Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2017
Volume 20
Issue 1
Topic:
Food Science and Technology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Pakulski T. , Bagnicka E. 2017. INFLUENCE OF MANUFACTURING TECHNOLOGY ON AMINO ACID COMPOSITION OF SOFT WHITE UNRIPENED CHEESE FROM SHEEP MILK, EJPAU 20(1), #04.
Available Online: http://www.ejpau.media.pl/volume20/issue1/art-04.html

INFLUENCE OF MANUFACTURING TECHNOLOGY ON AMINO ACID COMPOSITION OF SOFT WHITE UNRIPENED CHEESE FROM SHEEP MILK

Tadeusz Pakulski1, Emilia Bagnicka2
1 Experimental Station Kołuda Wielka, National Research Institute of Animal Production, Janikowo, Poland
2 Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzębiec, Poland

 

ABSTRACT

The aim of the study was to determine the effect of the production technology of soft white unripened cheeses from sheep milk on amino acid compositionof protein. Cheeses from the milk of Coloured Polish Merino sheep were made using four methods: (I) “traditional” rennet method – control; (II) calcium-rennet method; (III) acid-rennet method; (IV) acid-rennet method combined with heat treatment of the curd. The method of manufacturing influenced the content of solids, minerals, solids not fat, essential and non-essential amino acids, protein composition, and energy value of the product. Cheese I contained more solids, ash and solids not fat and had a higher energy value. Cheese III had the lowest mineral content. The highest content of essential and non-essential amino acids was obtained per kg of the cheese made by method II. In 100 g of protein from cheeses made by methods II and III there was more glycine, phenylalanine, tyrosine, lysine, methionine and cysteine, and less arginine compared to methods I and IV. Tryptophan was the limiting amino acid for WE and MH protein standards. EAA content of cheese protein was 41.66 (I), 45.26 (II), 44.41 (III) and 42.67 g/100 g (IV).

Key words: sheep milk, cheese, manufacturing method, composition, amino acids.

INTRODUCTION

The main protein in the milk of sheep and other ruminants is casein, which constitutes 76 to 80% of all proteins; the other proteins are β-lactoglobulin, α-lactalbumin, albumins and whey proteins [12]. The commonly used cheese making technologies involve rennet precipitation of casein from milk and, generally, the loss of other milk proteins with whey.Compared to other milk proteins, casein has a lower nutritive value; a much higher value is characteristic of whey proteins [7]. The methods were developed which allow cheeses to be produced from all cow milk proteins [9, 18], from sheep milk as well [10, 11]. However, no studies are available concerning the amino acid composition of cheeses produced from sheep milk.

The aim of the study was to determine the possibility of using different methods to produce soft unripened cheeses containing also whey proteins from sheep milk, and to determine if the applied technology has an effect on cheese composition and nutrititional value.

MATERIAL AND METHODS

The milk was obtained between February and April 2011 from Coloured Polish Merino sheep. The sheep were milked mechanically twice a day. Milk samples were taken and cheeses were manufactured four times at 7- to 10-day intervals. The soft unripened cheeses were made using four methods: (I) traditional rennet method, with milk pasteurization at 78°C  and coagulation of proteins generally confined to casein only (bundz making technology) [8]; the other methods allowed for all milk proteins to be coagulated: (II) calcium-rennet method, in which part of milk proteins is precipitated by a calcium chloride aqueous solution at 92–95°C and the rest by rennet (known as serwit) [3]; (III) acid-rennet method, involving long-term rennet coagulation of pre-acidified milk at reduced temperature (curd cheese) [based on the technology of sheep cheese Erlauftaler; 8]; (IV) acid-rennet method combined with heat treatment of the curd (curd cheese) (Patent No PL 405282-A1).

It was assumed that the experimental cheeses made with methods II, III and IV will also contain milk whey proteins. Control cheese was produced as follows: pasteurized milk was brought to 34–36°C and renneted after adding calcium chloride; the curdled milk was salted, the curd was broken and mixed; after whey was expelled, the resulting cheese mass was transferred to a cheese cloth and pressed for 20–24 h to remove whey. In method II, milk proteins were precipitated in two stages: in the first stage, part of milk proteins was precipitated as a result of their interaction with calcium chloride by heating the milk to 92–94°C, adding calcium chloride aqueous solution (2/3 of the dose, i.e. 4 g of CaCl2/10 kg milk) and mixing, and holding at this temperature for 2–3 minutes until the first signs of clotting (flocculent particles); in the second stage, milk was cooled to 36–32°C, adding the rest of calcium chloride, and renneted; the procedure continued as with method I. Acid-rennet cheese (III) was produced as follows: following pasteurization, milk was brought to ≤30°C, adding calcium chloride, and acidified for 2 h with homofermentative mesophilic cultures, adding rennet, and left for long-term coagulation (up to 20 h) at 20–30°C; The curd obtained was cut, mixed and transferred to a cheese cloth to drain the whey. No salt was added with this method. In method IV, pasteurized milk was brought to 28–30°C, adding calcium chloride, and acidified for 2 h with the same bacteria cultures as with method III, after which acidified milk was heated to 32°C and renneted. The curd obtained was salted, broken, and mixed, after which it was left at constant temperature for 30 minutes and gradually heated to 38°C until small, firm and elastic grains were obtained. Cheese mass was placed on a sieve, rinsed with ice-cold water (4–6°C), transferred to a cold store room for 2–3 h, adding pasteurized cream, and packed. Milk and cheese samples were collected to determine basic composition and amino acid content. Basic composition of milk was determined using a MilkoScan-133B device. Cheeses were analysed for the content of solids – by drying at 105°C; ash (minerals) – by burning in an electric furnace; nitrogen – by the Kjeldahl method; fat – using the Soxhlet method; and solids not fat – from the difference between solids and fat. The amino acid content of milk and cheese was determined chromatographically in acid hydrolysates; cystine/cysteine in acid hydrolysates after oxidation (procedure SOP M.004 ver. 6 of 4 June 2008); and tryptophan in alkaline hydrolysate (SOP M.006 ver. 5 of 21 February 2008) using INGOS AAA400 amino acid analyser [1, 14]. The nutritional quality of cheese proteins was evaluated by calculating the chemical score (CS) as described by Block and Mitchell [2] for whole egg (WH) and mature human (MH) standards [15]. The results obtained were analysed by one-way analysis of variance using STATISTICA ver. 8.0.

RESULTS

Sheep milk used to make different cheeses did not differ in basic composition, and in solids protein and fat content (Tab. 1). Some differences were found in protein composition of the milk used for cheese making according to method III, because it contained fewer amino acids threonine, glycine, alanine, lysine, tyrosine and phenylalanine in relation to the milk used in method IV as well as less serine and lysine compared to that used in method II (Tab. 2). However, there were no differences when the content of amino acids was expressed as their percentage in total amino acids (ΣAA = 100%) (Tab. 3). Moreover, the differences in several amino acids contents in processed milk have not been preserved in cheeses obtained from it. In cheeses the differences related to the content of the other amino acids.

Table 1. Composition of sheep milk processed into four type of cheeses [content in 100 g]
Trait
Type of  cheese:
SEM
I
II
III
IV
N
4
4
4
4
Solids [g]
20.43
20.75
20.34
20.73
0.008
Crude protein [g]
6.76
6.92
6.90
6.89
0.006
Fat [g]
8.23
8.50
7.97
8.49
0.022
Lactose [g]
4.75
4.63
4.74
4.66
0.006
SNF* [g]
12.20
12.25
12.37
12.24
0.004
Energy value [kcal]
119.5
122.0
117.8
121.9
0.013
Protein/fat ratio
0.824
0.823
0.870
0.817
0.022
Protein/solids [%]
33.08
33.38
33.95
33.26
0.009
Fat/solids [%]
40.23
40.87
39.18
40.87
0.014
Protein/SNF [%]
55.36
56.48
55.81
56.26
0.004
* SNF – solids not fat


Table 2. Amino acid composition of sheep milk processed into four type of cheeses [g/kg]
Amino acid
Type of  cheese :
SEM
I
II
III
IV
N
4
4
4
4
Asparagine [g/kg]
5.90
6.01
5.70
6.03
0.015
Threonine [g/kg]
3.07
3.17
2.94b
3.29a
0.018
Serine [g/kg]
4.09
4.32a
3.81b
4.28
0.020
Glutamine [g/kg]
16.85
17.65
16.09
17.80
0.017
Proline [g/kg]
8.22
8.68
7.87
8.76
0.018
Glycine [g/kg]
1.39
1.43
1.32b
1.48a
0.018
Alanine [g/kg]
2.90
3.00
2.77b
3.06a
0.016
Valine [g/kg]
4.97
5.25
4.76
5.25
0.018
Isoleucine [g/kg]
3.80
3.98
3.62
4.00
0.017
Leucine [g/kg]
7.65
7.99
7.26
8.04
0.018
Tyrosine [g/kg]
3.29
3.42
3.12b
3.44a
0.016
Phenylalanine [g/kg]
3.46
3.57
3.30b
3.62a
0.015
Histydine [g/kg]
1.85
1.86
1.79
1.93
0.024
Lysine [g/kg]
5.81
6.09a
5.53b
6.10a
0.016
Arginine [g/kg]
2.12
2.21
2.01
2.12
0.016
Cysteine [g/kg]
0.50
0.53
0.50
0.54
0.026
Methionine [g/kg]
2.18
2.24
2.14
2.19
0.029
Tryptophan [g/kg]
0.51
0.51
0.50
0.52
0.011
ΣAA sulphur [g/kg]
3.19
3.28
3.14
3.24
0.024
ΣEAA [g/kg]
39.21
40.82
37.47
41.04
0.016
a, b –  values in rows with different letters differ significantly (P≤0.05)
ΣAA – sum of sulphur amino acids = cysteine + methionine + tryptophan
ΣEAA – sum of essential amino acids


Table 3. Proportion of amino acids in their sum determined in milk processed into four type of cheeses [%]
Amino acid
Type of  cheese:
SEM
I
II
III
IV
N
4
4
4
4
Asparagine [%]
7.52
7.34
7.60
7.32
0.009
Threonine [%]
3.91
3.87
3.92
3.99
0.006
Serine [%]
5.21
5.27
5.08
5.18
0.007
Glutamine [%]
21.46
21.54
21.45
21.56
0.004
Proline [%]
10.46
10.60
10.48
10.61
0.004
Glycine [%]
1.76
1.74
1.75
1.80
0.008
Alanine [%]
3.68
3.66
3.69
3.72
0.005
Valine [%]
6.33
6.41
6.35
6.37
0.004
Isoleucine [%]
4.83
4.85
4.83
4.85
0.005
Leucine [%]
9.73
9.75
9.68
9.75
0.005
Tyrosine [%]
4.18
4.18
4.16
4.18
0.007
Phenylalanine [%]
4.41
4.36
4.41
4.39
0.007
Histidine [%]
2.36
2.27
2.38
2.36
0.024
Lysine [%]
7.40
7.43
7.37
7.39
0.004
Arginine [%]
2.70
2.71
2.67
2.58
0.013
Cysteine [%]
0.64
0.65
0.66
0.66
0.020
Methionine [%]
2.77
2.74
2.86
2.66
0.025
Tryptophan [%]
0.65
0.63
0.66
0.63
0.013
ΣAA sulphur [%]
4.06
4.02
4.18
3.95
0.020
Σ EAA [%]
47.21
47.14
47.17
47.23
0.002
ΣAA – sum of sulphur amino acids = cysteine + methionine + tryptophan
ΣEAA – sum of essential amino acids

Cheese making method affected the composition of cheeses (Tab. 4). Control cheese (I) contained more solids in relation to cheeses made by methods II, III and IV. Mineral (ash) content was the highest in cheese made by method II and the lowest by method III. There were no differences in protein and fat contents between cheeses. The solids not fat content was the highest in control cheese, slightly lower in cheese II, and much lower in cheeses III and IV. The energy value of cheeses was highest for control cheese, slightly lower and similar for cheeses II and III, and lowest for cheese IV. There was a tendency for lower protein to fat ratio in cheese made by method III, while in the others it was similar and close to unity.

Table 4. Composition of four type of cheeses [g in 100 g]
Component
Type of cheese:
SEM
I
II
III
IV
N
4
4
4
4
Solids [g]
43.03a
41.87
38.54
36.48b
0.027
Ash [g]
2.44aa
2.63A
1.28Bb
2.42a
0.085
Crude protein [g]
18.25
18.19
15.94
15.45
0.030
Fat [g]
18.95
18.63
18.89
15.87
0.031
SNF [g]
24.08a
23.24a
19.65b
20.61
0.031
Energy value [kcal]
255.46a
248.48
241.65
214.22b
0.028
Protein/fat ratio
0.966
0.982
0.847
0.977
0.026
Protein/solids [%]
42.42
43.42
41.39
42.42
0.012
Fat/solids [%]
44.01A
44.50b
48.92Ba
43.54A
0.016
Protein/SNF [%]
75.76A
78.20
81.05B
75.14A
0.010
a, b – values in rows with different letters differ significantly (P≤0.05)
A, B –  as a
bove for P≤0.01

Cheese made by method II contained more essential and non-essential amino acids then cheeses made by III and IV methods. The control cheese contained more arginine compared to cheeses III and IV, and more alanine, cysteine and methionine compared to cheese IV; a significant difference was also found in methionine level between cheeses III and IV. Cheese IV had a lower total content of sulphur amino acids compared to the other cheeses (Tab. 5).

Table 5. Amino acid content in curd cheeses [g/kg of cheese]
Amino acid
Type of cheese:
SEM
I
II
III
IV
N
4
4
4
4
Asparagine [g/kg]
11.47
12.35a
10.62
9.92b
0.032
Threonineg [g/kg]
6.07
6.50a
5.49b
5.23b
0.031
Serine [g/kg]
8.65
9.05
8.06
7.80
0.027
Glutamine [g/kg]
34.02
35.71
30.86
30.93
0.029
Proline [g/kg]
17.70
17.52
15.30
15.58
0.029
Glycine [g/kg]
2.97
3.24a
2.75
2.57b
0.033
Alanine [g/kg]
5.65ab
6.16a
5.07bc
4.58c
0.038
Valine [g/kg]
10.08
10.71a
8.98b
8.93b
0.031
Isoleucine [g/kg]
7.55
8.04a
6.90
6.72b
0.030
Leucine [g/kg]
15.71
16.85a
14.09b
13.70b
0.032
Tyrosine [g/kg]
6.95
7.67
6.80
6.70
0.027
Phenylalanine [g/kg]
7.31
8.28a
6.92b
6.78b
0.031
Histidine [g/kg]
4.08
4.39
3.82
3.74
0.029
Lysine [g/kg]
11.93
13.51Aa
10.98b
10.28B
0.035
Arginine [g/kg]
5.62A
4.96ABa
4.02BCb
3.74C
0.049
Cysteine g [g/kg]
1.19A
1.33Aa
0.85b
0.61B
0.091
Methionine [g/kg]
4.46a
4.85a
4.71a
3.41b
0.053
Tryptophan [g/kg]
1.22
1.10
1.26
0.97
0.054
ΣAA sulphur* [g/kg]
6.87a
7.28A
6.82a
4.99Bb
0.047
Σ EAA [g/kg]
76.55
83.23a
70.80b
67.07b
0.031
Σ AA [g/kg]
162.63
172.22a
147.48
142.19b
0.030
a, b, c – values in rows with different letters differ significantly (P≤0.05)
A, B, C – as above for P≤0.01
* sum of sulphur amino acids = cysteine + methionine + tryptophan
Σ EAA – sum of essential amino acids
Σ AA – sum of amino acids

The cheeses also differed in the amino acid composition of 100 g protein (Tab. 6). The experimental cheeses contained more phenylalanine and tyrosine than control cheese. Protein from cheese II contained more arginine, glycine, and lysine compared to control cheese. Of the sulphur amino acids which constitute whey proteins, most methionine was found in protein from cheese III, slightly less in cheese II, much less in control cheese, and the least in cheese IV; cysteine content was the lowest in protein from cheese IV. There were also some fluctuations in the level of tryptophan in 100 g of cheese protein. As a result, the total content of sulphur amino acids was highest in protein from cheese III, slightly lower in cheeses II and I, and the lowest in cheese IV. The essential amino acids (EAA) and total amino acids (AA) in 100 g of protein from experimental cheeses II were higher in relation to control. Tryptophan was the limiting amino acid for both whole egg (WH) and mature human (MH) protein standards in all cheeses; methione and cysteine was the second limiting amino acid for WE and threonine for MH.

Table 6. Amino acid composition (g/100 g protein) and nutritional value of protein in curd cheeses
Amino acid
Type of cheese:
SEM
I
II
III
IV
N
4
4
4
4
Crude protein [g/kg]
183.45a
184.13a
159.35b
157.28b
0.028
Asparagine [g/100 g]
6.24
6.71
6.66
6.32
0.014
Threonine [g/100 g]
3.30
3.54
3.45
3.33
0.014
Serine [g/100 g]
4.71
4.92
5.06
4.96
0.012
Glutamine  [g/100 g]
18.51
19.40
19.36
19.70
0.013
Proline  [g/100 g]
9.63
9.53
9.59
9.90
0.012
Glycine  [g/100 g]
1.61b
1.77a
1.72
1.63
0.015
Alanine  [g/100 g]
3.07
3.35
3.19
2.92
0.019
Valine   [g/100 g]
5.48
5.82
5.64
5.68
0.011
Isoleucine  [g/100 g]
4.11
4.37
4.33
4.27
0.011
Leucine  [g/100 g]
8.55
9.16
8.84
8.72
0.012
Tyrosine [g/100 g]
3.78A
4.17B
4.26B
4.26B
0.016
Phenylalanine  [g/100 g]
3.99Aa
4.50B
4.34b
4.31b
0.014
Phe+tyrosine [g/100 g]
7.77B
8.67A
8.60A
 8.57A
0.014
Histidine  [g/100 g]
2.22
2.39
2.39
2.38
0.013
Lysine  [g/100 g]
6.49A
7.35Ba
6.88b
6.54A
0.016
Arginine  [g/100 g]
3.06Aa
2.70b
2.52B
2.38B
0.030
Cysteine [g/100 g]
0.65a
0.72A
0.54
0.39Bb
0.075
Methionine [g/100 g
2.42bc
2.64ab
2.95Aa
2.17Bc
0.038
Met+cysteine [g/100 g]
3.07
3.36a
3.49A
2.56Bb
0.039
Tryptophan  [g/100 g]
0.67
0.60
0.79
0.62
0.049
ΣAA – sulphur [g/100 g]
3.74
3.96a
4.28A
3.18Bb
0.035
CS(WE) tryptophan [%]
39.13
35.13
46.35
36.20
0.049
CS(MH) tryptophan [%]
36.59
33.23
43.73
34.15
0.049
CS (WE) Met+Cys  [%]
53.83
58.90a
61.18a
44.95b
0.039
CS (MH) threonine [%]
70.28
75.25
73.48
70.90
0.014
Σ EAA [g/100 g]
41.66b
45.26a
44.41
42.67
0.012
ΣAA [g/100 g]
88.49
93.64
92.51
90.48
0.011
a, b – values in rows with different letters differ significantly (P≤0.05)
A, B – as above for P≤0.01
CS – chemical score of the limiting amino acid
WE – whole egg protein standard
MH – mature human (human milk protein)
Phe – phenylalanine
Met – Methionine
Cys – Cysteine

DISCUSSION

The composition of sheep milk depends on feeding, breed, lactation period, and udder health status [12, 15] and that determined in our study was similar to the levels reported by Park et al. [12] but differ from results showed by Raynal-Ljutovac et al. [2008] [15].

The effect of manufacturing method that we observed on basic composition of the cheeses is indirectly confirmed by the results of other authors, who showed that in “salami” cheeses made from cow milk by the thermal-calcium method, ash and solids content increased, and protein and fat content as well as calorific value decreased compared to the traditional method [18]. The increase in fat content of solids in cheese made by method III, observed in our study, should be associated with its higher retention on the one hand, and the lower retention of minerals on the other. These observations are supported by Chmura et al. [4], who reported that approximately 65% of minerals are lost in the acid coagulation of protein during curd cheese making. A similar relationship was shown in our earlier study [9] for curd cheese made by the acid-rennet method, in which higher protein and fat retention and low mineral retention were noted compared to control cheese. In turn, Szpendowski et al. [17] showed the ash content of curd cheeses made from milk supplemented with CaCl2 and pasteurized at 90°C to increase compared to cheeses made from unsupplemented milk pasteurized at 75°C. Also in our study, we observed a tendency for higher ash content in cheese II made by the method in which milk was heated to 92–95°C and CaCl2 was added, compared to control cheese.

Since the differences in amino acids composition of cheeses differ from their composition in milk it means that the method of cheese production has strong influence on their profile in final product. When making cheese by the calcium-rennet method (II), we observed elevated amino acid content in both cheese and in protein composition in relation to the other cheeses, which may indirectly show that also milk whey proteins were coagulated in addition to casein. Few studies appear in the literature on the effect of cheese making technology on amino acid composition in cheese [16, 17], but their results indicate that cheese made by the methods allowing for coagulation of all milk proteins had a higher nutritive value of protein and contained more essential amino acids [7]. Similar to Szpendowski et al. [18], we found increased proportion of essential amino acids in 100 g of protein from cheeses II and III, made by the methods based on coagulation of all milk proteins. It is especially worth noting the high proportion of sulphur amino acids (including methionine and cysteine) in the composition of protein from cheese III made by the acid-rennet method, which is linked to a higher degree of precipitation of whey proteins.

This is confirmed by the observations of Szpendowski et al. [16], showing a higher content of sulphur amino acids methionine and cysteine in cheeses that also contained whey proteins compared to those containing mainly casein. Also the results of other studies [18] for thermal-calcium making of cheese from all proteins of cow milk compared to the traditional method showed an increase in the proportion of essential amino acids (such as isoleucine, lysine, cysteine and tryptophan) in cheese protein and elevated nutritive value of protein as measured by the chemical score (CS) and the essential amino acid index (EAAI). A similar tendency, partly significant, towards higher content of these amino acids in cheese made by method II and, to a lesser extent in cheese III, was observed in our study.

In summary it may be stated that manufacturing method had an effect on the composition, energy value, amino acid composition and nutritive value of cheese protein.

Cheese made by the calcium-rennet method (II) contained most essential and total amino acids per unit of product.

Cheese made by the acid-rennet method (III) combined with long-term coagulation of milk proteins was characterized by lowest mineral content and highest fat concentration in solids, while protein from this cheese had the highest proportion of sulphur amino acids.

Curd cheese making by the acid-rennet method combined with heat treatment of the curd (method IV) resulted in a product in which the amino acid composition of protein was closer to that of control cheese, which indirectly shows a lower degree of whey protein precipitation compared to the calcium-rennet method and the acid-rennet method combined with long-term precipitation of milk proteins at reduced temperature.

The elevated proportion of sulphur amino acids in the protein composition of cheeses made by methods II and III compared to control cheese and cottage cheese points to the coagulation of milk whey proteins.

In all cheeses, tryptophan was found to be the limiting amino acid for whole egg and mature human protein standards.

CONCLUSION

There is possible to obtain unripened sheep cheeses containing whey proteins using proper method of cheese production. Depend on technology applied it is possible to obtain cheese with elevated amount of amino acids including essential and sulphur amino acids (method II) or cheese with decreased fat content and thus reduced energy value (method IV).

ACKNOWLEDGEMENT

Research was realised within the project ”BIOFOOD – innovative, functional products of animal origin” no. POIG.01.01.02-014-090/09 co-financed by the European Union from the European Regional Development Fund within the Innovative Economy Operational Programme 2007–2013.

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Accepted for print: 26.02.2017


Tadeusz Pakulski
Experimental Station Kołuda Wielka, National Research Institute of Animal Production, Janikowo, Poland
88-160 Janikowo
Poland

Emilia Bagnicka
Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzębiec, Poland
Jastrzębiec, ul. Postępu 36A
05-552 Magdalenka
Poland
email: e.bagnicka@ighz.pl

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