Confocal scanning laser microscopy and quantitative image analysis: application to cream cheese microstructure investigation

The naked eye observation of cream cheese confocal scanning laser microscopy images only provides qualitative information about its microstructure. Because those products are dense dairy gels, confocal scanning laser microscopy images of 2 different cream cheeses may appear close. Quantitative image analysis is then necessary to compensate for human eye deficiency (e.g., lack of precision, subjectivity). Two kinds of quantitative image analysis were performed in this study: high-order statistical methods and grayscale mathematical morphology. They were applied to study the microstructure of 3 different cream cheeses (same manufacturing process, same dry matter content, but different fat and protein contents). Advantages and drawbacks of both methods are reviewed. The way they may be used to describe cream cheese microstructure is also presented.     

The addition of calcium chloride in combination with a lower draining pH to change the microstructure and improve fat retention in Cheddar cheese

Calcium chloride addition and the whey draining pH are known to impact on cheese making. The effect of 100 or 300 mg kg⁻¹ calcium chloride (CaCl₂) and the whey draining pH (6.2 or 6.0) on the microstructure of Cheddar cheese was assessed using confocal and cryo scanning electron microscopy. The gel made with 300 mg kg⁻¹ CaCl₂ was found to have a denser protein network and smaller pores than the gel with lower or no CaCl₂ addition. CaCl₂ addition reduced fat lost to the sweet whey. The texture of the cheeses with a lower draining pH was harder and moisture content lower. Our results show that the combination of calcium addition and lower draining pH could be used to increase network formation at the early stages of cheese making to improve fat retention while maintaining a similar level of total calcium in the final cheese.     

Cold Stage Scanning Electron Microscopy Study of Meat Batters

Cryo scanning electron microscopy (Cryo SEM) was used to study the microstructure of raw and cooked meat batters and compared with conventional SEM and transmission electron microscopy (TEM). Cryo SEM revealed that raw batters consisted of an organized, highly interconnected protein matrix in which fat globules were dispersed. Cryo SEM resulted in much better preservation of protein gel structure compared to conventional SEM which caused shrinkage of the specimen, resulting in a closed gel matrix structure. TEM confirmed the differences between the matrices of raw and cooked batters shown by cryo SEM. Methods for preparation of specimen for cryo SEM were developed.     

The effect of varying fat content on the microstructure of Oaxaca cheese,a typical pasta filata cheese

The effect of varying fat content in Oaxaca cheese, a typical pasta filata, on microstructure was described. Microstructure of cheeses was analysed by scanning electron microscopy (SEM) and light microscopy (LM) in nondehydrated and dehydrated samples. In nondehydrated samples, protein fibres were wide and compact in fat‐free cheese, and big serum channels were approximately 100 μm in width. Width of protein fibres and size of channels decreased as fat content increased. Small channels seemed to be occupied only by fat, while in big channels, water and fat were observed. LM both confirmed and supplemented the observations made by SEM, particularly the presence and distribution of fat in channels.     

Microstructure of milk gel and cheese curd observed using cryo scanning electron microscopy and confocal microscopy

Cryo scanning electron microscopy (cryo SEM) and confocal laser scanning microscopy (CLSM) were used to visualise changes in the microstructure of milk, rennet-induced gel and curd during the manufacture of Cheddar cheese. Our results show that cryo preservation did not alter the microstructure of the sample when it was fixed by rapid freezing in slush liquid nitrogen due to the formation of amorphous ice. Artefacts such as the formation of ice crystals could be observed in samples when immersed directly into liquid nitrogen (−196 °C) at atmospheric pressure. These ice crystals changed the shape of sample pores increasing their size to >20 μm. The etching time, thickness of gold coating, accelerating voltage and type of detector used for cryo SEM observation were varied in order to minimise the formation of such artefacts and optimise conditions for imaging. Chains and clusters of casein micelles and fat globules were best observed in the gel and the cooked curd when the samples were freeze fractured and etched for 30 min, coated with a mixture of gold and palladium alloy approximately 6 nm thick at −140 °C and observed using a backscattered electron detector at 15 kV. The structure of the gel, curd and cheese was also observed using CLSM. Spherical fat globules were mostly present in the serum pores of the gel prepared from unhomogenised milk but were found embedded in the aggregated chains of the casein network within the gel prepared from homogenised milk when observed using CLSM. The porosity measurements obtained using cryo SEM were similar to those obtained using CLSM. These two complementary techniques can potentially be used to assist studies for the control of cheese texture and functionality.     

Light-induced formation of free radicals in cream cheese

Radicals were found, by electron spin resonance (ESR) spectroscopy, to accumulate in cream cheese (26% fat, 7% protein) and more significantly in low fat cream cheese (17% fat, 11% protein) upon light exposure. The decay of radicals following illumination (875 lux, with a strong UV-component for up to 80 min) followed first-order kinetics with a half-life at room temperature of around 0.5 h both for cream cheese and low fat cream cheese. The surprisingly long-lived radicals had a broad structureless ESR-spectrum (g-value of 2.006) which, for partly desiccated cream cheese, changed towards a nitrogen-centred ESR powder spectrum (g-value of 2.0014) typical for immobilised protein-based radicals. The protein oxidation product, dimethyl disulphide, and the lipid oxidation products, hexanal and 2-butanone, were detected by GC-analysis in higher concentrations in the outer 1 mm layer than in the second layer (of 1 mm thickness) of the product, in agreement with absorption of 99% of the UV-light intensity in the outer 1 mm layer. The low fat cream cheese had higher levels of both lipid and protein oxidation products, in agreement with the higher steady state concentration of radicals and confirming the role of proteins in oxidative changes also for lipids in cheese. The low fat cream cheese was initially more oxidised than was the cream cheese, as seen from the peroxide values, and oxidation products in lipid droplets could be visualised in three dimensions by confocal laser scanning microscopy, using the fluorescent probe C₁₁-Bodipy (581/591).     

The Potential Application of Rice Bran Wax Oleogel to Replace Solid Fat and Enhance Unsaturated Fat Content in Ice Cream

The development of structure in ice cream, characterized by its smooth texture and resistance to collapse during melting, depends, in part, on the presence of solid fat during the whipping and freezing steps. The objective of this study was to investigate the potential application of 10% rice bran wax (RBW) oleogel, comprised 90% high‐oleic sunflower oil and 10% RBW, to replace solid fat in ice cream. A commercial blend of 80% saturated mono‐ and diglycerides and 20% polysorbate 80 was used as the emulsifier. Standard ice cream measurements, cryo‐scanning electron microscopy (cryo‐SEM), differential scanning calorimetry (DSC), and transmission electron microscopy (TEM) were used to evaluate the formation of structure in ice cream. RBW oleogel produced higher levels of overrun when compared to a liquid oil ice cream sample, creating a lighter sample with good texture and appearance. However, those results were not associated with higher meltdown resistance. Microscopy revealed larger aggregation of RBW oleogel fat droplets at the air cell interface and distortion of the shape of air cells and fat droplets. Although the RBW oleogel did not develop sufficient structure in ice cream to maintain shape during meltdown when a mono‐ and diglycerides and polysorbate 80 blend was used as the emulsifier, micro‐ and ultrastructure investigations suggested that RBW oleogel did induce formation of a fat globule network in ice cream, suggesting that further optimization could lead to an alternative to saturated fat sources for ice cream applications.     

Microstructure of dairy foods. 2. Milk products based on fat

Cold-stage scanning electron microscopy and transmission electron microscopy of replicas of freeze-fractured samples have been suggested as the electron microscopic techniques best suited to study the microstructure of milk products based on fat. New developments in other techniques such as fixation of fat with imidazole-buffered osmium tetroxide and embedding in a resin also have been mentioned. Microstructure of various forms of cream, ice cream, cream cheese, cream cheese spread, and butter, established by the techniques mentioned, has been reviewed with respect to the specific properties of each of the fat-based milk products.     

Use of a β-glucan hydrocolloidal suspension in the manufacture of low-fat Cheddar cheese: manufacture, composition, yield and microstructure

Low‐fat Cheddar cheese was manufactured using a β‐glucan, hydrocolloidal fat replacer denoted as Nutrim. The composition, production efficiency, microstructure, and utility of replacing fat with Nutrim were examined. Cheese samples (designated as Nutrim‐I, and Nutrim‐II) containing Nutrim were produced with mean fat levels of 6.84 and 3.47%, respectively. A low‐fat cheese was also produced as a control with a mean fat level of 11.2%. Nutrim‐II cheese had significantly higher moisture, salt, and ash contents as compared with the low‐fat control cheese. The low‐fat control cheese had a higher yield normalized for 54% moisture and 1.5% salt content as compared with the Nutrim‐II cheese. Scanning electron microscopy revealed smaller and more uniform fat droplet voids in the Nutrim cheese than the low‐fat control, and a more dense, noncontinuous background protein matrix with globular clusters suggesting a physical buffering afforded by the presence of the β‐glucan hydrocolloid or its associated water.     

Development of Formulations and Processes to Incorporate Wax Oleogels in Ice Cream

The objective of this study was to investigate the influence of emulsifiers, waxes, fat concentration, and processing conditions on the application of wax oleogel to replace solid fat content and create optimal fat structure in ice cream. Ice creams with 10% or 15% fat were formulated with rice bran wax (RBW), candelilla wax (CDW), or carnauba wax (CBW) oleogels, containing 10% wax and 90% high‐oleic sunflower oil. The ice creams were produced using batch or continuous freezing processes. Transmission electron microscopy (TEM) and cryo‐scanning electron microscopy were used to evaluate the microstructure of ice cream and the ultrastructure of oleogel droplets in ice cream mixes. Among the wax oleogels, RBW oleogel had the ability to form and sustain structure in 15% fat ice creams when glycerol monooleate (GMO) was used as the emulsifier. TEM images revealed that the high degree of fat structuring observed in GMO samples was associated with the RBW crystal morphology within the fat droplet, which was characterized by the growth of crystals at the outer edge of the droplet. Continuous freezing improved fat structuring compared to batch freezing. RBW oleogels established better structure compared to CDW or CBW oleogels. These results demonstrate that RBW oleogel has the potential to develop fat structure in ice cream in the presence of GMO and sufficiently high concentrations of oleogel.     

超高压处理对再制奶油干酪质构、流变学特性及微观结构的影响

以低饱和脂肪酸的再制奶油干酪为研究对象,探究不同条件超高压处理（压力：150、300、450 MPa;保压时间：10 min;保压温度：25 ℃）对再制奶油干酪质构、流变学特性及微观结构的影响。通过SPSS软件分析压力变化与干酪水分质量分数、水分活度、pH值及质构特性的相关性,通过质构分析仪测定干酪质构特性（涂抹性、硬度、黏合性及黏聚力）的变化,并使用流变仪分析干酪流变学特性变化;同时采用扫描电子显微镜观察干酪微观结构的变化。结果表明,随着压力的增加,干酪的水分质量分数变化不明显,150 MPa处理组干酪的水分活度显著高于其他组干酪（P＜0.05）;压力越大,干酪pH值越高;压力与水分质量分数正相关,相关系数为0.646,与水分活度负相关,相关系数为－0.346,压力与pH值、涂抹性、硬度、黏合性、黏聚力呈显著正相关,相关系数分别为0.963、0.959、0.951、0.956、0.956;超高压处理可以降低干酪黏度对温度的依赖性,增加了干酪网络结构的稳定性; 150 MPa和450 MPa条件下的干酪弹性模量与黏性模量高于对照组,黏弹性较好;超高压处理影响干酪微观结构变化,压力越大,图像中孔洞数量越少,蛋白质基质更加光滑和均匀,结构更加紧密。综上,超高压处理与再制奶油干酪质构、流变性和微观结构关系密切,研究结果可为超高压干酪的工艺研发提供数据参考。     

Effect of buttermilk made from creams with different heat treatment histories on properties of rennet gels and model cheeses

Although many studies have reported negative effects on cheese properties resulting from the use of buttermilk in cheese milk, the cause of these effects has not been determined. In this study, buttermilk was manufactured from raw cream and pasteurized cream, as well as from a cream derived from pasteurized whole milk. Skim milks with the same heat treatments were also manufactured to be used as controls. Compositional analysis of the buttermilks revealed a pH 4.6-insoluble protein content approximately 10% lower than that of the skim milk counterparts. Milk fat globule membrane (MFGM) proteins remained soluble at pH 4.6 in raw cream buttermilk; however, when heat was applied to cream or whole milk before butter making, MFGM proteins precipitated with the caseins. Rennet gel characterization showed that MFGM material in the buttermilks decreased the firmness and increased the set-to-cut time of rennet gels, but this effect was amplified when pasteurized cream buttermilk was added to cheese milk. The microstructure of gels was studied, and it was observed that gel appearance was very different when pasteurized cream buttermilk was used, as opposed to raw cream buttermilk. Model cheeses manufactured with buttermilks tended to have a higher moisture content than cheeses made with skim milks, explaining the higher yields obtained with buttermilk. Superior retention of MFGM particles was observed in model cheeses made from pasteurized cream buttermilk compared with raw cream buttermilk. The results from this study show that pasteurization of cream and of whole milk modifies the surface of MFGM particles, and this may explain why buttermilk has poor coagulation properties and therefore yields rennet gels with texture defects.     

Effect of fat reduction on chemical composition, proteolysis, functionality, and yield of Mozzarella cheese

Mozzarella cheese was made from skim milk standardized with cream (unhomogenized, 40% milk fat) to achieve four different target fat percentages in the cheese (ca. 5, 10, 15, and 25%). No statistically significant differences were detected for cheese manufacturing time, stretching time, concentration of salt in the moisture phase, pH, or calcium as a percentage of the protein in the cheese between treatments. As the fat percentage was reduced, there was an increase in the moisture and protein content of the cheese. However, because the moisture did not replace the fat on an equal basis, there was a significant decrease in the moisture in the nonfat substance in the cheese as the fat percentage was reduced. This decrease in total filler volume (fat plus moisture) was associated with an increase in the hardness of the unmelted cheese. Whiteness and opacity of the unmelted cheese decreased as the fat content decreased. Pizza baking performance, meltability, and free oil release significantly decreased as the fat percentage decreased. The minimum amount of free oil release necessary to obtain proper functionality during pizza baking was between 0.22 and 2.52 g of fat/100 g of cheese. Actual cheese yield was about 30% lower for cheese containing 5% fat than for cheese with 25% fat. Maximizing fat recovery in the cheese becomes less important to maintain high cheese yield, and moisture control and the retention of solids in the water phase become more important as the fat content of the cheese is reduced.     

Effect of an Exopolysaccharide-Producing Strain of Streptococcus Thermophilus on the Yield and Texture of Mexican Manchego-Type Cheese

An exopolysaccharide-producing strain of Streptococcus thermophilus was evaluated in the production of Mexican manchego-type cheese. This ropy strain improved water and fat retention, and significantly increased cheese yield. Furthermore, the ropy strain cheese retained more moisture than control cheese during ripening, suggesting that exopolysaccharide strongly bound water within the protein matrix of the cheese. Scanning electron microscopy confirmed that exopolysaccharide bound to the protein matrix of the cheese, producing a dense network that helped to increase water and fat retention and leading to a more open structure of the cheese that gave a softer product, as confirmed by instrumental texture profile analysis and sensory evaluation. Comparison of scanning electron microscopy micrographs of the different sections of the cheese showed higher concentration of exopolysaccharide in the centre than in the outer sections, indicating that exopolysaccharide production continued during ripening and that the environment at the centre of the cheese (moisture and/or oxygen concentration) favoured exopolysaccharide production. Instrumental texture profile analysis also demonstrated that the ropy strain cheese was more cohesive and less elastic than the control; in contrast, exopolysaccharide did not affect chewiness. The changes in texture could be correlated to composition: hardness increased as water and fat decreased, while springiness decreased with increasing fat. The interactions of exopolysaccharide with the cheese protein matrix had an affect on the increase in cohesiveness of the ropy strain cheese.     

Microstructure of industrially manufactured goat cheese Queso de Murcia al Vino during synaeresis

The microstructural parameters of an industrially manufactured goat cheese curd (pore number, area and perimeter, strand thickness and porosity) were analysed by scanning electron microscopy and image analysis during synaeresis. The water‐holding capacity, whey fat, pH and moisture content were also determined to establish any relationship with the curd microstructure. The quantification of the different microstructural parameters made it possible to assign pitching and stirring as important processing steps because these steps impart different features to the curd microstructure. Higher pore number was related to reduced pore area, perimeter and strand thickness, but higher porosity and moisture.     

Characterization of particles in cream cheese

Cream cheese is used as a spread and as an ingredient in many food applications. A gritty or grainy mouthfeel is an undesirable textural defect that occurs in cream cheese. However, the factors that cause the textural defect are not well understood. The objectives of this study were to isolate and characterize particles from cream cheese and to study the effect of particles on cheese texture. Particles were isolated by washing cream cheese with water first at 25 degrees C and then at 50 degrees C repeatedly 4 to 5 times. The size of these particles was determined using a particle size analyzer. The particles as well as the original cheeses were analyzed for moisture, fat, protein, ash, and lactose. The particle size ranged of 0.04 to 850 microm. It was found that isolated particles were significantly higher in protein content as compared with the whole cheese. To study the effect on the cheese texture, particles were added at 5, 15, and 25% (wt/wt) levels to smooth cream cheese, and a sensory ranking test was done on the samples. Isolated particles were further separated into 2 size classes of 2.5 to 150 microm and > or =150 microm. These particles were then mixed with smooth cream cheese at 16 and 29% (wt/wt), and a sensory test was conducted on these samples. Smooth cream cheese with only 5% (wt/wt) added particles was perceived as significantly grittier than the control sample. This experiment also revealed that the perceived grittiness increased with increase in amount and size of particles.     

Effect of calcium on microstructure and meltability of part skim mozzarella cheese

The role of calcium in the microstructure of part skim Mozzarella cheese was evaluated using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Part skim Mozzarella cheeses with 4 calcium levels (control 0.65%, T1 0.48%, T2, 0.42%, and T3 0.35%) were manufactured and stored at 4 degrees C. Microstructure and meltability of cheeses were studied on d 1 and 30. The micrographs were analyzed for numbers, area, perimeter, roundness, and size of the fat particles. Reduced calcium cheeses had greater meltability and more hydrated protein matrix with greater number of fat particles (control=125, T1=193, T2=184, and T3=215 with SEM and control = 86, T1=87, T2= 125, and T3= 140 with CLSM). Further, area and perimeter of these fat particles were also greater in reduced calcium cheeses. Area, perimeter, and size of fat particles increased and their roundness decreased upon storage of 30 d. Decrease in free serum in the protein matrix of all cheeses upon refrigerated storage was evident from the CLSM. Hydrated protein network and better emulsified fat in low calcium cheeses might have improved melt properties of Mozzarella cheese.     

Effect of HPMC addition on the microstructure, quality and aging of wheat bread

The effect of hydroxypropylmethylcellulose (HPMC) addition on a basic bread formulation is described. The effect of HPMC as bread improver and antistaling agent was analysed in terms of microstructure. Bread quality was assessed by physical parameters (volume, width/height ratio, moisture content and hardness), crumb grain structure (number of air cells, cells area and the ratio between cells area and total area) and sensory evaluation (appearance, aroma, taste and texture). Bread staling was determined by following both the hardness increase and the starch retrogradation during storage. The microstructure was analyzed by cryo scanning electron microscopy (cryo-SEM). The results confirm the ability of the HPMC for improving fresh bread quality and for delaying staling. The presence of HPMC decreased the hardening rate of the bread crumb and also retarded the amylopectin retrogradation. The microstructure analysis revealed the possible interaction between the HPMC and the bread constituents, which could partially explain the antistaling effect of this hydrocolloid.     

Rheological properties and microstructure of Cheddar cheese made with different fat contents

Reduced- and low-fat cheeses are desired based on composition but often fall short on overall quality. One of the major problems with fat reduction in cheese is the development of a firm texture that does not break down during mastication, unlike that observed in full-fat cheeses. The objective of this investigation was to determine how the amount of fat affects the structure of Cheddar cheese from initial formation (2 wk) through 24 wk of aging. Cheeses were made with target fat contents of 3 to 33% (wt/wt) and moisture to protein ratios of 1.5:1. This allowed for comparisons based on relative amounts of fat and protein gel phases. Cheese microstructure was determined by confocal scanning laser microscopy combined with quantitative image analysis. Rheological analysis was used to determine changes in mechanical properties. Increasing fat content caused an increase in size of fat globules and a higher percentage of nonspherical globules. However, no changes in fat globules were observed with aging. Cheese rigidity (storage modulus) increased with fat content at 10°C, but differences attributable to fat were not apparent at 25°C. This was attributable to the storage modulus of fat approaching that of the protein gel; therefore, the amount of fat or gel phase did not have an effect on the cheese storage modulus. The rigidity of cheese decreased with storage and, because changes in the fat phase were not detected, it appeared to be attributable to changes in the gel network. It appeared that the diminished textural quality in low-fat Cheddar cheese is attributed to changes in the breakdown pattern during chewing, as altered by fat disrupting the cheese network.     

Microstructure and rheological properties of Iranian white cheese coagulated at various temperatures

The effect of milk coagulation temperature on the composition, microstructure monitored using scanning electron micrographs, opacity measured by a Hunter lab system, and rheological behavior measured by uniaxial compression and small amplitude oscillatory shear were studied. Three treatments of Iranian White cheese were made by applying coagulation temperatures of 34, 37, and 41.5°C during the cheese-making procedure. A higher coagulation temperature resulted in increased fat and protein contents, and decreased the moisture content and ratio of moisture to protein. The highest temperature (41.5°C) had a significant effect on the opacity of Iranian White cheese. Milk coagulation at this temperature decreased the whiteness index (Hunter L value) and increased the yellowness index (Hunter b value) of the aged product compared with cheeses coagulated at lower temperatures. Microstructure of the cheese coagulated at 41.5°C was more compact and undisturbed, reflecting the higher values of stress at fracture and storage modulus measured for this treatment.