Adherence as a method of differentiating virulent and avirulent strains of Vibrio parahaemolyticus.

Usually only Kanagawa-positive strains of Vibrio parahaemolyticus are considered virulent; yet, a significant portion of V. parahaemolyticus food poisonings appear to be caused by Kanagawa-negative strains. Therefore, additional and more accurate measurements of a strain's food-poisoning potential are needed. Adherence of V. parahaemolyticus to human fetal intestinal (HFI) cells in vitro seems to offer this information. All strains of V. parahaemolyticus adhered to the HFI cells, but the degree of adherence was related to a number of factors. These included the age of the culture, the strain's Kanagawa reaction and source, the length of time the bacteria were exposed to the HFI cells, and the composition of the growth medium. Cells harvested during the late log phase of growth adhered more intensely than those harvested from the late stationary phase. Virulent strains, i.e., those involved in food poisoning, were observed to have a high adherence ability regardless of their Kanagawa reaction, whereas Kanagawa-negative strains isolated from seafood exhibited weak adherence intensities. Kanagawa-positive strains isolated from seafood adhered strongly to the HFI cells. The difference between the virulent and avirulent strains was quantitative in nature, and the greatest differences in adherence intensities were observed after short (10 to 15 min) exposure times. The presence of ferric iron in the growth medium was found to increase the adherence intensities of the virulent strains.

Incidence of Vibrio parahaemolyticus in and the Microbiological Quality of Seafood in North Carolina 1.

Microbiological analyses of 716 seafood samples over a 3-year period revealed that the microbiological quality of fresh seafood in North Carolina was generally acceptable. The mean aerobic counts (APC) and fecal coliform counts were low as was the occurrence of enteric pathogens, except for Vibrio parahaemolyticus and coagulase-positive staphylococci. Salmonella species were isolated from three samples, but the fecal coliform counts of these samples far exceeded the shellfish standard of the United States Food and Drug Administration. Coagulase-positive staphylococci were isolated in low numbers from nearly all the different types of seafoods; unpasteurized crabmeat and head-peeled shrimp samples showed the highest counts (10% of these samples had numbers which exceeded 100/g). V. parahaemolyticus occurred frequently in fresh seafood (overall 46% of the samples were positive) and its numbers showed a definite seasonal variation. No positive statistical correlation was found between the numbers of V. parahaemolyticus and the bacteriological indices, such as coliforms, fecal coliforms, enterococci and APC. Processing practices were found to influence the occurrence of V. parahaemolvticus in seafood; for example, improperly cleaned flumes were found to be a reservoir for V. parahaemolyticus in mechanical scallop processing plants. Also 'picking' waste 'containers' were found to be sources of V. parahaemolyticus in crab processing plants. Some processing practices such as heat shocking of oysters to facilitate opening were found to reduce the numbers of V. parahaemolyticus . Fifty V. parahaemolyticus isolates from different seafoods were tested for their Kanagawa reaction and all were found to be negative.

Preparation of Lactobacilli for Dietary Uses.

Increased use of lactobacilli as dietary supplements has emphasized the need for preparations of those cultures that possess the attributes of cell viability. resistance to bile and capability of establishing in the intestinal tract. Incompatibility of different species of lactobacilli may limit development of products designed to overcome objectionable, or impart improved flavor to cultured foods. Stresses that are imposed on cultures during their preparation and distribution must minimize damage to cells which would detract from the foregoing characteristics.

Lactase and Starter Culture Survival in Heated and Frozen Yogurts 1.

Yogurt customarily contains large numbers of viable starter culture, although some yogurt is heat-treated to inactivate the starter. It was found that heating to inactivate the starter also inactivated lactase. This could have a disadvantageous effect on lactose-intolerant consumers. Manufacture of frozen yogurt did not have any appreciable damaging effect on the yogurt culture or lactase activity.

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.

Plating procedure for the enumeration of coliforms from dairy products.

A "repair-detection" procedure consisting of pour plating of food samples with Trypticase soy agar, followed by 1-h repair incubation at room temperature and subsequent overlay with violet red bile agar, was found to be an effective method for the detection of injuried and uninjuried coliforms from dairy products. This method was relatively less effective for the detection of coliforms in many semipreserved foods as compared with dairy products, but more effective than the most-probable-number method.

Enumeration of Vjable Lactobacillus acidophilus Organisms in Dairy Products.

Pasteurized low fat milk to which is added Lactobacillus acidophilus has now become a product of major volume on the U.S. market. The product contains millions of L. acidophilus per ml and is considered to be in the general category of cultured milk products for application of regulatory monitoring. There is a growing interest in developing standards for allowable minimum numbers of L. acidophilus in such products at point of sale. This can be accomplished best by use of a medium selective for lactobacilli such as Lactobacillus Selection Agar (LBS); non-selective media, such as APT agar, can also be used to enumerate L. acidophilus . The latter would provide less assurance that the counts were due to lactobacilli. The non-selectivity of APT, however, usually results in higher counts of L. acidophilus . There is need for application of accurate monitoring of such products to ensure the numbers as well as the identity of the culture used as the milk supplement.

Use of the Minitek system for characterizing lactobacilli.

Incubation of inoculated substrates of the Minitek system in anaerobic GasPak jars provided a method that produced results comparable to those of conventional tubed media for characterizing species of Lactobacillus. The use of sterile mineral oil to overlay inoculated substrate disks was responsible for erroneous results.

Deconjugation of bile acids by intestinal lactobacilli.

Lactobacillus species normally found in the intestinal tract of humans varied in the ability to deconjugate bile acids, whereas laboratory strains of Lactobacillus acidophilus deconjugated both glycocholate and taurocholate. All isolates of L. acidophilus from human feces deconjugated taurocholate, whereas only one of six deconjugated glycocholate. None of 13 isolates identified as L. casei deconjugated taurocholate, whereas 9 deconjugated glycocholate. The deconjugating system of L. acidophilus appeared to be constitutive, required low oxidation-reduction potential, and was most active at pH 6. No degradation beyond deconjugation was detected.

Effects of Freezing and Storage on Microorganisms in Frozen Foods: A Review.

The fate of bacteria contained in food during freezing, storage, and thawing is usually one that is detrimental. However, many microorganisms considered to have been killed by such treatments actually are only injured. Their viability can be determined by allowing the injury to repair in a non-selective medium before testing for their presence on selective media. Injured cells of pathogens have been found to be as pathogenic as uninjured ones; injured spoilage microorganisms can cause spoilage if permitted to repair and grow. Injured microorganisms and their potential importance in food safety and shelf-life constitute and important problem for the frozed food industry as well as the food sanitarian.

Antagonistic Action of Lactobacillus acidophilus Toward Intestinal and Foodborne Pathogens in Associative Cultures 1.

Lactobacillus acidophilus exerted antagonistic actions on growth of Staphylococcus aureus , Salmonella typhimurium , enteropathogenic Escherichia coli , and Clostridium perfringens when grown with each in associative cultures. S. aureus and C. perfringens were more sensitive to the inhibition than were S. typhimurium and E. coli . The amount of the antagonism produced varied among strains of L. acidophilus and could not be directly related to amounts of acid produced; hydrogen peroxide produced by the lactobacilli appeared to be partially responsible for the antagonistic interaction. The inhibitory effect was produced also under anaerobic conditions in a pre-reduced medium.

Enumeration and Identity of Lactobacilli in Dietary Products 1.

A medium was developed for selectively enumerating bile-resistant lactobacilli by adding 0.15% oxgall to lactobacillus selective agar (BBL). Lactobacilli were not found in many products sold as sources of Lactobacillus acidophilus , whereas others contained widely different numbers of lactobacilli capable of forming colonies on the bile medium. Of seven products from which lactobacilli were isolated, only three contained L. acidophilus . One of these products was from a pharmacy and the other two were from dairy processor(s).

Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at ultrahigh temperature.

The heat-stable extracellular protease of Pseudomonas sp. (isolate MC60) was investigated. Heat resistance of the enzyme in milk at sterilization temperature was dependent on the presence of Ca2+. The half-life of the enzyme at ultrahigh temperature (149 C) in skim milk or milk-salts buffer with Ca2+ was approximately 7.0 s. Treatment of milk with chelators completely removed the heatstabilizing effect of milk. The enzyme was partially purified by ammonium sulfate precipitation and column chromatography on Sephadex G-100. At 21 C the enzyme retained greater than 85% activity after exposure to pH values between 5 and 10. Enzyme activity was reduced by metal chelating agents. Both Ca2+ and Zn2+ were required for optimal enzyme activity. Molecular weight was estimated at 48,000 by gel filtration.

Effect of psychrotrophic bacteria from raw milk on milk proteins and stability of milk proteins to ultrahigh temperature treatment.

The effects of psychrotroph growth in raw milk on proteins of mils and on the response of milk proteins to heat treatments with ultrahigh temperature were studied. Ten gram-negative psychrotrophs isolated from raw milk readily attacked raw milk proteins. Kappa- and beta-casein were most susceptible although some of the isolates also attacked the whey proteins. Detectable proteolysis did not require large psychrotroph populations. A 10 to 20% decrease in kappa-casein during 2 days at 5 C accompanied growth of one isolate to a population of only 10,000/ml. Growth of psychrotrophs in raw milk predisposed the proteins to deleterious effects of ultrahigh temperature treatments. Ultrahigh temperature treatment by direct steam injection had little effect on raw milk caseins and decreased alpha-lactalbumin and beta-lactoglobulin by 21% and 34%, respectively. Milk that had undergone proteolysis exhibited decreased detectable kappa-, beta-, and alphas-caseins and increased loss of beta-lactoglobulin as a result of ultrahigh temperature treatment. Milk suffering extensive kappa-casein degradation coagulated during ultrahigh temperature treatment. Coagulation during or shortly after heating increased with severity of heat treatment and size of psychrotroph population.

Interactions among lactobacilli and man.

Lactobacilli and other lactic acid bacteria have been involved intimately with man for centuries. The associations have involved manufacture of various human foods as well as performance of various beneficial interactions in different parts of the human body. Renewed interests in the role of the intestinal microflora again have focused on the intestinal lactobacilli, particularly Lactobacillus acidophilus.

Detection of Lactobacillus acidophilus in feces of humans, pigs, and chickens.

Lactobacilli in fecal material from humans, pigs, and chickens were enumerated on lactobacillus selective agar (LBS). In all samples, higher numbers of lac-tobacilli were detected when plates were incubated in a system flushed with CO2 rather than in air. Much higher numbers of bacteria from human feces were detected when the LBS agar plates were incubated anaerobically in a hydrogen-carbon dioxide atmosphere (GasPak) than when incubated in CO2. The bacteria from human feces isolated on LBS agar incubated anaerobically were predominately bifidobacteria. Cultures from all three sources isolated on LBS agar incubated under CO2 were lactobacilli, including Lactobacillus acidophilus. Differences were observed in biochemical characteristics of some of the L. acidophilus isolated from all three sources. Guanine plus cytosine base ratios of deoxyribonucleic acid isolated from L. acidophilus cultures from humans were lower, in most cases, than those from pigs and chickens.

Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.

Production of heat resistant proteases by psychrotrophs growing in milk, resistance of such proteases to ultrahigh temperature treatments and action of these enzymes on milk were studied. All of the psychrotrophs obtained from raw milk produced proteases that survived 149 C for 10s. Seventy to ninety percent of the raw milk samples contained psychrotrophs capable of producing heat resistant proteases. The protease chosen as a model was resistant to heat treatments at 110 to 150 C, and the inactivation parameters suggested that thermal destruction of heat resistant proteases would damage the milk severely. The casein content and pH of normal milk were suitable for protease action, and the protease was quite active at normal and elevated room temperatures. The protease rapidly spoiled sterile milk with the development of bitter flavor, clearing, or coagulation; and the susceptibility of sterile milk to protease increased during storage of the milk.

Repair and enumeration of injured coliforms by a plating procedure.

Surface plating of coliforms on Trypticase soy agar, followed by 1 to 2 h of incubation at 25 C and subsequent overlay with violet red bile agar, was found to be a useful method for the repair and enumeration of coliforms injured by freezing.