Microstructural studies of imitation cheese with a shift in continuous phase.

When casein is replaced with starch in imitation cheese, the functionality changes. Three different microscopy methods were applied to understand the microstructural differences in the product depending on which component dominates the microstructure. Confocal Laser Scanning Microscopy (CLSM) for component identification. Scanning Electron Microscopy (SEM) and Cryogenic Scanning Electron Microscopy (Cryo-SEM) for studying surface structures. Differences in the surface structures were detected between SEM and Cryo-SEM. In SEM, starch appeared rough and protein smooth, while in Cryo-SEM no starch roughness of the surface was found. A change in starch modification and effects of protein prehydration was also analysed. Adding octenyl succinic anhydride (OSA) modified starch for emulsifying properties resulted in a microstructure with fragmented protein at a protein level of 7 %, but not at 9 or 12 %. Protein prehydration had limited effect on microstructure. On a macrostructural level, the change to an emulsifying starch increased hardness in imitation cheese with 7 and 9 % protein. Protein prehydration slightly decreased the hardness, but the difference was not significant at all concentrations. This research provides valuable information about the microstructure of imitation cheese at a 50/50 composition, how the microstructure changes with an emulsifying starch and what occurs after a protein prehydration was included in the production.

A sustainable and regioselective synthesis of Hemi-bis(monoacylglycero)phosphates and bis(diacylglycero)phosphates.

A sustainable and green approach was developed for the scalable synthesis of uncommon naturally occurring phospholipid species, Hemi-bis(monoacylglycero)phosphates (Hemi-BMPs) and bis(diacylglycero)phosphates (BDPs) via the phospholipase D (PLD) mediated transphosphatidylation. PLD from Streptomyces sp. showed great substrate promiscuity for both phospholipids from different biological sources, and alcohol donors with diverse regiochemistry; monoacylglycerols with diverse fatty acyl structures (C12-C22), affording 74-92 wt% yields in 2 h. Experimental results demonstrated that the reaction rate is rather independent of phosphatidyls but to a large extent governed by the size, shape and regiolocation of fatty acyls incorporated on the glycerol backbone, particularly for the regio-isomers of bulky diacylglycerols (Sn-1,3 or Sn-1,2), which displays great diversity. In addition, a plausible mechanism is proposed based on molecular simulations for an elaborated explanation of the reaction thermodynamic and kinetic favorability toward the synthesis of Hemi-BMPs and BDPs.

Cold ultrafiltered or microfiltered milk retentates: A systematic comparison of the effects of compositional differences on their gelation functionality.

The colloidal stability of casein micelles suspensions prepared using ultrafiltration (UF) and microfiltration (MF) was studied by testing acid- and rennet-induced destabilization. Skim milk and 4× (based on volume reduction) concentrates were obtained by processing under similar conditions, at temperatures below 10°C. Concentrates were subjected to different levels of diafiltration (DF), resulting in samples with comparable casein volume fractions but different amounts of proteins and ions in the serum phase. The novelty of the work is the systematic comparison of MF and UF concentrates of similar history. More specifically, concentrates similar in ionic composition but with or without serum proteins were compared, to evaluate whether whey proteins and ß-casein depletion from the micelles will play a role in the processing properties, or whether these are affected solely by the ionic balance. Microfiltered micelles' apparent diameter decreased by about 50 nm during the specific hydrolysis of κ-casein by chymosin, whereas those in skim milk control showed a decrease of about half that size. All concentrates subjected to extensive DF showed smaller hydrodynamic diameters, with reductions of ∼18 and 13 nm for MF and UF, respectively. Highly diafiltered UF retentates showed a delayed onset of rennet-induced gelation, due to low colloidal calcium, compared with other samples. Low-diafiltered samples showed weak storage modulus (∼1 Pa) after 60 min of onset of gelation. In addition, onset pH increased with diafiltration to ∼5.8 for UF and ∼6 for MF in high-diafiltered samples. These results clearly demonstrated that the functional properties of casein micelles change during membrane concentration, and this cannot be solely attributed to changes in ionic equilibrium.

Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteristics.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn2+-binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential.

Designer phospholipids - structural retrieval, chemo-/bio- synthesis and isotopic labeling.

Phospholipids are unique and versatile molecules, essential in a variety of biological systems. Moreover, their diverse structures and amphiphilic properties endorse their indispensable and unparalleled roles in research and industrial-related applications. However, in most cases of applications, naturally occurring phospholipids are either deficit in structural variety or insufficient in quantity; therefore, novel methods must be developed for the synthesis of new molecules or modification of natural structures. To identify sustainable and environmentally friendly approaches, this work reviews the latest progress in the acquisition of structurally defined phospholipids (designer phospholipids) from natural resources, including structural retrieval, redesign and synthesis of designer phospholipids via chemo-/enzymatic approaches. This review additionally highlights the opportunity to use biological systems to direct the production of specific phospholipid species through genetic engineering via defined metabolic pathways, and functionalization of natural phospholipids through synthetic modifications: substitutions, removals or additions of specific functional groups. A particular focus is given to the establishment of chemical and biological systems for the synthesis of isotopically labelled phospholipids for biomedical applications. The application of green chemistry principles in semi-synthesis of phospholipids including extended use of greener biocatalysts and diatomaceous earth and reduced use of hazardous and toxic solvents is also summarized.

Assessment of rheological methods to study crystallization of palm oil fractions.

This study provides an evaluation of the use of rheology to characterize soft, semi-hard, and hard fats in relation to determine the crystallization onset, crystallization behavior, as well as microstructure development using either a plate-plate or a starch pasting cell (SPC). The results from this study demonstrate that when applying rheology to study fat crystallization, the results must be interpreted with care. The application of a plate-plate geometry allowed for sensitive evaluation of the initial nucleation phase, which was not possible with an SPC. Both geometries could provide information on crystallization behavior in terms of one-step or two-step crystallization. However, in the late stage of the crystallization process, when the fat crystals form a strong network, the SPC could not describe differences in the rheology of the fat-crystal network, which was a possibility by the use of a plate-plate geometry. Thus, oscillatory rheology with a suitable geometry can be used to evaluate the entire crystallization process.

Freezing as a solution to preserve the quality of dairy products: the case of milk, curds and cheese.

When thinking of the freezing process in dairy, products consumed in frozen state, such as ice creams come to mind. However, freezing is also considered a viable solutions for many other dairy products, due to increasing interest to reduce food waste and to create more robust supply chains. Freezing is a solution to production seasonality, or to extend the market reach for high-value products with otherwise short shelf life. This review focuses on the physical and chemical changes occurring during freezing of milk, curds and cheeses, critical to maintaining quality of the final product. However, freezing is energy consuming, and therefore the process needs to be optimized to maintain product's quality and reduce its environmental footprint. Furthermore, the processing steps leading to the freezing stage may require some changes compared to traditional, fresh products. Unwanted reactions occur at low water activity, and during modifications such as ice crystals growth and recrystallization. These events cause major physical destabilizations of the proteins due to cryoconcentration, including modification of the colloidal-soluble equilibrium. The presence of residual proteases and lipases also cause important modifications to the texture and flavor of the frozen dairy product.

Effect of frozen and refrigerated storage on proteolysis and physicochemical properties of high-moisture citric mozzarella cheese.

High-moisture mozzarella is one of the most-exported Italian cheeses worldwide, but its quality is affected by storage. Freezing is regarded as a solution to decrease product waste, extend market reach, and increase convenience, but its effect on quality has to be estimated. In this study, the details related to proteolysis, physicochemical properties, and sensory quality parameters of high-moisture mozzarella as a function of frozen storage (1, 3, and 4 mo) and subsequent refrigerated storage after thawing (1, 3, and 8 d) were evaluated. Frozen cheeses stored at -18°C showed a higher extent of proteolysis, as well as different colorimetric and sensory properties, compared with the fresh, nonfrozen control. Sensory evaluation showed the emergence of oxidized and bitter taste after 1 mo of frozen storage, which supports the proteolysis data. The extent of proteolysis of frozen-stored cheese after thawing was greater than that measured in fresh cheese during refrigerated storage. These results help better understand the changes occurring during frozen storage of high-moisture mozzarella cheese and evaluate possible means to decrease the effect of freezing on the cheese matrix.

Applicability of Confocal Raman Microscopy to Observe Microstructural Modifications of Cream Cheeses as Influenced by Freezing.

Confocal Raman microscopy is a promising technique to derive information about microstructure, with minimal sample disruption. Raman emission bands are highly specific to molecular structure and with Raman spectroscopy it is thus possible to observe different classes of molecules in situ, in complex food matrices, without employing fluorescent dyes. In this work confocal Raman microscopy was employed to observe microstructural changes occurring after freezing and thawing in high-moisture cheeses, and the observations were compared to those obtained with confocal laser scanning microscopy. Two commercially available cream cheese products were imaged with both microscopy techniques. The lower resolution (1 µm/pixel) of confocal Raman microscopy prevented the observation of particles smaller than 1 µm that may be part of the structure (e.g., sugars). With confocal Raman microscopy it was possible to identify and map the large water domains formed during freezing and thawing in high-moisture cream cheese. The results were supported also by low resolution NMR analysis. NMR and Raman microscopy are complementary techniques that can be employed to distinguish between the two different commercial formulations, and different destabilization levels.

Disulfide bond formation is not crucial for the heat-induced interaction between ß-lactoglobulin and milk fat globule membrane proteins.

During heat treatment of milk, ß-lactoglobulin (ß-LG) associates with the milk fat globule membrane (MFGM). The objective of this study was to examine different binding types that could be involved in this process. First, we tested the thiol-disulfide bond interchange between ß-LG and MFGM by heating raw milk (87°C, 8 min) in the presence of different reagents capable of preventing this interaction, and then evaluated the presence of ß-LG in resulting MFGM preparations by sodium dodecyl sulfate-PAGE. Contrary to commonly accepted theory, ß-LG still associated with MFGM when milk was heated in the presence of 10 mM N-ethylmaleimide, dithiobis-nitrobenzoic acid, or dithioerythritol. This finding indicated that noncovalent binding could be involved in the interaction, and therefore these were studied next. Preventing noncovalent interactions by heating milk in the presence of 8 M urea (to inhibit formation of hydrogen bonds) or 2 M NaCl (to inhibit electrostatic and hydrophobic interactions) reduced the association of ß-LG and MFGM. Inhibiting both hydrogen and disulfide bond formation by addition of 8 M urea and 10 mM dithioerythritol or inhibiting hydrophobic interactions with 0.2% sodium dodecyl sulfate completely prevented the association. In contrast to the simple thiol-disulfide interaction model, the results suggest a more complex understanding of the interactions between ß-LG and MFGM during heating of milk. This indicates that disulfide formation between ß-LG and proteins in the MFGM is not required for the association, but that hydrophobic interactions and hydrogen bonding may be crucial. This novel insight into ß-LG and MFGM association is in contrast to the current literature and requires further study.

Impact of industrial cream heat treatments on the protein composition of the milk fat globule membrane.

The impact of cream processing on milk fat globule membrane (MFGM) was assessed in an industrial setting for the first time. Three creams and their derived MFGM fractions from different stages of the pasteurization procedure at a butter dairy were investigated and compared to a native control as well as a commercial MFGM fraction. The extent of cross-linking of serum proteins to MFGM proteins increased progressively with each consecutive pasteurization step. Unresolved high molecular weight aggregates were found to consist of both indigenous MFGM proteins and ß-lactoglobulin as well as αs1- and ß-casein. With regards to fat globule stability and in terms of resistance towards coalescence and flocculation after cream washing, single-pasteurized cream exhibited reduced sensitivity to cream washing compared to non- and double-pasteurized creams. Inactivation of the agglutination mechanism and the increased presence of non-MFGM proteins may determine this balance between stable and non-stable fat globules.

Herd factors influencing free fatty acid concentrations in bulk tank milk.

Free fatty acid (FFA) concentrations can be elevated in raw milk due to improper handling and management at the dairy farm, and high concentrations of FFA can lead to off flavors in milk. This study aimed to describe how the herd production system, milking system, feeding and technological factors impact on FFA concentrations in bulk tank milk. FFA concentrations in bulk milk samples from 259 organic and 3326 conventional herds were analyzed by FT-IR during one year. The FFA content was significantly lower in bulk milk from organic than conventional herds. This was most evident during the summer half-year when the organic cows graze pasture. Bulk milk from automatic milking systems (AMS) and tie-stalls contained greater concentrations of FFA than any other milking parlor systems. In AMS, high milking frequency was found to be the most significant contributor to elevated FFA content in milk. Moreover, a strong interaction was found between milking interval and production system (organic vs. conventional). The technical factors, pre-cooling, onset time for cooling after milk inlet, contact between milk and agitation also impacted on the FFA concentration, whereas other technical factors including centrifugal pump type, length and height of pumping line and type of AMS manufacturer were found to be without significant effect on FFA. Feeding variables, based on feeding plans and evaluation, only explained a small part of the variation in bulk milk FFA. Overall, this study demonstrated that AMS compared to other milking system contributes significantly to increased FFA concentration in bulk tank milk, and within AMS high milking frequency contributes to increased FFA concentration.

Effects of Different Fasting Durations on Glucose and Lipid Metabolism in Sprague Dawley Rats.

Overnight fasting of varying length is common practice when studying glucose and lipid metabolism in rats. However, prolonged fasting may influence insulin sensitivity, and it is unknown to which extent different fasting durations affect postprandial metabolism in rats. The purpose of this study was to investigate the effect of different fasting durations (6-, 12-, or 18-h) on fat tolerance and glucose tolerance in male Sprague Dawley rats. We also aimed to examine the effect of two test fats with different fatty acid composition on postprandial triglycerides. We conducted a fat tolerance test, where butterfat or rapeseed oil was administered in a crossover design (experiment 1), and an oral glucose tolerance test (experiment 2). Regarding the fat tolerance test, we found no effects of fasting duration on triglycerides or free fatty acids, whereas the 18-h fast resulted in reduced glucose and insulin area under the curves. We did not find differential effects of butterfat and rapeseed oil on the outcomes. We found decreased fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR), and increased beta-hydroxybutyric acid concentrations after 18-h fast compared with shorter fasting durations. Regarding the oral glucose tolerance test, both 12-h and 18-h fast resulted in greater peak insulin concentrations than 6-h fast, and peak glucose concentrations were higher after 18-h than 12-h fast. We found no effects of fasting on the insulin sensitivity index. In conclusion, extending the fasting duration had an impact on glucose metabolism in rats, but did not appear to influence fat tolerance.

Thermal effects on IgM-milk fat globule-mediated agglutination.

The process of agglutination causes firm cream layers in bovine milk, and a functioning agglutination mechanism is paramount to the quality of non-homogenized milks. The phenomenon is not well-described, but it is believed to occur due to interactions between immunoglobulins (Ig) and milk fat globules. For the first time, this paper demonstrates how the process of agglutination can be visualized using confocal laser scanning microscopy, rhodamine red and a fluoresceinisothiocynat-conjugated immunoglobulin M antibody. The method was used to illustrate the effect on agglutination of storage temperature and pasteurization temperature. Storage at 5 °C resulted in clearly visible agglutination which, however, was markedly reduced at 15 °C. Increasing storage temperature to 20 or 37 °C cancelled any detectable interaction between IgM and milk fat globules, whereby the occurrence of cold agglutination was documented. Increasing 20 s pasteurization temperatures from 69 °C to 71 °C and further to 73 °C lead to progressively higher inactivation of IgM and, hence, reduction of agglutination. Furthermore, 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that changes in storage temperature caused a redistribution of Ig-related proteins in milk fat globule membrane isolates. Poly-immunoglobulin G receptor was present in milk fat globule preparations stored at cold (4 °C) conditions, but absent at storage at higher temperature (25 °C). The findings provide valuable knowledge to dairy producers of non-homogenized milk in deciding the right pasteurization temperature to retain the crucial agglutination mechanism.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Impact of triacylglycerol composition on shear-induced textural changes in highly saturated fats.

This study demonstrates a strong interaction between triacylglycerol (TAG) composition and effects of shear rate on the microstructure and texture of fats. Cocoa butter alternatives with similar saturated fat content, but different major TAGs (PPO-, PSO-, SSO-, POP- and SOS-rich blends) were evaluated. Results show how shear can create a harder texture in fat blends based on symmetric monounsaturated TAGs (up to ∼200%), primarily due to reduction in crystal size, whereas shear has little effect on hardness of asymmetric monounsaturated TAGs. Such differences could not be ascribed to differences in the degree of supercooling, but was found to be a consequence of differences in the crystallisation behaviour of different TAGs. The fractal dimension was evaluated by dimensional detrended fluctuation analysis and Fourier transformation of microscopy images. However, the concept of fractal patterns was found to be insufficient to describe microstructural changes of fat blends with high solid fat content.

Effects of automatic cluster removal and feeding during milking on milking efficiency, milk yield and milk fat quality.

In order to increase milking efficiency, the effects of two different cluster take-off levels (200 and 800 g/min) and feeding vs. not feeding during milking were tested in a Latin square design study including 32 cows. Milk yield, milking time, milk flow and milking interval were measured and milk samples were analysed for gross composition, sodium and potassium concentration, free fatty acid (FFA) content, milk fat globule (MFG) size, MFG membrane (MFGM) material and fatty acid composition. Residual milk was harvested to evaluate udder emptying. Increasing the take-off level from 200 to 800 g/min at the whole udder level decreased milking time and increased harvest flow. Udder emptying decreased slightly, but there were no effects on milk yield, FFA content or MFGM. There were interactive effects of take-off level and feeding during milking on content of fatty acids C4:0, C6:0, C16:0, C18:3(n-3) and C20:0. Feeding during milking increased milk yield per day and decreased milking interval. Sodium and potassium concentrations in milk were unaffected by treatments, indicating no loss of tight junction integrity. From these results, it is clear that feeding during milking should be used to increase milk yield and improve milking efficiency, regardless of take-off level used, and that the effect of feeding is more pronounced when a low take-off level is used. Feeding seemed to counteract the effects of the low take-off level on milking time and milking interval. Low take-off levels can therefore be used in combination with feeding.

Covariance structures of fat and protein influence the estimation of IgG in bovine colostrum.

On-farm instruments for assessing colostrum quality are needed in order to ensure that the calf is supplied with enough IgG to avoid failure of passive transfer. The aim of this study was to evaluate methods for estimating the IgG concentration in cows' colostrum. This research included 126 colostrum samples from 21 Danish farms with different breeds, ensuring a broad variation pattern in IgG, total protein and fat concentration. Approximately one third of the samples did not fulfil the recommendation of >50 g IgG/l colostrum, and the IgG concentration decreased with time from calving to milking. The ratio of IgG to total protein varied from 6 to 61%, however IgG and total protein were correlated with r2 = 0.70. The variation in fat was independent of variations in protein and IgG. The IgG concentration was measured by ELISA and compared to fast measurements by specific gravity by colostrometer, Brix by refractometer and prediction from infrared spectroscopy. The three fast methods were all correlated to the total protein concentration of colostrum; however specific gravity was also influenced by the fat concentration. Furthermore, specific gravity generally overestimated the IgG concentration, and the cut-off level should be raised to 1050 in order to ensure adequate IgG in colostrum. None of the methods estimated IgG concentration better than the correlation of total protein and IgG, meaning that they all depended on the indirect correlation between total protein and IgG. The results suggest that using a refractometer for quality control of colostrum is an easy and feasible method, and a cut-off level of Brix 22 seems sufficient to assure adequate IgG concentration in colostrum fed to the calf.

Protein denaturation of whey protein isolates (WPIs) induced by high intensity ultrasound during heat gelation.

In this study, the impact of high intensity ultrasound (HIU) on proteins in whey protein isolates was examined. Effects on thermal behavior, secondary structure and nature of intra- and intermolecular bonds during heat-induced gelling were investigated. Ultrasonication (24 kHz, 300 W/cm(2), 2078 J/mL) significantly reduced denaturation enthalpies, whereas no change in secondary structure was detected by circular dichroism. The thiol-blocking agent N-ethylmaleimide was applied in order to inhibit formation of disulfide bonds during gel formation. Results showed that increased contents of α-lactalbumin (α-La) were associated with increased sensitivity to ultrasonication. The α-La:ß-lactoglobulin (ß-Lg) ratio greatly affected the nature of the interactions formed during gelation, where higher amounts of α-La lead to a gel more dependent on disulfide bonds. These results contribute to clarifying the mechanisms mediating the effects of HIU on whey proteins on the molecular level, thus moving further toward implementing HIU in the processing chain in the food industry.

The effect of butter grains on physical properties of butter-like emulsions.

Milk fat exists as globules in its natural state in milk. The potential of using globular fat to modulate the rheological properties and crystallization behavior in butter-like emulsions was studied in the present work. We conducted a comparative study of butter-like emulsions, with a fat phase consisting of 0, 10, 25, 50, or 100% anhydrous milk fat (AMF), the remaining fat being butter grains, and all samples containing 20% water, to obtain systematic variation in the ratio of globular fat. All emulsions were studied over 4wk of storage at 5°C. By combining small and large deformation rheology, we conducted a detailed characterization of the rheological behavior of butter-like emulsions. We applied differential scanning calorimetry to monitor thermal behavior, confocal laser scanning microscopy for microstructural analysis, and low-field pulsed nuclear magnetic resonance spectrometry to measure solid fat content. By combining these techniques, we determined that increasing the fraction of globular fat (by mixing with butter grains) decreases the hardness of butter-like emulsions up to an order of magnitude at d 1. However, no difference was observed in thermal behavior as a function of butter grain content, as all emulsions containing butter grains revealed 2 endothermal peaks corresponding to the high (32.7°C ± 0.6) and medium (14.6°C ± 0.1) melting fractions of fatty acids. In terms of microstructure, decreasing the amount of butter grains in the emulsions resulted in formation of a denser fat crystal network, corresponding to increased hardness. Moreover, microstructural analysis revealed that the presence of butter grains resulted in faster formation of a continuous fat crystal network compared with the 100% AMF sample, which was dominated by crystal clusters surrounded by liquid oil. During storage, hardness remained stable and no changes in thermal behavior were observed, despite an increase in solid fat content of up to 5%. After 28d of storage, we observed no difference in either microstructural or rheological properties, indicating that formation of primary bonds occurs primarily within the first day of storage. The rheological behavior of butter-like emulsions is not determined solely by hardness, but also by stiffness related to secondary bonds within the fat crystal network. The complex rheological behavior of milk fat-based emulsions is better characterized using multiple parameters.