Expression of Rice Glutamate Decarboxylase in <Emphasis Type="Italic">Bifidobacterium Longum</Emphasis> Enhances γ-Aminobutyric Acid Production

Bifidobacteria are important for the production of fermented dairy products and probiotic formulas but have a low capacity for γ-aminobutyric acid (GABA) production. To develop a Bifidobacterium strain with an enhanced GABA production, we transformed Bifidobacterium longum with a rice glutamate decarboxylase (OsGADC⁻) gene by electroporation. When the transformed strain was cultured in medium containing monosodium glutamate, the amount of GABA increased significantly compared with those of untransformed Bifidobacterium. Thus, by introducing a plant derived GAD gene, a Bifidobacterium strain has been genetically engineered to produce high levels of GABA from glutamate.     

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.     

Probiotics from an industrial perspective

Probiotic products have gained popularity with consumers that expect that the products they consume are healthy and help them maintain health. Hence, the need and preferences of the consumers are translated into a product format concept. Probiotics have been used for a long time as natural components in supplements and functional foods, mainly in fermented dairy products. Most of the strains used as probiotics belong to the genera Lactobacillus and Bifidobacterium. By definition, a strain has to have documented health benefits, in order to be called a probiotic. Although each bacterial strain is unique, there are some points that are essential when selecting a probiotic regarding the genetic stability, survival, and technical properties of a strain. Proper components, food matrices and production processes need to be selected since the matrices may affect the viability of the strain in the product and the intestine. Survival in the product is considered a requirement for the beneficial effects of probiotics.     

Probiotic Bacteria May Become Dormant during Storage

The determination of bacterial viability in probiotic products is of economic, technological, and clinical significance. We compared four methods to enumerate three Bifidobacterium strains in fermented oat products during storage. A subpopulation of nonculturable cells retained a functional cell membrane typical of viable cells, indicating that probiotic bacteria become dormant during storage.     

Assuring the continued safety of lactic acid bacteria used as probiotics

Lactic acid bacteria have long been used to improve the safety of foods through fermentation. Some fermented products were also early used for their perceived health benefits, which lead to the development of probiotics as we now know them. Probiotics mainly belong to the genera Lactobacillus and Bifidobacterium. Most members of these genera are not considered pathogens or even opportunistic pathogens. Nevertheless, rare cases of Lactobacillus and Bifidobacterium infection have been reported, possibly even associated with the consumption of probiotic products. Such cases are extremely rare and the subjects always had severe underlying conditions most often affecting the immune system. There does not seem to be any risk for the general population. Safety assessments can be performed and many possible tests exist. It is, however, not certain these tests will prevent rare case of Lactobacillus infection in certain high-risk patients. The benefits of probiotic use should be weighed against the possible small risk. Such an evaluation will, in most cases, be favourable and should therefore not discourage consumption of probiotics. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Polydisulfides of substituted phenols as effective protectors of peroxidase against inactivation by ultrasonic cavitation

Kinetics of inactivation of horseradish peroxidase (HP) induced by low-frequency ultrasonic (US) treatment (27 kHz) with the specific power of 60 W/cm² were studied in phosphate (pH 7.4) and acetate (pH 5.2) buffers within the temperature range of 36.0 to 50.0°C and characterized by effective first-order rate constants of US inactivation k _in (us) in min^–1. Values of k _in (us) depend on the specific ultrasonic power within the range of 20-60 W/cm², on the concentration of HP, and on pH and temperature of the solutions. The activation energy of US inactivation of HP is 9.4 kcal/mole. Scavengers of HO^· radicals, mannitol and dimethylformamide, significantly inhibit the US inactivation of HP at 36.0°C, whereas micromolar concentrations of polydisulfide of gallic acid (poly(DSG)) and of poly(2-aminodisulfide-4-nitrophenol) (poly(ADSNP)) virtually completely suppress the US inactivation of peroxidase at the ultrasonic power of 60 W/cm² on the sonication of the enzyme solutions for more than 1 h at pH 5.2. Various complexes of poly(DSG) with human serum albumin effectively protect HP against the US inactivation in phosphate buffer (pH 7.4). The findings unambiguously confirm a free radical mechanism of the US inactivation of HP in aqueous solutions. Polydisulfides of substituted phenols are very effective protectors of peroxidase against inactivation caused by US cavitation.     

Evaluation of Gambierdiscus survival after exposure to ballast water

The dinoflagellate Gambierdiscus was exposed to ballast water from a trans-oceanic vessel, and maintained at a variety of temperatures in the dark to determine if viability would be maintained. Logarithmically growing Gambierdiscus inocula were admixed (1:6, vol:vol) with ballast water, maintained in the dark at 22.6 °C, 24.6 °C, 26.8 °C and 29.0 °C and assessed for numerical abundance over six days. Calculated growth rates from the biomass time series showed no indication that ballast water negatively impacted Gambierdiscus viability; accompanying microscopic inspections supported this conclusion. Filtration of large volumes of collected ballast water failed to show the presence of any Gambierdiscus cells contained therein. Recovery and microscopic examination of the experimental inocula after 10 weeks in the dark, failed to show cyst development at any temperature regime. This examination of ballast water showed no evidence of cytotoxicity to Gambierdiscus spp.     

Effects of temperature on acaricidal and insecticidal activities of the benzoylureas flucycloxuron and diflubenzuron

Effects of temperature on the activity of flucycloxuron on larval stages of Panonychus ulmi (Koch), based on LC₅₀ values, were highly significant (P < 0.001) with temperature coefficients of-1.7 in both the ranges of 15° to 25°C and 20° to 30°C. The slopes of probit regression lines at 15° and 20°C were significantly steeper than those at 25° and 30°C. As a consequence the temperature coefficients based on LC₉₀ values were-4.4 and-2.2, for the 2 temperature ranges. The ovicidal activity of flucycloxuron on P. ulmi was low and was only statistically detectable at 20°C (LC₉₀ of 84 mg a.i./l). In studies with larvae of Aedes aegypti (Linnaeus), Leptinotarsa decemlineata (Say), Plutella xylostella (Linnaeus), Spodeptera exigua (Hübner) and Spodoptera littoralis (Boisduval) probit regression lines were parallel over temperature. The activity of flucycloxuron on these five insect species was not affected by temperature. Based on LC₅₀ values, diflubenzuron showed positive temperature coefficients on P. xylostella of + 2.1 at 15° to 25°C and + 2.5 at 20° to 30°C. For S. littoralis the temperature coefficient was positive (+ 2.4) at 15° to 25°C but negative (-1.9) at the 20° to 30°C range. Temperature coefficients of diflubenzuron were neutral for A. aegypti, L. decemlineata and S. exigua. In the design and analysis of these studies special allowance was made for date effects and variation in natural mortality over temperature.     

Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ~10⁸ bacteria/ml (equivalent to ~10⁷ CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

In vitro assay of the antimicrobial activity of kephir against bacterial and fungal strains

Kephir is a fermented carbonated refreshing milk, with a slightly acidic aromatic taste and creamy foam composition which contains lactobacilli, leuconostocci, acetic acid bacteria, lactostreptococci and yeasts. Recent studies have demonstrated its antibacterial, immunostimulating, antitumoral and cholesterol-lowering activities.

Purpose

The purpose of this study was to investigate the antimicrobial activity of kephir against Bacillus subtilis spp. spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739, Salmonella enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 9027 and Candida albicans ATCC 10231. The kephir fermented for 24 h and 48 h, as well and after 7 days preservation at 4–8 °C was tested by in vitro disk diffusion method. The intensity of the antimicrobial activity was interpreted by comparison with two antibiotics, i.e. ampicillin and neomycin.

Results

The antimicrobial activity of 24 h as well as 48 fermented kephir, fresh or after 7 days preservation at 4–8 °C was similar and observed against B. subtilis, S. aureus, E. coli, E. faecalis and S. enteritidis. For E. coli, E. faecalis and S. enteritidis the antimicrobial activity was superior to both tested antibiotics and for B. subtilis and S. aureus to one antibiotic. The tested products exhibited no activity against P. aeruginosa and C. albicans.

Conclusion

Kephir is exhibiting large spectrum and strong antibacterial activity probably due to the complex viable probiotic strains association producing antimicrobial substances.     

Seasonal Prevalence ofEntomophthora muscaeand Introduction ofEntomophthora schizophorae(Zygomycotina: Entomophthorales) inMusca domestica(Diptera: Muscidae) Populations on California Dairies

Two entomopathogenic fungi in theEntomophthora muscaespecies complex that infect house flies were used in this study:E. muscaeFresenius (16–17 nuclei/conidium) occurred naturally at four southern California dairies, whileEntomophthora schizophoraeKeller and Wilding (4–8 nuclei/conidium) did not. During the first year of the study, onset of measurableE. muscaeinfections occurred between September and November but varied among sites. At least 20% of the flies at all four dairies were infected by November, and infection at one site exceeded 70%. During the fall epizootic period, infection levels were inversely related to temperature. Average weekly temperatures higher than 17–20°C and maximum daily temperatures higher than 26–28°C were statistically correlated with low infection levels. In the second year,E. schizophoraewas introduced by releasing diseased flies at two dairies (four times at one dairy and three times at the other).E. schizophoraewas recovered for a brief time in the house fly population after the first two releases at one site but not at the second site.     

Effects of pH and deuterium oxide on the heat-inactivation temperature of chloroplasts

The inactivation temperature for Hill activity and for the long-lived delayed fluorescence of isolated Pisum sativum L. chloroplasts was found to depend on pH, the maximal value being in the pH region 5–7. Salts increase the inactivation temperature by 4–7°C. Effects of D₂O and some other substances that modify the thermostability of chloroplasts are dependent on pH. It is concluded that thermal denaturation of proteins is the most probable mechanism for heat inactivation of chloroplasts.Abbreviations Hepes 4-(2-hydroxymethyl)-1-piperazineethane sulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo–MGD) cofactor and one [4Fe–4S] iron–sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap_Sgel) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap_Sput) was examined at varied temperature. Irreversible deactivation of Nap_Sgel and Nap_Sput occurred at 54.5 and 65 °C, respectively. When Nap_Sgel was preincubated at 21–70 °C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 °C, which suggested that Nap_Sgel was poised for optimal catalysis at modest temperatures and, unlike Nap_Sput, did not benefit from thermally-induced refolding. At 20 °C, Nap_Sgel reduced selenate at 16% of the rate of nitrate reduction. Nap_Sput did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap_Sgel that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap_Sgel cold-adapted phenotype. Protein homology modeling of Nap_Sgel using a mesophilic template with 66% amino acid identity showed the majority of substitutions occurred at the protein surface distal to the Mo–MGD cofactor. Two mesophilic ↔ psychrophilic substitutions (Asn ↔ His, Val ↔ Trp) occurred in a region close to the surface of the NapA substrate funnel resulting in potential interdomain π–π and/or cation–π interactions. Three mesophilic ↔ psychrophilic substitutions occurred within 4.5 Å of the Mo–MGD cofactor (Phe ↔ Met, Ala ↔ Ser, Ser ↔ Thr) resulting in local regions that varied in hydrophobicity and hydrogen bonding networks. These results contribute to the understanding of thermal protein adaptation in a redox-active mononuclear molybdenum enzyme and have implications in optimizing the design of low-temperature environmental biosensors.     

Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu)

Eight different strains ofBacillus were isolated from fermented fish (Budu) and their proteolytic enzyme activities were determined after 18 h cultivation at room temperature (35° C). Four isolates possessed high protease activities. Optimum pH for these enzymes was between 7.0 and 8.0 and the optimal temperature was 55° C. The proteases retained 40% of their original activity after 20 min at 55° C but lost all activity at 65° C. Three of the four isolates were identified asBacillus subtilis, the fourth asBacillus licheniformis.     

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk. This revised version was published online in June 2006 with corrections to the Cover Date.     

New species of psychrophilic acetogens: Acetobacterium bakii sp. nov., A. paludosum sp. nov., A. fimetarium sp. nov.

Three strains of new acetogenic bacteria were isolated from several low temperature environments. Cells were gram-positive, oval-shaped flagellated rods. The organisms fermented H₂/CO₂, CO, formate, lactate, and several sugars to acetate. Strains Z-4391 and Z-4092 grew in the temperature range from 1 to 30°C with an optimum at 20°C; strain Z-4290 grew in the range from 1 to 35°C with an optimum at 30°C. The DNA G+C content of strains Z-4391, Z-4092, and Z-4290 was 42.1, 41.7, and 45.8 mol% respectively.     

Insights in starch acetylation in sub- and supercritical CO2

An in-depth study on the acetylation of starch with acetic anhydride (Ac₂O) and sodium acetate (NaOAc) as the catalyst in pressurized carbon dioxide (scCO₂) in a broad pressure range (8–25 MPa) and a temperature of 90 °C is provided. Highest degrees of substitution (DS) of 0.29 (1 h reaction time) and 0.62 (24 h reaction time) were found near the critical point of the mixture (15 MPa). The phase behavior of the system CO₂, starch and acetic anhydride (Ac₂O) was studied in a high pressure view cell. The critical points were a clear function of the temperature and increased from the range of 9.4–10 MPa to 14.5–14.8 MPa when going from 50 to 90 °C (Ac₂O mole fraction at the critical point in the range of 0.08–0.09). Acetylation experiments with a range of starch particles sizes showed a clear relation between the DS and the particle size.     

Flowering of Watsonia laccata as influenced by corm storage and forcing temperatures

The genus Watsonia, belonging to the family Iridaceae, is comprised of about 50 species including W. laccata (Jacquin) Ker Gawler that flowers from September to November following low temperature and winter rainfall. Therefore, we hypothesized that flowering would be favored by forcing at low greenhouse temperatures. Using clonal W. laccata corms, four experiments were designed to investigate the effect of temperatures during corm storage, forcing, and their interaction on growth and flowering. Corm formation is favored by growing plants at 18°–20°/15°–17 °C and 21°–23°/18°–20 °C, day/night temperatures. Flowering was earliest with corms produced at 24°–26°/18°–20 °C and forced at 18°–20/15°–17 °C, and was significantly delayed when forced at 27°–29°/24°–26 °C. Flowering was, however, favored by 2 or 4 weeks of high temperatures (27°–29°/24°–26 °C) prior to forcing at low temperatures (18°–20°/15°–17 °C). The number of florets was not significantly affected by corm storage, forcing temperatures, or their interaction, although forcing at high temperatures tends to reduce the floret number. Burn symptom at the tips of leaves was frequently observed, and further studies are required to understand the cause of the tip burn and how to correct the symptom.     

Temperature and seedling age affect susceptibility of perennial ryegrass seedlings to pathogenic fungi

Summary Effects of temperature and seedling age on survival of perennial ryegrass (Lolium perenne L.) seedlings grown on sand-wheat wholemeal cultures of different isolates ofFusarium spp. (9 isolates),Pythium spp. (9 isolates), andChaetomium spp. (1 isolate) are reported. Some isolates were virulent over the whole range of temperatures tested (7.5–27.5°C). The virulence of others depended on temperature. Most isolates were less virulent at intermediate temperatures (12.5–22.5°C) than at higher or lower temperatures. At 25°C ryegrass seedlings were susceptible to fungal attack for only a limited period after germination commenced. This period differed for different fungi, but for most isolates tested, seedlings were resistant after 2–3 days.     

Hormonal growth-promotant effects on grain-fed cattle maintained under different environments

Six steers (3/4 Charolais×1/4 Brahman) (mean body weight 314±27 kg) and six spayed heifers (3/5 Shorthorn×2/5 Red Angus) (mean body weight 478±30 kg) were used to determine the effects of climatic conditions and hormone growth promotants (HGP) on respiration rate (RR; breaths/min), pulse rate (beats/min), rectal temperature (RT; °C), and heat production (HP; kJ). Cattle were exposed to the following climatic conditions prior to implantation with a HGP and then again 12 days after implantation: 2 days of thermoneutral conditions (TNL) [21.9±0.9°C ambient temperature (T_A) and 61.7±22.1% relative humidity (RH)] then 2 days of hot conditions [HOT; 29.2±4°C (T_A) and 78.3±13.2% (RH)], then TNL for 3 days and then 2 days of cold conditions [COLD; 17.6±0.9°C (T_A) and 63.4±1.8% (RH); cattle were wet during this treatment]. The HGP implants used were: estrogenic implant (E), trenbolone acetate implant (TBA), or both (ET). Both prior to and following administration of HGP, RRs were lower (P<0.05) on cold days and greater (P<0.05) on hot days compared to TNL. On hot days, RTs, were 0.62°C higher after compared to before implanting. Across all conditions, RTs were >0.5°C greater (P<0.05) for E cattle than for TBA or ET cattle. On cold days, RTs of steers were >0.8°C higher than for the heifers, while under TNL and HOT, RTs of steers were 0.2–0.35°C higher than those of heifers. Prior to implantation, HP per hour and per unit of metabolic body weight was higher (P<0.05) for cattle exposed to hot conditions, when compared to HP on cold days. After implantation, HP was greater (P<0.05) on hot days than on cold days. Under TNL, ET cattle had the lowest HP and greatest feed intake. On hot days, E cattle had the lowest HP, and the highest RT; therefore, if the potential exists for cattle death from heat episodes, the use of either TBA or ET may be preferred. Under cold conditions HP was similar among implant groups.