Iron saturation and binding capacity of lactoferrin - development and validation of a colorimetric protocol for quality control.

Among the numerous biofunctional properties of lactoferrin, its ability to bind iron ions can be considered a core function. The saturation level with ferric iron affects the stability and functionality of the protein. To reliably quantify the iron saturation, an assay based on the color reagent Ferrozine was developed and validated concerning the lower detection (0.023 µg mL-1) and quantification limits (0.069 µg mL-1), as well as precision, recovery and accuracy values. The established assay was used to monitor iron uptake, comparing two commercially available bovine lactoferrin powders. Significant differences between the samples were observed. One sample exhibited nearly ideal binding behavior with a high affinity for ferric iron (saturation > 98 %), while the comparison sample did not exceed saturation values >80 %. This finding underscores the importance of assessing the iron status and binding capacity for the quality evaluation of lactoferrin products.

Reproducing high mechanical load during industrial processing of UHT milk: Effect on frothing capacity.

In this study, possible reasons for an increased level of free fatty acids (FFAs) in Ultra-High Temperature (UHT) treated full fat (3.5% wt/wt) milk and its effect on the frothing properties of milk were investigated. Lipolysis of raw milk from 2 different breeds of cattle (Holstein and Jersey) was induced by mechanical stress and kinetics of lipolysis were compared. Frothing capacity and foam stability of shelf stable milk with different concentrations of FFAs were determined, with a good to medium initial foam volume for up to 4 mEquiv FFA · (100 g fat)-1 fat and poor foam stability with >2 mEquiv FFA · (100 g fat)-1. A combination of mechanical stress and initial condition of fresh raw milk was found to trigger lipolysis and potential sources of mechanical stress during milk processing were identified.

Modelling of dynamic mastication physics of cheese/casein gels as function of milk-fat distribution and intensity of thermo-mechanical treatment.

Pasta filata-style cheese products are among the world's most famous cheese varieties. Thermo-mechanical processing of cheese curd results in stringy, fibrous, and anisotropic structures with pleasing texture attributes. A recent area of research focuses on improving yield during the manufacturing of pasta filata-type cheese products by homogenizing the milk. This process reduces the size of fat droplets, leading to better retention of milk fat during curd plasticization. As this sometimes results in texture deficits, this study aims to investigate the impact of thermo-mechanical processing on curd from homogenized and non-homogenized milk. The hypothesis is that increased thermo-mechanical processing, leading to more anisotropic structural elements, may offset texture deficits caused by homogenization. To assess textural and structural changes due to homogenization and thermo-mechanical processing, mechanical tests including rheology and texture analysis were conducted, along with confocal-laser-scanning microscopy. Additionally, sensory evaluation involving panelists consuming the samples and recording mastication properties such as muscle activity and jaw movement was carried out. Dynamic data modeling was used to derive connections between structure and texture. Results showed that homogenization alone did not yield significant differences between the samples, but plasticization and texturization properties differed significantly. Non-homogenized samples developed a distinct fibrous structure, and muscle activities and jaw movements increased significantly (p < 0.01) with longer thermo-mechanical processing.

Formation of Key Aroma Compounds During 30 Weeks of Ripening in Gouda-Type Cheese Produced from Pasteurized and Raw Milk.

Gouda-type cheeses were produced on a pilot-scale from raw milk (RM-G) and pasteurized milk (PM-G). Sixteen key aroma compounds previously characterized by the sensomics approach were quantitated in the unripened cheeses and at five different ripening stages (4, 7, 11, 19, and 30 weeks) by means of stable isotope dilution assays. Different trends were observed in the formation of the key aroma compounds. Short-chain free fatty acids and ethyl butanoate as well as ethyl hexanoate continuously increased during ripening but to a greater extent in RM-G. Branched-chain fatty acids such as 3-methylbutanoic acid were also continuously formed and reached a 60-fold concentration after 30 weeks, in particular in PM-G. 3-Methylbutanal and butane-2,3-dione reached a maximum concentration after 7 weeks and decreased with longer ripening. Lactones were high in the unripened cheeses and increased only slightly during ripening. Recent results have shown that free amino acids were released during ripening. The aroma compounds 3-methylbutanal, 3-methyl-1-butanol, and 3-methylbutanoic acid are suggested to be formed by microbial enzymes degrading the amino acid l-leucine following the Ehrlich pathway. To gain insight into the quantitative formation of each of the three aroma compounds, the conversion of the labeled precursors (13C6)-l-leucine and (2H3)-2-keto-4-methylpentanoic acid into the isotopically labeled aroma compounds was studied. By applying the CAMOLA approach (defined mixture of labeled and unlabeled precursor), l-leucine was confirmed as the only precursor of the three aroma compounds in the cheese with the preferential formation of 3-methylbutanoic acid.

Influence of Milk Pasteurization on the Key Aroma Compounds in a 30 Weeks Ripened Pilot-Scale Gouda Cheese Elucidated by the Sensomics Approach.

Gouda cheese was produced from pasteurized milk and ripened for 30 weeks (PM-G). By application of gas chromatography/olfactometry and an aroma extract dilution analysis on the volatiles isolated by extraction/SAFE distillation, 25 odor-active compounds in the flavor dilution (FD) factor range from 16 to 4096 were identified. Butanoic acid, 2- and 3-methylbutanoic acid, and acetic acid showed the highest FD factors, and 2-phenylethanol, δ-decalactone, and δ-dodecalactone were most odor-active in the neutral-basic fraction. Quantitations by stable isotope dilution assays followed by a calculation of odor activity values (OAVs) revealed acetic acid, 3-methylbutanoic acid, butanoic acid, and butane-2,3-dione with the highest OAVs. Finally, an aroma recombinate prepared based on the quantitative data well agreed with the aroma profile of the PM-G. In Gouda cheese produced from raw (nonpasteurized) milk (RM-G), qualitatively the same set of odor-active compounds was identified. However, higher OAVs of butanoic acid, hexanoic acid, and their corresponding ethyl esters were found. On the other hand, in the PM-G, higher OAVs for 3-methylbutanoic acid, 3-methylbutanol, 3-methylbutanal, and butane-2,3-dione were determined. The different rankings of these key aroma compounds clearly reflect the aroma differences of the two Gouda-type cheeses. A higher activity of lipase in the RM-G and higher amounts of free l-leucine in PM-G on the other side were responsible for the differences in the concentrations of some key aroma compounds.

Does the high biodiversity of lactococcal bacteriophages allow predictions about their different UV-C susceptibilities?

Fermentation processes can only succeed if intact and active starter cultures are present. Bacteriophages, which can lyse bacteria and thus bring entire fermentation processes to a standstill, therefore pose a major threat. Cheese production, for example, is often affected. The by-product whey can be highly contaminated with bacteriophages (≤109 plaque-forming units/mL) and in this state, further utilization is a quality and processing risk. Therefore, an orthogonal process consisting of membrane filtration followed by UV-C irradiation could be applied to eliminate bacteriophages and to generate "phage-free" whey. In order to define suitable process parameters, 11 lactococcal bacteriophages belonging to different families and genera and differing in their morphology, genome size, heat resistance, and other attributes, were screened for their UV-C resistance in whey. P369 was found to be the most resistant and could thus be well-suited as a biomarker. Starting from a 4 log unit bacteriophage reduction by membrane filtration, another 5 log unit decrease should be realized when applying a UV-C dose of 5 J/cm2. A clear correlation of UV-C sensitivity to the chosen attributes studied such as bacteriophage morphology and genome size was difficult and ambiguous, presumably because other yet unidentified parameters are important. Mutation experiments were performed with the representative bacteriophage P008 by multiple cycles of UV-C irradiation and propagation. A few mutational events were found, but could not be linked to an artificially generated UV-C resistance, indicating that the process used would probably not lose its effectiveness over time.

Enrichment of Lactoferrin and Immunoglobulin G from Acid Whey by Cross-Flow Filtration.

The production of cream cheese, curd, high-protein yogurt, or caseinate results in large amounts of acid whey as a by-product. So far acid whey is often disposed as animal feed or organic fertilizer. However, these approaches ignore the valorization potential that arises from the unique composition of the whey protein fraction. Whey contains the biofunctional proteins lactoferrin and immunoglobulin G, which possess immune-supporting, antibacterial, antiviral, and numerous further health-promoting functions. However, the concentration of these proteins in bovine milk or whey is below a physiologically relevant level. Based on literature research we specified a daily intake of 200 mg lactoferrin as the minimal functional dose. By means of cross-flow ultrafiltration, an attempt was made to increase the concentration of biofunctional proteins. Therefore, a membrane for the selective retention of lactoferrin and immunoglobulin G was identified, and the process parameters were optimized. Finally, a concentration experiment was conducted, whereby the concentration of biofunctional proteins was increased up to factor 30. The biofunctionality was assessed in a microbiological assay. Surprisingly, the antimicrobial growth inhibition of the produced concentrate was even higher than in pure lactoferrin. The presented approach offers a strategy to convert an abundant but underutilized by-product into valuable products for human nutrition.

The behavior of cathepsin D during milk processing and its contribution to bitterness in a model fresh cheese.

The bovine endopeptidase cathepsin D was investigated regarding its temperature-dependent inactivation and ability to form bitter peptides within a spiked model fresh cheese. Cathepsin D was found to be more susceptible than other milk endogenous peptidases to temperature treatments in skim milk. Inactivation kinetics revealed decimal reduction times of 5.6 min to 10 s in a temperature range from 60 to 80°C. High temperature and ultra-high temperature (UHT) treatments from 90 to 140°C completely inactivated cathepsin D within 5 s. A residual cathepsin D activity of around 20% was detected under pasteurization conditions (72°C for 20 s). Therefore, investigations were done to estimate the effect of residual cathepsin D activity on taste in a model fresh cheese. The UHT-treated skim milk was spiked with cathepsin D and acidified with glucono-δ-lactone to produce a model fresh cheese. A trained bitter-sensitive panel was not able to distinguish cathepsin D-spiked model fresh cheeses from the control model fresh cheeses in a triangle test. Model fresh cheese samples were also analyzed for known bitter peptides derived from casein fractions using a HPLC-tandem mass spectrometry (MS) approach. In accordance with the sensory evaluation, the MS analyses revealed that the bitter peptides investigated within the cathepsin D-spiked model fresh cheese were not found or were below the limit of detection. Even though cathepsin D may be present during the fermentation of pasteurized milk, it does not seem to be responsible for bitter peptide formation from milk proteins on its own.

Expansion of the concept of critical micelle concentration for the application of a saturated monoacylglyceride emulsifier in aerosol whipping cream.

The concept of critical micelle concentration (CMC) is an important principle for the application of emulsifiers in various fields, not least in the production of food emulsions. In general, the application of emulsifier concentrations > CMC, often 10-20 times CMC, is suggested, to ensure the generation of stable emulsions. Typically, the concept of CMC is only discussed for emulsions or foams, but not simultaneously for liquid emulsions that are to be foamed. This study investigates the CMC concept for aerosol whipping cream (AWC), a system in which emulsion stability has to be guaranteed during storage and foam build up and stability has to be enabled upon foaming. Model AWC (30 g·100 g-1 fat; 6/1 MPa) was processed in the presence of various concentrations of a medium chain saturated monoacylglyceride (cme-sMAG: 0 - 2.0 g·100 g-1). It was found that similarly to the CMC concept in emulsions, the application of me-sMAG in model AWC has to be divided into three sections: i) cme-sMAG < CMC; ii) cme-sMAG ≈ CMC; iii) cme-sMAG > CMC.

Technologies for sustainable heat generation in food processing.

The utilization of heat is one of the foundations of modern food processing. At present, boilers that operate on fossil fuels are still dominating the generation of hot water, steam, and hot air in the food industry. In light of sustainability goals and carbon taxes as well as international efforts to reduce the dependence on natural gas, new technologies are needed to lower the greenhouse gas emissions related to thermal processing of foods. This review discusses important technologies that could serve as a replacement for conventional fossil fuel boilers in the future. These technologies are based on electricity, geothermal energy (direct/indirect use), and electricity to hydrogen conversion and include fuel cells, microturbines, engines, electrical boilers, heat pumps, radiation, and use of geothermal energy. The majority of these technologies are already available for implementation at larger scales and emissions are generally lower compared to burning fossil fuels. At present, major obstacles, such as low fossil fuel prices, still exist that prevent the widespread adoption of more sustainable heating technologies. However, the direct transformation of electrical energy and utilization of geothermal energy for heating purposes seem promising and should be more frequently installed in the future, whereas the use of H2 obtained through electrolysis as a transportable source of energy may also serve as a source of thermal energy where it is useful to generate electricity and heat on the production site or where the availability of electricity is limited.

Tailoring the Textural Characteristics of Fat-Free Fermented Concentrated Milk-Protein Based Microgel Dispersions by Way of Upstream, Downstream and Post-Production Thermal Inputs.

There is a growing demand for new strategies to tailor the texture of fat-free fermented concentrated milk products, also referred to as milk protein-based (MPb) microgel dispersions. Methods should be easy to incorporate into the production scheme, offer labelling without added components and be cost-efficient. Thermal treatments are traditionally used upstream (milk heating) and downstream (pre-concentration heating) in the production of these dispersions, though there is little knowledge as to the effects that combinations of different thermal input levels have on final texture. Therefore, this study investigated combinations of thermal input at different intensities and steps in the production scheme at the pilot scale and the relationships with texture. We demonstrated that increasing the intensity of upstream milk heat treatment, in combination with downstream pre-concentration heating, increases gel firmness and apparent viscosity. Downstream pre-concentration heating produces final fat-free fermented concentrated MPb microgel particles that are resistant to post-heating aggregation. On the other hand, omission of downstream pre-concentration heating results in smaller particles that are sensitive to post-heating aggregation. Furthermore, gel firmness and apparent viscosity increase with post-heating. Consequently, combining different levels of thermal inputs upstream, downstream (pre-concentration) and post-production, can produce fat-free fermented concentrated MPb microgel dispersions with a range of different textures.

What a difference a gas makes: Effect of foaming on dynamic aroma release and perception of a model dairy matrix.

When we are trying to decrease caloric intake by reducing fat content, aroma perception of the food is changed as well. Since previous studies indicated that foaming changes the aroma release, it was our goal to understand the physico-chemical background of this effect. Therefore, ten aroma compounds were added to a foamed acidified dairy matrix (4% milk protein, 1% gelatin, 60% gas volume). We simulated oral temperature conditions in a simplistic way through incubation at 40 °C and analyzed aroma release using headspace-solid phase microextraction-gas chromatography-ion mobility spectrometry. Significantly more highly hydrophobic aroma compounds were released from the foamed matrix than the unfoamed matrix, while compounds of intermediate hydrophobicity were released more from unfoamed matrix. The effect was independent from foam collapse and persisted for hours afterwards. Analytical results were complemented by orthonasal and retronasal sensory perception studies, which confirmed significant differences between aroma release behavior from foamed foods.

Fat-free fermented concentrated milk products as milk protein-based microgel dispersions: Particle characteristics as key drivers of textural properties.

The popularity of fat-free fermented concentrated milk products, such as fresh cheeses and high-protein yogurt, has increased over the recent years, attributed to greater availability and improvements in taste and texture. These improvements have been achieved through modifications and new developments in processing technologies, for example, higher heat treatment intensities and incorporating different membrane filtration technologies. Though numerous processing parameters are discussed in the literature, as well as reasons behind the developments, detailed examinations of how process modifications affect the final textural attributes of these products are lacking. To draw links between processing parameters and texture, we review the literature on fat-free fermented concentrated milk products from the perspective of fermented milk protein-based microgel particles as the basic structural unit. At each main processing step, relationships between process parameters, micro- and macrostructural and sensory (textural) properties are discussed.An overview of particle characteristics that drive structural changes at each processing step is developed in relation to textural characteristics. Using this approach of assessing relationships between structural characteristics of concentrated dispersions of fat-free fermented milk protein-based microgel particles and processing parameters provides a basic context for the selection of optimal parameters to achieve a desired texture.

Effects of Protein, Calcium, and pH on Gene Transcription, Cell-Envelope Peptidase Activity of Lactococcus lactis Strains, and the Formation of Bitter Peptides.

Calcium- and protein-rich fermented milk products, such as concentrated yoghurts and fresh cheeses, may contain undesired bitter peptides, which are generated by the proteolytic cleavage of casein. Up to now, it is not clear whether this process is caused by endogenous milk enzymes, such as plasmin and cathepsin D, or whether proteolytic enzymes from applied starter cultures, such as the lactococcal cell-envelope peptidase PrtP, are involved. A sensory analysis of fresh cheese products made from milk concentrates fermented with prtP-negative and -positive Lactococcus lactis strains revealed bitterness in the products fermented with prtP-positive L. lactis strains. Two prtP-positive strains, LTH 7122 and LTH 7123, were selected to investigate the effect of increased calcium concentrations (additional 5 mM and 50 mM CaCl2) at neutral (pH 6.6) and acidic (pH 5.5) pH-values on the transcription of the prtP gene and its corresponding PrtP peptidase activity in milk citrate broth (MCB). For both strains, it was shown that prtP transcription was upregulated only under slightly elevated calcium conditions (5 mM CaCl2) after 5 h of growth. In concordance with these findings, PrtP peptidase activity also increased. When higher concentrations of calcium were used (50 mM), prtP expression of both strains decreased strongly by more than 50%. Moreover, PrtP peptidase activity of strain LTH 7123 decreased by 15%, but enzymatic activity of strain LTH 7122 increased slightly during growth under elevated calcium concentrations (50 mM CaCl2). Fermentations of reconstituted casein medium with 3.4% (w/v) and 8.5% (w/v) protein and different calcium concentrations using strain LTH 7122 revealed no clear relationship between prtP transcription and calcium or protein concentration. However, an increase in PrtP peptidase activity under elevated protein and calcium conditions was observed. The activity increase was accompanied by increased levels of bitter peptides derived from different casein fractions. These findings could be a possible explanation for the bitterness in fermented milk concentrates that was detected by a trained bitter panel.

Predictive modeling of the early stages of semi-solid food ripening: Spatio-temporal dynamics in semi-solid casein matrices.

A mechanistic, spatio-temporal model to predict early stage semi-solid food ripening, exemplary for semi-solid casein matrices, was created using software based on the finite element method (FEM). The model was refined and validated by experimental data obtained during 8 wk of ripening of a casein matrix that was inoculated by one single central injection of starter culture. The resulting spatio-temporal distributions of lactococci strains, lactose, lactic acid/lactate and pH allowed us to optimize a number of parameters of the predictive model. Using the optimized model, the agreement between simulation and experiment was found to be satisfactory, with the pH matching best. The predictive model unveiled that effective diffusion of substrate and metabolites were crucial for an eventual homogeneous distribution of the measured substances. Hence, while using the optimized parameters from the single injection model, an injection technology for starter culture to inoculate and ferment casein matrices homogeneously was developed by means of solving another optimization problem with respect to injection positions. The casein matrix inoculated by the proposed injection pattern (21 injections, distance = 19 mm) showed sufficient homogeneity (bacterial activity and pH distribution) after the early stages of ripening, demonstrating the potential of application of the injection technology for fermentation of casein-based foods e.g. cheese.

Off-flavor in soy drink: Development, optimization, and validation of an easy and fast method to quantify the key odorants.

A detailed molecular flavor profile was necessary to understand the low acceptance of soy drink by Western consumers. Accordingly, key odor-active compounds were detected by means of aroma dilution analyses coupled with gas chromatography-mass spectrometry-olfactometry after application of suitable solvent-free volatile extraction techniques. Four quantification methods (standard addition, external calibration, internal standard, and stable isotope dilution assay) were developed and validated to measure the concentrations after direct immersion-stir bar sorptive extraction. The quantitative methods provided correctness between 97% and 111% and precision ranging from 78% to 99% for the 21 key odorants. Considering the advantages to be efficient, easy to perform and cheap, internal standard method was further applied to four commercially available soy drinks in Germany. Correlated to a sensory acceptance test (n = 52) contents of 1-octen-3-one, (E,E)-2,4-decadienal and 2-methoxy-4-vinylphenol were suggested to be linked to the aversion of Western consumers to soy drink.

Assessing Whey Protein Sources, Dispersion Preparation Method and Enrichment of Thermomechanically Stabilized Whey Protein Pectin Complexes for Technical Scale Production.

Whey protein pectin complexes can be applied to replace fat in food products, e.g., pudding and yogurt, contributing to creaminess while adding a source of protein and fiber. Production of these complexes is usually conducted on the laboratory scale in small batches. Recently, a process using a scraped-surface heat exchanger (SSHE) has been employed; however, dispersion preparation time, feasibility of using different whey protein sources and enrichment of the complexes for subsequent drying have not been assessed. Preparing whey protein pectin dispersions by solid mixing of pectin and whey protein powders resulted in larger complexes than powders dispersed separately and subsequently mixed after a hydration time. Dispersions without hydration of the mixed dispersions before thermomechanical treatment had the largest particle sizes. The targeted particle size of d90,3 < 10 µm, an important predictor for creaminess, was obtained for five of the six tested whey protein sources. Dispersions of complexes prepared using whey protein powders had larger particles, with less particle volume in the submicron range, than those prepared using whey protein concentrates. Efficiency of complex enrichment via acid-induced aggregation and subsequent centrifugation was assessed by yield and purity of protein in the pellet and pectin in the supernatant.

Studying dynamic aroma release by headspace-solid phase microextraction-gas chromatography-ion mobility spectrometry (HS-SPME-GC-IMS): method optimization, validation, and application.

To understand aroma perception from complex food matrices' determination of dynamic aroma release during simulated oral processing is necessary. In this study optimization, validation and application of a novel method coupling headspace-solid phase microextraction (HS-SPME) with gas chromatography-ion mobility spectrometry (GC-IMS) is presented. Thirteen character impact compounds imparting different chemical properties are studied to understand capabilities and limitations of the method. It was shown for the first time that the temperature of the IMS sample inlet can be increased up to 200 °C without instrumental constraints. Linear calibration was possible for eleven of the thirteen compounds with one decade dynamic range. The limit of detection and quantitation were 2.1-63.0 ppb and 7.2-210.1 ppb, respectively. Diacetyl could be detected in negative polarity mode of IMS, however with lower precision compared to the compounds detected in positive mode. Limitations of the method were short HS-SPME extraction time, which in the case of caproic acid was not sufficient for reliable quantification. Additionally, δ-decalactone could not be detected due to maximum GC temperature of 200 °C. Application of the method to determine dynamic aroma release from a dairy matrix was successfully shown for nine compounds. Analysis of complex food matrix was performed with similar precision compared to analysis in aqueous solution, thus proving high robustness of the method towards matrix effects.

Cover Feature: Parameter and state estimation of backers yeast cultivation with a gas sensor array and unscented Kalman filter.

DOI: 10.1002/elsc.202000058 Successful operation, control and optimization of biotechnological process depend on reliable real-time available measurements of the process variables. Although some hardware sensors are readily available, they often have several drawbacks: cost, sample destruction, discrete-time measurements, processing delay, sterilization, disturbances in the hydrodynamic conditions inside the bioreactor, etc. It is therefore of interest to use software sensors [29, 30]. The central idea behind a soft sensor is to use easily accessible on-line data for the estimation of other process variables that are either difficult to measure or only measured at low frequency [30]. The figure illustrates a software sensor for on-line monitoring of substrate and biomass production in backers yeast cultivation. For details see article DOI 10.1002/elsc.202000058 on page 169.

Parameter and state estimation of backers yeast cultivation with a gas sensor array and unscented Kalman filter.

Real-time information about the concentrations of substrates and biomass is the key to accurate monitoring and control of bioprocess. However, on-line measurement of these variables is a challenging task and new measurement systems are still required. An alternative are software sensors, which can be used for state and parameter estimation in bioprocesses. The software sensors predict the state of the process by using mathematical models as well as data from measured variables. The Kalman filter is a type of such sensors. In this paper, we have used the Unscented Kalman Filter (UKF) which is a nonlinear extension of the Kalman filter for on-line estimation of biomass, glucose and ethanol concentration as well as for estimating the growth rate parameters in S. cerevisiae batch cultivation, based on infrequent ethanol measurements. The UKF algorithm was validated on three different cultivations with variability of the substrate concentrations and the estimated values were compared to the off-line values. The results obtained showed that the UKF algorithm provides satisfactory results with respect to estimation of concentrations of substrates and biomass as well as the growth rate parameters during the batch cultivation.