GENERAL AND FOOD MICROBIOLOGY PART II - L. V. Krasnikova - 2016

TOPIC 5. MICROBIOLOGICAL EXAMINATION OF CANNED MEAT PRODUCTS

Canned foods are products designed for long-term storage, packaged in hermetically sealed containers that protect them from the ingress of microorganisms.

The MICROBIOLOGICAL EXAMINATION OF canned meat involves analyzing raw Materials and semi-finished products, auxiliary materials, as well as the Contents of the cans both before and after sterilization.

Microbiological analysis of raw ingredients is performed when their microbial contamination levels are elevated following butchering and washing, whereas the analysis of semi-finished and auxiliary materials is conducted as they arrive at the canning facility.

5.1. Sampling

Samples for analyzing the contents of cans are collected depending on the type and consistency of the product. If the canned food contains a large amount of liquid medium or broth, the liquid portion of the product is taken directly for inoculation. If the liquid phase is absent or present in negligible quantities, sterile Water is added to the product in a 1:1 ratio, and either the undiluted washings from the product or its serial dilutions are plated. When packaging in containers with a volume of up to 0.5 L, the contents of the can are transferred to sterile glassware (1.5–2.0 L capacity) with the same amount of water, covered with a lid, and shaken for 3 minutes, after which a sample is taken for inoculation. Depending on the expected microbial load, the sample is diluted so that no more than 350 colonies grow on a Petri dish containing nutrient medium.

5.2. Bacteriological examination OF Canned Meat Prior to Sterilization

Microbiological examination of cans before sterilization is carried out immediately after seaming. The analysis includes determining the total microbial count in the can contents and detecting spores of obligate anaerobes and thermophilic Bacteria. Monitoring for the presence of thermophilic bacteria—the causative agents of flat-sour spoilage—is performed exclusively for canned foods containing products with a neutral pH value.

Determination of the total count of mesophilic aerobic and facultatively anaerobic microorganisms (Total Plate Count) is carried out According to the Procedure given in Section 1.3.1.

Detection of spores of obligate anaerobes. Using a sterile pipette or tube, 10 cm³ of the product is taken from the prepared sample, transferred to a sterile test tube, and placed in a boiling water bath for 20 minutes. After cooling, 0.5 cm³ of the heated product is inoculated into a test tube containing Kitt-Tarozzi enrichment medium. The cultures are incubated at 37 °C for 48 hours. Upon completion of incubation, the presence of gas production and anaerobic growth in the enrichment medium is recorded. If anaerobic growth is present, 1–2 drops from the enrichment cultures are plated into Petri dishes, which are then poured over with 30 cm³ of molten nutrient Agar supplemented with 1% glucose. Immediately after the agar solidifies, a sterile Microscope slide is placed on its surface using tweezers so that no air bubbles are trapped underneath. The dish is then inverted (lid down) and placed in an incubator at 37 °C for 48 hours.

Obligate anaerobes are detected on the agar plate beneath the Glass in the central zone as isolated colonies or confluent growth at a distance of 3–4 mm from the edge of the slide, occasionally forming gas bubbles underneath it. Facultative anaerobes grow not only beneath the slide but across the entire surface of the medium in the dish.

Detection of thermophilic bacterial spores. From a sample pre-heated to 80–85 °C, 5 cm³ of the suspension is taken and added to 25 cm³ of nutrient agar containing 1% glucose and 0.004% bromocresol purple (the medium should have a pale violet tint). The cultures are incubated at 55 °C for 24–48 hours. A color change of the medium from violet to yellow or the appearance of yellow halos around the colonies indicates the presence of thermophilic bacterial spores in the test sample.

The causative agents of flat-sour spoilage in canned goods are thermophilic spore-forming aerobic microorganisms belonging to the following species: Bacillus stearothermophilus, B. aerothermophilus, B. coagulans, and others. The characteristics of these microorganisms are summarized in Table 5.1.

Thermophilic microorganisms, multiplying within the product during storage at elevated temperatures (40–70 °C), can degrade CARBOHYDRATES to produce organic acids without gas formation. They do not cause container Swelling (bombaż), but the product develops a sour odor and an unpleasant acidic taste.

5.3. Microbiological Examination of Canned Meat After Sterilization

Finished canned products undergo microbiological testing after sterilization under the following circumstances:

✵ if an elevated count of microorganisms or obligate anaerobic spores is detected in a batch of cans prior to sterilization;

✵ when canned goods are placed into long-term storage;

✵ in the absence of pre-sterilization data regarding the acceptable level of bacterial contamination in the canned product.

Samples of canned goods selected for analysis are visually inspected and checked for hermetic sealing in baths of hot water or using specialized apparatus. Only hermetically sealed cans are subjected to microbiological analysis. To verify sterility and detect the METABOLIC ACTIVITY OF mesophilic and thermophilic microorganisms, the selected cans are held in an incubator at 37 °C for 3 days (to determine the presence of mesophilic microorganisms) or at 55 °C for 5 days (to determine the presence of thermophilic microorganisms).

The residual microflora in canned foods most frequently comprises spores of aerobic bacilli such as Bacillus subtilis, B. licheniformis, B. polymyxa, B. cereus, B. coagulans, B. stearothermophilus, alongside anaerobic clostridia (Clostridium perfringens, C. putrefaciens, and occasionally C. butyricum). C. botulinum is rarely detected in canned products. Because the spores of the botulinum bacterium possess lower heat resistance than the spores of other anaerobic clostridia, the destruction of C. botulinum serves as the minimum standard baseline when designing sterilization regimes for canned meat.

As a result of the metabolic activity of microorganisms that survive the sterilization process, canned foods undergo microbiological spoilage. The primary types of spoilage in canned products include swelling (bombaż), flat-sour spoilage, and sulfide spoilage.

Swelling (Bombaż) — the bulging of a can resulting from increased internal pressure driven by gas accumulation (CO₂, H₂S, NH₃) produced by gas-forming spore-forming bacteria such as C. sporogenes, C. thermosaccharolyticum, C. perfringens, C. thermoaceticum, and others. Can swelling may also be triggered by coliform bacteria and Yeasts entering the finished product if container integrity is compromised. The organoleptic Properties of the canned goods alter drastically due to the accumulation of microbial metabolic byproducts, presenting tissue maceration, a sour or putrid odor, and foaming. Typically, such alterations manifest at high microbial proliferation levels—10⁶ to 10⁸ Cells per 1 gram of product. Swollen cans are easily identified and rejected based on external visual inspection.

Flat-sour spoilage — an alteration of the organoleptic properties of canned foods occurring without gas production. The products acquire an unappealing sour odor and taste, and occasionally the color of the product shifts. The causative agents of this type of spoilage are thermophilic spore-forming aerobic bacilli: Bacillus subtilis, B. cereus, B. stearothermophilus, B. aerothermophilus, B. coagulans, and others (Table 5.1). These microorganisms break down carbohydrates to form various organic acids without releasing gas.

Sulfide spoilage — the accumulation of hydrogen sulfide in canned foods caused by thermophilic spore-forming bacteria of the species Clostridium nigrificans. They decompose Sulfur-Containing Amino Acids with The formation of H₂S. Sulfide spoilage is characterized by the swelling of the can ends, blackening of the product, and a putrid odor.

Class="center">Table 5.1. Characteristics of certain bacterial species causing flat sour spoilage in canned foods

To detect The activity of mesophilic facultatively anaerobic and anaerobic microorganisms, canned foods with pH >4.4 are thermostatted at 37±0.5 °C. Canned foods in containers with a capacity of 1 dm³ or less are kept in the thermostat for 5 days, while those with a capacity greater than 1 dm³ are kept for 10 days.

To establish sterility or detect the activity of thermophilic aerobic, facultatively anaerobic, and anaerobic microorganisms, canned foods in containers of any capacity are thermostatted at 55±0.5 °C for 3 days. Upon completion of thermostatting, the canned foods are kept for another 24 h at room Temperature, after which container defects are recorded.

Along with thermostat incubation, inoculations onto nutrient media are performed from the canned food samples selected for analysis to determine The Nature of the residual microflora.

Based on the results of the bacteriological analysis for commercial sterility, appropriate Conclusions are drawn in accordance with the data given in Table 5.2.

Table 5.2. Evaluation of commercial sterility of full canned foods (Group A)

To detect mesophilic aerobic microorganisms, 2 cm³ from each analyzed can of food is inoculated into two test tubes with meat-peptone broth (MPB) containing 1% glucose. The cultures are incubated at 37 °C for 5 days. If signs of aerobic Microbial growth appear by the end of incubation (turbidity of the MPB, formation of a wall ring or pellicle and sediment), smears are prepared, Gram-stained, and examined microscopically.

If large Gram-positive rods or cocci are found in the smears, the culture is subcultured from the MPB test tubes onto meat-peptone agar (MPA) plates. The plates are placed in an incubator at 37 °C for 48 h. Then, the colonies grown on the MPA are examined to determine whether the microorganisms belong to a specific group.

If small Gram-negative non-spore-forming rods are detected in the smears, material from the colonies is streaked onto Endo agar plates, into Shukevich slanted agar tubes, and onto MPA plates with 1% glucose to identify the microorganisms.

To detect anaerobes, 2 cm³ from the test can is inoculated into four test tubes containing Kitt-Tarozzi medium, previously heated in a boiling water bath for 20–30 min and then cooled to 50 °C. Two inoculated test tubes are heated at 80 °C for 20 min to detect all anaerobes. When testing for the presence of C. botulinum type E, one test tube is heated to 60 °C for 15 min (which preserves C. botulinum type E spores), while the other is left unheated.

To detect C. botulinum type E, the cultures are incubated at 30 °C, and to detect other anaerobes, at 37 °C. Incubation is carried out for 5–10 days, and culture growth is monitored daily.

Mesophilic anaerobic microbes cause medium turbidity with gas evolution and the appearance of an off-odor. When microbial growth is detected, smears are prepared, Gram-stained, and examined microscopically. Smears containing obligately anaerobic bacteria reveal Gram-positive spore-forming rods.

To confirm that the identified spore-forming mesophilic anaerobic bacteria belong to the genus Clostridium, their lack of catalase is verified. To determine the catalase enzyme in microorganisms, 1–2 cm³ of a 1% hydrogen peroxide solution is added to the culture. The appearance of gas bubbles indicates the formation of oxygen resulting from The breakdown of hydrogen peroxide by catalase. If catalase is not detected, it is considered that mesophilic obligately anaerobic microorganisms of the genus Clostridium are present in the cultures.

If the botulism pathogen is present in the medium, turbidity, gas formation, and a butyric acid odor are noted. Gram-stained smears reveal large Gram-positive rods with tennis racket-like spores. If C. botulinum was in vegetative form in the canned food, visible microbial growth can be observed in the unheated samples.

Note. If C. botulinum is detected, the canned food is considered unfit for consumption and is destroyed. If other species of clostridia are detected, the issue of using the canned food is decided by the Sanitary and epidemiological service authorities.

Topic Assignment:

1. Familiarize yourself with the Methods of sampling and microbiological analysis of canned foods before and after sterilization. Perform inoculations from sterilized canned food samples onto nutrient media.

2. Study the cultural and morphological properties of microorganisms grown on nutrient media. Describe the microbial colonies, prepare fixed smears from them, Gram-stain them, and sketch the microscopic field.

3. Draw a Conclusion regarding the quality of the analyzed canned food.

Control Questions

1. How is MICROBIOLOGICAL CONTROL OF canned meat conducted before and after sterilization?

2. Which microorganisms cause container bulging ( swells) in canned foods?

3. List the bacteria that cause flat sour spoilage in canned meat products.

4. How is the identification of isolated spore-forming mesophilic anaerobic microorganisms as belonging to the genus Clostridium confirmed?

5. What action is taken regarding the products if Clostridium botulinum is detected?

6. The presence of which microorganisms in finished canned goods indicates that they fail to meet commercial sterility requirements?