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Item The effect of milk processing on protein digestion and amino acid absorption in the gastrointestinal tract of pigs as a model human : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Sciences at Massey University, Manawatū, New Zealand(Massey University, 2024-06-19) Ahlborn, Natalie Gisela MarlisGlobally, milk is processed using heat and homogenisation to improve food safety and extend shelf life. These common processing techniques can alter the native structures present in milk, including protein structures. However, the impact of these processing-induced changes on the digestion of milk protein and subsequent absorption of amino acids in the human body is not yet fully understood. The overall objective of this research was to understand how heat treatment and homogenisation affect milk protein coagulation and digestion in the stomach, and to investigate how changes to gastric coagulation (curd formation) influence amino acid (AA) absorption in the small intestine and AA concentrations in blood circulation. Due to the limited accessibility of the human gastrointestinal tract, pigs were used as a model of the human. An initial study using raw bovine (cow), caprine (goat), and ovine (sheep) milk established the role of gastric curd formation in small intestinal AA absorption in piglets at a single postprandial time point. Specifically, differences in the retention of AA in the gastric curd were responsible for differences in the small intestinal AA absorption across milk of different species. A separate study using bovine milk as a milk model was then conducted to determine the effect of heat treatment and homogenisation on the kinetics of milk protein digestion and small intestinal AA absorption. The selected processing treatments were pasteurisation, ultra-high temperature treatment (UHT), and homogenisation. Raw milk was included as a comparator. In the stomach, heat treatment and homogenisation altered the strength and structure of the curd formed during gastric digestion, which in turn affected both milk protein hydrolysis and the rate of AA entering the small intestine. Differences in the release of digested protein and AA into the small intestine were reflected in the kinetics of AA absorption of the processed milk types. For example, UHT milk had both a faster rate of AA entering the small intestine and a faster rate of AA absorption. Processing also altered the appearance of some AA in blood circulation; however, these differences were not directly reflective of the differences observed in their small intestinal absorption kinetics. In conclusion, this PhD research demonstrated that the rate of small intestinal AA absorption was modulated by gastric curd formation, indicating that milk processing could be used as a strategy to modulate protein digestion and AA absorption in the gastrointestinal tract.Item Studies on heat-induced protein interaction and digestion behavior of sheep milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Riddet Institute, Massey University, Palmerston North, New Zealand(Massey University, 2023) Pan, ZhengSheep milk has low heat stability, which results in undesirable changes after heat treatment, such as separation of milk fat, sediment formation, and phase separation. However, the mechanism of low heat stability of sheep milk has not yet been elucidated. Additionally, the protein interactions in sheep milk during heating and the digestion behaviors of differently processed sheep milk are also unknown. Therefore, the aim of this thesis was to explore the protein interactions of sheep milk during heat treatment (67.5–90 °C and 140 °C), and the mechanism of heat coagulation of sheep milk. In addition, the effect of the commercial processing treatment [homogenization (200/50 bar) and thermal (75 °C/15 s and 95 °C/5 min) processing] on the digestion behavior of sheep milk were determined. Sheep skim milk (SSM) was heated under various conditions (including temperatures, heating times and pH values) and the denaturation of whey protein and protein interactions occurring during heating were characterized using high-performance liquid chromatography. Casein micelle diameter increased upon heating, depending on the temperature and time. The association of whey protein with casein micelles and the aggregation of casein micelles occurred simultaneously and contributed to the increase in casein micelle size in SSM. SSM was stable to heat (140 °C) at pH 6.8–6.9 but became unstable at higher or lower pH. The low heat stability of sheep milk was attributed to the low proportion of κ-casein surrounding the casein micelles, high ionic calcium levels and ready dissociation of κ-casein from casein micelles upon heating at pH 7.0. The Human Gastric Simulator was used for in vitro dynamic gastric digestion and pH-stat for simulated small intestinal digestion. Heat treatment of sheep milk resulted in the incorporation of MFGs into the curds through casein‒whey protein or whey protein‒whey protein interactions; this hindered the formation of the closely knitted protein network and led to the formation of fragmented curds. Homogenization of sheep milk resulted in looser and more fragmented curd in comparison with unhomogenized sheep milk; this accelerated the protein hydrolysis and increased the rate of release of protein, fat, and calcium from the curds into the digesta. Processing treatments affected the lipolysis rate but not the lipolysis degree during small intestinal digestion. In conclusion, the findings of this study have advanced our understanding of the heat-induced protein interactions in sheep milk and provided insights into the digestion behavior of differently processed sheep milk within the gastrointestinal tract. This may help to design and develop sheep milk-based products with desired digestive and functional properties.Item Characterisation and protein complexation of an anthocyanin-bound pectin extracted from New Zealand blackcurrant (Ribes nigrum) : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2022) Salleh, NurhazwaniThe main objective of this thesis was to investigate the cause of physical instability in blackcurrant juice-milk system. Poor phase stability in fruit juice-milk beverages is a major challenge for the clean-label beverage industry as milk protein can interact with fruit components, like polysaccharides and polyphenols, generating unwanted characteristics such as coagulation of milk proteins and phase separation. Hence, the principal step to understand the causes of poor phase stability was to identify and study the key interactive components of the juice, which was extracted from the New Zealand blackcurrant (Ribes nigrum), and then investigate their interactions with milk proteins. The key components of the blackcurrant juice were first isolated using mild extraction procedures, via ethanol precipitation and dialysis, and were identified as a complex fraction particularly rich in pectin and anthocyanins (Chapter 4). Proximate analysis revealed that the fraction contained carbohydrate (78% w/w), uronic acid (21% w/w), protein (4.8% w/w), anthocyanin (3.9% w/w) and calcium (2.2% w/w). The pectin-rich fraction had a net negative surface charge of -23.1 mV (at pH 4.8), a pKₐ value of 1.7 and a relatively high degree of esterification (65.2%). Constituent sugar analysis showed that the fraction was mostly made of galacturonic acid, rhamnose, arabinose and galactose, and NMR spectroscopic analysis revealed that it was rich in rhamnogalacturonans with arabinogalactan side chains. This pectic fraction was unique as it was highly pigmented, with cyanidin 3-O-rutinoside as its major anthocyanin. Liquid chromatography revealed that the anthocyanins were tightly bound to the fraction as methanol used in the technique failed to separate them. Results from size-exclusion chromatography coupled with multi-angle laser light scattering showed that the blackcurrant juice contained two major pectic fractions—≈283 kDa present at 14.6% w/w and ≈97 kDa at 85.5% w/w—with the latter producing higher UV₂₈₀ ₙₘ signal, signifying that proteins and/or polyphenols were present mainly in the second fraction. Association of anthocyanins to biopolymers like pectin and protein can occur via multiple interactive forces (electrostatic, hydrophobic and hydrogen bonding forces), and pH is known to play a significant role as it can affect the associative mechanisms of anthocyanins by changing their molecular configuration and ability to electrostatically interact. An attempt to dissociate blackcurrant anthocyanins from the blackcurrant biopolymers was carried out by disrupting electrostatic interactions and changing the planarity of anthocyanins via pH adjustments and ultra-filtration (Chapter 5). Lowering the juice pH to 2 did not result in anthocyanins dissociation, likely because anthocyanins were bound to the biopolymers by other interactive forces apart from the electrostatic bonds. Increasing the juice pH to 4.5 might have dissociated some anthocyanins from the biopolymers, but this was not reflected in the analysis of anthocyanins, probably because the freed anthocyanins had degraded before the analysis was carried out. Overall, size segregation of the juice components via ultra-filtration was relatively effective. Regardless of the pH, majority of the anthocyanins were still tightly associated with the large molecular weight biopolymers, confirming the involvement of multiple interactive forces. In order to uncover the cause of phase instability in blackcurrant juice-milk system, a complexation study between the isolated pectin-rich fraction and whey proteins was conducted (Chapter 6). The impact of bound anthocyanins on pectin-protein interactions was studied by exploring the effects of pH (pH 3.5 and pH 4.5), heating (85 °C, 15 min) and heating sequence (mixed-heated or heated-mixed). The pH was found to influence the colour, turbidity, particle size and surface charge of the mixtures, but its impact was most drastic when heating was introduced. Heating increased the amount of blackcurrant pectin within the complexes—especially at pH 3.5, where 88% w/w of the initial pectin was found in the sedimented (insoluble) fraction. Based on physical stability measurements, the mixed-heated system at pH 4.5 displayed better stability than at pH 3.5. A noteworthy finding was that heating sequence was found to be effective in preventing the destabilisation of the systems. Mixing of components before heating produced a more stable system with small complexes (<300 nm) and relatively low polydispersity. However, heating whey proteins before mixing with blackcurrant pectin prompted protein aggregation, producing large complexes (>400 nm) that worsened the destabilisation. The influence of bound anthocyanins on pectin-protein complexation was further studied by comparing two types of pectin-protein mixtures: (i) a mixture that is rich in anthocyanin (blackcurrant pectin-whey protein, BCP-WP) and (ii) a mixture that is free of anthocyanin (citrus pectin-whey protein, CP-WP) (Chapter 7). The mixtures were prepared at pH 4.5 with and without heat treatment at 85 °C. The study revealed that there was no direct relationship between anthocyanin presence and the destabilisation of mixtures. The Fourier-Transform Infrared (FTIR) spectrum of the heated and non-heated BCP-WP sedimented fractions showed the emergence of a peak at 800-1200 cm⁻¹, signifying the presence of anthocyanin-protein interactions. This peak, however, was absent in the spectrum of any of the anthocyanin-free CP-WP sedimented fractions, indicating that the bound anthocyanins of blackcurrant pectin provided the whey proteins with additional binding sites. The findings from FTIR analyses also indicated that non-electrostatic forces were most likely the governing forces of the heated BCP-WP mixture, via hydrophobic interactions and later reinforced by hydrogen bonds upon cooling. This thesis revealed that poor phase stability of the blackcurrant juice-milk system should not be attributed exclusively to the blackcurrant juice components, particularly the polyphenols. Environmental factors like pH and heat were likely the leading cause of phase instability as they could intensify the interactions that occurred in the mixed system, which eventually destabilised the mixture. This suggests that appropriate processing conditions can be applied to positively affect the blackcurrant juice-milk system.Item Characterisation of de-structured starch and its interactions in whey protein isolate gels : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2022) Ang, Cai LingStarch serves as an important additive to enhance the physico-chemical properties of many food products. With the increased pursuit of natural products, there is an increasing demand for “clean-label” starches. In this study, waxy potato starch was physically-modified at elevated temperatures of 120–150 °C for 30 min at 300 rpm, in a pressurised reactor. The treatment converted native starch granules into their macromolecular chains (denoted as de-structured waxy potato starch, DWPS). This doctoral thesis presents the: (i) method of modifying starch (i.e., the de-structuring process), (ii) the mechanism of starch de-structuring, (iii) the rheological changes in DWPS samples and the shear-thickening mechanism, and (iv) the interactions of these DWPS ingredients with whey protein isolate (WPI) in a protein-based gel system, at different pH and ionic strength. The molar mass (Mᵥᵥ), particle size, rheological properties, degree of branching (DB) and side-chain length distribution of DWPS samples were characterised to elucidate the starch de-structuring mechanism. DWPS treated at 120 °C DWPS showed similar Mᵥᵥ (~3.6 × 10⁸ Da) as its native form (~3.7 × 10⁸ Da) indicating that the treatment at 120 °C resulted in the disassembly of starch granules into their macromolecular chains. Reduction in viscosity, Mᵥᵥ and particle size was observed with an increase in temperature from 120 to 150 °C, suggesting a cleavage in amylopectin chains. The DB and side-chain distribution data suggest that the reduction in Mᵥᵥ is likely due to the cleavage at α-1,4 linkages near the middle of the main amylopectin backbone. Particle size analysis by laser diffraction measurements revealed the presence of large fragment particles (> 1 µm) in DWPS samples, indicating that the starch de-structuring process into its macromolecules was incomplete even at 150 °C for 30 min. The DWPS (5% w/w) samples were found to exhibit a wide range of rheological properties—Newtonian, shear-thinning, shear-thickening and anti-thixotropy behaviours—depending on their treatment temperature (120–150 °C). In particular, 120 °C DWPS exhibited interesting shear-thickening, anti-thixotropy and shear-induced gelation. These rheological properties are different from the shear-thinning and thixotropy behaviours observed in most conventionally gelatinised waxy potato starches treated at 95 °C. The complex shear-induced structures of 120 °C DWPS were attributed to a two-step process: (i) upon shear at the critical shear rate (~10–20 s⁻¹), the shear stress caused a size reduction in the starch fragments and (ii) the increased number of small fragments together with the amylopectin chains in very close proximity could lead to the formation of a complex network probably consisting of amylopectin chains and a large number of fragments (2–20 μm). Shear thickening properties were attributed largely to these soft fragment particles colliding and sliding past each other during shear. The data from this study has also shown that the hydrogen bonding, electrostatic, hydrophobic interactions, or the combination of these interactions did not cause the shear-thickening behaviour. The influence of 4% w/w DWPS on 13% w/w WPI gels was studied by characterising the phase stability of the liquid mixtures, and mechanical properties, microstructure, and water-immersion stability of fine-stranded polymeric and coarse-stranded particulate protein gels at pH 7 and pH 5, respectively. At neutral pH, synergistic gel hardness of WPI was obtained with the incorporation of 140 °C DWPS. The increased gel strength was attributed to the enhanced density of a very fine-stranded gel network. The ability of the gel to retain its shape when immersed in water for 40 h was most noticeable for the composite gels containing either gelatinised starch or DWPS samples (swollen gels but with intact shape). In contrast, pure WPI gel and composite gel containing maltodextrin turned into very weak fluid-like and disintegrated gels, respectively. At pH 5, WPI formed particulate gels. The addition of gelatinised starch or DWPS weakened the particulate protein gels, likely due to phase separation and interrupted protein network with starch polymers acting as inactive fillers. The effects of NaCl and CaCl₂ (i.e., type of salts and ionic strength) on the mechanical and microstructural properties of composite gels containing 13% w/w WPI and 4% w/w 140 °C DWPS were also evaluated. Thermodynamic incompatibility between WPI and 140 °C DWPS was observed upon the addition of NaCl (~75 mM) or CaCl₂ (10–75 mM). The combined effects of such thermodynamic incompatibility with the changes in protein connectivity induced by varied ionic strength led to the formation of distinctive gel structures (inhomogeneous self-supporting gels with a liquid centre and weak gels with paste-like consistency) that were different from thermodynamic compatible homogeneous self-supporting gels (pure WPI and WPI + maltodextrin gels). At ≥ 250 mM NaCl, instead of a paste-like texture, a recovered soft self-supporting gel structure was observed when using 140 °C DWPS. The ability to generate a range of textures in WPI gelation-based foods by using 140 °C DWPS under different ionic conditions, is a feasible strategy for structuring high-protein foods for dysphagia—aimed to be either thickened fluids or soft solids. Additionally, this acquired knowledge is also relevant when formulating food gels for 3-D printing. The desirable rheological properties of DWPS samples and their ability to alter WPI gel structure signify the potential of DWPS as a clean-label ingredient to structure foods of specific needs (e.g., whipping cream for enhanced structure upon shear and high-protein foods for dysphagia sufferers).Item Recovery of α-lactalbumin from whey protein isolate and osteopontin from milk by anion exchangers : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Chemistry at Massey University, New Zealand(Massey University, 1998) Chen, YishanA series of amines, DMEA, DMH, DMO, DMD, DMDo, and Do, were coupled to Sepharose, which was activated by epichlorohydrin first, to prepare amino anion exchangers DMEA-Seph, DMH-Seph, DMO-Seph, DMD-Seph, DMDo-Seph, and Do-Seph. The batch binding of α-lactalbumin and ß-lactoglobulin in 25 mM NaCl at around proteins' IEP to these exchangers and a commercial anion exchanger Q-Seph-ff were tested. Q-Seph-ff, DMEA-Seph, DMH-Seph, and DMO-Seph bound both of proteins at pH > IEP. Q-Seph-ff, DMEA-Seph, DMH-Seph did not bind either of the proteins at pH < IEP. DMO-Seph, DMD-Seph, DMDo-Seph, and Do-Seph bound both of the proteins, especially α-lactalbumin, by hydrophobic interaction at and below the proteins' IEP. The proteins bound by these exchangers except DMO-Seph could not eluted by HCl at low pH. HCI at pH 2.5 could be used to elute these proteins from DMO-Seph. Recovery of α-lactalbumin from WPI in 25 mM NaCl at pH 3.8-5 by DMO-Seph, DMD-Seph, DMDo-Seph, and Do-Seph were tested. These exchangers were able to bind α-lactalbumin in preference to ß-lactoglobulin at and below the proteins' IEPs. Thus DMD-Seph gave an α-lactalbumin yield and purity of 70 and 80% at pH 4.3, DMO-Seph 77 and 81% at pH 4.4. However DMD-Seph had difficulty in eluting all of the α-lactalbumin unless using ethanol. The batch binding of α-lactalbumin and ß-lactoglobulin in high concentration of NaCl at low pH (2.5) by DMO-Seph was tested. The exchanger showed strong hydrophobic affinity for a-lactalbumin but not ß-lactoglobulin in 200-500 mM NaCl. Recovery of native α-Iactalbumin from WPI in 400 mM NaCl at pH 2.5 by DMO-Seph was tested. This gave an α-lactalbumin yield and purity of 79 and 73% and a capacity of DMO-Seph 0.73 g/g in 400 mM NaCl at pH 2.5, compared to 67%, 84% and 16 mg/g of DMO-Cell. DMO-Cell was prepared. Cellulose was modified by propyl oxide and epichlorohydrin and then activated by ECH. The activated cellulose was then coupled with DMO. The effects of cellulose particle size, cellulose type and substitution level of DMO-Cell on binding of whey proteins were investigated. DMO-Cell, activated by 1,4-butanediol diglycidyl ether, with substitution level 0.55 meq/g was prepared. It showed a better binding capacity than DMO-Cell activated by epichlorohydrin. Recovery of native α-lactalbumin from different WPI in 400 mM NaCl at pH 2.5 by DMO-Cell was tested. DMO-Cell showed good selectivity for α-lactalbumin from all of three WPI. This gave an α-lactalbumin yield and purity of 13.5 mg/g from WPI (PT8253). From a low α-lactalbumin content WPI this gave an α-lactalbumin yield and purity of 70 and 91%.Item High protein Chinese steamed bread : physico-chemical, microstructural characteristics and gastro-small intestinal starch digestion in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand(Massey University, 2019) Mao, ShiyuanIn Asia, high protein low carbohydrate foods are in high demand because their consumption can provide improved nutritional benefits and help maintaining blood glucose levels close to normal. High protein versions of popular, highly consumed food products (staple foods) such as Chinese steamed bread (CSB) can be very useful to improve the health status of our populations. Thus, the objectives of this study were: to develop high protein Chinese steamed bread (HPCSB) using plant protein, dairy protein combinations. The high protein versions of the steamed breads were then compared with control 100% wheat flour based Chinese steamed bread for physico-chemical, microstructural, textural and in vitro starch digestion characteristics. In order to develop HPCSB, plant proteins (soy protein isolate) and dairy proteins (rennet casein and milk protein concentrate) were blended into wheat flour at two different levels. The addition of proteins has led to a change in colour characteristics (L*, a*, b*) and also resulted in a decreased specific volume of the breads. The textural characteristics measured through textural profile analysis of HPCSB showed an increased hardness and gumminess than control. The microstructure of HPCSB was observed to be more compact and had fewer air cells when observed through Scanning Electronic Microscopy. Furthermore, in vitro starch digestion of HPCSB depicted that the addition of proteins was capable of lowering the starch hydrolysis (%) and estimated glycaemic index (eGI), especially for RC I and RC II at significant levels. Addition of both proteins influenced the microstructure of HPCSBs, which in turn affected the textural and starch digestion properties. High protein Chinese steamed bread with low glycaemic properties can be prepared by critically selecting the protein sources with minimum changes in their physical and textural characteristics.Item Effect of air temperature on the thermal degradation of heat liable products in spray drying and monodisperse drying : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand(Massey University, 2018) Sang, XiaoqiThree heat liable protein-based materials β-galactosidase, whey protein isolate (WPI) and egg white (with 30-35% w/w total solids) were dried through conventional spray drying and monodisperse drying respectively with constant inlet air temperature 200 ℃ and different outlet air temperature. The purpose was to test the hypothesis that monodisperse droplet drying could produce more control over time-temperature experience during drying, resulting in reduced loss of structure or activity. The residual enzyme activity of the dried lactase product was determined by ONPG β-galactosidase assay, and the extent of denaturation of WPI and egg white was determined by differential scanning calorimetry (DSC). Particle size and morphology were also measured and observed. The results showed that for both spray drying and monodisperse drying, the extent of protein denaturation increased as outlet air temperature increased. In comparison with spray drying, monodisperse drying had a longer residence time using our particular apparatus and gave rise to higher extent of heat degradation for all three materials. The dried products from monodisperse drying showed a narrower particle size distribution but had larger particle size compared to the products from spray drying. The majority of monodisperse dried powders had a multivesicular hallow morphology due to high interior temperature and coalescence of neighbouring particle in flight. The feasibility of using monodisperse drying in real industry is still under investigation. Although the results obtained from this study denied the expectation that monodisperse drying can reduce the thermal degradation of product during drying process, they are still useful in developing the monodisperse drying system and optimizing the operating parameters.Item A comparative study of an aminopeptidase from lactic acid bacteria: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1992) Midwinter, Robyn GillianAminopeptidase enzymes from the proteolytic systems of S.salivarius subsp.thermophilus Lactococcus lactis subsp.cremoris and Lactococcus lactis subsp.lactis have been investigated. An aminopeptidase was purified to near homogeneity from a crude cell free extract of S.thermophilus 5109. The enzyme had a native molecular weight of approximately 96kDa determined by gel-filtration, and a subunit molecular weight of 98kDa, determined by denaturing polyacrylamide gel electrophoresis, showing the native enzyme to be a monomer. The aminopeptidase activity was optimal at pH 7.0 and 35°C. The enzyme was inactivated by p-chloromercuribenzoic acid, iodoacetic acid,the chelating agents EDTA and 1,10-phenanthroline and the divalent cations Cu2+, Zn2+ and Co2+. The aminopeptidase was not inhibited by the serine protease inhibitor PMSF and only minor inhibition occured with the inhibitor No:-p-tosyl-L-lysine chloromethyl ketone (TLCK). The aminopeptidase was capable of hydrolysing several amino-acyl amido methyl coumarin (AMC) and p-nitroanilide (pNA) derivatives, particularly those of lysine, arginine and leucine. The enzyme showed greatest activity with lysyl derivatives (and is therefore referred to in this thesis as a lys-aminopeptidase). The enzyme was able to degrade several oligopeptides by progressive cleavage of the peptide bond but did not hydrolyse peptides containing a proline or aspartic acid residue in the second position. The aminopeptidase activity was dependent on the size of the peptide in that generally only peptides with more than three amino acids were degraded. The aminopeptidase had no endopeptidase or dipeptidase activity. Five different amino-acyl p-nitroanilides derivatives and two amido methyl coumarin derivatives were used to determine the kinetic parameters of the aminopeptidase. The Km values obtained for all the substrates tested were similar, with the exception of ala-pNA, for which the Km value was significantly higher. On the basis of the distribution of activity between different cell-fractions the lys aminopeptidase appears to be localised intracellularly. An aminopeptidase was also partially purified from cell-free extracts from Lactococcus lactis subsp.cremoris AM2 and Lactococcus lactis subsp.lactis ML3. The aminopeptidase from L.cremoris AM2 was shown to have a molecular weight of 106kDa and was a monomer. It showed optimal activity at a pH of 7.0 and 450c. The aminopeptidase activity was inhibited by metal-chelators, SH group inhibitors and TLCK. The aminopeptidase hydrolysed lysyl-, arginyl- and leucyl-p-nitroanilide derivatives, but had little or no activity with other pNA substrates. The aminopeptidase from L.lactis ML3 had a molecular weight of 100-105kDa and was monomeric. The optimal activity for the aminopeptidase was at pH of 7.0 and 40°C. The enzyme was inactivated by metal-chelators, sulphydryl inhibitors and by TLCK. Like the aminopeptidases from the other two strains the ML3 aminopeptidase was very specific hydrolysing lysyl-, leucyl- and arginyl-pNA but with very little or no activity with other amino-acyl derivatives.Item A study of aminopeptidases from lactic streptococci : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1989) Lloyd, Richard JohnTwo arninopeptidase enzymes from the proteolytic system of Streptococcus lactis 4760 have been studied. An X-Prolyl dipeptidyl arninopeptidase has been purified and characterised. The enzyme has a native molecular weight of approximately 150 kDa determined by gel filtration, and a subunit molecular weight of 83 000, determined by denaturing polyacrylarnide gel electrophoresis, showing the native enzyme to be a dimer. It is inhibited by phenyl methyl sulphonyl fluoride and is active over a pH range of 6 - 9. A range of X-Prolyl-amido methyl coumarin (X-Pro-AMC) derivatives with different aminoacyl residues in the X position have been used to define the steady state kinetic parameters. The Km and kcat values obtained with all of the X-Pro-AMC substrates tested were similar, with the exception of Glu-Pro-AMC, which gave a somewhat higher Km value. The action of the enzyme in degrading small peptides has been studied. It was found to be capable of removing X-Proline residues from peptides, except where two proline residues are situated in consecutive positions. A Lysyl-arninopeptidase has been partially purified and its characteristics studied. This enzyme has been shown to have a native molecular weight of approximately 78 000. It hydrolyses lysyl-, arginyl-, and leucyl-arnido methyl coumarin derivatives, but has little or no activity with other arninoacyl-AMC substrates. It also catalyses the removal of lysine and arginine residues from the amino-terminus of short peptides. The partially purified arninopeptidase preparation also has endopeptidase activity which is probably due to contamination by a separate enzyme. The individual and combined effects of these two enzymes on -casein-derived oligopeptides (produced by proteolytic action of the S.lactis proteinase) have been studied. These results indicate that these enzymes may be important in degradation of some casein-derived peptides during cheese ripening, while other peptides are resistant to hydrolysis.Item Properties of recombined milk protein composite gels : effects of protein source, protein concentration and processing time : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2002) Findlay, Robyn RosemaryIncreased knowledge of the interactions involved in the manufacture of Milk Protein Composite Gels (MPCGs) is essential for the further development of dairy-based analogue and recombined products and the advancement of novel product development. This study investigated MPCG manufacture using four protein sources (Rennet Casein, skim milk cheese (SMC), milk protein concentrate (MPC 85), calcium-depleted milk protein concentrate (IX MPC 85)), three protein to water (P/W) ratios (0.4, 0.5, 0.6) and four processing times (0, 4, 8, 16 minutes). The properties of the products were investigated using confocal and transmission electron microscopy, as well as rheological and functional tests. Protein source was found to have the greatest impact on product characteristics, followed by P/W ratio with processing time having little, and often inconsistent, effects. Increased protein concentration resulted in a higher viscosity during manufacture, a decrease in fat droplet size, an increase in gel firmness, and a decrease in meltability. Increased processing time resulted in a decrease in fat droplet size, few significant changes in firmness (both small- and large-strain), and an increase in meltability Fracture property analysis showed that SMC produced softer, more elastic gels than Rennet Casein. The whey-containing samples produced softer, more brittle gels with little difference between them Small-strain analysis showed that all samples were weak gels but the results did not follow the same trend as the fracture properties. The samples increased in firmness in the following order: SMC < Rennet Casein < IX MPC 85 < MPC 85. Microstructure analysis showed the presence of whey protein aggregates in the MPC 85 and IX MPC 85 samples. These samples also demonstrated aggregation of the lipid droplets, which was attributed to the presence of whey proteins. Reduced levels of calcium resulted in lower levels of emulsification (larger lipid droplets) due to lower in-process viscosities. Correlations between large- and small-strain testing showed that the correlation coefficient was dependent on the protein source being used and that although the level of correlation was not high, there was a general positive trend The small-strain and UW Meltmeter tests did not agree on the order of increasing meltability except for the SMC samples, which were significantly more meltable than the other protein sources. The two tests were poorly correlated (R² = 0.446).