Inhibition of Listeria monocytogenes in a model cheese system based - - PowerPoint PPT Presentation

March 3, 2016

Inhibition of Listeria monocytogenes in a model cheese system based on pH, moisture, and acid type

Sarah Engstrom, Christie Cheng Principal Investigator: Dr. Kathleen Glass Food Research Institute, UW-Madison

July 10, 2018

ILSI 2005; Batz 2011; USDA-ERS 2016.

• Made via acid and/or rennet

addition (no starter)

• Standard of Identity not

established for some varieties

• Refrigeration is typically only

post-manufacturing hurdle

• 287 million lbs Latin-style

cheese produced in 2017 (316% increase from 1997)

Soft, unaged cheeses

• Ubiquitous organism with 20%

fatality

• Psychrotrophic (-0.4°C / 31.1°F

min)

• Main concern = post-

processing contamination

• $60M to $2B annual cost due

to illness from dairy foods

• L. monocytogenes

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 2018

CDC 2012-2018.

Acid pH Citric 5.25 Lactic 5.50 Acetic 5.75 Propionic 6.00 Moisture 50% 56%

To determine the effect of pH, moisture, and acid type on the growth of

• L. monocytogenes in a model cheese system

4 acid types 4 pH values 2 moistures Salt constant (1.25%) 32 cheeses

Objective

Methods – Model System

• Model soft cheese system:
• Cream (pasteurized, 40% fat)
• Micellar casein
• Sterile water
• Lactose
• Salt
• pH adjustment using organic acids

(citric, lactic, acetic, or propionic)

Methods – Inoculation

• Inoculated with 3-log CFU/g (0.5% v/w) 5-strain L. monocytogenes cocktail:

LM 108 - hard salami LM 301 - cheddar LM 310 - goat’s milk cheese R2-500 - food outbreak R2-501 - clinical isolate

• 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

+

  

1:2 dilution in 0.1% peptone 30 sec Serial dilutions in 0.1% peptone

Methods – Uninoculated Sample Enumeration

Duplicate samples Plate Count agar 2 days 35°C MRS agar 2 days 35°C Potato dextrose agar 5 days 21°C

+

  

pH measured weekly

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

Salt

Methods – Proximate Analysis

pH Moisture aw

Uninoculated 0-time samples of each treatment were analyzed to confirm target parameters

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

log CFU/g Weeks at 4°C

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

log CFU/g Weeks at 4°C

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

log CFU/g Weeks at 4°C

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

log CFU/g Weeks at 4°C

Citric Acid Lactic Acid Acetic Acid Propionic Acid

Data Handling – DMFit

64 curves fitted for Lag and Growth Rate

Full Model

• Independent variables: acid type, target pH, target moisture, target

pH*target moisture interaction, trial

• Dependent variables: Lag, Growth Rate

Reduced Model

• Independent variables: acid type, target pH
• Dependent variables: Lag, Growth Rate

Lag Phase (weeks)

Citric A Lactic B Acetic C Propionic C

Growth Rate (log CFU/g per week)

Citric A Lactic A Acetic B Propionic B

Tukey’s HSD

Example - cheese pH 5.6

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

Conclusions

• L. monocytogenes growth is dictated by acid type and

pH in model cheeses of 50-56% moisture

• Efficacy of organic acids follows pKa value
• Citric 3.13, Lactic 3.83, Acetic 4.75, Propionic 4.88
• Acetic and propionic model cheeses performed similarly
• Our model cheese system can be used to screen

antilisterial ingredients

Acknowledgements

• Funding: National Dairy Council
• Technical: Randy Brandsma (Schreiber);

Gina Mode, Mike Molitor, John Lucey (CDR)

• Modeling: Dennis Seman (Meat

Science)

• Lab: Makala Bach, Morgan Brown,

Christie Cheng, Max Golden, Quinn Huibregtse, Jie Yin Lim, Kori Scherer, Sam Trotter, Megan Wagner, and Brandon Wanless

16

THANK YOU

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

Mani-Lopez 2012.

TEST

Higher pKa, higher antimicrobial activity

Antimicrobial Activity of Organic Acids

Antimicrobial Activity of Organic Acids

Citric pKa’s: 3.13, 4.76, 6.40 0.7% Lactic pKa: 3.86 4.7% Acetic pKa: 4.76 25.0% Propionic pKa: 4.87 27.2%

TEST

Extracellular pH = 5.25