# Presentation: 696439

## Process Effects on  Microbiological Contaminant Levels

- Phares O. Okelo, Ph.D.
- Center for Veterinary Medicine/FDA
- 					AFSS Public Meeting
- 					May 22, 2007

## Outline

- Ingredient/feed manufacturing processes
- Process effects on contaminant levels
- Some examples

## Ingredients and Manufacturing Processes (swine diet)

| Ingredient Name | Ingredient
Origin | Manufacturing Processes |  |
| --- | --- | --- | --- |
|  |  |  |  |
| Corn
(ground) | Corn | Ground or Chopped (entire corn kernel) |  |
| Soybean meal
(solvent extracted) | Soybean | Grinding the flakes which remain after removal of most of the oil by solvent extraction |  |
| Fish meal
(select menhaden) | Fish | Clean, dried, ground tissue of undecomposed whole fish or fish cuttings.  May have part of oil extracted.  |  |
| Spray-dried animal blood cells | Whole 
animal blood | Separating and spray drying red and white blood cells |  |
| Spray-dried whey | Milk | Separating whey from coagulum Spray-drying of whey
(whey – fluid from the coagulum from milk, cream or
 skimmed milk.  A portion of the milk fat may have been removed) |  |
| ... | ... | ... |  |
| Fat | Animal, Beef | Rendering or extraction of beef tissue |  |

## Ingredient/Feed Manufacturing Process Categories

- Thermal
- Physical
- Chemical
- Other

## Ingredient-Contaminant-Process Interactions

## Ingredient-Contaminant-Process Interactions ‘cont.

- Manufacturing
- Process

- Final
- Contaminant
- Levels

- Initial
- Contaminant
- Levels

## Microbiological Contaminants

**Fungi****	****	****	****	****Bacteria**
*Penicillium* spp.			*Salmonella enterica*
*Aspergillus *spp.			*E. coli O157:H7*
*Fusarium* spp.			*Mycobacterium* spp.
- 						*Bacillus* spp.
**	****	****	****	****	****	***Clostridium* spp.
*	**	**	**	**	**	**Pseudomonas *spp.
- 						*Staphylococcus *spp.

## Manufacturing Processes - Thermal

- (Temp, time, moisture)
- Spray-drying
- Drum/tube-drying
- Fluid-bed drying
- Pelleting

## Manufacturing Processes – Thermal (spray-dried animal blood cells)

## Manufacturing Processes - Physical

- (psd, temp, press, time)
- Grinding
- Pressing
- Centrifugation
- Membrane separation
- Mixing

## Manufacturing Processes - Chemical

- (aw, time, pH, temp, conc)
- Preservatives
- Anti-microbial activity
- Fermentation

## Manufacturing Technologies and Inactivation Kinetics

- N	=	Noe-kt
- Where:
- N	=	number of viable bacterial cells after t seconds
- No	=	number of bacterial cells at the start of thermal
-           process
- t	=	duration of thermal process, s
- k	=	activation constant of the organism (death rate),
-           dimensionless

## Manufacturing Technologies and Inactivation Kinetics (DT value)

- DT value
- The time (min) required to kill 90% of viable
- vegetative cells or spores at T (oC)

- No

- 0.1No

- DT (min)

- T oC

## Manufacturing Technologies and Inactivation Kinetics (DT value ‘cont.)

- DT = [2.303/exp(ln ko – Ea / RT)]
- where:
- ko	=	the pre-exponential factor, dimensionless
- Ea 	=	activation energy of the organism, (J mol-1)
- R	=	the universal gas constant, (8.314 J mol-1 K-1)
- T	=	absolute temperature of thermal process, K

## Manufacturing Technologies and Inactivation Kinetics ‘cont.

- N	=	Noe-(ln ko–Ea/RT)t
- Where:
- N	=	# of viable bacterial cells after t seconds
- No	=	# of bacterial cells at start of thermal process
- ko	=	the pre-exponential factor, dimensionless
- Ea 	=	activation energy of the organism, (J mol-1)
- R	=	the universal gas constant, (8.314 J mol-1 K-1)
- T	=	absolute temperature of thermal process, K
- t	=	duration of thermal process, s

## Manufacturing Processes – Thermal (spray-dried animal blood cells)

## Inactivation of M. avium –  (spray-dried animal blood cells)

| T (oC) | T (K) | k | *DT oC (s) | *DT oC (min) | Spray Dryer Dwell Time (min) | Log Reduction (dimensionless) | Percent Reduction (%) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 45 | 318.15 | 3.08291E-05 | 7.5E+04 | 1.2E+03 | 10.0 | 0.01 | 1.8 |
| 50 | 323.15 | 0.000208655 | 1.1E+04 | 1.8E+02 | 10.0 | 0.05 | 11.8 |
| 55 | 328.15 | 0.001332263 | 1.7E+03 | 2.9E+01 | 10.0 | 0.35 | 55.0 |
| 60 | 333.15 | 0.008046048 | 2.9E+02 | 4.8E+00 | 10.0 | 2.10 | 99.2 |
| 65 | 338.15 | 0.046076483 | 5.0E+01 | 8.3E-01 | 10.0 | 12.01 | 100.0 |

- *predicted DT values for *M. avium *sp*. paratuberculosis*

## Manufacturing Technologies and Inactivation Kinetics (HHP)

| Microorganism | Medium | Temp   Press   DpValue

(oC)     (MPa)    (min) |  |
| --- | --- | --- | --- |
|  |  |  |  |
| 
Salmonella seftenberg  | 
Chicken 
 |     
    23a         340a         7.1a |  |
| Pseudomonas
     flourescens | Minced beef |     20b         150b         24b |  |

- aMetrik et al., 1989
- bCalrez et al., 1993

## Expert Opinion – Microbes (process effects)

- If the microbiological contaminant is present in the
- specified feed ingredient, will the processing step in
- question 1) increase, 2) reduce, or 3) have no effect
- on the level of the microbiological contaminant?
- (Please restrict your estimates to effects occurring only during the specific processing step.)

## Expert Opinion – Microbes (magnitude of effect)

- What is the estimated magnitude, in terms of
- percentages, of that effect (e.g., 50% reduction,
- 200% increase, etc.)?

## Expert Opinion – Microbes (expert’s certainty on estimate)

- How certain are you in your estimation?  Please
- use the following scale:
- 1 = very uncertain (just a guess)
- 2 = uncertain
- 3 = medium certainty
- 4 = fairly certain
- 5 = very certain (I know what I am talking about!)

## Microbes Inactivation (Expert Opinion Table)

- PES = Processing Effect Score (magnitude of effect,  e.g., 50% reduction, 200% increase, etc.)

| Contaminant | Thermal Processes (T) |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Cooking/
Rendering |  | Drying |  | Distillation |  | Pelleting |  | Notes |
|  | PES | Certainty | PES | Certainty | PES | Certainty | PES | Certainty |  |
| Fungi |  |  |  |  |  |  |  |  |  |
| Penicillium spp. |  |  |  |  |  |  |  |  |  |
| Aspergillus spp. |  |  |  |  |  |  |  |  |  |
| Fusarium spp. |  |  |  |  |  |  |  |  |  |
| Bacteria |  |  |  |  |  |  |  |  |  |
| Salmonella enterica |  |  |  |  |  |  |  |  |  |
| ... |  |  |  |  |  |  |  |  |  |
| Staphylococcus spp. |  |  |  |  |  |  |  |  |  |

## Manufacturing Processes - Thermal

- (Temp, time, etc)
- Spray-drying (~45-55 oC, few min)
- Drum/tube-drying (~35-40, few min)
- Fluid-bed drying (~35-40, few min)
- Pelleting (~66-100 oC, 30-140+ seconds)

## Post Processing Operations (Effects on Level of Microbes) 

- Storage
- Transportation
- Feeding Practices
- Criteria for estimation of effects of these operations
- on the levels of microbes in ingredients and feed is
- being developed.

## Final Level of Microbes in Feed 

- The overall estimate of levels of microbes in the
- feed will be based on the following factors:
- Initial levels in ingredients
- Composition of contributing ingredient in diet (%)
- Processing effects
- Daily consumption

## Microbe Inactivation -Levels After Processing (M. avium)

- Final level in feed:
- x cfu/g

- Final level in feed:
- 0 cfu/g

- C Effects

## Expert Opinion WorksheetMicrobiological Contaminant

| Contaminant | Thermal Processes (T) |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Cooking/
Rendering |  | Drying |  | Distillation |  | Pelleting |  |
|  | PES | Certainty | PES | Certainty | PES | Certainty | PES | Certainty |
| Salmonella  | 93±9.6  | 5±0.5 | 33±27.7  | 3 | 48±47.3   | 3±0.8 | 78±38.6  | 4±1.4 |
| E. coli | 93±9.6  | 5±0.5 | 33±27.7  | 3 | 48±47.3   | 3±0.8 | 78±38.6  | 4±1.4 |

- Process Effect Score (PES, %):  - Increase; - Decrease; NE - No Effect
- Certainty: 1–Very uncertain; 2-Uncertain; 3-Medium certainty; 4-Fairly certain; 5-Very certain

- DRAFT

**Notes:**
Mycotoxins - destruction a function of time, temp, MC and initial contamination level

## DRAFT

| 

Swine Diet Ingredients | 

Processes | Salmonella |  | E. coli |  |
| --- | --- | --- | --- | --- | --- |
|  |  | FCP
% pos, mean | EOW
(PES/C)  | FCP
% pos, mean | EOW
(PES/C) 
 |
| Corn, ground | Grind or chop | 7.69 | 4  | 0 | 4  |
| Soybean Meal,
solvent extracted | Solvent extraction, grind | 7.69 | 75  | 0 | 95  |
| Fish Meal,
select menhaden | Clean, dry, ground, mechanical extraction | 72 | 90 | 0 | 95 |
| Spray dried blood cells | Spray dry | 72 | 90 | 0 | 95 |
| Dried whey | Coagulate, separation,
dry | 72 | 75 | 0 | 75 |
| Choice white grease
&
Fat | Render | 72 | 95 | 0 | 95 |
| DDGS | Grind, ferment,, distill, dry | 7.69 | 75 | 0 | 75 |

**Notes:**
Regulatory limits:
Aflatoxins - 20 ppb
Malathion
Cadmium - 100% (n=2 for ingredients; n=9 for mixed feed)
Dioxin - (n=26, corn; n=10, soybean meal; n=55 choice white grease, fat)

## Summary

- Start with diet with known ingredients & composition,
- a list of list of identified microbiological contaminants
- Assess effects of key manufacturing processes on contaminant levels using established techniques (and available data on: process parameters, microbe levels, expert opinion, etc.)
- Use model to estimate final contaminant levels in mixed feed

## Questions ?