Indicators of Food Microbial Quality and Safety

Indicator organisms may be employed to reflect the microbiological

quality of foods relative to product shelf life or their safety from
foodborne pathogens. In general, indicators are most often used to assess
food safety/sanitation, and most of this chapter treats them in this
context. However, quality indicators may be used, and some general
aspects of this usage are outlined in the following section.

SOME INDICATORS OF PRODUCT QUALITY

Microbial product quality or shelf-life indicators are organisms

and/or their metabolic products whose presence in given foods
at certain levels may be used to assess existing quality or, better,
to predict product shelf life. When used in this way, the
indicator organisms should meet the following criteria:

1. They should be present and detectable in all foods whose quality (or lack
thereof) is to be assessed.
2. Their growth and numbers should have a direct negative correlation with
product quality.
3. They should be easily detected and enumerated and be clearly distinguishable
from other organisms.
4. They should be enumerable in a short period of time, ideally within a working
day.
5. Their growth should not be affected adversely by other components of the food
microbiota.
n Table 20–1 is a function of the number of organisms noted, and shelf
life can be increased by their control. In effect, microbial quality
indicators are spoilage organisms whose increasing numbers result in
loss of product quality.
Metabolic products may be used to assess and predict microbial quality
in some products; and some examples are listed in Table 20–2. The
diamines, histamine, and polyamines have been found to be of value for
several products. Diacetyl was found to be the best negative predictor of
quality in frozen orange juice concentrates, where it imparts a buttermilk
aroma at levels of 0.8 ppm or above.62 A 30-minute method for its
detection was developed by Murdock.61 Ethanol has been suggested as
a quality index for canned salmon, where 25–74 ppm were associated
with “offness,” and levels higher than 75 ppm indicated spoilage.35
Ethanol was found to be the most predictive of several alcohols in fish
extracts stored at 5◦C, where 227 of 241 fish-spoilage isolates produced
this alcohol.3 Lactic acid was the most frequently found organic acid in
spoiled canned vegetables, and a rapid (2-hour) silica-gel plate method
was developed for its detection.1 The production of trimethylamine
(TMA) from trimethylamine-N
oxide by fish spoilers has been used by a large number of investigators
as a quality or spoilage index.
Various procedures have been employed to measure total volatile
substances as indicators of fish quality, including total volatile bases
(TVB)—ammonia, dimethylamine, and TMA—and total volatile
nitrogen (TVN), which includes TVB and other nitrogen compounds
that are released by steam distillation of fish products.
INDICATORS OF FOOD SAFETY

Microbial indicators are employed more often to assess food safety and
sanitation than quality. Ideally, a food safety indicator should meet
certain important criteria. It should
1. be easily and rapidly detectable
2. be easily distinguishable from other members of the food biota
3. have a history of constant association with the pathogen
whose presence it is to indicate
4. always be present when the pathogen of concern is present
5. be an organism whose numbers ideally should correlate with
those of the pathogen of concern
The first fecal indicator was Escherichia coli. When the concept of fecal
indicators was applied to food safety, some additional criteria were
stressed, and those suggested by Buttiaux and Mossel in 196111 are still
valid:
1. Ideally, the bacteria selected should demonstrate specificity,
occurring only in intestinal environments.
2. They should occur in very high numbers in feces so as to
be encountered in high dilutions.
3. They should possess a high resistance to the extra
enteral environment, the pollution of which is to be assessed.
4. They should permit relatively easy and fully reliable detection
even when present in very low numbers.

Coliforms
While attempting to isolate the etiologic agent of cholera in 1885,
Escherich24 isolated and studied the organism that is now E. coli. It was
originally named Bacterium coli commune because it was present in the
stools of each patient he examined.
Fecal coliforms are defined by the production of acid and gas in EC
broth between 44◦C and 46◦C, usually 44.5◦C or 45.5◦C.

Distribution
The primary habitat of E. coli is the intestinal tract of most warm-
blooded animals, although sometimes it is absent from the gut of hogs.
The primary habitat of E. aerogenes is vegetation and, occasionally, the
intestinal tract.
It is not difficult to demonstrate the presence of coliforms in air and
dust, on the hands, and in and on many foods. The issue is not simply
the presence of coliforms but their relative numbers. For example, most
market vegetables harbor small numbers of lactose-fermenting,
Gram negative rods of the coliform type, but if these products have
been harvested and handled properly, the numbers tend to be quite low
and of no real significance from the standpoint of public health.

Coliform Criteria and Standards

1. Under proper conditions of harvesting, handling, storage,

and transport of foods by use of a hazard analysis critical control
point (HACCP) system, what is the lowest possible and feasible
number of coliforms to maintain?
2. At what quantitative level do coliforms or E. coli indicate
that a product is unsafe?

1. not over 10/ml for Grade A pasteurized milk and milk products,
including cultured products
2. not over 10/ml for certified raw milk and not over 1 for certified
pasteurized milk
3. not over 10/ml for precooked and partially cooked frozen foods
 4. not over 100/ml for crabmeat 5. not over 100/ml for custard-filled
items
Enterococci
Around 30 species of the genus Enterococcus are recognized; and 22 are
summarized in Table 20–5. Prior to 1984, the “fecal streptococci”
consisted of two species and three subspecies, and they, along with S.
bovis and S. equinus, were placed together because each contained
Lancefield group D antigens. The latter two species are retained in the
genus Streptococcus.

1. They generally do not multiply in water, especially if the

organic matter content is low.

2. They are generally less numerous in human feces than E. coli,

with ratios of fecal coliforms to enterococci of 4.0 or higher being
indicative of contamination by human waste. Thus, the classic
enterococcal tests presumably reflect more closely the numbers of
intestinal pathogens than fecal coliforms.

3. The enterococci die off at a slower rate than coliforms in

Waters and thus would normally outlive the pathogens whose
presence they are used to indicate.

Distribution
Although the two classic enterococcal species (E. faecalis and
E. faecium) are known to be primarily of fecal origin, the new
ones await further study of natural occurrence, especially
regarding fecal occurrence.

Relationship to Sanitary Quality of Foods

In this section, the enterococci discussed are those that were defined
prior to 1984. A large number of investigators found the classic
enterococci to be better than coliforms as indicators of food sanitary
quality, especially for frozen foods. In one study, enterococcal numbers
were more closely related to aerobic plate counts (APC) than to coliform
counts, whereas coliforms were more closely related to enterococci than
to APC.30 Enterococci have been found in greater numbers than
coliforms in frozen foods (Table 20–6). In a study of 376 samples of
commercially frozen vegetables, Burton9 found that coliforms were
more efficient indicators of sanitation than enterococci prior to freezing,
whereas enterococci were superior indicators after freezing and storage.
In samples stored at −20◦C for 1–3 months, 81% of enterococci and 75%
of coliforms survived. After 1 year, 89% of enterococci survived but
only 60% of coliforms. In another study, enterococci remained relatively
constant for 400 days when stored at freezing temperatures. Enterococci
were recovered from 57% of 14 samples of dried foods, whereas 87% of
13 different frozen vegetables yielded these organisms, many of which
were of the vegetation-resident types.57 The relative longevity of
coliforms and enterococci in frozen fish sticks is presented in Table 20-7.
Bifidobacteria
Around the year 1900, in the course of his research on the stools
of infants Tissier89 noted an organism that occurred with great
frequency and named it Bacillus bifidus; it was later named
Lactobacillius bifidus and is currently known as
Bifidobacterium bifidum. The common occurrence of the
bifidobacteria in stools led Mossel55 to suggest the use of these
Gram-positive anaerobic bacteria as indicators of fecal pollution,
especially of waters. Interestingly, some bifidobacteria are
employed in the production of fermented milks, yogurt, and
other food products, and they are believed to provide some
health benefits.
The genus Bifidobacterium consists of at least 25 species of
catalase-negative, nonmotile rods whose minimum and
maximum growth temperature ranges are 25 to 28◦C and 43 to
45◦C, respectively. They grow best in the pH range 5 to 8 and
produce lactic and acetic acids as the major end products of their
carbohydrate metabolism.