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

TOPIC 3. MICROBIOLOGICAL EXAMINATION OF SAUSAGE PRODUCTS

Sausage products provide a highly favorable environment for the growth of various groups of microorganisms that cause spoilage. These include thermophilic lactic acid Bacteria, proteolytic and lipolytic bacteria, Molds, and others.

Microbiological testing of sausage products is carried out:

✵ periodically to ensure compliance with sanitary and technological production standards, at least once every 10 days;

✵ upon request by regulatory authorities;

✵ whenever raw Materials and auxiliary ingredients of questionable quality are used;

✵ in cases of violations of the Temperature or sanitary-hygienic production regime.

Routine testing for the preventative control of sanitary and technological standards in sausage production is performed According to the following schedule:

✵ for stuffed, Liver, and Blood sausages of higher, 1st, and 2nd grades, and HEAD cheeses of higher, 1st, and 2nd grades — at least once every 15 days;

✵ for boiled sausages of higher, 1st, and 2nd grades, meat loaves, frankfurters, and wieners — at least once every 15 days;

✵ for liver and blood sausages of the 3rd grade, head cheeses of the 3rd grade, jellied meats, and pâtés — at least once every 5 days;

✵ for semi-smoked, cooked-smoked, and dry-cured sausages — at least once a month;

✵ for pork, beef, mutton, poultry, and other slaughter animal products (boiled, baked, or roasted) — at least once every 15 days; cooked-smoked, smoke-baked, and dry-cured products — at least once a month.

The storage stability of sausage products varies and depends on several factors: sodium chloride content, pH level, degree of dehydration, texture, Chemical composition of the forcemeat, and smoking components.

Dry-cured and air-dried sausage products are the most stable during storage, as they have a firm texture, the highest concentration of sodium chloride, and minimal Water activity.

Under improper storage conditions, residual microflora in sausage products rapidly multiply. Numerous microbes cause product spoilage: putrefaction, rancidity, acid Fermentation, and mold growth.

Bacteriological examination OF sausage products includes determining the total microbial count per 1 g of product (TVC) (not determined in dry-cured sausages), detecting bacteria of the genera *Escherichia*, *Salmonella*, and *Proteus*, as well as coagulase-positive staphylococci and sulfite-reducing anaerobes.

3.1. Sampling and Sample preparation

Samples for microbiological testing are taken in accordance with regulatory documentation from each batch (of the same type, grade, and name, produced during the same shift, etc.).

Samples must be stored at a temperature of 4 to 6 °C for no more than 4 hours from the time of sampling.

For microbiological analysis, a composite sample with a mass of 50 g is prepared as follows: the sampled products are placed in an enameled basin, which is wiped with an alcohol-soaked swab and flamed. The sausage sticks are cut in half with a sterile scalpel without cutting through the casing on the opposite side. Samples are taken from several spots in the central part and from beneath the casing of both halves of the sausage stick.

From the composite sample of each specimen, a 20±0.1 g analytical portion is weighed into sterile glassware, placed into a sterile homogenizer cup, mixed with 80 cm³ of sterile saline solution, and homogenized. If a homogenizer is not available, a sterile mortar is used to prepare the suspension by grinding

20 g of the product with 2-3 g of sterile sand, gradually adding 80 cm³ of sterile saline solution.

The suspension is allowed to settle at room temperature for 15 minutes. 1 cm³ of the prepared suspension contains 0.2 g of the tested product.

3.2. Determination of Total Viable Count (TVC)

A 10 g sample of the test product is placed into a sterile porcelain mortar and thoroughly ground with a sterile pestle, while gradually adding 90 cm³ of sterile saline to achieve a 1:10 dilution. After allowing it to settle at room temperature for 15 min, 1 cm³ of the prepared suspension is inoculated onto nutrient media.

The Determination of the total microbial count (TVC), coliform titer, and the detection of bacteria of the genera Salmonella and Proteus are carried out according to the Procedure described in Section 1.3.

3.3. Detection of Coagulase-Positive Staphylococci

From the initial dilution of the sausage products, plating is performed using Drigalski's method onto egg-yolk salt Agar (EYSA) containing 6.5% NaCl to detect the lecithinase activity of coagulase-positive staphylococci. The inocula are incubated at 37 °C for 24 h. Colonies of toxigenic staphylococci form an “iridescent halo” on EYSA medium. Smears are prepared from suspicious colonies and stained by Gram's method. To confirm the pathogenicity of the staphylococci, a plasma coagulation test is performed.

The presence of Gram-positive staphylococci that yield a positive plasma coagulation reaction and lecithinase reaction indicates the presence of toxigenic staphylococci.

3.4. Detection of Sulfite-Reducing Clostridia

Sulfite-reducing clostridia are widespread in nature and are frequently found in the intestines of humans and animals. Certain strains of sulfite-reducing clostridia (in particular, Clostridium perfringens) can cause intestinal diseases in humans characterized as foodborne toxicoinfections, the MECHANISM OF ACTION of which is due to the presence of enterotoxins in these microorganisms.

Clostridium perfringens are Gram-positive rods, occurring singly, in pairs, or in chains, non-motile, forming a capsule in vivo. Endospores are oval, located centrally, subterminally, or at The Cell pole. On solid media, they form S- and R-colonies; S-colonies are round, smooth, with even edges, resembling dew drops in the Cytology/cytology/16.html">Early stages of growth; R-colonies are irregular, bumpy, with rough, uneven edges, resembling clumps of cotton wool deep within the agar.

The identification of this group of bacteria is based on observing the specific growth of clostridia in media containing iron sulfite. When sodium sulfite reacts with iron chloride, iron sulfate is formed, which causes the medium to turn black (Fig. 3.1).

Class="center">Fig. 3.1. Colonies of sulfite-reducing clostridia on iron-sulfite medium

The detection of vegetative Cells of sulfite-reducing clostridia in meat products involves two stages:

✵ detection of the sulfite-reducing capacity of the microorganisms;

✵ determination of whether the isolated microorganisms belong to the genus Clostridium.

The sulfite-reducing capacity of microorganisms is determined by inoculating the test product onto Wilson-Blair medium or iron-sulfite agar. If, after incubating the Petri dishes on the medium, dark grey or black colonies grow and cause blackening of the medium, they are tested to confirm if they belong to clostridia. To do this, material from the colonies is subcultured into tubes containing Kitt-Tarozzi medium and incubated at 37 °C for 24–72 h. If turbidity of the medium and gas production are observed, culture from the bottom of the tube is taken, microscopic preparations are made, stained by Gram's method, spore formation is examined, and catalase activity is determined. Sulfite-reducing clostridia do not produce catalase.

To detect sulfite-reducing clostridia, 1 cm³ of decimal dilutions (from 10^-1 to 10^-7) of the test product suspension is added to test tubes containing 9 cm³ each of Wilson-Blair medium melted and cooled to 45 °C. The inoculum is thoroughly mixed, placed in an incubator, and cultivated at 46 °C for 8–12 h or at 37 °C for 20 h. The appearance of black colonies on the medium or blackening of the medium indicates the presence of sulfite-reducing clostridia in the test product.

The positive clostridial titer is taken as the maximum amount of suspension in the inoculation of which blackening of the medium occurred. For example, if characteristic changes are observed in tubes with a 10^-2 dilution, it is considered that 1 g of the test product contains 1 x 10² microbial cells.

In the absence of growth on selective media and in the presence of growth on enrichment media, cultures from these media are streaked onto Endo agar Petri dishes and incubated in an incubator at 37 °C for 24 h. Further analysis is carried out according to the bacteriological meat testing procedure.

Table 3.1 presents the microbiological parameters for sausage products.

Table 3.1. Microbiological parameters of sausage products (CU TR 034/2013)

Y. - yeasts; M. - molds.

Topic Assignment:

1. Familiarize yourself with the sampling Methods for sausage products for bacteriological testing.

2. Determine the total viable count (TVC) and coliform bacteria (coliforms) in sausage products.

3. Examine the cultural and morphological CHARACTERISTICS OF MICROORGANISMS grown on nutrient media. Describe the colonies, prepare fixed smears, Gram-stain them, and sketch the microscopic field.

4. Draw a Conclusion regarding the wholesomeness and safety of the tested sausage products.

Review Questions

1. What factors determine the storage stability of sausage products?

2. How is a composite sample prepared for the MICROBIOLOGICAL ANALYSIS OF sausage products?

3. What criteria are used to assess the microbiological safety of sausage products?

4. What features indicate the presence of sulfite-reducing bacteria in sausage products?

5. What is the coliform titer and a positive clostridial titer?

6. What characteristics are typical of coagulase-positive staphylococci?