THE INFLUENCE OF ASSIMILABLE NITROGEN COMPOUNDS IN WORT ON THE ABILITY OF YEAST TO REDUCE DIMETHYL SULPHOXIDE

Dimethyl sulphoxide (DMSO) reductase activity increased at the end of the growth cycle of Saccharomyces cerevisiae NCYC 240 on complex medium. On defined medium, DMSO reductase and thioredoxin reductase activities and thioredoxin were all elevated in cultures limiting in nitrogen, but not in nitrogen-sufficient cultures. In nitrogen limiting cultures it was also shown that the reduction of DMSO (measured as disappearance of [¹⁴C] from the medium) was much increased compared with conditions of nitrogen excess. This applied whether ammonium or glutamate was the limiting nitrogen source. When methionine was used as the limiting nitrogen source, DMSO reduction was higher still. Methionine-limited cells accumulated [¹⁷C] methionine much more rapidly than did cells from methionine-sufficient cultures, and [¹⁴C] methionine sulphoxide could be identified in the cells after 1 min incubation with labelled methionine. It is concluded that DMSO reductase activity is controlled by nitrogen catabolite repression and may be involved in methionine uptake by yeast. The amino nitrogen content of wort will in consequence have an important effect on levels of dimethylsulphide (DMS) produced during fermentation.     

Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate

The influence of dilution rate (D) and aeration on soluble and cell-bound nisin Z production was investigated during continuous free (FC) and immobilized cell (IC) cultures with Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in supplemented whey permeate. Maximum total bacteriocin titres during non-aerated continuous FC and IC cultures were obtained for low D, with 1490 and 1090 IU mL⁻¹ for 0.15 h⁻¹ or 0.25 and 0.5 h⁻¹, respectively. For both systems, aeration increased nisin total production with maximum titres of 2560 and 2430 IU mL⁻¹ for low D, respectively, as well as specific production. Volumetric productivity was the highest for an intermediate D of 0.4 h⁻¹ during FC cultures (460 IU mL⁻¹ h⁻¹ for both aerated and non-aerated cultures), while it increased continuously with D during IC cultures, reaching high values of 1090 and 1760 IU mL⁻¹ h⁻¹ at 2.0 h⁻¹ without and with aeration, respectively. In comparison with previous data for FC batch cultures, data from this study may indicate that during continuous fermentations at steady state, some steps in nisin biosynthesis are limiting. In these conditions, nisin production by immobilized cells is reduced.     

Antibacterial Activities of Chitosans and Chitosan Oligomers with Different Molecular Weights on Spoilage Bacteria Isolated from Tofu

Seven bacteria were isolated from spoiled tofu and identified as Bacillus sp. (S08), B. megaterium (S10), B. cereus (S17, S27, S28, S32), and Enterobacter sakazakii (S35). In a paper disc test with 6 chitosans and 6 chitosan oligomers of different molecular weights, chitosans showed higher antimicrobial activity than did chitosan oligomers at a 0.1% concentration. Results of inhibitory effects of 6 chitosans on growth of Bacillus sp. (S08) failed to detect viable cells after incubation for 24 hrs at 37 C, even at 0.02% concentration. With B. megaterium (S10) and B. cereus (S27), a 3 to 4 log cycle reduction was found in the chitosan‐treated group. The growth of Enterobacter sakazakii (S35) was completely suppressed in the presence of 0.04% chitosan except for 1 chitosan product. The minimum inhibitory concentration of chitosan differed with products and isolates, ranging from 0.005% to above 0.1%.     

Glucoamylase Biosynthesis by Cells of Aspergillus niger C58-III Immobilized in Sintered Glass and Pumice Stones

A simple method of A. niger C_58-III cell immobilization is described. This strain produces extracellular glucoamylase. According to the proposed method A. niger spores were first immobilized by adsorption in sintered glass Rasching rings (RR) or pumice stones (PS). Growing out from spores, A. niger cells produced extracellular glucoamylase. This technique facilitates the culture growth in a filamentous spongy structure of the supports with a continuous accumulation of biomass. After every 24 h it was possible to obtain culture liquid rich in glucoamylase. This procedure can be repeated 30 times using the same sample of immobilized A. niger culture without any loss of glucoamylase activity in the liquid medium. In a 96 h period immobilized A. niger cells produced 300 units × ml⁻¹ whereas a shake culture of this fungus produced only 186 units × ml⁻¹.     

Effect of microencapsulation on survival of Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration, and yogurt

In the present research the survival of free and microencapsulated cells of a new strain of Lactobacillus plantarum BL011 under stress conditions was tested in sodium alginate or pectin, coated with sodium alginate or chitosan. Results for the simulated gastrointestinal medium (SGT) showed no change in viability of cells in relation to the control. However, the simulated gastric medium (GM) drastically reduced the viability under the tested conditions, with no significant differences between free and immobilized cells. Under refrigerated storage viability of immobilized cells were greatly enhanced compared to the free microorganisms, and the treatments showing the lowest loss of viability were those of 4% (w/v) pectin, 3% (w/v) sodium alginate coated with chitosan and a mixture of 2% (w/v) sodium alginate and 2% (w/v) pectin, respectively. Loss of viability of immobilized L. plantarum in 3% alginate coated with chitosan in yogurt was of 0.55 log cycles during 38 days of storage. The results of this study suggest the efficiency of immobilization techniques to increase the survival of lactobacilli in yogurt under refrigerated storage.     

Shelf life extension of ground meat stored at 4 °C using chitosan and an oxygen absorber

The combined effect of chitosan (1%) and an oxygen absorber on shelf life extension of fresh ground meat stored at 4 °C was investigated. Parameters monitored over a 10‐day storage period were Total Viable Count (TVC), Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria, Enterobacteriaceae, pH, thiobarbituric acid (TBA), colour, odour and taste. Microbial populations were reduced by 0.4–2.0 log CFU g^?1 for a given sampling day using either chitosan or the oxygen absorber, with the more pronounced effect being achieved by the combination of two. Thiobarbituric acid values for all samples increased during storage with the exception of samples treated with both chitosan and the oxygen absorber in which Thiobarbituric acid values decreased. Changes in pH values with time/different treatments were statistically insignificant. Colour parameters were affected by most treatments. On the basis of microbiological and sensory evaluation, a shelf life extension of 5–6 days was obtained for samples treated with the chitosan and the oxygen absorber combination.     

Enhancement of Antimicrobial Activity of Chitosan by Irradiation

Antimicrobial activity of irradiated chitosan was studied against Escherichia coli B/r. Irradiation of chitosan at 100 kGy under dry conditions was effective in increasing the activity, and inhibited the growth of E coli completely. The molecular weight of chitosan significantly decreased with the increase in irradiation dose, whereas the relative surface charge of chitosan was decreased only 3% by 100 kGy irradiation. Antimicrobial activity assay of chitosan fractionated according to molecular weight showed that 1×10⁵–3×10⁵ fraction was most effective in suppressing the growth of E coli. This fraction comprised only 8% of the 100 kGy irradiated chitosan. On the other hand, chitosan whose molecular weight was less than 1×10⁵ had no activity. The results show that low dose irradiation, specifically 100 kGy, of chitosan gives enough degradation to increase its antimicrobial activity as a result of a change in molecular weight. © 1997 SCI.     

Effect of chitosan on incidence of brown rot,quality and physiological attributes of postharvest peach fruit

The effect of chitosan (5.0 and 10.0 mg ml ⁻¹) on the incidence of brown rot (caused by Monilinia fructicola), quality attributes and senescence physiology of peaches was investigated. It was found that both concentrations of chitosan reduced the incidence of brown rot significantly and delayed the development of disease compared with the control, but were less effective than the fungicide prochloraz. Chitosan‐treated peaches were firmer and had higher titratable acidity and vitamin C content than prochloraz‐treated or control peaches. Compared to control (water‐treated) peaches, chitosan‐treated peaches showed lower respiration rate, less ethylene and malondialdehyde (MDA) production, higher superoxide dismutase (SOD) activity and better membrane integrity. Hence it can be concluded that chitosan has the potential to control brown rot, preserve valuable attributes and prolong the shelf life of postharvest peaches, presumably because of its antifungal property and inhibition of the ripening and senescence process of postharvest peaches. © 2000 Society of Chemical Industry     

High nisin-Z production during repeated-cycle batch cultures in supplemented whey permeate using immobilized Lactococcus lactis UL719

Nisin-Z production was studied during repeated-cycle pH-controlled batch (RCB) cultures using Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 immobilized in κ-carrageenan/locust bean gum gel beads in supplemented whey permeate. After an initial colonization of gel beads during the first two cycles, nisin-Z production in bulk medium and gel beads was very similar for 1-h and 2-h cycle RCB cultures. A very high nisin-Z production (8200 IU mL⁻¹) was measured in the broth after the 1-h cycles, with a corresponding volumetric productivity of 5730 IU mL⁻¹ h⁻¹. This productivity is much higher than maximum nisin productivities reported in literature or maximum productivities obtained previously for free-cell batch cultures (850 IU mL⁻¹ h⁻¹), and free-cell (460 IU mL⁻¹ h⁻¹) or immobilized-cell (1760 IU mL⁻¹ h⁻¹) continuous cultures, using the same strain and fermentation conditions. The stability of RCB cultures was demonstrated for 24 and 36 1-h cycles carried out over 3 and 6-day periods, respectively. Changing environmental conditions during batch cultures resulted high nisin production.     

Study on the degradation of chitosan by pulsed electric fields treatment

Chitosan solution was processed by applying pulsed electric fields (PEF) with different strengths up to 25 kV cm^− 1. Changes of the physicochemical properties of chitosan, such as molecular weight and crystallinity degree, were measured by analyses of scanning electron microscopy (SEM), viscosity molecular weight (M_v), X-ray diffractometry (XRD), FTIR and UV spectra. The results showed that after being treated at 25 kV cm^− 1, the chitosan granules were significantly deformed with many pits and cracks appeared on the surface. M_v was decreased with the increasing electric field strength, for example, the M_v was decreased from 2.81 × 10⁵ Da (initial chitosan) to 1.54 × 10⁵ Da after the PEF treatment at 25 kV cm^− 1. Meanwhile, the crystalline region of the treated sample was significantly damaged from XRD patterns. All results showed that the PEF technique is a possible method to obtain low molecular-weight chitosan.

Industrial Relevance

Chitosan solution was treated by using pulsed electric fields (PEF) with different strengths up to 25 kV·cm^-1. Changes of the physicochemical properties of chitosan were measured by analyses of scanning electron microscopy (SEM), viscosity molecular weight (M_v), X-ray diffractometry (XRD), FTIR and UV spectra. The results showed that the PEF technique is a promising method for scale-up industrial manufacture of low-molecular-weight chitosan.     

Effects of chitosan coating on oxidative stress in bruised Yali pears (Pyrus bretschneideri Rehd.)

The protective effects of chitosan on oxidative stress in bruised Yali pears (Pyrus bretschneideri Rehd.) were investigated. The fruit were treated with 1.5% chitosan before or after damage, respectively, and then stored at 16 °C and 90% relative humidity. Postharvest quality, reactive oxygen species (ROS) and antioxidant enzymes were analysed. Results showed that bruise induced the accumulation of H₂O₂ and O^2?. However, chitosan treatments reduced the levels of ROS and delayed the decease of glutathione (GSH) content in bruised fruit. Meanwhile, activities of antioxidant enzymes, including catalase, peroxidases, superoxide dismutase, ascorbate peroxidase or GSH reductase was 40.7%, 98.1%, 62.3%, 127.8% or 23.8% higher in chitosan‐bruised fruit and 41.1%, 80.8%, 18.5%, 102.9% or 45.2%, respectively, higher in bruised‐chitosan fruit than untreated bruised fruit on the 15th day.     

Pyruvate decarboxylase: An indispensable enzyme for growth of Saccharomyces cerevisiae on glucose

In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate-decarboxylase-negative (Pdc⁻) mutant lacking all three PDC genes exhibited a three-fold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc⁻ strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc⁻ and wild-type S. cerevisiae were identical. However, Pdc⁻ S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0·10 h⁻¹) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.     

Dephytinization of food stuffs by phytase of Geobacillus sp. TF16 immobilized in chitosan and calcium-alginate

In this work, Geobacillus sp. TF16 phytase was separately immobilized in chitosan and Ca-alginate with the efficiency of 38% and 42%, respectively. These enzymes exhibited broad substrate specificity. Maximal relative phytase activity was measured at pH 5.0 and 95°C and pH 3.0 and 75°C for chitosan and Ca-alginate, respectively. The enzymes were highly stable in a wide pH and temperature range. Values of K_m and V_max were determined as 2.38 mM and 3401.36 U/mg protein for chitosan, and 7.5 mM and 5011.12 U/mg protein for Ca-alginate. The immobilized enzymes showed higher resistance to proteolysis. After 4 h incubation, hydrolysis capacities of chitosan- and Ca-alginate immobilized enzymes for soymilk phytate were calculated as 24% and 33%, respectively. The chitosan- and Ca-alginate immobilized phytases conserved its original activity after 8 and 6 cycles of reuse, respectively. The features of the enzymes were very attractive and they might be useful for some industrial applications.     

Enzymatic grafting of gallate ester onto chitosan: evaluation of antioxidant and antibacterial activities

The antioxidant octyl gallate (OG) has been successfully grafted onto chitosan by means of horseradish peroxidase biocatalyst. The maximum gallate incorporation onto chitosan determined by ¹H NMR spectroscopy was up to 16 molar%. The resulting materials displayed antioxidant capacities with DPPH radical inhibition percentage up to 23% upon the assay conditions. The grafting of the antioxidant on chitosan enhanced its antimicrobial activity. Minimal inhibitory concentration (MIC) for Escherichia coli was 0.5 g L^?1. The native medium molecular weight chitosan alone or grafted with the lowest OG incorporation (ca. 11 molar%) attained 100% inhibition of E. coli, whereas lower inhibition was observed for all other materials 50.7–68.9% corresponding a reduction in the counts from 10.6 to 5.23–3.30 Log CFU mL^?1. The inhibition of Listeria monocytogenes was significantly higher (59.8–100%) than that with the Gram negative bacterium reaching the MIC at 0.25 g L^?1 with a reduction in the counts from 12.6 Log CFU mL^?1 to 5.06–0 Log CFU mL^?1.     

Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.     

Antifungal activity of edible coating made from chitosan and lactoperoxidase system against Phomopsis sp. RP257 and Pestalotiopsis sp. isolated from mango

Lactoperoxidase system (LPOS) was incorporated into chitosan solutions (0.5, 1, and 1.5%) to protect mangoes against two strains of fungi. Coating solutions effectiveness in vitro (in Petri dish) and in vivo (on mango) were studied on fungal (Phomopsis sp. RP257 and Pestalotiopsis sp.) growth isolated from mango cv Amelie. In vitro, chitosan concentration at least 1% containing or not LPOS effectively inhibited Pestalotiopsis sp. growth at 100%. Presence of LPOS or Lactoperoxydase system with iodine (LPOSI) in chitosan at 0.5% increased the percentage of inhibition from 26 to 93%. Edible films with LPOS inhibited Phomopsis sp. RP257 particularly when LPOS was incorporated in chitosan concentrations of 1 and 1.5%. Iodine did not influenced antifungal activity of LPOS against Pestalotiopsis sp. but decreased activity antifungal toward Phomopsis sp. RP257. The properties (water vapor permeability and mechanical properties) of chitosan films were not significantly changed by the incorporation of the enzyme system. in vivo condition, chitosan coating at 1 and 1.5% with or without enzyme system was sufficient to inhibit totally (100%) Pestalotiopsis sp. and was 60% efficient against Phomopsis sp. with chitosan only at 1 and 1.5%. However, when coating solution mainly at 1 and 1.5% was enhanced by LPOS with or without iodine, it inhibited totally (100%) Phomopsis sp. RP257. The presence of iodine slightly reduced antifungal activity against Phomopsis sp. RP257.     

Antibacterial Effect of Tertiary Butylhydroquinone Against Two Genera of Gram Positive Cocci

The minimum inhibitory concentration (MIC) of tertiary butylhydroquinone (TBHQ) against Pediococcus pentosaceus in a bacteriological medium was 15 μg/ml for 10³ to 10⁵ cells/ml and 20 μg/ml for 10⁶ cells/ml. Heat stress and sodium chloride were each synergistic to the inhibitory effect of TBHQ. Production of both L and D lactic acid was inhibited by TBHQ. Fermentation of sucrose was totally inhibited by 5 μg/ml TBHQ as compared to 10 μg/ml TBHQ for L-arabinose, D-galactose and maltose and to 15 μg/ml TBHQ for glucose. TBHQ (50 μg/ml) did not inhibit growth of 1.2 × 10³ cells of S. aureus z-88, but 30 μg/ml totally inhibited growth of 3.0 × 10³ cells of S. aureus 100 for 36 hr. TBHQ (100 μg/ml) was bactericidal against the latter strain. TBHQ (10–50 μg/ml) slightly inhibited production of nonheat treated nuclease but had little or no effect on heat treated nuclease of S. aureus z-88.     

Effect of inulin on growth and bacteriocin production by Lactobacillus plantarum in stationary and shaken cultures

The prebiotic effect of inulin added to MRS medium on growth and bacteriocin production by L. plantarum ST16 Pa was investigated in stationary cultures in anaerobic jars with medium containing 0.025% sodium thioglycolate or in flasks shaken at 100 rpm. In the presence of 1% inulin in anaerobic stationary cultures, this strain produced lactic acid at a level that was 36.5% higher than in the absence of the polysaccharide. In shaken cultures without inulin, cell count was 54% higher than in the stationary ones. Under stationary conditions in anaerobic jars, the addition of inulin increased the maximum specific growth rate from 0.37 to 0.49 h^?1 and reduced the generation time from 1.85 h to 1.40 h. Consequently, the exponential phase was shortened from 12 to 9 h when the cells were grown in stationary cultures with the oxygen scavenger. Despite this effect of inulin on growth rate, stationary cultures without inulin displayed higher antimicrobial activity against Listeria monocytogenes L104 (3200 AU/mL) than cultures with inulin (1600 AU/mL); therefore, inulin behaved as a compound able to accelerate growth rather than to stimulate bacteriocin production. The results presented in this study are very promising, as L. monocytogenes is a well‐known foodborne pathogenic microorganism. Moreover, L. plantarum ST16 Pa has proven to be a potential producer of a natural food preservative at an industrial level.     

Conjugated linoleic acid production by immobilized cells of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus acidophilus

Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) and L. acidophilus (CCRC14079), immobilized with chitosan and polyacrylamide, were tested for CLA production. A 10-ml aliquot of L. delbrueckii ssp. bulgaricus cell suspension (3.59 × 10⁷ CFU/ml) was adsorbed to 0.5 g chitosan and polyacrylamide, mixed with 0.2 ml linoleic acid (0.9 g/ml), and incubated at 37 °C for 24 h at pH 5, 6, 7, and 8 for CLA production. CLA levels, produced by immobilized cells of L. delbrueckii ssp. bulgaricus and L. acidophilus with increasing cell counts to 1.08 and 1.28 × 10¹⁰ CFU/ml, respectively, at optimal reaction pHs were evaluated. More CLA was formed at pH 8 of chitosan and pH 7 of polyacrylamide-immobilized L. delbrueckii ssp. bulgaricus cell treatments. Increase in cell count resulted in higher CLA production. The adsorption of L. delbrueckii ssp. bulgaricus cells onto polyacrylamide at pH 7 showed significant improvement in total CLA level. Results demonstrated a potential for enhancing CLA production through immobilization.     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.