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    The relationship between daily mood and salivary immunoglobulin A : a thesis presented in partial fulfilment of the requirements for the degree of Master of Arts in Psychology at Massey University
    (Massey University, 1991) Binnie, Jan Elizabeth
    The aim of the present study was to examine the influence of daily positive and negative mood on secretory immunoglobulin A (S-lgA) concentrations in human saliva. An instrument was constructed for the measurement of daily mood, based on current theories in the psychobiology of affect, neuroendocrinology and behaviour. With this instrument the average intensity, peak intensity and duration of eight moods, two from each pole of positive and negative affect dimensions, were measured. From these scores three positive affect variables were created by combining scores on positive dimension moods, and three negative affect variables created by combining scores on negative dimension moods, and these variables were used for multivariate analysis. Twenty female subjects between the ages of 18 and 60 years were studied for 28 consecutive days. They were each required to capture 1.5 ml of free flowing parotid saliva, fill in the mood questionnaire, and record whether or not they had taken medication, exercise, alcohol, tobacco or menstruated on each evening of the study. These last variables were subsequently used as control variables in the multivariate analysis. Concentrations of S-lgA in the saliva were measured with an enzyme-linked immunosorbent assay (ELISA). No significant associations between S-lgA levels and positive or negative mood variables were detected. The lack of significant effects of mood variables on S-lgA is discussed in the context of the psychoneuroimmunological literature, and with particular emphasis on measurement issues.
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    A study of morphological and physiological changes in the mandibular gland of the sheep associated with eating and direct stimulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1981) Ariyakulkaln, Punnipa
    This study was undertaken to investigate relationships between the structure of the mandibular gland of the sheep and its secretory activity in response to feeding, direct stimulation of autonomic nerves, or pharmacological agents that mimic the action of autonomic transmitters. Forty-five crossbred Romney ewes and wethers were used in acute experiments and twenty-two in chronic experiments. Histochemical and electron microscopical examinations of the structure of mandibular glands confirmed that their secretory endpieces are composed of mucous tubulo-acinar cells and seromucous demilunes. The mucous acini contained a single type of electron lucent granules, whereas the granules of demilunes typically exhibited a tripartite structure. The intercalated ducts were relatively short and lined by non-secretory, simple cuboidal cells and occasional basal cells. Striated ducts were numerous and lined by four cell types, the most common of which (type-I) were tall, columnar, electron lucent cells with well developed membrane infoldings basally with associated mitochondria and small, dense, apical bodies. Myoepithelial cells were distributed densely around the secretory endpieces and within the basement membranes. Myoepithelial cells were also found embracing the intercalated duct cells. Both AChE-positive and biogenic-amine fluorescent nerve fibres were present around the secretory endpieces and the walls of blood vessels. Fewer biogenic-amine fluorescent fibres were seen in relation to duct cells. Electron microscopy showed unmyelinated fibres in both epilemmal and hypolemmal sites. The epilemmal axons were frequently found close to a variety of effector cells, while hypolemmal axons were observed occasionally in the intercellular space between adjacent striated duct cells and between intercalated duct and mucous cells. Axons containing large granular vesicles were also found within interstitial nerve bundles. Mandibular secretion was studied after cannulation of the mandibular duct in both acute and chronic experiments. In anaesthetized animals, stimulation of either the chorda lingual nerve (3-8V, 5-10Hz, 0.2 msec) or injection of carbachol (40 µg kg-1 body weight, iv) within 10-25 sec caused a copious secretion (0.33-0.74 g min-1) of low protein content (0.44-1.56 mg ml-1). This response was completely blocked by atropine (0.1 mg kg-1 body weight). In contrast, stimulation of cervical sympathetic trunk (3-8V, 5-10 Hz, 0.2 msec) after a latency of 35-102 sec caused a meagre secretion (0.01-0.06 g min-1) of high protein concentration (4.02-25.68 mg ml-1). Isoprenaline had similar effects. Secretory responses to sympathetic stimulation were blocked by propranolol (1.0 mg kg-1 body weight). Studies involving gel electrophoresis demonstrated major protein bands exclusively in the sympathetic nerve or isoprenaline stimulated saliva. These major protein components (both soluble and insoluble) were found by immunocytochemical studies to be localized in the demilunes and some striated duct cells of the resting gland. It was found that in sheep fed lucerne chaff (ca. 1,000 g daily) a rapid and sustained mandibular flow only occurred during eating, although, short term increases were seen, for example, during drinking. Flow was absent during rumination and slight (0.95 ± 0.09 g h-1) or absent at rest. Saliva produced during eating had its highest protein concentration almost immediately as eating commenced (1.65 ± 0.06 mg ml-1) and remained at a high level during the first hour of eating (1.55 ± 0.06 mg ml-1) Propranolol (1.0 mg kg-1 body weight, iv) caused significant reductions in protein secretion during eating (P<.001) without associated changes in flow. Gel electrophoretic studies confirmed the presence of a major protein band similar to soluble protein band X found in sympathetically evoked saliva. The intensity of this major protein band in saliva collected during eating was also reduced after propranolol treatment. Saliva collected during teasing had a high protein concentration (2.73 ± 0.20 mg ml-1). It is concluded that sympathetic activation was involved mainly early in the eating period and that parasympathetic nerves were active throughout. The latter was confirmed by a great reduction in flow after injection of atropine (0.1 mg kg-1, iv). Morphological studies of the glands of sheep whose food had been withheld for 20 hours revealed that both the mucous acini and seromucous demilunes were filled with secretory granules. Stimulation of the chorda lingual nerve for 2-4 hours caused acini to discharge their contents of secretory granules, but no appreciable changes in the demilunes. On the other hand, stimulation of the cervical sympathetic trunk produced varying degrees of degranulation in the demilunes, with, in some cells, vacuolation. Infusion of isoprenaline (2h; 0.3 µg kg-1 min-1) produced similar changes in demilunes. Striated duct cells showed reduced PAS-staining, and disruption of their basal regions, particularly after stimulation of sympathetic nerves. Concurrent stimulation of both sympathetic and parasympathetic nerves resulted in a combination of the above separate effects. Eating led to extensive degranulation and greater evidence of synthesis in the mucous acini than parasympathetic nerve stimulation, the changes increasing with the duration of eating, and a depletion of secretory granules in demilunes that could be prevented by propranolol. (1.2 mg kg-1 body weight, iv ). The morphological changes in demilunes were not proportional to the duration of eating but were greatest in its early phases. Evidence of small dense bodies which were apparently discharged via the apical membrane of striated duct cells and a loss of PAS-staining in these cells suggest that they secrete during eating. However, neither damage to striated duct cells nor secretory endpieces was evident. The results suggest that the sheep mandibular gland is naturally stimulated by both divisions of the autonomic nervous system, with acinar cells predominantly under the parasympathetic and demilunes under the sympathetic control. The sympathetic stimulation of salivary protein secretion appears to be mainly mediated via a β-adrenergic mechanism whereas the secretion of fluid and probably also mucus glycoproteins is an atropine-sensitive parasympathetic effect. On both morphological and physiological grounds it is suggested that in sheep mandibular glands, myoepithelial cell contraction is important in assisting the secretion of viscous saliva. Further studies on the following areas would seem appropriate: (i) systematic morphological studies using stereological analysis of changes in the acinar cells, demilunes, striated ducts and their cytoplasmic components; (ii) ultrastructural examinations of the innervation pattern in this gland under normal conditions, after specific denervation and reinnervation; (iii) studies of the nature and origin of the salivary proteins secreted during eating and nerve stimulation and (iv) the use of chronically cannulated animals for studies of the influence of different conditions of feeding.
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    Investigating astringency mechanism of WPI8855 in acidic condition : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2011) Sun, Xiaoli
    Whey protein isolate is used as a functional ingredient in acidic whey protein beverages, but the associated astringency is a big hurdle to introduce these beverages into the mainstream market. If we can solve the astringency issue, Fonterra would have big advantages over their competitors. Our hypothesis is that whey protein interacts with human saliva proteins and the subsequent precipitation causes astringency. In the present study, ion exchange whey protein isolates (WPI) 8855, and solutions of pure a-lac and ß-lg were used to determine which whey protein fractions are responsible for sedimentation in artificial or human saliva. It has been shown that sedimentation correlates to the level of astringency. Therefore only the level of sedimentation was investigated. The human saliva and artificial saliva were also compared in the astringency titration model in order to determine whether artificial saliva is representative of human saliva. Heat treatment (85°C, 30s) of whey protein solution was performed to mimic commercial beverage manufacture. The heated and non-heated whey protein solutions were titrated with artificial saliva, human saliva or sodium bicarbonate buffer in the range of pH 3 to 6. The sediment was recovered by centrifugation of the titrated samples, and analysed using liquid chromatography-mass spectrometry (LC-MS/MS) or one and two dimensional polyacrylamide gel electrophoresis (PAGE) with amido black and periodic acid Schiff stain. This study showed that ß-lg is the key sedimentation component in heated acidic WPI8855 beverages due to the heat aggregation, pH change through the isoelectric point and interaction with human saliva proteins, including mucin, proline-rich proteins (PRPS) and a-amylase. BSA also interacted with artificial and human saliva, whereas a-lac did not interact with either artificial or human saliva. Heat treatment caused extensive whey protein aggregation and precipitation. Artificial saliva and human saliva behaved differently in this astringency titration model, therefore it is not recommended to use artificial saliva in an in vitro model to predict astringency in vivo. Artificial saliva interacted with whey protein and caused additional precipitation compared to titration with sodium bicarbonate, whereas human saliva was able to hinder some whey protein sedimentation caused by titration with sodium bicarbonate. If astringency is caused by the amount of precipitation of protein, heat treatment would be a major factor in the astringency of whey proteins.
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    Interactions of whey protein isolate and human saliva, as related to the astringency of whey protein beverages : a thesis in partial fulfilment of the requirement of the degree of Master of Technology in Food Technology at Riddet Institute, Massey University, New Zealand
    (Massey University, 2010) Streicher, Christina
    Interactions between 3 different proteins (lactoferrin, beta-lactoglobulin and Whey Protein Isolate) and human saliva were determined. Lactoferrin and whey proteins are known to be astringent at low pH. Astringency is defined as the tactile sensation, mainly on the tongue, caused by astringent compounds when in contact with human saliva. Proline-rich proteins are already known to be directly involved in the astringency of polyphenols. Whey proteins do not contain polyphenols. However, because whey proteins at low pH develop an astringent sensation when consumed, it was expected to detect proline-rich proteins in the interaction between Whey Protein Isolate (WPI) and saliva as well. The protein solutions were adjusted to different pH-levels, ranging from neutral to high acidic, where a part of each protein solution was heat-treated. All solutions were mixed with human saliva in the same ratio (w/w). One part of all mixtures was pH-readjusted. Additionally, WPI model solutions were prepared, adjusted to different pH-levels, heat-treated and then consumed by voluntary participants, who swirled each solution in their mouth for at least 10 seconds. These mixtures of WPI and saliva were collected for further analysis. After consuming the WPI model solutions, followed by rinsing the mouth with water, tongue swabs were taken to determine the particle sizes and zeta-potentials of the remaining material on the tongue. Control tongue swabs of the clean tongue were taken by the participants before any consumption of the WPI model solutions. All mixtures as well as lactoferrin, beta-lactoglobulin (beta-lg), WPI and saliva on their own, were analysed for particle size, zeta-potential and turbidity, which may give an indication for possible aggregation/precipitation of the proteins as well as the analysis of the SDS-PAGE profile of the sediments of the sample mixtures. Saliva is negatively charged between neutral pH and 3.0, whereas lactoferrin has a positive charge below pH 8.0. WPI has a positive charge below pH 5.1; the same applies to beta-lg. None of the proteins themselves showed aggregation/precipitation at pH-levels 6.8, 3.6, 3.4, 3.0, 2.5 or 2.0. However, after the proteins were mixed with saliva, the pH of mixtures shifted towards neutral pH. The mixtures of lactoferrin (unheated/heat-treated) and saliva neither showed any significant increases in particle size nor the presence of turbidity. Salivary proteins were not detected in any mixtures at any observed pH either, despite the known fact that lactoferrin causes astringency. The mixtures of beta-lg (unheated/heated) and saliva displayed high particle sizes below final pH 3.6, whereas the high turbidities of both mixtures were measured between final pH 3.6 and 3.4. Furthermore, only at final pH 2.8 were salivary proteins (mainly glycosylated proline-rich proteins and alpha-amylase) detected. However, higher concentrations of salivary proteins were measured when heat-treated beta-lg was mixed with saliva. The mixtures of WPI and saliva presented the strongest interaction compared to lactoferrin and beta-lg. High aggregation/precipitation occurred in the mixtures between pH 4.3 and 3.0, where significantly high particle sizes and turbidities were detected. The pH-readjusted mixtures of lactoferrin/beta-lactoglobulin/WPI and saliva showed similar values in particle size and turbidity as the mixtures of the proteins and saliva without pH-readjustment at similar pH-values. Furthermore, the pH-readjusted mixtures of the proteins and saliva showed in their sediments the presence of alpha-amylase and glycosylated proline-rich proteins.The mixtures of heat-treated WPI and saliva, collected from the mouth after taking a sip (ratio unknown), revealed that the strongest interactions occurred when WPI-solutions were adjusted to pH 3.6 and 3.4. Similar observations were made for heat-treated WPI-solutions, which were adjusted to pH 3.6 and 3.4, when mixed with saliva 1:1 (w/w). However, additionally to the glycosylated proline-rich proteins and alpha-amylase, faint bands of mucin as well as basic proline-rich proteins were detected in the mixtures collected from the mouth. The proteins of the material remaining on the tongue followed the consumption of WPI-solutions and rinsing with water showed that the particle size measurementswere not reliable. However, pH-levels between 6.8 and 5.7 occurred and negative charges were measured on the tongue after rinsing the mouth twice with water. The strongest interactions between the proteins and human saliva occurred when the proteins, in particular beta-lg and WPI, were positively charged and then mixed with saliva (negative charge). Concluding from that it is suggested that electrostatic interactions may cause the astringent sensations. However, since no evidence could be found that salivary proteins were involved in the interaction between lactoferrin and saliva (without pH-readjustment), it is suggested that other interactions than electrostatic interactions cause the astringent sensation of lactoferrin.