Mid-infrared spectroscopy predictions as indicator traits in breeding programs for enhanced coagulation properties of milk

The aims of this study were to investigate variation of milk coagulation property (MCP) measures and their predictions obtained by mid-infrared spectroscopy (MIR), to investigate the genetic relationship between measures of MCP and MIR predictions, and to estimate the expected response from a breeding program focusing on the enhancement of MCP using MIR predictions as indicator traits. Individual milk samples were collected from 1,200 Brown Swiss cows (progeny of 50 artificial insemination sires) reared in 30 herds located in northern Italy. Rennet coagulation time (RCT, min) and curd firmness (a₃₀, mm) were measured using a computerized renneting meter. The MIR data were recorded over the spectral range of 4,000 to 900 cm⁻¹. Prediction models for RCT and a₃₀ based on MIR spectra were developed using partial least squares regression. A cross-validation procedure was carried out. The procedure involved the partition of available data into 2 subsets: a calibration subset and a test subset. The calibration subset was used to develop a calibration equation able to predict individual MCP phenotypes using MIR spectra. The test subset was used to validate the calibration equation and to estimate heritabilities and genetic correlations for measured MCP and their predictions obtained from MIR spectra and the calibration equation. Point estimates of heritability ranged from 0.30 to 0.34 and from 0.22 to 0.24 for RCT and a₃₀, respectively. Heritability estimates for MCP predictions were larger than those obtained for measured MCP. Estimated genetic correlations between measures and predictions of RCT were very high and ranged from 0.91 to 0.96. Estimates of the genetic correlation between measures and predictions of a₃₀ were large and ranged from 0.71 to 0.87. Predictions of MCP provided by MIR techniques can be proposed as indicator traits for the genetic enhancement of MCP. The expected response of RCT and a₃₀ ensured by the selection using MIR predictions as indicator traits was equal to or slightly less than the response achievable through a single measurement of these traits. Breeding strategies for the enhancement of MCP based on MIR predictions as indicator traits could be easily and immediately implemented for dairy cattle populations where routine acquisition of spectra from individual milk samples is already performed.     

Genetic parameters of coagulation properties, milk yield, quality, and acidity estimated using coagulating and noncoagulating milk information in Brown Swiss and Holstein-Friesian cows

The aim of this study was to estimate heritabilities of rennet coagulation time (RCT) and curd firmness (a₃₀) and their genetic correlations with test-day milk yield, composition (fat, protein, and casein content), somatic cell score, and acidity (pH and titratable acidity) using coagulating and noncoagulating (NC) milk information. Data were from 1,025 Holstein-Friesian (HF) and 1,234 Brown Swiss (BS) cows, which were progeny of 54 HF and 58 BS artificial insemination sires, respectively. Milk coagulation properties (MCP) of each cow were measured once using a computerized renneting meter and samples not exhibiting coagulation within 31 min after rennet addition were classified as NC milk. For NC samples, RCT was unobserved. Multivariate analyses, using Bayesian methodology, were performed to estimate the genetic relationships of RCT or a₃₀ with the other traits and statistical inference was based on the marginal posterior distributions of parameters of concern. For analyses involving RCT, a right-censored Gaussian linear model was used and records of NC milk samples, being censored records, were included as unknown parameters in the model implementing a data augmentation procedure. Rennet coagulation time was more heritable [heritability (h²) = 0.240 and h² = 0.210 for HF and BS, respectively] than a₃₀ (h² = 0.148 and h² = 0.168 for HF and BS, respectively). Milk coagulation properties were more heritable than a single test-day milk yield (h² = 0.103 and h² = 0.097 for HF and BS, respectively) and less heritable than milk composition traits whose heritability ranged from 0.275 to 0.275, with the only exception of fat content of BS milk (h² = 0.108). A negative genetic correlation, lower than −0.85, was estimated between RCT and a₃₀ for both breeds. Genetic relationships of MCP with yield and composition were low or moderate and favorable. The genetic correlation of somatic cell score with RCT in BS cows was large and positive and even more positive were those of RCT with pH and titratable acidity in both breeds, ranging from 0.80 to 0.94. Including NC milk information in the data affected the estimated correlations and decreased the uncertainty associated with the estimation process. On the basis of the estimated heritabilities and genetic correlations, enhancement of MCP through selective breeding with no detrimental effects on yield and composition seems feasible in both breeds. Milk acidity may play a role as an indicator trait for indirect enhancement of MCP.     

Influence of fatty acid chain length and unsaturation on mid-infrared milk analysis

Our first objective was to optimize center wavelengths and bandwidths for virtual filters used in a Fourier transform mid-infrared (MIR) milk analyzer. Optimization was accomplished by adjusting center wavelengths and bandwidths to minimize the size of intercorrection factors. Once optimized, the virtual filters were defined as follows: fat B sample, 3.508 μm (2,851 cm⁻¹), and bandwidth of 0.032 μm (26 cm⁻¹); fat B reference, 3.556 μm (2812 cm⁻¹), and bandwidth of 0.030 μm (24 cm⁻¹); lactose sample, 9.542 μm (1,048 cm⁻¹), and bandwidth of 0.092 μm (20 cm⁻¹); lactose reference, 7.734 μm (1,293 cm⁻¹), and bandwidth of 0.084 μm (14 cm⁻¹); protein sample, 6.489 μm (1,541 cm⁻¹), and bandwidth of 0.085 μm (20 cm⁻¹); protein reference, 6.707 μm (1491 cm⁻¹), and bandwidth of 0.054 μm (12 cm⁻¹); fat A sample, 5.721 μm (1,748 cm⁻¹), and bandwidth of 0.052 μm (16 cm⁻¹); fat A reference, 5.583 μm (1,791 cm⁻¹), and bandwidth of 0.050 μm (16 cm⁻¹). The bandwidth and its proximity to areas of intense water absorption had the largest effect on the intercorrection factors. The second objective was to quantify the impact of fatty acid chain length and unsaturation on fat B and fat A MIR measurements. Increasing the chain length increased the difference (i.e., MIR minus reference) between MIR prediction and reference chemistry by 0.0429% and by −0.0566% fat per unit of increase in carbon number per 1% change in fat, for fat B and fat A, respectively. Increasing the unsaturation decreased the difference (i.e., MIR minus reference) between MIR prediction of fat and chemistry for fat B by −0.4021% and increased fat A by 0.0291% fat per unit of increase in double bonds per 1% change in fat concentration.     

Mid infrared attenuated total reflection spectroscopy as a rapid tool to assess the quality of Sicilo-Sarde ewe’s milk during the lactation period after replacing soybean meal with scotch bean in the feed ration

This study investigates the potential of attenuated total reflection spectroscopy in the mid infrared (MIR) for monitoring changes in the quality of ewe’s milk as a function of lactation period and feeding systems. Twelve 5-year-old lactating Sicilo-Sarde ewes (third lambing) were kept in environmentally controlled sheepfolds and were divided into two homogenous weight matched groups (n = 6). Ewes were fed ad libitum with two iso-energetic diets (20% barley, 3% vitamin and mineral premix, and 77% soybean meal or scotch bean). Physico–chemical analyses and MIR (3000–900 cm⁻¹) were performed on milk samples after 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 weeks of lactation period. The inclusion of scotch bean in the diet resulted in a significant decrease (P ? 0.05) of fat content (7.85 g 100 g⁻¹ vs. 6.75 g 100 g⁻¹) and a significant increase (P ? 0.05) of lactose level (3.49 g 100 g⁻¹ vs. 3.61 g 100 g⁻¹). The principal component analysis (PCA) applied to the 1700–1500 cm⁻¹ spectral region showed only some discrimination between milk samples according to diet compositions. The best results were obtained in the 3000–2800 cm⁻¹ and 1500–900 cm⁻¹ spectral regions since a good discrimination between milk from ewes fed soybean meal from those fed scotch bean meal was observed. It can be concluded that these spectral regions could be considered as fingerprint, regions allowing a good identification of milk according to diet composition. However, the MIR failed to discriminate milk samples according to the lactation period for the two feeding systems.     

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10⁵ CFU mL⁻¹ of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL⁻¹) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10⁶–10⁷ CFU mL⁻¹ by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.     

A spectroscopic and chemometric study of virgin olive oils subjected to thermal stress

The paper describes a study of thermal stress of three different samples of virgin olive oil in terms of oxidative stability. Fatty acid composition, evaluation of oxidative stability under forced conditions (OSI), determination of UV-spectrophotometric oxidation indexes (k₂₃₂ and k₂₇₀) and spectral properties were explored along the thermal treatment. The samples were subjected to heating treatment at 180 °C and evaluated after 0, 30, 60, 90, 120, 150 and 180 min. Middle infrared (MIR) and visible–near infrared (Vis–NIR) spectra were elaborated by partial least squares modelling to individualise regions and bands where critical variations were present. Two bands were found as principal influential ones (1245–1180 cm⁻¹ and 1150–1030 cm⁻¹) on MIR while one primary region was identified on Vis–NIR (2200–1325 cm⁻¹).     

Utilisation of mid-infrared spectroscopy for determination of the geographic origin of Gruyère PDO and L’Etivaz PDO Swiss cheeses

The potential of mid-infrared spectroscopy (MIR), using an attenuated total reflectance (ATR) cell, was evaluated for the authentication of 25 Gruyère PDO and L’Etivaz PDO cheeses produced at different altitudes in Switzerland. In order to test the ability of MIR to authenticate the investigated cheeses, chemometric tools, such as principal component analysis (PCA) and factorial discriminant analysis (FDA), were applied to the three spectral regions of the MIR (e.g. 3000–2800 cm⁻¹, 1700–1500 cm⁻¹, and 1500–900 cm⁻¹). By applying the FDA to the first 10 principal components (PCs) of the PCA applied to each spectral regions, the best rate of correct classification was obtained in the 3000–2800 cm⁻¹ and 1500–900 cm⁻¹ spectral regions, since 90.5% and 90.9% were achieved, respectively. It can be concluded that these two spectral regions could be considered as valuable tools for the determination of the geographical origin of the investigated cheeses.     

Genetic parameters of milk coagulation properties and their relationships with milk yield and quality traits in Italian Holstein cows

Milk coagulation properties (MCP) are an important aspect in assessing cheese-making ability. Several studies showed that favorable conditions of milk reactivity with rennet, curd formation rate, and curd strength, as well as curd syneresis, have a positive effect on the entire cheese-making process and subsequently on the ripening of cheese. Moreover, MCP were found to be heritable, but little scientific literature is available about their genetic aspects. The aims of this study were to estimate heritability of MCP and genetic correlations among MCP and milk production and quality traits. A total of 1,071 Italian Holstein cows (progeny of 54 sires) reared in 34 herds in Northern Italy were sampled from January to July 2004. Individual milk samples were collected during the morning milking and analyzed for coagulation time (RCT), curd firmness (a₃₀), pH, titratable acidity, fat, protein, and casein contents, and somatic cell count. About 10% of individual milk samples did not coagulate in 31 min, so they were removed from the analyses. Estimates of heritability for RCT and a₃₀ were 0.25 ± 0.04 and 0.15 ± 0.03, respectively. Estimates of genetic correlations between MCP traits and milk production traits were negligible except for a₃₀ with protein and casein contents (0.44 ± 0.10 and 0.53 ± 0.09, respectively). Estimates of genetic correlations between MCP traits and somatic cell score were strong and favorable, as well as those between MCP and pH and titratable acidity. Selecting for high casein content, milk acidity, and low somatic cell count might be an indirect way to improve MCP without reducing milk yield and quality traits.     

Milk coagulation ability of five dairy cattle breeds

Samples of herd milk (506) were analyzed to assess sources of variation for milk coagulation properties (MCP) for 5 different dairy cattle breeds. Data were recorded in 55 single-breed dairy herds in the Trento province, a mountain area in northeast Italy. The 5 cattle breeds were Holstein-Friesian (8 herds), Brown Swiss (16 herds), Simmental (10 herds), Rendena (13 herds), and Alpine Gray (8 herds). Herd milk samples were analyzed for the MCP traits, milk rennet coagulation time (RCT), curd-firming time, and curd firmness (a₃₀), as well as protein and fat percentages, somatic cell count, Soxhlet-Henkel acidity, and bacterial count. An ANOVA was performed to study the effect of breed, herd within breed, DIM, month of lactation, protein and fat percentages, somatic cell score, titratable acidity, and log bacterial count within breed on MCP. Breed was the most important source of variation. In particular, the Rendena breed showed the best MCP traits at 13.5 min and 27.0 mm for RCT and a₃₀, respectively. The Holstein-Friesian breed had the worst coagulation properties at 18.0 min and 17.5 mm for RCT and a₃₀, respectively. The other 3 breeds showed intermediate coagulation properties. The RCT values were better at the beginning of lactation, whereas RCT and a₃₀ values were better in September and October (14.3 min and 25.7 mm, respectively). Among the composition traits, only the titratable acidity affected MCP traits of herd milk positively.     

Effectiveness of mid-infrared spectroscopy for prediction of the contents of calcium and phosphorus,and titratable acidity of milk and their relationship with milk quality and coagulation properties

Individual milk samples from Holstein Friesian cows were collected and analysed by inductively coupled plasma optical emission spectrometry (ICP-OES) and titration for the determination of calcium (Ca), phosphorus (P) and titratable acidity (TA) contents, respectively. Prediction models were obtained using partial least squares (PLS) regression analyses using two statistical packages. The average Ca, P and TA were 1156 mg kg⁻¹, 934 mg kg⁻¹ and 3.42 °SH 50 mL⁻¹, respectively. Pearson's correlations between Ca and P and other milk traits were significant (P < 0.05) and ranged from 0.16 to 0.53 for chemical composition traits and from 0.17 to −0.35 for milk coagulation properties (MCP). Results from the two statistical packages were comparable. Prediction models using MIR spectroscopy were satisfactory for Ca, P and TA, with coefficients of correlation of cross-validation greater than 0.73. Moreover, the study highlighted favourable relationships of these traits with milk coagulation properties.     

Mid infrared and fluorescence spectroscopies coupled with factorial discriminant analysis technique to identify sheep milk from different feeding systems

Mid infrared spectroscopy (MIR) combined with multivariate data analysis was used to discriminate between ewes milk samples according to their feeding systems (controls, ewes fed scotch bean and ewes fed soybean). The MIR spectra were scanned throughout the first 11 weeks of the lactation stage. When factorial discriminant analysis (FDA) with leave one-out cross-validation was applied, separately, to the three spectral regions in the MIR (i.e. 3000–2800, 1700–1500 and 1500–900 cm⁻¹), the classification rate was not satisfactory. Therefore, the first principal component (PCs) scores (corresponding to 3, 10 and 10 for, respectively, the 3000–2800, 1700–1500 and 1500–900 cm⁻¹) of the principal component analysis (PCA) extracted from each of the data sets were pooled (concatenated) into a single matrix and analysed by FDA. Correct classification amounting to 71.7% was obtained. Finally, the same procedure was applied to the MIR and fluorescence data sets and 98% of milk samples were found to be correctly classified. Milk samples belonging to control and soybean groups were 100% correctly classified. Regarding milk samples originating from the scotch bean group, only 2 out of 33 samples were misclassified. It was concluded that concatenation of the data sets collected from the two spectroscopic techniques is an efficient tool for authenticating milk samples according to their feeding systems, regardless of the lactation stage.     

Selection for milk coagulation properties predicted by Fourier transform infrared spectroscopy in the Italian Holstein-Friesian breed

Milk coagulation is based on a series of physicochemical changes at the casein micelle level, resulting in formation of a gel. Milk coagulation properties (MCP) are relevant for cheese quality and yield, important factors for the dairy industry. They are also evaluated in herd bulk milk to reward or penalize producers of Protected Designation of Origin cheeses. The economic importance of improving MCP justifies the need to account for this trait in the selection process. A pilot study was carried out to determine the feasibility of including MCP in the selection schemes of the Italian Holstein. The MCP were predicted in 1,055 individual milk samples collected in 16 herds (66 ± 24 cows per herd) located in Brescia province (northeastern Italy) by means of Fourier transform infrared (FTIR) spectroscopy. The coefficient of determination of prediction models indicated moderate predictions for milk rennet coagulation time (RCT = 0.65) and curd firmness (a₃₀ = 0.68), and poor predictions for curd-firming time (k₂₀ = 0.49), whereas the range error ratio (8.9, 6.9, and 9.5 for RCT, k₂₀, and a₃₀, respectively) indicated good practical utility of the predictive models for all parameters. Milk proteins were genotyped and casein haplotypes (α_S1-, β-, α_S2-, and κ-casein) were reconstructed. Data from 51 half-sib families (19.9 ± 16.4 daughters per sire) were analyzed by an animal model to estimate (1) the genetic parameters of predicted RCT, k₂₀, and a₃₀; (2) the breeding values for these predicted clotting variables; and (3) the effect of milk protein genotypes and casein haplotypes on predicted MCP (pMCP). This is the first study to estimate both genetic parameters and breeding values of pMCP, together with the effects of milk protein genotypes and casein haplotypes, that also considered k₂₀, probably the most important parameter for the dairy industry (because it indicates the time for the beginning of curd-cutting). Heritability of predicted RCT (0.26) and k₂₀ (0.31) were close to the average heritability described in literature, whereas the heritability of a₃₀ was higher (0.52 vs. 0.27). The effects of milk proteins were statistically significant and similar to those obtained on measured MCP. In particular, haplotypes including uncommon variants showed positive (B-I-A-B) or negative (B-A¹-A-E) effects. Based on these findings, FTIR spectroscopy-pMCP is proposed as a potential selection criterion for the Italian Holstein.     

Short communication: Influence of composite casein genotypes on additive genetic variation of milk production traits and coagulation properties in Holstein-Friesian cows

The aim of the study was to quantify the effects of composite β- and κ-casein (CN) genotypes on genetic variation of milk coagulation properties (MCP); milk yield; fat, protein, and CN contents; somatic cell score; pH; and titratable acidity (TA) in 1,042 Italian Holstein-Friesian cows. Milk coagulation properties were defined as rennet coagulation time (RCT) and curd firmness (a₃₀). Variance components were estimated using 2 animal models: model 1 included herd, days in milk, and parity as fixed effects and animal and residual as random effects, and model 2 was model 1 with the addition of composite β- and κ-CN genotype as a fixed effect. Genetic correlations between RCT and a₃₀ and between these traits and milk production traits were obtained with bivariate analyses, based on the same models. The inclusion of casein genotypes led to a decrease of 47, 68, 18, and 23% in the genetic variance for RCT, a₃₀, pH, and TA, respectively, and less than 6% for other traits. Heritability of RCT and a₃₀ decreased from 0.248 to 0.143 and from 0.123 to 0.043, respectively. A moderate reduction was found for pH and TA, whereas negligible changes were detected for other milk traits. Estimates of genetic correlations were comparable between the 2 models. Results show that composite β- and κ-CN genotypes are important for RCT and a₃₀ but cannot replace the recording of MCP themselves.     

Migration of α-tocopherol and resveratrol from poly(L-lactic acid)/starch blends films into ethanol

Poly(L-lactic acid) (PLLA)/starch blends with various concentrations of two natural antioxidants, α-tocopherol (α-TOC) and resveratrol, were fabricated by a melt blending and compression molding processes. The effects of the two antioxidants on the optical (color), thermal and mechanical properties of PLLA/starch blends with antioxidants were assessed. PLLA/starch blend films with α-TOC and resveratrol showed a yellowish color influenced by the combined effect of white starch and the brown color of the antioxidants. The glass transition and melting temperatures were significantly reduced with the addition of antioxidants while enhanced thermal stability was observed, which could be a benefit and important for processing and production. The enhanced mechanical properties could be attributed to not only a compatibilization effect based on the chemical linkage between PLLA and starch chains, but also restriction of the chain mobility by antioxidants. The release of resveratrol from PLLA and PLLA/starch blend films into ethanol followed Fickian behavior. The D values of α-TOC were in the range of 0.47–3.95 × 10⁻¹¹ cm² s⁻¹ for PLLA films and 0.70–6.83 × 10⁻¹¹ cm² s⁻¹ for PLLA/starch blend films at 13 °C, 5.67–13.0 × 10⁻¹¹ cm² s⁻¹ for PLLA films and 4.10–24.2 × 10⁻¹¹ cm² s⁻¹ for PLLA/starch blend films at 23 °C, and 89.0–118.0 × 10⁻¹¹ cm² s⁻¹ for PLLA films and 123–282 × 10⁻¹¹ cm² s⁻¹ for PLLA/starch blend films at 43 °C. The D values of resveratrol were in the range of 0.073–0.54 × 10⁻¹⁰ cm² s⁻¹ for PLLA films and 1.42–6.93 × 10⁻¹⁰ cm² s⁻¹ for PLLA/starch blend films at 13 °C, 0.90–3.44 × 10⁻¹⁰ cm² s⁻¹ for PLLA films and 4.16–22.3 × 10⁻¹⁰ cm² s⁻¹ for PLLA/starch blend films at 23 °C, and 24.8–74.1 × 10⁻¹⁰ cm² s⁻¹ for PLLA films and 40.1–309 × 10⁻¹⁰ cm² s⁻¹ for PLLA/starch blend films at 43 °C.     

Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: Purification and biochemical characterisation

A dimeric serine protease Neriifolin S of molecular mass 94 kDa with milk clotting activity has been purified from the latex of Euphorbia neriifolia by anion exchange and size-exclusion chromatography. It hydrolyses peptidyl substrates l-Ala-pNA with highest affinity (K_m of 0.195 mM) and physiological efficiency (K_cat/K_m of 144.5 mM s). Enzyme belongs to the class of neutral proteases with pI value of 6.8, optimal proteolytic activity displayed at pH 9.5 and temperature 45 °C. Its proteolytic activity is strongly stimulated in the presence of Ca⁺² ions and exclusively inhibited by serine protease inhibitors. Enzyme is fairly stable toward chemical denaturants, pH and temperature. The apparent T_m, was found to be 65 °C. Thermal inactivation follow first order kinetics with activation energy (Ea), activation enthalpy (ΔH∗), free energy change (ΔG∗) and entropy (ΔS∗) of 27.54 kJ mol⁻¹, 24.89 kJ mol⁻¹, −82.34 kJ mol⁻¹ and 337.20 J mol⁻¹ K⁻¹.     

Utilization of fourier transform infrared spectroscopy for measurement of organic phosphorus and bound calcium in cheddar cheese

The methods available for measuring organic P and bound Ca in cheese are either cumbersome or involve dilution of the cheese. Dilution of the cheese can lead to erroneous results, particularly in the case of bound Ca. Hence, the objective of this study was to evaluate the feasibility of Fourier transform infrared (FTIR) spectroscopy for direct measurement of organic P and bound Ca in Cheddar cheese. Two hundred sixteen samples of cheese were analyzed for protein-bound organic P, bound Ca using a water-extraction based method, and buffering curves. Additionally, the infrared spectra of the cheeses were collected between 4,000 and 650 cm⁻¹, at a resolution of 4 cm⁻¹, and 256 scans per sample. The spectral shifts in the infrared region from 1,050 to 900 cm⁻¹, in addition to the measured concentrations of organic P, bound Ca, and buffering peak area at pH 5.1, were used to develop calibration models using partial least squares (PLS) regression analysis. The spectral region of 956 to 946 cm⁻¹ correlated with the measured concentrations of organic P and the overall PLS model had a correlation (R²) of 0.76 between the predicted and measured concentrations. The spectral region at ∼980 cm⁻¹ was correlated with the measured concentrations of bound Ca, and the overall PLS model had a correlation (R²) of 0.70 between the predicted and measured concentrations. A similar spectral region at ∼980 cm⁻¹ was also correlated with the measured buffering peak areas and the overall PLS model had a correlation (R²) of 0.64 between the predicted and measured peak areas. A linear regression analysis between the bound Ca and buffering peak area demonstrated that bound Ca was correlated (R² = 0.73) with buffering peak area. This study demonstrates that FTIR can be used to measure organic P in cheeses. It also has the potential to be used for measuring bound Ca in undiluted cheeses, and for prediction of the buffering capacity of cheese.     

Thermal inactivation of lactoperoxidase in goat,sheep and bovine milk – A comparative kinetic and thermodynamic study

Using isothermal heating, inactivation of lactoperoxidase (LPO) in goat, sheep and cow milk was studied in the temperature range of 70–77 °C. Kinetic and thermodynamics studies were carried out at different time–temperature combination in order to evaluate the suitability of LPO as marker for the heat-treatment of milk and dairy products from different species. The thermal inactivation of LPO followed the first-order kinetics. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The influence of temperature on the inactivation rate constant was quantified using the Arrhenius and thermal death time models. The corresponding z-values were 3.38 ± 0.013, 4.11 ± 0.24 and 3.58 ± 0.004 °C in goat, sheep and cow milk, respectively. Activation energy values varied between milk species with 678.96 ± 21.43 kJ mol⁻¹ in goat milk, 560.87 ± 28.18 kJ mol⁻¹ in sheep milk and 641.56 ± 13.12 kJ mol⁻¹ in cow milk, respectively.     

Immobilization of pepsin on chitosan beads

In this study, chitosan beads were prepared by using a cross-linking agent and the resulting beads were employed in immobilization process. Studies on free and immobilized pepsin systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, storage stability and kinetic parameters were carried out. The optimum temperature interval for free pepsin and immobilized pepsin were 30–40 and 40–50 °C, respectively. Free and immobilized pepsin showed higher activity at pH 2.0–4.0. Apparent K_m = 12.0 g L⁻¹ haemoglobin (1.56 mM tyrosine) and V_max = 5220 μmol (mg protein min)⁻¹ values were obtained for free pepsin, while apparent K_m = 20.0 g L⁻¹ haemoglobin (2.16 mM tyrosine) and V_max = 2780 μmol (mg protein min)⁻¹ values were obtained for immobilized pepsin. Thermal stability and storage stability of immobilized pepsin were higher than that of free pepsin. Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process. Optimum milk clotting temperature was found as 40 °C for free pepsin and 50 °C for immobilized pepsin.     

Kinetic and thermodynamic study of thermal inactivation of the antimicrobial peptide P34 in milk

The knowledge on thermal inactivation of biopreservatives in a food matrix is essential to allow their proper utilisation in food industry, enabling the reduction of heating times and optimisation of heating temperatures. In this work, thermal inactivation of the antimicrobial peptide P34 in skimmed and fat milk was kinetically investigated within the temperature range of 90–120 °C. The inactivation kinetic follows a first-order reaction with k-values between 0.071 and 0.007 min⁻¹ in skimmed milk, and 0.1346 and 0.0119 min⁻¹ in fat milk. At high temperatures, peptide P34 was less resistant in fat milk, with a significant decrease in residual activity as compared with skimmed milk. At temperatures below 110 °C, the fat globules seem to have protective effect to the peptide P34. Results suggest that peptide P34 is heat stable in milk with activation energy of 90 kJ mol⁻¹ in skimmed milk and 136 kJ mol⁻¹ in fat milk.