In Vitro Antimicrobial Activity of Spices and Medicinal Herbs against Selected Microbes Associated with Juices

In the present investigation, comparison of antimicrobial activities of different spices, Curcuma longa, Zingiber officinale, and Mentha arvensis, and medicinal herbs, such as Withania somnifera, Rauvolfia serpentina, Emblica officinalis, Terminalia arjuna, and Centella asiatica, was evaluated. Different extraction solvents (acetone, methanol, ethanol, and water) were used and extracts were examined against Bacillus cereus, Serratia sp., Rhodotorula mucilaginosa, Aspergillus flavus, and Penicillium citrinum isolated from juices. Extracts from the medicinal herb and spices have significant activity. B. cereus was the most sensitive and R. mucilaginosa was the most resistant among the microorganisms tested. Ethanolic and methanolic extract of C. asiatica displayed maximum diameter of inhibition zone against bacteria and yeast and percentage mycelial inhibition against moulds. This study confirmed the potential of selected extracts of spices as effective natural food preservative in juices.


Introduction
Emergence of new technologies in food preservation leads to a reduction in the levels of preservatives and promotes the use of "naturally derived antimicrobials of animal, plants, and microbial origin [1]. Antimicrobial compounds derived from plants were used for centuries in food preservation. Egyptians, Chinese, and Indians used spices and essential oils since ancient time. Some of the spices such as mint, garlic, and ginger are still practiced in alternative health remedies in India [2,3]. About 250 to 500 thousand plant species are estimated to exist on the planet and only between 1 and 10% of them are used as food by humans and other animals [4]. Spices and herbs used in foods as flavoring agents, in addition to enhancing flavors, were used as folk medicines and food preservatives. Spices and herbs extend the shelf life of foods by restricting rancidity through their antioxidant activity or through their bacteriostatic and bactericidal activity [5].
Spices, herbs, and their constituents are generally recogonised as safe (GRAS) and approved by several regulatory agencies such as US Food and Drug Act, the European Union standards, Codex Alimentarius, and Food Safety and Standards Authority of India [3]. In India, the trend of consumption of spices and herbs in food or using them as medicine aims to maintain proper sanitation, health, and hygiene and to increase longevity of life. Several spices such as ajowan, clove, ginger, black pepper, cumin, and asafetida are commonly used in the Indian diet [3,6,7]. Literature cited the work of several authors on the antimicrobial activity of plants against wide range of bacteria, yeasts, and moulds [2,3,[8][9][10][11][12][13][14][15][16][17][18][19][20]. The general description of spices and herbs used in the present study is tabulated in Table 1.
Spices and herbs, owing to their natural origin, attract more attention of consumers that have doubt regarding the safety of chemical preservatives. Several plant extracts have gained momentum in recent years due to their bioactive principals and formed the basis of pharmaceutical and food processing industries [17,21].
Unpasteurized fruit juice consumption has increased in the last decades, which is attributed to the contents of antioxidants, vitamins, and minerals. Fresh fruit juices are highly vulnerable to spoilage, since fluid components are in contact with air and microorganisms form the environment while handling [22]. Fruit juices spoilage bacteria include acid 2 International Journal of Microbiology Certain common moulds such as Penicillium sp., Aspergillus sp., Eurotium, Alternaria, Cladosporium, Paecilomyces, and Botrytis have also been reported in spoilage of fruit juices. The presence of pathogenic bacteria and mycotoxin producing mould cannot be ruled out in fruit juice which has been responsible for the increase in food borne outbreaks with the consumption of fresh fruit juices during the last two decades [14,23,24]. Therefore, the main objective of this study was to examine the in vitro antimicrobial activity of different spices and herbs extracts and to compare the effect of different solvents in the extraction method for antimicrobial activity.

Extraction of Plant Material.
Four different solvents, namely, ethanol, methanol, acetone, and aqueous (hot and cold), were used for extraction and plant extracts were prepared according to the methods described by Sharma et al. [25].

Test Microorganisms.
In the previous study [24], microbiological analysis of fruit juices was done by serial dilution agar plate technique. On the basis of percentage of occurrence of microorganisms in juice samples, one Gram-positive bacterium, one Gram-negative bacterium, one yeast, and two moulds were selected for examining the antimicrobial activity of spices. Bacterial strains were identified on the basis of gram staining and biochemical and molecular characteristics (16S rRNA sequencing) [23]. Yeast was identified on the basis of staining, morphological, cultural characteristics, and molecular characteristics (28S rRNA sequencing). Moulds were identified on the basis of morphological and cultural characteristics and further identification was confirmed by CABI International Mycological Institute, UK.

Screening for Antimicrobial Activity against Bacteria and
Yeast. The acetone, methanol, ethanol, and hot and cold aqueous extracts of different plants were used for evaluation of antimicrobial activity by the agar well diffusion method. In this method, a pure isolate of bacteria and yeast was grown on NA and PDA plates and incubated at 37 ∘ C and 25 ∘ C for 24 h and 72 h, respectively. One plate of each microorganism was taken and colonies were transferred into normal saline (0.85%) under aseptic conditions. Density of each microbial suspension was adjusted to be equal to that of 10 6 cfu/mL (standardized by 0.5 McFarland standard) and to be used as the inoculum for performing an agar well diffusion assay. 100 L of the inoculum of each test organism was spread onto the agar plates so as to achieve a confluent growth. The agar plates were allowed to dry and 8 mm wells were made with a sterile borer in the inoculated agar plates. The lower portion of each well was sealed with molten agar medium. The dried extracts were reconstituted to 20% in dimethyl sulfoxide (DMSO) to the final concentration of 100 mg/mL for the bioassay analysis. A 100 L volume of each extract was propelled directly into the wells (in triplicate) of the inoculated agar plates for each test organism. The plates were allowed to stand for 1 h at room temperature (40 ∘ C) for diffusion of the extract into agar and incubated at 37 ∘ C and 25 ∘ C for 24 h and 72 h, respectively. Sodium benzoate (100 mg/mL) was used as positive reference standards to determine the sensitivity of each microbial species tested. Sterile DMSO served as the negative control. The antimicrobial activity, indicated by an inhibition zone surrounding the well containing the extract, was recorded if the zone was greater than 8 mm. The experiments were performed in triplicate and the mean values of the diameter of inhibition zones ± standard deviations were calculated [34].

Screening for Antimicrobial Activity against Moulds.
The antimould activity of plant extracts in different solvent was accessed by poison food technique. 100 L of plant extract with concentration of 100 mg/mL was poured into sterile Petri plate (90 mm diameter) and 15 mL of molten potato dextrose agar (PDA) was added to the Petri plate and swirled to achieve a uniform mixture and allowed them to solidify at room temperature. The solidified agar plates were inoculated at the centre with fungal disc (6 mm diameter), obtained from the actively growing one-week-old colony of the test fungus, placed with inoculums side down in the centre of each Petri plate aseptically and incubated at point 25 ∘ C for 7 days. DMSO was used as negative control and chemical preservative sodium benzoate served as the positive control. The antifungal activity of each extract was evaluated by measuring the radial growth of fungus in terms of diameter and expressed as percentage mycelia inhibition determined by applying the following formula [35][36][37]: inhibition of mycelia growth% = ( − ) × 100, (1) where is average diameter of fungal colony in negative control plates and is average diameter of fungal colony in extract added plates.

Determination of Minimum Inhibitory Concentration.
Minimum inhibitory concentration (MIC) for each test organism was determined by the broth macrodilution method [38].

Statistical Analysis.
The experimental results were repeated thrice in triplicate each time and expressed as mean ± SD and results were statistically evaluated using SPSS software version 16 at 5% significant level. Means were compared using Tukey's simultaneous test set at < 0.05.

Antimicrobial Activity of Plant Extracts.
The dietary herb and spices are used as food additives in foods not only to improve the sensory characteristics of food but also to increase the shelf life by reducing or eliminating survival of pathogenic bacteria [2].
In the present study, the antimicrobial activity of the different plant extracts in different solvents was examined. Perusal data in Table 2 revealed the mean diameters of the inhibition zones of all plant extracts against two bacteria and yeast and percentage mycelia inhibition against two moulds. There was significant variation ( < 0.05) observed between acetone, methanol, ethanol, cold aqueous, and hot aqueous solvents for the antimicrobial activities of each of the tested plant extracts and microorganisms.
For Serratia (KC67407), a total of 25 extracts (62.5%) exhibited inhibitory activity with DIZ values ranging from 11.3 mm to 22.6 mm, and 47 extracts had low activity. The remaining 15 extracts had no inhibitory activity ( Table 2). The sample with strongest antimicrobial activity was methanolic extract of T. arjuna (DIZ = 22.6 mm), followed by acetonic extract of R. serpentina (21.3 mm).
Of the two moulds tested, for A. flavus, the percentage mycelial inhibition of 8 extracts (20%) varied from 20.6 mm to 26.3 mm. Nine extracts (22.5%) had low inhibitory activity (percentage mycelial inhibition ranging between 11.6 mm and 19.3 mm). The other 23 extracts (57.5%) showed no inhibitory activity. Ethanolic extract of C. asiatica exhibited the strongest antimould activity (percentage mycelial inhibition = 26.3 mm), followed by methanolic extract of C. asiatica and T. arjuna (24.6 mm). For P. citrinum, 8 extracts exhibited high inhibitory activity (percentage mycelial inhibition = 19.6 mm-24.3 mm) and nine extracts possessed less inhibitory activity (percentage mycelial inhibition ranging between 10.3 mm and 19.3 mm), while the other 23 extracts had no inhibitory activity. Methanolic extract of C. asiatica (24.3 mm) showed the strongest inhibitory activity, followed by ethanolic extract of C. asiatica and W. somnifera (23.3 mm).
Literature search revealed the in vitro and in vivo antimicrobial activities of plant extracts and essential oils against food borne pathogens but they are difficult to compare owing to the use of different methods of extraction, solvents, microbial strains, and antimicrobial test methods [3,5,16,17,39,40]. In the present analysis, 4 plants, C. asiatica, E. officinalis, M. arvensis, and T. arjuna, exhibited broad spectrum activity against tested microbes. The present results were in line with the observation of earlier workers [5-7, 15-17, 41, 42]. Methanolic and ethanolic extract of C. asiatica exhibited maximum zone of inhibition against all tested microorganisms in the present investigation. The antimicrobial potential of C. asiatica was observed by Arumugam et al. [43] in a study to check the antimicrobial efficacy of C. asiatica against B. cereus, S. aureus, and Pseudomonas aeruginosa in four different solvents such as methanol, chloroform, water, and acetone and they found that methanolic extract of C. asiatica leaves at the concentration of 50 g per mL displayed activity (24-27 mm) against all tested microorganisms. The antimicrobial efficacy of C. asiatica is due to the presence of terpenoids [44]. Acetonic extracts of T. arjuna displayed maximum zone of inhibition (16-28 mm) against S. aureus, Acinetobacter, P. aeruginosa, and Proteus mirabilis in comparison to the other extracts (methanolic, ethanolic, and water extracts); however, all the extracts lacked activity against Candida albicans [32]. In the present investigation, methanolic and ethanolic extracts of T. arjuna exhibited antimicrobial activity against all tested microbes due to the presence of flavonoids (luteolin) [45,46].
Ethanolic extract of E. officinalis leaves showed more activity against P. aeruginosa, Proteus mirabilis, S. aureus, and B. cereus in comparison to ethanolic extract of M. arvensis leaves at the concentration of 100 mg/mL [47]. However, in the present investigation, the ethanolic extracts of M. arvensis displayed more activity in comparison to E. officinalis ethanolic extract at the concentration of 100 mg/mL. The antimicrobial potential of E. officinalis and M. arvensis is attributed to the presence of hydrolysable tannins and menthol, respectively [48]. Although, in present investigation, R. serpentina and W. somnifera revealed antibacterial and antimould activity but lacked antiyeast activity, several International Journal of Microbiology 5 authors confirmed antibacterial and antifungal potential of W. somnifera and R. serpentina [31,33,49,50]. Sunilson et al. [15] observed the antimicrobial activity of C. longa and Z. officinalis in four different solvents (petroleum ether, chloroform, methanol, and water) against food borne pathogens. The solvent extracts of C. longa and Z. officinalis displayed antibacterial and antiyeast activity; however, in present investigation, C. longa and Z. officinalis possessed antibacterial activity.
The extraction of biologically active compound from plant material is largely dependent on the type of the solvent used in the extraction procedure. The present study revealed that the organic extracts provided more powerful antimicrobial activity compared to aqueous extracts. Among 6 International Journal of Microbiology  [3,5,16,17,48,[51][52][53]. This is attributed to the differences in the outer layers of Gramnegative and Gram-positive bacteria. Gram-negative bacteria possess an outer membrane and a unique periplasmic space not found in Gram-positive bacteria [5,54,55].

Determination of Minimum Inhibitory Concentration.
The minimum inhibitory concentration was determined for 31 active plant extracts which show antimicrobial activity against microbes associated with juices (

Conclusion
The results of present work established that all the tested plant extracts possess antimicrobial activity against selected microbes associated with juices. Alcoholic extracts of medicinal herbs such as C. asiatica, T. arjuna, and R. serpentina displayed better antimicrobial activity than chemical preservative sodium benzoate; therefore these plant extracts have the potential to extend the shelf life or they are used as natural preservatives in fruit juices.