Challenges & Solutions for “Preservative-free,”

Microbial-Safe Foods

Kathleen Glass, Ph.D.Associate Director

Food Research InstituteUniversity of Wisconsin-Madisonkglass@wisc.edu; 608.263.6935

Clean Label Conference, 26 March 2019

March 26-27, 2019

Overview• Challenges: Preservative-free foods may pose

food safety risks• Developing Solutions

– Factors to control microbial growth– “Clean label” alternatives to synthetic preservatives– Factors affecting efficacy of antimicrobials

• Summary

2

Making Processed Foods Safe: The Ideal

3

Validated Cooking or Alternative Lethality

Rapid Chilling

Controlled temperatures during distribution/storage

Cleaning & Sanitation

Making Processed Foods Safe: Facing the Reality

4

Large quantities thermodynamically

difficult to chillHeat resistant

microbes

Temperature abuse at retail and at consumer levels

Recontamination at retail or at consumer

levels

Niches in equipment and processing

facilities

Extended Runs

Microbes Cause Foodborne Illness• 48 Million episodes of foodborne illness per year• 128,000 hospitalizations• 3,000 deaths

– Salmonella (<1% mortality)– Listeria monocytogenes (~20% mortality, 90% hospitalizations)– Toxoplasma gondii (84% mortality in AIDS patients)

• Economic impact: US$ 7billion annually – Medical costs & lost wages– Litigation, recalls, lost business, business closures

• Contributing factors: Lack of kill step, cross-contamination, temperature abuse, and lack of microbial growth inhibitors

Scallon et al., 2011; University of Florida Emerging Pathogens Institute, 2011

Case Study: RTE Meats with no growth inhibitorsL. monocytogenes, storage at 41°F (5°C)

6Glass and Doyle, Appl. Environ. Microbiol., 1989

Critical factors• pH, moisture• Nitrite•Competitive microbiota

-2

-1

0

1

2

3

4

5

6

0 2 4 6

Chan

ge L

og C

FU/g

Lis

teria

Weeks at 41F (5C)

Turkey

Ham

Frankfurters

Beef

Salami

Learning Lessons the Hard WayListeriosis in Ready-to-Eat Meats

7

1998 2002 2008 2018

US Hot dogsDeli meats50 cases8 deaths

US Deli-style Turkey meat46 cases10 deaths

CanadaSliced meats57 cases22 deaths

South AfricaPolony1060 cases216 deaths

US 2004: lactate, diacetate2013: propionate, benzoate

Canada 2012: lactate, diacetate

Outside North America: Approval??

Approval of antimicrobials

*Waiver for use prior to these dates.

Antimicrobial research fast tracked

Lessons learned: need sanitation, temperature control, antimicrobials

If we use GMPs, why need preservatives?

• Supplement current good manufacturing practices• Control microorganisms throughout the food chain,

production to consumption• Ability of disease causing bacteria to grow at

refrigeration temperatures• Susceptible consumers• Recontamination of cooked products• Improper holding temperature

8

Ecosure 2007: Retail Temperatures

9

0

10

20

30

40

50

60

Cottage Cheese Yogurt PrepackagedLunch Meat

Deli CounterMeat

PrepackagedDeli

Fresh Meat

Percent of Product Observed above 41°F at Retail

> 50°F > 45°F > 41°F

2.6% of prepacked lunch meat >54°F1.9% of deli meat >54°F

Ecosure 2007: Home Refrigerator

10

Mean product temp (°F) 38.2°F

% above 41°F 17%% above 45°F 5%% above 50°F 0.7%

Maximum temperature 63°F

Solution: Adjust pH and water activity to inhibit microbial growth

11

Critical pH

Critical AW 4.6 or less 4.6 to 5.6 >5.6

0.92 or less Growth inhibition Growth inhibition Growth inhibition

>0.92-0.95 Growth inhibition Growth inhibition ??

>0.95 Growth inhibition ?? ??

?? = Use antimicrobials; starter/protective cultures, etc. to improve safetyAdditive or synergistic effect allows lower level of each factor to be usedRequires time/temperature control unless product testing demonstrates otherwise Adapted from: IFT. 2001. Evaluation and Definition of Potentially Hazardous Foods, IFT/FDA Contract No. 223-98-2333.Water activity impacted by moisture, protein and salts rather than other humectants

Example: inhibiting sporeforming bacterial pathogens

Solution: “Preservatives”Synthetic/Conventional Clean Label

NaCl Salt (Reduces available water, aw)

Lactate, propionate Cultured sugar, cultured milk, cultured wheat*

Diacetate, acetic acid Vinegar* (dry vinegar, buffered vinegar)

Nitrite Cultured celery* (convert nitrate to nitrite)

Erythorbate, ascorbate Acerola cherry powder*

Sorbic acid None (derived from rowanberries)

Benzoic acid Cranberries, prunes, plums, cloudberries, cinnamon

Nisin (bacteriocins) Culture sugar/ dairy solids

Phenolics, flavonoids Fruit / spice extracts

Lactic acid bacteria starter cultures

Protective culturesCompetitive microfloraIn situ acid/bacteriocin production

* Clean label substitute with documented efficacy

Effectiveness as an antimicrobial• Concentration of active compounds• Solubility of antimicrobial• Dissociation constant• Food composition

– pH/water activity/moisture/salt – Fat, hydrophobic proteins, free iron

• Synergistic/additive effects between antimicrobials• Processing, cooling and storage temperature/time

13

Effect of Organic Acid Type

PropionicpKa 4.87

AceticpKa 4.76

LacticpKa 3.86

Citric pKa 3.13, 4.76, 6.40

Journal of Applied Bacteriology 1996, 81, 147-1 53, Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate., Y. Kouassi and L.A. Shelef , Department of Nutrition and Food Science, Wayne State University, Detroit, MI, USA

Foodborne Pathogens and Disease 2012, 9, 1126-1129. Comparing Organic Acids and Salt Derivatives as Antimicrobials Against Selected Poultry-Borne Listeria monocytogenes Strains In Vitro. , J.F.R. Lues and M.M. Theron.

23456789

0 2 4 6 8

2

3

4

5

6

7

8

9

0 2 4 6 82

3

4

5

6

7

8

9

0 2 4 6 8

23456789

0 2 4 6 8

L. m

onoc

ytog

enes

(log

CFU

/g)

Citric Lactic

PropionicAcetic

Solution: Effect of acid type, pH, and moisture on L. monocytogenes in “preservative-free” fresh cheese

Engstrom et al., 2018, manuscript in preparation

Solution: Fermentates• Labeled as cultured milk, cultured sugar• Commercially available proprietary ingredients• Likely active compounds

– Organic acids; single or blends (lactic, propionic)– Frequently blended with vinegar (acetic)– May or may not contain bacteriocin activity

• Fermentation byproducts depend on – Starter culture(s) used: Propionibacterium, Lactococcus, Pediococcus, etc.– Substrate– Controlled fermentation: Temperature, oxygen, nutrient availability

• Challenges: Activity may vary between suppliers, within portfolio, between lots

Solution: Cultured milk solidsMozzarella (50% moisture); L. monocytogenes

17

2

3

4

5

6

7

8

9

0 15 30 60

Log

CFU

/g

Days storage 4C (40°F)

Mozz, pH 5.8: 1.7% NaCl

Mozz, pH 5.8: 1.0% NaCl

Mozz, pH 5.8: 1.0% NaCl+FermB

Fermentate labeled : cultured milkGlass et al., unpublished data

1

2

3

4

5

6

7

8

0 2 4 6 8

L. m

onoc

ytog

enes

(log

CFU

/g)

Weeks storage at 4°C

Control

1.0% Fermentate E

1.0% Fermentate C

1.0% Fermentate D

Engstrom et al., 2018, manuscript in preparation

Solution: Cultured milk solids or cultured sugar-vinegarL. monocytogenes, pH 6.0, lactic acid, 56% moisture cheese

19

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8

L. m

onoc

ytog

enes

(log

CFU

/g)

Weeks at 4C

Listeria in 56% moisture cheese acidified with lactic acidSupplemented with 0.5% cultured sugar-vinegar Fermentate 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

Solution: Fine tuning formulations

Homemade cheese.org

Solutions: Protective cultures & starter cultures• Produce acids, bacteriocins• Compete for nutrients• Can be used effectively in conjunction with

other preservatives• Effective when product may be temperature

abused

20

fromagehomage.co.uk

Challenge: extended cooling of cultured dairy products• Dairy products are cultured at 75-110°F• Pasteurized milk ordinance (PMO) required

“immediate cooling” of grade A milk products• After packaging and casing, products may

take days to cool to 42°F– Specifically, cottage cheese (up to pH 5.2) takes 72

hours to cool to 42°F– Without preservative, Listeria grows quickly, even

with immediate cooling• 0.06% potassium sorbate inhibits growth

21

Solution: Protective culture + cultured milk

-1

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14

Chan

ge L

og C

FU/g

Days after Inoculation

pH 5.2, 80% moisture Cooling (55 to 42F) immediately or in 72 hStorage at 42F through 14 days

Control, 72 h coolControl, Immediate Cool0.1% Ferm E, 72 h cool0.1% Ferm E + Culture, Immediate Cool0.1% FermE+Culture, 72 H cool

Glass et al., 2010, unpublished dataSee M-A-97 for details 22

Labels:Protective Culture: Lactic acid starter cultureFermentate: Cultured skim milk, skim milk powder

Challenge: Inhibit Listeria in fresh cheese during shelf-life

23

1

2

3

4

5

6

7

8

9

10

0 1 2 3 4 5 6 7 8

L. m

onoc

ytog

enes

(log

CFU

/g)

Weeks storage at 4C

Effect of protective culture on L. monocytogenesLactic Acid pH 6.0, 56% H2O

Control

Culture C

Culture K

Culture D

Cultures were previously shown to be active at 10C, little activity at 4C

Lessons learned…protective cultures don’t always work if storage temperature doesn’t allow metabolism

Challenge: Outbreaks of listeriosis on caramel-coated apples (2014, 2017)

• Sanitation insufficient to kill Listeria in stem/calyx

• Listeria trapped under caramel• Rapid growth at room temperature• Response

– Addition of cultured sugar-vinegar or sorbate to caramel

– Insufficient concentration; did not sufficiently delay growth

• Potential Solution: application of protective culture to apple surface before dipping in caramel

24

Spray application: Beading along waxy surface

25

0

1

2

3

4

1 2 3

Chan

ges i

n Li

ster

ia m

onoc

ytog

enes

Log

CFU

/ml r

inse

Days at 25C

Spray treatment Control

Spray Treatment PC

Brown et al., 2018

foodsafety.wsu.edu

Targeted application: Stem/calyx only

26

-2

-1

0

1

2

3

4

1 2 3 7 10

Chan

ges i

n Li

ster

ia m

onoc

ytog

enes

Log

CFU

/ml r

inse

Days at 25C

Control Protective Culture

Brown et al., 2018

Lesson learned…protective cultures must be in proximity of pathogens

Challenge: Inhibition of Listeria on RTE meatsSolution: Buffered vinegar to inhibit ListeriaLessons Learned: affected by moisture and pH

27

-1

0

1

2

3

4

5

6

0 2 4 6

Chan

ge P

opul

atio

ns L.

mon

ocyt

ogen

es (L

og C

FU/g

)

Weeks storage at 40F (4C)

Turkey Control No AntimicrobialsBeef Control No AntimicrobialsTurkey - 2.0% buffered vinegarBeef - 2.0% buffered vinegar

Turkey: ~73% moisture, pH 6.15, 1.1% NaCl, no nitriteBeef: ~66% moisture, pH 5.75, 0.6% NaCl, no nitrite

Adapted from JFP 76:1366, 2013Similar results with 1.5% lemon/cherry/vinegar blend; 3.0% cultured sugar-vinegar blend

Solution: Cultured sugar to inhibit ListeriaLessons Learned: Spoilage difficult to control

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16

Log

CFU

/ ml o

f rin

se

Weeks at 4C

Leuc 4 (69.64, 6.24, 3.75)

Leuc 8 (69.86, 6.09, 3.75)

L.mono 4 (69.64, 6.24, 3.75)

L. mono 8 (69.86, 6.09, 3.75)

Model uncured turkey, 70% moisture, pH 6.1 or 6.25, 3.75% cultured sugar

Weyker et al, 2013

LeuconostocSpoilage microbe growth

Listeria monocytogenesPathogenic microbe controlled

28

Challenge: removal of synthetic antimicrobials allow Clostridium perfringens growth extended coolingSolution: Cultured celery, cherry powderLessons Learned: cure accelerator needed for activity

-2-10123456

0 5 10 15

Log

CFU

/g

Cooling Time (h)

No Cure

547 ppm purified ascorbate

100 ppm synthetic nitrite

100 ppm natural nitrite

100 ppm synthetic nitrite + 547 ppmascorbate100 ppm natural nitrite + 547 ppmnatural ascorbate

King et al., 201329

Sampling schedule Storage temp

Uncured Control0 ppm

Conv. +2% Lactate-

Diacetate

Cultured celery + 0.9% dry vinegar

Cultured celery +

2.2% CS-V

2, 4, 6, 8, 10, 12 wk 10°C 2 wk >12 wk >12 wk >12 wk

Challenge: removal of synthetic antimicrobials allow botulinum growth during mild temperature abuse Solution: Cultured celery and vinegar or CS-V blend

Glass et al., unpublished data, 2017

60 ppm ingoing nitrite + 250 ppm cure accelerator (erythorbate or cherry powder)60% moisture, pH 6.3, 2.2% salt

BEEF, WATER, MODIFIED FOOD STARCH, CONTAINS 2% OR LESSOF: SODIUM LACTATE, SALT, HYDROLYZED SOY PROTEIN,FLAVORINGS, PAPRIKA, POTASSIUM CHLORIDE, SODIUMDIACETATE,SODIUMERYTHORBATE,SODIUMNITRITE.

BEEF, WATER, CONTAINS 2% OR LESS OF: SALT, SPICE, DISTILLEDVINEGAR POWDER, PAPRIKA, CULTURED CELERY POWDER,GARLIC POWDER, CHERRY POWDER, SEA SALT, NATURALFLAVORINGS.

Challenges• Preservative-free foods may pose a health risk• Clean label “preservatives”

– Undefined activity between manufacturers; lots– Contain low concentrations of active compounds– Addition levels may negatively affect sensory attributes

• No single antimicrobial will control all microbes• Affected by processing, food components,

temperature control, intrinsic factors (aw, pH)• Must be validated in specific foods

– Formulations changes may be needed to optimize efficacy

31

Solutions• Clean label antimicrobials can be applied to wide

variety of foods• Ingredient companies are actively developing clean

label alternatives to synthetic preservatives• Optimization of ingredients can reduce usage levels,

improve sensory attributes and be cost-effective• Clean label growth inhibitors that are familiar to

consumer can inhibit microbial growth – Enhance the safety of foods– Enhance confidence by consumers

32

Thank you for your attention. Questions: kglass@wisc.edu 33