A

na Ref
N
ly Procedure eren
o
si ce
s
1 2.3 Samples were prepared and analysed for the total available sugar,
. total reducing sugar, cellulose, hemicellulose and glucose content as
Es described
tim elsewhere [22] by using DNSA method [31]; GOD-POD (glucose A
ati oxidase peroxidase) kit; PhenolSulphuric acid method [9,45]. com
on preh
of ensiv
car e
bo anal
hy ysis
dr of
ate algin
ate
2 2.4 One gram of dried sample was dissolved in 100 ml of 10% NaCl, cont
. stirred for 15 min and then filtered through Whatman filter paper No. ent
Es 1. Filtrateswere used as a crude extract for protein analysis and estimated and
tim by Bradford's [5] method. bioc
ati hemi
on cal
of com
pr positi
ote on of
in lefto
3 2.5 Total lipid was estimated as per Bligh and Dyer method [4]. For the ver
. determination of total lipid content, 10 g of sample was homogenised pulp
Es in chloroform:methanol (10:20 v/v) mixture. from
tim brow
ati n
on seaw
of eed
tot Sarg
al assu
lipi m
d wight
4 2.6 Samples were prepared as per Kumar et al. [22] and total phenolic ii
. compounds were estimated as per Malick and Singh [26]. Standard Savi
Es curve was prepared using pyro-catechol 10100 g/ml. ndra
tim Kum
ati ar,
on Dina
of band
tot hu
al Saho
ph o
en (201
oli 7)
c
co
mp
 ou
nd
s
5 2.7 Sample preparation and analysis for the total ash content, as well as
. various macro and micro element analysis were done as described
Es elsewhere
tim [22] by using Allen's [2] method.
ati
on
of
as
h
an
d
mi
ne
ral
co
nte
nt
5 To The TCP of the extracts was determined by means of the colori-metric Antio
tal method of Folin-Ciocalteu, with some modifications [16,17].Extracts (100 xida
co nt
nte L) diluted in EtOH (8 mg/mL) were mixed withthe Folin-Ciocalteu reagent capa
nt (750 city
of of
ph L, 10%, w/v aqueous solution),and maintained in darkness. After 5 min, Colo
en 750 mbia
oli n
c L of an aqueoussodium carbonate solution (6%, w/v) were added, and the seaw
co solutionwas allowed to stand for 90 min in darkness. Subsequently, eeds
mp theabsorbance at 765 nm was measured using a spectrophotometerUV- : 1.
ou Vis (Genesys 10, Thermo-Scientific). The results were expressedin mg Extra
nd gallic acid equivalents per gram of seaweed in dry basis (mg GAE/g db.). cts
s Gallic acid was used as standard for the calibration curve(y = 4.897x + obtai
(T 0.027, R2= 0.996), with concentrations between 0.02and 0.19 mg/mL. nedfr
CP om
) Grac
6 To The TCC of the extracts was evaluated following the method pro-posed by ilaria
tal Parsons and Strickland with some modifications [18,19]. Acetone at 90% mam
co (2 mL) was added to 5 mg extract, and the slurrywas centrifuged for 10 milla
nte min at 4C and 14,000 rpm. The precip-itate was washed successively ris
nt with acetone until discoloration,and all the supernatants were poured in a by
of volumetric flask, whosevolume was completed with acetone. The mea
car absorbance at 480 nmwas determined using a spectrophotometer UV-Vis ns of
ote (Genesys 10,Thermo-Scientific). The results were expressed as mg supe
ne carotenes pergram of seaweed in dry basis (mg C/g db.). -Carotene type I rcriti
s was used for the calibration curve (y = 70.524x 0.0053, R2= 0.996) cal
(T inconcentrations varying from 0.0005 to 0.0035 mg/mL. fluid
C extra
C) ction
Mni
ca
Ospi
na,
 Henr
y I.
Cast
ro-
Varg
as,
Fabi
n
Para
da-
Alfon
so
(201
7)
7 2.4 During the experiment, representative feed samples were collected, Effec
. milled through a 1 mm screen (Christy and Norris hammer mill, Ipswich, t of
Ch UK) and retained for chemical analysis. The proximate analysis dieta
em of the feed for dry matter (DM) was determined after drying overnightat ry
ica 105 C (16 h minimum). Ash was determined after ignition of a seaw
l knownweight of concentrate in amuffle furnace (Nabertherm, Bremen, eed
an Germany) at 550 C for 6 h. The neutral detergent fibre (NDF) content extra
aly was determined by the method of Van Soest, Robertson, and Lewis cts,
sis (1991) using the Ankom 200 Fibre Analyzer (Ankom technology, gala
of Macedon, NY, USA). The nitrogen content was determined as N 6.25 ctooli
the using the LECO TruSpec N instrument (LECO Corporation, MI, USA). gosa
fee ccha
din ride
g and
die vita
ts min
8 2.5 For antioxidant analysis, five gram of each meat sample was E
. homogenised (Stomacher 400 circulator, Steward Ltd., UK) with 50 ml supp
An phosphate buffer (0.05 M, pH 7) for 3 min. The resulting homogenate leme
tio was centrifuged at 4600 rpm at 4 C for 12 min (Rotanta 460 R, ntati
xid Zentrifugen, Hettich) and the supernatant was collected for further on
ant analysis. The collectedmeat supernatant and plasma sampleswere tested on
act for lipid peroxidation (LPO) assay and for the determination of total meat
ivitantioxidant status (TAS) by DPPH radical scavenging (1,1-diphenyl-2- quali
y picrylhydrazyl) and FRAP (ferric reducing antioxidant power) assays. ty
an A deproteinisation step was added in the case of plasma for DPPH para
aly analysis mete
sis where 0.1 ml of serum was added to 0.9 ml of methanol, vortexed rs in
for 30 s and centrifuged at 13,800 g for 60min (Eppendorf centrifuge, finish
5417R) to separate the proteins (Martinez, Valek, Rseti, & Rui, er
2006). pigs
9 2.6 The surface colour of the LD steaks was measured using a colorimeter Effec
. (CR-400 handheld Chroma meter, KonicaMinolta, Co., Oska, Japan). t of
Co The Chroma meter was calibrated on the CIE LAB colour system using a dieta
lou CR-A43 calibration plate (Dc: L* = 97.79, a* = 0.11, b* = 2.69). The ry
r L* value represents lightness and a* and b* values represent redness seaw
me and yellowness, respectively. Colour measurements of LD steaks were eed
as recorded on days 0, 4, 7, 11 and 14 of storage. These valueswere extra
ur recorded cts,
in duplicate (averaged from two locations) from each side of the cut gala
 em of LD for all pork samples. ctooli
ent gosa
1 2.7 Samples of LD steaks (10 g) were weighed out and stored under ccha
0 . MAP conditions (as described above) at 4 C for 14 days. Enumeration ride
En of total viability counts (TVC) was performed on day 0, 3, 5, 10 and 14 and
um of incubation. On each sampling day, 10 g sample of LD steak was vita
er stomached (400 lab blender, Steward and Co. Ltd. London, UK) with min
ati 90 ml maximum recovery diluent (MRD) in a sterile plastic filtered E
on stomacher bags for 3min. Enumeration of TVC was performed supp
of immediately, leme
tot where samples were serially diluted in MRD and 0.1 ml aliquots ntati
al were spread plated onto plate count agar media (PCA, Oxoid) in duplicate on
via and incubated at 30 C for 48 h. Following incubation, colonies on
bili were counted and log10 transformed. meat
ty quali
co ty
unt para
s mete
rs in
finish
er
pigs
1 2.6 The fatty acids were converted to their fatty acid methyl esters Multi
1 . (FAMES) by trans methylation of samples with, adding 1 ml of 2% varia
G methanolic HCl and heating for 1 h at 80 C te
C then adding 1 ml of anal
M 0.9% NaCl in H2O. It was followed by addition of 2 ml of hexane, ysis
S vortex mixing for 30 s and centrifugation at 2000 rpm for 5 min. of
FA The upper phase (hexane) was moved into fresh tube and dried fatty
M under N2 flow. 50 ll of hexane was added to dried FAMES and acid
ES 1 ll was injected to GCMS for analysis. The GCMS analysis of and
FAMES was carried out on a gas chromatographymass spectrometer bioc
(GC-7890B coupled with GCMS5977A MSD) equipped with hemi
an autosampler (G4513A) from Agilent Technologies (USA) using cal
a DB-Wax fused silica capillary column, 30 m cons
0.25 lm titute
0.25lm s of
(Agilent Technologies). Helium (99.9% purity) was used as the seaw
carrier gas with the column flow rate of 1.8 ml/min and the precolumn eeds
pressure of 20.90 psi. The column temperature regime to
was 50 C for 1 min, followed by a 25 char
C/min ramp up to 200 acter
C ize
followed by 18 min at 230 C. The mass their
spectrometer was operated pote
in electron compact mode with electron energy of 70 eV. Both the ntial
ion source temperature and the interface temperature were set at as
230 C. FAMES peaks were identified via biore
NIST (National Institute of sour
Standards and Technology) library. Post run analysis and quantified ce
by area normalization. for
biofu
el
and
 fine
che
mical
s
Priya
nka
Ver
ma
a,
Man
oj
Kum
ar a,
Giris
h
Mish
ra a,
Dina
band
hu
Saho
o
(201
7)
1 2.7 The amount of chlorophyll a, b, and total chlorophyll present in Multi
2 . seaweed was estimated by following the method of Seely et al. varia
Es (1972). The chlorophyll content was determined by using the te
tim Arnons (1949) equations: anal
ati Chlorophyll a lg=ml 12:7A663 ysis
on of
of 2:69A645 fatty
pig Chlorophyll b lg=ml 22:9A645 acid
me and
nts 4:68A663 bioc
Total chlorophyll lg=ml 20:2A645 8:02A663 hemi
The Carotenoids content was determined by using the following cal
equations Duxbury and Yentach (1956): cons
Carotenoids 4:2 OD452:5 titute
s of
0:0264 chl: a seaw
0:426 chl: b mg=g eeds
where, A = Absorbance at respective wave length = Volume of to
extract (ml), W= Fresh weight of the sample (g). char
acter
ize
their
pote
ntial
as
biore
sour
ce
for
biofu
el
and
fine
che
mical
s
Priya
nka
Ver
ma
a,
Man
oj
Kum
ar a,
Giris
h
Mish
ra a,
Dina
band
hu
Saho
o
(201
7)