Thermal resistance of vegetative thermophilic spore forming bacilli in skim milk isolated from dairy environments

Thirteen thermophilic bacilli, major contaminants of powdered milk products, were characterized regarding their heat sensitivity. All strains belong to the two species Geobacillus stearothermophilus and Anoxybacillus flavithermus. Five of the tested strains were classified as heat sensitive, one strain showed a low amount of vegetative cells and was not heat-treated. With the seven heat resistant strains, a pasteurization step (73 °C, 20 s) at laboratory-scale was applied. Two strains, both from the species G. stearothermophilus, could survive the heat treatment without an inactivation effect. One of those G. stearothermophilus strains was pasteurized on pilot-scale high-temperatures-short-time (HTST) and the cells were able to survive the treatment as well. For one A. flavithermus strain, thermal inactivation data was determined and modelled with two approaches. The work emphasizes, that milk pasteurization for the production of a low-spore powder regarding the vegetative cells is not sufficient. The adaptation of the process temperature is necessary.     

A survey on occurrence of thermophilic bacilli in commercial milk powders in China

Twenty-two milk powders including infant formula milk powders and whole milk powders collected from different manufacturers in China were examined for the presence of contaminated thermophilic bacilli. The thermophilic isolates were grouped by whole cell protein profiles, and representative strains of each group were further identified by analyzing partial 16S rDNA. 80.5% of all 801 isolates were identified as Bacillus licheniformis, Anoxybacillus flavithermus and Geobacillus stearothermophilus. The most dominant strain was B. licheniformis L1, which accounted for 27.8% of all isolates and appeared in eighteen of twenty-two milk powders. Furthermore, the inactivation effect of conventional heat treatments that were widely used in industry for milk powder was tested on ten representative strains. The three pasteurization processes did not show inactivation effect on most strains, but in some cases even activated thermophilic spores. The UHT process efficiently inactivated both thermophilic vegetative cells and spores.     

Engineering of operation condition in milk pasteurization with PEF treatment

We propose a new continuous pasteurization technique for milk that involves pulsed electric field (PEF) treatment with a voltage of 40 kVp and a frequency of 100 Hz. Escherichia coli with an initial concentration 10⁷ cells/mL that was suspended in commercial milk was used as the target microorganism. Without control of the milk temperature, an average holding time in PEF treatment of 20 s should be necessary for the efficient inactivation of E. coli suspended in milk. Thermal control was incorporated in our system to improve the productivity and stability of the pasteurization process. Sufficient inactivation of E. coli cells was found to be possible even with an average holding time in PEF treatment of 10 s at a milk temperature of 50 °C or higher. We proposed a system for the PEF pasteurization of milk, in which a preheating unit and postholding unit were incorporated. When the milk temperature at the inlet of the PEF treatment chamber was controlled at 60 °C using preheating unit and the milk temperature after the PEF treatment was held at 70 °C for 20 s using the postholding unit, a reduction in the CFU of E. coli cells suspended in milk of at least seven orders of magnitude was achieved in our continuous PEF inactivation system.     

Antimicrobial peptides derived from hen egg lysozyme with inhibitory effect against Bacillus species

In food industry, Bacillus species are encountered in deteriorating many food products thus shortening their shelf-life. Moreover, Bacillus cereus and the subtilis group (Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus) have been recognized as food poisoning agents. Lysozyme peptides preparation (LzP) is a commercially available as a natural food preservative. Although, LzP derived from lysozyme yet it showed only 11% of the lysozyme lytic activity. LzP at a concentration of 100 μg ml⁻¹ completely inhibited B. subtilis, B. licheniformis, B. megaterium, B. mycoides, B. pumilus, B. coagulans, B. amyloliquefaciens, B. polymexa and B. macerans. However, B. cereus and B. stearothermophilus showed a slightly higher resistance. Interestingly, LzP at concentration ⩾10 μg ml⁻¹ showed inhibitory effect on both vegetative and spore forms of B. subtilis. Moreover, LzP was stable at 95 °C for 30 min and at different pH values (4.5–7). In conclusion, LzP may be useful to control growth of Bacillus spoilage organisms.     

Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk

Bacillus sp. P34, a strain isolated from aquatic environments of Brazilian Amazon basin, produces a bacteriocin-like substance (BLS) which was encapsulated in nanovesicles prepared from partially purified soy lecithin. The efficiency of free and encapsulated BLS P34 to control the development of L. monocytogenes and maintenance of antimicrobial activity was assessed over time in milk. The antimicrobial activity of free and encapsulated BLS P34 decreased approximately 50% after 4 days of storage (<4 °C) in skim and whole milk. After this period there was not significant loss of activity up to 21 days. The viable counts of Listeria monocytogenes in skim and whole milk containing 3200 AU/ml of free or encapsulated BLS P34 were always lower than those observed in controls without bacteriocin at both 30 °C and 7 °C. At 1600 AU/ml concentration, free and encapsulated BLS P34 were inhibitory to L. monocytogenes in skim milk, when compared with the control at 7 days. Nanovesicle-encapsulated and free BLS P34 shows potential use as biopreservative for application in milk-derived products.     

Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink by 16S rDNA sequencing

Iranian Kefir type drink (IKTD) is a highly consumed, traditional Iranian, fermented milk product. To improve monitoring procedures for food safety 32 industrial Kefir type drinks from 4 brands and 8 different production dates as well as 32 samples from pasteurized milk of the same Kefir manufacturers and air of the production sites were analyzed for contaminations. 16S rDNA extraction from Kefir samples as well as 16S rDNA obtained from samples incubated on Columbia agar were analyzed using PCR/DGGE, cloning, sequencing and phylogenetic classification. Already DGGE analysis indicated contaminations including Bacillus strains. Subsequently analysis of cultured clones indicated contaminations with Bacillus sp. including Bacillus cereus, Bacillus thuringiensis and Paenibacillus sp. in 9 (28%) from all analyzed samples. Also 38% of pasteurized milk samples were contaminated with B. cereus. The average count of B. cereus was 74 ± 19 cfu/ml. B. cereus and B. thuringiensis were found as contaminant bacteria in the air of the all manufacturing sites. These results suggest that milk is one of the most important sources of contamination with Bacillus sp., especially B. cereus for Kefir products in Iran. But bacterial contamination in Kefir samples might also originate from the air of the production sites. 16S rDNA analysis accelerates monitoring strategies.     

Evaluation of the factors affecting the activity of sakacin C2 against E. coli in milk

Sakacin C2 is a novel, broad spectrum bacteriocin secreted by Lactobacillus sake C2 isolated from Chinese traditional fermented cabbage. Effects of milk fat, emulsifiers, preservatives and homogenization on the activity of sakacin C2 against Escherichia coli ATCC 25922 in milk were evaluated by determined the changes of viable cell counts of E. coli ATCC 25922 after inoculation (10⁵ CFU/ml) then storage at 4 °C. Milk fat in pasteurized and homogenized milk products (low fat milk and whole milk) decreased the activity of sakacin C2 against E. coli ATCC 25922. Addition of 5 μl/ml of Tween 80 decreased the effect of sakacin C2 against E. coli ATCC 25922, but 5 mg/ml of lecithin increased the effect against E. coli ATCC 25922 in pasteurized and homogenized whole milk. Nisin and ε-Polylysine significantly increased the effect of sakacin C2 against E. coli ATCC 25922 in pasteurized and homogenized whole milk. Homogenization interfered with the activity of sakacin C2 against E. coli ATCC 25922 in pasteurized skim milk and whole milk. This study might lay the groundwork for the application of sakacin C2 as bio-preservative in milk and dairy products.     

Susceptibility of Clostridium perfringens to antimicrobials produced by lactic acid bacteria: Reuterin and nisin

The effectiveness as antimicrobials of lactic acid bacteria produced compounds reuterin and nisin was assessed against vegetative cells and spores of Clostridium perfringens isolates (from ovine milk obtained in farms with diarrheic lambs) and C. perfringens CECT 486 (type A toxin producer). We also tested the inhibitory effect of lysozyme and sodium nitrite on Clostridium. Minimal inhibitory concentrations (MIC) of antimicrobials were determined in modified RCM (mRCM) and milk by using a broth microdilution method, after 7 d at 37 °C under anaerobic conditions. The sensitivity of C. perfringens to the tested antimicrobials was strain and culture medium-dependent. In general, vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 2.03–16.25 mM) inhibited the growth of vegetative cells and the outgrowth of spores of all tested C. perfringens, both in mRCM and milk, with higher resistance in milk. Nisin (MIC values 0.78–12.5 μg/ml) was also effective against vegetative cells and spores of tested C. perfringens in both culture media. However, lysozyme (up to 400 μg/ml) did not control the growth of any of the tested Clostridium. Sodium nitrite only inhibited the outgrowth of spores of two C. perfringens isolates at the maximum concentration assayed (300 μg/ml) exclusively in mRCM medium. These results suggest that reuterin and nisin have the potential to control the growth of C. perfringens, and might help to ensure safety at different stages of the food chain. Future studies in food/feed products would be necessary to further corroborate this hypothesis.     

Chemical composition of fermented milk (roub and mish) in Sudan

Two batches of whole milk, skim milk and yoghurt that used for processing of roub and mish from Khartoum Dairy Company (Butana factory) were analyzed. Similarly roub and mish (fermented milk), obtained from the same samples, were also collected and analyzed during this study. Similarly, a total of four experiments as an attempt to process “Roub and mish” in the factory’s laboratory were tried following the same procedures of the factory for two batches. While in the other two pasteurization for whole milk were done before manufacturing. Comparison of chemical constituents (total solids %, total proteins %, fat %, lactose %, ash %, and acidity), and pH were estimated in those products.The total solids %, the total proteins % and the fat % recorded lower means for the control samples, compared to those of the factory for whole milk; yoghurt; mixture; and roub. However, the control samples for mish revealed a higher mean than those produce commercially by the factory. Moreover, the higher values obtained for ash and acidity for skim milk and fermented products, in the control samples, reflected the adulteration done at the factory. Similarly, Lactose revealed higher means for whole milk; skim milk; and mixture for the control samples. While yoghurt; roub; and mish showed a higher mean values for the factory samples. This might be due to the fermentation action by the bacteria.The results indicated that there were significance differences in total solids %, ash %, and lactose % (P ⩽ 0.05) between the different groups. Similarly there is significant increase in pH, fat % (P ⩽  0.001) and acidity (P ⩽ 0.01) between the different groups. However, a non-significance difference was found for the other measurements.When comparing roub and mish a non significant differences were obtained for the measurements. Between mixture and roub significance variations were reported for ash %, lactic acid % (P ⩽ 0.05) and lactose (P  ⩽ 0.001). Also, significance variations were found in ash % (P  ⩽ 0.05), and lactose % (P ⩽ 0.001), when comparing mixture and mish. This could be due to fermentation and addition of spices.     

Antibacterial activity of bovine milk lactoferrin on the emerging foodborne pathogen Cronobacter sakazakii: Effect of media and heat treatment

Cronobacter sakazakii is a pathogen transmitted by food, with high osmotic resistance and tolerance to desiccation, which affects mainly to newborns, infants and immunocompromised adults. C. sakazakii infection in infants has been associated with consumption of powdered milk. The purpose of this study was to evaluate the antibacterial activity of native and iron-saturated bovine lactoferrin (bLF) (from 0.5 to 5 mg/ml) on non-desiccated and desiccated C. sakazakii (10⁴ CFU/ml) in different media (phosphate buffer, bovine skim milk and whey). In general, native bLF was the only effective form that inhibited growth of C. sakazakii in all media, its activity increasing with concentration and time of incubation. These results suggest that the antibacterial effect of bLF on C. sakazakii is mainly due to iron sequestration. However, iron-saturated bLF showed some effect by reducing the viability of C. sakazakii in whey. There has not been observed an increased sensitivity of desiccated bacteria to native bLF in phosphate buffer. However, although the antibacterial activity of native bLF against non-desiccated C. sakazakii was drastically reduced in milk or whey compared to phosphate buffer, there was a certain activity when it was assayed against desiccated cells in those media. The effect of some heat treatments on the antibacterial activity of native bLF was evaluated and only those of 72 °C for 15 s, 85 °C for 15 s, and 63 °C for 30 min maintained its whole activity.     

Surface conditioning of stainless steel coupons with skim milk,buttermilk, and butter serum solutions and its effect on bacterial adherence

In the dairy industry, dairy by-products such as skim milk, buttermilk and butter serum which possess different specific compositions, could contact with processing surfaces to form conditioning layers and subsequently alter bacterial attachment behavior of the surfaces. In order to simulate and elucidate this process, stainless steel coupons were conditioned with skim milk, buttermilk and butter serum solutions. Formed conditioning layers were examined under a confocal laser scanning microscope (CLSM) and the influence of surface conditioning on bacterial adherence was investigated. The results showed that different conditioning layers were formed by different dairy by-products. The layer formed by skim milk, buttermilk and butter serum was the thinnest, medium and the thickest, respectively. The treatment of stainless steel surfaces with skim milk, buttermilk and butter serum could reduce the adherence of dairy-related bacteria (Lactococcus lactis subsp. lactis NBRC 100933, Leuconostoc mesenteroides subsp. cremoris NBRC 107766 and Lactobacillus casei FIRI 108) at different levels. In the majority of cases, the adherence-reducing ability of buttermilk and butter serum was found better than skim milk. While skim milk could reduce bacterial adherence during shorter exposure time (almost of 30 min), buttermilk and butter serum could act during the longer period (up to 720 min). The result suggested that, bacterial adherence-reducing effect of buttermilk and butter serum may correlate to their substances associated with milk fat globule membrane. In order to decrease bacterial adherence, surface conditioning with skim milk, buttermilk and butter serum is recommended. Surface conditioning with skim milk is suitable for short bacterial exposure time (30 min), for a longer period of time (more than 180 min), only surface conditioning with buttermilk and with butter serum is advisable.     

Control of milk pH reduces biofilm formation of Bacillus licheniformis and Lactobacillus paracasei on stainless steel

Microbial biofilms present in dairy farms may contaminate milk during milk harvest and transfer diseases from the environment to cows. In order to reduce biofilm formation with respect to the role of pH, a study involving the control of milk pH during long-term biofilm formation of Bacillus licheniformis NBRC 12195 and Lactobacillus paracasei subsp. paracasei NBRC 15889 on stainless steel coupons in different dilutions of skim milk (0.1%, 1.0% and 5.0%) was conducted. During long incubation at 30 °C, pH decreased due to bacterial development in unadjusted samples. In pH-adjusted samples, pH was kept at around 7.0 by the addition of sterile sodium hydroxide. Biofilms formed on stainless steel coupons were daily stained by 0.1% Crystal Violet solution and assessed by the evaluation of optical density. The bacterial count of the suspensions showed that the control of pH enhanced the growth of bacteria in free-floating form. In contrast, optical densities of biofilms formed in the pH-adjusted samples were significantly lower than in the pH-unadjusted samples in all of three skim milk dilutions. Comparison of maximum OD values of adhered cells at different nutrient levels also implicated that for both tested strains, thicker biofilms were formed in milk dilutions at higher nutrient levels. These results suggested that, control of milk pH and milk residue level could significantly reduce biofilm formation of the tested bacteria.     

Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 10² CFU/mL, as low as 7.5 × 10⁰ CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 10¹ CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 10³ CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.     

Inhibition of spoilage and toxigenic Bacillus species in dough from wheat flour by the cyclic peptide enterocin AS-48

Enterocin AS-48 was tested against rope-forming Bacillus subtilis CECT 4002 and Bacillus licheniformis CECT 20, as well as on Bacillus cereus and Bacillus pumilus strains in broth and in experimental dough from wheat flour. Vegetative B. subtilis cells in liquid broth were rapidly inactivated by AS-48 (7 AU/ml). In wheat dough, higher bacteriocin concentrations of 14 and 23 AU/g were required for inactivation of B. subtilis vegetative cells and endospores activated to germinate, respectively. B. cereus LWL1 and B. licheniformis CECT 20 were inactivated by AS-48 (14 AU/g) in doughs stored at 22 °C much faster compared to doughs stored at 10 °C. Strains of Bacillus pumilus were partially inactivated in dough by bacteriocin addition (14 AU/g). Results from this study indicate that enterocin AS-48 can reduce the populations of spoilage and potentially-toxigenic bacilli in wheat dough, decreasing the risks for spoilage and food intoxication.     

Radio frequency heating of comminuted meats - considerations in relation to microbial challenge studies

Bacillus cereus and Clostridium perfringens vegetative cell and spore cocktails in maximum recovery diluent (MRD) were inoculated into pork luncheon meat to challenge a previously developed radio frequency (RF) cooking protocol. After RF cooking and cooling microbial enumeration results showed a reduction in B. cereus vegetative cell and spores of 5.4 and 1.8 log₁₀ cfu g⁻¹, respectively while the corresponding reduction for C. perfringens vegetative cells and spores were 6.8 and 4.1 log₁₀ cfu g⁻¹, respectively. However, post cooking temperatures within the product were lower than anticipated. Subsequent analysis of product thermal and dielectric properties indicated that MRD addition and compositional variations within meat ingredients altered thermal and dielectric properties which in turn contributed to reduced and less uniform temperatures. The study shows that for RF microbial challenge studies, adjustment of product formulation prior to MRD addition is critical to ensure a similar composition to the normal product and a true picture of microbial inactivation.     

Effects of blackberry juice on growth inhibition of foodborne pathogens and growth promotion of Lactobacillus

Berries such as blueberry, blackberry and raspberry possess several biological activities including antimicrobial and nutritional effects. In this study, the antimicrobial activities of blackberry (Rubus fruticosus) juice against foodborne pathogens including Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7 were investigated. Inhibition of growth of these foodborne pathogens was measured in broth (Luria–Bertani broth for E. coli O157:H7 and S. Typhimurium, and brain heart infusion broth for L. monocytogenes), skim milk and whole milk supplemented with 10% blackberry juice at different time points (0, 24, 48 and 72 h). The effects of blackberry juice on the growth of Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus were also investigated in Man–Rogosa–Sharpe (MRS) broth and skim and whole milk supplemented with blackberry juice. The growth of L. monocytogenes, S. Typhimurium and E. coli O157:H7 were significantly inhibited by blackberry juice by 1–3 logs in both milk and broth. We also observed that the growths of Lactobacillus strains were significantly stimulated (1–4 logs CFU/mL) by blackberry juice in both milk and MRS broth. These data clearly demonstrate that diluted blackberry juice can be used as a preservative in food processing and a preventive in foodborne infections as a natural antimicrobial.     

Microflora of traditional starter made from cassava for “attiéké” production in Dabou (Côte d’Ivoire)

Attiéké is a food made from cassava in Côte d’Ivoire by fermentation. The process uses a traditional starter. Studies on 81 starter samples from 3 villages showed that the dominant microflora consists of lactic acid bacteria (5.7 × 10⁷ cfu/g), yeasts (5.5 × 10⁷ cfu/g), Bacillus (3.8 × 10⁷ cfu/g), Enterococcus (3.0 × 10⁶ cfu/g), total coliforms (3.0 × 10⁶ cfu/g), thermotolerant coliforms (8.0 × 10³ cfu/g) and mould (2.0 × 10⁶ cfu/g). Lactic acid bacteria, Bacillus spp, yeasts, faecal Enterococci and moulds are organisms which could play a role in the cassava fermentation. Coliforms may indicate contamination from the environment during production.     

Development and application of a centrifugation-plating method to study the biodiversity of Bacillus species in rice products

A centrifugation-plating method was developed, using amylase and Tween 80 pre-treatment, for detection and enumeration of Bacillus spp. in rice products. The high sensitivity of this method improved detection of a variety of Bacillus species in rice products compared to the spread-plate method. Bacillus spp. were detected in 33 out of 35 raw rice samples with the centrifugation-plating method, but only 13 samples using the spread-plating method, even though 1 mL of a 10⁻¹ sample dilution was used, to increase sensitivity of the method. Known toxigenic or potentially toxigenic species (Bacillus cereus/Bacillus thuringiensis, Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus) were those most frequently found in both raw and cooked rice.     

Migration of two antioxidants from packaging into a solid food and into Tenax®

The migration of chemicals from packaging materials into foods is an important issue in food safety and quality. European legislation sets specific migration limits which compliance must be assessed, surveyed and controlled by industry and authorities. Recently, the new Regulation UE 10/2011 included a new simulant - the Modified Polyphenylene Oxide (Tenax^®) for solid dry foods. The objective of this work was to compare the migration kinetics of two antioxidants into Tenax^® as compared to rice at 3 temperatures: 23, 40 and 70 °C. The application of two different solutions of the 2nd Fick's law to describe and simulate the migration of the migrants to the present systems was studied. Diffusion coefficients ranged between 4.80E-13 and 2.84E-11 cm²/s for the migration into Tenax^® and between 6.90E-18 and 4.33E-17 cm²/s for the migration into rice. The partition coefficients ranged between 6 and 29 for Tenax^® and were over 1000 for rice. The activation energy for the migration into rice was half of that for Tenax^®. The models described relatively well the experimental data (ε < 12% and < 30% for rice and Tenax^®, respectively). Results indicate that the food simulant tends to overestimate migration values and thus can be safely used to assess materials compliance when materials are intended to contact with rice. However, results also indicate that Tenax^® is a much more severe simulant in representing rice.     

Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey

The aim of the present study was to investigate the biochemical characteristics, extracellular enzyme production and enterotoxigenic genes contents of 6 Bacillus cereus and 22 Bacillus thuringiensis strains, isolated from 100 cheese samples in Turkey. Crystal morphologies of B. thuringiensis strains were found either spherical (n = 12) or spherical and irregular-shaped (n = 10) by phase contrast microscopy. B. cereus and B. thuringiensis strains were found to produce extracellular enzymes, respectively: gelatinase (83% and 91%), DNase (83% and 77%), lecithinase (83% and 95%), protease on skim milk agar (100% and 100%), protease on milk agar (100% and 91%), protease on casein agar (83% and 77%), xylanase (100% and 45%), and cellulase (0% and 41%), and amylase (83% and 27%). All of the strains, except for Bt-D1, hydrolyzed Tween 20 (96%), but not Tween 80 or tributyrin. Pectinolytic activity was obtained to be the least frequent (4%). PCR analysis showed that all strains contained nheA, nheB, nheC and hblD genes. The hblA and hblC genes were present in 2 and 4 of B. thuringiensis strains, respectively. The bceT gene was detected in 1 B. cereus and 9 B. thuringiensis strains. The entFM gene was detected more frequently in B. thuringiensis (82%) than in B. cereus strains (50%). To our knowledge, this is the first report about the isolation and identification of enterotoxigenic B. cereus and B. thuringiensis strains from cheese samples in Turkey.