Food and Industrial Microbiology

Lesson 4. FACTORS AFFECTING GROWTH AND SURVIVAL OF MICROORGANISMS IN FOODS

Module 2. Microorganisms and food materials

Lesson 4
FACTORS AFFECTING GROWTH AND SURVIVAL OF MICROORGANISMS IN FOODS

4.1 Introduction

Food spoilage means the original nutritional value, texture, flavour, etc.,of the food are damaged, the food become harmful to people and unsuitable to eat. Food can deteriorate as a result of two main factors:

1) Growth of micro-organisms - usually from surface contamination - especially important in processed food

2) Action of enzymes - from within cells - part of normal life processes, (responsible for respiration, for instance). It is important to note that many plants - fresh vegetables and fruit - are still alive when bought and even when eaten raw, and meat from animals undergoes gradual chemical changes after slaughter.

The various sources through which microorganisms gain entry into the foods are shown in Table 3.1. Microorganisms involved in food spoilage (other than Canned Foods) with some examples of causative organisms are enlisted in Table 4.2

Table 4.1 Primary sources of microorganisms found in foods

Table 4.2 Microorganisms involved in food spoilage (other than canned foods) with some examples of causative organisms

A variety of intrinsic and extrinsic factors determine whether microbial growth will preserve or spoil foods, as shown in Table 3.3. Intrinsic or food related parameters are those parameters of plants and animal tissues which are inherent part of the tissue. e.g., pH, water activity (a _W ), oxidation-reduction potential (Eh), nutrient content, antimicrobial constituents and biological structures. Extrinsic or environmental parameters are properties of storage environments which affect both foods as well as microorganisms and include temperature of storage, relative humidity of storage environment, and concentration of gases in environment.

Table 4.3 Factors affecting the development of microorganisms in foods

4.2 Intrinsic Parameters

4.2.1 Nutrient content

Like all other living beings, microorganisms need water, a source of carbon, an energy source, a source of nitrogen, minerals, vitamins and growth factors in order to grow and function normally. Since foods are rich source of these compounds, they can be used by microorganisms also. It is because of these reasons that various food products like malt extracts, peptone, tryptone, tomato juice, sugar and starch are incorporated in microbial media. The inability to utilize a major component of the food material will limit its growth and put it at a competitive disadvantage compared to those that can. In general, molds have the lowest requirement, followed by yeasts, Gram-negative bacteria, and Gram-positive bacteria. Many food microorganisms have the ability to utilize sugars, alcohols, and amino acids as sources of energy. Few others are able to utilize complex carbohydrates such as starches and cellulose as sources of energy. Some microorganisms can also use fats as the source of energy, but their number is quite less. The primary nitrogen sources utilized by heterotrophic microorganisms are amino acids. Also, other nitrogenous compounds which can serve this function are proteins, peptides and nucleotides. In general, simple compounds such as amino acids are utilized first by a majority of microorganisms.

4.2.2 Water activity (a_W)

Water is often the major constituent in foods. Even relatively ‘dry’ foods like bread and cheese usually contain more than 35% water. The state of water in a food can be most usefully described in terms of water activity.

Water activity of a food is the ratio between the vapour pressure of the food, when in a completely undisturbed balance with the surrounding air, and the vapour pressure of pure water under identical conditions. Water activity, in practice, is measured as Equilibrium Relative Humidity (ERH) and is given by the formula:

Water Activity (a_W) = ERH / 100

Water activity is an important property that can be used to predict food safety, stability and quality. The various applications of water activity includes maintaining the chemical stability of foods, minimizing non enzymatic browning reactions and spontaneous autocatalytic lipid oxidation reactions, prolonging the desired activity of enzymes and vitamins in foods, optimizing the physical properties of foods such as texture.

Water activity scale extends from 0 (bone dry) to 1.00 (pure water). But most foods have a water activity in the range of 0.2 for very dry foods to 0.99 for moist fresh foods. Based on regulations, if a food has a water activity value of 0.85 or below, it is generally considered as non-hazardous. This is because below a water activity of 0.91, most bacteria including the pathogens such as Clostridium botulinum cannot grow. But an exception is Staphylococcus aureus which can be inhibited by water activity value of 0.91 under anaerobic conditions but under aerobic conditions, it requires a minimum water activity value of 0.86. Most molds and yeasts can grow at a minimum water activity value of 0.80. Thus a dry food like bread is generally spoiled by molds and not bacteria. In general, the water activity requirement of microorganisms decreases in the following order: Bacteria > Yeast > Mold. Below 0.60, no microbiological growth is possible. Thus the dried foods like milk powder, cookies, biscuits etc are more shelf stable and safe as compared to moist or semi-moist foods.

Factors that affect water activity requirements of microorganisms include the following- kind of solute added, nutritive value of culture medium, temperature, oxygen supply, pH, inhibitors, etc. Each microorganism has a minimal water activity for growth as shown in Table 4.4.

Table 4.4 Minimum water activity values of spoilage microorganisms

Water activity of some foods and susceptibility to spoilage by microorganisms is shown in Table 4.5. Water acts as an essential solvent that is needed for most biochemical reactions by the microorganisms. Water activity of the foods can be reduced by several methods: by the addition of solutes or hydrophilic colloids, cooking, drying and dehydration: (e.g. egg powder, pasta), or by concentration (e.g. condensed milk) which restrict microbial growth so as to make the food microbiologically stable and safe.

Table 4.5 Water activity of some foods and susceptibility to spoilage by microorganisms

A wide variety of foods are preserved by restricting their water activity. These include:

4.2.2.1 Dried or low moisture foods

These contain less than 25% moisture and have a final water activity between 0.0 and 0.60. e.g., Dried egg powder, milk powder, crackers, and cereals. These products are stored at room temperature without any secondary method of preservation. These are shelf stable and do not spoil as long as moisture content is kept low.

4.2.2.2 Intermediate moisture foods

These foods contain between 15% and 50% moisture content and have a water activity between 0.60 and 0.85. These foods normally require added protection by secondary methods such as pasteurization, pH control, refrigeration, preservatives, but they can also be stored at room temperature. These include dried fruits, cakes, pastries, fruit cake, jams, syrups and some fermented sausages. These products are usually spoiled by surface mold growth.

4.2.3 pH and buffering capacity

The pH, or hydrogen ion concentration, [H⁺], of natural environments varies from about 0.5 in the most acidic soils to about 10.5 in the most alkaline lakes. Since the pH is measured on a logarithmic scale, the [H⁺] of natural environments varies over a billion-fold and some microorganisms are living at the extremes, as well as every point between the extremes. The range of pH over which an organism grows is defined by three cardinal points: the minimum pH, below which the organism cannot grow, the maximum pH, above which the organism cannot grow, and the optimum pH, at which the organism grows the best. Microorganisms which grow at an optimum pH well below neutrality (7.0) are called acidophiles. Those which grow best at neutral pH are called neutrophiles and those that grow best under alkaline conditions are called alkalophiles. In general, bacteria grow faster in the pH range of 6.0- 8.0, yeasts 4.5-6.5 and filamentous fungi 3.5-6.8, with the exception of lactobacilli and acetic acid bacteria with optima between pH 5.0 and 6.0 (Table 4.6). The approximate pH ranges of some common food commodities are shown in Table 4.7.

Table 4.6 Approximate pH ranges of different microbial groups

Table 4.7 Approximate pH ranges of some common food commodities

4.2.4 Redox potential (Eh)

Microorganisms display varying degrees of similarity to Oxidation-Reduction potential of their growth medium. The O/R potential is the measure of tendency of a revisable system to give or receive electrons. When an element or compound looses electrons, it is said to be oxidized, while a substrate that gains electrons becomes reduced. Thus a substance that readily gives up electrons is a good reducing agent, while one that readily gains electrons is a good oxidizing agent. When electrons are transferred from one compound to another, a potential difference is created between the two compounds and is expressed in as milk volts (mV). If a substance is more highly oxidized, the more positive will be its electrical potential and vice versa. The O/ R potential of a system is expressed as Eh. Aerobic microorganisms require positive Eh values for growth while anaerobic microorganisms require negative Eh values (reduced). The redox potential we measure in a food is the result of several factors: redox couples present, ratio of oxidant to reductant, pH, poising capacity, availability of oxygen and microbial activity. Some redox couples typically encountered in food material and their standard redox potential (Eh) values are shown in Table 4.8.

With the exception of oxygen, most of the couples present in foods, e.g, glutathione, cysteine, ascorbic acid and reducing sugars tend to establish reducing conditions. pH of the food has a bearing on the redox potential and for every unit decrease in the pH the Eh increases by 58 mV (Table 4.8). As redox conditions change there will be some resistance to change in food’s Eh, and is known as poising and is similar to buffering of the medium. Poising is maximum when the two redox couples are present in equal amounts. Oxygen is the most powerful of redox couple present in food system and if the food is stored in the presence of air, high positive potential will result. Thus, increasing the exposure to oxygen in air by mincing, cutting, chopping, grinding of food will increase the Eh as evident by high positive Eh of minced meat as compared to raw meat ( Table 4.9). Finally microbial growth in the food reduces the Eh due to oxygen depletion. The decrease in Eh due to microbial activity forms the basis of some tests used frequently in raw milk such as platform MBRT test.

Table 4.8 Some important redox couples and standard redox potential (Eh) values

Table 4.9 Redox potential of some foods

4.2.5 Antimicrobial constituents and barriers

Some foods can resist the attack by microorganisms due to the presence of certain naturally occurring substances which possess antimicrobial activity such as essential oils in spices (eugenol in cloves and cinnamon, allicin in garlic, cinnamic aldehyde in cinnamon, thymol in sage); lactaferrin, lactoperoxidase and lysozyme in milk; and ovatransferrin, avidin, lysozyme and ovoflavoprotein in hen’s egg albumin. Similarly, casein as well as free fatty acids found in milk also exhibit antimicrobial activity. The hydroxycinnamic acid derivatives (p-coumaric, feluric, caffeic and chlorogenic acids) found in fruits, vegetables, tea and other plants possess antibacterial and antifungal activity. Also natural covering of foods like shell of eggs and nuts, outer covering of fruits and testa of seeds, hide of animals provide protection against entry and subsequent spoilage by microorganisms.

4.3 Extrinsic Parameters

4.3.1 Temperature of storage

Microorganisms have been found growing in virtually all temperatures. A particular microorganism will exhibit a range of temperature over which it can grow, defined by three cardinal points in the same manner as pH. Considering the total span of temperature where liquid water exists, the prokaryotes may be subdivided into several subclasses on the basis of one or another of their cardinal points for growth. For example, organisms with an optimum temperature near 37°C are called mesophiles. Organisms with an optimum temperature between about 45°C and 70°C are thermophiles e.g, Bacillus, Clostridium etc. Some archaebacteria with an optimum temperature of 80°C or higher and a maximum temperature as high as 115°C , are now referred to as extreme thermophiles or hyperthermophiles. The cold-loving organisms are psychrophiles defined by their ability to grow at 0°C. A variant of a psychrophile (which usually has an optimum temperature above 10°C) is a psychrotroph, which grows at below 7°C but displays an optimum temperature in the mesophile range, nearer room temperature. Psychrotrophs are the scourge of food storage in refrigerators since they are invariably brought in from their mesophilic habitats and continue to grow in the refrigerated environment where they spoil the food. Of course, they grow slower at 2°C than at 25°C. In food microbiology mesophilic and psychrotrophic organisms are of greatest importance.

4.3.2 Relative humidity of the storage environment

Relative humidity and water activity are interrelated. When foods with low a _W are stored in environment of high humidity, water will transfer from the gas phase to the food and thus increasing a_W of the food leading to spoilage by the viable flora. There is a relationship between temperature and humidity which should be kept in mind. In general, the higher the temperature, lower is the relative humidity and vice-versa. Foods that undergo surface spoilage from molds, yeasts, and some bacteria should be stored in conditions of low relative humidity to increase their shelf life. This can also be done by proper wrapping of the food material also. However, variations in storage temperature should be minimal to avoid surface condensation in packed foods.

4.3.3 Gaseous atmosphere

Oxygen is one of the most important gases which come in contact with food influence the redox potential and finally the microbial growth. The inhibitory effect of CO ₂ on the growth of microorganisms is applied in modified atmosphere packaging of foods. The storage of foods in atmosphere containing 10% of CO₂ is referred to as “Controlled Atmosphere”. This type of treatment is applied more commonly in case of fruits such as apples and pears. With regards to the effect of CO₂ on microorganisms, molds and Gram-negative bacteria are the most sensitive, while the Gram-positive bacteria, particularly the lactobacilli tend to be more resistant. Some yeasts such as Bretanomyces spp. also show considerable tolerance of high CO₂ levels and dominates the spoilage microflora of carbonated beverages. Some microorganisms are killed by prolonged exposure to CO₂ but usually its effect is bacteriostatic. Also, the presence of CO₂ tends to decrease the pH of foods and thereby inhibiting the microorganisms present in it by adversely affecting the solute transport, inhibition of key enzymes involved in carboxylation/ decarboxylation reactions.