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Microbial Cause of Calcium Lactate Defect in Cheddar Cheese

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Microbial Cause of Calcium Lactate Defect in Cheddar Cheese

  1. 1. 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
  2. 2. History of Cheese <ul><li>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. </li></ul><ul><li>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. </li></ul><ul><li>Cheesemaking was brought over to Europe from Asia, including northern Africa. </li></ul><ul><li>Cheesemaking was brought over to current day United States when the Pilgrims landed in 1620. </li></ul><ul><li>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. </li></ul><ul><li>“ Big Cheese” – 1801, an enterprising cheesemaker made a 1,225 lb wheel of cheese to Thomas Jefferson. </li></ul>
  3. 3. Cheese Industry <ul><li>2003 – 8.598 billion pounds of cheese in the U.S. market </li></ul><ul><li>Italian and American type cheeses dominate the market at 3.522 billion and 3.67 billion pounds, respectively. </li></ul><ul><ul><li>Cheddar cheese comprises most of the American type cheese produced at 2.749 billion pounds. </li></ul></ul><ul><li>Estimated 300 varieties of cheese in the U.S. </li></ul><ul><li>Cheese defects range from physical to chemical. </li></ul>
  4. 4. Calcium Lactate Crystal (CLC) Defect <ul><li>First described in 1930s as formation of white specks. Identified on Cheddar cheese. </li></ul><ul><li>1980s – White specks on cheddar cheese classified as calcium lactate crystal. </li></ul><ul><li>CLCs pose no health hazard to the consumer. </li></ul><ul><li>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. </li></ul><ul><li>Chemical Formula - C 6 H 10 CaO 6 </li></ul><ul><li>MW: 2182214 </li></ul><ul><li>Calcium lactate is sold as vitamin supplements and as chemical agents. </li></ul>
  5. 5. How do CLCs form in cheese? <ul><li>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). </li></ul><ul><li>Proposed by Dybing, et. al. (Land O’Lakes, Inc., Cheese Research Group, R&D) </li></ul>
  6. 6. How do CLCs form in cheese? (cont.) <ul><li>Dybing et al. (1988) proposed several causes to CLC formation. </li></ul><ul><ul><li>Physical causes – Packaging (temperature, CO 2 vs. vacuum), length of curing, seasonal effects </li></ul></ul><ul><ul><li>Chemical causes – D to L -lactic acid content, free Ca ion content, salt concentration, pH at milling, rate of acidification during manufacture. </li></ul></ul><ul><li>Several more studies showed non-starter lactic acid bacteria (NSLAB) and D -lactic acid as a possible underlying cause to CLCs. </li></ul><ul><ul><li>Johnson et al. (1990) used an unidentified strain of Lactobacillus . Found when used, crystals would form and noticed an increase of D -lactic acid. </li></ul></ul><ul><ul><li>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. </li></ul></ul><ul><ul><li>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 . </li></ul></ul>
  7. 7. Non-Starter Lactic Acid Bacteria (NSLAB) <ul><li>Generally defined as a lactic acid producing bacterium introduced post-manufacture. </li></ul><ul><li>One method of NSLAB classification is by the sugars and the metabolic pathway utilization. </li></ul><ul><ul><li>Obligately homofermentative – Glycolysis </li></ul></ul><ul><ul><li>Obligately heterofermentative – 6-phosphogluconate/phospoketolase (6-PG/PK) </li></ul></ul><ul><ul><li>Facultative heterofermentative – May use both Glycolysis and 6-PG/PK pathways </li></ul></ul><ul><ul><ul><li>Lb. casei, Lb. plantarum, Lb. sake, Lb. curvatus </li></ul></ul></ul><ul><li>Lb. curvatus – Gram positive single rods with a slight moon shape curve. Commonly found in fermented foods such as sausage and sauerkraut. </li></ul><ul><ul><li>Produces the enzyme lactate dehydrogenase that yield lactic acid. </li></ul></ul>
  8. 8. Lactate Dehydrogenase (Ldh) <ul><li>Dehydrogenases belong to the EC 1.1 class of enzymes . </li></ul><ul><ul><li>Transfer a hydride ion (H: - ) to an acceptor such as nicotinamide adenine dinucleotide (NAD + ), oxygen, quinone, or cytochrome. </li></ul></ul><ul><li>Lactate dehyrogenase catalyzes the reaction from pyruvate (glycolysis) to lactic acid (fermentation). </li></ul><ul><li>D and L lactate dehydrogenase have been identified and produces stereoisomers of lactic acid. </li></ul><ul><li>~140 kDa for both D and L forms of LDH </li></ul>
  9. 9. Lactic Acid D (-) -Lactic Acid L (+)-Lactic Acid
  10. 10. Lactic Acid <ul><li>Byproduct of lactic acid fermentation </li></ul><ul><ul><li>Skeletal muscle under strenuous exercise undergo lactic acid fermentation </li></ul></ul><ul><ul><li>Cheese ripening occurs under anaerobic conditions, allowing for lactic acid fermentation </li></ul></ul><ul><li>C 3 H 6 O 3 </li></ul><ul><li>MW: 90.0786 g/mole </li></ul><ul><li>Two isomers of lactic acid </li></ul><ul><ul><li>L (+)-lactic acid – produced normally from commercial cheese starter cultures (mesophillic Lactococci ) </li></ul></ul><ul><ul><li>D (-)-lactic acid – not normally produced, specifically seen if there is the presence of D -LDH. </li></ul></ul>L (+)-Lactic Acid L (+)-Lactic Acid
  11. 11. Objectives <ul><li>Isolate NSLAB from cheese with CLC defect. </li></ul><ul><li>Identify NSLAB isolated from cheese with CLC defect. </li></ul><ul><li>Use isolated organism as an adjunct to produce CLC defect in cheese. </li></ul><ul><li>Isolate the D(-)-ldh gene from the organism used to produce the CLC defect in cheese. </li></ul>
  12. 12. Materials and Methods – The Cheese <ul><li>Bulk Cheese from Wisconsin, Asiago Cheddar, Tillamook Cheddar, New Zealand and Iceland type Cheddar, and Edam Gouda were received. </li></ul><ul><li>The cheese from Wisconsin, Asiago Cheddar, and New Zealand and Iceland type Cheddar exhibited CLCs. </li></ul><ul><li>Tillamook Cheddar (store bought) and Edam Gouda did not show signs of CLCs. </li></ul><ul><li>Each cheese was cut into smaller blocks, grated, packaged and stored @2-3ºC for analysis. </li></ul>
  13. 13. Materials and Methods – Objective #1 <ul><li>Isolate NSLAB from cheese with CLC defect </li></ul><ul><li>10g from each cheese was added to 90 mL 2% trisodium citrate (TSC) solution (10 -1 ) and homogenized with a stomacher. </li></ul><ul><li>10 -2 – 10 -6 serial dilutions were made with 9 mL Butterfield’s buffer with corresponding pour plates (enumeration) and streak plates (isolated bacteria). </li></ul><ul><li>Rogosa SA and MRS agar were the primary agars used to isolate and enumerate bacteria. </li></ul><ul><ul><li>Rogosa SA – Incubated 3-4 days, anaerobic conditions, 37ºC </li></ul></ul><ul><ul><li>MRS agar – Incubated 1-2 days, anaerobic conditions, 37ºC </li></ul></ul><ul><li>Isolated bacteria were cultivated and further stored in MRS broth. </li></ul><ul><ul><li>For frozen storage, the bacteria was suspended in “S” buffer and glycerol. </li></ul></ul><ul><li>Averaged to 80 bacterial isolated </li></ul>
  14. 14. Materials and Methods – Objective #2 <ul><li>Identify NSLAB isolated from cheese with CLC defect </li></ul><ul><li>Gram staining – visual observation </li></ul><ul><li>16S rRNA Polymerase Chain Reaction (PCR) – rRNA isolation </li></ul><ul><ul><li>Genomic DNA purification of each isolated bacteria. Used a DNA isolation kit (MoBio Laboratories, Inc., Solana Beach, CA) </li></ul></ul><ul><ul><li>PCR – Used universal primers UF2 and UR523 for 16S rRNA isolation </li></ul></ul><ul><ul><ul><li>94ºC 2 min, (94ºC 15 sec, 55ºC 1 min, 72ºC 1.5 min) × 30, 72ºC 7 min. </li></ul></ul></ul><ul><ul><li>PCR product cleaned with a PCR clean up kit (MoBio Laboratories, Inc., Solana Beach, CA) </li></ul></ul><ul><li>PCR product sequencing </li></ul><ul><ul><li>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. </li></ul></ul><ul><ul><li>Sequences were sent back and used BLAST software (National Center of Biotechnology Information Center) to identify the bacteria. </li></ul></ul>
  15. 15. Materials and Methods – Objective #3 <ul><li>Use isolated organism as adjunct to produce CLC defect in cheese </li></ul><ul><li>BUT WAIT!! We needed to figure out what was the proper adjunct. </li></ul><ul><li>The proper adjunct must be: </li></ul><ul><ul><li>NSLAB </li></ul></ul><ul><ul><li>Evidence the NSLAB can produce D -LDH </li></ul></ul><ul><ul><li>Produces a large amount of D (-)-lactic acid </li></ul></ul><ul><li>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. </li></ul><ul><li>When the proper adjunct was chosen, the NSLAB was grown in 150 mL MRS broth for 16-20 hours before the day of cheesemaking. </li></ul>
  16. 16. Material and Methods - Cheesemaking Milk C (Control) 60 gal Experimental 60 gal Gas Flushed 20 lb block Vacuumed Packaged 20 lb block A (no CaCl 2 ) A+ (CaCl 2 ) Gas Flushed 10 lb block Vacuumed Packaged 10 lb block Gas Flushed 10 lb block Vacuumed Packaged 10 lb block
  17. 17. Materials and Methods – Cheese Sampling and Analysis <ul><li>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. </li></ul><ul><ul><li>Exception was if crystals were observed early, stop sampling. </li></ul></ul><ul><li>Cheese analysis includes: </li></ul><ul><ul><li>Composition – fat (%), moisture (%), pH, DL -lactic acid content. </li></ul></ul><ul><ul><li>Microbiology – Rogosa SA and MRS Agar bacterial enumeration </li></ul></ul>
  18. 18. Materials and Methods – Objective #4 <ul><li>Isolate the D -ldh gene from the organism used to produce the CLC defect. </li></ul><ul><li>We used specific custom primers to isolate a fragment of the D-ldh gene and used PCR to amplify the fragment. </li></ul><ul><li>PCR product sent to Utah State University – Biotechnology Center for sequencing. </li></ul><ul><li>Sequences were compared to published D -ldh gene sequences using Clustal W. </li></ul>
  19. 19. Results and Discussion <ul><li>~80 bacterial isolates – 60 isolates from cheeses with the CLC defect, 20 isolates from cheeses with no signs of CLCs. </li></ul><ul><ul><li>2/3 of the 60 isolates were identified as Lb. curvatus </li></ul></ul><ul><ul><li>Remaining isolates identified as Lb. casei, Lb. paracasei, Lb. coryniformus . </li></ul></ul><ul><li>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. </li></ul><ul><li>An isolate was selected at random from the 7 identified as Lb. curvatus to be used as an adjunct. </li></ul>
  20. 20. Results and Discussion <ul><li>Compositional Analysis of cheeses </li></ul><ul><li>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. </li></ul><ul><ul><li>Food and Drug Administration (FDA), Code of Food Regulation, Title 21, Section 133.113. </li></ul></ul>5.03 ± 0.03 5.01 ± 0.01 5.02 ± 0.02 pH 1.34 ± 0.02 1.16 ± 0.16 1.45 ± 0.04 Salt (%) 35.87 ± 0.72 35.90 ± 0.77 36.09 ± 0.85 Moisture (%) 33.0 33.0 33.0 Fat (%) n=2 n=2 N=2   w/ Adjunct + CaCl 2 w/ Adjunct Control   Mean composition of seven-day old Cheddar cheeses.
  21. 21. Results and Discussion <ul><li>NSLAB counts from preliminary trial and trial cheeses. </li></ul>
  22. 22. Results and Discussion <ul><li>When did crystals form? </li></ul><ul><ul><li>Preliminary trial – Control cheese showed no signs of CLCs. Vacuumed packaged cheese showed CLCs ~180 days of ripening. Gas flushed packaged cheese showed CLCs ~150-160 days of ripening. </li></ul></ul><ul><ul><li>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. </li></ul></ul>Cheddar cheese ( 6 0 day-old) showing calcium lactate crystals. Cheese manufactured with LbA2 starter adjunct and vacuum packaged. Cheddar cheese (60 day-old) showing calcium lactate crystals. Cheese manufactured with LbA2 starter adjunct and CO2 flushed packaging.
  23. 23. Results and Discussion <ul><li>Each cheese manufactured was screened for D and L -lactic acid, including in the different packaging conditions. </li></ul><ul><li>Statistics – ANOVA (Minitab 14) </li></ul><ul><ul><li>No significant difference of lactic acid content between A and A+ cheese (p=0.711-0.970). </li></ul></ul><ul><ul><ul><li>Expected result since addition of CaCl 2 was not expected to influence production of D (-)-lactic acid. </li></ul></ul></ul><ul><ul><li>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). </li></ul></ul><ul><ul><ul><li>Expected to observe an increased amount of D (-)-lactic acid, but whether a significant increase was in question. </li></ul></ul></ul><ul><ul><li>There was no significant difference of L (+)-lactic acid content in C and A/A+ cheeses over ripening time. </li></ul></ul><ul><ul><li>When looking at D (-)-lactic acid content on sampling days between C and A/A+ cheese, there is a significant difference. </li></ul></ul><ul><ul><li>Type of packaging showed a significant difference on control cheese, however, no difference was observed with the A/A+ cheeses. </li></ul></ul>
  24. 24. What does this mean? <ul><li>There seems to be a correlation of Lb. curvatus cell density to D (-)-lactic acid content. </li></ul><ul><ul><li>However, a direct enzyme activity wasn’t done to confirm this. </li></ul></ul><ul><ul><li>Observation: Preliminary trial bacterial counts and lactic acid content compared to experimental trials. </li></ul></ul><ul><li>High correlation of D (-)-lactic acid to CLC development. </li></ul><ul><ul><li>Based on observation and lactic acid analyses. </li></ul></ul><ul><li>Packaging influences CLC development. </li></ul>
  25. 25. Polymerase Chain Reaction (PCR) <ul><li>PCR is a technique for amplifying the amount of a specific segment of DNA. </li></ul><ul><li>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. </li></ul><ul><li>Basic strategy of PCR: </li></ul><ul><ul><li>Denaturation of template DNA </li></ul></ul><ul><ul><li>Annealing of primers </li></ul></ul><ul><ul><li>Extension of DNA </li></ul></ul><ul><li>For this project, needed to figure out the D-ldh gene for Lb. curvatus. </li></ul><ul><ul><li>Template DNA </li></ul></ul><ul><ul><li>Primers </li></ul></ul><ul><ul><li>PCR </li></ul></ul><ul><ul><li>Did we get the gene? </li></ul></ul>
  26. 26. PCR General priming sites for FCPBY12, FCPBY15, FCPBY19, and FCPBY19D. FCPBY18D General priming sites for RCPBY15, RBYCP24, and RCPBY20 RCPBY20D RCPBY21D
  27. 27. PCR 5'-TTTGCTTACGCTATTCGTG-3' FCPBY19D 5'-ACCATGTTACGTACAGCATGA-3' RCPBY21D 5'-ATCTTTAAGTGCGCTTGTCA-3' RCPBY20D 5'-AGGGTGCTGACCGAGAAT-3' FCPBY18D 5'-GCAGTATGTGGAGTTACCAA-3' RCPBY20 5'-GCTTACGCTATTCGTAAAG-3' FCPBY19 5'-AGTGTAGAAAGCAGTGTGTGGAGT-3' RBYCP24 5'-GTAGAAAGCAGTGTG-3' RCPBY15 5'-ATTTTTGCTTACGCT-3' FCPBY15 5'-ATTTTTGCTTACGCTATTCGT-3' FCPBY12 Primer Sequence Primer Name Primers used for D-Lactate Dehydrogenase Gene Isolation.
  28. 28. PCR 1000bp, 2000 bp 72ºC 2.5 min 45ºC 1 min 94ºC 15 sec RCPBY15 FCPBY15 1000bp, 2000 bp 72ºC 2 min 35ºC 1 min 94ºC 15 sec RCPBY15 FCPBY15 no product 72ºC 2 min 35ºC, 55º C 1 min 94ºC 15 sec RCPBY21D FCPBY19D no product 72ºC 2 min 35ºC, 55º C 1 min 94ºC 15 sec RCPBY20D FCPCY18D no product 72ºC 1.5 min 55ºC 1 min 94ºC 30 sec RCPBY20 FCPBY19 no product 72ºC 1.5 min 45ºC 1 min 94ºC 30 sec RBYCP24 FCPBY19 no product 72ºC 1.5 min 45ºC 1 min 94ºC 1 min RCPBY21 FCPBY19 no product 72ºC 1.5 min 40ºC, 50º C 1 min 94ºC 15 sec RBYCP24 FCPBY21 1500 bp 72ºC 1.5 min 35ºC 1 min 94ºC 15 sec RBYCP24 FCPBY15 no product 72ºC 1.5 min 35ºC 1 min 94ºC 15 sec RCPBY15 FCPBY15 500 bp 72ºC 1.5 min 35ºC 1 min 94ºC 15 sec RBYCP24 FCPBY21 400 bp 72ºC 1.5 min 35ºC 1 min 94ºC 15 sec RCPBY15 FCPBY21 Results Elongation Annealing Denaturation Reverse Forward Primers pairs with their respective PCR cycles and results. Final PCR cycle chosen
  29. 29. Did we get the D -LDH gene? <ul><li>Yes and no. We believe we have a fragment of the gene. </li></ul><ul><li>2 fragments were obtained. LB2-5C12 and LB2-5C-10 (920bp and 1132bp, respectively) </li></ul><ul><ul><li>Clustal W to compare with published D -ldh genes. Fragment reside together and towards the middle of the published sequence. </li></ul></ul>>LB25C10 ATCTGGCGTTTAACGAATCGCCCTTGTAGAAGCAGTGTGCCCATTGGCTAACTTCTTCTTTTCGATGACGTATTCGTAAATTTTAGCGGCACTACGCGTGACCGTTTCTTGTGATAAGCGATCTTTGTTAGCTTGAATAAATGCTTGGACATCGGAATCGTTCAAGGCATCTTCGACCAATTTATTAAACTGTTGGTTTAACTTTTGCCGATTCATGTAATCCGTCAAATCTTTACCCATATTTTCCATTACGATTCCCGCCCCTCACGAATTTTTTTCAATGCCGCTTCTAGTTCATGCTGATGCTCAGGTGTGGTTGtTTCTTTTGGCGCTTGATAATCTGGTTTAGCCCATTTAGGTACTGGTTCTTTTTTAGTTTTGTTCTGATAACGCGTTTGGCGATTGTTTTGCACCTTTGCCGYCTGTTTGGTTTGGAAATCGSGAATCTGCAAAATCGCATCCGCTGCCGTCTTAACCCCTTGTTGAGCCCATTGGTTCGCAATCCGATCGACTAGCGCCTGAGTGAGCCCATCATATTGCGTAATGATATAAACGACCAAAATATTGAGCACATCATTGTTAAAGATATAGCGGTTTTGCAAGTCCTTTAACGCCCGGATTTCATTTTTAGCGACAAAGCCGTGGTTCTTTTGTTTCAAGTATTCGAGATAATCAACTGGCAAGTAGCCCTGACTTTCTGTTAGCCACTGCAATTCCTGTTCGTTAAAGCCGGCTTTTTGCCATTCTTTTTGKAACTCGGCTAACGGTTTTtGCGTTGTTGTAgCASCTGGCGTTTGCCGGTtACTCTGACGTCSTGKGKAATTGKTCAAgACGTTCTGTTCgAGCGCTGCCATATTAATCGTACTGTCAGCAACATTCATCGTCAAGCCAATCAAACGCGCTAAGTCCATCTCATCTAGACCATAGAAATAATGGAGATTAAACAAGCCCTGTTCATTTTTGAGAATTTGATCCGTATCAATCTGGTATTGCGCCGTTGCATCTTGTAATAGGTCCCAATCAAAAGTCCGCAACTCAACCGAGGTAAACTGCGGTGTGCTAGCTGCTTTTTGTTGATACATTGCCTTAGTTTCAGCCACACGCTTCTACAAGGGCGAATCGGGCGTATCAG
  30. 30. Conclusions <ul><li>Strong correlation between high bacterial density of Lb. curvatus and CLC formation. </li></ul><ul><li>CO 2 packaging seem to allow faster CLC formation versus vacuum packaging. </li></ul><ul><li>Lb. curvatus produces the enzyme D -lactate dehydrogenase yielding D (-)-lactic acid. </li></ul><ul><li>Correlation of D (-)-lactic acid content and CLC formation. </li></ul>
  31. 31. Recommendation for Future Work <ul><li>Produce a mutant Lb. curvatus with the D -LDH gene deleted or partially deleted. </li></ul><ul><li>Complete sequence of Lb. curvatus genome and its D -LDH gene. </li></ul><ul><li>Bacteriocins or cross-inhibition studies to Lb. curvatus and other D (-)-lactic acid producing bacteria. </li></ul>
  32. 32. Recognition <ul><li>Dr. Shakeel-Ur Rehman and Dr. Vedamuthu, E. </li></ul><ul><li>Agriculture Research Initiative </li></ul><ul><li>Staff and colleagues of the Dairy Products Technology Center </li></ul><ul><li>Mr. and Mrs. Chong Nam Yim and family </li></ul><ul><li>Jocelyn Fagar – Rest in peace. </li></ul>
  33. 33. References <ul><li>Agarwal, S. C., S.; Swanson, B.G.; Yuksel, G.U. 2003. Non starter lactic acid bacteria and calcium lactate crystal formation in Cheddar cheese. Institute of Food Technologists. </li></ul><ul><li>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. </li></ul><ul><li>Coyne, V. E., M. D. James, S. J. Reid, and E. P. Rybicki. 2001. PCR Primer Design and Reaction Optimisation. Molecular Biology Techniques Manual. 3:1-11. </li></ul><ul><li>Dorn, F. L. and A. C. Dahlberg. 1941. Identification of the White Particles Found on Ripened Cheddar Cheese. New York Agricultural Experiment Station.31-36. </li></ul><ul><li>Dybing, S. T., J. A. Wiegand, S. A. Brudvig, E. A. Huang, and R. C. Chandan. 1988. Effect of Processing Variables on the Formation of Calcium Lactate Crystals on Cheddar Cheese. Journal of Dairy Science. 71:1701-1710. </li></ul><ul><li>Johnson, M. E., B. A. Riesterer, C. Chen, B. Tricomi, and N. F. Olson. 1990. Effect of Packaging and Storage Conditions on Calcium Lactate Crystallization on the Surface of Cheddar Cheese. Journal of Dairy Science. 73:3033-3041. </li></ul><ul><li>Kochhar, S., H. Hottinger, N. Chuard, P. G. Taylor, T. Atkinson, D. Scawen, and D. J. Nicholls. 1992b. Cloning and Overexpression of Lactobacillus helveticus D-Lactate Dehydrogenase Gene in Escherichia coli . European Journal of Biochemistry. 208:799-805. </li></ul><ul><li>Marshall, R. T. 1992. Standard Methods for the Examination of Dairy Products. 16th ed. </li></ul><ul><li>Roux, K. H. 1995. Optimization and Troubleshooting in PCR. Cold Spring Harbor Laboratory. 4:S185-S194. </li></ul><ul><li>Severn, D. J., M. E. Johnson, and N. F. Olson. 1986. Determination of Lactic Acid in Cheddar Cheese and Calcium Lactate Crystals. Journal of Dairy Science. 69:2027-2030. </li></ul><ul><li>Templeton, N. A. 1992. The Polymerase Chain Reaction. Diagnostic Molecular Pathology. 1(1):58-72. </li></ul><ul><li>Thomas, T. D. and V. L. Crow. 1983. Mechanism of D(-)-Lactic Acid Formation in Cheddar Cheese. New Zealand Journal of Dairy Science and Technology. 18:131-141. </li></ul>

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