INFLUENCE OF RAW MILK QUALITY ON LACTOBACILLUS ACIDOPHILUS MULTIPLICATION AND PROBIOTIC YOGHURT PRODUCTION

Universitatea de ?tiin?e Agricole ?i Medicin? Veterinar? Ia?i
Lucr?ri ?tiin?ifice - vol. 52, Seria Zootehnie

INFLUENCE OF RAW MILK QUALITY ON
LACTOBACILLUS ACIDOPHILUS MULTIPLICATION
AND PROBIOTIC YOGHURT PRODUCTION

Eva Csutak

University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania
e-mail: szkkeva@yahoo.com

Abstract
Considering that the quality of raw milk is a prerequisite condition to obtain a good quality
probiotic yoghurt, our studies aimed the measurement of milk factors which can affect the
multiplication of probiotic lactic acid bacteria (LABs) Lactobacillus acidophilus (LA-5. The
probiotic strains Bifidobacterium BB-12 and Lactobacillus acidophilus LA-5-we used for trials- are
tested probiotics by Christian Hansen company. We studied comparatively raw and pasteurized
milk, their chemical composition and the correlations between the spontaneous microbial flora
(NTG) found in milk samples and the impact of this flora on the multiplication of LABs. We
investigated as well the effect milk proteins, added prebiotics( lactose, molasses) on pH and LAB
development, the influence of NTG (number of total germs), NCS (somatic cells number) in raw milk
before and after pasteurization, on lactic fermentations and LA-5( Lactobacillus acidophilus)
Generally multiplication of LA-5 strains was reversely correlated with NTG values. There is a
direct correlation between presence of prebiotics and probiotic bacteria activity.

Key words: yoghurt, probiotics, lactic acid, NTG, pasteurization

INTRODUCTION
replication, and/or pathogenic action of
Yoghurt is a long time known and certain enteropathogenes. To improve
appreciated dairy product, obtained continuously the quality of yoghurts,
traditionally by the spontaneous or induced preservation of probiotic characteristics and
lactic fermentation of milk. The the shelf-life of live LABs, with improved
microbiology of lactic-producing bacteria capacity of fermentation, are needed [9],
and the fermentation biochemistry and [10], [12], [17], [18].
technology of yoghurt is well documented
Among many strains, Lactobacillus
[1], [2], [3], [6], [13].
acidophilus is best candidates to be used,
The term “probiotic” is known since 1903
alone or in combinations as lactic fermenting
when the benefic actions of Lactobacillus
microorganisms with high probiotic activity
acidophilus strains were observed in human (Kailaspathy, 1997). An important factor
intestine, and the term of “prebiotic” is which influence the development and survival
known since 1961, and define the substances,
rate of probiotic LAB is the milk quality and
generally natural ingredients or
its bacterial flora. It is known that the quality
microorganisms which improve the intestinal
of raw milk in Romania is still an unsolved
equilibrium and defense against pathological
problem, since the number of total germs and
bacteria [4], [7], [11], [15].
of somatic cells found in milk is higher than
Yoghurt, by its high content in lactic acid
the permitted level in European Union (NTG
bacteria (LABs) possesses antimicrobial <100000/ml, NCS<400000/ml) [2], [3].
activity in vitro against a wide variety of
Considering that the quality of raw milk is
Gram-positive and Gram-negative bacteria, a prerequisite condition for obtaining a good
as well as some fungi. The exact cause of quality probiotic yoghurt, our studies aimed
inhibition is not known, but may be due to the measurement of main milk factors which
the antagonist action of LAB species which can affect the multiplication of probiotic-
prevent the adherence, establishment, forming bacteria Lactobacillus acidophilus

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Universitatea de ?tiin?e Agricole ?i Medicin? Veterinar? Ia?i
Lucr?ri ?tiin?ifice - vol. 52, Seria Zootehnie

(LA-5) We studied comparatively the raw and
with a pipette we measure 50 µl to a test-tube
pasteurized milk, the correlations between the
with bung. To the diluated yoghurt sample we
spontaneous microbial flora found in milk add 4 ml 3:1 H2SO4: H2O, and 100µl hydro-
samples and the impact of this flora on the kynon solution with etanol 12.5%.The test-
multiplication of probiotic bacteria. Added tube must be agitated ( shaked), and boiled in
prebiotics are influencing the multiplication of
a water-bath about 20 minutes (together with
LA-5, and in this way may be decreased raw
an etalon and a standard sample), until the
milk qualities effects.
color of sample will become yellow.

After cooling the content of test-tube, by
MATERIALS AND METHODS
photometry-on 405 nm- we determine the
The samples of cow milk originated from
lactic acid content.
the region of Harghita county and the tests
Etalon sample is prepaired from 50µl of
were made at NIZO, Netherlands and at S.C.
distillated water, and the standard solution
Gordon Prod company’s authorized lab. For
from 50-50 micro-liter of standard.The lactic
experiments we used Lactobacillus
acid content of product was tested a University
acidophilus (LA-5) strain provided by of Kaposvar, and at Gordon Prod’s lab.
Christian Hansen company. The media used
Bactocount IBCm is an automated
for the storage and determinations of instrument using flow cytometry (FCM) for
bacterial multiplication were MRS agar for the rapid enumeration of individual bacteria
LA-5 the nutritive, sorbitol agar (Sanimed) in raw milk. The milk is sampled and
used for the determination of NTG .
dispensed into individual vials manually. An
To make measurements, the raw milk, incubation reagent made up with a
after cooling, was inoculated with both clarification buffer, a proteolytic enzyme, and
bacterial strains at three dilutions (10-1, 10-2
a fluorescent marker are then added in
and 10-3) and incubated for 72 hrs. The manually in order to lyse the somatic cells,
counting of bacteria was made after 48 and solubilize the fat globules and proteins,
72 hrs of incubation. For LA-5 the incubation
permeabilize the bacteria and stain their
was made at 43°C. All samples were done in
DNA. The vials are then placed on an
duplicate. For preparing of probiotic yogurt incubator at 50 °C. The fluorescence marker
pasteurized milk on 95°C was cooled to intercalates rapidly and selectively into all
43°C, and inoculated with LA-5 probiotic the bacteria double-stranded nucleic acid.
strain. After 3 hours of incubation were The mixture is then sonicated manually
measured the parameters presented in results.
during the incubation on two different
To determine the NTG we used the occasions. The sonication process is used to
bacterial counter IBCM Bactocount and to help the chemical breakdown of the
count the NCS, we used the Somatos tester interfering particles, disrupt die remaining
NCS. Somatos tester used for NCS bacteria colonies to improve the detection of
determination is an equipment, based on individual bacteria and reduce the
difference of viscosity of milk with different background fluorescence. The cell debris,
number of somatic cells. The equipment was devoid of nucleic acid, are excluded from the
bought from Viola company, from Bucharest.
analysis.
The pH was determined using the lab pH
After the incubation period the mixture is
meter WTW315i. PH meter WTW 315i is then transferred manually from the
imported by Viola company, produced by incubation plate to the sample intake pipette.
WTW company. By photometry we The mixture is then transferred to the flow
determined the lactic acid and lactates from cytometer where the bacteria are aligned and
our samples (yoghurt, yoghurt with molasses).
exposed to an intense laser beam and
With added phosphoric acid the lactates fluoresce. The fluorescence signal is
are transformed to lactic acid.
collected by the optics, filtered, and detected
Form the prepaired samples we obtain a with a photo multiplier. The fluorescence
dilution 10-2, and from this diluated sample pulses intensity and height are recorded and
used as gating parameters. The sorted pulses

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Universitatea de ?tiin?e Agricole ?i Medicin? Veterinar? Ia?i
Lucr?ri ?tiin?ifice - vol. 52, Seria Zootehnie

are dien translated into individual bacteria
The main characteristics of raw milk
count after instrument calibration.
comparing with the pasteurized milk used in

experiments are presented in Table 1.and the
RESULTS AND DISCUSSION
composition of native proteins found in whey
The composition of milk samples (raw
originating from raw and pasteurized milk
and pasteurized) before to be inoculated are presented in Table 2. The fat, protein,
with probiotic strains:
lactose content of milk are measured in
percent, the microorganisms number in
CFU/ml(colony formating unit).


Table 1.
Main chemical parameters which characterize the milk samples (raw and pasteurized)

Aerobic
Total
Milk
Lactose
Fat
Density
psyhotrophic
TSR9
Phosphatase
protein
samples
%
%
Kg/l
microorganisms
(CFU/ml)
activity
%
(CFU/ml)
Raw milk
3.28
4.41
3.80
1.029
1.4*107 1.7*104 positive
Pasteurized
3.27 4.53 3.77
1.029 9.6*104 1.3*104 negative
milk

No significant differences between density, proteins, lactose and fat contents were
observed for raw vs pasteurized milk, but a significant decrease of aerobic psyhotropic
microorganims, TSR9 density.

Table 2.
Analysis of native protein fractions found in native whey originating from raw and pasteurized milk.

De-
?-
ß-
Native whey
BSA
Immuno
Total whey
natured
lactalbumin lactoglobulin
Proteins
(g/l)
globulin (g/l)
protein (g/l)
whey-
(g/l)
(g/l)
protein %
Raw milk
0.97
3.19
0.29
0.50
4.96
0
Pasteurized milk
0.95
2.96
0.18
0.27
4.35
12.2

Regarding the native whey proteins, found out that pasteurization at 95°C was not
significant decreases in BSA (bovine serum enough efficient and even after 95°C
albumin), immunoglobulin and total protein pasteurization, the presence of
were observed for pasteurized milk, as well microorganisms can affect the probiotic
the increase of denatured proteins (up to development.
12.2%) (Table 2).
Samples of raw milk we used for trials
Relations between the NTG found in are presented in table 3.
raw and pasteurized milk and their effects
The influence of the NTG found in raw
on the multiplication probiotic strain of and pasteurized milk and their effects on the
LA-5:
multiplication (expressed in % ) of LA-5 at
We observed that the initial milk NTG, concentrations of 0.025g/l is presented on fig.
and as well NCS influenced significantly the
1. and table 4. Incubation temperature of
probiotic bacteria evolution (Table 3). We milk: 43ºC.







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Universitatea de ?tiin?e Agricole ?i Medicin? Veterinar? Ia?i
Lucr?ri ?tiin?ifice - vol. 52, Seria Zootehnie

Table 3.
Determination of raw milk parameters used for evaluation of NTG influence on probiotic bacteria
multiplication

Fat
Nr. of
Name of collector or
NCS,
Protein
Lactose content,
content,
sample
collecting center
CFU/ml
content, %
%
%
1 Torok
Antal
76300
3.42
4.14
4.69
2 Turdeni
103000
3.46
4.08
4.71
3 Gordon’s
Farm
92000
3.38
4.08
4.67
4 Hadnagy
Antal
116000
3.44
4.09
4.69
5 Dobeni
96000
3.45
4.11
4.71
6 Farm
3
13200
3.42
4.16
4.72
7 Cobatesti
84000
3.41
4.08
4.68
8 Kiss
Antal
123000
3.39
4.09
4.71

Table 4.
Influence of raw milk NTG on LA-5 bacteria multiplication

NTG of
Number of LA-
NTG of milk saple by
Titrable
Nr. of
pasteurized
Lactic acid
5, by
IBCm BactoCount,
acidity,
pH,
sample
milk,
content, %
inoculation on
CFU/ml
Th°
CFU/ml
MRS agar
1 10621099
10200
71
0.69
2360000
4.79
2 1589781
8100
78
0.72
2640000
4.68
3 357856
6400
84
0.78
2730000
4.56
4 1702388
7600
81
0.73
2620000
4.65
5 989569
6350
82
0.76
2700000
4.59
6 91392
4850
88
0.88
2830000
4.55
7 272785
6100
76
0.72
2690000
4.67
8 114321
5150
84
0.87
2838000
4.61

Influence of raw milk total bacteria number on LA-5
multiplication
120

100
NTG of milk saple by
IBCm BactoCount,
80
CFU/ml/100000
NTG of pasteurized milk,
60
CFU/ml/10000
40
Titrable acidity, Th°
20
Lactic acid content,
%*100
0
1
2
3
4
5
6
7
8
9
pH*10
Variables value mentioned in
legend

Fig.1. Influence of raw milk total bacteria number on LA-5 multiplication

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Universitatea de ?tiin?e Agricole ?i Medicin? Veterinar? Ia?i
Lucr?ri ?tiin?ifice - vol. 52, Seria Zootehnie

Lactic acid content of probiotic yogurt with LA-5 is in direct relation with number of LA-5
bacteria, how it is presented on fig.2.

Relation betw een lactic acid content and
num ber of LA-5 from probiotic yoghurt
30
f

20
e
r
o
b
m
i
a
*
100000
10
er
Nu
act
0
b
0.69 0.72 0.78 0.73 0.76 0.88 0.72 0.87 0.68
Lactic acid, %

Fig.2. Relation between lactic acid content and number of LA-5 from probiotic yoghurt

For determining of total bacteria number
initial contamination of milk and the
bacteria number influence on LA-5 effectiveness of LA multiplication.
multiplication it was important selecting of

raw milks with different total bacteria CONCLUSION
number. It was important that chemical
The quality of raw milk in Romania
properties of milk ( fat content, protein needs to be improved, in order to obtain
content, lactose content) and its somatic cell
higher quality probiotic yoghurts. At his
number to be similar and low (lower than moment the number of total germs (NTG) is
200000 CFU/ml). For inoculation we used over the limits accepted by EU legislation,
pure probiotic strains of LA-5. Influence of and it can induce technological problems
total bacteria number on LA-5 multiplication
during production.
was measured off by determining of acidity,
In this context, the Gordon Prod company
pH,NTG, lactic acid content of final product. where our experiments were done, tried to
Raw milk’s NTG influences directly total
investigate the impact of existing NTG and
bacteria number of pasteurized milk. Acidity
NCS in raw and pasteurized milk on LAB
,NTG, pH, lactic acid content of probiotic probiotics (Lactobacillus acidophilus ). We
yoghurt obtained from raw milk with high found out that pasteurization at 95°C was not
NTG will be lower. Samples with NTG > enough efficient and even after 98°C
500000 CFU/ml after pasteurizing process pasteurization, the presence of
will have a total bacteria number > 6000 microorganisms can affect the probiotic
CFU/ml, and this concurrent microflora will
development. Small increases of milk protein
affect the multiplication of LA-5 probiotic content seem not to influence the LABs
strains.
development.
Generally, the NTG in raw milk at the
We investigated as well the effect milk
beginning of experiments were around 20 proteins on pH and LAB development, the
times higher than in pasteurized milk (where
influence of NTG, NCS in raw milk before
even after pasteurization at 95ºC, not all and after pasteurization, on lactic
microorganisms were destroyed). In time, fermentations and LA activities.
after inoculation with LA-5 the NTG
Our further studies will be directed on the
decreased significantly, around 23 times (for
addition of milk powder (industrial), alone or
raw milk) and 5 times (for pasteurized milk).
in combination with other prebiotics such as
It is obvious the competition between the inulins, or mono-and oligo-carbohydrates
from molasses.

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Universitatea de ?tiin?e Agricole ?i Medicin? Veterinar? Ia?i
Lucr?ri ?tiin?ifice - vol. 52, Seria Zootehnie

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