Effect of commercial bacterial fermentates and protective cultures on - PowerPoint PPT Presentation

Effect of commercial bacterial fermentates and protective cultures on Listeria monocytogenes growth in a refrigerated model high-moisture cheese Sarah Engstrom Principal Investigator: Dr. Kathleen Glass Food Research Institute, UW-Madison July 23, 2019 March 3, 2016

Roos Forever ??? Miller’s Blue Bell Vulto Foods Organic Cheese Creamery Deli- Creameries Farm sliced 16 Latin- Ricotta Raw milk, Ice cream Raw milk style Salata soft-ripened 2012 2019 Crave Karoun Dairies Oasis La Nica Deutsch Brothers Panera Brands Products Queso Kase Haus Washed rind, Cream 6 Latin- fresco/blanco, 2 Latin- 7 hard soft-ripened cheese feta, mozzarella style style varieties CDC 2012-2019.

• Two-thirds of 2006-2014 Listeria cheese outbreaks linked to soft Latin-style varieties • 287 million lbs Latin-style cheese produced in 2017 ( 316% increase from 1997) Roos Forever ??? Miller’s Blue Bell Vulto Foods Organic Cheese Creamery Deli- Creameries Farm sliced • Refrigeration typically only post- 16 Latin- Ricotta Raw milk, Ice cream Raw milk style Salata manufacturing hurdle for fresh soft cheeses soft-ripened 2012 2019 Crave Karoun Dairies Oasis La Nica Deutsch Brothers Panera Brands Products Queso Kase Haus Washed rind, Cream 6 Latin- fresco/blanco, 2 Latin- 7 hard soft-ripened cheese feta, mozzarella style style varieties CDC 2012-2019, Jackson 2018, Ibarra-Sánchez 2017.

Biopreservation: “The use of microorganisms and/or their metabolites to improve the safety of foods without altering their sensory attributes” Bacterial Fermentates Protective Cultures • Industrial-scale fermentation • Added as starter or adjunct byproducts of culture, e.g. Lactococcus lactis , Propionibacterium , Lactococcus , Lactobacillus plantarum Pediococcus , etc. • Ideal qualities: GRAS status, • Likely active compounds: bacteriocin production, organic acids, bacteriocin, acidification ability, vinegar (as blend) growth/survival in product Holzapfel 1995, Stiles 1996, Melero 2013, Favaro 2015.

Previous Studies Listeria (log CFU/g) 7.5 - Glass 1995 Queso blanco (pH 5.2, 52% moisture) made 6.5 with ( ● ) or without ( ○ ) 0.6% fermentate 5.5 0 42 Days at 4°C Listeria (log CFU/g) 5 - Mendoza-Yepes 1999 Queso fresco (pH 6.5) made with ( ● ) or without ( ○ ) protective culture (10 7 CFU/g) 4 3 0 23 Days at 3 ° C 4 - McAuliffe 1999 Listeria (log CFU/g) Cottage cheese (pH 5.2) made with control ( ○ ) or bacteriocin-producing ( ● ) protective 2.5 culture (10 5 CFU/g) 1 0 7 Days at 4°C

Objective: To determine the effect of 3 commercial fermentates and 3 commercial protective cultures on the growth of L. monocytogenes in a model soft cheese Fermentate Label Format Level CM-1 “Cultured milk” Powder 0.5%, 1.0% CM-2 “Cultured milk powder” Powder 0.5%, 1.0% CSV-1 Powder 0.5%, 1.0% “Cultured sugar, vinegar” Culture Label Format Level 10 6 CFU/g PC-1 “Culture” ( Lactococcus lactis ) Pelleted 10 6 CFU/g PC-2 “Culture” ( Lactococcus lactis ) Concentrate 10 6 CFU/g PC-3 “Culture” ( Lactobacillus plantarum ) Powder

Methods – Model Soft Cheese System Targets: pH 6, 56% H 2 O, 1.25% salt -Cream (pasteurized, 40% fat) -Micellar casein -Sterile water -Lactose -Salt -Lactic acid

Methods – Biopreservative Incorporation Bacterial Fermentates Protective Cultures Target: 10 6 CFU/g Target: 0.5%, 1.0% <0.1% to 0.5% 0.25% (v/w) MRS broth 30°C 18-22 h, peptone serial dilutions

Methods – Inoculation • Inoculated with 3-log CFU/g (0.5% or 0.25% v/w) 5-strain L. monocytogenes cocktail: LM 108 - hard salami (1/2b) LM 301 – cheddar (1/2a) LM 310-goat’s milk cheese (4b) R2-500 - food outbreak (4b) R2-501 - clinical isolate (4b) • Mixed 2 minutes to distribute • 25 g portions vacuum-sealed • Each formulation replicated in duplicate trials

Methods – L. monocytogenes Enumeration Samples stored at 4 ° C and plated at time 0 and weekly through 8 weeks + Serial dilutions in Triplicate samples 1:2 dilution in 0.1% 30 sec 0.1% peptone peptone Modified Oxford agar 2 days 35 ° C

Other Analyses pH Moisture Salt a w Weeks 0-8 Day 0 Day 0 Day 0 Lactic ic Acid id Ba Bacteria Yeasts & Molds Aerobi bic P Pla late C Count Weeks 0-8 Day 0, Week 4, Week 8 Day 0, Week 4, Week 8

L. monocytogenes in pH 6 / 56% moisture cheese + 0.5% fermentate (n=3 per trial, 2 trials) 9 L. monocytogenes (log CFU/g) 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 Weeks at 4 ° C Control CM-1 CM-2 CSV-1

L. monocytogenes in pH 6 / 56% moisture cheese + 0.5% fermentate (n=3 per trial, 2 trials) 9 L. monocytogenes (log CFU/g) 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 Weeks at 4 ° C Control CM-1 CM-2 CSV-1

L. monocytogenes in pH 6 / 56% moisture cheese + 1.0% fermentate (n=3 per trial, 2 trials) 9 L. monocytogenes (log CFU/g) 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 Weeks at 4 ° C Control CM-1 CM-2 CSV-1

L. monocytogenes in pH 6 / 56% moisture cheese + 1.0% fermentate (n=3 per trial, 2 trials) 9 L. monocytogenes (log CFU/g) 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 Weeks at 4 ° C Control CM-1 CM-2 CSV-1

L. monocytogenes in pH 6 / 56% moisture cheese + 10 6 CFU/g protective culture (n=3 per trial, 2 trials) 9 L. monocytogenes (log CFU/g) 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8 Weeks at 4°C Control Lc. lactis 1 Lc. lactis 2 Lb. plantarum

9 9.0 L. monocytogenes (log CFU/g) 8 8.5 7 8.0 6 7.5 Minimum pH values: PC-1 5.52 5 7.0 PC-2 5.90 4 PC-3 5.74 6.5 Cheese pH 3 6.0 2 5.5 1 5.0 0 1 2 3 4 5 6 7 8 Weeks at 4°C Control Lc. lactis 1 Lc. lactis 2 Lb. plantarum Control cheese pH PC-1 cheese pH PC-2 cheese pH PC-3 cheese pH

Conclusions • Protective cultures were ineffective in controlling L. monocytogenes growth in our application • Commercial fermentates delayed growth • 0.5% level – 1.5 to 2 weeks • 1.0% level – 4.5 to 7+ weeks • Fermentate containing vinegar most effective • Our model cheese system can be used to screen antilisterial ingredients

Acknowledgements • Funding: Innovation Center for US Dairy Listeria Research Consortium • Technical: Dr. John Lucey, Gina Mode, Mike Molitor (WI Center for Dairy Research); Dr. Randy Brandsma (Schreiber) • Lab: Kory Anderson, Christie Cheng, Max Golden, Jieyin Lim, Quinn Huibregtse, Brandon Wanless

THANK YOU 20

Fine-tuning formulations L. monocytogenes in 56% moisture cheese acidified with lactic acid Supplemented with 0.5% cultured sugar-vinegar Fermentate C 10 pH 6.0 9 8 pH 5.75 L. monocytogenes (log CFU/g) 7 pH 5.5 6 pH 5.25 5 pH 6.0, 0.5% Cultured Sugar-Vinegar 4 pH 5.75, 0.5% Cultured Sugar-Vinegar 3 pH 5.5, 0.5% Cultured Sugar-Vinegar 2 pH 5.25, 0.5% Cultured Sugar-Vinegar 1 0 Anderson et al., 2019, unpublished data 21 0 2 4 6 8 Weeks at 4 ° C Anderson, et al., 2019, unpublished data

Example - cheese pH 5.6 1-log L. monocytogenes growth at: • 1 week – citric • 3.5 weeks – lactic • >8 weeks – acetic or propionic Citric Lactic Acetic Propionic 8 7 6 log CFU/g 5 4 3 2 0 2 4 6 8 Weeks at 4°C Engstrom, et al., 2018, manuscript in preparation

Phase I Results Cheese Acid % Total Acid (mM) Undissociated pH Undissociated Acid (mM) 5.25 Citric 0.7 29.93 0.21 TEST Lactic 4.7 140.40 6.87 Acetic 25.0 99.09 24.76 Propionic 27.2 117.70 32.00 Acid pKa 5.50 Citric 0.4 18.52 0.08 Citric 3.13 Lactic 2.4 72.14 1.73 Acetic 15.8 60.61 9.57 Lactic 3.83 Propionic 19.5 80.65 15.69 Acetic 4.75 5.75 Citric 0.3 10.23 0.03 Lactic 1.3 41.99 0.56 Propionic 4.88 Acetic 10.6 38.38 4.08 Propionic 11.7 40.11 4.68 6.00 Citric 0.1 3.67 0.01 Lactic 0.7 14.08 0.10 Acetic 5.6 12.41 0.70 Propionic 6.8 14.82 1.00