A Linkam microscopy stage has been used to analyse a potential carrier for probiotic bacteria in a low saturated fat ice cream formulation. Duncan Stacey, Sales and Marketing Director for Linkam Scientific Instruments, explains
Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.1 They have been promoted for their role in maintaining or improving digestive health and are also thought to deliver a “health halo,” potentially helping consumers to improve their general well-being.
Consumer awareness of these possible health benefits has led to a huge increase in demand for probiotic-enriched food and beverage products. The global probiotics market was valued at US$42.55 Billion in 2017 and is projected to reach $74.69bn by the end of 2025 at a CAGR of 7.3%.2
For nutritional food and beverage manufacturers, tapping into this burgeoning market is not always easy, particularly in the European Union (EU), where health claims are tightly regulated and there are no approved health claims for probiotics.
Probiotic bacteria are susceptible to damage from processing, manufacturing and storage procedures, as well as from the acidic environment of the stomach
However, there is a wide body of research that points to the potential benefits of specific species of probiotic bacteria, although the positive effects can vary depending on environmental factors and on each individual’s intestinal microflora.
For these so-called friendly bacteria to have a positive impact, a sufficient amount of live probiotic bacteria needs to be delivered to the small or large intestine (the most common site of action). Probiotic bacteria are susceptible to damage from processing, manufacturing and storage procedures, as well as from the acidic environment of the stomach.
So, not only are the specific health benefits of probiotics sometimes in question, it can also be difficult to ensure that enough of the probiotic bacteria are delivered live to the site of action to have any positive effect at all.
Duncan Stacey, Sales and Marketing Director, Linkam Scientific Instruments
Because of these factors, significant research efforts are being put into designing probiotic delivery systems that are able to protect probiotic bacteria against the adverse conditions they face during food production, handling and following consumption.
One possible delivery option is via monoglyceride-structured emulsions (MSEs), which have recently shown promising potential. In one study, the inclusion of a probiotic Lactobacillus rhamnosus strain in MSEs containing skim milk, sunflower oil or anhydrous milk fats, and saturated monoglycerides as structurants, resulted in a high cell viability during storage at 4 °C for up to two months.3
Following this finding, a group from Udine University in Italy recently investigated the potential of saturated monoglyceride structured emulsions as a delivery system for probiotics in ice cream.4 Here, we summarise key findings from their research and explain how the Linkam Optical Shearing System CSS450 played a key part in the study.
Ice cream is a complex food colloid that contains fat globules, air bubbles and ice crystals dispersed in a freeze-concentrated dispersion/solution of proteins, salts, polysaccharides and sugars. Because of its multiphase composition and low-temperature storage, ice cream has been proposed as a potential carrier for probiotic bacteria.
The high level of total solids in ice cream (including fat and milk solids) may protect the probiotic bacteria. However, the freezing process can damage the cell membrane of the probiotic bacteria, leading to injuries that compromise cell function and metabolic activity.5 Thus, strategies have to be developed to protect probiotic bacteria during ice cream processing and storage.
The high level of total solids in ice cream (including fat and milk solids) may protect the probiotic bacteria
The challenge of this study was to demonstrate the effectiveness of MSEs to deliver and protect probiotic bacteria in artisanal ice cream. MSEs containing a probiotic Lactobacillus rhamnosus strain were prepared using anhydrous milk fat (AMF-MSE) or sunflower oil (Oil-MSE) as the lipid phase and were added to the ice cream mix as a substitute for milk cream just before freezing.
Results highlighted the good capacity of MSEs to protect probiotic bacteria cells against the stresses suffered during processing and storage.
The microstructure of ice cream was characterised using a temperature-controlled optical shear stage (Linkam CSS450, Linkam Scientific Instruments, UK). One drop of ice cream was placed in the middle of a glass slide and a glass coverslip, previously cooled at –7 °C, was centred above the drop between a stationary and moving glass plate (also cooled at –7 °C).
The sample was then observed using a polarised light optical microscope (Leica DM 2000, Leica Microsystems, Heerburg, Switzerland) connected with a Leica EC3 digital camera.
Monoglyceride crystalline structures formed in the MSE played both a probiotic protective and structuring role
The Linkam Optical Shearing System (CSS450) allows the structural dynamics of complex fluids to be directly observed via a standard optical microscope while they are under precisely controlled temperature conditions and various shear modes.
Using the optical shearing cell, it is possible to mimic rheological test procedures, such as cyclical or oscillatory shearing, shear strain or shear relaxation, to study and image the microstructural evolution of complex fluids in great detail for many physical processes.
Figure 1: An oil–water pre-emulsion with a lauryl sulphate surfactant taken with a stationary CSS450 stage using an optical microscope with a 10x polarised objective
The amount of air and the size and distribution of air bubbles can affect ice cream’s physical properties. Figure 1 shows how polarised microscopy highlights the surfactant, giving the cells a halo effect.
Air cells in each image were subdivided into three classes, depending on the maximum length of the longest line joining two points of the cell’s outline and passing through the centroid (average size): class 1 consisted of 0–20 μm air cells, class 2 consisted of 20–40 μm air cells and class 3 consisted of air cells larger than 40 μm. The percentage ratio between the number of air cells belonging to each class and the total number of air cells in the image was calculated.
Oil-MSE ice cream exhibited the lowest number of air cells that were uniformly distributed. By contrast, AMF-MSE ice cream displayed the highest number of air cells … but they were non-uniformly distributed and closely packed, leading to a structure similar to that of a foam.
Finally, the control sample showed air bubbles with a distribution size that enabled comparisons to be made between the ice cream prepared with MSE containing oil and that containing anhydrous milk fat. The researchers concluded that the presence of crystalline monoglycerides in oil-MSE containing samples probably allowed partial fat coalescence during whipping and freezing, leading to air bubble stabilisation that resulted in a higher overrun.6
The meltdown stability of ice cream is another quality parameter that is affected by the lipid phase structure.7 In this study, the control and the Oil-MSE ice creams showed similar meltdown profiles, whereas the sample containing AMF-MSE had the lowest meltdown resistance.
Based on observations of optical microscopic images, it is likely that the high overrun value together with the very close distribution of air cells may have increased the melting rate of AMF-MSE ice cream.
The oil-MSE structured emulsion demonstrated the ability to create a melt-resistant fat network structure in ice cream when milk fat was replaced with sunflower oil. It was concluded that the monoglyceride crystalline structures formed in the MSE played both a probiotic protective and structuring role.
The use of MSE containing sunflower oil makes it possible to not only to successfully protect probiotic bacteria, but also to formulate a low saturated fat ice cream — which could deliver added appeal for health-conscious consumers.
The use of the CSS450 Optical Shearing System from Linkam enabled the researchers to capture high definition images of the microstructure of the ice cream samples at specific temperatures to evaluate how each ingredient combination would affect the quality of the end product.