The Role of Microbial Aspartic Protease Enzyme in Food and Beverage Industries

Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total global enzymes sale. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases are classified in enzymes of class 3, the hydrolases, and the subclass 3.4, the peptide hydrolases or peptidase. Proteases are generally grouped into two main classes based on their site of action, that is, exopeptidases and endopeptidases. Protease has also been grouped into four classes based on their catalytic action: aspartic, cysteine, metallo, and serine proteases. However, lately, three new systems have been defined: the threonine-based proteasome system, the glutamate-glutamine system of eqolisin, and the serine-glutamate-aspartate system of sedolisin. Aspartic proteases (EC 3.4.23) are peptidases that display various activities and specificities. It has two aspartic acid residues (Asp32 and Asp215) within their active site which are useful for their catalytic activity. Most of the aspartic proteases display best enzyme activity at low pH (pH 3 to 4) and have isoelectric points in the pH range of 3 to 4.5. They are inhibited by pepstatin. The failure of the plant and animal proteases to meet the present global enzyme demand has directed to an increasing interest in microbial proteases. Microbial proteases are preferred over plant protease because they have most of the characteristics required for their biotechnological applications. Aspartic proteases are found in molds and yeasts but rarely in bacteria. Aspartic protease enzymes from microbial sources are mainly categorized into two groups: (i) the pepsin-like enzymes produced byAspergillus,Penicillium,Rhizopus, andNeurosporaand (ii) the rennin-like enzymes produced byEndothiaandMucorspp., such asMucor miehei,M. pusillus, andEndothia parasitica. Aspartic proteases of microbial origin have a wide range of application in food and beverage industries. These include as milk-clotting enzyme for cheese manufacturing, degradation of protein turbidity complex in fruit juices and alcoholic liquors, and modifying wheat gluten in bread by proteolysis.

A Preliminary Preparation of Endophytic Bacteria CE3 Wettable Powder for Biological Control of Postharvest Diseases

Recently, there has been an increasing interest among researchers in using combinations of biological control agents to exploit potential synergistic effects among them. In the present study, there were investigated commercially acceptable formulations of Bacillus cereus CE3 wetting powder with long storage life and retained efficacy to control chestnut and other fruit rot caused by Endothia parasitica (Murr) and Fusarium solani. The study sought to develop a new B. cereus formulation that would be more effective and better suited to the conditions of field application. By a series of experiments, the formulation was confirmed as follows: 60% B. cereus freeze-dried powder, 28.9% diatomite as carrier, 4% sodium lignin sulfonate as disperser, 6% alkyl naphthalene sulfonate as wetting agent, 1% K2HPO4 as stabilizer, 0.1% β-cyclodextrin as ultraviolet protectant. The controlling experiments showed that the diluted 100 times of 60% B. cereus wetting powder had 79.47% corrosion rate to chestnut pathogens; and this result is comparable to the diluted 1,000 times of 70% thiophanate-methyl. Safety evaluation results showed that rats acute oral lethal dose 50% was 5,000.35, therefore application of B. cereus wettable powder could not cause a person or animal poisoning. This work illustrated that 60% B. cereus wetting powder had commercial potential; however, to apply this formulation as a biological pesticide in the field, masses production processes need to be further studied.

The Morphology and the Biological Control of Cryphonectria parasitica

Cryphonectria parasitica  (Murr.) Bar [syn. Endothia parasitica (Murr. And.] (anamorf: Endothiella sp .) is the causal agent of chestnut bark disease or chestnut blight, disease which produced great damages throughout the world, for example, in Europe, the European chestnut tree ( Castanea sativa (P.) Mill) was heavily affected. Environmental concerns have focused attention on natural forms of disease control as an effective alternative to chemical pesticides. In the chestnut blight fungus, Cryphonectria parasitica deals with a natural form of biological control in which the virulence of a fungal pathogen is attenuated by an endogenous viral RNA genetic element- the hypovirulent strain. In our researches we picked samples of chestnut bark from different areas in Maramures county. We’ve isolated the fungus on PDA medium and we’ve studied the morphological characteristics of the usual virulent strain and we looked for a possible hypovirulent strain in order to study its capacity for biological control. The fungus develops in the bark and in cambium where forms a yellowish or brownish stroma and produces both conidia and ascospores. The pycnidia stromata break through the lenticels producing conidia and later in the same stroma develop the perithecia which produce ascospores. Both strains of the fungus were found in the research area. The hypovirulent strain had a slower development, showed no sporu lation and pigmentation “white cultural strain” and was tested in vitro for the capacity to convert the virulent isolates by dual culture tests.

In Vitro Studies Regarding the Control of Cryphonectria parasitica with Hydroalcoholic

Cryphonectria parasitica  (Murr.) Bar [syn. Endothia parasitica (Murr. And.] (anamorf: Endothiella sp .) is the causal agent of chestnut bark disease or chestnut blight. This disease produced great damages throughout the world, it has nearly eliminated the American chestnut tree ( Castanea dentata (Marsh.) Borkh.) from its natural range and has heavily affected the European chestnut tree ( Castanea sativa (P.) Mill). Successful protection against Cryphonectria parasitica is a very difficult problem because the conventional control methods against the fungus are not applicable with a great success because of the extreme fungus pathogenity and on the other hand because of the characteristics of sites and host plants. In our experiments the isolates of C. parasitica were picked from the Baia Mare area and for the control with plant extracts the isolates were inoculated on PDA medium that contained the extract using the poison food technique. The different extracts in three different concentrations were tested in 80 mm diameter Petri plates incubated at 25ºC and assessed after three, six and nine days of incubation. The fungistatic effect of the extracts was established by examining the presence or the absence of the colony growth. The colonies were measured establishing the colony area that appeared on the treated plates using the ellipse area formula. The results were expressed as the inhibition percent of the tested plant extracts.

New finds of Cryphonectria parasitica and the first record of chestnut blight on red oak Quercus rubra L. in the Czech Republic

The causal agent of the chestnut blight, the fungus Cryphonectria parasitica (Murrill) M. E. Barr (syn. Endothia parasitica (Murrill) P. J. Anderson et H. W. Anderson), was found out at new localities in the Czech Republic. The chestnut blight was observed for the first time in the Czech Republic in 2002. Two new localities were discovered in southern Moravia in May and June 2004. The disease was identified both on the sweet chestnut (Castanea sativa Mill.) and on the red oak (Quercus rubraL.). Infected trees were treated according to the order of the State Phytosanitary Administration of the Czech Republic. 

Detection of natural infection of Quercus spp. by the chestnut blight fungus (Cryphonectria parasitica) in Hungary

The chestnut blight fungus Cryphonectria parasitica (Murrill) Barr [syn.: Endothia parasitica (Murr) Anderson] caused almost total destruction of the American chestnut (Castanea dentata) and widely spread on European chestnut (Castanea saliva) in many European countries. In Hungary, because this fungus threatens most of the Hungarian chestnut stands, great efforts have been made to delay its spread. Biological control with Hungarian hypovirulent strains of the pathogen seems to be an effective method for saving the affected chestnut trees. Until 1998 the fungus was detected on Castanea saliva only, then on some trees of young Quercus petrea in mixed chestnut forests, which also showed the typical symptoms of blight (Kőszeg and Zengővárkony). Although blight symptoms are not so serious in Quercus spp. than in Castanea spp., it seems that C. parasitica threatens the young Quercus spp. in Hungary, mainly in heavily infected chestnut forests. This is the first report of C. parasitica cankers on oak in Hungary.