The scientific community is currently facing a more than exponential increase of knowledge in all areas and disciplines. In the last 10 years, the contribution of the journal Applied Food Biotechnology was eminent in distributing that knowledge by providing a tribune for researchers from different countries all over the world to share their ideas, observations, and hypotheses. With a focus on different aspects of applied and fundamental biological sciences, the journal Applied Food Biotechnology was established as a reference of the Iranian scientific community. In the last 10 years, the journal has been covering several topics related to exploring the beneficial properties of lactic acid bacteria (LAB) and their role in modern food technologies. LAB are already proven as a realization of Hippocrates' vision for the potential role of food in human and other animals' health. However, what will be the next frontier? What are the challenges in understanding the interactions between microbiota and host microorganisms? What will be the novel analytical tools, facilitating a new era of probiotic research? These were only a few research topics presented and discussed in Applied Food Biotechnology in the last decade. This editorial overview aims to celebrate the scientific contribution of Applied Food Biotechnology in the area of research associated with the beneficial properties of LAB, summarizing some of the studies published in the journal.

In this editorial article, nationality of the authors from establishment of Applied Food Biotechnology from 2014 to the present time has been overviewed. The editorial board, especially chief editor, wish to make a broad global audience to spread knowledge of food biotechnology via publication of outstanding articles in this journal. At the beginning of the activity of journal office, a limited number of non-Iranian authors submitted their manuscripts to this journal; however immediately after the publication of the first issue, number of the foreign authors increased further, while they showed their satisfaction with the acceleration in peer-review processes of their manuscripts. From the published articles, probiotics has been the major scope; therefore, screening of the beneficial probiotics from various natural sources and their uses in prevention of diseases have been introduced by various authors. Thus, the most interesting findings of the authors have been introduced; through which, readers are further adapted to the journal priorities and preferences in probiotics and postbiotics. It is believed that invitation of prestigious authors and carrying out rapid peer-review processes are a key success to achieve high article citations and authors’ satisfactions.

The researchers are fronting the increasing of knowledge in extraction, application, and also antioxidant and antimicrobial properties of plant extracts and essential oils. In recent decade, the journal "Applied Food Biotechnology" has been established a channel for scientists all around the world to share their own hypotheses, results, and conclusions. As a peer-reviewed multi-disciplinary biotechnological publication, it covers several scopes which one important one is food microbiology. In this context, the journal has published several reports on food application of plant extracts and essential oils. The aim of this text is to determine the main categories of published articles in this context in the Journal of "Applied Food Biotechnology" and so on by editors. It seems that research tend to show the effective function of essential oils, as well as comparison of free and encapsulated forms as antimicrobial and antioxidant agents in food. With the aim of holding the potential to alleviate certain complexities, enhance yield, and simplify the isolation process of bioactive metabolites or their individual components, research has played a significant role in reducing production cost of essential oil and herbal extract.

 

Abstract

 

Background and Objective: Use of natural ingredients is a safe efficient approach to overcome various diseases. Encapsulated ginger extract has shown improved physicochemical characterizations, compared with that, the ginger extract has. In this study, a natural system integrated with ginger bioactive compounds (6-gingerol) and green microalgae of Chlorella vulgaris was reported to increase bioactive compounds medicinal effectiveness and introduce a novel food supplement.

Material and Methods: First, nanoparticles of microalgae were produced using ball-milling technique. Ethanolic ginger extract, loaded on microalga nanoparticles, was investigated at various pH values (2-7.4) to effectively release the active agents. Various analytical techniques (e.g., Fourier transform infrared, thermogravimetric analyses) were used to characterize the nanocomposite and investigate its anticancer and antimicrobial effects.

Results and Conclusion: Dynamic light scattering showed a medium size of 20.9 nm for the microalga nanoparticles. The release assay of ginger polyphenols showed a releasing process controlled by the pH. Fourier transform infrared, thermogravimetric analysis and differential thermal analysis revealed adsorption of ginger extract on nano Chlorella vulgaris surface. Moreover, 2,2-diphenyl-picrylhydrazyl bioassay results on the nanocomposite (GE@nano C.v) verified its significant antioxidant, antibacterial and anticancer activities. The nanocomposite has the minimum inhibitory effect on human breast adenocarcinoma cells and bacterial growth at 1 and 6.25 mg ml^-1 concentrations, respectively. In brief, adsorption of ginger extract on the microalga nanoparticle surfaces enhanced physical and chemical characteristics of the ginger extract, compared to its free form. Bioactive compounds in Chlorella vulgaris and ginger extract strengthen their reported activities. Furthermore, microalgal nanoparticles could act as a safe carrier for the controlled release of 6-gingerol in addition to their nutraceutical characteristics. 

Conflict of interest: The authors declare no conflict of interest.

 

Article Information

 

Article history:

- Received

19 Nov 2023

- Revised

17 Jan 2024

- Accepted

17 Feb 2024

 

Keywords:

▪ Antitumor

▪ Food supplement

▪ Ginger

▪ Microalgae

▪ Natural nanomedicine

 

*Corresponding author:

 

Mahdi Rahaie *

Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran 14399-57131, Iran.

 

Tel: +98-21-86093408

 

E-mail:

mrahaie@ut.ac.ir  

 

How to cite this article

Rajabi M, Rahaie M, Sabahi H. Design and Synthesis of a New Anticancer and Antimicrobial Nanocomposite by Microalgae Based on an Up-down Approach. Appl Food Biotechnol. 2024; 11 (1): e15. http://dx.doi.org/10.22037/afb.v11i1.43923

 

1. Introduction

 

By decreasing consumption of essential nutrition, various chronic diseases emerge. This dilemma needs a reliable effective solution to prevent future malnutrition illnesses. Nowadays, plant and algal-derived supplies as functional foods are attracting public attentions due to healthful bioactive components and their therapeutic effects [1,2]. One of the wide subgroups of algae includes microalgae. Algal biomass includes diverse health-beneficial bioactive compounds such as fibers, carotenoids, polysaccharides, polyphenols and peptides that are resulted from various metabolic pathways and effective on cardiovascular diseases (CVDs), types of cancers, atherosclerosis, neurodegenerative diseases, obesity, gut health, bone health, inflammation, type II diabetes and antioxidant and antiviral activities [3,4]. In developing seaweeds and microalgal foods, aspects such as consumer awareness and demands, bioactive compound bioavailability and stability, cost-effectiveness and life durability needs further attentions. Moreover, only a few species of microalgae are approved for the human consumption as foods due to strict food safety regulations. These include Arthrospira platensis (spirulina), Chlorella spp., Aphanizomenon flosaquae, Schizochytrium spp., Scenedesmus spp., Dunaliella salina, Tetraselmis chuii, Haematococcus pluvialis and Porphyridium purpureum as resources of human nutrition within the microalgal species [5,6]. It has been reported that bioactive agents are critical for better health conditions. Therefore, plant phytochemicals have been selected as they include generally favorable characteristics such as less toxicity and well bearing by normal body cells. Frequently assessed bioactive compounds in plant extracts include curcumin, gingerol, β-carotene, quercetin and linamarin, which are developed as anticancer drugs. These compounds are rich in active agents such as phenols, alkaloids, flavonoids and tannins, which include high anti-inflammatory, antioxidant, antimicrobial, antica-ncer and antiaging activities [7,8].

One of the active agents of the ginger (Zingiber officinale) rhizome is found in the polyphenol group of 6-gingerol, which is popular for the protection of several cancers. The 6-gingerol mechanism includes interfering with a number of cell signaling pathways that affect balances between the cell proliferation and apoptosis [9]. These lead to effective approaches majorly for liver carcinoma and breast cancer. In addition, antiviral, antimicrobial, antihyperglycemic, antilipidemic, cardioprotective and immunomodulatory activities have been shown. However, 6-gingerol disadvantages such as pH and oxygen sensitivities, temperature lability, light instability and poor aqueous solubility limit its potential administrations. Thus, novel approaches for efficient delivery of 6-gingerol in a targeted controlled manner is critically important [10]. Drug delivery paths have been improved with developing of nanotechnology science. Appropriate drug-delivery nanosystems are used to enhance drug stability, specificity and durability in human blood circulatory system. Various drug vehicles have been introduced to improve drug efficiency and therapeutic effects. Some nanotechnology systems used for targeted drug delivery include dendrimers, micelles, carbon nanotubes, nanoparticles and liposomes, which are extensively used in various industries such as cosmetic, food, medicine, health, energy, electronics and environment industries [11,12]. Additionally, nanote-chnology has provided a path; in which, well-qualified and practical forms of foods with nutrient bioavailability can be produced. Moreover, most of the recent studies is dedicated to crop and food processing developments through nanotechnology [13].

One of the nanotechnology approaches in food technology includes a top-down approach that uses nanostructures from bulk materials by size decreasing via milling, nanolith-ography or accurate-engineering techniques. In contrast to nanostructures with a large surface area-to-volume ratio, this nano-approach induces a higher activity and enhances performance. Factors such as non-specificity, low stability and aggregation can delay these nanotechnologies' uses because of decreased functions. To extend stability of nanosize structures, associating a host material (as a matrix or support) can be an alternative way to overcome the former issues [13-15]. Ginger is an old additive and traditional medicine; previous studies on using ginger extract for therapeutic approaches have been less effective, compared with the loaded ones in a carrier due to less stability and low solubility. Therapeutic characteristics of this natural medicine are limited as previously stated. Moreover, Chlorella (C.) vulgaris is approved by the Food and Drug Administration (FDA) with nutritional and medicinal characteristics and potential of uses as a natural carrier. To enhance physiochemical characteristics of ginger extract, containing health-promoting bioactive compounds, as well as natural ingredient demands in food nutraceutical and medical industries, a nanocomposite was designed that was a novel approach to overcome disadvantages of using ginger extract alone. In this study, a nano-microalga was synthetized using up-down path as a carrier for ginger extract. Moreover, composite was assessed using several bioassays.

1. Materials and Methods

2.1. Materials

Ginger rhizome was purchased from a local market in Tehran, Iran. Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, Gallic acid and sodium carbonate were purchased from Sigma-Aldrich, USA. Methanol (purity>99%) and ethanol (purity≥99.7%) were provided by Merck, Germany. Fresh C. vulgaris microalgae was provided by the Faculty of New Sciences and Technologies, University of Tehran, and cultured before use. All the chemicals included analytical grades and deionized water was used to prepare solutions.

2.2. Microalgae culture

Briefly, BG-11 liquid media were used to propagate C. vulgaris. Microorganism was inoculated at 10% using 500-ml flasks of 200 ml of BG-11. Culture flasks were incubated using rotary incubator at 25 ℃ ±2 and 100 rpm. Cells were harvested by centrifugation (Awel, model MF 20-R, France) at 8000 rpm for 10 min at 4 ℃. These were freeze-dried (Operon, model FDB 5503, South Korea) [16].

2.3. Ginger extraction

Ginger rhizomes were dried at room temperature (RT) and grounded using blender and then the fine powder was stored at -20 ℃ for further use. Extraction method (maceration technique) reported by Ali, et al. was used with modifications [17]. Generally, 5 g of the ginger powder were transferred into a 50-ml tube and extracted with 25 ml of 70% ethanol at 26 ℃ for 72 h using shaker-incubator. The hydroalcoholic extract was filtered through Whatman filter papers (Whatman, UK) to separate from the solid phase. Concentrated extract, achieved using rotary evaporator and freeze-drier, was stored at 4 °C until use.

 

2.4. Characterization of the ginger extract

2.4.1. Total phenol content of the ginger extract

To investigate total phenolic content of the extract, Folin–Ciocalteu method was used [18]. Gallic acid calibration curve was plotted using mixture of ethanolic solution of Gallic acid (1 ml) and Folin–Ciocalteu reagent (200 µl, 10× diluted) and sodium carbonate (160 µl, 0.7 M). Absorbance ratio of the solution was read using UV-Vis spectrophotometer (Thermo, WPA, Germany) at 760 nm. To assess the total phenolic content, ginger extract was mixed with the highlighted reagent and the absorbance ratio was measured with three replicates. Equation 1 was used for the calculation of total phenolic compounds as follows.

T = C ^ V/M                                                                                          Eq. 1

Where, T was the total phenolic content [mg g^-1 sample extract in Gallic acid equivalents; C was the concentration of Gallic acid established from the calibration curve (mg ml^-1 )]; V was volume of the extract (ml); and M was mass of the sample extract (g) [19]. Results were expressed as µg of ginger extract ml^-1 of supernatant based on the calibration equation. Calibration equation of the ginger polyphenols (6-gingerol) was achieved via UV [absorbance value = 0.0507 (ginger polyphenols concentration in mg ml^-1) - 0.0636 (R² = 0.991)].

2.4.2. High-performance liquid chromatography analysis

Waters liquid chromatography apparatus, including a separation module (Waters 2695, USA) and a photodiode array detector (PDA) (Waters 996, USA) was used for high-performance liquid chromatography (HPLC) analysis. Data acquisition and integration were carried out using Millennium 32 software. Injection was carried out using auto-sampler injector. Chromatographic assay was carried out on a 15 cm × 4.6 mm with pre-column, Eurospher 100-5 C18 analytical column provided by Waters (sunfire) reversed-phase matrix (5 μm) (Waters, USA) and eluting was carried out in a gradient system with ACN as the organic phase (Solvent A) and distilled water (DW) (Solvent B) with a flow-rate of 1 ml min^-1. Injection volume was 20 µl and temperature was set at 25 °C (run time, 40 min and columns size, 2.1 mm) [20].

2.5. Size decreases in nanoscale, dynamic light scattering and zeta potential

Nano-microalgae powder with nanometer dimensions was produced using ball-milling technique (600 rpm, 6 h). Ball-milling process was carried out at 27 ℃, to avoid excessive heating. Temperature was controlled using air-cooling system. After the process, samples were transferred into a closed container to prevent moisture. The most common technology for assessing particle sizes based on particle-light interactions is dynamic light scattering (DLS) technique. Assessment of the nano C.v size was carried out using particle size analyzer (Horiba, SZ100 model, Japan). A critically physical parameter to identify surface charges of a particle (microalgae, GE, nano C.v and GE@nano C.v) in suspensions, which could anticipate interactions, is zeta potential analysis (ζ). Technically, ions around the particles dispersed in the fluid regulate charges of the particle surface layer. In this study, zeta potential was assessed using Smoluchowski formula (Eq. 2) as follows.

Ζ = µ                                                                                                     Eq. 2

Where, η was viscosity of the media, ε was the permittivity and μ was the electrophoretic mobility [21]. In this study, zeta potential of the samples (ginger extract, micro C. vulgaris cell, nano C. vulgaris, GE@micro C.v and GE@nano C.v) homogenized in deionized water via ultrasound technique was assessed (Malvern, model ZEN 3600, UK).

2.6. Adsorption experiment

Ethanolic ginger extract was adsorbed onto C. vulgaris surface as a function of stirring time and ginger extract dosage. A suspension of 5 mg ml^-1 nano C.v and 0.5 mg ml^-1 ginger extract was stirred for 1, 2, 4, 6, 8, 10 and 12 h and then centrifuged (5000 rpm, 15 min). The harvested GE@ nano C.v (ginger extract on nanoparticles of C. vulgaris) was freeze-dried. As the adsorption yield curve (%) reached a plateau pattern after 1 h, this time was selected as the stirred time. Various quantities of GE (0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg ml^-1) were suspended in flasks containing 5 mg ml^-1 nano C.v (dissolved in 70% ethanol), stirred for 1 h and then centrifuged (5000 rpm, 15 min). The harvested GE@nano C.v was freeze-dried. Polyphenol assessment (especially 6-Gingerol) of ginger extract adsorbed onto nano C.v was carried out as follows: 1 ml of the solution was collected and centrifuged (5000 rpm, 15 min). Supernatant was separated from the sediment and re-centrifuged (12,000 rpm, 10 min). Concentration of the GE was calculated at 760 nm, using UV-visible spectroscopy (Thermo, WPA, Germany) [16]. Encapsulation efficiency (EE%) and encapsulation yield (EY%) were calculated using Eqs. 3 and 4:

Encapsulation efficiency (%) =  × 100                                                Eq. 3

Encapsulation yield (%) =  × 100                                                Eq. 4

2.7. Verification of adsorption

To verify adsorption of ginger extract on nano Alg surface, Fourier transform-infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTA) were carried out.

2.7.1. Thermogravimetric analysis

The TGA was carried out at a heating rate of 10 ℃ min^-1 from RT to 600 ℃ with the inert nitrogen gas of 50 ml min^-1 using thermal gravimetric analyzer (TGA/DSC/1, Mettler Toledo, Singapore).

2.7.2. Differential thermogravimetric analysis

The DTA analysis of the GE, nano C.v and nano Alg/GE was carried out to assess physical state of the extract in this carrier and possibility of the interactions between the GE and the microalga.

2.7.3. Fourier transform-infrared spectroscopy

Chemical characteristics of the material were investigated using FTIR spectroscopy (Perkin-Elmer Frontier, USA) using KBr disks in the range of 400–4000 min^-1.

2.7.4. Antioxidant assay (total antioxidant capacity)

Photochemical stabilities of the nanocomposite (GE@ nano C.v) and ginger extract were assessed via a procedure described by Paramera et al. [22] with minor modifications. Free GE (200 mg) and a quantity of each nanocomposite (containing 200 mg of ginger polyphenols) were exposed to sunlight for a month using enclosed glass Petri dishes. Time of the light exposure included 12 h day^-1 with a total of 720 h. The average daily temperature within the month was 25 ℃. After exposure for 7, 14 and 30 d, samples were collected and bioactivities of their 6-gingerol were assessed as follows: antioxidant activity of dispersed GE and nanocomposite (GE@nano C.v) were assessed using DPPH free radical scavenging method [23]. Inhibition of DPPH radicals by the samples was calculated using Eq. 5 as follows.

DPPH inhibition (%) =  × 100              Eq. 5

Where, A _control was the absorbance spectrum without GE and A _sample was the absorbance of ginger polyphenol nanocomposite.

2.8. In vitro assays

2.8.1. Antibacterial assay

Staphylococcus aureus ATCC 33591, Pseudomonas aeruginosa ATCC 9027, Salmonella enterica ATCC 9270 and Escherichia coli ATCC 10536 were selected for the antibacterial susceptibility assay. Minimal inhibitory concentrations (MICs) of the ginger extract and nanocomposite were assessed using microtube dilution assay as described by Acharya and European Committee for the highlighted bacterial strains. Ethanolic extract concentrations of 2.5, 1.25 and 0.625 mg ml^-1 and nanocomposite concentrations of 1-4 mg ml^-1 (consisting of ginger extract) were prepared using serial dilution in LB broth and 0.5 McFarland standard (10⁸ CFU ml⁻¹) of the bacterial suspensions was added to each tube. All tubes were incubated at 30 °C for 14 h at 100 rpm. Absorption of each sample was measured at 600 nm using UV-vis spectroscopy (Thermo, WPA, Germany). The lowest extract concentration that inhibited bacterial growth was recorded as MIC (in three independent experiments) [24]. Inhibition of the bacterial growth was calculated using Eq. 6 as follows.

Antibacterial activity (%) =  × 100                                                                                                                         Eq. 6

Where, A_{positivecontrol} was the absorbance spectrum without samples (ginger extract and nanocomposite) and A_sample was the absorbance of bacteria with certain concentrations of ginger extract and nanocomposite. Culture media was used as blank.

2.8.2. Cytotoxicity assay

In this study, human breast adenocarcinoma MCF-7 cell line was cultured using T-25 cell culture flasks containing Hi-Gluta XL Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg ml^-1) and penicillin (100 U ml^-1). Cells were propagated using cell culture incubator supplied with 5% CO₂ at 37 ℃. cells were seeded overnight using 96-well cell culture plates and RPMI media suspension of three treatments (ginger extract, nano C.v and GE@nano C.v) was added to the wells at a confluence of 70% to avoid possible interferes of DMEM media with the samples (200 µl total volume). The control wells included no agents. Experiments were carried out with three replicates. Effects of each treatment on cancer cell growth were assessed after 24, 48 and 72 h, separately. After treatment of cells with various concentrations of the samples (0.5, 0.75 and 1 µg ml^-1) in each well for 24, 48 and 72 h, cells were rinsed with 1× PBS buffer and incubated with 100 µl of 0.5 mg ml^-1 MTT at 37 ℃. After 4 h of incubation, 100 µl of DMSO were added to dissolve the dark-blue crystals of formazan (MTT metabolites) and incubated for 30 min at 37 ℃. Then, absorbance of the reduced MTT was measured at 560 nm using plate reader device (Convergys ELR 96×, Germany) [25].

2.9. Statistical analysis

Data represent mean ±SD (standard deviations) of three replicates. The mean comparisons were carried out using one-way variance analysis (ANOVA). Antioxidant activity data of nanocomposite and ginger extract were analyzed in a completely randomized block design with three replicates and three treatments (exposure to sunlight for 0, 14, 30 and 60 d). Duncan multiple range test (p<0.05) was used to assess significance of the difference within the treatment means. Data for each analysis were represented as the mean ±SE (standard error) of the mean.

1. Results and Discussion

3.1          High-performance liquid chromatography analysis

The HPLC analysis was carried out to quantify 6-gingerol as a bioactive compound (polyphenol) with therapeutic characteristics as well as antioxidant activities. Chromatograms in Fig. 1b show spectra associated to ginger extract as well as the standard spectra of 6-gingerol (Fig. 1a). Peaks linked to 6-gingerol at 14.38 min were clearly present in the two spectra, showing 6-gingerol in the ginger extract. Using data, standard curve of 6-gingerol was first plotted and then quantity of 6-gingerol in the ginger extract was calculated as 96 mg g^-1 DW (dry weight) extract (Fig. 1 c). This value was higher than that by Yamprasert et al. (2020), where concentration of 6-gingerol in the extract was calculated as 71.13 mg g^-1 using soaking method [26]. Compared to the method by Simonati et al. (2009) that calculated 170 mg g^-1, the former value was lesser, which could be due to the use of supercritical fluid^[1] extraction technique and high pressure for extraction in that study [27].

3.2. Release assay

Release assessment of 6-gingerol in the ginger extract was carried out based on the absorbance at 760 nm in the simulated environment of body conditions with pH 7.4 using Folin-Ciocalteu reagent at various times. Since extract was loaded on nanoparticles of the microalgae, further releases were expected at the beginning. At t = 1 h in the acidic conditions similar to the stomach with pH 2, the release rate was 16% and at pH 7.4, the rate was 14%. Up to 72 h, the release rate was constant with mild changes and reached 18% (Fig. 2). Complete release of the ginger extract was not observed at no pH conditions. However, release of the extract in acidic conditions of the stomach was higher, which was due to the effects of acidic pH on interactions between the algal nanoparticles and the extract as well as the surface load. According to Zarei et al., release rates of gingerol loaded in pegylated and non-pegylated nanoliposome respectively were 3.1 and 4.8% within 48 h [28]. Release rate in report of Jafari et al. was nearly 39% within 2 h and 59% within 4 h [16]. In another study by Shateri et al., release comparison of two synthetic systems (BCE-Spirulina and BCE-nanosized Spirulina) was investigated in physiological (pH 7.4) and acidic (pH 1.2) conditions within 96 h. Release rates of the extract were respectively calculated as 50 and 40% within the first 12 h. Based on the results, release of extract from the nanocomposite was faster and higher at the two pH conditions because of loading further extracts on the Spirulina surface, compared with the microalgal whole cell. The plateau curve was seen after 48 h, which reached 50% in acidic pH [29].

3.3. Dynamic light scattering and zeta potential assessments

To assess sizes of the ground C. vulgaris nanoparticles, an appropriate quantity of the microalgal nanoparticles was first dissolved in DW and sizes of the particles were assessed after sonication for 15 min. Sizes of Chlorella nanoparticles included 20.9 nm. Sharp peak and high height of the peak ndicated the high number of the particles (90%) in this size range (Fig. 3). Furthermore, C. vulgaris included three growth phases of log, stationary and lag phases. In the early log phase, a fragile unilamellar layer with 2-nm thickness could be demonstrated. Through maturation (log phase), cell wall thickness gradually increased to 17-20 nm, forming a microfibrillar layer of glucosamine. Due to the variation of cell wall rigidity of C. vulgaris within the growth phase, appropriate digestion and absorption of the valuable nutritional substances were limited [30]. Therefore, nano-sized microalgae were used not only to overcome problems of the ginger extract adsorption onto the Chlorella surface, but also to enhance drug loading efficiency of the synthetic nanocomposite.

Naturally, surface groups, extracellular products and cell structures affect electric characteristics of cells. In physiological pH, zeta potentials of the ginger extract (GE), C. vulgaris (microalgae), nanoparticles of C. vulgaris (nano C.v), microcomposite (GE@micro C.v) and nanocomposite (GE@nano C.v) included -22.8, -16.8, -27.2, -4.3 and -9.3 mV, respectively (Fig. 4). Based on the results, the lowest quantity of the extract could be loaded on the particles of C. vulgaris, this occurred due to the negative surface charge and repulsion in loading process. The whole cell of C. vulgaris included a negative surface charge of -16.8 mV; similar to that of Hao, et al., which linked to cell wall compositions of majorly cellulose, hydroxyl(-OH), carboxyl(-COOH) and aldehyde(-CHO) groups with negative charges [31]. Surface charge of the nanocomposite was -9.35 mV, becoming further positive and closer to the charge of the extract that indicating better adsorptions of the ginger extract on the microalgal nanoparticles. Zeta potential of the synthesized nanocomposite could be addressed as the reason for the low adsorption of ginger on the microalgal surfaces. Results showed that by shrinking the size of C. vulgaris particles from micrometers to 20 nm (less than 100 nm) with effects on physical characteristics, its surface charge became further negative and closer to -30 mV; thus, its stability increased due to the increases in the ratio of surface to volume [32]. Moreover, surface charge of the ginger extract was -22.8 mV, similar to that reported by Min Ho et al. This was because of the presence of carbohydrates and numerous anion sites, lipids and polyphenols due to possible groups in the extract [33].

3.4. Fourier transform-infrared spectroscopy analysis

Figure 5 shows FTIR graphs of ginger extract, microalgae and nanocomposite (GE@nano C.v), to indicate chemical groups in each treatment individually and characterize their interactions in synthetized nanocomposite.

 

 

A

 

 

 

B

 

 

 

C

 

 

 

Figure 1. High-performance liquid chromatography peaks corresponding to 6-gingerol standard (260 ppm) (a), ginger extract (b) and 6-gingerol content of the ginger extract (c).

 

 

Figure 2. Release rate curves of the ginger extract from nanocomposite at two acidic (red line) and neutral (black line) pH values.

Figure 3. The size measurement of Chlorella vulgaris particles after milling by DLS technique. Most of the particles' sizes are in the average of 20.9 nm.

 

Figure 4. Surface charges (zeta potentials) of the four samples. GE (orange), Chlorella vulgaris whole cell (green), Chlorella vulgaris nanoparticles (green pattern), microcomposite (blue) and nanocomposite (light blue pattern).

 

 

3.4.1. The ginger extract

 

The strong band located in the area of 3527.24 was linked to alcohol groups (O-H) and the presence of intramolecular and intermolecular hydrogen bonds; which could be associated to carbohydrates and polyphenols in the extract. The peak in the area of 2916.69 showed asymmetric stretching vibration of the C-H group and possibly the carboxyl group [34]. In addition, FTIR spectrum of the ginger included a relatively strong peak at 1606.5 , which was in the range of 1600–1609 and linked to the ring of polyphenols. The absorption spectrum in 1515.8 belonged to the nitro-aromatic groups of ginger extract. The broadening of the peak in the area of 1500–1800 indicated double and amide bonds. Moreover, C=O stretching bonds and N-H bending bonds when showing a broad peak in the range of 1516-1863.86 centered on the 1638  area. Presence of a 1268.13 was associated to the vibrations of alkyl ether and ester groups, demonstrating presence of triglycerides in the extract [35]. Furthermore, peak of 1040.10  included bending vibrations of the C-C bond of cellulose extract [33]. Absorption bands in the area of 924.45 were linked to carbon-carbon double bonds [36].

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 5. Fourier transform-infrared spectroscopy spectra of the ginger extract (GE, orange), nanocomposite (GE@nano C.v, blue) and nano Chlorella vulgaris (nano C.v, green).

 

 

3.4.2. Microalgae

Spectrum of C. vulgaris microalgae showed several peaks in various areas. The band of 3316.01  corresponded to the O-H stretching group, revealing presence of a strong alcohol group. Area of 1659.46  showed a strong band, sign of the presence of the first type of amide group within the proteins. Band of 2927.12 demonstrated presence of lipids in microalgae [37]. Band in the area of 2855.44 showed the bending CH₂ group linked to carbohydrates and lipids. Band in the area of 1738.96 cm^-1 highlighted ester bond of the carbonyl group. Peak in the area of 1547.97 belonged to the proteins and C=O stretching group. Presence of peaks in the areas of 1406.05 and 1242.77 was linked to the carbon-carbon bending group and the carboxylic acid group of microalgae, respectively. Peak of 1072–1099  was associated to -O-C group of carbohydrates, nucleic acids and other phosphate-containing compounds. Moreover, peak of 980–1072 belonged to the group (C-O-C) of polysaccharides [16,38].

3.4.3 Nanocomposite

Nanocomposite spectrum was located between the FTIR spectra of extract and microalga with further similarity to the extract, which could be attributed to loading of the extract on the microalgae. In FTIR spectra of the nanocomposite, bands were removed, replaced or decreased in depth. Band in the range of 3500–3300  of the spectra of the extract and microalga nanoparticles shifted to the left (3667.23 ) in the spectra of the nanocomposite, revealing connections of the extract with microalga. Lack of 3527 peak of the extract could be due to the interactions of O-H group of phenol with microalga. In contrast, 2927 peak of Chlorella became wider in the present nanocomposite, which was possibly due to the interactions of microalga with the extract. The microalga peak shifted from 1738 to 1728.65 and the extract from 1515 to 1534.94 in the nanocomposite could be due to the interactions of microalgal proteins with the extract through C=O and N-H groups, respectively. Peak in 1047  area of the extract could be seen in the spectra of the nanocomposite. Peaks between 924 and 564 cm^-1 in the extract and peaks between 1242 and 582 cm^-1 of Chlorella in the nanocomposite were removed, demonstrating interactions between the extract and microalga and the formation of C-C and C-O single bonds.

3.5. Thermogravimetric analysis assay

Investigating thermal stability (Fig. 6), graph of microalga showed its three-stage mass losses due to the pyrolysis process (25-600 ℃), which ultimately led to 70% decreases in the mass of microalga. Biomass's type and composition could change the TGA curve. In the first stage, mass losses occurred due to dehydration [16]. Nearly 4.65% of the microalga mass decreased as water evaporation up to 117 °C occurred (moisture loss). Small changes in the slope of the graph at 117-201 °C could be due to the beginning of the decomposition of compounds such as hemicellulose and carbohydrates. By increasing the temperature to values higher than 117 ℃, the second stage of biomass degradation began with a sharp slope (major zone).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6. Thermogravimetric analysis of the three samples: nano C.v (green), nanocomposite (blue) and ginger extract (orange).

 

 

Hence in the temperature range of 201–379 °C, nano C.v biomass quickly decreased by 46%, which could be caused by decomposition of the components. Volatile compounds such as organic materials, proteins, lipids, carbohydrates and cellulose and at higher temperatures (up to 379 °C), lignin, carbon materials and minerals (nearly 12% wt), were decomposed slowly [16,39,40]. With a low slope of the graph (third stage of 400–600 ℃), all volatile components of nano C.v were removed and non-volatile components were destroyed left as amorphous carbon forms [39]. In ginger extract graph, the mass curve decreased gradually. At 100 ℃, the first peak corresponded to the water loss (6.46%) [41]. The second weight loss occurred at 142.8–172.4 ℃, which represented decomposition of several aromatic compounds [42]. In the third stage (172.4-241.6 ℃), organic compound degradation occurred in decomposition of organic materials [43]. The last and the major step of mass loss occurred at 241.6-600 ℃ (36.6%) due to organic compound oxidation, lignin and cellulose degradation and complete decomposition [43]. The second and the third stages of GE degradation occurred at 142–241 ºC, while this range for GE@nano C.v was 210-300 ºC, meaning that synthesize of nanocomposite (GE@nano C.v) led to thermal stability of the ginger extract up to 300 ℃. In contrast, onset of the destruction temperature of microalgae was at 200 ℃. In nanocomposite graph, degradation temperature began at 150 ℃, showing that when the extract was loaded on C. vulgaris, degradation began earlier. As previously reported by Shetta et al. [44], green tea extract on the surface of chitosan nanoparticles was destroyed faster. This higher thermal stability of GE after adsorption on nano C.v is similar to that of Jafari et al. study, which demonstrated that thermal stability of curcumin was improved after its encapsulation into C. vulgaris [16]. Therefore, thermal degradation of organic compounds (the major step) in the nanocomposite occurred at higher temperatures, compared to those in ginger extract and nanomicroalga (two degradation steps). This could address improved intramolecular interactions between the ginger extract chemical groups and nanomicroalga that suggested a natural nanocomposite with enhanced thermal characteristics.

To calculate the quantity of ginger extract adsorbed on C. vulgaris surface, Eq. 7 by Shateri et al. [29] was used as follows.

Calculated mass residue =    Eq. 7

Where, x was the load quantity (%). Based on the formula, quantity of the extract loaded on the microalga was 31.64%. Table 2 shows weight decompositions (%) of the three samples.

3.6. Differential thermal analysis

The DTA curve shows thermal difference analysis. The technique calculated differences between the sample temperature and the reference temperature. The DTA graph shows a better temperature difference, compared with the TGA curves [49]. As shown in Fig. 7, DTA curves showed degradation peaks of nano CV, GE and GE@nano CV composite. In nano C.v, a calorific peak was seen at 100 °C, revealing dehydration and water loss. A strong second peak and a weak peak at 200 °C specify thermal decomposition of various Chlorella compounds and beginning of their degradation.

 

 

 

Table 1. Comparison of the current study with similar studies using various drug carriers and their effects.

Carrier

Cargo

Loading (%)

Positive effects

Reference

Spirulina platensis

Doxorubicin

85

+ Anticancer efficacy on lung metastasis

Biodegradable

Improved fluorescence imaging

[45]

Spirulina platensis

Black cumin

81.94

+ Antibacterial activities

+ Antioxidant activities

+ Anticancer activities

+ Thermal stability

[29]

Chlorella vulgaris

Curcumin

55

+ Thermal stability

+ Photostability

[16]

Chlorella pyrenoidosa cells

Lycopene

96.31

+ Stability

+ Antioxidant activity

[46]

Polymeric micelles (TPGS/PEG-PCL)

6- Gingerol

79.68

+ Solubility of 6-gingerol

+ Oral bioavailability

+ Improved brain distribution

[47]

Nanostructured lipid carriers

6- Gingerol

76.71

Higher drug concentrations in serum

+ Solubility of 6-gingerol

+ Oral bioavailability

[48]

Chlorella vulgaris

Ginger extract

31.64

+ Antibacterial effect

+ Antioxidant activities

+ Anticancer activities

+ Thermal stability

This work

 

Table 2. Thermogravimetric analysis assay with weight decreases as the temperature changes for the three samples (nanocomposite, nano C.v and GE).

Samples

Temperature range (℃)

Weight loss (%)

Nanocomposite

25.00-157.88

5.77

 

157.88-600.70

63.43

Nano Chlorella vulgaris

24.50-117.31

5.46

 

117.31-201.39

4.90

 

201.39-379.55

46.65

 

379.55-600.77

12.94

Ginger extract

24.60-142.8

6.46

 

142.8-172.4

6.39

 

172.4-241.62

18.26

 

241.62-600

36.69

 

 

 The fourth strong heating peak was observed at 300 °C, which included almost destruction of the microalga [39]. In DTA curve of GE, three strong peaks were seen in the temperature range of 100-200 °C (respectively from left to right: endothermic, endothermic and exothermic); which belonged to dehydration, destruction and decomposition of the major compounds in the ginger extract. Other exothermic and endothermic peaks were seen in the range of 200-300 °C. Residual and volatile compounds and carbohydrate depolymerization of the extract were almost completely decomposed, based on the studies by Kuk et al. [50].

Diagrams of DTA of GE@nano C.v were similar to diagrams of nano C.v, showing low loads of the extract. The two peaks of nano C.v degradation at 200 and 300 °C decreased to one peak at 250 °C. This major change in the decomposition peak of nano C.v showed that GE absorption on microalga nanoparticles was so strong that caused major changes in the rate and point of its decomposition. Diagram of the nanocomposite is plotted to the left, compared to that of the extract. Interactions between the extract and the microalga decreased thermal resistance in the range of 200-300 °C, which could be caused by weak interactions. In contrast, heat flow of the microalgae and its mild changes could be due to the low superficial loading of the extract on surface of the microalga as well as weak connections caused by it. It could be interpreted at a heating rate of 10 min/℃ in the first stage of the heating process with a peak at 55 °C with a molecular weight (MW) of 0.09 and a second peak of heat removal at 114 °C with an MW of 0.05 possibly due to the water loss. Thermal decomposition in the second stage was exothermic with two weak peaks at 236 °C and a strong peak at 297 °C with MWs of 0.2 and 0.35. This is the maximum heat loss due to protein decomposition at the top of the peak and carbohydrates, where bonds included O-O, N-O, C-N, C-C, C-O, N-H, C-H, N=N, H=H, O-H, O=O, C=C, C=N and C=O.

 

 

Figure 7. Differential thermogravimetric analysis diagram of nano C.v, ginger extract and nanocomposite.

 

 

Following the thermal decomposition curve, gasification occurred in the third stage with a weak peak at 370 °C and MW of 0.1. These results were similar to the results of Wang et al. [40]. In the third stage, only non-condensable gases such as CO, CO₂, H₂ and CH₄ were released with a little weight loss. This might reflect that the necessary heat was directly proportional to the increased temperature, as almost all the volatiles in the microalga were released in the thermal decomposition zone. This could be understood due to the soft structure of microalga with a relatively low lignin concentration (7.33-9.55% wt), thus volatile substances were easily separated from the tissues. Compared to hardwoods with high lignin contents (25%) and complex textures, thermal decomposition of hardwoods over 600 °C still produced non-condensable gases, explaining its relatively high weight losses.

3.7. The 2,2-diphenyl-1-picrylhydrazyl  test

Ginger increases blood plasma antioxidant capacity and decreases lipid peroxidation and renal nephropathy in rats. Gunathilake et al. detected that 6-gingerol in ginger extract removed peroxide radicals and hence could be used as a natural food additive and a substitute for artificial antioxidants [51]. Antioxidant activities of the ginger extract and nanocomposite (GE@nano C.v, loaded with 0.5 mg mg ml^-1 GE) were investigated for one month. As shown in Fig.8, antioxidant activity of the nanocomposite on the first day was nearly 60% at a concentration of 1.5 mg ml^-1. This increased by ~2% within one month as a result of antioxidant characteristics of Chlorella, when GE adsorbed on the surface within the first week and then made the antioxidant agents of C. vulgaris available. Karaman et al. detected that the higher activity of inhibiting free radicals for the sample encapsulated in yeast cells was associate to the higher anti-radical contents of the yeast cells [52].

In a study by Ghasemzadeh et al., antioxidant activity of the methanol extract of ginger was reported as 51-58% at a concentration of 40 µg ml^-1[53], possibly due to differences in the extraction method and various bioactive contents. In a study of Stoyanova et al., antioxidant activity of the ginger extract at 20 µg ml^-1 was calculated as 90% [54]. In the study of Abdel Karim et al., C. vulgaris showed a 50% antioxidant activity with a concentration of 1.95 mg ml^-1 due to the presence of plant chemicals (e.g., phenols, flavonoids, etc.) [55].

3.8. Antibacterial assay

Antibiotics are the compounds, which are used for human, animal and aquaculture treatments. Recently, their residues and degraded products in environment have included potential risks and toxicity worldwide. Antimicrobial activity against pathogenic and food-spoiling microorganisms is due to the bioactive compounds. Phenolic compounds such as gingerol and shogaol and their relationships majorly with other compounds such as β-sesquiphalendrene, cis-caryophyllene, zingiberene and α-farnesin are responsible for their antimicrobial activities in ginger essential oil and extract [56]. Hydrophobic residues of the ginger extract may interact with the lipophilic part of the cell membrane, disrupting their membrane integrity and function (e.g., electron transfer, nutrient absorption, protein and nucleic acid synthesis and enzymatic activity). In this study, results of the MIC assay showed that the ginger extract (Fig. 9a) and nanocomposite (Fig. 9b) included antibacterial charact-ristics and rates of their inhibition depended on their doses. By decreasing concentrations of the extract and nanocomposite, their antibacterial activities decreases. Concentrations of 1, 2 and 4 mg ml^-1 of the extract and 6.25, 12.5 and 25 mg ml^-1 of the nanocomposite were investigated.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 8. Antioxidant diagrams of the ginger extract (GE) and nanocomposite at concentrations of 0.5 and 1.5 mg ml^-1, respectively. All data are expressed as the mean ±SEM. Statistical analysis was carried out using ANOVA (letters, p < 0.05). Charts with similar letters include no significant differences.

 

 

 

 Results of the antibacterial assay showed that absorption of the extract on the nanoparticle significantly decreased its antibiotic characteristics, which could be linked to the slow releases of the extract active compounds from the surface of the nanoparticles; however, this could be active for a longer time. Antibacterial activity of the ginger extract was due to its polyphenol compounds, especially zingibrene, gingerol and terpenoids [57]. Based on the study of Hussein et al., E. coli and S. aureus were susceptible to the active compounds of C. vulgaris at a high concentration of 100 mg ml^-1. This could be due to the fatty acid contents, bioactive compounds, effects on cell cycle and protein and DNA syntheses or hydrophobic interactions that ultimately lead to cell leakage and death [58]. Inhibitory effects of the ginger extract on S. aureus, Pseudomonas spp. and E. coli was estimated as 90%. Inhibitory effects of nanocomposite on Salmonella spp. was higher than 60% and its effects on Pseudomonas spp. reached the maximum, increasing by ~6% for S. aureus to 50%. These differences could occur due to the structural differences in these bacteria [59].

3.9. Cytotoxicity assay

Ginger compounds (6-gingerol and its derivatives) have been shown to decrease hazards of several diseases, majorly in the gastrointestinal tract (GIT) and cancers such as carcinogenesis in the skin and breasts [60]. To assess cell viability of the breast cancer cell line (MCF-7) for their ability to decrease tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide], various concentr-ations of GE, nano C.v and GE@nano C.v within 24, 48 and 72 h were investigated (graphs of 24 and 48-h assays not shown). Results showed outstanding cancerous-cell inhibitions by the nanocomposite (GE@nano C.v), compared to ginger extract and nano C.v. Based on the growth inhibition chart of the MTT assay in Fig. 10,

growth inhibitions of 67, 73 and 86%, were respectively reported after 24 h of incubation (37 ℃, 98% humidity, 5% CO₂), with ginger extract at concentrations of 0.5, 0.75 and 1 mg ml^-1,. After 48 h, this value reached 28, 46 and 84%, respectively, which indicated dose and time-dependent cytotoxicity of the three treatments, as the more concentration, the higher growth inhibition. Cell viabilities were inhibited by the ginger extract treatment during 48 h. After 72 h of adding GE, cytotoxicity effects on the breast cancer cells for the concentrations of 0.5, 0.75 and 1 mg ml^-1 were 47, 48 and 47%, respectively. It was demonstrated that anticancer activity of the ginger extract decreased within 72 h. Various studies have shown effectiveness of the ginger extract on tumors. However, the extract not only includes therapeutic characteristics (due to 6, 8 and 10-gingerol and 6, 8 and 10-shogaol agents) but also decreases vomiting and nausea of the patients after chemotherapy [61]. In a study by Mohammed et al., further gingerol concentrations caused higher effects on cancer cell growth inhibition as 0.1 mg ml^-1 gingerol included significant cytotoxicity for 24 h and 0.4 mg ml^-1 gingerol showed 80% inhibition on MCF-7 cell line [62].

As previously stated, microalgae include bioactive compounds such as phytochemicals and carotenoids (lutein in C. vulgaris) with verified anticancer activities [63,64]. Cytotoxicity rates included 82, 75 and 76% after 24 h of treatment with nano C.v at concentrations of 0.5, 0.75 and 1 mg ml^-1 that reached to 96, 77 and 67% after 48 h, respectively.

 

A

B

 

 

Figure 9. Graphs of the average inhibitory proportions of the ginger extract (GE) (a) and nanocomposite (b) on four bacterial strains of Salmonella typhi (black), Staphylococcus aureus (gray dotted black pattern), Pseudomonas aeruginosa (light gray-black pattern) and Escherichia coli (gray-black pattern) using MIC assay in three various concentrations with controls. Significant differences (mean ±SE) of the effects of concentrations on the bacteria is illustrated with letters at p <0.05.

 

 

 

 Growth rates of the cancer cells at 0.5, 0.75 and 1 mg
ml^-1 respectively included 62, 62 and 67% after 72 h. This showed mild increases at 72 h, which could be due to the limited anticancer compounds in the cell wall of C. vulgaris microalga after grinding. Anticancer activities of the nanocomposite included 63, 63 and 71% after 24 h at 0.5, 0.75 and 1 mg ml^-1, respectively. After 48 h from the addition of nanocomposite, these values included 74, 71 and 59% for the concentrations of 0.5, 0.75 and 1 mg ml^-1 with significant changes, compared to those after 24 h, respectively. After 72 h at the concentrations of 0.5, 0.75 and 1 mg ml^-1, growth rates of the cancer cells included 51, 54 and 53%, respectively. The maximum inhibitory effect could be seen at 1 mg ml^-1 concentration of the nanocomposite due to the increases of ginger extract concentration adsorbed on nano C. vulgaris.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 10. Cell viabilities of the cancer cells (MCF-7 cells) after incubation in 0.5, 0.75 and 1 mg ml^-1 ginger extract, nanoparticles of Chlorella vulgaris and nanocomposite (GE@nano C.v) with different significant values at p<0.05.

 

 

 

Anticancer activity of the ginger extract alone decreased after 72 h. However, activity of the nanocomposite increased and was set constantly, demonstrating preservation of the activity of ginger extract adsorbed on the surface of microalga. This is a significant advantage for the use of nano C.v as a carrier for GE.

1. Conclusion

In this study, the synthesized nanocomposite included promising characteristics, which could be used in medicinal and nutritional industries. The HPLC assay showed bioactive compounds, especially 6-gingerol, in the extract (0.48 mg in 5 mg total extract). Furthermore, FTIR, TGA and DTA results verified adsorption of ginger extract on nano Chlorella surface. Due to intramolecular interactions of ginger with Chlorella chemical groups, improved thermal stability in range of 200-300 ℃ was observed. In addition, nanocomposite included a mild improved antioxidative activity within a month with 1.5 mg ml^-1 concentration. Antimicrobial and anticancer assays indicated good effects on microbial and breast-carcinoma cell-line (MCF-7) growth at concentrations of 6.25 and 1 mg ml^-1, respectively. In conclusion, combination of ginger and C. vulgaris could improve further antioxidant, antimicrobial and antitumor effects of ginger extract, compared to ginger extract alone. Further studies are needed to focus on novel methods for increasing adsorption of ginger extract on C. vulgaris cell wall to enhance nanocomposite stability.

1. Acknowledgements

The authors acknowledge Prof. Ali Reza Zomorodipour from NIGEB, Iran, for his support to provide research instruments of cell culture. In addition, authors acknowledge the instrumental supports of the University of Tehran.

1. Conflict of Interest

The authors declare no conflict of interest.

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Research Article

Applied Food Biotechnology, 2024, 11(1):e15

Journal homepage: www.journals.sbmu.ac.ir/afb

pISSN: 2345-5357

eISSN: 2423-4214

طراحی و ساخت  یک نانوکامپوزیت جدید ضد سرطان و ضد میکروب با استفاده از ریزجلبک‌ و مبتنی بر یک رویکرد بالا به پایین

مرجان رجبی، مهدی رهایی*، حسین صباحی

گروه مهندسی علوم زیستی، دانشکده علوم و فناوری های نوین، دانشگاه تهران، تهران، ایران

تاریخچه مقاله

دریافت 19 نوامبر 3202

داوری 17 ژانویه 2024

پذیرش 17 فوریه 2024

 

چکیده

سابقه و هدف: استفاده از مواد طبیعی روش کارآمد و ایمنی برای غلبه بر بیماری­های گوناگون است. نشان داده شده است که ویژگی­های فیزیکوشیمیایی عصاره زنجبیل ریزپوشانی شده در مقایسه با عصاره آزاد، بهبود یافته­ است. این مطالعه، یک سامانه طبیعی حاوی ترکیبات زیست فعال زنجبیل (6 -جینجرول) و ریزجلبک سبز کلرلا ولگاریس، ترکیبات زیست فعال با اثربخشی دارویی بیشتر و مکمل غذایی جدید را معرفی می­کند.

مواد و روش ها: ابتدا، نانوذرات ریزجلبک با روش آسیاب گلوله­ای یا گوی­آس^[2] تولید شدند. عصاره اتانولی زنجبیل، بارگذاری شده بر روی نانوذرات ریز جلبک، در pH  های گوناگون (4/7-2) مورد بررسی قرار گرفت تا عوامل فعال رهایش موثری داشته باشند. روش­های تحلیلی گوناگونی (به­عنوان مثال، تبدیل فوریه مادون قرمز^[3]، تجزیه و تحلیل گرما وزن­سنجی^[4]) برای توصیف نانوکامپوزیت و بررسی اثرات ضد سرطانی و ضد میکروبی آن استفاده شد.

یافته‌ها و نتیجه­گیری: اندازه نانوذرات ریزجلبک با روش پراکندگی نور دینامیک به­طور متوسط 9/20 نانومتر تعیین شد. بررسی رهایش پلی­فنول­های زنجبیل نشان داد pH فرآیند رهایش را کنترل می­کند. تبدیل فوریه مادون قرمز، تجزیه و تحلیل گرما وزن­سنجی و آنالیز حرارتی افتراقی^[5]، جذب عصاره زنجبیل بر سطح نانو کلرلا ولگاریس نشان داد. علاوه بر این، نتایج تعیین مقدار زیستی^[6] 2،2-دی فنیل-پیکریل هیدرازیل بر روی نانوکامپوزیت (GE@nano C.v) فعالیت­های قابل­توجه ضداکسایشی، ضد باکتریایی و ضد سرطانی آن را تأیید کرد. این نانوکامپوزیت به­ترتیب در غلظت­های 1 و 25/6 میلی­گرم بر میلی­لیتر، کمترین اثر بازدارندگی را بر روی سلول­های آدنوکارسینومای پستان انسان و رشد باکتری­ها دارد. به­طور خلاصه، جذب عصاره زنجبیل بر سطوح نانوذرات ریز جلبک، ویژگی‌های فیزیکی و شیمیایی عصاره زنجبیل را در مقایسه با فرم آزاد آن افزایش داد. ترکیبات زیست فعال موجود در کلرلا ولگاریس و عصاره زنجبیل فعالیت­های آنها را تقویت می­کند. علاوه بر این، نانوذرات ریزجلبک می‌توانند علاوه بر ویژگی‌های تغذیه‌ای، به عنوان یک حامل امن برای رهایش کنترل‌شده 6-جینجرول عمل کنند.

تعارض منافع: نویسندگان اعلام می‌کنند که هیچ نوع تعارض منافعی مرتبط با انتشار این مقاله ندارند.

واژگان کلیدی

▪ ضدتومور

▪  مکمل غذایی

▪ زنجبیل

▪ ریزجلبک

▪ نانو داروی طبیعی

 

^*نویسنده مسئول

مهدی رهایی

گروه مهندسی علوم زیستی، دانشکده علوم و فناوری های نوین، دانشگاه تهران، تهران، ایران

تلفن: 811118583-62+

پست الکترونیک:

mrahaie@ut.ac.ir  

 

 

 

[1] SC-CO2

[2] Ball-milling آسیابی که در آن از گلوله‏های فولادی یا سرامیکی برای خرد و نرم کردن مواد غیرفلزی استفاده کنند

[3] Fourier transform infrared (FT IR)

[4] Thermogravimetric   روشی تحلیلی بر مبنای اندازه‏گیری تغییرات وزن ترکیب یا آمیزه با افزایش دما 

[5] Differential thermal analysis

[6] Bioassay   تعيين قدرت يا غلظت يك مادة دارويي ازطريق سنجش اثرات آن بر بافت‌هاي زنده

 

Abstract

 Background and Objective: Lactic acid bacteria are well known as beneficial microorganisms and most of them are probiotic distributed widely, especially in fermented dairy products e.g. yogurt. This study aimed to isolate, characterize and assess antimicrobial effects of lactic acid bacteria producing bacteriocin-like inhibitory substances against foodborne pathogenic bacteria.

Material and Methods: In the present study, 17 lactic acid bacteria strains were isolated from 10 commercial yogurt samples and the antibacterial effects of lactic acid bacterial cell culture, cell-free supernatant and neutralized cell-free supernatant was assessed against standard foodborne pathogenic bacteria of Escherichia coli, Listeria monocytogenes, Klebsiella pneumonia and Salmonella typhimurium using agar well diffusion assay. Although various treatments were used, most of the lactic acid bacterial isolates showed antimicrobial activity against the foodborne pathogenic bacteria. Moreover, Lactiplantibacillus pentosus (SY1), Lacticaseibacillus rhamnosus (SY5), Lactiplantibacillus plantarum (SY8) and Lactiplantibacillus plantarum (SY9) showed significantly the best antimicrobial activity against the foodborne pathogens and thus were further identified using 16S rRNA gene molecular method.

Results and Conclusion: Results showed that four isolates could produce bacteriocin-like inhibitory substances, which was significantly effective to inhibit growth of the pathogens. Primary screening for antimicrobial activity showed that 10 lactic acid bacterial strains inhibited Escherichia coli. The results revealed that Listeria monocytogenes and Salmonella typhimurium was inhibited by six and one lactic acid bacterial isolates. Moreover, results showed that Klebsiella pneumoniae was not affected by the isolates or treatment methods. It is concluded that a bacteriocin-like inhibitory substance of lactic acid bacterial isolates was effective; hence, it could be used as a natural food additive to prevent foodborne infections and improve the food quality.

Conflict of interest: The authors declare no conflict of interest.

Background and Objective: Lactose intolerance is a prevalent clinical syndrome due to consumption dairy products. Development of low-lactose products may help consumers to receive nutrition of dairy products without negative health effects. The aim of this study was to investigate survival, physicochemical and sensory characteristics of low-lactose yogurt drinks produced with probiotic Lactiplantibacillus plantarum subsp. plantarum Dad-13 during cold storage.

Material and Methods: Low-lactose milk was inoculated with probiotics in fermentation process of yogurt at 37, 39 and 42 °C while the growth of lactic acid bacteria and pH were monitored. Prepared yogurt drink was stored at 4 °C for 35 days and characterized for microbial and physicochemical aspects.

Results and Conclusion: Lactiplantibacillus plantarum subsp. plantarum Dad-13 showed high count at all the three temperature and reached to 7.64-8.96 log CFU.ml^-1 at 37 °C within 36 h when cultured with yogurt. During the 35-d storage, cell viability of Lactiplantibacillus plantarum Dad-13 was 7.79 log CFU.ml^-1. Furthermore, sensory assessment significantly increased (p≤0.05) for odor, viscosity, taste and total acceptability, compared to the non-probiotic yogurt.

Conflict of interest: The authors declare no conflict of interest.

Abstract

Background and Objective: Ganoderma lucidum, with its medicinal characteristics, is one of the most beneficial fungi in traditional Asian medicine. This fungus low efficiency of ganoderic acid production has limited its use as a valuable secondary metabolite. Environmental stresses and elicitors such as microbial volatile organic compounds in co-cultures can increase ganoderic acid production. To investigate effects of variables of co-culture time and volume on Ganoderma lucidum growth and ganoderic acid production, Bacillus subtilis and Aspergillus niger were aerially co-cultured with Ganoderma lucidum.

Material and Methods: To investigate fungus growth and production of ganoderic acid using bubble column bioreactor, effects of independent variables of temperature, initial inoculation, length-to-diameter ratio (L: D) and aeration were investigated using Taguchi method. Then, effects of co-culture of Ganoderma lucidum with Bacillus subtilis and Aspergillus niger under optimum conditions were investigated.

Results and Conclusion: Optimizing effects of co-culture time and volume variables led to 2.9-fold increases in production of ganoderic acid, compared to the control sample. Optimization of biomass production in the bioreactor showed that biomass production increased significantly by increasing the initial inoculation percentage and temperature. These two variables significantly affected ganoderic acid production and its optimum production point was 10% of initial inoculation, temperature of 25.6 °C, L: D of 4:8 and aeration rate of 0.64 vvm. Gas holdup investigation for air-water and air-fermentation media systems showed that the presence of suspended solids and aeration rate affected gas holdup. Microbial volatile organic compounds in co-culture of microorganisms can increase ganoderic acid production by Ganoderma lucidum.

Conflict of interest: The authors declare no conflict of interest.

 

Abstract

 

Background and Objective: Glucansucrases from GH70 family are effective transglucosylases, able to use non-carbohydrate acceptors. Glycosylation of flavonoids or terpenoids increases their water-solubility and bioavailability. Enzymatic glycosylation by glucansucrases can be improved by addition of organic solvents to the reaction media. Thus, the aim of the study was to assess effects of menthol, carvacrol and thymol solubilized in organic solvents on the activity of glucansucrase URE 13-300 and transferase reaction.

Material and Methods: Several organic solvents were assessed for their effects on glucansucrase activity using DNS method. Kinetic parameters in presence of the most appropriate solvents were evaluated as well. Thymol, carvacrol and menthol were solubilized in DMSO and their effects on the enzyme activity was assessed. Dynamic of oligosaccharides synthesis in aqueous-organic media was investigated using high-performance liquid chromatography.

Results and Conclusion: Maltose-derived oligosaccharides synthesized by glucansucrase URE 13-300 showed degrees of polymerization from 3 to 6 in presence of organic solvents, as well as in presence of buffer alone. Their concentrations did not differ significantly in each of the reactions in aqueous-organic media. Furthermore, kinetic parameters showed adjacent K_m values with 5% solvents compared to the control reaction in buffer. These findings revealed that the overall synthesis of glucooligosaccharides was not altered by the organic solvents, nevertheless they changed the product distribution throughout the transferase reactions. These moderate effects of the selected organic solvents were important requirement for the glycosylation of biologically active compounds for use in the food industry.

Conflict of interest: The authors declare no conflict of interest.

Abstract

 

Background and Objective: Nowadays, there is a growing interest for use of plant-based products such as extracts in various industrial sectors. Therefore, optimization of conditions for ideal extraction of bioactive compounds is highly important. Olive leaves and fruits include biophenols, which can be used as natural antimicrobial and antioxidant agents. Therefore, extraction of these bioactive compounds can create value-added products, which can be used as natural preservatives in food industries. The aim of this study was to investigate effects of various extraction parameters (type of solvent, solvent volume, temperature, time and pH) on in-vitro antioxidant and antifungal activities of Iranian olive leaf and fruit extracts against five Candida species.

Material and Methods: Olive fruit and leaf extracts were achieved using maceration method at various extraction conditions. Antioxidant activity of the prepared extracts was assessed by cupric reducing antioxidant capacity method. The phenolic profile in olive leaf extract was assessed using high performance liquid chromatography. Antifungal activity of the olive leaf extract was assessed using disk diffusion method and minimum inhibitory concentration and minimum fungicidal concentration values.

Results and Conclusion: Results showed that the highest antioxidant activity was recorded in olive leaf extract prepared by 100 ml of 96% ethanol at pH 7 and 80 °C for 6 h. Moreover, HPLC analysis of the ethanolic olive leaf extract showed that oleuropein was the major compound of the extract. Antioxidant activity of the olive leaf extract was higher than that of the fruit extract in various conditions. Regarding antifungal activity, the olive leaf extract showed a higher activity, compared to olive fruit extract at all concentrations. In olive leaf extract, the highest (62.5 µg ml^-1) and the lowest minimum fungicidal concentration (15.6 µg.ml^-1) values were reported for Candida tropicalis and Candida albicans, respectively. The minimum fungicidal concentration of the olive leaf extract was 250 µg ml^-1 for Candida albicans, Candida parapsilosis, Candida glabrata and Candida krusei and 500 µg ml^-1 for Candida tropicalis. It can be concluded that olive leaf extract is a source of antioxidant and antifungal substances with potential uses in food industries.

Conflict of interest: The authors declare no conflict of interest.

Abstract

 

Background and Objective: Use of bacterial cellulose has been interested in various industries, especially medical and pharmaceutical industries, due to its unique characteristics, compared to plant cellulose. However, bacterial cellulose production costs have limited its industrial uses, compared to plant cellulose. Decreasing costs of the culture media is one of the effective parameters for the industrial production of bacterial cellulose. This is the first report on combination of vinasse and glucose syrup as a bacterial cellulose culture medium.

Material and Methods: Two inexpensive culture media were developed for high-level production of bacterial cellulose based on food industrial wastes of corn steep liquor-glucose and vinasse-glucose syrups. Concentrations of glucose syrup and corn steep liquor as a culture medium and concentrations of vinasse and glucose syrup as another culture medium were optimized using response surface method with central composite design to maximize bacterial cellulose production yields.

Results and Conclusion: Under the optimal conditions after seven days, 14.8 and 13.3 g.l^-1 dry bacterial cellulose were achieved in corn steep liquor-glucose syrup and vinasse-glucose syrup respectively. Yield of produced bacterial cellulose from these two cost-effective culture media was one of the highest values reported for bacterial cellulose. Furthermore, the produced bacterial cellulose was characterized using Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy.

Conflict of interest: The authors declare no conflict of interest.

Background and Objective: Principles of osmo and thermoanabiosis are used to produce sweetened condensed milks. Regarding their extended shelf lives, there are demands for their export to countries with various climates. However, high-positive and low-negative ambient temperatures during sweetened condensed milks transportation can affect their quality. Hence, it is important to study effects of critical storage temperatures on microbiological, physicochemical and sensory indicators of sweetened condensed milks.

Material and Methods: This investigation included a comprehensive study of the physicochemical, microbiological and sensory characteristics of sweetened condensed milks after storage under conditions involving multiple-stage and single-stage temperature changes within various ranges (from 5 to 50 °C; from 5 to -50 °C, from 50 to -50 °C and reverse cycles.).

Results and Conclusion: Analysis of samples subjected to cyclic changes, including multiple-stage heating for 9 d followed by multiple-stage cooling for 11 d, revealed that only viscosity changed relative to the control samples. In the reverse similar cycle (cooling to heating), formation of destabilized fat was observed. Moreover, changes of cycles and subsequent storage of the samples for 6 m led to increased viscosity, compared to control samples. It was established that single-stage freezing with a 14-d storage did not critically affect its quality. In contrast, rapid heating of the sweetened condensed milk up to 50 °C and storage under such critical conditions outside a cooled storage area were unacceptable. Further storage of samples subjected to cycles of single-stage freezing and heating for 6 m demonstrated a complete non-compliance with control samples for all parameters. Thus, sweetened condensed milk can be subjected to single-stage freezing to -50 °C and storage for 14 d, as well as multiple-stage cooling/freezing to -50 °C and multiple-stage heating to 50 °C following by cooling to 5 °C without loss of quality and safety during 6 m.

Conflict of interest: The authors declare no conflict of interest.

 

Background and Objective: Inactivated probiotics provide various health and technological benefits, making them appropriate for the production of functional dairy products. The aim of this study was to investigate effects of adding nonviable probiotics (Lactobacillus acidophilus LA-5 and Lacticaseibacillus casei 431) to doogh (a typical Iranian fermented milk drink).

Material and Methods: Probiotics were inactivated by heat or sonication and added to the samples before or after fermentation. Various parameters such as pH, titratable acidity, redox potential, antioxidant capacity, color, viscosity, and phase separation, viability of traditional starter bacteria and probiotics and sensory characteristics were assessed during fermentation and refrigerated storage at 5 °C.

Results and Conclusion: Sonicated probiotic-containing treatments included the highest pH decrease rate (0.011 pH min^-1) during fermentation, as well as the highest antioxidant capacity (16.45%) and viscosity (35.15 mPa.s), while heat-inactivated probiotic- containing treatments included the lowest viscosity (17.60 mPa.s). Treatments with viable probiotics reasonably included the highest post-acidification rate during storage (4.14 °D d^-1), compared to those containing nonviable cells, as well as the minimum phase separation rate. The b* and L* values of color did not differ significantly within treatments, but the highest a* value was observed in the treatments with sonication. The highest populations of Lactobacillus delbrueckii ssp. bulgaricus (log 11,891 cfu ml^-1) and Streptococcus thermophilus (log 14,977 cfu ml^-1) at the end of the storage were observed in treatments with heated probiotics (compared to viable probiotics) and treatments with sonicated probiotics, respectively. In addition, Lactobacillus acidophilus was more susceptible than Lacticaseibacillus case and included lower viability. Taste, mouth feeling and total acceptance of all samples did not differ significantly within treatments. The present study suggests that inactivated probiotics can successfully be used for the production of fermented milk beverages with appropriate sensory characteristics and higher antioxidant capacity, compared to the control group.

Conflict of interest: The authors declare no conflict of interest.

 

1. Introduction

 

Recently, it has been suggested that probiotics, viable or non-viable, are bacterial cells that include positive effects on human health. By this general definition, probiotics are divided into two categories of viable and non-viable probiotics [1, 2]. The idea of using non-viable probiotics in food industries is originated from the fact that probiotic bacteria are susceptible to environmental conditions during passage through the gastrointestinal tract (GIT), include limited stability over a wide range of pH and temperature, include a shorter shelf-life and need refrigerated storage. Therefore, their use in various industries is further technologically and economically feasible [3-6]. Additionally, it has been verified that non-viable probiotics include beneficial effects for humans such as immunostimulating activity [7], cholesterol decrease [8], anticancer characteristics [9], healing gastrointestinal disorders [10] and suppression of pro-inflammatory cytokine production [11]. There are several available methods to inactivate probiotics, including heat treatment, ultraviolet (UV) irradiation, irradiation, sonication (ultrasound), high pressure, ionizing radiation, pulsed electric field (PEF), supercritical CO₂, drying and changes in pH [8,12]. Sonication and heating are the most commonly used methods for inactivating probiotics, majorly because they are cost-effective and time-efficient. Ultrasound at frequencies of 20–40 kHz can be lethal to microorganisms by creating acoustic cavities on their cell membrane (CM), leading to the release of their contents [13]. In contrast, during heating, intracellular contents are not released.

Doogh is a fermented beverage whose major ingredients include yogurt, water, salt and flavoring agents [14]. However, studies on adding non-viable probiotics to fermented foods are limited. Parvayi et al. studied effects of inactivated Lactobacillus acidophilus ATCC SD 5221 and Bifidobacterium lactis BB-12 on yogurt characteristics and reported that incorporation of heat-inactivated probiotics to yogurts included less technological challenges and could be deliberated as an appropriate alternative for probiotics in functional yogurts [15]. Overall, there is still a research gap in the development and commercialization of inactivated probiotic dairy products in food industries. While interests in probiotics and prebiotics are increasing, inactivated probiotics have not received much attention for product development and market availability. In addition, knowledge on specific inactivated probiotic compounds in dairy products and their potential effects on human health is limited. Further research are needed to identify and characterize these compounds and assess their potential health benefits and uses in functional foods [16,17]. Moreover, there is a lack of standardized methods for the production and quality control of inactivated probiotic dairy products, which limits their widespread commercialization. Research in this area is essential to establish industry standards and guidelines for the production and commercialization of inactivated probiotic dairy products. The aim of this study was to assess effects of adding non-viable forms of Lacticaseibacillus casei 431 and lactobacillus acidophilus LA-5 probiotics inactivated by heating or sonication on the quality characteristics of doogh, a traditional fermented milk beverage from Iran. Probiotics were added before or after the milk fermentation processes.

1. Materials and Methods
   • Materials

Skim milk powder was purchased from Pegah, Tehran, Iran. Starter culture included Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus (YF-3331) and the probiotics (Lacticaseibacillus casei 431 and Lactobacillus acidophilus LA-5) were provided by Chr. Hansen, Copenhagen, Denmark. De Man-Rogosa-Sharpe (MRS) agar and M17 agar were purchased from Quelab, Montreal, Canada, and salt from a local market.

• Preparation of nonviable probiotics

Probiotic suspension was subjected to thermal inactivation by heating at 121 °C for 15 min [18].

To achieve ultrasound inactivation, probiotic suspension was exposed to ultrasound waves at a frequency of 250 kHz for 25 min [19].

• Preparation of doogh

To prepare doogh, skim milk powder was reconstituted and diluted to a total solid content of 3.5%. Mixture was heated to 90 °C and set for 15 min before cooling down to 45 °C. Probiotics in viable or nonviable form were added before heat treatment (B) or after fermentation (A). Mixture was incubated at 42 °C until the pH reached 4.5, cooled down to 5 °C and stored in refrigerator for 28 d, as presented in Fig. 1.

• Assessment of pH, redox potential and titratable acidity

The pH, RP (redox potential) and titratable acidity of the doogh samples were checked every 30 min during fermentation. After fermentation, doogh samples were cooled and stored in refrigerator for 28 d, during which, pH, RP (redox potential) and titratable acidity were assessed every 7 d to monitor the shelf life. The pH and RP were assessed using pH meter at room temperature (RM). Titratable acidity was assessed by titrating with 0.1 M NaOH solution and 0.5% phenolphthalein indicator [20]. Increase in acidity, decrease in pH value (pH value min^-1) and increase in redox potential (mV min^-1) were calculated using Eqs. 1, 2 and 3:

Figure 1. Study design of the present study.

 

• Serum separation analysis

After cooling down, samples were stored in 10 ml vials and incubated at 5 ºC to assess serum separation. During the shelf-life period, height of the supernatant was assessed every 7 d to assess degrees of serum separation that were expressed as proportions using the following Eq. 4 [21]:

 

                Eq. 4

• Rheological assessment

Rheological assessments were carried out using Brookfield viscometer at refrigerator temperature, one day after the samples were prepared [22]. Briefly, no. 2 cylindrical spindle and spindle speeds of 0.3, 0.6, 1.5, 3, 6, 12, 30 and 60 rpm were used during 90 s if the torque to rotate the spindle in the samples was between the 15.0 and 85.0% of the maximum torque.

• Assessment of antioxidant capacity

To assess antioxidant capacity of the samples, a method was used based on the ability of antioxidants to scavenge the stable radical DPPH (1,1-diphenyl2-picrylhydrazyl). This method was described by Farahmandfar et al. essentially, sample ability to reduce the concentration of DPPH was assessed by measuring absorbance of the solution before and after exposure to the samples [23]. Antioxidant capacity of all samples and inactivated bacterial suspension were assessed on two occasions. The first assessment was carried out on the day of production, while the second assessment was carried out on Day 28 of the shelf-life.

       Eq. 5

• Color assessment

Color characteristics of doogh were assessed using Hunter Lab Color Flex EZ explained by Milovanovic et al. [24]. Color parameters were L* (brightness, white = 100, black = 0), a* (+, red; -, green) and b* (+, yellow; -, blue).

• Bacterial enumeration

Pour plate method was used to count numbers of L. delbrueckii subsp. bulgaricus, S. thermophilus and L. casei [25]. The L. bulgaricus, starter bacteria of doogh, was cultured in MRS-bile agar at 42 ºC for 72 h under anaerobic conditions using Gas Pac system. Enumeration of S. thermophilus was carried out using M17 agar at 37 ºC for 24 h under aerobic conditions [26]. Lactobacillus acidophilus LA-5 and L casei were cultured in MRS agar with added bile (0.15% w w^-1) to prepare selective media of probiotic enumeration at 37 ºC for 72 h under aerobic conditions [27, 28]. The initial counts of L. acidophilus and L. casei were 10⁷ CFU ml^-1. To calculate the viability proportion index, final cell population of the microorganisms was divided into the initial cell population based on the Eq. 6 [25].

                                                           Eq. 6

• Sensory evaluation

Taste, mouth feel and overall acceptance of doogh were assessed using 5-point hedonic scale rating test (with 5 excellent, 4 good, 3 acceptable, 2 bad and 1 very bad) [29]. Twenty consumers assessed the sensory attributes of doogh samples after the first day of preparation.

• Statistical analysis

All experiments were carried out in triplicate and expressed as mean ±SD (standard deviation) (n = 3). Data were analyzed using univariate analysis of variance (Tukey test) AND SPSS statistical software v.26 (SPSS, Chicago, USA). Generally, p < 0.05 was addressed as the significance threshold.

1. Results and Discussion

• Assessments of pH, redox potential and titratable acidity

During milk fermentation, growth of starter bacteria leads to the conversion of lactose into various compounds such as lactic acid, acetate, formate, acetaldehyde and ethanol. This process results in lactic acid production, causing decreases in pH and increases in redox potential and titratable acidity [30]. Figure 2 illustrates changes in pH, redox potential and titratable acidity during the fermentation process. The initial pH of milk at the beginning of fermentation was 6.8, dropping to 4.5 by the end of fermentation. As shown in Fig. 2, and Table 1 fermentation process included three distinct phases of lag, log and constant phases. During the first 30 min, the lag phase, no significant changes were seen, possibly due to the adaptation of the starter bacteria and buffering characteristics of milk [31]. The fastest decrease in pH and increase in redox potential were observed in sample with ultrasound-inactived L. casei. This might be attributed to the ultrasound treatment, which caused puncturing of the membrane of the probiotics, resulting in the release of their cell contents into doogh [5,13]. Feeding the starter bacteria resulted in decreases in the rate of pH and pH of BUC reached 4.5 as the fastest rate (after 210 min). However, BUA included the highest titratable acidity, indicating that the type of probiotic bacteria included major effects on the rate of pH drop and acidity increase. Similarly, Tian and colleagues (2017) reported that the type of bacteria included effects on the quantification of organic acids [32]. In addition, postbiotics produced from L. acidophilus LA-5, L. casei 431 and L. salivarius included 62 vrious components, including alcohols, terpenes, norisoprenoids, acids, ketones and esters [33]. Hence, these compounds were available in the environment and might improve the fermentation stage.

Based on Fig. 2, BHC included similar rates of pH decrease and increase in redox potential through the fermentation process as the sample without probiotics. However, BHA showed the lowest rate of acid increase at the end of the fermentation, indicating that the starter bacteria alone were responsible for lactic acid production and the intact cells of the probiotic bacteria included no significant effects on acid production. Furthermore, heat-inactivated L acidophilus demonstrated the antibacterial activity [34]. Samples containing live probiotics needed longer times (240 min) to reach pH 4.5. This finding was similar to the finding of Parvayi (2021), who reported that live probiotic samples needed longer times to reach pH 4.5, compared to paraprobiotic samples [15]. Based on a study by Vinderola et al. (2002), adding L. casei and L. acidophilus to the media with the starter bacteria included negative effects on the growth of the starter bacteria, resulting in decreases in lactic acid production [35].

Statistical analysis showed no significant differences in redox potential between various types of bacteria (p>0.05). However, L. acidophilus resulted in further decreases in pH and increases in titratable acidity during the storage, compared to that L. casei did (p<0.05) as represented in Table 2. These results suggested that the selection of probiotic bacteria should carefully be considered based on the specific goals of the fermentation process [36]. Throughout the storage, the highest level of titratable acidity was seen in sample containing live probiotics of L. acidophilus (181AD∘), which could be attributed to the ongoing acid production by the live probiotics at the refrigerated storage. In contrast, BUC sample included the lowest acidity (117A^∘), suggesting that the addition of probiotics after the fermentation process could lead to uncontrolled increases in acidity and continued fermentation during cold storage [15]. Moreover, samples containing sonicated and live probiotics included the maximum and the minimum RP increasing rates because of producing the minimum and the maximum lactic acid quantities during storage (p<0.05).

• Serum separation

The study detected that the activity of starter bacteria and their ability to generate acids included significant effects on the separation of serum in the samples [37]. Data of Table 3 show increases in serum separation values for all samples during the storage. The initial and the final separation rates of BUC were the highest (32.4%), suggesting that the released intracellular contents were heavier than the whole bacterial cells, causing further sedimentations. In addition, Samples containing live probiotics included smaller serum separation ratios at the end of storage, indicating that they frequently produced lactic acid and their pH was further different from the isoelectric pH [38].

Relatively, Amani et al. reported effects of the activity of starters during storage due to their protein hydrolyzing characteristics on phase separation [37]. In addition, L. casei was reported to include lower serum separation ratios than that L. acidophilus did (p<0.05). This suggested that the type of bacteria in the samples played important roles in the serum separation rate because various strains of probiotic bacteria included various abilities to ferment and break down organic compounds and producing exo-endo polysaccharides as discussed in viscosity section [39]. However, np statistically significant differences were detected between the sequences of probiotic additions (p> 0.05).

• Viscosity

Naturally, acidification and lowering of pH during fermentation cause milk casein proteins to clump, affecting viscosity of the final products. Figure 3 shows assessed viscosity of the samples. Sonicated probiotic-containing treatments (BUC and BUA) included the highest viscosity (3.083 ±0.6 and 3.515 ±0.5, respectively). Additionally, addition of live probiotics during fermentation led to increased viscosity, compared to samples without probiotics. It was reported that the release of exopolysaccharides and intracellular polysaccharides from the probiotics significantly increased viscosity [40, 41]. Exopolysaccharides secreted by Lactobacillus spp. during their growth affect viscosity of dairy products [42]. Moreover, "intracellular polysaccharides" are polysaccharides that accumulate within cells. The intracellular biosynthetic process involves transferring sugar residues into the cell, converting them into various monomeric units, partially polymerizing them and attaching them to isoprenoid lipid carriers [43]. Viscosity of heat-inactivated treatments was similar to that of control treatment, possibly because intact cells of probiotic bacteria did not release biopolysaccharides into doogh samples. Furthermore, type of bacteria significantly affected the viscosity (p<0.05). It was previously reported that variations in the viscosity values could be affected by characteristics of the probiotics cultures as well as adaptability of the bacteria [44].

• Antioxidant activity assessment

The DPPH radical scavenging method, widely used to assess antioxidant activities, is simple, rapid, sensitive and reproducible compared to other methods [45]. Figure 4 shows antioxidant capacities of the samples on Days 1 and 28. Antioxidant capacity of the samples decreased significantly during the storage due to inappropriate sealing, oxygen entry into the samples and uncontrolled bacterial activity. Sonicated probiotic-containing treatments increased the antioxidant capacity, as the intracellular content of lactic acid bacteria (LAB) demonstrated greater antioxidant characteristics than that the whole cell or the extracellular metabolites did [46,47]. Antioxidant activity of the intracellular contents of LAB was linked to the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), nicotinamide adeninedinucleotide (NADH)-oxidase, NADH-peroxide and glutathione (GSH) enzymes [48]. In addition, use of live or heat-inactivated probiotics did not result in significant differences (p>0.05). This was due to the cell contents that were not released. Relatively, lyophilized cells of Lactococcus lactis subsp. cremorishave included the highest antioxidant capacity, compared to those the heat-killed and intact cells did [49].

In contrast, use of L. acidophilus rather than L. casei significantly increased the antioxidant capacity (p<0.01). Amdekar et al. assessed antioxidant and anti-inflammatory potentials of L. casei and L. acidophilus in in-vitro models of arthritis. Results indicated that arthritic rats treated with L. acidophilus included higher glutathione peroxidase and decreased glutathione concentration, compared to that arthritic rats treated with L. casei did [50]. Additionally, adding probiotics before fermentation improved the antioxidant capacity of doogh samples (p>0.05).

• Color analysis

 

Color is a critical characteristic in assessing quality of products such as yogurts and doughs. The L* parameter indicates lightness or darkness of the color, the a* parameter shows redness or greenness of the color and the b* parameter represents yellow or blueness of the color [51]. The color values are shown in Fig. 5. Integration of probiotics inactivated using ultrasound resulted in increases in a* value, indicating release of probiotic contents into the doogh sample (p<0.05) and showing that green pigment substances such as thiamine were present in intracellular probiotics [52]. However, no significant differences were reported between the paraprobiotics and probiotics in a* value (p>0.05). Additionally, no significant differences were demonstrated between the sequential additions of probiotics in a* value (p > 0.05). Type of the probiotics in doogh samples included significant effects on a* value (p < 0.05). It has previously been suggested that various types of bacteria with special characteristics can affect color of the products [53]. In L* and b* values, no differences were observed within the addition of active/inactivated probiotics into doogh samples (p>0.05). Furthermore, types of probiotic bacteria (L. casei or L. acidophilus) and probiotic adding sequences did not include significant effects on L* and b* values (p > 0.05).

• Viable counts of the starter bacteria and probiotics

The L. bulgaricus and S. thermophilus are critical for acidification and production of doogh [54]. Survival of the starter and probiotic bacteria in yogurts depends on various factors such as the specific strains, interactions between the species, chemical compositions of the yogurts, the culture conditions, production of hydrogen peroxide during bacterial metabolism, final acidity of the yogurts, rates of lactic and acetic acids, nutrient availability and the storage temperature [46]. Table 4 shows the number of L. delbrueckii subsp. bulgaricus and S. thermophilus for all samples during the storage. The BUA included the highest number of L. delbrueckii subsp. bulgaricus on the first and the last days of enumeration (logs 11.89 and 10.91 cfu ml^-1, respectively), while BC included the lowest number (logs 11.35 and 9.68 cfu ml^-1, respectively). Additionally, BA and BUA included the lowest and the highest L. delbrueckii subsp. bulgaricus counts at the end of storage (logs 7.84 and 10.91 cfu ml^-1, respectively). Type of probiotics (viable or non-viable) in doogh samples included significant effects on the viability of L. delbrueckii subsp. bulgaricus (p<0.05) through competitive interactions, metabolic activities, cell-cell interactions and protective effects. However, a study by Parvayi et. al (2021) showed that addition of viable or non-viable probiotics did not affect L. delbrueckii subsp. bulgaricus when used as the starter bacteria [15].

As a statistical result, significant differences were observed between using L. casei and L. acidophilus probiotic bacteria (p<0.05). Selection of probiotic strains such as L. casei and L. acidophilus in doogh could affect viability of L. delbrueckii subsp. bulgaricus through species-specific interactions, metabolic compatibility, competition for resources, synergistic or antagonistic effects and stability of the microbial community. In addition, inhibition of L. delbrueckii subsp. bulgaricus growth by L. acidophilus has previously been reported. Vinderola reported L. casei strains did not include effects on the growth of L. delbrueckii subsp. bulgaricus [35]. Furthermore, no statistical differences were reported between the sequences of adding probiotics (before or after fermentation) into doogh samples (p>0.05). In contrast, viability of S. thermophilus decreased significantly (p<0.05) during the storage. On the first day of storage, BUC included the highest number of S. thermophilus (log 14.67 cfu ml^-1), whereas BA included the lowest number of S. thermophilus (log 13.54 cfu ml^-1). In addition, AA and AUC included the lowest and the highest S. thermophilus counts at the end of storage (logs 10.11 and 12.85 cfu ml^-1, respectively). The highest rate of decrease in S. thermophilus viability was associated to AA (0.73), while AUA included the lowest rate of decrease in S. thermophilus viability (0.88). Addition of non-viable probiotics caused significant differences in the bacterial population, compared with that addition of live probiotics did (p<0.05). This could be due to the indirect antagonistic effects of live probiotics [4, 35].

Doogh samples with inactivated probiotic cells showed significantly higher starter proliferation, compared to those treated with probiotic bacteria due to the cell wall structure of L. acidophilus and L. casei in their ruptured cells (p<0.05). Naturally, cell wall majorly consists of teichoic acids, cell structural protein (S-layer), peptidoglycan and polysaccharides [10]. Additionally, LAB intracellular contents include GABA, B-vitamin complex, polysaccharides, biopeptides, polysaccharides and lipoteichoic acids [4,47,55]. Therefore, fermentation in the environment is strengthened when the intracellular contents are released into doogh. It is possible that choosing the right time for inoculation can significantly affect growth of starter bacteria. Results showed significant differences (p < 0.01) between adding probiotics before or after the fermentation process, affecting viability and activity of the starter bacteria due to its adaptation to the culture medium during fermentation and storage. Moreover, it was reported that L. acidophilus included stronger growth inhibitory effects on S. thermophilus than that L. casei did (p <0.01). In a study by Vinderola et. al (2002), L. casei strains inhibited growth of S. thermophilus while L. acidophilus did not affect the growth of S. thermophilus [35]. Sample inoculated with live probiotics before fermentation included the lowest count of S. thermophilus due to the potential antagonism effects of the probiotics (p<0.01).

Generally, live probiotics included side effects on the growth and viability of the starter bacteria. For nonviable probiotic cells, these inhibitory effects are rarely observed and can surprisingly promote starter bacterial activity by providing various nutritious (e.g., amino acids, minerals, B vitamins and saccharides) and growth stimulatory elements [56]. Moreover, live probiotic bacteria include side effects on the starter bacterial growth because of their antimicrobial secretion and competition. Probiotic bacteria included more inhibitory effects on LAB than that LAB did when probiotics were not present [35]. For inactivated probiotics, there are no severe competitions for nutrition between the starter bacteria that may nourish them and enhance their growth due to the release of cytoplasmic contents. It is noteworthy that addition of live probiotics to media before fermentation increased the number of probiotic cells during storage due to better adaptation (p < 0.05). Furthermore, L. acidophilus was more susceptible than L. casei and statistically significant differences were recorded in viability of the probiotic bacteria (p < 0.05). Therefore, it can be concluded that selection of an appropriately adaptable strain may play critical roles in preserving viability of probiotics during the shelf life of the products [36].

• Sensory evaluation

Sensory attributes play key roles in attracting consumers. Daily probiotic products may include distinct tastes that are not be accepted by the consumers [57]. Therefore, studies have focused to improve consumer acceptance of probiotic beverages. In this study, taste, mouthfeel and overall acceptability of sensory aspects were assessed on the first day of fermentation (Figure 6). The AHC included the highest score (4.1/5) for taste, while AC included the lowest score (2.6/5) due to uncontrolled lactic acid formation. Nevertheless, no significant differences were seen for taste, mouthfeel and total acceptance of doogh samples (p > 0.05). A study demonstrated that probiotic beverages containing L. casei included high acceptance, compared with beverages containing L. acidophilus due to desirable flavors [58].

1. Conclusion

These non-viable probiotics have been shown to eleminate technological limitations by enhancing rates of titratable acidity and fermentation, texture, viability of starter bacteria including S. thermophilus and L. bulgaricus and decreasing post-acidification rate as well as potentially improving gut health and immunity by increasing antioxidant capacity of doogh samples. Further studies are needed to fully understand mechanisms of these effects and optimize formulation of non-viable probiotics in fermented milk products. Overall, findings suggest that incorporating non-viable probiotics into fermented milks can be a valuable strategy for enhancing functional characteristics of dairy products.

1. Acknowledgements

Please write one paragraph

1. Conflict of Interest

The authors report no conflicts of interest.

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Background and Objective: The objective of this study was to assess antimicrobial effects of silver nanoparticles on Gram-positive and Gram-negative bacteria that used in preparing silver nanocomposite with the antibacterial characteristics using solution method. Moreover, the aim of the current study was to produce antimicrobial silver nanocomposites for food coating with their effects on a wide range of bacteria.

Material and Methods: To assess antibacterial characteristics of silver nanoparticles, several steps were carried out. First, nanoparticles were synthesized through a chemical reduction method using NaBH4 and then analyzed using x radiation diffraction, ultraviolet and visible spectroscopic analysis, dynamic light scattering and scanning electron microscopy nanometric assays. Then, Staphylococcus aureus and Escherichia coli were used as Gram-positive and Gram-negative bacterial indicators. Minimum inhibitory concentration, minimum bactericidal concentration and inhibition zone levels were measured. Nanocomposite was produced using solution blending method and its antibacterial characteristics were assessed using inhibition zone method.

Results and Conclusion: Results indicated that silver nanoparticles with 20 and 50 µg.l^-1 concentrations included inhibitory effects on Staphylococcus aureus and Escherichia coli, respectively. Furthermore, concentrations of 40 to 60 mg.l^-1 included lethal effects on Staphylococcus aureus and Escherichia coli, respectively. Based on the results, the highest antibacterial effects were observed on Gram-positive Staphylococcus aureus. In inhibition zone assays, a 3-5 mm zone was seen around the silver nanoparticle discs in cultures of the microorganisms. In the inhibition zone assay of the produced nanocomposites, the zone was expected regarding the concentrations. Results were calculated in three repetitions and the value estimated through ANOVA was significant when p<0.0001. It has been concluded that silver nanoparticles are useful in Gram-positive and Gram-negative bacteria for the inhibition and destruction. Moreover, it has been verified that using the method includes great effects on antibacterial characteristics of the nanocomposites.

Conflict of interest: The authors declare no conflict of interest.

 

 

*Corresponding authors:

 

Hamed Ahari *

Food Biotechnology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran

 

Tel: +98-9121872334

 

E-mail:

dr.h.ahari@gmail.com

 

 

1. Introduction

 

In recent years, use of metal nanoparticleshas increased due to the resistance of pathogenic microorganisms (bacteria, fungus and viruses) against conventional antimicrobials. General concerns on the safety and quality of foods, particularly marine foods, during storage and stocking have led the microbial growth control a fundamental part of the distribution and storage chain of such products [1-4]. Based on various studies and assessment of silver nanoparticle function mechanism against microorganisms, their use as antimicrobial agents, especially in the food and medical industries, can be one of the novel solutions for conquering problems caused by the pathogenic microorganisms. Food products are infected by various microbial agents during production processesss. Infections may occur in formulation of ingredients, using chemical additives and posing high pressure and flash pasteurization. Harmful materials likely enter food formulation during this process, or chemical reactions may occur with dangerous outcomes to humans.

If food products are in contact with contaminated surfaces or surfaces with metal ions during the production processes, this can endanger consumers’ health. Therefore, use of proper antimicrobial packaging based on bio-polymers is nowadays highly interested due to being biodegradable, lack of collection of various synthesized materials in the natural ecosystem and improvement of mechanical and viscoelastic characteristics as well as appropriate antimicrobial characteristics [5]. Liao et al. studied the antibacterial activity and action mechanisms of silver nanoparticles (AgNPs) against Pseudomonas aeruginosa resistant to several medicines. In that study, use of morphological changes and assessment of active oxygen and activity of enzymes in the bacteria when exposed to AgNPs as well as reporting minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) showed the potential antibacterial effects of AgNPs on the bacteria [6]. Jo et al. investigated antibacterial characteristics of polyethylene and polypropylene nanocomposite films using AgNPs. First, nanocomposites were prepared using melting method and extruder. Based on the results, these nanocomposites included a 99.9% destructive effect on Staphylococcus aureus and Escherichia coli bacteria. This result showed well that using these nanocomposites could be effective and efficient in food packaging [7]. Furthermore, researchers synthesized degradable films with mixed clay and polyvinyl alcohol (PVA). They used these films against essential food pathogens such as Salmonella typhimurium and Staphylococcus aureus. Their results could reflect high antimicrobial effects of these nanoparticles, their mechanical characteristics and appropriate flexibility caused by PVA, as well as biodegradability of these films, which were verified through burring assaessment of them in the ground. To show efficiency of the highlighted packaging bags, shelf life of the chicken sausage samples was compared with that of regular polyethylene bags. Results showed enhancements in shelf life by decreasing microbial loads [7].

Mathew et al. synthesized nanocomposites, combining clay and biodegradable PVA. Based on their findings, the combined nanocomposite films included sufficient antimicrobial characteristics against food pathogens such as S. typhimurium and S. aureus and higher mechanical characteristics such as resistance against water and light transmission, compared to control films. The soil burial assay revealed that the nanocomposites degraded within 110 d and hence were considered biodegradable. Then, nanocomposite combined films were included in the bags used for keeping chicken sausages, which resulted in decreases in microbial loads compared to the control polyethylene bags and were much more effective in increasing shelf life of the chickens [8]. Findings from their study were similar with those of the the current study.

Liao et al. studied antibacterial characteristics and mechanisms of AgNPs against P. aeruginosa resistant to drugs. In this study, antimicrobial effects of AgNPs on resistant clinical isolates against P. aeruginosa with MIC and MBC were investigated. Morphological changes were observed in P. aeruginosa resistant against drugs under transmission electron microscopy (TEM). Distinct protein highlighted in the proteomics approach was studied quantitatively and production of reactive oxygen species was assessed using 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) coloring. Activity of superoxide dismutase (SOD), catalase and peroxidase was chemically assessed and apoptosis effects were studied through flow cytometry. Findings revealed that AgNPs included strong inhibitory effects on P. aeruginosa resistant against the antimicrobials with MIC of 1.406-5.625 mg.ml^-1 and MBC of 2.81-5.62 mg.ml^-1. Results of TEM revealed that AgNPs could penetrate resistant bacteria and disrupt their structure. Furthermore, quasi-apoptosis in bacteria affected by AgNPs was significantly higher. General findings and the estimated p-value (p<0.01) revealed strong antibacterial effects of AgNPs on multiresistant P. aeruginosa [9]. Active packaging incorporating AgNPs becomes popular due to its efficacy in combating foodborne pathogens. This technology uses AgNPs directly embedded in the packaging materials or adsorbed as ions, offering a safe effective antimicrobial shield. Recent approvals by the European Food Safety Authority (EFSA) for specific silver compounds further facilitates broader implementations [10].

This study investigated antibacterial effects of AgNPs on Gram-positive and Gram-negative bacteria and produced silver nanocomposites with appropriate antibacterial characteristics using solution blending. As previously stated, the present experimental study investigated use of AgNPs synthesized via chemical resuscitation method by assessing their MIC, MBC and inhibition zone against S. aureus, E.coli and Candida albicans, leading to decreases of food spoilage and enhancement of food shelf life. Moreover, their antimicrobial effects were studied as alternatives to antimicrobials.  The aim of this study was to investigate antibacterial effects of AgNPs on Gram-negative and Gram-positive bacteria and produce silver nanocomposites with appropriate antibacterial characteristics using solution blending method.

1. Materials and Methods

2.1. Synthesis and characterization of silver nanoparticles

The AgNPs were synthesized using chemical reduction method with sodium borohydride (NaBH₄). Dynamic light scattering (DLS) verified the particle sizes within the desired range of 25-40 nm. The X-ray diffraction (XRD) analysis revealed crystal structure of the materials, while UV-VIS spectroscopy provided information on nanoparticle size and homogeneity. Additionally, scanning electron microscopy (SEM) visualized morphology of the synthesized nanoparticles.

2.2. Antimicrobial activity assessment

Culture media and autoclaves were sterilized. Each experimental tube included 5 ml of culture media, 100 mg of bacteria/fungi and calculated concentrations of AgNPs. Triplicate experiments were carried out.

2.3. Minimum inhibitory concentration and minimum bactericidal concentration assessments

Microdilution assay in gamma tubes was used. Nutrient broth was used for S. aureus and E. coli and Sabouraud dextrose (SD) broth for C. albicans. Standardized inocula (100 μl) were added to the broths and incubated at 37 °C for 24 h. The MIC was assessed as the lowest concentration inhibiting visible growth. The MBC included plating 100-μl aliquots onto agar media (nutrient agar for bacteria and SD agar for fungi) and incubating at 37 °C for 24 h. Moreover, MBC was defined as the lowest concentration demonstrating no microbial growth or less than three colonies (99-99.5% killing).

2.4. Nanoparticle synthesis method

First, sodium borohydride was dissolved in water (ice bath) and mixed with polyvinylpyrrolidone (PVP). Silver nitrate solution was then added to the mixture, which changed the color from yellow to orange, brown and then black. This was then stirred quickly (~1500 rpm) at 50–60º C, which created clods. Drops of silver nitrate (0.001 M) were added to sodium borohydride (0.002 M) set in the ice bath, which changed color of the final product to yellow. This color became darker over time. To increase stability of the product, 1% PVP was added to the solution, which changed color of the product to pale orange-red. Concentration of the colloidal nanosilver was 6 mg.ml^-1 and based on the UV-VIS assay, size of the particle was 25–40 nm. The quantity of PVP used for increasing stability was 3 mg.ml^-1.

2.5. Nanocomposite production through solution blending

In brief, 500 ml of the polymer PVA were divided into five beakers with volume of 0.28, 0.42, 0.83, 1.67 and 2.5 ml. Then, AgNPs were added to 12.5 25, 50, 100 and 150 ml with a concentration of 6 mg.ml^-1. These were stirred on a stirrer heater at 50 ºC for 24 h until volume of the solution reached 20 ml. The final product was poured into a Petri dish and set in the oven for 24 h to dry. Then, the final composite with similar thickness and appropriate level of flexibility was ready.

2.6. Analysis of the size of nanoparticles using dynamic light scattering method

In general, DLS is a technique used to assess particle sizes in solutions and suspensions. In this method, specialized devices analyze the motion of particles while they are suspended in a liquid. It provides a rapid and non-destructive way to assess particle sizes, ranging from nano to micrometers. For example, researchers transferred nanosilver colloids into the DLS device cell. The subsequent analysis estimated the particle size. Specifically, 5 ml of nanosilver colloid were analyzed at 25 °C with a laser strength of 60%.

2.7. UV-VIS analysis

Characteristics of photons on samples and measuring the rate of passage or absorption (rate of adsorption or reflectance of light) in various wavelengths ranging 200-1100 nm. Results of the assay were presented in a typical surface absorption plasmon at 420 nm  achieved from the AgNPs.

2.8. Scanning electron microscopy analysis

The SEM is an exceptionally well-suited method for the study of nanoparticle structure and it depicts the size of AgNPs in ranges from 10 to 100 nm. No agglomeration was seen in nanoparticles, showing stabilization of the nanoparticles.

1. Results and Discussion

3.1. Analysis of the nanoparticle characteristics

The DLS results from Fig. 1 (a, b, c) revealed essential information on AgNPs. Based on the figure, AgNPs demonstrated the following characteristics. Number distribution, approximately 41% of the particles within specific size ranges; volume distribution, nearly 48% of the particles contributing to the overall volume; intensity distribution, significantly 92% of the scattered light intensity originating from the specific particle sizes.

Additionally, Fig. 2 shows a SEM image of AgNPs synthesized through chemical reduction. These nanoparticles exhibited spherical shapes and included a size range of approximately 25 to 40 nm.

Furthermore, Fig. 3 presents the UV-VIS diagram, providing further characterizations of the AgNPs. To assess structural characteristics of the nanocomposite films, XRD analysis was used. Nanosilver samples were irradiated with Cu-Kα radiation (λ = 54.1 Å) using X-ray spectrometer operating at 40 kV and 30 mA. The resulting XRD pattern provided valuable information on the crystalline phases and crystallographic orientation within the nanocomposite films (Fig. 4).

 

 

 

 

1. Nanoparticles with a size of 145.8 nm

Figure 1. Dynamic light scattering diagram of the produced silver nanoparticles with various sizes: a, 121.9, b, 145.8 and c, 150.3

 

 

 

 

 

Figure 2. Scanning electron microscopy of the silver nanoparticle synthesis using NaBH₄

 

Figure 3. The UV-VIS diagram of the produced silver nanoparticles

 

 

 

 

 

 

Figure 4. The X-ray diffraction diagram of the produced silver nanoparticles

 

 

 

3.2. Minimum inhibitory concentration and minimum bactericidal concentration assessments

The MIC and MBC of the biosynthesized nanoparticles were assessed against various pathogens. Nanoparticles showed potential antibacterial activities against E. coli (MIC, 50 µg.ml^-1 and MBC, 70 µg.ml^-1) and S. aureus (MIC, 25 µg.ml^-1 and MBC, 45 µg.ml^-1). However, nanoparticles demonstrated weaker antifungal activities against C. albicans (MIC, 350 µg.ml^-1 and MBC, 380 µg.ml^-1). Results suggested potentials of these nanoparticles as broad-spectrum antimicrobials. Although further optimizations may be necessary for the enhanced antifungal efficacies (Fig. 5).

3.3. Antimicrobial susceptibility assay for the assessment of inhibition zone diameters (disk diffusion)

To assess antibacterial and antifungal activities of the AgNPs, inhibition zone assay was used via diffusion disks impregnated with various concentrations (200 and 6000 µg.ml⁻¹). Three microorganisms were assessed, including S. aureus, E. coli and C. albicans. Isolated bacterial colonies of S. aureus and E. coli were suspended in sterile serum, creating a homogenous solution. This solution was then streaked onto agar plates using sterile swabs. Blank disks loaded with either AgNPs or control antibiotics (amikacin for bacteria and itraconazole for fungi) were transferred onto the inoculated plates. Following incubation at 37 °C for 24 h, diameters of the resulting inhibition zones around the disks were measured using caliper. For the nanocomposite disks, another experiment was carried out, where blank disks were punched out and loaded with various nanoparticle concentrations. These disks were then added to the bacterial cultures and the inhibition zones were measured as described. Results of this study provided information on the potentials of the AgNPs as antimicrobial agents against various pathogens (Table 2).

Findings of MBC and MIC assays verified that by prohibiting microorganisms, AgNPs could increase the shelf life of foods (Table 1). Results revealed the higher effects of AgNPs on Gram-positive S. aureus, compared to Gram-negative E. coli. According to Abbaszadegan et al., the major reason for differences in antibacterial effects of Gram-positive and Gram-negative bacteria included the quantity of peptidoglycan in the bacteria cell wall. Ggram-positive strains included further peptidoglycans in their cell walls, compared to those Gram-negative strains did, allowing AgNPs to include extended inhibition zones for these strains.

This study demonstrated effectiveness of AgNPs in extending food shelf life by inhibiting microbial growth. Studies, including those by Abbaszadegan et al. and Eslami et al., highlighted the nanoparticle efficacy against various bacteria, with Gram-positive strains such as S. aureus exhibiting a greater susceptibility, compared to that Gram-negative E. coli doing. This difference was attributed to the thicker peptidoglycan layer in Gram-positive bacterial cell walls, offering a larger target for AgNPs.

Researchers investigated the potential of AgNPs to combat microbes in various settings, including food preservation. In a study by Eslami et al. (2016), effectiveness of AgNPs in preserving saffron was investigated. Various concentrations of nanoparticles were incorporated into packaging materials and the microbial loads on the saffron were monitored over time.

 

Figure 5. Statistical findings of the minimum inhibitory concentration assay using ANOVA

 

Table 1. Estimated nanosilver concentrations for MBC, MIC and MFC

 

Table 2. Results of the inhibition zone assay for silver nanoparticles and nanocomposites

 

 

Results showed significant decreases in microbial growth, particularly at higher nanoparticle concentrations, highlighting the potential of this technology for extending the shelf life of food products [10]. Antibacterial activity of the AgNPs against hospital-acquired antibiotic-resistant strains of P. aeruginosa was assessed by Salomoni et al. [11]. Commercial nanoparticles effectively inhibited bacterial growth at specific concentrations, suggesting their potentials as tools to combat challenging infections. Further studies such as that by Alsharqi et al. deeper investigated mechanisms; by which, AgNPs exert their antimicrobial effects. Their in-vitro experiments demonstrated that nanoparticles interacted with bacterial cell membranes, ultimately suppressing bacterial growth. Significantly, this effect was observed against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, although various degrees of susceptibility were observed [12]. Similar to the current findings, these studies collectively present encouraging evidence for the use of AgNPs as a novel antimicrobial strategy. Further studies are needed to assess their potential uses and safety profiles.

The SEM images of the treated bacteria cells revealed significant morphologic changes in the cell membranes after processing with AgNPs. Results indicated strong antibacterial reactivities in AgNPs that could inactivate harmful and pathogenic microorganisms [12]; similar to results of the present study.  Previous findings showed that AgNPs directly attacked the cell membrane of bacteria, causing significant morphological changes [12]. This verified strong antibacterial activities of these nanoparticles, further supporting their potentials to combat harmful microorganisms.

Yan et al. [13] investigated broadly mechanisms of action using proteomics approaches, revealing 59 proteins affected by silver interactions. Interestingly, silver interacted with several membrane proteins and triggered production of ROS within the bacteria. This ROS production ultimately damaged the cell membrane, leading to bacterial death. These findings were perfectly similar to findings from the current study and other studies, highlighting the potential antimicrobial roles of AgNPs. Additionally, Pooyamanesh et al. [14] successfully incorporated AgNPs into food packaging materials, demonstrating their effectiveness against various foodborne bacteria such as E. coli and S. aureus. This evidence strongly suggests that AgNPs include tremendous potentials in combating harmful bacteria, opening doors for novel uses in food preservation. However, further studies are necessary to fully understand their safety and optimize their effectiveness for various uses.

1. Conclusion

This study has validated potentials of AgNPs and their nanocomposites for antimicrobial uses in food packaging. Findings from MIC, MBC and inhibition zone assays consistently have demonstrated their effectiveness against various bacteria, especially Gram-positive strains. These provide direct benefits for food preservation, extending shelf life while eliminating needs of harmful chemical additives. Integrating AgNPs into food packaging offers more than a chemical-free alternative; it presents a multifaceted solution with far-reaching benefits. First, it develops organic food production by effectively combating bacteria without conventional preservatives, fostering trust and enhancing food quality. Second, their significant antibacterial ability originates from their high surface areas and positive charges, disrupting the bacterial membranes and significantly extending food shelf lives. Third, the chemical resuscitation method allows for precise control of nanoparticle sizes, tailoring their interaction with specific bacteria for optimized performance. Fourth, the suggested solution blending method improves cost-effectiveness, making this innovative technology readily accessible. While further studies are critical to understand long-term effects and ensure responsible implementation, these diverse advantages offer AgNPs as a promising solution for the challenges of food preservation. While Gram-positive bacteria have demonstrated greater susceptibilities due to their cell wall structures, effectiveness of AgNPs even at authorized low concentrations and their minimal risks of release into foods further highlight their potentials as safe sustainable alternatives to the available antimicrobial agents. This study pioneers further investigations and optimization of silver nanoparticle-based food packaging solutions, offering a promising path towards enhanced food safety and decreased environmental adverse effects. However, it is important to acknowledge needs of continuous studies to comprehensively understand potential long-term effects of the current technology.

1. Acknowledgements

Special thanks to the Nano Research Laboratory (Ultrasonic Section), Science and Research Branch of Islamic Azad University.

1. Conflict of Interest

The authors declare no conflict of interest.

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Background and Objective: The antihyperglycemic effect is associated with the pre-hispanic fruit xoconostle or tunillo (Stenocereus stellatus, Pfeiffer and Riccobono). This fruit includes in various varieties, distinguished by color. Xoconostle fruits are highly perishable. Therefore, the aim of this study was to assess antihyperglycemic effects of xoconostle juice before (fresh) and after pasteurization. The study focused on the white and red varieties of xoconostle.

Material and Methods: In this study, the method involved collecting juice from xoconostle fruits, followed by pasteurization. Chemical, physical and microbial parameters were assessed for the juice and the ability to decrease capillary glucose levels (antihyperglycemic effect) was assessed in male Wistar rats.

Results and Conclusion: Pasteurization process led to decreases in total phenolic content of the red variety of xoconostle fruit, while the white variety showed increases in malic acid content. Despite these changes, fresh and pasteurized juices of the two varieties showed lower blood glucose levels, compared to the control group. Red variety demonstrated a stronger antihyperglycemic effect. In conclusion, pasteurization did not affect pharmacological effects of xoconostle juice, making it a viable preservation method without compromising the antihyperglycemic charac-teristics. Results of this research suggest a conservation method which preserve the antihyperglycemic effects while extending its shelf life.

Conflict of interest: The authors declare no conflict of interest.

Background and Objective: Meat can be contaminated by Leptospira species. This bacterial pathogen causes severe leptospirosis disease in humans and animals. The major aims of this study were to assess seroepidemiological prevalence of leptospirosis in employees of a slaughterhouse in Guilan Province, Iran, using microscopic agglutination test and further investigate the positive samples using nested polymerase chain reaction method.

Material and Methods: In this study, 150 employees of a slaughterhouse in Guilan Province, Iran, were participated after completing written consents and personal questionnaires. This sample size was calculated based on the mean prevalence of the pathogen in the region. After assessing sera of the participants for Leptospira antibody using microscopic agglutination test, urine samples were collected from the positive participant for further assessments using nested polymerase chain reaction.

Results and Conclusion: Based on the results, microscopic agglutination test was positive for 10.7% of the participants. However, Nested-PCR was negative for the positive microscopic agglutination tests on sera collected from the participants with antibodies against Leptospira antigens. The current results demonstrate that Leptospira can occur in asymptomatic humans in slaughterhouses and highlight the high potential of the disease transmission to humans in the province. Therefore, further extended control and prevention measures for slaughterhouse workers are recommended to guarantee the food safety.

Conflict of interest: The authors declare no conflict of interest.

Abstract

Background and Objective:

Black grass jelly served in sweet syrup is one of the Chinese and East and Southeast Asian traditional beverages. An innovative enrichment can make it a better functional food. This study innovatively enriched the black-jelly food with formulas of probiotic Lactobacillus plantarum Mar8, honey, D-allulose and mangosteen pericarp extract. The probiotic viability, antioxidant and hypoglycemic potential were investigated as well.

Material and Methods: Ready-to-drink functional beverages included mangosteen pericarp extract varied in concentrations of 0.1, 0.2 and 0.4 mg ml^-1, D-allulose in honey and encapsulated probiotic Lactobacillus plantarum Mar8 in black grass jelly containing konjac and carrageenan. The probiotic viability, antioxidant activity and hypoglycemic potential were the selective parameters for the functional beverage formulas. The viability of probiotic Lactobacillus plantarum Mar8 was assessed using total plate count method. Antioxidant activity was assessed based on the reaction of 2,2-Diphenyl-1-picrylhydrazyl radical scavenging. Hypoglycemic potential was investigated by counting petite yeast cells after treating with black grass jelly formulas. Significant differences were reported using one-way analysis of variance and Duncan's test. Statistically significance included p-values≤0.05.

Results and Conclusion: The probiotic Lactobacillus plantarum Mar8 encapsulated in black grass jelly survived well in the honey, D-allulose and mangosteen pericarp extract formulated beverages. Honey supported the probiotic viability better, producing further antioxidants and high potentials in hypoglycemia than that those of other formulas did. Mangosteen pericarp extract enriched the functionality of the black grass jelly probiotic beverages. However, further studies are needed to assess favorability and stability of this functional food.

Conflict of interest: The authors declare no conflict of interest.