Slit Defect in Cheddar Cheese

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Slit Defect in Cheddar Cheese

• Catherine Donnelly, P.I., UVM
• Cecilia Golnazarian, UVM
• Kathryn Boor, co-P.I., Cornell
• 13th Annual Cornell Conference on Dairy Markets
  and Product Research

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Slit defect

• A structural defect in long-hold Cheddar cheese.
• No specific sensory defects have been observed in
  defective cheeses.
• Defect can increase cutting losses from 10
  (non-defective) up to 50.
• This problem costs the cheese industry a lot of
  money.

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Project hypotheses

• Gas-producing non-starter lactic acid bacteria
  are responsible for the slit defect in long-hold
  Cheddar cheese.
• Identification of strains responsible for slit
  defect will allow development of specific control
  strategies to reduce the incidence of the defect.

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Where could gas come from?

• CO2 evolution from fermentation of residual
  lactose by heterofermentative lactobacilli
• CO2 from fermentation of residual citrate

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Residual lactose

• Initial lactose level 1, decreasing slowly over
  a few weeks with a parallel rise in L-lactate
  (lactic acid)
• Non Starter Lactics (NSLAB) reach high levels by
  50 days (107-108cfu/g)total
• Pediococci
• Lactobacilli
• Lactobacillus casei Lactobacillus plantarum
• Lactate remains 1.5, but NSLAB transform
  L-lactate to D-lactate

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Non-starter lactic acid bacteria

• Some strains can
• Oxidize lactate to acetate CO2 H20
• Oxidize citrate to acetate CO2 H20

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Citrate fermenting organisms

• Lactobacillus plantarum
• Lactobacillus casei
• Streptococcus lactis ssp. diacetylactis

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Project Objectives

• Isolate and characterize heterofermentative
  lactic acid bacteria associated with the slit
  cheese defect
• Use phenotypic and genetic methods to classify
  isolates into coherent subsets

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Experimental approach

• Isolation of lactic acid bacteria from Cheddar
  cheese and from plant environments using
  Petrifilm Aerobic Count Plate for Lactic Acid
  Bacteria
• Characterization of isolates by API 50 CH, Biolog
  and automated ribotyping (RiboPrinterTM,
  Qualicon)
• Test lactic acid bacteria representative for
  Cheddar cheese exhibiting the slit defect in
  cheese making experiments

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Results

• Obtained 83 and 27 lactic acid bacterial isolates
  from Cheddar cheese with and without the slit
  defect, respectively
• Characterization of isolates from slit cheese by
  API 50CH

Species Slit cheese Normal Cheese Lb.
curvatus 27 5 Lb. paracasei subsp.
paracasei 1 3 0 Lb. paracasei subsp.
paracasei 3 21 19 Unidentified 32 3
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Biolog Characterization

• Bacterial identification system that uses
  oxidation patterns of 95 different substrates for
  the identification of bacterial species
• In preliminary experiments, the standard protocol
  as suggested by Biolog gave unsatisfactory
  results
• We developed a modified Biolog protocol using
  anaerobic incubation which will provide better
  phenotypic characterization and differentiation
  of lactic acid bacteria
• Biolog data allowed clustering of isolates into
  subsets

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Ribotype Characterization

• 11 different ribotypes among 23 isolates
• Ribotyping shows a discriminatory index
  (Simpsons Index) of 0.848, i.e. this method can
  differentiate two unrelated strains 84.8 of the
  time

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Summary

• The API 50CH system identifies heterofermentative lactic acid bacteria isolates
  from Cheddar cheese to the species level
• The standard Biolog protocol does not allow good
  differentiation of heterofermentative lactic acid
  bacteria isolated from Cheddar cheese
• Automated ribotyping shows good discriminatory
  ability of lactic acid bacteria isolated from
  Cheddar cheese

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Summary (continued)

• Lb. curvatus and heterofermentative lactic acid
  bacteria which could not be identified by API
  50CH appear to be more common in Cheddar cheese
  exhibiting the slit defect as compared to normal
  cheeses

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Project Objectives

• Determine whether specific lactic acid bacteria
  strains are responsible for slit defect through
  cheese making experiments.
• Chemically analyze cheeses, including gas
  production.
• Monitor development of the slit defect using
  Magnetic Resonance Imaging.

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Strains used in cheese making trials

• L. curvatus
• L. paracasei ssp. paracasei 1
• L. paracasei ssp. paracasei 3 (4 strains)
• Cocktail of all 6 strains
• uninoculated control

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Time until slit development

• L. paracasei ssp. paracasei 3
• API-4 6mo
• 626-19 6mo
• 1033 4 mo
• 815 no slits

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Time until slit development

• L. paracasei 1 API-11 4 mo.
• L. curvatus 626-17 4 mo
• cocktail no slits
• control no slits

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• Cheese making experiments using Lb. curvatus
  (n3), Lb. paracasei subsp. paracasei 1(n2) and
  Lb. paracasei subsp paracasei 3 (n6) showed that
  some strains of each species appear to be able to
  cause the slit defect, although two isolates did
  not cause slit defect

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Project Objective

• Characterize strains for salt and thermotolerance
  and ability to form biofilms.

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Strains Chosen for Pasteurization Salt Tolerance

• Two L. para. ssp. para. 1
• 74-S-4 (2)
• Five L. para. ssp. para. 3
• 74-S-1 (2) 74-S-2 (1) 74-S-3 (2)
• Three L. curvatus
• 74-S-2 (1) 74-S-7 (2)

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HTST Pasteurization

• Cultured strains in MRSB (30oC/24h)
• Inoculated 2 sterile milk
• 105 cfu/ml
• Sealed and submerged in water bath
• 71.7oC 16 sec.
• Incubated 2 hr. at 31.1oC
• Plated serial dilutions on MRSA

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Pasteurization Results

• 2 isolates survived pasteurization
• L. paracasei ssp. paracasei 3
• 74-S-1
• 74-S-2

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Thermotolerance

• 105cfu/ml suspended in milk
• Sealed and submerged in water bath
• 62.8oC
• Removed at intervals
• 0, 10, 20, and 30 minutes
• Incubated 2 hr at 31.1oC
• Plated serial dilutions on MRSA

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Thermotolerance Results (62.8C)
4 11 12 13 15 17 18 19 103 815
strain
0 10 20 30 minutes
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Conclusions - Pasteurization

• Two strains survived HTST pasteurization
  (71.7oC)
• Four strains survived pasteurization at 62.8oC

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Salt Tolerance in MRS

• Isolates cultured in MRSB 18 hrs 30oC
• 20 ul aliquots delivered to MRSB NaCl
• 0, 1, 2, and 5
• Incubated 24hrs 30oC
• Plated on MRSA
• Incubated 48 hrs

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Salt Tolerance in Milk

• Isolates cultured in MRSB 18 hrs 30oC
• 20ul aliquots to 2 milk w/NaCl
• 0, 1, 2, and 5
• Incubated 24 hrs 30oC
• Plated on MRSA
• Incubated 48 hrs

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Conclusions - Salt Tolerance

• Numbers increased up to 2 added NaCl, then
  decreased at 5 added NaCl for
• L. curvatus (3)
• L. paracasei ssp. paracasei 3 (3)
• Numbers decreased with added NaCl for
• L. paracasei ssp. paracasei 3 (2)
• L. paracasei ssp. paracasei 1 (2)

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Cleaner And Sanitizer Efficacy Against Biofilm

• Develop biofilm of Lactobacilli
• an attachment of the bacteria to a surface
• development of glycocalyx
• Compare abilities of cleaners and sanitizers to
  remove biofilm

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Biofilm Formation

• Stainless steel coupons type 304/2b added to 2
  UHT milk 104 cfu/ml culture
• Shaking water bath 31oC 10 days
• milk refreshed at day 5
• Confirm presence of biofilm with SEM

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SEM - 8 day biofilm 7,500x
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SEM - 8 day biofilm 7,500x
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Cleaning/Sanitizing Protocol

• Designed to simulate cleaning of processing
  pieces by hand
• Rinse vigorously with PBS (3x)
• Swab with cleaner Rinse (3x)
• Alkaline detergent
• Place into sanitizer Rinse (3x)
• Quaternary Ammonium

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Survival (Presence/Absence)

• Controls
• 1.Uninoculated milk, not cleaned/sanitized
• 2. Inoculated, not cleaned/sanitized
• Rinse in PBS (3x)
• Swab with PBS Rinse (3x)
• 3. Uninoculated milk, cleaned/sanitized

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Biofilm Results

• Control - uninoculated milk coupon
• Rinsed and swabbed only
• positive
• Control - uninoculated milk coupon
• Cleaned and sanitized
• negative

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Biofilm Results

• Control - Inoculated
• Untreated
• positive
• Cleaned and Sanitized - Inoculated
• positive

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Conclusions

• Heterofermentative lactic acid bacteria can play
  a role in the genesis of the slit defect
• Species identification of lactic acid bacteria
  isolated from Cheddar cheese using phenotypic
  methods is often unsatisfactory
• Automated ribotyping shows promise as a highly
  discriminatory tool for tracking the spread and
  origin of non-starter lactic acid bacteria

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Conclusions

• Heterofermentative lactobacilli isolated from
  defective Cheddar cheeses have the capacity to
  form biofilms that are resistant to hand cleaning
  and sanitizing procedures.
• These biofilms are likely to contaminate cheeses
  during manufacturing processes.
• Plant sanitation practices are therefore likely
  to play an important role in the occurrence of
  this defect.