Influence of harvesting strategy on nutrient supply and production of dairy cows consuming diets based on grass and red clover silage

The main objective of this thesis was to elucidate the effects of regrowth grass silage and red clover silage on nutrient supply and milk production of dairy cows as compared with primary growth grass silages. In the first experiment (publication I), two primary growth and four regrowth grass silages were harvested at two stages of growth. These six silages were fed to 24 lactating dairy cows with two levels of concentrate allowance. Silage intake and energy corrected milk yield (ECM) responses, and the range in these response variables between the diets, were smaller when regrowth silages rather than primary growth silages were fed. Milk production of dairy cows reflected the intake of metabolizable energy (ME), and no differences in the ME utilization were found between the diets based on silages harvested from primary growth and regrowth. The ECM response to increased concentrate allowance was, on average, greater when regrowth rather than primary growth silages were fed. In the second experiment (publication II), two silages from primary growth and two from regrowth used in I were fed to rumen cannulated lactating dairy cows. Cows consumed less feed dry matter (DM), energy and protein, and produced less milk, when fed diets based on regrowth silages rather than primary growth silages. Lower milk production responses of regrowth grass silage diets were mainly due to the lower silage DM intake, and could not be accounted for by differences in energy or protein utilization. Regrowth grass silage intake was not limited due to neutral detergent fibre (NDF) digestion or rumen fill or passage kinetics. However, lower intake may be at least partly attributable to plant diseases such as leaf spot infections, dead deteriorating material or abundance of weeds, which are all higher in regrowth compared with primary growth, and increase with advancing regrowth. In the third experiment (publications III and IV), red clover silages and grass silages harvested at two stages of growth, and a mixed diet of red clover and grass silages, were fed to five rumen cannulated lactating dairy cows. In spite of the lower average ME intake for red clover diets, the ECM production remained unchanged suggesting more efficient utilisation of ME for red clover diets compared with grass diets. Intake of N, and omasal canal flows of total non-ammonia N (NAN), microbial and non-microbial NAN were higher for red clover than for grass silage diets, but were not affected by forage maturity. Delaying the harvest tended to decrease DM intake of grass silage and increase that of red clover silage. The digestion rate of potentially digestible NDF was faster for red clover diets than for grass silage diets. Delaying the harvest decreased the digestion rate for grass but increased it for red clover silage diets. The low intake of early-cut red clover silage could not be explained by silage digestibility, fermentation quality, or rumen fill but was most likely related to the nutritionally suboptimal diet composition because inclusion of moderate quality grass silage in mixed diet increased silage DM intake. Despite the higher total amino acid supply of cows fed red clover versus grass silage diets, further milk production responses on red clover diets were possibly compromised by an inadequate supply of methionine as evidenced by lower methionine concentration in the amino acid profile of omasal digesta and plasma. Increasing the maturity of ensiled red clover does not seem to affect silage DM intake as consistently as that of grasses. The efficiency of N utilization for milk protein synthesis was lower for red clover diets than for grass diets. It was negatively related to diet crude protein concentration similarly to grass silage diets.

Digestion capasity, nutrient digestibilities and physico-chemical conditions in the intestine influenced by the age of growing turkeys

Six experiments have been conducted to examine digestibility and feeding value of domestic Finnish fibre-rich cereals (barley and oats as compared to maize and wheat) and protein sources (rapeseed meal and cake, peas, faba beans, lupin seeds) for growing turkeys and to investigate effects of age of the birds (from 3 to 12 weeks of age) on digestion process and estimated nutrient digestibility and energy values. Besides, an objective of the study was to test applications of digestibility research methodology for turkeys. Total tract digestibility and apparent metabolizable energy (AME) was assayed in experimental cages using excreta collection, and a slaughter method was applied to sample small intestinal digesta for determination of apparent ileal crude protein digestibility (AICPD), jejuno-duodenal digesta viscosity and caecal volatile fatty acid (VFA) concentration. Digesta viscosity decreased and caecal VFA production increased with age of growing turkeys. Digesta retention times in the small intestine were generally longer in the older birds than in the younger ones. Crude fat digestibility and AME increased with age of growing turkeys, especially with viscous diets. AICPD seemed to decrease with age in most cases. Supplementation with β-gucanase-xylanase decreased viscosity, improved crude fat digestibility and metabolizable energy value and increased VFA production especially in barley-fed turkeys and especially in the young birds. Poor protein digestibility and low energy value of rapeseed meal and rapeseed cake decreased their feeding value for turkeys. In addition, a typical goitrogenic effect of rapeseed feeding was detected. Use of legume seeds as feed for growing turkeys is limited mostly by the low energy value in lupin seeds and the low ileal protein and amino acid digestibility in faba beans. Digestibility of fibre-rich protein sources was not improved with age of the turkeys. Euthanizing the turkeys for AICPD determination by carbon dioxide and bleeding led to lower digestibility values than mechanical stunning and cervical dislocation, suggesting inferiority of carbon dioxide stunning in experimental use. Comparison of AICPD and AME results obtained using different markers showed that considerable differences may occur, especially on total tract level, when acid-insoluble ash gave considerably lower AME values than titanium dioxide and chromic oxide.

Effect of phenolic-rich plant materials on protein and lipid oxidation reactions

The antioxidant activity of natural plant materials rich in phenolic compounds is being widely investigated for protection of food products sensitive to oxidative reactions. In this thesis plant materials rich in phenolic compounds were studied as possible antioxidants to prevent protein and lipid oxidation reactions in different food matrixes such as pork meat patties and corn oil-in water emulsions. Loss of anthocyanins was also measured during oxidation in corn oil-in-water emulsions. In addition, the impact of plant phenolics on amino acid level was studied using tryptophan as a model compound to elucidate their role in preventing the formation of tryptophan oxidation products. A high-performance liquid chromatography (HPLC) method with ultraviolet and fluorescence detection (UV-FL) was developed that enabled fast investigation of formation of tryptophan derived oxidation products. Byproducts of oilseed processes such as rapeseed (Brassica rapa L.), camelina (Camelina sativa) and soy meal (Glycine max L.) as well as Scots pine bark (Pinus sylvestris) and several reference compounds were shown to act as antioxidants toward both protein and lipid oxidation in cooked pork meat patties. In meat, the antioxidant activity of camelina, rapeseed and soy meal were more pronounced when used in combination with a commercial rosemary extract (Rosmarinus officinalis). Berry phenolics such as black currant (Ribes nigrum) anthocyanins and raspberry (Rubus idaeus) ellagitannins showed potent antioxidant activity in corn oil-in-water emulsions toward lipid oxidation with and without β-lactoglobulin. The antioxidant effect was more pronounced in the presence of β-lactoglobulin. The berry phenolics also inhibited the oxidation of tryptophan and cysteine side chains of β-lactoglobulin. The results show that the amino acid side chains were oxidized prior the propagation of lipid oxidation, thereby inhibiting fatty acid scission. In addition, the concentration and color of black currant anthocyanins decreased during the oxidation. Oxidation of tryptophan was investigated in two different oxidation models with hydrogen peroxide (H2O2) and hexanal/FeCl2. Oxidation of tryptophan in both models resulted in oxidation products such as 3a-hydroxypyrroloindole-2-carboxylic acid, dioxindolylalanine, 5-hydroxy-tryptophan, kynurenine, N-formylkynurenine and β-oxindolylalanine. However, formation of tryptamine was only observed in tryptophan oxidized in the presence of H2O2. Pine bark phenolics, black currant anthocyanins, camelina meal phenolics as well as cranberry proanthocyanidins (Vaccinium oxycoccus) provided the best antioxidant effect toward tryptophan and its oxidation products when oxidized with H2O2. The tryptophan modifications formed upon hexanal/FeCl2 treatment were efficiently inhibited by camelina meal followed by rapeseed and soy meal. In contrast, phenolics from raspberry, black currant, and rowanberry (Sorbus aucuparia) acted as weak prooxidants. This thesis contributes to elucidating the effects of natural phenolic compounds as potential antioxidants in order to control and prevent protein and lipid oxidation reactions. Understanding the relationship between phenolic compounds and proteins as well as lipids could lead to the development of new, effective, and multifunctional antioxidant strategies that could be used in food, cosmetic and pharmaceutical applications.

Microfiltration in cheese and whey processing

Milk microfiltration (0.05-0.2 um) is a membrane separation technique which divides milk components into casein-enriched and native whey fractions. Hitherto the effect of intensive microfiltration including a diafiltration step for both cheese and whey processing has not been studied. The microfiltration performance of skimmed milk was studied with polymeric and ceramic MF membranes. The changes caused by decreased concentration of milk lactose, whey protein and ash content for cheese milk quality and ripening were studied. The effects of cheese milk modification on the milk coagulation properties, cheese recovery yield, cheese composition, ripening and sensory quality as well as on the whey recovery yield and composition by microfiltration were studied. The functional properties of whey protein concentrate from native whey were studied and the detailed composition of whey protein concentrate powders made from cheese wheys after cheese milk pretreatments such as high temperature heat treatment (HH), microfiltration (MF) and ultrafiltration (UF) were compared. The studied polymeric spiral wound microfiltration membranes had 38.5% lower energy consumption, 30.1% higher retention of whey proteins to milk retentate and 81.9% lower permeate flux values compared to ceramic membranes. All studied microfiltration membranes were able to separate main whey proteins from skimmed milk. The optimal lactose content of Emmental cheese milk exceeded 3.2% and reduction of whey proteins and ash content of cheese milk with high concentration factor (CF) values increased the rate of cheese ripening. Reduction of whey protein content in cheese milk increased the concentration of caseinomacropeptide (CMP) of total proteins in cheese whey. Reduction of milk whey protein, lactose and ash content reduces milk rennet clotting time and increased the firmness of the coagulum. Cheese yield calculated from raw milk to cheese was lower with microfiltrated milks due to native whey production. Amounts of a-lactalbumin (a-LA) and b-lactoglobulin (b-LG) were significantly higher in the reference whey, indicating that HH, MF and UF milk pretreatments decrease the amounts of these valuable whey proteins in whey. Even low CF values in milk microfiltration (CF 1.4) reduced nutritional value of cheese whey. From the point of view of utilization of milk components it would be beneficial if the amount of native whey and the CMP content of cheese whey could be maximized. Whey protein concentrate powders made of native whey had excellent functional properties and their detailed amino acid composition differed from those of cheese whey protein concentrate powders.

Redox-linked proton transfer by cytochrome c oxidase

The respiratory chain is found in the inner mitochondrial membrane of higher organisms and in the plasma membrane of many bacteria. It consists of several membrane-spanning enzymes, which conserve the energy that is liberated from the degradation of food molecules as an electrochemical proton gradient across the membrane. The proton gradient can later be utilized by the cell for different energy requiring processes, e.g. ATP production, cellular motion or active transport of ions. The difference in proton concentration between the two sides of the membrane is a result of the translocation of protons by the enzymes of the respiratory chain, from the negatively charged (N-side) to the positively charged side (P-side) of the lipid bilayer, against the proton concentration gradient. The endergonic proton transfer is driven by the flow of electrons through the enzymes of the respiratory chain, from low redox-potential electron donors to acceptors of higher potential, and ultimately to oxygen. Cytochrome c oxidase is the last enzyme in the respiratory chain and catalyzes the reduction of dioxygen to water. The redox reaction is coupled to proton transport across the membrane by a yet unresolved mechanism. Cytochrome c oxidase has two proton-conducting pathways through which protons are taken up to the interior part of the enzyme from the N-side of the membrane. The K-pathway transfers merely substrate protons, which are consumed in the process of water formation at the catalytic site. The D-pathway transfers both substrate protons and protons that are pumped to the P-side of the membrane. This thesis focuses on the role of two conserved amino acids in proton translocation by cytochrome c oxidase, glutamate 278 and tryptophan 164. Glu278 is located at the end of the D-pathway and is thought to constitute the branching point for substrate and pumped protons. In this work, it was shown that although Glu278 has an important role in the proton transfer mechanism, its presence is not an obligatory requirement. Alternative structural solutions in the area around Glu278, much like the ones present in some distantly related heme-copper oxidases, could in the absence of Glu278 support the formation of a long hydrogen-bonded water chain through which proton transfer from the D-pathway to the catalytic site is possible. The other studied amino acid, Trp164, is hydrogen bonded to the ∆-propionate of heme a3 of the catalytic site. Mutation of this amino acid showed that it may be involved in regulation of proton access to a proton acceptor, a pump site, from which the proton later is expelled to the P-side of the membrane. The ion pair that is formed by the ∆-propionate of heme a3 and arginine 473 is likely to form a gate-like structure, which regulates proton mobility to the P-side of the membrane. The same gate may also be part of an exit path through which water molecules produced at the catalytically active site are removed towards the external side of the membrane. Time-resolved optical and electrometrical experiments with the Trp164 to phenylalanine mutant revealed a so far undetected step in the proton pumping mechanism. During the A to PR transition of the catalytic cycle, a proton is transferred from Glu278 to the pump site, located somewhere in the vicinity of the ∆-propionate of heme a3. A mechanism for proton pumping by cytochrome c oxidase is proposed on the basis of the presented results and the mechanism is discussed in relation to some relevant experimental data. A common proton pumping mechanism for all members of the heme-copper oxidase family is moreover considered.