Mycelial

Ling Zhi-8 (LZ-8) is an immunomodulatory protein (29,30) isolated from the mycelial extract of Ganoderma lucidium, that has been purified and shown to stimulate mouse spleen and human peripheral blood lymphocytes. LZ-8 is able to inhibit antibody production and prevent the development of autoimmune type I diabetes in NOD mice. 

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). 

Polyethers are usually found in both the filtrate and the mycelial fraction, but in high yielding fermentations they are mosdy in the mycelium because of their low water-solubiUty (162). The high lipophilicity of both the free acid and the salt forms of the polyether antibiotics lends these compounds to efficient organic solvent extraction and chromatography (qv) on adsorbents such as siUca gel and alumina. Many of the production procedures utilize the separation of the mycelium followed by extraction using solvents such as methanol or acetone. A number of the polyethers can be readily crystallized, either as the free acid or as the sodium or potassium salt, after only minimal purification. 

Polyethers such as monensin, lasalocid, salinomycin, and narasin are sold in many countries in crystalline or highly purified forms for incorporation into feeds or sustained-release bolus devices (see Controlled-RELEASE technology). There are also mycelial or biomass products, especially in the United States. The mycelial products are generally prepared by separation of the mycelium and then drying by azeotropic evaporation, fluid-bed driers, continuous tray driers, flash driers, and other types of commercial driers (163). In countries allowing biomass products, crystalline polyethers may be added to increase the potency of the product. 

Following the discovery of penicillins, an extensive program for the screening of culture fluids and residual mycelial material commenced which resulted in the discovery of a large number of pyrazinones and related 1-hydroxy-2-pyrazinones with pronounced antibiotic character. Some examples are shown in Table 4. One of the earliest substances to be isolated, aspergillic acid (110 = OH, = Me, R = Et, R = R = H, R = Pr ), was found... 

This was inoculated with a spore suspension of P. patu/um (1 liter containing 3-5 x 10 spores/ml) and grown at 25°C in 100 gallon tank. The inoculum is transferred at 40 hours or when the mycelial volume (after spinning 10 minutes at 3,000 rpm) exceeds 25%. The fermentation is conducted as near to the ideal pH curve as possible by addition of crude glucose, according to U.S. Patent 3,069,328. 

Inoculation is by conidia of A. niger or alternatively using pre-cultured mycelial pellets. Broken mycelial masses are slow to grow initially and are unsuitable here. Two to three days are required for germination during which heat input to maintain 30°C is required. During dtric add formation, cooling is necessary. 

Medium containing up to 35% glucose is steam sterilised by a steam jacket around a conventional stirred tank reactor. pH adjustment starts as the medium cools and is maintained at 65. Vigorous aeration is again required. Inoculation is usually by using a mycelial suspension. Under these conditions a 30% glucose solution can be almost quantitatively converted to sodium gluconate within 36 hours. 

Fig. 9.2 Changes in the luminescence intensity, the contents of luciferin and SOD, and medium pH during the mycelial growth of five kinds of bioluminescent fungi Panellus stipticus, Armillaria mellea, Mycena citricolor, Pleurotus japonicus, and Omphelotus olearius. The ordinate readings of the curves marked 1/3 should be multiplied by 3. For the SOD activity, see Table 9.2, footnote e. From Shimomura, 1992, with permission from Oxford University Press.

Bermudes, D., Gerlach, V. L., and Nealson, K. H. (1990). Effects of culture conditions on mycelial growth and luminescence in Panellus stypticus. Mycologia 82 295-305. 

Filter aids are widely used in die fermentation industry to improve the efficiency of filtration. It is a pre-coated filter medium to prevent blockage or blinding of the filter by solids, which would otherwise wedge diemselves into the pores of the cloth. Filter aid can be added to the fermentation broth to increase the porosity of the cake as it formed. This is only recommended when fermentation product is extracellular. Filter aid adds to the cost of filtration. The minimum quantity needed to achieve the desired result must be established experimentally. Fermentation broths can be pretreated to improve filtration characteristics. Heating to denature proteins enhances the filterability of mycelial broths such as in penicillin production. Alternatively, electrolytes may be added to promote coagulation of colloids into larger, denser particles, which are easier to filter. The filtration process is affected by the viscosity and composition of the broth, and the cell cake.5... 

A typical penicillin broth contains 20-35 mg/1 of antibiotic. Filtration is used to remove mycelial biomass from fermentation broth. The filtration may be subjected to filter aided polymers. Neutralisation of penicillin at pH 2-3 is required. Amyl acetate or butyl acetate is used as an organic solvent to remove most of the product from the fermentation broth. Finally, penicillin is removed as sodium penicillin and precipitated by a butanol-water mixture. 

A fermentation broth of Streptomyces tsukubaensis No. 9993 is filtered and the mycelial cake is extracted with acetone. The filtrate is combined with the acetone extract and passed through a column of Diaion HP-20. The dilution with 75 % aqueous acetone, by evaporation gives an oily residue that is extracted with ethyl acetate and submitted to column chromatography over silica gel. 

The perceived sensitivity of plant cells to the hydrodynamic stress associated with aeration and agitation conditions is typically attributed to the physical characteristics of the suspended cells, namely their size, the presence of a cell wall, the existence of a large vacuole, and their tendency to aggregate. Table 1 illustrates some of the differences between plant cells and other biological systems. Chalmers [19] attributed shear sensitivity in mammalian cultures at least in part to the fact that these cells occur naturally as part of a tissue, surrounded by other cells. The same is true for plant cells. The more robust microbial systems, on the other hand, exist in nature as single organisms or mycelial structures, very close to the forms they assume in submerged culture. 

A similar approach has been suggested in other studies of plant cells [57] and protein precipitates [133]. However, information on the rate of the size distribution shift process cannot be inferred from chain-length measurements made only at the beginning and end of the experiment. To date, there have been no reports on the progressive modification of the size distribution of plant cells subjected to continued exposure to turbulent forces. There are, however, a number of studies which address the break-up of mycelial hyphae in agitated vessels... 

Contaminants of high molecular weight (considered to have arisen from mycelial residues frxm the fermentation process) may be responsible for the induction of allergy to penicillins their removal leads to a marked reduction in the antigenicity of the... 

A, discoloured, oil-depleted, aqueous phase B, oil globule-rich creamed layer, C, coalesced oil layer limi cracked emulsion D, liingal mycelial growth on surface. Also present are a foul taste and evil smell ... 

Culture conditions The exo-1 strain was cultivated in two-stages (I) pre-cultivation for 24 hours in Vogel s medium [7] supplemented with 2% glucose, and (II) transfer of the mycelial... 

The isolates were cultured in 100 ml Erlenmeyer flasks containing 20 ml liquid medium (12) with different carbon sources. The cultures were inoculated with mycelial disks cut from the margins of 7 day-old-colonies and incubated at 25 ( under static conditions. 

Strains requirement sponilation growlh e.xtracel. mycelial age time yield... 

Estrogen enhances Candida adherence to vaginal epithelial cells and yeast-mycelial transformation this is supported by the fact that infection rates are lower before menarche and after menopause (except in women taking hormone replacement therapy), while rates are higher during pregnancy... 

Rhizopus nigricans, Mucor vesiculosis mycelial exudates inhib d. R japonicum T. veridi inhib d. nodulation M. vesiculosis inc d nodule no. 121... 

One of the best methods to synthesize cyclopentenone derivatives is the Pauson-Khand procedure. However, Shindo s group have recently developed a domino process consisting of a [2+2] cycloaddition of a ketone with anynolate, followed by a Dieckmann condensation to give a 3-lactone as 4-190 which is decarboxylated under reflux in toluene in the presence of silica gel to afford cyclopentenones [64a]. Thus, the reaction of 4-188 and 4-189 led to 4-190, which on heating furnished the linear cucumin 4-191 (Scheme 4.41). This natural product has been isolated from the mycelial cultures of the agaric Macrocystidia cucumis [65, 66]. The domino procedure described was also used to synthesize dihydrojasmone and a-cuparenone. Moreover, the [2+2] cycloaddition can be combined with a Michael reaction [64b]. 

Shibata, N., Kobayashi, H., Tojo, M. and Suzuki, S. (1986) Characterization of phosphomannan-protein complexes isolated from viable cells of yeast and mycelial forms of Candida albicans NIH B-792 strain by the action of Zymolyase-IOOT. Archives of Biochemistry and Biophysics, 251 (2), 697-708. 

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