Antilisterial and antioxidant exopolysaccharide from Enterococcus faecium PCH.25 isolated from cow butter: characterization and probiotic potential.

Exopolysaccharides produced by lactic acid bacteria have gained attention for their potential health benefits and applications in functional foods. This study explores the isolation and characterization of a novel exopolysaccharide-producing strain from dairy products. The aim was to evaluate its probiotic potential and investigate the properties of the produced exopolysaccharide. A strain identified as Enterococcus faecium PCH.25, isolated from cow butter, demonstrated exopolysaccharide production. The study's novelty lies in the comprehensive characterization of this strain and its exopolysaccharide, revealing unique properties with potential applications in food, cosmetic, and pharmaceutical industries. The E. faecium PCH.25 strain exhibited strong acid tolerance, with a 92.24% viability rate at pH 2 after 2 h of incubation. It also demonstrated notable auto-aggregation (85.27% after 24 h) and co-aggregation abilities, antibiotic sensitivity, and absence of hemolytic activity, suggesting its probiotic potential. The exopolysaccharide produced by this strain showed bactericidal activity (MIC and MBC = 1.8 mg/ml) against Listeria monocytogenes and antioxidant properties (22.8%). Chemical analysis revealed a heteropolysaccharide composed of glucose and fructose monomers, with various functional groups contributing to its bioactivities. Physical characterization of the exopolysaccharide indicated thermal stability up to 270 °C, a negative zeta-potential (-27 mV), and an average particle size of 235 nm. Scanning electron microscopy and energy dispersive X-ray analysis revealed a smooth, nonporous structure primarily composed of carbon and oxygen, with an amorphous nature. These findings suggest that the exopolysaccharide from E. faecium PCH.25 has potential as a natural antibacterial and antioxidant polymer for use in functional foods, cosmetics, and pharmaceuticals.

Study of the bacterial profile of raw milk butter, made during a challenge test with Listeria monocytogenes, depending on cream maturation temperature.

Bacteria can play different roles and impart various flavors and characteristics to food. Few studies have described bacterial microbiota of butter. In this study, next-generation sequencing was used to determine bacterial content of raw milk butter, processed during a challenge test, depending on cream maturation temperature and on the presence or not of L. monocytogenes. Two batches were produced. pH and microbiological analyses were conducted during cream maturation and butter storage. DNA was also isolated from all samples for 16S rRNA amplicon sequencing analysis. For butter made from cream matured at 14 °C, a growth potential of L. monocytogenes of - 1.72 log cfu/g was obtained. This value corresponds to the difference between the median of counts at the end of storage and the median of counts at the beginning of storage. This butter (pH value of 4.75 ± 0.04) was characterized by a dominance of Lactococcus. The abundance of Lactococcus was significantly higher in inoculated samples than in control samples (p value < 0.05). Butter made from cream matured at 4 °C (pH value of 6.81 ± 0.01) presented a growth potential of 1.81 log cfu/g. It was characterized by the abundance of psychrotrophic bacteria mainly Pseudomonas. This study demonstrated that cream maturation temperature impacts butter microbiota, affecting thus product's characteristics and its ability to support or not the growth of pathogens like L. monocytogenes.

Longer Contact Times Increase Cross-Contamination of Enterobacter aerogenes from Surfaces to Food.

Bacterial cross-contamination from surfaces to food can contribute to foodborne disease. The cross-contamination rate of Enterobacter aerogenes on household surfaces was evaluated by using scenarios that differed by surface type, food type, contact time (<1, 5, 30, and 300 s), and inoculum matrix (tryptic soy broth or peptone buffer). The surfaces used were stainless steel, tile, wood, and carpet. The food types were watermelon, bread, bread with butter, and gummy candy. Surfaces (25 cm2) were spot inoculated with 1 ml of inoculum and allowed to dry for 5 h, yielding an approximate concentration of 107 CFU/surface. Foods (with a 16-cm2 contact area) were dropped onto the surfaces from a height of 12.5 cm and left to rest as appropriate. Posttransfer, surfaces and foods were placed in sterile filter bags and homogenized or massaged, diluted, and plated on tryptic soy agar. The transfer rate was quantified as the log percent transfer from the surface to the food. Contact time, food, and surface type all had highly significant effects (P < 0.000001) on the log percent transfer of bacteria. The inoculum matrix (tryptic soy broth or peptone buffer) also had a significant effect on transfer (P = 0.013), and most interaction terms were significant. More bacteria transferred to watermelon (∼0.2 to 97%) than to any other food, while the least bacteria transferred to gummy candy (∼0.1 to 62%). Transfer of bacteria to bread (∼0.02 to 94%) was similar to transfer of bacteria to bread with butter (∼0.02 to 82%), and these transfer rates under a given set of conditions were more variable than with watermelon and gummy candy. IMPORTANCE: The popular notion of the "five-second rule" is that food dropped on the floor and left there for <5 s is "safe" because bacteria need time to transfer. The rule has been explored by a single study in the published literature and on at least two television shows. Results from two academic laboratories have been shared through press releases but remain unpublished. We explored this topic by using four different surfaces (stainless steel, ceramic tile, wood, and carpet), four different foods (watermelon, bread, bread with butter, and gummy candy), four different contact times (<1, 5, 30, and 300 s), and two bacterial preparation methods. Although we found that longer contact times result in more transfer, we also found that other factors, including the nature of the food and the surface, are of equal or greater importance. Some transfer takes place "instantaneously," at times of <1 s, disproving the five-second rule.

Influence of water activity on inactivation of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in peanut butter by microwave heating.

This study evaluated the efficacy of a 915 MHz microwave with 3 different electric power levels to inactivate three pathogens in peanut butter with different aw. Peanut butter inoculated with Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Listeria monocytogenes (0.3, 0.4, and 0.5 aw) were treated with a 915 MHz microwave with 2, 4, and 6 kW for up to 5 min. Six kW 915 MHz microwave treatment for 5 min reduced these three pathogens by 1.97 to >5.17 log CFU/g. Four kW 915 MHz microwave processing for 5 min reduced these pathogens by 0.41-1.98 log CFU/g. Two kW microwave heating did not inactivate pathogens in peanut butter. Weibull and Log-Linear + Shoulder models were used to describe the survival curves of three pathogens because they exhibited shouldering behavior. Td and T5d values were calculated based on the Weibull and Log-Linear + Shoulder models. Td values of the three pathogens were similar to D-values of Salmonella subjected to conventional heating at 90 °C but T5d values were much shorter than those of conventional heating at 90 °C. Generally, increased aw resulted in shorter T5d values of pathogens, but not shorter Td values. The results of this study can be used to optimize microwave heating pasteurization system of peanut butter.

Exploring microbial diversity in Kermanshah province's Kermanshahi oil through DGGE and sequencing analysis.

BACKGROUND: Ghee, known as "roghane heiwâni," or "Kermanshahi oil" is a traditional fermented butter-like product highly esteemed for its nutritional value. Ghee is prepared using traditional methods and has substantial potential as a reservoir of probiotic microorganisms. Previous research delved into isolating and identifying lactic acid bacteria (LAB) in Kermanshahi through culture and PCR sequencing. This study seeks to elucidate the microbial profiles and diversity within Kermanshahi using culture, Denaturing Gradient Gel Electrophoresis (DGGE), and sequencing methodologies. METHODS: Twenty samples of Kermanshahi oil were meticulously gathered from diverse locales across Kermanshah province. These samples were cultivated under specialized conditions in MRS and M17 environments spanning 24 to 72 h. Following DNA extraction, amplification of the 16SrRNA gene sequences was performed, culminating in sequencing for conclusive identification of the isolates. Furthermore, the DGGE technique was directly employed to separate and identify various species present in the oil samples utilizing bioinformatics software. RESULTS: Sequencing outcomes revealed a diverse array of microorganisms among the isolates, with Lactobacillus constituting 43%, Streptococcus comprising 27.6%, Enterococcus at 4.61%, and yeasts at 7.6%. Other species exhibited lower frequencies, encompassing Rhizobium, Bacillus coagulans, and Staphylococcus hominis. CONCLUSIONS: The isolation of a diverse spectrum of probiotic microorganisms underscores their potential utility in the realm of industrial dairy product production. These findings allude to the possibility of integrating these valuable microorganisms, which have historically been associated with traditional products, into the contemporary dairy industry. As consumer interest in probiotic-enriched products surges, the insights gained from this study pave the way for harnessing the benefits of Kermanshahi-derived probiotics.

Conditions associated with Clostridium sporogenes growth as a surrogate for Clostridium botulinum in nonthermally processed canned butter.

The objective of this study was to better understand the effect of butter composition and emulsion structure on growth and survival of Clostridium sporogenes, used as a surrogate for C. botulinum in canned butter. The lack of a thermal process step in commercially available canned butter raises questions of potential safety, because it is hermetically sealed and generally exhibits anaerobic growth conditions, which are optimal for Clostridium botulinum growth. Without thermal processing, low-acid canned foods must have inhibitory factors present to prevent C. botulinum growth. Some potential intrinsic inhibitory factors, or hurdles, within butter include: reduced water activity, acidity in cultured products, elevated salt content, and the micro-droplet nature of the aqueous phase in the butter emulsion. It was hypothesized that a normal, intact butter emulsion would have sufficient hurdles to prevent C. botulinum growth, whereas a broken butter emulsion would result in a coalesced aqueous phase that would allow for C. botulinum growth. Batch-churned butter was inoculated with C. sporogenes; butter samples with varying salt contents (0, 0.8, 1.6, and 2.4% wt/wt NaCl) were prepared and stored in coated steel cans for varying times (1 or 2 wk) and temperatures (22 or 41°C) to determine temperature and emulsion structure effects on C. sporogenes growth. Samples stored at 41°C showed a significant increase in C. sporogenes growth compared with those stored at 22°C. Furthermore, NaCl addition was found to have a significant effect on C. sporogenes growth, with 0.8% NaCl promoting more growth than 0%, but with decreases in growth observed at 1.6 and 2.4%. Uninoculated control plates were also found to have bacterial growth; this growth was attributed to other anaerobic bacteria present within the cream. It was concluded that removal of the hurdle created by the micro-droplet size of the emulsion aqueous phase could result in C. botulinum growth even at elevated salt levels and, therefore, home preparation of canned butter is not advisable. It is also possible that commercially canned butter, if heat abused, could potentially allow for C. botulinum growth and, therefore, consumption is not recommended.

Survival and heat resistance of Salmonella enterica and Escherichia coli O157:H7 in peanut butter.

Significant differences (P < 0.05) were found between the survival rates of Salmonella enterica and Escherichia coli O157:H7 in peanut butter with different formulations and water activity. High carbohydrate content in peanut butter and low incubation temperature resulted in higher levels of bacterial survival during storage but lower levels of bacterial resistance to heat treatment.

Transesterification of used edible and non-edible oils to alkyl esters by Aspergillus sp. as a whole cell catalyst.

Aspergillus sp. (MTCC 5436), isolated from contaminated clarified butter was used as a whole cell catalyst for transesterification of oils from different sources. The strain was observed to be tolerant and grow in 90% oil as carbon source. Oils of Jathropa, karanj and spent cottonseed were used as carbon sources in the study. The product, alkyl ester, was characterized and quantified using (1) H-NMR. The strain was observed to facilitate transesterification in an oil:minimal medium with the ratio of 70:30 resulting in a 98% conversion of oil to ethyl esters within 48 h at 28 °C and 120 rpm. The physico-chemical characteristics of the ethyl ester (>98%) at 70% oil as carbon source were similar to the standards specified for biodiesel as per standards of American Society for Testing and Materials (ASTM) and Bureau of Indian Standards (BIS), India.

Assessment of growth and survival of Listeria monocytogenes in raw milk butter by durability tests.

Butter is a complex matrix characterized by a high fat content. Existing publications on the behavior of Listeria monocytogenes in this type of food reported contrasted results. This study was performed to provide further information and data about raw milk butter's ability to support survival or growth of L. monocytogenes. Durability tests were performed on naturally contaminated samples of raw milk butter with various physico-chemical characteristics. At the end of shelf life, no growth of L. monocytogenes was observed in the studied butters, regardless of their physico-chemical characteristics (pH, aw, water dispersion index and salt concentration) and the initial level of contamination. The number of positive samples and the colony counts of L. monocytogenes were even decreased at the end of the storage period.

Bacterial and oxidative control of local butter with smart/active film based on pectin/nanoclay/Carum copticum essential oils/ß-carotene.

In this study, Pectin/Nanoclay (montmorillonite)/Carum copticum Essential oils/ß-Carotene (Pec/Clay/CCE/ßC) composite film was prepared. The effect of ß-Carotene (ßC) and Carum copticum Essential oils (CCE) concentration on the antioxidant activity and color/mechanical properties of Pec/Clay/CCE/ßC film was studied. The morphology and antibacterial activity of film were studied. The films containing maximum essential oil and ß-carotene showed the best antibacterial activity, antioxidant activity, flexibility and firmness. So the Pec/Clay/CCE0.5%/BC0.03% film as the optimum film was used for packaging of local butter. Oxidative stability, microbial count, and color properties (L*, a* and b*) of butter packaged with active film (Pec/Clay/CCE/ßC) were investigated. Results showed that Pec/Clay/CCE/ßC film was effective against Bacillus cereus (B. cereus) more than Escherichia coli (E. coli). According to the results active packaged butter had the highest oxidative stability, lowest microbial load, and the least color change during storage. In the packaging process, Pec/Clay/CCE/ßC film color was changed from orange to light yellow and this color change was used as a smart color indicator to detect the oxidation of butter and expiration time of butter.

Yeasts and filamentous fungi associated with some dairy products in Egypt.

This work aimed to identify and evaluate the diversity of yeasts and filamentous fungi that contaminate some dairy products (fresh buffalo milk, plain yoghurt, butter and Kareisk cheese) in Assiut city, Egypt. The identification was based on phenotypic characteristics, and genotypically in case of yeasts. The pHs of all dairy products lie within the acidic range, but yoghurt registered the highest value. A total of 41 genera and 89 species+3 varieties were recovered from all dairy products investigated on DRBC (26 genera representing 59 species+1 variety), DG18 (32 genera, 56 species+2 varieties) and MY50G (31 genera, 64 species+2 varieties). From these, filamentous fungi were represented by 21 genera and 59 species+2 varieties, and yeasts by 20 genera and 30 species+1 variety. In all products except yoghurt, the xerophilic media (DG18 and MY50G) supported more diversity of fungal species than the general medium (DRBC). Reversely, DRBC supported more propagules originated from all products than DG18 and DRBC. On the other hand, the highest numbers of propagules were recovered from Kareish cheese and butter on MY50G, while the lowest numbers were recorded from yoghurt and raw milk on MY50G. Moreover, yeasts constituted the greatest part of propagules from the four products on the three isolation media except the raw milk on MY50G. From yeasts, Candida, Cyberlindnera, Debaryomyces, Galactomyces, Kazachstania, Kluyveromyces, Myerozyma, Pichia, Rhodotorula, Trichosporon; and from filamentous fungi, only Aspergillus, Cladosporium, Mucor and Penicillium were found contaminating all dairy products. Possible medical problems related to the presence of these fungi or to their metabolic products are discussed.

Prevalence of Listeria monocytogenes and other Listeria species in butter from United Kingdom production, retail, and catering premises.

Two recent listeriosis outbreaks involving butter prompted this first cross-sectional study on the prevalence, levels, and types of Listeria species in 3229 samples of butter from production, retail, and catering premises in the United Kingdom during May and June 2004. When the criteria of the Microbiological Guidelines were used, 99.4% of samples were found to be of satisfactory microbiological quality, 0.5% were of acceptable quality, and 0.1% were of unsatisfactory quality as a result of high levels (>100 CFU/g) of Listeria spp. The butter samples with Listeria spp. present at more than 100 CFU/g were negative for L. monocytogenes. L. monocytogenes was detected in 0.4% (n=13) of samples, all at levels of less than 10 CFU/g, and were therefore of acceptable quality. Butter was contaminated more frequently with Listeria spp., including L. monocytogenes, when packed in plastic tubs, when in pack sizes of 500 g or less, when stored or displayed above 8 degrees C, when a hazard analysis system was not in place, and when the manager had received no food hygiene training. This study demonstrates that although butter is regarded as a low-risk product, it may provide an environment for the persistence and growth of Listeria spp., including L. monocytogenes. The control of L. monocytogenes in food processing and supply systems is critical in order to minimize the potential for this bacterium to be present in foods at the point of consumption at levels hazardous to health.

Sensory, functional, and analytical comparisons of whey butter with other butters.

The objective of this work was to characterize the sensory attributes of whey (WB), cultured (CB), and regular sweet cream (SB) unsalted butters produced at the Dairy Products Technology Center (experimental; n = 3) or obtained from commercial sources (n = 6). Nine judges were trained for nine 1-h sessions; they then rated samples on a 15-cm line scale in triplicate using descriptive analysis. Data obtained were analyzed using SAS statistical software. Significant differences between the 3 types of butters were obtained on yellow, shiny, acidic odor, melt rate, porous, hard, spreadable, cheese odor, mouthcoating, nutty, cardboard odors, acidic, nutty, diacetyl, and grassy flavors. Cultured butter and SB were significantly shinier than WB. Whey butter was more yellow than CB, which in turn was more yellow than SB. Whey butter was more porous, and had higher scores on nutty flavor and cardboard odor than SB and CB. Sweet cream butter was significantly harder than CB but not WB. Cultured butter had more mouthcoating, acidic odor and flavor, and grassy flavor than SB and WB. The commercial samples were more porous, crumbly, and had significantly more artificial butter odor, rancid odor, and flavor. Results from principal component analysis indicated that experimental WB and SB were similar and were characterized by a sweet taste. Whey butter's characteristics compared favorably with commercial CB and were very similar to sweet cream butter. No major significant differences were obtained for triangle tests, with the exception of that for WB and CB in pound cake. No significant differences were obtained for the acceptability of the different versions of any of the 3 foods.

Effect of cream fermentation on microbiological, physicochemical and rheological properties of L. helveticus-butter.

The primary objective of this study was to evaluate the physicochemical and rheological properties of butter produced by Lactobacillus helveticus fermented cream. The incorporation of putative probiotic - the L. helveticus, to ferment cream prior to butter production was anticipated to alter the nutritional composition of butter. Changes in crude macronutrients and the resultant modification relating to textural properties of butter induced upon metabolic activities of L. helveticus in cream were focused in this research. Fermented butter (LH-butter) was produced by churning the cream that was fermented by lactobacilli at 37 °C for 24 h. Physicochemical analysis, proximate analysis and rheology properties of LH-butter were compared with butter produced using unfermented cream (control). LH-butter showed a significantly (P<0.05) higher fat content and acid value; lower moisture and ash; and was softer than the control. Cream fermentation modified nutritional and textural properties of butter in which LH-butter contained higher health beneficial unsaturated fatty acids than the control and thus rendered the product softer. Its enrichment with probiotics could thus further enhance its functional property.

Enhancement of infrared spectral images for maximizing chemical information by minimizing baseline interferences.

The popularity of spectral images in many areas of analysis has greatly increased during the last decade due to the development of charge-coupled device (CCD) and infrared sensitive cameras. Large amounts of spatial information can be obtained in short periods of time. The general goal in analytical chemistry is to convert spectral images into chemical images, which show the spatial locations of various chemical components. Self-modeling multivariate curve resolution methods can be used to extract pure component spectra from the mixture spectra in images and produce chemical images. However, there is a difficulty in processing infrared spectral images due to large pixel-to-pixel baseline variations. Herein, a method for minimizing baseline interferences using fast Fourier transform (FFT) filtering in both the spectral and spatial domains is discussed. The methodology is demonstrated on a microscopic sample of butter contaminated with non-pathogenic E. coli and on a cross-sectional sample of rabbit aorta containing plaque. The processing to reduce baseline effects improved the spatial resolution without compromising the spectral resolution.

Ability of Lactobacillus plantarum MON03 to mitigate aflatoxins (B1 and M1) immunotoxicities in mice.

Aflatoxin B1 (AFB1) and M1 (AFM1) are mycotoxins produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. AFB1 and AFM1 display a potent economic loss in livestock and also cause severe immunological problems. The aims of this study were to: evaluate a new AFB1 and AFM1-binding/degrading micro-organism for biological detoxification; examine its ability to degrade AFB1 and AFM1 in liquid medium; and evaluate its potential for in vivo preventative effects against AFB1- and AFM1-induced immunomodulation in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFB1 and AFM1 in PBS (i.e. 82% and 89%, respectively) within 24 h of incubation and able to tolerate gastric acidity, have strongly hydrophilic cells surface properties, and adhere efficacy to Caco-3 cells in vitro. The in vivo study was conducted using Balb/c mice that received by oral gavage vehicle (control), LP only (2 × 10(9) CFU/L, ~2 g/kg BW), AFB1 or AFM1 alone (0.25 and 0.27 mg/kg, respectively), or AFB1 + LP or AFM1 + LP daily for 15 days. Compared to in control mice, treatments with AFB1 and AFM1 led to significantly decreased body weight gains, histopathological changes, and decrements in all hematologic and immune parameters assessed. Co-treatment with LP strongly reduced the adverse effects of each mycotoxin. In fact, the mice receiving AFB1 + LP or AFM1 + LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria alone had no adverse effects in the mice. From these data, it is concluded that the tested bacteria could be beneficial in biotechnology detoxification of contaminated food and feed for humans and animals.

Potential preventive role of lactic acid bacteria against aflatoxin M1 immunotoxicity and genotoxicity in mice.

Aflatoxin M1 (AFM1) is a mycotoxin produced by numerous Aspergillus species in pre- or post-harvest cereals and milk. Exposure to AFM1 imparts potent economic losses in the livestock industry. Toxicologically, it also causes severe immune system problems. The aims of this study were to evaluate a new AFM1-binding/degrading microorganism for biologic detoxification, to examine its ability to degrade AFM1 in liquid medium, and to evaluate its potential for in vivo preventative effects against AFM1-induced immunotoxicity and genotoxicity in mice. Lactobacillus plantarum MON03 (LP) isolated from Tunisian artisanal butter was found to display significant binding ability to AFM1 in PBS (93%) within 24 h of incubation. Further, the LP was able to tolerate gastric acidity, bile salts, and adhere efficiently to Caco-3 cells in vitro. The in vivo study used Balb/c mice that received either vehicle (control), LP only (at 1 × 10(9)CFU/L, ∼1 mg/kg bw), AFM1 (100 mg/kg bw), or AFM1 + LP daily for 15 days (by gavage); two other groups received a single dose of colchicine (4 mg/kg) or mitomycin C (1 mg/kg) as positive controls for induction of micronuclei and chromosomal aberrations, respectively. The results showed that, compared to in control mice, AFM1 treatment led to significantly decreased body weight gains, and caused cytotoxic/genotoxic effects as indicated by increases in frequencies of polychromatic erythrocytes, as well as those with micronucleation (PCEMN) and chromosomal aberrations, among bone marrow cells. The concurrent administration of LP with AFM1 strongly reduced the adverse effects of AFM1 on each parameter. Mice receiving AFM1 + LP co-treatment displayed no significant differences in the assayed parameters as compared to the control mice. By itself, the bacteria caused no adverse effects. Based on the data, it is concluded that the test bacteria could potentially be beneficial in the detoxification of AFM1-contaminated foods and feeds for humans and animals.

Further observations on ghee as a risk factor for neonatal tetanus.

BACKGROUND: Previous case-control studies of neonatal tetanus (NNT) in the North West Frontier Province of Pakistan indicated that clarified butter (ghee) applied to the umbilical wound of newborns was a significant risk factor for NNT. However, the mechanisms underlying the risk remained undisclosed. METHODS: A hospital-based case-control study was undertaken to evaluate further ghee and other factors possibly associated with risk of NNT. Mothers of several recent ghee-associated cases were visited in their homes, asked to simulate the procedures used in preparing the ghee, and samples of ghee were collected for culture. RESULTS: Topical application of ghee to the umbilical wound was again shown to pose a significant risk for NNT. In-use contamination of ghee was documented as mothers repeatedly heated and manipulated samples of ghee set aside in special containers for this purpose. Ghee was usually applied to the umbilical wound of the baby several times each day for the first few days of life. Mothers of cases were again confirmed to be substantially more likely to report prior NNT cases than mothers of controls. CONCLUSIONS: Educational interventions to reduce umbilical ghee use or to wash hands before each manipulation might reduce the risk of NNT in babies exposed to ghee who are born to non-immunized mothers. Increased efforts to immunize women of childbearing age with tetanus toxoid are also needed, with special priority for mothers known to have been associated with a previous NNT case. Topical antibiotics should be further evaluated for protective effects in non-immunized mothers.

PIP: Previous investigations in Pakistan revealed that the application of ghee (clarified butter) to umbilical wounds is a risk factor for neonatal tetanus (NNT), but the underlying mechanisms remained unknown because multiple cultures of ghee obtained from relevant households failed to culture Clostridium tetani. This study used a case-control approach to continue the evaluation of the risk of ghee applications through within-household observations of patterns of use of ghee and further microbiological tests. 100 physician-diagnosed cases who were hospitalized with NNT from September 1990 to January 1991 were compared with 300 controls matched as nearly as possible in age and sex. Data collected through questionnaires were submitted to descriptive analyses, matched analysis with single variables, stratified analysis, and other tests of statistical significance. Conditional logistic regression and Pearson correlation coefficients were also assessed. The only significant factors discovered were delivery by an academically trained attendant (which had a protective effect against NNT) and use of ghee on the umbilical wound (which was a risk factor for NNT). The household investigations revealed that the ghee for use on the newborn is kept in a separate container than that for general household use. The newborn's ghee is reheated and manipulated frequently by the mother. 25% of the samples from the secondary pots of ghee were contaminated. Heating is likely to activate rather than kill the spore-bearing bacteria which causes NNT. The influence of maternal practices is also seen in the fact that the incidence of NNT among previous births was statistically higher for mothers of cases than for mothers of controls. Since use of ghee is unlikely to be abandoned for sociocultural reasons, the added use of topical antibiotics should be evaluated for their impact on the risk factor posed by ghee. Also, special priority should be paid to the immunization of mothers of NNT cases.

Exposure of Listeria monocytogenes within an epidemic caused by butter in Finland.

Data on the levels of bacteria and the amounts of food consumed in food-borne outbreaks provides an excellent opportunity to study the effects of exposure to Listeria monocytogenes. Between June 1998 and April 1999, an outbreak caused by L. monocytogenes serotype 3a in butter occurred in Finland. The majority of the cases were immunocompromised and hospitalized at the Helsinki University Central Hospital (HUCH), where 7-g butter packages produced by a dairy plant were used as the only butter brand. The butter had also been sold to 10 other central hospitals as well as to the retail market. Based on the data on hospital stay, butter consumption and the qualitative and quantitative analyses of L. monocytogenes in butter, the attack rates and exposure were estimated. Incubation studies on the naturally contaminated small butter packages showed that the levels found in the packages at the time of detection of the outbreak could reliably be used for these estimations. However, the levels of L. monocytogenes in 500-g packages increased. The attack rate among HUCH patients varied from 70 to 117 cases per 1000 patients at risk, depending on which estimate of the contamination level of butter (100-60%) was used. The highest single dose (7.7 x 10(4) CFU in one meal) could have been sufficient to cause the listeriosis cases at HUCH. However, this data also supports another hypothesis, according to which these listeriosis cases were caused by a prolonged daily consumption of contaminated butter during the hospital stay. The estimated daily dose, based on the hospital kitchen data or the highest detected level in a wholesale sample (11,000 CFU/g), would have varied from 1.4 x 10(1) to 2.2 x 10(3) CFU/day or from 2.2 x 10(4) to 3.1 x 10(5) CFU/day, respectively. The choice of the hypothesis has a crucial impact on the interpretation of this data for the dose-response estimations as well as for the discussion on Food Safety Objectives. Due to the susceptibility of hospital patients, special care must be taken in order to avoid even low levels of L. monocytogenes in food served.

The effects of environmental conditions on the lipolytic activity of strains of Penicillium roqueforti.

The lipolytic activity of 30 strains of Penicillium roqueforti was investigated by agar diffusion tests on tributyrin (esterase activity) and olive oil agar (lipase activity), by titration of the free fatty acids (FFA) produced and by gas chromatographic analysis of the individual FFA released after growth at 25 or 10 degrees C in butterfat emulsions containing 0, 2 or 7% NaCl. All strains investigated by the agar diffusion tests possessed esterase activity and 23 strains were also able to hydrolyse olive oil, but differences in esterase activity were seen. The agar diffusion tests and the titration of FFA showed that the amount of FFA released by a strain of P. roqueforti is determined by both esterase and lipase activity. A large release of FFA was only seen for strains with the ability to hydrolyse both short- and long-chained fatty acids, while strains with esterase activity produced smaller amounts of FFA. Between 7 and 14 days of incubation a steep increase in the release of FFA was observed both by the titration and by GC analysis, and then a decline from 14 to 21 days, probably caused by conversion of FFA to methyl ketones. Identical FFA profiles were found for two strains with different lipolytic activity. Long-chained fatty acids dominated the profile, while the short-chained fatty acids only were detected in small amounts and mainly in the end of incubation. Both strains were stimulated by NaCl in the emulsions.