title: Aspects of the chemistry of milk protein films formed on stainless steel

authors: Nisbet, Trevor John

abstract: The literature on the properties of stainless steel surfaces, the physico-chemical properties of milk and the surface chemistry of proteins was reviewed to gain an insight into the formation, of protein-mineral films (“milkstone”) on the contact surfaces of dairy milk lines. Protein adsorption processes were considered fundamental to the formation of such deposits. Standard methods of protein separation and purification were employed to obtain purified αₛ₁, β, and K-casein. 
Radiochemical methods provided a convenient means of studying protein adsorption on to low surface area systems. Of the various methods for labelling proteins, radioiodination by a slightly modified version of Hunter and Greenwood Chloramine T method proved to be the most satisfactory. By this method, stable labelled milk proteins of high specific activity containing not more than one iodine atom per protein molecule and having properties that could not be distinguished from those of unlabelled proteins were prepared.
In the course of the work several further aspects of the iodination reaction and the reactions of iodine species with milk components were investigated. Iodination efficiencies were found to be sensitive to the storage time of the radioactive iodine stock solutions (supplied free of reducing agent at pH 8-11). The drop in iodination efficiency was correlated with an increase in the concentration of oxidised iodine species in the radioactive stock solutions. Studies of the iodination of whole milk showed that with the exception of K-casein, the tyrosyl residues of all proteins in milk were iodinated with approximately the same iodination efficiency. The iodination efficiencies of K-casein tyrosyls were significantly lower. A theoretical examination of the effect of the hydrophobicity of the molecular environment on tyrosyl iodination efficiency, indicated the likelihood that the affect observed was due to the incorporation of the K-casein in inaccessible regions of the micelle rather than a molecular environment effect.
Iodinated caseins, when added to whole skim milk became bound to the micelle fraction. In the case of β and K-casein, the binding was temperature dependent and at least partially reversible. αₛ₁-casein binding to micelle was largely independent of temperature and irreversible. The stabilising effect of calcium ions on diluted micelle suspensions was demonstrated. The variation in the K-casein content of casein micelles with micelle size was also demonstrated. When inorganic iodine was added to milk, part of this became encapsulated by milk micelles. However, there was virtually no chemical binding of the iodine to the milk proteins of cooled milk. 
Techniques were developed to study the surface chemistry of radioactively labelled milk proteins at stainless steel-water interfaces. The amount adsorbed was calculated according to the principle of isotope dilution. Autoradiography and high resolution autoradiography, using nuclear track emulsion and scanning electron microscopy confirmed the uniform distribution of the adsorbed protein. Studies of adsorption from whole skim milk, whey and milk serum, indicated that of the various milk proteins serum caseins had the greatest affinity for stainless steel surfaces. Adsorption data for purified caseins and mixtures of casein indicated that β-casein was the species preferentially adsorbed. Generally it was observed that adsorption was largely irreversible and providing adequate time was allowed, proceeded to form a relatively constant close packed film of adsorbed protein molecules. The amount of protein in the films ranged from 2.0 mg m⁻² to 3.0 mg m⁻² irrespective of protein concentration, except at high concentrations of β-casein and to some extent, αₛ₁-casein, where a second adsorption process seemed to be present. This effect also seemed to be present in studies of adsorption from whole milk. 
The kinetics of adsorption and desorption were monitored by tracer techniques. The data for adsorption from unstirred solutions indicated the process was diffusion controlled. A diffusion model was developed which accounted for the rate of adsorption and the amount adsorbed in terms of the size, shape and hydration of the protein molecule, the viscosity of the medium and the effective surface area of the stainless steel surface. The model was used to estimate hydration and axial ratio values for β-casein. Values of 1.9 grams of water per gram of protein for the  hydration, and 11 for the axial ratio (a/b) were obtained. 
Desorption of adsorbed protein is a complex process that does not obey simple rate laws. Desorption was facilitated by hydroxyl ions and anionic surfactants but no simple relationship between rate of desorption and species concentration was obtained.
Simple adsorption process cannot account for the formation of macroscopic milk protein films on milk contact surfaces. After the surface has been covered by the initial adsorbed layer, no further protein could be adsorbed until the surface of this layer was modified in some way, for example, by the adsorption of positively charged colloids of iron oxide, chromium oxide or calcium phosphate. Deposition by evaporation of solution films, or precipitation from solution are other mechanisms by which macroscopic films may be formed. The film thicknesses of the drained milk films formed after immersions of clean stainless steel surfaces into dilute milk solutions remained independent of dilution at 3.8 ± 0.6 x 10⁻⁴ cm. The properties of protein films formed at surfaces can be altered by thermal denaturation, chemical modification and also presumably by bacterial action. 
Analytical and microscopic examination of actual milkstone deposits generally supported a previous milkstone classification based upon chemical analysis, i.e. fat deposits, high protein, deposits, calcium phosphate milkstone and iron milkstone. As a result of the present investigation the further classification of high protein deposits into chlorine-induced (from sodium hypochlorite) and phosphate-induced (from phosphoric acid) was suggested. It does not seem likely that milkstone deposits are formed by a single mechanism. This conclusion was supported by an analysis of data for deposits formed in simulator trials where evaporation of milk films (in one simulator) and precipitation induced by hypochlorite (in two simulators) were major factors. The variety of possible mechanisms for deposition is due to the range of conditions that can occur in milk lines. The results of the present work will assist in determining the mechanism operative in a particular case and will allow the development of better procedures for the removal and prevention of such deposits.

text: Aspects of the chemistry of milk protein films formed on stainless steel The literature on the properties of stainless steel surfaces, the physico-chemical properties of milk and the surface chemistry of proteins was reviewed to gain an insight into the formation, of protein-mineral films (“milkstone”) on the contact surfaces of dairy milk lines. Protein adsorption processes were considered fundamental to the formation of such deposits. Standard methods of protein separation and purification were employed to obtain purified αₛ₁, β, and K-casein. 
Radiochemical methods provided a convenient means of studying protein adsorption on to low surface area systems. Of the various methods for labelling proteins, radioiodination by a slightly modified version of Hunter and Greenwood Chloramine T method proved to be the most satisfactory. By this method, stable labelled milk proteins of high specific activity containing not more than one iodine atom per protein molecule and having properties that could not be distinguished from those of unlabelled proteins were prepared.
In the course of the work several further aspects of the iodination reaction and the reactions of iodine species with milk components were investigated. Iodination efficiencies were found to be sensitive to the storage time of the radioactive iodine stock solutions (supplied free of reducing agent at pH 8-11). The drop in iodination efficiency was correlated with an increase in the concentration of oxidised iodine species in the radioactive stock solutions. Studies of the iodination of whole milk showed that with the exception of K-casein, the tyrosyl residues of all proteins in milk were iodinated with approximately the same iodination efficiency. The iodination efficiencies of K-casein tyrosyls were significantly lower. A theoretical examination of the effect of the hydrophobicity of the molecular environment on tyrosyl iodination efficiency, indicated the likelihood that the affect observed was due to the incorporation of the K-casein in inaccessible regions of the micelle rather than a molecular environment effect.
Iodinated caseins, when added to whole skim milk became bound to the micelle fraction. In the case of β and K-casein, the binding was temperature dependent and at least partially reversible. αₛ₁-casein binding to micelle was largely independent of temperature and irreversible. The stabilising effect of calcium ions on diluted micelle suspensions was demonstrated. The variation in the K-casein content of casein micelles with micelle size was also demonstrated. When inorganic iodine was added to milk, part of this became encapsulated by milk micelles. However, there was virtually no chemical binding of the iodine to the milk proteins of cooled milk. 
Techniques were developed to study the surface chemistry of radioactively labelled milk proteins at stainless steel-water interfaces. The amount adsorbed was calculated according to the principle of isotope dilution. Autoradiography and high resolution autoradiography, using nuclear track emulsion and scanning electron microscopy confirmed the uniform distribution of the adsorbed protein. Studies of adsorption from whole skim milk, whey and milk serum, indicated that of the various milk proteins serum caseins had the greatest affinity for stainless steel surfaces. Adsorption data for purified caseins and mixtures of casein indicated that β-casein was the species preferentially adsorbed. Generally it was observed that adsorption was largely irreversible and providing adequate time was allowed, proceeded to form a relatively constant close packed film of adsorbed protein molecules. The amount of protein in the films ranged from 2.0 mg m⁻² to 3.0 mg m⁻² irrespective of protein concentration, except at high concentrations of β-casein and to some extent, αₛ₁-casein, where a second adsorption process seemed to be present. This effect also seemed to be present in studies of adsorption from whole milk. 
The kinetics of adsorption and desorption were monitored by tracer techniques. The data for adsorption from unstirred solutions indicated the process was diffusion controlled. A diffusion model was developed which accounted for the rate of adsorption and the amount adsorbed in terms of the size, shape and hydration of the protein molecule, the viscosity of the medium and the effective surface area of the stainless steel surface. The model was used to estimate hydration and axial ratio values for β-casein. Values of 1.9 grams of water per gram of protein for the  hydration, and 11 for the axial ratio (a/b) were obtained. 
Desorption of adsorbed protein is a complex process that does not obey simple rate laws. Desorption was facilitated by hydroxyl ions and anionic surfactants but no simple relationship between rate of desorption and species concentration was obtained.
Simple adsorption process cannot account for the formation of macroscopic milk protein films on milk contact surfaces. After the surface has been covered by the initial adsorbed layer, no further protein could be adsorbed until the surface of this layer was modified in some way, for example, by the adsorption of positively charged colloids of iron oxide, chromium oxide or calcium phosphate. Deposition by evaporation of solution films, or precipitation from solution are other mechanisms by which macroscopic films may be formed. The film thicknesses of the drained milk films formed after immersions of clean stainless steel surfaces into dilute milk solutions remained independent of dilution at 3.8 ± 0.6 x 10⁻⁴ cm. The properties of protein films formed at surfaces can be altered by thermal denaturation, chemical modification and also presumably by bacterial action. 
Analytical and microscopic examination of actual milkstone deposits generally supported a previous milkstone classification based upon chemical analysis, i.e. fat deposits, high protein, deposits, calcium phosphate milkstone and iron milkstone. As a result of the present investigation the further classification of high protein deposits into chlorine-induced (from sodium hypochlorite) and phosphate-induced (from phosphoric acid) was suggested. It does not seem likely that milkstone deposits are formed by a single mechanism. This conclusion was supported by an analysis of data for deposits formed in simulator trials where evaporation of milk films (in one simulator) and precipitation induced by hypochlorite (in two simulators) were major factors. The variety of possible mechanisms for deposition is due to the range of conditions that can occur in milk lines. The results of the present work will assist in determining the mechanism operative in a particular case and will allow the development of better procedures for the removal and prevention of such deposits.

year: 1979

title: Investigation of glucose and energy metabolism in eumenorrheic female ultra-runners across the menstrual cycle

authors: Dole, Andrew

subjects: blood glucose

abstract: Female athletes, ultra-running, and the utilization of continuous glucose sensors in endurance sports are each individually underrepresented in current literature. When these areas are examined collectively, a substantial gap in knowledge becomes evident. Therefore, the purpose of this PhD study was to collect glucose and energy metabolism data in regularly menstruating female athletes within the complex sporting environment of ultra-running. Initially, a narrative review of the current study methodologies in female performance research was completed. The narrative review aided in the identification of gaps in study design and validation methodologies within female performance research, put forth a suggestive framework for standardizing terminology, and provides a comprehensive list of options for a combined methods approach to menstrual tracking. Subsequently, a randomized, cross-over experimental design was performed with twelve experienced female ultra-runners (age 39 ± 6 y) acting as their own control across menstrual cycle phases during an ultra-marathon simulation. Participants completed a 3-hour fasted outdoor run (FASTED) followed by a one-hour treadmill run (TREAD) where three standardized oral glucose doses were provided, in the mid-follicular (FOL) and mid-luteal phases (LUT). Using a mixed linear model, the menstrual phase was identified as being statistically significant for differences in glucose measurements between CGM and capillary glucose during TREAD (p = 0.02) but not FASTED. Also, the CGM reported glucose levels an average of -0.43 mmol·L-1 (95% CI -0.86, -0.005) and -1.02 mmol·L-1 (95% CI -1.63, -0.42) lower in fasted and fed scenarios respectively when compared to capillary glucose. Median glucose was higher in the LUT (Capillary 0.25-0.29 mmol·L-1, CGM 0.38-0.4 mmol·L-1) both when fasted or supplemented with carbohydrate. Of note, this is the first study to show that glucose is on average higher in the mid-luteal phase as well as observe the avoidance of clinically significant non-diabetic hypoglycemia (< 3.0 mmol·L-1) during 3-hours of fasted running following an 8-hour fast among female ultra-runners. Additionally, higher individual responses of low blood sugar were observed in the follicular phase. Further, there were no significant differences in the respiratory exchange ratio (RER) between the FOL (0.805 ± 0.028) and LUT phases (0.809 ± .035); t (10) -0.401, p = 0.697 during comparable submaximal running intensity (57% ± 7.3% and 57.3% ±7.9% VO2MAX respectively). Post-hoc analysis revealed no significant main effect of RER on glucose levels.

text: Investigation of glucose and energy metabolism in eumenorrheic female ultra-runners across the menstrual cycle Female athletes, ultra-running, and the utilization of continuous glucose sensors in endurance sports are each individually underrepresented in current literature. When these areas are examined collectively, a substantial gap in knowledge becomes evident. Therefore, the purpose of this PhD study was to collect glucose and energy metabolism data in regularly menstruating female athletes within the complex sporting environment of ultra-running. Initially, a narrative review of the current study methodologies in female performance research was completed. The narrative review aided in the identification of gaps in study design and validation methodologies within female performance research, put forth a suggestive framework for standardizing terminology, and provides a comprehensive list of options for a combined methods approach to menstrual tracking. Subsequently, a randomized, cross-over experimental design was performed with twelve experienced female ultra-runners (age 39 ± 6 y) acting as their own control across menstrual cycle phases during an ultra-marathon simulation. Participants completed a 3-hour fasted outdoor run (FASTED) followed by a one-hour treadmill run (TREAD) where three standardized oral glucose doses were provided, in the mid-follicular (FOL) and mid-luteal phases (LUT). Using a mixed linear model, the menstrual phase was identified as being statistically significant for differences in glucose measurements between CGM and capillary glucose during TREAD (p = 0.02) but not FASTED. Also, the CGM reported glucose levels an average of -0.43 mmol·L-1 (95% CI -0.86, -0.005) and -1.02 mmol·L-1 (95% CI -1.63, -0.42) lower in fasted and fed scenarios respectively when compared to capillary glucose. Median glucose was higher in the LUT (Capillary 0.25-0.29 mmol·L-1, CGM 0.38-0.4 mmol·L-1) both when fasted or supplemented with carbohydrate. Of note, this is the first study to show that glucose is on average higher in the mid-luteal phase as well as observe the avoidance of clinically significant non-diabetic hypoglycemia (< 3.0 mmol·L-1) during 3-hours of fasted running following an 8-hour fast among female ultra-runners. Additionally, higher individual responses of low blood sugar were observed in the follicular phase. Further, there were no significant differences in the respiratory exchange ratio (RER) between the FOL (0.805 ± 0.028) and LUT phases (0.809 ± .035); t (10) -0.401, p = 0.697 during comparable submaximal running intensity (57% ± 7.3% and 57.3% ±7.9% VO2MAX respectively). Post-hoc analysis revealed no significant main effect of RER on glucose levels.

year: 2024-12-20

title: Is there evidence that traditional mōteatea (Māori laments) were composed from a common stock of oral formulae?

authors: Roa, Raukura

subjects: New Zealand

abstract: Oral formulaic composition, which involves the use of communally owned formulae of various kinds, is a common feature of verbal arts produced in many different languages. It is particularly associated with pre-literate cultures and tends to be gradually replaced by more individualistic verbal art forms when societies become literate. There are very few publications in which the analysis of mōteatea (Māori laments) is linked explicitly to oral formulaic theory. Nevertheless, there is sufficient evidence in published sources to indicate that traditional mōteatea (defined here as mōteatea that are not fundamentally influenced by European cultural beliefs and practices) exhibit evidence of regularly recurring, conventional themes (such as death, separation, loss and travel) and motifs (such as the setting sun, the presence of rain or mist and sleeplessness). The research reported here set out to test the hypothesis that traditional mōteatea were made up of verbal formulae, that is, of the same or very similar groups of words derived from a common store of poetic resources. Based on the analysis of the mōteatea included in Ngā mōteatea, a collection initially established by Sir Apirana Ngata, I conclude that this hypothesis must be rejected.

text: Is there evidence that traditional mōteatea (Māori laments) were composed from a common stock of oral formulae? Oral formulaic composition, which involves the use of communally owned formulae of various kinds, is a common feature of verbal arts produced in many different languages. It is particularly associated with pre-literate cultures and tends to be gradually replaced by more individualistic verbal art forms when societies become literate. There are very few publications in which the analysis of mōteatea (Māori laments) is linked explicitly to oral formulaic theory. Nevertheless, there is sufficient evidence in published sources to indicate that traditional mōteatea (defined here as mōteatea that are not fundamentally influenced by European cultural beliefs and practices) exhibit evidence of regularly recurring, conventional themes (such as death, separation, loss and travel) and motifs (such as the setting sun, the presence of rain or mist and sleeplessness). The research reported here set out to test the hypothesis that traditional mōteatea were made up of verbal formulae, that is, of the same or very similar groups of words derived from a common store of poetic resources. Based on the analysis of the mōteatea included in Ngā mōteatea, a collection initially established by Sir Apirana Ngata, I conclude that this hypothesis must be rejected.

year: 2007