Indicators of Food Microbial Quality and Safety

 WRITTEN BY VINOD KUMAR KUSHWAHA  MSC IN MICROBIOLOGY ON AUG 2018

Indicators of Food Microbial
Quality and Safety

1.    Indicator organisms may be employed to reflect the microbiological quality of foods relative to product shelf life or their safety from
food  borne pathogens. In general, indicators are
most often used to assess food 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.

2.    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:
• They should be present and detectable in all
foods whose quality (or lack thereof) is to
be assessed.
• Their growth and numbers should have a
direct negative correlation with product
quality.
• They should be easily detected and enumerated and be clearly distinguishable from
other organisms.
• They should be enumerable in a short period of time, ideally within a working day.
• Their growth should not be affected adversely by other components of the food
flora.
In general, the most reliable indicators of product quality tend to be product specific; some
examples of food products and possible quality
indicators are listed in Table 20-1.The products
noted have restricted biota, and spoilage is typically the result of the growth of a single organism. When a single organism is the cause of spoilage, its numbers can be monitored by selective
culturing or by a method such as impedance with
the use of an appropriate selective medium. The
overall microbial quality of the products noted
in Table 20-1 is a function of the number of organisms noted, and the 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; some
examples are listed in Table 20-2. The diamines
(cadaverine and putrescine), histamine, and
polyamines have been found to be of value
for several products (discussed further in Chapter 4). 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.54 A 30-minute
method for its detection was developed by
Murdock.53 Ethanol has been suggested as a qualIndicators of

3.    Food Microbial
Quality and Safety

Table 20-1 Some Organisms That Are Highly
Correlated with Product Quality

Organisms	Products
Acetobacter spp.	Fresh ciaer
Bacillus spp.	Bread dough
Byssochlamys spp.	Canned fruits
Clostridium spp.	Hard cheeses
Flat-sour spores	Canned vegetables Fruit cannery sanitation Beers, wines
Geotrichum spp.
Lactic acid bacteria
Lactococcus lactis	Raw milk (refrigerated) Sugar (during refinery) Beers
Leuconostoc mesenteroides Pectinatus cerevisiiphilus
"Pseudomonas putrefaciens" Yeasts	Butter
Fruit juice concentrates Mayonnaise, salad dressing
Zygosaccharomyces bailii

ity index for canned salmon, where 25-74 ppm
were associated with "offness," and levels higher
than 75 ppm indicated spoilage.32 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-7Voxide 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 offish quality, including total volatile bases
(TVB)—ammonia, dimethylamine, andTMA—
and total volatile nitrogen (TVN), which includes
TVB and other nitrogen compounds that are released by steam distillation offish products.
Total viable count methods have been used to
assess product quality. They are of greater value
as indicators of the existing state of given products than as predictors of shelf life because the
portion of the count represented by the ultimate
spoilers is difficult to ascertain.
Overall, microbial quality indicator organisms
can be used for food products that have a biota
limited by processing parameters and conditions
where an undesirable state is associated consistently with a given level of specified organisms.
Where product quality is significantly affected
by the presence and quantity of certain metabolic
products, they may be used as quality indicators.
Total viable counts generally are not reliable in
this regard, but they are better than direct microscopic counts.

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
• be easily and rapidly detectable
• be easily distinguishable from other members of the food biota

Table 20-2 Some Microbial Metabolic
Products That Correlate with Food Quality
Applicable

Metabolites	Food Product
Cadaverine and putrescine Diacetyl	Vacuum-packaged beef Frozen juice concentrate Apple juice, fishery products Canned tuna
Ethanol
Histamine
Lactic acid	Canned vegetables
Trimethylamine (TMA)	Fish
Total volatile bases (TVB), total volatile nitrogen (TVN) Volatile fatty acids	Seafoods
Butter, cream

·      
• have a history of constant association with
the pathogen whose presence it is to indicate
• always be present when the pathogen of concern is present
• be an organism whose numbers ideally
should correlate with those of the pathogen
of concern (Figure 20-1)
• possess growth requirements and a growth
rate equaling those of the pathogen
• have a die-off rate that at least parallels that
of the pathogen and ideally persists slightly
longer than the pathogen of concern
(Figure 20-1)
• be absent from foods that are free of the
pathogen except perhaps at certain minimum numbers
These criteria apply to most, if not all, foods
that may be vehicles of foodborne pathogens,
regardless of their source to the foods. In the historical use of safety indicators, however, the
pathogens of concern were assumed to be of intestinal origin, resulting from either direct or indirect fecal contamination. Thus, such sanitary
indicators were used historically to detect fecal
contamination of waters and thereby the possible
presence of intestinal pathogens. The first fecal
indicator was Escherichia coll. When the concept of fecal indicators was applied to food safety,
some additional criteria were stressed, and those
suggested by Buttiaux and Mossel10 are still valid:
• Ideally the bacteria selected should demonstrate specificity, occurring only in intestinal environments.
• They should occur in very high numbers in
feces so as to be encountered in high dilutions.
• They should possess a high resistance to the
extraenteral environment, the pollution of
which is to be assessed.
Figure 20-1 Idealized relationship between an indicator organism and the relevant pathogen(s). The indicator
should exist in higher numbers than the pathogen during the existence of the latter.
TIME
NUMBERS
• They should permit relatively easy and fully
reliable detection even when present in very
low numbers.
Following the practice of employing E. coli as
an indicator of fecal pollution of waters, other
organisms were suggested for the same purpose.
In time, most of these were applied to foods.
Coliforms
While attempting to isolate the etiologic agent
of cholera in 1885, Escherich21 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. Schardinger68 was the first to suggest
the use of this organism as an index of fecal pollution because it could be isolated and identified more readily than individual waterborne
pathogens. A test for this organism as a measure
of drinking water potability was suggested in
1895 by T. Smith.73 This marked the beginning
of the use of coliforms as indicators of pathogens in water, a practice that has been extended
to foods.
Strains
In a practical sense, coliforms are gram-negative asporogeneous rods that ferment lactose
within 48 hours and produce dark colonies with
a metallic sheen on Endo-type agar.4 By and
large, coliforms are represented by four genera
of the family Enterobacteriaceae: Citrobacter,
Enterobacter, Escherichia, and Klebsiella. Occasional strains ofArizona hinshawii andHafnia
alvei ferment lactose but generally not within 48
hours, and somePantoea agglomerans strains are
lactose positive within 48 hours.
Since E. coli is more indicative of fecal pollution than the other genera and species noted (especially E. aerogenes), it is often desirable to
determine its incidence in a coliform population.
The IMViC formula is the classic method used,
where I = indole production, M = methyl red reaction, V = Voges-Proskauer reaction (production of acetoin), and C = citrate utilization. By
this method, the two organisms noted have the
following formulas:
I M V C
E. coli + +
E. aerogenes — — + +
The IMViC reaction + + — designates E. coli
type I; E. coli type II strains are - + - -. The MR
reaction is the most consistent for E. coli.
Citrobacter spp. have been referred to as intermediate coliforms, and delayed lactose fermentation by some strains is known. All are MR+
and VP-. Most are citrate +, whereas indole production varies.Klebsiella isolates are highly variable with respect to IMViC reactions, although
K. pneumoniae is generally MR-, VP+, and C+,
but variations are known to occur in the MR and
I reactions. Fluorogenic substrate methods for
differentiating between E. coli and other
coliforms are discussed in Chapter 11.
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. (EC broth, for
 
Growth
Like most other nonpathogenic gram-negative
bacteria, coliforms grow well on a large number
of media and in many foods. They have been
reported to grow at temperatures as low as -20C
and as high as 500C. In foods, growth is poor or
very slow at 5°C, although several investigators
have reported the growth of coliforms at 3-6°C.
Coliforms have been reported to grow over a pH
range of 4.4-9.0. E. coli can be grown in a minimal medium containing only an organic carbon
source such as glucose and a source of nitrogen
such as (NH4)2S04and other minerals. Coliforms
grow well on nutrient agar and produce visible
colonies within 12-16 hours at 37°C. They can
be expected to grow in a large number of foods
under the proper conditions.
Coliforms are capable of growth in the presence of bile salts, which inhibit the growth of
gram-positive bacteria. Advantage is taken of
this fact in their selective isolation from various
sources. Unlike most other bacteria, they have
the capacity to ferment lactose with the production of gas, and this characteristic alone is
sufficient to make presumptive determinations.
The general ease with which coliforms can be
cultivated and differentiated makes them nearly
ideal as indicators, except that their identification may be complicated by the presence of atypical strains.The aberrant lactose fermenters, however, appear to be of questionable sanitary
significance.26
One of the attractive properties of E. coli as a
fecal indicator for water is its period of survival.
It generally dies off about the same time as the
more common intestinal bacterial pathogens, although some reports indicate that some bacterial pathogens are more resistant in water. It is
not, however, as resistant as intestinal viruses.
Figure 20-2 Summary of most probable number methods for total coliforms, fecal coliforms, and Escherichia
coli. Source: Reprinted from Ref. 34, p. 542,by courtesy of Marcel Dekker. Jay, J.M. Indicator organisms in
foods. In Foodborne Disease Handbook, Vol. 1, eds.Y.H.Hui, IR. Gorham, K.D.Murrell, and D.O. Cliver,
537-546. Marcel Dekker, Inc.,N.Y, 1994.
MPN confirmed test for total
coliforms
Record gas + BGB tubes and
calculate MPN from table.
From all gas + tubes, inoculate
BGB broth; incubate at 35°C for
48 h.
MPN presumptive test for total
coliforms
GAS-POSITIVE TUBES
Inoculate LST broth
tubes; incubate at
35°C for 48 h.
Serial dilutions of
food homogenates
Fecal coliforms
Inoculate LST + MUG
broth; incubate at
35°C for 24-28 h.
From all gas + tubes, inoculate
EC medium and incubate at
44.5°C for 24 h.
Record gas + tubes and
calculate MPN for fecal
coliforms.
Record fluorescent-positive
tubes and calculate E. coli MPN
from table.
Examine tubes under long-wave
(365 nm) UV light.
Buttiaux and Mossel10 concluded that various
pathogens may persist after E. coli is destroyed
in foods that are frozen, refrigerated, or irradiated. Similarly, pathogens may persist in treated
waters after E. coli destruction. Only in acid food
does E. coli have a particular value as an indicator organism due to its relative resistance to a
lowpH.10
Detection and Enumeration
A large number of methods have been developed for the detection and enumeration of
E. coli and coliforms, and some are discussed in
Chapters 10 and 11. One of the standard references listed in Table 10-1 should be consulted
for an appropriate method to use under specified conditions.
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
Although the presence of large numbers of
coliforms and E. coli in foods is highly undesirable, it would be virtually impossible to eliminate all from fresh and frozen foods. The basic
questions regarding numbers are as follows:
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 has become
unsafe?
In the case of water and dairy products, there
is a long history of safety related to allowable
coliform numbers. Some coliform and E. coli
criteria and standards for water, dairy products,
and other foods covered by some regulatory
agencies are as follows:
• not over 10/mL for Grade A pasteurized
milk and milk products, including cultured
products
• not over 10/mL for certified raw milk and
not over 1 for certified pasteurized milk
• not over 10/mL for precooked and partially
cooked frozen foods
• not over 100/mL for crabmeat
• not over 100/mL for custard-filled items
Low numbers of coliforms are permitted in
sensitive foods at numbers ranging from 1to not
over 100/g or 100 mL.These criteria reflect both
feasibility and safety parameters.
Some products for which coliform criteria have
been recommended by the International Commission on the Microbiological Specifications
for Foods (ICMSF)33 are listed inTable 20-3.The
values noted are not meant to be used apart from
the total suggested criteria for these products.
They are presented here only to show the acceptable and unacceptable ranges of coliforms or
E. coli for the products noted. Implicit in the recommendations for the first four products is that
one or two of five subsamples drawn from a lot
may contain up to 103 coliforms and yet be safe
for human consumption.

Some Limitations for Food Safety Use
Although the coliform index has been applied
to foods for many years, there are limitations to
the use of these indicators for certain foods. As
a means of assessing the adequacy of pasteurization, a committee of the American Public
Health Association in 1920 recommended the
use of coliform,45 and this method was well established in the dairy industry around 1930.59
Coliform tests for dairy products are not intended
to indicate fecal contamination but do reflect
overall dairy farm and plant sanitation.65 For frozen blanched vegetables, coliform counts are of
no sanitary significance because some, especially
Enterobacter types, have common associations
with vegetation.74 However, the presence of E.
coli may be viewed as an indication of a processing problem. For poultry products, coliforms
are not good sanitary indicators because salmonellae may exist in a flock prior to slaughter, and
thus positive fecal coliform tests may be unrelated to postslaughter contamination.79 The standard coliform test is not suitable for meats because of the widespread occurrence of
psychrotrophic enterics and Aeromonas spp. in
meat environments, but fecal coliform tests are
of value.55
Coliform tests are widely used in shellfish
sanitation, but they are not always good predictors of sanitary quality. The U.S. National Shellfish Sanitation Program was begun in 1925, and
the presence of coliforms was used to assess the
sanitation of shellfish-growing waters. Generally,
shellfish from waters that meet the coliform criteria ("open waters") have a good history of sanitary quality, but some human pathogens may still
exist in these shellfish. In oysters, there is no
correlation between fecal coliforms and Vibrio
cholerae15'35or between is. coli and either Vibrio
parahaemolyticus or Yersinia enterocolitica.44
 

 Enterococci
Over 22 species of the genusEnterococcus are
recognized; 22 of the species are summarized in
Table 20-4. 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.
Historical Background
Escherich was the first to describe the organism that is now E.faecalis, which he named Micrococcus ovalis in 1886. E.faecium was recognized first in 1899 and further characterized in
1919 by OrIa-Jensen.56 Because of their existence in feces, these classic enterococci were suggested as indicators of water quality around 1900.
Ostrolenk et al.57 and Burton8 were the first to
compare the classic enterococci to coliforms as
indicators of safety. Pertinent features of the classic enterococci that led to their use as pollution
indicators for water are the following:
• They generally do not multiply in water, especially if the organic matter content is low.
• 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.
• 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.
The simultaneous use of enterococci and
coliforms was advocated in the 1950s by
Buttiaux,9 as in his opinion the presence of both
suggested the occurrence of fecal contamination.
In his review of the literature, Buttiaux noted that
100% of human and pig feces samples contained
enterococci, whereas only 86-89% contained
coliforms.9

Classification and Growth Requirements
Although the classic enterococci never
achieved the status of coliforms as sanitation
indicators for water or foods, their current classification in an expanded genus could, on one
hand, make them more attractive as indicators
or, on the other hand, make them less attractive
and meaningful. E. faecalis is found most frequently in the feces of a variety of mammals and
E. faecium largely in hogs and wild boars 4872;
the natural distribution of some other members
of the new genus is less well understood. Prior
to 1984, enterococci and "fecal strep" were essentially synonymous and consisted principally
of only E. faecalis, E. faecium, and E. durans.
Currently, a test for enterococci is of less significance as fecal, sanitary, or quality indicators
than the classic species.An inspection of the features in Table 20-4 reveals that E. cecorum does
not grow at 100C or in 6.5% NaCl. Although E.
pseudoavium grows at 100C, it does not grow in
the presence of 6.5% NaCl.14 With the exception of E. cecorum, apparently all grow at 100C,
and some strains of E. faecalis and E. faecium
have been reported to grow between 00C and 6°C.
Most of the enterococci grow at 450C and some,
at least E.faecalis and E.faecium, grow at 500C.
The phylogenetic relationship of enterococci,
  E. columbae, E. dispar, and
E. saccharolyticus do not react with serologic
group D antisera. In addition to reacting with
group D antisera, E. avium alone reacts with
group Q.12 The murein type possessed by E. faecalis is Lys-Ala2_3, whereas the other species contain the Lys-D-Asp murein. The mol% G + C
content of DNA of the enterococci is 37-45.
Regarding biochemical characteristics, esculin
is hydrolyzed by all species. Four species produce a yellow pigment (E. casseliflavus, E. flavescens, E. mundtii, and E. sulfureus); two produce H2S (E. casseliflavus and E. malodoratus)\
and all known strains of E. gallinarum13 and
E.flavescens are motile.
As is typical of other gram-positive bacteria,
enterococci are more fastidious in their nutritional requirements than gram negatives but differ from most other gram positives in having requirements for more growth factors, especially
B vitamins and certain amino acids.The requirement for specific amino acid allows some strains
to be used in microbiological assays for these
compounds. They grow over a much wider range
of pH than all other foodborne bacteria (see
Chapter 3). Although they are aerobes, they do
not produce catalase (except a pseudocatalase by
some strains when grown in the presence of O2),
and they are microaerophiles that grow well under conditions of low oxidation-reduction potential (Eh).
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. E. hirae andE. durans
have been found more often in poultry and cattle
than in six other animals, whereas E. gallinarum
has been found only in poultry.19 E. durans and
E.faecium tend to be associated with the intestinal tract of swine more than does E. faecalis.
The last appears to be more specific for the human intestinal tract than are other species.
E. cecorum was isolated from chicken cecae,
E. columbae from pigeon intestines, and E. saccharolyticum from cows. E. avium is found in
mammalian and chicken feces  E. gallinarium in the intestines of fowls.
It is well established that the classic enterococci exist on plants and insects and in soils.The
yellow-pigmented species are especially associated with plants, and E. cecorum appears to be
closely associated with chicken cecae. In general, enterococci on insects and plants may be
from animal fecal matter. Such enterococci may
be regarded as temporary residents and are disseminated among vegetation by insects and wind,
reaching the soil by these means, by rain, and by
gravity.50 Although E. faecalis is often regarded
as being of fecal origin, some strains appear to
be common on vegetation and thus have no sanitary significance when found in foods. Mundt51
studied E. faecalis from humans, plants, and
other sources and found that the nonfecal indicators could be distinguished from the more fecal types by their reaction in litmus milk and their
fermentation reactions in melizitose and melibiose broths. In another  
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.27
Enterococci have been found in greater numbers
than coliforms in frozen foods .
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