Effect of transglutaminase on milk proteins : a thesis presented in partial fulfilment of the requirements for the degree of Master in Technology at Massey University, New Zealand

In this study transglutaminase was used to achieve ε-γ-(glutamyl) lysine cross-linking of milk proteins, in Trim™ and Full Fat milks, the same milks with a variety of added protein concentrates, and finally in yogurt and Petite Suisse acid gel systems. The effects of a preheat treatment, enzyme incubation temperature, enzyme inactivation after the enzyme incubation period and homogenization on the cross-linking of the three major casein and two whey proteins were also studied. The degree of cross-linking was established by the use of SDS PAGE gel electrophoresis. The results indicated that cross-linking of the major casein and whey proteins was maximized if the milk was preheated for 10 minutes at 90°C and then cooled before addition of the transglutaminase. However, the preheat treatment was not always advantageous in Trim™ milk systems, but was essential for Full Fat milk systems. Maximal cross-linking of milk proteins occurred if the enzyme/milk system was incubated at 37°C for two hours rather than at 55°C for the same period. The extent of cross-linking increased in an almost linear fashion with increasing transglutainase concentration in most milk systems, with maximal cross-linking occurring when the enzyme concentration was 100 U/mL. Studies on one milk system showed that whey loss and gel strength deteriorated if more than 100 U/mL of enzyme was used. The study demonstrated that homogenization was an essential step for protein cross-linking if the system contained any fat. Casein and whey protein transglutaminase mediated cross-linking was maximized in Full Fat milk systems if the milk was homogenized before transglutaminase was added. Maximal cross-linking, particularly of whey proteins, occurred in Full Fat milk systems if the milk was preheated for 10 minutes at 90°C, cooled to 60°C and then homogenized at 50/150, cooled further to 37°C and then incubated with 100 U/mL of enzyme for two hours. Addition of sodium caseinate or milk protein isolate to Trim™ and Full Fat milk systems was shown to significantly improve protein cross-linkage by up to 50% for β-casein and whey protein respectively. Transglutaminase addition to milk systems containing the previously mentioned protein concentrates further enhanced cross-linking compared to the non-enzyme controls, particularly when the enzyme concentration was 100 U/mL Addition of transglutaminase to acid milk gels dramatically improved the whey holding and gel properties of the products, particularly when the enzyme concentration was 100 U/mL. The reduction in whey loss was proportional to transglutaminase concentration up to 100 U/mL. A 100% reduction in whey syneresis and a 10g F improvement in gel strength improvement were obtained when 0.5 % sodium caseinate and 100 U/mL of transglutaminase were added to a gel milk system compared to a control sample with no enzyme. The physical properties of the milk acid gels were further improved if the transglutaminase in the acid gel systems was not inactivated prior to the addition of the enzyme. The addition of milk protein concentrates such as sodium caseinate and total milk proteinate were shown to have dramatic effects on the whey holding and gel properties of acid gels. Moreover, the properties showed little reduction over a two week storage period compared to yogurt with no added protein. The addition of transglutaminase at a concentration of 100 U/mL further enhanced the above physical characteristics of the acid milk gels. Variations in cross-linking within systems containing either sodium caseinate, milk protein concentrate and milk protein isolate were observed. These variations need to be examined in further work. The addition of NaCNTMP further enhanced the gel and whey-holding properties compared to systems containing either sodium caseinate or total milk proteinate. The final study was conducted on Petite Suisse, a high fat acid milk gel, and here the addition of transglutaminase at 100 U/mL dramatically improved the gel strength of the system by 500% compared to the control. Finally, this research confirmed that transglutaminase effectively cross-linked milk proteins, and in particular β- and κ-casein and β-lactuglobulin. Transglutaminase addition to milk and acid milk systems clearly improved some of the physical properties of the systems. However, much work is needed before it could be recommended for use by industry. The effect of adding transglutaminase to acid milk gels and milk systems should be evaluated by consumer panels to ensure that the sensory properties of these systems have in no way been compromised. Furthermore the economic costs of adding transglutaminase should be determined to ensure that the process would not be uneconomic. If the above evaluations prove to be beneficial then the process could be investigated and further studies carried out to see whether improvements could result by addition of transglutaminase to such milk products as yogurts, desserts, cheese etc, and to create new products with different textural and water holding characteristics. Further work is needed on a scientific front to assess the effects of transglutainase and added proteins on the structure of milk gels and the precise mechanism of filament formation in these gels. Some questions were also raised concerning the exact mechanism that was responsible for removal of monomeric forms of whey protein in the various milk systems evaluated in this study, and these should be determined by further research work

Effects of high pressure processing on carrot tissue : a microstructure approach : 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

High pressure processing (HPP) has the potential of extending the shelf life of fruits and vegetables whilst preserving nutrients and, importantly, many sensory attributes. Although there is a developing body of literature identifying the advantages of this technology for specific products under specific conditions, it is important to gain further understanding of why undesirable quality changes can also be enhanced by this process. For this reason, this work focused on the changes that HPP promotes within the microstructure of the product (carrots, Daucus carota L.) considering that macroscopic quality is determined at a cellular level. This project was part of a government funded flagship programme at CSIRO Australia, where carrots were chosen as a model product of study. The effects of HPP on this commodity were studied for a range of pressures (100-600 MPa) applied for different holding times (2, 10 and 30 minutes) at ambient temperatures (20 °C). The effects were measured qualitatively and quantitatively by using several microscopy techniques, textural, physiological, biochemical and sensory analysis and through comparison with unprocessed (raw), frozen and heat processed (boiled, steamed and sous vide) carrots. The information collected provided understanding of how different pressure levels affected the physical and physiological responses of carrots based on cellular changes. It also allowed HPP to be positioned within the range of other preservation techniques and to identify relationships between quantitative and sensory quality attributes. The key findings of the study can be divided into HPP effects below and above 200 MPa, as near this pressure a “tissue break point” was identified. Pressures below 200 MPa only slightly affected the cellular structure arrangement according to microscopy techniques, which explained small textural changes, but there was an interesting shift in the metabolic response from aerobic to anaerobic metabolism, presumably due to stress. Above 200 MPa, cell structures became less organized and more disrupted resulting in significant loss of textural characteristics such as hardness and cutting forces compared to raw carrots. This texture loss was related to cellular leakage and loss of turgidity. Considering that ii texture is one of the most important quality attributes in carrots, this study searched for ways of ameliorating the impact of pressure by manipulating turgidity before and after the HPP process. One possibility was by weight loss prior to high pressure processing, but this approach did not help to overcome texture losses after HP treatments above 200 MPa, as structures were irreversibly damaged. Below 200 MPa, cells were still able to regain some turgor pressure (pressure of the cell content against the cell wall); however changes in cell permeability were evident. The addition of calcium chloride solutions in samples high pressure treated at above 200 MPa showed no quantitative texture improvements, confirming membrane damage as the principle mechanism and limited influence of biochemical reactions (pectin degradation by pectin methylesterase) affected cell walls at the conditions studied. Sensory perception by a trained panel showed a positive response toward HPP carrots treated at 600 MPa for 2 minutes. It was interesting to observe no significant differences in many sensory attributes in comparison to raw and sous vide samples, while boiled carrots showed low acceptability due to loss of most volatiles, texture and colour attributes. Storage trials confirmed that high pressure treated samples retained higher quality after 14 days at 4°C by supporting a lower count of lactic acid bacteria and consequently having less ethanol and acetic acid production in the pack. Overall, this research has provided a greater understanding of the application of high pressure on whole vegetable pieces by following microstructural changes. Based on this work, HPP can be considered equivalent to other ‘lightly processed’ technologies such as sous vide and may offer benefits as a complementary process to this or other similar preservation techniques. Future opportunities could be investigated taking advantage of the changes observed in cell permeability (< 200 MPa) for diffusion processes such as salting and candying. Health benefits arising from nutrients being more exposed and preserved after pressure treatments should be further studied by following nutrient availability and body absorption. Furthermore, studies on altering rates of compression or decompression and various pressure cycling effects could assist in optimisation of future commercial HPP applications

Microstructural characterization of commercial kiwifruit cultivars using X-ray micro computed tomography

The skin is the physical barrier between the fruit and the environment in which it develops. Environmental conditions during fruit development have a large influence on fruit quality, both at the time of harvest and during subsequent storage. It is hypothesized that some features of the skin and sub-epidermal tissues could provide information about the past growing conditions to which the fruit was exposed and therefore be of predictive value for storage quality. In this study, five commercial kiwifruit cultivars (‘Hayward’, ‘Hort16A’, ‘G3’, ‘G9’ and ‘G14’) were studied, and ‘Hayward’ fruit were manipulated during growth with different light or cultural practices. After harvest at horticultural maturity, X-ray micro computed tomography (µCT) was used to investigate features of the skin and the immediate parenchyma tissue. Despite orchard management practices (crop load and girdling) being observed to effect macro fruit quality parameters (mass, firmness, SSC, and DM), differences in microstructure (e.g. porosity) caused by these practices were not observed. However, porosity and pore size were found to be highly variable between cultivars. The thickness of dense sub-epidermal tissue could be readily measured and the 3-D distribution of raphide bundles was visible as high density particles distributed within the parenchyma. Overall, µCT was found to be a powerful technique to explore fruit epidermal and sub-epidermal structures in three dimensions at a micro level. However, the length of time required for data capture and analysis and the large number of samples required to overcome natural variation within horticultural products need to be considered. Future work may define the impact of differences in porosity or sub-epidermal anatomy on kiwifruit physiology (e.g. firmness change or sensitivity to low oxygen storage atmospheres). With this information, µCT could be used as a screening tool during plant breeding, or to determine the response to agronomic treatments, without conducting lengthy storage trials.publisher: Elsevier articletitle: Microstructural characterisation of commercial kiwifruit cultivars using X-ray micro computed tomography journaltitle: Postharvest Biology and Technology articlelink: http://dx.doi.org/10.1016/j.postharvbio.2014.01.012 content_type: article copyright: Copyright © 2014 Elsevier B.V. All rights reserved.status: publishe