IMPROVEMENTS IN PROCEDURES FOR THE PRODUCTION OF EICOSAPENTAENOIC ACID BY MORTIERELLA

Modifications of growing conditions including time, light exposure, extraction methods, and use of shaker flask versus fermentor were used to increase the production of eicosapentaenoic acid (EPA, 20:5w3) by Mortierella elongata ATCC 16271. The highest productivity for EPA (66 mg/g dry mycelia) was obtained when M. elongata ATCC 16271 was grown at 12C, in yeast malt broth, pH 5.1, and harvested after 12 d. The Bioflo C-30 fermentor increased the EPA content 1.8 times and the fungal biomass 3.2 times when compared with the shaker flask. The extraction method of Sasser (1990) extracted higher amounts of EPA than the method of Shimizu et al. (1988b). Mortierella mycelial extract is of particular interest because it has fewer extraneous fatty acids than fish oil, which is presently an important source of EPA. The procedures developed in this study resulted in the production of a greater amount of EPA containing mycelia than previously reported, and suggest that commercialization of this process may be economically advantageous.     

过量积累γ-亚麻酸的深黄被孢霉突变株MI-33的发酵研究

对过量积累γ-亚麻酸的深黄被孢霉突变株MI-33的发酵条件作了初步研究。通过试验不同接种方式对发酵的影响,确定了接种菌丝体为合理的接种方式;通过试验不同碳源对发酵的影响,确定了葡萄糖为产生γ-亚麻酸的最适碳源;菌体生长动态的研究表明,合适的种子液培养时间为48h;发酵动态的研究表明,合适的发酵终止时间为96h,此时干菌体收率为26.51g/L、油脂产率为14.08g/L、GLA产率为901.16mg/L。     

Submerged culture conditions for mycelial yield and polysaccharides production by Lyophyllum decastes

The effects of various carbon and nitrogen sources, their concentrations, initial pH and fermentation duration on the production of mycelia in terms of dry weight, exo-polysaccharide (EPS) and inner polysaccharide (IPS) by Lyophyllum decastes, a culinary-medicinal mushroom, were investigated in shake-flask cultures. Lactose, glucose and fructose were the top three best carbon sources for mycelial growth with corresponding yields of 6.73 g/l, 6.36 g/l and 6.10 g/l, respectively. Glucose was the best for production of EPS and IPS with 1.65 g/l and 317 mg/g dry mycelia, respectively. Maltose also performed well for EPS production. Yeast extract was the best nitrogen source for the production of mycelia (7.03 g/l) and IPS (325 mg/g dry mycelia), whereas EPS was improved further by increasing the yeast extract concentration (2.46 g/l at 2%). Similarly, initial pH 7 and 8 were best for polysaccharides production (EPS 1.73 g/l and IPS 320 mg/g) and mycelial growth (7.10 g/l), respectively. Maximum mycelial growth peaked at 15 days of cultivation whereas polysaccharides peaked at 10 days, and then tapered off. A concentration of glucose 3% and yeast extract 1% (mycelial yield and IPS) were found to be a suitable condition for submerged culture.     

Optimum conditions for S-allyl-(L)-cysteine accumulation in aged garlic by RSM

The objective of this study was to determine the optimum conditions for accumulating SAC [S-allyl-(L)- cysteine]. Although SAC is one of the bioactive compounds in garlic that has been reported to be beneficial for good health, it is found only in trace amounts in raw garlic bulbs and there are no clear research results indicating the accumulation method. We discovered a new method for accumulating SAC by adding a freezing and thawing process. Furthermore the aging process was undertaken at a lower temperature to reduce energy consumption. A 1.64-fold increase in the SAC content of thawed garlic surveyed was noted compared to fresh garlic (2.6391±0.0022 mg/dry g vs. 1.7363±0.2180 mg/dry g). Maximum accumulation (8.0212 mg/dry g) was achieved after 15 days of aging at 40°C, and response surface analysis was performed to determine the optimum conditions including aging period and temperature.     

Optimization of Selenium-Enriched Mycelium of Lentinula edodes (Berk.) Pegler as a Food Supplement

Our goal was to optimize the growth conditions of submerged mycelial cultures of Lentinula edodes (Shiitake mushroom) in order to obtain a new dietary supplement enriched in selenium We designed a process technology in which mycelial cultures were cultivated in media composed of beet molasses, 10%; liquid stillage, 5%; corn steep liquor, 0.15%; and KH₂PO₄, 0.3%, enriched with selenium in concentrations ranging from 0 to 100 μg/mL by the addition of sodium selenite of selenic acid. Se concentrations in mycelial dry mass rose from 0.001 mg/g (mycelia cultivated in media containing no selenium) to 50 mg/g (mycelia cultivated in medium containing 100 μg Se/mL). The highest mycelial specific growth rate (0.46/day) was recorded when the concentration of selenium in the medium was lower than 20 μg/mL. In vitro, estimated Se bioavailabilities from selenized mycelium strongly depended on its preparation in a proper manner and were 60% and 82% for dried mycelium and mycelial lyophilizate, respectively. Our results suggest that these optimized culture conditions could be applied to obtain a new Se-enriched dietary supplement.     

Culture of Aspergillus parasiticus in apple juice. I. The influence of sodium benzoate and potassium sorbate on fungal growth and aflatoxin biosynthesis

Sodium benzoate or potassium sorbate (100, 200, 300 and 400 mg/l) were added to cultures of Aspergillus parasiticus NRRL 2999 on apple juice (from syrup) and incubated quiescently at 25 degrees C for 3, 6, 9, 12 or 15 days. The cultures were analyzed for pH, mycelial dry weight and accumulation of aflatoxin B1 and G1. The initial pH of 2.5 remained constant in all instances throughout the incubation period. Sodium benzoate, at all concentrations, suppressed fungal growth and stimulated the biosynthesis of G1, whereas little influence was exerted upon the accumulation of B1. Potassium sorbate stimulated fungal growth at 100 mg/l, while at all concentrations it considerably inhibited toxin production (no detectable amounts of B1 and 3 to 5 times less G1 than in controls). The concentration of G1 surpassed that of B1 without exception.     

Effect of lycopene on biomarkers of oxidative stress in rats supplemented with ω−3 polyunsaturated fatty acids

Eicosapentaenoic (C20:5 n−3, EPA) and docosahexaenoic (C22:6 n−3, DHA) fatty acids are highly susceptible to lipid oxidation and may contribute to oxidative stress. The purpose of this study was to evaluate if a diet rich in EPA and DHA could promote higher oxidation in both plasma and brain tissue of rats and if lycopene supplementation could revert the oxidation biomarkers to their baseline levels. Wistar rats were divided into four groups (7 rats/group): SOY group received AIN93M diet containing 8% soybean oil for 21 days; OMEGA group received 8% fish oil containing 2% EPA and 1% DHA instead of soybean oil for 21 days; OMEGA-L0.5 and OMEGA-L50 groups were fed a diet containing 8% fish oil for 11 days followed by lycopene supplementation (5 and 500 mg/kg diet, respectively) for 10 more days. The brain homogenate oxidation measured by TBARS suggested that supplementation with EPA and DHA reduced the oxidation rate and this effect was suppressed by lycopene at both concentrations. Antioxidant capacity measured by DPPH method in plasma of groups supplemented with EPA and DHA was nil while in SOY group it was 21.4%. Lycopene was not detected in the plasma of the animals. It is likely that antioxidants present in plasma have been depleted to keep malondialdehyde concentration measured by TBARS unchanged. Both plasma malondialdehyde concentration and antioxidant activity of groups with and without lycopene supplementation did not differ. Hence, diets rich in EPA and DHA did not promote higher oxidation in rat brain homogenate but reduced plasma antioxidant capacity. Lycopene did not show antioxidant plasma protection at both doses. Taking into account that plasma α-tocopherol concentration did not change among the groups and that rats synthesize ascorbic acid, additional research should be carried out to identify the oxidative results observed in this study.     

HEAT RESISTANCE IN DIFFERENT HEATING MEDIA OF LISTERIA MONOCYTOGENES ATCC 15313 GROWN AT DIFFERENT TEMPERATURES

This paper examines the influence of growth temperature on thermal resistance of Listeria monocytogenes ATCC 15313. Regardless of the incubation temperature, the heat resistance of Listeria monocytogenes increased during incubation until the stationary phase of growth was reached. The maximum heat resistance of cells grown at 4C, 20C or 37C was attained after 14 days, 36 h and 18 h of incubation, respectively. After longer incubation times the heat resistance of cells grown at 4C and 20C did not change but that of cells grown at 37C decreased. The maximum heat resistance was usually greater at higher incubation temperatures. However, the magnitude of these differences depended on the pH and composition of heating media. By raising the incubation temperature from 4C to 37C, the D₆₂ value in pH 7 citrate-phosphate buffer increased from 0.13 to 0.34 min. However, when skimmed milk was used as menstruum this difference was not observed. Cells grown at 37C attained maximum heat resistance at pH 7 but those grown at 4C, at pH 6. The magnitude of the effect of the incubation temperature on heat resistance was constant at all heating temperatures tested (z = 6 ± 0.8C). The higher heat resistance of cells grown at higher temperatures was not due to a greater capacity of heat damage repair     

Screening for yeasts incorporating the exogenous eicosapentaenoic and docosahexaenoic acids from crude fish oil

The screening for yeasts incorporating exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into their cellular lipids was conducted. Two percent of oil from fish scraps was added to a yeast isolation medium as a sole carbon source. From 143 soil samples, we isolated 23 yeast strains, nine of them were found to be capable of quickly assimilating the scrap fish oil. These nine strains, in addition to four previously isolated triglyceride-assimilating and lipase-producing strains, Yarrowia lipolytica and three Geotrichum species (FO274A, FO347-2 and FO401B), were cultured in a growth medium at 30 degrees C. Strains FO726A, FO765A and FO347-2 were selected on the basis of dry cell weight production and ability to store EPA and DHA in their cells, and their performance was further compared by varying cultivation temperature and time. From 1 g of the scrap fish oil, FO726A yielded 620 mg of dry cells, containing 47.1% lipid, 38.1% triglyceride, 3.3% EPA, and 4.9% DHA, when cultured at 25 degrees C for 36 h. Strain FO726A apparently has the highest ability to incorporate EPA and DHA into its cellular lipids. Results from further experiments showed that the incorporated EPA and DHA mainly existed in the form of triglyceride in the FO726A cell. These results suggest that FO726A is suitable for the production of cell mass rich in EPA and DHA for feed.     

Maximizing the production of γ-linolenic acid in Mortierella ramanniana var. ramanniana as a function of pH,temperature and carbon source,nitrogen source,metal ions and oil supplementation

A locally isolated (Perryvale, Alta., Canada) fungal genus, Mortierella ramanniana var. ramanniana, was evaluated as a potential industrial producer of γ-linolenic acid (GLA). Six growth variables (pH, temperature, carbon source, nitrogen source, and metal ions and oil supplementation) were systematically manipulated. The results indicated that the GLA production for this particular strain, could be maximized by using a basal growth medium consisting of 5% dextrose and 1% yeast extract, supplemented with 5 mg/L Mn²⁺ with incubation at 20 °C. The lipid yield under optimum conditions was 54.2% of the total dry biomass and consisted of 84.3% unsaturated fatty acids. The yield of GLA per gram biomass represented 13.3% of the total lipid content, slightly higher than that of the typical evening primrose oil GLA yield. The study establishes this variety as an effective intermediate fungal source of GLA.     

蛹虫草液体种制备及发酵生产菌丝体和虫草菌素工艺优化

为获得蛹虫草液体种制备和液体发酵生产菌丝体和虫草菌素的最佳工艺,以蛹草拟青霉Peacilomyces militarisBCEC07菌株为菌种,通过对接种量(孢子浓度)的考察,探索不同孢子浓度对蛹虫草液体母种制作效果的影响;通过单因素和正交试验,优化生产虫草菌素各营养因子的最佳种类和配比。结果表明:在1.5×1010孢子数的接种量时制作的母种最适合用于蛹虫草液体发酵产菌丝体和虫草菌素;蔗糖、蛋白胨、MgSO4.7H2O、K2HPO4和NAA是最适合的碳源、氮源、无机盐及生长因子;工艺优化后得出蛹虫草液体发酵产菌丝体的最佳配方为30g/L蔗糖、25g/L蛋白胨、1.5g/L KH2PO4、0.5g/L MgSO4.7H2O和4.0mg/L NAA;产虫草菌素的最佳配方为:30g/L蔗糖、25g/L蛋白胨、1.5g/L KH2PO4、0.5g/L MgSO4.7H2O和3.0mg/L NAA。优化后生物量和虫草菌素总产量分别提高了43.00%(达31.60g/L)和31.60%(达645.12mg/L)。为进一步提高蛹虫草菌丝体及虫草菌素的单位产量提供参考。     

Growth and biosynthesis of aflatoxin by Aspergillus parasiticus in cultures containing nisin

Nisin, 200 or 5000 Reading units/ml, was added to Aspergillus parasiticus cultures. The cultures were incubated at 28 degrees C for 3, 7 or 10 days and analyzed for mycelial dry weight, pH and accumulation of aflatoxin B1 and G1. During the first 3 days of incubation, dry weight, pH decrease and aflatoxin accumulation were suppressed by nisin, when compared with similar values for the nisin-free control. After longer incubation, differences in dry weight nd pH values decreased, whereas accumulation of aflatoxin in the nisin-containing cultures surpassed that of the control.     

Antibacterial activity of eicosapentaenoic acid (EPA) against foodborne and food spoilage microorganisms

Eicosapentaenoic acid (EPA), a long-chain polyunsaturated fatty acid of ω-3 type was evaluated for its antimicrobial action against the range of foodborne and food spoilage pathogens, using agar disc diffusion assay in Luria broth (LB) media. The EPA exhibited antimicrobial activity against Bacillus subtilis ATCC 6633, Listeria monocytogenes ATCC 19166, Staphylococcus aureus ATCC 6538, S. aureus KCTC 1916 and Pseudomonas aeruginosa KCTC 2004. The minimum inhibitory concentrations (MICs) of the EPA against the tested bacterial strains were found in the range of 500-1350 μg/ml using broth dilution method. EPA reduced the viability of S. aureus at 250, 125 and 62.5 μg/ml after 15 min exposure and a steep decline in colony forming units (CFUs) was observed at 125 μg/ml after 30 min exposure, while similar reduction in CFU rate was exhibited by EPA when treated with 62.5 μg/ml after 180 min. EPA also reduced the CFU numbers of P. aeruginosa at all the concentrations used in this study after 15 min exposure. On the other hand, scanning electron microscopy (SEM) study of bacterial cells clearly exhibited the antibacterial effect of EPA as evidenced by the damages found in the outer membrane of the cells when treated with EPA. The results demonstrated that EPA exerted significant bactericidal and bacteriostatic effects against both P. aeruginosa and S. aureus.     

BACTERIAL FORMATION OF HISTAMINE IN JACK MACKEREL (TRACHURUS SYMMETRICUS)

Peak histamine concentrations of 0.023, 0.031 and 0.027 g histamine/100 g muscle and maximal bacteria concentrations of 1.75, 1.59 and 0.423 g dry cells/100 g muscle were observed in muscles of jack mackerel stored at 25, 15 and 5C, respectively. Incubated fish homogenates suggest rate and transport limitations in histamine formation in muscle. The Mulchandani model predicted bacterial growth in muscle. The Luedeking and Piret expression fitted histamine formation in muscle; α values were 3.0 × 10⁻³, 1.23 × 10⁻² and 4.17 × 10⁻² g histamine/g dry cells, while β-values were 4.5 × 104, 8.0 × 10⁻⁵ and 0 g histamine/g dry cells × h at 25, 15, and 5C, respectively. The model predicts that jack mackerel could be stored from 4.5 to 5.5 days in ice, from 1 to 2 days at 15C and from 17 h to 2 days at 25C before fishmeal quality might be affected.     

初始pH值对细脚拟青霉小试发酵过程及胞内核苷积累的影响

在30L发酵罐中,采用液体深层发酵的方法,考察培养液初始pH7、6.5、6、5.5、5、4.5时对细脚拟青霉菌体生长及胞内核苷类物质积累的影响。结果表明：初始pH6.5时,对菌体生长和胞内核苷的积累最为有利,最大生物量为28.7g/L,最高胞内核苷类物质产量为5.94mg/g;起始pH7、6、5.5、5、4.5时,最大生物量分别在发酵的第9、10、11、11、13天获得,依次为26.4、27.3、23.6、21.2、18.5g/L;最大胞内核苷类物质的产量分别在发酵的第11、11、11、13、13天获得,依次为5.27、5.51、4.89、4.29、4.05mg/g。细脚拟青霉以菌体或者胞内代谢物为发酵目标进行优化控制时,其发酵初始pH值应调节到相应的最佳值。     

Factors affecting patulin production by Penicillium expansum

Patulin, a mycotoxin produced by Penicillium spp. during fruit spoilage, is a major concern with regard to human health because exposure can result in severe acute and chronic toxicity, including carcinogenic, mutagenic, and teratogenic effects. In this study, we investigated the effects of Penicillium expansum isolate, apple cultivar, storage temperature and time, and pH on the production of patulin. Patulin was analyzed by a previously developed micellar electrokinetic capillary electrophoresis method. P. expansum isolates originating from across Ontario produced widely differing levels of patulin, ranging from 0 to >6 mg/g by dry mycelial weight. The highest patulin levels were those for isolates displaying aggressive growth (characterized by rapidly increasing acidity) accompanied by profuse mycelial development. Distinct patterns in fungal growth rates and patulin production were evident among isolates grown in McIntosh, Empire, and Mutsu ciders. Extensive fungal growth and higher patulin levels (538 to 1,822 microg/ml on day 14) in apple ciders were associated with incubation at room temperature (25 degrees C), although potentially toxic patulin levels (75 to 396 microg/ml on day 24) were also found in refrigerated ciders (4 degrees C) inoculated with P. expansum.     

Submerged Production of Agaricus campestris Mycellum in Peat Extracts

The edible mushroom Agaricus campestris has been grown using peat extracts as the only substrate source. Shaker flask fermentations were conducted with Sphagnum peat extracts obtained by autoclaving peat mixed with 1.5% (v/v) H₂SO₄ for 2 hr at 121°C. Inoculum ratio, temperature, initial pH, fermentation time and agitation were tested in order to evaluate the effect of those factors on the mycelial growth, expressed as final dry mycelium concentration and biomass yield (g of dry mycelium produced per g of carbohydrates consumed). The best combination of operating variables, in the range of values investigated, are: 4% (v/v) inoculum ratio, 24°C, pH 6.0, 6 days and 150 rpm.     

真菌发酵法产油脂的研究

本文以深黄被孢霉(Mortierellaisabellina)为试验菌株,采用发酵法生产油脂。采用单因素试验设计,研究了产脂培养基中碳源、氮源种类及其浓度对菌体生长和油脂积累的影响。试验确定酶法水解的玉米淀粉水解糖为最佳碳源,其最适添加浓度为100g/L;酵母膏为最适氮源,其最适添加浓度为3g/L。优化培养基条件下获得菌体生物量为12.350g/L,油脂含量为52.19%,油脂产量为6.4376g/L。但还有待于进一步优化培养基中的其它成分,以有效提高菌体油脂产量。     

Optimization of bioprocess for production of copper-enriched biomass of industrially important microorganism Saccharomyces cerevisiae

The production of Saccharomyces cerevisiae cells enriched with copper and the effects of adding copper ions to different media on yeast cell growth and ethanol production were studied. In the media Cu(2+) concentrations of up to 0.094 mM had no effect on alcoholic fermentation, whereas higher Cu(2+) concentrations markedly decreased yeast cell growth rate and ethanol production. Under static conditions, the maximum amounts of copper uptake (i.e., 1.16 mg/g, 1.2 mg/g and 0.81 mg/g dry matter yeast biomass for glucose, sucrose and molasses media, respectively) were obtained after 8 h of fermentation, whereas under dynamic conditions smaller amounts of copper uptake (i.e., 0.98 mg/g, 1.02 mg/g and 0.7 mg/g dry matter yeast biomass for glucose, sucrose and molasses media, respectively) were obtained after 6 h of fermentation.     

INHIBITION OF ASPERGILLUS GROWTH AND EXTRACELLULAR AFLATOXIN ACCUMULATION BY SORBIC ACID AND DERIVATIVES OF FATTY ACIDS

A study was conducted to determine the effects of sorbic acid and several derivatives of fatty acids (amides, aminimides, and monoglycerides) upon toxigenic cultures of Aspergillus flavus and A. parasiticus. A synthetic medium was inoculated with spores, incubated for 48 h at 27°C, and then supplemented with sorbic acid and fatty acid derivatives. Cultures were then incubated for an additional 5 days. Aflatoxins were extracted, separated, and quantitated. Mycelial mats were dried, weighed, and analyzed for lipid and mineral content. Cerulenin (8 μg/ml) was the most effective fatty acid derivative examined, reducing mycelial growth by 37% and completely inhibiting extracellular accumulation of aflatoxins. Other derivatives, in decreasing order of effectiveness, included M-20 (an aminimide), lauribic, and lauricidin. Mycelia grown in the presence of fatty acid derivatives contained less phosphorus, potassium, magnesium, phosphatidyl ethanolamine, phosphatidyl serine, cholesterol, and triglycerides, but more cardiolipin, phosphatidyl choline, free fatty acids, fatty acid esters, and diglycerides. Levels of monoglycerides and cholesterol esters remained essentially unchanged. Inhibition by sorbic acid was nonspecific, affecting both mycelial growth and extracellular aflatoxin accumulation to approximately the same extent. Utilization of fatty acid derivatives for determining mechanisms of aflatoxin accumulation and lipid biosynthesis appears promising.