MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 5. Techniques for microbiological analyses

Lesson 23
ENUMERATION OF HYGIENE INDICATOR ORGANISMS

23.1 Introduction

Indicator organisms are bacteria that are used as a sign of quality or hygienic status in a food, water or environment. The definition of the word ‘indicator,’ in fact, includes the concept of the indicator organism, i.e. something ‘so strictly associated with particular conditions that its presence is indicative of the existence of these conditions’. Historically, these conditions have been related to in-sanitation and public health concerns. Over the years, however, the use of indicator organisms has been extended to provide evaluations of the quality, in addition to the safety, of particular commodities.

Indicator organisms are important components of microbiological testing programs conducted both by regulatory agencies and the food industry. They may signify the potential presence of pathogens, a lapse in sanitation as required in good manufacturing practices (GMPs), or a process failure. They may reflect quality attributes that can influence con-an indicator organism alone is cause for concern; in other cases, it is the quantity that is significant. Many foods provide an environment conducive to microbial growth, and indicator counts in such foods may reflect the time and conditions of storage. The microbiological snapshot that is the indicator test must always be assessed in an appropriate context, taking into account the natural microbial ecology, intrinsic and extrinsic chemical and physical factors that might influence microbial growth, process history, and storage conditions of the product. The dual goals of safety and quality often overlap in the water, food, and environmental arenas, and it is important to choose the type of indicator organism that best fits a particular system. This is not an easy task, and the question of indicator selection has generated much discussion and debate. Perhaps adding confusion to the discussion are attempts that have been made over the years to apply various terms so as to distinguish the different functions of indicators, e.g. index, marker, model, sentinel, and surrogate organisms. It seems reasonable to view two general categories of indicators, i.e. safety and quality indicators. Safety indicators suggest that a microbial hazard may exist, and their use is intended to minimize the risk of exposure to the hazard. Quality indicators are used to assess issues important to product acceptability, e.g. shelf life, organoleptic characteristics, spoilage, etc. The International Commission on Microbiological Specifications for Foods (ICMSF) has noted that selection of an indicator must be considered carefully with an understanding of how to interpret the results of indicator testing. Indicators are a compromise, representing an analytical substitute for detection of the target hazard or concern directly. They can never be used to prove the presence or absence of the target.

23.2 Selection Criteria for Indicator Organisms

The ICMSF has listed the factors that should be considered when selecting an indicator organism for a particular purpose:

1. Presence of the indicator should suggest a faulty process or practice or a potential for spoilage.
2. Survival or stability of the indicator should be similar to or greater than the hazard or spoilage organism.
3. Growth characteristics of the indicator should be similar to or faster than the hazard or spoilage organism.
4. Identifiable characteristics of the indicator should be stable.
5. Method for detection and/ or quantitation should be easy, rapid, inexpensive, reliable, sensitive, and validated; does not risk analyst health; and is suitable for in-plant use.
6. Quantitative results should show a correlation between the concentration of the indicator and the level of the hazard or spoilage organism.
7. Results should be applicable to process control.

23.3 Hygiene Indicators

The microbial composition of a product significantly determines its quality. The types and number of microorganisms present influence the sensory properties (taste, aroma, texture, color) and shelf life of the product. Among these microbial populations, a particular one may be useful as an indicator to reflect quality changes in the product. Such quality indicators are often used to ensure that the product is microbiologically stable and aesthetically acceptable. The primary attribute of a quality indicator is that its growth and numbers should be inversely related to acceptable product quality. The indicator should be present in all products whose quality is to be assessed, its growth is unaffected by other microbial populations present and there should be relatively simple methods available for detection, differentiation, and quantization. A good example is the yeast and mould determination, which can serve as a quality indicator for cereal grains. Growth of yeasts and moulds in cereal grains is generally not influenced by the presence of many other microorganisms, which would be inhibited by the low water activity of the commodity.

23.4 Commonly Used Indicator Organisms

Many different types of indicators have been advocated for use in particular applications; however, this is limited to the most common indicators used for foods and drinking water, i.e. the aerobic plate count; coliforms; E. coli; Enterobacteriaceae, enterococci and the yeasts and moulds.

23.4.1 Aerobic plate count

The aerobic plate count (APC) is one of the most widely used indicator tests. Although the applications of the APC are diverse, on one thing there is agreement: it cannot be used as a safety indicator, as there is generally no correlation between APCs and the presence of pathogens or their toxins. The APC may be a quality indicator, and then only when used in an appropriate context. It has no indicator value for some products, for example, vegetable sprouts, which naturally have high APCs in the range of 10⁸–10⁹ CFU/g, or for fermented products, such as yogurt, which yield high APCs due to the starter cultures incorporated. The APC of a product may reflect the microbial load of raw materials and ingredients, or its age and storage history. Assays for specific spoilage microorganisms may be necessary and more reliable than APCs for determining the acceptability of certain products. Nevertheless, in the appropriate context, the APC can indicate adherence to sanitation GMPs and product acceptability.

23.4.1.1 Detection and enumeration

The method recommended by the International Organization for Standardization (ISO 4833) calls for aerobic incubation on plate count agar at 30°C for 72 hours. The FDA’s BAM recommends 35°C for 48 hours for non-dairy foods. The Standard Plate Count, which is used for estimating bacterial populations in dairy products, strictly specifies 32°C for 48 hours. The ‘pour plate’ method for the APC is officially recognized (AOAC 966.23C; ISO 4833). The ‘spread plate’ technique is generally easier to perform and may have other advantages: different colony morphologies may be recognized, translucent media are not required, and microorganisms are not exposed to the heat of the molten agar. Other rapid methods have been officially recognized, including use of the hydrophobic grid membrane filter (HGMF; AOAC 986.32), pectin gel (AOAC 988.18), and dry rehydratable film (AOAC 990.12). SimPlate® Total Plate Count, which uses colorimetric detection of growth in micro wells to determine the most probable number (MPN) of the microorganisms, is the most recent method to receive official status (AOAC 2002.07).

23.4.2 Coliforms and E. coli

The coliform group is not a valid taxonomic distinction, but is defined functionally, i.e., by the fermentation of lactose in the coliform test. Coliforms may be defined as Gram-negative, oxidase-negative, aerobic or facultative anaerobic non-spore-forming rods, able to grow in the presence of bile salts, and which ferment lactose to produce acid and gas within 48 hours at 37°C. Genera that fit this description are Citrobacter, Enterobacter, Escherichia and Klebsiella. However, Citrobacter, Enterobacter, Klebsiella and include species that are normal inhabitants of plants and the environment; thus, a positive coliform test does not necessarily indicate fecal contamination, as originally proposed by the PHS in 1914 for evaluation of drinking water. This realization discredited the coliform test as an indicator of faecal pollution and prompted development of the faecal coliform test. Sometimes referred to as thermotrophic, thermoduric, or thermotolerant coliforms, the faecal coliforms have the same properties as the coliform group, except that the fermentation is able to proceed at 44.5–45.5°C. However, species that have this capacity also are known to be present naturally in the environment; thus the faecal coliforms are not specific indicators of faecal pollution of water, either. E. coli is present in all mammalian faeces at high concentrations; it does not multiply appreciably but can survive in water for 4–12 weeks, and so it is useful as an indicator of faecal pollution of drinking water systems. The case for E. coli as an indicator in foods and the processing environment is not as clear, however. Certainly, the organism can survive, but it can also grow, in certain foods. It can become established in the food processing environment and contaminate foods in the facility; thus, recent faecal contamination cannot be concluded when it is detected in foods or food manufacturing plants. The coliform groups and E. coli are most widely applied in the food industry as sanitation and process integrity indicators and for Hazard Analysis Critical Control Point (HACCP) verification.

Both quantitative and presence/absence methods are described for determining total coliforms and faecal coliforms. Methods for coliform testing generally incorporate the distinguishing physiological characteristics of the group, i.e. lactose fermentation and resistance to bile salts (or a similar surfactant, such as Sodium Lauryl sulfate). Colony counts of the coliform group are obtained from violet red bile lactose (VRBL) agar (ISO 4832; ISO 5541/1). Injured coliform populations may be recovered by first inoculating the sample onto a non-selective agar medium, incubating for several hours to allow resuscitation, followed by a VRBL agar overlay for selection The MPN method for enumeration of coliforms uses Lauryl sulfate tryptose (LST) broth as a first step, with confirmation of positive tubes, indicated by gas production, in brilliant green bile lactose broth (BGBLB).

Because certain strains of E. coli known to exist in some foods, e.g. meat products, do not produce gas in LST, the ISO method 4831 recommends transfer of all turbid LST tubes, regardless of gas production, to BGBLB for confirmation. Membrane filtration, which allows analysis of a larger sample volume than other methods, is recommended for coliform counts in 100 ml water (ISO 9308-1). Appropriate enzymatic treatments for foods are necessary to allow filtration of 0.5–2.0 ml sample volumes in the application of HGMF for coliform determinations (AOAC 983.25). The huge number of samples that are routinely tested for coliforms spurred development of rapid methods for these determinations.

23.4.3 Enterobacteriaceae

The family Enterobacteriaceae encompasses approximately 20 genera, including E. coli and the other members of the coliform group; food-borne pathogens Salmonella, Shigella, Yersinia and other related genera. The family was originally proposed as an indicator alternative to the coliform group, because testing for the entire family would be more inclusive for the pathogenic genera. Lactose, the carbohydrate specified in the coliform test, is not fermented by Salmonella, Shigella, or Yersinia, so their presence would not be detected by the test. But substituting glucose for the lactose in the test would allow detection of all members of the Enterobacteriaceae, including the pathogens, as well as variant strains that do not show the typical lactose fermentation trait. The rationale for the use of the Enterobacteriaceae as indicators was advanced by reports noting low or negative coliform test results despite detection of Salmonella in certain foods, by a shigellosis outbreak in a nursing home in which Enterobacteriaceae tests might have indicated a cause for concern, and by a cheese-associated outbreak caused by an enteropathogenic E. coli strain that was a slow lactose fermenter. These reports notwithstanding, the Enterobacteriaceae are no more indicative of faecal contamination in foods than are the coliforms, i.e. not indicative at all. Nevertheless, they are useful, like the coliforms, as process integrity indicators.

The Enterobacteriaceae may be superior to the coliforms as indicators of sanitation GMPs because they have collectively greater resistance to the environment than the coliforms, can colonize where sanitation has been inadequate, and are sensitive to sanitizers. Thus, the Enterobacteriaceae are useful for monitoring sanitation in food manufacturing plants, although they are more widely used as indicators in Europe than in the United States.

23.4.4 Detection & enumeration

Like the coliforms, members of the Enterobacteriaceae family demonstrate bile resistance, but unlike the coliforms, they do not universally demonstrate lactose fermentation. However, they all ferment glucose. A simple switch of the carbohydrate from lactose to glucose in the coliform selective medium formulation provides a way to test for all of the members of the family, including the pathogens. The Enterobacteriaceae are enumerated on violet red bile glucose (VRBG) agar or by MPN determination using brilliant green bile glucose (BGBG) broth (ISO 7402). A petrifilm method for determining Enterobacteriaceae counts has recently received AOAC Official Method SM status (AOAC 2003.01). Given that the Enterobacteriaceae are commonly used as sanitation indicators, and as such may be subjected to various environmental stresses, a method for their recovery by pre-enrichment in non-selective buffered peptone water, followed by selective growth in BGBG broth and isolation on VRBG agar, has been validated (ISO 8523).

23.4.5 Enterococci

Monitoring the microbiological quality of milk relies largely on examination for indicator bacteria such as coliforms, E. coli and Enterococci. The presence of Enterococcus group, which is a subgroup of the faecal Streptococci, serves as a valuable bacterial indicator for determining the extent of faecal contamination and it is more specific than the detection of coliforms, which may originate from non-faecal. Enterococci have different useful applications in the dairy industry. Nevertheless, they also have been described as spoilage micro-organisms and cross-contaminants during food processing, when their initial numbers in raw milk are high, pasteurisation is poor, or the pasteurized milk is not stored properly indicates poor hygiene during milk handling and processing. There are no standards set for the minimum and maximum count of enterococci because their counts vary with product handling, time of storage and other factors and are not normally counted in microbiological analyses. But, being a severe problem in dried milk products and infant feed as enterococcal counts in milk-based infant foods are as high as 19 × 102 cfu/g, PFA Rules 1956 have given standards for malted and infant milk food, according to which faecal streptococci should be absent in 0.1 gram. Enterococci can enter the milk chain either primarily from human or animal faeces but also secondarily from contaminated water sources, the exterior of the animal or other contaminated milking equipment or bulk storage tanks handled in the processing plant. The HTST pasteurisation (72°C/15 seconds) followed by proper storing conditions ensures no enterococcal re-growth and thus enterococcal presence is extremely unlikely to occur in the pasteurised milk. In principal dairy products – milk powder and butter – their presence is unlikely, since their manufacture involves one or several heating stages that will effectively inactivate any enterococcal bacterium. Therefore, if final products are contaminated with enterococci, these would not have originated from the raw milk, but from post-heat re-contamination and growth.

23.4.5.1 The genus Enterococcus

The genus Enterococcus was carved out of the earlier larger genus, Streptococcus, and ‘faecal streptococci’ or ‘Lancefield’s group D streptococci’ are still maintained in this genus. Out of the 20 species of this genus, only two (E. faecalis and E. faecium) are suggested to be responsible for nosocomial infections. The Enterococcus spp. is regular Gram-positive, on-spore forming, non motile, facultative anaerobic, gamma-haemolytic on blood agar, catalase negative, homofermentative ovoid cocci (pairs to short chains).These bacteria can grow between temperature ranges of 5 to 50°C with an optimum growth temperature of 30 to 37°C. Typical pH ranges for growth is 4.6 to 9.9 with an optimum growth pH at neutral condition i.e. at 6.0-7.0.

23.4.5.2 Enumeration of enterococci from dairy products

Principle: The enumeration of enterococci in dairy products is based on its extraordinary physiological characteristics which differentiates them from other microorganisms and include:

1. Bile tolerance up to 40%
2. Sodium chloride tolerance up to 6.5%
3. Survive heating at 60°C for 30 minutes
4. Hydrolyse esculin in the presence of bile salts by the enzymatic action of β-glucosidase/ esculinase
5. High level of endemic antibiotic resistance
6. Reduces 2,3,5-triphenyltetrazolium chloride (TTC)
7. Tolerance to sodium azide
8. Enzymatic action of β-glucosidase on various chromogenic and fluorogenic substrates.

23.4.5.3 Conventional methods

Exploiting above parameters several media have been devised for enterococci isolation and enumeration from dairy products. Even though several media have been advocated for the selective isolation and quantification of enterococci, several protocols have been published for diverse purposes. Till date, there is no single method alone that universally meets all requirements as all have one or more shortcomings. The typical culture media employed for the estimation of enterococci in water, foods, feeds and clinical specimens such as the Slanetz-Bartley (membrane Enterococcus agar), kanamycin esculin azide (KAA) medium, citrate azide agar (CAA) and bile esculin azide agar (BEA) are advantageously applied in the case of selective enumeration of enterococci as single components, i.e. if enterococci are the only microbial component in the product. However, like any other members of the LAB, enterococci are often found associated with a micro-flora of considerable diversity and this is reflected in a much more complicated situation when samples containing such a mixed micro-flora have to be examined for enterococcal recovery. Consequently, a number of selective agents, incubation conditions, and combinations and modifications thereof have to be used; taking into account various advantages but also drawbacks. The use of media containing either selective chromogenic dyes or selectively inhibitory substances (e.g. antibiotics) may, however, enable some differential bacteriological enumeration. In spite of the large variety of suggested media and methods with their modifications, the citrate azide agar and the bile esculin azide agar, are the most recommended media for enterococcal isolation in dairy products. It is always practical to bear in mind, when examining enterococci in dairy products a higher incubation temperature (45°C) may be necessary to suppress the growth of the background micro-flora.

23.4.5.4 List of various selective agents commonly used for enterococci and their mode of action

23.4.5.5 Various media used for selective isolation of enterococci and their mode of action

23.4.5.5.1 Citrate azide agar

It is a selective agar used for the identification of enterococci in meat, meat products, dairy products and other food stuffs. The high concentrations of citrate and azide almost completely inhibit the growth of the accompanying microbial flora. Enterococci reduce the colorless 2,3,5-triphenyltetrazolium chloride dye present in medium to a red formazan, their colonies thus become red in color (Fig. 23.1).

Fig. 23.1 Red coloured colonies on citrate azide agar

23.4.5.5.2 Bile esculin azide agar

Bile esculin azide agar is a modification of the earlier medium bile esculin agar. This formula modifies Bile Esculin Agar by adding sodium azide and reducing the concentration of bile. Organisms positive for esculin hydrolysis, hydrolyze esculin to esculetin and dextrose (glucose). The esculetin reacts with the ferric citrate to form a dark brown or black complex (Fig. 23.2 & 23.3). Oxbile is used to inhibit Gram-positive bacteria and other streptococci, while sodium azide inhibits Gram-negative bacteria. Enzymatic digest of casein and yeast enriched meat peptone are the carbon, nitrogen, and vitamin sources used for general growth requirements in bile esculin agar. Sodium chloride maintains the osmotic balance of the medium. Sodium citrate acts as a preservative.

Fig. 23.2 Reaction catalysed



Fig. 23.3 Black color colonies on bile esculin azide agar

23.4.5.5.3 Kanamycin-esculin-azide (KAA) medium

It is a selective isolation and enumeration medium for enterococci in foods. Sodium azide and kanamycin provide the selective inhibition required whilst esculin and iron salts form an indicator system for the presumptive identification of enterococci as explained above. Incubation at 42°C will increase the medium’s selectivity.

23.4.5.6 Slanetz-bartley (Membrane enterococcus agar)

Enterococci reduce 2, 3, 5-triphenyltetrazolium chloride to the insoluble red dye formazan, producing colonies which are dark red or maroon on the surface of the membrane or agar. This reaction is not exclusive to enterococci, and the count at this stage should be considered presumptive. Colonies may be confirmed as enterococci by demonstrating esculin hydrolysis using kanamycin esculin azide agar.

23.4.5.7 Conventional procedure for isolation and enumeration of enterococci in milk

23.4.5.8 Enzyme/ substrate based detection method

There are unique biochemical pathways that characterize each genus, species or strain and are based on unique key enzymes that participate in such metabolisms. These enzymes are referred to as ‘marker enzymes’. The chromogenic or fluorogenic substrate complex is hydrolyzed by marker enzyme and free chromogen or fluorogen is released which can be detected either colorimertrically or fluorometerically. It is therefore useful to assay directly for the activity of these enzymes for detection of micro organisms.

23.4.5.8.1 Hi chrome rapid enterococcus agar (Chromogenic based)

It is recommended for rapid detection of enterococci from water samples sources. The enzyme β-glucosidase which act as marker enzyme for enterococci cleaves the chromogenic substrate X-GLU (5-bromo-4-chloro-3-indolyl-ß-D-glucopyranoside), resulting in an intensive bluish green color colonies due to formation of indigo (Fig. 23.4 & 23.5). The medium contains special peptone, which provides nitrogenous compounds and other essential nutrients. Sodium chloride maintains the osmotic balance of the medium. Sodium azide inhibits the accompanying micro-flora, especially gram negative organisms. Tween 80 (Polysorbate80) acts as a source of fatty acids.

Fig. 23.4 Bluish green color colonies on Hi chrome rapid enterococcus agar

Fig. 23.5 Reaction catalysed

23.5 Yeasts and Moulds

Yeasts and moulds are commonly enumerated in foods as quality indicators. They have no predictive value for the occurrence of toxigenic fungi or other pathogens. As a group, the yeasts and moulds are diverse and can grow on virtually any type of food. They survive a wide range of environmental conditions: pH 2–9; temperatures of 5^o–35⁰C; and water activity (a_w) of 0.85 or less. As quality indicators, they can be used to assess ingredient acceptability, organoleptic characteristics, stability, and shelf life of a product. Osmophilic yeasts, commonly members of the genus Zygosaccharomyces, can grow down to Aw of 0.65 and are used as indicators in low aw foods, e.g., jams, syrups, juice concentrates.

23.5.1 Detection and enumeration

Although they have a diverse growth habit, yeasts and moulds grow slowly in laboratory culture when compared with bacterial groups. Thus, yeasts and moulds are enumerated by a plate count procedure that uses agar supplemented with agents inhibitory to bacteria. Chloramphenicol, rose bengal, and dichloran are common selective agents. Spread or pour plates, incubated at 25^oC for 3–7 days, are recommended (ISO 7954). If osmophilic types are suspected, care must be taken to decrease the Aw of both the plating media and diluents as appropriate and to allow extended incubation times (31). Rapid official methods using HGMF (AOAC 995.21) and dry rehydratable film (AOAC 997.02) recommend 50 hours or 5-day incubation, respectively. A method using the SimPlate colorimetric format determines yeast and mould counts in 56–72 hours (AOAC 2002.11). Despite the improvements provided by the rapid methods, a relatively lengthy time of analysis still is required for yeast and mould determinations, compared with other microbial groups. Significant economic consequences can result if product release is delayed until assay results are obtained. Clearly, there is a need for more research to improve methods for determining yeasts and moulds in foods.