Cellobiohydrolase from Trichoderma reesei.

The secondary structure pattern of a cellobiohydrolase from Trichoderma reesei was predicted using three different algorithms. The relative amounts of the different secondary structure classes derived by this procedure were in good agreement with the values determined by circular dichroism measurements. A twofold internal sequence homology with a repeat distance of approximately 200 residues is observed and possibly also a third, partial, repetition in the C-terminal region. The predicted secondary structure and hydrophobicity pattern show a similar repeat.     

Safety evaluation of alkaline cellulase.

A programme of studies was conducted to establish the safety of alkaline cellulase, an enzyme used as a processing aid in the food industry. Laboratory animal studies were used to assess general, inhalation and reproduction toxicity, eye irritation and skin irritation and sensitization. Mutagenic potential was assessed in microbial and animal in vivo studies. The pathogenicity of Humicola insolens, the micro-organism used in the production of alkaline cellulase, was also assessed in laboratory animals. Basic ecotoxicity in a variety of test species was studied. General toxicity by a variety of routes was low; there was no evidence of reproductive toxicity. There was evidence of mild skin irritation and some eye conjunctival reddening. There was no evidence of skin sensitization, mutagenic potential, ecotoxicity or notable pathogenicity. When these results are considered along with levels of human exposure and previously published data, it appears that alkaline cellulase is safe for consumers in the given applications, requires no special occupational health precautions in manufacture, and is of low environmental impact. Furthermore, the micro-organism used in production of alkaline cellulase has no notable pathogenicity.     

A new cyclotetrapeptide from marine fungus Trichoderma reesei

A new cyclotetrapeptide, trichoderide A, was isolated from the marine fungus Trichoderma reesei. The structure was identified by spectral methods, and the stereochemical assignments were made by chiral HPLC of the hydrolyzed compound. Trichoderide A showed moderate cytotoxity against human A375-S2 melanoma cell line.     

Safety evaluation of phosphodiesterase derived from Leptographium procerum.

5'-Phosphodiesterase is produced by fermentation of the fungus Leptographium procerum and is used to hydrolyse yeast RNA to produce flavour enhancers. To establish the safety in use of this enzyme preparation a number of studies have been performed: analysis for the potential of the production strain to produce toxic secondary metabolites, 28-days oral toxicity study of the preparation in the rat, bacterial mutation assay and in vitro mammalian chromosome aberration test in human lymphocytes. The production strain did not produce any secondary metabolites that may be of significance in food. Administration of dosage levels of 1250, 2500 and 5000 mg/kg body weight/day to rats for 28 day did not result in any toxicological significant changes. The enzyme preparation showed no mutagenic activity in the bacterial mutation assay and no clastogenic potency in an in vitro test. These results together with existing knowledge of the production organism and the chemical and microbiological characterisation of the enzyme preparation lead to the conclusion that the enzyme preparation containing 5'-phosphodiesterase activity from Leptographium procerum can safely be used for the production of flavour enhancers from bakers yeast at the anticipated intake levels for these uses.     

海洋真菌Trichoderma reesei的次级代谢产物的分离与鉴定

目的系统研究海洋真菌Trichoderma reesei的次级代谢产物。方法利用硅胶柱色谱、高效液相色谱等手段进行分离,根据理化性质及波谱数据对所得结构进行了鉴定。结果从海洋真菌Trichoderma reesei发酵液的乙酸乙酯提取物中共分离得到6个成分,分别鉴定为cyclonerodiol(1)、8,9-dihydro-dihydroxymegastigmatrienone(2)、harzialactone A(3)、3,6-二苄基哌嗪-2,5-二酮(4)、3-异丁基-8-羟基吡咯并哌嗪-2,5-二酮(5)、3-苄基-8-羟基吡咯并哌嗪-2,5-二酮(6)。结论所分得的化合物均为从海洋真菌Trichoderma reesei的次级代谢产物中首次分离得到。     

Production of toxic metabolites in Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei: justification of mycotoxin testing in food grade enzyme preparations derived from the three fungi

Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei are three important production organisms used in industrial fermentations. Several of the fungal secondary metabolites produced by selected strains of these three fungi are capable of eliciting toxicity in animals. Among those toxic substances are the well-known mycotoxins 3-nitropropionic acid and ochratoxin A. However, many others, such as kojic acid, may not be true mycotoxins. The production, extraction, chemical structure, and the toxicity (expressed as LD(50)) of these substances are reviewed. Production of toxic secondary metabolites in A. niger, A. oryzae, and T. reesei is strain-specific and environment-dependent. Considering all of the safety measures taken in the industrial production process, these three fungal species are safe to use. The recently revised JECFA specification for mycotoxins in food enzyme preparations is also discussed. The extent of mycotoxin tests in food enzyme preparations should be judged on a case-by-case basis, through a careful evaluation based on knowledge of taxonomy, biochemistry, and genetics. In many cases, the testing scope at the level of genus should be sufficient. In other cases, the scope can even be further narrowed based on scientific knowledge and assessment.     

Safety evaluation of a lactase enzyme preparation derived from Kluyveromyces lactis.

Neutralact(R), the DSM brand name of a lactase enzyme preparation, obtained from a homologous rDNA strain of Kluyveromyces lactis, was subjected to a series of toxicological tests to document the safety for use as a processing aid in the dairy industry. The enzyme preparation was examined for subacute oral toxicity and mutagenic potential. As a result of these tests, no evidence of oral toxicity, mutagenicity or clastogenicity was found. Administration of the lactase enzyme preparation at doses of 500, 3000 and 10,000 mg/kg body weight/day for 28 days did not induce noticeable signs of toxicity. The no-observed-adverse-effect level (NOAEL) of the enzyme preparation in the acute toxicity study was 10,000 mg/kg body weight/day (equivalent to 114,000 NL units/kg body weight/day). It can be concluded that no safety concerns were identified in the studies conducted with this lactase enzyme preparation derived from Kluyveromyces lactis under controlled fermentation conditions.     

Safety evaluation of a natural tomato oleoresin extract derived from food-processing tomatoes

Experimental and epidemiological studies indicate that consumption of tomato products containing high amounts of lycopene is associated with lowered cancer risk. The protective effects of lycopene may be related to its antioxidant potential. Lycopene has been demonstrated to inhibit oxidation. A proprietary, natural tomato oleoresin extract (NTOE), is a purified tomato oleoresin containing 6% lycopene produced from tomatoes. NTOE was evaluated for toxicological effects, and found the 50% lethal dose (LD(50)), derived from the acute oral toxicity study, was greater than 5000 mg/kg body weight. The no-observed-adverse-effect level (NOAEL) derived from the 13-week study was 4500 mg/kg/day. Acute dermal toxicity study of NTOE found no toxicity at 2000 mg/kg body weight. NTOE lacked dermal irritation in the rabbit model, but was found to have moderate eye-irritant capabilities. NTOE tested at 5% (w/w) in petroleum jelly was a moderate sensitizer in the guinea pig model. There was no evidence of mutagenic potential up to 5000 microg/plate, as determined by the Ames assay. These results demonstrate the inability of NTOE to produce oral, dermal or mutagenic toxicity in animal models at doses greater than 300 times the normal human consumption of lycopene. Consumption analysis of lycopene-containing foods estimated mean daily intake of lycopene at 8.2mg/day.     

Safety evaluation of an extract from Salacia oblonga.

Plant extracts from the Salacia genus have been found to have intestinal alpha-glucosidase inhibitor activity, which may have application to the development of medical foods for people with diabetes. We evaluated the safety of a hot water extract of S. oblonga (salacinol extract) supplemented to or processed into a medical food. Thirty male Sprague-Dawley rats were assigned among one of three treatments: (1) EN-0178 (control, liquid diet), (2) EN-0178+salacinol (as 1 plus 500 mg of salacinol extract per 253 g diet, which was added to product immediately prior to feeding), (3) EN-0195 (as 1 plus 500 mg of salacinol extract per 253 g diet, which was added during product manufacture). After 14 days of free access to dietary treatments, rats were sacrificed, blood collected and organs weighed. Rats consuming salacinol extract had reduced (P <0.05) weight gain and feed intake. The relative (% of body weight) testicular weight was higher (P<0.05) for rats consuming salacinol extract, whereas, the relative liver and spleen weight was lower (P<0.05) for rats consuming salacinol extract. Of the serum chemistries analyzed, blood urea nitrogen and alkaline phosphatase was lower (P<0.05) for rats consuming salacinol extract. No differences in blood hematology were found. We conclude that salacinol extract, in a medical food consumed for 2 weeks in amounts estimated at 10-fold greater than proposed for human intake, did not result in clinical chemistry or histopathologic indications of toxic effects in male Sprague-Dawley rats.     

Safety evaluation of an alpha-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I

BD5088 alpha-amylase derived from archaeal sources has characteristics of pH and temperature tolerance that are well suited to hydrolysis of starch in food processing applications. The production microorganism recipient strain, Pseudomonas fluorescens biovar I, strain MB101, was avirulent after oral administration to mice and does not represent an infectious threat to humans. Repeated dose gavage studies with BD5088 enzyme preparation, up to 13 weeks in duration, showed no systemic toxicity due to the oral route with an NOAEL of 890 mg/kg/day as Total Organic Solids. Some irritation occurred in the respiratory tract, which was considered to be a consequence of reflux and aspiration of test material that contained lipopolysaccharide from the Pseudomonas production strain. A 2-week dietary study (0 and 310 mg/kg/day) confirmed that there were no respiratory tract effects related to oral ingestion. There was no genotoxic activity based on Ames, mouse lymphoma, mouse micronucleus, and rat lymphocyte chromosomal aberration tests. There was no evidence of allergenic potential based on a comparison of the primary sequence of BD5088 with sequences in an allergen database. The enzyme was labile to pepsin digestion. Based on these data, BD5088 alpha-amylase preparation may be considered safe for use in food production such as corn wet milling.     

Safety evaluation of alpha-lipoic acid (ALA)

The safety of the antioxidant alpha-lipoic acid (racemic form) (ALA), also called thioctic acid (CAS RN 1077-28-7) was assessed in acute and subchronic toxicity studies as well as in in vitro and in vivo mutagenicity/genotoxicity studies. ALA was not acutely toxic to rats (LD(50)>2000mg/kg bw, OECD method 425). Administration of 31.6 or 61.9mg ALA/kg bw/day for 4 weeks to male/female Wistar rats did not show any adverse effects. Specifically, there was no significant difference between control and treated animals at 31.6 or 61.9mg ALA/kg bw with regard to body weight gain, feed consumption, animal behaviour, or haematological and clinical chemistry parameters. Only the high-dose of 121mg ALA/kg bw was associated with slight alterations in liver enzymes as well as histopathological effects on the liver and mammary gland. ALA did not possess any mutagenic activity in the Ames assays conducted with various bacterial strains of Salmonella typhimurium. Moreover, there was no evidence of genotoxic activity in a mouse micronucleus assay. The results of these studies support the safety of ALA. The no-observed-adverse-effect level (NOAEL) is considered to be 61.9mg/kg bw/day.     

Safety evaluation of an alpha-cyclodextrin glycosyltranferase preparation

Alpha-cyclodextrin glucosyltransferase (alpha-CGTase, EC 2.4.1.19) is an amylolytic enzyme used for the production of alpha-cyclodextrin (alpha-CD), a novel, soluble dietary fiber, from food-grade starch. The safety of an alpha-CGTase preparation obtained by batch fermentation from a recombinant strain of Escherichia coli K12 harboring the alpha-CGTase gene from Klebsiella oxytoca strain M5a1 was examined. In a 13-week subchronic toxicity study in rats, the administration by gavage of the alpha-CGTase preparation at levels of up to 20 ml/kg bw/day, corresponding to a total organic solids dosage of 260 mg/kg bw/day, did not cause any systemic toxic effect. Some signs of irritation were observed in the respiratory tract which occurred, however, in one sex only and/or were not dose-related. Accordingly, these changes were considered to be an unspecific consequence of the reflux and aspiration of the dosing solution. There was no evidence of a genotoxic activity in Ames tests and a chromosome aberration test in cultured human lymphocytes. It is concluded that the examined alpha-CGTase preparation is safe when used for the production of alpha-CD.     

Two acid sorbicillin analogues from saline lands-derived fungus Trichoderma sp

Two new acid sorbicillin analogues, (E)-6-(2,4-dihydroxyl-5-methylphenyl)-6-oxo-2-hexenoic acid (1) and 6-(2,4-dihydroxyl-5-methylphenyl)-6-oxohexanoic acid (2), together with 12 known compounds, were isolated from a saline lands-derived fungus Trichoderma sp. The structures of the new compounds were established by interpretation of their spectroscopic data. Their cytotoxic effects on P388 and HL-60 cell lines were preliminarily evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) method. Furthermore, the new compounds exhibited weak radical scavenging activity against DPPH (1,1-diphenyl-2-picrylhydrazyl).     

Synthesis, biological evaluation and molecular modeling studies of Schiff bases derived from 4-methylsalicylic acid as potential immunosuppressive agents

A series of Schiff bases derived from 4-methylsalicylic acid (4a–4s) were synthesized, 14 of which (4a–4h, 4j–4l, 4n, 4q, and 4s) were reported for the first time. All the synthesized compounds were evaluated for their immunosuppressive activities for the first time. Among them, compound 4o displayed the most potent biological activity against lymph node cells (IC₅₀ = 1.02 μM for lymph node cells and IC₅₀ = 2.17 μM for PI3Kγ). The results of apoptosis and western-blot assays demonstrated that the immunosuppressive activity of compound 4o might be mediated by the inhibition of PI3K/AKT signaling pathway. Docking simulation was performed to position compound 4o into the PI3Kγ structure active site to determine the probable binding model.     

Action-mechanism of trichoderin A, an anti-dormant mycobacterial aminolipopeptide from marine sponge-derived Trichoderma sp

In the course of our search for anti-dormant mycobacterial substances from marine organisms, we previously isolated three new aminolipopeptides, named trichoderins A, A1 and B, from the culture of the marine sponge-derived fungus of Trichoderma sp. and determined their chemical structures. To identify the gene that could confer a resistance to trichoderin A, we prepared transformants of Mycobacterium (M.) smegmatis, which were transformed with the genomic DNA library of M. bovis BCG constructed in the multi-copy shuttle cosmid pYUB145. Then, the transformant of M. smegmatis, which over-expressed a part of genes that coded mycobacterial ATP synthase, was found to exhibit a resistance to trichoderin A. In addition, trichoderin A reduced ATP contents in M. bovis BCG. These findings elucidated that the anti-mycobacterial activity of trichoderins comes from the inhibition of ATP synthesis.     

Development of an exposure system for the toxicological evaluation of particles derived from coal-fired power plants

To investigate the toxicity of particles originating from coal-fired power plants it is necessary to consider the effects of both primary particles and secondary components formed in the air through atmospheric reactions. This report describes a new exposure system that can be used to expose animals to both directly emitted particles and to secondary particles. The system consists of three main components. The first is a sampling system to continuously collect and dilute power plant stack emissions. The second is a reaction laboratory that contains reaction chambers to simulate atmospheric reactions. The following atmospheric reactions were simulated: (1) the oxidation of sulfur dioxide to form sulfuric acid, (2) the neutralization of sulfuric acid by ammonia, and (3) the reaction of alpha-pinene with ozone to form secondary organic aerosol. Using these chambers with the diluted emissions, different typical atmospheric scenarios can be simulated. The final component is a mobile toxicology laboratory where animals are exposed to the resulting test aerosols. We report here the characteristics of the test aerosol exposures obtained at a coal-fired electric power plant. Particle exposures were characterized for concentrations of mass, elements, elemental carbon, organic species, inorganic ions, strong acidity, particle number, and size distributions. Mass concentrations ranged from a few micrograms per cubic meter for a scenario of primary emissions only, to about 250 microg m(-3) for the most complex scenario. We show that the different scenarios produced a large variation in the composition of the test aerosol, thus potentially changing the toxicity of the emissions.