Tunable aryl imidazolium ionic liquids (TAIILs) as environmentally benign catalysts for the esterification of fatty acids to biodiesel fuel

Article abstract of DOI:10.1016/j.catcom.2020.106243

Herein, we describe the synthesis of tunable aryl imidazolium ionic liquid catalysts and tested for esterification of fatty acids to biodiesel. In this work, six tunable aryl imidazolium ionic liquids (TAIILs) 1a-1f were prepared. These ionic liquids were used as the economical and reusable catalysts for the synthesis of biodiesel fuels. The reaction has been preceded in a monophase at 80 °C for 4 h, after which the product was separated from the catalyst system by a simple liquid/liquid phase separation at room temperature with excellent yields. With the simple post-process, the catalyst is reusable at least 6 times. This novel method offers a short reaction time, good yields, and environmentally benign characteristics.

Full text of DOI:10.1016/j.catcom.2020.106243

J. Dairy Sci. 100:991–1003
https://doi.org/10.3168/jds.2016-11314
®
©
American Dairy Science Association , 2017.
The effect of storage conditions on the composition
and functional properties of blended bulk tank milk
1
A. O’Connell,*† A. L. Kelly,‡ J. Tobin,§ P. L. Ruegg,† and D. Gleeson*
*
Teagasc, Livestock Systems Research Department, Animal and Grassland Research and Innovation Centre, Moorepark, Fermoy,
Co. Cork, Ireland
†
‡
§
Department of Dairy Science, University of Wisconsin-Madison, Madison 53706
Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland
Food Chemistry and Technology Department, Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
ABSTRACT
most likely due to the dissociation of β-casein from the
casein micelle, which can be reversed upon pasteuriza-
tion. Thus, this study suggests that blended milk can
be stored for up to 96 h at temperatures between 2°C
and 6°C with little effect on its composition or func-
tional properties.
Key words: raw milk, milk storage, storage
temperature, proteolysis
The objective of this study was to investigate the
effects of storage temperature and duration on the
composition and functional properties of bulk tank
milk when fresh milk was added to the bulk tank twice
daily. The bulk tank milk temperature was set at each
of 3 temperatures (2, 4, and 6°C) in each of 3 tanks
on 2 occasions during two 6-wk periods. Period 1 was
undertaken in August and September when all cows
were in mid lactation, and period 2 was undertaken
in October and November when all cows were in late
lactation. Bulk tank milk stored at the 3 temperatures
was sampled at 24-h intervals during storage periods of
INTRODUCTION
Due to ongoing expansion of the dairy industry in
Ireland following the abolition of the milk quota system
in the European Union in 2015, it is anticipated that
on-farm storage of raw milk may be extended from
0
to 96 h. Compositional parameters were measured for
all bulk tank milk samples, including gross composition
and quantification of nitrogen compounds, casein frac-
tions, free amino acids, and Ca and P contents. The
somatic cell count, heat stability, titratable acidity, and
rennetability of bulk tank milk samples were also as-
sessed. Almost all parameters differed between mid and
late lactation; however, the interaction between lacta-
tion, storage temperature, and storage duration was
significant for only 3 parameters: protein content and
concentrations of free cysteic acid and free glutamic
acid. The interaction between storage temperature and
storage time was not significant for any parameter mea-
sured, and temperature had no effect on any parameter
except lysine: lysine content was higher at 6°C than at
4
8 h to up to 96 h to improve logistical efficiency for
milk processors. Current European Union legislation
dictates that milk produced and stored on-farm must
be cooled to at least 8°C when a daily milk collection
regimen is in place, and to at least 6°C when collection
is less frequent (Annex A, Directive 92/46; European
Commission, 1992). However, many milk processors in
Ireland request that milk be cooled to 2 to 4°C within
2
to 3 h after milking. There may be an economic
incentive for farmers to store milk at higher tempera-
tures (e.g., at 6°C compared with 2°C) when milk is
stored for longer durations on-farm, as milk cooling is
a significant operational expenditure at the farm level
(
Upton et al., 2013). However, cooling milk to higher
2
°C. During 96 h of storage, the concentrations of some
temperatures, in conjunction with longer on-farm stor-
age intervals, may have implications for milk quality,
with possible deleterious effects on milk functionality
at milk processing facilities.
free amino acids (glutamic acid, lysine, and arginine)
increased, which may indicate proteolytic activity dur-
ing storage. Between 0 and 96 h, minimal deterioration
was observed in functional properties (rennet coagula-
tion time, curd firmness, and heat stability), which was
Milk processing as a generic term covers the typi-
cal unit operations and processes applied to milk in
an integrated milk processing facility. Such processes
can include thermal processing—HTST pasteurization
and UHT or high temperature treatments. Separation
technologies can include centrifugal separation, micro-
filtration, ultrafiltration, nanofiltration, and reverse
Received April 13, 2016.
Accepted October 21, 2016.
1
Corresponding author: david.gleeson@teagasc.ie
991

992
O’CONNELL ET AL.
osmosis, whereas concentration technologies include often because of poorer environmental conditions and
evaporation and spray drying.
increased prevalence of subclinical mastitis within the
Ireland’s established reputation for efficient produc- herd (O’Connell et al., 2015). Alterations in diet, stage
tion of high-quality products has allowed the Irish dairy of lactation, and SCC can alter the composition of milk
industry to become a major exporter of premium dairy produced by cows. With a reduction in milk yield, the
products. The manufacture of such products requires fat and protein contents of milk increase (Quinn et al.,
the supply of the highest quality raw milk, highlight- 2006); however, the β-CN and α -CN contents of milk
S
ing the importance of on-farm milk storage conditions decrease and the γ-CN content increases because of
as a critical point in the dairy supply chain. On-farm higher plasmin activity during late lactation (Lucey,
storage of raw milk provides conditions suitable for the 1996; O’Brien et al., 2001). Late-lactation milk used for
growth of microorganisms, with refrigerated conditions cheese production is associated with longer coagulation
preferentially selecting for psychrotrophic bacteria, times and weaker gel structures, reducing its suitability
which can deleteriously affect the quality of raw milk for use (Lucey, 1996). Due to the changes in milk com-
through production of heat-stable proteinases and li- position in late lactation, the milk produced during this
pases (Sorhaug and Stepaniak, 1997; Haryani et al., period may be particularly susceptible to compositional
2
003; Hantsis-Zacharov and Halpern, 2007).
Proteolysis can reduce the economic value of milk by ditions of raw milk on farms due to less-frequent milk
negatively affecting its performance within milk pro- collections from the farm.
and functional changes related to extended storage con-
cessing facilities. In particular, the hydrolysis of casein
Milk with high SCC is often associated with elevated
can reduce cheese yield (Barbano et al., 1991; Klei et indigenous enzyme activity, which contributes to in-
al., 1998). Lipolysis is the enzymatic conversion of lip- creased proteolysis and lipolysis during storage (Bas-
ids into free fatty acids (FFA) and partial glycerides. tian and Brown, 1996; Deeth, 2006). The 2 most signifi-
An increase in FFA in milk can result in undesirable cant indigenous enzymes for milk spoilage are plasmin
off-flavors and altered functionality (Ma et al., 2000; and lipoprotein lipase. Plasmin cleaves polypeptide
Deeth, 2006), such as increased churning time during chains after a lysine or, to a lesser extent, an arginine
the production of butter (Deeth and Fitzgerald, 1995). residue (Ueshima et al., 1996); α -, α -, and β-CN
S1
S2
Due to enzymatic and microbial activity, both the are all susceptible to hydrolysis by plasmin (Andrews,
microbial and functional quality of raw milk deterio- 1983; Le Bars and Gripon, 1989; McSweeney et al.,
rates with time. After storing raw milk obtained from 2 1993a). Schroeder et al. (2008) showed that milk stored
milkings at 4°C for 6 d, Guinot-Thomas et al. (1995a) at lower temperatures (2.2°C compared with 4.4°C)
reported declines in pH, casein nitrogen, β-CN, and for 24 h resulted in less plasmin-induced proteolysis.
colloidal Ca and P contents, whereas levels of NPN However, Leitner et al. (2008) reported a 4% loss in
and γ-CN increased. A reduction in the β-CN level of curd yield from milk sourced from an uninfected cow
milk may also cause increased rennet coagulation time (SCC of 25,000 cells/mL) after 48 h of storage at 4°C.
(
RCT); de Moura Maciel et al. (2015) reported an This loss in yield was likely linked to continued casein
increase in RCT after 24 h of storage of milk at 4°C. proteolysis during cold storage, resulting in impairment
The concentration of FFA in milk has been shown to of curd formation (Crudden et al., 2005a). Although
increase during storage (Wiking et al., 2002). Muir et plasmin activity in raw milk stored at low temperature
al. (1978) observed a temperature-dependent increase (5°C) is reduced due to autolysis and low temperature
in concentrations of FFA in milk after 96 h of storage, inhibition (Crudden et al., 2005b), the high thermal
with higher concentrations linked to milk stored at 8°C stability of the enzyme allows it to survive conventional
compared with 6°C or 4°C.
pasteurization, which can limit the shelf life of resulting
Seasonal milk production is the dominant milk dairy products (Alichanidis et al., 1986). Lipoprotein
production system in Ireland (O’Connell et al., 2015), lipase catalyzes the hydrolysis of ester bonds of tria-
resulting in a large proportion of the national dairy cylglycerols, resulting in the release of FFA, with the
cow population (1.127 million cows) approaching late subsequent accumulation of short-chain FFA in milk
lactation within the same period (during October and the development of off-flavors (Ma et al., 2000;
and November). Due to adverse weather conditions Dickow et al., 2011). Lipoprotein lipase is relatively
and minimal grass growth during this period, cows heat-sensitive and can be completely inactivated by
are typically housed indoors. Consequently, cow’s di- pasteurization for 10 s at 85°C (Driessen, 1989).
ets are altered from a pasture-based grazed grass to
On Irish farms, milk is stored in bulk milk tanks
a grass silage system. During late lactation the total between milk collections, typically for 48 h, and fresh
bacterial count (TBC) and SCC of milk also increase, milk is added to the bulk tank at each milking. Given
Journal of Dairy Science Vol. 100 No. 2, 2017

EFFECT OF STORAGE CONDITIONS ON BULK TANK MILK QUALITY
993
the addition of fresh milk from each milking to the bulk period. Valves in the milk-line were used to divide the
tank between milk collections, only the milk harvested milk flow in equal proportions to each of the 3 tanks.
at the first milking is stored for the maximum duration. Equal volumes of fresh milk (approximately 300 L)
Thereafter, there is a stepwise reduction in the storage were collected in each tank at each milking over 96 h.
time of milk collected at each subsequent milking oc- The milk passed through a plate cooler and was cooled
casion. Thus, deterioration in milk quality is limited to approximately 14.5°C before entering each tank,
and likely to be lesser in magnitude compared with and the milk was subsequently cooled to the desired
that considered in other studies in which the addition temperature within the tank. After each 96-h storage
of fresh milk during the storage period was not studied period, the milk was removed from each tank. Dupli-
(
Guinot-Thomas et al., 1995a,b). Most of the current cate bulk tank milk samples for each test were taken
literature has documented laboratory-based experi- from each tank immediately after the initial morning
ments that aim to simulate on-farm storage conditions. milking once milk was cooled to the desired tempera-
However, no study has accounted for the sequential ad- ture and subsequently at 24, 48, 72, and 96 h, before
dition of fresh milk to stored milk within the on-farm morning milking, when there was milk from 2, 4, 6,
storage period. With the possibility of large increases in and 8 milkings, respectively, in each tank for analysis
on-farm milk storage periods (up to 96 h), particularly of composition and functional properties. To assess the
after peak milk production (mid lactation), there is a quality of milk entering the bulk tanks at each milking,
need to establish the real-time effects of extended stor- a milk sample was taken from the milk-line before the
age periods of raw milk on-farm, while also account- milk was diverted into each of the 3 tanks. A sample
ing for the effect of continued addition of fresh milk tap fitted to the milk-line, which recovered a constant
throughout the storage period.
stream of milk throughout milking, was used to collect
Thus, the objective of this study was to investigate this sample. The milk was collected in a sterile Dur-
the effects of bulk tank storage temperature and storage ham flask that was surrounded by ice. These milk-line
time on the composition and functionality of bulk tank samples were analyzed for SCC and gross composition.
milk during mid and late lactation, when fresh milk
was added twice daily throughout the storage period.
SCC and Gross Composition
Duplicate bulk tank milk samples and duplicate milk
line samples were analyzed daily for fat, protein and
MATERIALS AND METHODS
Expanded details outlining the complete experimen- lactose composition and SCC using a Milkoscan 203
tal design and milk sampling procedures can be found (Foss Electric, Hillerød, Denmark).
in the companion paper (O’Connell et al., 2016). Brief-
Duplicate bulk storage tank milk samples were sub-
ly, spring-calving dairy cows (n = 280) were milked mitted for wet chemistry analysis of nitrogen fractions
over two 6-wk periods: August 11 to September 26, after storage for 0, 48 and 96 h. The percentage to-
2
014 (period 1; mean DIM of 172; mid lactation) and tal protein, NPN, and noncasein nitrogen content of
October 13 to November 21, 2014 (period 2; mean DIM the milk samples were determined using the Kjeldahl
of 235; late lactation). During period 1 and the first 4 method [methods 20-3 (IDF, 2004b), 20-4 (IDF, 2001),
wk of period 2, the cows were outdoors and consumed and 29-1 (IDF, 2004a), respectively] using a Tecator
a diet of grass. During the remaining 2 wk of period 2, Digestor Auto and Kjeltec 8400 distiller (Foss Electric).
the cows were partially housed indoors during times of
The protein composition of duplicate bulk storage
heavy rainfall in cubicles fitted with rubber mats that tank milk samples was quantified daily (0, 24, 48, 72,
were bedded with lime, and consumed a diet consist- and 96 h) by HPLC using the method described by
ing of approximately 50% grazed grass and 50% grass Mounsey and O’Kennedy (2009). Briefly, 200 μL of
silage.
milk was diluted in 3,800 μL of dissociating buffer (7
Three identical bulk milk tanks each with a capacity M urea and 20 mM Bis-Tris propane, pH 7.5), to which
of 4,000 L (Swiftcool, Dairymaster, Causeway, Ireland) 5 μL of 2-mercaptoethanol was added, before filtering
were used in this study. Each tank was set at 1 of 3 through a 0.22-μm filter. Separation of the milk protein
temperatures (2, 4, or 6°C) on 2 occasions during mid fractions was achieved in reverse-phase mode, using an
lactation and on 2 occasions in late lactation, result- Agilent Poroshell 300SB C18 column (2.1 × 75 mm;
ing in 6 test periods within each lactation period [3 Agilent Technologies, Santa Clara, CA). The HPLC
treatments, 2 replicates of each temperature (n = 3) equipment consisted of an Agilent 1200s with quater-
at each tank] during which milk was stored for up to nary pump and multi-wavelength detector. Gradient
9
6 h. The 3 bulk tanks were set to cool milk to the elution and peak detection were performed according
different temperatures at the beginning of each test to the method of Mounsey and O’Kennedy (2009).
Journal of Dairy Science Vol. 100 No. 2, 2017

994
O’CONNELL ET AL.
Free Amino Acid Analysis
11700, Foss Electric). Ten milliliters of milk was heated
to 35°C and chymosin (Chr. Hansen, Cork, Ireland;
diluted 1:20 with deionized water) was added accord-
ing to the following calculation: (% protein in milk ×
Bulk tank milk samples were analyzed daily in dupli-
cate for free amino acid content. Briefly, samples were
deproteinized by mixing equal volumes of 24% (wt/
vol) trichloroacetic acid and milk sample, followed by
equilibration for 10 min before centrifugation at 14,400
3
6)/3.5. The samples were then incubated at 35°C in
the Formagraph and the coagulation properties moni-
tored over a 30-min period.
×
g for 10 min. Supernatants were removed and diluted
The following parameters were obtained from the
bifurcated displacement/time output signal: RCT (i.e.,
the time in seconds for the onset of gelation), curd firm-
ing rate (k ; time in seconds required for the bifurcated
with 0.2 M sodium citrate buffer (pH 2.2), and then
diluted 1 in 2 with internal standard norleucine. Amino
acids were quantified using a Jeol JLC-500/V amino
acid analyzer (Jeol UK Ltd., Garden City, UK) fitted
2
0
signal to reach a width of 20 mm), and curd firmness
a ; width in millimeters of the signal at 30 min).
+
with a Jeol Na high-performance cation-exchange col-
(
3
0
umn.
Statistical Analysis
Mineral Analysis
The study was conducted using a multiple Latin
square design with repeated measures (sampling bulk
tank milk every 24 h) whereby each temperature (2, 4,
and 6°C) and each bulk tank (n = 3) were included each
week (n = 6) for each lactation period (n = 2). Each
Latin square was repeated 4 times throughout both lac-
tation periods. Least squares means for the main effects
of storage time, temperature, and their interaction were
calculated using the MIXED procedure in SAS version
Bulk tank milk samples collected from each tank at
, 24, and 48 h were frozen at −20°C before analysis
of Ca and P contents, which was completed within 6
wk of collection. Five grams of each milk sample was
0
diluted with 5 g of HNO (69%, Trace Select for Trace
3
Analysis, Fluka Analytical, Sigma-Aldrich Ireland Ltd.,
Arklow, Co. Wicklow, Ireland) and 0.5 g of HCl (37%,
Fluka Analytical). Samples were digested at 1,600 W
using a microwave digester (Marsxpress CEM Micro-
wave Technology Ltd., Dublin, Ireland), and digested
samples were diluted to 100 mL with Milli-Q water.
One milliliter of the 100-mL digest was diluted to 10
9
.3 (SAS Institute, 2011). Storage time was defined as
the total number of hours each tank had been cool-
ing since the first addition of milk. Tank within week
was the experimental unit. Response variables included
milk gross composition, N, casein, and AA concentra-
tions, renneting and heat stability properties, Ca and P
concentrations, pH, and titratable acidity.
mL with HNO (5%, Fluka Analytical) and samples
3
were assessed for Ca and P contents by inductively
coupled plasma mass spectroscopy using an Agilent
ASX 500 series auto-sampler (Agilent Technologies).
The software used was Mass Hunter software (version
A. 01. 02 patch 4; Agilent Technologies).
The following general mixed model was offered to all
parameters:
Y = μ + tank + time + temp + week + lact + time
Assessment of Functional Properties
×
temp + lact × week + time × temp × lact + e,
The thermal stability of bulk storage tank milk sam-
ples was measured daily in duplicate. The heat coagula- where Y = response variable; tank = fixed effect of
tion time (HCT) was determined following the method each bulk tank (1 to 3); time = repeated effect of stor-
described by Davies and White (1966). Milk (2.5 mL) age time (0, 24, 48, 72, 96 h); temp = fixed effect of
was added to glass test tubes, which were placed in a storage temperature (2, 4, and 6°C); week = fixed effect
rocker and immersed in an oil bath containing mineral of sampling period (1 to 6); lact = fixed effect of lacta-
oil at a temperature of 140°C (the pH of milk samples tion period (mid or late); time × temp = interaction
was not adjusted). The HCT was recorded as the time between storage temperature and time; lact × week =
between immersing the sample in the oil bath and the interaction between lactation period and week; time ×
appearance of protein aggregates within the test tubes. temp × lact = interaction between storage time, stor-
Titratable acidity was measured daily on all bulk age temperature, and lactation period; and e = residual
tank milk samples by titrating milk samples to an end- component.
point of pH 8.3 (Metrohm automatic titrator) using 0.1
M NaOH (VWR, Dublin, Ireland).
When the interaction between lactation period, stor-
age time, and storage temperature was not significant
Rennet coagulation time (RCT) was measured in for a particular parameter, the fixed effect of lactation
duplicate daily using a Formagraph instrument (Type period, the interaction between lactation period and
Journal of Dairy Science Vol. 100 No. 2, 2017

EFFECT OF STORAGE CONDITIONS ON BULK TANK MILK QUALITY
Table 1. Composition and SCC of milk-line samples in mid and late lactation
995
Percentile
50th
Composition/
lactation period
No. of
samples
Mean ± SD
Maximum
Minimum
10th
25th
75th
90th
Protein (%)
Mid
Late
b
42
46
3.89 ± 0.17a
4.29
4.53
3.58
4.07
3.65
4.13
3.76
4.20
3.89
4.31
3.98
4.39
4.11
4.44
4.30 ± 0.11
Fat (%)
Mid
Late
b
42
46
4.97 ± 0.86a
6.54
6.86
3.32
4.94
3.84
5.19
4.17
5.60
5.08
5.84
5.71
6.14
5.90
6.47
5.86 ± 0.46
Lactose (%)
Mid
a
42
46
4.57 ± 0.11
4.77
4.59
4.38
4.17
4.43
4.39
4.47
4.48
4.58
4.52
4.66
4.54
4.69
4.58
b
Late
4.49 ± 0.08
3
SCC (×10 cells/mL)
b
Mid
Late
40
46
260 ± 110a
573
782
121
136
141
176
184
214
240
288
315
366
420
604
323 ± 154
a,b
Means between lactation periods within a composition parameter with different superscripts differ significantly (P < 0.05).
week, and the interaction between storage temperature Guinee et al. (2007), who reported that the decrease in
and storage time were removed from the model. The milk yield with advancing stage of lactation coincided
fixed effect of sampling period (week) was then coded 1 with increases in total protein and casein levels, and
to 12 rather than 1 to 6.
a reduction in that of lactose. Within each week, the
The unstructured covariance structure was used in differences in percentage protein of milk added to the
all models. Residual checks were made to ensure that bulk tanks (i.e., the difference between the milk-line
the assumptions of the analysis model were met. Treat- sample with the highest percentage protein and that
ment means were compared using the Tukey test at a with the lowest percentage protein within each week)
5
% error probability.
ranged from 0.12 to 0.31% in mid lactation and from
.20 to 0.40% in late lactation. Within each week, the
0
differences in percentage fat of milk added to the bulk
tanks ranged from 2.01 to 2.53% in mid lactation and
RESULTS AND DISCUSSION
In this study, we investigated the effect of storage from 0.95 to 1.86% in late lactation (data not shown).
conditions on the composition and functional proper-
The median SCC of milk entering the bulk tanks was
ties of blended bulk tank milk. Although we did not 240,000 and 288,000 cells/mL in mid and late lactation,
investigate the effects of storage of final product per- respectively. In each lactation period, more than 25%
formance, the parameters we measured are commonly of samples had an SCC >300,000 cells/mL. It is well
used within the dairy industry to predict the suitability documented that bulk milk SCC increases toward the
of milk for processing. Assessing key performance indi- end of lactation (Berry et al., 2006; Hagnestam-Nielsen
cators, such as thermal stability, titratable acidity, and et al., 2009; O’Connell et al., 2015), which is related to
rennetability, among others, can identify milk that is increased prevalence of cows with chronic subclinical
suitable for processing and that will not affect prod- mastitis. Herds with higher bulk tank SCC (>200,000
uct quality or process performance. The level of FFA cells/mL) tend to have greater prevalence of IMI (Fen-
in stored milk was not measured in this study due to lon et al., 1995; Barkema et al., 1998; Rodrigues et al.,
daily time constraints, and the microbiology of the milk 2005), and thus the SCC of milk stored in this experi-
stored in this experiment is described in a companion ment indicates the presence of IMI within the herd.
paper (O’Connell et al., 2016).
The fat and protein contents of the milk-line samples
milk sampled at each milking before entry into each
tank) were higher in late lactation than mid lacta-
Lactation Period
(
Lactation period significantly affected the protein
tion (Table 1). In contrast, the lactose level of milk content of bulk tank milk samples, with significantly
entering the tanks was greater in mid lactation than higher contents observed in late lactation than in mid
in late lactation. Daily milk yields decreased as cows lactation (Table 2). The mean protein level in bulk tank
entered late lactation and, because of reduced dilution milk increased from 3.65% in wk 1 (average DIM =
by milk volume, the concentrations of protein and fat 172) of the mid-lactation period to 4.29% in wk 4 (av-
in milk were higher in late lactation compared with erage DIM = 263) of the late-lactation period, before
mid lactation. Similar trends have been described by decreasing to 4.17% in the final week of the experiment.
Journal of Dairy Science Vol. 100 No. 2, 2017

996
O’CONNELL ET AL.
The decrease in protein from wk 4 of the late-lactation the level of β-CN declined in late lactation, whereas
period is likely due to the inclusion of silage in the Ostersen et al. (1997) reported that β-CN increased
diet at this time. Similar trends have been reported throughout lactation. In the latter study, cows had a
for seasonal variation in the protein content of an Irish low SCC (<200,000 cells/mL) and were dried off before
spring-calving herd (Guinee et al., 2007) and reflect daily milk yield declined to <10 L, which the authors
the effect of stage of lactation of cows on the casein suggested might account for the difference in results.
and protein contents in milk (White and Davies, 1958; In this study, although β-CN levels were higher in late
Kefford et al., 1995; Auldist et al., 1996). Similarly, the lactation, the weekly means for β-CN content in late
fat content of bulk tank milk was significantly higher lactation declined in the last 3 wk of the study, as cows
during late lactation than mid lactation, as reported approached the end of lactation. The β-CN content
by O’Brien et al. (1999). This increase in fat content decreased from 13.72 to 9.05 g/L between wk 1 and
for milk produced during late lactation reflects dietary 6 within the late-lactation test period. Although the
changes such as increased fiber content of grazed grass plasmin activity in bulk tank milk was not measured in
and physiological changes relating to stage of lactation this study, it is postulated that the greater SCC in late-
(
Phelan et al., 1982). Although statistically significant, lactation milk compared with mid-lactation milk could
minimal changes were seen in percentage lactose be- lead to elevated plasmin activity in the milk stored and
tween mid- and late-lactation milk, at levels of 4.57 and thus increased proteolysis (Bastian and Brown, 1996).
4
.52%, respectively.
Casein number, percentage casein, and concentra- tation increased significantly compared with the values
tion of each casein fraction increased in late lactation for mid-lactation milk (Table 2). Conversely, the curd
Table 2). Interestingly, lactation period had a sig- aggregation rate (k ) was higher for mid-lactation milk
The curd firmness (a ) of milk samples in late-lac-
30
(
2
0
nificant effect on whey protein fractions, with the level compared with late-lactation milk, whereas RCT was
of β-LG-b increasing between mid and late lactation, not affected by lactation period (Table 2). It is known
whereas β-LG-a was not affected, a result that requires that protein content is a confounding variable in terms
additional research to elucidate. There have been con- of the curd firmness of rennet-induced milk gels (Guinee
flicting reports on the distribution of β-CN through- et al., 1997). Thus, as protein levels increased in late
out lactation. Barry and Donnelly (1980) found that lactation, a30 is expected to increase. An improvement
Table 2. The effect of lactation period on the composition and functional properties of bulk tank milk samples
Mid
lactation
Late
lactation
Item
SEM
P-value
Fat (%)
4.72
3.90
4.57
0.13
78.59
0.036
3.06
0.96
1.26
836
5.13
4.23
0.049
0.004
0.004
0.001
0.118
0.001
0.011
0.003
0.005
9.0
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.3251
Protein (%)
Lactose (%)
4.52
Noncasein nitrogen (%)
Casein number (%)
NPN (%)
0.15
77.35
0.044
3.33
Casein (%)
Phosphorus (g/L)
Calcium (g/L)
Heat coagulation time (s)
pH
Rennet coagulation time (s)
Curd firming rate (k20; s)
Curd firmness (a30; mm)
Cysteic acid (μg/mL)
Glutamic acid (μg/mL)
Histidine (μg/mL)
Lysine (μg/mL)
Glycine (μg/mL)
Cysteine (μg/mL)
Arginine (μg/mL)
κ-CN (g/L)
01.00
1.38
678
6.77
1,367
367
6.69
1,405
472
0.003
26
16
0.680
0.23
0.0002
19.39
13.71
25.84
4.02
2.47
4.83
7.73
2.97
4.17
3.23
12.14
9.82
0.90
3.11
1.90
22.18
9.30
0.0088
<0.0001
<0.0001
<0.0001
0.0026
12.88
2.60
0.290
0.062
0.047
0.083
0.158
0.065
0.055
0.032
0.159
0.116
0.018
0.088
0.313
2.24
2.98
<0.0001
<0.0001
<0.0001
<0.0001
0.2774
<0.0001
<0.0001
0.1766
5.68
2.50
4.92
α
α
S2-CN (g/L)
S1-CN (g/L)
3.28
13.40
11.45
0.87
β-CN (g/L)
α-LA (g/L)
β-LG-a (g/L)
β-LG-b (g/L)
3.27
2.58
0.1989
<0.0001
Journal of Dairy Science Vol. 100 No. 2, 2017

EFFECT OF STORAGE CONDITIONS ON BULK TANK MILK QUALITY
997
in k was also observed by O’Brien et al. (1999), when contrast to the results reported here, those authors
20
milk from spring-calving cows was compared at 164 (k20 reported increased concentrations of lysine in late-
11.8 min) and 234 DIM (k = 8.2 min). The greater lactation milk compared with mid-lactation milk.
=
2
0
concentration of Ca in late-lactation milk agrees with
the observations of Auldist et al. (1996) and Ostersen
et al. (1997) and may contribute to the increased curd
firmness observed compared with mid-lactation milk
Interaction Between Lactation Period and Storage
Temperature and Duration
(
Guinee et al., 1997).
As demonstrated in this study, stage of lactation af-
The HCT of milk samples was greater in mid lac- fects milk quality at both a gross nutritional level and a
tation than in late lactation (Table 2), in agreement functional level. Given the seasonal nature of Irish milk
with the results of Kelly et al. (1982). The differences production, this study included the interaction between
in heat stability between mid- and late-lactation milk lactation period, storage temperature, and storage du-
reflects the challenges typically encountered in dairy ration at farm level to assess whether stage of lactation
processing facilities relative to thermal processing and would cause different outcomes during storage on the
concentration of late-lactation milk. Milk produced parameters studied. The interaction between lactation
during late lactation, or when cows have mastitis, has period, storage temperature, and storage duration was
a higher plasmin activity (Lucey, 1996; O’Mahony and only significant for protein content (P < 0.001; Fig-
Fox, 2013), promoting hydrolysis of casein, which may ure 1), cysteic acid content (P = 0.03; Figure 2), and
contribute to reduced thermal stability (Crudden et al., glutamic acid content (P = 0.047; Figure 3) in bulk
2
005a). Observed differences in thermal stability be- tank milk. However, the interactions observed mainly
tween mid- and late-lactation milk are most likely due reflect large differences relative to stage of lactation,
to seasonal influences, such as changes in diet, coupled with lesser effects observed within each discreet lacta-
with stage of lactation. Variations in the natural level tion period. For example, the range of values for pro-
of urea in milk because of changes to the cows’ diet tein content was between 3.86 and 3.92% and between
(
from grass to grass silage and supplements) appear to 4.18 and 4.27% in mid and late lactation, respectively.
be a major factor influencing thermal stability (Kelly However, smaller differences were seen in mid lactation
et al., 1982). where the protein content of bulk tank milk stored at
The concentration of free amino acids in bulk tank 2°C and 4°C decreased between 72 and 96 h from 3.91
milk (cysteic acid, glutamic acid, histidine, lysine, to 3.88% (P = 0.0027) and from 3.91 to 3.88% (P =
glycine, cysteine and arginine) decreased (P < 0.01) 0.0373), respectively; however, the industrial relevance
between mid and late lactation (Table 2). Davis et al. of this difference is questionable. Similarly, no sig-
(
1994) reported similar changes in amino acid concen- nificant changes in cysteic acid contents were observed
trations between mid and late lactation; however, in within each lactation period.
Figure 1. The effect of storage temperature (2°C: ■; 4°C: ▲; 6°C: ●) and duration on the protein concentration of bulk tank milk in mid
lactation (solid lines and symbols) and late lactation (dashed lines and open symbols). Each bar represents the 95% CI for that mean.
Journal of Dairy Science Vol. 100 No. 2, 2017

998
O’CONNELL ET AL.
Figure 2. The effect of storage temperature (2°C: ■; 4°C: ▲; 6°C: ●) and duration on the concentration of cysteic acid in bulk tank milk
in mid lactation (solid lines and symbols) and late lactation (dashed lines and open symbols). Each bar represents the 95% CI for that mean.
The amount of glutamic acid in bulk tank milk was Sweeney et al. (1993b) postulated that an increase in
greater in mid lactation than in late lactation (Figure free glutamic acid concentration in raw milk cheese
3
). Between 0 and 24 h, the glutamic acid content of compared with pasteurized milk cheese was linked to
milk increased from 22.04 to 26.18 g/L when mid- higher levels of proteolysis in raw milk cheese, resulting
lactation milk was stored at 6°C (P = 0.011). In late from greater peptidase activity from both indigenous
lactation, the glutamic acid content of milk increased and endogenous sources. Because glutamic acid is the
significantly (P < 0.05) between 0 and 24 h when most abundant free amino acid in bovine milk (Rassin
stored at 2°C (10.50–14.18 g/L), 4°C (10.02–14.61 g/L), et al., 1978; Lindmark-Månsson et al., 2003), it is per-
and 6°C (10.76–14.91 g/L). The detection of changes haps not surprising that changes in glutamic acid were
in the free amino acid content of dairy products has most apparent in this study, compared with other free
previously been used as an indicator of proteolysis amino acids, possibly indicative of proteolytic activity
(McSweeney et al., 1993b; Doolan et al., 2014). Mc- during storage.
Figure 3. The effect of storage temperature (2°C: ■; 4°C: ▲; 6°C: ●) and duration on the concentration of glutamic acid in bulk tank milk
in mid lactation (solid lines and symbols) and late lactation (dashed lines and open symbols). Each bar represents the 95% CI for that mean.
Journal of Dairy Science Vol. 100 No. 2, 2017

EFFECT OF STORAGE CONDITIONS ON BULK TANK MILK QUALITY
999
Effect of Storage Temperature and Duration
on the Composition of Bulk Tank Milk
olysis, was not observed in this study, and any changes
in percentage casein due to proteolysis that may have
occurred during storage are likely to be masked by the
Because of the sequential addition of milk through- addition of fresh milk throughout the storage period.
out storage, only milk from the first milking was stored Significant proteolytic activity was seen in milk stored
for the full 96 h. Thus, storage time was reduced for at 4°C after 6 d of storage by Guinot-Thomas et al.
milk added at subsequent milkings throughout storage. (1995a) when the psychrotrophic count increased from
3
6
7
This addition of fresh milk throughout storage in the 10 cfu/mL (d 0) to 10 to 10 cfu/mL (d 6). In the cur-
5
current study is different from other studies that have rent study, the psychrotrophic count did not exceed 10
investigated the effect of storage conditions on compo- cfu/mL (O’Connell et al., 2016), which may account for
sition (Guinot-Thomas et al., 1995a,b; O’Brien et al., the difference in the results reported herein.
1
999). Thus, unlike other studies, the significance of
Similar to the findings of Guinot-Thomas et al.
both indigenous and endogenous enzymatic activity in (1995a), we found no difference in the P or Ca concen-
milk may not be detected in this study due to the addi- trations of milk between 0 and 96 h when stored at 2,
tion of fresh milk throughout storage. The addition of 4, or 6°C; however, the partition of salts between the
milk with varying composition and bacterial quality is soluble and colloidal phases was not investigated.
also likely to limit detection of changes in milk quality
The β-CN concentrations of milk were greater at 48 h
due to storage. As indicated earlier, the composition of than at 0 h (P = 0.038; Table 4). Likewise, the concen-
milk-line samples differed in each lactation period and trations of β-CN were greater in milk stored at 48 and
values varied within week. However, as outlined in the 72 h compared with milk stored at 24 h (P = 0.008 and
companion paper, O’Connell et al. (2016), even though P = 0.034, respectively; Table 4). The increase in β-CN
the TBC of milk entering the tanks from each milking content throughout storage is most likely due to the
were numerically different, 75% of samples had TBC addition of fresh milk throughout storage. We observed
≤
4,800 cfu/mL, indicating that the bacterial count no effect of temperature on the total levels of β-CN in
of the milk added was consistent. Despite these chal- bulk tank milk; however, the effect of temperature on
lenges, this study best reflects milk storage conditions β-CN depletion from the casein micelle was not inves-
experienced on commercial dairy farms. Moreover, the tigated. Decreases in κ-CN and β-CN contents of raw
findings from this study can be used to extrapolate milk were observed by Guinot-Thomas et al. (1995a)
guidelines on the expected deterioration of milk com- between 96 and 144 h of storage at 4°C. Coincidentally,
position during storage for milk processors and farmers this reduction was only observed as psychrotrophic bac-
6
7
alike.
terial counts exceeded 10 to 10 cfu/mL, highlighting
We detected no effect of temperature or interaction the importance of microbial growth relative to protein
between temperature and time on the percentage of fat quality during storage.
and lactose in bulk tank milk (Table 3). The concentra-
tion of fat in milk increased between 0 and 24 h (P < between 0 and 24 h (P < 0.0001) and remained similar
.0001) of storage and remained unchanged thereafter thereafter (Table 4). These findings are in contrast to
Table 4). Milk sampled at 0 h contained milk from the those of Gandolfi et al. (1992), who measured contents
The levels of arginine in bulk tank milk increased
0
(
first morning milking of the trial period. Thereafter, of alanine, aspartic acid, and glutamic acid of milk be-
milk sampled was a combination of milk obtained from fore and after storage of raw milk samples (n = 2) at
morning and evening milkings. Because of the milk- 4°C for 7 d, and reported that ꢀ-alanine was the only
ing interval, cows produced more milk at the morning amino acid to increase during storage, again attributed
milking than the evening milking and thus, due to dilu- to growth of psychrotrophic bacteria during storage.
tion, the fat content of milk was typically lower in milk As outlined in the companion paper (O’Connell et al.,
collected from the morning milking than the evening 2016), we detected limited growth of bacteria during
milking (Walstra et al., 1999). This would account for storage in this study, which may account for the limited
the lower fat content of milk observed at 0 h, which was changes in amino acid concentrations observed. The lev-
not observed at any other sampling point.
els of lysine increased between 0 and 96 h (P = 0.0001)
In the current study, we detected no main effect of and as storage temperature increased from 2°C to 6°C
temperature on casein content in bulk tank milk; how- (P < 0.05). Levels of lysine tended (P = 0.11) to be
ever, over time, the casein content of bulk tank milk greater in milk stored at 6°C for periods greater than 48
increased from 3.19 to 3.22% between 0 and 48 h and h compared with milk stored at 2 or 4°C (Table 5). The
then decreased to 3.18% between 48 and 96 h (Table slight increase in levels of total bacteria and psychro-
2
). A reduction in casein content, often due to prote- trophic bacteria as storage temperature and duration
Journal of Dairy Science Vol. 100 No. 2, 2017

1000
O’CONNELL ET AL.
Table 3. The significance of the main effects of storage time, temperature, and their interaction on the
composition and functional properties of bulk tank milk samples
P-value
1
2
Item
Time
Temperature
Time × Temperature
Fat (%)
<0.01
<0.01
<0.01
0.74
0.44
0.13
0.72
0.10
0.21
0.80
0.63
0.43
0.23
0.84
0.83
0.97
0.92
0.36
0.35
0.40
0.41
0.80
<0.01
0.32
0.47
0.39
0.40
0.81
0.85
0.46
0.50
0.63
0.91
0.43
0.45
0.99
0.67
0.73
0.83
0.96
0.70
0.68
0.92
0.78
0.36
0.71
0.62
0.64
0.05
0.35
0.26
0.11
0.65
0.33
0.26
0.99
0.89
0.83
0.93
0.91
0.85
0.93
Protein (%)
Lactose (%)
Noncasein nitrogen (%)
Casein number (%)
NPN (%)
0.22
0.16
Casein (%)
Phosphorus (g/L)
Calcium (g/L)
Heat coagulation time (s)
pH
Rennet coagulation time (s)
Rate of curd aggregation (s)
Curd firmness (mm)
Titratable acidity (mL)
Cysteic acid (μg/mL)
Glutamic acid (μg/mL)
Histidine (μg/mL)
Lysine (μg/mL)
Glycine (μg/mL)
Cysteine (μg/mL)
Arginine (μg/mL)
κ-CN (g/L)
0.02
0.57
0.54
<0.01
0.03
<0.01
0.02
<0.01
0.08
0.02
<0.01
0.21
<0.01
0.39
0.13
<0.01
0.03
α
α
S2-CN (g/L)
S1-CN (g/L)
0.06
0.05
β-CN (g/L)
α-LA (g/L)
β-LG-a (g/L)
β-LG-b (g/L)
0.01
0.22
0.07
0.01
1
Milk was stored for 96 h and sampled every 24 h. Fresh milk was added to the tank at each milking throughout
storage.
2
Milk was stored at 2, 4, or 6°C throughout storage.
increased (O’Connell et al., 2016) and the subsequent Assessment of Functional Properties
production of microbial proteases in milk throughout
storage may have contributed to increased proteolytic
The HCT of bulk tank milk was affected by stor-
activity, which may account for the increased concen- age time (P < 0.01; Table 3) but not by temperature
tration of particular amino acids, as observed by Albert or the interaction between temperature and time. The
et al. (2009).
HCT decreased between 0 and 72 h and then increased
1
Table 4. The effect of storage time on the composition and functional properties of bulk tank milk
Storage time (h)
Item
0
24
48
72
96
P-value
b
a
a
a
a
Fat (%)
4.52 ± 0.07
5.00 ± 0.05
5.02 ± 0.031
5.02 ± 0.03
5.06 ± 0.02
<0.01
<0.05
<0.05
<0.05
<0.05
<0.05
<0.01
<0.01
<0.01
<0.05
<0.02
ab
ab
b
ab
b
a
Lactose (%)
4.53 ± 0.010
3.19 ± 0.016
4.53 ± 0.007
4.55 ± 0.004a
4.55 ± 0.004
4.56 ± 0.002
3.18 ± 0.001
b
Casein (%)
Heat coagulation time (s)
pH
3.22 ± 0.008
a
a
ac
bc
bc
803 ± 26
803 ± 9
739 ± 8
704 ± 7
736 ± 7
b
ac
bc
bc
bc
6.71 ± 0.01
6.74 ± 0.01
6.74 ± 0.01
6.73 ± 0.01
6.72 ± 0.01
b
ac
bc
bc
ac
Rennet coagulation time (s)
Curd firmness (a30; mm)
Lysine (μg/mL)
Arginine (μg/mL)
β-CN (g/L)
1,319 ± 9
1,428 ± 35
1,367 ± 32
1,400 ± 32
1,417 ± 12
a
bc
ac
b
b
24.02 ± 0.52
19.70 ± 0.77c
20.93 ± 0.75
19.55 ± 0.82a
2.80 ± 0.08a
3.04 ± 0.11
19.62 ± 0.74a
2.97 ± 0.05a
2.77 ± 0.18
c
b
1.60 ± 0.11
1.99 ± 0.09
2.40 ± 0.09a
b
a
2.30 ± 0.08
2.73 ± 0.06
2.82 ± 0.05
bcd
ac
bd
a
ac
ad
10.22 ± 0.28
2.25 ± 0.06
9.81 ± 0.29a
11.20 ± 0.12
11.12 ± 0.23
2.29 ± 0.04
10.87 ± 0.12a
bc
ac
β-LG-b (g/L)
2.13 ± 0.08
2.21 ± 0.03
2.35 ± 0.03
a–d
Means within a row with different superscripts differ significantly (P < 0.05).
1
Values at each storage temperature represent the least squares means of 12 replicates ± SEM.
Journal of Dairy Science Vol. 100 No. 2, 2017

EFFECT OF STORAGE CONDITIONS ON BULK TANK MILK QUALITY
1001
Table 5. Least squares means analysis of lysine concentration (μg/mL; mean ± SE ) in milk samples stored at
different temperatures (2°C, 4°C, 6°C) and for different durations (0, 24, 48, 72, and 96 h)
Temperature
(
°C)
0 h
24 h
48 h
72 h
96 h
bc
bcd
bd
ad
bc
bc
ac
a,B
2
4
6
1.53 ± 0.18d
1.64 ± 0.18d
1.62 ± 0.18
1.80 ± 0.16
1.88 ± 0.16
2.31 ± 0.16
2.19 ± 0.09
2.39 ± 0.09
2.61 ± 0.09
2.40 ± 0.14a
2.99 ± 0.14
2.61 ± 0.09
2.82 ± 0.09
3.49 ± 0.09
ac,B
a,A
bcd
ac
3.02 ± 0.14
a–d
Means within a row with different superscripts differ significantly (P < 0.05).
Means within a column with different superscripts differ significantly (P < 0.05).
A,B
between 72 and 96 h. The HCT at 0 and 24 h were content of milk did not change during storage, but the
different (P < 0.04) from those at 48, 72, and 96 h methodology used to measure the casein contents of
(
7
Table 4). The limited improvement in HCT between milk in this study was unable to differentiate between
2 and 96 h was probably due to the addition of fresh intact and dissociated β-CN. The decrease in a30 may
milk throughout the sampling period. Davies and Law thus be linked to the dissociation of β-CN from the
1983) and Dalgleish and Law (1989) reported that ex- casein micelle during refrigerated storage, which can be
(
tensive dissociation of β-CN occurs during cold storage, reversed by heating (Puhan, 1988). A correlation exists
with a concomitant increase in the level of β-CN in between decreases in a value and curd yield (Hurtaud
3
0
ultracentrifugal supernatants, which may account for et al., 1995; Mara et al., 1998) and this change may be
the reduction in HCT. Indeed, Crudden et al. (2005b) of industrial significance; however, larger studies are
reported an increased dissociation of β-CN into the su- needed to evaluate the significance of this effect.
pernatant during storage of skim milk at 5°C for 2 and 5
Though statistically significant, the effect of stor-
d, as measured using urea–PAGE. This was associated age time on the pH of bulk tank milk was minimal
with a reduction in HCT of ~50% after 5 d of storage. (Table 4). Proliferation of lactic acid bacteria during
The findings of the current study are in agreement with storage increases titratable acidity, with values above
those of Crudden et al. (2005b), although the reduction 0.18% being regarded as indicative of spoilage (Lu et
in HCT was not as significant in this study, most likely al., 2013). In the current study, titratable acidity never
due to the sequential addition of fresh milk.
exceeded this critical spoilage value. In a similar study
Between 0 and 24 h, the RCT increased from 1,260 in which milk was stored for 5 d at 5.5°C, the titratable
and 1,428 s (P < 0.03) and remained similar thereafter acidity increased from 0.15 to 0.17%, which was at-
(
RCT at 96 h = 1,416 s; Table 4). Previously, after tributed to an increase in microbial population within
storing milk from cows classified as producing milk the storage period (560 to 120,000 cfu/mL; Schmidt et
with poor coagulation properties for 72 h at 4°C, de al., 1996). Given that the majority of milk in this study
Moura Maciel et al. (2015) observed very similar trends was stored at temperatures below 5.5°C for a maximum
in RCT, in agreement with the current study, whereby of 96 h, coupled with the sequential addition of fresh
a longer RCT was observed after 24 h of storage (in- milk throughout the storage period, it is unlikely that
creased from ~1,020 to 1,200 s) with no further increase sufficient lactic acid bacteria would be present to cause
being observed during a further 48 h of storage. During spoilage.
cold storage, β-CN and colloidal calcium phosphate
dissociate from the casein micelle, resulting in changes
to the micelle structure that result in poor rennetabil-
ity (Raynal and Remeuf, 2000). These changes do not
CONCLUSIONS
Few researchers have examined the effect of storage
occur immediately on cooling but are more or less com- conditions on milk stored in bulk tanks located on
plete after 24 h of storage at 4°C (Walstra et al., 1999), farms. Such studies simulate the conditions experienced
as observed in the current results.
on commercial farms. The composition and functional
The a30 decreased between 0 and 96 h (P < 0.0001; properties of milk differed between mid and late lacta-
Table 4). Similar decreases in curd firmness were re- tion. Milk storage temperature had a negligible effect
ported by O’Brien et al. (2001) after 72 and 144 h of on gross composition and functional properties of milk
storage of milk at 4°C. In that study, the deterioration and only influenced the concentration of some free
in curd firmness was attributed to a decrease in the rela- amino acids in milk. Between 0 and 96 h, minimal dete-
tive proportion of casein and a concomitant increase in riorations in some functional properties were observed,
the loss of protein in the whey fraction during storage and were most likely due to the dissociation of β-CN
because of proteolysis. In the current study, the casein from the casein micelle, which can be reversed upon
Journal of Dairy Science Vol. 100 No. 2, 2017

1002
O’CONNELL ET AL.
Driessen, F. M. 1989. Inactivation of lipases and proteinases (indig-
enous and bacterial). Pages 71–93 in Bulletin of the International
Dairy Federation 238. International Dairy Federation, Brussels,
Belgium.
pasteurization. Thus, blended milk can be stored for
up to 96 h at temperatures between 2°C and 6°C with
little effect on its composition or functional properties.
European Commission. 1992. Annex A Council Directive 92/46/EEC.
Health rules for the production and placing on the market of raw
milk, heat-treated milk and milk-based products. European Com-
mission, Brussels, Belgium.
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