Nutrition of Coryneform Bacteria from Milk and Dairy Sources

S ummary . Growth requirements were determined for 112 cultures of coryneform bacteria including strains of Corynebacterium bovis, C. ulcerans and C. lacticum ; in general they were quite distinctive. Of 42 C. bovis strains all had an unsaturated fatty acid requirement fulfilled in chemically defined media by Tween 80, and, but for an atypically nonureolytic strain, all utilized ammonia and urea nitrogen. Whereas vitamins were unessential for the majority of C. bovis. 18 strains failed to grow in the absence of nicotinic acid; however, for 11 of these Casamino Acids replaced nicotinic acid in supporting growth. Thirteen C. ulcerans strains from aseptically drawn milk, as well as two Type Culture strains (NCTC 7907 and 7908) from the human throat required amino acid nitrogen sources, nicotinic acid, pantothenate and biotin. Some nutritional heterogeneity was shown by 46 strains collectively described as C. lacticum obtained from market milk, milk products and dairy utensils; nevertheless the nutritional differences between the groups of these strains correlated broadly with other distinguishing properties. A group of 9 markedly caseolytic strains required amino acid nitrogen sources, thiamine and biotin, whereas a second group of 30 strains, some utilizing inorganic nitrogen sources, required thiamine and pantothenate, with or without biotin. The remaining C. lacticum strains together with a number of unidentified strains from similar sources were generally slow growing and had widely differing growth requirements.     

Production of blastospores by three strains of metarhizium anisopliae (metch.) sorokin in submerged culture

Studies on blastospore production in different liquid media were conducted with three strains of Metarhizium anisopliae var. anisopliae (M. a.) derived from various countries (M. a. 43: Austria, M. a. 57: Brazil, M. a. 97: Philippines). Variation of six fermentation parameters (cornsteep products, carbohydrates, pH values, temperature, Tween 80, and polyethyleneglycol (PEG) 200) disclosed that the three strains of M. anisopliae differed in their growth pattern and physiology. In standard medium and in all tests, M. a. 57 produced the highest number of blastospores invariably amounting to > 10⁸ per ml, while mycelial pellets were never formed. The preferred carbohydrates were glucose and fructose. Blastospore production of M. a. 43 was increased by growth at 30°C, at pH 6.5 or by addition of 5% PEG 200. However, it was impaired by different concentrations of Tween 80 or higher concentrations of PEG 200 (10–15%). M. a. 97 produced most blastospores at 30°C, and the strain preferred basic (pH 8.0) as well as acid (pH 4.5) media. Blastospore production was increased by the addition of 5% PEG 200 or 0.4–1.2% Tween 80. Moreover, PEG 200 suppressed pellet formation effectively. Altogether, our results showed that for optimal blastospore production of Metarhizium anisopliae, suitable strain‐specific parameters have to be evaluated.     

Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid’s pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.     

A comparison of factors affecting the production of two bacteriocins from lactic acid bacteria

The effect of tryptone, yeast extract, Tween 80 and initial pH on the production of enterocin 1146 and lactocin D, two bacteriocins produced by lactic acid bacteria, was studied in a basal buffered medium (tryptone-yeast extract-tween, TYT) using factorial experiments and empirical modelling. Production of enterocin 1146 was affected by pH, yeast extract and Tween 80 and to a lesser degree, by the initial pH of the medium. On the basis of the predictions of the models developed, three TYT media (TYT10, TYT11 and TYT30) were designed to maximize bacteriocin production while minimizing the amount of peptides in the medium. Growth and bacteriocin production by Enterococcus faecium DPC 1146 (enterocin 1146), Lactococcus lactis subsp. lactis biovar diacetylactis DPC 3286 (lactocin D) and Lact. lactis subsp. cremoris LMG2130 (lactococcin A) was compared in TYT media and seven other culture media (Elliker lactic broth, M17, M17 dialysate, MRS, tryptose phosphate, tryptone yeast extract broth, yeast glucose Lemco broth). Bacteriocin production in TYT media was comparable with that in M17 and MRS, which had a higher peptide content. TYT30 allowed good production of enterocin 1146 and lactocin D while TYT11 proved acceptable for all the strains tested.     

Cholesterol requirement of mycoplasmas

Cholesterol requirement for growth of mycoplasmas was tested in a serum-free medium supplemented with albumin, l-arginine, palmitic acid, and various concentrations of cholesterol dissolved in Tween 80. In cases in which Tween 80 was shown to inhibit growth, the test medium was supplemented with cholesterol dissolved in ethanol. Of the 31 species examined, all but Mycoplasma laidlawii, M. granularum, and Mycoplasma species strain S-743 exhibited a growth response to cholesterol. No requirement for cholesterol could be shown with the stable L-phase variants of Streptobacillus moniliformis and Proteus species. The results provide experimental support for the view that the large majority of the established Mycoplasma species require cholesterol for growth.     

Analysis of Tween 80 as an esterase/ lipase substrate for lipolytic activity assay

The Tween 80 assay to detect lipolytic activities in agar media was evaluated. A spectrophotometric assay for Tween 80 hydrolysis was established. The specific activities with Tween 80, as well as with some conventional lipase-type and esterase-type substrates, were measured using several lipases and esterases. The activity with Tween 80 was similar to that obtained with p-nitrophenyl butyrate; the enzyme activities with both substrates were between the esterase and lipase categories. © Rapid Science Ltd. 1998     

Tween 80 effect on glucosyltransferase synthesis by Streptococcus salivarius.

Streptococcus salivarius (ATCC 25975) produced very low or nondetectable amounts of the extracellular enzyme glucosyltransferase (GTase) when grown in a chemically defined medium. The addition of Tween 80 to this medium resulted in the production of markedly enhanced levels of the enzyme. Oleic acid, the methyl ester of oleic acid, and sucrose each could not substitute for Tween 80 in this regard. The surfactant had no direct activating effect on performed enzyme activity. Tween 80 also stimulated the production of GTase by concentrated cells suspended in defined medium during a time when no measurable growth occurred. Under these conditions, the stimulatory effect of Tween 80 was blocked by chloramphenicol. It was further found that the surfactant dramatically stimulated the differential rate of GTase synthesis. These and other data strongly suggest that Tween 80 stimulates the production of extracellular GTase by acting either directly or indirectly at the level of enzyme synthesis.     

Effect of Agitation on Ligninase Activity and Ligninase Production by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium produces extracellular ligninases as part of its idiophasic ligninolytic system. Agitation has been widely reported to suppress both ligninase production and lignin degradation. Results show that mechanical inactivation of ligninase is possibly the reason why ligninase accumulation is low or absent in agitated shake-flask cultures. Agitation seems to affect the catalytic activity of ligninase and has no apparent effect on either the rate of ligninase production or the physiology of P. chrysosporium. The detergents Tween 20, Tween 40, Tween 60, Tween 80, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) are able to protect both purified ligninase and extant ligninase in culture fluids (free of biomass) against mechanical inactivation due to agitation. Addition of Tween 80 at the end of primary growth to agitated shake flasks containing either pelleted or immobilized mycelial cultures results in production and maintenance of high levels of ligninase activity over several days under conditions of high agitation. Possible mechanisms by which the detergents could protect ligninase are discussed.     

Optimized culture conditions for bacteriocin production by Pediococcus acidilactici LAB 5 and its characterization

A strain of Pediococcus acidilactici LAB 5 was isolated from vacuum-packed fermented meat product, in order to obtain a novel bacteriocin from food-grade organisms. Optimized culture conditions for bacteriocin production in different media (viz., MRS, TGE, TGE + buffer, TGE + Tween 80, and TGE + Tween 80 + buffer) and at different temperatures and pH conditions were reported. TGE + Tween 80 + buffer medium was found to be most effective for bacteriocin production (about 2400 AU/ml) by this strain, when incubated at 37 degrees C for 24 h. Bacteriocin, partially purified by adsorption-desorption method showed molecular mass of 10.3 kDa and produced prominent inhibition zone in activity gel. It showed significant storage stability both at high as well as in low temperatures for up to 6 months and retained its activity in a number of organic solvents, except in 2-mercaptoethanol. The treatment with amylase and lysozyme did not change its activity, but it lost its activity on proteinase K treatment. Antibacterial efficacy of bacteriocin was proved against some food spoilage and human pathogenic bacteria like Enterococcus, Leuconostoc, Listeria, Staphylococcus and Streptococcus.     

Effects of Tween 80 and sodium fluoride on extracellular glucosyltransferase production and membrane lipids of Streptococcus mutans

1. Both Tween 80 and sodium fluoride significantly enhanced total extracellular glucosyltransferase activities of Streptococcus mutans. 2. Water-insoluble and water-soluble glucan formation were uniformly increased by Tween 80, whereas fluoride stimulated only water-soluble glucan formation. 3. Elevated glucan formation was due to an increase in enzymes secreted from bacterial cells. 4. Fatty acid composition and phospholipid content in bacterial membrane were changed by Tween 80, although sodium fluoride scarcely showed these changes. 5. Comparative results suggest that modulation of membrane lipids participates in mutansucrase production but not in dextransucrase production of S. mutans.     

Optimization of lactic acid production in SSF by Lactobacillus amylovorus NRRL B-4542 using Taguchi methodology

Lactic acid production parameter optimization using Lactobacillus amylovorus NRRL B-4542 was performed using the design of experiments (DOE) available in the form of an orthogonal array and a software for automatic design and analysis of the experiments, both based on Taguchi protocol. Optimal levels of physical parameters and key media components namely temperature, pH, inoculum size, moisture, yeast extract, MgSO4 . 7H20, Tween 80, and corn steep liquor (CSL) were determined. Among the physical parameters, temperature contributed higher influence, and among media components, yeast extract, MgSO4 . 7H20, and Tween 80 played important roles in the conversion of starch to lactic acid. The expected yield of lactic acid under these optimal conditions was 95.80% and the actual yield at optimum conditions was 93.50%.     

Tween 80 effect on bacteriocin synthesis by Lactococcus lactis subsp. cremoris J46

E. HUOT. C. BARRENA-GONZALEZ AND H. PETITDEMANGE. 1996. Sorbitan polyoxyethylene monooleate (Tween 80) suppressed bacteriocin cell adhesion. Within the range 0–1% (v/v), there was an increase in bacteriocin production in regulated (pH 5.5 or 6.0) batch cultures with increasing Tween 80 concentration. For example, at pH 5.5 and in the presence of 1% Tween 80, bacteriocin production was about fourfold higher than in its absence. However, further increase in Tween 80 concentration did not result in a significant modification of the bacteriocin titre. It was shown that the increase was not linked to an activating effect of the surfactant on preformed enzyme, to an increase of bacteriocin availability or to a sensitization of the target cell, demonstrating that Tween 80 promoted bacteriocin production.     

云芝深层培养生产木质素降解酶的研究

木质索降解本质上是氧化反应,参与木质素降解的酶都是非专一性的,目前人们认识到的参与木质素降解的酶主要有多酚氧化酶(Polyphenol oxidase)、锰过氧化物酶(Maganese peroxidase)和木质素酶(Ligninase)。后者是近来新发现在木质素降解过程中起作用的过氧化物酶。本文研究一种对木质素降解能力很强的云芝(Polyporus versicolor)在摇瓶培养条件下,培养方式、营养条件以及添加诱导剂藜芦醇和表面活性剂Tween80等因素对木质素降解酶生产的影响。     

Effect of Tween 80 on the production of curdlan by Alcaligenes faecalis ATCC 31749

This study aims to investigate the effects of Tween 80 on curdlan production, cell growth, and glucosyltransferase activity. The addition of Tween 80 to the culture medium increased curdlan production. However, curdlan production did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. The addition of Tween 80 to the culture medium did not affect cell growth. The glucosyltransferase activity involved in the curdlan synthesis increased with the increase of Tween 80 concentration. The glucosyltransferase activity did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. Maximum curdlan was observed at day 5 and then levelled off. The biomass continued to increase until the end of the experimental period (6 d). Maximum glucosyltransferase activity was also observed at day 5 and decreased thereafter. The results indicate that the enhanced curdlan production by Tween 80 is highly correlated with glucosyltransferase activity.     

Preparation and use of lipid microemulsions as nutritional supplements for culturing mammalian cells

Summary Cells grown in vitro generally have a requirement for an exogenous source of lipid. This requirement is often met by the addition of serum lipoproteins, or lipids complexed to albumin. To overcome the disadvantages of using lipoproteins or albumin for culturing cells in serum-free media, a method has been devised to provide necessary lipids. This report describes the preparation and use of protein-free lipid microemulsions suitable for use in tissue culture. The microemulsions are prepared from purified, synthetic lipids to produce a homogeneous, water-soluble, stable suspension that can be sterile-filtered. The best results were obtained using a sonicate of cholesterol oleate, dipalmitoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol, sphingomyelin, alpha-tocopherol, alpha-tocopherol acetate, and Tween 80. Using Chinese hamster ovary (CHO) cells in a protein-free medium, cell growth was 222% vs. control (no microemulsion) in a 5-d assay. Incluction of the microemulsion to protein-free media also increased the growth rate of murine hybridomas, H9 transformed T lymphoblasts, and human skin keratinocytes.     

Comparative media studies in the isolation ofCandida albicans from pregnant women

Summary Rice agar and corn meal agar, with and without Tween 80, were evaluated clinically as directly inoculable selective and differential media for the isolation ofC. albicans from vulvovaginal specimens taken from pregnant women. Chlamydospore formation on these media was investigated as a criterion for the identification ofC. albicans.Of 301 patients cultured, 118 (39.2 %) gave positive cultures for yeast-like fungi of the genusCandida. Of 118 strains for which fermentation patterns were determined, 69 (58.5 %) gave the pattern forC. albicans. Of these, 56 (81.1 %) formed chlamydospores.Tween 80 was found to exert a very stimulating influence on chlamydospore production. Rice agar with Tween 80 appeared to be the most efficient medium for eliciting chlamydospores. However, since strains of 4 out of 6 species ofCandida isolated were found to sporulate it was concluded that chlamydospore formation is not a reliable criterion for the speciation ofC. albicans.Each of the 4 media served satisfactorily as a directly inoculable selective medium for the isolation of yeast-like fungi of the genusCandida from vulvovaginal specimens. None of the media appeared to preferentially stimulate chlamydospore production inC. albicans.Dr.Smith is Associate Professor in the Department of Microbiology; Dr.Taubert is a Fellow in the Department of Obstetrics and Gynecology; Mr.Towns is Laboratory Assistant in the Department of Microbiology.Supported in part by a grant from the Lederle Medical Faculty Awards Committee and in part by United States Public Health Service Grant E-3068.     

Effect of surfactants on ethanol fermentation using glucose and cellulosic hydrolyzates

The effect of the non-ionic surfactants on the ethanol fermentation was greatly dependent on the surfactant added. While Tween 20 and Tween 80 slightly enhanced ethanol fermentation, Triton X-100 which exhibited the inghest increase in the enzymatic saccharification had a negative effect on the ethanol fermentation. The negative effect of Triton X-100 on ethanol production was the most pronounced when the cellulosic hydrolyzates were used. Tween 80 showed the best performance for the ethanol production from steam exploded wood hydrolyzate.     

Effect of surfactants on cellulase production by Nectria catalinensis

The effect of different nonionic surfactants (Tween 80, Tween 20, Triton X-100) and polyethylene glycol (PEG 6000) was tested on cellulolytic enzyme system production. Tween 80 gave the highest yield of endoglucanase, exoglucanase, and cellobiase at the 20th day of growth, presumably by causing increased permeability of cell membranes and/or by promoting the release of cell-bound enzymes. Maximal yield of endoglucanase was achieved with 1.7 mM Tween 80, whereas exoglucanase and cellobiase were at 0.85 mM. In the same way, this compound increased fungal growth. On the other hand, Tween 20 and Triton X-100 inhibited growth and cellulolytic enzyme production. High yields of endoglucanase and exoglucanase were achieved with PEG 6000 in comparison with the control, presumably by increasing enzyme stability. Received: 22 January 1996 / Accepted: 28 March 1996     

Comparative testing of skin reactions to PPD mycobacterins from Mycobacterium tuberculosis and Mycobacterium scrofulaceum in school-age children

Comparative skin tests with 2 TU PPD-RT 23 with Tween 80 (prepared from M. tuberculosis) and 5 TU PPD-RS 95 with Tween 80 (prepared from M. scrofulaceum) were intradermally given to a total of 1,140 7-year-old children in two towns of Karviná district (340 and 255 children) and in Teplice (267 children) and Prague (278 children). In the two groups of Karviná district children the percentages of small-sized reactions (6-9 mm) to PPD-RT 23 were 13.6 and 22.3% compared to 7.1% in Teplice and 5.7% in Prague. The prevalence of small reactions to the PPD-RS 95 test in district of Karviná was 14.4 and 16.9%, in Teplice 4.5% and in Prague 6.8%. In the two towns of Karviná district the percentages of children whose reaction to PPD-RS 95 was larger than to PPD-RT 23 were 56.4 and 42.8%, in Teplice 24% and in Prague 24%. The hypothesis is advanced that the higher degree of skin hypersensitivity to the M. scrofulaceum mycobacterin which was found among the Karviná district children tested is due to sensitization with environmental mycobacteria which are common in this area.     

The standardization of a 'spot-test' ELISA for the rapid screening of sera and hybridoma cell products I. The determination of the optimum buffering system

Using the different commercially available enzyme-linked immunosorbent assay (ELISA) plates, several sources of albumin were tested along with Tween 20 as supplements to the diluting buffer in ELISA for their ability to minimize non-specific reactions. There was an obligate requirement for Tween 20 (0.05%) but the different albumin sources had varied effectiveness on each of the different ELISA plates. In general, however, the optimum buffering system was concluded to be phosphate-buffered saline supplemented with 0.05% (v/v) Tween 20 and 3% (w/v) lactalbumin yeast hydrolysate or bovine serum (plasma) albumin fraction V.