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 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Cheese Industry ,[object Object],[object Object],[object Object],[object Object],[object Object]

Calcium Lactate Crystal (CLC) Defect ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

How do CLCs form in cheese? ,[object Object],[object Object]

How do CLCs form in cheese? (cont.) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Non-Starter Lactic Acid Bacteria (NSLAB) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Lactate Dehydrogenase (Ldh) ,[object Object],[object Object],[object Object],[object Object],[object Object]

Lactic Acid D (-) -Lactic Acid L (+)-Lactic Acid

Lactic Acid ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],L (+)-Lactic Acid L (+)-Lactic Acid

Objectives ,[object Object],[object Object],[object Object],[object Object]

Materials and Methods – The Cheese ,[object Object],[object Object],[object Object],[object Object]

Materials and Methods – Objective #1 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Materials and Methods – Objective #2 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Materials and Methods – Objective #3 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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

Materials and Methods – Cheese Sampling and Analysis ,[object Object],[object Object],[object Object],[object Object],[object Object]

Materials and Methods – Objective #4 ,[object Object],[object Object],[object Object],[object Object]

Results and Discussion ,[object Object],[object Object],[object Object],[object Object],[object Object]

Results and Discussion ,[object Object],[object Object],[object Object],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.

Results and Discussion ,[object Object]

Results and Discussion ,[object Object],[object Object],[object Object],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.

Results and Discussion ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

What does this mean? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Polymerase Chain Reaction (PCR) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

PCR General priming sites for FCPBY12, FCPBY15, FCPBY19, and FCPBY19D. FCPBY18D General priming sites for RCPBY15, RBYCP24, and RCPBY20 RCPBY20D RCPBY21D

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.

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

Did we get the D -LDH gene? ,[object Object],[object Object],[object Object],>LB25C10 ATCTGGCGTTTAACGAATCGCCCTTGTAGAAGCAGTGTGCCCATTGGCTAACTTCTTCTTTTCGATGACGTATTCGTAAATTTTAGCGGCACTACGCGTGACCGTTTCTTGTGATAAGCGATCTTTGTTAGCTTGAATAAATGCTTGGACATCGGAATCGTTCAAGGCATCTTCGACCAATTTATTAAACTGTTGGTTTAACTTTTGCCGATTCATGTAATCCGTCAAATCTTTACCCATATTTTCCATTACGATTCCCGCCCCTCACGAATTTTTTTCAATGCCGCTTCTAGTTCATGCTGATGCTCAGGTGTGGTTGtTTCTTTTGGCGCTTGATAATCTGGTTTAGCCCATTTAGGTACTGGTTCTTTTTTAGTTTTGTTCTGATAACGCGTTTGGCGATTGTTTTGCACCTTTGCCGYCTGTTTGGTTTGGAAATCGSGAATCTGCAAAATCGCATCCGCTGCCGTCTTAACCCCTTGTTGAGCCCATTGGTTCGCAATCCGATCGACTAGCGCCTGAGTGAGCCCATCATATTGCGTAATGATATAAACGACCAAAATATTGAGCACATCATTGTTAAAGATATAGCGGTTTTGCAAGTCCTTTAACGCCCGGATTTCATTTTTAGCGACAAAGCCGTGGTTCTTTTGTTTCAAGTATTCGAGATAATCAACTGGCAAGTAGCCCTGACTTTCTGTTAGCCACTGCAATTCCTGTTCGTTAAAGCCGGCTTTTTGCCATTCTTTTTGKAACTCGGCTAACGGTTTTtGCGTTGTTGTAgCASCTGGCGTTTGCCGGTtACTCTGACGTCSTGKGKAATTGKTCAAgACGTTCTGTTCgAGCGCTGCCATATTAATCGTACTGTCAGCAACATTCATCGTCAAGCCAATCAAACGCGCTAAGTCCATCTCATCTAGACCATAGAAATAATGGAGATTAAACAAGCCCTGTTCATTTTTGAGAATTTGATCCGTATCAATCTGGTATTGCGCCGTTGCATCTTGTAATAGGTCCCAATCAAAAGTCCGCAACTCAACCGAGGTAAACTGCGGTGTGCTAGCTGCTTTTTGTTGATACATTGCCTTAGTTTCAGCCACACGCTTCTACAAGGGCGAATCGGGCGTATCAG

Conclusions ,[object Object],[object Object],[object Object],[object Object]

Recommendation for Future Work ,[object Object],[object Object],[object Object]

Recognition ,[object Object],[object Object],[object Object],[object Object],[object Object]

References ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Microbial Cause of Calcium Lactate Defect in Cheddar Cheese

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Microbial Cause of Calcium Lactate Defect in Cheddar Cheese