# Effect of Differing Ingredients and Packaging Technologies on the Color of High-Pressure Processed Ground Beef

Authors: Jhinuk Gupta; Chad G. Bower; Gary Sullivan; George Cavender
Journal: Journal of Food Quality
 (2018)

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## Abstract

High-pressure processing (HPP) is a nonthermal pasteurization technique to control pathogens, likeEscherichia coli. However, color changes in raw beef induced by HPP restrict its use within the beef industry. The objectives of this study were to investigate the effects of adding curing agents (nitrite) and packaging with or without reducing compounds (ascorbic acid/erythorbate) on color retention in high-pressure processed ground beef. Color was measured (CIE L∗a∗b∗) before HPP and on days 3, 7, 12, 14, 19, and 21 after HPP. Statistical analysis (SAS GLIMMIX) was run to identify the main effects of adding curing agents, packaging, and reducing agents on color retention. HPP resulted in a detrimental effect on the color of the beef patties for all treatments. Lightness and yellowness increased (P<0.001) and redness decreased (P<0.001) after high-pressure processing. The effect remained the same throughout the course of the study. However, there were less color changes in samples treated with reducing compounds. Both synthetic and natural sources of nitrite and ascorbic acid/erythorbate performed similarly in terms of their ability to maintain redness. Treatments leading to formation of nitrosylmetmyoglobin (Fe3+) had less severe color change compared to the treatments leading to the generation of nitrosylmyoglobin (Fe2+).


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## Body

## 1. Introduction

A major challenge faced by the ground beef processors is microbial contamination such asE coli O157:H7 and other Shiga toxin producing E. coli (STEC). Sanitary handling, preharvest washing, and spraying the carcass with organic acids reduce the risk but do not completely eliminate contamination [1]. In ground beef and other nonintact beef products, STECs are considered an adulterant by the USDA [2]. These products are at a greater food safety risk as pathogens can be introduced throughout the product, rather than just on the surface. High-pressure processing (HPP) is a nonthermal pasteurization technique where pressure treatment between 300 and 800 MPa kills bacteria [3] by cell wall/spore coat rupture [4] or denaturation of critical proteins/enzymes [5, 6]. The process is most effective on Gram negative bacteria followed by yeasts/molds, Gram positive bacteria, and spores [4]. Salmonella, Listeria, and E. coli are the major meat pathogens which can be effectively controlled by HPP. A 2.0 to 6.0 log CFU/g reduction of these pathogens is achievable by HPP treatment [7–9].Several HPP-treated food products are in the market including fruit jellies and jams, fruit juices, pourable salad dressings, raw squid, rice cakes, foie gras, ham, and guacamole [3]. However, the use of HPP on raw meat products is uncommon due to high-pressure-induced protein denaturation and discoloration [10–15]. Therefore, it is important to find ways to stabilize the bright red color (oxymyoglobin) of fresh meat to develop a HPP-based pasteurization techniques for raw ground beef products. Effectiveness of HPP to control pathogens in cooked and uncooked meat and poultry items has been studied in great detail [7–9, 12, 14]. However, there are only very few reports available about the effect of HPP on the appearance of raw beef [3, 13, 16, 17].Use of curing agents, such as nitrite salts, is well known to retain the bright red meat color. Nitrite salt generates HNO2 and is reduced to NO in the presence of reducing agents or by other endogenous pathways [18, 19]. NO reacts with myoglobin in anaerobic and aerobic conditions to generate bright red nitrosylmyoglobin and nitrosylmetmyoglobin, respectively. These two are more stable than oxymyoglobin and thus impart greater color stability. Reducing agents, such as erythorbate or ascorbic acid, increase the nitrosylation rate and have shown to improve color stability in raw ground beef [20]. Excess reducing agent plays a dual role by inhibiting lipid oxidation and by increasing stability of cured meat color through shifting the equilibration between nitrosylmyoglobin and oxymyoglobin [21].With the recent trend of using natural food ingredients, synthetic nitrite salt has been greatly replaced by plant based nitrite sources, such as celery juice powder (CJP), swiss chard, spinach, and broccoli [22]. CJP is advantageous due to absence of any strong color or flavor [23]. Similarly, cherry powder (CP) is a rich source of ascorbic acid and potential alternative to sodium ascorbate/erythorbate [24, 25]. The objective of this study was to study the effects of adding different nitrosylating agents in the presence and absence of reducing agents and aerobic and anaerobic packaging on the color stability of HPP-treated ground beef patties.


## 2. Materials and Methods

### 2.1. Patty Preparation

Boneless, denuded USDA Select beef top rounds were ground through 1.27 cm and 0.32 cm grinding plates (Model 4732, Hobart Manufacturing, Troy, OH) and subdivided into six batches of 2.27 kg. The fine ground beef was mixed using a commercial kneader-mixer (RM-20, Manica USA, St. Louis, MO) with the following ingredients to convert myoglobin to different nitrosylmyoglobin states with or without the addition of reducing compounds (sodium erythorbate or ascorbic acid from cherry powder). The treatments (T1–T6) are as follows:T1: sodium nitrite 156 ppm/vacuum packaging (VP; anaerobic packaging)T2: sodium nitrite 156 ppm + sodium erythorbate 547 ppm/VPT3: celery juice powder (VegStable 506, Florida Food Products, Inc., Eustis, FL; to add 100 ppm sodium nitrite equivalent)/VPT4: celery juice powder (equivalent to 100 ppm nitrite) + 0.43% cherry powder (VegStable 515, Florida Food Products, to add 469 ppm ascorbic acid)/VPT5: sodium nitrite 156 ppm/oxygen permeable wrap (OPW; aerobic packaging)T6: sodium nitrite 156 ppm + sodium erythorbate 547 ppm/OPW.Four 113 g patties were formed from each portion. Patties were formed using a 10.92 cm diameter hand operated patty press. All T1, T2, T3, and T4 patties were vacuum packed (Clarity 3 mil standard barrier nylon/polyethylene pouches, Bunzl Processors Division, North Kansas City, MO; OTR = 0.007 ml/cm2/24 hr at 23°C and 0% relative humidity) using the vacuum sealer (Multivac Model C500; Multivac Inc., Kansas City, MO). Treatments T5- and T6-treated patties were placed on foam trays (13.3 × 25.6 × 1.4 cm, Styro-Tech, Denver, CO) and overwrapped with oxygen permeable polyvinyl chloride (Prime Source PSM 18 #75003815, Bunzl Processors Division, North Kansas City, MO; oxygen transmission rate = 2.25 ml/cm2/24 hr at 23°C and 0% relative humidity; water vapor transfer rate = 496 g/m2/24 hr at 37.8°C and 90% relative humidity). All patties were stored at 4°C for two days to allow for conversion to nitrosylmyoglobin (T1–T4) and nitrosylmetmyoglobin (T5-T6). After 48 hours, T5 and T6 were vacuum packaged just prior to HPP treatment. Three independent replications were produced.


### 2.2. High-Pressure Processing Treatment

Samples were processed using a 55 L HPP unit (Hiperbaric 55, Miami, FL) located in the food grade lab of the Food Processing Center, University of Nebraska Lincoln [26]. Processing of HPP-treated samples was performed at three different combinations of pressure, and the hold time (600 MPa/3 minutes, 600 MPa/6 minutes, and 450 MPa/3 minutes) that were chosen based on their effectiveness to reduce pathogens, according to previous research. During the course of the study, all samples were stored at 4°C to better simulate commercial refrigerated storage.


### 2.3. Colorimetry

Color of the patties was measured (CIEL∗a∗b∗) through the vacuum pouch prior to HPP and on days 3, 7, 12, 14, 19, and 21 after HPP [26]. A colorimeter (CR-300, MINOLTA, Japan) was used to determine the instrumental color which uses diffuse D65 illumination, 8 mm viewing port, and 0° viewing angle (specular component included). The system was calibrated to the included white calibration plate covered in the vacuum pouch before each measurement period. The average of at least three measurements was taken from randomly selected areas on the patty surface. Change in color, ΔE, was calculated with respect to the control samples (non-HPP treated) within each of the six treatments:(1)ΔE=Lf−Li2+af−ai2+bf−bi21/2,where subscripts i and f represent before and after HPP treatment, respectively.


### 2.4. Statistical Analyses

Statistical analyses were run on color data (L, a∗, b∗, ΔE) using a statistical software package (SAS 9.4, SAS Cary, NC) to see the main effects of ingredient/packaging conditions (T1–T6) and HPP treatment and their interactions within each day of storage [26]. Treatment interaction and main effects were determined using the mixed mode general linear model (PROC GLIMMIX). When significant (P<0.05) interactions or main effects were identified, separation of least square means was conducted.


## 2.1. Patty Preparation

Boneless, denuded USDA Select beef top rounds were ground through 1.27 cm and 0.32 cm grinding plates (Model 4732, Hobart Manufacturing, Troy, OH) and subdivided into six batches of 2.27 kg. The fine ground beef was mixed using a commercial kneader-mixer (RM-20, Manica USA, St. Louis, MO) with the following ingredients to convert myoglobin to different nitrosylmyoglobin states with or without the addition of reducing compounds (sodium erythorbate or ascorbic acid from cherry powder). The treatments (T1–T6) are as follows:T1: sodium nitrite 156 ppm/vacuum packaging (VP; anaerobic packaging)T2: sodium nitrite 156 ppm + sodium erythorbate 547 ppm/VPT3: celery juice powder (VegStable 506, Florida Food Products, Inc., Eustis, FL; to add 100 ppm sodium nitrite equivalent)/VPT4: celery juice powder (equivalent to 100 ppm nitrite) + 0.43% cherry powder (VegStable 515, Florida Food Products, to add 469 ppm ascorbic acid)/VPT5: sodium nitrite 156 ppm/oxygen permeable wrap (OPW; aerobic packaging)T6: sodium nitrite 156 ppm + sodium erythorbate 547 ppm/OPW.Four 113 g patties were formed from each portion. Patties were formed using a 10.92 cm diameter hand operated patty press. All T1, T2, T3, and T4 patties were vacuum packed (Clarity 3 mil standard barrier nylon/polyethylene pouches, Bunzl Processors Division, North Kansas City, MO; OTR = 0.007 ml/cm2/24 hr at 23°C and 0% relative humidity) using the vacuum sealer (Multivac Model C500; Multivac Inc., Kansas City, MO). Treatments T5- and T6-treated patties were placed on foam trays (13.3 × 25.6 × 1.4 cm, Styro-Tech, Denver, CO) and overwrapped with oxygen permeable polyvinyl chloride (Prime Source PSM 18 #75003815, Bunzl Processors Division, North Kansas City, MO; oxygen transmission rate = 2.25 ml/cm2/24 hr at 23°C and 0% relative humidity; water vapor transfer rate = 496 g/m2/24 hr at 37.8°C and 90% relative humidity). All patties were stored at 4°C for two days to allow for conversion to nitrosylmyoglobin (T1–T4) and nitrosylmetmyoglobin (T5-T6). After 48 hours, T5 and T6 were vacuum packaged just prior to HPP treatment. Three independent replications were produced.


## 2.2. High-Pressure Processing Treatment

Samples were processed using a 55 L HPP unit (Hiperbaric 55, Miami, FL) located in the food grade lab of the Food Processing Center, University of Nebraska Lincoln [26]. Processing of HPP-treated samples was performed at three different combinations of pressure, and the hold time (600 MPa/3 minutes, 600 MPa/6 minutes, and 450 MPa/3 minutes) that were chosen based on their effectiveness to reduce pathogens, according to previous research. During the course of the study, all samples were stored at 4°C to better simulate commercial refrigerated storage.


## 2.3. Colorimetry

Color of the patties was measured (CIEL∗a∗b∗) through the vacuum pouch prior to HPP and on days 3, 7, 12, 14, 19, and 21 after HPP [26]. A colorimeter (CR-300, MINOLTA, Japan) was used to determine the instrumental color which uses diffuse D65 illumination, 8 mm viewing port, and 0° viewing angle (specular component included). The system was calibrated to the included white calibration plate covered in the vacuum pouch before each measurement period. The average of at least three measurements was taken from randomly selected areas on the patty surface. Change in color, ΔE, was calculated with respect to the control samples (non-HPP treated) within each of the six treatments:(1)ΔE=Lf−Li2+af−ai2+bf−bi21/2,where subscripts i and f represent before and after HPP treatment, respectively.


## 2.4. Statistical Analyses

Statistical analyses were run on color data (L, a∗, b∗, ΔE) using a statistical software package (SAS 9.4, SAS Cary, NC) to see the main effects of ingredient/packaging conditions (T1–T6) and HPP treatment and their interactions within each day of storage [26]. Treatment interaction and main effects were determined using the mixed mode general linear model (PROC GLIMMIX). When significant (P<0.05) interactions or main effects were identified, separation of least square means was conducted.


## 3. Results and Discussion

Regardless of the ingredients/packaging treatment, HPP had a detrimental effect on the color of the beef patties for all three pressure and time combinations (Table1). Lightness (L∗) and yellowness (b∗) increased and redness (a∗) decreased (P<0.001) due to HPP treatment for all days of storage. Within each day, color change with respect to control samples (ΔE) was similar (P>0.05) for all three HPP conditions. Table 2 represents the effect of different ingredients/packaging on the color parameters. Within a particular day, all six differently treated samples had similar lightness (L∗, P>0.05, except for day 21) and yellowness (b∗, P>0.05, except for day 3 and day 21) but showed differences in redness (a∗, P<0.001). Samples treated with reducing compounds (T2, T4, and T6) showed greater redness (higher a∗) than the counterparts without reducing compounds (T1, T3, and T5), and this pattern was maintained throughout the course of the study. Reduction of oxidized myoglobin (nitrosylmetmyoglobin) to nitrosylmyoglobin may be responsible for increasing the redness. Among the color parameters evaluated, a∗ had an interaction of treatment (T1–T6) × HPP effects (P≤0.004) for days 14 and 19 only (data not shown) and b∗ had an interaction of treatment (T1–T6) × HPP effects (P=0.012) for days 21. On these days, treatments with reducing compounds had redness values that were more similar to the non-HPP treated control samples than treatments without reducing compounds which matches the significant main effect identified for a∗ for all other days. On day 21, HPP-treated samples were more yellow than non-HPP treated samples. Others have reported that the addition of antioxidants containing cherry powder, a natural source of ascorbic acid, to ground beef resulted in greater red color in patties in simulated retail display [20]. Similar a∗ values of T2 and T4 within a particular day signifies that both inorganic and plant-based sources of nitrite and reducing compounds had a similar influence on color. T1 had significantly higher a∗ than T5 on day 3, but the difference became less profound during storage. Although immediately after HPP, nitrosylmyoglobin is more red, and it became less red and approached that of T5, likely due to the fact that nitrosylmetmyoglobin in T5 had already oxidized and it started with less red color. ΔE of T6 was significantly higher than ΔE of T2 immediately after HPP but gradually decreased during storage. This suggests that T6 changes color after HPP, but color changes lessened during shelf storage. This is most likely due to the reduction of nitrosylmetmyoglobin (brown) to nitrosylmyoglobin (red) by sodium erythorbate. In the absence of reducing agents (T1 versus T5), ΔE was similar throughout the course of the study.Table 1
Least square means (±SE) for main effect of high-pressure processing on color (L∗, a∗, b∗) and change in color (ΔE) during storage of ground beef patties.
Color traits
HPP (MPa/min)
Color values1
Day 3
Day 7
Day 12
Day 14
Day 19
Day 21
L
∗
0/0
40.78 ± 0.48b
42.20 ± 0.34c
43.13 ± 0.30c
43.73 ± 0.38c
42.06 ± 0.43b
43.31 ± .034c
450/3
53.58 ± 0.50a
54.49 ± 0.36b
54.87 ± 0.31b
55.09 ± 0.39b
55.30 ± 0.45a
55.26 ± 0.36b
600/3
54.53 ± 0.48a
55.62 ± 0.34a
56.66 ± 0.30a
56.2 ± 0.38a
56.45 ± 0.43a
55.61 ± 0.34ab
600/6
53.47 ± 0.48a
55.84 ± 0.34a
55.98 ± 0.30a
56.20 ± 0.38a
56.36 ± 0.43a
56.29 ± 0.34a
P value
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
a
∗
0/0
22.50 ± 0.55a
21.98 ± 0.56a
19.46 ± 0.46a
19.33 ± 0.44†
21.04 ± 0.50†
19.39 ± 0.62a
450/3
21.33 ± 0.57ab
18.18 ± 0.58b
16.56 ± 0.48b
14.91 ± 0.46†
16.02 ± 0.52†
16.38 ± 0.64b
600/3
18.17 ± 0.55b
16.35 ± 0.56c
14.31 ± 0.46c
14.67 ± 0.44†
14.62 ± 0.50†
14.68 ± 0.62bc
600/6
20.43 ± 0.55c
15.81 ± 0.56c
14.46 ± 0.46c
14.76 ± 0.44†
13.77 ± 0.50†
13.21 ± 0.62c
P value
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
b
∗
0/0
8.95 ± 0.17d
9.50 ± 0.18c
8.70 ± 0.14c
8.34 ± 0.16c
9.20 ± 0.20b
8.90 ± 0.17†
450/3
11.14 ± 0.18c
10.67 ± 0.19b
10.67 ± 0.15b
10.44 ± 0.16b
11.39 ± 0.20a
11.00 ± 0.18†
600/3
11.67 ± 0.17b
11.32 ± 0.18a
11.27 ± 0.14a
11.39 ± 0.16a
11.95 ± 0.20a
11.55 ± 0.17†
600/6
12.34 ± 0.17a
11.12 ± 0.18ab
11.44 ± 0.14a
11.49 ± 0.16a
11.72 ± 0.20a
11.65 ± 0.17†
P value
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
Δ
E
450/3
13.14 ± 0.89
13.29 ± 0.54
12.84 ± 0.62
12.95 ± 0.52
15.07 ± 0.73
13.17 ± 0.62
600/3
15.00 ± 0.85
14.90 ± 0.52
14.92 ± 0.59
14.01 ± 0.50
16.54 ± 0.71
13.98 ± 0.60
600/6
13.90 ± 0.85
15.32 ± 0.52
14.34 ± 0.59
14.14 ± 0.50
16.57 ± 0.71
14.94 ± 0.60
P value
0.322
0.026
0.056
0.209
0.259
0.134
a–cLS means in a column and within a color trait with a common superscript are similar (P>0.05). †A significant myoglobin state by HPP treatment interaction (P<0.05) for the color trait within the day. 1Each experiment was carried out in triplicate.Table 2
Least square means (±SE) for main effect of myoglobin state (Mb) on color (L∗, a∗, b∗) and change in color (ΔE) during storage of ground beef patties.
Color traits
Mb state1
Color values2
Day 3
Day 7
Day 12
Day 14
Day 19
Day 21
L
∗
T1
50.64 ± 0.62
52.25 ± 0.44
53.55 ± 0.39
53.06 ± 0.49
53.32 ± 0.56
53.40 ± 0.44a
T2
51.17 ± 0.58
52.72 ± 0.42
52.63 ± 0.37
52.83 ± 0.46
51.59 ± 0.53
52.65 ± 0.42a
T3
49.74 ± 0.58
51.61 ± 0.42
52.58 ± 0.37
53.49 ± 0.46
52.49 ± 0.53
52.84 ± 0.42a
T4
50.37 ± 0.58
51.86 ± 0.42
52.09 ± 0.37
52.23 ± 0.46
52.01 ± 0.53
52.28 ± 0.42ab
T5
51.86 ± 0.58
52.08 ± 0.42
53.04 ± 0.37
52.99 ± 0.46
53.63 ± 0.53
53.17 ± 0.42a
T6
49.75 ± 0.58
51.70 ± 0.42
52.07 ± 0.37
52.25 ± 0.46
52.20 ± 0.53
51.37 ± 0.42b
P value
0.095
0.455
0.065
0.357
0.073
0.023
a
∗
T1
20.51 ± 0.72b
17.28 ± 0.72b
13.42 ± 0.60c
14.49 ± 0.57†
13.99 ± 0.65†
13.88 ± 0.80b
T2
23.29 ± 0.68a
19.94 ± 0.68a
18.34 ± 0.57ab
18.67 ± 0.54†
19.42 ± 0.61†
17.80 ± 0.76a
T3
21.38 ± 0.68ab
17.28 ± 0.68b
14.23 ± 0.57c
13.21 ± 0.54†
14.13 ± 0.61†
14.47 ± 0.76b
T4
23.13 ± 0.68a
20.53 ± 0.68a
19.83 ± 0.57a
19.23 ± 0.54†
18.90 ± 0.61†
18.14 ± 0.76a
T5
14.31 ± 0.68c
14.45 ± 0.68c
13.71 ± 0.57c
12.50 ± 0.54†
13.20 ± 0.61†
13.28 ± 0.76b
T6
21.03 ± 0.68b
19.00 ± 0.68ab
17.65 ± 0.57b
17.42 ± 0.54†
18.52 ± 0.61†
17.93 ± 0.76a
P value
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
b
∗
T1
11.00 ± 0.23a
10.47 ± 0.24
10.41 ± 0.18
10.60 ± 0.20
11.52 ± 0.25
11.27 ± 0.22†
T2
11.34 ± 0.21a
10.88 ± 0.23
10.58 ± 0.17
10.33 ± 0.19
10.89 ± 0.24
10.10 ± 0.21†
T3
11.45 ± 0.21a
10.72 ± 0.23
10.42 ± 0.17
10.26 ± 0.19
11.51 ± 0.24
10.89 ± 0.21†
T4
11.48 ± 0.21a
10.97 ± 0.23
10.84 ± 0.17
10.59 ± 0.19
10.70 ± 0.24
10.39 ± 0.21†
T5
9.91 ± 0.21b
10.46 ± 0.23
10.39 ± 0.17
10.41 ± 0.19
11.06 ± 0.24
11.42 ± 0.21†
T6
10.96 ± 0.21a
10.41 ± 0.23
10.47 ± 0.17
10.31 ± 0.19
10.71 ± 0.24
10.60 ± 0.21†
P value
<0.001
0.362
0.431
0.724
0.056
<0.001
Δ
E
T1
14.10 ± 1.30
14.03 ± 0.79ab
13.3 ± 0.90bc
14.54 ± 0.76b
18.75 ± 1.08a
16.85 ± 0.91a
T2
12.49 ± 1.20
12.24 ± 0.73b
12.46 ± 0.84c
12.28 ± 0.70c
16.07 ± 1.00ab
13.13 ± 0.84bc
T3
12.65 ± 1.20
15.97 ± 0.73a
15.20 ± 0.84ab
17.60 ± 0.70a
16.63 ± 1.00ab
13.80 ± 0.84b
T4
13.88 ± 1.20
14.17 ± 0.73ab
13.08 ± 0.84bc
11.78 ± 0.70c
15.29 ± 1.00bc
14.25 ± 0.84b
T5
13.36 ± 1.20
15.26 ± 0.73a
16.51 ± 0.84a
13.34 ± 0.70bc
16.56 ± 1.00ab
14.92 ± 0.84ab
T6
17.61 ± 1.20
15.34 ± 0.73a
13.67 ± 0.84bc
12.65 ± 0.70bc
13.05 ± 1.00c
11.24 ± 0.84c
P value
0.055
0.015
0.015
<0.001
0.015
0.003
a–cLS means in a column and within a color trait with a common superscript are similar (P>0.05). †A significant myoglobin state by HPP treatment interaction (P<0.05) for the color trait within the day. 1T1: sodium nitrite 156 ppm/vacuum packaging (VP; anaerobic packaging); T2: sodium nitrite 156 ppm + sodium erythorbate 547 ppm/VP; T3: celery juice powder to add 100 ppm sodium nitrite equivalent/VP; T4: celery juice powder (equivalent to 100 ppm nitrite) + 0.43% cherry powder to add 469 ppm ascorbic acid)/VP; T5: sodium nitrite 156 ppm/oxygen permeable wrap (OPW; aerobic packaging); T6: sodium nitrite 156 ppm + sodium erythorbate 547 ppm/OPW. 2Each experiment was carried out in triplicate.


## 4. Conclusions

While the addition of nitrite compounds alone did not stabilize ground beef color during HPP treatment, reducing compounds decreased the color change associated with HPP treatment of ground beef. These findings may allow processors to progress toward the development of technologies that allow for the HPP treatment of raw ground beef without the negative color changes typically associated with the application of HPP.



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*Source: 4590143-2018-06-05.xml*