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

March 3, 2016

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

Forever Cheese Ricotta Salata Roos Foods 16 Latin- style Oasis Brands 6 Latin- style Karoun Dairies Queso fresco/blanco, feta, mozzarella La Nica Products 2 Latin- style Panera Cream cheese 2012 2019

CDC 2012-2019.

Crave Brothers Washed rind, soft-ripened Blue Bell Creameries Ice cream Miller’s Organic Farm Raw milk Vulto Creamery Raw milk, soft-ripened ??? Deli- sliced Deutsch Kase Haus 7 hard varieties

Forever Cheese Ricotta Salata Roos Foods 16 Latin- style Oasis Brands 6 Latin- style Karoun Dairies Queso fresco/blanco, feta, mozzarella La Nica Products 2 Latin- style Panera Cream cheese 2012 2019

CDC 2012-2019, Jackson 2018, Ibarra-Sánchez 2017.

Crave Brothers Washed rind, soft-ripened Blue Bell Creameries Ice cream Miller’s Organic Farm Raw milk Vulto Creamery Raw milk, soft-ripened ??? Deli- sliced Deutsch Kase Haus 7 hard varieties

• Two-thirds of 2006-2014 Listeria cheese
• utbreaks linked to soft Latin-style varieties
• 287 million lbs Latin-style cheese produced

in 2017 (316% increase from 1997)

• Refrigeration typically only post-

manufacturing hurdle for fresh soft cheeses

Biopreservation:

“The use of microorganisms and/or their metabolites to improve the safety of foods without altering their sensory attributes”

Bacterial Fermentates

• Industrial-scale fermentation

byproducts of Propionibacterium, Lactococcus, Pediococcus, etc.

• Likely active compounds:
• rganic acids, bacteriocin,

vinegar (as blend)

Protective Cultures

• Added as starter or adjunct

culture, e.g. Lactococcus lactis, Lactobacillus plantarum

• Ideal qualities: GRAS status,

bacteriocin production, acidification ability, growth/survival in product

Holzapfel 1995, Stiles 1996, Melero 2013, Favaro 2015.

Previous Studies

• Glass 1995

Queso blanco (pH 5.2, 52% moisture) made with (●) or without (○) 0.6% fermentate

• Mendoza-Yepes 1999

Queso fresco (pH 6.5) made with (●) or without (○) protective culture (107 CFU/g)

• McAuliffe 1999

Cottage cheese (pH 5.2) made with control (○) or bacteriocin-producing (●) protective culture (105 CFU/g)

3 4 5 23 Listeria (log CFU/g) Days at 3°C 1 2.5 4 7 Listeria (log CFU/g) Days at 4°C 5.5 6.5 7.5 42 Listeria (log CFU/g) Days at 4°C

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 “Cultured sugar, vinegar” Powder 0.5%, 1.0%

To determine the effect of 3 commercial fermentates and 3 commercial protective cultures on the growth

• f L. monocytogenes in a model soft cheese

Objective:

Culture Label Format Level PC-1 “Culture” (Lactococcus lactis) Pelleted 106 CFU/g PC-2 “Culture” (Lactococcus lactis) Concentrate 106 CFU/g PC-3 “Culture” (Lactobacillus plantarum) Powder 106 CFU/g

Methods – Model Soft Cheese System

Targets: pH 6, 56% H2O, 1.25% salt

• Cream (pasteurized, 40% fat)
• Micellar casein
• Sterile water
• Lactose
• Salt
• Lactic acid

<0.1% to 0.5% MRS broth 30°C 18-22 h, peptone serial dilutions

Methods – Biopreservative Incorporation

Bacterial Fermentates Target: 0.5%, 1.0% Protective Cultures Target: 106 CFU/g

0.25% (v/w)

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

Triplicate samples 1:2 dilution in 0.1% peptone 30 sec Serial dilutions in 0.1% peptone Modified Oxford agar 2 days 35°C

Samples stored at 4°C and plated at time 0 and weekly through 8 weeks

+

Salt Day 0

Other Analyses

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

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8

• L. monocytogenes (log CFU/g)

Weeks at 4°C

• L. monocytogenes in pH 6 / 56% moisture cheese

+ 0.5% fermentate (n=3 per trial, 2 trials)

Control CM-1 CM-2 CSV-1

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8

• L. monocytogenes (log CFU/g)

Weeks at 4°C

• L. monocytogenes in pH 6 / 56% moisture cheese

+ 0.5% fermentate (n=3 per trial, 2 trials)

Control CM-1 CM-2 CSV-1

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8

• L. monocytogenes (log CFU/g)

Weeks at 4°C

• L. monocytogenes in pH 6 / 56% moisture cheese

+ 1.0% fermentate (n=3 per trial, 2 trials)

Control CM-1 CM-2 CSV-1

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8

• L. monocytogenes (log CFU/g)

Weeks at 4°C

• L. monocytogenes in pH 6 / 56% moisture cheese

+ 1.0% fermentate (n=3 per trial, 2 trials)

Control CM-1 CM-2 CSV-1

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8

• L. monocytogenes (log CFU/g)

Weeks at 4°C

• L. monocytogenes in pH 6 / 56% moisture cheese

+ 106 CFU/g protective culture (n=3 per trial, 2 trials)

Control

• Lc. lactis 1
• Lc. lactis 2
• Lb. plantarum

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8

Cheese pH

• L. monocytogenes (log CFU/g)

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 Minimum pH values: PC-1 5.52 PC-2 5.90 PC-3 5.74

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

20

THANK YOU

21

1 2 3 4 5 6 7 8 9 10 2 4 6 8

• L. monocytogenes (log CFU/g)

Weeks at 4°C

pH 6.0 pH 5.75 pH 5.5 pH 5.25 pH 6.0, 0.5% Cultured Sugar-Vinegar pH 5.75, 0.5% Cultured Sugar-Vinegar pH 5.5, 0.5% Cultured Sugar-Vinegar pH 5.25, 0.5% Cultured Sugar-Vinegar

Anderson et al., 2019, unpublished data

Anderson, et al., 2019, unpublished data

Fine-tuning formulations

• L. monocytogenes in 56% moisture cheese acidified with lactic acid

Supplemented with 0.5% cultured sugar-vinegar Fermentate C

Example - cheese pH 5.6

2 3 4 5 6 7 8 2 4 6 8

log CFU/g Weeks at 4°C

Citric Lactic Acetic Propionic

1-log L. monocytogenes growth at:

• 1 week – citric
• 3.5 weeks – lactic
• >8 weeks – acetic or propionic

Engstrom, et al., 2018, manuscript in preparation

Phase I Results

TEST

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

Acid pKa Citric 3.13 Lactic 3.83 Acetic 4.75 Propionic 4.88