1/2006 - Potravinárstvo

cheesemaking properties of milk for A β-Lg. Several
reserchers found β-Lg AA genotype to be more suitable
for cheese production from the point of better renetability,
clotting capability and cheese yield. Milk with β-Lg
A allele showed a positive effect on curd formation and
curd firming. Curd from milk of β-Lg BB genotype was
evaluated as significantly softer (Rampilli et al., 1997).
While β-Lg BB genotype is associated with higher milk
yield, β-Lg AA and AB genotypes produce milk
with higher protein and casein content as well as cheese
yield (Garzon a Martinez, 1992).
On contrary, Pilla et al. (1995) indicate a positive effect
on the B variant β-Lg of clotting time. The highest yield
of curd saw Anton et al. (2005) with BB genotype in
sheep breeds Awassi and Lacaune. Korman et al.
(2002) argue that the productions of whey cheeses were
obtained better results with the AB genotypes than in milk
from sheep with homozygous genotypes.
The goal of the work was study the quality of sheep milk
as raw material for the dairy industry, characterize the
genetic polymorphism of milk proteins Slovak domestic
Improved Valachian sheep breed and to determine an
effect of β-lactoglobulin genetic variants on the total
milk protein, nitrogen distribution in the total protein
and technological properties of milk.
MATERIAL AND METHODS
Individual milk and blood samples were collected from
Improved Valachian sheep (75). The sampling was
performed once a month during pasture milking season
from May to September. Finally, the effects of lactation
stage were evaluated.
Individual milk samples were collected at the morning
milking and were divided into two parts. The first one
(30ml), used for the study of milk composition performed
by Milko Scan apparatus was collected into tubes
containing preservative agents Acidiol and the second one
was only cooled and held at a maximum temperature
of 10°C during the transport and was analyzed
immediately after arrival to the laboratory with the
aim to determine physico-chemical and technological
properties of milk.
The protein content was stated by infrared technique
using a Milko Scan FT 120 (FOSS Electric, Milcom
servis, Czech Republic) according to the Slovak
technical norm STN 57 05 36 „Determination of milk
composition by infrared absorbance analyzator“.
True protein nitrogen (TPN) and whey protein
nitrogen (WPN) were determined by the method of
Cvak et al. (1992). For determination of TPN, 10
grams of whole milk were precipitated with 18%
NaCl and 20-25% Almen’s Solution (4g of tannin
solubilised in 190 ml of 50% ethanol et 8 ml of 25%
acetic acid) and filtered through a nitrogen free KA5
filter (Reachem, Bratislava); three times washed with
distilled water and the precipitate retained on the
filter was dried at room temperature. The dried
precipitate retained on the filter was used for the
mineralization stage of the Kjeldal method. For
determination of WPN, 20 grams of whole milk were
Potravinárstvo
diluted with distilled water (40 ºC) in the proportion
of 1:3.3 (v:v), and precipitated with 10% acetic acid
and sodium acetate. A precipitant consisting of the
whey proteins passed through a filtrate paper. About
50 ml of the filtrate containing whey proteins was
evaporated using a mineralization digestion unit
Bloc-Digest 12 (J.P. Selecta, Spain) to obtain a final
volume of 15 ml used for nitrogen analyses. The
nitrogen content was measured using the Kjeldal
method following the EN ISO 8968-1:2001 (2002).
The casein content [g.100g -1 ] was calculated as
follows: Casein (CN) = true proteins (TP) [g.100g -1 ] –
whey proteins (WP) [g.100g -1 ]. The casein number (CNu)
was stated according to the following equation: Casein
number (CNu) [%] = CN [g.100g -1 ] / TP [g.100g -1 ] x 100.
The non-protein nitrogen (NPN) [g.100g -1 ] was expressed
as the difference between crude protein nitrogen (CPN)
and true protein nitrogen (TPN).
Total calcium was stated by the complexometric method of
Cvak et al. (1992). One ml of milk was diluted in the
distilled water in the proportion of 1:70; 5 ml of 5 M KOH
was added in the presence of the fluorexon indicator. The
titration was made with 0.01 M Normanal Chelaton III
(Reachem, Bratislava). The total calcium content was
calculated by the equation present in the method.
The pH of milk samples was checked at 20 ºC with the pH
meter MS 22 (Laboratory equipments, Praha).
Heat stability (alcohol number) was determined by the
titration with 96% ethanol according to Gajdůšek (1998).
Alcohol number expresses an ethanol consumption of
given concentration for fixed bulk of milk (2 cm 3 ) till
protein coagulation under the terms of the method.
Rennetability was determined by the method of Kažimír
a Gemeri (1993). 20 cm 3 of sample was equilibrated at 35
ºC when 1 ml of rennet (power of 1:400) (Chr. Hansen’s
hannilase powder, MG 2080, Denmark) was added. The
milk was stirred. The time till the creation of first curd
flakes was measured in seconds.
Genotyping of β-lactoglobulin by Restriction
Fragment Length Polymorphism- Polymerase Chain
Reaction (RFLP-PCR)
Genetic polymorphism of β-lactoglobulin gene
(alleles A and B) was analyzed by the RLFP-PCR
method by Anton et al. (1999) in the laboratory of
Department of Botany and Genetics, Constantine the
Philosopher University in Nitra.
The following primers were used for the
amplification of DNA:
LGB1 forward primer: (5'- CTTCCCACCCCCAGAG
TGCAAC-3')
LGB2 reverse primer: (5'- TGGGGAGTGGGGGTTC
CATGTT-3').
PCR reaction was performed in a 20-µl reaction
mixture containing 1x PCR reaction buffer, 2 mM
MgCl2, 0.2 mM dNTP mix, 0.5 µM primers, 0.5 U
Taq DNA polymerase (Invitrogen) and 1 µl of DNA
sample (± 100 ng) in thermocycler Primus. The PCR
conditions were applied as follows: 94°C 4 min., 31x
(94°C 30 s, 65°C 30 s, 72°C 30 s) and 72°C 10 min. The
length of the PCR product was verified on the 1% agarose
gel. Finally, a 217 bp DNA fragment was amplified.
ročník 4 59 1/2010