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Food Nutrition & Health Team, Food and Bio-based Products Group, AgResearch Grasslands, Palmerston North, New Zealand
Riddet Institute, Massey University, Palmerston North, New Zealand
c
AgResearch Grasslands, Palmerston North, New Zealand
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 13 October 2014
Received in revised form 19 January 2015
Accepted 21 January 2015
Available online 9 March 2015
Goat milk contains oligosaccharides that are structurally similar to human milk, which suggests that
caprine milk oligosaccharides (CMO) could mimic the benecial physiological effects described for
human milk oligosaccharides for infant health. This study aimed to characterise the nutrient
composition of New Zealand Saanen goat colostrum, regular milk and whey samples and to develop
an easily scalable approach to produce an enriched CMO product for use in in vivo experimentation. Goat
milk whey was processed by a combination of ultraltration, enzymatic hydrolysis of the lactose, solidphase extraction and rotary vacuum evaporation. An 80% recovery of the oligosaccharide fraction with
an enrichment of 24-fold was obtained when compared to the starting whey. Lactose was reduced to
2.5% of its initial concentration by enzymatic treatment. From 8 batches (approximately 1200 mL per
batch) of whey, 19 g of product were generated of which around 8% were oligosaccharides, 44%
monosaccharides, 44% lactose and 4% galacto-oligosaccharides.
2015 Elsevier Inc. All rights reserved.
1. Introduction
Goat milk production is a dynamic and growing industry that
is fundamental to the wellbeing of millions of people worldwide
Abbreviations: CMO, caprine milk oligosaccharides; HMO, human milk oligosaccharides; BMO, bovine milk oligosaccharides; GOS, galacto-oligosaccharides; HPLC,
high performance liquid chromatography; LCMS, liquid chromatographymass
spectrometry.
* Corresponding authors at: AgResearch Grasslands Research Centre, Tennent
Drive, Private Bag 11008, Palmerston North, New Zealand. Tel.: +64 6 356 8019;
fax: +64 6 351 8032.
E-mail addresses: caroline.thum@agresearch.co.nz (C. Thum),
nicole.roy@agresearch.co.nz (N.C. Roy).
http://dx.doi.org/10.1016/j.jfca.2015.01.022
0889-1575/ 2015 Elsevier Inc. All rights reserved.
31
Ten litres of goat whey were processed in 8 batches (approximately 1200 mL per batch). Each batch was subject to lactose
32
Table 1
Optimisation of adsorption and elution conditions utilised on porous graphitic
carbon chromatography.
Solutiona
Elution
Recovery (%)
Oligosaccharides
Contaminants
(glucose,
galactose
and lactose)
Ethanol
1 V, 20%
1 V, 40%
1 V, 50%
0.5 V, 50%
2 V, 50%
42
70
75
59
80
13
24
25
18
36
Acetonitrile
1 V, 2%
1 V, 5%
1 V, 40%
1 V, 40%
1 V, 40%
1 V, 50%
0.5 V, 40%
2 V, 40%
70
70
80
87
55
80
10
12
5
16
5
16
Adsorption
a
The adsorption and elution conditions were optimised with different solutions
of aqueous acetonitrile (2 or 5% adsorption; 40 or 50% elution) and ethanol (20%,
40%, 50%) and different elution volumes (0.5, 1.0, 2.0 column volumes (V)).
33
Table 2
Dry matter, lipid, protein and carbohydrate composition of goat colostrum, milk and whey.
Sample
pH
Lipid
Protein
Lactose
7
36
107
30
58
47
Colostrum
Milk
6.2
6.5
175
115
Whey
Ultraltration permeate
Permeate hydrolysed and centrifuged
Solid phase extracted nal product
4.7
4.2
7.5
58
29
28
2
12
<0.1
<0.1
<0.1
7
0.1
<0.1
<0.1
36
25
3
0.9
Glucose
Galactose
0.23
0.15
0.0025
0.0015
2
2
14
0.5
0.2
0.3
11
0.4
GOSa
Oligosaccharidesb
0
0
0.32
0.26
0
0
0.09
0.08
0.20
0.20
0.18
0.16
Milk and colostrum values are the average of single analysis of 4 different samples.
a
GOS concentration is based on the sum of the ion intensities (m/z 665 and m/z 827).
b
Oligosaccharide concentrations are based on the sum of ion intensities (m/z 503, m/z 632, m/z 648, and m/z 923).
c
Values are expressed in grams per litre and are the average of single analyses of eight different batches (whey to solid phase extract).
both bound to, and eluted from, the porous graphitised carbon
under the adsorption/desorption conditions used to bind and elute
the oligosaccharides and thus could not be separated from the
oligosaccharides during this step. The b-galactosidase successfully
converted 92% of the lactose to monosaccharides, reducing the
lactose level from 25.6 g L1 in the whey permeate to
0.9 g L1. However, the level of lactose was still greater compared
to the oligosaccharide levels (0.16 g L1), thus the need for further
purication. The inhibitory effect of higher concentrations of
galactose in batch systems on lactose hydrolysis have been
reported previously (Albrecht et al., 2014; Marino et al., 2011).
Higher conversion rates may be achieved by continuous systems
using immobilised b-galactosidase.
No GOS was identied in the goat colostrum, milk and whey
samples based on LC/MS characterisation of typical GOS masses
commonly detected by negative ion electrospray MS (at m/z 665,
827 and 989). However, low levels of GOS were detected by LCMS
in the hydrolysed permeate and nal product after 24 h of bgalactosidase incubation (Table 3). These may have been formed by
the galactosyl-transferase activity associated with b-galactosidase. An additional potential GOS ion at m/z 503 may have also
been produced by b-galactosidase activity, however it was not
possible to distinguish this ion from one already present in the
whey (retention time of 4.09 min, m/z 503.16). Optimisation of
hydrolysis time and b-galactosidase concentration was needed to
obtain a balance between lactose hydrolysis and GOS production.
Tests showed that if lactose hydrolysis was carried out for longer
periods, or with higher concentrations of enzyme, GOS was
produced at a higher rate than lactose was hydrolysed. Whey
incubated with b-galactosidase for 36 h, for example, increased
the lactose hydrolysis by 5% but also increased the production of
GOS by 50%.
b-galactosidase activity was stopped by raising the hydrolysed
permeate pH to 7.5 and decreasing the temperature to 4 8C. During
refrigeration a portion of the galactose (7.5%) produced by lactose
hydrolysis was precipitated and this was removed by centrifugation using Sorvall RC5 plus (Thermo Fisher, Langenselbold,
Germany) at 25,000 g for 10 min at 4 8C. After loading the
Table 3
Recovery rate of lipid, protein and carbohydrate during whey ultraltration, hydrolysis and solid phase extraction.
Sample
Volume (L)
Whey
Ultraltration permeate
Permeate hydrolysed and centrifuged
Solid phase extracted nal product
9.6
8.5
8.2
9.6
Protein
Lactose
Oligosaccharides
100
<0.1
<0.1
<0.1
100
1
<0.1
<0.1
100
62
7
2.5
100
85
76
80
a
Values are expressed in grams per litre and are the average of single analyses of eight different batches (whey to solid phase extract). Milk and colostrum values are the
average of single analysis of 4 different samples.
34
Fig. 2. LC/MS extracted ion chromatograms of (A) goat whey and (B) puried product, showing the ions with m/z 503.1 (3-galactosyl-lactose); m/z 632.3 (3-sialyl-lactose and
6-sialyl-lactose); m/z 648.3 (N-glycolylneuraminyl-hexosyl-lactose); 923.3 (disialyl-lactose); m/z 665.2 (GOS) and m/z 827.3 (GOS).
35
Table 4
Oligosaccharides concentration (g L1 in goat colostrum, regular milk, whey, hydrolysed whey and nal product using the LCMS method and standards.
Oligosaccharidesa
m/z 503
m/z 632
(30 -SL)
m/z 632
(60 -SL)
m/z 648
m/z 923
m/z 487
(20 -FL)
m/z 1071
m/z 665
m/z 827
Colostrum
Regular milk
Whey
Hydrolysed whey
Final product
4
4
8
8
8
0.08 0.02
0.08 0.01
0.05 0.02
0.04 0.01
0.03 0.02
0.05 0.02
0.06 0.01
0.04 0.02
0.04 0.01
0.04 0.02
0.07 0.02
0.06 0.02
0.04 0.02
0.03 0.01
0.03 0.01
0.04 0.02
0.04 0.01
0.06 0.02
0.05 0.04
0.05 0.03
0.05 0.004
0.01 0.008
0.002 0.001
0.001 0.001
Traces
Traces
0.002 0.001
Traces
Traces
Traces
0.03 0.02
Traces
Traces
Traces
Traces
0.04 0.01
0.05 0.01
0.05 0.01
0.03 0.01
a
CMO are represented by m/z 503, m/z 632, m/z 648, and m/z 923; m/z 487 and GOS by m/z 665 and m/z 827. Due to the lack of commercial standard, N-glycolylneuraminyl
lactose (m/z 648) concentrations were estimated based on 30 -sialyllactose (m/z 632) standard.
oligosaccharides in the samples was performed using commercially available standards of oligosaccharides reported in goat
(Martinez-Ferez et al., 2006b; Urashima et al., 1994, 1997) and
bovine milk (Aldredge et al., 2013) (Table 4). To illustrate the
prole of the main oligosaccharides present in the enriched
product, a typical MS spectrum is provided in Fig. 3. In this
spectrum, the most abundant ions (m/z 503, m/z 632 and m/z 648)
corresponded to the masses of the oligosaccharides galactosyllactose (a-30 - or b-60 -), sialyl-lactose (30 - or 60 -) and N-glycolylneuraminyl-lactose. Adduct ions, consistent with the covalent
linking of the neutral sugar galactosyl-lactose (m/z 503) to a
molecule of formic acid (46 u), at 549 m/z were also observed. It is
interesting to note that the formic acid adduct is only detected for
the neutral sugars, not the acidic ones. Traces of other possible
CMOs, previously described (Albrecht et al., 2014), lacto-Nneotetraose (m/z 706.27), lacto-N-novo pentaose (m/z 868.36)
and diglycolyl-lactose (m/z 955.33), were also observed in the
colostrum, regular milk, whey and the nal puried product.
Another feature of the MS prole were ions consistent with lactose
phosphate (m/z 421), another compound produced during lactose
and galactose metabolism (Benthin et al., 1994). Lactose
phosphate was detected in the regular milk (0.01 0.002 g L1),
whey (0.007 0.001 g L1) and in the puried product
(0.006 0.002 g L1). Lactose phosphate was rst detected in bovine
milk (Cumar et al., 1965) and, recently reported in caprine milk
(Albrecht et al., 2014).
A recent study has identied 40 different oligosaccharide
structures in goat milk and described the relative abundance of
both acidic and neutral oligosaccharides using ultra-performance
liquid chromatography (Albrecht et al., 2014). The results of this
current study use the relative intensities (peak areas) of extracted
mass peaks from the mass spectrometric analysis for the
quantication of CMO compared against the specic standards.
While it is recognised that the intensity of each ion is depended on
the ability of that particular molecule to ionise in solution,
Fig. 3. Mass spectrum of the product collected with a 40% acetonitrile solution from solid-phase medium. The spectrum, showing the main chromatogram peaks between the
retention time 512 min, was recorded using an LTQ linear ion trap mass spectrometer with electrospray ionisation in negative mode.
36
4. Conclusions
The present study is the rst to report NZ Saanen goat
colostrum, milk and whey composition. It also describes a multistep approach to process goat whey containing 0.3% oligosaccharides and produce an enriched CMO product with a nal CMO
content of 8% (w/w) and with an oligosaccharides prole similar to
the initial goat milk whey. Using this method eight, batches of
approximately 1.2 L of goat cheese whey produced approximately
19 g of product containing approximately 1.6 g CMO for use for in
vivo experiments. The major purication contaminants were
lactose, glucose and galactose. The method recovered approximately 80% of the original oligosaccharides present in the starting
material, cheese whey. These results suggest that goat whey from
camembert-type cheese manufacture is a rich source of oligosaccharides and the LCMS data suggested that there are some
common oligosaccharides between New Zealand Saanen goat and
human milk. Six out of seven predominant oligosaccharides,
previously described for goat milk, were putatively identied and
quantied. The high concentrations of sialyl-oligosaccharides
found in Saanen goat milk in New Zealand provide a useful source
for in vivo experimentation of their modes-of-action and to further
explore the potential health benets. Future studies are needed to
conrm and compare the oligosaccharides concentrations for
different goat breeds, stage of lactation and diet.
Acknowledgements
We thank Pauline Hunt for drawing Fig. 1 and Drs Karl Fraser,
Jolon Dyer and Alison J. Hodgkinson (AgResearch) for proof-reading
the manuscript. Caroline Thum acknowledges the Ministry of
Business, Innovation and Employment, New Zealand (C10X0907),
the Riddet Institute Core of Research Excellence (Core) and
AgResearch for the funding and the PhD Scholarship.
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