Sugar matrices in stabilization of bioactives by dehydration

Development of functional foods with bioactive components requires component stability in foods and ingredients. Stabilization of sensitive bioactive components can be achieved by entrapment or encapsulation of these components in solid food matrices. Lactose or trehalose was used as the structure-forming material for the entrapment of hydrophilic ascorbic acid and thiamine hydrochloride or the encapsulation of oil particles containing hydrophobic α-tocopherol. In the delivery of hydrophobic components, milk protein isolate, soy protein isolate, or whey protein isolate were used as emulsifiers and, in some cases, applied in excess amount to form matrices together with sugars. Dehydrated amorphous structures with bioactives were produced by freezing and freeze-drying. Experimental results indicated that: (i) lactose and trehalose showed similar water sorption and glass transition but very different crystallization behavior as pure sugars; (ii) the glass transition of sugar-based systems was slightly affected by the presence of other components in anhydrous systems but followed closely that of sugar after water plasticization; (iii) sugar crystallization in mixture systems was composition-dependent; (iv) the stability of bioactives was better retained in the amorphous matrices, although small losses of stability were observed for hydrophilic components above glass transition and for hydrophobic components as a function of water activity; (v) sugar crystallization caused significant loss of hydrophilic bioactives as a result of the exclusion from the continuous crystalline phase; (vi) loss of hydrophobic bioactives upon sugar crystallization was a result of dramatic change of emulsion properties and the exclusion of oil particles from the protecting structure; (vii) the double layers at the hydrophilic-hydrophobic interfaces improved the stability of hydrophobic bioactives in dehydrated systems. The present study provides information on the physical and chemical stability of sugar-based dehydrated delivery systems, which could be helpful in designing foods and ingredients containing bioactive components with improved storage stability.

Evaluation of microbial adjuncts and their effect on the ripening of cheddar cheese

A bacteriocin-producing strain of Lactobacillus paracasei DPC 4715 was used as an adjunct culture in Cheddar cheese in order to control the growth of “wild” nonstarter lactic acid bacteria. No suppression of growth of the indicator strain was observed in the experimental cheese. The bacteriocin produced by Lactobacillus paracasei DPC 4715 was sensitive to chymosin and cathepsin D and it may have been cleaved by the rennet used for the cheese manufactured or by indigenous milk proteases. A series of studies were performed using various microbial adjuncts to influence cheese ripening. Microbacterium casei DPC 5281, Corynebacterium casei DPC 5293 and Corynebacterium variabile DPC 5305 were added to the cheesemilk at level of 109 cfu/ml resulting in a final concentration of 108 cfu/g in Cheddar cheese. The strains significantly increased the level of pH 4.6-soluble nitrogen, total free amino acids after 60 and 180 d of ripening and some individual free amino acids after 180 d. Yarrowia lipolytica DPC 6266, Yarrowia lipolytica DPC 6268 and Candida intermedia DPC 6271 were used to accelerate the ripening of Cheddar cheese. Strains were grown in YG broth to a final concentration of 107 cfu/ml, microfluidized, freeze-dried and added to the curd during salting at level of 2% w/w. The yeasts positively affected the primary, secondary proteolysis and lipolysis of cheeses and had aminopeptidase, dipeptidase, esterase and 5’ phosphodiestere activities that contributed to accelerate the ripening and improve the flavor of cheese. Hafia alvei was added to Cheddar cheesemilk at levels of 107 cfu/ml and 108 cfu/ml and its contribution during ripening was evaluated. The strain significantly increased the level of pH 4.6-soluble nitrogen, total free amino-acids, and some individual free amino-acids of Cheddar cheese, whereas no differences in the urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms of the cheeses were detected. Hafia alvei also significantly increased the level of some biogenic amines. A low-fat Cheddar cheese was made with Bifidobacterium animalis subsp. lactis, strain BB-12® at level of 108 cfu/ml, as a probiotic adjunct culture and Hi-Maize® 260 (resistant high amylose maize starch) at level of 2% and 4% w/v, as a prebiotic fiber which also played the role of fat replacer. Bifidobacterium BB-12 decreased by 1 log cycle after 60 d of ripening and remained steady at level of ~107 cfu/g during ripening. The Young’s modulus also increased proportionally with increasing levels of Hi-maize. Hencky strain at fracture decreased over ripening and increased with increasing in fat replacer. A cheese based medium (CBM) was developed with the purpose of mimicking the cheese environment at an early ripening stage. The strains grown in CBM showed aminopeptidase activity against Gly-, Arg-, Pro- and Phe-p-nitroanalide, whereas, when grown in MRS they were active against all the substrates tested. Both Lb. danicus strains grown in MRS and in CBM had aminotransferase activity towards aromatic amino acids (Phe and Trp) and also branched-chain amino acids (Leu and Val). Esterase activity was expressed against p-nitrophenyl-acetate (C2), pnitrophenyl- butyrate (C4) and p-nitrophenyl-palmitate (C16) and was significantly higher in CBM than in MRS.

Physico-chemical properties and component interactions in high solids food systems

The present study aimed to investigate interactions of components in the high solids systems during storage. The systems included (i) lactose–maltodextrin (MD) with various dextrose equivalents at different mixing ratios, (ii) whey protein isolate (WPI)–oil [olive oil (OO) or sunflower oil (SO)] at 75:25 ratio, and (iii) WPI–oil– {glucose (G)–fructose (F) 1:1 syrup [70% (w/w) total solids]} at a component ratio of 45:15:40. Crystallization of lactose was delayed and increasingly inhibited with increasing MD contents and higher DE values (small molecular size or low molecular weight), although all systems showed similar glass transition temperatures at each aw. The water sorption isotherms of non-crystalline lactose and lactose–MD (0.11 to 0.76 aw) could be derived from the sum of sorbed water contents of individual amorphous components. The GAB equation was fitted to data of all non-crystalline systems. The protein–oil and protein–oil–sugar materials showed maximum protein oxidation and disulfide bonding at 2 weeks of storage at 20 and 40°C. The WPI–OO showed denaturation and preaggregation of proteins during storage at both temperatures. The presence of G–F in WPI–oil increased Tonset and Tpeak of protein aggregation, and oxidative damage of the protein during storage, especially in systems with a higher level of unsaturated fatty acids. Lipid oxidation and glycation products in the systems containing sugar promoted oxidation of proteins, increased changes in protein conformation and aggregation of proteins, and resulted in insolubility of solids or increased hydrophobicity concomitantly with hardening of structure, covalent crosslinking of proteins, and formation of stable polymerized solids, especially after storage at 40°C. We found protein hydration transitions preceding denaturation transitions in all high protein systems and also the glass transition of confined water in protein systems using dynamic mechanical analysis.

Novel structured emulsions for delivering of food flavours

Flavour release from food is determined by the binding of flavours to other food ingredients and the partition of flavour molecules among different phases. Food emulsions are used as delivery systems for food flavours, and tailored structuring in emulsions provides novel means to better control flavour release. The current study investigated four structured oil-in-water emulsions with structuring in the oil phase, oil-water interface, and water phase. Oil phase structuring was achieved by the formation of monoglyceride (MG) liquid crystals in the oil droplets (MG structured emulsions). Structured interface was created by the adsorption of a whey protein isolate (WPI)-pectin double layer at the interface (multilayer emulsion). Water phase structured emulsions referred to emulsion filled protein gels (EFP gels), where emulsion droplets were embedded in WPI gel network, and emulsions with maltodextrins (MDs) of different dextrose-equivalent (DE) values. Flavour compounds with different physicochemical properties were added into the emulsions, and flavour release (release rate, headspace concentration and air-emulsion partition coefficient) was described by GC headspace analysis. Emulsion structures, including crystalline structure, particle size, emulsion stability, rheology, texture, and microstructures, were characterized using differential scanning calorimetry and X-ray diffraction, light scattering, multisample analytical centrifuge, rheometry, texture analysis, and confocal laser scanning microscopy, respectively. In MG structured emulsions, MG self-assembled into liquid crystalline structures and stable β-form crystals were formed after 3 days of storage at 25 °C. The inclusion of MG crystals allowed tween 20 stabilized emulsions to present viscoelastic properties, and it made WPI stabilized emulsions more sensitive to the change of pH and NaCl concentrations. Flavour compounds in MG structured emulsions had lower initial headspace concentration and air-emulsion partition coefficients than those in unstructured emulsions. Flavour release can be modulated by changing MG content, oil content and oil type. WPI-pectin multilayer emulsions were stable at pH 5.0, 4.0, and 3.0, but they presented extensive creaming when subjected to salt solutions with NaCl ≥ 150 mM and mixed with artificial salivas. Increase of pH from 5.0 to 7.0 resulted in higher headspace concentration but unchanged release rate, and increase of NaCl concentration led to increased headspace concentration and release rate. The study also showed that salivas could trigger higher release of hydrophobic flavours and lower release of hydrophilic flavours. In EFP gels, increases in protein content and oil content contributed to gels with higher storage modulus and force at breaking. Flavour compounds had significantly reduced release rates and air-emulsion partition coefficients in the gels than the corresponding ungelled emulsions, and the reduction was in line with the increase of protein content. Gels with stronger gel network but lower oil content were prepared, and lower or unaffected release rates of the flavours were observed. In emulsions containing maltodextrins, water was frozen at a much lower temperature, and emulsion stability was greatly improved when subjected to freeze-thawing. Among different MDs, MD DE 6 offered the emulsion the highest stability. Flavours had lower air-emulsion partition coefficients in the emulsions with MDs than those in the emulsion without MD. Moreover, the involvement of MDs in the emulsions allowed most flavours had similar release profiles before and after freeze-thaw treatment. The present study provided information about different structured emulsions as delivery systems for flavour compounds, and on how food structure can be designed to modulate flavour release, which could be helpful in the development of functional foods with improved flavour profile.

The biodiversity, control and genetic characterisation of the lactococcal 936 group phages

Phages belonging to the 936 group represent one of the most prevalent and frequently isolated phages in dairy fermentation processes using Lactococcus lactis as the primary starter culture. In recent years extensive research has been carried out to characterise this phage group at a genomic level in an effort to understand how the 936 group phages dominate this particular niche and cause regular problems during large scale milk fermentations. This thesis describes a large scale screening of industrial whey samples, leading to the isolation of forty three genetically different lactococcal phages. Using multiplex PCR, all phages were identified as members of the 936 group. The complete genome of thirty eight of these phages was determined using next generation sequencing technologies which identified several regions of divergence. These included the structural region surrounding the major tail protein, the replication region as well as the genes involved in phage DNA packing. For a number of phages the latter genomic region was found to harbour genes encoding putative orphan methyltransferases. Using small molecule real time (SMRT) sequencing and heterologous gene expression, the target motifs for several of these MTases were determined and subsequently shown to actively protect phage DNA from restriction endonuclease activity. Comparative analysis of the thirty eight phages with fifty two previously sequenced members of this group showed that the core genome consists of 28 genes, while the non-core genome was found to fluctuate irrespective of geographical location or time of isolation. This study highlights the continued need to perform large scale characterisation of the bacteriophage populations infecting industrial fermentation facilities in effort to further our understanding dairy phages and ways to control their proliferation.

Infant milk formula manufacture: process and compositional interactions in high dry matter wet-mixes

Infant milk formula (IMF) is fortified milk with composition based on the nutrient content in human mother's milk, 0 to 6 months postpartum. Extensive medical and clinical research has led to advances in the nutritional quality of infant formula; however, relatively few studies have focused on interactions between nutrients and the manufacturing process. The objective of this research was to investigate the impact of composition and processing parameters on physical behaviour of high dry matter (DM) IMF systems with a view to designing more sustainable manufacturing processes. The study showed that commercial IMF, with similar compositions, manufactured by different processes, had markedly different physical properties in dehydrated or reconstituted state. Commercial products made with hydrolysed protein were more heat stable compared to products made with intact protein, however, emulsion quality was compromised. Heat-induced denaturation of whey proteins resulted in increased viscosity of wet-mixes, an effect that was dependant on both whey concentration and interactions with lactose and caseins. Expanding on fundamental laboratory studies, a novel high velocity steam injection process was developed whereby high DM (60%) wet-mixes with lower denaturation/viscosity compared to conventional processes could be achieved; powders produced using this process were of similar quality to those manufactured conventionally. Hydrolysed proteins were also shown to be an effective way of reducing viscosity in heat-treated high DM wet-mixes. In particular, using a whey protein concentrate whereby β-Lactoglobulin was selectively hydrolysed, i.e., α-Lactalbumin remained intact, reduced viscosity of wet-mixes during processing while still providing good emulsification. The thesis provides new insights into interactions between nutrients and/or processing which influence physical stability of IMF both in concentrated liquid and powdered form. The outcomes of the work have applications in such areas as; increasing the DM content of spray drier feeds in order to save energy, and, controlling final powder quality.

Drinking from arboreal water sources by mantled howling monkeys (Alouatta palliata Gray).

Despite occasional trips to the ground and feeding in trees whose canopies touched the river, mantled howling monkeys were never seen to drink from any ground water. Drinking from arboreal cisterns was observed, but only during the wet season (meteorologically the less stressful season but phenologically the more stressful season). The lack of sufficient new leaves during the wet season forced the howlers to ingest more mature leaves which contained significantly less water. To compensate for the lowered amount of water in their food, the monkeys utilized arboreal water cisterns. The cisterns dried up during the dry season, but the howlers maintained their water balance by altering their time of actiivity and selecting a diet comprised largely of succulent new leaves. The effect of plant-produced secondary compounds on drinking also was discussed.

Mechanical defenses in leaves eaten by Costa Rican howling monkeys (Alouatta palliata).

Primate species often eat foods of different physical properties. This may have implications for tooth structure and wear in those species. The purpose of this study was to examine the mechanical defenses of leaves eaten by Alouatta palliata from different social groups at Hacienda La Pacifica in Costa Rica. Leaves were sampled from the home-ranges of groups living in different microhabitats. Specimens were collected during the wet and dry seasons from the same tree, same plant part, and same degree of development as those eaten by the monkeys. The toughness of over 300 leaves was estimated using a scissors test on a Darvell mechanical tester. Toughness values were compared between social groups, seasons, and locations on the leaves using ANOVA. Representative samples of leaves were also sun-dried for subsequent scanning electron microscopy and energy dispersive x-ray (EDX) analyses in an attempt to locate silica on the leaves. Both forms of mechanical defense (toughness and silica) were found to be at work in the plants at La Pacifica. Fracture toughness varied significantly by location within single leaves, indicating that measures of fracture toughness must be standardized by location on food items. Monkeys made some food choices based on fracture toughness by avoiding the toughest parts of leaves and consuming the least tough portions. Intergroup and seasonal differences in the toughness of foods suggest that subtle differences in resource availability can have a significant impact on diet and feeding in Alouatta palliata. Intergroup differences in the incidence of silica on leaves raise the possibility of matching differences in the rates and patterns of tooth wear.

Suppression of the Hepatitis C viral load by Phyllanthus niruri extracts

Gemstone Team ANTIDOTE

Cross-sectional study of 168 patients with hepatorenal tyrosinaemia and implications for clinical practice

Background: Hepatorenal tyrosinaemia (Tyr 1) is a rare inborn error of tyrosine metabolism. Without treatment, patients are at high risk of developing acute liver failure, renal dysfunction and in the long run hepatocellular carcinoma. The aim of our study was to collect cross-sectional data. Methods. Via questionnaires we collected retrospective data of 168 patients with Tyr 1 from 21 centres (Europe, Turkey and Israel) about diagnosis, treatment, monitoring and outcome. In a subsequent consensus workshop, we discussed data and clinical implications. Results: Early treatment by NTBC accompanied by diet is essential to prevent serious complications such as liver failure, hepatocellular carcinoma and renal disease. As patients may remain initially asymptomatic or develop uncharacteristic clinical symptoms in the first months of life newborn mass screening using succinylacetone (SA) as a screening parameter in dried blood is mandatory for early diagnosis. NTBC-treatment has to be combined with natural protein restriction supplemented with essential amino acids. NTBC dosage should be reduced to the minimal dose allowing metabolic control, once daily dosing may be an option in older children and adults in order to increase compliance. Metabolic control is judged by SA (below detection limit) in dried blood or urine, plasma tyrosine (<400 μM) and NTBC-levels in the therapeutic range (20-40 μM). Side effects of NTBC are mild and often transient. Indications for liver transplantation are hepatocellular carcinoma or failure to respond to NTBC. Follow-up procedures should include liver and kidney function tests, tumor markers and imaging, ophthalmological examination, blood count, psychomotor and intelligence testing as well as therapeutic monitoring (SA, tyrosine, NTBC in blood). Conclusion: Based on the data from 21 centres treating 168 patients we were able to characterize current practice and clinical experience in Tyr 1. This information could form the basis for clinical practice recommendations, however further prospective data are required to underpin some of the recommendations.

Maíz rae y suero de queso en la alimentación de cerdos en crecimiento, terminación

p.29-33

Suero de queso en la alimentación de cerdos Duroc Jersey y Cruza Landrace X Large White

p.39-42

In vitro release of bovine serum albumin from alginate/HPMC hydrogel beads

In recent years, the use of swelling polymeric matrices for the encapsulation and controlled release of protein drugs has received significant attention. The purpose of the present study was to investigate the release of albumin, a model protein from alginate/hydroxypropyl-methylcellulose (HPMC) gel beads. A hydrogel system comprised of two natural, hydrophilic polymers; sodium alginate and HPMC was studied as a carrier of bovine serum albumin (BSA) which was used as a model protein. The morphology, bead size and the swelling ratio were studied in different physical states; fully swollen, dried and reswollen using scanning electron microscopy and image analysis. Finally the effect of different alginate/HPMC ratios on the BSA release profile in physiological saline solution was investigated. Swelling experiments revealed that the bead diameter increases with the viscosity of the alginate solution while the addition of HPMC resulted in a significant increase of the swelling ratio. The BSA release patterns showed that the addition of HPMC increased the protein-release rate while the release mechanism fitted the Peppas model. Alginate/HPMC beads prepared using the ionic gelation exhibited high BSA loading efficiency for all formulations. The presence of HPMC increased the swelling ability of the alginate beads while the particle size remained unaffected. Incorporation of HPMC in the alginate gels also resulted in improved BSA release in physiological saline solution. All formulations presented a non-Fickian release mechanism described by the Peppas model. In addition, the implementation of non-parametric tests showed significant differences in the release patterns between the alginate/HPMC and the pure alginate beads, respectively.

An investigation into the subambient behavior of aqueous mannitol solutions using differential scanning calorimetry, cold stage microscopy, and X-ray diffractometry

The subambient behavior of aqueous mannitol solutions is of considerable relevance to the preparation of freeze dried formulations. In this investigation the properties of 3% w/v mannitol solutions were investigated using differential scanning calorimetry (DSC), cold stage microscopy (CSM), and X-ray diffraction (XRD) to identify the thermal transitions and structural transformations undergone by this system. It was found that on cooling from ambient the system formed ice at circa -20°C while a further exotherm was seen at approximately -30°C. Upon reheating an endotherm was seen at circa -30°C followed immediately by an exotherm at circa -25°C. Temperature cycling indicated that the thermal transitions observed upon reheating were not reversible. Modulated temperature DSC (MTDSC) indicated that the transitions observed upon reheating corresponded to a glass transition immediately followed by recrystallization, XRD data showed that recrystallization was into the ß form. Annealing at -35°C for 40 min prior to cooling and reheating resulted in a maximum enthalpy being observed for the reheating exotherm. It is concluded that on cooling 3% w/v aqueous mannitol solutions an amorphous phase is formed that subsequently recrystallises into the ß form. The study has also shown that DSC, CSM, and XRD are useful complementary techniques for the study of frozen systems

Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis.

Raman microscopy, based upon the inelastic scattering (Raman) of light by molecular species, has been applied as a specific structural probe in a wide range of biomedical samples. The purpose of the present investigation was to assess the potential of the technique for spectral characterization of the porcine outer retina derived from the area centralis, which contains the highest proportion of cone:rod cell ratio in the pig retina. METHODS: Retinal cross-sections, immersion-fixed in 4% (w/v) PFA and cryoprotected, were placed on salinized slides and air-dried prior to direct Raman microscopic analysis at three excitation wavelengths, 785 nm, 633 nm, and 514 nm. RESULTS: Raman spectra of each of the photoreceptor inner and outer segments (PIS, POS) and of the outer nuclear layer (ONL) of the retina acquired at 785 nm were dominated by vibrational features characteristic of proteins and lipids. There was a clear difference between the inner and outer domains in the spectroscopic regions, amide I and III, known to be sensitive to protein conformation. The spectra recorded with 633 nm excitation mirrored those observed at 785 nm excitation for the amide I region, but with an additional pattern of bands in the spectra of the PIS region, attributed to cytochrome c. The same features were even more enhanced in spectra recorded with 514 nm excitation. A significant nucleotide contribution was observed in the spectra recorded for the ONL at all three excitation wavelengths. A Raman map was constructed of the major spectral components found in the retinal outer segments, as predicted by principal component analysis of the data acquired using 633 nm excitation. Comparison of the Raman map with its histological counterpart revealed a strong correlation between the two images. CONCLUSIONS: It has been demonstrated that Raman spectroscopy offers a unique insight into the biochemical composition of the light-sensing cells of the retina following the application of standard histological protocols. The present study points to the considerable promise of Raman microscopy as a component-specific probe of retinal tissue.