Prediction of Pectin Yield and Quality by FTIR and Carbohydrate Microarray Analysis

Pectin production is complex, and final product quality assessment is generally accomplished at the end of the process using time-consuming off-line laboratory analysis. In this study, pectin was extracted from lime peel either by acid or by enzymes. Fourier transform infrared spectroscopy and carbohydrate microarray analysis were performed directly on the crude lime peel extracts during the time course of the extractions. Multivariate analysis of the data was carried out to predict final pectin yields. Fourier transform infrared spectroscopy (FTIR) was found applicable for determining the optimal extraction time for the enzymatic and acidic extraction processes, respectively. The combined results of FTIR and carbohydrate microarray analysis suggested major differences in the crude pectin extracts obtained by enzymatic and acid extraction, respectively. Enzymatically extracted pectin, thus, showed a higher degree of esterification (DE 82 %) than pectin extracted by acid (DE 67 %) and was moreover found to be more heterogeneously esterified when probed with the monoclonal antibodies JIM5, JIM7, and LM20. The data infer that enzymatic pectin extraction allows for extraction of complex, high DE pectin, and that FTIR and carbohydrate microarray analysis have potential to be developed into online process analysis tools for prediction of pectin extraction yields and pectin features from measurements on crude pectin extracts.     

Prediction and validation of residual feed intake and dry matter intake in Danish lactating dairy cows using mid-infrared spectroscopy of milk

The present study explored the effectiveness of Fourier transform mid-infrared (FT-IR) spectral profiles as a predictor for dry matter intake (DMI) and residual feed intake (RFI). The partial least squares regression method was used to develop the prediction models. The models were validated using different external test sets, one randomly leaving out 20% of the records (validation A), the second randomly leaving out 20% of cows (validation B), and a third (for DMI prediction models) randomly leaving out one cow (validation C). The data included 1,044 records from 140 cows; 97 were Danish Holstein and 43 Danish Jersey. Results showed better accuracies for validation A compared with other validation methods. Milk yield (MY) contributed largely to DMI prediction; MY explained 59% of the variation and the validated model error root mean square error of prediction (RMSEP) was 2.24 kg. The model was improved by adding live weight (LW) as an additional predictor trait, where the accuracy R² increased from 0.59 to 0.72 and error RMSEP decreased from 2.24 to 1.83 kg. When only the milk FT-IR spectral profile was used in DMI prediction, a lower prediction ability was obtained, with R² = 0.30 and RMSEP = 2.91 kg. However, once the spectral information was added, along with MY and LW as predictors, model accuracy improved and R² increased to 0.81 and RMSEP decreased to 1.49 kg. Prediction accuracies of RFI changed throughout lactation. The RFI prediction model for the early-lactation stage was better compared with across lactation or mid- and late-lactation stages, with R² = 0.46 and RMSEP = 1.70. The most important spectral wavenumbers that contributed to DMI and RFI prediction models included fat, protein, and lactose peaks. Comparable prediction results were obtained when using infrared-predicted fat, protein, and lactose instead of full spectra, indicating that FT-IR spectral data do not add significant new information to improve DMI and RFI prediction models. Therefore, in practice, if full FT-IR spectral data are not stored, it is possible to achieve similar DMI or RFI prediction results based on standard milk control data. For DMI, the milk fat region was responsible for the major variation in milk spectra; for RFI, the major variation in milk spectra was within the milk protein region.     

Effect of exogenous phytase on feed inositol phosphate hydrolysis in an in vitro rumen fluid buffer system

Three in vitro experiments using a rumen fluid buffer system were performed to investigate the effect of addition of 4 experimental phytases (Phy1, Phy2, Phy3, and Phy4) compared with no addition of phytase on feed inositol phosphate hydrolysis in wheat and rapeseed cake to determine which of the 4 phytases was most suitable under rumen-like conditions. The feedstuffs were incubated with a mixture of physiological buffer, ruminal fluid, and exogenous phytase at pH 6.2, after which the samples were incubated for different periods. Incubations were stopped using HCl, and the samples were analyzed for inositol phosphates via high performance ion chromatography. Addition of phytase (Phy1) resulted in enhanced degradation of myo-inositol hexakisphosphate (InsP₆) in rapeseed cake, whereas addition of exogenous phytase did not improve the degradation of InsP₆ in wheat. Only rapeseed cake was therefore used subsequently. All 4 phytases increased degradation of InsP₆ in rapeseed cake in the in vitro system, and degradability of InsP₆ increased with higher incubation time and higher phytase dosages, independent of phytase. Addition of 2 units of phytase per gram of substrate of the phytases Phy1, Phy2, Phy3, and Phy4 led to an undegraded InsP₆ content of 56, 49, 70, and 18%, respectively, when incubated with rapeseed cake for 6 h, indicating that Phy2 and Phy4 were the most effective phytases. However, Phy2 had a higher specific activity than Phy4, as 60% of the original InsP₆ content was remaining after 3 h when 5 mg of enzyme protein per gram of substrate of Phy2 was added to rapeseed cake, whereas 150 mg of enzyme protein per gram of substrate of Phy4 was necessary to achieve a similar result. Therefore, Phy2 appeared to be most applicable under rumen-like conditions.     

Water properties and structure of pork sausages as affected by high-pressure processing and addition of carrot fibre

The effects of high-pressure processing (HPP) and addition of carrot fibre on pork sausages have been studied using NMR T? relaxometry and measurements of water-binding capacity (WBC) by centrifugation. Significant effects of temperature (raw, 40, 50, or 60 °C), holding time (1s, 3, 6, or 9 min), and addition of carrot fibre on the distribution and mobility of water were found. However, the effect of carrot fibre could not be explained by structural changes in the sausages when examined by confocal laser scanning microscopy (CLSM). Correlations between T? relaxation measurements and WBC determined by centrifugation revealed that T? relaxation times were able to explain more than 90% of the variation in WBC for both non-pressure and pressure-treated sausages. However, only 49% of the variation was explained for pressure-treated sausages with carrot fibre, indicating that combining addition of fibre and high pressure treatment causes non-coherent changes in T? NMR relaxation times.     

Composition and effect of blending of noncoagulating, poorly coagulating, and well-coagulating bovine milk from individual Danish Holstein cows

The aim of the present investigation was to study the underlying causes of noncoagulating (NC) milk. Based on an initial screening in a herd of 53 Danish Holstein-Friesians, 20 individual Holstein-Friesian cows were selected for good and poor chymosin-induced coagulation properties; that is, the 10 cows producing milk with the poorest and best coagulating properties, respectively. These 20 selected cows were followed and resampled on several occasions to evaluate possible changes in coagulation properties. In the follow-up study, we found that among the 10 cows with the poorest coagulating properties, 4 cows consistently produced poorly coagulating (PC) or NC milk, corresponding to a frequency of 7%. Noncoagulating milk was defined as milk that failed to form a coagulum, defined as increase in the storage modulus (G′) in oscillatory rheometry, within 45 min after addition of chymosin. Poorly coagulating milk was characterized by forming a weak coagulum of low G′. Milk proteomic profiling and contents of different casein variants, ionic contents of Ca, P and Mg, κ-casein (CN) genotypes, casein micelle size, and coagulation properties of the 4 NC or PC samples were compared with milk samples of 4 cows producing milk with good coagulation properties. The studies included determination of production of caseinomacropeptide to ascertain whether noncoagulation could be ascribed to the first or second phase of chymosin-induced coagulation. Caseinomacropeptide was formed in all 8 milk samples after addition of chymosin, indicating that the first step (cleavage of κ-CN) was not the cause of inability to coagulate. Furthermore, the effect of mixing noncoagulating and well-coagulating milk was studied. By gradually blending NC with well-coagulating milk, the coagulation properties of the well-coagulating samples were compromised in a manner similar to titration. Milk samples from cows that consistently produced NC milk were further studied at the udder quarter level. The coagulation properties of the quarter milk samples were not significantly different from those of the composite milk sample, showing that poor coagulation traits and noncoagulation traits of the composite milk were not caused by the milk quality of a single quarter. The milk samples exhibiting PC or NC properties were all of the κ-CN variant AA genotype, and contained casein micelles with a larger mean diameter and a lower fraction of κ-CN relative to total CN than milk with good coagulation properties. Interestingly, the relative proportions of different phosphorylation forms of α-CN differed between well-coagulating milk and PC or NC milk samples. The PC and NC milk samples contained a lower proportion of the 2 less-phosphorylated variants of α-CN (α_S1-CN-8P and α_S2-CN-11P) compared with samples of milk that coagulated well.     

Flexible and fixed partitions in freestalls--effects on lying behavior and cow preference

The objective was to investigate the effect of stall partition design on total lying time, lying position, and stall cleanliness, and to evaluate the preferences of cows regarding stalls with traditional fixed stall dividers or flexible stall dividers. Using a crossover design, 16 nonlactating dairy cows were housed singly for 9 d in pens with 2 freestalls, 1 with fixed cantilever dividers and 1 with flexible dividers. The cows were first given access to one stall type, and then to the other type of stall, and finally to both in a preference test. Type of stall divider did not influence lying behavior (13.5 h for fixed versus 14.0 h for flexible, ± 0.4 h), lying positions, or stall cleanliness; however, the cows showed a preference for lying in the flexible stalls (65.2 for flexible vs. 34.8 for fixed ± 8.2%). This indicated that cows are able to distinguish between type of stall divider and that it is important to them; however, it is not clear if the reason for this is the shape or the properties of the dividers. We concluded that cattle chose a flexible stall divider over a fixed one, but the long-term consequences of this preference are not clear, because no obvious changes in stall usage were observed when cows were only given access to one type of divider.     

Blood plasma sample preparation method for the assessment of thyroid hormone-disrupting potency in effect-directed analysis

A sample preparation method combining solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was developed to be used in Effect-Directed Analysis (EDA) of blood plasma. Until now such a method was not available. It can be used for extraction of a broad range of thyroid hormone (TH)-disruptors from plasma with high recoveries. Validation of the method using spiked cow plasma showed good recoveries for hydroxylated polybrominated diphenyl ethers (OH-PBDEs; 93.8 ± 19.5%), hydroxylated polychlorinated biphenyls (OH-PCBs; 93.8 ± 15.5%), other halogenated phenols (OHPs; 107 ± 8.1%), and for short-chain (<8 C-atoms) perfluoroalkyl substances (PFASs; 85.2 ± 24.6%). In the same extracts, the potency of the compound classes spiked to the cow plasma to competitively bind to transthyretin (TTR) was recovered by 84.9 ± 8.8%. Furthermore, the SPE-LLE method efficiently removed endogenous THs from the extracts, thereby eliminating their possible contribution to the binding assay response. The SPE-LLE method was applied to polar bear plasma samples to investigate its applicability in future EDA studies focusing on TH-disrupting compounds in this top predator species that is exposed to relatively high levels of bioaccumulating pollutants. A first screening revealed TTR-binding potency in the polar bear plasma extracts, which could be explained for 60-85% by the presence of OH-PCBs.     

Detection of seven virulence and toxin genes of Campylobacter jejuni isolates from Danish turkeys by PCR and cytolethal distending toxin production of the isolates

A total of 117 Campylobacter jejuni isolates from Danish turkeys were tested for the presence of seven virulence and toxin genes by PCR. One hundred seventeen (100%) isolates were positive for flaA, cadF, and ceuE gene primers. One hundred three (88%) isolates were positive for cdt gene cluster PCR detection (cdt gene cluster-PCR), whereas 101 (86.3%), 102 (87.2%), and 110 (94%) isolates were positive for cdtA-, cdtB-, and cdtC-PCR, respectively. Only 39 (33.3%) isolates were positive for virB11. Of 117 isolates, 114 (97.4%) produced cytolethal distending toxin (CDT) in Vero cell assays, 105 (89.7%) in Colon 205 assays, and 109 (93.2%) in chicken embryo cell assays. The CDT titers were determined in Vero cell assays. Of 117 isolates, 50 (42.7%) produced a CDT titer of 1:100, 29 (24.8%) of 1:50, and 27 (23%) of 1:5 to 1:10; 8 (6.8%) produced a CDT titer at undiluted supernatants and 3 (2.6%) produced no toxin. Twenty-nine C. jejuni isolates that were PCR negative for one or more individual cdt toxin genes also produced low or no CDT toxin. The high prevalence of the seven virulence and toxin genes demonstrates that these putative pathogenic determinants are widespread among Campylobacter isolates from turkeys and calls for further investigation for the elimination of Campylobacter infection in industrial turkey production and in industrial food chains.     

Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase

Peptides from hydrolysates of fish proteins and from cheeses were analysed for inhibition of prolyl endopeptidase (PE) isolated from porcine muscle. Muscles of cod, salmon, and trout were homogenised and incubated at pH 4.0 with pepsin and then at pH 7.5 with trypsin to obtain fish protein hydrolysates. Homogenates were incubated without exogenous enzymes at pH 4.0 and 7.5 to obtain fish protein autolysates. Water-soluble extracts from "rakfisk" (a Norwegian fermented/autolysed trout muscle dish) and water-soluble extracts from Cheddar, Norvegia, Jarlsberg, and Blue cheese were also prepared. Peptides in the supernatants obtained after heat-treatment of fish hydrolysates, autolysates and water-soluble extracts of rakfisk and cheeses at 95 degrees C for 15 min were analysed for inhibition of PE. Inhibition was also measured in peptide fractions separated by reversed-phase high-performance chromatography and by gel permeation chromatography. The peptide fractions from fish hydrolysates, fish autolysates, and water-soluble extracts of cheeses inhibited PE in hydrolysing Z-Gly-Pro-amidomethylcoumarin. Inhibition by peptides from rakfisk was negligible. Pepsin + trypsin hydrolysates from the three fish species contained PE inhibitory peptides with a broad range of apparent hydrophobicity and apparent molecular mass. Autolysates from muscles of the 3 fish species contained narrow peptide peaks of different molecular mass and different apparent hydrophobicity with strong PE inhibitory activity. The content of hydrophilic inhibitory peptides was lower in cheeses than in pepsin + trypsin hydrolysates of fish muscle.