The Antifungal Effect of Pyroligneous Acid on the Phytopathogenic Fungus Botrytis cinerea.

In recent years, climate change has intensified harsh periods of rain alternating with periods of drought, leading to an increase in the presence of phytopathogenic fungi. In this study, we want to analyse the antifungal properties of pyroligneous acid against the fungal phytopathogen Botrytis cinerea. Through the inhibition test, we observed that the application of different dilutions of pyroligneous acid rarefied the growth of the fungal mycelium. Furthermore, we have seen through the metabolic profile that B. cinerea is not able to use pyroligneous acid as a resource or even grow in close contact with this resource. Moreover, we observed that the pre-incubation of the fungus in pyroligneous acid leads to a reduction in biomass production. These results give us hope for the possible use of this natural substance as a possible substance to protect plantations from pathogen attacks.

Soil-Borne Neosartorya spp.: A Heat-Resistant Fungal Threat to Horticulture and Food Production-An Important Component of the Root-Associated Microbial Community.

Soil-borne Neosartorya spp. are the highly resilient sexual reproductive stage (teleomorph) of Aspergillus spp. Fungi of this genus are relevant components of root-associated microbial community, but they can also excrete mycotoxins and exhibit great resistance to high temperatures. Their ascospores easily transfer between soil and crops; thus, Neosartorya poses a danger to horticulture and food production, especially to the postharvest quality of fruits and vegetables. The spores are known to cause spoilage, mainly in raw fruit produce, juices, and pulps, despite undergoing pasteurization. However, these fungi can also participate in carbon transformation and sequestration, as well as plant protection in drought conditions. Many species have been identified and included in the genus, and yet some of them create taxonomical controversy due to their high similarity. This also contributes to Neosartorya spp. being easily mistaken for its anamorph, resulting in uncertain data within many studies. The review discusses also the factors shaping Neosartorya spp.'s resistance to temperature, preservatives, chemicals, and natural plant extracts, as well as presenting novel solutions to problems created by its resilient nature.

Microbial Community, Metabolic Potential and Seasonality of Endosphere Microbiota Associated with Leaves of the Bioenergy Tree Paulownia elongata × fortunei.

The microbial structure and metabolic function of plant-associated endophytes play a key role in the ecology of various environments, including trees. Here, the structure and functional profiles of the endophytic bacterial community, associated with Paulownia elongata × fortunei, in correlation with seasonality, were evaluated using Biolog EcoPlates. Biolog EcoPlates was used to analyse the functional diversity of the microbiome. The total communities of leaf endophyte communities were investigated using 16S rRNA V5-V7 region amplicon deep sequencing via Illumina MiSeq. Community level physiological profiling (CLPP) analysis by the Biolog EcoPlate™ assay revealed that the carboxylic acids (19.67-36.18%) and amino acids (23.95-35.66%) were preferred by all by all communities, whereas amines and amides (0.38-9.46%) were least used. Seasonal differences in substrate use were also found. Based on the sequencing data, mainly phyla Proteobacteria (18.4-97.1%) and Actinobacteria (2.29-78.7%) were identified. A core microbiome could be found in leaf-associated endophytic communities in trees growing in different locations. This work demonstrates the application of Biolog EcoPlates in studies of the functional diversity of microbial communities in a niche other than soil and shows how it can be applied to the functional analyses of endomicrobiomes. This research can contribute to the popularisation of Biolog EcoPlates for the functional analysis of the endomicrobiome. This study confirms that the analysis of the structure and function of the plant endophytic microbiome plays a key role in the health control and the development of management strategies on bioenergy tree plantations.

Trichoderma-Based Biopreparation with Prebiotics Supplementation for the Naturalization of Raspberry Plant Rhizosphere.

The number of raspberry plants dying from a sudden outbreak of gray mold, verticillium wilt, anthracnosis, and phytophthora infection has increased in recent times, leading to crop failure. The plants suffer tissue collapse and black roots, symptoms similar to a Botrytis-Verticillium-Colletotrichum-Phytophthora disease complex. A sizeable number of fungal isolates were acquired from the root and rhizosphere samples of wild raspberries from different locations. Subsequent in vitro tests revealed that a core consortium of 11 isolates of selected Trichoderma spp. was the most essential element for reducing in phytopathogen expansion. For this purpose, isolates were characterized by the efficiency of their antagonistic properties against Botrytis, Verticillium, Colletotrichum and Phytophthora isolates and with hydrolytic properties accelerating the decomposition of organic matter in the soil and thus making nutrients available to plants. Prebiotic additive supplementation with a mixture of adonitol, arabitol, erythritol, mannitol, sorbitol, and adenosine was proven in a laboratory experiment to be efficient in stimulating the growth of Trichoderma isolates. Through an in vivo pathosystem experiment, different raspberry naturalization-protection strategies (root inoculations and watering with native Trichoderma isolates, applied separately or simultaneously) were tested under controlled phytotron conditions. The experimental application of phytopathogens attenuated raspberry plant and soil properties, while Trichoderma consortium incorporation exhibited a certain trend of improving these features in terms of a short-term response, depending on the pathosystem and naturalization strategy. What is more, a laboratory-scale development of a biopreparation for the naturalization of the raspberry rhizosphere based on the Trichoderma consortium was proposed in the context of two application scenarios. The first was a ready-to-use formulation to be introduced while planting (pellets, gel). The second was a variant to be applied with naturalizing watering (soluble powder).

Chemical Transformation of Humic Acid Molecules under the Influence of Mineral, Fungal and Bacterial Fertilization in the Context of the Agricultural Use of Degraded Soils.

The main goal of this work was to study the structural transformation of humic acids (HAs) under the influence of selected strains of fungi (Aspergillus niger and Paecilomyces lilacinus) and bacteria (Bacillus sp., Paenibacillus polymyxa and Bacillus amyloliquefaciens) with/without the presence of NPK fertilizers. Two-year experiments were conducted on two different soils and HAs isolated from these soils were examined for structure, humification degree, and quantity using fluorescence and UV-Vis spectroscopy, elemental analysis, and extraction methods. Results showed that the applied additives contributed to the beneficial transformation of HAs, but effects differed for various soils. HAs from silty soil with higher organic carbon content showed simplification of their structure, and decreases in humification, molecular weight, and aromaticity under the influence of fungi and bacteria without NPK, and with NPK alone. With both fungi and NPK, increases in O/H and O/C atomic ratios indicated an increase in the number of O-containing functional groups. HAs from sandy soil did not show as many significant changes as did those from silty soil. Sandy soil exhibited a strong decline in HA content in the second year that was reduced/neutralized by the presence of fungi, bacteria, and NPK. Periodically observed fluorescence at ~300 nm/450 nm reflected formation of low-molecular HAs originating from the activity of microorganisms.

Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi.

The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to generate reliable results for an emerging approach in the field of environmental control using microbial diversity biosensors. This work reports a method under development for determining soil microbial diversity using high efficiency Multiplex PCR-Terminal Restriction Fragment Length Polymorphism (M-T-RFLP) for the simultaneous detection of bacteria, archaea and fungi. Three different primer sets were used in the reaction and the analytical conditions were optimized. Optimal analytical conditions were achieved using 0.5 µM of primer for bacteria and 1 µM for archaea and fungi, 4 ng of soil DNA template, and HaeIII restriction enzyme. Comparative tests using the proposed analytical approach and a single analysis of each microorganism group were carried out to indicate that both genetic profiles were similar. The Jaccard similarity coefficient between single and multiplexing approach ranged from 0.773 to 0.850 for bacteria and fungi, and 0.208 to 0.905 for archaea. In conclusion, the multiplexing and pooling approaches significantly reduced the costs and time required to perform the analyses, while maintaining a proper effectiveness.

Triplex Real-Time PCR Approach for the Detection of Crucial Fungal Berry Pathogens-Botrytis spp., Colletotrichum spp. and Verticillium spp.

Phytopathogens cause undeniably serious damage in agriculture by harming fruit cultivations and lowering harvest yields, which as a consequence substantially reduces food production efficiency. Fungi of the Botrytis, Colletotrichum and Verticillium genera are a main concern in berry production. However, no rapid detection method for detecting all of these pathogens simultaneously has been developed to date. Therefore, in this study, a multiplex real-time PCR assay for this purpose was established. Universal fungal primers for the D2 region of the large subunit ribosomal DNA and three multiplexable fluorogenic probes specific for the chosen fungi were designed and deployed. The triplex approach for the molecular detection of these fungi, which was developed in this study, allows for the rapid and effective detection of crucial berry pathogens, which contributes to a more rapid implementation of protective measures in plantations and a significant reduction in losses caused by fungal diseases.

Searching for New Beneficial Bacterial Isolates of Wild Raspberries for Biocontrol of Phytopathogens-Antagonistic Properties and Functional Characterization.

The threat caused by plants fungal and fungal-like pathogens is a serious problem in the organic farming of soft fruits. The European Commission regulations prohibit some commercially available chemical plant protection products, and instead recommend the use of natural methods for improving the microbial soil status and thus increasing resistance to biotic stresses caused by phytopathogens. The solution to this problem may be biopreparations based on, e.g., bacteria, especially those isolated from native local environments. To select proper bacterial candidates for biopreparation, research was provided to preliminarily ensure that those isolates are able not only to inhibit the growth of pathogens, but also to be metabolically effective. In the presented research sixty-five isolates were acquired and identified. Potentially pathogenic isolates were excluded from further research, and beneficial bacterial isolates were tested against the following plant pathogens: Botrytis spp., Colletotrichum spp., Phytophthora spp., and Verticillium spp. The eight most effective antagonists belonging to Arthrobacter, Bacillus, Pseudomonas, and Rhodococcus genera were subjected to metabolic and enzymatic analyses and a resistance to chemical stress survey, indicating to their potential as components of biopreparations for agroecology.

How Do Trichoderma Genus Fungi Win a Nutritional Competition Battle against Soft Fruit Pathogens? A Report on Niche Overlap Nutritional Potentiates.

We present a case study report into nutritional competition between Trichoderma spp. isolated from wild raspberries and fungal phytopathogenic isolates (Colletotrichum sp., Botrytis sp., Verticillium sp. and Phytophthora sp.), which infect soft fruit ecological plantations. The competition was evaluated on the basis of nutritional potentiates. Namely, these were consumption and growth, calculated on the basis of substrate utilization located on Biolog® Filamentous Fungi (FF) plates. The niche size, total niche overlap and Trichoderma spp. competitiveness indices along with the occurrence of a stressful metabolic situation towards substrates highlighted the unfolding step-by-step approach. Therefore, the Trichoderma spp. and pathogen niche characteristics were provided. As a result, the substrates in the presence of which Trichoderma spp. nutritionally outcompete pathogens were denoted. These were adonitol, D-arabitol, i-erythritol, glycerol, D-mannitol and D-sorbitol. These substrates may serve as additives in biopreparations of Trichoderma spp. dedicated to plantations contaminated by phytopathogens of the genera Colletotrichum sp., Botrytis sp., Verticillium sp. and Phytophthora sp.

The Status of Soil Microbiome as Affected by the Application of Phosphorus Biofertilizer: Fertilizer Enriched with Beneficial Bacterial Strains.

Regarding the unfavourable changes in agroecosystems resulting from the excessive application of mineral fertilizers, biopreparations containing live microorganisms are gaining increasing attention. We assumed that the application of phosphorus mineral fertilizer enriched with strains of beneficial microorganisms contribute to favourable changes in enzymatic activity and in the genetic and functional diversity of microbial populations inhabiting degraded soils. Therefore, in field experiments conditions, the effects of phosphorus fertilizer enriched with bacterial strains on the status of soil microbiome in two chemically degraded soil types (Brunic Arenosol - BA and Abruptic Luvisol - AL) were investigated. The field experiments included treatments with an optimal dose of phosphorus fertilizer (without microorganisms - FC), optimal dose of phosphorus fertilizer enriched with microorganisms including Paenibacillus polymyxa strain CHT114AB, Bacillus amyloliquefaciens strain AF75BB and Bacillus sp. strain CZP4/4 (FA100) and a dose of phosphorus fertilizer reduced by 40% and enriched with the above-mentioned bacteria (FA60). The analyzes performed included: the determination of the activity of the soil enzymes (protease, urease, acid phosphomonoesterase, ß-glucosidase), the assessment of the functional diversity of microorganisms with the application of BIOLOGTM plates and the characterization of the genetic diversity of bacteria, archaea and fungi with multiplex terminal restriction fragment length polymorphism and next generation sequencing. The obtained results indicated that the application of phosphorus fertilizer enriched with microorganisms improved enzymatic activity, and the genetic and functional diversity of the soil microbial communities, however these effects were dependent on the soil type.

The Effect of Cultivation Method of Strawberry (Fragaria x ananassa Duch.) cv. Honeoye on Structure and Degradation Dynamics of Pectin during Cold Storage.

The high quality and long shelf life of strawberry fruit are largely dependent on the cultivation method. The goal of this experiment was to study the effect of different cultivation methods on molecular structure and rheological properties of pectin extracted from strawberry quality parameters during cold storage. Three methods of cultivation of strawberry cv. Honeoye were tested: organic cultivation on raised beds, organic cultivation with the flat-planted method and conventional cultivation with the flat-planted method. The nanostructure of pectin (AFM), its chemical structure (FT-IR) and rheological properties were studied. The fruits were also tested by size, dry matter, firmness, acidity and the content of soluble solids, anthocyanin, phenolics, vitamin C and galacturonic acid. Pectin isolated from organic strawberries was more rapidly degraded than conventional strawberry pectin, which limits the possibilities for their processing and use as gelling or stabilizing agents at 20 °C. The differences in fruit quality were particularly noticeable with respect to the anthocyanin content, which was significantly higher for organic strawberry. The organic fruit also had better sensory properties because of its lower acidity and higher soluble solid content (SSC). These and other results from this experiment showed that strawberries produced by organic farming methods had better biochemical properties compared to conventional fruit; however, pectin transformation undergone faster limits their further technological applications.

Alternative Molecular-Based Diagnostic Methods of Plant Pathogenic Fungi Affecting Berry Crops-A Review.

Increasing consumer awareness of potentially harmful pesticides used in conventional agriculture has prompted organic farming to become notably more prevalent in recent decades. Central European countries are some of the most important producers of blueberries, raspberries and strawberries in the world and organic cultivation methods for these fruits have a significant market share. Fungal pathogens are considered to be the most significant threat to organic crops of berries, causing serious economic losses and reducing yields. In order to ameliorate the harmful effects of pathogenic fungi on cultivations, the application of rapid and effective identification methods is essential. At present, various molecular methods are applied for fungal species recognition, such as PCR, qPCR, LAMP and NGS.

The Ability of a Novel Strain Scheffersomyces (Syn. Candida) shehatae Isolated from Rotten Wood to Produce Arabitol.

Arabitol is a polyalcohol which has about 70% of the sweetness of sucrose and an energy density of 0.2 kcal/g. Similarly to xylitol, it can be used in the food and pharmaceutical industries as a natural sweetener, a texturing agent, a dental caries reducer, and a humectant. Biotechnological production of arabitol from sugars represents an interesting alternative to chemical production. The yeast Scheffersomyces shehatae strain 20BM-3 isolated from rotten wood was screened for its ability to produce arabitol from L-arabinose, glucose, and xylose. This isolate, cultured at 28°C and 150 rpm, secreted 4.03 ± 0.00 to 7.97 ± 0.67 g/l of arabitol from 17-30 g/l of L-arabinose assimilated from a medium containing 20-80 g/l of this pentose with yields of 0.24 ± 0.00 to 0.36 ± 0.02 g/g. An optimization study demonstrated that pH 4.0, 32°C, and a shaking frequency of 150 rpm were the optimum conditions for arabitol production by the investigated strain. Under these conditions, strain 20BM-3 produced 6.2 ± 0.17 g/l of arabitol from 17.5 g/l of arabinose after 4 days with a yield of 0.35 ± 0.01 g/g. This strain also produced arabitol from glucose, giving much lower yields, but did not produce it from xylose. The new strain can be successfully used for arabitol production from abundantly available sugars found in plant biomass.

Microbial biodiversity of meadows under different modes of land use: catabolic and genetic fingerprinting.

The main goal of the study was to find differences in the bacterial community structure resulting from different ways of meadow management in order to get the first insight into microbial biodiversity in meadow samples. The next generation sequencing technique (454-pyrosequencing) was accompanied with the community level physiological profiling (CLPP) method in order to acquire combined knowledge of both genetic and catabolic bacterial fingerprinting of two studied meadows (hayland and pasture). Soil samples (FAO: Mollic Gleysol) were taken in April 2015 from the surface layer (0-20 cm). Significant differences of the bacterial community structure between the two analyzed meadows resulted from different land mode were evidenced by pyrosequencing and CLPP techniques. It was found that Alpha- and Gammaproteobacteria dominated in the hayland, whereas Delta- and Betaproteobacteria prevailed in the pasture. Additionally, the hayland displayed lower Firmicutes diversity than the pasture. Predominant bacterial taxa: Acidobacteria, together with Chloroflexi and Bacteroidetes seemed to be insensitive to the mode of land use, because their abundance remained at a similar level in the both studied meadows. The CLPP analysis confirmed much faster degradation of the carbon sources by microorganisms from the hayland rather than from the pasture. Amino acids were the most favoured carbon source groups utilized by microorganisms in contrast to carbohydrates, which were utilized to the lowest extent. The study clearly proved that the consequences of even moderate anthropogenic management are always changes in bacterial community structure and their metabolic activity. Bacterial taxa that are sensitive and resistant on modes of land use were determined.

Enhanced Phytoremediation of Crude Oil-Polluted Soil by Four Plant Species: Effect of Inorganic and Organic Bioaugumentation.

A field experiment investigating the removal and/or uptake of Polycyclic Aromatic Hydrocarbons (PAHs) and specific metals (As, Cd, Cr) from a crude oil polluted agricultural soil was performed during the 2013 wet season using four plant species: Fimbristylis littoralis, Hevea brasilensis (Rubber plants), Cymbopogom citratus (Lemon grass), and Vigna subterranea (Bambara nuts). Soil functional diversity and soil-enzyme interactions were also investigated. The diagnostic ratios and the correlation analysis identified mixed petrogenic and pyrogenic sources as the main contributors of PAHs at the study site. A total of 16 PAHs were identified, 6 of which were carcinogenic. Up to 42.4 mg kg(-1) total PAHs was recorded prior to the experiments. At 90 d, up to 92% total PAH reduction and 96% As removal were achieved using F. littoralis, the best performing species. The organic soil amendment (poultry dung) rendered most of the studied contaminants unavailable for uptake. However, the organic amendment accounted for over 70% of the increased dehydrogenase, phosphatase, and proteolytic enzymes activities in the study. Overall, the combined use of soil amendments and phytoremediation significantly improved the microbial community activity, thus promoting the restoration of the ecosystem.

Laccase production and metabolic diversity among Flammulina velutipes strains.

Twelve Flammulina velutipes strains originating from Poland were identified using internal transcribed spacer (ITS) region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was determined. All F. velutipes strains were also characterized using Biolog FF MicroPlates to obtain data on C-substrate utilization and mitochondrial activity. The ability to decompose various substrates differed among the F. velutipes strains up to five times. The highest catabolic activities were characteristic for only two strains with capabilities to decompose up to 22 carbon sources. The correlation between carbon repression and laccase production by F. velutipes was analyzed based on glucose assimilation by these strains. Moreover, the influence of metal ions (Cu(2+), Cd(2+)), veratric and ferulic acids, and temperature on laccase activities in the analyzed strains was determined. The results obtained proved that all the inducers influenced laccase expression in almost all the analyzed strains. However, the degree of induction depended not only on the strain used but also on the day of the induction.

The influence of ecological and conventional plant production systems on soil microbial quality under hops (Humulus lupulus).

The knowledge about microorganisms-activity and diversity under hop production is still limited. We assumed that, different systems of hop production (within the same soil and climatic conditions) significantly influence on the composition of soil microbial populations and its functional activity (metabolic potential). Therefore, we compared a set of soil microbial properties in the field experiment of two hop production systems (a) ecological based on the use of probiotic preparations and organic fertilization (b) conventional-with the use of chemical pesticides and mineral fertilizers. Soil analyses included following microbial properties: The total number microorganisms, a bunch of soil enzyme activities, the catabolic potential was also assessed following Biolog EcoPlates®. Moreover, the abundance of ammonia-oxidizing archaea (AOA) was characterized by terminal restriction fragment length polymorphism analysis (T-RFLP) of PCR ammonia monooxygenase α-subunit (amoA) gene products. Conventional and ecological systems of hop production were able to affect soil microbial state in different seasonal manner. Favorable effect on soil microbial activity met under ecological, was more probably due to livestock-based manure and fermented plant extracts application. No negative influence on conventional hopyard soil was revealed. Both type of production fulfilled fertilizing demands. Under ecological production it was due to livestock-based manure fertilizers and fermented plant extracts application.

Effect of microbial biostimulants on the antioxidant profile, antioxidant capacity and activity of enzymes influencing the quality level of raspberries (Rubus idaeus L.).

The influence of four microbial biostimulants containing various strains of Bacillus subtilis and/or Paenibacillus sp. on the quality of raspberries cv. Delniwa, Poemat, and Enrosadira cultivated in two consecutive seasons was investigated. The biostimulants influenced the antioxidant level, antioxidant capacity, phenolic acids and flavonoids profiles, enzymatic activity, and the degree of methylation and acetylation of the pectin in the raspberry fruits. The biostimulants had the greatest effect on the antioxidant content (16% - 20% increase) and capacity in the Delniwa raspberry fruits from the first season. A positive correlation was found between the activity of the ß-galactosidase enzyme and ferric reducing power. In the second season, a decrease in the activity of pectin esterase and α-L-arabinofuranosidase and an increase in the degree of methylation of pectin were noted. Our results suggest that the changes in raspberry quality were related to the type of biostimulant applied.

Comprehensive antifungal investigation of natural plant extracts against Neosartorya spp. (Aspergillus spp.) of agriculturally significant microbiological contaminants and shaping their metabolic profile.

Fungi belonging to the genus Neosartorya (teleomorph of Aspergillus spp.) are of great concern in the production and storage of berries and fruit-based products, mainly due to the production of thermoresistant ascospores that cause food spoilage and possible secretion of mycotoxins. We initially tested the antifungal effect of six natural extracts against 20 isolates of Neosartorya spp. using a traditional inhibition test on Petri dishes. Tested isolates did not respond uniformly, creating 5 groups of descending sensitivity. Ten isolates best representing of the established sensitivity clusters were chosen for further investigation using a Biolog™ MT2 microplate assay with the same 6 natural extracts. Additionally, to test for metabolic profile changes, we used a Biolog™ FF microplate assay after pre-incubation with marigold extract. All natural extracts had an inhibitory effect on Neosartorya spp. growth and impacted its metabolism. Lavender and tea tree oil extracts at a concentration of 1000 µg mL-1 presented the strongest antifungal effect during the inhibition test, however all extracts exhibited inhibitory properties at even the lowest dose (5 µg mL-1). The fungal stress response in the presence of marigold extract was characterized by a decrease of amino acids and carbohydrates consumption and an uptake of carboxylic acids on the FF microplates, where the 10 studied isolates also presented differences in their innate resilience, creating 3 distinctive sensitivity groups of high, average and low sensitivity. The results confirm that natural plant extracts and essential oils inhibit and alter the growth and metabolism of Neosartorya spp. suggesting a possible future use in sustainable agriculture as an alternative to chemical fungicides used in traditional crop protection.

The role of food preservatives in shaping metabolic profile and chemical sensitivity of fungi - an extensive study on crucial mycological food contaminants from the genus Neosartorya (Aspergillus spp.).

Food preservatives are crucial in fruit production, but fungal resistance is a challenge. The main objective was to compare the sensitivity of Neosartorya spp. isolates to preservatives used in food security applications and to assess the role of metabolic properties in shaping Neosartorya spp. resistance. Sodium metabisulfite, potassium sorbate, sodium bisulfite and sorbic acid showed inhibitory effects, with sodium metabisulfite the most effective. Tested metabolic profiles included fungal growth intensity and utilization of amines and amides, amino acids, polymers, carbohydrates and carboxylic acids. Significant decreases in the utilization of all tested organic compound guilds were observed after fungal exposure to food preservatives compared to the control. Although the current investigation was limited in the number of predominately carbohydrate substrates and the breadth of metabolic responses, extensive sensitivity panels are logical step in establishing a course of action against spoilage agents in food production being important approach for innovative food chemistry.