SOP: Detection of Thermophilic Anaerobic Sporeformers (Non-Hydrogen Sulfide Producers) in Canned Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP detects non-hydrogen sulfide-producing thermophilic anaerobic sporeformers in canned foods and their ingredients following the APHA Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 26, 4th Edition. The type species is Clostridium thermosaccharolyticum: obligately anaerobic, strongly saccharolytic rods producing acid and abundant gas from glucose, lactose, sucrose, salicin and starch. Spoiled containers swell and may burst, and the product usually has a cheesy odor. Spores show extreme heat resistance, with D-values at 121 °C of 3 to 4 minutes or higher and a z-value of about 6 to 7 °C. Optimum growth is 55 to 60 °C; the organisms seldom grow below 32 °C but can produce spoilage in 14 days at 37 °C if the spores are first germinated at a higher temperature. Growth pH optimum is 6.2 to 7.2, and growth occurs readily in products at pH 4.7 or higher. The organisms are of spoilage but not of public health significance.

2. Apparatus required

• Blender
• Incubator that maintains a uniform temperature of 55 °C ± 2 °C
• Microscope with 1000× oil immersion objective
• Pipettes of 10, 1.0 and 0.1 mL capacity
• 18 × 150 mm tubes with venting caps
• Wide-bore pipette (for finished product subculture)

3. Reagents

• A. PE-2 medium (commercially available; peptone yeast extract broth containing whole peas)
• Per the chapter, supplement the medium to contain 0.3% yeast extract for the detection of severely heat-stressed spores
• Bromcresol purple 2% ethanol solution: dissolve 2.0 g of the dye in 10.0 mL of ethanol and dilute to 100.0 mL with distilled water; add 2.0 mL of this solution to 1.0 L of medium
• To prepare tubes: dissolve the ingredients in distilled water by heating if necessary and dispense 19 mL portions into 18 × 150 mm screw-cap tubes. Add 8 to 10 untreated peas to each tube. Allow the ingredients to stand 1 hour to permit hydration. Autoclave at 121 °C for 15 min
• B. Liver broth (commercially available; the AOAC International detection procedure specifies liver broth, but non-commercially prepared liver broth is difficult to make and may contain metabolic inhibitors)
• C. Sterile 2% agar (20.0 g of agar per litre of distilled water) or Vaspar, for stratifying tubes
• D. Vaspar: combine one part mineral oil with two parts petroleum jelly, and sterilize in an oven at 191 °C for 3 hours
• E. Sterile distilled water

4. Sample preparation

• Ingredients: take 200 g samples aseptically from five different bags or barrels per shipment or lot — for lot sizes of 50 or fewer containers, from 10% of the containers for lot sizes of 50 to 100, and from a number of containers equal to the square root of the lot size for shipments greater than 100 containers. Sample liquid sugar by drawing five 200 g portions per tank during transfer or at the refinery during tank filling. Place samples in sterile sealed containers; increase the number of samples if preliminary analyses show considerable variability in a lot.
• Dry sugar and powdered milk: place 20 g of sample in a sterile flask and add sterile distilled water to a final volume of 100 mL. Stir or swirl to dissolve, bring rapidly to a boil, boil for 5 min, cool in cold water, and bring the volume back to 100 mL with sterile distilled water.
• Liquid sugar: place a sample containing 20 g of dry sugar, determined on the basis of degrees Brix (29.411 g of 68 °Brix liquid sugar is equivalent to 20 g of dry sugar), in a sterile flask, and proceed as for dry sugar.
• Fresh mushrooms: homogenize 200 g of mushrooms in a sterile blender jar until finely chopped, shaking the jar frequently. Place 20 g of the blended sample in a sterile flask and proceed as for dry sugar.
• Starches and flours: place 20 g of sample in a sterile flask containing a few glass beads and add sterile distilled water to a final volume of 100 mL. Shake well for a uniform suspension.
• Cereals and alimentary pastes: place 50 g of well-mixed sample in a sterile blender jar and add 200 mL of sterile distilled water. Blend for 3 min for a uniform suspension. For calculations, assume that 10 mL of the blended material contains 2 g of the original sample.
• Product in process: obtain a 200 g sample periodically, timed to coincide with the introduction of a new batch of ingredients or a shutdown that may have permitted an incubation period. Cool immediately but slowly to room temperature and analyze as soon as possible; refrigeration is not recommended.
• Equipment and systems: sample accumulated food materials from locations providing elevated temperatures in a relatively microaerophilic environment containing nutrients, using a sterile spatula or similar device, into sterile sealed containers; analyze as soon as possible. Sample foams and their residues.
• Finished product: obtain representative containers reflecting the condition of the entire population of containers in a production period, of the order of one container per thousand produced. If immediate post-process cooling to 43 °C is not achievable, monitoring of surviving thermophiles is extremely important.

5. Procedure

5a. Culture medium preparation

Unless freshly prepared medium is used, subject previously sterilized tubes to flowing steam for 20 min to exhaust oxygen, and cool to 55 °C before use
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After inoculation, stratify the tubes with 3 mL of sterile 2% agar or Vaspar and allow the agar to harden before tempering the tubes to 55 °C for incubation. Use venting caps as a safety precaution because of the abundant gas production

5b. Dry sugar and powdered milk

Divide 20 mL of the boiled solution equally among 6 freshly exhausted tubes of PE-2 medium
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Stratify each tube with 3 mL of sterile 2% agar or Vaspar, allow the agar to solidify, preheat the tubes to 55 °C, and incubate at 55 °C for 72 hours
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Positive tubes show gas production with the peas rising to the top of the liquid medium. Thermophilic flat sour bacteria may change the color from purple to yellow without gas

5c. Liquid sugar

Place the Brix-determined sample in a sterile flask and proceed exactly as for dry sugar

5d. Fresh mushrooms

Place 20 g of the blended mushroom sample in a sterile flask and proceed as for dry sugar

5e. Starches and flours

Divide 20 mL of the suspension equally among 6 freshly exhausted tubes of PE-2 medium
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Spin 3 tubes at a time in the hands immediately after adding the sample
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Place the tubes in a boiling water bath and continue to spin the tubes for the first 5 min of heating. Continue heating for an additional 10 min
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Remove the tubes and place them in cold water
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Stratify the tubes with 3 mL of sterile 2% agar or Vaspar, allow to solidify, preheat the tubes to 55 °C, and incubate at 55 °C for 72 hours

5f. Cereals and alimentary pastes

Proceed as for starches and flours, using the blended suspension and assuming 10 mL contains 2 g of the original sample

5g. Product in process

Place 100 g of product in a sterile blender jar and blend for 3 min
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Distribute 20 mL or 20 g of the blended sample equally among 6 freshly exhausted tubes of PE-2 medium and proceed as for starches and flours

5h. Finished product

Incubate representative containers of finished product at 55 °C for 5 to 7 days and observe daily for evidence of loss of vacuum or container distortion
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Remove samples showing signs of spoilage such as gas formation or cloudy brine from incubation and open aseptically
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Place 3 g of the contents in each of 2 tubes of freshly exhausted PE-2 medium by means of a wide-bore pipette
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Make smears of the product for morphological confirmation

5i. Spore suspensions (for thermal inactivation studies)

Place 10 mL of the desired dilution of the spore suspension in an 18 × 150 mm screw-cap tube and immerse in boiling water for 8 min, followed by rapid cooling in cold water
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Prepare a conventional 5-tube most probable number (MPN) dilution series of the boiled suspension in freshly exhausted PE-2 medium
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Treat the inoculated tubes as for dry sugar and compute the population of the original spore suspension from MPN tables

6. Calculation

• The procedures are not truly quantitative; the objective in surveying ingredients is to detect spores in a known quantity of the ingredient.
• For spore suspensions, compute the population from MPN tables.
• Interpretation standard for ingredients for canners’ use: spores of non-hydrogen sulfide-producing thermophilic anaerobes should not be found in more than 60% of the samples tested or in more than 66% of the tubes for any single sample. Canned foods with a pH below 4.0 are not susceptible to spoilage by thermophilic anaerobes.
• Low numbers of spores in processed canned foods are not unusual given their extreme resistance. If cans are cooled immediately to a center-can temperature of 43 °C or less and stored below 35 °C, the presence of spores is of no consequence; however, temperature abuse creates spoilage potential. Detectable thermophilic anaerobes in canned foods destined for hot-vend service or tropical distribution constitute an unacceptable spoilage hazard.

7. Precautions

• Ensure the ingredients of the detection medium are free from growth inhibitors; test each new lot of ingredients (for example peas) for growth inhibitors with a known suspension of a thermophilic anaerobe to avoid false negatives.
• In preparing PE-2, soak the dried peas in the peptone solution for 1 hour before autoclaving to ensure the proper sterilizing effect. Repeated steaming of unused tubes of medium does not reduce its effectiveness.
• Do not refrigerate spoiled canned food suspected of thermophilic growth: vegetative cells of thermophilic anaerobes usually die under refrigeration, and spores are generally not produced in canned foods.
• Omitting the heating step allows vegetative cells to be propagated by these procedures; the standard procedures detect spores only.

8. References

• APHA, Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 26, 4th Edition.