Nut Handling and Processingfor Confectioners and Small Nut Roasters

Session 1: Microbiology of Nuts

Dr. Michael Doyle, University of Georgia

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Microbiology of Nuts

Michael Doyle

Microbiology of Nuts

Examples of Types of Microbes Potentially Found on Nuts

• Bacteria

– Not likely harmful• Pseudomonas• E. coli• Xanthomonas• Clostridium

– Human pathogens• Salmonella• Enterohemorrhagic E. coli (e.g., E. coli O157:H7)

Examples of Types of Microbes Potentially Found on Nuts

• Molds

– Aspergillus spp.• Aspergillus flavus/parasiticus - Aflatoxin

– Penicillium spp.– Fusarium spp.– Eurotium spp.

Sources of Microbial Contaminants

• Primary sources of salmonellae are intestinal tracts of animals (domestic and wild) and humans; shed in feces

– Feces can contaminate soil and water (irrigation and processing)

– Salmonellae can survive in some soils for months to years; in water for weeks to months

• Primary sources of Enterohemorrhagic E. coli (e.g., E. coli O157:H7) are intestinal tract of ruminants (e.g., cattle, goats, sheep, deer)

Sources of Microbial Contaminants

• Primary source of molds such as aspergilli and penicillia is soil; not directly related to fecal contamination

Peanuts

Cultivation, Harvest, Storage

Cultivation• Seed is planted in April & May• Approximately 130 days from planting to harvesting• Cultivated in Virginia, North & South Carolina, Georgia, Florida,

Alabama, Mississippi, Texas, Oklahoma, and New Mexico

Harvesting• Peanuts are harvested from Aug-Nov• Peanuts are dug, inverted, and left in the field to partially dry

for about 3 days before being combined which separates the vine from the inshell peanut

Grading/Storage

• After the peanuts are combined, these “farmer stock” peanuts are taken to a local buying point for grading, drying, and storage until needed for shelling

Storage

• Peanuts are stored in specially-designed farmers stock warehouses until shelled. The raw shelled peanuts are put in one ton tote bags and then go into cold storage warehouses until shipped to manufacturer

Examples of Potential Sources of Pathogen Contamination of Peanuts During Production• Field fertilized with untreated manure or sewage as a soil amendment

– Salmonella can survive in soil for months or years

• Field irrigated with water contaminated with animal waste

– Untreated surface water (ponds, rivers) with runoff from livestock operations

• Wildlife grazing on or near fields

Examples of Potential Sources of Pathogen Contamination of Peanuts During Storage

• Leaks in roof on which birds congregate

• Rodent and insect activity, especially if facility is near livestock operations

• Forklift and transport equipment exposed to mud, water or contaminated soil outdoors brought into sheds and warehouses without prior cleaning and disinfection

Examples of Potential Sources of Pathogen Contamination of Tree Nuts in Orchards

• Cattle, livestock or wildlife grazing in orchards especially near trees

• Fertilizing soil in orchards with untreated fecal waste

• Shells of many tree nuts such as pecans are porous and can become contaminated by contact with soil

– Exposure to water (rain or processing) provides conditions for growth of salmonellae

Essential Conditions for Microbial (Salmonella) Growth

• Food/Nutrients

• Water/Moisture

• Temperature

Characteristics of Human Pathogens That May be Associated with Nuts

• Salmonella

– Can survive for months to years in low moisture foods such as nonfat dry milk, peanut butter, chocolate

– Small numbers of this bacterium can produce illness when consumed in high-fat foods such as chocolate (< 1 Salmonella/g), peanut butter, cheese

Characteristics of Human Pathogens That May be Associated with Nuts

• Salmonella (Cont)

– Heat resistance increases with decreased moisture content/water activity

Example

165°F (instantaneous) kills > 10,000,000 (>7 log) Salmonella/g in ground beef, milk, poultry

194°F for 50 min kills 100,000 (5 log) Salmonella/g in peanut butter

305°F (oven dry heat) for 15 min kills 100,000 (5 log) Salmonella/g on peanuts

Characteristics of Human Pathogens That May be Associated with Nuts

• Salmonella (Cont)

– Growth prevented by low moisture content (water activity < 0.95)

– Growth typically prevented by temperature less than 5°C (41°F) or greater than 46°C (115°F)

• Optimum temperature for growth is 35 - 37°C (95 - 99°F)

Characteristics of Human Pathogens That May be Associated with Nuts

• Enterohemorrhagic E. coli (E. coli O157:H7)

– Can survive in soil and water for months

– Exceptional tolerance to acidic conditions• Can persist in acidic foods for exceptionally long periods of

time

– Have a low infectious dose (< 100 cells)

– Cause severe symptoms in children

Determination of Pathogen Contamination of Nuts and Nut Products

• Requires a microbiological assay that has been specifically validated to detect the target pathogen (such as Salmonella) at a minimum level of sensitivity (e.g., 1 CFU/25 grams)

• Methods in the FDA Bacteriological Analytical Manual are the “gold standard” for detecting pathogen contamination in food products regulated by FDA (Dec 2007 edition)

(http://www.foodsafety.gov/~ebam/bam-5.html)

Determination of Pathogen Contamination of Nuts and Nut Products

• Methods validated to be equivalent to the FDA BAM protocols for detecting the target pathogen in specific products at the minimum level of sensitivity indicated by FDA, are acceptable alternatives

Determination of Salmonella Contamination of Nut Products by FDA BAM Protocol (Cultural Method)

• Preenrichment

– Add 25-gm sample to 225 ml trypticase soy broth, mix well and incubate for 24 ± 2 h at 35°C

• Selective enrichment

– Incubate 0.1-ml portion of preenrichment culture in Rappaport-Vassiliadis (RV) medium and tetrathionate broth, and incubate for 24 h ± 2 h at 42°C

Determination of Salmonella Contamination of Nut Products by FDA BAM Protocol (Cont)

• Selective Isolation

– Plate onto bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar, and incubate at 35°C for 24 ± 2 h; BS agar also for 48 h if no suspect colonies at 24 h

• Identification/Confirmation

– Streak suspect colonies onto MacConkey agar, HE agar or XLD agar, and incubate at 35°C for 24 ± 2 h

• Biochemical and serological assays

Rapid Detection of Salmonella Contamination of Nuts and Nut Products

• Acceptable methods for rapidly detecting (< 8 hrs) Salmonella in nut products should soon (within 2 years) be available

– Based on shortened enrichment and molecular detection (e.g., Gen-Probe rRNA-based automated pathogen detection system) or Raman spectroscopy real-time (< 5 min) assay

Symptoms and Consequences of Salmonellosis

• Symptoms

– Fever, abdominal pain, diarrhea

• Chronic disease consequences

– Arthritis (Reiter’s syndrome)

• Onset time – usually 12 - 72 hrs (can be a week)

• Duration of illness – usually resolved within 5 - 7 days

• Most susceptible populations generally made ill by lower infectious dose and experience more severe symptoms or death than general population

Symptoms and Consequences of Enterohemorrhagic E. coli (E. coli O157:H7) Infection

• Symptoms

– Severe abdominal cramping (like appendicitis), bloody diarrhea (all blood, in stools), vomiting, kidney failure

• Onset time – usually 3 - 4 days• Duration of illness – usually 5 - 7 days

• Most susceptible populations are children and elderly (5 - 10% of those diagnosed with EHEC infection) who develop renal failure (hemolytic uremic syndrome, HUS) which may require dialysis (can be permanent) and kidney transplant; can lead to death

Most Susceptible Populations to Salmonella

• Newborns, infants, the elderly and immunocompromised individuals are more susceptible to Salmonella infections than healthy adults

– Incompletely developed immune system in newborns or infants

– Weak or delayed immune response in the elderly and debilitated persons

– Low gastric acid production in infants and seniors

High Fat Content of Food InfluencesInfectious Dose

• High fat content is common factor among foods associated with low infectious dose (e.g., cocoa butter in chocolate, milkfat in cheese and nut butter in nut-containing foods)

• Suggested that entrapment of salmonellae within hydrophobic lipid micelles affords protection against the bactericidal action of gastric acidity

• Rapid emptying of fat-based gastric contents could also provide alternate mechanism

Principles Used in Mitigating Risks of Pathogen Contamination of Nuts

• Preventing contamination throughout the entire cycle, from production to mouth

• Applying control interventions from production to mouth

– Moisture, temperature, vermin controls– Detection of pathogens and indicators of pathogen

contamination by testing– Chemical antimicrobials– Thermal treatments– Non-thermal physical treatments (e.g., irradiation,

high pressure) • Responding rapidly to pathogen contamination and taking

effective corrective action

The principles used in mitigating risks of pathogen

contamination of foods (nuts) are applied by the food

industry using a systems approach that includes:

• Sanitation Standard Operating Procedures (SSOP)

• Current Good Manufacturing Practices (cGMP)

• Hazard Analysis Critical Control Point Systems (HACCP)

HACCP for Nut and Nut Product Processors

• Thermal processing is a common treatment (CCP) employed by the nut industry for mitigating pathogenic bacterial hazards

Theoretical Thermal Inactivation Curve for Salmonella in Milk Chocolate at 160°F (71°C)

5 10 15 20 25 30

1

2

3

4

5

6

log

Sal

mon

ella

/g

Time (hours)

5 D = 33 hr

Thermal Inactivation of Pathogens

• Thermal processes are designed to kill a minimum number of a target pathogen (e.g., Salmonella or E. coli O157:H7)– Heat ground beef to internal temperature of 160°F to kill

100,000 (5 log) E. coli O157:H7/g– Heat canned foods to kill 12 log (12D) of Clostridium

botulinum spores/g– Heat poultry to internal temperature of 165°F to kill 5 log

Salmonella/g– Heat almonds (peanuts) to kill at least 10,000 (4 log)

Salmonella/g

What Should Be the Targeted Log Reduction

for Salmonella on Nuts/Nut Products to

Ensure Safety?

Thermal Inactivation of Salmonella in Peanut Buttera

Internal

Temperature Mean maximum time (min)

(°C / °F) 3-log reduction 5-log reduction 7-log reduction

71 / 160 107 402 965

77 / 170 62 197 423

83 / 181 33 110 227

90 / 194 21 49 120

a Commercial, creamy-style peanut butter; aw = 0.45, pH = 5.1

Ma et al., J. Food Protect. July 2009

Considering the high temperature and

extended heating time needed to kill 5 log of

Salmonella in peanut butter (49 min at 194°F

or 3.3 h at 170°F), this is not likely a suitable

treatment to render a product of acceptable

quality.

Therefore, the key critical control point to

ensure safety from Salmonella contamination

from raw nuts in peanut butter/product

manufacture is the whole nut roaster.

If the roaster conditions are designed to kill

100,000 (5 log) Salmonella/g, then the

incoming load of Salmonella on peanuts

must be less than 5 log or greater than 1

Salmonella will be present per gram of

peanuts.

– Areas of localized Salmonella growth,

as occurs with A. flavus for aflatoxin

production, can be a confounding factor.

Thermal Inactivation of Salmonella on Unblanched Virginia Peanuts by Dry Roasting

Oven Temp

(°C / °F)

Time

(min) log reduction

129 / 264 45 4.3

146 / 295 15 4.9

163 / 325 10 5.8

S. Goodfellow (Deibel Lab)

Conclusions

• Peanuts and tree nuts are now considered high-risk foods with regard to Salmonella contamination

• The low-moisture, high-fat content of nuts contributes to the:– High temperature, extended time required to kill Salmonella– Lower infectious dose of Salmonella– Long-term persistence of Salmonella in nut products

Conclusions

• Mitigation of Salmonella contamination of nuts should be addressed from production to consumption.

• Nuts should be produced and stored under conditions to prevent Salmonella growth.

• The nut roaster is critical to ensuring the safety of nuts– Temperature-time conditions for nut roasting must be

validated to ensure efficacy in killing the targeted number of Salmonella.

– Roaster conditions, including nut bed depth and uniform loading, and accurate monitoring of temperatures and time, must be properly controlled.

Questions?

For Q&As and Food & Nut Safety Resource Guide:

http://www.ecandy.com/Content.aspx?ContentID=7134