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Degradation of aflatoxin M1 in skim milk using UVC or cold plasma : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, the School of Food and Advanced Technology, Massey University, Manawatu, New Zealand
(Massey University, 2021) Nguyen, Thi Thu
Contamination of aflatoxin M1 (AFM1) in milk and milk products has been an issue for decades as it is a food safety risk, classified as a Group 1 carcinogen. Cows consuming feed contaminated with fungi (Aspergillus flavus and Aspergillus parasiticus) that produce aflatoxin B1 (AFB1), convert AFB1 to AFM1 that is released into the milk. The best way of controlling AFM1 contamination in milk is to keep the feed dry to prevent the growth of fungi to avoid the production of AFB1. However, this is challenging in some tropical countries where the weather is hot and humid all year around. Treating milk contaminated with AFM1 is an alternative method of control. The aim of this study was to investigate two methods for milk treatment - UVC and cold plasma to reduce AFM1 in milk, investigate the factors influencing these treatments and identify the degradation products after treatment. UVC (254 nm) reduced AFM1 in skim milk to below MRL (0.5 μg/L) from an initial level of 1 μg/L after 20 min treatment. Treatment time (min), depth of samples (mm) and the stirring of the milk sample during treatment were found to significantly (P < 0.05) enhance the reduction of AFM1 in milk. The contamination level (μg/L) and fat content in milk did not significantly (P > 0.05) effect the UVC efficacy. A change in milk colour was observed but the pH of the milk samples did not change. The degradant of AFM1 after UVC treatment was identified as an oxidation product which resulted in hydroxylation occurring at the double bond of the furan ring of AFM1 molecules. High voltage atmospheric cold plasma (HVACP) was used to reduce AFM1 in skim milk and explore the effect of treatment times (5, 10 and 20 min), operating gases (air and MA65 - 65% O2, 30% CO2, 5% N2), three voltages (60, 70 and 80 kV), using direct and indirect treatment, AFM1 contamination levels (0.1; 1 and 50 μg/L) and the volume of the sample (10, 20 and 30 mL). A reduction of 64.99 and 78.86% of AFM1 in skim milk after 20 min HVACP treatment using air and MA65, respectively, was achieved with the initial level of 1 μg/L. HVACP did not change the milk colour after 20 min treatment but a slight change in pH was observed. Different treatment times, different operating gases and voltages, direct and indirect treatments were found to have the most effect on AFM1 reduction. While AFM1 contamination levels (0.1; 1 and 50 μg/L) had an insignificant (P > 0.05) effect on AFM1 reduction in milk. A dielectric barrier discharge (DBD) cold plasma set up with small capacity high voltage generator was used to investigate the effects of other operating gases with different mixtures (5, 10 and 20% of air, pure oxygen and nitrogen in helium) and the effect of milk components (casein, lactose and whey protein) on AFM1 reduction. The degradation products of AFM1 after cold plasma treatment were determined. Although this small capability system reduced approximately 70-100% of AFM1 in water after 3 and 10 min treatment by using air/helium (10/90), the reduction of AFM1 in skim milk, whey and casein was much less, although 70% of AFM1 was reduced in lactose. The reduction of AFM1 in water was significantly (P < 0.05) improved by cold plasma with the increase in the concentration of air/pure oxygen in helium but it was unchanged regardless of the ratio of nitrogen in helium. The structure of three degradants of AFM1 after cold plasma treatment was elucidated with the confirmation of two of them resulting from damage to the furan ring of AFM1 molecules. The structure of the third one was proposed but another analysis technique is required to confirm.
Magnesium enrichment of skim milk : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Auckland, New Zealand
(Massey University, 2019) Begum, Noorzahan
Effective magnesium enrichment of dairy products depends on the amount of magnesium salt added and the nature of its distribution between the serum and micellar phases. Thus, this study firstly aimed to profile the magnesium distribution in skim milk as a function of pH (pH 5.50 to 7.20), preheating temperatures (20 to 80 ± 1 °C) and concentration of added magnesium chloride (0 to 40 mmol L-1). The second aim was to investigate the rheological properties of magnesium-induced skim milk gels as a function of different concentrations of magnesium chloride (0 to 20 mmol L-1) added to heated skim milk. The total concentration of magnesium in skim milk and serum was determined using a complexometric titration (EDTA titration) and the EDTA results were validated by atomic absorption spectroscopy (AAS). The measurement of ionic magnesium (Mg2+) in milk is of importance for understanding the bioavailability of magnesium-enriched dairy products. Hence, the concentration of ionic magnesium (Mg2+) in the serum phase was measured using a novel magnesium fluorescence dye (Magnesium 510 probe). In all the samples, a reduction in the pH increased the total soluble magnesium and ionic magnesium (Mg2+) concentrations in the serum phase, regardless of whether magnesium chloride (15 mmol L-1) was added or not to skim milk at 20 ± 1 °C. At pH 5.50, more than 92% magnesium was found in the serum phase for both added (15 mmol L-1) and no added magnesium chloride samples. The concentration of magnesium in the serum phase remained unchanged as the preheating temperature was increased from 20 to 80 ± 1 °C. The addition of magnesium chloride to skim milk reduced the milk pH and increased the ionic magnesium (Mg2+) and total soluble magnesium concentration in the serum phase at 20 ± 1 °C. The pH values are important for new magnesium-enriched dairy products as the distribution of added magnesium chloride in the serum phase was different dependent on pH adjustment between the pH 6.50 and natural pH 6.70. Rheological measurements using cone and plate geometry at constant strain showed that the addition of 5 mmol L-1 magnesium chloride induced the gelation of skim milk after 22 min of heating at 80 °C in the rheometer. The time and temperature for reaching the gelation in skim milk depended on preheating and concentration of added magnesium chloride. A higher concentration of added magnesium chloride achieved gelation at a lower temperature in the rheometer. With the magnesium-induced gels, G’ values obtained were found to increase with increasing concentrations of added magnesium chloride (0 to 20 mmol L-1), increasing holding times (10 to 60 min) and increasing heating temperatures (70 to 80 °C). Compared with samples with 5 mmol L-1 magnesium chloride, those with 15 mmol L-1 magnesium chloride showed a more rapid increase in G’. The G’ value obtained with 15 mmol L-1 magnesium chloride was 15.01 Pa at 80 °C during 10 min of holding and cooling to 20 °C rapidly increased the final G’ value to 81.44 Pa. In contrast, the addition of 5 mmol L-1 magnesium chloride resulted in the formation of a weak gel with a final G’ value of 6.87 Pa after cooling to 20 °C. The oscillation stress of milk gels also increased with increasing magnesium chloride concentration, heating temperatures and holding times in the rheometer. Preheating significantly (P < 0.05) affected the strength of magnesium-induced skim milk gels. The addition of 20 mmol L-1 magnesium chloride to preheated skim milk followed by heating at 85 °C then cooling to 20 °C formed strong skim milk gels. In parallel, samples with no added magnesium chloride did not undergo gelation. In conclusion, the distribution of magnesium was influenced by pH and magnesium concentration added and the strength of magnesium-induced gels was influenced by magnesium concentration added, preheating and the heating time and temperature in the rheometer. The technology of making magnesium-induced skim milk gels can be exploited commercially for the formation of non-fermented dairy products supplemented with magnesium.
A study on the mechanisms of calcium-induced gelation in skim milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Albany, New Zealand
(Massey University, 2019) Lin, Liangjue
The destabilisation and aggregation of milk proteins is the first step towards the gelation of milk. The addition of calcium to milk is known to destabilise milk proteins and may result in gelation on heating. However, the mechanisms involved in gelation induced by heating calcium-added milk was not well understood. Therefore, this project aimed to determine the fundamental mechanisms involved in the development of a calciuminduced skim milk gel. Skim milk was selected as the model system and gelation was induced in-situ by heating the calcium-added skim milk at the rheometer. The changes in the storage modulus, G′, were monitored to study the development of the gel network. This project examined the impact of the following factors on the rheological properties of a calciuminduced skim milk gel: the type of soluble calcium salt added (calcium chloride, calcium lactate, calcium gluconate, calcium lactobionate and calcium iodide), pH, holding temperature during gelation (70°C to 90°C), preheat treatment, ionic strength (by addition of sodium chloride) and the contribution of casein and whey proteins. A higher calcium ion activity (aCa 2+), which indicated a higher calcium ion (Ca2+) concentration, and a lower pH favoured the formation of a stronger gel. An increase in ionic strength by addition of sodium chloride decreased the final G′ of the calciuminduced skim milk gel due to reduced calcium bridging and increased hydration repulsion. A higher heating temperature also resulted in gels with higher final G′ due to more frequent particle collisions. Casein micelles and whey proteins were both responsible for the structure of the gel network. The contribution of whey proteins towards the gel network was dependent on if they were denatured prior to heating, on the concentration of calcium ions available, and on the ratio between the casein and whey proteins present. At lower added calcium concentrations (10 mmol L-1) where the available calcium ions were limited, interactions and aggregation amongst denatured whey proteins via hydrophobic and disulphide bonds may have resulted in the formation of a stronger gel. However, at higher added calcium concentrations (20 and 40 mmol L-1), where sufficient calcium ions may be available for binding, interactions between casein and calcium dominated over the self-aggregating effect of denatured whey proteins. In conclusion, the results demonstrated that the final gel properties of a calcium-induced skim milk gel were dependent on the net effect of all the factors involved in the stability and interactions of the milk proteins, including the calcium salt concentration, pH, preheat treatment, ionic strength, and the protein composition in solution. These findings provide alternative methods for texture modification in milk.
Fate of hydroxyapatite nano particles during in vitro gastrointestinal digestion : a thesis presented in partial fulfilment of the requirements for the degree of Master in Food Technology at Massey University, Riddet Institute and Massey Institute of Food Science and Technology, Palmerston North, New Zealan
(Massey University, 2018) Choki, Kinley
There is an increasing change in population demographics towards an aging population in the world, which had led to the availability of various commercial nutritionally supplemented products. Hydroxyapatite (HA), with chemical formula Ca10(PO4)6(OH)2, is an insoluble calcium salt used for calcium supplementation because of its similarity to the minerals found in human bone and teeth. The insoluble calcium salts are preferred over the soluble ones because of their high heat stability during milk processing under high heat treatment. However, the drawback of insoluble calcium salts is the tendency to sediment during storage resulting in unfavourable gritty texture. Thus, reduction in particle sizes into micron to nano-size improves the dispersion of these insoluble salts. However, the application of nano-sized particles in food products have raised concerns from both the regulatory organizations and consumers on the implications related to both the environmental and health safety aspects. Thus, the objective of the study is to determine the digestion behaviour of nano-sized needle/rod shaped HA (nHA) when added into skim milk during in vitro gastrointestinal digestion. Determination of calcium such as soluble and ionic calcium was conducted to determine the dissolution of nHA. The structural changes and the crystallographic changes of nHA were determined using electron microscopy and x-ray diffraction techniques. The results of in vitro gastric digestion showed presence of undissolved nHA particles even after 240 min of gastric and 120 min of intestinal digestion when examined under TEM, while the XRD analysis detected the presence of crystalline nHA in the first 120 min of gastric digestion. Thus, the possible mechanisms leading to the incomplete dissolution of nHA under acidic conditions of the stomach are discussed subsequently.
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
(Massey University, 2017) Smialowska, Alice Małgorzata
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.
The effect of heat treatment on lysine availability and dye binding capacity of skim milk : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology at Massey University
(Massey University, 1976) Reeves, Malcolm John
The reported work on changes in lysine content in milk and dried milk is examined. The cause of these losses, the Maillard reaction, and the methods of lysine determination are discussed. All methods have recognised faults. Little information is available to the food processor regarding the kinetics of these losses, and the methods of their determination are not simple enough for routine quality control application. Although the lysine content of milk products determined after acid hydrolysis is known to be higher than nutritional studies indicate the causes of this are being established. Therefore acid hydrolysis in conjunction with a GLC method of amino acid analysis was adopted after some modification. (It was found that dialysis of the milk prior to hydrolysis resulted in cleaner chromatograms and that as the recovery of several amino acids, such a proline, leucine, and isoleucine, was not affected by heat treatment then these were used as internal 'internal standard'.) No simple rate expression could be found to fit the kinetics of the loss of acid released lysine. A first order model requiring the losses to be increased by a factor of 3.43 was devised and this could be used to satisfactorily predict values for acid available lysine in the heat treated milk. The possibility of the 3.43 factor being due to the regeneration of lysine by acid from Maillard intermediates, although requiring assumptions, was found to be not unreasonable. The energy of activation of the reaction leading to a loss in acid released lysine at 31.5 Kcal/mole is similar to literature values while the model value of 37.2 Kcal/mole is rather higher. The literature findings of little or no loss of lysine during pasteruization, evaporation, and sterilization of milk are supported. The technique of protein determination by dye binding was examined and applied to following changes in lysine in heat treated milk. The inconsistencies in reported work on dye binding is of little consequence as relative changes only are required. Changes in dye binding using amido black did not follow simple order kinetics, even when allowance was made for the constant binding by arginine and histidine. A first order model requiring the changes to be increased by a factor of 3.68 was developed. About 46% of this factor can be explained by assuming constant binding by arginine and histidine, the remainder of the factor possibly being due to Maillard intermediates binding dye, and/or a change in binding stoichiometry occurring. From the model it is possible to predict the observed changes in dye binding. Literature findings were supported. The energy of activation for the dye binding changes is 28.6 Kcal/mole, and for the model, 30.8 Kcal/mole. Ancillary investigations showed that the concurrent colour changes due to heat treatment have an energy of activation of about 30 Kcal/mole, and that there is a relationship between colour and dye binding capacity in heat treated milk. The relationship between the Pro-Milk and a typical absorbance spectrophotometer was determined, and an expression found which would enable a spectrophotometer to be used for protein determination.
Heat-induced interactions of [beta]-lactoglobulin, [alpha]-lactalbumin and casein micelles : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Education in Food Technology at Massey University
(Massey University, 1996) Chiweshe, Martha Chogugudza
The denaturation and aggregation of β-lactoglobulin and α-lactalbumin were studied in the following mixtures, designed to simulate the protein concentrations and ionic environment in normal skim milk. 1. β-lactoglobulin (0.4% w/v), 2. α-lactalbumin (0.15% w/v), 3. β-lactoglobulin (0.4% w/v) and casein micelles (~ 2.6% w/v), 4. α-lactalbumin (0.15% w/v) and casein micelles (~ 2.6% w/v), 5. β-lactoglobulin (0.4% w/v) and α-lactalbumin (0.15% w/v) and 6. β-lactoglobulin (0.4% w/v), α-lactalbumin (0.15% w/v) and casein micelles (~ 2.6% w/v) Proteins were dissolved in SMUF, pH 6.7, and heated at 80 and 95°C for various times and centrifuged at 100,000 g for 60 min. The supernatants and pellets obtained were analysed using gel electrophoresis under non-dissociating (Native-PAGE in the absence of dissociating and reducing agents), dissociating but non-reducing (SDSNR-PAGE) and dissociating and reducing conditions (SDSR-PAGE). When β-lactoglobulin was heated alone and examined by native-PAGE, the quantity of native protein decreased with increasing heating time at 80°C. Addition of α-lactalbumin to the β-lactoglobulin solution increased the loss of β-lactoglobulin during the initial stages of heating. Addition of casein micelles to the β-lactoglobulin solution markedly increased the loss of native β-lactoglobulin throughout the heating period. The loss of β-lactoglobulin from the mixture containing β-lactoglobulin, α-lactalbumin and casein micelles was similar to that from the mixture of β-lactoglobulin and casein micelles. The loss of β-lactoglobulin from these protein mixtures could be described by second-order reaction kinetics. Heating these mixtures at 95°C caused very rapid loss of native β-lactoglobulin, but the effects of the addition of casein micelles and α-lactalbumin were generally similar to those observed at 80°C. When α-lactalbumin was heated at 80°C either alone or in the presence of casein micelles, there was only a slight loss of the native α-lactalbumin. However the corresponding losses of native α-lactalbumin were considerable greater on heating at 95°C. At both temperatures, the addition of β-lactoglobulin increased the rate of loss of α-lactalbumin substantially. The addition of casein micelles to the mixture of α-lactalbumin and β-lactoglobulin had little further effect on the loss of native α-lactalbumin. The rates of loss of α-lactalbumin at 95°C in all mixtures could be adequately described by first-order kinetics. When β-lactoglobulin was heated either alone or in the presence of casein micelles and examined by SDSNR-PAGE, the loss of SDS-monomeric β-lactoglobulin was less than the loss of native β-lactoglobulin. In contrast, when α-lactalbumin was added to β-lactoglobulin or β-lactoglobulin and casein micelles mixture, the loss of SDS-monomeric β-lactoglobulin was comparable to that of native β-lactoglobulin. The difference between native and SDS-monomeric β-lactoglobulin represents aggregates that are linked by non-covalent (hydrophobic) interactions. Thus the protein mixtures containing α-lactalbumin, contain no or little non-covalently linked β-lactoglobulin aggregates, and consequently, all the β-lactoglobulin aggregates would be disulphide linked. The results for the loss of SDS-monomeric and native α-lactalbumin at 95°C showed that both non-covalent and disulphide-linked aggregates of α-lactalbumin were present in all the protein mixtures studied. When β-lactoglobulin solution was heated at 95°C, large aggregates were formed which could be sedimented at 100,000 g for 60 min. Addition of casein micelles to β-lactoglobulin solution caused greater sedimentation of β-lactoglobulin. Similar results were obtained when the mixture containing β-lactoglobulin, α-lactalbumin and casein micelles was heated at 95°C. In contrast, the mixture containing β-lactoglobulin and α-lactalbumin behaved in a similar manner to β-lactoglobulin alone. When α-lactalbumin was heated at 95°C alone or in the presence of casein micelles, it did not interact to form large sedimentable aggregates. However when β-lactoglobulin was added to the above protein solutions, there was a considerable increase in sedimentation of α-lactalbumin.
Milk separation and pasteurisation : the impact of separating temperature, and order of separation and pasteurisation, on the composition of skim milk, cream and separator sludge : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology of Massey University, Palmerston North, New Zealand
(Massey University, 2014) Brooks, Evonne Hilary
A principal purpose of the present study was to determine whether the order in which separation and pasteurisation of whole milk occurs has an effect on the composition of skim milk and cream, and thus potentially of products made using these streams. The study also sought to determine the effect of separating temperature on the composition and microbiological quality of skim milk and cream. In addition, a survey of whole milks and separator sludges at four Fonterra manufacturing sites across New Zealand was carried out to determine whether there was regional variation in minerals content. This related to the suspected involvement of sludge minerals content in the incidence of desludging port erosion found in some separators, particularly in Northland. Trials to study the effects of order of separation and pasteurisation, and of separating temperature, were first carried out in an ideal environment in the pilot plant at what is now Fonterra Research and Development Centre. Commercial-scale trials of the same kind were then carried out at Fonterra Kauri. The minerals survey was conducted by collecting and analysing whole milk and separator sludge samples collected at Fonterra Kauri, Fonterra Whareroa, Fonterra Clandeboye and Fonterra Edendale. This study has identified that dairy manufacturing plants have a larger operating window in terms of separating temperature and equipment configuration than previously thought. The ANOVA analysis may have found significant effects, but the compositional changes were minor. The mineral survey work showed that there were significant batch differences for all minerals. The calcium and phosphate contents explained most of the variability in the composition. The milk at the Kauri plant was different to milk in other parts of the country. Calcium content could be used to differentiate between the different sites tested. The phosphate content could be used to distinguish between separators.
An immobilised cell system for the delivery of functional Lactobacillus reuteri DPC16 cells to their target site in a simulated gastrointestinal tract : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Science at Massey University, Albany, New Zealand
(Massey University, 2012) Zhao, Qian
The objective of this study was to design and produce calcium alginate beads that can deliver immobilised cells of Lactobacillus reuteri DPC16 to a target site of the colon in the gastrointestinal (GI) tract, without any diminution of their important physiological characteristics. Several factors that might affect the effectiveness of calcium alginate beads for the cell delivery were investigated, using an in vitro GI tract model to simulate the conditions within the tract. Firstly, by varying the concentration of alginate at a constant concentration of CaCl2, and combining the system with gelatin, chitosan or skim milk, the survival of immobilised DPC16 cells in simulated gastric fluid (SGF) was observed. Secondly, the physical stability of calcium alginate beads containing skim milk was observed during sequential incubation in the GI fluids using optimal concentrations of alginate. Finally, the survival of DPC16 cells immobilised within alginate beads containing skim milk was monitored when the beads were incubated for different times during sequential exposure to the simulated fluids. The results demonstrated that non-encapsulated DPC16 cells were sensitive to an acidic environment, and no viable cells were detected after 90 min exposure in SGF (pH 1.2). After appropriate experimentation, an alginate concentration of 3% (w/v) was deemed to be the optimum value and was used in subsequent investigations. When skim milk (8% (w/v)) was added to the alginate solution, the cell survival in SGF was improved markedly. The optimal concentration of calcium chloride was 0.3 M, based on the beads maintaining their integrity in SGF and simulated intestinal fluid (SIF) while disintegrating in simulated colonic fluid (SCF) to release viable cells. Hence, the beads made from 3% alginate, 8% skim milk and 0.3 M CaCl2 proved to be an effective delivery and release system for DPC16 cells. L. reuteri DPC16 has strong antimicrobial activities against pathogens, due mainly to its ability to produce reuterin. Hence this and other functional properties of the bacterial cells were studied before and after passage through the GI tract. The cells that were recovered after release from the alginate beads in the SCF showed no diminution in functional properties, including their growth kinetics, ability to adhere to epithelial cells and ability to inhibit the adhesion of E. coli to epithelial cells. However, the bacteriostatic and bactericidal properties of the recovered cells against some pathogens were significantly greater (P<0.05) than those of the original cells. Production of reuterin by the recovered cells was significantly greater (P<0.05) than that of the original cells when cultured in MRS medium in the absence of its metabolic precursor, glycerol. The results demonstrate significant (P<0.05) consequences for the application of the encapsulation technique to protect and/or enhance the functional properties of the probiotic cells. Subsequently, an investigation was carried out to find the reason for the antimicrobial activity enhancement. By recovering cells from different stages of the immobilisation and delivery process and examining them for their antimicrobial properties, it was found that it was the immobilisation process per se, rather than passage through the simulated gastrointestinal fluids, that caused the enhancement of antimicrobial activity, and that this was related to increased activity of the enzyme (diol dehydratase) that is responsible for reuterin production from glycerol. Finally, it was demonstrated that freeze-drying of the alginate beads was not an appropriate storage technique as it resulted in a significant (P<0.05) diminution of the antimicrobial activities. Based on these findings it is confirmed that the alginate-skim milk-CaCl2 immobilisation system is an effective and efficient method, not only for protecting the viability of DPC16 cells, but also for maintaining the physiological characteristics.
High-pressure-induced starch gelatinisation and its application in a dairy system : a thesis presented in partial fulfilment of the requirements for the Doctor of Philosophy in Food Science at Massey University, Auckland, New Zealand
(Massey University, 2009) Oh, Hyunah Eustina
This study investigated pressure-induced starch gelatinisation in water and milk suspensions. A rheological method, termed ‘pasting curves’, provided an objective and analytical means to determine the degree of pressure-induced starch gelatinisation. In addition, a polarised light microscope was used to observe birefringence of the starch granules and the degree of starch swelling was measured. The preliminary investigation into pressure-induced gelatinisation of six different starches showed that potato starch was the most pressure resistant and was not gelatinised after a pressure treatment of 600 MPa for 30 min at 20 °C. Waxy rice, waxy corn and tapioca starches showed complete gelatinisation after the same treatment while normal rice and normal corn starches were only partially gelatinised despite the disappearance of birefringence. Based on the preliminary study, two starches (normal and waxy rice starches) were selected for more detailed studies. The effects of treatment conditions (pressure, temperature and duration) on the gelatinisation were investigated with these selected starches. The degree of gelatinisation was dependent on the type of starch and the treatment conditions. The results also indicated that different combinations of the treatment conditions (e.g. high treatment pressure for a short time and low treatment pressure for a longer time) could result in the same degree of gelatinisation. Both starch types exhibited sigmoidal-shaped pressure-induced gelatinisation curves and there was a linear correlation between the degree of swelling and the apparent viscosity of the starch suspension. After treatments at =500 MPa for 30 min at 20 °C, both starches lost all birefringence although the apparent viscosity and the degree of swelling of normal rice starch did not increase to the same extent as observed in waxy rice starch. Pressure-induced gelatinisation of starch was retarded when starch was suspended in skim milk. This was attributed to the effect of soluble milk minerals and lactose present in the milk whereas milk proteins (casein and whey) did not affect the degree of gelatinisation at the levels present in 10% total solids skim milk. The presence of soluble milk and/or lactose may lead to less effective plasticising of starch chains by the suspension medium. Interactions between milk components and starch molecules may also play a role in retarding gelatinisation by reducing the mobility of starch chains. The functionality of starch in a dairy application was tested using acid milk gels as a model system. Skim milk with added starch (waxy rice or potato starch) was either pressure treated (500 MPa, 20°C, 30 min) or heat treated (80°C, 30 min) and subsequently acidified to form acid milk gels. The addition of waxy rice starch resulted in firmer acid milk gels, and increasing the amount of starch caused an increase in the firmness of both pressure-treated and heat-treated samples. However, pressure-treated samples with added potato starch did not show significant changes in the firmness whereas the heat-treated counterparts showed a marked increase in the firmness as the level of potato starch increased. The difference between the effects of the two different starches can be explained by the extent of starch gelatinisation in skim milk. Starch granules absorb water during gelatinisation whether induced by pressure or heat which effectively increases milk protein concentration in the aqueous phase to form a denser protein gel network on acidification. The firmness of acid milk gels can be increased by adjusting the pH at pressure or heat treatment to higher than the natural pH of milk. The effect of pH at pressure or heat treatment and addition of starch on the acid milk gel firmness was additive and independent of each other up to a starch addition level of 1%. This study provided an insight into pressure-induced gelatinisation of starch by showing gelatinisation properties of starches of different botanical origins and the effects of the treatment conditions (treatment pressure, treatment temperature and duration) on the degree of gelatinisation. Furthermore, the results from the pressure treatments of starch in dairy-based suspensions showed that pressure-induced gelatinisation was affected by other components in the system. These results demonstrate the importance of understanding the gelatinisation properties of starch in complicated food systems in which a number of other components are present. In terms of the application of starch in dairy systems, when starch was added to milk and gelatinised by pressure treatment, the acid milk gel produced by subsequent acidification was firmer than the acid milk gel made from skim milk alone.

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