Applied microbiology

Which heat process value F should a particular food receive to make it safe and shelf stable?
* Section 1 lists reported sterilization values F0 = F zero (reference temperature 121.1 oC; z = 10 oC) for commercial food preservation processes of all types of food products, for several package sizes and types.
* Section2 contains reported pasteurization values F, or P, of a great variety of foods. The required storage conditions of the pasteurized foods, either at ambient temperature, or refrigerated (4 - 7 °C), are indicated.
* Section 3 shows a decision scheme: should a particular food be pasteurized or sterilized? This depends on the intended storage temperature (refrigerated or ambient) after heating, the required shelf life (7 days to 4 years), the food pH (high acid, acid, or low acid), the food water activity aW, and on the presence of preservatives such as nitrite (E250) mixed with salt NaCl, or nisin.
* In Section 4, two worked examples are presented on how to use an F value when calculating the actual sterilization time Pt:
- C.R. Stumbo’s (1973) calculation method has been manually applied, verified by computer program STUMBO.exe, to find the sterilization time and the thiamine retention of bottled liquid milk in a rotating steam retort;
- O.T. Pham’s (1987; 1990) formula method,  incorporated in Excel program “Heat Process calculations according to Pham.xls”, has been used  to find the sterilization time and the nutrient retention of canned carrot purée in a still steam retort.
* The worked example in Section 5 illustrates the use of the pasteurization value F of apple juice, in calculating the required (average) residence time in the holding tube of a heat exchanger. The Excel program Build-Heat-Exchanger.xls next calculates the juice’s spoilage rate by a mold, a yeast and an enzyme, and the nutrient retention of vitamins C and folic acid in the pasteurized juice.
* Section 6 explains how F values can be calculated when a microbial analysis of the food is available. The actual pasteurization or sterilization time calculations should be based on all micro-organisms of concern present, and rather NOT on the highest F value only!
An example calculation shows that the required F value for a food, to be sold in a moderate climate country, usually differs considerably from the F, required  for the same food, to be sold in tropical areas.

Aims:  To determine the counts and/or prevalence in fresh bovine faeces of Escherichia coli, enterococci, Campylobacter, Salmonella, shiga toxin-producing E. coli (STEC), Giardia and Cryptosporidium, as inputs to numerical models designed to estimate microbial loadings on pasture grazed by cattle in New Zealand.Methods and Results:  In each season over one year, samples of freshly deposited bovine faeces were collected from four New Zealand dairy farms (n = 155), and enumerated for E. coli, enterococci, Campylobacter, Giardia and Cryptosporidium. They were also tested for the presence of Salmonella and STEC. The overall median bacterial counts (g−1 wet weight) were E. coli– 5·9 × 106; enterococci – 1·3 × 104; Campylobacter– 3·9 × 105. All counts were highly variable within and between samplings, and few seasonal or regional patterns emerged. However, mean Campylobacter counts were consistently higher in spring. No Salmonella spp. was detected, and only two samples were positive for STEC. Cryptosporidium and Giardia were isolated from 5·2% and 4·5% of the samples, respectively, yielding low numbers of (oo)cysts (1–25 g−1 and 1–17 g−1, respectively).Conclusions:  Fresh bovine faeces are a significant source of E. coli, enterococci and Campylobacter on New Zealand pastures, although numbers are likely to vary markedly between faecal samples.Significance and Impact of the Study:  The study provides the first significant set of indicator and pathogen counts for one of the largest sources of faecal contamination of natural waters in New Zealand, and will be used to model these inputs.

Aims:  The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species.Methods and Results:  The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2·5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10−5 TCID50 ml−1. The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot).Conclusions:  The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish.Significance and Impact of the Study:  The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.

In this work, a brewer’s yeast strain was used to remove heavy metals from a synthetic effluent. The solid–liquid separation process was carried out using the flocculation ability of the strain. The yeast strain was able to sediment in the presence of Cu2+, Ni2+, Zn2+, Cd2+ and Cr3+, which evidences that the flocculation can be used as a cheap and natural separation process for an enlarged range of industrial effluents. For a biomass concentration higher than 0.5 g/l, more than 95% of the cells were settled after 5 min; this fact shows that the auto-aggregation of yeast biomass is a rapid and efficient separation process. Cells inactivated at 45 °C maintain the sedimentation characteristics, while cells inactivated at 80 °C lose partially (40%) the flocculation.The passage of metal-loaded effluent through a series of sequential batches allowed, after the second batch, the reduction of the Ni2+concentration in solution for values below the legal limit of discharge of wastewater in natural waters (2 mg/l); this procedure corresponds to a removal of 91%. A subsequent batch had a marginal effect on Ni2+ removal (96%).Together, the results obtained suggest that the use of brewing flocculent biomass looks a promising alternative in the bioremediation of metal-loaded industrial effluents since the removal of the heavy metals and cell separation are simultaneously achieved.

Aims:  The presented study was aimed to reveal transcriptional regulation of genes involved in SDS degradation (sdsA and sdsB) in Pseudomonas sp. ATCC19151. In addition, the ability of Pseudomonas sp. ATCC19151 to degrade anionic surfactants present in commercial detergent and septic tank drain was analysed.Methods and Results:  Strain ATCC19151, at 30°C, degrades all SDS present in the liquid medium (up to 4% w/v of SDS) within 48 h. ATCC19151 grows in the presence up to 15% (v/v) ‘Fairy’ commercial detergent and mineralizes 35% of present anionic surfactants. Analysis of the sdsA (PsdsA) and divergent sdsB (PsdsB) gene promoter activities revealed that SdsB acts as a positive regulator of sdsA and sdsB transcription. PsdsA and PsdsB activities rose significantly in the presence of the SDS, indicating inducibility of sdsA and sdsB transcription. DNA-binding assay indicated that SdsB directly regulates the transcription of sdsA and sdsB genes. Strain ATCC19151 grew in a sterile septic tank drain and on commercial detergent as sole source of carbon.Conclusions:  SdsA enables Pseudomonas sp. ATCC19151 to utilize SDS as a sole carbon source. SdsB is positive transcriptional regulator of sdsA and sdsB genes.Significance and Impact of the Study:  Ability of ATCC19151 to degrade anionic surfactants makes Pseudomonas sp. ATCC19151 a good candidate for bioremediation.

An isolated gene from Neosartorya fischeri NRRL181 encoding a β-glucosidase (BGL) was cloned, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,467 bp, capable of encoding a polypeptide of 488 amino acid residues. The gene was over-expressed in Escherichia coli, and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified recombinant BGL showed a high level of catalytic activity, with V max of 886 μmol min−1 mg-protein−1 and a K m of 68 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature for enzyme activity was about 40°C, and the optimal pH was about 6.0. A homology model of N. fischeri BGL1 was constructed based on the X-ray crystal structure of Phanerochaete chrysosporium BGLA. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose shed light on the unique substrate specificity of N. fischeri BGL1 only towards pNPG.

Pseudomonas putida KT2440 strain was investigated for biosynthesis of the valuable xanthophyll zeaxanthin. A new plasmid was constructed harboring five carotenogenic genes from Pantoea ananatis and three genes from Escherichia coli under control of an l-rhamnose-inducible promoter. Pseudomonas putida KT2440 wild type hardly tolerated the plasmids for carotenoid production. Mating experiments with E. coli S17-1 strains revealed that the carotenoid

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10(3) bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.

In Mass cell producing Factories like Bio fertilizer/Bio pesticide manufacturing factories, Probiotic manufacturing units, aerobic bacteria is produced in submerged fermentation process supplying air through air filters to the fomenters.  In this process there is always a tendency of contamination that crepts in owing to the failure of these air filters. Moreover, filters of considerable quality are very expensive.
In these circumstances, it is conceived to study the possibilities of growing aerobic bacteria without aeration in a closed cycle, which minimizes contamination problems.

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l−1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l−1 and 99 mg l−1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l−1 and 79 mg l−1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg−1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.

In the human large intestine bifidobacteria are a numerically important group of micro-organisms which are considered to exert a range of biological activities related to host health. One aspect is the inhibitory effect of these bacteria on other species, possibly excluding long term colonization by invasive pathogens. It has been suggested that the mechanism of inhibition carried out by bifidobacteria is related to the fermentative production of acids such as acetate and lactate. Experiments reported in this paper attemptedto address this theory. Co-culture experiments whereby Bifidobacterium infantis was incubated with Escherichia coli and Clostridium perfringens, in a varietyof fermentation systems, indicated that the bifidobacterium was able to exert an inhibitory effect not necessarily related to acid production. Further studies showed that eight species of bifidobacteria could variously excrete an anti-microbial substance with a broadspectrum of activity. Species belonging to the genera Salmonella, Listeria, Campylobacter and Shigella, as well as Vibrio cholerae, were all affected. These results show that bifidobacteria are able to exert more than one mechanism of inhibition, which may be of some importance with regard to protection against gastroenteritis.