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.     

Diversity of lactic acid bacteria and yeasts in Airag and Tarag,traditional fermented milk products of Mongolia

We used culture- and molecular-biology-based methods to investigate microbial diversity in the traditional Mongolian fermented milks “Airag” (fermented mare’s milk) and “Tarag” (fermented milk of cows, yaks, goats, or camels). By rRNA or functional gene sequencing, we identified 367 lactic acid bacteria (LAB) strains and 152 yeast strains isolated from 22 Airag and 31 Tarag samples. The total concentration of LAB in Airag (10^{7.78 ± 0.50} c.f.u. ml^–1; mean ± SD) was significantly lower (P < 0.01) than in Tarag (10^{8.35 ± 0.62} c.f.u. ml⁻¹), whereas the total concentration of yeasts in Airag (10^{7.41 ± 0.61} c.f.u. ml^-1) was significantly higher (P < 0.01) than in Tarag (10^{5.86 ± 1.29} c.f.u. ml^-1). Lactobacillus helveticus and Lactobacillus kefiranofaciens were isolated from Airag as the predominant LAB strains at levels of about 10⁷ c.f.u. ml⁻¹, whereas Lactobacillus delbrueckii subsp. bulgaricus, L. helveticus, and Streptococcus thermophilus were the predominant isolates from Tarag at about 10⁷ c.f.u. ml⁻¹. The lactose-fermenting Kluyveromyces marxianus was isolated predominantly from Airag as its major alcoholic fermentation component. Non-lactose-fermenting yeasts such as Saccharomyces cerevisiae, Issatchenkia orientalis, and Kazachstania unispora were the predominant isolates from Tarag, at about 10⁵ c.f.u. ml⁻¹. The apparent geographic differences in the L. kefiranofaciens and S. thermophilus contents of Tarag strongly suggested that differences among the animal species from which the milk was sourced, rather than geographic distances, were the most important factors influencing the diversity of the microbial composition of traditional fermented milks in Mongolia.     

Effects of Acetic Acid on the Regrowth of Heterotrophic Bacteria in the Drinking Water Distribution System

Summary Three laboratory-scale water pipe systems were set up to study the effects of adding two levels of acetic acid (10 and 50 μg acetate eq-C l⁻¹) on the bacterial regrowth in water pipes. The results of the water pipe test showed that nearly all carbon in the acetic acid could be readily utilized by bacteria and resulted in an increase in biomass concentration. The maximum heterotrophic plate counts in biofilm were equal to 3.5 × 10⁴, 8.9 × 10⁵ and 2.9 × 10⁷ c.f.u. cm⁻² while the maximum heterotrophic plate counts of free bacteria were equal to 1.2 × 10³, 5.0 × 10³ and 6.8 × 10⁴ c.f.u. ml⁻¹ for the blank and with addition of 10 and 50 μg acetate eq-C l⁻¹. These results showed that addition of acetic acid to drinking water has a positive effect on the assimilable organic carbon content of drinking water and bacterial regrowth in the distribution system. This effect is enhanced with addition of high-level acetic acid. Batch tests were also conducted using water samples collected from a Taiwanese drinking water distribution system. The bacterial regrowth potentials of the blank were equal to 4.3 × 10³, 1.5 × 10⁴, 4.9 × 10⁴ and 7.5 × 10⁴ c.f.u. ml⁻¹ for water samples collected from treatment plant effluent, commercial area, mixed area, and residential area, respectively. These results showed that the biological stability of drinking water is the highest in treatment plant effluent, followed by distributed water of the commercial area, distributed water of the mixed area, and then the distributed water of residential area.     

Detection of Viable Toxigenic Vibrio cholerae from Environmental Water Sources by Direct Cell Duplex PCR Assay

Summary Environmental monitoring is important to enable effective resource management and public health protection as well as rapid and accurate identification of Vibrio cholerae in drinking-water sources. Traditional methods employed in identification are laborious, time-consuming and practically not viable for screening of a large number of samples. In this study, a direct cell duplex PCR assay for the detection of viable toxigenic V. cholerae in environmental water samples was developed. In the PCR assay, two gene sequences were amplified together, one of outer membrane protein (ompW), which is species-specific and another of cholera toxin (ctxAB). The detection limit of duplex PCR was 5 × 10⁴ V. cholerae/reaction. Different environmental water samples were artificially spiked with V. cholerae O1 cells and filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly in the duplex PCR assay. The PCR procedure coupled with enrichment could detect as few as 1.2 c.f.u./ml in ground water, 1.2 × 10² c.f.u. ml⁻¹ in sewer water and 1.2 × 10³c.f.u. ml⁻¹ in tap water. The assay was successfully applied directly for screening of environmental potable water samples collected from a cholera-affected area. The proposed method is simple and can be used for environmental monitoring of toxigenic as well as non-toxigenic V. cholerae.     

Production,formulation and antagonistic activity of the biocontrol like-yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> against <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Aureobasidium pullulans (de Bary) Arnaud (Ach 1-1) was grown in a glucose fed-batch fermentor to 106 g dry wt l⁻¹ in 48 h. The cells were dried in a fluidized bed dryer with a final viability of 62%. After 7 months at 4°C, the viability was 28% of the initial value (= 2.3 × 10¹⁰ c.f.u. g⁻¹ dry matter). A protection level of 89% was achieved with the biomass preparation at 1 × 10⁸ c.f.u. ml⁻¹ after 28 and 7 days for apples stored respectively at 5 and 25°C against Penicillium expansum. Our process is suitable to produce large quantities of the strain Ach 1-1 as biological control agent for apple preservation.     

Aerobic heterotrophic bacteria and petroleum-utilizing bacteria from cow dung and poultry manure

In this study, enumeration and identification of total aerobic heterotrophic bacteria and petroleum-utilizing bacteria as well as the degradative potential of petroleum-utilizing bacterial isolates were carried out. The average counts of total aerobic heterotrophic bacteria in cow dung and poultry manure were 74.25 × 10⁵ c.f.u. g⁻¹ and 138.75 × 10⁵ c.f.u. g⁻¹ respectively. Acinetobacter sp, Bacillus sp, Pseudomonas sp, and Serratia spp. occurred as aerobic heterotrophs in both cow dung and poultry manure. However, Alcaligenes spp. occurred only in cow dung while, Flavobacterium sp, Klebsiella sp, Micrococcus sp, and Nocardia spp. occurred only in poultry manure as aerobic heterotrophs. The average counts of petroleum-utilizing bacteria in cow dung and poultry manure were 9.25 × 10⁵ c.f.u. g⁻¹ and 17.25 × 10⁵ c.f.u. g⁻¹ respectively. Pseudomonas spp. occurred as petroleum utilizer in both cow dung and poultry manure. However, Bacillus spp. occurred only in cow dung while Acinetobacter spp. and Micrococcus spp. occurred only in poultry manure as petroleum utilizers. Relative abundance of petroleum utilizers in total aerobic heterotrophs ranged from 6.38% to 20.00% for cow dung and from 9.38% to 17.29% for poultry manure. Introduction of pure cultures of petroleum-utilizing bacteria from cow dung and poultry manure into sterile oil-polluted soil revealed oil degradation in one week period.     

<Emphasis Type="Italic">In vitro</Emphasis> assessment of immunomodulating activity of the two <Emphasis Type="Italic">Lactobacillus</Emphasis> strains isolated from traditional fermented milk

The objective of this study was to evaluate the in vitro immunomodulating capacity of Lactobacillus coryniformis subsp torquens T3L (L. coryniformis T3L) isolated from traditional fermented yak’s milk in Tibet, China, and Lactobacillus paracasei supsp. paracasei M5L (L. paracasei M5L)isolated from kumiss in Sinkiang, China was used as control. The effects of live bacteria, cell wall and genomic DNA of the two Lactobacillus strains on human peripheral blood mononuclear cells (PBMCs) proliferation, production of interleukin-12 (IL-12 p70), interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and natural killer (NK) cell activity were assessed. The live bacteria, cell wall and genomic DNA of the two lactobacilli exerted proliferative effects on PBMCs. Live bacteria at 1 × 10⁶ c.f.u. ml⁻¹, cell wall at 20 μg protein ml⁻¹ and DNA at 50 μg DNA ml⁻¹ of the strainS induced the secretion of IL-12 (p70), IFN-γ and TNF-α by PBMCs. NK cell activities increased after cultivation of PBMCs with live bacteria, cell wall and DNA of the strains. Overall, these results demonstrate that the live bacteria, cell wall and genomic DNA of the two Lactobacillus strains exhibit immunomodulating activity.     

Ginsenoside F1 production from ginsenoside Rg1 by a purified β-glucosidase from Fusarium moniliforme var. subglutinans

Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F₁ from ginsenoside Rg₁. The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg⁻¹. It hydrolysed glucose-linked ginsenosides Rb₁, Rd and Rg₁ but not for other monosaccharide-linked ginsenosides, Rb₂, Rc, R₁, and Re. Under the optimum conditions of pH 6.0, 50°C, 30 U l⁻¹ of enzyme, and 5 mg Rg₁ ml⁻¹, 4 mg F₁ ml⁻¹ was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l⁻¹ h⁻¹. This represents the highest productivity and conversion yield of F₁ yet reported.     

Effect of Abiotic Stress on Phosphate Solubilization by Biocontrol Fungus <Emphasis Type="Italic">Trichoderma</Emphasis> sp.

This study was undertaken to explore the role of Trichoderma sp. in phosphate (P) solubilization and antagonism against fungal phytopathogens. All fungal isolates (SE₆, KT₆, KT₂₈, and BRT₁₁) and a standard culture of T. harzianum (Th-std) were able to antagonize two fungal phytopathogens (Sclerotium rolfsii and Rhizoctonia solani) of chickpea (Cicer arietinum L.) wilt complex. Transmission electron microscopic studies (TEM) further confirmed ultra-cytological changes in the sclerotia of S. rolfsii parasitized by Trichoderma sp. All fungal cultures exhibited production of NH₃ and siderophore, but only BRT₁₁, SE₆, and Th-std could produce HCN. Among all the cultures tested, isolate KT₆ was found to be most effective for solubilization of ferric phosphate releasing 398.4 μg ml⁻¹ phosphate while isolates BRT₁₁ and SE₆ showed more potential for tricalcium phosphate (TCP) solubilization releasing 449.05 and 412.64 μg ml⁻¹ phosphate, respectively, in their culture filtrates. Part of this study focused on the influence of abiotic stress conditions such as pH, temperature, and heavy metal (cadmium) on phosphate (TCP) solubilizing efficiency. Two selected cultures KT₆ and T. harzianum retained their P solubilizing potential at varying concentrations of cadmium (0–1000 μg ml⁻¹). Isolate KT₆ and standard culture of T. harzianum released 278.4 and 287.6 μg ml⁻¹ phosphate, respectively, at 1000 μg ml⁻¹cadmium. Maximum solubilization of TCP was obtained at alkaline pH and at 28°C temperature. Isolate BRT₁₁ was found most alkalo-tolerant releasing 448.0 μg ml⁻¹ phosphate at pH 9.     

Carob pulp as raw material for production of the biocontrol agent <Emphasis Type="Italic">P. agglomerans</Emphasis> PBC-1

Large-scale production has been the major obstacle to the success of many biopesticides. The spreading of microbial biocontrol agents against postharvest disease, as a safe and environmentally friendly alternative to synthetic fungicides, is quite dependent on their industrial mass production from low-cost raw materials. Considerable interest has been shown in using agricultural waste products and by-products from food industry as nitrogen and carbon sources. In this work, carob pulp aqueous extracts were used as carbon source in the production of the biocontrol agent Pantoea agglomerans PBC-1. Optimal sugar extraction was achieved at a solid/liquid ratio of 1:10 (w/v), at 25°C, for 1 h. Batch experiments were performed in shake flasks, at different concentrations and in stirred reactors at two initial inoculums concentrations, 10⁶ and 10⁷ cfu ml⁻¹. The initial sugar concentration of 5 g l⁻¹ allowed rapid growth (0.16 h⁻¹) and high biomass productivity (0.28 g l⁻¹ h⁻¹) and was chosen as the value for use in stirred reactor experiments. After 22 and 32 h of fermentation the viable population reached was 3.2 × 10⁹ and 6.2 × 10⁹ cfu ml⁻¹ in the fermenter inoculated at 10⁶ cfu ml⁻¹ and 2.7 × 10⁹ and 6.7 × 10⁹ cfu ml⁻¹ in the bioreactor inoculated at 10⁷ cfu ml⁻¹. A 78% reduction of the pathogen incidence was achieved with PBC-1 at 1 × 10⁸ cfu ml⁻¹, grown in medium with carob extracts, on artificially wounded apples stored after 7 days at 25°C against P. expansum.     

Endocrinological investigation into the reproductive cycles of two sympatric skate species, Malacoraja senta and Amblyraja radiata, in the western Gulf of Maine

The smooth skate, Malacoraja senta, and thorny skate, Amblyraja radiata, are two commercially exploited batoids found within the Gulf of Maine. During the past five years, we conducted a large study to accurately describe important biological life history parameters previously lacking for these species. As part of that project, the current study reports our findings on the hormonal profiles associated with the reproductive cycles of M. senta and A. radiata. Blood samples were obtained from mature M. senta and A. radiata of both sexes from all months of the year, and plasma testosterone (T), estradiol (E₂) and progesterone (P₄) concentrations were determined using radioimmunoassay (RIA). In female M. senta and A. radiata, monthly T concentrations ranged from 4,522 pg ml⁻¹ to 1,373 pg ml⁻¹ and 31,940 pg ml⁻¹ to 14,428 pg ml⁻¹, E₂ concentrations from 831 pg ml⁻¹ to 60 pg ml⁻¹ and 8,515 pg ml⁻¹ to 2,902 pg ml⁻¹, and P₄ concentrations from 3,027 pg ml⁻¹ to 20 pg ml⁻¹ and 3,264 pg ml⁻¹ to 331 pg ml⁻¹, respectively. No statistical differences were detected between any months for any hormone. Estradiol concentrations were not correlated with ovary weight, shell gland weight, or diameter of the largest follicles in either species. Monthly T concentrations in male M. senta and A. radiata ranged from 23,146 to 12,660 pg ml⁻¹ and from 57,500pg ml⁻¹ to 24,737 pg ml⁻¹, while E₂ concentrations ranged from 7.5 pg ml⁻¹ to undetectable and 103 to 30 pg ml⁻¹, respectively. No statistical differences were observed between months for either steroid. Testosterone concentrations were weakly correlated with testes weight and percent of stage VI spermatocysts in A. radiata, however, no correlation was detected between T and stage VI spermatocysts in M. senta. Collectively, these data support the previous conclusion that M. senta and A. radiata of both sexes are capable of reproducing year round in the western Gulf of Maine.     

Identification and characterization of a novel xylanase derived from a rice straw degrading enrichment culture

A metagenomic library containing ca. 3.06 × 10⁸ bp insert DNA was constructed from a rice straw degrading enrichment culture. A xylanase gene, umxyn10A, was cloned by screening the library for xylanase activity. The encoded enzyme Umxyn10A showed 58% identity and 73% similarity with a xylanase from Thermobifida fusca YX. Sequence analyses showed that Umxyn10A contained a glycosyl hydrolase family 10 catalytic domain. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically. Recombinant Umxyn10A was highly active toward xylan. However, the purified enzyme could slightly hydrolyze β-1,3/4-glucan and β-1,3/6-glucan. Umxyn10A displayed maximal activity toward oat spelt xylan at a high temperature (75°C) and weak acidity (pH 6.5). The K _m and V _max of Umxyn10A toward oat spelt xylan were 3.2 mg ml⁻¹ and 0.22 mmol min⁻¹ mg⁻¹ and were 2.7 mg ml⁻¹ and 1.0 mmol min⁻¹ mg⁻¹ against birchwood xylan, respectively. Metal ions did not appear to be required for the catalytic activity of this enzyme. The enzyme Umxyn10A could efficiently hydrolyze birchwood xylan to release xylobiose as the major product and a negligible amount of xylose. The xylanase identified in this work may have potential application in producing xylobiose from xylan.     

Coinoculation of Bacillus thuringeinsis-KR1 with Rhizobium leguminosarum enhances plant growth and nodulation of pea (Pisum sativum L.) and lentil (Lens culinaris L.)

Nodulation and the subsequent nitrogen fixation are important factors that determine the productivity of legumes. The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote plant growth of field pea (Pisum sativum L.) and lentil (Lens culinaris L.) under Jensen’s tube, growth pouch and non-sterile soil, respectively, when co-inoculated with Rhizobium leguminosarum-PR1. Coinoculation with B. thuringiensis-KR1 (at a cell density of 10⁶ c.f.u. ml⁻¹) provided the highest and most consistent increase in nodule number, shoot weight, root weight, and total biomass, over rhizobial inoculation alone. The enhancement in nodulation due to coinoculation was 84.6 and 73.3% in pea and lentil respectively compared to R. leguminosarum-PR1 treatment alone. The shoot dry-weight gains on coinoculation with variable cell populations of B. thuringiensis-KR1 varied from 1.04 to 1.15 times and 1.03 to 1.06 times in pea and lentil respectively, while root dry weight ratios of coinoculated treatments varied from 0.98 to 1.14 times and 1.08 to 1.33 times in pea and lentil respectively, those of R. leguminosarum-PR1 inoculated treatment at 42 days of plant growth. While cell densities higher than 10⁶ c.f.u. ml⁻¹ had an inhibitory effect on nodulation and plant growth, lower inoculum levels resulted in decreased cell recovery and plant growth performance. The results of this study indicate the potential of harnessing endophytic bacteria of wild legumes for improving the nodulation and growth of cultivated legumes.     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Characterization of a recombinant endo-type alginate lyase (Alg7D) from <Emphasis Type="Italic">Saccharophagus degradans</Emphasis>

A gene, alg7D, from Saccharophagus degradans, coding for a putative alginate lyase belonging to the family of polysaccharide lyase-7, was overexpressed in Escherichia coli. The properties of the recombinant Alg7D were characterized. The enzyme endolytically depolymerized alginate by β-elimination into oligo-alginates with degrees of polymerization of 2–5. Its activity was maximal at 50°C and pH 7 and was slightly increased in the presence of Na⁺. The K _M , V _max , k _cat , and k _cat /K _M values were: 3 mg ml⁻¹, 6.2 U mg⁻¹, 1.9 × 10⁻² s⁻¹, and 6.3 × 10⁻³ mg⁻¹ ml s⁻¹, respectively.     

Inhibition of galectin-3 mediated cellular interactions by pectic polysaccharides from dietary sources

Pectic polysaccharides from dietary sources such as Decalepis hamiltonii—swallow root (SRPP), Hemidesmus indicus (HPP), Nigella sativa—black cumin (BCPP), Andrographis serpyllifolia—(APP), Zingiber officinale—ginger (GRPP) and, citrus pectin (CPP) were examined for galectin inhibitory activity. Inhibition of (a) galectin-3 of MDA-MB-231 cells induced hemagglutination of red blood cells; (b) galectin-3 mediated interaction between normal/metastatic human buccal cells (NBC)/(MBC) and; (c) invasion of MDA-MB-231 and MBC in the invasive chamber was assessed. Results indicated that SRPP inhibited hemagglutination at Minimum Inhibitory Concentration (MIC) of 1.86 μg ml⁻¹ equivalent of carbohydrate as apposed to those of BCPP (130 μg ml⁻¹), APP (40 μg ml⁻¹), HPP (40 μg ml⁻¹) and CPP (25 μg ml⁻¹). GRPP even at concentration >1–6 mg ml⁻¹ did not inhibit agglutination. Also SRPP showed ∼15 and 2 fold potent anti hemagglutination activity relative to that of galectin-3 specific sugars—galactose (MIC-27.1 μg ml⁻¹) and lactose (MIC-4.16 μg ml⁻¹) respectively. Further, SRPP at 10 μg ml⁻¹ inhibited agglutination of NBC by galectin-3 of MDA-MB-231 cells. Modified swallow root pectic polysaccharide (MSRPP) of 50 kDa retained anti hemagglutination activity (MIC of 1.03 μg ml⁻¹) and inhibited MDA-MB-231 and MBC invasion by 73 and 50% with an IC₅₀ of 136 and 200 μg ml⁻¹ respectively. Both SRPP and MSRPP induced apoptosis up to 80% at 100 μg ml⁻¹ concentration by activating ∼2 and 8 folds of Caspase-3 activity. Sugar composition analysis and its correlation with the galectin inhibitory property indicated that pectic polysaccharides with higher arabinose and galactose content—arabinogalactan inhibited hemagglutination significantly.     

Isolation of sensitive nisin-sensing GFP<Subscript>uv</Subscript> bioassay <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains using FACS

Fluorescence-activated cell sorting (FACS) was used to isolate mutants of Lactococcus lactis LAC275, an indicator strain in GFP_uv nisin bioassay. It harbors the GFP_uv encoding gene under the nisA promoter and the nisin signal transduction nisRK genes whereby nisin concentration can be correlated to GFP_uv fluorescence. The sorted L. lactis cells, which showed higher fluorescence intensities at low inducer concentration, were analysed for higher responsiveness to low concentration of nisin. Two strains showed lower detection limits (0.2 pg ml⁻¹) for nisin than the parent strain (10 pg ml⁻¹). This showed that mutants of LAC275 could successfully be isolated using FACS.     

Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes

In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml^–1) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml^–1 dropped to 10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml^–1 probably reflects a loss of cell division capability rather than cell death.     

Characterization of a thermostable endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from <Emphasis Type="Italic">Caldicellulorsiruptor saccharolyticus</Emphasis>

A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg⁻¹ by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K _m, k _cat, and k _cat/K _m values were 18 mg ml⁻¹, 50 s⁻¹, and a 2.8 mg ml⁻¹ s⁻¹, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440, 254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases.