Mechanisms behind protein-protein interactions in a ß-lg-legumin co-precipitate.

Interactions between pea protein and whey protein isolates in co-precipitates and blends consist of a combination of disulphide bonds, hydrophobic and electrostatic interactions. The present study aims to clarify if the two proteins with free thiols, ß-lactoglobulin (ß-lg) and legumin, played a significant role for these interactions. This study used different reagents to modify the conditions of interactions: N-ethylmaleimide (NEM) was used to block reactive thiols, while NaCl and SDS were used to prevent electrostatic or hydrophobic interactions, respectively. The effects of treatments were studied on protein solubility, structure and stability. SDS had no effect, while NEM and NaCl both had great effect, especially in combination. The results showed that interactions of ß-lg and legumin in both co-precipitates and blends are a synergism of electrostatic interactions and disulphide bonds. Thus, ß-lg and legumin are the main proteins responsible for previously observed interactions in protein isolates of whey and pea.

Influence of type of dairy matrix micro- and macrostructure on in vitro lipid digestion.

Recent research indicates that the food matrix can influence digestion kinetics and uptake of nutrients, thus affecting human health. The aim of this study was to obtain knowledge on how variations in microstructure and texture of foods represented by four dairy products; (i) cheddar cheese, (ii) a homogenized cheddar cheese, (iii) a micellar casein and cream drink or (iv) a micellar casein and cream gel, all of identical nutrient ratios of protein : fat and calcium : fat, affect the in vitro digestibility kinetics of lipids. Rheology of the four dairy structures was measured at 10 °C and 37 °C before digestion, and during the gastric phase of in vitro digestion. During digestion cheddar cheese was most resistant to enzymatic and mechanical disintegration, followed by homogenized cheese, while both the drink and gel had low resistance and dissolved in the gastric juice. Particle size, fat droplet size and microstructure were assessed by light scattering and confocal microscopy during digestion. Significantly larger fat droplets were observed during digestion of the cheddar cheese sample. The release of free fatty acids during the initial intestinal digestion showed cheddar cheese to provide a significantly lower release than homogenized cheese, whereas the drink and gel both had significantly higher free fatty acid release. The results suggest that the cheese matrix resistance to degradation and its large fat droplets were responsible for a slower fat digestion.

Effect of heating strategies on whey protein denaturation--Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry.

Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of ß-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5-soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment.

Identification of important mechanical and acoustic parameters for the sensory quality of cocoa butter alternatives.

In this study, the correlation between sensory attributes and the mechanical and acoustic properties of cocoa butter alternatives was elucidated. Needle penetration, cone penetration and compression tests were used to characterise mechanical properties and acoustic properties were evaluated by simultaneous texture and sound analyses. Results were correlated with a descriptive sensory evaluation. A significant correlation was found between hardness (needle penetration) and sensory hardness evaluated upon biting (r=0.91, p<0.05) and between Hencky strain (compression test) and the sensory toughness (r=0.94, p<0.05). In contrast, no significant correlation was found between brittleness (cone penetration) and the sensory brittleness. The use of different mechanical methods shed light on a complex rheological behaviour of fat which demonstrates the importance of not simply relying on results from penetration tests when evaluating fat texture. For instance, a hard fat was perceived very differently depending on the degree of elasticity. A significant correlation was found between sound pressure level (simultaneous sound and texture analyses) and the sensory evaluation of the sound intensity upon breakage (r=0.96 and 0.97, p<0.05). Both hardness and elasticity were found to be of great importance for the intensity of the sound emission i.e. a hard texture with a low degree of flexibility (less elastic) is more likely to provide a rapid energy release upon breakage and thus a high intensity sound emission.

Distinct composition of bovine milk from Jersey and Holstein-Friesian cows with good, poor, or noncoagulation properties as reflected in protein genetic variants and isoforms.

The objective of this study was to examine variation in overall milk, protein, and mineral composition of bovine milk in relation to rennet-induced coagulation, with the aim of elucidating the underlying causes of milk with impaired coagulation abilities. On the basis of an initial screening of 892 milk samples from 42 herds with Danish Jersey and Holstein-Friesian cows, a subset of 102 samples was selected to represent milk with good, poor, or noncoagulating properties (i.e., samples that within each breed represented the most extremes in regard to coagulation properties). Milk with good coagulation characteristics was defined as milk forming a strong coagulum based on oscillatory rheology, as indicated by high values for maximum coagulum strength (G'(max)) and curd firming rate (CFR) and a short rennet coagulation time. Poorly coagulating milk formed a weak coagulum, with a low G'(max) and CFR and a long rennet coagulation time. Noncoagulating milk was defined as milk that failed to form a coagulum, having G'(max) and CFR values of zero at measurements taken within 1h after addition of rennet. For both breeds, a lower content of total protein, total casein (CN) and κ-CN, and lower levels of minerals (Ca, P, Mg) were identified in poorly coagulating and noncoagulating milk in comparison with milk with good coagulation properties. Liquid chromatography/electrospray ionization-mass spectrometry revealed the presence of a great variety of genetic variants of the major milk proteins, namely, α(S1)-CN (variants B and C), α(S2)-CN (A), ß-CN (A(1), A(2), B, I, and F), κ-CN (A, B, and E), α-lactalbumin (B), and ß-lactoglobulin (A, B, and C). In poorly coagulating and noncoagulating milk samples of both breeds, the predominant composite genotype of α(S1)-, ß-, and κ-CN was BB-A(2)A(2)-AA, which confirmed a genetic contribution to impaired milk coagulation. Interestingly, subtle variations in posttranslational modification of CN were observed between the coagulation classes in both breeds. Poorly coagulating and noncoagulating milk contained a lower fraction of the least phosphorylated α(S1)-CN form, α(S1)-CN 8P, relative to total α(S1)-CN, along with a lower fraction of glycosylated κ-CN relative to total κ-CN. Thus, apparent variation was observed in the milk and protein composition, in the genetic makeup of the major milk proteins, and in the posttranslational modification level of CN between milk samples with either good or impaired coagulation ability, whereas the composition of poorly coagulating and noncoagulating milk was similar.

The effects of kale (Brassica oleracea ssp. acephala), basil (Ocimum basilicum) and thyme (Thymus vulgaris) as forage material in organic egg production on egg quality.

1. In organic egg production, forage material as part of the diet for laying hens is mandatory. The purpose of the present study was to examine the effect of feeding with forage materials including maize silage, herbs or kale on egg production and various egg quality parameters of the shell, yolk colour, egg albumen, sensory properties, fatty acid and carotenoid composition of the egg yolk. 2. A total of 5 dietary treatments were tested for 5 weeks, consisting of a basal organic feed plus 120 g/hen.d of the following forage materials: 1) maize silage (control), 2) maize silage incl. 15 g/kg basil, 3) maize silage incl. 30 g/kg basil, 4) maize silage incl. 15 g/kg thyme, or 5) fresh kale leaves. Each was supplied to three replicates of 20 hens. A total of 300 hens was used. 3. Feed intake, forage intake and laying rate did not differ with treatment, but egg weight and egg mass produced increased significantly with the kale treatment. 4. The egg shell strength tended to be higher with the kale treatment, and egg yolk colour was significantly more red with the kale treatment and more yellow with basil and kale treatments. The albumen DM content and albumen gel strength were lowest with the thyme treatment. By sensory evaluation, the kale treatment resulted in eggs with less sulphur aroma, higher yolk colour score, and more sweet and less watery albumen taste. Furthermore, the eggs of the kale treatment had significantly higher lutein and ß-carotene content. Also, violaxanthin, an orange xanthophyll, tended to be higher in kale and eggs from hens receiving kale. 5. In conclusion, forage material, especially basil and kale, resulted in increased egg production and eggs of high and differentiable quality.

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 45min 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.

Cooling causes changes in the distribution of lipoprotein lipase and milk fat globule membrane proteins between the skim milk and cream phase.

Lipoprotein lipase (LPL) activity and free fatty acid levels were studied in freshly milked, uncooled milk from individual Danish Holstein or Jersey cows, or after storage for up to 24h at either a cooling temperature (4°C) or at the milking temperature (31°C). Upon cooling for up to 24h, LPL activity increased in the cream phase, whereas the activity in the skim milk was steady, as observed for Jersey cows, or increased, as seen for the Holsteins. Storage at 31°C decreased the LPL activity in both the cream phase and the skim milk phase. The increase in free fatty acid levels was found to depend on LPL activity, incubation temperature, substrate availability, and incubation time. Furthermore, the migration of milk proteins between the skim milk phase and the cream phase upon cooling of milk from Jersey cows or from Danish Holstein cows was studied using proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry. Proteins associated with the milk fat globules were isolated from all milk fractions and analyzed. Major changes in the distributions of proteins between the skim milk phase and the cream phase were observed after cooling at 4°C for 4h, where a total of 29 proteins between the 2 breeds was found to change their association with the milk fat globule membrane (MFGM) significantly. Among these, the MFGM proteins adipophilin, fatty acid-binding protein, and lactadherin, as well as the non-MFGM proteins ß-casein, lactoferrin, and heat shock protein-71, were identified. Adipophilin, lactadherin, and lactoferrin were quantitatively more associated with the MFGM upon cold storage at 4°C, whereas ß-casein, fatty acid-binding protein, and heat shock protein-71 were found to be less associated with the MFGM upon cold storage.

Fermented feed for laying hens: effects on egg production, egg quality, plumage condition and composition and activity of the intestinal microflora.

1. An experiment with a total of 480 hens (Babcock) was carried out from 16 to 38 weeks of age to evaluate the suitability of wet fermented feed (feed water ratio, 1:1.2-1:1.4) for layers, taking aspects of nutrition and gastrointestinal health into consideration. The production performance, egg shell quality, plumage condition, litter dry matter (DM) content, as well as the composition and activity of the intestinal microbial flora were analysed. 2. Fermented feed was characterised by a high concentration of lactic acid (160-250 mmol/kg feed) and a moderate level of acetic acid (20-30 mmol/kg feed), high numbers of lactic acid bacteria (log 9-10 CFU/g feed) and a pH of approximately 4.5. Feed fermentation reduced the concentration of dietary sugar from 32.1 to 7.3 g/kg DM and the phytate bound phosphorus from 2.7 to 1.9 g/kg DM. 3. Fermented feed seemed to loose attractiveness for the birds quite rapidly, resulting in a more aggressive behaviour and a poorer plumage condition than in birds given dry feed. The use of fermented feed reduced the litter DM content. 4. During the experimental period, the body weight gain of hens receiving fermented feed was 80 g higher than of hens fed the dry mash. Presumably because of an extended adaptation time to the feed, the onset of lay occurred later when hens were fed on fermented feed, resulting in non-significantly reduced total egg production (75 vs. 82%). 5. There was no significant difference between groups with respect to the total egg mass production (g/d/hen, 42 and 45 for fermented feed and dry mash, respectively). Throughout the experimental period, the feed DM intake of hens fed with fermented feed was lower than that of hens receiving the dry mash (110 vs. 125 g). From week 26 to 37, fermented feed improved the feed conversion as compared with the dry mash (g feed DM/g egg mass, 2.28 vs. 2.53). 6. The use of fermented feed increased egg weight in the period from 34 to 37 weeks (61.4 vs. 60.0) and increased shell weight (g/100 g egg weight, 10.2 vs. 9.9) and shell stiffness (N/mm, 161 vs. 150) of eggs collected at 37 weeks. 7. The feeding of fermented feed increased intestinal health by acidification of the upper digestive tract, forming a natural barrier towards infection with acid sensitive pathogens, e.g. E. coli, Salmonella and Campylobacter. 8. It was concluded that fermented wet feed offers potential benefits for health and nutrition, but may become suitable for layers only after the practical problems related to this feeding form have been overcome. However, an early adaptation of the birds during the rearing period seems to be necessary.

Effects of blue lupin (Lupinus angustifolius) in organic layer diets and supplementation with foraging material on egg production and some egg quality parameters.

Six groups of organically fed hens were studied for egg production, feed parameters, and egg quality from 20 to 31 wk of age. Treatments were 3 diets (0, 15, and 25% blue lupin) with or without supplements of foraging material (whole carrots and corn silage). Increased lupin content increased nonstarch polysaccharides content and reduced methionine content below the hens' requirement. Egg production at 31 wk was lower with the 25% lupin diet without (69%) and with foraging material (76%) compared with the other diets (approximately 90%). Egg weight was highest with the 0% lupin diet (64 g), and 15% lupin diet (60 g), whereas the 25% lupin diet without and with foraging material resulted in egg weights of 58 and 56 g, respectively. Feed intakes were approximately 113 g of diet/ hen per d and 113 g of supplement/hen per d in 0 and 15% lupin treatments, respectively. Feeding the 25% lupin diet significantly reduced diet intake to approximately 91 g, and increased supplement intake to 118 g for the treatment with foraging material. Eggs from treatments with foraging material had significantly higher sulfur-like aftertaste in sensory evaluation. Yolk color became significantly lighter and more yellow with lupin content, but darker and less greenish with foraging material. Increased lupin levels decreased albumen DM, whereas foraging material had no effect. Inclusion of 25% lupin in layer diets is only recommended when supplying some methionine source, as egg production and quality parameters are dramatically impaired. However, supplement of foraging material significantly improves egg production.

Influence of aromatic components from pig manure on odour and flavour of cooked chicken meat.

An experiment with 96 broilers distributed across six treatments was carried out. Each treatment consisted of four cages each with four broilers. The six treatments were: the control (treatment 1), broilers placed in cages with a welded wire bottom hanging over smelling pig manure (treatment 2), or placed on a pig manure mat (treatment 3), combined with two intervals, i.e. 1 week (group a) and 2 weeks (group b) before slaughter. The content of skatole in the abdominal fat was measured, and the odour and flavour impressions after cooking were evaluated by a taste panel. The treatments were compared with two control treatments: broilers produced traditionally in cages either without (treatment 1a) or with (treatment 1b) contact with their own manure for a fortnight. A higher skatole level in abdominal fat of broilers in physical contact with pig manure (treatment 3) was measured, compared with broilers without physical contact with pig manure for at least 1 week (treatments 1 and 2) (p<0.05). The consumer taste panel evaluation was not affected by the fact that the broilers had been lying on pig manure. However, the evaluation of the odour of the cooked meat, when opening the cooking bag, was to some extent negatively affected by the experimental treatments of broilers, which had physical contact with the pig manure (p<0.05). Furthermore, physical contact with the manure, regardless of whether it was broiler or pig manure, proved to affect the odour of the meat (p<0.01). For broilers placed in cages without physical contact with the pig manure (treatments 2a and 2), the smell had no negative influence on either the odour or on the flavour of the meat, and the content of skatole was not different from that of the broilers on the control treatment. A taste panel evaluation performed on four broilers of the treatments 1a, 2b, and 3b showed no flavour differences among treatments. Whether the broilers had been exposed to a given treatment for 1 or 2 weeks made no difference, either to the skatole concentration or to the odour or flavour impression of the newly cooked chicken meat.