, Germany) equipped with a 10× objective and 64 × 1.4 or 100 × 1.4 oil-immersion objective was applied to acquire images. The fluorescence in samples was excited by the 488 nm (20 mV) line of an Argon laser. The fluorescence light emitted by NR and FITC was detected at 595–648 nm and 500–536 nm, respectively.[12]
Particle size distribution observation
After being stored at 4°C for 24 h, the cream was whipped at 4°C for various durations (0, 15, 30, 60, 90, 120, 130, 160, and 200 s). The size distribution of the fat clumps was determined on a Malvern MasterSizer 3000 (Malvern Instruments Co., Ltd., Worcestershire, UK). The refractive index, dispersed phase adsorption, and continuous phase refractive index were set to 1.52, 0.1, and 1.33, respectively.[16,17]
Overrun measurement
After being stored at 4°C for 24 h, the cream was whipped at 4°C for various durations (0, 15, 30, 60, 90, 120, 130, 160, and 200 s). Overrun is a common indicator that represents the amount of air introduced in cream and can be determined by using the following equation[18–21]:
where Munwhipped and Mwhipped are the mass of unwhipped and whipped cream with the same volume, respectively. Each sample was measured in triplicate.
Determination of firmness
After being stored at 4°C for 24 h, the cream was whipped at 4°C for various durations (60, 90, 120, 130, 160, and 200 s). The 15 and 30 s durations were left out of this test as the fluidity of samples was too large to determine the firmness. Firmness was determined using a Texture Analyzer (TA-XT2i, Lloyd Instruments, UK) equipped with an acrylic probe (HDP/FE3).[19,20,22] Measurement was performed at the rate of 1 mm s−1 over a distance of 30 mm in the sample. The trigger value for the start of the measurement was set to 5 g. Tests were carried out in triplicate.
Statistical analysis
Statistical analysis (one-way analysis of variance) was conducted in SPSS 23. The difference between means was determined by Bonferroni’s test and significance was defined at P < 0.05.
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