Valorization of fish processing by-products for protein hydrolysate recovery: Opportunities, challenges and regulatory issues.

Protein-rich fish processing by-products, often called rest raw materials (RRM), account for approximately 60% of the total fish biomass. However, a considerable amount of these RRM is utilized for low-value products such as fish meal and silage. A promising and valuable approach for maximizing the utilization of RRM involves the extraction of bioactive fish protein hydrolysate (FPH). This review assesses and compares different hydrolyzation methods to produce FPH. Furthermore, the review highlights the purification strategy, nutritional compositions, and bioactive properties of FPH. Finally, it concludes by outlining the application of FPH in food products together with various safety and regulatory issues related to the commercialization of FPH as a protein ingredient in food. This review paves the way for future applications by highlighting efficient biotechnological methods for valorizing RRM into FPH and addressing safety concerns, enabling the widespread utilization of FPH as a valuable and sustainable source of protein.

Antifeedant, antifungal and nematicidal compounds from the endophyte Stemphylium solani isolated from wormwood.

The continuous search for natural product-based biopesticides from fungi isolated from untapped sources is an effective tool. In this study, we studied a pre-selected fungal endophyte, isolate Aa22, from the medicinal plant Artemisia absinthium, along with the antifungal, insect antifeedant and nematicidal compounds present in the extract. The endophyte Aa22 was identified as Stemphylium solani by molecular analysis. The antifungal activity was tested by broth microdilution against Fusarium solani, F. oxysporum, F. moniliforme and Botrytis cinerea, the insect antifeedant by choice bioassays against Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi and the in vitro mortality against the root-knot nematode Meloiydogyne javanica. The structures of bioactive compounds were determined on the basis of 1D and 2D NMR spectroscopy and mass spectrometry. The ethyl acetate extract obtained from the solid rice fermentation showed mycelial growth inhibition of fungal pathogens (EC50 0.08-0.31 mg/mL), was antifeedant to M. persicae (99%) and nematicidal (68% mortality). A bioguided fractionation led to the isolation of the new compound stempholone A (1), and the known stempholone B (2) and stemphol (3). These compounds exhibited antifeedant (EC50 0.50 mg/mL), antifungal (EC50 0.02-0.43 mg/L) and nematicidal (MLD 0.5 mg/mL) activities. The extract activities can be explained by 3 (antifungal), 1-3 (antifeedant) and 1 (nematicidal). Phytotoxicity tests on Lolium perenne and Lactuca sativa showed that the extract and 1 increased L. sativa root growth (121-130%) and 1 reduced L. perenne growth (48-49%). These results highlight the potential of the endophytic fungi Aa22 as biotechnological source of natural product-based biopesticides.

Marine diatom algae cultivation in simulated dairy wastewater and biomass valorization.

Liquid byproducts and organic wastes generated from dairy processing units contribute as the largest source of industrial food wastewater. Though bacteria-mediated treatment strategies are largely implemented, a more effective and innovative management system is needed of the hour. Thus, the current study involves the cultivation of centric diatoms, Chaetoceros gracilis, and Thalassiosira weissflogii in simulated dairy wastewater (SDWW) formulated using varying amounts of milk powder with artificial seawater f/2 media (ASW). The results revealed that cell density and biomass productivity were highest in the 2.5% SDWW treatment cultures of both the strains, the maximum being in C. gracilis (7.5 × 106 cells mL - 1; 21.1 mg L-1 day-1). Conversely, the total carotenoid, chrysolaminarin, and phenol content were negatively impacted by SDWW. However, a considerable enhancement in the total lipid content was reported in the 2.5% SDWW culture of both species. Furthermore, the fatty acid profiling revealed that though the total polyunsaturated fatty acid (PUFA) content was highest in the control setups, the total mono polyunsaturated fatty acid (MUFA) content was higher in the 5% SDWW setups (30.66% in C. gracilis and 33.21% in T. weissflogii). In addition to it, in the cultures utilizing energy from external carbon sources provided by SDWW, the biodiesel produced was also enhanced owing to the heightened cetane number. Thus, the current study evidently highlights the organic carbon acquisition potential of marine diatoms with the scope of providing sustainable biorefinery.

Comparison of nutritional quality of fourteen wild Linum species based on fatty acid composition, lipid health indices, and chemometric approaches unravelling their nutraceutical potential.

Fatty acid profiles of 14 Linum species was determined by GC-MS analysis to study the nutritional quality of Linum species based on fatty acid composition, lipid health indices, and chemometric approaches. L. lewisii and L. marginale found to have the highest content of ALA i.e., 65.38 % and 62.79 %, respectively, L. tenuifolium recorded the highest linoleic acid content (69.69 %), while, L. catharticum recorded highest oleic acid (27.03 %). Health indices viz. polyunsaturated fatty acids/saturated fatty acids ratio, n-6/n-3 fatty acids ratio, atherogenicity, thrombogenicity, oxidability, oxidative stability, hypocholesterolemic/hypercholesterolemic fatty acids, and peroxidisability calculated based on the fatty acid composition revealed that all the linseed species except L. aristatum, L. tenuifolium and L. hudsoniodes have healthy fatty acid composition. L. lewisii clearly emerges as a promising species followed by L. bienne with great values across multiple indices, making them as a potential candidate for dietary or nutritional interests. The lipid profile of Linum species could be well distinguished by two principal components by Principal Component Analysis (PCA).

Optimization of single-step gelatin extraction from pink perch (Nemipterus japonicus) skin and bone obtained from surimi industry using a green solvent.

Surimi industry produces a large quantity of byproducts that are currently being utilized to produce low-value commodities. This study aims to extract gelatin from pink perch skin and bone obtained from the surimi industry using a green single-step extraction method. In addition to using a green solvent, that is, acetic acid, the new method combines the multiple steps of pre-treatment and hydrolysis into one single-step extraction process. Response surface methodology was used to optimize extraction parameters (pH, temperature, and time) to maximize yield and l-hydroxyproline (l-hyp) content. The optimum condition for gelatin extraction was obtained at pH 3, 75°C, and 30 min. At optimum conditions, gelatin yield and l-hyp content were observed to be 16.07% and 41.26 mg g-1 , respectively. The gelatin obtained at optimized condition was further compared with commercial bovine gelatin (BG) in terms of chemical composition and textural, functional, and rheological properties. The results suggested that the optimized pink perch gelatin had higher protein content (92.06%), better gel strength (251.08 g), higher imino acid (18.01%), and improved textural and functional properties than the commercially available BG. The optimized single-step gelatin extraction method from pink perch skin and bones is a promising, rapid, and efficient method for the production of good-quality gelatin, which can be further used for the development of high-value products such as food formulations. PRACTICAL APPLICATION: Fish gelatin is widely used in food product development. Most of the existing methods of the development of high-value product such as gelatin, use multi-step process and harsh mineral acid, therefore, are time-consuming and harmful to the environment. This study provides a green single-step gelatin extraction method that provides an efficient, rapid, and convenient method of gelatin extraction and a sustainable solution for fish industry byproduct utilization. The data obtained with this laboratory-scale study provides a strong basis for scale-up studies.

Novel biogenic silver nanoconjugates of Abrus precatorius seed extracts and their antiproliferative and antiangiogenic efficacies.

Biogenic silver nanoconjugates (AgNCs), derived from medicinal plants, have been widely explored in the field of biomedicines. AgNCs for the first-time were synthesized using ethyl acetate seed extracts of Abrus precatorius and their antiproliferative and antiangiogenic efficacies were evaluated against cervical and oral carcinoma. Ultraviolet-Visible spectrophotometry, dynamic light Scattering (DLS), and scanning electron microscopy (SEM) were used for characterization of AgNCs. Antiproliferative activity was investigated using MTT, DNA fragmentation and in-vitro antioxidant enzyme activity assays. In-vivo chick chorioallantoic membrane (CAM) model was used to evaluate antiangiogenic activity. A total of 11 compounds were identified in both the extracts in GCMS analysis. The synthesized AgNCs were spherical shaped with an average size of 97.4 nm for AgAPE (Sox) and 64.3 nm for AgAPE (Mac). AgNCs possessed effective inhibition against Hep2C and KB cells. In Hep2C cells, AgAPE (Mac) revealed the highest SOD, catalase, GST activity and lower MDA content, whereas AgAPE (Sox) showed the highest GSH content. On the other hand, in KB cells, AgAPE (Sox) exhibited the higher SOD, GST activity, GSH content, and least MDA content, while AgAPE (Mac) displayed the highest levels of catalase activity. Docking analysis revealed maximum binding affinity of safrole and linoleic acid with selected targets. AgAPE (Sox), AgAPE (Mac) treatment profoundly reduced the thickness, branching, and sprouting of blood vessels in the chick embryos. This study indicates that A. precatorius-derived AgNCs have enhanced efficacies against cervical and oral carcinoma as well as against angiogenesis, potentially limiting tumour growth.

HPLC-DAD fingerprinting coupled with chemometric analysis can successfully differentiate Indian Berberis species and its plant parts.

Identification of the plant parts and genuine species is one of the most important steps for the quality control of raw material used in herbal medicines. In the present study, comparative analysis of TLC, HPLC and FTIR-ATR fingerprinting in conjunction with chemometric method was done to select the most appropriate fingerprinting method for quality control of Berberis aristata and its related species. To achieve this, extracts were first utilized to obtain TLC, HPLC and FT-IR fingerprinting data. The data was analyzed by chemometric methods and similarity analysis. The PCA model obtained with HPLC fingerprinting method could successfully classify the Berberis species as well plant parts viz. root, stem and leaves which was not possible with the PCA model of TLC or FT-IR fingerprints. Similarity analysis based on the mean HPLC fingerprinting chromatogram could also appropriately assess the chemical variability and identification of different Berberis species. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03644-6.

Effect of Pink Perch Gelatin on Physiochemical, Textural, Sensory, and Storage Characteristics of Ready-to-Cook Low-Fat Chicken Meatballs.

In recent years consumer demand for low-fat convenience food is increasing rapidly. This study was designed to develop low-fat ready-to-cook (RTC) chicken meatballs using pink perch gelatin. Meatballs were prepared using different concentrations of fish gelatin (3%, 4%, 5%, and 6%). The effect of fish gelatin content on the physico-chemical, textural, cooking, and sensory properties of meatballs was studied. Further, the shelf-life of meatballs was also studied at 4 °C for 15 days and -18 °C for 60 days. The addition of fish gelatin to meatballs decreased the fat content by 67.2% and 79.7% and increased the protein content by 20.1% and 66.4% in comparison to control and Branded Meatballs, respectively. As compared to the Control Meatballs, the addition of fish gelatin also reduced hardness by 26.4% and increased yield and moisture retention in the RTC meatballs by 15.4% and 20.9%, respectively. Sensory analysis suggested that a 5% fish gelatin addition in meatballs has the best acceptability among all tested treatments. Storage study indicated that the addition of fish gelatin to RTC meatballs delayed lipid oxidation during both refrigerated and frozen storage. The results suggested that pink perch gelatin can be used as a fat replacer in chicken meatballs and can potentially increase their shelf-life.

Nutritionally Enriched Muffins from Roselle Calyx Extract Using Response Surface Methodology.

Hibiscus sabdariffa, often called Roselle, is a flowering plant with a variety of traditional medicinal uses. Its calyx, with a bright and attractive red color, produces a tart and pleasant acidic taste. The purpose of this study was to develop a Roselle muffin and assess the acceptability, nutrition, and shelf life of the muffin using its ingredients. The muffin was developed using different formulations in different proportions resulting from Response Surface Methodology (RSM). Sensory parameters were used to assess the muffin's acceptability. According to the findings, the combination of extract volume 45.37 mL, citric acid 1.11 g, and sodium bicarbonate 1.67 g produces the best muffin, with the panelist's sensory scores reaching up to 84%. The outcome of the study suggests muffins baked with the Roselle calyx extract have high antioxidant (12.53 ± 0.13)%, anthocyanin (126.63 ± 1.96) mg Cyn-3-glu/100 g, phenolic (12.91 ± 0.69) mg GAE/100 g, and ascorbic acid (12.10 ± 0.89) mg/100 g contents. The microbial shelf life of the developed muffin is estimated to be 6 days at room temperature. The study findings can therefore be utilized in the development of foods containing Roselle calyx extract.

Exploring the potential of endophytes and their metabolites for bio-control activity.

In the current scenario, extensive use of synthetic chemicals in agriculture is creating notable problems such as disease and pest resistance, residues, yield loss, and soil unproductiveness. These harmful chemicals are eventually reaching our food plate through bioaccumulation and biomagnification in a crop. As a result, beneficial microorganisms are regularly being explored as a safer option in the agriculture sector for their ability to produce valuable bioactive secondary metabolites, particularly for crop protection. Such natural (bio) products are harmless to plants, humans, and the environment. In our quest for the search of the sources of bioactive constituents from the microorganisms, endophytes are the front-runner. They mutually reside inside the plant providing support against phytopathogens by releasing an array of bioactive secondary metabolites building climate reliance of the host plant. The purpose of this review is to examine the biocontrol potential of endophytes against bacterial and fungal pathogens in sustainable agriculture. We also attempt to explain the structure and activity of the secondary metabolites produced by bacterial and fungal endophytes in conjunction with their biocontrol function. Additionally, we address potential future research directions for endophytes as biopesticides.

Distribution, biosynthesis and therapeutic potential of lignans.

Lignans have long been known for their abundant therapeutic properties due to their polyphenolic structure. Linseed is the richest plant source of lignans and has been studied widely for their properties. The most prevalent lignan, secoisolariciresinol diglucoside (SDG), is consumed with linseed and converted into mammalian lignans, enterodiol (END) and enterolactone (ENL), by the gut microbiota. SDG can easily be assessed using HPLC and its deglycosylated form viz secoisolariciresinol can be asses using GC-MS techniques. Variety of extraction and analysis methods has been reported for plant lignans. SDG is known to have therapeutic properties including anti-oxidant, anti-cancerous, anti-inflammatory, modulation of gene expression, anti-diabetic, estrogenic and anti-estrogenic. Despite a large number of bioactivities, strong evidences for the underlying mechanisms for most of the properties are still unknown. SDG is most studied for its anti-cancerous properties. But the use of lignans as anti-carcinogenic agent is limited and commercially not reported due to challenges of purification at commercial level, rapid metabolism, untargeted delivery and toxic compounds associated with lignans. Exploration of more prominent and active derivatives of SDG and their targeted drug delivery should be an important research toward the use of bioactive lignans of linseed.

Management of bakanae disease of rice using biocontrol agents and insights into their biocontrol mechanisms.

Bakanae disease is an emerging problem for the Basmati rice cultivation in India. Forty-seven endophytes isolated earlier along with three Talaromyces flavus isolates evaluated against Fusaium fujikuroi [Nirenberg] bakanae pathogen [isolate F250] through dual culture and enzymatic assays. Out of 50 isolates, 6 isolates namely, Tf1, Tf2, Tf3, Fusarium equiseti, Fusarium sp. and Trichoderma sp. produced good inhibitory results under in vitro conditions and were proceeded with in planta studies and conducted microscopic studies and real-time PCR assays. Microscopic studies revealed that the defense response system of plants was activated to a longer extent in bioagent treatments, since the number of live nuclei (DAPI staining) and green stained live plant cells (FDA staining) were higher as seen in treated plants when compared to pathogen-inoculated and uninoculated control when observed under confocal laser scanning microscopy. The analysis of cell cycle-related genes expressed during the ROS activity showed increased expression of the cell cycle-related genes involved. The selected isolates were also tested under glasshouse for disease inhibition studies. F. equiseti, Fusarium sp. and Trichoderma sp. gave a disease inhibition of, 87%, 66% and 94%, respectively. Tf2 and Tf1 isolate dominantly inhibited the disease with 95% whereas Tf3 also inhibited successfully with 70%. Through the results of our study, we can deduce that the T. flavus (Tf1, Tf2, Tf3) isolates and the endophytes F. equiseti, Fusarium sp. and Trichoderma sp. may represent an important biocontrol agent to control the bakanae disease of rice and also implicated that could further be befitting to capitalize them for field evaluations.

Screening of Tomato Seed Bacterial Endophytes for Antifungal Activity Reveals Lipopeptide Producing Bacillus siamensis Strain NKIT9 as a Potential Bio-Control Agent.

The current study investigates the diversity pattern and fungicidal potential of bacterial endophytes isolated from two different organic varieties of tomato plants (V1 and V2). A total of seventy-five bacterial isolates identified by 16S rRNA gene sequencing revealed a majority of genus as Bacillus and one Planococcus, which were grouped into eight different species. The Shannon diversity H' (1.56), Simpson's index of diversity (0.93), Magalef' index (2.23), Evenness (0.96), and Species richness (7) indicated the high endophytic bacterial diversity in the V1 variety of the tomato. Bacterial endophytes isolated from both of the varieties were screened for their antifungal activity against five economically critical fungal pathogens (viz., Botrytis cinerea, Rhizoctonia solani, Fusarium solani, Verticillium lateritium, and Alternaria solani) of tomato crop through dual culture assay. The data revealed B. siamensis strain NKIT9 as the most potent antagonist, significantly (p < 0.05) inhibiting the mycelial growth between 75 to 90% against selected fungal pathogens. High bioactivity of lipopeptide extract of strain NKIT9 was recorded against R. solani with minimum IC50 value of 230 µg/ml. The Ultra Performance Liquid Chromatography-High Definition Mass Spectrometry (UPLC-HDMS) analysis of this lipopeptide extract revealed the presence of Surfactin and Bacillomycin D. Furthermore, in-vitro results showed that the selected bacterial strain significantly minimized the disease incidence in damping-off assay which makes this strain a promising antifungal bio-control agent. Moreover, in the pot experiment the NKIT9 increased the fruit yield by 59.2% compared with the untreated R. solani infested control.

Effect of Different Drying Methods on the Nutritional Value of Hibiscus sabdariffa Calyces as Revealed by NMR Metabolomics.

Red mature calyces of Hibiscus sabdariffa were collected from 16 different locations in Meghalaya, India. Samples were processed using shade drying (SD) and tray drying (TD). NMR spectroscopy was used to assess the metabolic composition of the calyces. In this study, 18 polar metabolites were assigned using 1D and 2D NMR spectra, and 10 of them were quantified. Proximate analysis showed that the TD method is more efficient at reducing moisture and maintaining the ash content of the Hibiscus biomass. NMR metabolomics indicates that the metabolite composition significantly differs between SD and TD samples and is more stable in TD plant processing. The differences in post-harvest drying has a greater impact on the metabolite composition of Hibiscus than the plant location.

Harnessing the Phytotherapeutic Treasure Troves of the Ancient Medicinal Plant Azadirachta indica (Neem) and Associated Endophytic Microorganisms.

Azadirachta indica, commonly known as neem, is an evergreen tree of the tropics and sub-tropics native to the Indian subcontinent with demonstrated ethnomedicinal value and importance in agriculture as well as in the pharmaceutical industry. This ancient medicinal tree, often called the "wonder tree", is regarded as a chemical factory of diverse and complex compounds with a plethora of structural scaffolds that is very difficult to mimic by chemical synthesis. Such multifaceted chemical diversity leads to a fantastic repertoire of functional traits, encompassing a wide variety of biological activity and unique modes of action against specific and generalist pathogens and pests. Until now, more than 400 compounds have been isolated from different parts of neem including important bioactive secondary metabolites such as azadirachtin, nimbidin, nimbin, nimbolide, gedunin, and many more. In addition to its insecticidal property, the plant is also known for antimicrobial, antimalarial, antiviral, anti-inflammatory, analgesic, antipyretic, hypoglycaemic, antiulcer, antifertility, anticarcinogenic, hepatoprotective, antioxidant, anxiolytic, molluscicidal, acaricidal, and antifilarial properties. Notwithstanding the chemical and biological virtuosity of neem, it has also been extensively explored for associated microorganisms, especially a class of mutualists called endophytic microorganisms (or endophytes). More than 30 compounds, including neem "mimetic" compounds, have been reported from endophytes harbored in the neem trees in different ecological niches. In this review, we provide an informative and in-depth overview of the topic that can serve as a point of reference for an understanding of the functions and applications of a medicinal plant such as neem, including associated endophytes, within the overall theme of phytopathology. Our review further exemplifies the already-noted current surge of interest in plant and microbial natural products for implications both within the ecological and clinical settings, for a more secure and sustainable future.

Chemical Composition of an Aphid Antifeedant Extract from an Endophytic Fungus, Trichoderma sp. EFI671.

Botanical and fungal biopesticides, including endophytes, are in high demand given the current restrictive legislations on the use of chemical pesticides. As part of an ongoing search for new biopesticides, a series of fungal endophytes have been isolated from selected medicinal plants including Lauraceae species. In the current study, an extract from the endophytic fungus Trichoderma sp. EFI 671, isolated from the stem parts of the medicinal plant Laurus sp., was screened for bioactivity against plant pathogens (Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum and Botrytis cinerea), insect pests (Spodoptera littoralis, Myzus persicae, Rhopalosiphum padi) and plant parasites (Meloidogyne javanica), with positive results against M. persicae. The chemical study of the neutral fraction of the active hexane extract resulted in the isolation of a triglyceride mixture (m1), eburicol (2), ß-sitostenone (3), ergosterol (4) and ergosterol peroxide (5). The free fatty acids present in the acid fraction of the extract and in m1 (oleic, linoleic, palmitic and stearic) showed strong dose-dependent antifeedant effects against M. persicae. Liquid (potato dextrose broth, PDB and Sabouraud Broth, SDB) and solid (corn, sorghum, pearl millet and rice) growth media were tested in order to optimize the yield and bioactivity of the fungal extracts. Pearl millet and corn gave the highest extract yields. All the extracts from these solid media had strong effects against M. persicae, with sorghum being the most active. Corn media increased the methyl linoleate content of the extract, pearl millet media increased the oleic acid and sorghum media increased the oleic and linoleic acids compared to rice. The antifeedant effects of these extracts correlated with their content in methyl linoleate and linoleic acid. The phytotoxic effects of these extracts against ryegrass, Lolium perenne, and lettuce, Lactuca sativa, varied with culture media, with sorghum being non- toxic.

Pharmacological evaluation of Convolvulus pluricaulis as hypolipidaemic agent in Triton WR-1339-induced hyperlipidaemia in rats.

OBJECTIVE: The aim of the study was to evaluate the effect of Convolvulus pluricaulis (CP; C. pluricaulis) methanolic extract on Triton WR-1339-induced hyperlipidaemia in rats. METHODS: The study comprised of six groups namely normal control, experimental control and treatment groups (100, 200 and 400 mg/kg of C. pluricaulis, and 65 mg/kg of Fenofibrate). Hyperlipidaemia was induced by a single intraperitoneal injection of Triton WR-1339 400 mg/kg in rats. Parameters such as lipid profile, oxidative stress, histological analysis and atherogenic index were evaluated. The plant extract was further studied by HPLC and LCMS, for analyses of active phytochemicals. KEY FINDINGS: The result of the study showed that C. pluricaulis significantly decreased total cholesterol, triglycerides, LDL-c, MDA levels and atherogenic index while the levels of HDL-c and GSH were found to be raised. Plant extract at the dose of 400 mg had a consistent effect on all lipid profile parameters. Lower doses (100 and 200 mg) did not produce a statistically significant reduction in LDL-c. In addition, the protective effect of C. pluricaulis was confirmed by histological analysis. Further, the findings of the study were found to be comparable with fenofibrate. CONCLUSIONS: Therefore, the present study suggests that C. pluricaulis has the potential for the treatment of hyperlipidaemia.

Fungal and Bacterial Diversity Isolated from Aquilaria malaccensis Tree and Soil, Induces Agarospirol Formation within 3 Months after Artificial Infection.

Aquilaria malaccensis Lam, commonly known as Agarwood, is a highly valuable species used in production of agar oil from its infected wood, which is utilized in pharmaceutical and perfumery industry. Agar oil formation in agarwood takes years through the natural process which is induced by natural or artificial injury or microbial infection. The role of soil fungi and bacteria in artificial induction is still an unexplored area. In the present study, we isolated the fungal and bacterial community residing inside the stem of A. malaccensis tree and circumventing soil, samples collected from 21 different sites of the north-eastern state Assam of India and explored their potential in induction of Agarospirol (2-(6,10-Dimethylspiro[4,5]dec-6-en-2-yl)-2-propanol) production by artificially infecting the trees with these microorganisms. A total 340 fungi and 131 bacteria were isolated from 50 stem samples, and 188 fungi and 148 bacteria were isolated from 50 soil samples. Highest Shannon (H' = 2.43) and Fisher (α = 5.57) diversity index was observed in the stem isolates. The dominant fungal genus was Trichoderma in stem with Pi value of 0.18; while in soil, Aspergillus showed dominance with Pi value 0.73. In bacteria, Bacillus genera showed dominance in both stem and soil samples with Pi = 0.62 and 0.51, respectively. Forty fungal and bacterial isolates were used to assess their potential to induce formation of agarwood in A. malaccensis by artificial infection method. Gas chromatography mass spectroscopy (GC-MS) analysis confirmed development of Agarwood by the presence of Agarospirol compound in samples collected after 3 months of the artificial infection. Only 31% of bacterial and 23% of fungal isolates showed their ability in production of Agarospirol by artificial infection method. Bacteria Pantoea dispersa and fungi Penicillium polonicum showed the highest production in comparison to other isolates.