International Dairy Journal 85 (2018) 153e158

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International Dairy Journal

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Rennet coagulation properties of milk in the presence of MFGM

fragments isolated from raw e and pasteurised-cream buttermilk
Marie-Pierre Gauvin a, Yves Pouliot a, Michel Britten a, b, *
a
STELA Dairy Research Centre, Institute of Nutrition and Functional Foods (INAF), Universit

e Laval, Quebec City, Quebec, G1V 0A6, Canada
b
Saint-Hyacinthe Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Quebec, J2S 8E3, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Milk fat globule membrane (MFGM) fragments were isolated from raw- and pasteurised-cream butter-
Received 1 February 2018 milks to determine their impact on the rennet coagulation properties of milk. These MFGM fragments
Received in revised form were recovered by ultracentrifugation after casein micelle dissociation using sodium citrate. This pro-
9 May 2018
cedure was also applied to raw skim milk as a control. More protein was recovered from the two types of
Accepted 5 June 2018
Available online 18 June 2018
buttermilk than from the skim milk upon centrifugation. This protein was mostly MFGM, but significant
amounts of caseins and whey proteins were also recovered. This suggests that the churning of cream
induces changes in these proteins, favouring their sedimentation upon ultracentrifugation. The isolated
material was suspended in reconstituted skim milk, and rennet coagulation kinetics and gel contraction
capacity were measured. The MFGM fragments isolated from buttermilk impaired rennet gel formation
and reduced gel contraction capacity, but these effects were not related to the cream pasteurisation
treatment.
Crown Copyright © 2018 Published by Elsevier Ltd. All rights reserved.

1. Introduction formation either by becoming physically entrapped within the

Traditional buttermilk is the by-product obtained by phase
inversion upon churning of cream into butter. Because of its simi-
larity in composition to skim milk, traditional buttermilk has
attracted attention in cheese manufacture (Govindasamy-Lucey, Lin,
Jaeggi, Johnson, & Lucey, 2006; Govindasamy-Lucey et al., 2007;
Hickey et al., 2018; Mistry, Metzgeer, & Maubois, 1996; Morin,
Pouliot, & Britten, 2008; Poduval & Mistry, 1999; Turcot, Turgeon, &
St-Gelais, 2001). However, the use of buttermilk in cheesemaking is
limited by technological problems, such as poor milk coagulation
and excessive moisture in cheese. The main compositional differ-
ence between buttermilk and skim milk is buttermilk's higher
concentration of milk fat globule membrane (MFGM) fragments,
which are released during cream churning. The problems encoun-
tered with the use of buttermilk in cheesemaking have therefore
mainly been attributed to these constituents. Moreover, the thermal
history of cream before churning was shown to influence the
cheesemaking aptitude of buttermilk (Morin et al., 2008). In that
study, the authors suggested that heat treatments could induce the
formation of large MFGM fragments that would impair rennet gel
* Corresponding author. Tel.: þ1 450 768 7981.
E-mail address: michel.britten@agr.gc.ca (M. Britten).
casein matrix or by interacting directly with casein micelles.
In a previous study, buttermilk serum components were frac-
tionated by centrifugation (Gauvin, Pouliot, & Britten, 2018). A large
proportion of the MFGM components were concentrated in a
fraction that also contained casein micelles. This situation compli-
cated the determination of the impact of MFGM fragments on the
rennet gel formation properties of milk, since the casein micelles
present in this fraction also participated in gel formation. To
circumvent this problem, casein micelles can be dissociated with
sodium citrate before centrifugation (Corredig & Dalgleish, 1997).
Morin, Jime
nez, and Pouliot (2007) used a similar approach to
isolate MFGM to characterise the changes that it undergoes during
the processing of buttermilk (pasteurisation, evaporation, and
spray drying). Those authors found that the pasteurisation of cream
resulted in the highest impact on the composition of MFGM iso-
lates, with a significant increase in whey protein concentration.
Those authors also systematically observed a higher concentration
of caseins, which they attributed to the incorporation of caseins
into MFGM as a result of the partial homogenisation of the fat
globules during the churning process.
The objectives of the present study were to isolate MFGM
fragments from buttermilk made from raw cream and from pas-
teurised cream and to characterise their protein content and their

https://doi.org/10.1016/j.idairyj.2018.06.006
0958-6946/Crown Copyright © 2018 Published by Elsevier Ltd. All rights reserved.
154 M.-P. Gauvin et al. / International Dairy Journal 85 (2018) 153e158
impact on rennet gel formation and properties in comparison with
an isolate obtained from raw skim milk.
2. Material and methods
2.1. Buttermilk and skim milk
Raw cream and whole milk were obtained from local dairy
plants (Agropur, Saint-Hyacinthe, QC, Canada; Laiterie Chalifoux,
Sorel-Tracy, QC, Canada). The whole milk was skimmed at 45  C
using a bench-top milk separator (Elecrem 1; Fromagex, Rimouski,
QC, Canada) and rapidly cooled to 4  C. Eight litres of the raw cream
were standardised to 35% milk fat, and a half portion was batch-
pasteurised at 68  C for 30 min (excluding come-up time) in a
culture incubator used as a thermostatic bath (Laboratum Wesby,
Niebüll, GmbH & Co, Germany). The pasteurised and unpasteurised
creams were kept at 4  C overnight before they were churned
separately in a Hobart mixer (Troy, OH, USA). The two types of
buttermilk were filtered through cheesecloth and skimmed by
centrifugation (1000  g, 20 min, 4  C) (Avanti J-26 XPI centrifuge;
Beckman Coulter, Mississauga, ON, Canada). Four batches each of
raw-cream buttermilk (RCB), pasteurised-cream buttermilk (PCB),
Protein content in dry pelletð%Þ  mass of dry pelletðgÞ
Protein recovery ð%Þ ¼
Protein content of substrateð%Þ  mass of substrate centrifugedð22gÞ
and raw skim milk (RSM) were obtained, aliquoted and frozen
(20  C) until use (within 4 months).
2.2. Isolation of MFGM fragments
Fragments of MFGM from the two types of buttermilk and the
skim milk were isolated according to the method of Corredig and
Dalgleish (1997) with slight modifications. Trisodium citrate
(Na3C6H5O7$2H2O) was added to the two types of buttermilk and
the skim milk (2%, w/v), which were then kept at 4  C overnight to
dissociate casein micelles. Sodium azide (0.02%, w/v) was also
added to prevent microbial growth. The trisodium citrate increased
the pH of the substrates, so it was standardised to 7.3 ± 0.1 at 34  C
with minute amounts of 10% lactic acid (v/v). The two types of
buttermilk and the skim milk were then weighed (22 ± 0.005 g) in
ultracentrifuge tubes (Beckman Coulter) and centrifuged
(100,000  g, 60 min, 34  C). After centrifugation, the supernatant
was discarded, and the interior of each tube was carefully dried
with a paper towel. The pellets were suspended in milk ultrafil-
tration permeate to the initial volume using a dispersing tool
(model S25N-19G, Ultra-Turrax; IKA, Staufen, Germany) at
8000 rpm and centrifuged a second time (100,000  g, 60 min,
34  C) to wash out the residual sodium citrate.
2.3. Compositional analyses
The ultracentrifugation pellets were lyophilised, and their pro-
tein content was determined using the Kjeldahl method with a
nitrogen-to-protein conversion factor of 6.38 (AOAC, 2005).
The two types of buttermilk, the skim milk, and the MFGM iso-
lates were analysed by sodium dodecyl sulphateepolyacrylamide
gel electrophoresis (SDS-PAGE) in reducing conditions. Two fresh
MFGM pellets from each substrate were suspended in 9 mL deion-
ised water using a tissue homogeniser (Wheaton, Millville, NJ, USA)
before being lyophilised and kept at 20  C for further protein
characterisation by SDS-PAGE. This procedure was used to obtain
homogeneous samples that would be easier to dissolve in the
electrophoresis buffer. The samples were diluted to 2e3 mg protein
mL1 in Laemmli buffer (Bio-Rad, Mississauga, ON, Canada) with 5%
b-mercaptoethanol as a reducing agent and boiled for 5 min.
Samples, prepared in duplicate, were loaded (10 mL) onto 4e15%
polyacrylamide precast gels (Bio-Rad) and run at 150 mV for
approximately 45 min at ambient temperature. The gels were
stained with Coomassie Brilliant Blue R-250 (Bio-Rad) and
destained with a mixture of 10% acetic acid and 40% methanol in
deionised water. Relative proportions of each band were deter-
mined using QuanTL densitometry software (GE Healthcare Life
Sciences, Mississauga, ON, Canada) and no correction was made for
difference in dye binding among proteins. The protein content was
reported on the basis of molecular mass as belonging to one of
three main categories, the first containing mostly whey proteins
(<20 kDa), the second containing mostly caseins (20e35 kDa), and
the third containing mostly MFGM proteins (>35 kDa).
The proportions of proteins recovered in the pellet were esti-
mated using the following equation:
 100 (1)
2.4. Dispersion of MFGM isolates in reconstituted skim milk
After isolation, the fresh pellets were immediately dispersed to
their initial concentration in reconstituted skim milk (Agropur)
adjusted to 3.5% protein (w/w) with milk protein concentrate (MPC
70; Idaho Milk Products, Jerome, ID, USA). The dispersions were
homogenised using a tissue homogeniser (Wheaton) and agitated for
30 min at room temperature before being stored at 4  C overnight.
2.5. Aggregation kinetics of casein micelles in presence of MFGM
fragments
The MFGM dispersions (see section 2.4) were diluted 50-fold in
Ca-imidazole buffer (20 mM imidazole/HCl, 50 mM NaCl, 5 mM CaCl2,
pH 6.5). The initial apparent diameter (Z-average) of casein micelles
was measured 30 min after equilibrium at 34  C by means of a
Zetasizer Nano ZS instrument (Malvern Instruments, Worcester-
shire, UK) as described by Gauvin et al. (2018). Rennet (Chy-Max
Extra; Chr. Hansen, Milwaukee, WI, USA) was added (0.001%, v/v),
and the Z-average was recorded every 3 min at 34  C as long as the
cumulant fit was good (error < 0.005). The results were analysed as
described by Gauvin et al. (2018), with apparent diameters
expressed as the differential apparent diameter (DDapp), which is
the difference between the apparent diameter at time t and the
initial apparent diameter (t ¼ 0). The lag time corresponds to the
time to reach a DDapp of 50 nm, and the aggregation rate corre-
sponds to the rate of increase in DDapp between 100 and 300 nm.
2.6. Coagulation kinetics of casein micelles in presence of MFGM
fragments
2.6.1. Optical density
The MFGM dispersions (see section 2.4) were adjusted to pH 6.5
using 10% lactic acid (v/v), and 0.01% rennet (Chy-Max Extra; Chr.
 M.-P. Gauvin et al. / International Dairy Journal 85 (2018) 153e158 155

Hansen) was added at 34  C. Aliquots (4  20 mL) were pipetted Table 1

onto a microplate cover and covered with a microscope cover glass. Protein concentration and molecular mass distribution in raw-cream buttermilk
(RCB), pasteurised-cream buttermilk (PCB), and raw skim milk (RSM).a
The optical density at 650 nm was monitored every 30 s for 90 min
according to the method of Robitaille, Giroux, and Britten (2004). Parameter RCB PCB RSM S.E.
The results were expressed as an optical density ratio relative to the Total protein content (g 100 g1) 2.91a 2.96a 3.39b 0.04
initial optical density (ODi) and were fitted to a five-parameter <20 kDa fraction (whey proteins) (%) 31 31 33 1
sigmoidal model (Pandey, Ramaswamy, & St-Gelais, 2003). Lag 20e35 kDa fraction (caseins) (%) 46.7a 47.1a 59.4b 0.9
>35 kDa fraction (MFGM) (%) 22.0a 21.7a 7.2b 0.4
time (Tlag), maximum gelation rate (Vmax), and OD90min/ODi were
a
the parameters extracted from the model. Protein concentration and molecular mass distribution were as determined by
densitometric analysis of sodium dodecyl sulphateepolyacrylamide gel electro-
phoresis, expressed as the percentage of total protein. Values are means (n ¼ 4);
2.6.2. Storage modulus means with different lowercase superscript letters in the same row differ (P < 0.05);
The dispersions were renneted as described in section 2.6.1. The S.E., standard error; MFGM, milk fat globule membrane.
coagulation kinetics was monitored using dynamic rheology as
described by Perreault, Turcotte, Morin, Pouliot, and Britten (2016)
(1 Hz frequency, 0.01% strain) using a rheometer equipped with that in the three isolates, the largest proportion (52%e58%) of the
concentric cylinders (Physica MCR301; Anton Paar GmbH, Graz, total protein pelleted had a molecular mass greater than 35 kDa
Austria). The storage modulus (Gʹ) was recorded every 30 s for (considered to be mainly MFGM proteins). The SDS-PAGE profiles of
100 min. the isolates are presented in Fig. 2. The major bands resolved were
ascribed, in accordance with Mather (2000, 2011), to xanthine ox-
idase (XDH/XO), CD36, butyrophilin (BTN), and PAS 6/7 or adipo-
2.7. Gel syneresis
philin (ADPH) (which may be confounded with PAS 6/7 bands).
Protein recovery in the isolates was estimated for total protein
Rennet-gel syneresis at the cooking temperature was monitored
and for each protein class. The results are reported in Table 2 (lower
using a simplified version of the method proposed by Giroux,
Bouchard, and Britten (2014). The pH of the dispersions (see sec-
tion 2.4) was adjusted to 6.5 with 20% lactic acid (v/v) at 34  C.
Rennet (0.01%, v/w, Chy-Max Extra; Chr. Hansen) was added, and
the dispersions were aliquoted in 5 g portions in triplicate and
incubated for 30 min at 34  C. The gels were then incubated at 42  C
for 80 min. The weight of whey expelled after this period was
recorded and reported relative to the initial weight of the disper-
sion (% syneresis). Total solids were determined on the curds after
drying in an oven at 100  C for 18 h. Protein concentration in the
whey was determined using the Kjeldahl method (AOAC, 2005),
and the protein retention coefficient (Kp) was calculated using the
following equation:

%Protein in whey
Kp ¼ 1  : (2)
%Protein in renneted dispersion

2.8. Statistical analysis

The experiment, including the preparation of MFGM extracts,

was repeated four times from independent batches of cream and
milk. Data were analysed with the SAS software package (SAS
Institute Inc., Cary, NC, USA) using ANOVA. Differences were
considered significant at P < 0.05.

3. Results and discussion

3.1. Composition and characterisation of buttermilk and skim milk

isolates

The protein composition of the RCB, PCB, and RSM is reported in

Table 1, and their SDS-PAGE profiles are shown in Fig. 1. As generally
reported in the literature (Vanderghem et al., 2010), protein con-
tent was lower in the RCB and the PCB than in the RSM. In com-
parison with the RSM, the two types of buttermilk contained 30%
less casein but, as expected, 2.6 times more membrane proteins. No
significant impact of cream pasteurisation was observed on total
protein content (w/w) in buttermilk, and this finding is consistent
Fig. 1. Sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS-PAGE)
with the results reported by Morin et al. (2008). profiles of raw skim milk (RSM), raw-cream buttermilk (RCB), and pasteurised-cream
The protein composition of the isolates is reported in Table 2 buttermilk (PCB). Whey proteins, <20 kDa; caseins, 20e35 kDa; milk fat globule
(upper part). Densitometric analysis of the SDS-PAGE gels showed membrane proteins, >35 kDa.
156 M.-P. Gauvin et al. / International Dairy Journal 85 (2018) 153e158

Table 2 (Dalgleish & Banks, 1991; Houlihan, Goddard, Nottingham, Kitchen,

Composition of milk fat globule membrane (MFGM) isolates from raw-cream & Masters, 1992; Kim & Jimenez-Flores, 1995; Ye, Singh, Taylor, &
buttermilk (RCB), pasteurised-cream buttermilk (PCB), and raw skim milk (RSM).a
Anema, 2004). However, the higher whey protein proportion
Protein proportions and recovery RCB PCB RSM S.E. recovered from the RCB in comparison with the RSM suggests that
Proportions the churning process in itself had more impact on whey protein
<20 kDa (whey proteins) (%) 21b 23b 6a 1 sedimentation than the heat treatment did. In the present study,
20e35 kDa (caseins) (%) 26a 25a 37b 2 whey protein accounted for 21% of the RCB isolate. Other authors
>35 kDa (MFGM) (%) 53 52 58 3
(Corredig & Dalgleish, 1996, 1998b; Morin, Jime
nez-Flores, &
Recovery 11.4b 13.2c 0.8a 0.2
<20 kDa (whey proteins) (%) 7.7b 9.9c 0.1a 0.5 Pouliot, 2007) also observed the presence of whey proteins in
20e35 kDa (caseins) (%) 6.4b 7.1b 0.5a 0.3 their raw MFGM isolates. In a study by Corredig and Dalgleish
>35 kDa (MFGM) (%) 27b 32b 7a 1 (1998a), the proportion of whey proteins (b-lactoglobulin and a-
a
Protein proportions were as found in the isolate (% of total protein in the lactalbumin) in the isolate obtained from buttermilk made from
isolate); protein recovery is that from the substrate (% of initial protein in RCB, PCB, raw cream was approximately 15%. Morin et al. (2007) obtained
or RSM). Values are means (n ¼ 4); means with different lowercase superscript comparable results (approximately 13%).
letters in the same row differ (P < 0.05); S.E., standard error.
The proportions of caseins recovered from the RCB and the PCB
were comparable to each other and were also significantly higher
part). Total protein recovery in the isolates was significantly higher than the proportions recovered from the RSM (P < 0.05). The
in both types of buttermilk (11.4% and 13.2% of total protein in the presence of caseins in buttermilk isolate obtained after dissociation
RCB and the PCB, respectively) than in the RSM (0.8%) (P < 0.05). with sodium citrate was also reported by Morin et al. (2007) and
Interestingly, significant amounts of caseins and whey proteins was attributed to partial homogenisation of the fat globules upon
were recovered in the RCB and PCB isolates along with MFGM churning and adsorption of proteins at the oil-water interface. The
proteins. Care was taken to wash out the residual sodium citrate churning process also introduces air bubbles in the cream, which
using milk ultrafiltration permeate, and this step should also have increases protein exposure to the airewater interface. Individual
washed out caseins and whey proteins that could have been caseins, casein micelles and whey proteins are known to adsorb to
“entrapped” within the MFGM pellet during the first centrifugation. the airewater interface and undergo structural changes (Kamath,
The recoveries of proteins with a molecular mass greater than Webb, & Deeth, 2011; Zhang, Dalgleish, & Goff, 2004; van Aken,
35 kDa from the RCB and the PCB were similar (around 30%) 2001). Fat globules also adsorb at the interface of the air bubbles
(Table 2). The recovery percentage from the RSM was significantly (Walstra, Geurts, Noomen, Jellema, & Van Boekel, 1999) and the
lower (7%). The MFGM proteins found in the RSM may be the result membrane components of fat globules are likely to interact with
of relatively mild mechanical treatments, such as pumping and adsorbed proteins.
centrifugal separation. These treatments are not expected to break
the MFGM like churning does, and only loosely attached peripheral 3.2. Aggregation kinetics in diluted condition
membrane proteins may have been shed. This type of MFGM pro-
teins (peripheral proteins) has been found in milk supernatant after The lag time and the aggregation rate (rate of increase in DDapp
ultracentrifugation (Mather, 2000, 2011). between 100 and 300 nm) of renneted casein micelles in the
The protein recovery from the PCB was slightly higher than from presence of the RCB, PCB, and RSM isolates are presented in Table 3.
the RCB, and that difference was attributed to higher whey protein The lag time was slightly shorter (approximately 3 min) in the
sedimentation. Interactions between skim milk proteins (mainly b presence of either type of buttermilk isolate than in the presence of
lactoglobulin but also a lactalbumin) and MFGM proteins can occur the skim milk isolate (P < 0.05). However, the aggregation rate was
in whole milk upon heating, as a result of disulphide bonding similar for the three isolates (P > 0.05). The first phase of rennet-

Fig. 2. Sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDS-PAGE) profiles of raw-cream buttermilk (RCB), pasteurised-cream buttermilk (PCB), and raw skim milk
(RSM) isolates. Whey proteins, <20 kDa; caseins, 20e35 kDa; milk fat globule membrane (MFGM) proteins, >35 kDa.
 M.-P. Gauvin et al. / International Dairy Journal 85 (2018) 153e158 157

Table 3 120
Lag time and aggregation rate of renneted micelles in the presence of raw-cream
buttermilk (RCB), pasteurised-cream buttermilk (PCB), and raw skim milk (RSM)
isolates.a
100
Substrate Lag time (min) Aggregation rate (nm min1) 80
a

G' (Pa)
RCB 28.9 14.8
PCB 28.8a 14.1 60
RSM 32.1b 14.7
S.E. 0.6 0.3
40
a
Definitions are: lag time, time needed to increase micelle apparent diameter
(DDapp) by 50 nm; aggregation rate, average rate of DDapp increase measured be- 20
tween 100 and 300 nm. Values are means (n ¼ 4); means with different lowercase
superscript letters in the same column differ (P < 0.05); S.E., standard error.
0
0 20 40 60 80 100
induced aggregation is limited by the reactivity of casein micelles
Time (min)
and starts only when a critical portion of the k-casein is hydrolysed
(Horne & Lucey, 2017). Our results suggest that buttermilk isolates Fig. 3. Rheological coagulation kinetics in the presence of raw-cream buttermilk ,
alter the reactivity of casein micelles, allowing aggregation at a pasteurised-cream buttermilk , and raw skim milk isolates (n ¼ 4). Gʹ, storage
lower degree of k-casein hydrolysis. Once triggered, the aggrega- modulus.
tion proceeds through a diffusion-limited process (Corredig &
Salvatore, 2016), which explains why the buttermilk or skim milk
isolates showed no influence on the aggregation rate (Table 3). Consistent with previous results, no significant distinction in
behaviour was made between the suspensions containing the RCB
3.3. Coagulation kinetics isolate and those containing the PCB isolate. This means that the
pasteurisation treatment applied to the cream did not induce changes
3.3.1. Optical density in the isolated buttermilk constituents that significantly influenced
The change in optical density was used to monitor the coagulation rennet gel formation. The fact that a similar heat treatment was
kinetics of non-diluted milk. The Tlag, Vmax, and OD90min/ODi are previously proven to negatively influence rennet gel formation
reported in Table 4. The time at which the optical density started to (Morin et al., 2008) can suggest that this negative impact of pasteur-
increase (Tlag) was the same for all the treatments. Despite an effect isation resides, as is the case with milk, in the properties of either
of buttermilk isolates on the lag time for diluted milk aggregation micellar casein or the serum fraction of buttermilk (Kethireddipalli,
(Table 3), gelation time was not significantly affected. This could Hill, & Dalgleish, 2010; Vasbinder, Rollema, & de Kruif, 2003).
mean that the impact of the buttermilk isolates on early aggregation
is not large enough to influence the early coagulation process. 3.4. Contraction capacity of rennet gels
Nevertheless, the Vmax was lower in the presence of the RCB isolate
than in the presence of the RSM isolate. This difference suggests that The extent of syneresis and the curd moisture after 80 min of
the structure of the gel formed more slowly in the presence of the cooking at 42  C are reported in Table 5. The mass of the expelled
buttermilk isolate. The extent of the increase in optical density at serum was lower in the gels containing the buttermilk isolates than
90 min was significantly lower in the presence of the RCB or PCB in those containing the RSM isolate. However, the difference was
isolate than in the presence of the RSM isolate, suggesting that the not large enough to induce statistically significant effect on curd
structure of the gel was less dense with the buttermilk isolates. No moisture (Table 5).
distinction was made between the results obtained with the RCB Interestingly, the protein retention coefficients were higher in
isolate and those obtained with the PCB isolate. the presence of the RCB and PCB isolates in comparison with the
RSM isolate. The buttermilk isolates were therefore well retained in
3.3.2. Storage modulus the curd and may have prevented the gel from contracting and
The development of gel strength was assessed by measuring the expelling the whey. Given that the aggregation step was not overly
changes in the storage modulus over time after renneting. Fig. 3 influenced, the gel may have been prevented from rearranging (van
shows how the storage modulus (Gʹ) changed after rennet addi- Vliet, van Dijk, Zoon, & Walstra, 1991). Rearrangements are also
tion. The observations made by measuring optical density to responsible for gel strengthening after aggregation (Mellema,
determine the coagulation kinetics were confirmed by these Walstra, van Opheusden, & van Vliet, 2002). These rearrange-
rheological measurements. ments could be prevented either by impeding the breaking of
strands within the gel network or by creating physical obstacles to
stop the strands from getting closer to each other. Buttermilk
Table 4
Coagulation kinetics parameters obtained by optical density measurements of
reconstituted skim milk (3.5% protein) in the presence of raw-cream buttermilk
(RCB), pasteurised-cream buttermilk (PCB), and raw skim milk (RSM) isolates at the Table 5
concentration initially found in their respective substrate.a Syneresis, curd moisture, and protein retention after 80 min of cooking at 42  C in
the presence of raw-cream buttermilk (RCB), pasteurised-cream buttermilk (PCB),
Substrate Lag time (min) Vmax (min1) OD90min/ODi and raw skim milk (RSM) isolates.a
RCB 6.7 0.163a 4.00a
Substrates Syneresis (%) Curd moisture (%) Protein retention, Kp (%)
PCB 6.6 0.170ab 4.05a
RSM 6.7 0.187b 4.28b RCB 47a 87.5 75.7b
S.E. 0.1 0.006 0.03 PCB 44a 88.4 75.2b
a RSM 55b 86.6 73.6a
Values are means (n ¼ 4); means with different lowercase superscript letters in
S.E. 1 0.6 0.3
the same column differ (P < 0.05); Vmax, maximum gelation rate; OD90min/ODi, ratio
a
of the optical density at 650 nm at 90 min over the initial optical density; S.E., Values are means (n ¼ 4); means with different lowercase superscript letters in
standard error. the same column differ (P < 0.05); S.E., standard error.
158 M.-P. Gauvin et al. / International Dairy Journal 85 (2018) 153e158
isolates may also impede syneresis by causing flow resistance,
which is another factor that influences gel syneresis (Walstra, van
Dijk, & Geurts, 1985).
4. Conclusions
The main objective of this study was to determine the impact of
buttermilk MFGM fragments, independent from the impact of
casein micelles, on the rennet coagulation of milk. Isolates from
raw- and pasteurised-cream buttermilk and raw skim milk that
were composed of at least 50% proteins with a molecular mass
greater than 35 kDa (mainly MFGM proteins) were obtained.
Our results show that the RCB and PCB isolates interacted with
renneted casein micelles at an early stage and did not influence
their aggregation rate but that gel formation was impeded by these
components. However, more investigation is needed to explain the
mechanism of interference.
In parallel, we aimed to determine the influence of the heat
treatment of cream on the impact of MFGM fragments on rennet gel
formation. Our results did not show a significant impact of heat
treatment on the parameters measured. This finding may suggest
that the detrimental effect, observed by Morin et al. (2008), of
cream pasteurisation on the rennet gel formation aptitude of
buttermilk was instead the result of other factors, such as the
functionality of casein micelles or changes in the serum fraction of
buttermilk.
Interestingly, higher concentrations of caseins and whey pro-
teins were observed in the buttermilk isolates in comparison with
the RSM isolate. Conformational changes or aggregation of these
proteins may explain their sedimentation with the MFGM frag-
ments upon ultracentrifugation after sodium citrate treatment. At
this time, it is still unclear whether these components interact with
or simply sediment simultaneously with MFGM fragments.
Nevertheless, more investigation is needed to assess the impact of
churning on the functionalities of these proteins.
Acknowledgements
This work was funded by Novalait Inc., the Ministe re de l’Agri-
^cheries et de l'alimentation du Que
culture, des pe
bec (MAPAQ), and
the Fonds de recherche du Que
bec e Nature et technologies
(FRQNT). The first author would also like to thank the Canadian
Dairy Commission and Novalait Inc. for the PhD scholarship and
Andre
anne Lemay (trainee) for her help with some of the
experiments.
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