Food and Nutritional Sciences - Doctoral Theses

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3D printing of dairy-based ingredients and investigation into Irish consumer acceptance of 3D food printing applications
(University College Cork, 2022-05-05) Ross, Megan M.; Kelly, Alan; Morrison, Alan P.; Crowley, Shane; Mccarthy, Mary; The Lauritzson Foundation
Three-dimensional (3D) food printing is a type of additive manufacture in which foods with certain rheological characteristics are mechanically layered to create 3D structures from a digitally model. 3D food printing can potentially offer consumers a range of benefits, such as personalised nutrition, customisable textures and unique structures and shapes. This multidisciplinary thesis outlines research completed in three key areas: engineering, food science and consumer science, reflecting the importance of evaluating 3D food printing in this holistic manner. The primary objectives of this work was three-fold: (1) to develop a practical 3D food printer design suitable for printing dairy-based ingredients; (2) to identify and develop suitable dairy-based recipes and investigate factors affecting their printability; and (3) to explore determinants of Irish consumer willingness to try 3D food printing applications. The rationale behind two selected 3D food printer designs (Cartesian and Delta), as well as a brief comparison between the functionality of both designs, are reported. The effects of printing parameters (i.e., nozzle diameter, distance to print bed, print speed etc.) on print quality are also discussed. Certain physicochemical factors such as pH and structural protein content were found to significantly affect the printability and texture of a basic processed cheese recipe. Samples with a higher pH (pH 5.8) tended to print less accurate grids and were significantly softer and less gummy, chewy and resilient than those with lower pH (≤ pH 5.6). Printed processed cheese recipes formulated with fresh curd (high structural protein content) resulted in significantly harder prints, yet printed grids inaccurately due to the material dragging during printing. Viscosity profiles were created for each processed cheese recipe using rheological methods and correlated with absolute printing precision values to identify a range of suitable recipes for accurate printing. The effect of various factors on the printability and functionality of Micellar Casein Concentrate (MCC) suspensions was also investigated. Increasing calcium chloride concentration (5 mM) of suspensions lead to significantly harder printed samples, which had lower dissolution and solubility rates than control or printed samples with lower calcium chloride concentration (1 mM). MCC suspensions printed in porous lattice structures were found to dissolve at a quicker rate than those printed in a hemisphere structure due to a higher surface area to volume ratio. An example of possible 3D-printed product concepts demonstrating the potential of MCC as a printing material is presented. In order to achieve a balanced and comprehensive understanding of 3D food printing technology from a number of perspectives, consumer acceptance research was incorporated into this study to compliment the food science and engineering narrative. Using qualitative methods, a series of themes were identified as forming Irish consumers’ perceptions of 3D food printing applications. Consumers’ affinity for naturalness and a strong association for unprocessed, homemade meals were considered barriers to acceptance of 3D-printed foods. As an extension of this study, data from quantitative research further revealed perceived personal relevance as a significant determinant affecting the dependent variable (i.e., willingness to try 3D food printing applications in the food service sector). Trust in science was found to diminish the negative effects of novel food technology neophobia on willingness to try. Potential solutions for negating factors affecting consumer acceptance are also discussed, which may be of benefit to those looking to market 3D food printing applications in the Irish marketplace. The findings from these studies present an opportunity for food sector stakeholders to utilise this knowledge as part of their foundation to build upon and create novel 3D food printer designs and printable formulations which are suitable for acceptance in the consumer market.
Aggregation and gelation characteristics of high protein dairy ingredient powders
(University College Cork, 2017) Lin, Yingchen; O'Mahony, Seamus Anthony; Kelly, Alan; Guinee, Timothy P.; Dairy Research Trust, Ireland
Milk protein-containing dairy ingredient powders are used extensively in the formulation of a wide range of foods and beverages. Protein is an essential component in such food systems, where it provides nutrition and a range of functionalities including rennet gelation, heat stability, ethanol stability and acid gelation, which influence the manufacture and stabilization of the foods. The protein dispersed in water is typically subjected to various processes, including concentration, homogenization, heating, acidification, in the presence of other formulation constituents (e.g., salts, sugars, ethanol, and acids) during food processing. The exposure of proteins to different processing steps and other constituents can significantly alter their aggregation behaviour, and hence, stability. The current study investigated the changes in compositional, physico-chemical and functional properties of skim milk and protein-based dispersions such as reconstituted skim milk, protein-fortified milk, and reconstituted milk protein concentrate, which resulted from seasonal variations, fortification of milk with different high protein ingredients, processing operations (heat treatment, evaporation and spray-drying) during the manufacture of skim milk powder, at pH during milk heat treatment followed by restoration of pH after heat treatment. Seasonal variations in proportion of spring- and autumn-calving milk in mixed-herd milk and in milk composition were characterized. Key factors influencing the functionality of the milk protein included the calcium-phosphate content, heat treatment of milk during powder manufacture, pH adjustment prior to heat treatment, and the composition of the solvent. These factors exerted their effects by altering one or more physicochemical parameters of the reconstituted ingredients, including denaturation of whey protein, complexation of denatured whey proteins with dissociated κ-casein or casein micelles, partitioning of individual caseins and minerals between the serum and the casein micelle, casein micelle size and charge. The results of the current studies have expanded our knowledge of the factors affecting key functional parameters of reconstituted milk protein powders, and provide mechanistic bases for understanding how these factors exert their effects.
Antioxidant activity of plant-derived extracts as assessed through dietary supplementation of chicken diets and via active packaging applications employing commercial chicken packaging formats
(University College Cork, 2019) Mohd Razali, Noorul Syuhada; Kerry, Joseph P.; O'Grady, Michael; Ministry of Higher Education, Malaysia; Universiti Kebangsaan Malaysia
Phenolic compounds, antioxidant activity (in vitro and in chicken muscle homogenates) and antimicrobial activity of grape seed (GS) extract and two mixtures of grape seed, olive leaf and sweet chestnut bark (GS/OL/CB1 and GS/OL/CB2) extracts were examined. Both extract mixtures contain diverse phenolic compounds and possessed greater (p< 0.05) antioxidant activities than GS alone. Both extract mixtures exhibited moderate antimicrobial activities against Gram positive bacteria. Chicken diets were supplemented with GS extracts (100, 200 and 300 mg/kg) and extract mixtures (50 mg/kg) for 35 days pre-slaughter. Quality parameters of fresh chicken breast meat stored in aerobic and modified atmosphere packaging (MAP) (40% C02 : 60% N2) at 4°C were compared to controls. GS-enriched diets inhibited lipid oxidation in chicken breast (300 mg/kg) and thigh (100 mg/kg) muscle homogenate systems. Supplementation did not influence the antioxidant status of blood plasma, lipid stability of chicken organs, proximate composition of chicken breast and thigh muscles and the majority of shelf-life parameters of chicken breast meat. GS extract (300 mg/kg) improved the sensorial attributes of chicken breast meat in both packaging condition. The effect of GS-enriched diets on the shelf-life parameters of cooked chicken breast and thigh meat patties stored in aerobically and in MAP (40% CO2 : 60% N2) at 4°C was evaluated. GS-supplementation reduced the redness of cooked chicken breast and thigh patties stored in both packaging treatments. Dietary supplementation decreased the levels of lipid oxidation in cooked chicken patties and improved sensory qualities of cooked chicken patties stored in both packaging treatments. The ability of both extract mixtures to improve the shelf-life parameters of cooked chicken breast stored in aerobic and MAP (40% CO2 : 60% N2) at 4°C, in comparison to tea catechin (TC) pads and films (positive control) was examined. TC, GS/OL/CB1 and GS/OL/CB2 active pads (1.9 and 3.8 mg/cm2 ) and films (0.7 and 1.5 mg/ml) were developed and characterised. Both active pads and films improved the lipid stability of cooked breast meat during refrigerated storage.
The application of next generation sequencing to profile microbe related cheese quality defects
(University College Cork, 2015) O'Sullivan, Daniel; Giblin, Linda; McSweeney, Paul L. H.; Cotter, Paul D.; Sheehan, Diarmuid; Teagasc; Department of Agriculture, Food and the Marine, Ireland
High throughput next generation sequencing, together with advanced molecular methods, has considerably enhanced the field of food microbiology. By overcoming biases associated with culture dependant approaches, it has become possible to achieve novel insights into the nature of food-borne microbial communities. In this thesis, several different sequencing-based approaches were applied with a view to better understanding microbe associated quality defects in cheese. Initially, a literature review provides an overview of microbe-associated cheese quality defects as well as molecular methods for profiling complex microbial communities. Following this, 16S rRNA sequencing revealed temporal and spatial differences in microbial composition due to the time during the production day that specific commercial cheeses were manufactured. A novel Ion PGM sequencing approach, focusing on decarboxylase genes rather than 16S rRNA genes, was then successfully employed to profile the biogenic amine producing cohort of a series of artisanal cheeses. Investigations into the phenomenon of cheese pinking formed the basis of a joint 16S rRNA and whole genome shotgun sequencing approach, leading to the identification of Thermus species and, more specifically, the pathway involved in production of lycopene, a red coloured carotenoid. Finally, using a more traditional approach, the effect of addition of a facultatively heterofermentative Lactobacillus (Lactobacillus casei) to a Swiss-type cheese, in which starter activity was compromised, was investigated from the perspective of its ability to promote gas defects and irregular eye formation. X-ray computed tomography was used to visualise, using a non-destructive method, the consequences of the undesirable gas formation that resulted. Ultimately this thesis has demonstrated that the application of molecular techniques, such as next generation sequencing, can provide a detailed insight into defect-causing microbial populations present and thereby may underpin approaches to optimise the quality and consistency of a wide variety of cheeses.
Application of novel processing technologies for salt reduction in meat products
(University College Cork, 2019-12) Inguglia, Elena S.; Kerry, Joseph; Tiwari, Brijesh K.; Burgess, Catherine M.; Teagasc
Currently, daily sodium intake is approximately three-times the recommended daily allowance for an adult. Processed meat products are generally high in salt and 20% of the total sodium dietary intake is linked to this category. This thesis assesses the feasibility of employing novel processing technologies to develop reduced-salt meat products. To achieve these objectives, a series of six studies were carried out, proposing three different strategies: i) enhancement of salt diffusion by power ultrasound (US), ii) improvement of natural saltiness by high pressure processing (HPP) and iii) use of plasma technology as an alternative curing source. The first four chapters are focus on the use of US alone or in combination with salt blends (NaCl/KCl), to enhance salt diffusion and distribution. Initially, in Chapter 2 the operation parameters of the US system influencing salt uptake are investigated; modifications in moisture and salt content were used to evaluate salt distribution in the samples. Results of this chapter identify US has a faster processing tool and suggest that geometric parameters of US systems can have a strong influence on the efficiency of US-assisted brining. Subsequently, to address possible concerns related to salt reduction and safety of the product, the efficacy of power US for the inactivation of Escherichia coli and Listeria innocua was investigated, first in broth (Chapter 3), and then on reduced salt meat (Chapter 4). Inactivation curves of inoculated bacterial suspensions were fitted to the Weibull and Biphasic models. In meat, total viable counts, lactic acid and inoculated bacteria were monitored during storage at 4 and 10oC, along with quality parameters. US-brining achieved 25% sodium reduction, with a processing time almost four-times shorter than standard curing, with minor impact on key meat quality parameters. Whilst no differences were observed in the growth parameters of inoculated bacteria on the meat surfaces, inactivation of bacteria in broth subjected to US treatment suggest the potential of US as a hurdle-technology to prevent cross-contamination during processing and to minimize processing losses by extending the shelf life of the brines. Additionally, the role of US-processing to achieve salt reduction was further investigated in Chapter 5, where US bath operating at a frequency of 25, 45 or 130 kHz was used to assist the marination of chicken samples. Significant decreases in lipid oxidation values was observed in salt-reduced samples treated with US. However, US processing alone, without the functional support of other salts, could not compensate for texture loss caused by further salt reduction. In Chapter 6, another technological strategy to increase product saltiness was asses by using micro salt (<30 µm), incorporated into Lyoner sausages at 1.5% and 1.0% levels and compared to standard salt at 2.0% salt. Reduced salt samples passed unrecognized by the sensory panels, suggesting advantages in the use of micronized salt over regular salt to intensify saltiness. Furthermore, additional processing with HPP (200 MPa), led to sensorial acceptance of sample with 1% micro salt, suggesting that further development of this method could allow for up to 50% salt reduction in emulsified meat products. Finally, in Chapter 7, the use of plasma-activated brine (PAB) as a novel alternative source of nitrite for meat curing was proposed. Both, air and nitrogen (N2) gas were used to generate plasma; results showed that when air plasma was used, higher levels of nitrites were formed in PAB, with no significant differences in the texture and lipid oxidation of the beef jerky produced. Overall, the research conducted in this thesis contributed to increase the range of technologies and applications in the area of non-thermal processing. Further optimization of these processes could find successful uses for the development of healthier processed meat products.
Application of ultrasound technology for functional meat products
(University College Cork, 2017) Ojha, K. Shikha; Kerry, Joseph P.; Teagasc
With increasing consumer awareness of health and nutrition, the meat processing industry is eager to engage in a search for innovative ways of processing and developing novel meat products with potential health benefits. Novel processing technologies including ultrasound (US) technology can offer several benefits for versatile applications in food processing. In relation to meat processing, US technology has previously been investigated for many potential applications including accelerated brining, tenderisation, rapid cooking, faster thawing and decontamination of muscle-based food products. This thesis assesses the feasibility of employing US technology to assist in the development of functional meat products. To achieve this objective, a series of five studies were carried out. The first study investigated the efficacy of high intensity US on the fermentation profile of Lactobacillus sakei (a starter culture used for meat fermentation) in a meat model system. This study observed that both stimulation and retardation of L. sakei is possible, depending on the ultrasonic power and sonication time employed. Furthermore, to underline the fundamental mechanism influencing the behaviour of microorganisms subjected to ultrasonic frequencies of 20, 45, 130 and 950 kHz on growth kinetics, phenotypic behaviour and cell morphology were assessed in study 2. Results presented in this study showed that the physiological response of L. sakei to US is frequency-dependent and US can influence metabolic pathways. The influence of US frequency on the behaviour of L. sakei culture in a meat matrix was also studied. Additionally, this study investigated possible synergistic effects between US and L. sakei addition on drying kinetics, moisture mobility and key nutritional (fatty acid profile, protein, amino acids, and organic acids) in beef jerky samples. Given the complexity and number of parameters involved, a reliable multivariate statistical strategy was adopted. The results presented in this study showed a significant effect of US pre-treatment on various physicochemical properties as a result of employing both L. sakei and US. The individual effects of L. sakei and US were prominent compared to interactive effects. Conversely, the effect of US frequency on L. sakei culture observed in model systems (Chapter 2 and 3) was not evident when assessed in a solid meat matrix. Two specific studies were carried out to investigate US-assisted diffusion of ingredients into meat matrices with the objective of improving the nutritional profile and healthy image of meat. Results from salt diffusion studies showed improved diffusion rates for sodium salt compared to a static brining system can be obtained. However, no significant differences were observed in the case of sodium salt replacers. A second aspect of US-assisted diffusion focused on the incorporation of essential fatty acids (FA) to improve the lipid profile of pork meat. This study has demonstrated the positive effect of US application in improving diffusion of encapsulated FA into meat, thereby resulting in higher levels of essential FA compared to the static diffusion system. It can be concluded from the thesis work conducted that the effectiveness of US technology is application dependent. The commercial utilisation of US technology remains challenging for solid food applications, especially for meat products due to the complex nature of meat. However, further research is needed prior to commercial uptake of US technology for a range of applications.
Assessment of blue whiting protein hydrolysate bioactivities using cell culture models
(University College Cork, 2022-07-21) Heffernan, Shauna; O'Brien, Nora M.; Giblin, Linda; Department of Agriculture, Food and the Marine, Ireland
Low-value underutilised blue whiting fish represents a potential profitable source of protein for the generation of high-value, health-enhancing fractions. The research described in this thesis assessed the bioactive potential of blue whiting soluble protein hydrolysates (BWSPHs) using cellular model systems. Minced, deboned blue whiting was initially hydrolysed at laboratory scale to develop a protocol for the reproducible generation of eleven different BWSPHs. Cellular bioactivity analysis of these eleven hydrolysates indicated that none of the BWSPHs tested exhibited satiating activity, antioxidant activity, immunomodulatory activity or anti-obesity activity as measured using specific cellular models. However, six BWSPHs did exhibit anti-diabetic activity in vitro, therefore, these six BWSPHs were prepared at semi-pilot scale and all of the above bioactivities were reassessed in greater detail. The antioxidant and immunomodulatory activities of the six industrial-scale BWSPHs before simulated gastrointestinal digestion (SGID) (BW-SPH-A to BW-SPH-F) and after SGID (BW-SPH-A-GI to BW-SPH-F-GI) were assessed in stimulated murine RAW264.7 macrophage. Hydrolysate BW-SPH-A (0.5%, w/v dw), both pre- and post-SGID, increased the endogenous antioxidant, reduced glutathione (GSH), in tert-butylhydroperoxide (tBOOH)-treated cells and reduced reactive oxygen species (ROS) in H2O2-challenged RAW264.7 cells compared with stimulated controls (p<0.05). In vitro digested hydrolysate BW-SPH-F-GI (0.5%, w/v dw) induced immunostimulating effects in lipopolysaccharide (LPS)-activated RAW264.7 macrophages though increasing pro-inflammatory cytokine interleukin (IL)-6 and tumour necrosis factor (TNF)-α levels compared with the LPS-stimulated control (p<0.05). The satiating potential of the six BWSPHs was then assessed in murine enteroendocrine STC-1 cells. The ability of BWSPHs and SGID BWSPHs to modulate the secretion and/or production of satiety hormones glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK) and peptide YY (PYY) in STC-1 cells was studied as well as the signalling pathway activated by BWSPHs to modulate the secretion of these hormones. All BWSPHs (BW-SPH-A to BW-SPH-F) (1.0%, w/v dw) increased active GLP-1 secretion and proglucagon production in STC-1 cells compared to the basal control (Krebs-Ringer buffer) (p<0.05), possibly via intracellular calcium signalling, however this activity was lost following SGID. In addition, neither pre- nor post-SGID hydrolysates affected epithelial barrier integrity or stimulated IL-6 secretion in differentiated Caco-2/HT-29MTX co-cultured cells. The anti-obesity effects of BWSPHs and SGID BWSPHs was investigated using the murine 3T3-L1 cell line. Before SGID, hydrolysates BW-SPH-A, -B, -C and -F (1.0%, w/v dw) reduced triglyceride accumulation during preadipocyte differentiation (p<0.05), however none of the hydrolysates hydrolysed triglycerides in fully mature adipocytes. Interestingly, after SGID, all hydrolysates reduced triglyceride accumulation during differentiation and all except one BWSPH increased glycerol levels in mature adipocytes compared with the differentiated controls (p<0.05). Two anti-adipogenic hydrolysates, BW-SPH-A and BW-SPH-F, and their corresponding in vitro digests were observed to modulate triglyceride accumulation during preadipocyte differentiation via down-regulating the expression of key adipogenic transcription factors (peroxisome proliferator activated receptor (PPAR)-γ and CAAT (controlled amino acid therapy)/ enhancer binding protein (C/EBP)-α) compared with the differentiated controls (p<0.05). These SGID hydrolysates also exhibited anti-obesity activities following simulated intestinal permeation. After a 4 h exposure of specific SGID BWSPHs to 21-day differentiated Caco-2/HT-29MTX co-cultured cells, cell basolateral was subsequently observed to exhibit anti-adipogenic and adipolytic activities in 3T3-L1 cellular models. In addition, exposure of 3T3-L1 preadipocytes to hydrolysate BW-SPH-A during differentiation also increased GSH concentration upon stimulation with antioxidant tBOOH compared with the tBOOH control (p<0.05). Specific BWSPHs were also observed to reduced adiponectin production in LPS-stimulated cells compared with the LPS control (p<0.05). To conclude, certain BWSPHs exhibited significant bioactivities before SGID, after SGID, and after simulated intestinal absorption, therefore may have potential as health-enhancing functional food ingredients.
Assessment of dietary background exposure of the Irish adult population to dioxins and PCBs particularly taking into account additional exposure due to the 2008 Irish dioxin food contamination incident
(University College Cork, 2014) Tlustos, Christina L. T.; Flynn, Albert; Anderson, Wayne; Food Safety Authority of Ireland
Irish monitoring data on PCDD/Fs, DL-PCBs and Marker PCBs were collated and combined with Irish Adult Food Consumption Data, to estimate dietary background exposure of Irish adults to dioxins and PCBs. Furthermore, all available information on the 2008 Irish pork dioxin food contamination incident was collated and analysed with a view to evaluate any potential impact the incident may have had on general dioxin and PCB background exposure levels estimated for the adult population in Ireland. The average upperbound daily intake of Irish adults to dioxins Total WHO TEQ (2005) (PCDD/Fs & DLPCBs) from environmental background contamination, was estimated at 0.3 pg/kg bw/d and at the 95th percentile at 1 pg/kg bw/d. The average upperbound daily intake of Irish adults to the sum of 6 Marker PCBs from environmental background contamination ubiquitous in the environment was estimated at 1.6 ng/kg bw/d and at the 95th percentile at 6.8 ng/kg bw/d. Dietary background exposure estimates for both dioxins and PCBs indicate that the Irish adult population has exposures below the European average, a finding which is also supported by the levels detected in breast milk of Irish mothers. Exposure levels are below health based guidance values and/or Body Burdens associated with the TWI (for dioxins) or associated with a NOAEL (for PCBs). Given the current toxicological knowledge, based on biomarker data and estimated dietary exposure, general background exposure of the Irish adult population to dioxins and PCBs is of no human health concern. In 2008, a porcine fat sample taken as part of the national residues monitoring programme led to the detection of a major feed contamination incidence in the Republic of Ireland. The source of the contamination was traced back to the use of contaminated oil in a direct-drying feed operation system. Congener profiles in animal fat and feed samples showed a high level of consistency and pinpointed the likely source of fuel contamination to be a highly chlorinated commercial PCB mixture. To estimate additional exposure to dioxins and PCBs due to the contamination of pig and cattle herds, collection and a systematic review of all data associated with the contamination incident was conducted. A model was devised that took into account the proportion of contaminated product reaching the final consumer during the 90 day contamination incident window. For a 90 day period, the total additional exposure to Total TEQ (PCDD/F &DL-PCB) WHO (2005) amounted to 407 pg/kg bw/90d at the 95th percentile and 1911 pg/kg bw/90d at the 99th percentile. Exposure estimates derived for both dioxins and PCBs showed that the Body Burden of the general population remained largely unaffected by the contamination incident and approximately 10 % of the adult population in Ireland was exposed to elevated levels of dioxins and PCBs. Whilst people in this 10 % cohort experienced quite a significant additional load to the existing body burden, the estimated exposure values do not indicate approximation of body burdens associated with adverse health effects, based on current knowledge. The exposure period was also limited in time to approximately 3 months, following the FSAI recall of contaminated meat immediately on detection of the contamination. A follow up breast milk study on Irish first time mothers conducted in 2009/2010 did not show any increase in concentrations compared to the study conducted in 2002. The latter supports the conclusion that the majority of the Irish adult population was not affected by the contamination incident.
Assessment, development, and optimisation of packaging systems for cheese products using smart packaging technologies
(University College Cork, 2016) O'Callaghan, Karen A. M.; Kerry, Joseph P.; Food Research Industry Measure; Irish Research Council
The principle objective of this thesis was to develop a package to improve packaging function for end use with cheese products. This objective was undertaken by focussing on the use of smart technology, inclusive of the areas of active, intelligent and nanotechnology. Research commenced by conducting a survey to evaluate consumer attitudes towards smart technologies as it was deemed important to gauge acceptance prior to development. Overall, respondents were accepting of the application of smart packaging technologies to cheese products. Intelligent oxygen sensor technology was employed on an industrial scale to evaluate the technical performance of commercial cheese packaging. Sensors demonstrated a high level of package containment failure, particularly with packs subjected to distribution. Natural substances (nanoparticled and non-nanoparticled) were assessed against cheesederived cultures to determine antimicrobial activity for potential use as active packaging agents. Subsequently, the best performing agents, which were those that exhibited antimicrobial activity or an increased solubility, were combined to determine if synergistic relationships could be achieved. From this work, it was apparent that nanoparticled rosemary extract and non-nanoparticled chitosan (both low- and medium-molecular weights) demonstrated the greatest microbial inhibition. The success of non-nanoparticled chitosan led to the laboratory synthesis of nanoparticled chitosan. Manufactured nano-chitosan displayed similar antimicrobial effects to non-nanoparticled chitosan. Finally, agents possessing the greatest antimicrobial activity (non-nanoparticled chitosan, nanoparticled chitosan and nanoparticled rosemary) were individually incorporated into HPMC-based films and the efficacy of these films determined via cheese application. An inhibitive microbial response was achieved, particularly when nanoparticled films were employed. This thesis successfully demonstrated the acceptance, need, operation and viable application of smart technologies to cheese products.
Bioactivity of protein hydrolysates derived from casein, bovine blood and lung tissues in cultured cells
(University College Cork, 2017) O'Sullivan, Siobhán M.; O'Brien, Nora M.; Department of Agriculture, Food and the Marine
Bioactive peptides are short amino acid sequences produced from food and food waste-derived proteins and are believed to be capable of exhibiting a variety of physiological functions. Further studies are needed to identify novel bioactive peptides and better understand mechanisms of action. The bioactivities of seven casein hydrolysates generated using different enzymes (mammalian, plant, fungal and bacterial) were compared. All hydrolysates demonstrated significant anti-inflammatory activity; decreasing interleukin (IL)-6 production in RAW264.7 macrophages and Jurkat T cells. There were no differences in bioactivities across the seven samples. Additionally, three of the hydrolysates decreased IL-1β production in RAW264.7 cells. None of the hydrolysates demonstrated antioxidant activity in cells despite showing antioxidant activity in non-cellular assays. Sodium caseinate treatment produced an inflammatory response in 3T3-L1 adipocytes, significantly increasing IL-6 production. This response was not seen when cells were treated with SGID of casein or the casein hydrolysates. The bioactivities of bovine blood and bovine lung protein hydrolysates were also assessed. Papain hydrolysis of blood γ-globulin or fibrinogen generated hydrolysates with significant non-cellular antioxidant activity and these hydrolysates also significantly decreased proliferation in several cancer cell lines, including U937 lymphocytes. Hydrolysates did not demonstrate cellular antioxidant activity. The γ-globulin hydrolysate induced apoptosis in U937 cells, determined by cell morphological changes, DNA fragmentation and flow cytometry analysis. Colony formation in MCF-7 cells was also significantly reduced after hydrolysate treatment. An alcalase hydrolysate from bovine lung demonstrated significant anti-inflammatory activity in RAW264.7 macrophages by decreasing cellular IL-6 and IL-1β production. Bioavailability of bioactive hydrolysates (from casein, bovine blood or bovine lung) was determined in the Caco-2 transwell model and all hydrolysates were transported across the monolayer. In conclusion, casein hydrolysates and novel hydrolysates generated from bovine blood and lung proteins had significant bioactivities and were bioavailable, therefore they show potential as anti-inflammatory and anti-cancer agents.
Biochemistry, physicochemical properties, and processing of human milk and comparison to bovine milk
(University College Cork, 2021-06) Meng, Fanyu; Uniacke-Lowe, Therese; Kelly, Alan; Department of Agriculture, Food and the Marine, Ireland
Human milk is a valuable biofluid that provides essential nutrients and immunological protection to newborns. In the thesis, levels and properties of macronutrients in human milk, their influencing factors, and physico-chemical properties were studied, and in some cases compared to those of bovine milk. A longitudinal analysis of human milk composition showed a decrease in protein level and plasmin activities in both term and preterm human milk over six months of lactation after the due date. Infant gender and pre-pregnancy maternal body mass index (BMI) influenced total fat, energy density, and fatty acid profiles in human milk. Concerning the physical stability of human milk, this is the first study to show that creaming rates of human milk increased with creaming temperatures in the range 5 to 40°C. There was no significant impact of freezing at -20°C for 1 week on creaming rates, while pre-heating at 70°C for 10 min slowed the creaming of human milk; the results may help to improve human milk handling methods. Compared to human milk creaming, bovine milk exhibited a lag phase at the beginning of creaming, followed by a much higher creaming rate at 5°C, suggesting the clustering of bovine milk fat globules through cold agglutination, which could occur in a greater extent than in human milk. Casein stability in human milk, in relation to ethanol stability, heat stability and heat-induced changes, chymosin-/pepsin-induced coagulation, chaotropic agent induced-dissociation, and cooling-induced dissociation were also examined in this thesis. Human milk showed higher resistance to heat-induced coagulation compared with bovine milk. Proteolysis occurred during incubation at 37°C; in human milk with low plasmin activity, proteolysis still occurred due to the action of aspartyl peptidases such as cathepsin D. In separate studies, novel processing methods that could be applied to human milk as alternatives to holder pasteurization were evaluated. High-pressure treatment at 400 MPa for 10 min was shown to be a promising preservation method to compromise between maintaining nutritional quality and ensuring microbial safety. Other non-thermal treatments showed better performance in preserving lysozyme and lactoperoxidase activities compared to holder pasteurization. However, addition of nisin A to human milk could not prevent the proteolysis by bacterial proteases and addition of lactose oxidase decreased the pH of human milk during storage at 4°C for 1 week. These preserving methods required further investigation. Overall, this thesis provides valuable data on nutrients in human milk provided by Irish mothers and expands the knowledge of stability of milk from different species. This study will help improve the handling and storage methods in homes, hospitals, or human milk banks, therefore, helping infants to obtain better human milk and benefit their growth in the future.
Cancer associated malnutrition: prevalence of cachexia, sarcopenia and impact on treatment outcomes, survival and health related quality of life
(University College Cork, 2015) Cushen, Samantha J.; Ryan, Aoife
Malnutrition, sarcopenia and cancer cachexia (CC) are prevalent among cancer patients and can have detrimental effects on clinical outcomes such as quality of life (QoL) and overall survival. Cachexia is associated with lower tolerance for chemotherapy, which limits the total dose that can be delivered, the number of symptomatic responses and any survival advantage that might be accrued. Moreover, for the majority who do not respond, cachexia may be exacerbated by systemic chemotherapy, thus increasing the net symptom burden experienced by patients. The multitude of interactions between cancer location, treatments, nutritional status and QoL has never been thoroughly explored in an Irish cancer cohort. The objectives of this thesis were to further understand nutritional status, especially body composition in ambulatory cancer patients and determine the relationship between nutritional status using different assessment criteria and QoL, chemotherapy toxicity and survival among cancer patients undergoing chemotherapy. Results aimed to identify baseline factors that may be predictive of poor outcome, toxicities to chemotherapy and disease-free and overall survival. This thesis broadly divides into two sections. The first section (Chapters 3 & 4) focuses on improving our knowledge of the nutritional status of Irish cancer outpatients using a cross sectional study design. A study of 517 patients referred for chemotherapy was conducted using computed tomography (CT) imaging (body composition) and a survey that documented oncologic data, weight loss (WL) data and QoL data. We revealed that a significant proportion of Irish cancer patients undergoing chemotherapy experience unintentional WL over the previous 6 months (62%), sarcopenia (45%) and CC (43%), and the distribution of WL and nutritional risk were associated with site of primary tumour and treatment intent. Patients that had sarcopenia, nutritional risk, or CC had significantly reduced functional abilities, more symptoms and adverse global QoL. In the second section of this thesis (Chapters 5 & 6) the potential link between developing toxicity to antineoplastic regimens in patients with sarcopenia was conducted by way of retrospective studies. A retrospective serial CT analysis defined the prevalence of sarcopenia in patients with metastatic renal cell carcinoma (mRCC) and metastatic castrate resistant prostate cancer (mCRPC), which was then correlated with dose limiting toxicities of sunitinib and docetaxel respectively. Sarcopenia was prevalent in patients with mRCC and mCRPC, was an occult condition in patients with normal/high BMI, was associated with less treatment days, was a significant predictor of DLT in patients receiving sunitinib and a significant predictor of neutropenia and neurosensory toxicities in patients receiving docetaxel. This thesis attempted to address the underlying research deficiencies in Irish oncology nutritional data at national level. The findings from this thesis have implications for the planning of cancer care interventions and indicate that further research is required to improve nutritional screening, in particular for CC and sarcopenia, in the hope that timely intervention can improve both patient-centered and oncologic outcomes.
Cellular and in-vitro models to assess antioxidant activities of seaweed extracts and the potential use of the extracts as ingredients
(University College Cork, 2013) O'Sullivan, Anthoney Michael Naim; Kerry, Joseph P.; O'Brien, Nora M.; Marine Institute; Department of Agriculture, Food and the Marine, Ireland
Seaweeds contain a range of antioxidant compounds such as polyphenols, carotenoids, sulphated polysaccharides and vitamins and have the potential to be used as ingredients in neutraceuticals. The antioxidant activity of crude 60% methanol extracts prepared from five Irish seaweeds, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus were examined using in-vitro assays and a cell model system to determine the antioxidant activity of the extracts and their ability to protect against H2O2 and tert-BOOH-induced DNA damage and alterations in cellular antioxidant status in the human adenocarcinoma, Caco-2 cell line. To optimise the extraction of antioxidant compounds from seaweeds, an accelerated solvent extraction (ASE®) was used in combination with food grade solvents. The antioxidant activity of these extracts against H2O2 and tert-BOOH-induced DNA damage and alterations in cellular antioxidant status was also assessed. Extracts that exhibited the highest antioxidant activity, A. nodosum (100% water and 80% ethanol extracts) and F. vesiculosus (60% ethanol extract) were selected as ingredients for incorporation into fluid milk and yogurt at concentrations of 0.25% and 0.5%. The addition of the seaweed extracts to milk and yogurt did not affect the pH or shelf-life properties of the products. Seaweed addition did however significantly influence the colour properties of the milk and yogurt. Yellowness values were significantly higher in yogurts containing F. vesiculosus at both concentrations and A. nodosum (80% ethanol) at the 0.5% concentration. In milk, the F. vesiculosus (60% ethanol) and A. nodosum (80% ethanol) at both the 0.25% and the 0.5% concentrations had higher greenness and yellowness values than the milk containing A. nodosum (100% water). Sensory analysis revealed that appearance and flavour governed the overall acceptability of yogurts with the control yogurt, and yogurts containing A. nodosum (100% water) were the most preferred samples by panellists. However, in the milk trial the perception of a fishy taste was the determining factor in the negative perception of milk. The unsupplemented control and the milk containing A. nodosum (100% water) at a concentration of 0.5% were the most overall accepted milk samples by the sensory panellists. The antioxidant activity of the extracts in milk and yogurt remained stable during storage as determined by the in-vitro assays. Seaweed supplemented milk and yogurt were also subjected to an in-vitro digestion procedure which mimics the human digestive system. The milk and yogurt samples and their digestates were added to Caco-2 cells to investigate their antioxidant potential however neither the undigested or digested samples protected against H2O2-induced DNA damage in Caco-2 cells.
Cereal products for specific dietary requirements. Evaluation and improvement of technological and nutritional properties of gluten free raw materials and end products
(University College Cork, 2013) Hager, Anna-Sophie; Arendt, Elke K.; Irish Research Council
Coeliac disease is one of the most common food intolerances worldwide and at present the gluten free diet remains the only suitable treatment. A market overview conducted as part of this thesis on nutritional and sensory quality of commercially available gluten free breads and pasta showed that improvements are necessary. Many products show strong off-flavors, poor mouthfeel and reduced shelf-life. Since the life-long avoidance of the cereal protein gluten means a major change to the diet, it is important to also consider the nutritional value of products intending to replace staple foods such as bread or pasta. This thesis addresses this issue by characterising available gluten free cereal and pseudocereal flours to facilitate a better raw material choice. It was observed that especially quinoa, buckwheat and teff are high in essential nutrients, such as protein, minerals and folate. In addition the potential of functional ingredients such as inulin, β-glucan, HPMC and xanthan to improve loaf quality were evaluated. Results show that these ingredients can increase loaf volume and reduce crumb hardness as well as rate of staling but that the effect diverges strongly depending on the bread formulation used. Furthermore, fresh egg pasta formulations based on teff and oat flour were developed. The resulting products were characterised regarding sensory and textural properties as well as in vitro digestibility. Scanning electron and confocal laser scanning microscopy was used throughout the thesis to visualise structural changes occurring during baking and pasta making
Characterisation and application of fruit by-products as novel ingredients in gluten-free products
(University College Cork, 2014) O'Shea, Norah L.; Gallagher, Eimear; Arendt, Elke K.; Department of Agriculture, Food and the Marine, Ireland; Teagasc
The physicochemical and nutritional properties of two fruit by-products were initially studied. Apple pomace (AP) contained a high level of fibre and pectin. The isolated AP pectin had a high level of methylation which developed viscous pastes. Orange pomace also had high levels of fibre and pectin, and it was an abundant source of minerals such as potassium and magnesium. Due to the fibrous properties of orange pomace flour, proofing and water addition were studied in a bread formulation. When added at levels greater than 6%, the loaf volume decreased. An optimised formulation and proofing time was derived using the optimisation tool; these consisted of 5.5% orange pomace, 94.6% water inclusion and with 49 minutes proofing. These optimised parameters doubled the total dietary fibre content of the bread compared to the original control. Pasting results showed how orange pomace inclusions reduced the final viscosity of the batter, reducing the occurrence of starch gelatinisation. Rheological properties i.e. the storage modulus (G') and complex modulus (G*) increased in the orange pomace batter compared to the control batter. This demonstrates how the orange pomace as an ingredient improved the robustness of the formulation. Sensory panellists scored the orange pomace bread comparably to the control bread. Milled apple pomace was studied as a potential novel ingredient in an extruded snack. Parameters studied included apple pomace addition, die head temperature and screw speed. As screw speed increased the favourable extrudate characteristics such as radical expansion ratio, porosity and specific volume decreased. The inclusion of apple pomace had a negative effect on extrudate characteristics at levels greater than 8% addition. Including apple pomace reduced the hardness and increased the crispiness of the snack. The optimised and validated formulation and extrusion process contained the following parameters: 7.7% apple pomace, 150°C die head temperature and a screw speed of 69 rpm.
Characterisation of pulse protein ingredients produced using different protein sources and processing technology, and potential for application in milk alternatives
(University College Cork, 2022-02-25) Vogelsang-O'Dwyer, Martin; Arendt, Elke K.; Horizon 2020
In the face of the growing global population along with climate and food security concerns, there is currently much interest in developing plant protein ingredients from sustainable sources. Pulses are a valuable source of protein which could play an increased role in reducing our reliance on animal protein. Pulse protein ingredients can potentially provide the required functional properties (e.g., solubility, emulsifying, gelling, foaming) for a range of food applications. However, further development is necessary in order to increase our understanding and maximise the utility of pulse proteins as demand increases. This thesis addresses how protein source and processing can impact various properties of ingredients, including functional, nutritional and environmental aspects. In addition, the application of pulse proteins in high protein milk alternatives is examined. Furthermore, the impact of enzymatic hydrolysis on physicochemical and functional properties is explored. Protein isolates from blue lupin (Lupinus angustifolius) and white lupin (L. albus) were compared to assess the influence of protein source on various properties. Electrophoresis revealed major differences in molecular weight distribution. Solubility and foaming properties were similar for both isolates, while major differences in thermal gelling behaviour were found, with white lupin protein gelling at a lower concentration (7%) compared to blue lupin (23%). Both isolates were low in FODMAPs (fermentable mono-, di, oligosaccharides and polyols). In addition, the life cycle assessment (LCA) showed generally lower environmental impacts for the lupin isolates compared to milk powder per kg protein. Faba bean protein-rich flour (FPR) produced using dry fractionation was compared to faba bean protein isolate (FPI) produced using wet fractionation. Protein content of dry matter was lower for FPR (64.1%) compared to FPI (90.1%) Overall, superior functionality was found for FPR, including solubility and foaming properties. Wet processing (FPI) was found to be effective for removal of vicine, and trypsin inhibitors and FODMAPs. In the LCA, lower environmental impacts were found for FPR than FPI; however, both FPR and FPI had much lower impacts compared to milk powder. Milk alternatives were produced with blue lupin (BL) or white lupin (WL) protein isolate, with similar protein and fat content to low-fat cow’s milk. Both BL and WL milk alternatives had good colloidal stability and were suitable for low FODMAP diets. Increased homogenisation pressure (780 bar vs 180 bar) resulted in smaller particle size and increased stability for both BL and WL milk alternatives. WL milk alternative homogenised at 780 bar had the lowest separation rate, comparable to low fat cow’s milk, indicating good resistance to creaming. The effects of enzymatic hydrolysis on the physicochemical and functional properties of lentil protein isolate were assessed, in a comparison of three proteases. Hydrolysis with Alcalase or Novozym 11028 resulted in considerably higher solubility from pH 4–6, where the control showed poor solubility. Hydrolysis with Flavourzyme resulted in moderately increased solubility in this pH range, but slightly decreased solubility at neutral pH. Foaming properties were not impacted significantly by hydrolysis. Hydrolysis with Alcalase resulted in larger particle size and higher viscosity compared to the control. Overall, these studies provide useful insight into the properties of pulse protein ingredients which depend on pulse type and ingredient processing, modification with enzymatic hydrolysis, as well as their suitability for high protein milk alternatives.
Cold microfiltration as an enabler of innovative dairy protein ingredient composition and techno-functionality
(University College Cork, 2022-08) France, Thomas C.; O'Mahony, Seamus Anthony; Crowley, Shane; Dairy Processing Technology Centre
Microfiltration (MF) of skim milk at warm temperatures (i.e., 45-55 °C) is common practise in the dairy industry; however, high rates of fouling and short run times due to fouling and microbial quality can occur when operating at these temperatures. The adoption of cold temperatures (i.e., ≤20 °C) provides opportunities to improve efficiency and overall sustainability of the process. However, it can affect the protein, mineral and enzyme partitioning during MF which can ultimately impact the functional properties of the final ingredients, although this has not been sufficiently studied within the cold MF range. This thesis systematically studies the effects of different temperatures within the cold MF range on processing performance and fouling, protein, mineral and enzyme partitioning and some of the consequences on the physicochemical properties of the resulting process streams. Lower processing temperatures, in the range from 20 to 4 °C, resulted in significantly higher concentrations of β-casein in the permeate, with κ-casein, α-lactalbumin and β-lactoglobulin not significantly impacted by alterations in processing temperature. Lower processing temperature also resulted in a lower rate of permeate flux decline during MF although higher mechanical (pump) and thermal energy consumption were measured for the lower processing temperature (i.e., 4 °C). Altering processing temperature had minor effects on plasmin activity in permeate streams although extensive plasmin-mediated hydrolysis was observed on storage; such plasmin-mediated hydrolysis had significant impacts on the temperature-dependent self-association of β-casein. Extensive diafiltration (DF) with water was performed at 4 °C, resulting in a β-casein-depleted micellar casein retentate which displayed superior heat stability when compared to a micellar casein retentate produced at 50 °C. The learnings obtained were applied to the final study in which different DF media were used, i.e., water and two simulated milk ultrafiltrates (SMUF) designed to resemble the mineral profile of ultrafiltration permeate produced at 10 and 50 °C. The use of both SMUFs resulted in similar protein partitioning and significantly improved the heat stability of the micellar casein retentates generated, compared to when water was used as the DF medium. The findings presented in this thesis constitute a significant advance in the understanding of the effects of processing temperature within the cold MF range on parameters of relevance in the optimisation of next generation protein ingredient development in the dairy industry.
Composition and eating quality of beef from young male dairy cattle derived from different production systems assessed by standard and rapid spectroscopic methods
(University College Cork, 2017) Nian, Yingqun; Kerry, Joseph P.; Allen, Paul; Teagasc
The objective of this thesis was to assess the eating quality of beef from young male Holstein-Friesian (HF) and Jersey×Holstein-Friesian (JEX) dairy cattle from different production systems. Bulls were slaughtered at 15, 19 and 22 months, after finishing on diets differing in energy content. Longissimus thoracis (LT), Semitendinosus (ST) and Gluteus medius (GM) muscles were assessed primarily from bulls. Effects of carcass suspension method and ageing time on eating quality were also investigated. Standard eating quality variables assessed were post-mortem pH-temperature window, ultimate pH, colour, WarnerBratzler variables (WBSF, WB-slope, WB-area), cooking loss, intramuscular fat (IMF), moisture and protein content, collagen characteristics, fatty acid (FA) composition and sensory attributes. Raman spectroscopy (RS) and near infrared spectroscopy (NIRS) were explored as rapid methods to predict eating quality characteristics. The JEX breed type had superior eating quality to HF, while their FA profiles were similar. Slaughter age affected collagen characteristics. Higher energy diets produced beef with relatively better eating quality, but a less healthy FA profile than mainly forage diets. However, amount of concentrates in the first or second grazing season had limited effects on meat quality traits. Quality varied between muscles and the interaction between muscle and production system was significant for IMF content and FA profile. Eating quality of LT was superior but FA profile was inferior to GM and ST. Steer beef had superior eating quality to bull beef. Compared to Achilles tendon suspension (AS), Pelvic suspension (PS) improved tenderness up to 7 days of ageing and accelerated ageing processes. Both RS (1300-2800 cm-1) and NIRS (400-1900 nm) showed considerable potential for assessing physico-chemical quality traits from young male dairy beef, with RS demonstrating enhanced prediction performance. Consistent and significant correlations were also obtained among technological, compositional, sensorial and nutritional quality traits for dairy beef.
Controlling the denaturation and aggregation of whey proteins using κ-casein and caseinomacropeptide
(University College Cork, 2019-12) Gaspard, Sophie J.; O'Mahony, Seamus Anthony; Kelly, Alan; Brodkorb, Andre; Teagasc; Dairy Research Ireland
Whey proteins ingredients are extensively used in a variety of product formulations such as dairy beverages, infant formula and sport nutritional beverages, due to their nutritional and functional properties. Dairy protein-containing beverages are thermally processed, typically to ensure microbiological safety. However, whey proteins denature and aggregate at temperatures greater than 60°C, which can lead to fouling of industrial equipment and/or uncontrolled gelation, depending on formulation and heating conditions. The presence of caseins has been previously reported to limit the extent of aggregation of whey proteins. The objective of this study was to investigate the effect of κ-casein and caseinomacropeptide (CMP) on the denaturation and aggregation of whey proteins, with a view to developing practical strategies for controlling whey protein denaturation and aggregation for ingredient applications. This study demonstrated that both κ-casein and CMP have the ability to improve the heat stability of whey proteins. The inclusion of κ-casein reduced the size of the aggregates of whey protein after a first heat treatment (90°C for 25 min at pH 7.2) and enhanced their solubility during subsequent heating (90°C for 1 h at pH 7.2). The presence of CMP during heating increased the temperatures of denaturation and gelation of whey proteins and prevented the formation of solid whey protein gels when combined with a low heating rate. The presence of CMP also resulted in a lower turbidity of whey protein solutions after heating and an enhanced solubility of whey protein aggregates. The effect of glycosylation of CMP on the denaturation and aggregation of whey proteins was pH-dependent; a transition occurred at pH 6, below which the glycosylation of CMP reduced its stabilizing properties. This thesis provides new insights into the interactions of whey proteins with κ-casein and CMP, with potential for novel applications in improving the heat-stability and solubility of whey proteins. The outcomes of this study have applications for the manufacture of clear, heat-stable beverages containing whey proteins.
Cytochemical assessment of brewers’ spent grain (BSG) extract bioactivities
(University College Cork, 2017) Crowley, Damian; O'Brien, Nora M.
Brewers’ spent grain (BSG) is a useful source of protein and phenolic compounds which can be obtained via different extraction methods. The aim of this thesis was to assess protein hydrolysates and phenolic extracts generated from BSG using various extraction procedures, for their potential as functional food ingredients with anti-inflammatory, antioxidant, anticancer and neuroprotective properties. Firstly, the anti-inflammatory effects of an alkaline-extracted BSG protein rich fraction and three ultrafiltration-generated fractions added to milk and subjected to simulated gastrointestinal digestion (SGID) was shown in Jurkat T cells. Enzyme extraction methods were then used to produce phenolic fractions with strong bioactivities. The cellular antioxidant potential of both black and pale BSG phenolic extracts isolated using carbohydrases was demonstrated in U937 cells and HepG2 cells. Following this, the ability of the most active phenolic extracts to enhance the antioxidant potential of flavoured water drinks before and after SGID was investigated. However, none of the phenolic extracts added to water drinks significantly increased antioxidant activity. In other studies the anti-cancer potential of a pale BSG phenolic extract was demonstrated, through the increase of apoptosis in U937 cells. Additionally, the neuroprotective potential of both black and pale BSG phenolic extracts was shown in SK-N-BE(2) neuronal cells, where extracts protected against hydrogen peroxide (H2O2)-induced cytotoxicity, H2O2-induced apoptosis and H2O2-induced lipid peroxidation. Finally, the cytotoxicity, anti-inflammatory activity and antioxidant potential of BSG protein hydrolysates generated using different extraction procedures was assessed and compared. Direct enzymatic hydrolysates demonstrated anti-inflammatory activity in Jurkat T cells and RAW 264.7 cells. Alkaline-extracted BSG protein hydrolysates, as well as a 10kDa permeate showed anti-inflammatory effects in RAW 264.7 cells. As well as this, unhydrolysed fractions, a direct enzymatic hydrolysate, alkaline-extracted BSG protein hydrolysates, as well as a 10kDa permeate also displayed cellular antioxidant effects in U937 cells and HepG2 cells.

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