To date, aquatic feeds have depended heavily on fishmeal and fish oil as their source of protein and lipid. However, the feed industry is encountering shortfalls in the availability of these ingredients because of a decline in the number of fish captured in the wild and the increased human demand for some of the species currently being used for fishmeal and oil production. Therefore, efforts are now being directed in different parts of the world to finding alternative quality ingredients, which ideally are less expensive and readily available for use in practical diets. The data accrued have shown that a large proportion of both fishmeal and fish oil can be replaced by other protein and lipid sources. However, it is emphasized that an optimal essential amino acid balance be maintained and that the n-3 highly unsaturated fatty acid requirement be satisfied when combining economical protein and lipid ingredients. Newly developed feeds should aim at being nutrient-dense in order to reduce the output of solid, P and N waste. This can be done through improving nutrient availability, optimizing the digestible protein to energy balance of the diet, and replacing dietary fishmeal with alternate ingredients. These diets should also be effective for maintaining good health and improving disease resistance in fish through enhancement of immune responses. A wholesome approach to culturing fish would be to use appropriate feeding standards that are aimed not only at improving economic returns but also at developing a lasting cohabitation of sustainable aquaculture and a cleaner environment. Furthermore, in the 2lst century aquaculture would still retain its place as a prominent source of food protein, signifying that fish feed research remains a forceful area in discovering better feeds for the industry.

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Stress and immune responses were studied in Nile tilapia Oreochromis niloticus under both conditions of self-feeding and scheduled feeding. Self-feeding fish had a significantly lower cortisol level in their blood plasma than that of scheduled feeding fish. Furthermore, skin color was found to be paler in the self-feeding fish compared with that of the scheduled feeding fish. Thus, the self-feeding fish had a less stressful physiological status than the other experimental fish. Immunological analyses revealed that the self-feeding fish significantly exceeded the scheduled feeding fish in the phagocytic activity of their macrophages, antibody production, and the number of blood-circulating lymphocytes. The higher immune responses of self-feeding fish seem to be attained through a less stressful environment. This self-feeding behavior could be exploited to create reduced stress for disease control in fish farming.

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In order to clarify the influence of mariculture on the benthic fauna, samples of the macrobenthos were collected from Gokasho Bay, where intensive fish culture and pearl oyster culture have been carried out. Monthly samples collected from the fish farm and pearl farm sites during June 1995 to July 1996 revealed that the community structure of the two sites showed distinct differences with seasonal fluctuations. At the fish farm site, azoic conditions were found from July to November; after December, the diversity increased markedly through successive recruitments of small-sized species such as the polychaetes Capitella sp. and Pseudopolydora paucibranchiata, and the amphipods Aoroides spp.; macrofaunal density, biomass and species richness peaked from March to April. At the pearl farm site, a higher diversity, including larger-sized species, and no clear seasonal fluctuations in abundance was found, and the community structure was similar to that at the control site. These results show the large impact by fish farming on the macrofauna, whereas pearl farming causes less effect on the benthic fauna. It is suggested that the difference in the level of organic input between the two sites results in the differences in the dissolved oxygen content of the bottom water, sulfide content of the sediments and, subsequently, the macrobenthic assemblages.

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The effects of salmon calcitonin (sCT) on osteoblasts and osteoclasts were histologically and histomorphometrically investigated in the pharyngeal bone of fed and starved goldfish Carassius auratus. The thickness of the osteoid and the height of osteoblasts were also measured. Fish were given sCT intraperitoneally at a dose of 10 ng/g bodyweight four times every other day under either fed or starved conditions. The sCT treatment induced the retraction of osteoclasts and their disengagement from the bone surface. Salmon calcitonin had no effect on the histomorphometric parameters of bone formation activity, but suppressed the parameter (OcP/EP) that showed osteoclast activity. Salmon calcitonin did not affect the thickness of the osteoid, but increased the height of osteoblasts in starved fish. Histological and histomorphometric results demonstrated that osteoclast activity was suppressed when sCT was given to starved estrogenized goldfish five times for 10 days. Overall, results suggest that calcitonin is involved in bone assimilation by suppressing osteoclast activity and by enhancing osteoblast activity in the pharyngeal bone of goldfish.

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A new method for monitoring spinal motoneuron activities during fictive swimming in teleost fish was developed. Enamel-insulated copper wire electrodes were implanted in the trunk muscle of goldfish or carp. For each freely moving fish, an electromyogram (EMG) was recorded using the electrodes. In those fish paralyzed with curare, using the same electrode set, bursts of electrical activities consisted of spikes of smaller amplitude and of shorter duration compared with those recorded by EMG. Simultaneous recording of the extracellular activity and intracellular recording from the muscle revealed that the bursts of spikes recorded in the paralyzed fish were motor nerve impulses innervating the muscle and are considered to be fictive swimming activity. The method developed in the present study provides a useful tool with which to investigate neuronal mechanisms underlying swimming activity in teleost fish.

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The contribution of the thyroid hormone to the metamorphosis of the coral trout Plectropomus leopardus was examined. Juveniles, aged 35 days, were exposed to thyroxine (T4, 0.1 p.p.m) and thiourea (TU; inhibitor of thyroid hormone synthesis, 30 p.p.m) for 13 days. All the fish in the T4-treated group had completed metamorphosis (second metamorphosis, pigmentation and resorption of fin spines) within three days after the beginning of the experiment (day 3), whereas it took 13 days for this to occur in the control fish. Moreover, the fish in the TU group had not completed metamorphosis even by day 13. Settling behavior was significantly stimulated by T4 treatment, as was the resorption of the dorsal and pelvic fin spines, the reduction of serration on the spines, and the transition of body color into an opaque reddish hue. Fish in the TU group had longer dorsal and pelvic spines, retarded pigmentation and an abnormal black coloration. These results suggest that thyroid hormone accelerates most changes during the second metamorphosis in the coral trout.

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Repeated injection of salmon gonadotropin (sGTH) can induce ovarian maturation in the female Japanese eel Anguilla japonica. The participation of endogenous GTH in this process remains unclear. The steady-state mRNA levels of pituitary GTH Iβ and GTH IIβ subunits in individual fish was investigated using a real-time quantitative reverse transcription-polymerase chain reaction assay based on the TaqMan^TM fluorogenic detection system. Pituitary GTH Iβ mRNA levels were high when the fish were immature, particularly before being injected with sGTH, but levels decreased with the progression of ovarian development and remained very low from the late vitellogenic stage until postovulation. In contrast, mRNA levels of GTH IIβ were very low until the previtellogenic stage after injection with sGTH, and increased markedly in accordance with ovarian maturation and ovulation. These results suggest that sGTH injection inhibits GTH I synthesis, whereas it induces ovarian maturation and stimulates GTH II synthesis. Thus, the differential expression of GTH Iβ and GTH IIβ genes during artificial maturation suggests that the two GTH are synthesized sequentially and have separate functional roles relating to reproductive events in the Japanese eel.

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The allelic inheritance mode of microsatellite DNA markers was examined using seven copulated wild females and their offspring. Five microsatellite loci, CSPJ002*, CSPJ010*, CSPJ012*, CSPJ014*, and CSPJ015*, were used in the study. At almost all family/locus combinations, one sire was determined and distributions of genotypes in offspring were consistent with the Mendelian segregation ratio. Distributions of genotypes were consistent with the ratio after assuming a null allele at some loci. Consequently, the alleles of CSPJ002* and CSPJ012* were inherited following the Mendelian inheritance mode in every family; however, the null allele was expected in CSPJ010*, CSPJ014*, and CSPJ015* in some families. Thus, these loci should be used carefully in population genetic analysis, but siblings could be detected in the dendrograms based on unweighted pair-group method using arithmetic averages (UPMGA).

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The validity of using the RNA:DNA ratio, glycogen content, and C : N ratio in the columellar muscle as indicators of the nutritional condition in the Japanese turban shell Turbo cornutus was examined. These biochemical indices were compared between fed and starved animals. The fed animals were fed brown algae to satiation for 108 days as a control, whereas the starved animals were not fed for the first 75 days and then fed for next 33 days. All three indices declined during the starvation period, and the values for the starved group were significantly lower than those for the fed group during days 20-75. At day 108, after the re-feeding period, the indices for the starved group were found to have increased. Among the indices, only the RNA:DNA ratio for the starved males responded rapidly to starved conditions and became significantly lower even at day 4, and only the RNA:DNA ratio for the starved males and females recovered to those levels of the fed animals at day 108. The results indicated that the RNA:DNA ratio is the most rapid indicator of nutritional stress among the three indices. However, it is recommended that glycogen content and C : N ratio be used in addition to the RNA:DNA ratio for monitoring the health of T. cornutus as the RNA:DNA ratio showed large variations.

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Effects of the chromosome set manipulation on early embryonic development were examined in goldfish and crucian carp. Both pressure- and heat-shock treatments of fertilized eggs induced developmental disorders such as thin blastodisc formation, delay of epiboly, and deficiency of dorso-anterior structures. The longer the treatments were applied to the eggs, the more frequently severe dorsal deficiencies were induced in the embryos. No and reduced signals of goosecoid mRNA, a dorsal mesodermal marker, were observed in embryos treated with heat-shock at 40°C for 1 min at 5 min postfertilization. These results suggested that such treatments should affect not only the retention of the second polar body, but also the suppression of dorso-ventral differentiation.

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Developmental changes of swimming speed were analysed in the chub mackerel Scomber japonicus raised on two distinct dietary regimens. One group was fed only zooplankton such as rotifers and Artemia nauplii (R group) and the other group was fed rotifers plus red sea bream Pagrus major larvae (Pm group). Fish from the Pm group grew faster than those from the R group, and both the cruise and burst swimming speeds were faster for the Pm group even when match-sized comparisons were made. Fish in the Pm group attained a standard length (SL) of 17.5 mm by day 14 and their cruise and burst swimming speeds were 5.39 SL/s and 18.45 SL/s, whereas the R group attained 17.35 mm SL by day 17 and their cruise and burst swimming speeds were 4.28 SL/s and 13.98 SL/s. The results suggest that the swimming speed of chub mackerel in the wild would develop differently depending on the food resources they happen to encounter.

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The absorption of phosphorus (P) contained in several feed ingredients by rainbow trout was compared during the growing stages of the fish. The ingredients tested were pollock meal, sardine meal, poultry feather meal (PFM), meat and bone meal (MBM), defatted soybean meal (SBM), soy protein concentrate (SPC), corn gluten meal (CGM), and defatted soybean meal extruded at 150°C (exSBM). Phosphorus absorption was determined for fish of sizes 2 g, 5 g, 10 g, 20 g, 50 g, 100 g, and 200 g. Absorption of P from the two fishmeals slightly decreased as bodyweight increased. Conversely, P absorption from PFM and the plant ingredients was very low by the 2 g fish, but increased during the early growth stages and seemed to have reached a plateau in fish bigger than 10 g. Absorption from exSBM was much higher than that from SBM at all bodyweights examined. Phosphorus absorption from the different ingredients were: pollock meal 31-46%; sardine meal 49-59%; MBM 36-46%; PFM 36-84%; SBM 3.4-37%; exSBM 25-58%; SPC 3.7-34%; and CGM, 0-17%. The results demonstrate that, depending on the ingredient P absorption in rainbow trout changes as the fish grows. Extrusion cooking at 150°C improved the availability of P in SBM.

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Stranded organisms were observed on the sandy beach of Kuroshima Island, Ryukyu Archipelago, in the early morning of 28 January 1993. Stranded organisms of 525 individuals were classified into 74 species. These organisms included 13 individuals of Panulirus pueruli, which were identical to P. longipes bispinosus. Kuroshima Island is located near Ishigakijima and Iriomote islands, Ryukyu Archipelago, where coral reefs develop. Coral reefs are common and develop in the Ryukyu Archipelago, southern Japan, with the exception of Yakushima and Tanegashima islands. Panulirus japonicus in Japanese waters has not been found in coral reef regions where P. longipes bispinosus are mainly found. The present study describes and identifies to species level puerulus specimens that were stranded on the beach of Kuroshima Island.

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The gut and gut contents of ayu Plecoglossus altivelis caught in the Ohta River in Hiroshima prefecture, Japan were examined. Relative intestine length (intestine length/body length) was almost constant. Except for relative intestine length, characteristics of the gut of wild ayu were not significantly different from those of cultured ayu. The height of intestinal folds increased and their width declined from stomach to anus. Cyanobacteria (Homoeathrix sp. and Calothrix sp.) and diatoms (Cymbella sp., Gomphonema sp., Melosira sp., Navicula sp., and Synedra sp.) were well digested during passage through the gut despite a lack of apparent destruction. In contrast, green algae (Dictyosphearium sp., Coelastrum sp., and Pediastrum sp.) exhibited little degradation. The pH of stomach contents ranged from 2.8 to 7.4 (mean ± SD=4.1 ± 1.0), whereas that of the posterior intestine was 7.0-8.5 (7.9 ± 0.4). Gut contents of wild ayu increased with bodyweight, and were more than threefold greater than those of cultured ayu, suggesting that wild ayu compensate for low nutrient content of algae through the continuous ingestion of large quantities of feed organisms.

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To examine the involvement of chloride cells in the uptake of Ca²⁺ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca²⁺ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na⁺, K⁺-ATPase. Chloride cells, located mostly on the afferent-vascular edge of the filaments, were larger at lower Ca²⁺ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca²⁺ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca²⁺ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca²⁺ groups. Plasma osmolality decreased with decreasing ambient Ca²⁺ concentration, suggesting that environmental Ca²⁺ affects the permeability of the body surfaces. Gill Na⁺, K⁺-ATPase activity in the 0.1 mM and 0.5 mM Ca²⁺ groups were significantly higher than that in the 2.5 mM Ca²⁺ group, implying the involvement of the Na⁺-Ca²⁺ exchanger in Ca²⁺ uptake in the gills. These findings suggest that chloride cells function as the site for Ca²⁺ uptake in killifish acclimated to low Ca²⁺ environments.

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Temperature and salinity ranges in which Gymnodinium catenatum (Hiroshima Bay strain) showed specific growth rates higher than 0.2/day were approximately 20-30°C and 20-32. The specific growth rate (μ), expressed as a polynomial equation as functions of temperature (T; °C) and salinity (S) were μ=(-6.84×10^-4T²+0.0354T-0.213)×(-1.03×10^-3S²+0.0579S-0.548)/0.31; the maximum growth rate (0.31/day) was obtained at 25°C and 30. From a comparison with field data recording temperature, salinity and light intensity, this species may be expected to bloom from summer to autumn in Hiroshima Bay.

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To clarify the characteristics of the larval and juvenile fish community in a Japanese estuary, monthly collections were conducted in the shallow waters of the Shimanto Estuary, Japan from July 1985 to June 1987. A total of 49 101 fishes of more than 100 species from 44 families were sampled over the 2-year period. Numerically, Gerres equulus was most common, accounting for 20.02% of the total abundance. Rhyncopelates oxyrhynchus was ranked second, followed by Acanthopagrus latus, Acanthogobius flavimanus, Mugil cephalus cephalus, Parioglossus dotui, and Acanthopagrus schlegeli. Thus, fishes observed in the estuary were mostly marine species that spawn in the sea. The majority of these fish first appeared within the estuary at late postlarval and/or early juvenile stages. Based on their utilization of the shallow waters in the estuary, the majority of species could be categorized as residents. In the Shimanto Estuary, shallow waters are studded with shelters, especially eelgrass beds, which serve as important nursery grounds for coastal and euryhaline fishes.

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Tensile and knot fatigue property of high-performance polyethylene (HPPE), regular polyethylene (PE), nylon multifilament (PAmu), and nylon monofilament (PAmo) under cyclic loading conditions were investigated. The durability of HPPE under tensile fatigue testing was largest, followed by PAmo, PAmu and PE. In knot fatigue, the largest was PAmo, followed by HPPE, PAmu, and PE. High resistance by HPPE to the cyclic loads was also confirmed by scanning electron microscopy, which showed that HPPE suffered the least damage from abrasion compared with the other twines. From the measurement of elongation changes during the cyclic loading, HPPE had very small changes. The low elongation value of HPPE was probably the result of its better fatigue property because there was less damage from abrasion.

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Changes in the freshness indices and extractive components in the foot muscle of live small abalone Haliotis diversicolor during storage at 5°C, 15°C, and 25°C were investigated. The pH values declined with storage time. Volatile basic nitrogen and the K-value increased gradually with storage time at 15°C and 25°C, but changes were small at 5°C. The onset of initial decomposition of samples was observed after 3.5 days at 5°C, after 2.5 days at 15°C, and after one day at 25°C. Adenosine triphosphate and adenosine diphosphate degraded rapidly within the early days of storage. In contrast, levels of adenosine monophosphate increased and exhibited prolonged accumulation throughout the storage period. The total amount of free amino acids increased markedly during storage. The dominant free amino acids, such as taurine, glutamic acid, glycine, alanine, and arginine, also increased after storage.

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Differences in taste preference and the levels of extractive components and glycogen were compared between cooked meats of small abalone Haliotis diversicolor fed either gracilar (G-small abalone) or an artificial diet (A-small abalone). Using sensory tests, taste preference of cooked meats was significantly higher for A-small abalone than for G-small abalone. Results of clustering analysis and principal component analysis of chemical data also revealed that the two cooked meats differed from each other in terms of their measured constituent compositions irrespective of sampling periods. Compared with G-small abalone, A-small abalone meats were lower in taurine and arginine, but higher in glycine (Gly), glutamic acid (Glu), alanine, serine, proline, adenosine monophosphate (AMP), and glycogen. It is concluded that the discrepancy in the levels of taste-active components of abalone, such as Gly, Glu, and AMP, is likely to be responsible for the differences in taste preference between G- and A-small abalone cooked meats.

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Protoplast fusion between different species of Streptomyces was performed using a liquid regeneration method developed for a rapid and simple preparation of the fusants. Consequently, new clones, which could not be obtained using the conventional agar regeneration method, were obtained. In the crosses between S. griseus and S. durhamensis, and between S. californicus and S. catenulae, eight and two recombinants, respectively, were obtained using the liquid regeneration method. Conversely, in the case of crosses between S. ornatus and S. catenulae, and between S. ornatus and S. vendargensis, seven recombinants each were obtained using only the agar method. The physiological characteristics, such as the assimilation of carbohydrate and antibiotic resistance, of these fusants differed considerably from those of their parental strains. Using the proposed liquid regeneration method, a simpler and quicker procedure for protoplast fusion is described.

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It has been reported that the amino acid sequences of striated and catch muscle myosin heavy chains from two scallop species (Argopecten irradians and Placopecten magellanicus) are almost identical, but that the ATPase activities between these myosins vary several-fold. These myosin sequences have been useful for identifying the region that modulates the ATPase activity of scallop myosin. In the present study, a cDNA encoding a myosin heavy chain was isolated from the mantle tissue of scallop Patinopecten yessoensis. The cDNA is composed of 6067 base pairs (bp) including an open-reading frame of 5841 p, which encodes an amino acid sequence of 1947 residues. The deduced amino acid sequence of P. yessoensis mantle myosin had a high identity of 90%, 92%, and 91% to P. magellanicus, A. irradians, and Pecten maximus striated muscle myosins, respectively. Interestingly, while the deduced amino acid sequences of around adenosine triphosphate-binding and actin-binding sites of the mantle myosin are homologous to those of A. irradians striated muscle myosin, the subfragment 2 hinge region and the non-helical tail region are similar to those of catch muscle myosin.

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The utilization of dissolved organic phosphorus (DOP) by the two toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Gymnodinium catenatum Grahamm which were isolated from Hiroshima Bay, Japan, was studied. Alexandrium tamarense grew poorly on fructose-6-phophate, glucose-1-phosphate, glycerophosphate, and ribose-5-phosphate with a phosphomonoester bond, although it grew well on the nucleotides adenosine-5-diphosphate (ADP) and adenosine triphosphate (ATP), as well as on dissolved inorganic phosphorus (DIP; as metaphosphate, pyrophosphate, tripolyposphate and orthophosphate). The results imply that A. tamarense was able to utilize DOP and DIP from ambient water using nucleotidase, pyrophosphatase and poly-phosphatase, which hydrolyze phosphodiesters. In contrast, G. catenatum was able to utilize DOP compounds of various molecular weights and structures as well as DIP. In time-course experiments, alkaline phosphatase activity (APA) was induced at orthophosphate concentrations of 0.43 mmol/L and 3.3 mmol/L for A. tamarense and G. catenatum, respectively, and APA increased with orthophosphate depletion. The experiments also demonstrated that APA was maximum at the optimum temperatures for the growth of A. tamarense and G. catenatum; that is, 15°C and 25°C, respectively. These results suggest that the DIP-depleted conditions in Hiroshima Bay might have led to the outbreaks of noxious dinoflagellates in recent years.

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To elucidate the physiological role of the calcitonin gene-related peptide (CGRP) in teleosts, the expressions of calcitonin gene-related peptide receptor (CGRPR) and CGRP mRNA were examined using the reverse transcription-polymerase chain reaction method in the gills of flounder during adaptation from seawater to freshwater. Another related receptor mRNA, which is estimated to be a calcitonin receptor (calcitonin receptor-like receptor; CTRLR), was also monitored in the gills. The expressions of CGRPR mRNA in the gills decreased after transferring flounder from seawater to dilute seawater and became undetectable in freshwater conditions. In contrast, CTRLR mRNA expression in the gills did not change during acclimation. Interestingly, CGRP mRNA was not detected in gills when flounder were transferred from seawater to dilute seawater or freshwater. Therefore, CGRP secreted from other tissue, such as the intestine, may act on the gills and play a role in the seawater adaptation of flounder.

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Protease XXIII, from Aspergillus oryzae, was used to hydrolyze tuna cooking juice at 37°C for up to 6 h. The hydrolyzate obtained at the degree of hydrolysis of 25.68% (after hydrolysis for 2.5 h) displayed the highest 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging effect, reaching 82.19%. Six major fractions (A, B, C, D, E, and F) of this hydrolyzate were obtained by Sephadex G-25 column chromatography using a 0.05 M phosphate buffer (pH 6.5) as the mobile phase. All six fractions displayed a scavenging effect for the DPPH radical, but the scavenging effect was only obvious in two fractions (B and C). After the solid content of hydrolyzates was concentrated from one to five times, the scavenging effect of the DPPH radical increased from 17% to 75% for fraction B, and from 13% to 66% for fraction C. Seven anti-oxidative peptides were isolated from the hydrolyzates (mixture of B and C fractions) by reversed-phase HPLC. The peptide sequences comprised four to eight amino acid residues, including Val, Ser, Pro, His, Ala, Asp, Lys, Glu, Gly, or Tyr.

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Four bacteria with degrading activity against the cell walls of Pythium porphyrae were successfully isolated from Porphyra-culturing environments. The crude enzymes from these bacterial isolates, Bacillus sp. BE1, Bacillus sp. FE1, Pseudomonas sp. PE1, and Pseudomonas sp. PE2, degraded the mycelial cell walls of Pythium sp. N-Acetylglucosamine and glucose were detected in the supernatants of Pythium cell walls treated with the enzymes from Bacillus sp. BE1, Bacillus sp. FE1, and Pseudomonas sp. PE2 as the final degrading products by high-performance liquid chromatography, whereas only glucose was detected in the supernatant of the cell walls treated with the enzyme from Pseudomonas sp. PE1. Moreover, the activities of β-1, 3-glucanase, β-1, 3-1, 4-glucanase, and chitinase were observed by polysaccharolytic analysis using the enzymes from Bacillus sp. BE1, Bacillus sp. FE1, and Pseudomonas sp. PE2, whereas only the activities of β-1, 3-glucanase and β-1, 3-1, 4-glucanase were found during analysis using Pseudomonas sp. PE1. β-1, 4-Glucanase, β-1, 6-glucanase, and mannanase activities were not detected in any of the crude enzymes obtained from these isolates. From the four isolates, the molecular weights (MW) of β-1, 3-glucanase and chitinase were estimated to be approximately 50000-100000 by the ultrafiltration method. Two Pseudomonas spp. were also suggested to have β-1, 3-1, 4-glucanases with MW of 50000-100000. However, the MW of β-1, 3-1, 4-glucanases from the two Bacillus spp. might be close to 50000 or they produce at least two β-1, 3-1, 4-glucanases with MW of 30000-50000 and 50000-100000, respectively.

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