title: Thermophilic enzymes and their impact on milk powder during storage

authors: Chen, Lin

abstract: Milk powder quality and storage life can be compromised by functionality and flavour defects. These defects can be the result of chemical or biochemical reactions, such as the Maillard (browning) reaction, proteolysis and lipolysis. The New Zealand dairy industry has set specifications for thermophile numbers in milk powders that have been used for many years as an indicator of microbial quality. However, any correlation between the numbers of these bacteria and product quality is anecdotal, and there is little supportive scientific evidence. 

This research has set out to: (1) survey the cause of the problem for off-flavours in milk powder storage, mainly whole milk powders (WMPs) because they are the largest powder-product, and most likely to have problems associated with flavour; (2) develop assay methods for detecting possibly very low levels of protease and lipase in the WMPs; and finally (3) determine enzymatic effects in the WMPs during storage. 

The study has focused on the detection of protease and lipase activities. Several sensitive and commonly used assay methods were applied in reconstituted milk made from different batches of commercial WMPs. Both protease and lipase activities were detected, and the lipase activity was quantified at 0.7-1.1 U/g powder (pNP caproate unit), but the level of protease activity is yet to be accurately determined. 

A lipase was semi-purified from a WMP using Phenyl Sepharose hydrophobic chromatography. The enzyme is most active at 60°C under the conditions used, and has similar substrate specificity to the lipases produced by seven Bacilli isolated from a milk powder production stream. Although the source of the powder lipase is yet to be identified, data suggest that it is most likely from a thermophilic bacterium. 

Preliminary studies on the characteristics of the proteases and the lipases produced by the seven Bacilli showed that the proteases differ between isolates in term of pH optima and heat-stabilities, so do the lipases. The lipases are more heat-stable than the proteases in a buffer system. Under the protection of milk proteins, these enzymes can survive the heat-treatments applied in the milk powder manufacture process, and retain activities in the powder. 

Further studies on WMPs spiked with thermophilic proteases and lipases, or taken directly from the commercial process have confirmed that the proteases and the lipases are active in the powder (at moisture < 5%) during storage. Proteolysis was observed as the increase of 1% trifluoroacetic acid (TFA) soluble protein fragments and the decrease of solubility, and lipolysis was shown as the release of free fatty acids (FFA). The lipases showed the same specificities in a buffer system and in the powder. The levels of enzymatic products increase with increasing storage temperatures and time. Proteolysis resulted in poor solubility of WMP within one week of storage at 37°C. Lipolysis resulted in the levels of FFA, especially short chain butyric and caproic acids, exceeding the organoleptic threshold values under the same storage conditions. 

Nevertheless, none of the defects observed showed any correlation with thermophile numbers - one of the current specifications for quality control of WMP. Therefore, it is not thermophiles per se that affect milk powder quality during storage, it is the heat-stable enzymes, from whatever source.

text: Thermophilic enzymes and their impact on milk powder during storage Milk powder quality and storage life can be compromised by functionality and flavour defects. These defects can be the result of chemical or biochemical reactions, such as the Maillard (browning) reaction, proteolysis and lipolysis. The New Zealand dairy industry has set specifications for thermophile numbers in milk powders that have been used for many years as an indicator of microbial quality. However, any correlation between the numbers of these bacteria and product quality is anecdotal, and there is little supportive scientific evidence. 

This research has set out to: (1) survey the cause of the problem for off-flavours in milk powder storage, mainly whole milk powders (WMPs) because they are the largest powder-product, and most likely to have problems associated with flavour; (2) develop assay methods for detecting possibly very low levels of protease and lipase in the WMPs; and finally (3) determine enzymatic effects in the WMPs during storage. 

The study has focused on the detection of protease and lipase activities. Several sensitive and commonly used assay methods were applied in reconstituted milk made from different batches of commercial WMPs. Both protease and lipase activities were detected, and the lipase activity was quantified at 0.7-1.1 U/g powder (pNP caproate unit), but the level of protease activity is yet to be accurately determined. 

A lipase was semi-purified from a WMP using Phenyl Sepharose hydrophobic chromatography. The enzyme is most active at 60°C under the conditions used, and has similar substrate specificity to the lipases produced by seven Bacilli isolated from a milk powder production stream. Although the source of the powder lipase is yet to be identified, data suggest that it is most likely from a thermophilic bacterium. 

Preliminary studies on the characteristics of the proteases and the lipases produced by the seven Bacilli showed that the proteases differ between isolates in term of pH optima and heat-stabilities, so do the lipases. The lipases are more heat-stable than the proteases in a buffer system. Under the protection of milk proteins, these enzymes can survive the heat-treatments applied in the milk powder manufacture process, and retain activities in the powder. 

Further studies on WMPs spiked with thermophilic proteases and lipases, or taken directly from the commercial process have confirmed that the proteases and the lipases are active in the powder (at moisture < 5%) during storage. Proteolysis was observed as the increase of 1% trifluoroacetic acid (TFA) soluble protein fragments and the decrease of solubility, and lipolysis was shown as the release of free fatty acids (FFA). The lipases showed the same specificities in a buffer system and in the powder. The levels of enzymatic products increase with increasing storage temperatures and time. Proteolysis resulted in poor solubility of WMP within one week of storage at 37°C. Lipolysis resulted in the levels of FFA, especially short chain butyric and caproic acids, exceeding the organoleptic threshold values under the same storage conditions. 

Nevertheless, none of the defects observed showed any correlation with thermophile numbers - one of the current specifications for quality control of WMP. Therefore, it is not thermophiles per se that affect milk powder quality during storage, it is the heat-stable enzymes, from whatever source.

year: 2000

title: The effects of intensive grazing on nitrogen fixation by white clover

authors: Menneer, John Christopher

abstract: The effects of the grazing animal (treading, defoliation, and excreta) on N2 fixation were investigated in an intensively managed legume-based dairy pasture in the Waikato region, North Island, New Zealand. Two major 12-month field experiments were conducted in a long-term (>30 years) permanent mixed pasture of perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) to determine the full effect of grazing animals on legume growth and N2 fixation. Each study was commenced in spring ( 1998 and 1999), when pasture was taken out of grazing and the relevant treatments applied in a randomised block design. Subsequently, pasture was defoliated by mowing at regular intervals and the herbage dry matter yield and pasture species composition determined. N2 fixation was estimated using the 15N isotope dilution technique to give an integrated estimate of the proportion of clover N derived from atmospheric N2 (%Ndfa) at each harvest. 
The first experiment (initiated in October 1998) investigated the effects of urine additions on N2 fixation in pasture managed with light and moderately severe cutting treatments. Urine application (equivalent to 746 kg N ha-1) increased average annual production by 85%, but did not significantly impact on clover production, except in the light cutting treatment at days 50-100. Urine application reduced %Ndfa to 25% of the control (as measured by 15N methods), with recovery taking almost a year, thus reducing total fixed N in clover herbage from 232 to 145 kg N ha-1 yr-1. Four methods of estimating %Ndfa from 15N data were used in the urine treated plots. The use of 15N-labelled urine gave estimates of  %Ndfa that were 20-30% below values calculated using conventional 15N-labelling during the first 161 days. These differences were probably due to differences in the rooting depth between ryegrass and white clover in conjunction with treatment differences in 15N distribution with depth. The 15N natural abundance technique gave highly variable estimates of %Ndfa (-56 to 24%) during the first 23 days after urine application, but thereafter estimates of %Ndfa were similar to those using 15N-labelling methods. Defoliation severity had no immediate effect on N2 fixation, but during summer and autumn %Ndfa inmoderately-severe plots was consistently higher than under light defoliation. The greater abundance and productivity of vigorous growing weed and summer-grass species recorded in moderately-severe plots during summer and autumn indicate that these species were probably regulating N2 fixation by changing soil N availability. Overall, total N fixed increased by 36% under moderately-severe defoliation compared to light defoliation. Defoliation severity had no significant effect on clover production. 
The second major experiment (initiated in September 1999), examined the effects of pugging intensity on pasture growth and N2 fixation. A single pugging event at stocking rates of 4.5 cows 100 m-2 for 1.5 or 2.5 hours on a wet silt loam soil initiated the experiment, and was equivalent to a moderate or severe pugging event in spring. Fixed N in clover herbage decreased significantly from 76 kg N ha-1 yr-1 in the non-pugged control, to 66 kg N ha-1 yr-1 and 36 kg N ha-1 yr-1 under moderate and severe pugging, respectively. The decrease in fixed N was mainly due to treading damage to clover plants and an associated reduction in clover production which persisted for up to 259 days under severe pugging. Detailed clover morphology measurements revealed an increase in stolon fragmentation and a decrease in plant density, which heightened the vulnerability of plants to dry soil conditions following pugging. A medium-term reduction in grass growth under pugging probably led to increased soil inorganic N and would account for an observed decline in %Ndfa that occurred during the first 3-months of the study .Although pugging caused a major disturbance and rearrangement of the soil surface layers this only had a marginal short-term effect on soil physical properties. Soil aeration was reduced for approximately 21 days and did not measurably affect N2 fixation. However, an increase in denitrification during the first 21 days after pugging suggested biological process were affected by the reduction in soil aeration. 
A conceptual model is proposed to describe the principal processes that affect N2 fixation by grazing animals in intensively managed clover-grass pastures. The use of a whole farm dairying model of N cycling to further investigate animal impacts in legume-based pastures highlighted the need to consider pasture management strategies and interactions between treading, defoliation and excreta. In the future, more complex models specifically dealing with legume-based pasture ecosystems will be needed to integrate detailed animal-plant-soil components and thus assist in identifying how N2 fixing efficiency can be improved.

text: The effects of intensive grazing on nitrogen fixation by white clover The effects of the grazing animal (treading, defoliation, and excreta) on N2 fixation were investigated in an intensively managed legume-based dairy pasture in the Waikato region, North Island, New Zealand. Two major 12-month field experiments were conducted in a long-term (>30 years) permanent mixed pasture of perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) to determine the full effect of grazing animals on legume growth and N2 fixation. Each study was commenced in spring ( 1998 and 1999), when pasture was taken out of grazing and the relevant treatments applied in a randomised block design. Subsequently, pasture was defoliated by mowing at regular intervals and the herbage dry matter yield and pasture species composition determined. N2 fixation was estimated using the 15N isotope dilution technique to give an integrated estimate of the proportion of clover N derived from atmospheric N2 (%Ndfa) at each harvest. 
The first experiment (initiated in October 1998) investigated the effects of urine additions on N2 fixation in pasture managed with light and moderately severe cutting treatments. Urine application (equivalent to 746 kg N ha-1) increased average annual production by 85%, but did not significantly impact on clover production, except in the light cutting treatment at days 50-100. Urine application reduced %Ndfa to 25% of the control (as measured by 15N methods), with recovery taking almost a year, thus reducing total fixed N in clover herbage from 232 to 145 kg N ha-1 yr-1. Four methods of estimating %Ndfa from 15N data were used in the urine treated plots. The use of 15N-labelled urine gave estimates of  %Ndfa that were 20-30% below values calculated using conventional 15N-labelling during the first 161 days. These differences were probably due to differences in the rooting depth between ryegrass and white clover in conjunction with treatment differences in 15N distribution with depth. The 15N natural abundance technique gave highly variable estimates of %Ndfa (-56 to 24%) during the first 23 days after urine application, but thereafter estimates of %Ndfa were similar to those using 15N-labelling methods. Defoliation severity had no immediate effect on N2 fixation, but during summer and autumn %Ndfa inmoderately-severe plots was consistently higher than under light defoliation. The greater abundance and productivity of vigorous growing weed and summer-grass species recorded in moderately-severe plots during summer and autumn indicate that these species were probably regulating N2 fixation by changing soil N availability. Overall, total N fixed increased by 36% under moderately-severe defoliation compared to light defoliation. Defoliation severity had no significant effect on clover production. 
The second major experiment (initiated in September 1999), examined the effects of pugging intensity on pasture growth and N2 fixation. A single pugging event at stocking rates of 4.5 cows 100 m-2 for 1.5 or 2.5 hours on a wet silt loam soil initiated the experiment, and was equivalent to a moderate or severe pugging event in spring. Fixed N in clover herbage decreased significantly from 76 kg N ha-1 yr-1 in the non-pugged control, to 66 kg N ha-1 yr-1 and 36 kg N ha-1 yr-1 under moderate and severe pugging, respectively. The decrease in fixed N was mainly due to treading damage to clover plants and an associated reduction in clover production which persisted for up to 259 days under severe pugging. Detailed clover morphology measurements revealed an increase in stolon fragmentation and a decrease in plant density, which heightened the vulnerability of plants to dry soil conditions following pugging. A medium-term reduction in grass growth under pugging probably led to increased soil inorganic N and would account for an observed decline in %Ndfa that occurred during the first 3-months of the study .Although pugging caused a major disturbance and rearrangement of the soil surface layers this only had a marginal short-term effect on soil physical properties. Soil aeration was reduced for approximately 21 days and did not measurably affect N2 fixation. However, an increase in denitrification during the first 21 days after pugging suggested biological process were affected by the reduction in soil aeration. 
A conceptual model is proposed to describe the principal processes that affect N2 fixation by grazing animals in intensively managed clover-grass pastures. The use of a whole farm dairying model of N cycling to further investigate animal impacts in legume-based pastures highlighted the need to consider pasture management strategies and interactions between treading, defoliation and excreta. In the future, more complex models specifically dealing with legume-based pasture ecosystems will be needed to integrate detailed animal-plant-soil components and thus assist in identifying how N2 fixing efficiency can be improved.

year: 2003

title: Rethinking English in Māori-medium education

authors: Hill, Richard Kenneth

subjects: Māori-medium

abstract: English language instruction in New Zealand's Māori-medium schools is controversial, with many schools either excluding it from their curriculum or adopting a tokenistic approach. Yet, how Māori-medium educators can best support their students' academic English language growth is still an under-researched and unresolved question. This paper reports on a project involving the English language transition programmes of three Māori-medium primary schools. Interviews and observations of key personnel within these schools revealed the components of their programmes, their perceptions of the need for English transition education and the issues they were facing in implementing English language instruction. The project found a direct relationship between levels of student language attainment and the quantity and quality of exposure to English instruction. Where an English programme occupied a significant place in a school's timetable and was staffed by teachers knowledgeable about the learning needs of bilingual students and how best to attend to these, the result was higher literacy scores and more satisfied students. This paper argues that planning English language outcomes for Māori-medium students is essential if becoming biliterate is an important aim. This planning must also be long-term, across all 13 years of the students' education.

text: Rethinking English in Māori-medium education English language instruction in New Zealand's Māori-medium schools is controversial, with many schools either excluding it from their curriculum or adopting a tokenistic approach. Yet, how Māori-medium educators can best support their students' academic English language growth is still an under-researched and unresolved question. This paper reports on a project involving the English language transition programmes of three Māori-medium primary schools. Interviews and observations of key personnel within these schools revealed the components of their programmes, their perceptions of the need for English transition education and the issues they were facing in implementing English language instruction. The project found a direct relationship between levels of student language attainment and the quantity and quality of exposure to English instruction. Where an English programme occupied a significant place in a school's timetable and was staffed by teachers knowledgeable about the learning needs of bilingual students and how best to attend to these, the result was higher literacy scores and more satisfied students. This paper argues that planning English language outcomes for Māori-medium students is essential if becoming biliterate is an important aim. This planning must also be long-term, across all 13 years of the students' education.

year: 2011