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Fruits and vegetables are important for the nutrition and health of individuals. They are highly perishable in nature because of their susceptibility to microbial growth. Foodborne pathogens create a significant problem for consumers, food businesses, and food safety. Postharvest factors, including transportation, environment, and preservation techniques, cause a reduction in product quality. The present world is using synthetic preservatives, which have negative impacts on consumer health. Food safety and demand for healthy foods among consumers, the scientific community, and the food industry resulted in the exploitation of natural preservatives, which play an important role in their effectiveness, prolonged shelf life, and safety. Natural preservatives include plants, animals, and microbiological sources with polymers to extend shelf life, improve quality, and enhance food safety. This review specifically focuses on mechanism of action of natural preservatives, spoilage of fruit and vegetables, the importance of edible film and coating on fruits and vegetables.

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Yeasts are the usual contaminants in fruit juices and other beverages, responsible for the decrease in the quality and shelf-life of such products. Preservatives are principally added to these beverages to enhance their shelf-life. With the increasing consumer concern towards chemical food additives, plant-derived antimicrobials have attracted the attention of researchers as efficient and safer anti-yeast agents. However, the methods currently used for determining their anti-yeast activity are time- and material-consuming. In this study, the anti-yeast effect of plant phenolic compounds in apple and orange juice food models using microtiter plates has been evaluated in order to validate the modified broth microdilution method for screening the antimicrobial activity of juice preservative agents. Among the twelve compounds tested, four showed a significant in vitro growth-inhibitory effect against all tested yeasts (Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces rouxii) in both orange and apple juices. The best results were obtained for pterostilbene in both juices with minimum inhibitory concentrations (MICs) ranging from 32 to 128 µg/mL. Other compounds, namely oxyresveratrol, piceatannol, and ferulic acid, exhibited moderate inhibitory effects with MICs of 256-512 µg/mL. Furthermore, the results indicated that differences in the chemical structures of the compounds tested significantly affected the level of yeast inhibition, whereas stilbenes with methoxy and hydroxy groups produced the strongest effect. Furthermore, the innovative assay developed in this study can be used for screening the anti-yeast activity of juice preservative agents because it saves preparatory and analysis time, laboratory supplies, and manpower in comparison to the methods commonly used.

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This literature review explores cutting-edge microencapsulation techniques designed to enhance the antimicrobial efficacy of essential oils in dairy products. As consumer demand for natural preservatives rises, understanding the latest advancements in microencapsulation becomes crucial for improving the shelf life and safety of these products. The bibliometric analysis utilized in this review highlighted a large number of documents published on this topic in relation to the following keywords: essential oils, AND antimicrobials, AND dairy products, OR microencapsulation. The documents published in the last 11 years, between 2013 and 2023, showed a diversity of authors and countries researching this topic and the keywords commonly used. However, in the literature consulted, no study was identified that was based on bibliometric analysis and that critically evaluated the microencapsulation of essential oils and their antimicrobial potential in dairy products. This review synthesizes findings from diverse studies, shedding light on the various encapsulation methods employed and their impact on preserving the quality of dairy goods. Additionally, it discusses the potential applications and challenges associated with implementation in the dairy industry. This comprehensive analysis aims to provide valuable insights for researchers, food scientists, and industry professionals seeking to optimize the use of essential oils with antimicrobial properties in dairy formulations.

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Ensuring the microbiological safety of food products is majorly important to regulatory agencies, producers, and consumers. This study aimed to examine the effects of three different antimicrobial agents, including chitosan (CH), mastic oil (M), and citric acid (CA), individually or as a combination, against Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes (artificially inoculated) in Guacamole, a ready-to-eat (RTE) avocado-based salad. The Guacamole samples included untreated samples, designated as CNL, and samples treated as follows: CA 0.15% and CA 0.30% with citric acid added at 0.15% and 0.30% v/w; CH 0.5% and CH 1% with chitosan at 0.5 and 1% v/w; M 0.2% and M 0.4% with mastic essential oil (EO) at 0.2% and 0.4% v/w; CACH with CA 0.30% and CH 1% v/w; CAM with CA 0.30% and M 0.4% v/w; CHM with CH 1% and M 0.4% v/w; and CACHM with CA 0.30%, CH 1%, and M 0.4% v/w. Microbiological evaluation, monitoring of the pH values, and proximate analyses (moisture, fat, protein, ash, and water activity) were performed at different time intervals (days 0, 1, 3, 5, and 7) at two storage temperatures (4 and 10 °C). Antimicrobial treatments, particularly CH 1% and CACHM, effectively (p < 0.05) reduced Salmonella spp. and E. coli O157:H7 populations at 4 °C, while CACHM showed the most efficacy against L. monocytogenes. However, at 10 °C, antimicrobials had limited impact, and the bacterial counts exhibited an increasing trend during storage. The pH values in the avocado-based salad samples showed, in general, higher decreases at 10 compared to 4 °C, with the CHM combination showing the highest antimicrobial effect.

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The study aims to explore bee venom (honey-BV) as a potential natural preservative for "Tallaga" soft cheese. Characterization of the active compounds in honey-BV was conducted via chromatographic analyses. Antimicrobial efficacy against pathogenic bacteria and fungi was evaluated, and minimum inhibitory concentration (MIC) was determined. Subsequently, honey-BV was applied to Tallaga cheese at 15 mg/g concentrations. The main active ingredients identified in bee venom were apamin (2%) and melittin (48.7%). Both concentrations of bee venom (100 and 200 mg/mL) exhibited significant antifungal and antibacterial properties against tested organisms, with MIC values varied from 0.2 to 0.5 mg/mL for bacteria to 3-13 mg/mL for fungi. Application of honey-BV in Tallaga cheese resulted in complete elimination of Staphylococcal populations after 2 weeks of cold storage, with no detectable growth of molds or yeasts throughout the storage period. Additionally, a steady decrease in aerobic plate count was observed over time. In summary, honey-BV holds promise as a natural preservative for soft cheese, however, more investigation is required to optimize the concentration for economic viability, taking into account health benefits and safety considerations.

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The use of natural raw substances for food preservation could provide a great contribution to food waste reduction, circular economy enhancement, and green process application widening. Recent studies indicated that the use of porous materials as adsorbents for natural essential oils provided nanohybrids with excellent antioxidant and antimicrobial properties. Following this trend in this work, a thymol oil (TEO) rich SBA-15 nanohybrid was prepared and characterized physiochemically with various techniques. This TEO@SBA-15 nanohybrid, along with the pure SBA-15, was extruded with low-density polyethylene (LDPE) to develop novel active packaging films. Results indicated that TEO loading was higher than other porous materials reported recently, and the addition of both pure SBA-15 and TEO@SBA-15 to the LDPE increased the water/oxygen barrier. The film with the higher thyme-oil@SBA-15 nanohybrid content exhibited a slower release kinetic. The antioxidant activity of the final films ignited after 48 h, was in the range of 60-70%, and was almost constant for 7 days. Finally, all tests indicated a sufficient improvement by the addition of thyme-oil@SBA-15 nanohybrids in the pure LDPE matrix and the concentration of wt. 10% of such nanocarriers provided the optimum final LDPE/10TEO@SBE-15 active packaging film. This material could be a potential future product for active packaging applications.

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This study delves into the production and antimicrobial characteristics of cell-free supernatants from Pediococcus acidilactici (CFSs-Pa). Antimicrobial activity was initially observed in CFS-Pa harvested after 12 h of incubation and increased up to the late stationary phase at 48 h. The increase in antimicrobial activity did not align with total protein content, pointing to other factors linked to the accumulation of organic acids, particularly lactic acid. The SDS-PAGE analysis also indicated that the expected proteinaceous compound (pediocin) was not observed in CFS-Pa. Further investigations suggested that the antimicrobial properties of CFS-Pa were exclusively due to organic acids. The MIC values confirmed potent antimicrobial activity, particularly at a 10% dilution of CFS-Pa in MRS broth. The time-kill assays demonstrated bactericidal activity against EHEC, Listeria monocytogenes, and Staphylococcus aureus by 12 h, 18 h, and 24 h using a 10% dilution of CFS-Pa. Additionally, CFS-Pa exhibited dose-dependent antioxidant activity, requiring a 70% (v/v) concentration to inhibit DPPH scavenging activity by 50%. All the experimental results suggested potential applications of CFS-Pa in food preservation. An attempt to incorporate CFS-Pa into bacterial cellulose (BC) for edible food packaging demonstrated promising antimicrobial results, particularly against L. monocytogenes and S. aureus, with room for optimization.

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The comparative metabolic profiling and their biological properties of eight extracts obtained from diverse parts (leaves, flowers, roots) of the medicinal plant Flourensia fiebrigii S.F. Blake, a chemotype growing in highland areas (2750 m a.s.l.) of northwest Argentina, were investigated. The extracts were analysed by GC-MS and UHPLC-MS/MS. GC-MS analysis revealed the presence of encecalin (relative content: 24.86 %) in ethereal flower extract (EF) and this benzopyran (5.93 %) together sitosterol (11.35 %) in the bioactive ethereal leaf exudate (ELE). By UHPLC-MS/MS the main compounds identified in both samples were: limocitrin, (22.31 %), (2Z)-4,6-dihydroxy-2-[(4-hydroxy-3,5-dimethoxyphenyl)methylidene]-1-benzofuran-3-one (21.31 %), isobavachin (14.47 %), naringenin (13.50 %), and sternbin, (12.49 %). Phytocomplexes derived from aerial parts exhibited significant activity against biofilm production of Pseudomonas aeruginosa and Staphylococcus aureus, reaching inhibitions of 74.7-99.9 % with ELE (50 µg/mL). Notably, the extracts did not affect nutraceutical and environmental bacteria, suggesting a selective activity. ELE also showed the highest reactive species scavenging ability. This study provides valuable insights into the potential applications of this chemotype.

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There is a burgeoning focus on utilizing the antifungal and antioxidant properties of essential oils derived from various plants as a modern and natural approach to combat the growth of fungi that contaminate food. In this study, we used essential oils extracted from Thymus daenensis Celak. subsp. daenensis to address three mycotoxin-producing species of Aspergillus, specifically A. flavus, A. parasiticus, and A. niger, all of which are recognized contaminants of food and agricultural products. Concurrently, the antioxidant properties of the essential oils were evaluated, revealing their noteworthy role in the antifungal activity. Essential oils were derived from T. daenensis subsp. daenensis was observed to have a significant inhibitory effect on all three species of Aspergillus, as evidenced by the minimum inhibitory concentration (MIC) ranging from 575 to 707 ppm and the half-maximal inhibitory concentration (IC50) ranging from 237 to 280 ppm. These results confirm the strong antifungal activity of the essential oils. Furthermore, the essential oil exhibited free radical scavenging activity, resulting in an EC50 value of 37.1 µg/ml. In summary, T. daenensis subsp. daenensis essential oil demonstrated a competitive advantage over other similar plants and synthetic antibiotics. This indicates the promising potential of this essential oil as a natural antifungal agent to control Aspergillus growth and mycotoxin contamination. It offers an alternative or complementary approach to conventional antifungal agents and could be a valuable addition to the arsenal of natural remedies to address fungal contamination in food and agricultural products.

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Antimicrobial resistance is a major global health concern, threatening the effective prevention and treatment of infections caused by microorganisms. These factors boosted the study of safe and green alternatives, with hydrosols, the by-products of essential oils extraction, emerging as promising natural antimicrobial agents. In this context, four hydrosols obtained from Cupressus leylandii A.B. Jacks & Dallim, Eucalyptus globulus Labill., Aloysia citrodora Paláu and Melissa officinalis L. were studied. Their chemical composition comprises neral, geranial, 1,8-cineole, terpinen-4-ol, and oplopanonyl acetate, compounds with recognised antimicrobial activity. Concerning antimicrobial activity, significant differences were found using different hydrosol concentrations (10-20% v/v) in comparison to a control (without hydrosol), showing the potential of the tested hydrosols to inhibit the microbial growth of Escherichia coli, Staphylococcus aureus, and Candida albicans. A. citrodora hydrosol was the most effective one, inhibiting 90% of E. coli growth and 80% of C. albicans growth, for both hydrosol concentrations (p < 0.0001). With hydrosol concentration increase, it was possible to observe an improved antimicrobial activity with significant reductions (p < 0.0001). The findings of this work indicate the viability of reusing and valuing the hydrosols, encouraging the development of green applications for different fields (e.g., food, agriculture, pharmaceuticals, and cosmetics).

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Consumers expect safe, healthy, natural, and sustainable food. Within the food industry, ingredient use is changing due to these consumer demands. While no single agreed-upon definition of clean label exists, a "clean label" in the context of food refers to a product that has a simplified and transparent ingredient list, with easily recognizable and commonly understood components to the general public. Clean-label products necessitate and foster a heightened level of transparency between companies and consumers. Dairy products are vulnerable to being contaminated by both pathogens and spoilage microorganisms. These microorganisms can be effectively controlled by replacing conventional antimicrobials with clean-label ingredients such as protective cultures or bacterial/fungal fermentates. This review summarizes the perspectives of consumers and the food industry regarding the definition of "clean label," and the current and potential future use of clean-label antimicrobials in dairy products. A key goal of this review is to make the concept of clean-label antimicrobial agents better understood by both manufacturers and researchers.

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In this study, Salvia officinalis L. and Salvia sclarea essential oils (EOs) were investigated using gas chromatography-mass spectrometry (GC-MS) to describe their chemical composition. The obtained results show, for both EOs, a profile rich in terpene metabolites, with monoterpenes predominating sesquiterpenes but with significant qualitative and quantitative differences. The main compound found in the Salvia officinalis EO (SOEO) was camphor (19.0%), while in Salvia sclarea EO (SCEO), it was linalyl acetate (59.3%). Subsequently, the in vitro antimicrobial activity of the EOs against eight pathogenic strains was evaluated. The disc diffusion method showed a significant lysis zone against Gram-positive bacteria. The minimum inhibitory concentrations (MICs) ranged from 3.7 mg/mL to 11.2 mg/mL, indicating that each EO has specific antimicrobial activity. Both EOs also showed significant antiradical activity against DPPH radicals and total antioxidant activity. In addition, the preservative effect of SOEO (9.2%) and SCEO (9.2%), alone or in combination, was tested in ground beef, and the inhibitory effect against Listeria monocytogenes inoculated into the raw ground beef during cold storage was evaluated. Although the effect of each individual EO improved the biochemical, microbiological, and sensory parameters of the samples, their combination was more effective and showed complete inhibition of L. monocytogenes after 7 days of storage at 4 °C. The results show that both EOs could be used as safe and natural preservatives in various food and/or pharmaceutical products.

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The microbial quality of raw milk artisanal cheeses is not always guaranteed due to the possible presence of pathogens in raw milk that can survive during manufacture and maturation. In this work, an overview of the existing information concerning lactic acid bacteria and plant extracts as antimicrobial agents is provided, as well as thermisation as a strategy to avoid pasteurisation and its negative impact on the sensory characteristics of artisanal cheeses. The mechanisms of antimicrobial action, advantages, limitations and, when applicable, relevant commercial applications are discussed. Plant extracts and lactic acid bacteria appear to be effective approaches to reduce microbial contamination in artisanal raw milk cheeses as a result of their constituents (for example, phenolic compounds in plant extracts), production of antimicrobial substances (such as organic acids and bacteriocins, in the case of lactic acid bacteria), or other mechanisms and their combinations. Thermisation was also confirmed as an effective heat inactivation strategy, causing the impairment of cellular structures and functions. This review also provides insight into the potential constraints of each of the approaches, hence pointing towards the direction of future research.

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Consumer demand for natural and healthier products has led to an increasing interest in the bioactive and therapeutic properties of plant extracts. In this study, we evaluated the phenolic compounds profile, bioactivities, and toxicities of plant extracts from eight European flora species, including Calendula officinalis L., Calluna vulgaris (L.) Hull, Hippophae rhamnoides L., Juglans regia L., Mentha cervina L., Rubus idaeus L., Sambucus nigra L., and Vitis vinifera L. The aim was to identify potential preservatives of natural origin. Phenolic compounds were identified by HPLC-DAD-ESI-MS. Caffeic acid derivatives, ellagitannins, flavonols, and flavones were the major phenolic compounds identified. The total phenolic content varied from 16.0 ± 0.2 (V. vinifera) to 123 ± 2 mg/g (H. rhamnoides) of dry extract. All extracts showed antioxidant potential and exhibited activity against some of the microorganisms tested. S. nigra showed the highest activity in the inhibition of oxidative hemolysis (OxHLIA) assay and H. rhamnoides, notably, had the lowest IC50 values in TBARS and DPPH assays, as well as the lowest minimum inhibitory concentration (MIC) values. Regarding in vitro cytotoxicity, in tumor and non-tumor cell lines, although some extracts revealed toxicity against normal cells, it was found that the samples C. vulgaris, V. vinifera and R. idaeus might be used against tumor cells since the active concentration is much lower than the one causing toxicity. In vivo acute toxicity tests using Artemia franciscana suggest low toxicity for most extracts, with LC50 > 400 mg/L. These results showed the potential of the studied extracts as natural preservatives, given their richness in compounds with bioactive properties, highlight their potential value to the production chain.

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Opportunities for the valorisation of agro-industrial residues of the chestnut (Castanea sativa Mill.) production chain have been fostered with the production of multifunctional polyphenol-rich extracts with the potential to be introduced as natural additives or active components in several products. Nonetheless, it is crucial to explore the feasibility of different extracts from the various by-products for these applications through the exhaustive study of their composition and bioactivities without losing sight of the sustainable character of the process. This work aimed at the screening of the phenolic compound composition and bioactivities of different green extracts of chestnut burs, shells and leaves, as the first step to establish their potential application as natural ingredients, primarily as food preservatives. To this end, maceration (MAC) as a conventional extraction method besides ultrasound and microwave-assisted extractions (UAE and MAE) was employed to obtain the extracts from chestnut by-products using water (W) and hydroethanolic solution (HE) as solvents. Phenolic compounds were analysed by HPLC-DAD-(ESI-)MS/MS; the antioxidant capacity was assessed by colourimetric assays, and the antimicrobial activity was evaluated against several strains of food-borne bacteria and fungi. The leaf extracts obtained by MAC-HE and UAE-HE presented the highest concentration of phenolic compounds (70.92 ± 2.72 and 53.97 ± 2.41 mg.g-1 extract dw, respectively), whereas, for burs and shells, the highest recovery of total phenolic compounds was achieved by using UAE-HE and UAE-W (36.87 ± 1.09 and 23.03 ± 0.26 mg.g-1 extract dw, respectively). Bis-HHDP-glucose isomers, chestanin and gallic acid were among the most abundant compounds. Bur extracts (MAC-HE and UAE-HE) generally presented the highest antioxidant capacity as measured by TBARS, while the best results in DPPH and reducing power assays were found for shell extracts (MAE-W and MAC-HE). Promising antibacterial activity was noticed for the aqueous extracts of burs, leaves and hydroethanolic extracts of shells, with emphasis on the MAE-W extract of burs that showed bactericidal activity against E. cloacae, P. aeruginosa and S. aureus (MBC 5 mg.mL-1). Overall, it can be concluded that chestnut by-products, including burs, shells and leaves, are sources of polyphenolic compounds with significant antioxidant and antimicrobial activities. The choice of extraction method and solvent greatly influenced the composition and bioactivity of the extracts. These findings highlight the potential of chestnut by-products for the development of natural additives, particularly for food preservation, while also emphasizing the importance of sustainable utilization of agricultural waste materials. Further research is warranted to optimize extraction techniques and explore additional applications for these valuable bioactive compounds.

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Campylobacter jejuni is a Gram-negative bacterium which is considered as the most reported cause of foodborne infection, especially for poultry species. The object of this work is to evaluate the occurrence of C. jejuni in chicken meat as well its control via three types of sorghum extracts (white sorghum (WS), yellow sorghum (YS), and red sorghum (RS)); antibacterial activity, antioxidant power, and cytotoxicity of sorghum extracts were also assessed. It was found that C. jejuni is very abundant in chicken meat, especially breast and thigh. WS extract showed more effectiveness than both yellow and red ones. Lyophilized WS extract offered high total phenolic compounds (TPCs) and total flavonoid compounds (TFCs) of 64.2 ± 0.8 mg gallic acid equivalent (GAE/g) and 33.9 ± 0.4 mg catechol equivalent (CE)/g, respectively. Concerning the antibacterial and antioxidant activities, WS showed high and significant antibacterial activity (p < 0.001); hence, WS displayed a minimum inhibitory concentration (MIC) of 6.25%, and revealed an inhibition zone of 7.8 ± 0.3 mm; it also showed an IC50 at a concentration of 34.6 µg/mL. In our study, different samples of chicken fillet were collected and inoculated with pathogenic C. jejuni and stored at 4 °C. Inoculated samples were treated with lyophilized WS extract at (2%, 4%, and 6%), the 2% treatment showed a full reduction in C. jejuni on the 10th day, the 4% treatment showed a full reduction in C. jejuni on the 8th day, while the 6% treatment showed a full reduction in C. jejuni on the 6th day. Additionally, 2%, 4%, and 6% WS extracts were applied on un-inoculated grilled chicken fillet, which enhanced its sensory attributes. In sum, WS extract is a promising natural preservative for chicken meat with accepted sensory evaluation results thanks to its high antibacterial and antioxidant potentials.

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Raw beef patties were treated with either 450 ppm of Sodium metabisulphite (SMB), or Kakadu plum powder (KPP) (0.2%, 0.4%, 0.6%, 0.8%) or no additive (negative control) and stored under Modified Atmosphere Packaging at 4 ± 1 °C for 20 days. Lipid oxidation, microbial growth rate, pH, instrumental color, and surface myoglobin were studied. Total phenolic compounds (TPC) and vitamin C of the KPP were also measured. The TPC was 13.9 g GAE/ 100 g dry weight (DW) and for vitamin C, the L-AA (l-ascorbic acid) and DHAA (dehydroascorbic acid) were 12.05 g/100 g and 0.5 g/ 100 g DW, respectively. The experimental results indicated that lipid oxidation was significantly delayed throughout the storage period for KPP-treated samples compared to both the negative control and SMB-treated samples. KPP at levels of 0.2% and 0.4% in the raw beef patties were efficient in slowing down the microbial growth rate compared to the negative control; however, SMB had a higher antimicrobial activity. The pH, the redness as well as metmyoglobin formation in the raw beef patties were reduced by the inclusion of the KPP in treated samples. A correlation (r = -0.66) was noted between KPP treatments and lipid oxidation, but there was no correlation (r = -0.006) between KPP treatment and microbial growth. This study demonstrates that KPP could be used as natural preservative for shelf-life extension of raw beef patties.

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Nisin is a cationic antimicrobial peptide used as a natural food preservative against gram-positive bacteria. However, nisin is degraded following interaction with food components. Here, we report the first use of Carboxymethylcellulose (CMC), a versatile and affordable food additive, to protect nisin and extend its antimicrobial activity. First, we optimized the methodology by considering the effect of nisin:CMC ratio, pH, and, especially, the degree of substitution of CMC. In particular, we show here how these parameters affected the size, charge, and, notably, the encapsulation efficiency of these nanomaterials. This way, optimized formulations contained over 60 % w/w in nisin while encapsulating ∼90 % of the nisin used. We then show that these new nanomaterials inhibited the growth of Staphylococcus aureus, a major foodborne pathogen, using milk as a representative food matrix. Remarkably, this inhibitory effect was observed with one-tenth of the concentration of nisin currently used in dairy products. We believe that the combination of the affordability of CMC, flexibility and simplicity of preparation, and the ability to inhibit the growth of food pathogens, makes these nisin:CMC PIC nanoparticles an ideal platform to underpin new nisin formulations.

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The study was carried out to investigate cricket protein hydrolysates' (CPH) potential to enhance the storage stability of cheddar cheese. The cricket protein (CP) samples pre-processed with microwave (T1), ultrasonication (T2) or without pre-treatment (T0) were used for developing the CPH using alcalase enzyme (3%). Freeze-dried CPH were incorporated in the cheese samples (CPH-T1, CPH-T2 and CPH-T0) at the maximum level of 1.5% and were analysed for quality during 3 months of storage (4 ± 1 °C) compared to the control samples without CPH. The pre-treatments significantly improved the antimicrobial and antioxidant potential of the CPH. The CPH exhibited a significant positive effect on antioxidant potential, lipid stability, protein oxidation, microbial growth, and sensory quality of the cheddar cheese during storage. Digestion simulation showed a significant positive impact on the antioxidant activity of the cheddar cheese. Our results indicate the potential of CPH to enhance the quality of fat-rich foods during storage.

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BACKGROUND: Guava (Psidium guajava Linn.) has been traditionally used in the treatment of a wide range of diseases due to its rich content of secondary metabolites. AIM: This study was aimed to evaluate the effect of altitude and solvent systems on guava leaves crude extract's phenolics and flavonoid content, antioxidant, antimicrobial, and toxicity nature. METHODS: Guava leaves were collected from three different geographical locations in Nepal while solvents with an increasing polarity index were used for extraction. The yield percentage of extracts was calculated. Total Phenolic Content, Total Flavonoid Content, and antioxidant activity were determined by the Folin-Ciocalteu method, Aluminium chloride colorimetric method, and DPPH (2,2'-Diphenyl-1-picrylhydrazyl) assay respectively. The quantification of fisetin and quercetin was performed using the HPLC with method validation. The antimicrobial activity of the extracts was tested against bacteria and fungus isolated from spoiled fruits and vegetables and identified through 16s and 18s rRNA sequencing. Finally, Brine Shrimp Lethality Assay (BSLA) was used for testing the toxicity of the extracts. RESULTS: The phenolic and total flavonoid content was found to be higher in ethanol extract (331.84 mg GAE/g dry extract) and methanol extract (95.53 mg QE/g dry extract) from Kuleshwor respectively. Water extract of guava leaves from Kuleshwor (WGK) did not show significantly different antioxidant activity when compared to methanol and ethanol extracts. Fisetin and quercetin were higher in WGK (1.176 mg/100 g) and (10.967 mg/100 g) dry extract weight respectively. Antibacterial activity against food spoilage bacteria was dose-dependent and found to be highest for all the extracts from different solvents and altitudes at higher concentrations (80 mg/ml). Similarly, methanol and ethanol guava extracts from all locations showed antifungal activity against Geotrichum candidum RIBB-SCM43 and Geotrichum candidum RIBB-SCM44. WGK was found to be non-toxic. CONCLUSION: Our study concludes that the antioxidant and antimicrobial activity of WGK was found to be similar statistically to that of methanol and ethanol extracts of Bishnupur Katti and Mahajidiya. These results suggest the possibility of using water as a sustainable solvent to extract natural antioxidant and antimicrobial compounds which can further be used as natural preservatives to extend the shelf life of fruits and vegetables.