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Effect of processing on muscle structure and protein digestibility in vitro : 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
Full text linkFigures 2-1, 2-2, 2-3, 2-4 and Table 2-2 are re-used with publishers' permission.The objective of this thesis was to investigate the effect of processing on meat protein properties, muscle structure and in vitro protein digestibility of beef. Meat processing techniques including pulsed electric field (PEF), shockwave (SW) processing, exogenous enzyme (actinidin) treatment, and sous vide (SV) cooking were explored, either alone or in combination, in this project. This thesis also aimed to study the diffusion of enzymes (actinidin from kiwifruit and pepsin in the gastric juice) into the meat.
The first experiment investigated the effect of PEF processing alone on the ultrastructure and in vitro protein digestibility of bovine Longissimus thoracis, a tender meat cut (Chapter 3). It was observed that the moisture content of the PEF-treated samples (specific energy of 48 ± 5 kJ/kg and 178 ± 11 kJ/kg) was significantly lower (p < 0.05) by 1.3 to 4.6 %, compared to the untreated samples. The pH, colour, and protein thermal profile of the PEF-treated muscles remained unchanged. Pulsed electric field treatment caused the weakening of the Z-disk and I-band junctions and sarcomere elongation (25 to 38 % longer) of the muscles. The treatment improved in vitro meat protein digestibility by at least 18 %. In this thesis, the protein digestibility was determined in terms of the ninhydrin-reactive amino nitrogen released during simulated oral-gastro-small intestinal digestion. An enhanced proteolysis of the PEF-treated meat proteins (such as α-actinin and β-actinin subunit) during simulated digestion was also observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The improvement in protein digestibility of the PEF-treated meat was supported by more severe disruption of Z-disks and I-bands observed in PEF-treated samples, at the end of simulated digestion.
In the second experiment, PEF treatment (specific energy of 99 ± 5 kJ/kg) was applied to bovine Deep and Superficial pectoral muscles in conjunction with SV cooking (60 ℃ for 24 h) (Chapter 4). This muscle cut was tested as it is a tough cut and requires slow cooking. There was no significant difference detected in the specific activities of the sarcoplasmic cathepsins present in the cytosol between the control and PEF-treated samples, both before and after cooking. In addition, similar micro- and ultrastructures were observed between the control SV-cooked and PEF-treated SV-cooked pectoral muscles. The combined PEF-SV treatment increased the in vitro protein digestibility of the pectoral muscles by approximately 29 %. An improvement in proteolysis of the treated meat proteins (e.g. myosin heavy chains and C-protein) during simulated digestion was also observed using SDS-PAGE. More damaged muscle micro- and ultrastructures were detected in PEF-treated SV-cooked muscles at the end of in vitro oral-gastro-small intestinal digestion, showing its enhanced proteolysis compared to the control cooked meat.
Next, the effect of SW processing and subsequent SV cooking on meat protein properties, muscle structure and in vitro protein digestibility of bovine Deep and Superficial pectoral muscles were investigated (Chapter 5 and 6). Shockwave processing (11 kJ/pulse) alone decreased the enthalpy and thermal denaturation temperature of the collagen (p < 0.05) when compared to the raw control, studied using a differential scanning calorimeter. The purge loss, pH, colour, and the protein gel electrophoresis profile of the SW-treated raw muscles remained unaffected. Shockwave processing led to the disorganisation of the sarcomere structure and also modified the protein secondary structure of the myofibres. After subsequent SV cooking (60 ℃ for 12 h), more severe muscle fibre coagulation and denaturation were observed in the SW-treated cooked meat compared to the cooked control. An increase in cook loss and a decrease in the Warner-Bratzler shear force were detected in the SW-treated SV-cooked meat compared to the control cooked meat (p < 0.05). The in vitro protein digestibility of the SW-treated SV-cooked meat was improved by approximately 22 %, with an enhanced proteolysis observed via SDS-PAGE, compared to the control SV-cooked meat. These results were supported by the observation of more destruction of the micro- and ultrastructures of SW-treated cooked muscles, observed at the end of the simulated digestion.
The effect of the kiwifruit enzyme actinidin on muscle microstructure was studied using Picro-Sirius Red staining (Chapter 7). Meat samples were subjected to two different conditions, simulating meat marination (pH 5.6) and gastric digestion in humans (pH 3). Actinidin was found to have a greater proteolytic effect on the myofibrillar proteins than the connective tissue under both conditions. When compared with pepsin under simulated gastric conditions, actinidin had a weaker proteolytic effect on the connective tissue of cooked meats. Nevertheless, incubating the cooked meat in a solution containing both actinidin and pepsin resulted in more severe muscle structure degradation, when compared to muscles incubated in a single enzyme system. Thus, the co-ingestion of kiwifruit and meat could promote protein digestion of meat in the stomach. In addition, both actinidin and pepsin were successfully located at the edges of the muscle cells and in the endomysium using immunohistofluorescence imaging. The observations suggest that the incubation solutions penetrate into the muscle through the extracellular matrix to the intracellular matrix, enabling the proteases to access their substrates.
Overall, the present work demonstrated that there were strong interactions between processing, muscle protein properties and structure, and in vitro protein digestibility of the meat. Processing induces changes in meat protein properties and muscle structure, which in turn affects the digestion characteristics of muscle-based foods
Plant-based meat analogues and hybrid meats produced by high-moisture extrusion and high-temperature shear processing : 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. EMBARGOED until 15 November 2027.
No full textPlant-based meat analogues (PBMAs) are primarily made from plant proteins as the foundational ingredients, which undergo thermomechanical processing (TMP) techniques to mimic the fibrous texture and flavour of meat but face issues such as a lack of fibrous structure, low digestibility and deficiencies in essential amino acids. High-moisture extrusion (HME) is the primary processing technique for preparing fibrous meat analogues. This study developed a novel high-temperature shear processing (HTSP) method for producing uniformly consistent hybrid meat analogues and PBMAs. A systematic comparison was conducted between meat analogues prepared via HME and HTSP, focusing on the impact of these TMP on protein conformational changes at the molecular level. It was found that these two techniques distinctly affect the secondary and tertiary conformations of soy, pea, and rice proteins. This study also found that the hybrid meat analogues demonstrated improved protein digestibility and bioavailability compared to PBMAs
Characterization of mānuka and rosemary oils as antimicrobial and antioxidant agents for meat applications : 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
Full text linkFigures 2.1, 2.3 A, 2.4 & 2.5 are reproduced with permission. Figures 2.2 & 2.3 B are reproduced under a CC BY 4.0 DEED license.The usage of chemical preservatives in meat products has been associated with adverse health effects, which is driving consumers' preferences towards natural preservatives. Raw and processed meats have been linked to cancers due to the presence of nitrate/nitrite as a chemical preservative. In recent years, extensive innovative and promising approaches have been exploited to entirely or partially replace synthetic preservatives. Plant-based natural preservatives possessing antioxidant and antimicrobial characteristics can be ideal for food applications. Essential oils are aromatic liquids extracted from different plant parts, such as leaves, bark, roots, and seeds, and are rich sources of bioactive compounds like monoterpenes, sesquiterpenes, and oxygenated sesquiterpenes and phenolic compounds. As per the literature, essential oils possessing antioxidant and antimicrobial characteristics have been reported to decline the rate of oxidative reactions and microbial growth. This study aims to harness the potential of essential oil obtained from the indigenous plant of New Zealand, i.e., mānuka, as a natural antioxidant and antimicrobial agent for meat preservation than chemical preservatives like nitrates/nitrites. As per the available literature, β-triketones are responsible for the antimicrobial characteristics of mānuka oil. We hypothesise that using antioxidants and antimicrobial bioactive compounds of Leptospermum scoparium (mānuka) will improve the shelf life and stability of the meat products.
The first research objective characterised and compared the antioxidant and antimicrobial potential of mānuka oil with different triketone contents (5, 25, and 40 %) and kānuka oil with a commonly used natural preservative, i.e., rosemary oil. In chemical composition, kānuka oil possessed higher levels of α-pinene, while rosemary oil exhibited higher amounts of 1,8 cineole and α-pinene as primary compounds. In mānuka oils, the concentration of other compounds decreased as triketone content increased from 5 to 40 %. A comparison of the antioxidant characteristics of these oils was also made with chemical antioxidants, i.e., butylated hydroxytoluene (BHT). It was observed that mānuka oils possess higher antioxidant properties than rosemary and BHT (at both the lowest tested concentrations of 0.1 % and 1 %). In the antimicrobial efficacies assay results, all mānuka oils showed more effectiveness against Listeria monocytogenes and Staphylococcus aureus than Salmonella and Escherichia coli. However, the inhibition effect of rosemary oil was greater against Salmonella and Escherichia coli than mānuka oil (Chapter 3). The minimum inhibitory concentration of all mānuka oils required to inhibit Listeria monocytogenes and Staphylococcus aureus was below 0.04 %, while kānuka and rosemary oil inhibited these microbes at 0.63 and 2.5 %, respectively. On the other hand, a minimum 2.5 % concentration of all oils was needed to inhibit Salmonella and Escherichia coli. These results indicated that mānuka oil can be used as an antimicrobial agent, particularly against tested Gram-positive microbes (at a very concentration of 0.04 %) in meat products, while rosemary oil can be used against all tested microbes at 2.5 %. However, meat constituents such as fats have a significant effect on the efficacies of added bioactive compounds, therefore, it is essential to have insights into the lipophilicity of added essential oils and their bioactive compounds.
In the next research experiment, to confirm the lipophilic behaviour of chemical compounds present in mānuka oil, the octanol-water partition coefficient of beta-triketones (leptospermone, isoleptospermone, and flavesone), α-pinene and γ-terpinene were elucidated using shake flask method (Gas chromatography and mass spectrometry) and predicted using EPI software (Chapter 4). High values of the octanol-water partition coefficient of these compounds indicate their more affinity towards the fat than the water. Further, when the concentration of the compounds separated in 3 and 12 % beef-fat and water systems was determined, all compounds showed higher concentrations in water of the low-fat system than in the high-fat. The findings pointed out that essential oils may exert an antioxidant effect in the high-fat system to prevent lipid oxidation; however, their antimicrobial effect may be reduced due to the presence of fat, and higher concentrations of these oils may be needed to achieve an antimicrobial effect against selected microbes.
In the third research experiment, selected mānuka and rosemary oils were used as natural antioxidants and antimicrobial agents in low and high-fat meat pastes prepared from commercial-breed and wagyu beef tenderloins, respectively (Chapter 5). These effects were compared against the chemical preservative sodium nitrate and butylated hydroxytoluene during refrigerated storage of meat pastes at 4 °C. In commercial and wagyu beef pastes, a lower number of Listeria monocytogenes and Staphylococcus aureus were observed in mānuka and rosemary oil treatments than in the sodium nitrate and control samples (without added preservative). Rosemary oil also delayed the growth of Salmonella and Escherichia coli more than mānuka oil added and control samples. In terms of oxidative stability, mānuka oil added wagyu beef pastes were more stable and showed the lowest lipid oxidation values than all treatments. In commercial beef samples, no significant difference between essential oils added samples, either mānuka or rosemary oil and control samples was observed. There was a significant change in pH values of all wagyu and commercial beef samples, whilst these changes were greater in untreated samples (controls) than in the essential oils-treated samples. Despite the promising antioxidant and antimicrobial characteristics of essential oils, these are rarely utilised in food products owing to their easy degradation, low water solubility, low stability, and unwanted odour and flavour. The application of essential oils in encapsulated form is an effective and innovative approach to overcome these limitations by covering the core materials (oil droplets) in carrier materials. In addition, it improves stability and provides controlled release and targeted delivery of essential oils in foods.
In the next research objective, mānuka and rosemary oils-containing nonentities (nanoemulsions and nanocapsules) made of sodium alginate and whey protein were fabricated and compared for their thermal stability and release characteristics (Chapter 6). The particle size and zeta potential of prepared nanoentities were between 100 -600 nm and -10 to -40 mV, confirming that the obtained nanoemulsions and nanocapsules were stable and in the nano range. The obtained nanoentities were observed to be more thermostable, sustained release profile than the free form of oils while showing a lower in vitro antioxidant effect. The release mechanism of the essential oil from nanoemulsions and nanocapsules was also studied using different mathematical models. The release mechanism of essential oil from nanoemulsions and nanocapsules followed Higuchi’s law, which indicates that the solvent first penetrates the encapsulated matrix and then dissolves the embedded oil droplets through the diffusion process. The delayed or sustained release from encapsulated oil might influence the antioxidant and antimicrobial activity of essential oils in meat pastes. However, a food matrix made up of different constituents can affect the partitioning and release of essential oils from the carrier material, and consequently, their preservative effect may vary according to the meat paste.
An improvement in the antioxidant activity of oils after emulsification was observed as nanoemulsions of both oils had the lowest TABRS values in crossbred and wagyu pastes (Chapter 7). Mānuka oil and its nanoentities had more antioxidant effects than rosemary oil. In wagyu pastes, there was a significant difference in nanoemulsions added pastes than the other treatments, while in crossbred pastes, no significant differences were noted between free oils and nanoentities containing beef pastes. Despite the antioxidant efficacies, the antimicrobial activity of free, nanoemuslfisied and nanoencapsulated oils was also determined in the wagyu and crossbred beef pastes during refrigerated storage (4 °C) of two weeks. These antimicrobial effects were compared against controls (without added preservatives) and sodium nitrite-added paste samples. There was a significant increase in microbial counts of all inoculated-paste samples, whilst this increase was lower in preservatives added samples than in the controls. In wagyu and crossbred beef pastes, mānuka oil and its nanoentities delayed the growth of Listeria monocytogenes and Staphylococcus aureus, and mānuka-nanoemulsions exhibited the lowest number of these microbes than all other treatments. However, rosemary oil and its nanoforms effectively inhibited Salmonella and Escherichia coli during refrigerated storage at 4 °C. To better understand the mechanism for the antimicrobial activity of essential oils against selected pathogens, cell viability membrane integrity and the release of intracellular compounds and proteins through fluorescence-based assays were determined. In all these assay results, mānuka and rosemary oils treatment of Listeria monocytogenes, Staphylococcus aureus, Salmonella and Escherichia coli exhibited a decline in cell viability, disrupted cell-wall permeability and enhanced release of intracellular compounds and proteins from cells than the untreated cells. Scanning electron micrographs also confirmed that these mechanisms were responsible for the antibacterial efficacy of mānuka and rosemary oil. To correlate the effect of fat content on varied antimicrobial characteristics of essential oils in meat pastes, the partitioning of essential oils in different phases, such as octanol, beef and water, was determined.
Overall, the work showed that mānuka oil has the potential to be used in meat pastes as an antimicrobial agent, especially against tested Gram-positive (Listeria monocytogenes and Staphylococcus aureus). In addition, this oil can be used to completely replace synthetic antioxidants like butylated hydroxytoluene to inhibit lipid oxidation in high-fat meat systems. Due to the lipophilic nature of oils, the fat content of meat systems significantly affects the partitioning of these oils in water and fat phases, which in turn affect their antimicrobial efficacies
Structure of potato starch
No full textPotato starch granules consist primarily of two tightly packed polysaccharides, amylose and amylopectin. Amylose, which amount for 20-30%, is the principal linear component, but a fraction is in fact slightly branched. Amylopectin is typically the major component and is extensively branched, containing short chains with an average length of 22-25 glucosyl residues. The branching pattern is not well known, but branch point clustering guides chains to determine the overall starch granule architecture and starch functionality. The clusters consist of 5-10 grouped short chains, which are interconnected by long chains with more than 36 residues. The clusters consist of still smaller, very tightly branched building blocks. The clusters direct the semicrystalline structures found inside the starch granules. The crystals, which are ~5.2. nm thick, contain double helices formed from the external chains extending from the clusters. A range of enzymes is involved in the biosynthesis of the cluster structures and linear segments. These are required for sugar activation, chain elongation, branching, and trimming of the final branching pattern. As an interesting feature, potato amylopectin is substituted with low amounts of phosphate groups monoesterified to the C-3 and the C-6 carbons of the glucose units. They seem to align well in the granular structure and have tremendous effects on starch degradation in the potato and functionality of the refined starch. A specific dikinase catalyzes the phosphorylation of amylopectin.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Multimodal and multispectral imaging approaches to study the diffusion of pepsin during in vitro digestion of meat
No full textMeat and fish are high-protein foods with high nutritional value, since all the essential amino acids are represented. The most nutritious proteins are the myofibrillar proteins that are contained in the cytoplasm of muscle cells. In meat, the muscle cells are wrapped by a sheath of connective tissue (the endomysium) and grouped in bundles also wrapped with connective tissue (the perimysium). This degree of muscle organization is likely to inhibit the penetration of pepsin into myofibrillar proteins during gastric digestion and consequently affect their degradation.
To characterize the effect of muscle structure on the diffusion of pepsin and to better understand the mechanisms of digestion of meat and meat products, we have implemented a series of experiments involving imaging and complementary approaches.
Samples of meat were incubated for 2 hours in a pepsin solution at pH 2 and slowly stirred to simulate gastric digestion. Serial histological sections of 6 to 10 μm thick (depending on the type of targeted imaging) were prepared from the entire surface of the block. The diffusion of pepsin was assessed indirectly, through its proteolytic action on the sample (histology, infrared microscopy, MALDI-TOF imaging), and directly, using immunohistochemistry and Secondary Ion Mass Spectroscopy (SIMS ).
The results show the diffusion of pepsin with a relatively constant migration front. To a lesser extent, some of the pepsin also appears to migrate along the perimysium sheaths.
For the first time, we have been able to demonstrate the diffusion of pepsin in meat digested in vitro. Implementation of complementary imaging techniques seems to be the most useful approach to acquiring new information on the mechanisms of diffusion of digestive enzymes in food
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