Effect of Fermentation Oyster Mushrooms (Pleurotus ostreatus) on Grain Barly contaminated with Fungus exudate toxins Fusarium sporotrichioides and the inhibition growth and Degradation Toxins produced by it

 

Hussein. S. Naji. Kermasha

Department of Biological Sciences, College of Education for Girls, University of Kufa, Iraq

 

ABSTRACT:

Different derivatives of 4-hydroxy-3-methoxy benzoic acid were synthesized and evaluated for their antioxidant, α-amylase inhibition and urease inhibition ability. Antioxidant evaluation was performed by DPPH radical scavenging assay and the results revealed that compounds 8, 10 and 13 as most active antioxidant agent with IC₅₀ values of 43.09µg/ml, 44.59µg/ml and 43.43µg/ml respectively. α-Amylase inhibition study was performed using diastase by colorimetric method. Compound 9 showed maximum inhibition with IC₅₀ value of 33.26 µg/ml. Compound 4 was found to possess maximum urease inhibition ability with IC₅₀ value of 35.82µg/ml. Molecular docking study was performed using autodock software.

 

 

INTRODUCTION:

Pleurotus ostreatus is a fungus that has medicinal benefits for containing effective compounds (Dutta et al., 2013). It has many antifungal and toxin substances (Oei, 2005) and has a clear effect as an anti-tumor and in stimulating cellular immunity (Hatvani et al., 2008). It also has a distinct role in the destruction of toxic substances in the circles where this mushroom grows through the enzymes produced (Lacina and his group, 2003). 2000), the species of Pleurotus ostreatus have high susceptibility and rapid growth of fungal spawn to different irrigated media with different environmental conditions (Kong, 2004). In Ahmed (2015), it has been shown that the fungus is highly competitive against a number of fungus fungi, and now occupies the second place after white Agaricus bisporus, 25% of the global production of food fungi Zhang and others (2014) and has high nutritional value because it is rich in protein, which represents 40% - 20 of dry weight ( Ahmed and others, 2009).

 

T2-toxin toxins are important toxins. The partial formula of these toxins is C24H34O9 and its partial weight is 466.5. The main productive fungi include Fusarium sporotrichioides (Y. Konishi and K. Sugiyama, 2008).

 

The fungus is one of the plant pathogens that cause scabies mainly in wheat, barley, etc. (Food and Agricultural Inspection Center, 2009), and damage to grain by contamination in the field (JSFA, 2010).

 

This type of toxin decreases the amount of food intake and vomiting at the lowest concentration. In addition, blood disorders such as aleukia (ATA) and anemia are also observed in addition to gastroenteritis and dermatitis (National Society for Animal Health, 2010) and also reduce the immune function (Y. Konishi and K. Sugiyama, 2008). These toxins act on neurons by serotonin and induce loss of appetite and vomiting. In addition, they inhibit protein synthesis by Linking to ribosome 60S subunit (CODEX, 2003). The effect appears significantly in the bone marrow, the mucous membrane of the digestive tract, and the skin, which are the areas of active cell division. Furthermore, they stimulate apoptosis in immune system cells, stimulating the production of cytokines infections (JAP, 2012).

 

Natural pollution often occurs in cultivated grains in large areas of the world. As for the feed material, it has been discovered in corn, wheat, barley, corn gluten feed, bran (Japan Ministry of Agriculture, 2012).

Daily T2-toxin intake is equal to or equal to 0.06μg/kg bw/day. The fungal toxins are stable at high temperature and are not less toxic in normal treatment and cooking procedures T2-toxin persisted in milk, meat and eggs (MAFF, 2008).

Barley beans are of major importance for their contribution to the provision of a major food source for humans to meet the needs of animal protein. Poultry farming has a number of problems, including the availability of fodder in the growing broiler population and its rising prices (US Cereal Council, 2003). The availability of suitable types of fodder at a reasonable price is the key to success in the meat production process. In order to compete with local markets, it is important to use non-traditional feeds and incorporate them into broiler mix to reduce production costs (Basak et al., 2002).). International companies began to specialize in manufacturing Meat breeds produce global strains with high productivity to provide minimum food security requirements (Billard, 2004). These specifications have increased their nutritional, administrative and health requirements (Naji, 2006). The presence of fungal toxins in fodder crops is one of the most important problems facing poultry farming in developing countries. This is due to the lack of modern scientific foundations used in agricultural operations, including the technology of drying and storing seeds. Yellow maize is the first of this problem as a feedstock The strategy in poultry diets is 50-60% of its components and increase its exposure to pollution, especially those planted in the autumn loin, as it is harvested by the end of the winter months, thus prolonging the drying time and thus increasing the possibility of contamination of fungal toxins.  

 

Many countries have introduced maximum limits for the availability of fungal toxins in human food and animal feed at 20μg/kg (Adler, 2002), meaning that the majority of barley grains marketed from Grain stores in Iraq are not suitable for human consumption or forage materials, which are included in the industry of livestock according to the results of recent studies in the country (Hadith, 2005). T-2 toxin has been an important place among the toxins of importance, as it is the most dangerous natural substance in its complex health effects in the low levels of pollution and spread in large ranges of heat so that the contamination of feed crops and fodder is difficult to avoid, despite all the modern methods and means (Gratz et al., 2004). The absence of fodder crops and domestic feces has become a priority for the success of poultry projects, and this study has been conducted to eliminate the negative effects of fungal toxins from diets and their erosion.

 

The most important themes of this study are:

1    Test the ability of the oyster mushroom Pleurotus ostreatus against a number of fungi contaminated barly grain.

2    The use of oyster mushroom Pleurotus ostreatus in the remediation of contaminated barly grain industrially certain level of poison T-2 toxin secreted by the fungus Fusarium sporotrichioides

 

MATERIALS AND METHODS:

This study included conducting laboratory experiments  It was obtained pure isolation from oyster mushroom (Pleurotus ostreatus) mushrooms producer of mycotoxins Fusarium sporotrichioides fungi in the laboratory of the Plant Protection Department - Faculty of Agriculture, University of Kufa. The media of agricultural used in testing: Used in the study media of agricultural to isolate the fungus agricultural development and diagnosis for the purpose of testing and are as follows: The media Potato Dextrose Ager P.D.A. Attended by dissolving 39g per liter of distilled water, according to the manufacturer instructions and then added to the antibiotic Chloramphenicol by 250mg for per liter of distilled water. Distributed media in flasks glass 250ml and blocked pores plugs cotton and sterilized locked to a temperature of 121°C and pressure of 15pounds/Lang 2 for 20 minutes, after the sterilization jugs were left to cool to the pre-sclerosis then pour in the dishes, or storing it in the refrigerator degree 4°C until use. This is the center and used to classify and growth of oyster mushrooms and some fungi isolated and contrast with one another as a compromise record.

 

The test the ability of the oyster mushroom P. ostreatus against a number of fungi contaminated barly grain:

Used double culture technique in Petri dishes and food containers on the center P.D.A. Sterilized, To test the ability of the oyster mushroom against fungi isolated from the diet, the dish is divided into two equal parts and Vaccinate Disc first section center diaeter (0.5)cm of mushroom isolated. reasonable by piercing Corky sterile from near the edges of the colonial center developing on PDA also the age of 7 days, while the second section of a petri dish center has been vaccinated disk (0.5)cm from the edges of the oyster mushroom colony-old 7 days, repeated three times each transaction and apply the same steps with all fungi isolated. Treated as implemented to control and then inoculating the first section of the center of the dish oyster mushrooms and fungi isolated and each individually (Dewan, 1989). All the dishes were incubated in the incubator at a temperature (25 ± 2)°C, and took the results after 7days of double culture, was estimated by contrast ladder standardization quintet prepared by (Bell el al ,1982).

 

Isolation, purification and diagnosis of fungi associated with barly grain for Dilutions Plate method:

In order to know the fungus in the diet poultry have been taking random samples from different parts of the barly grain bag comparison was mixed uniformly and then take 10g of which was added to a beaker containing 90 ml sterile distilled water and mixing brokered an electric mixer to break up its parts and uniformly, then taking one ml of it was added to the test tube containing 9ml sterile distilled water to get the concentration of 10×2. Then, we worked dilution series until reaching dilution fourth and fifth 10×4 and 10×5 then took one ml of each of the two preceding the tubes and put all of them in a petri sterile dish diameter of 9cm and then added a 20ml of culture-food sterile PDA added to the antibiotic Chloramphenicol by 250mg per litter before intransigence. Repeated three times for each dilution and stirred dishes capstan movement, and left the dishes in order to harden and then incubated in incubator upside down at a temperature of 25±2°C for four days.   Fungus was isolated with high frequency and then purified individually on Petri dishes container 20ml of sterile food center P.D.A. Depending on the qualities that I mentioned in taxonomic keys and with the help of Dr. Majid Diwan it was then estimated the frequency of isolated fungi ratios according to the following equation:

 

                                              The number of colonies of fungus

The percentage of frequency (%) =-------------------------------------x 100

                                         The total number of colonies

(Booth et al., 1988).

 

Fermentation processes that took place on the barly grain:

The following operations conducted respectively, in the laboratory of Advanced fungus to the Department of Biological Sciences/College of Education/University of Kufa for the period from 01/07/2016 to 10/01/2016. The creation of diet poultry growth stage (15-35 day) (Titus and Fritz, 1971 and Ibrahim, 1987 and Fayad and Nagy, 1989), has been taken into account in these diets that adequacy the bird needs of crude protein and energy metabolic ratio energy: protein at every stage and according to the (Ibrahim, 1987), as well as adequacy  the bird need of essential amino acids was according to the (NRC, 1994), minerals, vitamins and fatty acid linoleic it according to the (NRC, 1984), it was the amount of metabolic energy calculated to equal the diets exactly as crude protein and energy ratios calculated: protein are equal to a very large extent.

 

Method of (Fermentation solid state) is adopted in the fermentation of the diet, the process according to the method cited by (Semeniuk et al, 1970), with added water to the diet by 60% to get the moisture required for the development of mushroom oyster Pleurotus ostreatus and fungus product poison aflatoxin Aspergillus flavus were fill in diet  in transparent plastic bags with dimensions 30 × 51cm where to put 1kg of diet on the basis of wet weight in each bag closed bags in court and were sterilized by (Autoclave) at a temperature of 121°C and pressure of 15 pounds/Ang 2 for 20 minutes and after that cooled the bags have been added The vaccine Fungal for both two fungi 5% of the weight culture (Oei, 2005) and in multiple layers (Balakrishnan and Nair, 1995) and then was shut down the barrel of the bag floss tightly. Repeated three times, and then transferred the bags inoculated into the incubator has been installed temperature at 25±2°C leaving the fungus to grow on a diet for 21, 28 days where control mycelium growth speed and biodegradable materials in the diet with a shake sacks process from time to time to ensure oyster mushrooms distributed uniformly, conducted a chemical analysis of the diet before and after the development of the fungus as it took a diet, and mixed well and the use of electric grinding mill to conduct analyzes on them. Transactions were as follows and as depicted by Figures (1-4):

 

T1: control treatment barly grain sterile

T2: treatment fermentation of the diet to add fungus Fusarium sporotrichioides for 21 days.

T3: treatment fermentation T2 by oyster mushroom Pleurotus ostreatus for 21 days

T4: treatment fermentation T2 by oyster mushroom Pleurotus ostreatus for 28 days

 

 

Figure (1) Inoculation of local barley grains with fungus Pleurotus ostreatus

 

 

Figure (2) Barley grain with Pleurotus ostreatus in the incubator

 

Figure (3) Fermentation of P.ostreatus on barley grain for 28 days

 

Figure (4) Drying and peeling processes of barley grain fermented with Pleurotus ostreatus

 

Detection of the T-2 toxin using the ELISA test:

It was added to each sample 1 ml of methanol alcohol and then samples mixed well by Vibrating Vortex. It used the standard kit of Neogen Corporation Analytical System for detecting T-2 toxin. Developed the kit standard Filed under temperature of 4°C in the laboratory for one hour in order to become a degree lab temperature by the company producing the standard for several instructions have been grinding model are soft which is a diet poultry relay growth by electric grinder and took him 5grams, was then preparation 70% of alcohol Methanol and took him to 25ml, were mixing 5 grams of the bush to be estimating the toxins in with 25 ml of alcohol are well were mixed for 3 minutes to a rocking after which he was nominated by nomination watman type. no.1 to get rid of impurities and to obtain clear solution Paper then take him to 5ml and then softened with distilled water and 1: 1 ratio is considered at this stage is ready for testing, then add 100 Microlietter of enzyme linked (enzyme Conjugate) in thickly glass dish and then followed by the addition of 50 microlitter of samples T1, T2, T3, T4 to the canyons and added the same amount of standard solution poison the concentrations T-2 toxin 0, 25, 50, 100, 250g/l in the side grooves of the dish and shake the dish helicopter movement of the sample in order to mix with enzyme connectivity solution. Add 100 microlitter antibodies Solution to the canyons and mixed ingredients helicopter movement of the dish and then Vaccinate of the dish for 5 minutes at the laboratory temperature. Neglected components of the dish and then washed gullies using 4- 5 times washing solution and dry the dish so light means the tissue paper to get rid of any trace of the washing solution. Add 100 microliter initiator of Substrate for each gully solution and incubated for 5 minutes degree laboratory temperature. Add 1 Ayari HCL solution to stop the reaction and then read the results Bakarye ELISA on the wavelength of 650 nanometers, according to the following equation can concentrate the toxin in the sample account tested using a standard curve.

 

            Absorption standard solution

             containing the poison or sample

Absorption % = ----------------------------------------- × 100

              Absorption standard solution of

                                      non-Hawi on poison

 

Statistical analysis:

The percentage of the appearance and frequency of fungi isolated, toxic and pathogenicity experiments using a randomized complete design data analysis (Completely Randomized Design) according to international experience with two factors along with the averages were compared by the way less significant difference average L.S.D. At the level of probability of 0.05 (Narrator and Khalaf Allah, 2000).

 

RESULTS AND DISCUSSION:

The isolates and diagnosis of 9 fungal species of chicken barley samples were isolated as in Table 1. Fusarium sporotrichioides were the most frequent, with a frequency of 22.9 % higher than all fungi other isolates. This gives a strong indication of the ability of fungi F. sporotrichioides to be successful in such environments, stability and growth to confront many other microorganisms. The percentage of other high frequencies of species A. niger and Penicillium. sp and A. parasiticus and A. terreus were 10.2, 16.6, 11.2 and 9.4% respectively. It is also noted that some fungi have low percentages of frequencies of 7.7, 9.4, 5.8 and 4.3 for Rhizopus sp, Alternaria sp, Aspergillus flavus and Mucor. sp, respectively. The results of the study showed the high resistance of P. ostreatus to the fungus. The fungus covered the entire area of ​​the dish without allowing the other fungus to grow, except Fusarium sporotrichioides where it covered one third of the dish and the mushroom P. ostreatus covered two thirds of the dish. The species of Pleurotus sp have a high susceptibility to colonization and rapid growth of fungal spawn on different irrigated media with different environmental conditions, and agree with Ahmed (2015) that fungi have high competitiveness against the polluted fungi.

The barly grain contaminated with fungi and toxins are a very important problem in the process of poultry raising, which may extend to the threat of production and the consumer. The disposal of fungi found in contaminated feed is a very difficult obstacle. In the light of this, the results showed the protective effect of P. ostreatus on the fungus and therefore control of the toxin and its toxins, in addition to the high competitiveness of the mentioned fungi against many of the fungus fungi. As shown in this study, many studies have shown the effectiveness of P. ostreatus In the absorption of many fungal toxins where Mutani and Alani (2009) found that the fungus is very effective in reducing the toxin T-2 toxin which is one of the most dangerous toxins belonging to the group Tricothicin by 68%, Motomura and others (2003) to the ability of mushrooms to destroy the venom AFB1 has Extract an enzyme from the fungus that has the property of destroying the poison. Table (1) depicts percentage frequency fungi isolated from barly grain and the degree of antagonism with oyster mushrooms pleurotus ostreatus.

 

 

Table (1) the percentage frequency fungi isolated from barly grain and the degree of antagonism with oyster mushrooms pleurotus ostreatus

 

Table (2) has shown susceptibility oyster mushrooms p. ostreatus to break down toxins contaminated with T-2 toxin to barly grain in the treatment of T4 where oyster mushrooms used the duration of fermentation 28 days where the highest percentage bresking down 5.9 micrograms/kg, followed by treatment T3 wher of oyster mushrooms the duration of fermentation 21 days amounted to 13.0 micrograms/kg in comparison with untreated T2 oyster mushrooms, which showed the highest contamination rate was 23.7 micrograms/kg either the control treatment were free of contamination and mycotoxin which gave 0.0 micrograms/kg.

 

Table (2) Test the effect of oyster mushrooms on diet poultry contaminated sorter fungus toxins Aspergillus flavus and recognize the effect of mushrooms to break down Aflatoxin Productivity

 

Slope: 2.077 ,9Corr Coeff: 0.99 ,Units: ppb Method: Direct Competitive,

The contaminated feed and fungal toxins very important problem in the process of raising poultry, where this influence may extend to the threat of production as well as the consumer.     And make up to get rid of the fungus found in contaminated feed process extremely difficult obstacle. The protective effect of the fungus P. ostreatus against contaminated fungi, and thus control over it and their toxins, in addition to the highly competitive mentioned fungicide against many of the contaminated mushrooms in a diet and as many of the studies the effectiveness of fungus P. ostreatus showed the adsorption of many of mycotoxins where he found (Mtunai and Ani 2009) that is very effective mushrooms in shorthand T-2 toxin, one of the most dangerous toxins belonging to the group trichothecens by 68%. The results consistent with (Motomura et al, 2003) to the ability of oyster mushrooms to break down the poison AFB1. It was concluded enzyme from mushrooms poison him smashing the said property.

 

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Received on 20.06.2019           Modified on 07.08.2019

Accepted on 10.09.2019         © RJPT All right reserved