Dehydroalanine and Lysinoalanine in Thermolyzed Casein do not Promote Colon Cancer in the Rat

Thermolysis of proteins produces xenobiotic amino-acids such as the potentially toxic lysinoalanine, and the alkylating agent, dehydro¬alanine, which have been considered possible health hazards. We observed that thermolysed casein promoted aberrant crypt foci (ACF) and colon cancer growth in rats initiated with azoxymethane and speculated that promotion might be due to the formation of these compounds. To test this notion we first measured the concentration of the modified amino acids as a function of thermolysis time. The concentration of dehydroalanine in the casein paralleled the degree of promotion, that of lysinoalanine did not. We then tested diets containing foods with high levels of dehydroalanine (thermolysed sodium-caseinate, cooked Swiss cheese) for their effect on ACF promotion. They decreased the number and/or size of ACF significantly, indicating that dehydroalanine did not promote, but protected rats against colon carcinogenesis. These results do not support the notion that lysinoalanine or dehydroalanine are a hazard with respect to colon carcinogenicity

Behaviour of milk protein ingredients and emulsions stabilised by milk protein ingredients in the simulated gastrointestinal tract : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology, Massey University, Manawatu, New Zealand

Milk clotting behaviours in the stomach impact the digestion rates of protein and fat. A variety of milk protein products are applied as functional ingredients in many foods. This research was conducted to investigate the digestion behaviours of various commercial dairy ingredients and lipids in emulsions stabilised by these ingredients using a dynamic in vitro digestion model, i.e., a human gastric simulator (HGS), with a focus on the effect of different structures of clots formed in dairy ingredients during gastric digestion on hydrolysis of proteins and/or lipids. Skim milk powder (SMP), milk protein concentrate (MPC) 4851, MPC 4861, sodium caseinate, whey protein isolate (WPI) and heated (90°C, 20 min) WPI were used in the present study. Results showed that SMP and MPC 4851, which contained casein micelles, formed ball-like clots with a relatively dense network after 10 min of gastric digestion. These clots did not disintegrate after 220 min of digestion. MPC 4861 and sodium caseinate generated clots at around 40 min, and a loose, fragmented structure was observed at the end of the gastric digestion due to a lacking micellar structure of caseins. No clot was observed in WPI or heated WPI after 220 min gastric digestion, although aggregation occurred at around 40 min in heated WPI. These differences in coagulation behaviours apparently affected the rate of gastric emptying and protein hydrolysis by pepsin in the gastric system. In SMP and MPC 4851, the gastric emptying and hydrolysis of caseins was much slower than that observed in MPC 4861 and sodium caseinate. The most rapid gastric emptying of proteins was observed in the WPI samples both with and without heating. This is attributed to the formation of varied structured clots at different times under the gastric conditions. The effect of protein concentration on the gastric behaviour of these dairy ingredients in solution was then examined, with a particular emphasis on the structure of clots. SMP and MPC 4851 have been selected as model protein ingredients. Their gastric behaviours were investigated over a protein concentration range of 0.5-5.0% (w/w). The results showed that the digestion behaviour of SMP and MPC 4851 followed a similar pattern. The rate of pH changes in the emptied digesta during digestion was protein concentration dependent. With an increase in protein concentration, the decrease in pH slowed. The protein concentration had no apparent impact on the casein clotting time. Clots were formed in the first 10 min of digestion in all samples. However, in both SMP and MPC 4851, when protein concentration was lower than 2.0% (w/w) the clots consisted of small protein pieces with a loose, porous and open structure after a 220 min digestion. Whereas a cheese ball-like clot with a denser network was observed at the end of gastric digestion when the protein concentration varied from 2.0% to 5.0% (w/w). Such a difference in the structure apparently affected the rate of protein hydrolysis. A more rapid hydrolysis (P < 0.05) of the clotted protein was observed when protein concentration was lower than 2.0% (w/w) compared to the samples containing a higher proportion of protein (2.0%-5.0%, w/w). To study the effect of different coagulation behaviours on the digestion of oil droplets in oil-in-water emulsions, these dairy ingredients (with the exception of SMP) were used to prepare an oil-in-water emulsion (20.0% soy oil and 4.0% protein, w/w). They were digested under the dynamic gastric conditions using the HGS. The gastric digesta was emptied at 20 min intervals. Then all digesta were mixed to investigate the lipid digestion under the small intestinal conditions. Changes in physicochemical properties of emulsions, involving the particle size, the microstructure, the oil content of the emptied gastric digesta and the amount of free fatty acids (FFAs) released during the small intestine stage, were determined using an in vitro small intestinal digestion model. Aggregation of MPC 4851-stabilised emulsion took place after 5 min of digestion in the HGS with the largest size. The aggregates remained in the stomach and did not disappear during the whole gastric digestion. The hydrolysis of the aggregated network by pepsin was largely slowed by the reduced ability of the simulated gastric fluid (SGF, containing pepsin) to diffuse into the larger sized aggregates. MPC 4851-stabilised emulsion thus resulted in the slowest release of oil droplets into the small intestine. In comparison, MPC 4861 and sodium caseinate-stabilised emulsions aggregated in the stomach at approximately 40 min, forming smaller sized aggregates. These aggregates disintegrated at the mid and late-stages of digestion in these two emulsions. Therefore, MPC 4861 and sodium caseinate-stabilised emulsions had a more rapid delivery of oil droplets into the small intestine. In relation to the WPI-stabilised emulsions both with and without heating, the aggregations formed at a similar time to that which was observed in MPC 4861 and sodium caseinate-stabilised-emulsions; i.e., at approximately 40 min. However, they had the smallest sized aggregates amongst all samples and they disintegrated quickly with further digestion. WPI-stabilised emulsions both with and without heating had the fastest gastric emptying and hydrolysis by pepsin in the early and mid-stages of the gastric digestion process. Thus, the highest level of oil content contained in the emptied gastric digesta was produced from both WPI-stabilised emulsions. In the mixed gastric digesta, which were subjected to the small intestinal digestion, the oil contents contained in the different emulsion samples varied. This difference impacted the extent of lipid digestion by pancreatic lipase. The sample with a higher oil content released a greater amount of FFAs compared to the sample with a lower oil content. The extent of lipid digestion of different emulsion samples adhered to the following pattern: MPC 4851-stabilised emulsion < MPC 4861-stabilised emulsion < sodium caseinate-stabilised emulsion, WPI-stabilised emulsions both with and without heating. Overall, the gastric behaviours of dairy ingredients either in solutions or emulsions were affected by the formation of structured clots/aggregates. The differences in clotting/aggregation times and their structures were greatly dependent on the component and structure of protein, the processing prior to digestion and the susceptibility to proteases. These differences in protein coagulation/aggregation behaviour impacted the rates of protein hydrolysis and gastric emptying. The oil content and protein composition of the gastric digesta transferred into small intestine and the extent of lipid digestion in small intestine were also affected. These results are important in an application perspective. They provide useful information for the design and development of healthier food products by allowing greater control over the manipulation of protein bioavailability, which subsequently provides greater control over lipid metabolism

Preparation, characterisation and application of naturally derived polar lipids through lipolysis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

Monoglycerides are lipid based emulsifiers extensively used for their broad technical function in the food industry. Commercial monoglycerides are generally manufactured through chemical synthesis; however, lipolysis of triglycerides by lipase enzyme provides a biochemical pathway by which monoglycerides may be produced. This is particularly appealing for consumers for whom all natural and clean labelled food products are a particular driver. Accordingly, rather than replacing monoglycerides from formulations with other types of emulsifiers (and that may lack the requisite functionality), an alternative approach may be to develop a non-chemical and more natural pathway to produce the emulsifier, thereby allowing the particular monoglyceride functionality to be retained within products. Therefore, this study was conducted to investigate the feasibility of using lipase enzyme as a processing tool to synthesise polar lipids, namely monoglycerides, in situ of the manufacture of whippable food emulsions, such as cream and ice cream. This concept idea was initially proven viable through interfacial tension (IFT) measurements obtained using a straight-forward surface characterisation technique. R. miehei lipase was found to competitively bind at the interface of vegetable oils-water and that the adsorbed protein (sodium caseinate and whey protein isolate) or surfactant layer (lecithin and Tween 80) did not act as a barrier to lipase adsorption at the oil-water interface. IFT measurements were also able to demonstrate the progressive accumulation of polar lipids at the oil-water interface arising from lipolysis, and were additionally used to indicate how thermal treatment of the enzyme could be used to terminate activity. In considering how the requisite functionality could be achieved for whippable emulsion formulations, emulsion droplet size, type of emulsifiers used as well as lipase concentration were shown to be key variables by which the extent and rate of lipolysis could be manipulated and controlled. The results showed that formulation (emulsifier types and oil content) and processing conditions (Microfluidizer® pressure and number of passes) had significant effects on the emulsion droplet size. As part of controlling the extent of lipolysis, the conditions by which the reaction could be terminated were investigated by measuring the viability of R. miehei lipase against thermal treatment. Results showed that the R. miehei lipase was thermostable up to temperatures of 70 °C. Above this temperature, substantial reduction of the residual activity occurred. However, even elevated temperature of between 90 and 100 oC did not immediately inactivate the lipase, with heating for ~ 2 min required before activity was no longer detected. In terms of emulsion stability, the palm oil emulsion tested in this study was found to be thermostable up to 100 oC, thus allowing development of a thermalisation step that was able to inactivate the enzyme without compromising the stability of the emulsion. The shear stability analyses on lipolysed O/W emulsion showed the lipolysed emulsions were susceptible to shear-induced aggregation, and that the degree of aggregation could be manipulated as a consequence of controlling the extent of lipolysis through either enzyme concentration or holding time. The drastic increase in the viscosity curve between the nonlipolysed and lipolysed emulsion suggested that the shear–induced partial coalescence was primarily due to the lipolysis reaction and was not as a result of the high fat content (30 %). The findings elucidate the ability of the generated polar lipids in the emulsion to displace the existing sodium caseinate adsorbed layer, thus compromising emulsion stability upon shearing. The quantification of synthesised polar lipids from the triglyceride component of fat droplets by the lipolysis reaction showed a mixture of fatty acids, di- and mono-glycerides being produced. Palmitic acid was observed to be the main liberated fatty acids. While, monoolein and monopalmitin were the most prominent monoglycerides, with measured concentrations of 3.755 ± 0.895 and 1.660 ± 0.657 mg / g fat respectively after 15 min with lipase concentration of 50 mg /g fat. The relative concentration of polar lipids produced was found to be dependent on the lipase concentration as well as time of lipolysis. Furthermore, up to 30 min of lipolysis (concentration 50 mg /g fat) were seen to have no observable effect on the droplet size distribution of the emulsion suggesting that quiescently stable emulsions could be produced. The results show the importance of controlling reaction conditions (both enzyme concentration and reaction time) in order to provide requisite functionality without excessively destabilising emulsions such that droplet structuring can occur under quiescent conditions. The generation of monoglycerides at quantum satis levels able to impart critical functionality was demonstrated in whipped cream and ice cream. The addition of R. miehei lipase at very low concentration of 5 mg /g fat was able to produce a rigid and stable whipped cream with overrun exceeding 100 %. However, good stability of the whipped cream over time was achievable with concentration above 10 mg /g fat. Similarly, ice cream made with the addition of 5 mg /g fat exhibited good melt stability and firmness. The findings proved the feasibility of in situ production of polar lipids, namely monoglycerides and fatty acids, in replicating the functionality imparted by commercial monoglycerides in whippable emulsions. Thus, the findings in this thesis offer an alternative biochemical pathway for the generation of polar lipids to that of commercially available monoglycerides, which are currently produced synthetically. The potential for using this approach as part of the processing step for food emulsion manufacture has also been demonstrated. The concept can be tailored for various emulsion based food products

Food-grade Pickering stabilisation of foams by in situ hydrophobisation of calcium carbonate particles

© 2016 Elsevier Ltd The aim of this study was to investigate the possibility of stabilising foam bubbles in water by adsorption of calcium carbonate (CaCO 3 ) particles. Because CaCO 3 is hydrophilic and not surface-active, particles were hydrophobised in situ with several emulsifiers. The used emulsifiers were food-grade and negatively charged at the pH employed. The effect of particle addition on foamability and foam stability of solutions containing either β-lactoglobulin, sodium caseinate, Quillaja, sodium dodecanoate (SD) or sodium stearoyl-2-lactylate (SSL) was studied. It was found that the ability of the emulsifiers to induce surface activity such that the particles are able to adsorb to the air-water interface is related to their structure. The structure needs to consist of a well-defined hydrophobic part and a charged part. Large emulsifiers with a complex structure, such as β-lactoglobulin, sodium caseinate and Quillaja, were able to partially hydrophobise the particles but were not able to act synergistically with the particles to increase the foam stability. Low molecular weight emulsifiers, however, consisting of a single tail with one charged group, such as SD and SSL, adsorbed at the particle surface rendering the particles partially hydrophobic such that they adsorb to the air-water interface. In a subsequent investigation, the pH was changed to a value typical for food products (pH 6–7) and the addition of milk salts on the foamability and foam stability was assessed. Based on these results, the use of food-grade CaCO 3 particles hydrophobised in situ with food-grade surfactants (SD or SSL) to prepare ultra-stable aqueous foams is demonstrated

The effect of a meat extract on iron absorption in young women : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Human Nutrition at Massey University, Palmerston North Campus, New Zealand

Iron deficiency is a global problem for which determinants and solutions need to be investigated. The first part of this study assessed the iron status and dietary intakes of 85 non vegetarian women aged 18-40 years living in the Manawatu region. Exclusion criteria included pregnancy or breastfeeding in the past 12 months, smoking, excess alcohol consumption and recent blood donation. Dietary intakes were estimated using a 24 hour recall and a non validated food frequency questionnaire. Serum ferritin (SF), haemoglobin (Hb), C-reactive protein, height, weight and supplement use were measured. Two women (2.4%) had iron deficiency anaemia (SF20µg/L). The daily mean and median iron intakes were 12.7±6.2mg and 10.8mg. 71 women (83.5%) consumed less than the Recommended Dietary Intake (RDI) of 18mg iron per day and 21.2% consumed less than the Estimated Average Requirement (EAR) of 8mg iron per day. Serum ferritin was positively associated with age and total dietary iron intake. No statistically significant relationship was found between serum ferritin and Body Mass Index or exercise, or daily intakes of energy, protein, haem iron, red meat, total meat, vitamin C, vitamin A, total tea, coffee, alcohol, fibre or calcium (p>.05). Eighteen women who had low iron stores (SF<30µg/L) were selected to take part in a second study to investigate the effect of a meat extract (<0.5kDa sarcoplasmic fraction) on non haem iron absorption. Each subject consumed a sodium caseinate meal, a meat meal or a sodium caseinate meal containing the meat extract. Each meal was labeled with 8.5mg 57 Fe and each subject received 0.5mg 58 Fe administered by intravenous infusion. Fourteen days later iron absorption from these meals was determined using ratios of stable isotopes of iron incorporated into the red blood cells. Iron status was significantly inversely related to iron absorption. After adjusting to a serum ferritin of 40µ/L, iron absorption was 3.8% from the sodium caseinate meal, 3.9% from the meat meal and 5.1% from the meal containing the meat extract. These values were not significantly different from one another (p>.05)

Wine Fining with Plant Proteins

: Fining treatments involve the addition of a substance or a mixture to wine, and are generally carried out in order to clarify, stabilize or modify the wine\u2019s organoleptic characteristics. Usually these fining agents will bind the target compound(s) to form insoluble aggregates that are subsequently removed from the wine. The main reasons to perform wine fining treatments are to carry out wine clarification, stabilization and to remove phenolic compounds imparting unwanted sensory characteristics on the wine, which is an operation that often relies on the use of animal proteins, such as casein, gelatin, egg and fish proteins. However, due to the allergenic potential of these animal proteins, there is an increasing interest in developing alternative solutions including the use of fining proteins extracted from plants (e.g., proteins from cereals, grape seeds, potatoes, legumes, etc.), and non-proteinaceous plant-based substances (e.g., cell wall polysaccharides and pomace materials). In this article, the state of the art alternative fining agents of plant origins are reviewed for the first time, including considerations of their organoleptic and technological effects on wine, and of the allergenic risks that they can pose for consumer

Goat and cow casein derived ingredients and their interactions with iron : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, Massey University, Palmerston North, New Zealand

The objective of this study was to gain a fundamental understanding of how goat casein micelles and the products of casein proteins behave when fortified with iron. Iron fortified skim milk was characterised by analysing the mass balance of micellar/non micellar fractions, chemical changes, micellar size changes and internal structure. Two treatments were examined to determine where in the processing line the addition of iron might best be added to a milk system. On average, at least 72% of the iron is bound to the micellar phase across the treatments and iron concentrations. Small angle X-ray scattering (SAXS) indicated that internal changes, mainly at the location of the colloidal calcium phosphate, occurred with iron addition. Casein was extracted from goat milk using isoelectric precipitation however the extraction was more difficult than using cow milk. Iron fortification of the caseinates resulted in a tendency for oxidation and precipitation of the proteins to occur causing the formation of large aggregates. The caseinates could not stabilise the same amounts of iron to that of an intact casein micelle. Phosphopeptides were isolated by adding calcium and ethanol to caseinate digests. There was an increase in serine, glutamic acid and isoleucine residues compared to caseinate. There was an increase in phosphorus from 7.8 ± 0.3 mg P/ g solids to 45.4 ± 2.4 mg P/ g solids in the isolate. The phosphopeptides were composed of smaller, more hydrophilic peptides compared to the full digest prior to precipitation. Ferrous sulfate was then investigated for use as the precipitant, instead of calcium. The peptides produced similar trends in terms of amino acid profile changes, phosphorus concentration increase and yield. Immobilised metal affinity chromatography was also investigated however this had a low throughput that may not be effective at process scale. The effect of heating, cooling, ionic strength of the solution, holding time, iron loading, processing order and use in a model milk system were investigated to simulate potential industrial processing conditions using the calcium - extracted phosphopeptides. It was found that goat peptide isolates were able to bind 54.4 ± 0.5 mg Fe/ g protein compared to goat milk of 4.3 ± 0.1 mg Fe/ g protein. The optimal conditions for binding were found to be at pH 6.7 in a low ionic strength solution, around 37 oC. There was a potential synergistic effect of adding the peptides to milk in terms of iron binding capacity. There were few differences in the amount of iron that could be bound comparing cow and goat derived phosphopeptides under the tested conditions. The oxidation potential of ingredients was determined using malondialdehyde (MDA) as an oxidation product marker. There was a reduction in oxidation when iron was bound to milk or peptides compared to free ferrous sulfate in solution with intact goat milk performing the best producing 0.46 ± 0.04 μg MDA/mL after 3 days at 30 oC compared to the blank of 1.25 ± 0.16 μg MDA/mL. The goat peptides produced non-significantly different levels of MDA compared to the blank containing no ferrous sulfate. Caco-2 cell lines are a way of approximating how systems may function in an intestine in terms of nutrient absorption. Iron absorption was improved in the order of casein hydrolysates > caseinate > skim milk for goat milk. In contrast, cow milk appeared to perform better without any modifications to the proteins. On an equal iron filtrate basis after the digestion and intestinal phase, calcium- precipitated goat phosphopeptides produced a response of 9.64 ± 0.94 ng ferritin/ nM iron. This response was greater than all other treatments with the exception of goat milk fortified with 5 mM iron and ascorbic acid with 12.30 ± 1.23 ng ferritin/ nM iron. This work covers a wide range of milk products and iron interactions and has helped to build a fundamental understanding of goat milk protein functionality. The underpinning considerations to a manufacturing setting may allow further development of large scale ingredient production for the improved stability of iron fortified systems