Role of Microorganisms in Cereal Processing

Several processing technologies and techniques have been widely applied in enhancing the nutritional properties of fermentable cereal products. This includes cooking, sprouting, milling, and fermentation. Microorganisms play both essential and deleterious roles in food products.

In the fermentation industry, the attributes of the food products produced are largely due to the type, age, and composition of the microorganism employed. Typically, the microbial load gradually increases from the first day (0 hours) and attains optimum at 24–48 hours of fermentation, before beginning to decline from 72 to 96 hours.

The density of the microbes for lactic acid bacteria culture using MRS agar is second to aerobic culture using plate count agar or nutrient agar. Like in the assessment of beverages, samples are collected and cultured to identify contamination of indicator microorganisms. The total plate counts, as well as the most probable number of identified organisms, are done to establish a baseline of safety.

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Overview of Laboratory Methods for Microbiological Assessment

For quality, safety of consumers, and longer shelf life of foods and beverages, they must meet certain microbiological criteria deemed safe. Because safety is very important, quality assurance cannot be limited to inspection of the final product alone; instead, continuous inspection of incoming raw materials and in-process quality control tests must be performed throughout production.

Although there is clearly a place for the direct examination of beverages for microorganisms, a full microbiological examination usually requires that individual viable cells are encouraged to multiply in broth or culture media.

Contaminating indicator organisms grow in the media. By ensuring optimal growth in the elective medium for one organism, it is desirable that conditions are sub-optimal, or even inhibitory, to others.

Enumeration Methods for Microbial Assessment

Enumeration methods of microbiological assessment involve different direct cell counts using direct or cell counter. These techniques are particularly useful for determining the total numbers of microbes, including those that cannot be grown in culture. Unfortunately, they generally do not distinguish between living and dead cells in the specimen.

Microscopic Count Technique

The microscopic count is a direct enumeration method. It is one of the most rapid methods of determining the cell concentration in a suspension. Here, a liquid specimen is added to a special glass slide designed specifically for counting cells.

The slide has a thin chamber that holds a known volume of liquid atop a microscopic grid. The contents of the chamber can be viewed under the light microscope, so the number of cells in a given volume can be counted precisely. At least 10 million bacteria (10^7) per milliliter are usually required for enough cells to be seen in the microscope field.

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Plate Counts for Viable Cell Measurement

Plate counts measure the number of viable cells in a sample by taking advantage of the fact that an isolated microbial cell on a nutrient agar plate will give rise to one colony. A simple count of the colonies determines how many cells were in the initial sample. Plate counts are generally only done if a sample contains more than 100 organisms/ml.

Otherwise, few if any cells will be transferred to the plates. In these situations, alternative methods give more reliable results. When counting colonies, the ideal number on a plate is between 30 and 300. Numbers outside of that range are more likely to be inaccurate. Samples usually contain many more cells than that, so they generally must be diluted by a stepwise process called serial dilution.

This is done using a sterile liquid called the diluent, often physiological saline (0.85% NaCl in water). Dilutions are normally done in 10-fold increments, making the resulting math relatively simple. Two techniques can be used to plate samples spread-plate and pour-plate. In the spread-plate method, 0.1 to 0.2 ml of the diluted sample is transferred onto a plate of a solidified agar medium.

It is then spread over the surface of the agar with a sterilized bent glass rod that resembles a miniature hockey stick. In the pour-plate method, 0.1 to 1.0 ml of the diluted sample is transferred to a sterile Petri dish and then overlaid with a melted agar medium cooled to 50°C. At this temperature, agar is still liquid. The dish is then gently swirled to mix the microbial cells with the liquid agar.

When the agar hardens, the individual cells become fixed in place; they form colonies when incubated. Colonies that form on the surface will be larger than those embedded in the medium.
In both methods, the plates are incubated, and then the number of colonies is counted.

From that number, the concentration of colony-forming units (CFUs) in the sample can be determined. This measure of viable cells accounts for the fact that microbial cells often attach to one another and then grow to form a single colony. When calculating CFUs, three things must be considered: the number of colonies, the amount the sample was diluted before being plated, and the volume plated.

Most Probable Number (MPN) Method

The most probable number (MPN) is a method for estimating the concentration of cells in a specimen. The procedure uses a series of dilutions to determine the point at which subsequent dilutions receive no cells. To determine the MPN, three sets of three or five tubes containing a growth medium are prepared. Each set receives a measured amount of a sample such as water, soil, or food.

The amount added is determined, in part, by the expected microbial concentration in that sample. What is important is that the second set receives 10-fold less than the first, and the third set 100-fold less.

In other words, each set is inoculated with an amount 10-fold less than the previous set. After incubation, the presence or absence of turbidity or other indication of growth is noted; the results are then compared against an MPN table, which gives a statistical estimate of the cell concentration.

Frequently Asked Questions (FAQs)

1. What role do microorganisms play in cereal processing?
   Microorganisms play both essential and deleterious roles in food products. In the fermentation industry, the attributes of the food products produced are largely due to the type, age, and composition of the microorganism employed.
2. How does microbial load change during cereal fermentation?
   The microbial load gradually increases from the first day (0 hours) and attains optimum at 24–48 hours of fermentation, before beginning to decline from 72 to 96 hours.
3. Why is continuous inspection necessary for cereal safety?
   Quality assurance cannot be limited to inspection of the final product alone; instead, continuous inspection of incoming raw materials and in-process quality control tests must be performed throughout production to ensure safety.
4. What are enumeration methods used for in microbiological assessment?
   Enumeration methods involve different direct cell counts using direct or cell counter and are particularly useful for determining the total numbers of microbes, including those that cannot be grown in culture.
5. How does the microscopic count method work?
   The microscopic count is a direct enumeration method where a liquid specimen is added to a special glass slide with a thin chamber that holds a known volume of liquid atop a microscopic grid, allowing the number of cells in a given volume to be counted precisely under a light microscope.
6. What is the purpose of plate counts in assessing cereal microbiology?
   Plate counts measure the number of viable cells in a sample by counting colonies formed from isolated microbial cells on a nutrient agar plate, determining how many cells were in the initial sample.
7. What is the difference between spread-plate and pour-plate techniques?
   In the spread-plate method, 0.1 to 0.2 ml of the diluted sample is spread over the surface of a solidified agar medium, while in the pour-plate method, 0.1 to 1.0 ml of the diluted sample is mixed with melted agar cooled to 50°C in a Petri dish, which is then swirled to mix the microbial cells with the liquid agar.
8. How does the most probable number (MPN) method estimate microbial concentration?
   The MPN method uses a series of dilutions in three sets of tubes containing growth medium, with each set receiving 10-fold less sample than the previous one. After incubation, the presence or absence of growth is compared against an MPN table to estimate the cell concentration statistically.

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