THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.     

Bacteriological quality control in human milk-banking.

The bacteriological quality of pooled human milk donated to the Oxford milk bank was analysed and the effects on bacteriology of sterilisation of the milk-collecting vessels in the home with hypochlorite solution and of Holder pasteurisation in a purpose-built human-milk pasteuriser were studied. Collecting milk in hypochlorite-sterilised vessels resulted in a significantly lower bacterial count of both pathogens and species of unlikely pathogenicity before pasteurisation and significantly increased the chance of pasteurisation giving a sterile product. Potentially pathogenic organisms grown in untreated milk were Escherichia coli, Staphylococcus aureus, and group B beta-haemolytic streptococci. Seven species of organisms of unlikely pathogenicity were also identified. Pasteurisation eliminated all potential pathogens from milk but did not reliably remove any of the species of unlikely pathogens. Banked human milk may be contaminated with bacteria which are known to be capable of producing lipases, proteases, and decarboxylases. Accurate pasteurisation, together with attention to the sterility of the collecting vessels, results in a bacteriologically safe product that retains many of the protective properties of raw milk.     

Collecting and banking human milk: to heat or not to heat?

Data on human breast milk and its handling when fed to babies who cannot be breast-fed were reviewed to determine whether the method of processing and storage affected the properties of the milk. Breast milk is normally contaminated by potential pathogens, which seem to produce no ill effects, but it also contains antimicrobial properties which protect against infection. The evidence suggests that pasteurisation not only eliminates pathogenic bacteria but also damages bacteriostatic mechanisms, so making the milk more susceptible to later contamination. Pasteurisation also affects the nutritional properties of milk. Freezing has little effect on milk proteins, while a study on the effect of refrigeration showed that there was little bacterial growth at temperatures below 8 degrees C. Several years'' experience of feeding donated raw milk to newborn infants has confirmed that it produces no ill effects. These findings suggest that pasteurisation of donated breastmilk is unnecessary, and it is not recommended, while the decision whether or not to freeze the milk may be made on practical grounds. Raw breast milk can be safely stored at 4-6 degrees C for 72 hours.     

Selective oxidation of primary alcohol groups of beta-cyclodextrin mediated by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)

Beta-cyclodextrin (beta-CD) was reacted with catalytic amounts of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), sodium hypochlorite and sodium bromide at 2 degrees C and a pH value of 10 in water. The primary alcohol groups were selectively oxidized into carboxylate groups within a few minutes, and mono- and dicarboxy-beta-cyclodextrin sodium salts were isolated and characterized by 1H, 13C NMR and mass spectroscopy. With this reaction system, the degradation of the cyclodextrin was limited, provided the oxidation was performed at 2 degrees C, at constant pH value of 10, with catalytic amounts of TEMPO and controlled quantities of sodium hypochlorite and sodium bromide for the continuous regeneration of the oxoammonium salt.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Microbial Destruction by Low Concentrations of Hypochlorite and Iodophor Germicides in Alkaline and Acidified Water

Hypochlorite and iodophor germicides were evaluated for their ability to destroy a variety of organisms at levels approximating those used for final sanitizing rinse for dairy and food equipment and beverage bottles (3 to 50 ppm). Test organisms included Escherichia coli, Streptococcus lactis, Lactobacillus plantarum, Pediococcus cerevisiae, and Saccharomyces cerevisiae. The hypochlorites and iodophors demonstrated approximate rates of destruction at equivalent concentrations for the bacterial species tested, except where the hypochlorite contained excess alkalinity. The hypochlorite responded more readily to a downward shift to a pH of 5.0 than did the iodophor. Excess alkalinity of the hypochlorite significantly affected its bactericidal activity. The iodophor exhibited a consistently greater rate of destruction of yeast cells than the hypochlorite. Successive treatment with low levels of iodophor (6 ppm) followed by a hypochlorite (12 to 25 ppm) resulted in a high level of destruction of all test organisms. Possibilities for employing these measures in a sanitizing rinse of bottles for maximal destruction of organisms were discussed. Among the test organisms, S. lactis showed a comparatively high resistance and was a useful organism for comparing the halogen preparations.     

Multiprofessional training for breastfeeding management in primary care in the UK

Upon hospital discharge it is not unusual for mothers of preterm infants to continue to meet all or most of their infants' nutritional needs through bottle feedings of expressed breast milk (EBM) because of infants' physiological immaturity and maternal concerns with an inadequacy of milk supply. Although for some mothers the challenge of transitioning the infant to feeding at the breast may be beyond their ability and resources, for others it appears to be based on a conscious choice. Mothers are often unaware of the advantages of breastfeeding at the breast. The purpose of this article is to examine some of the factors that may contribute to the inability and resistance of mothers to transition their preterm infants, and to report on the potential short and long-term advantages associated with feeding at the breast as opposed to feeding bottles of EBM.     

A flow-cytometric gram-staining technique for milk-associated bacteria

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50 degrees C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18 degrees C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5 degrees C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.     

Antimicrobial activity of a nisin-activated plastic film for food packaging

AIMS: To determine the effectiveness of a packaging film coated with nisin to inhibit Micrococcus luteus ATCC 10240 in tryptone soya broth (TSB) and the microbiota of raw milk during storage. A further aim was to examine the release of nisin from the activated film. METHODS AND RESULTS: An active package, obtained from nisin-treated film, was filled with 1 l of M. luteus ATCC 10240 (ML) suspension in TSB and stored at 4 and 25 degrees C for 2 days. After 24 h at 25 degrees C there was a remarkable reduction of M. luteus ATCC 10240 compared with the control, while at 4 degrees C a slight reduction was observed. Moreover, microbial growth was controlled when 1 l of three different kinds of milk was poured into the active package and stored at 4 degrees C for 7 days. The most significant results were observed in raw milk and pasteurized milk with a reduction of 0.9 log and 1.3 log, respectively. The release experiments showed that nisin release from the film was unpredictable, but it was favoured by low pH and high temperature. CONCLUSIONS: It appears that nisin-coated films were effective in inhibiting M. luteus ATCC 10240 in TSB and the bacterial flora in milk, and the release of nisin was pH and temperature dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin-activated film may control bacterial growth, maintaining food quality, safety and extending the shelf-life of food products.     

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water

The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9-12 months. The plate counts in R2A medium incubated at 22 degrees and 37 degrees C were low initially, increasing to 10(4)-10(5) cfu/ml within a few days of bottling. The number of bacteria recovered at 22 degrees C from PVC bottles was fairly constant during the storage period, but the population isolated at 37 degrees C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis. Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.     

Available lysine content in human milk: stability during manipulation prior to ingestion

Available lysine content is an indicator of protein quality and nutritional value of milk. Many studies have examined the effects of extraction, treatment and storage of human milk upon its components, though no references are found regarding the possible changes in milk quality as defined by its content in essential amino acids such as lysine. The present study investigates the available lysine content in human milk and the variations in lysine resulting from milk manipulation as follows: (a) Cold storage (refrigeration at 4 degrees C for 48 hours, and frozen for 15 days at -20 degrees C); (b) Thermal treatment under conditions of low (Holder)(63 degrees C/30 minutes) and high pasteurization (75 degrees C/15 seconds). The results obtained show a decrease in milk lysine concentration after storage in both refrigerated and frozen samples. Pasteurization causes a highly significant loss of available lysine. The lysine losses were greater on applying low pasteurization versus the more gentle conditions of high pasteurization. CONCLUSIONS: While manipulation through cold storage or thermal treatment does not affect the protein content of human milk, its protein quality is modified. When human milk must be subjected to hygienization, it is preferable to apply high temperature treatment (75 degrees C, 15 seconds) than habitual pasteurization (63 degrees C, 30 minutes).     

Sensitivity of Escherichia coli O157:H7 to commercially available alkaline cleaners and subsequent resistance to heat and sanitizers

The effects of seven commercially available alkaline cleaners used in the food processing industry, 0.025 M NaOH, and 0.025 M KOH on viability of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 in logarithmic and stationary phases of growth were determined. Cells were treated at 4 or 23 degrees C for 2, 10, or 30 min. Cleaners 2, 4, 6, and 7, which contained hypochlorite and <11% NaOH and/or KOH (pH 11.2 to 11.7), killed significantly higher numbers of cells than treatment with cleaner 3, containing sodium metasilicate (pH 11.4) and <10% KOH, and cleaner 5, containing ethylene glycol monobutyl ether (pH 10.4). There were no differences in the sensitivities of logarithmic and stationary-phase cells to the alkaline cleaners. Treatment with KOH or NaOH (pH 12.2) was not as effective as four out of seven commercial cleaners in killing E. coli O157:H7, indicating that chlorine and other cleaner components have bactericidal activity at high pH. Stationary-phase cells of strain EDL 933 that had been exposed to cleaner 7 at 4 or 23 degrees C and strain FRIK 816-3 exposed to cleaner 7 at 23 degrees C had significantly higher D(55 degrees C) (decimal reduction time, minutes at 55 degrees C) values than control cells or cells exposed to cleaner 5, indicating that exposure to cleaner 7 confers cross-protection to heat. Cells of EDL 933 treated with cleaner 7 at 12 degrees C showed significantly higher D(55 degrees C) values than cells of FRIK 816-3, indicating that rpoS may play a role in cross-protection. Stationary-phase cells treated with cleaner 5 or cleaner 7 at 4 or 12 degrees C were not cross-protected against subsequent exposure to sanitizers containing quaternary ammonium compounds or sodium hypochlorite, or to cetylpyridinium chloride and benzalkonium chloride.     

Toxin production by Bacillus cereus dairy isolates in milk at low temperatures

A total of 136 strains of Bacillus cereus isolated from milk and cream were evaluated for toxin production based on HeLa S3, Vero, and human embryonic lung (HEL) cell cytotoxicity in vitro. HEL cell monolayers were more susceptible than the other two cell lines. The percentage of isolates exhibiting HEL cytotoxicity was similar (43.0 and 48.4%) when the strains were grown in brain heart infusion broth containing 0.1% glucose (BHIG) at 7 and 24 h, respectively, at 30 degrees C. In milk, only 21.8% of isolates showed HEL cytotoxicity at 7 h, and the number increased significantly to 73.2% at 24 h at 30 degrees C. Further, 102 toxin-positive isolates were acclimatized to grow at 8 degrees C in milk. Ninety-four (92.2%) of the strains produced HEL cytotoxicity of various degrees with no strict correlation to bacterial cell numbers and also elicited vascular permeability reaction in rabbit skin. Under aerated growth conditions (agitation, 200 rpm) B. cereus elicited cytotoxicity in BHIG and in milk at temperatures of 30, 15, and 8 degrees C. However, in nonaerated (stagnant) cultures toxin production was diminished (BHIG) or completely lost (milk) at all temperatures. Toxin production at 8 degrees C was evaluated in two different types of commercial cardboard milk packages by inoculation with a potent toxigenic dairy isolate. No detectable HEL cytotoxicity was observed in milk in any of the packages either at stagnant conditions or during mechanical shaking. However, the same strain produced cytotoxin in whipped cream at 8 degrees C.     

Toxin production by Bacillus cereus dairy isolates in milk at low temperatures.

A total of 136 strains of Bacillus cereus isolated from milk and cream were evaluated for toxin production based on HeLa S3, Vero, and human embryonic lung (HEL) cell cytotoxicity in vitro. HEL cell monolayers were more susceptible than the other two cell lines. The percentage of isolates exhibiting HEL cytotoxicity was similar (43.0 and 48.4%) when the strains were grown in brain heart infusion broth containing 0.1% glucose (BHIG) at 7 and 24 h, respectively, at 30 degrees C. In milk, only 21.8% of isolates showed HEL cytotoxicity at 7 h, and the number increased significantly to 73.2% at 24 h at 30 degrees C. Further, 102 toxin-positive isolates were acclimatized to grow at 8 degrees C in milk. Ninety-four (92.2%) of the strains produced HEL cytotoxicity of various degrees with no strict correlation to bacterial cell numbers and also elicited vascular permeability reaction in rabbit skin. Under aerated growth conditions (agitation, 200 rpm) B. cereus elicited cytotoxicity in BHIG and in milk at temperatures of 30, 15, and 8 degrees C. However, in nonaerated (stagnant) cultures toxin production was diminished (BHIG) or completely lost (milk) at all temperatures. Toxin production at 8 degrees C was evaluated in two different types of commercial cardboard milk packages by inoculation with a potent toxigenic dairy isolate. No detectable HEL cytotoxicity was observed in milk in any of the packages either at stagnant conditions or during mechanical shaking. However, the same strain produced cytotoxin in whipped cream at 8 degrees C.     

Inactivation of Bacillus anthracis spores by liquid biocides in the presence of food residue

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H(2)O(2)) for 10 min at 10, 20, or 30 degrees C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H(2)O(2) concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20 degrees C, the minimum concentrations of peroxyacetic acid, H(2)O(2), and NaOCl (as total available chlorine) predicted to inactivate 6 log(10) CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10 degrees C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log(10) CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H(2)O(2) sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log(10) CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.     

Effects of sample handling temperatures on bovine skim milk progesterone concentrations

The effects of incubation of whole milk at various temperatures and times on the concentration of progesterone in the skim milk fraction was determined. For the study, milk samples were collected from 10 pregnant Holstein cows. The milk from each cow was transferred to culture tubes to provide 32 replicates of 3 ml volume. To begin the incubation study, all samples were placed in a 37 degrees C water bath for 4 h. The end of this incubation was designated as time 0 and a sample from each cow was centrifuged to harvest skim milk. At time 0, samples from each cow were divided among incubation temperatures of 0 degrees, 4 degrees, 20 degrees and 37 degrees C. Samples were removed from each incubation group at 30, 60, 90 and 120 min. After 120 min, all remaining samples were returned to the 37 degrees C incubation and skim milk was collected at 30, 60 and 90 min. Progesterone was measured in skim milk by radioimmunoassay. The mean +/- SE concentration of progesterone in skim milk at time 0 was 10.9 +/- 1.1 nmol/L. The mean concentration of progesterone in skim milk was higher (P < 0.05) in all samples incubated at 0 degrees and 4 degrees C, with incremental increases ranging from 34% to 67% above time 0. Progesterone in skim milk returned to time 0 concentrations in milk samples transferred from 0 degrees or 4 degrees C to 37 degrees C. There was no change in skim milk progesterone in whole milk samples incubated at 20 degrees or 37 degrees C. From this study, it can be concluded that the concentration of progesterone in skim milk is temperature dependent. Inconsistency in handling whole milk samples can have a profound effect in the concentration of progesterone on skim milk. The temperature-dependent effect was reversible and may be related to solubility of progesterone in milk fat.     

Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions

The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures.     

Einwirkung des immobilisierten Präparates „Sirenin”︁ aus Bacillus mesentericus auf Milch als Substrat

Comparative investigations for the possibilities of milk coagulation by the action of soluble and immobilized (on DEAE-cellulose by adsorption) bacterial enzyme preparation “syrenin” in periods from 30–120 minutes at 5°C are carried out. The milk clotting activities of both preparations are equalized after 40 minutes. The laboratory experiments for producing a bulgarian white brine cheese by the immobilized “syrenin” show a tendency to decrease the losses of substance into the whey and to increase the curdle yields in comparison with these made by soluble preparation.     

In vitro conidial germination in Arthrinium aureum and Arthrinium phaeospermum

This paper describes the microscopic details of conidial germination and the influence of pH, sodium chloride concentration, 3% glucose, 3% saccharose and ultraviolet irradiation on the conidial germination in Arthrinium species. Under laboratory conditions, germination started after an incubation period of 90 minutes in 2% malt extract broth at 25 degrees C. In vitro, the conidia of Arthrinium species have a very low percentage of germination (A. phaeospermum: 7.9%; A. aureum: 15.8%). Conidia of this genus have a characteristic equatorial slit. Conidia may break spontaneously at this slit, releasing their cytoplasmic contents. Arthrinium phaeospermum attains its optimum germination percentage when its conidia are suspended in a sterile saline solution (pH 3.5) in a water bath at 20 degrees C for 15 minutes before being inoculated on 2% malt extract agar. Conidial suspensions of A. aureum may be held in the same conditions, but for 30 minutes.     

The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): II. The effect of human milk and infant formula preparations on binding of Clostridium perfringens to epithelial cells

Breast feeding is known to protect an infant against gastrointestinal pathogens and epidemiological studies indicate that compared to breast fed infants, formula fed infants are at a greater risk of dying from sudden infant death syndrome (SIDS). Many SIDS infants have symptoms of gastrointestinal infections prior to death and one gastrointestinal pathogen associated with SIDS is Clostridium perfringens. Studies have found that a significantly higher number of formula fed SIDS infants have C perfringens and its enterotoxin in their faeces compared to breast fed infants. The aim of the study was to compare the effects of human milk and infant formula on binding of C perfringens to epithelial cells. Two protocols were used to assess the effect of human milk and infant formula to inhibit binding of C perfringens to epithelial cells. Binding was assessed by flow cytometry. For the in vivo protocol which more closely represents interactions on the mucosal surface, breast milk enhanced bacterial binding but infant formula caused inhibition of binding; however for the in vitro method, both human milk and infant formula resulted in consistent enhancement of binding. Flow cytometry studies indicated that enhancement of binding was due to the formation of bacterial aggregates. Lewis(a) and Lewis(b) antigens, found in both breast milk and infant formula, inhibited C. perfringens binding in a dose dependent manner. The Lewis(a) and Lewis(b) antigens in human milk and infant formula can inhibit C. perfringens binding to epithelial cells. While infant formula reduced binding of C. perfringens to epithelial cells in the experiments carried out with the in vivo protocol, the protective effects of breast feeding in relation to colonisation with C. perfringens are more likely to be due to formation of bacterial aggregates. These findings have implications for improving infant formula preparations.