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Item An investigation into the use of starch-gel-urea electrophoresis as a technique for studying the proteolysis occuring during cheese curing : a thesis presented to the Massey University College of Manawatu in partial fulfilment of the requirements of the degree of Master of Agricultural Science (Dairy Technology)(Massey University, 1963) Fenwick, Robin MilsonThe protean of Cheddar Cheese makes up a quarter of its bulk, supplies its high biological value and is a major factor in regulating the characteristics of its body. Knowledge of the agents involved in converting milk casein into typical cheese protein must have value in indicating ways by which cheese quality can be improved, or alternatively indicate ways to accelerate or control the rather haphazard process of cheese curing. Years of study into the subject of cheese protein degradation have shown the existence of a number of proteolytic agents present in cheese, viz: 1. The natural enzymes of milk. 2. The rennet enzymes. 3. Enzymes originating from the starter. 4. Enzymes originating from the adventitious flora of the cheese. Enquiry as to the relative importance of each enzyne system has been a long and confusing process employing a variety of techniques. Sherwood (1935) studied the changes in the various nitrogen fractions of cheeses in which bacterial numbers had been reduced by use of chloroform, but he was not able to completely eliminate the bacteria, neither distinguish between the activities of the various bacteria present in cheese, nor eliminate the effect of starter in the early period of manufacture.[FROM INTRODUCTION]Item Multiple proteolytic enzyme production in keratinophilic fungi (preliminary investigations) : a thesis presented in fulfilment of the requirement for the degree of Master of Science in Microbiology, Massey University(Massey University, 1998) Burrows-Anderson, DamarisSuperficial fungal infections can be acquired from a number of sources, e.g. animals, humans or from the soil. Many of the fungal species commonly associated with human disease arise from infection by species known commonly as dermatophytes, although infection from other non-dermatophytic keratinophilic fungi is becoming more common. Other species not commonly regarded as pathogenic have on occasion been found in human infection. Many of these opportunistic species are commonly found in soils. Isolation procedures employed in these studies were the hairbrush technique for small animals and the keratin-baiting technique for soil with samples being cultured on SDA containing antibiotics. Soil samples yielded 3 keratinophilic genera found in human infection (Microsporum spp.. Trichophyton spp. Aphanoascus sp.) while fungi isolated from animals yielded 3 fungal species. Microsporum canis. Microsporum cookei and Scopulariopsis brevicaulis. In these studies, various culture parameters e.g. pH, spore numbers and various hydrolysis techniques were examined in order to asses the production of proteolytic enzymes in vitro. Also in the course of these studies, the use of lactrimel medium as a suitable recovery agent for strains presenting atypical colony morphology and reduced proteolytic enzyme production was trialed with excellent results. The gelatin SDS-PAGE technique, mode of culture (shake and stationary) and the effect of substrate were analysed to compare the effects that these have on a range of keratinophilic fungi. Both pathogens and saprophytes were examined in an attempt to detect similarities in enzyme production which could be associated with the ability of various species to invade skin in vivo. A large body of data has been gathered demonstrating that the proteolytic enzymes produced by most keratinophilic fungi encompass a wide range of MW sizes and are not entirely predictable. This strongly suggests that when these fungi come into contact with a particular substrate, the ability of the strain to adapt may depend on the strains ability to produce a proteolytic enzyme capable of breaking down the substrates in the external environment providing nutrients for the growing fungi.Item Actinidin treatment and sous vide cooking : effects on tenderness and in vitro protein digestibility of beef brisket : 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, 2017) Zhu, XiaojieActinidin from kiwifruit can tenderise meat and help to add value to low-value meat cuts. Compared with other traditional tenderisers (e.g. papain and bromelain) it is a promising way, due to its less intensive tenderisation effects on meat. But, as with other plant proteases, over-tenderisation of meat may occur if the reaction is not controlled. Therefore, the objectives of this study were (1) finding a suitable process to control the enzyme activity after desired meat tenderisation has been achieved; (2) optimising the dual processing conditions- actinidin pre-treatment followed by sous vide cooking to achieve the desired tenderisation in shorter processing times. The first part of the study focused on the thermal inactivation of actinidin in freshly-prepared kiwifruit extract (KE) or a commercially available green kiwifruit enzyme extract (CEE). The second part evaluated the effects of actinidin pre-treatment on texture and in vitro protein digestibility of sous vide cooked beef brisket steaks. The results showed that actinidin in KE and CEE was inactivated at moderate temperatures (60 and 65 °C) in less than 5 min. However, the enzyme inactivation times increased considerably (up to 24 h at these temperatures) for KE/CEE-meat mixtures, compared with KE/CEE alone. The thermal inactivation kinetics were used as a guide for optimising actinidin application parameters during the second phase of the study. For the final experiments, beef steaks were injected with 5 % (w/w, extract/meat) of CEE solution (3 mg/mL) followed by vacuum tumbling (at 4 °C for 15 min) and cooking (at 70 °C for 30 min) under sous vide conditions. This cooking time was considerably less than usual sous vide cooking times used in the meat industry. The actinidin-treated meat had no change in pH and colour, but showed a lower instrumental shear force; and improved sensory scores for tenderness, juiciness and flavour than the untreated meat steaks when tested by a sensory panel. Improved tenderness agreed well with the Transmission Electron Microscopy (TEM) results that showed considerable breakdown of the myofibrillar structure, particularly around the Z line. The addition of actinidin enhanced the rate of breakdown of muscle proteins, as shown by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and led to an increase in both protein solubility and ninhydrin-reactive free amino N release, during simulated gastric digestion. These results demonstrate the positive effects of actinidin on meat tenderness and meat protein digestibility during gastric digestion in vitro.Item Influence of commercial proteases on the proteolysis of enzyme modified cheese : 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, 2000) Chen, Emily Yi ChuanThe influence of four commercial proteases, Protease A, Protease B, Protease C and a two enzyme blend Protease DE, on proteolysis in an enzyme modified cheese (EMC) base has been investigated. Also, a series of preliminary experiments to determine the basic characteristics of the four enzyme preparations in buffer systems has been undertaken. Generally, the exopeptidase activity of the four enzyme preparations was more stable than the endopeptidase activity of the preparations. The highest enzyme activity for all preparations was given at pH 6.5 and Protease B was found to be sensitive to chelating agents. In addition, Protease B was found to contain at least two exopeptidases. Residual protease activities in EMC using a 55% moisture cheese base were found to be 0.005%, 0.009%, 0.007% and 0.004% (w/v) for Protease A, Protease B, Protease C and Protease DE, respectively, following inactivation by heating at 95°C heating for 30 minutes. Under the same incubation conditions (0.15% enzyme at 40°C for 24 h), Protease DE gave greater proteolysis than the three other enzymes and Protease B was the weakest protease. EMC digestion with a combination of proteases was different from that obtained with individual proteases. The combinations of Protease A/Protease C, Protease DE/Protease C, Protease B/Protease C and Protease DE/Protease A showed that the higher the proportion of the former protease in the combinations, the higher the amounts of total amino acids produced in the EMC. The combinations of Protease A/Protease B and Protease B/Protease DE gave greater amounts of total amino acids with the ratio of each enzyme close to 50:50 than with the individual enzymes. With respect to the molecular mass distribution of peptides in the various EMC digestions, Protease DE produced the greatest amount of peptides of 3 or fewer residues and Protease C gave the greatest amount of more medium sized peptides with 11-20 residues. Compared with Protease C, Protease A was more efficient in giving small peptides, while Protease B gave the lowest levels of medium and small peptides, but a high level of free amino acids. In sensory testing, Protease DE produced EMC with a strong pungent and astringent flavour, Protease C gave bitterness, Protease A gave a sweet flavour at a low concentration but bitter flavours with a high concentration and Protease B produced more savoury flavour without bitterness.Item Aspects of proteolysis in cheese : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Food Technology at Massey University(Massey University, 1994) Coker, Christina JuneThe purpose of the present study was to elaborate methods for the detailed examination of proteolysis pathways in cheese (reviewed in Chapter 1) and to demonstrate their usefulness. Many techniques, including solvent fractionation, chromatographic separation and electrophoresis have been used previously and were revisited in this study. Gel electrophoresis can be a powerful technique and was examined in detail. The methods investigated were: 1) a slab gel system using the apparatus of the E-C Apparatus Corporation and a polyacrylamide gel in a Tris-EDTA-borate buffer at alkaline pH and containing urea; 2) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide stacking and resolving gel with a discontinuous (Tris-chloride/Tris-EDTA-borate) buffer system that contained urea; 3) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide stacking and resolving gel and acetic acid-ammonium acetate buffers at acidic pH that contained urea; 4) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide gel with a stacking and resolving gel in Tris-HCl buffers containing sodium dodecyl sulphate (SDS) and a Tris-chloride-glycine electrode buffer. The mini-slab alkaline urea polyacrylamide gel electrophoresis (PAGE) method was considered to be the most suitable for monitoring the loss of intact casein during cheese ripening. However, SDS-PAGE gave good resolution of para-κ-casein, β-lactoglobulin and α-lactalbumin and it could therefore be used for the analysis of cheese in which whey proteins have been incorporated or for monitoring the breakdown of para-κ-casein (Chapter 4) in cheese. Two-dimensional PAGE revealed the presence of more bands than were visible using any single method of electrophoresis. Traces of protein were found to lie beneath the α31-casein band and this explained why, even after considerable proteolysis, some α31-casein appeared to remain. Storing cheese samples in such a way that there is a minimum of further change was examined using several different storage methods and temperatures, including storage as: freeze-dried powder at 4°C in the dark, frozen at -9, -16, -35,-75 and -100°C, and dissolved in 6 M urea solution and stored at 4 and -16°C. The trial ran for 6 months and involved the multiple sampling and detailed analysis of three Cheddar cheeses by reversed phase fast protein liquid chromatography (RP-FPLC) for the water-soluble fraction (WSF) and alkaline urea-PAGE for the protein fraction. None of the methods used to store the cheese samples was completely satisfactory. Cheese stored at temperatures of -9 and -16°C was unstable, with proteolysis discernible after 66 days. Storage of cheese samples at these temperatures is, therefore, not recommended. Cheese stored at temperatures of -35, -75 and -100°C was unstable after 94 days, although the samples stored at -100°C were more stable. This lack of stability probably arose during thawing as well as during storage of the frozen cheese samples. Storage of freeze-dried samples at4°C in the dark was equivalent to storing the frozen cheese at -100°C. Storage of samples in alkaline urea sample buffer was better at -16°C than at 4° but should be for no longer than 1 month. An indication of the differences to be expected within the normal range of Cheddar cheese was determined using three very similar Cheddar cheeses ripened at5 and 13°C (Chapter 3, part II). Cheeses ripened at 5°C for 6 months were similar to those ripened at 13°C for 2 months and the proteolytic pathways appeared to the same at both temperatures. The proteolytic pathways in Cheddar and Mozzarella cheeses, manufactured according to standard protocols, ripened at 13°C and sampled at regular intervals over a six month period were examined using a variety of techniques: total nitrogen (TN), non- protein nitrogen (NPN), water-soluble nitrogen (WSN), alkaline urea-PAGE, low molecular weight (LMW) SDS-PAGE, RP-FPLC and size exclusion high performance liquid chromatography (SE-HPLC). The TN and NPN analyses were done at the time of sampling whereas the other assays were done on samples that had been stored at <-75°C so that they could be analysed simultaneously. The increase in WSN and NPN was greater in Cheddar cheese than in Mozzarella cheese and reflected the greater microbial enzyme activity in this cheese type. Alkaline urea-PAGE revealed that there was more α31-casein hydrolysis (with the formation of α31-casein-I) in Cheddar cheese than in Mozzarella cheese, indicating that rennet activity was greater in Cheddar cheese. The presence of peptides believed to be β-I- (β-casein fl-189/192) and β-II-casein (β-casein fl-165) indicated that rennet may have hydrolysed β-casein. The amount of β-casein hydrolysis (and γ-casein formation) was greater in Mozzarella cheese, reflecting the greater plasmin activity in this cheese type. Both LMW SDS-PAGE and SE-HPLC of the whole cheese provided little additional information. Examination of the WSF of each cheese by PAGE analysis showed that many of the larger peptides may have been present in both cheese types. The different concentrations of these peptides in each cheese type were consistent with different rennet and plasmin activities and suggested that they may have been products of these enzymes. RP-FPLC and SE-HPLC analysis of the WSF of Cheddar cheese revealed that, although the larger peptides continued to accumulate during ripening, there was also a large increase in the amount of small peptides and amino acids in the cheese. In the Mozzarella cheese, the larger peptides accumulated and there was little evidence of their further hydrolysis to small peptides and amino acids. The present studies indicate that SE-HPLC using a Toyo-Soda SW 2000 column and a 36% acetonitrile/0.1 % trifluoroacetic acid solvent system is a promising new technique that may be useful in determining cheese type and maturity and in relating changes in the molecular weight distribution of the peptides to changes in the textural, functional and flavour characteristics of cheese. It was concluded that the results are consistent with the concept that differences in the manufacture of Cheddar and Mozzarella cheeses result in the formation of two cheeses, each with different amounts of similar enzymes (rennet, plasmin, and the enzymes of the starter and non-starter lactic acid bacteria), and that these differences in enzyme concentration, combined with the modifying effect of pH, temperature, moisture content and S/M, result in different enzyme activities and patterns of proteolysis in the two types of cheese and these,in turn, result in cheeses with different functional properties.Item The multiple proteolytic enzymes of two microsporum species : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University(Massey University, 1995) Palmer, Jon StuartDermatophyte infections can be contracted from animals, humans or from the soil. In the genus Microsporum some species commonly are associated with cats & dogs but also often cause infections in humans. Others are regarded as non-pathogenic & are commonly isolated from the soil. The present studies investigated the production of proteolytic enzymes by the zoophilic species M.canis & the geophilic species M.cookei, in various cultural conditions which might affect expression of such enzymes, in an attempt to detect differences between the two that could be associated with the ability of M.canis to invade skin in vivo. Biochemical assays showed M.canis produced higher azocollytic & elastase activity in a keratin containing medium(BSW) than in Sabourauds Broth(SDB). In contrast, azocollytic & elastase activity of M.cookei in the two media was relatively similar. Azocollytic & elastase activity of both species peaked in the pH range 7-10 & azocollytic activity demonstrated highest activity around 45°C in both media. Both species produced some keratinolytic activity in BSW but not in SDB. Inhibition studies of azocollytic & elastase activity revealed the presence of an aspartic elastase with little or no azocollytic activity, which also was not detected using a substrate(gelatin) SDS-PAGE technique. Other proteinase types found were serine, cysteine & metalloproteinases. Using the gelatin-SDS-PAGE technique, the mode of culture(shake & stationary) & the effect of substrate, time & temperature were analysed to compare the effects these factors may have on proteolytic enzyme expression between the two species.Substrate proved to be the most important factor in the expression of gelatinases. Mode of culture in SDB demonstrated that some proteinases were expressed in shake culture sooner than in stationary cultures. M.canis in both SDB & BSW produced 6 bands between 85,000 Da & 13,000 Da. M.cookei in SDB produced 7 bands between 64,000 Da to 19,000 Da but in BSW only 5 bands between 61,500 Da to 19,000 Da. Inhibition studies revealed that both species expressed several metalloproteinases & serine proteinases in BSW which were not expressed in SDB cultures. It is suggested that these proteinases may be important factors in the ability of dermatophytes to colonise keratin & possibly, in the case of M.canis, to invade skin in vivo.