2nd International Conference

„CONFERENCE ON ADVANCES IN SCIENCE AND TECHNOLOGY“ COAST 2023

31 May - 03 June 2023 HERCEG NOVI, MONTENEGRO

USE OF OIL RESIDUES FROM OILSEEDS PROCESSING IN FUNGAL PROTEOLYTIC

ENZYMES PRODUCTION

Marko Zeljko, Nemanja Špirić, Ida Zahović, Zorana Trivunović, Jelena Dodić
Faculty of Technology Novi Sad, University of Novi Sad, Novi Sad 21000, Serbia

Corresponding author e-mail address: ida.zahovic@uns.ac.rs (I. Zahović)

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ABSTRACT:
Cold-pressed oils from various oilseeds have become very popular at global food market lately, due to its high
quality and beneficial effects on human health. Oilseeds are widely grown in Vojvodina Province and further
processed by regional enterprises causing significant amounts of effluents with no adequate method for its
utilization. Production of industrial microbial enzymes might be a promising solution for sustainable
valorisation of these effluents. Advanced developments in detergent industry require greener technology and
cost-effective production and one of potential alternatives is the utilization of microbial enzymes. Proteolytic
enzymes have found a very significant role in detergent industry for cleaning purposes. The aim of this study was
to examine the possibility of proteolytic enzymes production by cultivation of reference strain Trichoderma
reesei QM 9414 and wild-type isolate of Trichoderma sp. on medium containing oil residue from sunflower,
pumpkin seed and soybean processing. The success of the bioprocess was estimated based on the proteolytic
activity of enzymes in crude enzyme preparations in the temperature conditions of detergent application, i.e. at
30 °C and 60 °C. According to the obtained results maximal proteolytic activity of enzymes at 30 °C was
achieved when wild-type isolate Trichoderma sp. was cultivated in medium with oil residues from soybean
processing and at 60 °C when wild-type isolate Trichoderma sp. was cultivated in medium containing oil
residues from pumpkin seed processing. The results obtained in this study represent valuable information for
future investigations related to the utilization of oil residue through the production of value-added products.

Keywords: biotechnological production, cold-pressed oil residue, fungal enzymes, proteolytic activity,
Trichoderma
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1. INTRODUCTION

Modern nutrition trends favor the use of food that is minimally chemically processed and that is less detrimental
to the environment. As a result, the usage of cold-pressed oils in the diet as a "healthier" variant of oil has
recently expanded dramatically [1]. Oilseeds, such as soybean and sunflower, are grown in large quantities in the
territory of Vojvodina Province [2] and mostly used for oil production. However, due to the previously
mentioned reasons, there is a strong tendency to develop processes for the production of cold-pressed oils from
these oilseeds. New practice of certain number of small factories to produce such oils resulted in effluent
generation. One of these effluents is oil residues which contain oil and solid compounds originating from plants.
Considering the fact that oil residues from cold-pressed oils production are rich in organic matter, a promising
solution for sustainable valorisation of these effluents might be a production of industrial microbial enzymes [3].

Although the exact composition of these effluents is unknown, it can be assumed that it primarily consists of
lipid fractions and also contains all other substances found in oilseeds. Even though the amount of effluents
generated by the production of cold-pressed oils from oilseeds in Vojvodina Province is small, the amount of
raw materials in the media for the biotechnological production of enzymes is in a range of 5-10% [4].

Many molds have the ability to absorb large molecules because they can create enzymes that break down these
molecules and are therefore widely used as productive microorganisms in the biotechnological production of
enzymes. Most industrially important enzymes are produced by molds of the genera Aspergillus, Penicillium,
Rhizopus, Mucor and Trichoderma, bacteria of the genus Bacillus, actinomycetes of the genus Streptomyces and
yeasts of the genus Saccharomyces [5, 6]. The ability of Trichoderma strains to produce extracellular proteases
has been known for a long time [7], therefore in this paper the possibility of protease production by cultivating
Trichoderma strains on a medium containing residues from oilseed processing was investigated. Different strains
 2nd International Conference
„CONFERENCE ON ADVANCES IN SCIENCE AND TECHNOLOGY“ COAST 2023
31 May - 03 June 2023 HERCEG NOVI, MONTENEGRO

of Trichoderma mold can produce different amounts of proteases with different proteolytic activities, therefore,
for the purpose of comparison, 2 strains were used, one of which is a reference strain (Trichoderma reesei QM
9414), and the other is an wild-type isolate (Trichoderma sp.).

Certain enzymes are of great commercial importance and are used as biocatalysts in numerous processes at the
industrial level. They have found applications in the food and beverage industry, textile industry, detergent
industry, paper industry, waste management, leather processing, biofuel industry, biotechnology industry and
pharmaceuticals, as well as in agriculture and medicine [8]. Major classes of enzymes in detergents include
proteases, lipases, amylases, and cellulases. Proteases were the first to be used in this branch of industry and are
the most common group of enzymes in detergents, which is why today they have the largest number of
commercial forms on the market. Alkaline proteases in detergents break peptide bonds in stains of protein origin
and hydrolyze proteins into soluble polypeptides and amino acids [8].

By using detergents in water at a high temperature with vigorous mixing, it is possible to remove most of the
stains, the origin of which can be different (proteins, fats, starch). However, these conditions damage the laundry
and shorten its lifespan, and the economic costs arising from heating the water and washing the laundry for a
long time cannot be ignored. The addition of enzymes to detergents enables the use of lower temperatures and
shorter washing times with better stain removal results [9].

The aim of this study was to examine the possibility of proteolytic enzymes production by cultivation of
reference strain T. reesei QM 9414 and wild-type isolate of Trichoderma sp. on medium containing oil residue
from sunflower, pumpkin seed and soybean processing. The success of the bioprocess was estimated based on
the proteolytic activity of enzymes in crude enzyme preparations in the temperature conditions of detergent
application, i.e. at 30 °C and 60 °C.

2. MATERIALS AND METHODS

2.1. Producing microorganisms

The reference strain T. reesei QM 9414 and one Trichoderma strain isolated from the natural environment were
used as the producing microorganisms in these experiments. The strains were stored for a many years in the
Microbial Culture Collection of the Faculty of Technology Novi Sad at 4 °C on agar slant (Sabouraud Maltose
Agar ®, HiMedia, India) and subcultured every six months.

2.2. Cultivation media

The commercial semi-synthetic medium (Sabouraud Maltose Agar ®, HiMedia, India) was used in the initial
stages of production. Enzyme production was performed on medium containing oil residue obtained from local
oilseeds processing factories. Oil residue from sunflower, pumpkin seed and soybean processing were
investigated as sources of this nutrient and used in a quantity of 15 g/L. The cultivation media also contained
(NH4)2SO4 and K2HPO4 in quantity of 5 g/L and 2 g/L, respectively. The pH value of used media was adjusted
to 4.5±0.2 and then sterilized by autoclaving (121 °C, 2.1 bar, 20 min).

2.3. Inoculum preparation

Producing microorganisms were subcultured on agar slant and incubated at 28-30 °C until intensive sporulation.
Further, inoculum preparation procedure has included suspending of spores of producing microorganisms in
sterile physiological saline in order to obtain suspension with concentration in a range of 106 spores/mL.
Inoculation was performed by adding 10% (v/v) of inoculum, prepared as previously described, in cultivation
medium.

2.4. Enzyme production

The enzyme production was carried out in 100 mL Erlenmeyer flasks with 30 mL of the cultivation medium with
the appropriate composition. The biosynthesis was performed by submerged cultivation technique under aerobic
conditions at 28-30 °C and 150 rpm (Laboratory shaker KS 4000i control, Ika® Werke, Germany) for 168 h. At
the end of biosynthesis, the solid and liquid phases of cultivation medium were separated by filtration through
qualitative filter paper. The obtained filtrates were used for further analysis as crude enzyme preparations.
 2nd International Conference
„CONFERENCE ON ADVANCES IN SCIENCE AND TECHNOLOGY“ COAST 2023
31 May - 03 June 2023 HERCEG NOVI, MONTENEGRO

2.6. Protease activity assay

Protease activity of crude enzyme preparations was determinated according to modified Hagihara method [8]
where 1% solution of casein in a 0.05 M phosphate buffer with pH value of 7.5 was used as a substrate. Casein
solution and crude enzyme preparation (blank: distilled water) were mixed in a ratio of 5:1. After
homogenization, hydrolysis was performed at temperatures of 30 °C and 60 °C for 10 min in thermostatic water
bath. After hydrolysis, 0.1 М CCl3COOH was added and the mixture was homogenized and left for 30 min at
30 °C in thermostatic water bath, and then centrifuged for 20 min at 10,000 rpm at a temperature of 30C.
Proteolytic activity of crude enzyme preparations was evaluated based on the content of amino acids, expressed
through the content of tyrosine, in supernatants obtained after centrifugation, which was determined by a
spectrophotometric method at 275 nm, compared to a blank test. The L-tyrosine albumin standard was used for
the construction of the calibration curve [10, 11].

3. RESULTS AND DISCUSSION

In accordance with the defined aim of this research, the possibility of proteolytic enzymes production by
cultivation of reference strain T. reesei QM 9414 and wild-type isolate of Trichoderma sp. on media containing
oil residue from sunflower, pumpkin seed and soybean processing was examined. Taking into account the origin
of applied oil residiues it is most likely that the synthesis of lipolytic enzymes will occur on such media,
however, in this study, the possibility of Trichoderma strains to synthesize certain amounts of proteolytic
enzymes with satisfactory activity was examined. Bioprocess success in the applied experimental conditions was
evaluated based on the activity of crude enyzme preparations according to casein. Determination of the activity
of produced fungal proteases was carried out in the temperature conditions of detergent application, i.e. at 30 °C
and 60 °C.

The obtained results of determining the proteolytic activity of the crude enzyme preparations, obtained by
cultivation of reference strain T. reesei QM 9414 and wild-type isolate of Trichoderma sp. on medium
containing oil residue from sunflower, pumpkin seed and soybean processing, after hydrolysis at temperatures of
30 °C and 60 °C are presented in Table 1. Their activity is expressed through the content of amino acids, i.e.
through the content of tyrosine in the hydrolyzed samples.

Table 1. Proteolytic activity of crude enzyme preparations at 30°C and 60°C

Proteolytic activity of crude enzyme preparations after hydrolysis at 30 °C [mg/mL]

Medium
Trichoderma reesei QM 9414 Trichoderma sp.
Soybean 0.301 0.380
Sunflower 0.027 0.067
Pumpkin seed 0.029 0.032
Proteolytic activity of crude enzyme preparations after hydrolysis at 60 °C [mg/mL]
Medium
Trichoderma reesei QM 9414 Trichoderma sp.
Soybean 0.021 0.028
Sunflower 0.027 0.085
Pumpkin seed 0.029 0.105

The obtained results shown in Table 1 suggest that measurable values of enzymes activity indicate that the
tested microorganisms have the ability to biosynthesize proteolytic enzymes. This is in agreement with findings
from previous research where it is confirmed that T. reesei have the ability to produce proteolytic enzymes on
various carbon sources [12]. According to the results presented in Table 1, the proteolytic activity of crude
enzyme preparations obtained by cultivation of both producing strains on a medium containing oil residues from
the soybean processing is higher when hydrolysis is performed at 30°C, and the proteolytic activity of crude
enzyme preparations obtained by cultivation of Trichoderma sp. on the other two media is higher when
hydrolysis is performed at 60 °C. As it can be seen from the results presented in Table 1, cultivation of reference
strain T. reesei QM 9414 on medium containing oil residue from pumpkin seed and soybean processing results
in production of enzymes with similar proteolytic activity at both temperatures. The main difference in the
 2nd International Conference
„CONFERENCE ON ADVANCES IN SCIENCE AND TECHNOLOGY“ COAST 2023
31 May - 03 June 2023 HERCEG NOVI, MONTENEGRO

ability of applied producing strains to produce enzymes with different activity on the same medium may be due
to metabolic diversity of applied Trichoderma strains [13]. Results from previous studies indicate that reference
strain T. reesei QM 9414 has the ability to produce proteolytic enzymes on synthetic or semi-synthetic media of
optimal composition [14-16], however, in this research, the possibility of production of proteolytic enzymes on
natural media based on oil residues from the processing of oilseeds has been proven.

In order to conduct mutual comparison of the obtained results and to easier interpret results, experimental data
were relativized. The obtained results were relativized by assigning the largest obtained value to be maximally
successful (100%) while the other values were expressed in a proportion of the maximum. Graphic
representation of the relativized values of the proteolytic activity of crude enzyme preparations after hydrolysis
at 30 °C and at 60 °C is given in Figure 1 and Figure 2, respectively. The same enzymes can exhibit different
activities at different temperatures [17], and taking this into account, the activities of proteolytic enzymes of
crude enzyme preparations are shown through two graphs, one for each hydrolysis temperature.

100
Relativized values of proteolytic activity

80

60

40
[%]

20

0
Soybean Sunflower Pumpkin seed
Medium

Trichoderma
Trichodermareesei
reeseiQM
QM9414
9414 Trichodermasp.
Trichoderma sp.

Figure 1. Relativized values of the proteolytic activity of crude enzyme preparations at 30 °C

As it can be seen in Figure 1, the crude enzyme preparation obtained by cultivation of the Trichoderma sp. strain
on medium containig oil residues from soybean processing exhibited the highest proteolytic activity after
hydrolysis at 30 °C. The main reason for this may be due the fact that soybeans have the highest protein content
of all oilseeds [18, 19] and due to the fact that the temperature optimum for the growth and development of the
largest number of species from the genus Trichoderma is in the range of 27-30 °C [20]. In relation to this value,
the other values were relativized and it can be noticed that after the hydrolysis at 30 °C, proteolytic activity of
crude enzyme preparations obtained by cultivation of both strains on a medium containing oil residues from
soybean processing is significantly higher comparing to the proteolytic activity of crude enzyme preparations
obtained by cultivation of both of the strains on other two media. However, the obtained results indicate that
Trichoderma sp. strain proved to be more suitable for production of enzymes with higher proteolytic activity for
all the used media in the applied experimental conditions and after hydrolysis at 30 °C.

Relativized values of the proteolytic activity of crude enzyme preparations obtained by cultivation of reference
strain T. reesei QM 9414 and wild-type isolate of Trichoderma sp. on medium containing oil residue from
sunflower, pumpkin seed and soybean processing,producing strains after hydrolysis at 60 °C are presented in
Figure 2.
 2nd International Conference
„CONFERENCE ON ADVANCES IN SCIENCE AND TECHNOLOGY“ COAST 2023
31 May - 03 June 2023 HERCEG NOVI, MONTENEGRO

100

Relativized values of proteolytic activity

80

60
[%]

40

20

0
Soybean Sunflower Pumpkin seed
Medium

Trichoderma
Trichodermareesei
reeseiQM
QM9414
9414 Trichoderma
Trichodermasp.sp.

Figure 2. Relativized values of the proteolytic activity of crude enzyme preparations at 60 °C

Observing the results presented in Figure 2 it can be noticed that under the hydrolysis conditions of 60 °C, higher
proteolytic activity was exhibited within the crude enzyme preparation obtained by cultivation of the strain
Trichoderma sp. comparing to the reference strain T. reesei QM 9414 for all three medium used. Also, it can be
noted that during hydrolysis at 60 °C the proteolytic enzymes of the reference strain T. reesei QM 9414 reach
maximum relativized values of activity of around 20%, regardless of medium which was used for cultivation.
This is probably due the fact that the species from the genus Trichoderma isolated from natural environment
usually compete with pathogens for nutrients, ecological niches or infection sites on plant roots, easier adapt to
the non-standard content of the media, so they have the ability to grow and metabolize different carbon sources
in extreme conditions [21].

The highest proteolytic activity was exhibited within the crude enzyme preparation obtained by cultivation of the
strain Trichoderma sp. on the medium containing oil residues from the processing of pumpkin seeds and in
relation to this value, the other values of the proteolytic activity of crude enzyme preparations were relativized. It
can be noticed that the lowest proteolytic activity was exhibited within the crude enzyme preparation obtained by
cultivation of the both producing strains on the medium containing oil residues from the processing of soybean.
Despite the fact that the highest proteolytic activity at 30 °C was exhibited within the crude enzyme preparation
obtained by cultivation of the producing strain on the medium containing oil residues from the processing of
soybean, at 60 °C this activity was the lowest. This indicates that temperature of 60 °C have negative effect not
only the producing strain but substrate e. i. oil residue from soybean processing too.

By analyzing all the results given above it can be concluded that strain Trichoderma sp. isolated from natural
environment have a great potential for production of enzymes with proteolytic activity in the temperature
conditions of detergent application, i.e. at 30 °C and 60 °C. On the other side, in this study it is confirmed for the
first time ever that Trichoderma species possess ability to produce enzymes with proteolytic activity on medium
containing oil residue from sunflower, pumpkin seed and soybean processing. Despite the fact that some of
experiments in this research resulted in production of enzymes with poor proteolytic activity, the obtained results
are important for purpose of minimizing the negative impact of effluents from oilseeds processing on the
environment. Future research related to fungal enzymes production on oil residues from oilseed processing
should encompass optimization of bioprocess conditions, as well as screening of different producing strains and
residues from processing of other substrates.
 2nd International Conference
„CONFERENCE ON ADVANCES IN SCIENCE AND TECHNOLOGY“ COAST 2023
31 May - 03 June 2023 HERCEG NOVI, MONTENEGRO

4. CONCLUSIONS

In accordance with the defined aim, in this study the possibility of proteolytic enzymes production by cultivation
of reference strain T. reesei QM 9414 and wild-type isolate of Trichoderma sp. on medium containing oil residue
from sunflower, pumpkin seed and soybean processing was examined. The results of this study have confirmed
that the production of fungal proteolytic enzymes may be suitable solution for the usage of oil residues generated
in the production of cold-pressed oils.
The results of this study have a great importance from an ecological point of view, considering the fact that the
biotechnological production of enzymes with proteolytic activity on cultivation medium containing oil residue
from sunflower, pumpkin seed and soybean processing represents a promising solution for sustainable
valorization of this effluent. Moreover, the results obtained in this study represent valuable information that can
be used in future investigation research related to the optimization of bioprocess in the term of increasing the
bioprocess success and estimation of possible application of obtained enzymes.

5. ACKNOWLEDGEMENT

This study is part of the project (451-03-47/2023-01/ 200134) funded by the Ministry of Education, Science and
Technological Development of the Republic of Serbia and project 142-451-3187/2022-01/01 “Development of
industrial symbiosis in the AP Vojvodina through valorization of fruit processing by-products using green
technologies” financed by the Provincial Secretariat for Higher Education and Scientific Research, the
Autonomous Province of Vojvodina, Republic of Serbia.

5. LITERATURE

[1] Chandra, S., Kumar, M., Dwivedi, P., Shinde, L.P. (2020), “Functional and nutritional health benefit of
cold-pressed oils: a review“, Journal of Agriculture and Ecology, Vol. 9, 21-29.
[2] Nikolić, S., Mutavdžić, B., Novković, N., Sredojević, Z., Bjegović, M., Tekić, D. (2021), „Tendencies and
prediction of industrial plant production in Serbia“, Economics of Agriculture, Vol. 69, No. 2, 317-329.
[3] Gaca, A., Kludská, E., Hradecký, J., Hajšlová, J., Jeleń, H.H. (2021), “Changes in Volatile Compound
Profiles in Cold-Pressed Oils Obtained from Various Seeds during Accelerated Storage“, Molecules, Vol.
26, No. 2, 285.
[4] de Souza, P.M., Bittencourt, M.L., Caprara, C.D., de Freitas, M.M., de Almeida, R.P., Silveira, D.,
Fonseca, Y.M., Ferreira, E.X., Pessoa, A., Magalhães, P.D. (2015), “A biotechnology perspective of
fungal proteases“, Brazilian Journal of Microbiology, Vol. 46, 337 - 346.

[5] Dinmukhamed, T., Huang, Z., Liu, Y., Lv, X., Li, J. Du, G. (2021), “Current advances in design and
engineering strategies of industrial enzymes“, Systems Microbiology and Biomanufacturing, Vol. 1, 15-23.
[6] Patel, A. K., Dong, C., Chen, C., Pandey, A. Singhania, R. R. (2023), “Chapter 3 - Production,
purification, and application of microbial enzymes“, in Brahmachari, G. (Ed.) Biotechnology of Microbial
Enzymes (Second Edition), Cambridge, Massachusetts, USA: 25-57.
[7] Kredics, L., Antal, Z., Szekeres, A., Hatvani, L., Manczinger, L., Vagvolgyi, Cs. Nagy, E. (2005),
“Extracellular Proteases of Trichoderma Species“, Acta Microbiologica et Immunologica Hungarica, Vol.
52, No. 2, 169-184.
[8] Gürkök, S. (2019), „Microbial Enzymes in Detergents: A Review“, International Journal of Scientific &
Engineering Research, Vol. 10, No. 9, 75-81.
[9] Singh, S., Mangla, J. & Singh, S. (2021), “Evaluation of Aspergillus fumigatus NTCC1222 as a source of
enzymes for detergent industry“, Resources, Environment and Sustainability, Vol. 5, 1-5.
[10] Hagihara, B., Matsubara, H., Nakai, M. & Okunuki, K. (1958), “Crystalline bacterial proteinase:
I. Preparation of crystalline proteinase of Bac. subtilis“, The Journal of Biochemistry,
Vol. 45, 185-194.
[11] Jayashree, S., Annapurna, B., Jayakumar, R., Sa, T., Seshadri, S. (2014), “Screening and characterization
of alkaline protease produced by a pink pigmented facultative methylotrophic (PPFM) strain, MSF 46“,
Journal of Genetic Engineering and Biotechnology, 12, 111-120.
[12] Sun, Y., Qian, Y., Zhang, J., Wang, Y., Li, X., Zhang, W., Wang, L., Liu, H., Zhong, Y. (2021),
“Extracellular protease production regulated by nitrogen and carbon sources in Trichoderma reesei“,
Journal of Basic Microbiology, Vol. 61, 122 - 132.
 2nd International Conference
„CONFERENCE ON ADVANCES IN SCIENCE AND TECHNOLOGY“ COAST 2023
31 May - 03 June 2023 HERCEG NOVI, MONTENEGRO

[13] de Azevedo Silva, F.D., Vieira, V.D., Silva, R.L., Pinheiro, D.G., & Soares, M.A. (2021), “Introduction of
Trichoderma spp. biocontrol strains against Sclerotinia sclerotiorum (Lib.) de Bary change soil microbial
community composition in common bean (Phaseolus vulgaris L.) cultivation“, Biological Control, Vol.
163, 104755.
[14] Dienes, D., Börjesson, J., Hägglund, P., Tjerneld, F., Lidén, G., Réczey, K. Stålbrand, H. (2007),
Identification of a trypsin-like serine protease from Trichoderma reesei QM9414, Enzyme and Microbial
Technology, Vol. 40, No. 5, 1087-1094.
[15] Daranagama, N. D., Shioya, K., Yuki, M., Sato, H., Ohtaki, Y., Suzuki, Y., Shida, Y. Ogasawara, W.
(2019), Proteolytic analysis of Trichoderma reesei in celluase-inducing condition reveals a role for
trichodermapepsin (TrAsP) in cellulase production, Journal of Industrial Microbiology and Biotechnology,
Vol. 46, No. 6, 831-842.
[16] Qian, Y., Zhong, L., Sun, Y., Sun, N., Zhang, L., Liu, W., Qu, Y., Zhong, Y. (2019), Enhancement of
Cellulase Production in Trichoderma reesei via Disruption of Multiple Protease Genes Identified by
Comparative Secretomics, Frontiers in Microbiology, Vol. 10, 2784.
[17] Chapman, R., Stenzel, M.H. (2019), “All Wrapped up: Stabilization of Enzymes within Single Enzyme
Nanoparticles“, Journal of the American Chemical Society, Vol. 141, No. 7, 2754-2769 .
[18] Jithender, B., Upendar, K., Nickhil, C. Rathod PJ, “Nutritional and anti-nutritional factors present in oil
seeds: An overview“, International Journal of Chemical Studies, Vol. 7, No. 2, 1159-1165.
[19] Syed, Q. A., Akram, M. & Shukat, R. (2019), “Nutritional and Therapeutic Importance of the Pumpkin
Seeds“, Biomed J Sci & Tech Res, Vol. 21, No. 2, 15798-15803.
[20] Qiu, Z., Wu, X., Zhang, J. & Huang, C. (2017), “High temperature enhances the ability of Trichoderma
asperellum to infect Pleurotus ostreatus mycelia“, PloS ONE, Vol 12, No. 2, e0187055.
[21] Błaszczyk, L., Siwulski, M., Sobieralski, K., Lisiecka, J., Jędryczka, M. (2014), “Trichoderma spp. –
application and prospects for use in organic farming and industry“, Journal of Plant Protection Research,
Vol. 54, 309-317.