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

TOPIC 7. MICROBIOLOGICAL EXAMINATION OF EGGS AND EGG PRODUCTS

7.1. Microflora of Shell Eggs

Due to their chemical composition, bird eggs serve as an excellent substrate for the growth of various groups of microorganisms and are classified as perishable foods. Therefore, eggs must be stored under conditions that slow down their physical, chemical, and microbiological processes.

The shell protects eggs from microbial invasion. Egg components exhibit varying degrees of resistance to microbes. The albumen is more resistant to spoilage, which is attributed to the presence of bactericidal substances: Lysozyme, Avidin, conalbumin, ovomucin, ovomucoid, and carbon dioxide. Microorganisms can enter eggs via both endogenous and exogenous pathways.

Endogenous contamination occurs when birds are infected with tuberculosis or salmonellosis, the causative agents of which penetrate the interior of the egg from the Ovary or oviduct. Waterfowl eggs pose a particularly high risk regarding salmonellosis; Bacteria of the paratyphoid group are frequently found in their oviducts: Salmonella pullorum, S. gallinarium, S. enteritidis, S. dublin, S. newport, S. typhimurium, S. livingstone, etc. Consequently, the sale of duck and goose eggs and their use in public catering establishments are prohibited.

Exogenous contamination of the egg surface occurs after laying. The primary sources of microorganisms are droppings, soil, and feathers. The degree of microbial contamination of eggs depends on poultry housing conditions. Specifically, clean eggs contain no more than 10¹-10² microorganisms per 1 cm² of surface area, whereas contaminated eggs can reach up to 10⁵-10⁶ microorganisms. The COMPOSITION OF THE surface microflora of eggs is highly diverse. The most frequently detected microorganisms include coliforms (coliform bacteria), Proteus species, pseudomonads, aerobic spore-forming rods, micrococci, and mold fungal spores. These microorganisms primarily reach the egg as it passes through the cloaca.

Bird eggs can also be contaminated with pathogenic microorganisms. The causative agents of salmonellosis, avian tuberculosis, coccal intoxications, campylobacteriosis, and pseudomoniasis are transmitted through eggs and egg products.

Eggs with contaminated surfaces are rejected for sale and must be washed. The rate of microbial penetration into eggs depends on their freshness, ambient Temperature, humidity, and the species of microorganisms. Failure to maintain proper temperature and humidity during storage causes the surface microflora to penetrate through the eggshell pores, first reaching the shell membranes and subsequently the albumen and yolk. The protective bactericidal factors become inactivated, leading to egg spoilage. The primary types of egg spoilage are putrefaction and molding.

Putrefaction is most frequently caused by Gram-negative non-spore-forming bacteria of the genera Pseudomonas, Proteus, Aeromonas, Enterobacter, Serratia, Escherichia, Acinetobacter/Moraxella, and several others. As a result of the proliferation of fluorescent pseudomonads (Pseudomonas fluorescens), the egg white becomes turbid, liquefied, and eventually acquires a greenish hue that shifts to dark green (green rot). When other bacteria with high proteolytic activity, such as Aeromonas, Proteus, and Escherichia, penetrate the egg interior, the albumen liquefies and the yolk turns black (black rot). Due to the accumulation of gases produced by putrefactive bacteria, the shell ruptures, and the egg contents leak out, emitting a foul odor. The proliferation of putrefactive aerobic Gram-positive bacilli (Bacillus subtilis, B. mycoides, etc.) within the egg causes the yolk membrane to rupture, mixing the yolk with the albumen to form a turbid liquid mass. Such eggs are opaque during candling, a type of spoilage known as "bacterial rot" (tumak). Bacteria that produce a red or pink pigment (Serratia marcesscens, Micrococcus roseus) and certain Yeasts impart a pink coloration to the egg contents (pink rot). Mixed rot is caused by coliforms, aerobic bacilli, Proteus species, pseudomonads, and other microorganisms possessing active proteases. In this case, the egg albumen liquefies, turns gray, and develops a putrid odor.

Molding. Storing eggs in high-humidity environments leads to mold growth on their surface. Egg mold is most commonly caused by Fungi of the genera Penicillium, Cladosporium, and Sporotrichum. Fungi of the genus Penicillium form punctate yellow, green, or blue spots; Cladosporium produces dark green and black spots; and Sporotrichum causes red and pink spots. Mycelial fungi proliferate on the shell membrane and near the air Cell. The initial stage of mold growth is characterized by The formation of a dark spot visible during candling. Subsequently, the fungal mycelium spreads, the spot enlarges, the albumen and yolk mix, and the egg completely loses its transparency. This type of spoilage is referred to as "moldy rot" (molding tumak).

7.2. Microflora of Melange and Egg Powder

Melange is a well-blended mixture of egg white and yolk in their natural proportions, shelled and frozen in specialized containers. Only grade I eggs are used to prepare melange. Duck, goose, and lime-preserved chicken eggs, as well as eggs originating from farms affected by infectious avian diseases, must not be used.

Melange is a perishable product; therefore, Sanitary and hygienic conditions must be strictly observed during its production, and it must be processed within 2-3 hours after thawing.

The microflora of melange is represented by micrococci, sarcinae, Proteus species, pseudomonads, coliform bacteria, aerobic bacilli, and mold spores. Pathogenic microorganisms—such as Salmonella and Staphylococcus aureus—are occasionally present in melange. In addition to Salmonella, egg contamination by psychrotrophic pathogens such as Listeria monocytogenes and Yersinia enterocolitica is a major concern. These organisms can multiply at 3-5 °C in both raw and pasteurized melange. The presence of L. monocytogenes in egg products is considered potentially hazardous due to the relative thermotolerance of these bacteria.

To minimize the risk of pathogen Introduction and reduce the overall bacterial load in melange, it is recommended to treat eggs with disinfectants prior to breaking. Brief pasteurization of melange at 65-70 °C with a 1-2 minute holding time is sometimes applied.

Egg powder has a moisture content ranging from 4% to 8% immediately after drying. Viable aerobic bacilli, micrococci, staphylococci, and mold fungal spores remain within the product.

During the storage of egg powder, microorganisms that survived the drying process do not multiply and gradually die off. The rate of mortality depends on storage conditions. For instance, a greater reduction in microbial population occurs at 18-20 °C than at 1-2 °C. The most intensive microbial die-off is observed During the first 2-3 months of storing dried egg products. However, complete elimination of all vegetative bacterial forms does not occur even after 2-3 years of storage.

7.3. Sampling

Sanitary and microbiological examination of eggs is performed based on epidemiological indications during outbreaks of avian infectious diseases, when contamination with pathogens is suspected, and upon the request of the State Sanitary and epidemiological Surveillance.

For egg analysis, a random sample of 30 eggs is collected from various locations within the lot, or at least 5-10 eggs in certain cases. Eggs intended for microbiological testing are packed in clean containers and transported in a manner that prevents secondary contamination.

For the microbiological examination of melange, 1% of the total number of cans in a lot is sampled, with a minimum of 6 cans. Before opening a sealed metal can, an alcohol-soaked swab is placed on its surface and ignited. Following aseptic Procedures, pieces of frozen melange are collected from each sampled can and transferred to sterile glassware (flasks, jars). After thawing, the Contents of the flask are thoroughly mixed, and a composite sample of melange weighing 50-55 g is collected for analysis.

When inspecting egg powder packaged in bags, three packaging units are sampled from various locations within each lot, whereas one unit is sampled from canned products. A composite sample weighing 0.5 kg is prepared for each lot, thoroughly mixed, placed into sterile Glass jars with ground-glass stoppers, and sent to the laboratory for analysis.

7.4. Bacteriological examination OF Eggs and Egg Products

The bacteriological examination of eggs and egg products includes determining the total viable count (TVC), the presence of coliforms, Proteus species, Salmonella, Staphylococcus aureus, and, when necessary, Bacillus cereus.

The total bacterial contamination of the egg surface is determined by plating a specific volume of the wash-off or its tenfold dilutions into Petri dishes containing nutrient Agar. After incubating the dishes at 30 °C for 72 h, all surface and subsurface colonies that have grown are counted, the arithmetic mean number of colonies from two plates of the same dilution is determined, multiplied by the dilution factor, and divided by the egg surface area:

TVC = n 10^m/ S, CFU/cm²,

where n is the arithmetic mean number of colonies grown on the Petri dishes; m is the number of tenfold dilutions; S is the egg surface area (cm²), determined by the formula

S = 3.14 BP/2,

where B is the egg width, cm; P is the circumference, cm.

Determination of TVC in the internal content of the egg. The shell surface is washed using a small brush with warm soapy Water, then the egg is immersed in ethanol for 10 min and flamed over a burner flame. An opening 1 cm in diameter is made at the pointed end of the egg by puncturing the shell with a flamed instrument (tweezers or scalpel). The contents of one or several eggs, placed in a flask with glass beads, are homogenized into a uniform mass, which is warmed in a water bath at 20 °C. Then, 10 cm³ of the egg mass is added to a flask containing 90 cm³ of sterile saline to obtain a 10^-1 dilution, from which subsequent dilutions are prepared, and the TVC is then determined as described in section 1.3.1.

Determination of coliforms (coli-form bacteria). To detect coliforms, 1 cm³ of the 10 dilution of the test sample is inoculated into 5 cm³ of Kessler medium. The cultures are placed in an incubator at 37±1 °C for 24 h, after which the presence or absence of signs of growth of these bacteria is established.

To determine coliforms in dry dairy products, dilutions up to 10^-3 are prepared; for egg melange, up to 10^-4, which are also inoculated into test tubes with Kessler medium.

Determination of Staphylococcus aureus. The METHOD FOR DETERMINING Staphylococcus aureus in egg products is based on inoculating the product itself or its dilutions into selective nutrient media. The characteristics of Staphylococcus aureus include its ability to grow on media with an increased sodium chloride concentration, coagulate Blood Plasma, and produce acid during the Fermentation of mannitol and maltose under aerobic conditions.

A 1 g sample of a dry egg product (or 1 cm³ for liquid egg products) is inoculated into a test tube containing 9 cm³ of salt broth. The cultures are incubated in an incubator at 37±1 °C for 24 h, after which the material from the test tubes is subcultured into Petri dishes containing dried yolk-salt agar (see Appendix, cl. 1.8). The dishes are kept in an incubator at 37±1 °C for 18–24 h. Then, to reveal pigments, the dishes are kept in the light at room temperature for another 18–24 h. Staphylococcus aureus colonies on yolk-salt agar appear as convex disks 2–4 mm in diameter, yellow, cream, lemon, or golden in color, surrounded by an iridescent ring. From colonies characteristic of S. aureus, smears are prepared, Gram-stained, and examined microscopically. If Gram-positive cocci arranged in grape-like clusters are present in the preparation, material from suspicious colonies is subcultured into Petri dishes containing nutrient agar. The cultures are placed in an incubator at 37±1 °C for 18–24 h. The colonies grown that are suspected of being S. aureus are subjected to the plasma coagulation test. For this purpose, 0.5 cm³ of diluted rabbit plasma is added to each of two test tubes; material from a colony is introduced into one test tube using a loop, while the other test tube serves as a control and is left uninoculated. The test tubes are placed in an incubator at 37±1 °C; after 2–4 h, the tubes are examined, the result is recorded, and they are left overnight at room temperature for the final evaluation. The plasma coagulation reaction depends on the activity level of the coagulase enzyme produced by Staphylococcus aureus and is characterized as follows:

✵ clot is dense, motionless when the test tube is tilted (four-plus rating);

✵ clot has a small supernatant fraction, mobile when the test tube is tilted, with dense plasma coagulation (three-plus rating);

✵ clot is in the form of a suspended sac, incomplete plasma coagulation with the formation of a mobile clot in the center of the plasma (two-plus rating).

Material from colonies grown on yolk-salt agar and suspected of being S. aureus is subcultured with a loop into Petri dishes containing an agar medium with mannitol (or maltose) and phenol red. The result is considered positive if a yellow zone forms around the colony on the mannitol medium, sharply contrasting with the purple-red Background of the medium.

If smears from characteristic colonies on yolk-salt agar reveal Gram-positive cocci arranged in clusters, a positive plasma coagulation reaction is established, and mannitol is fermented with acid production, a positive result is reported for the presence of Staphylococcus aureus in 1 g (or 1 cm³) of the egg product.

Methods for detecting salmonellae and bacteria of the genus Proteus are described in sections 1.3.3 and 1.3.4.

Table 7.1 presents the microbiological safety criteria for egg products.

Class="center">Table 7.1. Microbiological parameters of eggs and egg products

For each batch of eggs and egg products, the manufacturing enterprise issues a sanitary and veterinary certificate regarding their safety and quality.

Topic Assignment:

1. Determine the TVC of egg powder.

2. Determine the fermentation titer of coliforms in egg melange.

3. Determine the presence of Proteus in egg melange.

Review Questions

1. Which microorganisms cause egg spoilage?

2. What determines the resistance of the egg's interior to various microbes?

3. What microbiological parameters are used to test eggs and egg products?

4. How are samples collected for the MICROBIOLOGICAL ANALYSIS OF eggs, melange, and egg powder?

5. How is S. aureus detected in egg products?

6. What are the Characteristic Features of Staphylococcus aureus?

7. What types of egg spoilage are you familiar with?