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Effects Of Milk Preservation Using The lactoperoxidase System On Processed

Yogurt And cheese Quality

Article in African Journal of Food, Agriculture, Nutrition and Development · September 2008
DOI: 10.4314/ajfand.v8i3.19198 · Source: OAI

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 Volume 8 No. 3 2008
September 2008

EFFECTS OF MILK PRESERVATION USING THE

LACTOPEROXIDASE SYSTEM ON PROCESSED YOGURT AND
CHEESE QUALITY

Ndambi OA1, Kamga PB2, Imelé H2, Mendi SD2 and FA Fonteh3

Asaah Ndambi

Corresponding author Email: ndamboa@yahoo.com

1
IFCN Dairy Research Centre at the Department of Agricultural Economics,
University of Kiel. Schauenburger Str. 116, 24118 Kiel, Germany.
2
Institute of Agricultural Research for Development (IRAD) - Bambui. Department
of Food Technology and Post Harvest, B.P. 51 Bamenda, Cameroon.
3
University of Dschang, Department of Animal Production, B.P. 222, Dschang,
Cameroon.

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ABSTRACT
The lactoperoxidase system (LP-system) is an acceptable chemical method for raw
milk preservation, especially in rural areas where refrigeration facilities are absent to
farmers. Milk production in most African countries is dominated by small-scale
traditional production systems using low yielding local breeds. Therefore, processors
who operate in such situations must rely on small volumes of milk from many
farmers. Application of the LP-system prolongs the shelf life of raw milk and also
encourages grouping of farmers hence facilitating milk collection by processors. The
application of the LP-system is a recent preservation method for milk in Cameroon
whose efficiency has been proven. Therefore, need arose for further studies on the
influence of this method on milk processing as well as the quality dairy products.

The LP-system was activated by adding 10 ppm sodium thiocyanate and 8.5 ppm
sodium percarbonate to fresh milk. Yoghurt and Bambui cheese were processed
separately from treated and untreated (control) milk samples. Yogurt was produced
from both the treated and the control milk samples at 2%, 3%, 4% and 5% (v/v)
culture levels. Yogurt samples were analysed for acidity, protein content and dry
matter content while cheese was analysed for butterfat and moisture content.
Statistical tests were conducted by Analysis of Variance using the Fisher’s test.
Simple organoleptic assessments were conducted to compare yogurt and cheese from
the treated and the control milk. Activation of the LP-system delayed lactic acid
formation in yogurt during incubation and storage leading to increased energy
consumption during processing and an improved keeping quality during storage. LP-
system treatment reduced the overall organoleptic quality of yogurt while it improved
on that of Bambui cheese. Dry matter content and fat content of yogurt were not
significantly affected by LP-system treatment (P<0.05). LP-system treatment did not
also affect the moisture and fat content of cheese but slightly improved on its yield.

Key words: Cheese, lactoperoxidase system, milk, yogurt

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INTRODUCTION

Dairy production in tropical countries is hindered by accelerated milk spoilage due to

poor production and transportation facilities as well as high ambient temperatures [1,
2]. Special care needs to be taken in order to keep microbial activity of the milk down
to a minimum so as to improve its keeping quality as well as the quality of its
products [3]. The lactoperoxidase system (LP-system) has been found useful in
prolonging the shelf life of raw milk in countries where refrigeration facilities are
absent and its efficiency has been proven [4, 5].

The LP-system is a natural anti-microbial system in milk which results from

interaction between three components; the enzyme lactoperoxidase, thoicyanate ion
(SCN-) and hydrogen peroxidase (H2O2) [4, 6, 7, 8]. It has been shown to exert
bacteriostatic effects on both gram- positive and gram-negative bacteria including
psychrotrophes, which decrease shelf life of liquid milk at refrigeration temperatures
[9, 10, 11, 12]. It also exhibits antiviral properties and plays a role in degrading
carcinogens and in protection of animal cells against peroxidative effects [10].The
natural LP-system phenomenon in fresh milk is short-lived and a prolongation of its
effects is done by addition of SCN- and H2O2. The LP-system, when properly applied,
is harmless to mammalian cells. Instead, oxidation products of SCN- may protect
these cells against toxic effects of H2O2 [8]. The LP-system also occurs in saliva, milk
and tears [4, 13], where it is involved in the natural host defence system against
invading microbes [8], and in the human airway, where it plays a defensive role
against some bacteria like Pseudomonas aeruginosa, Burkholderia cepacia and
Haemophilus influenzae [14]. In addition to the LP-system, other non-specific factors
like lactoferrin and lysosyme also exist in milk. Lactoferrin plays an anti-microbial
role in depriving bacteria from iron ions and may protect the dry udder from infection
[10, 15].

Before now, very little work has been done on the LPS in Cameroon compared to
other tropical countries like China and Kenya. Some studies were done by Imele and
others in 2000 and by Fonteh in 2001 [16, 17]. Research by Fonteh was done using
different doses of activators and on individual farmers’ milks. With the FAO’s
activators where a recommended dosage is set for a fixed quantity of bulk milk,
further trials on this system were inevitable. Initial trials carried out by Imele and
others using the FAO activators were had shortcomings due to financial limitations
and shortage of laboratory equipment and chemicals. There was therefore need for a
more detailed study first of all on the effectiveness of the lactoperoxidase system as a
preservative for raw milk under Cameroonian conditions and secondly, to see how
this preservation would affect the dairy industry. A previous study in Cameroon
showed that the LP-system did not only improve on milk quality but also encouraged
the grouping of farmers and stimulated milk production among them and thereby
encouraging collection by processors [5].

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Due to the dominance of small-scale traditional production systems coupled with the
poor productivity of local breeds, the quantity of milk brought in for sale by each
farmer is usually very little [18, 19]. For dairy processing units to operate under such
conditions, a well structured dairy system must be built, such that it can collect small
quantities of milk from many farmers.

Therefore, need arose to study the quality of dairy products resulting from milk
collected from several small producers and preserved using the LP-system. Though
the effects of the LP-system has been broadly studied in some countries, very few
studies have gone further to show the effect of this preservation on the quality of
processed milk. Khalid and Mathur showed that yoghurt processed from milk with
smaller concentrations of activators (10 ppm for both) had a higher acceptability than
yoghurt from control milk, meanwhile yoghurt resulting from treated milk at higher
concentrations (20 ppm and 30ppm for both activators respectively) showed negative
results with regards to acceptability [20]. Hirano et al. showed that the gelation pH of
milk was increased by the LP-system which probably resulted from an increase in
protein hydrophorbicity [21].

This study was aimed at finding out the effects of milk preservation using the
lactoperoxidase system on the quality of yogurt and cheese prepared in the Western
Highlands of Cameroon.

METHODOLOGY
Milk collection and LP-system activation
Milk samples were collected in Sabga village in the North West Province of
Cameroon. This village falls in the Western Highland agro-ecological zone of
Cameroon. The Western highlands lie between latitudes 5o20’ and 7o North and
longitude 9o40’ and 11o10’ East of the Equator where two main seasons exist: the
rainy season (when this study was conducted), which runs from mid March to mid
November, and the dry season, which runs from mid November to mid March.
Rainfall ranges from 1500 – 2500mm while minimum and maximum temperatures
have means of 15.5oC and 24.5oC [22].

Laboratory analyses were conducted in the Food Technology and Post Harvest
laboratory of the Institute of Agricultural Research for Development (IRAD) Bambui,
Cameroon.

Milk was obtained from over 60 local cows (white Fulani, red Fulani and Gudali
breeds) belonging to 32 herders. Collection was done in the rainy season at a common
collection site where all farmers brought their milk. Individual farmers’ milks were
tested for spoilage and adulteration before bulking. This was done using the specific
gravity, clot-on-boiling test, alcohol test, acidity test and sensory tests. Milk which
failed to meet the required standards following the above mentioned tests was
discarded, while that of acceptable quality was bulked into a sterilised metal churn,
after filtering through a sterile cloth. Part of the bulk milk was kept for analysis of

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specific gravity, acidity, butterfat, protein and dry matter content. Bulk milk was
divided in two parts; one part was treated by activation of the LP-system while the
remaining part was kept untreated (control) under ambient temperatures. Activation of
the LP-system was done by first of all pouring sodium thiocyanate (10 ppm) solution
(supplied as such by distributors), followed by mixing for 30 seconds and then adding
sodium percarbonate (8.5 ppm) and mixing again for 2 minutes. Sodium thiocyanate
and sodium percabonate used were produced by BIO SERAE, France and obtained
from the FAO. The treated and the control bulk milks were processed separately into
yogurt and cheese.

Analysis of physico-chemical properties of milk

Bulk milk was analysed for butterfat, casein, dry matter, protein and solids-not-fat as
described below. All samples were analysed in duplicates.

(i) Specific gravity

A lactometer was used to measure the specific gravity of the bulk milk at room
temperature. Milk was poured into the lactometer jar and the lactometer was allowed
to slide gently into the milk until it reached equilibrium. The reading was taken
directly on the lactometer to the nearest 0.1 on the lactometer [23].

(ii) Butterfat (BF)

Butterfat was determined using the Gerber method [23]..

(iii) Protein and casein

Protein and casein were determined using the Formol Titration method [24].

(iv) Dry matter (DM)

Each milk sample (3mL) was pipetted into previously dried and weighed steel dishes.
The total weight of the dish with sample was taken and the samples dried in an oven
at 100oC for five hours. The samples were removed, cooled, weighed and dried again
in the oven for one hour. This procedure was repeated until successive recordings
differed by less than 0.004g. The dry matter was then calculated as follows:

% Dry matter = Final weight of dish + milk – Weight of empty dish x 100
Initial weight of dish + milk – Weight of empty dish

(v) Solids-not-fat (SNF)

The value for solids-not-fat was obtained by subtracting the butterfat (BF) value from
the dry matter (DM) value as follows:
SNF = DM – BF

Yogurt Manufacture and Analysis

Milk was pasteurised in a steel pot over gas flame at 85oC for 15 seconds and rapidly
cooled to 43oC. Dry original yogurt culture obtained from BIONIC Biotechnologishes,
Niebülle- Germany, containing lactic acid culture of Lactobacillus yoghurtii,
Lactobacillus acidophilus, Lactobacillus bulgaricus and Streptococcus thermophilus

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was used. The mother culture was prepared using 0.5g of powdered culture in 200ml
of milk, which was previously sterilised at 100oC for 30 minutes and cooled to 43oC.
Milk samples were inoculated at 2, 3, 4 and 5% (v/v), using the mother culture and
then incubated at 43oC until gelation occurred (between 4-6 hours) [25]. Extra yogurt
was prepared using 3% (v/v) ratios, from both the treated and the control milks, which
was used in the organoleptic assessment of yogurt. Both yogurt types were divided
into two main sample groups before incubation (Figure 1). One of the groups
consisted of five samples at each culture level and intended for analysis during
incubation, while the second group consisted of three samples at each culture level
and was intended for analysis during storage. During incubation, each sample was
only analysed once and then discarded. This was to prevent any effects from the
shaking of samples by repeated sampling. The yogurt samples were stored in a
refrigerator at 6 - 8oC and analysed weekly for acidity. All analyses were done in
duplicates.

Figure 1: Distribution of yogurt samples for analysis

Yoghurt
Yogurt from
from control
treated milk
milk Yogurt from control milk

2% 3% 4% 5% 2% 3% 4% 5%

I(5) S(3) I(5) S(3) I(5) S(3) I(5) S(3) I(5) S(3) I(5) S(3) I(5) S(3) I(5) S(3)

32 samples 32 samples

I = Yoghurt used for analysis during incubation.

S = Yoghurt used for analysis during storage.
Figures in brackets represent the number of samples

The Amount of lactic acid in milk and yogurt was estimated using the titratable
acidity method [26], while the protein and dry matter content of yogurt were obtained
in the same manner as for milk.

Organoleptic assessment of yogurt

A simple organoleptic assessment was done to compare consumers’ preference for
either yogurt from the treated milk or the untreated milk. Only yogurt inoculated at
3% level was used for both the treated and the control samples. Since commercial
yogurt in this area is usually sweetened, 60g of powdered sugar was added per litre of
yogurt from both treatments and carefully mixed, prior to the organoleptic
assessment. Thirty-two panellists were selected at random and offered both types of
yogurt to choose one which they preferred.

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Cheese Manufacture and Analysis

Bambui cheese from the treated and the control milk was manufactured (separately)
as follows:
Milk was pasteurised by heating to 72-74oC for 15 seconds and cooled rapidly to 30–
33oC [27]. Mesophilic culture containing Streptococcus lactis, Streptococcus cremoris
and Streptococcus diacetilactis was obtained from Laboratoire de ferments bacterie,
Ukraine and used at 1% (v/v) ratio. Powdered rennet obtained from Hansen
laboratory, Denmark was dissolved in distilled water and used at 3% concentration.
Curdling occurred within one hour. The curd was cut manually with a flat metal
spoon, cooked at 32oC for 40 minutes and then drained. Pressing was done using local
cheese presses and metal moulds, lined with sterilised cheesecloth. After about 18
hours of pressing, salting was done by immersing in 20% brine solution for 6-8
hours/kg of cheese. The cheese was left to dry in a refrigerator at (6-8 oC) for 4 days
and ripened for 40-60 days in cheese shelves under ambient conditions (temperatures
of 22-25oC and 85% relative humidity).

The fat content of cheese was determined using the Gerber method using 3g of cheese
[23]. while its dry matter was obtained using 2g of cheese in the same manner as for
milk.

Organoleptic assessment of cheese

Panelists were selected for the organoleptic assessment of cheese, based on their
familiarity with the product. Eighteen panelists who had the habit of consuming
cheese in their homes were selected. This is because cheese consumption was not as
common as yoghurt consumption and hence, random sampling was not possible. The
test was simpler than standard cheese tests and did not require trained cheese experts.
It differed from the tests by Kameni et al, for example where a grading scale of 1-10
was used to assess cheese in the same region [28].

Statistical analysis
Data was submitted to Analysis of Variance using the Fisher’s test. Means of cheese
yield and composition were compared using Fisher’s least significant difference
(LSD).

RESULTS
Physico-chemical properties of bulk milk

The results of the physico-chemical analysis of bulk milk are shown on Table 1.

Effects of LP-system on acid development in yogurt during incubation

All yogurt samples from the control milk had higher acidity values than those of the
treated milk (Figure 2). At the sixth hour of incubation the lactic acid content in

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yogurt from the treated milk was 11.36, 10.71, 18.18 and 9.68% lower, as compared
to the control yogurt samples at 2, 3, 4 and 5% culture levels, respectively.

Figure 2: Effect of LP-system on acid development in yogurt during incubation

Yogurt acidity during incubation

0,6

0,55

Ctr-2% LPS-2%

0,5
Ctr-3% LPS-3%

Ctr-4% LPS-4%
0,45
Ctr-5% LPS-5%
Acidity (% lactic acid)

0,4

0,35

0,3

0,25

0,2

0,15
1 2 3 4 6
Incubation duration (hrs)

NB: The commas on the Y-axis replace the decimal point.

Effects of LP-system on Acid development in yogurt during storage

There was already a difference in acidity of yogurt during the first hour of incubation,
which varied from 0.18% lactic acid in the LPS-2% sample to 0.25% lactic acid in the
Ctr-5% sample. During storage, the acidity of individual yogurt samples increased
progressively during the first three weeks, as shown in Figure 3. However, there was
no significant difference (P<0.05) in acidity between the treated and the control
samples. At the end of the second week of storage, nine out of twelve (75%) control
samples produced off-flavours and gases indicating spoilage. Meanwhile all the
treated samples were still good. At the end of the third week of storage, only two of
the treated samples were spoilt whereas all the control samples were already spoilt.

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Figure 3: Effects of LP-system on Acid development in yogurt during storage

Yogurt acidity during storage

1,30

1,25

Ctr-2% LPS-2%

1,20
Ctr-3% LPS-3%

1,15 Ctr-4% LPS-4%

Ctr-5% LPS-5%
Acidity (% lactic acid)

1,10

1,05

1,00

0,95

0,90

0,85

0,80
1 2 3
Storage duration (days)

NB: The commas on the Y-axis replace the decimal point.

Effects of LP-system on protein and dry matter of yogurt

Table 2 shows the protein and dry matter content of yogurt samples. The percentage
of protein and dry matter were not significantly different (P< 0.05) between yogurt
samples from the treated milk and those from the control milk. However, the dry
matter content of yogurt from the treated milk samples was slightly higher than that
from the control milk samples at each level of culture concentration.

Organoleptic assessment of yogurt

From the simple organoleptic assessment, 18 (56.25%) panellists preferred yogurt
from the control milk, 5 (15.63%) didn’t find any difference between both types,
while the remaining 9 (28.12%) panellists preferred yogurt from the treated milk. This

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indicates that the consumer preference was higher for yoghurt from control milk than
that from treated milk.

Effects of LP-system on Bambui cheese

From the Table 3, we see that the average yield of Bambui cheese was 12.77kg/100L
of milk and 13.62kg/100L for the control and the treated milk, respectively. After
pressing (before ripening), the yield of cheese obtained from the treated milk was
significantly higher (P<0.05) than that of cheese from the control milk. After ripening,
there was no significant difference between the yield of cheese from the treated milk
and that from the control milk (P<0.05), though the yield of treated milk was higher
by 6.39 %.

The moisture and fat content of cheeses from the treated and the control milks were
not statistically different (P< 0.05). The organoleptic grades of cheeses are illustrated
in Figures 4 and 5.

Figure 4: Organoleptic assessment on texture of cheese

Cheese from control milk Cheese from treated milk

Soft Soft
17% 17%
Granular
Hard
33%
33%

Pasty
33%
Pasty
Granular 50%
17%

Figure 4 shows that 33 % of panelists (n = 18) graded the texture of cheese from the
control milk as pasty, 33 % graded it as “soft”, 17 % graded it “granular” and 17 %
graded it “hard”.

Figure 5: Organoleptic assessment on odour of cheese

With respect to odour, 50 % of panellists found cheese from the control milk to have a
strong odour (Figure 5), while 33 % found it not pronounced and 17 % found it mild.
Generally, the panellists appreciated the milder odour in the treated samples than in
the control samples.

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Finally, 61% of panellists gave “EXCELLENT” as the overall grade of cheese from
the treated milk while only 39% graded the cheese from the control milk as excellent.

DISCUSSIONS
The dry matter and fat content of 13.4% and 4.2% respectively were similar to those
of the lower acid development in the treated yogurt samples during incubation shows
that LP-system has an inhibitory effect on lactic acid bacteria which retarded acid
formation in the treated milk. These results conform to those of Khalid and Musad
[30], who also found reduced acidity and prolonged incubation period for yogurt
produced from the treated milk. Hirano and others suggested that this effect on
gelation was due to protein hydrophobicity [21]. The delayed acid development
causes an increase in production time which entails higher use of energy and hence
higher cost of production. This effect is undesirable to processing plants especially as
energy is becoming limited and more expensive.

The delayed acid development during storage of yogurt from the treated milk was also
noticed by Nakada and others [31]. These authors showed treatment of milk by the
LP-system resulted to yoghurt retained its acceptable quality for at least two weeks,
during storage.

Though there was no significant difference in protein and dry matter content of yogurt
from the treated and the control milk, these values were higher for yogurt from the
treated milk than for yogurt from the control milk at all culture levels. These results
conform to those obtained by Kumar and Mathur [32], who also found a higher dry
matter content in the treated yogurt. The higher dry matter content probably results
from the inhibitory effects of the LP-system on bacteria that degrade milk solutes.

The reduced “thickness” in yogurt was also noticed in previous studies, where it was
shown that milk treatment with the LP-system resulted to a reduction in hardness and
apparent viscousity of yogurt. The same authors suggested that this effect was caused
by the action of OSCN- (hypocyanite ion) on milk proteins [30]. Unfortunately, due to
lack of appropriate apparatus, the viscosity of the yogurt could not be determined in
this analysis. In order to improve on yogurt “thickness” little amounts of powdered
milk could be added to the treated milk during processing, for a better yogurt
viscosity. The simultaneous used of reconstituted powdered milk and fresh milk is
already practiced by processing units especially in the dry season when fresh milk is
limited in supply. Therefore this might not pose a problem with regards to technical
feasibility.

The moisture and fat content of Bambui cheese from the treated and the control milk
were not significantly different, showing that LP-system treatment has no influence
on the chemical composition of cheese. Other authors also found out that LP-system
treatment did not affect cheese composition [33]. Organoleptic assessment of cheese
showed that the milder odour (which was also found in other studies) and pastier
texture of cheese from the treated milk improved its quality and hence consumer

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preference [33]. This milder odour of cheese was of much importance and could
encourage consumption in such areas where cheese consumption is less common
because people do not appreciate its strong odour. Unlike in Cameroon and probably
many other African countries where locally produced cheeses are not standardised,
grading is a crucial issue in USA and standards are set for different cheeses. The
grading process is more complicated and takes several aspects into consideration.
Chedder cheese for example, is graded in Wisconsin following four major criteria;
flavour, texture, colour and appearance. The flavour is graded using 17 different
characteristics: feed, acid, flat, bitter, fruity, metallic, sour, whey-taint, yeasty, malty,
old milk, onion, weedy, sulphide, barny, and rancid. The texture of cheddar cheese is
graded on 13 characters, the colour on eight and the appearance on 18 characters.
Grading is done by filling tables where each of the above characters is judged using
the following grade attributes: (a) “Definite” meaning that the trait is not intense but is
detectable in the cheese being graded. (b) “Pronounced” means the trait is sufficiently
intense as to be easily identified. (c) “Slight” means the trait is detected only upon
critical examination. (d) “Very slight” means the trait is detected only upon very
critical examination [34]. In Ireland, the maximum scores for flavour, texture and
appearance of cheddar cheese are 45, 40 and 5 respectively. Meanwhile, for cheese to
be acceptable for commercial purpose, it must meet a minimum grade of 38 and 31
for flavour and texture respectively [35].

CONCLUSION

Activation of the LP-system system delayed lactic acid formation in yogurt during
incubation and storage leading to longer incubation duration during processing and a
longer yogurt shelf life during storage. LP-system treatment reduced the organoleptic
quality of yogurt while it improved on that of Bambui cheese. The moisture content
and fat content of cheese were not affected by LP-system treatment. Application of
the LP-system could be beneficial to the processor since his products will last longer.
However the delayed acid development during yogurt incubation might increase his
processing cost.

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Table 1: Physico-chemical properties of bulk milk

Parameter Value

Specific gravity 31.8 (SD 0.2)

Butterfat (%) 4.20 (SD 0.06)

Protein (%) 4.42 (SD 0.05)

Casein (%) 3.51 (SD 0.03)

Dry matter (%) 13.40 (SD 0.91)

Solids-not-fat (SNF) (%) 9.20 (SD 0.50)

Table 2: Effects of LP-system on protein and dry matter of yogurt

Percentage Culture
2% 3% 4% 5%
Control LP-S Control LP-S Control LP-S Control LP-S

Protein 4.61 5.18 4.99 4.80 4.80 4.42 4.03 4.80

(%) (SD 0.02) (SD 0.07) (SD 0.05) (SD 0.03) (SD 0.08) (SD 0.04) (SD 0.02) (SD 0.03)

Dry 11.89 12.25 12.28 13.00 12.59 12.66 12.30 12.58

matter (%) (SD 1.25) (SD 1.54) (SD 1.46) (SD 1.67) (SD 1.55) (SD 1.32) (SD 1.39) (SD 1.34)

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Table 3: Effects of LP-system on yield, moisture content and fat content of cheese

Parameter Control Treated

Yield before ripening

12.77a (SD 1.72) 13.62b (SD 2.04)
(kg cheese/100L milk)
Yield at consumption
10.80 a (SD 1.02) 11.49 a (SD 1.43)
(kg cheese/100L milk)
Moisture content (%) 33.67 a (SD 2.12) 33.59 a (SD 2.15)

Fat content (%) 20.25 a (SD 1.24) 20.38 a (SD 1.32)

a,b = Letter superscripts bearing different letters are significantly different (P<0.05)

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