STUDIES TO CHARACTERIZE METABOLOME SIGNATURE OF MILK COLOSTRUM OF MURRAH BUFFALOES USING NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY
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Date
2023
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ICAR-NDRI, KARNAL
Abstract
The present study was conducted with an aim to generate the baseline composition, to assess the
antioxidant and mineral level and metabolic signatures of colostrum, transition and mature milk
of Murrah buffalo (Bubalis bubalis). The composition data of fat, Protein, Lactose and SNF was
generated in 48 samples at 0- day colostrum, 2-, 4-, and 6-day transition milk and 50- and 100-
day mature milk of Murrah buffalo. The fat, SNF and protein (P<0.05) level were significantly
higher in colostrum as compared to other lactation days. In contrary, lactose percentage was
lowest in colostrum than transition and mature milk. To determine the antioxidant activity,
Colostrum, transition and mature milk samples of Murrah buffaloes representing 0-day, 2-day, 4-
day, 6-day and 100-day, were processed in duplicate for various antioxidant parameters such as
DPPH, FRAP, Catalase and GSH. The analysis revealed that all the antioxidants studied in the
present study have significantly (P<0.05) higher values in colostrum samples as compared to
transition and mature milk. The analysis of major (Na, K and Mg) and trace (Fe, Cu, Zn and
Mn) mineral elements through Atomic Absorption Spectroscopy has revealed that Na is present
significantly (P<0.05) in higher concentration as compared to the other mineral elements
included in the present study. For the metabolomics, total of 60 milk samples collected at 0, 2-,
4-, 6-, 50 and 100-days post calving representing colostrum, early transition and mature milk of
Murrah buffaloes were collected. The samples were freeze dried and subjected to 1D 1H 800
MHz NMR spectrometer equipped with a Cryoprobe (at 300 K) for the metabolic profiling of
colostrum, transition and mature milk. The NMR spectra revealed a total of 26 metabolites. The
metabolites such as lactose, guanidoacetate, glycerophosphocholine, glycine, betaine, choline,
NAG, O-PCh, UDP-Galactose, UDP-Glucose, Myo-Inositol, UDP-NAG, creatine, carnitine,
creatine phosphate, creatinine, O- acetylcarnitine, fucose, glutamate, threonine, lactate, lactulose,
leucine, malonate and maltose etc. could be identified with varying concentration in colostrum,
transition and mature milk. Partial least square discriminate analysis (PLS-DA) showed distinct
grouping of colostrum, transition and mature milk samples. The ranking of metabolites
responsible for distinct separation of three sample types (colostrum, transition and mature milk)
from each other was done based on variable importance in projection (VIP) score. The hierarchal
clustering demonstrates that the metabolome signature of colostrum samples (0 day) is distinctly
different from that of the rest of the samples. The analysis revealed that branched chain amino
acids leucine, isoleucine, and valine were significantly (P<0.05) high in colostrum samples.
Additionally, metabolic pathway analysis showed that the different metabolites were enriched
underamino acid metabolism, citrate cycle (TCA cycle), aminoacyl-tRNA biosynthesis, valine,
leucine and isoleucine biosynthesis. In summary, our study could successfully utilize NMR
technique to establish the metabolome signature of colostrum and milk samples of Murrah
buffaloes. Further, the study highlighted the higher abundance of metabolites in colostrum vis a
vis mature milk sample. Such information could provide tangible information on type of
metabolites available in colostrum and mature milk of Murrah buffaloes for further commercial
application.