Introduction to the subject
(Microbiology of Frozen Foods)
It is widely assumed that frozen foods do not pose a microbiological threat to consumers and, in general, this confidence is justified. Nevertheless (in spite of this), success does not depend on the fact that the initial foodstuffs are of a sound hygiene quality before freezing, and, furthermore, that the thawing/cooking operations reflects the properties of the frozen product. The achievements of these twin aims; without adverse effect on the organoleptic characteristics of the food, has been the essential aim of the manufacturers of frozen foods, and the increased usages of home freezers is a tribute to their success.
Some of the problems associated with the freezing of different commodities, together with the techniques for overcoming them, are to be discussed thoroughly in this subject, and if this knowledge will help to further the safety of frozen foods, then its collation (all together) will have been fully justified.
Normal flora of fish:
Outline
* Background
* Microbiology of fish
* Eggs, skin, gills microflora
* Intestinal microflora
* Diseases (pathogens)
Background:
The flora of living fish depends on the microbial content of the waters in which they live. The slime of fish contains species of Pseudomonas, Acinetobacter, Moraxella, Alcaligenes, Micrococcus, Flavobacterium, Corynebacterium, Saracia, Vibrio, and Bacillus. Fish from northern waters are mostly contains psychrophiles, but fish from tropical waters carry more mesophiles. Freshwater fish carry freshwater bacteria, e.g. species of Aeromonas, Lactobacillus, Brevibacterium, Alcaligenes, and Streptococcus.
In the intestines of fish from both sources contain species of Alcaligenes, Pseudomonas, Flavobacterium, Vibrio, Bacillus, Clostridium, and Escherichia.
Boats, boxes, bins, fish houses, and fishers soon become heavily contaminated with those bacteria and transfer them to the fish during cleaning.
Slime and the skin of newly caught ocean fish showed bacterial counts 102 to several million/cm2, and the intestinal fluid may contain from 103 to 108 /mm2. Gill tissue give shelter for 103 to 106/g. Washing reduces the surface count.
Oysters and other shellfish pick up soil and water microorganisms including pathogens, e.g. species of Alcaligenes, Flavobacterium, Moraxella, Acinetobacter, and some Gram-positive bacteria.
Shrimps, crabs, lobsters, and similar seafood have a bacteria-loaded slime on their surfaces that species of Bacillus, Micrococcus, Pseudomonas, Acinetobacter, Moraxella, Flavobacterium, Alcaligenes, and Proteus.
The numbers of microorganisms on the skin of fish can be influenced by the method of catching. For example, trawling fish nets kept at the bottom for long periods results bacteria of sediment in fish.
Natural microflora of mussels and oysters
A majority of isolates are Gram-negative (68%) and aerobic (76%) bacteria. Predominant microflora are species of Vibrio, Pseudomonas, Shewanella, Aeromonas, Acinetobacter, and Flavobacterium.
Amongst Gram-positive bacteria species of Staphylococcus, Bacillus, and Streptococcus are predominant organisms.
Predominant Vibrio species include V. alginolyticus, V. splendidus, and V. (Listonella) anguillarum.
Eggs microflora
Fish embryos secret inorganic and low molecular weight organic compound, which can diffuse out through the shells that attract bacteria for utilizing these compounds and colonize egg surface. Normal healthy eggs include microflora such as Species of Cytophaga and , Pseudomonas. But dead eggs showed fluorescent Pseudomonas spp., which do not involve in causing of dead, but rather attracting to nutrient leaching. Overgrown of bacteria can hamper eggs development., as for e.g. Leucothrix mucor on cod eggs and Flavobacterium ovolyticus on halibut eggs.
Skin Microflora
Skin microflora reflect that of surrounding water, which may have bacterial counts from 102 to 104 / cm2, for examples Gram negative bactewria: Species of Pseudomonas, Moraxella, Vibrio, Flavobacterium, Acinetobacter, and Aeromonas. Gram positive: Species of Micrococcus, and Bacillus.
Gill Microflora
Gill microflora may contain 102 to 106 bacteria/ g of fish and form extensive colonization of certain types of bacteria (e.g. Flavobacterium spp.). Other common examples are Gram negative: Species of Pseudomonas, Flavobacterium, Vibrio, Moraxella, Cytophaga and Gram positive: Species of Micrococcus, and Bacillus (in warmer water).
Intestinal microflora
Intestinal microflora have been established at the larval stage. These are developed into a persistent flora at the juvenile stage.
Population of microorganisms in fish tends to increase along the length of the gestrointestinal tract. Largest number of bacteria in the intestines (up to 108 CFU/g) includes Gram negative: Pseudomonas spp., Vibrio spp., Achromobacter spp., Flavobacterium spp., Corynebacterium spp., Aeromonas spp. and Gram positive: Bacillus spp., Micrococcus spp. Vibrio dominates in seawater and Aeromonas dominates in freshwater.
Development of the intestinal microbiology
At the time of hatching, the digestive tract of most fish species is an undifferentiated straight tube. Prior to first feeding, microbiology reflects that of the rearing environment. Once feeding begins, microbiology is derived from live feed ingested rather than water.
As the digestive tract becomes more developed, the intestinal microbiology becomes more stable and more complex for the followings:
pH change (lower)
O2 tension (more anaerobic)
Receptors for bacteria
Criteria for testing whether or not microorganism is indigenous to the intestinal tract of fish:
* are found associated with the epithelial
mucosal in the stomach,
* small found in healthy individuals
* colonize early stages and persist throughout
life
* are found in both free-living and hatchery-
cultured fish
* can grow anaerobically in intestine or large
intestine
Roles of intestinal microflora
Accumulate nutrition such as Polyunsaturated fatty acids, amino acids and vitamins. Produce extracellular enzymes: chitinase. Preventing infection from fish pathogens. Competitive attachment. Neutralization of toxins, and Bacteriocidal activity.
Survival and growth:
Bacterial load impacts on survival & digestive organ development. Presence of certain species influence survival of intestinal microflora.
Stimulation of the immune system:
Provide antigens to trigger development of immune responses in the gut
Pathogenesis
Pathogenesis = the origin and development of a disease
Pathogenicity = the ability of a parasite to inflict damage on the host
Entry of the pathogen into the host:
Exposure to pathogens
Adherence to skin or mucosal surface
Invasion through epithelium
Colonization and growth:
Localization (boil, ulcer, etc)
Systematic infection
Production of virulence factors:
Tissue damage via toxins or invasiveness
Types of pathogens
Obligate pathogens: Cause disease in healthy organisms and cause Contagious disease.
Aeromonas salmonicida: Causes Salmonids and other fishes (Furunculosis, skin lesions).
Opportunistic pathogens
Found in the environment
Do not cause disease unless the host immune response is suppressed (stress, environmental factor, etc)
Listonella anguillarum (Fish, mollusks, shrimp, crabs)
Vibriosis
Selective Questions
- (a) Write down the introduction to Microbiology of frozen
foods.
(b) Describe briefly the background of normal flora of fish.
2. (a) Write down natural microflora of mussels and oysters.
(b) illustrate the activity of bacteria on mucosal surface.
3. Write an essay on microflora of skin, gills, and eggs of fish.
4. (a) What are the intestinal microflora of fish?
(b) Give an account of the development of intestinal
microflora and roles of intestinal microflora in fish.
5. (a) Give an outline of pathogenesis and describe its major
steps.
(b) Categorize fish pathogens.
Factors affecting the types and loads of microflora in fish include: Part-1
* Bacterial flora on different parts of live fish,
* Types of fish
* Effect of the environment:
Temperature
Tropical vs temperate water and/or warm water vs cold
water;
• Place (fresh water vs marine water),
Part-2
*Water quality (Clean vs polluted water),
* Seasonal variation,
* Media for culturing,
* Chemicals:
Ammonia, Oxygen, Toxic materials, Pesticides,
insecticides and herbicides
Bacterial flora on different parts of live fish:
Microorganisms are found on skin and gills and in the intestines of live and newly caught fish. The total number of organisms vary enormously ranged from 102 to 107 cfu cm-2 on the skin surface. The gills and the intestines both contain between 103 and 109 cfu g-1. Photobacterium phosphoreum normally surface flora can also be isolated in high numbers from the intestinal tract of some fish species.
The microflora in the slime of fish depends on the species of fish. Liston (1955, 1956) found markedly different bacterial loads on the skin, and gills of skate (a kind of ray-fish) when compared with sole caught in the same place at the same time.
The flora on the skin and gills may derive largely from the surrounding water, or from the bottom mud. Anaerobes e.g. Clostridium spp. are usually absent in slime and gills, but are always present in the gut. Clostridium botulinum naturally occurs in the marine environment. The pychrophilic types E, F and non-proteolytic B contaminate fish from localities.
Clostridium botulinum type E, involved in outbreaks of botulism and can grow at temperatures as low as 3.3°C. This type of organism could cause problems in aquaculture systems, especially in natural earth ponds.
Types of fish:
Indian shark showed no Pseudomonas even though it is largely present in the sea water. Remarkable variations have been observed between flatfish versus round fish, or cartilaginous fish versus bony fish. However, significant differences were found between similar hake caught on the
Gram-positive organisms as Bacillus, Micrococcus, Clostridium, Lactobacillus and coryneforms can also be found in varying proportions, but in general, Gram-negative bacteria dominate the microflora in water.
In tropical warmer waters, higher numbers of mesophiles can be isolated. Gram-positive Bacillus and Micrococcus dominate on fish from tropical waters. However, this conclusion has later been challenged by several studies which have found that the microflora on tropical fish species is very similar to the flora on temperate species. Species of Pseudomonas, Acinetobacter, Moraxella and Vibrio has been found on newly-caught fish in several Indian studies.
The microflora on tropical fish often carries a slightly higher load of Gram-positives and enteric bacteria but otherwise is similar to the flora on temperate-water fish.
Tropical species caught in water around 20 to 25°C tend to have largely mesophilic floras, which able to grow at 34-37°C. Counts as high as 106-107 g-1 have been obtained on shrimp directly out of the nets of trawlers in
Deep water mollusks such as scallops or queens usually have lower counts of E. coli (103 to 106 g-1 at 20°C) than those of estuarine species, e.g. oysters, cockles or mussels (103-108 cm-3).
The main groups of bacteria of crustacean shellfish are coryneforms, Species of Micrococcus, Achromobacter and Pseudomonas, Flavobacterium/Cytophaga and Bacillus. Cold-water shellfish include largely Pseudomonas and Achromobacter, while warmer waters include Micrococcus and coryneforms e.g. Indian prawns. Gulf shrimp contained largely Achromobacter, Micrococcus, Pseudomonas and Bacillus.
Place (Fresh vs marine water):
Aeromonas sp. is typical of freshwater fish, as well as typical of marine waters. These include species of Vibrio, Photobacterium and Shewanella. However, although Shewanella putrefaciens is
characterized as sodium-requiring; it can also be isolated from freshwater environment.
Marine fish from cold or temperate waters have been found to have skin counts at 20°C of 101-107 cm-2, gill counts of 103-107 g-1 and gut content counts of 103-108 cm-3.
Higher figures have been reported for Indian sardine (30-37°C). While 37°C counts give lower recoveries than 20°C counts for cold-water fish. For cold-water fish, counts at 0oC are almost as high as for 20°C. The microflora on the skin and gill surfaces of cold-water marine species consists largely of Gram-negative rods e.g. species of Pseudomonas, Moraxella and Acinetobacter, Flavnbacterium and Vibrio. The microflora on warm-water marine fish,
consists mainly Gram-positive bacteria e.g. Micrococcus, Corynebacteriurn, Brevibacterium and Bacillus in varying proportions.
Freshwater fish tend to have lower counts than marine species, viz. 102 to 105 cm-2 for skin and 101 to107 g-1 or ml-1 for gut content of temperate fish, and 103 to 105 cm-2 and 104 to 106 g-1 or ml-1 for skin and guts of tropical species.
The microflora of freshwater fish includes most salt-water genera, including Aeromonas, Lactobacillus, Alkaligenes, Streptococcus and Enterobacteriaceae. Warm-water fish often have large numbers of coryneforms and Salmonella
Freshwater, immature fish had 100% terrestrial bacteria in the gut, while transplant fish in 50% sea
water had a 49/51% ratio of terrestrial to marine types. Freshwater fish are contaminated by faecal organisms and pathogens than marine fish. Higher contamination of fish was recorded during caught in areas close to sewage outfalls or other polluted streams.
In
Selective questions:
- Write down the bacterial flora on different parts
of live fish.
2.Elucidate the effect of the environment on
microflora of fish.
3.What are the microflora in fresh water and marine water fish?
Seasonal variation:
Evidence suggests that bacterial loads on skin slime and gills of fish show seasonal variations linked with changes in environment.
Skate (a ray fish), and cod (Sea-fish, a source of cod liver oil) showed two peaks, in late spring and autumn, following on plankton blooms. On the other hand, a three-year study of the skin flora of hake (a kind of cod fish) caught within a 15-hour fishing, showing considerable variations in both numbers and types of microorganisms.
For example-1, the dominant genus found on freshly caught hake (Sea-fish, related to cod fish) in 15 out of the 17 catches examined in one study was Pseudomonas, whereas a few years previously Achromobacter spp. was found to predominate. The major genus in cod has been variously quoted as Pseudomonas, Achromobacter, or Micrococcus. Some examples are given below.
Example-2:
A study on seasonal quantitative and qualitative analyses of the bacterial flora associated with the intestine of hybrid tilapia cultured in earthen ponds in Saudi Arabia showed total viable counts (cfu g-1) of bacteria in the intestine varied between 6.8±1.9×106 to 7.5±1.4×107 in early summer, 1.6±2.0×106 to 5.1±2.5×107 in summer, 3.1±1.4×108 to 1.3±2.2×109 in autumn, and 8.9±1.8×105 to 1.3±0.9×107 in winter. Altogether, 17 bacterial genera were identified from the intestine of tilapia. The bacteria were predominantly Gram-negative rods (77%). Aeromonas hydrophila, Shewanella putrefaciens, Corynebacterium urealyticum, Escherichia coli and Vibrio cholerae were the most abundant species with a prevalence of >10% in most cases except V. cholerae. Considerable numbers of Pseudomonas spp. were found only in winter. Photobacterium damselae, and species of Pasteurella, Cellulomonus and Bacillus were present in some seasons of the year.
Example-3:
The seasonal bacterial flora of fish ponds were quantitatively and qualitatively examined atquarterly intervals for one year from April 2001 to March 2002 for the first time in Saudi Arabia showing total viable count (cfu ml-1) of bacteria in pond water ranged from 7.8±0.9 x 103 to 1.3±1.1 x 104 in Summer (33.0±2.3oC); 5.1±1.7 x 103 to 2.2±1.0 x 104 in Fall (24.1±1.9oC); and 6.7±2.1 x 102 to 2.5±0.6 x 103 in Winter (14.5 ±1.5oC). In total, 21 different species of bacteria from 19 different genera were isolated. The predominant microflora consisted of Gram-negative rods.
Aeromonas hydrophila, Shewanella putrefaciens, Corynebacterium urealyticum, and Escherichia coli were the most prevalent bacteria in all seasons. Species of Flavobacterium and Pseudomonas were dominant only in the winterPasteurella spp. were also consistently isolated throughout the sampling period. Bacteria present seasonally were species of Pseudomonas, Flavobacterium, Cellulomonus, and Micrococcus. Ambient seasonal temperature variation could account for some of the bacterial population variation. Presence of fecal coliform bacteria in the fish-culture waters suggests that care should be practiced during processing the fish caught from these waters to prevent contamination of edible meat.
Example-4:
Seasonal variation in the number of halophilic histamine-forming bacteria on/in marine fish and changes in their number on marine fish stored at25oC were studied. Psychrophilic halophilic histamine forming bacteria were detected from viscera and skin of fish throughout one year. Besides these mesophilic halophilic histamine-forming bacteria were isolated from mackerel during May and July, and from horse mackerel (a sea fish) in June, July, September, and November. The occurrence of these bacteria showed similar seasonal variations to that in sea water reported earlier by the same group of investigators. During storage of mackerel at 25oC, both types of halophilic histamine- forming bacteria increased in the fish muscle, they could be detected at a cell count of 101 to 103 g-1 after 16 h of storage. Histamine concentration in the muscle after 24 h increased to become 70-120 mg g-100. This suggested that these new mesophilic halophilic histamine-formers were responsible for the scombroid poisoning.
Medium:
The numbers and types of bacteria recovered can also be influenced by the medium used and the incubation temperature. There was a debate whether marine bacteria are really psychrophilic or even truly marine. Zobell (1946) expressed that marine bacteria are not psychrophiles because their range of optimum growth temperatures is between 18°C and 22°C. But Jay (1978) defined that marine bacteria are psychrophiles capable of growing at temperatures
between 0°C and 7°C.
Many marine microorganisms will grow readily at -3°C and some strains will grow as low as -7.5°C. They can survive almost indefinitely at -3°C to 5°C, though many of them die rapidly at 37°C. Marine bacteria grow better on sea-water-based (SWB) media than on those based on fresh tap water.
More recent studies have suggested that there is little difference in the counts obtained whether or not sea water is used in the media. The proportions of the different genera are, however, affected by the medium used.
Cape hake stored in ice, showed no significant difference was found between total counts in SWB medium or in distilled water based (DWB) medium with 0.5% sodium chloride, at either 37°C or 20°C. Incubation temperature at 37°C showed higher counts in SWB medium than the other. But the counts in SWB medium at 20°C were from 10 to over 80 times greater than those at 37°C. The proportions of the different genera recovered were affected by both the incubation temperature and the medium used. Whereas, Micrococcus was more prolific at 37°C and Bacillus was recovered only at 37°C. Flavobacterium was recovered in small numbers, only in DWB medium at 37°C. This probably reflects the general scarcity of Flavo-bacterium as a component of the microflora of
Growth of Achromobacter, Pseudomonas and Corynebacterium was greater at 20°C, while Micrococcus and Brevibacterium were more prolific at 25°C. The preference of Achromobacter and Pseudomonas for lower temperature, or their relatively greater ability to grow in the cold than other organisms is noted at 0°C and even lower.
Water quality (Clean and polluted water):
Water quality is one of the primary factors affecting the spread of parasites and diseases. Many abnormal behaviors exhibited by fish can be attributed to poor water quality.
Upon determining that the fish has a problem, the first thing to suspect is to be water quality.
It is generally accepted that the flesh of freshly caught healthy fish is sterile. The skin and gills, however, may carry high loads of bacteria.
In polluted waters, high numbers of Enterobacteriaceae may be found. In clean temperate waters, these organisms disappear rapidly, but it has been shown that Escherichia coli and Salmonella can survive for very long periods in tropical waters and once introduced may almost become indigenous to the environment (Table 1).
Table 1. Bacterial flora on fish caught in clean, unpolluted
waters
| Gram-negative | Gram-positive |
| Pseudomonas | Bacillus |
| Moraxella | Clostridium |
| Acinetobacter | Micrococcus |
| Shewanella putrefaciens | Lactobacillus |
| Flavobacterium | Coryneforms |
| Cytophaga | |
| Vibrio, | |
Japanese studies have shown very high numbers of microorganisms in the gastrointestinal tract of fish than in the surrounding water.
Mollusks caught in unpolluted waters probably have floras similar to those of crustaceans or fish living in the same area. Certain shellfish, however, when harvested from waters subject to contamination, can become a health hazard. Oysters and other bivalves allow passing of large quantities of water over a filter system during feeding that can carry with them organisms of faecal origin. Mollusks grown in sewage-contaminated water can be dangerous. The large outbreak of food poisoning due to oysters in
Apart from contamination by pollution, shellfish can carry food poisoning organisms of marine origin.
C. botulinum has already been mentioned; another widespread species is V. parahaemolyticus. This organism found on fish or shellfish, has been responsible for about 40% of the incidents of food poisoning in
A further class of microorganism causing food poisoning in filter-feeding shellfish is the dinoflagellates (Gonyaulex and Gymnodinium). These could produce the most potent human toxins. Moulds, yeasts and viruses do not play a large partin the microbiology of fish. Rhodotorula yeasts are occasionally responsible for pink discoloration in oysters.
Food poisoning microbes are rarely found in fish caught in unpolluted waters and spoilage of fish is caused by their naturally occurring microflora. These are generally more or less psychrophilic and are capable of growing down to 0°C and, for some strains, several degrees lower.
Fish caught in very cold, clean waters carry the lower numbers whereas fish caught in warm waters have slightly higher counts. Very high numbers, i.e., 107 cfu cm-2 are found on fish from polluted warm waters.
Chemicals:
Ammonia
75% of the total ammonia present in a pond is from one of the bi-products of fish respiration. Ammonium (NH4), is the ionized form of ammonia. If the pH of the pond water is acid, the ammonium molecule remains intact and non toxic. If the pH of the pond water is alkaline, the ammonium molecule releases one hydrogen ion and becomes ammonia (NH3), the non ionized form. Ammonia is toxic to your fish. The amount of toxicity depends on how alkaline the water is. As pH increases above 7, the amount of ammonium transformed into ammonia is exponentially related to the pH.
Oxygen
Oxygen is needed for the normal day to day functions of a fish and by the bacteria necessary for the breakdown of the fish's waste products in the nitrification process. Factors affecting the amount of oxygen in the water are temperature, fish load, organic load, medications, and the turn over rate. All of these factors affect oxygen inversely except the turn over rate. Minimum levels of oxygen should be 5 PPM.
Toxic Metals
Most natural waters contain chloride, sulfate, carbon, calcium, magnesium, sodium and potassium. These ions serve a vital purpose in the mineral metabolism of all animals. If these ions are found in high concentrations, their toxicity is dependent on water hardness, pH, temperature and the presence of other dissolved substances.
The solubility and toxicity of zinc, lead, aluminum and copper have a direct relationship to increases of pH and water hardness.
Elevated levels of heavy metals can alter the qualitative and quantitative structure of microbial communities.
Pesticides & Insecticides & Herbicides
These are usually introduced into the water by runoff, precipitation or accidental spills, which make water changes. Water contaminated with these chemicals make water adverse for fish breeding resulting reduction of fish.
Use of antibiotics or dip
Antibiotics also have been tried experimentally, usually in a dip or in ice. Of those tested, , pediocin, nicin, natamycin, chlortetracycline and oxytetracycline seemed best, and now their use is permitted. Chloramphenicol is fairly effective, and penicillin, streptomycm, and subtilin are poor or useless.
Selective ouestion:
1.Elucidate seasonal variations of microflora in fish linked with changes in environment. Cite examples. (6.0)
2. The numbers and types of bacteria in fish can also be influenced by the medium used and the incubation temperature. Explain. (6.0)
3. Water quality is one of the primary factors affecting the spread of parasites and diseases in fish. Explain. (6.0)
4. Describe the effect of chemicals on microflora in fish. (3.0)
5. Describe fish-borne diseases and their causative agents.
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