Download Microbial Cause of Calcium Lactate Defect in Cheddar Cheese

Microbial Cause of Calcium Lactate
Defect in Cheddar Cheese
Boorus Yim
California Polytechnic State University
San Luis Obispo
Dairy Product Technology Center
December 8, 2005
History of Cheese
•
Cheese was known 6000 years ago by the ancient Sumarians. The ancient Greeks
accredited Aristaeus, son of Apollo and Cyrene, with its discovery. The ancient
Romans claim cheese came on its own.
•
Ancient legend first describes cheese when an Arabian merchant traveling through
the desert was carrying milk in an animal’s stomach. The combination of the heat
and rennet in the stomach separated the milk into curds (cheese) and whey.
•
Cheesemaking was brought over to Europe from Asia, including northern Africa.
•
Cheesemaking was brought over to current day United States when the Pilgrims
landed in 1620.
•
Cheesmaking was a local farm industry until in 1851 a cheese cooperative with local
cheese makers and dairy farmers was formed by Jesse Williams in Oneida, New
York.
•
“Big Cheese” – 1801, an enterprising cheesemaker made a 1,225 lb wheel of cheese
to Thomas Jefferson.
Cheese Industry
•
2003 – 8.598 billion pounds of cheese in the U.S. market
•
Italian and American type cheeses dominate the market at 3.522 billion and 3.67
billion pounds, respectively.
–
Cheddar cheese comprises most of the American type cheese produced at 2.749 billion
pounds.
•
Estimated 300 varieties of cheese in the U.S.
•
Cheese defects range from physical to chemical.
Calcium Lactate Crystal (CLC) Defect
•
First described in 1930s as formation of
white specks. Identified on Cheddar cheese.
•
1980s – White specks on cheddar cheese
classified as calcium lactate crystal.
•
CLCs pose no health hazard to the
consumer.
•
CLCs detract from the appearance of the
cheese since they look like mold to the
general consumer. Crystals obvious against
the background of yellow colored Cheddar
cheese.
•
Chemical Formula - C6H10CaO6
MW: 2182214
•
Calcium lactate is sold as vitamin
supplements and as chemical agents.
How do CLCs form in cheese?
•
In theory, CLCs form when there’s a racemic mixture of L(+)-lactic acid and the less soluble D(-)lactic acid with free calcium in the cheese serum (moisture expulsion).
•
Proposed by Dybing, et. al. (Land O’Lakes, Inc., Cheese Research Group, R&D)
How do CLCs form in cheese? (cont.)
•
Dybing et al. (1988) proposed several causes to CLC formation.
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–
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Physical causes – Packaging (temperature, CO2 vs. vacuum), length of curing, seasonal
effects
Chemical causes – D to L-lactic acid content, free Ca ion content, salt concentration, pH at
milling, rate of acidification during manufacture.
Several more studies showed non-starter lactic acid bacteria (NSLAB) and D-lactic
acid as a possible underlying cause to CLCs.
–
–
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Johnson et al. (1990) used an unidentified strain of Lactobacillus. Found when used,
crystals would form and noticed an increase of D-lactic acid.
Thomas and Crow (1983) found mature Cheddar cheese contained a racemic mixture of
lactic acid. But the Lactococci produces L-lactic acid. They found L and D lactate
dehydrogenase produced by Pediococci and some Lactobacillus species.
Generally accepted that NSLAB causes CLCs. Agarwal et al. (2003) and Chou et al. (2003)
narrowed the possible NSLAB to Lb. curvatus and P.acidlactici.
Non-Starter Lactic Acid Bacteria (NSLAB)
•
Generally defined as a lactic acid producing bacterium introduced post-manufacture.
•
One method of NSLAB classification is by the sugars and the metabolic pathway
utilization.
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–
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Obligately homofermentative – Glycolysis
Obligately heterofermentative – 6-phosphogluconate/phospoketolase (6-PG/PK)
Facultative heterofermentative – May use both Glycolysis and 6-PG/PK pathways
•
•
Lb. casei, Lb. plantarum, Lb. sake, Lb. curvatus
Lb. curvatus – Gram positive single rods with a slight moon shape curve. Commonly
found in fermented foods such as sausage and sauerkraut.
–
Produces the enzyme lactate dehydrogenase that yield lactic acid.
Lactate Dehydrogenase (Ldh)
•
Dehydrogenases belong to the EC 1.1
class of enzymes.
–
Transfer a hydride ion (H:-) to an acceptor
such as nicotinamide adenine dinucleotide
(NAD+), oxygen, quinone, or cytochrome.
•
Lactate dehyrogenase catalyzes the
reaction from pyruvate (glycolysis) to lactic
acid (fermentation).
•
D and L lactate dehydrogenase have been
identified and produces stereoisomers of
lactic acid.
•
~140 kDa for both D and L forms of LDH
Lactic Acid
D(-)-Lactic Acid
L(+)-Lactic
Acid
Lactic Acid
•
Byproduct of lactic acid fermentation
–
–
Skeletal muscle under strenuous exercise undergo
lactic acid fermentation
Cheese ripening occurs under anaerobic conditions,
allowing for lactic acid fermentation
•
C3H6O3
MW: 90.0786 g/mole
•
Two isomers of lactic acid
–
–
L(+)-Lactic
Acid
acid – produced normally from commercial
cheese starter cultures (mesophillic Lactococci)
D(-)-lactic acid – not normally produced, specifically
seen if there is the presence of D-LDH.
L(+)-lactic
L(+)-Lactic
Acid
Objectives
•
Isolate NSLAB from cheese with CLC defect.
•
Identify NSLAB isolated from cheese with CLC defect.
•
Use isolated organism as an adjunct to produce CLC defect in cheese.
•
Isolate the D(-)-ldh gene from the organism used to produce the CLC defect in
cheese.
Materials and Methods – The Cheese
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Bulk Cheese from Wisconsin, Asiago Cheddar, Tillamook Cheddar, New Zealand and
Iceland type Cheddar, and Edam Gouda were received.
•
The cheese from Wisconsin, Asiago Cheddar, and New Zealand and Iceland type
Cheddar exhibited CLCs.
•
Tillamook Cheddar (store bought) and Edam Gouda did not show signs of CLCs.
•
Each cheese was cut into smaller blocks, grated, packaged and stored @2-3ºC for
analysis.
Materials and Methods – Objective #1
Isolate NSLAB from cheese with CLC defect
•
10g from each cheese was added to 90 mL 2% trisodium citrate (TSC) solution (10-1)
and homogenized with a stomacher.
•
10-2 – 10-6 serial dilutions were made with 9 mL Butterfield’s buffer with corresponding
pour plates (enumeration) and streak plates (isolated bacteria).
•
Rogosa SA and MRS agar were the primary agars used to isolate and enumerate
bacteria.
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–
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Isolated bacteria were cultivated and further stored in MRS broth.
–
•
Rogosa SA – Incubated 3-4 days, anaerobic conditions, 37ºC
MRS agar – Incubated 1-2 days, anaerobic conditions, 37ºC
For frozen storage, the bacteria was suspended in “S” buffer and glycerol.
Averaged to 80 bacterial isolated
Materials and Methods – Objective #2
Identify NSLAB isolated from cheese with CLC defect
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Gram staining – visual observation
•
16S rRNA Polymerase Chain Reaction (PCR) – rRNA isolation
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–
–
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Genomic DNA purification of each isolated bacteria. Used a DNA isolation kit (MoBio
Laboratories, Inc., Solana Beach, CA)
PCR – Used universal primers UF2 and UR523 for 16S rRNA isolation
• 94ºC 2 min, (94ºC 15 sec, 55ºC 1 min, 72ºC 1.5 min) × 30, 72ºC 7 min.
PCR product cleaned with a PCR clean up kit (MoBio Laboratories, Inc., Solana Beach, CA)
PCR product sequencing
–
–
Cleaned PCR products were sent to Utah State University – Biotechnology Center for
sequencing. Used an ABI Prism 3730 DNA analyzer and Taq FS Terminator Chemistry.
Sequences were sent back and used BLAST software (National Center of Biotechnology
Information Center) to identify the bacteria.
Materials and Methods – Objective #3
Use isolated organism as adjunct to produce CLC defect in cheese
BUT WAIT!! We needed to figure out what was the proper adjunct.
•
The proper adjunct must be:
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–
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NSLAB
Evidence the NSLAB can produce D-LDH
Produces a large amount of D(-)-lactic acid
•
A DL-Lactic Acid Assay Kit (Biopharm, Inc., Marshall, MI) was used to determine how
much D(-) and L(+)-lactic acid each isolated bacteria produced.
•
When the proper adjunct was chosen, the NSLAB was grown in 150 mL MRS broth for
16-20 hours before the day of cheesemaking.
Material and Methods - Cheesemaking
Milk
C (Control)
60 gal
Gas Flushed
20 lb block
Experimental
60 gal
Vacuumed
Packaged
20 lb block
Gas Flushed
10 lb block
A (no CaCl2)
Vacuumed
Packaged
10 lb block
A+ (CaCl2)
Gas Flushed
10 lb block
Vacuumed
Packaged
10 lb block
Materials and Methods – Cheese Sampling and
Analysis
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Aseptic sampling of each cheese block on day 1 (before first packaging), day 7, day
30, day 60, and day 90 from date of manufacture. Half the sample was grated and
the other half stored frozen.
–
•
Exception was if crystals were observed early, stop sampling.
Cheese analysis includes:
–
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Composition – fat (%), moisture (%), pH, DL-lactic acid content.
Microbiology – Rogosa SA and MRS Agar bacterial enumeration
Materials and Methods – Objective #4
Isolate the D-ldh gene from the organism used to produce the CLC defect.
•
We used specific custom primers to isolate a fragment of the D-ldh gene and used
PCR to amplify the fragment.
•
PCR product sent to Utah State University – Biotechnology Center for sequencing.
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Sequences were compared to published D-ldh gene sequences using Clustal W.
Results and Discussion
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~80 bacterial isolates – 60 isolates from cheeses with the CLC defect, 20 isolates
from cheeses with no signs of CLCs.
–
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2/3 of the 60 isolates were identified as Lb. curvatus
Remaining isolates identified as Lb. casei, Lb. paracasei, Lb. coryniformus.
•
23 isolates were screened for D and L-lactic acid. 7 of the isolates later identified as
Lb. curvatus were the isolates positive for D(-)-lactic acid content.
•
An isolate was selected at random from the 7 identified as Lb. curvatus to be used as
an adjunct.
Results and Discussion
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Compositional Analysis of cheeses
Mean composition of seven-day old Cheddar cheeses.
•
Control
w/ Adjunct
w/ Adjunct + CaCl2
N=2
n=2
n=2
Fat (%)
33.0
33.0
33.0
Moisture (%)
36.09 ± 0.85
35.90 ± 0.77
35.87 ± 0.72
Salt (%)
1.45 ± 0.04
1.16 ± 0.16
1.34 ± 0.02
pH
5.02 ± 0.02
5.01 ± 0.01
5.03 ± 0.03
Average cheese fat content met the requirement of 50% of the cheese solids and
cheese moisture met the requirement of up to a maximum of 39% by weight.
–
Food and Drug Administration (FDA), Code of Food Regulation, Title 21, Section 133.113.
Results and Discussion
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NSLAB counts from preliminary trial and trial cheeses.
Experimental NSLAB count (log10) of Cheddar cheese made with starter
only (C), starter + adjunct (A) and starter + adjunct + CaCl2 (A+).
Preliminary NSLAB count (log10) of Cheddar cheese made with starter only
(control), starter + adjunct and starter + adjunct + CaCl2.
Preliminary
Trial
Cheese ID
1 day
7 day
30 day
90 day
1C
3.531
3.906
3.929
4.724
1A
6.580
6.653
6.848
7.419
1A+
6.585
6.667
6.839
7.303
Type of Cheese
7 day
30 day
90 day
180 day
Control
0.000
0.000
0.000
4.326
1C Gas Flushed
n/a
3.842
3.845
5.371
w/ Adjunct
1.943
2.690
5.068
7.188
1A Gas Flushed
n/a
6.637
6.699
8.443
w/ Adjunct + CaCl
2.151
3.097
5.445
7.152
1A+ Gas Flushed
n/a
6.638
6.676
8.173
n/a
0.000
0.000
5.321
2C
0.000
0.000
5.283
5.438
n/a
4.091
4.929
7.205
2A
7.097
7.860
8.441
8.460
n/a
3.720
5.158
7.238
2A+
7.130
7.527
8.563
8.575
2C Gas Flushed
n/a
2.739
5.633
6.109
2A Gas Flushed
n/a
8.029
8.710
8.720
2A+ Gas Flushed
n/a
8.092
8.692
8.674
Control Gas
Flushed
w/ Adjunct Gas
Flushed
w/ Adjunct + CaCl
Gas Flushed
Results and Discussion
•
When did crystals form?
–
Preliminary trial – Control cheese showed no signs of CLCs. Vacuumed packaged cheese
showed CLCs ~180 days of ripening. Gas flushed packaged cheese showed CLCs ~150160 days of ripening.
–
Experimental trials – Control cheese showed signs of CLC ~90 days of ripening. Gas
flushed packaged cheese showed CLCs ~30 days of ripening. Vacuumed packaged cheese
showed CLCs ~60-65 days of ripening.
Cheddar cheese (60 day-old) showing calcium
lactate crystals. Cheese manufactured with LbA2
starter adjunct and CO2 flushed packaging.
Cheddar cheese (60 day-old) showing calcium
lactate crystals. Cheese manufactured with LbA2
starter adjunct and vacuum packaged.
Results and Discussion
•
Each cheese manufactured was screened for
different packaging conditions.
•
Statistics – ANOVA (Minitab 14)
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–
–
–
–
D
and L-lactic acid, including in the
No significant difference of lactic acid content between A and A+ cheese (p=0.711-0.970).
• Expected result since addition of CaCl2 was not expected to influence production of D(-)lactic acid.
When looking at D(-)-lactic acid content of C and A/A+ cheeses over ripening time, there is a
significant difference (C p=0.00, A p=0.08, A+ p=0.04).
• Expected to observe an increased amount of D(-)-lactic acid, but whether a significant
increase was in question.
There was no significant difference of L(+)-lactic acid content in C and A/A+ cheeses over
ripening time.
When looking at D(-)-lactic acid content on sampling days between C and A/A+ cheese, there
is a significant difference.
Type of packaging showed a significant difference on control cheese, however, no difference
was observed with the A/A+ cheeses.
What does this mean?
•
There seems to be a correlation of Lb. curvatus cell density to D(-)-lactic acid content.
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–
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High correlation of D(-)-lactic acid to CLC development.
–
•
However, a direct enzyme activity wasn’t done to confirm this.
Observation: Preliminary trial bacterial counts and lactic acid content compared to
experimental trials.
Based on observation and lactic acid analyses.
Packaging influences CLC development.
Polymerase Chain Reaction (PCR)
•
•
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PCR is a technique for amplifying the
amount of a specific segment of DNA.
First conceived by Kary Mullis in the
1980s by manipulating DNA
polymerase, which received the Nobel
Prize in Chemistry in 1993 along with
Michael Smith for his contribution in
oligonucleotide, site-directed
mutagensis.
Basic strategy of PCR:
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–
–
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Denaturation of template DNA
Annealing of primers
Extension of DNA
For this project, needed to figure out
the D-ldh gene for Lb. curvatus.
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–
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Template DNA
Primers
PCR
Did we get the gene?
PCR
General priming sites for FCPBY12, FCPBY15, FCPBY19, and FCPBY19D.
General priming sites for RCPBY15, RBYCP24, and RCPBY20
RCPBY20D
FCPBY18D
RCPBY21D
PCR
Primers used for D-Lactate Dehydrogenase Gene Isolation.
Primer Name
Primer Sequence
FCPBY12
5'-ATTTTTGCTTACGCTATTCGT-3'
FCPBY15
5'-ATTTTTGCTTACGCT-3'
RCPBY15
5'-GTAGAAAGCAGTGTG-3'
RBYCP24
5'-AGTGTAGAAAGCAGTGTGTGGAGT-3'
FCPBY19
5'-GCTTACGCTATTCGTAAAG-3'
RCPBY20
5'-GCAGTATGTGGAGTTACCAA-3'
FCPBY18D
5'-AGGGTGCTGACCGAGAAT-3'
RCPBY20D
5'-ATCTTTAAGTGCGCTTGTCA-3'
RCPBY21D
5'-ACCATGTTACGTACAGCATGA-3'
FCPBY19D
5'-TTTGCTTACGCTATTCGTG-3'
PCR
Primers pairs with their respective PCR cycles and results.
Forward
Reverse
Denaturation
Annealing
Elongation
Results
FCPBY21
RCPBY15
94ºC 15 sec
35ºC 1 min
72ºC 1.5 min
400 bp
FCPBY21
RBYCP24
94ºC 15 sec
35ºC 1 min
72ºC 1.5 min
500 bp
FCPBY15
RCPBY15
94ºC 15 sec
35ºC 1 min
72ºC 1.5 min
no product
FCPBY15
RBYCP24
94ºC 15 sec
35ºC 1 min
72ºC 1.5 min
1500 bp
FCPBY21
RBYCP24
94ºC 15 sec
40ºC, 50º C 1 min
72ºC 1.5 min
no product
FCPBY19
RCPBY21
94ºC 1 min
45ºC 1 min
72ºC 1.5 min
no product
FCPBY19
RBYCP24
94ºC 30 sec
45ºC 1 min
72ºC 1.5 min
no product
FCPBY19
RCPBY20
94ºC 30 sec
55ºC 1 min
72ºC 1.5 min
no product
FCPCY18D
RCPBY20D
94ºC 15 sec
35ºC, 55º C 1 min
72ºC 2 min
no product
FCPBY19D
RCPBY21D
94ºC 15 sec
35ºC, 55º C 1 min
72ºC 2 min
no product
FCPBY15
RCPBY15
94ºC 15 sec
35ºC 1 min
72ºC 2 min
1000bp, 2000 bp
FCPBY15
RCPBY15
94ºC 15 sec
45ºC 1 min
72ºC 2.5 min
1000bp, 2000 bp
Final PCR cycle chosen
Did we get the D-LDH gene?
•
Yes and no. We believe we have a fragment of the gene.
>LB25C10
ATCTGGCGTTTAACGAATCGCCCTTGTAGAAGCAGTGTGCCCATTGGCTAACTTCTTCTTTTCGATGACGTATTCGTAAATTTTAGC
GGCACTACGCGTGACCGTTTCTTGTGATAAGCGATCTTTGTTAGCTTGAATAAATGCTTGGACATCGGAATCGTTCAAGGCATCTTC
GACCAATTTATTAAACTGTTGGTTTAACTTTTGCCGATTCATGTAATCCGTCAAATCTTTACCCATATTTTCCATTACGATTCCCGCC
CCTCACGAATTTTTTTCAATGCCGCTTCTAGTTCATGCTGATGCTCAGGTGTGGTTGtTTCTTTTGGCGCTTGATAATCTGGTTTAGC
CCATTTAGGTACTGGTTCTTTTTTAGTTTTGTTCTGATAACGCGTTTGGCGATTGTTTTGCACCTTTGCCGYCTGTTTGGTTTGGAAA
TCGSGAATCTGCAAAATCGCATCCGCTGCCGTCTTAACCCCTTGTTGAGCCCATTGGTTCGCAATCCGATCGACTAGCGCCTGAGT
GAGCCCATCATATTGCGTAATGATATAAACGACCAAAATATTGAGCACATCATTGTTAAAGATATAGCGGTTTTGCAAGTCCTTTAAC
GCCCGGATTTCATTTTTAGCGACAAAGCCGTGGTTCTTTTGTTTCAAGTATTCGAGATAATCAACTGGCAAGTAGCCCTGACTTTCT
GTTAGCCACTGCAATTCCTGTTCGTTAAAGCCGGCTTTTTGCCATTCTTTTTGKAACTCGGCTAACGGTTTTtGCGTTGTTGTAgCAS
CTGGCGTTTGCCGGTtACTCTGACGTCSTGKGKAATTGKTCAAgACGTTCTGTTCgAGCGCTGCCATATTAATCGTACTGTCAGCAA
CATTCATCGTCAAGCCAATCAAACGCGCTAAGTCCATCTCATCTAGACCATAGAAATAATGGAGATTAAACAAGCCCTGTTCATTTT
TGAGAATTTGATCCGTATCAATCTGGTATTGCGCCGTTGCATCTTGTAATAGGTCCCAATCAAAAGTCCGCAACTCAACCGAGGTAA
ACTGCGGTGTGCTAGCTGCTTTTTGTTGATACATTGCCTTAGTTTCAGCCACACGCTTCTACAAGGGCGAATCGGGCGTATCAG
•
2 fragments were obtained. LB2-5C12 and LB2-5C-10 (920bp and 1132bp,
respectively)
–
Clustal W to compare with published D-ldh genes. Fragment reside together and towards the
middle of the published sequence.
Conclusions
•
•
Strong correlation between high bacterial density of Lb. curvatus and CLC formation.
CO2 packaging seem to allow faster CLC formation versus vacuum packaging.
•
•
Lb. curvatus produces the enzyme D-lactate dehydrogenase yielding D(-)-lactic acid.
Correlation of D(-)-lactic acid content and CLC formation.
Recommendation for Future Work
•
Produce a mutant Lb. curvatus with the D-LDH gene deleted or partially deleted.
•
Complete sequence of Lb. curvatus genome and its D-LDH gene.
•
Bacteriocins or cross-inhibition studies to Lb. curvatus and other D(-)-lactic acid
producing bacteria.
Recognition
•
Dr. Shakeel-Ur Rehman and Dr. Vedamuthu, E.
•
Agriculture Research Initiative
•
Staff and colleagues of the Dairy Products Technology Center
•
Mr. and Mrs. Chong Nam Yim and family
•
Jocelyn Fagar – Rest in peace.
References
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formation in Cheddar cheese. Institute of Food Technologists.
Chou, Y. E. E., C.G.; Luedecke, L.O.; Bates, M.P.; Clark, S. 2003. Nonstarter lactic acid bacteria and aging
temperature affect calcium lactate crystallization in cheddar cheese. Journal of Dairy Science. 86:2516-2524.
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