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    The effect of microwave-assisted heating on bioactive and immunological compounds in donor human milk
    (Elsevier Ltd, 2022-05-01) Leite JAS; Robinson RC; Salcedo J; Ract JNR; Quintal VS; Tadini CC; Barile D
    Low-Temperature Long-Time pasteurization (LTLT) is normally applied in donor human milk from Human Milk Banks (HMBs) to guarantee microbiological safety; however, this treatment can modify the protein structures, decreasing their beneficial effects. Thus, this study aimed to determine the impact of microwave-assisted heating on the concentration of key biological compounds in donor human milk to verify whether a microwave heating technique can be used as an alternative to LTLT pasteurization in Human Milk Banks. The concentrations of oligosaccharides, immunoglobulins, lactoferrin and fatty acids were monitored in raw donor milk and after processing to assess the impact of the microwave and LTLT treatments. The concentration of oligosaccharides was determined by HPAEC-PAD, immunoglobulins and lactoferrin were quantified using ELISA kits and fatty acids were quantified by gas chromatography. Oligosaccharides and fatty acids were not significantly affected (p > 0.05) by LTLT and microwave processes; however, immunoglobulins and lactoferrin concentrations were better preserved when microwave-assisted heating was applied. For this reason, microwave-assisted heating can be considered a promising alternative to LTLT pasteurization of donor human milk in Human Milk Banks.
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    Functional protein display on the surface of biobeads produced by recombinant Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Chen, Shuxiong
    Polyhydroxyalkanoic acids (PHAs) are biopolyesters produced by various bacteria. They are deposited as spherical water-insoluble cytoplasmic inclusions (beads) containing an amorphous hydrophobic polyester core and surrounded by a phospholipid monolayer and embedded proteins, including PHA synthase (PhaC), the key enzyme required for PHA bead formation. Although inactive PhaC cannot produce PHA beads, fusing inactive PhaC to green fluorescent protein (GFP) leads to GFP protein bead formation. Both PHA and protein beads could serve as a versatile platform for display of desired proteins suitable for various biotechnological and medical applications. The tuberculin skin test (TST) for diagnosing bovine tuberculosis (TB) in cattle uses the purified protein derivative (PPD) that is prepared from Mycobacterium bovis. However, some antigens in the PPD are also present in environmental mycobacteria. Therefore, the TST lacks specificity if animals are exposed to non-pathogenic environmental mycobacteria. In this study, three specific TB antigens, CFP10, ESAT6, and Rv3615c — which are present in pathogenic but absent in most non-pathogenic mycobacteria — were displayed on the surface of PHA beads. The results demonstrated that these triple antigen-displaying PHA beads can differentiate TB-infected from non-infected cattle, making this an attractive alternative to current skin test diagnostic reagents. IgG binding domains displayed on GFP protein beads have a higher IgG binding ability when compared to their counterpart displayed on PHA beads. However, it is unclear whether an enhancement of IgG binding ability due to GFP protein beads could be achieved by immobilization on other fluorescent protein (FP) beads. The results showed that other FP (including yellow, red and cyan) beads displaying IgG binding domains have an approximately 1.5–2 fold greater IgG binding ability when compared to PHA beads displaying the same binding domains. To investigate whether protein beads displaying iron-binding peptides could be magnetized while maintaining IgG binding function, an iron binding peptide was displayed. The results demonstrated that protein beads displaying both IgG and iron binding peptides can be magnetised by iron oxide and retain a strong IgG binding ability. Finally, this study revealed that different cell disruption techniques could affect the morphology and functionality of FP protein bead.
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    Provision of immunoglobulins to suckling piglets can enhance post-weaning growth performance : a thesis presented in partial fulfilment of the requirements for the degree of Master of Applied Science in Animal Science at Massey University
    (Massey University, 1998) Vanavichial, Boonkasem
    In this experiment the hypothesis that providing a supplementary source of bovine milk immunoglobulin G (IgG) to suckling piglets increases post-weaning growth performance was tested. The litters from eight multiparous Large White x Landrace sows received oral supplements by syringe. Three piglets in each litter received oral doses of whey globulin concentrate (WGC) which contained 6% IgG. A second group of three piglets per litter received oral doses of whey protein isolate (WPI) to approximate the amino acids supplied in WGC but without IgG's. A third group of three piglets per litter received oral doses of water (CONT) to simulate the oral dosing procedure. The daily supplement of WGC and WPI provided 0.7 g per day of age of ideal protein during the first week and 1.4 g per day of age thereafter. The oral doses were provided twice daily at 09.00h and 15.00h from day 2 to day 24 of lactation. For the statistical analysis, a linear model including sex, sow and treatment as fixed effects, and live weight at birth as covariate was fitted to the data. The average daily gains measured over the suckling period (24d) were not atatistically significantly different between the three groups with the control gaining 249gd-1, WGC gaining 259gd-1 and WPI gaining 264gd-1. The provision of either WGC or WPI did not increase the average daily gain up to weaning, possibly because the piglets reduced their intake of sow's milk. To determine the effect of supplemental IgG, the most valid comparison is between WPI and WGC because the supply of ideal protein, and the time taken to provide each oral dose, were similar. Piglets receiving WGC grew 12% faster than WPI from transfer (62d) to slaughter (85kg) (P < 0.05), and 8% faster from birth to slaughter (P < 0.05). These findings indicate that the provision of IgG during early life can lead to long term advantages in growth rate.
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    Comparison of the passive prophylactic effect of bovine milk immunoglobulin fed either as a bolus or continuously against diarrhoea caused by Escherichia coli K88 using piglets as models : a thesis presented in partial fulfilment of the requirements for the degree of Master of Animal Science at Massey University, Palmerston North, New Zealand
    (Massey University, 1999) Kandiah, Geetha
    The overall aim of the study was to determine the passive prophylatic effect of bovine milk immunoglobulin administered orally either as a bolus (once a day) or as continuously (three times a day) against diarrhoea caused by Escherichia coli K 88. As the piglets have gastrointestinal structural and physiological similarities to humans they were assumed to be an appropriate model for the study. The first part of the study was done to evaluate the quantity of undigested immunoglobulin G in the gastrointestinal tract of the piglet. This was conducted in two experiments. One was a pilot study and the other was to estimate the quantity of undigested immunoglobulin G in the gut. A pilot study was conducted to evaluate the rate of movement and the quantities of digesta that can be collected in the gastrointestinal tract regions at varying time intervals. Ten piglets were randomly divided into five groups of two in each. One from each group was selected, fed with an experimental diet which contained blue colour glass beads and dye and slaughtered at either 1, 5, 9, 16 and 24 hrs after feeding. Digesta was collected from various regions of the gastrointestinal tract. Faeces were collected using ostomy bags from those slaughtered at 9, 16 and 24 hrs only. The dye movement and the glass beads recovered were monitored. The movement of the dye was observed up to the small intestine at 1 hr. the caecum at 5 hrs, the beginning of the colon at 9 hrs, the two third of the colon at 16 hrs, and in the faeces at 24 hrs. Most of the beads were found in the stomach between 1 and 5 hrs, spread throughout the small intestine at 9 hrs, in the caecum at 16 hrs and in the colon at 24 hrs. The results confirmed that a sufficient amount of digesta could be collected from the various regions over a 24 hr period. The data facilitated the planning of the immunoglobulin digestibility trial which is the second part of the experiment. To measure the IgG digestibility, the piglets were fed on a large dose of an experimental diet (10% of their metabolic body weight kg0.75 contain 30% immunoglobulin) and the digesta, faeces and blood were collected. On the slaughter day, a group of five animals were fed on an experimental diet and digesta and blood were collected 1, 5, 9, 16, and 24 hours after feeding. Faeces were collected from those killed at 16 and 24 hours. Blood was analysed for immunoglobulin G for all piglets. Digesta and faeces were analysed for chromium and immunoglobulin G. There was no evidence of the presence of immunoglobulin G in the blood and faeces. A larger quantity of immunoglobulin G was found in the stomach (p<0.001) with a less in the first and second third of the small intestine (p<0.05) 24 hours post-prandially. This demonstrated that immunoglobulin G could resist digestion in the gut. The second part of the study was conducted to compare the effect of feeding bovine immunoglobulin as a bolus versus continuous against Escherichia coli K88 diarrhoea. Twenty-four piglets, four-week-old, were randomly allocated to three treatment groups, namely continuous (fed a diet containing 10% immunoglobulins three times a day), control (fed an immunoglobulins free diet), and bolus (fed a 30% immunoglobulins diet in the morning and control diet the other two feeds). On Day 9, 30 minutes before the morning feed, the piglets were inoculated with 1 x 109 cfu Escherichia coli by a syringe into their throat, and observed for nine days. On day 17, all piglets were fed on control diet (no Ig) to evaluate if Escherichia coli K88 would have any effect on recolonisation and diarrhoea and observed for further three days. Finally they were all treated with antibiotics, Biosol M and Tylan 200. The observations include faecal culture, faecal consistency, the percent of free water content in the faeces, weight and feed intake. Faecal culture was done twice before inoculation, three times during treatment, and once after all were fed on control diet and once after the antibiotics treatment. The free liquid content in the faeces was highest in the control group (37.5%) and lower in the continuous immunoglobulin group (25.0%) and least in the bolus immunoglobulin group (17.5%). Bolus immunoglobulin feeding (11.25%) lessened the severity of diarrhoea (classified by consistency) compared with the control group (26.2%) and continuous group (25.0%). Hence bolus immunoglobulin feeding had a better effect in controlling water loss and the severity of diarrhoea. A higher dosage of immunoglobulin in bolus feeding may also have prevented bacterial shedding. From this study, it can be concluded that feeding immunoglobulin as a bolus could be used as a prophylactic treatment for diarrhoea.