1 Introduction

In recent years, the growing popularity of kombucha as a non-toxic, quick-growing fungus has served as a useful tool for scientists. “Kombucha” is a beverage made by fermenting tea and commonly known as “tea fungus” which has been mixed with a culture that contains a symbiotic partnership of yeasts and bacteria which is called SCOBY (Symbiotic colony of bacteria and yeasts). Increasingly popular foods include antibacterial and anticancer qualities, which help maintain the gut flora’s balance and have also been utilized as medicine for various ailments [1]. The probiotic microorganisms (acetic and lactic bacteria as well as yeast), antibiotics, amino acids, polyphenols from tea, sugars, organic acids, ethanol, water-soluble vitamins, and a range of micronutrients generated during fermentation are all thought to contribute to the health benefits of kombucha [2]. Lactic, acetic, glucuronic, gluconic and usnic acids, ethanol, and some antimicrobial compounds were reported as major compounds in the fermented liquid of Kombucha [3]. The distinct symbiotic association of bacteria and yeast with their secondary metabolic byproducts prevents the growth of other contaminating microbes [4].

On the other hand, the accumulation of organic acids causes the acidity of the beverage to rise as the fermentation rate increases. The amount of viable microbial cells in Kombucha tea declines as a result of oxygen shortage brought on by excessive acidity [5]. Nevertheless, throughout the manufacturing process, harmful microbes have the potential to contaminate the Kombucha tea [6]. According to some studies, Kombucha can become infected with possible harmful microbes [7, 8]. Some results by researchers demonstrated that the addition of antibiotics and other preservative material might help to prevent microbial contamination of Kombucha [9, 10]. Furthermore, microbial resistance is a crucial drawback which has increased the demand for the development of new antibacterial agents that pose a serious threat to human health [11]. The expense of therapy increases when infections are brought on by bacteria resistant to antibiotics. Bacteria that are resistant to antibiotics have these genes and may transfer them horizontally. The number of resistant microorganisms has increased due to the widespread misuse of antibiotics [12]. Ampicillin, ceftriaxone, cephalexin, amoxicillin, amoxicillin-clavulanate, ampicillin-sulbactam, several narrow-spectrum cephalosporins and ciprofloxacin are antibiotics that commonly used to treat urinary tract diseases and nosocomial infections. They impede protein synthesis by interfering with ribosomal subunits [13]. Many bacterial strains were found to be resistant to the previous routine antibiotics such as Staphylococcus aureus [14], Escherichia coli [15], Klebsiella pneumoniae [16], Pseudomonas aeruginosa [17] and Serratia sp. [18]. Serratia liquefaciens is one of the serious causes of nosocomial urinary tract infection (UTI) since it influences the adhesion of microbes to host epithelial surfaces. Beta-lactamase-mediated carbapenem resistance is uncommon among Enterobacteriaceae, however, it has been reported recently for S. liquefaciens. Aminoglycosides displayed a strong antibacterial action against S. marcescens, although recently discovered resistant strains [19].

It is necessary to obtain alternative antibacterial agents to find a solution of the growing problem of antibiotic resistance. There is a trend to combine metal or metal oxide with polymers or other materials to improve their antibacterial properties [20, 21]. Zinc (Zn) and zinc oxide (ZnO) were used as strong antibacterial agents in several pharmaceutical and industrial applications including paper, cosmetics, plastics, paint, building materials and ceramics due to their high stability, antimicrobial potential and anticorrosion properties [22]. They were added and combined with different materials to enhance their antibacterial activity [23, 24]. They can damage bacterial cell walls, prevent bacterial cell formation, block protein synthesis, reduce membrane permeability, and obstruct bacterial metabolic processes due to the interaction between Zn ions and biological macromolecules [25].

The zinc oxide nanoparticles (ZnO NPs) have higher antibacterial action and are more reactive than Zn in their bulk due to their unique chemical, physical, and biological properties such as mechanical properties, magnetic, piezoelectric, strong adsorption ability, high catalytic efficiency, biocompatibility, high isoelectric point, fast electron transfer kinetics, solubility and cellular uptake [26, 27]. Because of their very small size, high surface area to volume ratio and high oxidative effects, ZnO NPs have unique features that distinguish them from their bulk counterparts. In addition, investigations have shown that inorganic nanoparticles (NPs) are smaller particles that can easily enter cells through small holes in plasma membranes due to their immense surface area [28, 29]. In comparison to other NPs, ZnO NPs were the most effective and potent antibacterial agent that could completely inhibit Bacillus subtilis, E. coli, and P. fluorescens [30]. The action of the NPs and their bulk counterparts on the enzymatic activity such as dehydrogenases differed greatly. According to Joko et al. [31], ZnO NPs showed 2-fold superior activity of alkaline phosphatase activity than bulk Zn. Also, ZnO NPs have been reported as anti-fungal, anti-angiogenesis, anti-inflammatory, anti-platelet, and anti-viral agents [32, 33]. Many approaches were used and developed to synthesize NPs including physical, biological, and chemical techniques. Specialized equipment is needed since physical procedures require high pressure and temperature. Chemical approaches have the benefit of producing huge amounts of NPs in a short amount of time, but they also produce toxic and unfriendly by-products since they require capping agents to stabilize the NPs [1]. Currently, green synthesized NPs have attracted dramatic interest in themselves due to their superior biocompatibility and eco-friendly nature. Green protocols aim to reduce or eliminate dangerous substrates. On the other hand, biological approaches to NP synthesis do not necessitate the use of hazardous chemicals. From these green protocols, the biological synthesis of NPs is a single-step bio-reduction method, and less energy is used to synthesize eco-friendly [34]. Biological molecules themselves can serve as capping agents and particularly decrease the toxic quality caused by the conventionally integrated NPs. Utilizing intracellular or extracellular synthesis techniques, plants, and microorganisms, including fungi and bacteria, are employed to produce NPs in a green manner [35]. The extracellular biosynthesis of NPs is more prioritized than the intracellular biosynthesis which requires extraction and purification of NPs from the plant or microbial growth [36]. Hens, Kombucha extract was applied as a bio-reducing agent to fabricate ZnO NPs in an extracellular model benefiting from all the advantages of Kombucha and its effective active bio-compounds.

To our knowledge, this study is the first report to use Kombucha extract to biosynthesize ZnO NPs in a simple, fast, cheap, and environmentally friendly manner. This approach does not require specialized and complex procedures like isolation, maintenance of cultures, and many steps of purification. In addition, the antibacterial potential of the biosynthesized ZnO NPs solo or incorporation to Kombucha SCOBY against different Gram-positive and Gram-negative pathogenic bacterial strains was investigated.

2 Materials and methods

2.1 Materials

2.1.1 Microbial strains

Kombucha SCOBY, Serratia liquefaciens (AC: OQ071699.1), Staphylococcus saprophyticus (AC: OQ071703.1) and Lysinibacillus fusiformis (AC: OQ071701.1) were provided by the Microbiology Lab Culture Collection (MLCC), Damietta University, Egypt. Four American-type culture collection (ATCC) strains of pathogenic bacteria (S. aureus ATCC25923, E. coli ATCC25922, K. pneumoniae ATCC33495 and P. aeruginosa ATCC27853) were used in the antibacterial activity test.

2.2 Methods

2.2.1 Preparation of Kombucha extract

The Kombucha SCOBY sample was activated according to the procedure described by Chen & Liu [19]. Broth growth medium consisting of black tea (5 g/l) and sucrose (8% v/v) was prepared, distributed in 250 ml Erlenmeyer flasks (100 ml per flask), and boiled for 5 min. After cooling to 30 °C, tea broth was inoculated with Kombucha pellicle fragments (3.7%, w/v on wet weight basis) and liquid broth from the activated tea fungus (20%, v/v) and then incubated under dark and static conditions at 30 °C for 7 days in static Memmert incubator (Memmert, Germany). After the incubation period, the Kombucha biomass was collected by filtration using Whatman filter paper No.1 and repeatedly washed with sterile distilled water to remove medium components. About 10 g of biomass was added into 100 ml of sterile distilled water in a 250 ml Erlenmeyer flask and kept in the orbital shaker (LSI-3016R, Daihan Lab Tech Co., Ltd., Namyangju, Kyonggi, South Korea) at 150 rpm for 3 days at 35 °C and in dark conditions. After incubation time, Kombucha biomass extract was obtained through filtration and passing the filtrate through Millipore filters to get rid of any microbial spores [5, 37].

2.3 Green synthesis and optimization of ZnO NPs

About 10 ml of cell-free Kombucha biomass extract was mixed with 90 ml of zinc nitrate (Zn(NO3)2.6H2O, crystallized, ≥ 99.0%, Sigma Aldrich) solution (5 mM, by dissolving 0.1339 g in 90 ml of distilled water) at pH 7 ± 0.2 and agitated (150 rpm) in the dark at 35 °C. The colour change of the solution from pale yellow and white precipitate formation indicated the formation of ZnO NPs. Nanoparticles were collected using centrifugation at 8000 rpm for 15 min (Eppendorf 3H24RI intelligent high-speed refrigerated centrifuge, Herexi Instrument & Equipment Company, China) and then dried at 80 °C followed by calcination at 600 °C for 4 h in a muffle furnace (FN 100, Nüve, Turkey) [38].

The optimal conditions for the synthesis of ZnO NPs were chosen after evaluation of various Zn(NO3)2.6H2O solution concentrations (1, 5, 10, 15, 20, 25, and 30 mM, pH 7.2, 35 °C for one day), various concentrations of cell-free Kombucha biomass extract (10, 20, 30, 40, and 50%, pH 7.2, 35 °C for one day) and different incubation periods (3–24 h.). The pH value (1–10) and different temperatures (20–45 °C) were also studied.

2.4 Characterization of the synthesized ZnO NPs

The synthesis of ZnO NPs was confirmed by UV–visible spectroscopy (Beckman DU-40, USA), Fourier transform infrared spectroscopy (JASCO FTIR, FT/IR-4100typeA, USA) and X-ray diffraction (XRD, Shimadzu X-ray diffractometer, LabX XRD-6000, Japan) spectra. The Zeta potential of ZnO NPs in the solution was determined using a Zeta analyser (Malvern Zetasizer, Nano-ZS90, UK). Transmission electron microscope (TEM, JEOL JEM-2100, Japan) analysis of ZnO NPs was also recorded and compared to XRD results after analysing by the Debye-Scherrer equation: D = 0.9λ/β cos θ [39]. The presence of zinc and oxygen in the particles was confirmed using a scanning electron microscope (SEM, JEOL JSM-6510, Japan) outfitted with an EDX Genesis energy dispersive x-ray elemental analysis system (EDS).

2.5 Antibacterial activity of ZnO NPs using agar well diffusion method

The agar well diffusion method [40] was used to test the antibacterial activity of the green synthesized ZnO NPs against different bacterial strains. Bacterial slants were sub-cultured on nutrient agar (Oxoid) plates and incubated at 37 °C for 24 h. After incubation time, 3 single colonies were inoculated aerobically in a nutrient broth medium (Oxoid) and incubated at 37 °C and 150 rpm for 24 h. 0.5 MacFarland (1–2 × 108 CFU/ml) from each strain was prepared and inoculated on sterile Mueller-Hinton agar (MHA) plates using a spread plating method. Then, 5 mm wells were punched. 100 µl from three concentrations (50, 100 and 150 µg/ml) of ZnO NPs, Zn(NO3)2.6H2O, and ciprofloxacin (Sigma-Aldrich, as standard antibiotics) were prepared in dimethyl sulfoxide (DMSO) and added into the wells separately. The plates were incubated at 37 °C for 24 h. and then inhibition zones were measured in millimetres (mm).

2.6 Minimum inhibition concentration (MIC)

The broth dilution method was demonstrated to determine the MIC values for ZnO NPs, Zn(NO3)2.6H2O, and ciprofloxacin against the tested bacteria [41]. 0.5 MacFarland (1–2 × 108 CFU/ml) of each strain was inoculated in Mueller-Hinton broth media (Oxoid) supplemented with 2–30 mg/ml from the antibacterial agent and incubated at 37 °C and 150 rpm for 24 h. Bacterial growth rates were measured using a spectrophotometer (JASCO V-730 UV-Vis double beam spectrophotometer, USA) at 600 nm to determine the MIC values (Zero absorbance).

2.7 Minimum microbicidal concentration (MBC)

MIC tubes with no apparent bacterial growth were inoculated on MHA plates and incubated at 37 °C for 24 h. The MBC values of the antibacterial agents were recorded with no apparent growth plates.

2.8 Ultrastructure study of ZnO NPs-treated bacteria

S. liquefaciens and L. fusiformis were used as models for the treatment of Gram-negative and Gram-positive bacteria by ZnO NPs, respectively. 0.5 MacFarland (1–2 × 108 CFU/ml) of each bacterial strain was prepared, inoculated in Mueller-Hinton broth media and incubated at 37 °C and 150 rpm for 24 h. After incubation time, the developed bacterial growth was treated with the MIC concentration of ZnO NPs for 2 h. Bacterial pellets were collected by centrifugation at 5000 rpm for 15 min under aseptic conditions and repeatedly washed with sterile distilled water to remove excess media and particles. The bacterial cells were fixed using 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7 and left for 20 min at room temperature. A graded series of ethanol was used in the dehydration of the fixed cells. Double-stained with uranyl acetate and lead citrate samples were observed on carbon-coated copper grids (Type G 200, 3.05 µM diameter, TAAP, USA) using TEM (JEOL JEM-2100, Japan).

2.9 Antibacterial activity of Kombucha pellicle disc impregnated with ZnO NPs

Kombucha SCOBY was prepared in sweetened tea broth as previously mentioned then the SCOBY biomass was collected by filtration and washed well using sterile distilled water. Pellicle discs of Kombucha SCOBY were punched using a cork-borer (10 mm) and impregnated with ZnO NPs solution (MIC concentration) for different impregnating times (3, 6, 12 and 24 h.). The antibacterial activity of Kombucha SCOBY pellicle disc impregnated with ZnO NPs was tested against S. liquefaciens, L. fusiformis and K. pneumoniae ATCC33495 by standard disc diffusion method [42]. One pellicle disc of Kombucha SCOBY from each different impregnating time was added to the surface of inoculated bacterial agar plates. The plates were incubated at 37 °C for 24 h. and then inhibition zones were measured in millimetres (mm).

2.10 Statistical analysis

ANOVA test was applied to analyse the findings using SPSS software version 18. Fixed at 0.05 was the significance level. The experiments were conducted three times. All results were presented as the mean and standard deviation (SD) [43].

3 Results

3.1 Synthesis, optimization, and characterization of ZnO NPs

Kombucha extract produced ZnO NPs within 24 h. at 35 °C at dark conditions. Different conditions were applied to optimize the biosynthesis rate of ZnO NPs including Zn(NO3)2.6H2O solution concentrations, cell-free Kombucha biomass extract volume, incubation periods, and pH value at different temperatures (Fig. 1). The bio-formation of ZnO NPs was very good at 25 mM of Zn(NO3)2.6H2O solution compared to other concentrations (Fig. 1A). It also increased during the mixing of the previous concentration with 40% cell-free Kombucha biomass extract of the total mixture (Fig. 1B). It was observed that the progress of the biosynthesis process was increased gradually by increasing of the incubation time (Fig. 1C). The pH9 was the optimum pH value for the production of ZnO NPs followed by pH8, pH7 and pH6 (Fig. 1D). Other higher or lower pH values were affected negatively by the formation of ZnO NPs. Temperature increase increases the biosynthesis of ZnO NPs rate while it was decreased by raising the temperature to 45 °C (Fig. 1E).

Fig. 1
figure 1

Optimization of ZnO NPs biosynthesis. a Different concentrations of Zn(NO3)2.6H2O solution; mM. b Volume of cell-free Kombucha biomass extract (v/v%). c Incubation times; hr. d pH value. e Temperatures; °C

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The optimized ZnO NPs were characterized by UV–visible spectroscopy, XRD, Zeta analysis, FTIR, TEM and EDX (Fig. 2). Yellowish-white precipitates were deposited at the bottom of the tube compared to the control; it was possible to discern the early detection of ZnO NPs formation. The ZnO NPs’ surface plasmon resonance (SPR) resulted in the formation of this yellowish-white colour of the precipitate [44]. It was reported that several biofabricated ZnO NPs displayed comparable absorption peaks in the UV-vis absorption spectra ranging from 320 to 360 nm [44,45,46]. The absorption peak of the biosynthesized ZnO NPs showed SPR characteristic maximum absorption peak at 322 nm (Fig. 2A). XRD results displayed different characteristic peaks for ZnO at 2θ = 32.2°, 36.78°, 57.5°, and 64.4° which corresponds to crystallographic planes of (100), (101), (110), and (103), respectively (Fig. 2B). The size of the NPs can be used to explain the XRD pattern broadening, and the presence of strong, narrow peaks indicates that the product has good crystallinity [47]. According to Zeta analysis, ZnO NPs had a positive charge equal to 19 ± 3 mV (Fig. 2C). The size distribution homogeneity is described by the polydispersity index (PDI) value, which ranges from 0 to 1. The term “monodisperse” usually refers to a polydispersity index that is less than 0.1. By using photon correlation spectroscopy (PCS), the average Zeta size of ZnO NPs was determined to be 25 ± 13 d.nm, and Zeta analysis revealed that the PDI value ranged from 0.351 to 0.401 (Fig. 2D). As PCS measures the entire diameter size (diameter of NPs plus capping agents’ diameter), but TEM measures only NPs size, the previous diameters are greater than those reported by TEM (23 ± 1.5 nm). FTIR spectra results demonstrated the presence of proteins in the biosynthesis of ZnO NPs (Fig. 2E). Metal-oxygen stretching vibrations located between 400 and 600 cm−1 substituted the presence of ZnO [48]. Broad stretching −OH groups appeared at 3291.9, 3271.6 cm−1. The 2° amines vibration band is located at 2922.6 cm−1. Vinyl and cis-tri vibrations appeared at 1657.5 cm−1. 1° amines stretching vibrations substituted at 1529.3, 1457.3, 1406.8, 1242.9, and 1259.3 cm−1. The C−O bond stretching is located at 1065.5 cm−1 while the C–H bond stretching is presented at 873.5 and 600.7 cm−1. TEM micrograph of ZnO NPs confirmed the formation of homogeneous spherical-shaped NPs with small sizes (Fig. 2F). ZnO-NPs’ elemental zinc and oxygen signals were detected by EDS analysis (Fig. 2G). The production of ZnO NPs is indicated by distinct peaks obtained for zinc and oxygen atoms. The fabrication of ZnO NPs was confirmed by the elemental profile’s prominent upper peak at 1 keV, which is characteristic of Zn. It was found that zinc and oxygen, in terms of weight%, were 85.14 and 34.57, respectively [13].

Fig. 2
figure 2

Characterization of the optimized ZnO NPs. a UV–visible spectroscopy. b XRD. c Zeta potential. d Zeta distribution average size. e FTIR. f TEM with bar scale = 100 nm. g EDX

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3.2 Antibacterial activity

The antibacterial activity of ZnO NPs’ was established and compared with that of Zn(NO3)2.6H2O and ciprofloxacin. All of the bacterial strains that were evaluated responded negatively to treatment with ZnO NPs, Zn(NO3)2.6H2O, and ciprofloxacin, as indicated in Table 1. According to the results, ZnO NPs exhibited a stronger bactericidal effect against Gram-negative bacteria than against Gram-positive bacteria. E. coli ATCC25922 and P. aeruginosa ATCC27853 were the targets of the potent antibacterial action, with inhibition zones measuring 25 ± 0.03 mm and 14 ± 0.14 mm, respectively, at a concentration of 50 µg/ml. These inhibitory zones increased when the ZnO NPs concentration was raised, reaching 32 ± 0.03 mm and 25 ± 0.06 mm, respectively, at a concentration of 150 µg/ml. Although S. aureus ATCC25923 showed resistance to standard antibiotics, ciprofloxacin, and ZnO NPs had a bactericidal action with an inhibition zone equal to 19 ± 0 mm, at a concentration of 150 µg/ml.

Table 1 Antibacterial activity of ZnO NPs, Zn(NO3)2.6H2O, and ciprofloxacin at different concentrations, 50, 100 and 150 µg/ml, using agar well diffusion method
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The results were recorded as the average diameter of the inhibition zone (mm) ± standard errors.

MIC values were determined for ZnO NPs, Zn(NO3)2.6H2O, and ciprofloxacin against the different tested bacteria as shown in Fig. 3. ZnO NPs completely inhibited E. coli ATCC25922 at 25 µg/ml, S. aureus ATCC25923 and P. aeruginosa ATCC27853 at 30 µg/ml, S. liquefaciens and S. saprophyticus at 35 µg/ml, L. fusiformis and K. pneumoniae ATCC33495 at 40 µg/ml. On the other hand, the bulk zinc salt, Zn(NO3)2.6H2O, revealed complete inhibition against E. coli ATCC25922 at 35 µg/ml, S. aureus ATCC25923 at 40 µg/ml, and P. aeruginosa ATCC27853 at 45 µg/ml, S. liquefaciens, S. saprophyticus, L. fusiformis and K. pneumoniae ATCC33495 at 50 µg/ml. While ciprofloxacin inhibited L. fusiformis and P. aeruginosa ATCC27853 at 25 µg/ml, S. liquefaciens at 35 µg/ml, E. coli ATCC25922 and K. pneumoniae ATCC33495 at 40 µg/ml. S. aureus ATCC25923 and S. saprophyticus were inhibited at 50 µg/ml of ZnO NPs showing resistance to the standard antibiotic.

Fig. 3
figure 3

Minimum inhibition concentration of ZnO NPs; (A), Zn(NO3)2.6H2O; (B), and ciprofloxacin; (C)

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The MBC is the minimum concentration of an antibacterial agent needed to eradicate a specific bacterium over a long period, for example, 18 or 24 h. Antimicrobial agents are regarded as bactericidal if the MBC is no greater than 4 times the MIC. The MBC results are shown in Fig. 4. In comparison to ciprofloxacin, ZnO NPs showed greater bactericidal activity against the Gram-negative bacteria E. coli ATCC25922, S. liquefaciens and P. aeruginosa ATCC27853 as well as the Gram-positive bacteria L. fusiformis OQ071701.1. ZnO NPs demonstrated moderate to strong antibacterial activity when compared to other antibacterial agents, according to the findings of antibacterial activity tests against the tested bacterial strains.

Fig. 4
figure 4

The minimal bactericidal concentration of ZnO NPs, Zn(NO3)2.6H2O, and ciprofloxacin against the tested bacteria

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3.3 Ultrastructural changes in ZnO NPs-treated bacteria

S. liquefaciens, control images of untreated cells are shown in Fig. 5, in which the cells exhibited a uniformly dense and homogeneous microstructure as well as a serrate-shaped cell wall, indicating that the cells were intact. In contrast, the cells affected by Zn ONPs exhibited marked changes in the bacterial cell contents, including a separation between the cell wall and cell membrane with lysed cell walls accompanied by vacuole formation.

Also, L. fusiformis cells changed in response to their treatment with ZnONPs. Native L. fusiformis was rod-shaped, with intact cell walls. After ZnO NPs treatment, the cell walls of L. fusiformis became wrinkled and damaged, resulting in the rupture of the bacterial cell membrane. It shows that Zn ONPs revealed good impacts on the cell membranes and cell walls of treated bacteria including the appearance of extensively damaged cells with vacuole formation.

Fig. 5
figure 5

The bactericidal effect of ZnO NPs on the ultrastructure of S. liquefaciens and L. fusiformis. Negative untreated controls (without ZnONPs) showed normal cell structure including cell wall (CW), cytoplasm (CY) and nucleoid (NU). Treated bacterial samples (at MIC values) showed a separation between the cell wall and cytoplasmic membrane (white arrows) with lysed cell walls (Ly), and complete cell lysis (Cl). Note the visible vacuoles (V) and lipids (L) formation

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3.4 Antibacterial activity of Kombucha pellicle disc impregnated with ZnO NPs

The current study investigated the effect of the synergistic antibacterial action of Kombucha SCOBY in combination with ZnO NPs as a suggested solution to prevent the growth of contaminating microbes and to improve Kombucha’s antibacterial potential. Kombucha SCOBY pellicle discs were impregnated with ZnO NPs for different impregnating times (3, 6, 12 and 24 h.) and tested against S. liquefaciens, L. fusiformis and K. pneumoniae ATCC33495 using disc diffusion method. The Kombucha pellicle discs impregnated with ZnO NPs solution showed moderate to strong inhibition zones as shown in Fig. 6; Table 2. These inhibition zones increased by increasing the impregnated time for Kombucha pellicle discs in the ZnO NPs solution. Kombucha pellicle discs impregnated with ZnO NPs for 24 h. showed higher inhibition zones against the tested bacteria ranging between 41 and 45 mm than shorter times. This increase in the antibacterial activity might be explained by the fact that ZnO NPs need sufficient time to penetrate and embed the kombucha SCOBY in order to exhibit this synergistic antibacterial activity. In addition, the biosynthesis of ZnO NPs occurred outside the kombucha SCOBY cells using kombucha extract, which makes them act as foreign agents and might take a long time to recognize and uptake them. Overall, the incorporation of ZnO NPs into Kombucha SCOBY was more effective for increasing their antibacterial activity than single SCOBY.

Fig. 6
figure 6

Antibacterial action of Kombucha pellicle discs impregnated with ZnO NPs solution at different times, 3, 6, 12 and 24 h. against L. fusiformis; (A) S. liquefaciens; (B), and K. pneumoniae ATCC33495; (C)

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Table 2 Antibacterial activity of Kombucha SCOBY pellicle discs impregnated with ZnO NPs for different impregnating times, 3, 6, 12 and 24 h
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The results were recorded as the average diameter of the inhibition zone (mm) ± standard errors.

4 Discussion

Kombucha is a well-known example of probiotics with notable antioxidant effects. A thin coating of various yeasts and bacteria is added to sweetened tea to create the fermented beverage kombucha [49]. It has a range of acids, vitamins (B and C), carotenoids, polyphenols, catechins, theophylline, and other substances with high antioxidant activity as documented by Chen and Liu [19]. Beigmohammadi et al. [29] reported another essential ingredient in this beverage is glucuronic acid, specifically UDP-glucuronic acid, which is used to mix with toxins and eliminate them from the body. Also, recent research has shown that Kombucha might lessen the cellular damage brought on by oxidative stress [51, 52]. In addition, products made from kombucha were suggested as dietary supplements or functional beverages that may have advantageous impacts on allergies’ immune systems [53, 54]. ZnO NPs have unique and better advantages than other NPs moreover their antimicrobial and antioxidant properties. It is one of the essential mineral elements for human and exhibit strong activity even when administrated in small amount [55]. Herein, the current study used Kombucha as a bio-nano-factory agent for the synthesis of ZnO NPs to combine all their beneficial effects in one product and investigate its antibacterial activity.

The ability of microorganisms to biosynthesize various NPs through a variety of methods has been shown in previous studies [51, 55, 56]. Both external and intracellular processes can be used for this biosynthesis [57]. The advantages of the extracellular approaches outweigh the disadvantages of the intracellular ones, which necessitate additional processes including cell disintegration, NP extraction, and purification [58]. At 35 °C and in the dark, kombucha extract was able to biosynthesize ZnO NPs extracellularly in 24 h. Several methodologies were used to confirm and characterize the production of ZnO NPs. The kombucha extract-induced biotransformation of zinc ions into ZnO NPs, which was attributed to the excitation of ZnO NPs surface plasmon resonance, caused the colour to change to a pale yellow (SPR) [55]. The UV-visible spectrometer’s absorption peak, which is unique to ZnO NPs, was seen at 295 nm. Its absorption peak matches the ZnO NPs absorption band reported by Chekroun et al. [38] and Chatterjee et al. [39]. On the other hand, the biosynthesis of ZnO NPs was carried out by Al-Kordy et al. [40] using Alkalibacillus sp.W7, which had a wide absorption with peak maxima at about 310 nm. Also, ZnO NPs were produced by using Petroselinum crispum extract and revealed an absorption peak at approximately 338 nm as documented by Fadhel et al. [41].

Different parameters were investigated and studied to optimize the production of the ZnO NPs process. It was found that the mixing of 25 mM of Zn(NO3)2.6H2O solution with 40% cell-free Kombucha biomass extract at pH9 and 45 °C gives the best production rate for ZnO NPs formation. Agglomeration and aggregation are common problems which confront the use of NPs in different medical and industrial applications. The presence of proteins in the production of NPs that could serve as stabilizing agents was established by measuring the FTIR spectra of ZnO NPs [63]. According to Zeta size results, high amounts and thick layers of protein capping agents were found attached to the ZnO NPs surface. The Zeta potential test revealed that ZnO NPs had a surface positive charge of 19 ± 3 mV which also increased the repulsion rate between ZnO NPs which increased their stability. The current findings supported the high stability of the biosynthesized ZnO NPs due to the significant repulsion forces between the particles caused by their high surface positive charge values, which prevent their aggregation and agglomeration. The FTIR data also revealed the presence of various proteins that will serve as capping agents [64]. The crystalline nature of the ZnO NPs was confirmed by XRD. The Debye-Scherrer equation was used to determine the size of ZnO NPs. The results from the TEM were consistent with the average size, which was reported to be 23 ± 1.5 nm. Particle size distribution of the nanostructured system is shown by PDI values, which range from 0.0 (perfect homogeneity) to 1.0 (high heterogeneity). Further, PDI values of ZnO NPs ranged from 0.3 to 0.401 (values of < 0.5) also indicating a stable system and a narrow particle size distribution [55]. The biosynthesized spherical ZnO was well dispersed in the TEM image. Rehman et al. [44] demonstrated B. haynesii to produce spherical-shaped ZnO NPs with a 50 ± 5 nm range. While B. subtilis biosynthesized ZnO NPs with spherical and without aggregate with the size of 16–20 nm as reported by Sabir et al. [45].

According to our results of antimicrobial activity, ZnO NPs were efficient against both Gram-positive and Gram-negative bacteria. Because Gram-negative and Gram-positive bacteria have different cell wall compositions, it was discovered by Fang et al. [46] that ZnO NPs had greater antibacterial ability against the former than the latter. Thick walls and high amounts of peptidoglycan found in Gram-positive bacteria’s cell walls decreased the negative effects of ZnO NPs compared to Gram-negative bacteria [68]. Also, Kombucha pellicle discs impregnated with ZnO NPs solution had higher bactericidal action than the sole Kombucha pellicle. Based on these findings, novel kombucha products combined with ZnO NPs might be recommended as functional beverages or nutraceuticals with potential antibacterial benefits in different food and pharmaceutical industries.

Several studies have demonstrated the bactericidal effects of ZnO NPs, including the production of reactive oxygen species, alterations in cell wall permeability, and cell wall destruction [56, 60, 69]. It has been also reported by Saha et al. [49] that ZnO NPs bind to proteins and DNA, generating reactive oxygen species (ROS). ZnO NPs may interact with the cell wall of microorganisms, causing cell bursts, among other possible reasons for their alleged antimicrobial activity [71]. After being exposed to ZnO NPs that were effortlessly captured and absorbed through cell membranes, S. liquefaciens and L. fusiformis both changed. The modifications included a low DNA content as well as the inhibition of L. fusiformis growth. The bacterial cells displayed a normal cell wall, compact cytoplasm, cell membrane and normal nucleoid in their untreated state. On the other hand, numerous modifications were seen following the administration of ZnO NPs, including the emergence of large vacuoles, and the production of lipids. In addition to the accumulation inside the bacterial cells, ZnO NPs in the cytoplasmic membrane and cytoplasm may be the primary cause of the significant morphological abnormalities [72]. According to Ma et al. [52], this buildup may be related to the interaction of ZnO NPs with DNA. Xie et al. [53] concluded that ZnO NPs cause disturbance of the processes of bacterial DNA amplification, and alteration (more often, down-regulation) of expression in a wide range of genes. Additionally, according to Gold et al. [54], the smallest ZnO NPs can pass through cell membranes and interact with proteins inside of them, including thiol groups in enzymes, causing blockage, deactivation, and cell death. Thus, the direct bactericidal action of ZnO NPs was confirmed against both Gram-negative and Gram-positive bacteria [76].

The current study also investigated the synergistic antibacterial potential of ZnO NPs in combination with Kombucha SCOBY. Kombucha contains organic acids, active enzymes, amino acids, and polyphenols produced by these microbes. From these components, antibacterial acids were produced such as acetic acid, oxalic acid, gluconic acid, and malic acid [77]. It was reported that Zn ions are released from ZnO NPs [78] that could react with acetic acid and produce zinc acetate [79]. Sreeramulu et al. [80] demonstrated that there was no antimicrobial activity for Kombucha acetic acid only and gluconic acid only. When the two acids were mixed, there was a synergistic effect against Gram-negative bacteria. Similarly, the Kombucha SCOBY pellicle discs combined with ZnO NPs might be the main reason for the increase in their antibacterial activity. It could help to overcome the harmful microbial contamination problems (act as a preservative material), moreover, enhancing Kombucha’s antibacterial activity, especially against resistant microbial strains.

5 Conclusion

The biosynthesis of the ZnO NPs was carried out in a single batch phase using the Kombucha extract as a reducing agent and stabilizer. XRD, UV-visible spectrophotometry, Zeta potential, TEM, and other techniques were tested to verify ZnO NPs’ biogenesis. The Zeta average size, Zeta potential, PDI values, and FTIR of ZnO NPs revealed long-term particle stability. The biosynthesized NPs showed substantial antibacterial effectiveness against both Gram-positive and Gram-negative bacteria with MIC values ranging from 30 to 40 µg/ml. Regarding MIC, MBC, and the ultrastructure of the treated bacterial cells, Gram-negative bacteria are more sensitive to ZnO NPs than Gram-positive bacteria. According to the study, biosynthesized ZnO NPs have a high level of efficiency and stability, making them potentially useful as bacterial growth inhibitors alone or in combination with Kombucha.