Effect of NaOH (caustic wash) on the viability, surface characteristics and adhesion of spores of a Geobacillus sp. isolated from a milk powder production line

Aim: To investigate the viability, surface characteristics and ability of spores of a Geobacillus sp. isolated from a milk powder production line to adhere to stainless steel surfaces before and after a caustic (NaOH) wash used in clean‐in‐place regimes. Methods and Results: Exposing sessile spores to 1% NaOH at 65°C for 30 min decreased spore viability by two orders of magnitude. The zeta potential of the caustic treated spores decreased from ?20 to ?32 mV and they became more hydrophobic. Transmission electron microscopy revealed that caustic treated spores contained breaks in their spore coat. Under flow conditions, caustic treated spores suspended in 0·1 mol l^?1 KCl were shown to attach to stainless steel in significantly greater numbers (4·6 log₁₀ CFU cm^?2) than untreated spores (3·6 log₁₀ CFU cm^?2). Conclusions: This research suggests that spores surviving a caustic wash will have a greater propensity to attach to stainless steel surfaces. Significance of Study: The practice of recycling caustic wash solutions may increase the risk of contaminating dairy processing surfaces with spores.     

Inhibitory Effects of High Pressure and Heat on Alicyclobacillus acidoterrestris Spores in Apple Juice

The effectiveness of combined high pressure and heat treatment for reducing spore levels of Alicyclobacillus acidoterrestris, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated. Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90°C).     

Evaluation of alcohol wipes used during aseptic manufacturing

Aim: During aseptic manufacturing and specifically during the transfer of items into an isolator, disinfection of surfaces is essential for reducing the risk of final product contamination. Surface disinfection can be carried out by a variety of methods, however the most accepted current practice is a combination of spraying with 70% alcohol and wiping. The aim of this study was to evaluate the effectiveness of two wipe systems by determining their ability to remove, kill and transfer bacterial contaminants from standardized surfaces. Methods and Results: The protocol used to achieve these objectives was based on a newly published method specifically designed to test wipes. Alcohol impregnated wipes performed better at reducing microbial bioburden than the alcohol spray/dry wipe applications. Impregnated wipes drastically reduced (1–2 log₁₀ reduction) a small bioburden (approx. 2 log₁₀) of spores of Bacillus subtilis and methicillin‐resistant Staphylococcus aureus from the surface, but failed to remove (<0·2 log₁₀ reduction) Staphylococcus epidermidis. The alcohol spray/dry wipes did not manage to remove (<0·2 log₁₀ reduction) spore or bacterial bioburden from surfaces and was able to transfer some viable micro‐organisms to other surfaces. Both wipe types showed poor antimicrobial efficacy (<1 log₁₀ reduction) against the test bacteria and spores. Conclusions: As far as the authors are aware this is the first time that such a practical study has been reported and our results suggest that the best wipes for surface disinfection in aseptic units are the alcohol (IPA) impregnated wipes when compared with the dry wipes sprayed with alcohol. Significance and Impact of the Study: The impregnated wipes performed better than the dry wipes sprayed with alcohol and should be used for surface disinfection in aseptic units.     

Characterization of bacteriocin bificin C6165: a novel bacteriocin

Aims

To purify and primarily characterize an anti‐Alicyclobacillus bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, suggested to be named bificin C6165.

Methods and Results

During purification of the bificin C6165, optimal recovery was achieved with ammonium sulfate precipitation followed by two chromatographic steps. Mass spectrometry analyses revealed a distinctive peak corresponding to a molecular mass of 3395·1 Da. This bacteriocin was heat stable, effective after refrigerated storage and freeze–thaw cycles. The primary mode of action of bificin C6165 is most probably due to pore formation, as indicated by the efflux of K⁺ from metabolically active cells of Alicyclobacillus acidoterrestris. In the presence of 10 mmol l^?1 gadolinium, bificin C6165 did not affect cells of Alicyclobacillus acidoterrestris. This suggests that the mode of action of bificin C6165 relies on a net negatively charged cell surface.

Conclusions

Bificin C6165 is indeed a novel bacteriocin and it exhibited remarkable potency for Alicyclobacillus control.

Significance and Impact of the Study

Application of bacteriocins in preservation of fruit juices has seldom been studied. Bificin C6165 may be an alternative method to control juice spoilage by this Alicyclobacillus acidoterrestris and meet increasing consumer demand for nature and artificial chemical additive‐free food products.     

The inactivation and removal of airborne Bacillus atrophaeus endospores from air circulation systems using UVC and HEPA filters

Aims: To (i) evaluate the UV radiation in the ‘C’ band/high efficient particulate air (UVC/HEPA) instrument’s potential to inactivate spores of Bacillus atrophaeus and selected Bacillus species and (ii) test whether a titanium dioxide coating inside the cylindrical HEPA filter improves the system's efficacy. Methods and Results: Known amounts of dried spore preparations of B. atrophaeus, Bacillus cereus, Bacillus megaterium, Bacillus stearothermophilus and Bacillus thuringiensis were exposed to the UVC lamp within a cylindrical HEPA filter for different time lengths (30 min to 48 h) and with different air flow speeds (0–235 l s^?1). The log₁₀ reduction (range 5–16 logs) of colony forming units for spores exposed to the UVC compared with the unexposed spores was significant (P < 0·0001). The addition of a titanium dioxide (TiO₂) veneer to the interior surface of the HEPA filter significantly increased the inactivation of spores (P < 0·0001). Conclusions: The UVC/HEPA unit could inactivate spores of B. atrophaeus, B. cereus, B. megaterium, B. stearothermophilus and B. thuringiensis. Significance and Impact of the Study: The UVC/HEPA unit represents an effective method of decontaminating circulating air within an air‐duct system as found in a building.     

Effects of foliar apple trichomes on Galendromus occidentalis (Nesbitt) (Acari: Phytoseiidae): preferences,fecundity and prey consumption

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Blühfördernde Wirkung von 2-Chloräthylphosphonsäure bei Apfelbäumen

Experiments with the growth regulator ethephon (2-chloroethylphosphonic acid) were made to stimulate the flower differentiation in the ‘Carola’ and ‘Golden Delicious’ apple cultivars in Pillnitz (GDR) and ‘Jonathan’ and ‘Golden Delicious’ in Ujfehértó (Hungaria). A twofold application of 150 ppm ethephon in June proved most effective. Our results indicate that it is possible to promote flower differentiation in apple trees without growth retardation and fruit thinning.     

Temperature, soluble solids and pH effect on Alicyclobacillus acidoterrestris viability in lemon juice concentrate

Alicyclobacillus acidoterrestris is a thermoacidophilic, non-pathogenic, spore-forming bacterium detected in spoiled commercial pasteurized fruit juice. Apple, white grape and tomato are particularly susceptible. A. acidoterrestris spores are resistant to lemon juice pasteurization (2 min at 82°C), and they can germinate and grow causing spoilage. This contamination is characterized by a medicinal or disinfectant smell attributed to guaiacol (o-dihydroxybenzene) production and other taint chemicals. The aim of this work was to study the influence of temperature (82, 86, 92 and 95 °C), total soluble solids (SS) (6.20, 9.8, 50 and 68°Brix) and pH (2.28, 2.45, 2.80, 3.25, 3.5) on decimal reduction time (D) of the A. acidoterrestris in clarified and non-clarified concentrated lemon juice. Once D-value was determined, the resistance of A. acidoterrestris at the assayed temperatures was confirmed. SS and pH influence spore viability, because spore resistance increases with higher SS (50°Brix 22 min 82 °C–68°Brix 28 min 82 °C) and pH values (pH 2.28, 17 min–pH 4.00, 22 min). Bacterial growth was lower in clarified lemon juice, 26 min at 82 °C, than in non-clarified lemon juice, 51 min at 82 °C. Temperature was the parameter that had the greatest influence on the D value.     

High pressure homogenization inactivation of Escherichia coli and Staphylococcus aureus in phosphate buffered saline,milk and apple juice

High pressure homogenization (HPH) offers new opportunities for food pasteurization/sterilization. Escherichia coli and Staphylococcus aureus suspended in phosphate buffered saline (PBS) buffer, milk and apple juice at initial concentration of ~10⁶ log₁₀ CFU per ml were subjected to HPH treatments up to 200 MPa with inlet temperatures at 4–40°C. After HPH at 200 MPa with the inlet temperature at 40°C, the count of E. coli suspended in PBS, milk and apple juice reduced by 3·42, 3·67 and 3·19 log₁₀ CFU per ml respectively while the count of S. aureus decreased by 2·21, 1·02 and 2·33 log₁₀ CFU per ml respectively suggesting that S. aureus was more resistant. The inactivation data were well fitted by the polynomial equation. Milk could provide a protective effect for S. aureus against HPH. After HPH at 200 MPa with the inlet temperature at 20°C, the cell structure of E. coli was destroyed, while no obvious damages were found for S. aureus.     

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.     

Inhibition of the growth of apple pollen tubes by EDTA, surfactants and fungicides

The effects of EDTA (ethylene diamine tetraacetic acid), Kresoxim‐methyl, Wettable Sulphur 80 WP and the surfactants Triton X‐100, Triton X‐114, Dehydol TA 5 and Dehydol TA 29 on pollen tube growth of Malus domestica, Borkh. cvs ‘Golden Delicious’ and ‘Mondial Gala’ were determined by image analysis. The ED₅₀ ‐ and ED₉₀ ‐ values (effective doses giving 50% and 90% reduction of growth) of the chemicals, except for the surfactants, were determined. The selected test substances showed an inhibiting effect on pollen tube growth, with Kresoxim‐methyl being the most active. Surfactants are components of pesticides and they also caused inhibition of pollen tube growth but only at application rates more then 10‐fold higher than typically recommended. Pollen of both apple cultivars were almost equally sensitive to the chemicals.     

Immunomagnetic Separation Combined with Polymerase Chain Reaction for the Detection of Alicyclobacillus acidoterrestris in Apple Juice

A combination of immunomagnetic separation (IMS) and polymerase chain reaction (PCR) was used to detect Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice. The optimum technological parameters of the IMS system were investigated. The results indicated that the immunocapture reactions could be finished in 60 min and the quantity of IMPs used for IMS was 2.5 mg/mL. Then the combined IMS-PCR procedure was assessed by detecting A. acidoterrestris in apple juice samples. The agarose gel electrophoresis results of 20 different strains showed that the IMS-PCR procedure presented high specificity to the A. acidoterrestris. The sensitivity of the IMS-PCR was 2×10¹ CFU/mL and the total detection time was 3 to 4 h. Of the 78 naturally contaminated apple juice samples examined, the sensitivity, specificity and accuracy of IMS-PCR compared with the standardized pour plate method were 90.9%, 97.0% and 96.2%, respectively. The results exhibited that the developed IMS-PCR method will be a valuable tool for detecting A. acidoterrestris and improving food quality in juice samples.     

Susceptibility of selected apple cultivars to the Mediterranean fruit fly

Ceratitis capitata (Wiedemann), the Mediterranean fruit fly, is one of the key pest species affecting deciduous fruit orchards along the Mediterranean coasts. Because of global warming, C. capitata is gradually spreading north and is becoming a major pest of apples. Determining the susceptibility of the main apple varieties grown in the region will serve as a cornerstone to the management of this pest. In this study, we show the results of a field and laboratory no‐choice test conducted to determine the Medfly preferences on different apple cultivars. The seven main varieties of apples (Gala, Red Delicious, Golden Delicious, Granny Smith, Kanzi, Morgen Dallago and Fuji) were tested. The results demonstrate that C. capitata lays eggs on all apple cultivars in both field and laboratory conditions. The Granny Smith, Red Delicious and Morgen Dallago varieties showed the lowest susceptibility in laboratory conditions, (0.75, 1.55, 2 oviposition punctures/fruit, respectively), with significant differences in oviposition compared to the Golden Delicious, Kanzi and Fuji (3.27, 3.31, 3.1 oviposition punctures/fruit, respectively) varieties, which were shown to be the most susceptible to Medfly attack in laboratory conditions. On the other hand, only slight and not statistically significant differences emerged from the field trials. In relation to the physico‐chemical characteristics, the apple cultivars showing the lowest susceptibility (Granny Smith, Red Delicious and Morgen Dallago) had harder peels and pulps and lower sugar contents than the most susceptible cultivars (Golden Delicious, Fuji and Kanzi). These results were also confirmed through evaluation of larval development on different varieties. In fact, Granny Smith, Red Delicious and Morgen Dallago were the three varieties that did not allow adequate larval and adult development and reduced the possibility of the emergence of a new generation.     

Identification of thermo-acidophilic bacteria isolated from the soil of several Japanese fruit orchards

Aims: To investigate the occurrence and distribution of thermo‐acidophilic bacteria (TAB) associated with various commercial fruit crop soils in Japan and to assess their ability to produce the odorous phenolic compound, guaiacol. Methods and Results: Phylogenetic analysis based on the 5′ end of the 16S rRNA gene (approximately 500 bp), was performed on 62 TAB isolated from the soil of several Japanese fruit orchards. The results suggested that 60 of the bacterial strains analysed belonged to the genus Alicyclobacillus, while the remaining two belonged to the genus Bacillus. The majority of strains (58%) were identified as Alicyclobacillus acidoterrestris. This group partitioned into three phylogenetically distinct subgroups (A–C). Isolates identified as A. acidiphilus (two strains), A. acidoterrestris (36 strains), and A. hesperidum subsp. aigle (one strain), produced guaiacol from vanillic acid. Levels of guaiacol production varied significantly among strains. The guaiacol producing phenotype was conserved among certain species, however no correlation was observed between levels of guaiacol production and 16S rRNA gene‐based phylogenetic relatedness. Conclusions: Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans were widely distributed among various fruit orchards in Japan. Guaiacol production was common at the species/subspecies level; however the amount of guaiacol produced by each strain varied significantly. Significance and Impact of the Study: This study provides a comprehensive phylogenetic survey of Alicyclobacillus species in Japanese fruit orchards. Quality control standards for guaiacol producing Alicyclobacillus have also been described.     

Control of Alicyclobacillus acidoterrestris in fruit juices by a newly discovered bacteriocin

Alicyclobacillus acidoterrestris is one of the most spoilage-causing bacteria in fruit juices. Control of A. acidoterrestris in fruit juices by bificin C6165 (Pei et al. in J Appl Microbiol 114(5):1273–1284, 2013), a bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, was described in this study. Activity spectrum of bificin C6165 was investigated and sixteen strains of A. acidoterrestris were sensitive to bificin C6165 in diluted Apple Juices. In the commercial fruit juices, vegetative cells of A. acidoterrestris were inactivated by bificin C6165 at 40 μg/ml. The inhibitory effect of bificin C6165 was better at lower pH (pH 3.5) and at a higher temperature of 45 °C. Furthermore, electron microscopy examination of the vegetative cells treated with bacteriocin revealed substantial cell damage and bacterial lysis. The result suggested that primary mode of action of bificin C6165 was most probably due to pore formation. Although no significantly activity of bificin C6165 was observed against the endospores of A. acidoterrestris in commercial apple juice, the addition of bacteriocin contributed to the reduction of the thermal resistance of A. acidoterrestris spores. Additionally, encapsulation of bificin C6165 with Ca-alginate gel was investigated. Encapsulation of bificin C6165 provided a promising method to control A. acidoterrestris in food juice industry.     

Prevention of the accumulation of Alicyclobacillus in apple concentrate by restricting the continuous process running time

Aims: To study the accumulation of vegetative cells and endospores of Alicyclobacillus, as well as viable aerobic counts during the continuous production of apple juice concentrate. Methods and Results: Apples were processed for a continuous process running time of 108 h (processing rate 1·8–2·0 t h^?1) without clean‐in‐place (CIP) procedures in‐between different batches. Samples from single‐strength apple juice, concentrate after evaporation (±30°Brix), the final product (concentrate pasteurized at 102–104°C for 90 s) and condensate water (by‐product of the juice concentration process) were collected every 12 h. From 12 to 84 h of processing, vegetative Alicyclobacillus counts in single‐strength apple juice increased significantly (P < 0·05) from 1 to 3·15 log₁₀ CFU ml^?1. Accumulation patterns of vegetative cells in apple concentrate and the final product were similar from 24 to 84 h of processing, with the respective counts increasing from 0·13 to 1·63 and 0·01 to 1·69 log₁₀ CFU ml^?1. The highest Alicyclobacillus endospore counts in single‐strength juice, concentrate and the final product was at 84 h of processing with 1·32, 1·59 and 1·64 log₁₀ CFU ml^?1, respectively. Conclusions: Alicyclobacillus vegetative cells and endospores accumulate in fruit concentrates during a continuous process running time of 108 h. Significance and Impact of the Study: In conjunction with good manufacturing practices, fruit concentrate manufactures can minimize Alicyclobacillus accumulation in fruit concentrates by limiting the continuous process running time between clean‐ups to under 84 h.     

Holographic microscopy provides new insights into the settlement of zoospores of the green alga Ulva linza on cationic oligopeptide surfaces

Interaction of zoospores of Ulva linza with cationic, arginine-rich oligopeptide self-assembled monolayers (SAMs) is characterized by rapid settlement. Some spores settle (ie permanently attach) in a ‘normal’ manner involving the secretion of a permanent adhesive, retraction of the flagella and cell wall formation, whilst others undergo ‘pseudosettlement’ whereby motile spores are trapped (attached) on the SAM surface without undergoing the normal metamorphosis into a settled spore. Holographic microscopy was used to record videos of swimming zoospores in the vicinity of surfaces with different cationic oligopeptide concentrations to provide time-resolved insights into processes associated with attachment of spores. The data reveal that spore attachment rate increases with increasing cationic peptide content. Accordingly, the decrease in swimming activity in the volume of seawater above the surface accelerated with increasing surface charge. Three-dimensional trajectories of individual swimming spores showed a ‘hit and stick’ motion pattern, exclusively observed for the arginine-rich peptide SAMs, whereby spores were immediately trapped upon contact with the surface.     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

Spore lipids and germination in Agaricus bisporus

Summary The effects of a number of organic compounds on the germination of spores of Agaricus bisporus (J. Lange) Pilat has been investigated and a preliminary analysis of spore lipids carried out. Germination was stimulated by isocaproic acid but not by straight-chain C₅ to C₁₁ fatty acids or by the amino acids leucine and iso-leucine. Cholesterol at a concentration of 1 ppm was inhibitory. The lipid reserve of the spore comprised mono-, di- and tri-glycerides, free fatty acids and sterols. The phospholipid fraction was unusually small and contained a lecithin and cephalin fraction, phosphatidylinositol and cardiolipin phosphatidic acid; phosphatidylcholine being the most prominent component. The role of lipids and various germination stimulants in the physiology of A. bisporus spores is discussed.