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Antiinflammatory Activity of Slime Extract from Giant African Snail (Lissachatina fulica)

The present study aims to investigate the antiinflammatory activity of the slime extracts from the giant African snail, Lissachatina fulica. In this study, aqueous and ethanol extracts of the snail slime were evaluated for antiinflammatory activities in vitro. The aqueous extract showed greater antiinflammatory activities than the ethanol extract. The aqueous extract was further tested for antiinflammatory activity in vivo. For acute inflammation, the ear oedema assay was used to screen the slime extract. The results revealed that application of 2 mg/ear extract or phenylbutzone could significantly reduce oedema when compared with ethanol-treated ears. For chronic inflammatory test, the cotton pellet granuloma model was employed to determine the activity of the slime extract. It was found that 2.5 and 6.25 % extract could significantly reduce transudative and granuloma weights. Moreover, the extracts also reduced malondialdehyde and increased superoxide dismutase in the granuloma tissue of the induced rats. In conclusion, the slime extract showed antiinflammatory activity both in vitro and in vivo. The antiinflammatory activity was also observed against acute and chronic inflammations, which suggested that the slime extracts has the potential to be developed into antiinflammatory products.

Inflammation is a common response to detrimental stimuli such as irritants, pathogens or damaged cells. The outcomes of inflammatory reaction are stimuli elimination and tissue repair [1] . Although inflammation is a beneficial strategy, the progression of the inflammatory process often causes unwanted outcomes such as pain, swelling, redness and loss of function of inflamed cells or even oxidative stress. Non-steroidal antiinflammatory drugs (NSAIDs) were developed to relieve these inflammatory symptoms, especially pain. Unfortunately, treatment of inflammation with NSAIDs produces negative side effects, for instance stomach irritation and cardiovascular problems [2,3] . Recently, natural products that have antiinflammatory properties have become an alternative way to relieve these symptoms. There are several scientific reports about antiinflammatory properties of natural products, mainly from medicinal plants, but there are not many about animal product.
Snail slime is a secretory fluid produced from mucus glands located in the skin including the foot plate. It is mainly used to reduce friction and protect its foot during locomotion. Moreover, carnivorous slug or shell-less snail secrete slime to maintain moisture and to help their mating and hunting activities [4] . Snail slime has become a remarkable ingredient in cosmetic products because it contains some biochemical substances, which have properties useful to skin care, such as alantoin, elastin, collagen and hyaluronic acid [5] . Furthermore, the slime has also been reported to have pharmacological activities including antibacterial activity [6,7] , antioxidant and regenerative properties [8] and anticancer property [9] . Interestingly, the slime extracted from Achatina fulica has been reported to heal incised wounds faster than self-healing can [10] . In the wound healing process, the healing stages start after inflammatory products and dead cells were cleared out of the wound site [10,11] . Therefore, inflammation is an important stage, which directly affects the healing process. Due to the ability to accelerate wound healing, snail slime was assumed to have antiinflammatory activity. However, there is no scientific evidence for the antiinflammatory activity of snail slime. Therefore, the aim of this study was to evaluate the antiinflammatory activity of slime from the giant African snail (Lissachatina fulica) using various in vitro and in vivo methods.

Snail collection, identification, Slime collection and extraction:
Thirty African snails (adult stage) were collected from Chiang Mai, Thailand. These land snails were morphologically identified according to Bouchet et al. [12] . Snails were stimulated to secrete slime by putting them on a plate, which was immersed in warm water (32°). The snails were scrubbed and then the slime was squeezed out from the snail body. The slime was collected and extracted with distilled water and ethanol. The slime was mixed with each solvent and stirred for 1 h and then centrifuged at 3500 rpm for 10 min. Supernatants of the slime were collected, lyophilized as aqueous and ethanol extracts (yield 7.0 and 1.4 mg extract/ml slime, respectively).

Evaluation of in vitro antiinflammatory activity, antiproteinase activity:
This test was done according to a modified method of Oyedepo et al. [13] and Sakat et al. [14] . Two millilitres reaction mixture (0.06 mg trypsin, 1 ml of 20 mM pH 7.4 Tris HCl buffer and 1 ml of the sample) was incubated at 37° for 5 min, then 1 ml of 0.8 % casein was added and incubated again for 20 min, 2 ml of 70 % perchloric acid was added and then the cloudy suspension was centrifuged. The absorbance of the supernatant was measured at 210 nm, and sodium salicylate was used as a reference standard. The obtained absorbance was converted to percent antiproteinase activity using the Eqn. 1, % inhibition = [(Abs control-Abs test)/(Abs control)]×100.

Antilipoxygenase activity:
Antilipoxygenase activity of extracts was evaluated using the method described by Shinde et al. [15] . The sample was prepared by dissolving the extract in 2 M borate buffer (pH 9.0), 0.25 ml of prepared sample was mixed with 0.25 ml of lipoxidase (20000 u/ml) and incubated at 25° for 5 min. After that, 1.0 ml of 0.6 mM linoleic acid was added, mixed well and absorbance was measured at 234 nm and % antilipoxygenase activity was calculated using Eqn. 1. In this test, indomethacin was used as a reference standard.

Inhibition of protein denaturation:
This assay was conducted according to the method of Kumari et al. [16] . The reaction mixture contained 0.2 ml of egg albumin, 2.8 ml PHS (pH 6.4) and 2 ml of extract was incubated at 37° for 15 min and heated at 70° for 5 min. After cooling to room temperature, absorbance of a sample was measured at 660 nm on a spectrophotometer. Percent inhibition of protein denaturation was calculated using Eqn, 1. Sodium salicylate was used as a reference standard.

Membrane stabilization:
Antihaemolytic activity was determined to find out the ability of the extracts to stabilize membranes. The procedure used was as described by Shinde et al. [15] . In this assay, red blood cells (RBC) suspension was prepared from the blood collected from rats (without NSAIDs), washed 3 times by centrifugation at 3000 rpm for 30 min with normal saline. The pellet was resuspended to make a 10 % RBC suspension for the experiment. In the antihaemolytic test, 1 ml of 10 % RBC suspension was mixed with 1 ml of the sample, heated at 56° for 30 min, cooled down under running tap water and centrifuged at 2500 rpm for 10 min. The absorbance of the supernatant was measured at 560 nm. Percent antihaemolysis activity was calculated from Eqn. 1. In this study, indomethacin was used as a reference drug.

In vivo antiinflammatory study:
Male Wistar rats (Rattus norvegicus) were purchased from the National Laboratory Animal Center, Salaya, Nakhon Pathom, Thailand. The rats were housed in a controlled room, 25±1° under 12 h light/dark cycle, with free access to water and food, for 7 d to acclimate. The experimental protocol used in this study was reviewed and approved by the Institutional Animal Care and Use Committee of the Biology Department, Faculty of Science, Chiang Mai University, Thailand (ID: Re 009/17).

Ethyl phenyl propiolate (EPP)-induced ear edema assay:
The procedure used in this assay was modified from the method of Kale et al. [17] . Male Wistar rats, weighing 35-40 g, were used in this study. Before treating, their ear thickness was measured with automatic Vernier calipers. Then, 2 doses of aqueous extract of snail slime (1 mg/ear and 2 mg/ear) and phenylbutazone (1 mg/ear), dissolved in 20 µl of 50% ethanol (vehicle) were applied on both inner and outer left ears. Also, the vehicle was topically applied to the right ears (the negative control) in the same manner. Ear edema was then immediately induced by topical application of 5 % EPP and ear thickness was measured after induction at 15, 30, 60 and 120 min. Percent inhibition of edema (% EDI) = (EDC-EDT)/EDC×100, where, EDC is the oedema of control group obtained from ear thickness at X min-ear thickness at 0 min, EDT is the oedema of test, ear thickness at X min-ear thickness at 0 min.

Cotton pellet-induced granuloma model:
The method used in the study was the method reported by Agrawal et al. [18] with a slight modification. Twenty male Wistar rats, weighing 180-200 g, were randomly divided into 4 groups (5 rats per group). The rats were anesthetized with intramuscular injections of thiopental. Four sterilized cotton pellets (50±1 mg) were subcutaneously implanted on the back of the anesthetized rats (axillae and groin regions) with an aseptic technique. Two hours after the implantation, those rats were treated topically with aqueous extract of snail slime, 50 % ethanol (vehicle) and diclofenac (standard). The treatment was given once a day for 7 d. On day 8, the pellets were dissected and weighed (wet weight), then they were dried at 60° for 24 h and weighed again (dry weight). Percent inhibition of transudate and granuloma formations were calculated from wet and dry weights, respectively. Furthermore, the granuloma tissue was processed into histological slides stained with H&E and Masson's trichrome to observe leukocyte infiltration and fibrosis. The tissue was also examined for oxidative stress through measurement of malondialdehyde (MDA) level and superoxide dismutase (SOD) activity, which represent lipid peroxidation and antioxidant enzyme, respectively.

Evaluation of oxidative stress of granuloma tissue:
The granuloma tissue (20 mg) was homogenized in 1 ml of 0.1 M Tris-HCl buffer pH 7.4 and centrifuged at 3000 rpm for 10 min at 4°. The supernatant was collected for MDA and SOD measurement.

MDA level measurement:
The procedures followed the method of Uchiyama and Mihara [19] . About 100 µl of the sample (collected supernatant) was pipetted into a screw cap tube, then 450 µl of normal saline, 100 µl thiobarbituric acid and 1000 µl of 10 % trichloroacetic acid we collected and mixed them well. The mixture was incubated at 100° for 30 min and then cooled down under running tap water. Distilled water was added into the incubated solution, mixed well, and the mixture was centrifuged at 3000 rpm for 10 min. The supernatant was measured for absorbance at 532 nm. The obtained absorbance was compared with the standard curve of tetramethoxypropane, and the MDA level was calculated from the standard curve and represented it as nmol/mg protein.

Measurement of SOD activity:
The SOD activity was examined with a method modified from Takada et al. [20] . About 100 µl of the sample (collected supernatant) was combined with 1 ml reaction solution (0.1 mM Na 2 -EDTA, 0.025 mM nitroblue tetrazolium, and 3 mM xanthine and 60 mM sodium carbonate buffer). Then 100 µl of xanthine oxidase was added and mixed well. The absorbance was measured at 560 nm every minute for 5 min. The SOD activity was calculated from the absorbance and standard curve, and represented as unit/mg protein.

Statistical analysis:
Statistical analysis was performed using Statistical Package for the Social Sciences version 17. P value <0.05 was considered statistically significant.

RESULTS AND DISCUSSION
The slime extract showed antiproteinase activity, and the aqueous extract had a significantly higher effect than the ethanol extract. The aqueous extracts (1000 µg/ml) could inhibit proteinase activity up to 70.81±0.40 %, while the ethanol extract, at the same concentration, could inhibit only 43.27±0.23 %. Sodium salicylate (standard) was quite effective and caused 72.65 ±0.44 % inhibition at 70 µg/ml concentration.
In this study, it was found that the snail slime extracts had antilipoxygenase activity. The aqueous and ethanol extracts (1000 µg/ml) exhibited 37.19±3.15 and 33.88±2.77 % inhibition, respectively, which was not significant, while indomethacin inhibited up to 68.60±0.96 % at 70.93 µg/ml concentration.
The protein denaturation inhibitory activity of snail slime extracts was detected in both aqueous and ethanol extracts. Both extracts displayed inhibitory activities, though the aqueous extract exhibited significantly better performance than the ethanol extract (41.99±2.37 and 12.76±3.61 % at the concentration of 1000 µg/ml). Indomethacin also inhibited denaturation; it could produce 41.82±13.85 % inhibition at the concentration of 1000 µg/ml concentration. Both snail slime extracts had a slight effect on membrane stabilization. The aqueous and ethanol extracts could reduce haemolysis of red blood cells, this was reflected in the percentage of inhibitions (7.01±0.54 and 13.42±0.28 %, respectively, at the concentration of 1000 µg/ml). Indomethacin displayed highly effective antihemolytic activity; 100 µg/ml of indomethacin generated 23.15±4.13 % of inhibition.
From in vitro study, it was found that the aqueous extract showed better activity than the ethanol extract. The aqueous extract was therefore selected to study further in in vivo models of acute and chronic inflammation. Snail slime extract reduced EPP-induced ear oedema at all time points studied as shown in Table 1. The slime extract (2 mg/ear) at 60 and 120 min after oedema induction and phenylbutazone (1 mg/ear) at 30, 60 and 120 min after oedema induction showed significant inhibition relative to vehicle-treated ears. The slime extract at a dose of 1 mg/ear could also reduce ear oedema, but this was not significant compared with the vehicle-treated ears.
The slime extract could significantly decrease both transudate and granuloma weights when compared to negative control group (p<0.05, Table 2). Diclofenac also significantly decreased the transudate and granuloma weights and compared to negative control group.
H and E staining showed dense leukocyte infiltration and fibroblast and collagen formation in the granuloma tissue of untreated rats ( fig. 1). These features normally occur during inflammation. Granuloma tissues treated with diclofenac showed only a few scattered leukocytes and fibroblasts. Treatment with the slime extract showed decreased leukocyte infiltration compared with untreated rats, and showed fibroblast cells and collagen deposition. From Masson's trichrome stain, there was plenty of collagen formulated in the granuloma tissue of untreated rats as shown in fig. 2a. Treatment of slime extracts and diclofenac could reduce collagen deposition in granuloma tissue, as shown in fig. 2b-d. From MDA measurement of granuloma tissue, it was found that the MDA level of granuloma tissue from rats treated with vehicle, diclofenac, 2.5 % and 6.25 % slime extract were, 1.13±0.052, 0.88±0.053, 0.96±0.010 and 0.92±0.175 nmol/mg protein, respectively. These results showed that the extracts significantly reduced MDA levels compared to the negative control group (p<0.05; fig. 3a). SOD measurement indicated that the SOD activity of granuloma tissue from rats treated with vehicle, diclofenac, 2.5 % and 6.25 % slime extract were 20.37±10.25, 52.48±12.57, 44.90±7.35 and 61.11±7.91 unit/mg protein/min, respectively. These results showed that the slime extract and diclofenac significantly increased SOD activity when compared to untreated rats (fig. 3b).
The inflammatory process involves a complex series of events, for instance the release of mediators and enzymes; leukocyte migration; and extravascular fluid accumulation [21] . The results of this study indicated that snail slime extracts could ameliorate these inflammatory    of the inflammatory signals, leading to decreased inflammation. Slime extract and phenylbutazone generated highest % inhibition at 60 min, raising the possibility that the main mechanism of action of the slime extract is related to inhibition of cyclooxygenase.
Chronic inflammation occurs when the acute inflammatory response cannot eliminate the harmful stimuli. The chronic response is divided into two phases; the transudative/exudative phase and the proliferative phase. The transudative phase is the initial phase, characterized by increased vascular permeability and vascular protein leakage [24] . The proliferative is the later phase, in which granuloma tissue is formed that is characterized by leukocyte infiltration, fibroblast proliferation, collagen formation and angiogenesis [24,25] .
In the cotton pellet-induced granuloma assay, the wet weight of the cotton pellet was used to determine transudate volume, and the dry weight of the cotton pellet was used to evaluate granuloma formation [26] . In present study, slime extract showed antiinflammatory activity in both phases. In the transudative phase, the result indicated an inhibitory effect of the slime extract on vascular permeability, vasodilation and accumulation of extravascular fluid. In the proliferative phase, the snail slime extract could inhibit granuloma formation, which was shown by reduction in granuloma weight. Histopathology of granuloma showed the decreasing of inflammatory cell and collagen deposition in the granuloma tissues of the slime extract treated groups. It was clear that the slime extract affected to the formation of granuloma tissue. This suggested the inhibitory activity of the extract on leukocyte migration and collagen formation at site of inflammation. This might have been due to the ability of the slime extract to decrease the inflammatory signals that enhance the granuloma formation. It was reported that extracts from snails could decrease plasma IL-6, an inflammatory cytokine in osteoarthritis-induced dog [27] and decrease COX level induced in inflammatory keratinocytes [28] . In the study, the slime extracts also improved the oxidative status of cotton-pellet induced granuloma in rats. This effect might have been due to its antioxidant properties. The previous study found that this snail slime had SOD activity and had antilipidperoxidation activity [29] . Thus, it is possible that antioxidant activity might be another mechanism of action in the slime extract to improve inflammatory status.
The results of the present study demonstrated the in vitro and in vivo antiinflammatory activity of slime extracted from giant African snails (Lissachatina fulica). Thus it could be concluded that the snail slime extracts events due to thier antiinflammatory activity. From in vitro study, the slime extracts showed mechanisms of action through different pathways, including inhibitions of inflammatory enzymes, membrane stabilization and antiprotein denaturation. Diclofenac used as standard references in in vitro, also displayed antiinflammatory activity in every assay.
An EPP-induced ear edema test was used to verify antiinflammatory activity against acute inflammation in the aqueous extract, in vivo. Topical application of EPP causes release of the inflammatory mediators, histamine, serotonin, bradykinin and prostaglandins. These mediators promote vascular permeability, vasodilation and accumulation of fluid in tissues, which lead to ear edema [22,23] . In this study, high dose of slime extract could significantly reduce ear thickness indicating that the slime extract had antiinflammatory properties and the mechanism of action might be through inhibiting the mediators of the acute phase of inflammation. Phenylbutazone used in the experiment, also inhibited the induced ear oedema and showed higher efficacy than both doses of the extract. Phenylbutazone is an inhibitor of cyclooxygenase, which decreased amplification have antiinflammatory activity with potential to be developed as antiinflammatory products. Although this study revealed some mechanisms of action of the slime extracts, further study is necessary to probe it's activity in different models.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Isolation and Screening of Phosphate Solubilizing Bacteria from Paddy Rhizosphere Soil

Rice (Oryza sativa L.) is one of the most important cereal crops in the world belongs to the family Poaceae. Rice is the important food grain and staple food for over the billions of people in most of the countries particularly in East Asia. It’s a major part of balanced diet and rich source of energy and carbohydrate. It is extensively grown food crop in 114 countries across the world, occupying area of about 163.20 million hacters of farm lands with annual production of over 758.9 million tonnes with a productivity of 4448 kg ha -1 per hectare.

Introduction
Rice (Oryza sativa L.) is one of the most important cereal crops in the world belongs to the family Poaceae. Rice is the important food grain and staple food for over the billions of people in most of the countries particularly in East Asia. It's a major part of balanced diet and rich source of energy and carbohydrate. It is extensively grown food crop in 114 countries across the world, occupying area of about 163.20 million hacters of farm lands with annual production of over 758.9 million tonnes with a productivity of 4448 kg ha -1 per hectare.
Phosphorus is one of essential macro-minerals for the growth and development of plant Phosphorus is one of essential macro-minerals for the growth and development of plant. The main of this study to isolate the PSB and screened for its solubilization efficiency on Pikovskaya's agar and broth at different incubation period. Totally 40 PSB were isolated from paddy rhizosphere soils of Raichur and Koppal districts. PSB isolates were studied for zone of solubilization, solubilization efficiency, solubilization index, ph change titrable acidity and phosphatase activity. The solubilization zone, efficiency and index were highest on 6 th day of incubation period. From that 10 isolates were found to be good phosphate solubilizer. Among 10 isolates, isolate PPSB-21 showed highest zone of solubilization (18.4 mm) phosphate solubilization efficiency (253.84%) and solubilization index (3.53) on Pikovsakaya's medium. These efficient isolates can be used as biofertilizer. (Schachtman et al., 1998). It is a major component in ATP, the molecule that provides energy to the plant for such processes as photosynthesis, protein synthesis, nutrient translocation, nutrient uptake and respiration. In addition, P has been observed to increase root growth and influence early maturity, straw strength, crop quality and disease resistance (Deepak Kumar, 2011). The available phosphorus in soils of India is generally poor. Efficiency of P fertilizer is around 10-25 % throughout the world (Isheword, 1998).
India, majority of the phosphorus provided in the form of chemical fertilizer. Inorganic P occurs in soil, mostly in insoluble mineral complexes, these insoluble, precipitated forms cannot be absorbed by plants (Rengel and Marschner, 2005). Large amount of phosphorus applied as fertilizes enter into immobile pool through chelating action with highly reactive Al 3+ and Fe 2+ in acidic soil (Gyaneshwar et al., 2002). In nature, wide range of microbial biosolubilization mechanisms exist which are necessary to maintain global cycle (Whitelaw, 2000).
Phosphorus solubilizing bacteria a group of beneficial bacteria play a key role in soil Phosphate solubilization (Abd-Alla, 1994) there by increasing the bioavailability of soil P for plants (Zhu et al., 2011). Phosphate solubilization occurs by production of organic acids and released by microorganisms; this release also decreases soil pH (Rodriguez et al., 2006). Organic acids solubilize insoluble P either by decreasing the pH or by the complexing the cation which is bound to the P (Vassilev et al., 2006). The organic acids such as succinic acid, malic acid, propionic acid and oxalic acid are released by Phosphorus solubilizing bacteria (Panhwar et al., 2012), which chelate the cation bound to phosphate and being converted to soluble forms through their hydroxyl and carboxyl groups. Large number of bacteria including species of Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus, Rhizobium and Serratia have reported to enhance plant growth by with their different plant growth promoting activities including phosphate solubilization (Kumar et al., 2012).

Collection of soil sample
A total of forty soil samples from rhizosphere of paddy were collected from different locations of Raichur and Koppal district of Karnataka by adopting standard soil sampling methods described by Jackson (1973). Soil samples were collected in sterilized polythene bags. The Polythene bags were properly tied; labeled and at most care was taken to avoid contamination. Soil samples were stored in refrigerator at 4 o C for the isolation of phosphate solubilizing bacterial isolates.

Analysis of soil chemical properties
Air dried samples were analysed for pH, EC, OC, N, P, K and Zn content estimation by following standard procedures.

Isolaton of PSB from paddy rhizosphere soil
One gram of soil from each sample was suspended in 9 ml blank sterile distilled water and serially diluted up to 10 -6 homogenization of soil was carried out by keeping it on shaker. The dilutions were plated on Pikovskaya's agar medium Pikovaskaya's medium (Pikovskaya's, 1948) contains (grams/liter); Glucose, 10.0gm; Ca 3 (PO 4 ) 2 , 5.0 gm; (NH 4 ) 2 SO 4 , 0.5 gm; NaCl, 0.2 gm; MgSO 4 .7H 2 O, 0.1gm, pH 7.3±2. These plates were kept at 28±2 º C for 7 days in order to isolate the PSB. The bacterial colonies exhibiting the clearance zone around the colonies were selected, purified, sub-cultured and stored on the slants of Pikovskaya's agar for further studies.

Screening of PSB
Each of the isolates was screened for their ability to solubilize Tri phosphate Phosphate (TCP) present in the Pikovaskaya's medium. A loopful of bacterial culture was placed on the centre of the same agar plates and incubated for 28±2 o C for 6 days. The solubilization zone, efficiency and index were calculated at different incubation period. The phosphate solubilization efficiency was calculated (Kannapiran and Ramkumar, 2011).
Solubilization index on the solid media was calculated considering the ratio of the total (colony + halo zone) diameter and colony diameter (Edi-Premono et al., 1996).

Quantitative estimation of available phosphorous in Pikovsakaya's broth in vitro
The PSB isolates were tested invitro by estimating available phosphorus in the Pikovskaya's broth supplemented with TCP as a substrate. Cultures of PSB were inoculated to 100 ml of Pikovskaya's broth in 150 ml conical flask in triplicates with uninoculated controls and incubated for 10 days at 30 0 C and centrifuged at 10000 rpm for 10 minutes. Then separate the supernatant from cell growth and insoluble phosphate. The clear supernatant was collected in 100 ml volumetric flasks. The available phosphorus in the filtrate was estimated by method of Olsen, 1954.

pH change
Estimation of change in pH of the broth due to the growth of PSB was measured with a pH meter at different incubation period of 3 rd , 6 th , and 9 th day ( Parimal et al., 2015).

Titrable acidity
In order to study the titrable acidity of culture medium, 5 days old cultures were centrifuged at 1000 rpm for 10 min. 10 ml culture filtrate was taken in a 50 ml conical flask. 5ml of supernatant was added with few drops of phenolphthalein indicator and titrated against 0.01 N NaOH solution. The end point of titration was determined as pink colour. The titrable acidity was expressed as ml of 0.01 N NaOH consumed per 5 ml of culture filtrate ( Ponmurugan and Gopi, 2006).

Phosphatase activity
In order to study the phosphatase activity in response to the phosphorus enrichment, culture filtrate were centrifuged and subjected to estimate phosphatase activity following the standard procedure (Tabatabai and Bremner, 1969).

Results and Discussion
Total 40 PSB were isolated from 40 rhizospheric sample of rice from different paddy growing regions of Raichur and Koppal districts. Among 40 isolates, ten isolates showed the remarkable zone of solubilisation (table 1). Based on clear zone formation around colonies on Pikovsakaya's medium the solubilization efficiency and index calculated (table 1) Hallow zone of solubilization was ranged from 18.5 mm to 12.5mm Plate-1 shows maximum zone of solubilization. There was a correlation with incubation time and zone size. It was also observed that increasing in the incubation time, increases the in the zone size of each isolates. Percent Solubilization efficiency was ranging from 80.95 to 253.84% from 3 rd to 9 th day of incubation period. The SE% increased with incubation period. The solubilization index based on colony diameter and halo zone for each PSB isolate is presented in Table 1. The results showed that, among the 10 efficient isolates, the solubilization index varied from 1.80 to 3.22. The solubilization efficiency index enhanced with incubation period. The diameter of clear halo zone formed by the bacterial isolates has the direct correlation with the phosphate solubilization efficiency.
All the ten isolates were able to solubilize the insoluble phosphorus in Pikovaskaya's broth at different incubation period and were represented in table 2. Among ten isolates, available phosphorous content was ranged from 31.92 to 171.84mg/L. All the isolates were recorded decreased pH with increased incubation period. Among ten isolates, the reduction in the pH varied from 6.12 to 3.15. The drop of pH was due to the production of organic acids.
The titrable acidity of the culture medium was measured. All ten isolates showed values in the range of 0.30% to 0.65%. The titrable acidity increased with incubation period. The variability in the phosphatase activity of soil ranged from minimum of 16.28µmoles/g/hr to maximum of 42.40µmoles/g/hr with the application of PPSB-21 and PPSB-5 respectively (table 3). There was a positive correlation between the phosphate solubilizing capacity and phosphatase activity.  Rice is the important food grain and staple food for over the billions of people in most of the countries particularly in East Asia. It's a major part of balanced diet and rich source of energy and carbohydrate. Phosphorus is one of the essential macro-minerals for the growth and development of plant (Schachtman et al., 1998). It is a major component in ATP, the molecule that provides energy to the plant for such processes as photosynthesis, protein synthesis, nutrient translocation, nutrient uptake and respiration (Deepak kumar, 2011). But after application, a considerable amount of them are rapidly transferred into less available forms by forming complexes with Fe, Ca and Al cations before roots have a chance to absorb it (Alam and Ladha, 2004).
Under such conditions PSB play fundamental role in biogeochemical phosphorous cycling in natural and agricultural ecosystem. Extensive use of chemicals as fertilizer improves the plant health and productivity but disturbed the ecological balance of soil and resulted in nutrient depletion. This has necessitated the search for alternate source of this element. The use of PSB in agriculture practice is not only offset high cost of manufacturing phosphatic fertilizers but also make availability of insoluble P fertilizer.
The maximum halo zone was found with PPSB-21 with zone diameter of 18.5 mm. The results showed that, among the 10 isolates, the PSB-21 isolate showed a maximum solubilization efficiency and index of 253.84 and 3.53 respectively on 9 th day of incubation period over other bacterial strains. The solubilization efficiency enhanced with incubation period. The diameter of clear halo zone formed by the bacterial isolates has the direct correlation with the phosphate solubilization efficiency. The percent solubilization increased with incubation period. Similarly, Ngomle et al., (2014) isolated ninteen PSB. The P-solubilization efficiency was found maximum in UBPS-22 (28 mm) followed by UBPS-19 (25 mm) and UBPS-18(24 mm) respectively within 72 hrs of incubation in Pikovskaya's medium. Similar outcomes have been reported by many workers viz. Frateme et al., (2014), Deepak et al., (2018). Among ten isolates PPSB-21 recorded the highest available phosphorous content of 171.84mg/L on 9 th day of incubation. All the ten isolates were able to solubilize the insoluble phosphorus in Pikovaskaya's broth at different incubation period it may be due the abilities of the isolates to solubilize inorganic phosphate by the production of organic acids. Similarly, Karpagam and Nagalakshmi (2014) isolated 8 potent isolates, 3 strains showed high soluble phosphate production of 0.37mgL -1 , 0.30mgL -1 and 0.28mgL -1 in broth culture. Similar work was carried out by Manivannan et al., (2011), Buddhi andMin-Ho (2013), Gandhi et al., (2014), Manouchehr et al., (2016), Amit et al., (2017) and Deepak et al., (2018).
PPSB-21 showed highest reduction in pH of 3.15 on 9 th day of incubation. The drop of pH was due to the production of organic acids. Other reason like microbial respiration may also be involved for drop in pH. Tensingh and Jemeema (2015) observed the pH change of upto 4.6 by the Bacillus sp. Amit et al., (2017) isolated 8 PSB isolates, among 3 isolates showed lower pH ranging 3.08 ±0.08 to 3.82 ± 0.12. Similar results were obtained by Oliveira et al., (2009), Buddhi andMin-Ho (2013). Those bacteria that produced halo zones around colonies in PVK medium were able to produce organic acids in broth culture. This result is in accordance with Ogut et al., (2010). This is also in agreement with Mehta et al., (2001), Chen et al., (2006), Ponmurugan and Gopi (2012), Studies related to the production of organic acids have shown that citric and oxalic acids were two major organic acids produced by PSB (Alam et al., 2002).
Among all ten isolates, highest titrable acidity of 0.65% was expressed by PPSB-21. This might be due to reduction in the pH and secretion of organic acids. Results showed that strong positive correlation was found between titrable acidity and P solubilization. PSB produce phosphatase enzymes in soil, the activity of enzymes leads phosphate solubilization. There was a positive correlation between the phosphate solubilizing capacity and phosphatase activity. Phosphate solubilization occurs by production of organic acids and released by microorganisms; this release also decreases soil pH (Rodriguez et al., 2006). Organic acids solubilize insoluble P either by decreasing the pH or by the complexing the cation which is bound to the P (Vassilev et al., 2006). The organic acids such as succinic acid, malic acid, propionic acid and oxalic acid are released by Phosphorus solubilizing bacteria (Panhwar et al., 2012), which chelate the cation bound to phosphate and being converted to soluble forms through their hydroxyl and carboxyl groups. Phosphatesolubilizing bacterial strains isolated were identified by biochemical tests; the isolates PPSB-21 and PPSB-5 were identified as Pseudomonas sp. and Bacillus sp.
In conclusion, our study demonstrated that many of the bacteria had P solubilizing properties and the ability was not exclusive to specific genera, suggesting the importance of preliminary screening in vitro for a wide range of bacteria to characterize their potent P-solubilizing or mineralizing trait. The PSB population was found higher in rhizo-sphere when compared with non-rhizosphere soil. Isolated PSB strains were able to solubilize P, produce organic acids, and enzymes. These beneficial characteristics would be considered as potential biofertilizer of isolated PSB for rice production.


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Natural Plant-Derived Chemical Compounds as Listeria monocytogenes Inhibitors In Vitro and in Food Model Systems

Listeria monocytogenes is a foodborne pathogen, sporadically present in various food product groups. An illness caused by the pathogen, named listeriosis, has high fatality rates. Even though L. monocytogenes is resistant to many environmental factors, e.g., low temperatures, low pH and high salinity, it is susceptible to various natural plant-derived antimicrobials (NPDA), including thymol, carvacrol, eugenol, trans-cinnamaldehyde, carvone S, linalool, citral, (E)-2-hexenal and many others. This review focuses on identifying NPDAs active against L. monocytogenes and their mechanisms of action against the pathogen, as well as on studies that showed antimicrobial action of the compounds against the pathogen in food model systems. Synergistic action of NDPA with other factors, biofilm inhibition and alternative delivery systems (encapsulation and active films) of the compounds tested against L. monocytogenes are also summarized briefly.

Listeria Monocytogenes
Listeria monocytogenes is a foodborne pathogen, causative agent of listeriosis, which is a disease with high hospitalization and fatality rates, especially in pregnant women, newborns, the elderly and immune-compromised individuals [1][2][3]. Once ingested, the pathogen is able to cross the intestinal barrier, disseminating to the spleen and liver. It can also cross the blood-brain barrier, leading to meningitis, or the placental barrier, causing miscarriages and stillbirths of the fetus [1,[3][4][5]. Many virulence factors were determined for L. monocytogenes, out of which listerial hemolisin, called also listeriolysin O (LLO) was the first one [5] The pathogen can be found in various environments, such as soil, manure and water [6] and in variety of raw foods, such as uncooked meats and vegetables, as well as in ready-to-eat (RTE) foods, contaminated during or after processing [1,6]. L. monocytogenes can survive and proliferate over a wide range of environmental conditions, including refrigeration temperatures, low pH and in high salt concentrations, what makes it especially hard to eliminate from food products [2]. The ability to form biofilms is also a crucial factor in the survival of L. monocytogenes. Biofilms are composed of numerous cells attached to a biotic or an abiotic surface and to each other, surrounded by an extracellular matrix predominantly made up of polysaccharide material, but containing also proteins and extracellular DNA [2,7]. Presence of the pathogen in food is strictly regulated by legal authorities and in many countries, including European Union. Either complete absence of the pathogen in food product is required or the pathogen may be present, but it may not exceed 100 CFU/g (colony forming units per gram) throughout the shelf-life, depending on the food product properties. In the United States, for example, so-called "zero tolerance policy" requires absence of the pathogen in all food products. Complying with the legal microbiological criteria is still a great challenge for food manufacturers [8,9]. Natural of plant sources of the compounds most often mentioned throughout this publication are listed in Table 1.  [13,22] Anethum graveolens L. dill seed carvone S 45.5 [22,46] Cinnamomum zeylandicum cinnamon cinnamaldehyde transcinnamaldehyde 65 [13,22] Cymbopogon citratus lemongrass citral 65-85 [22,47] Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae) clove eugenol 88.6 [48] Lavandula angustifolia lavender linalool 5-57 [49] Thymus vulgaris, Origanum vulgare thyme, oregano thymol 10-64, trace-64 [22] Psidium guajava L. guava fruits (E)-2-hexenal 7.4 [50] In latter study performed by Friedman et al. [22], 96 EOs and 23 oil compounds were tested against two L. monocytogenes strains. However, the authors stated that standard diffusion methods based on measurement of inhibition zone of bacterial growth on solid medium is not accurate for EOs, as zones of inhibition may vary, depending on the ability of oil to diffuse through an agar medium and also vapors of the oil may influence bacterial growth. Thus, according to authors, the method is not suitable for comparing bactericidal activities of EOs and their compounds. Authors used a microtiter plate bactericidal assay and determined bactericidal activity, which was defined as the percentage of the sample in the assay mixture that resulted in a 50% decrease in colony forming units relative to a buffer control (BA50). According to the authors, the oil compounds that were most active against L. monocytogenes were cinnamaldehyde, eugenol, thymol, carvacrol, citral, geraniol, perillaldehyde (naturally occurring in mandarin peel), carvone S, estragole (naturally occurring in tarragon), and salicylaldehyde (naturally occurring in almonds). Substances with BA50 values lower than 0.5% (for at least one strain tested) are listed in Table 2. In other study also BA50 values of epigallocatechin gallate (naturally occurring in green tea) and oleuropein (naturally occurring in olives) were determined against L. monocytogenes RM2199 and resulted with 1.42% and 1.43%, respectively [40].
Bagheri et al. [51] made an attempt to create a mathematical model of minimal inhibitory concentrations (MIC) of essential oils against various microorganisms (including L. monocytogenes), depending of the chemical composition of the oils. The authors tested 38 essential oils, determined their chemical composition and inhibitory activity against microorganisms. The most significant compounds of essential oils affecting the inhibition of L. monocytogenes LMA1045 were phenols and aldehydes, although sesquiterpenes and esters are also included in the created model. Created linear model is statistically significant (with the F value of 5.8 and p value of 0.0012) and is as follows: MIC of L. monocytogenes LMA1045 = 15,989.3 − 13.8*Sesquiterpene − 15.3*Aldehyde − 13.0*Phenol − 30.6*Esters.
Minimal inhibitory concentrations and minimal bactericidal concentrations (MBC) for natural antilisterial compounds are listed in Tables 3 and 4, respectively. However, MIC or MBC values of the compounds cannot be directly compared within different references, as experiment conditions, e.g., temperatures of incubation, chosen medium or pathogen strain, as well as method used for determination of inhibitory or bactericidal concentration may influence the inhibitory or bactericidal values. What is more, MIC or MBC against L. monocytogenes has not always been determined for natural compounds, even though the components are proven to be antilisterial (usually by creating zone of inhibition on agar medium during diffusion tests). For example cymene (naturally present in oregano and thyme) [52], berberine [53], S-carvone, decanal [54], iberin, erucin, sulforaphane, benzylisothiocyanate and 2-phenylethylisothiocyanate [55], quercetin [36], also many acid compounds, i.e., rosmaric acid, caffeic acid (both naturally occurring in Melissa officinalis), hydroxyphenyllactic and trans-cinnamic acids [56], p-coumaric acid [36,56], gallic acid, ferulic acid [36], as well as peptides derived from rice or palm kernel cake [11] are antilisterial, however their MIC or MBC values against L. monocytogenes remain undetermined.    3 Depending on the level of the initial contamination-from 2 to 6 log CFU/mL (higher contamination resulted in higher MICs); 4 Authors described the isolate as "Dutch field isolate obtained from cheese"; 5 Depending on the time of the experiment (0-72 h)-longer inhibition required higher doses; 6 Lower range was obtained by microdilution method, higher by agar dilution method; 7 25 strains of food origin (from meat, vegetables, dairy and fish) were described as: 45,46,47,48,49,50,51,52,53,79,85,89,96,98,99,100,101,102,103,104,105,106,107,112 and 118 in the reference study; 8 MIC was determined at 2 temperatures (5 • C and 30 • C) and 3 levels of pH (5.0, 6.0 and 7.0); the lowest MIC was achieved at 5 • C at pH 5, whereas the highest at 30 • C at pH 7; 9 5 strains of food origin (from raw chicken meat, infant food, and ready-to-eat meals) were marked as: A17, A24, B9, B19, C6, and C34.  1 Values were converted for ease of comparison; density was assumed as 1 g/mL for all of the compounds, and molecular weights were obtained from PubChem [78]; 2 Presented range of the values, if not specified otherwise, refers to results obtained for different strains used in the study; 3 Depending on the level of the initial contamination-from 2 to 6 log CFU/mL (higher contamination resulted in higher MICs).

Mechanisms of Action
Only some of the substances known to be inhibitory against L. monocytogenes have been determined for their exact mechanism of action against the pathogen. However, some of the experiments were performed with Listeria innocua, which is closely related to L. monocytogenes, though not pathogenic to humans [82,83]. Traditionally, L. innocua have been used as a model for predicting L. monocytogenes behavior in food processing environments, as the two microorganisms share the ecological cohabitation and are similar physiologically. Existing differences between the two species and results obtained by examination of one of them may not be prudent to the other [83]. Similarly, as an antibiotic resistance differ for various strains of the same species, the same phenomenon can to be expected for NPDA susceptibility. Therefore, screening of numerous strains can provide truly informative overview. Nonetheless, Silva-Angulo et al. [84], after their experiments comparing the growth and injury of the two bacteria caused by citral, suggested that L. innocua could be used as surrogate for L. monocytogenes when testing the effect of the antimicrobial, as L. innocua due to its shorter lag phase, represents a worst case scenario. However, the mechanisms of action of most of the antilisterial compounds has not been examined for neither L. monocytogenes, nor L. innocua, as authors focused mostly on mechanisms of action of carvacrol, eugenol, cinnamaldehyde, thymol and citral.
One of the first studies aiming to determine mechanisms of the NPDA action against L. monocytogenes was performed with eugenol and cinnamaldehyde. Presence of the compounds prevented an increase in the cellular ATP levels upon glucose addition in nonenergized cells. Authors concluded, that effects of energy generation are crucial at bactericidal concentrations of eugenol and cinnamaldehyde [81]. During further experiments on isolated L. monocytogenes membranes, the membrane bound ATPase activity was significantly inhibited by eugenol (5 or 10 mM), carvacrol (10 mM) and cinnamaldehyde (10 mM) [85]. In other study the group focused on cell membrane disruption caused by the plant EO aromatic compounds. Eugenol and carvacrol, unlike cinnamaldehyde (up to 10 mM), increased uptake of propidium iodide by L. monocytogenes [86]. Increased propidium iodide internalization is an indicator of large pore formation, usually leading to the cell death [87]. All three compounds inhibited L. monocytogenes motility, as well as caused rapid decrease of the cellular ATP. Eugenol and carvacrol caused release of ATP. Experiments indicated that the primary mechanism of action of eugenol and carvacrol at bactericidal concentration is disruption of the cell membrane, leading to increased nonspecific permeability, whereas in cinnamaldehyde evidence for membrane disruption was less evident [86]. However, in other studies, the transmission electron microscopy of the L. monocytogenes ATCC 13932 cells after treatment with cinnamaldehyde (0.1%) at 37 • C for 6 min, revealed that the compound damages cell membrane, leading to disruption of its integrity and leakage of the cytoplasmic content [88]. Presence of trans-cinnamaldehyde caused perturbations in the cytoplasmic membrane fluidity, potential and intracellular pH in L. innocua LRGIA01. As hypothesized by the authors the changes happened due to accumulation of the trans-cinnamaldehyde in the hydrophobic core of the cytoplasmic membrane of the cells. However, similarly to the Gill and Holly [86] results, the membrane changes did not increase propidium iodide uptake, suggesting that cells survived the treatment and entered viable but not culturable state (VBNC) [87]. Flow cytometric assessment showed that the membrane permeabilization occurred when L. monocytogenes Scott A was exposed to the sub-inhibitory and MIC values of citral, carvacrol and (E)-2-hexenal. Esterase activity remained unchanged after treatments [89].
Sub-lethal concentrations of carvacrol, thymol and citral caused changes in the membrane fatty acid composition of L. monocytogenes Scott A, depending on the compound and its concentration. Carvacrol caused an increase in general unsaturation level and chain length of the membrane fatty acid composition, citral mainly induced an increase of unstaturation level, whereas in thymol changes in the parameters were less evident, however for example C 18:1trans9 concentration in the presence of 100 ppm of thymol increased from 2.1% (control sample) to 16% [90]. Similarly, Rogiers et al. [91] concluded that the membrane fatty acid composition is a determinant of L. monocytogenes sensitivity or resistance to trans-cinnamaldehyde, as L. monocytogenes mutant lacking in IlvE gene was hypersensitive to the antimicrobial. IlvE encoded by the gene is a branched-chain amino acid aminotransferase, that reversibly catalyzes the transamination of the branched-chain amino acids to the corresponding short-chain α-ketoacids. Whole-cell fatty acid composition of the hypersensitive mutant revealed significant reduction of anteiso branched-chain fatty acids which were replaced by unbranched saturated fatty acids and iso-branched-chain fatty acids.
Cell membrane composition changes may also be a factor determining temperature dependence of the activity of the antimicrobials. Usually at higher temperature, the compounds exhibit higher antimicrobial properties, for example reduction of L. monocytogenes achieved with 3 mmol/L carvacrol reached 0.6 log units at 1 • C and 1.8 log units at 20 • C [92], and in other study the highest activity of carvacrol was observed at 30 • C (compared to 10 • C and 20 • C) [63]. Karatzas et al. [54] performed an experiment, where exponential phase cultures grown at 8 • C, 35 • C or 45 • C were subjected S-carvone at 45 • C for 30 min. Only cells pre-grown at 8 • C showed reduction in viable counts. Authors concluded that changes in phospholipid composition of the cytoplasmic membrane may be the reason for the phenomenon. Cells pregrown at lower temperatures maintain membrane fluidity by changes in fatty acid composition and if these cells are transferred to higher temperatures, fluidity of their membranes is higher than would be if the cells had been cultured at higher temperature. As a consequence, S-carvone dissolves into lipid bilayer of the cells pregrown in lower temperatures in higher amounts. However, cells from sta-tionary phase were not susceptible to S-carvone at 45 • C, whether they were pregrown at 8 • C or 45 • C. Authors suggested that this phenomenon is due to the physiological changes such as decreased membrane fluidity and permeability and synthesis of stress proteins, as bacteria undergo these type of changes when they enter stationary phase [54].

Gene Expression Responses and Proteome Changes
Plant-derived substances present in the environment influence level of gene expression of L. monocytogenes cells. Consequently, they also influence bacterial proteome.
Trans-cinnamaldehyde, carvacrol and thymol at the sub-inhibitory and inhibitory concentrations statistically significantly down-regulated L. monocytogenes virulence genes (including among others internalins, listeriolysin O, phospholipases, actin polymerization protein as well as proteins associated with motility) expression levels in all three strains of the bacteria that were used in the study (ATCC 19115, Scott A and Presque-598) [59]. Down regulation of virulence factors of the same three strains of L. monocytogenes was also confirmed for eugenol [93].
L. monocytogenes exposed to sub-inhibitory concentrations of trans-cinnamaldehyde, carvacrol, thymol and eugenol showed changes in the expression of 13 (out of 13 targeted) critical biofilm associated genes, namely flagellar proteins, transcriptional regulators and quorum sensing genes. Relative fold change in the expression of these genes was negative in all cases, suggesting clear down-regulation. However, in two and three genes encoding flagellar proteins in trans-cinnamaldedyhe and eugenol treatment, respectively, the fold change was not significantly different [79].
Relative gene expression analysis was also performed with a sub-inhibitory concentrations of (E)-2-hexenal, citral, carvarcol on L. monocytogenes Scott A. Targeted genes selected in the study were responsible for various cell functions, including cell division and DNA repression and modulation, catabolic processes, synthesis of RNA and proteins, stress response and virulence and motility. Citral and carvacrol showed the same mechanism of action, different from (E)-2-hexenal. Citral and carvacrol caused an overexpression of cpsL and bsh genes, involved in stress response and motility, respectively. (E)-2-hexenal caused imbalance in catabolic processes, interpreted as a hypothetical change from oxidation to fermentation, executed by down-regulation of pgm and pdhD involved in glycolysis. (E)-2-hexenal was inhibitory towards the expression of genes responsible for stress response, protein synthesis and DNA protection and repair [58].
Thymoquinone at concentration of 1.56 µg/mL (whereas MIC was of 12.5 µg/mL) significantly down-regulated expression level of L. monocytogenes ATCC 19115 all seven targeted genes associated with motility, biofilm formation, hemolysin secretion, and adhesion and invasion of the host cells. A concentration of 0.78 µg/mL also caused down-regulation of all analysed genes, however in two cases the decrease was not significant [77].
L. monocytogenes Scott A treated with sub-MIC concentrations of citral, carvacrol and (E)-2-hexenal showed increased or decreased (≥ or ≤2-fold) levels of 223 protein spots (compared to profile of cells grown under optimal conditions), which affect many major cellular processes, including cell cycle control, cell division, chromosome, motility and regulatory related proteins, metabolism of carbohydrates, pyruvate, nucleotides and nitrogen, vitamins cofactors and also stress response proteins. Citral and (E)-2-hexenal adapted cells increased survival under acid stress [94].
Presence of carvacrol in tyrosine decarboxylase broth significantly changed production of the biogenic amines and ammonia by L. monocytogenes ATCC7677. Carvacrol at all 3 tested concentrations (0.1%, 0.5% and 1%) when compared to the control reduced production of: ammonia, putrescine, 2-phenyl-ethylamine, spermine, tyramine and agmatine. Interestingly, higher dose not always corresponded with greater reduction, e.g., ammonia and putrescine production were higher at 1% carvacrol than at 0.5% carvacrol. Production of caraverine, spermidine, tryptamine, histamine and dopamine were increased by 0.1% carvacrol as compared to the control, however reduced compared to the control at higher concentrations (0.5% or 1%). Trimethylamine production was reduced by 0.1% carvacrol and 0.5%, however 1% caused increased production. Serotonin was the only biogenic amine tested that lowest levels of production were achieved in the absence of carvacrol [95].

Bacterial Adaptation
Apolónio et al. [68] evaluated the impact of continuous exposure of three L. monocytogenes strains (Scott A, EGD, C882) to eugenol and citral. To determine ability of the pathogen to gain resistance to these compounds, the bacterial strains were subjected to sequential passages in the presence of sub-inhibitory to lethal concentrations. Following the four sequential passages at the desired concentration of the antilisterial compound, bacteria were transferred to the medium with higher concentration of the compound. The process was repeated until bacteria stopped growing. The L. monocytogenes EGD and Scott A adapted cells were not able to overcome the first passage at the MIC concentration of eugenol (after four passages at sub-inhibitory concentration). The L. monocytogenes C882 adapted cells overcame four passages at sub-inhibitory and MIC concentration but failed to overcome the first passage at the MBC concentration of eugenol. Identically, the L. monocytogenes EGD and C882 adapted cells overcame the four passages at MIC concentration of citral, but failed the first passage at MBC concentration. The L. monocytogenes Scott A adapted cells were only able to overcome three passages at MBC concentration of citral. In conclusion, none of the strains was able to mount a resistance response after the adaptation exposure to sub-inhibitory concentrations of both compounds. Similarly, in other experiment L. monocytogenes Scott A cells were cultured in presence of S-carvone at sub-inhibitory concentration and further combined treatment with mild heat was equally effective in adapted and unadapted cells [54].
Influence of the adaptation to eugenol and citral at their sub-inhibitory concentrations (0.05 mg/mL) on the susceptibility to 5 antibiotics (penicillin, chloramphenicol, kanamycin, vancomycin and erythromycin) was also evaluated. The sequential passages of all tested strains (L. monocytogenes-EGD, Scott A and C882) to eugenol or citral did not induce resistance to the antibiotics tested [68].

Virulence in In Vitro and In Vivo Models
As described earlier, studies are consistent that NPDA down-regulate expression of genes related to bacterial virulence [58,59,77,79,93]. These finding suggest the decreased virulence of L. monocytogenes in presence of that compounds, which was verified by actual in vivo and in vitro tests.
L. monocytogenes ATCC 19115, Scott A and Presque-598 in the presence of sub-inhibitory concentration or MIC of trans-cinnamaldehyde, carvacrol and thymol showed significantly lowered adhesion and invasion of Caco-2 (human enterocyte like) and human brain microvascular endothelial cell lines. Additionally, motility was significantly reduced compared to the untreated controls, as well as hemolysis of 3% sheep red blood cells and phospholipase activity [59]. Similar results were obtained for sub-inhibitory concentration of eugenol, as the antimicrobial reduced hemolysis of 3% sheep red blood cells and adhesion and invasion rate of Caco-2 cells [93]. Similar results were achieved for sub-inhibitory concentrations of thymoquinone present in L. monocytogenes ATCC 19115 environment. The treated bacteria showed reduced ability to adhere and invade Caco-2 cells. Thymoquinone at concentrations of 0.39, 0.78, and 1.56 µg/mL (compared to MIC of 12.5 µg/mL), reduced adhesion rate of the cells to 88.36, 80.03, and 74.02% compared to the control, whereas the invasion rate was reduced to 81.59, 52.06, and 46.08%, respectively. Listeriolysin O secretion rates of the pathogen dropped to 42.52, 30.25, and 25.69% in the presence of thymoquinone at concentrations of 0.39, 0.78, and 1.56 µg/mL, respectively [77].
Also in vivo tests determining influence of NPDA on L. monocytogenes infection were performed. Injection of 5 log CFU of L. monocytogenes per Galleria mellonella larvae resulted in 100% mortality by day 5 of the experiment. Pre-treatment with eugenol injection significantly enhanced the survival rates of infected larvae by at least 40% on day 7 [93].
Similar experiment was performed with trans-cinnamaldehyde, carvacrol and thymol. All of the compounds increased survival rate of the larvae by at least 20% at day 5. Carvacrol was the most effective and approximately 80% survival rate was observed at day 5 [96].
Apolónio et al. [68] on the other hand determined the impact of adaptation to eugenol on bacterial virulence, using Galleria mellonella model. The larvae were injected with two L. monocytogenes strains (Scott A or C882) adapted to eugeneol by sequential exposure to increasing concentrations of the compound. Larvae treated with Scott A and C882 adapted cells, survived at significantly higher level than the larvae injected with nonadapted cells along first four days and three days, respectively. No significant differences (p < 0.05) were found between survival rates of larvae treated with adapted or nonadapted L. monocytogenes at fifth day for larvae treated with C882 and at from fouth to fifth day for larvae treated with Scott A.
Silva et al. [97] evaluated effect of possible virulence changes of L. monocytogenes CECT 4032 untreated cells or treated with either citral or carvacrol using Caenorhabditis elegans as a model. C. elegans showed no loss in life span (p < 0.05) when it was fed in a lawn of L. monocytogenes previously treated with citral compared untreated L. monocytogenes, however the pathogen previously treated with carvacrol showed loss in a life span with last 5 percentile of worms. Within 75, 50 and 25 percentiles differences were not observed. Citral treated L. monocytogenes showed no effect on egg laying by C. elegans, compared to untreated L. monocytogenes, whereas carvacrol treated bacteria reduced statistically significantly the number of eggs.

Food Model Systems
Efficacy of NPDAs against L. monocytogenes was evaluated in various groups of food products. Attempts of inhibiting L. monocytogenes inoculated in food products are summarized in Table 5. However, the table focuses only on purified chemical compounds (used either alone or in combination with other substances or methods) and other treatments applied to the contaminated products within the references, even more effective than those incorporating NPDAs, may not be included. Supplementary information to Table 5 (including other, less effective treatments incorporating NPDAs tested in the studies, used bacterial strains and comments) are presented in Table 6. Both tables are connected by the common column with heading "Row", where in each row a unique two-letter symbol is placed. Corresponding rows within the two tables were marked with identical row symbol.           Even though L. monocytogenes reductions were achieved in most of the experiments presented in Table 5, it has been suggested that a 6-log reduction in foodborne pathogens is required for minimally processed foods [52] and reductions in this order of magnitude has only been achieved in some cases. According to strict legal criteria in many countries [8,9] it would be most desirable to achieve complete inhibition of the pathogen in food products or to reduce its presence to the maximum of 100 CFU/g. Notably, in most of the cases of reducing L. monocytogenes to undetectable levels NDPA were not used as sole factors. In raw meat combining carvacrol with high hydrostatic pressure (HHP) treatment [98] or immersing meat in teriyaki sauce with thymol [99] resulted in complete pathogen inhibition. The pathogens in teriyaki sauce itself [99] or in soy sauce [101] were inhibited by carvacrol or thymol. In raw fish complete inhibition to undetectable levels was achieved with carvacrol [100]. In meat products, such as frankfurters a successful pathogen eradication was achieved by immersing them in solution containing trans-cinnamaldehyde or carvacrol combined with 0.1% hydrogen peroxide along with mild-heat treatment (55-60 • C) for up to 1 min [102]. Additionally, in hams covered with antimicrobial films containing carvacrol [105] or carvacrol or gallic acid paired with chitosan [111] the pathogen was no longer detectable after storage. In milk, although many experiments were performed, only combined treatment with thymol, nisin and lactobionic acid eradicated the pathogen and only in reduced-fat milk [70]. In salad combination of enterocin with either thymol, terpineol or tyrosol [109], in mixed shredded leafy vegetables addition of carvacrol [65] and on cabbage leaves linalool [69] eradicated the pathogen, as well as carvacrol or 1,8-cinole or their combinations in vegetable model broth [65]. On cantaloupes also washing treatments and coating treatments incorporating thymol, carvacrol or caprylic acid combined with hydrogen peroxide eliminated L. monocytogenes [110].
Even if the pathogen was not eradicated, successful reduction of the pathogen counts has been achieved in almost all cases. However, in the study of Veldhuizen et al. [63], at end of 4 week storage at 10 • C, contaminated steak tartare treated with 5 mmol/g carvacrol was equally contaminated as the control sample and L. monocytogenes counts reached 10 8 CFU/g. Authors proved that the presence of food components interfered with the activity of carvacrol. Both egg yolk and bovine serum albumin (which are ingredients for tartare steak) inhibited carvacrol activity at >0.2% (wt/vol) in growth medium. Explanation of the phenomenon hypothesized by authors was that that the food product provides more nutrients to bacteria, what leads to quicker cell regeneration. Another explanation is that because EOs and their components are mostly hydrophobic, the antimicrobials are more likely to dissolve in lipid or fat fractions and their access to microorganisms is limited [63,112]. In two experiments comparing antimicrobial action of NPDA in milk, lower reductions were achieved in whole milk compared to reduced fat milk [70,74] and in one case final bacterial counts in whole milk were indifferent from the control sample [74]. In other study the effect of carvacrol combined with HHP was at least two orders of magnitude lower in milk inoculated with L. monocytogenes (3.2 log reduction) compared with buffer (>6 log reduction) [92], what leads to conclusion that fat inhibit action of those antimicrobials, as suggested earlier. However even in carrot juice, it was necessary to increase the concentrations of carvacrol and cymene (used in combination) more than 3-times compared to HEPES buffer to achieve desired reduction of L. monocytogenes [52]. Additionally, the antimicrobial activity of carvacrol in fresh cheese, chicken breast, fresh-cut pumpkin, and fresh-cut melon and of eugenol in chicken breast and fresh-cut pumpkin against L. innocua was less remarkable than in in vitro tests [113]. Concentration of the components not always can be increased in real foods, as the compounds generally reduce the sensory acceptability of food and can be an important limitation for their use as antimicrobial agents [61,112,114]. Many authors suggest that solution for this problem is utilization of the antimicrobials in a system of agents used together in a form of the hurdle technology [81,114]. One author clearly stated that citral and carvacrol can be used as preservatives to control the growth of L. monocytogenes only in combinations with other hurdles, as concentrations capable of having lethal effects are incompatible with the food sensory features due to their low sensory threshold [89].
Sensory evaluation of treated samples was performed in only few studies. Sensory evaluation of fillets marinated in solution containing 0.25 mg/mL thymol and 5% (w/v) salt, showed no significant difference when compared to control sample in both parameters tested, i.e., odor and taste. The maximum on the hedonic scale was 5 (corresponding to most-liked sample) and marinated fillets mean score were of 4.6 and 4.8 for odor and taste, respectively [72]. Soy sauce with up to 1 mM of thymol or carvacrol was scored statistically indifferent of the control soy sauce, reaching approximately 4.5 points on 9 point hedonic scale, where 9 corresponded to the most pleasant odor [101]. Sensory analysis of a shredded leaf mix after treatment with the combination of carvacrol and 1,8-ceineole or the components alone, showed that general perception and taste parameters after 72 h of storage was not significantly different (p > 0.05) among all 3 treated samples and the control. In case of the odor parameter after 72 h, only carvacrol sample was different compared to the control and was less desirable. Evaluation was done using 5 point hedonic scale, where the highest score corresponded to like very much [65]. Sensory analysis was also performed on catfish fillets treated with 2% carvacrol. Results indicated, that the aroma compared to the untreated sample is different (p < 0.05) in raw and cooked samples. Panelists stated that samples had a menthol or piney aroma in comparison to the control sample, what indicated chemical changes in the compound [100]. Apart from last study, sensory analysis was performed using a hedonic scale of personal preferences. However, ideally, treated product should not only be acceptable or desirable on the same level as untreated product, but it should not be different from untreated product. Thus, antimicrobial treatment would lead not to a final product ready to enter a market, but to a flavor-neutral product base, that could be used to create diverse versions of food products by adding various seasonings and would not limit manufacturers to provide only one flavor version of their product. In one study with L. innocua, analysis based on determining if the product is distinguishable from the untreated sample was performed. Addition of 203 ppm carvacrol to turkey breast ham was undetectable by the panelists and thus carvacrol at concentration of 200 ppm was used in the study to determine combined treatment with carvacrol and HHP in turkey breast ham. Complete inhibition of the bacterium was not achieved, as after 60 days of storage at 4 • C the control sample had contamination of approximately 6.5 log CFU/g, whereas treated sample 5.5 log CFU/g [115]. There are no other studies of this kind, where the effect of NPDA applied at a sensory undetectable level would be measured in food products contaminated with L. monocytogenes and such experiments would be valuable.
In other experiment, not included in Table 5, impact of activated plastic films with thymol and enterocin AS-48 was evaluated on vacuum-packed sea bream fillets by measuring changes in bacterial load and bacterial diversity under refrigerate storage for 10 days. Samples however were not intentionally inoculated, but natural microflora present was analyzed. Bacteria of Listeria genus had the greatest relative abundance (24.90%) of all the microorganisms at day 0 in untreated samples. In samples treated with the activated film, bacteria were reduced to 0.00% of all the microorganisms at day 0. After 10-day storage relative abundance of Listeria genus in the treated and untreated samples were of 0.09% and 0.79%, respectively [116].

Combining NPDA and Other Antilisterial Factors
Synergistic or additive effects are used in food perseveration in the "hurdle technology", when two or more preserving techniques are used in combination, to achieve gentle but effective preservation of foods. Antimicrobial technologies are used in lower doses to control simultaneously food safety and other food quality aspects such as sensory quality and nutritional values. Synergistic effects may occur among many factors such as pH control, water activity reduction, heat processing, incorporating natural antimicrobials and other [117,118]. Synergism in reducing L. monocytogenes between NPDA and other compounds or methods is described later in the text. Some of the experiments were per-formed with L. innocua, which as mentioned above, can be a L. monocytogenes surrogate. As suggested by many authors [81,89,114], combined treatment with natural compounds and other methods is necessary for antilisterial treatments of food products.

Plant Antimicrobials Used in Combination
Many compounds showed synergism when they were used in combination. Carvacrol with cymene used together allowed to achieve the same growth delay of L. monocytogenes when much lower concentrations of the antimicrobials were used [52]. Carvacrol and 1,8-cineole have also shown synergistic activity against L. monocytogenes, which allowed to reduce concentrations of the compounds by 4-folds compared when they were used separately [65]. Fruit extracts (lime, lemon or calamansi) also showed synergism with thymol or carvacrol. When antimicrobials were used in pairs, L. monocytogenes counts were reduced by up to 7 log CFU/mL, indicating complete elimination of the pathogen, even though antimicrobials alone were not effective [119]. Binary carvacrol-thymol and thymol-eugenol combinations and ternary combinations of carvacrol, thymol, and eugenol also showed synergistic action against L. innocua [114]. Synergy between carvacrol and thymol against L. innocua was further confirmed by another author [120]. Combination of monolaurin and Zataria multiflora Boiss. essential oil acted synergistically against L. monocytogenes under every combination of conditions tested, including 2 levels of temperature (5 • C and 30 • C) and 3 levels of pH (5.0, 6.0 and 7.0) [76]. Ternary combination of natural antimicrobials: grapefruit seed extract, cinnamanaldehyde and nisin showed synergistic effect, reducing L. monocytogenes growth on lettuce and on pork loin [41]. Other ternary combination tested against L. monocytogenes was carvacrol and thymol in combination with mild heat treatment (55 • C for 30 min). Treatment resulted in reduction of more than 99.99% of L. monocytogenes CECT 4031, though the results were statistically indifferent to those obtained when carvacrol or thymol separately were used in combination with heat. However, when shorter heat treatments were applied (5 and 10 min), differences were statistically significant and combination of 3 factors (heat and two antimicrobials together) was more effective than using heat and only one compound [121].

Bacteriocins
Combining plant-derived antimicrobials with bacteriocins, mostly nisin, was thoroughly investigated. Carvacrol and nisin showed synergism at both tested temperatures (8 • C and 20 • C), as L. monocytogenes was completely inhibited in the presence of a 16-fold lower concentration of nisin (compared to when nisin was applied as the sole preservative), when it was used along with 1.25 mmol/L of carvacrol [23]. Thymol with nisin Z showed synergy, as 0.02% thymol combined with 40 IU/mL of the nisin Z (even though separately these antimicrobials were ineffective even at higher concentrations) resulted in a great reduction of the growth of L. monocytogenes, as indicated by the decrease in optical density value [122]. Nisin A and Nisin V in combination with carvacrol, thymol or trans-cinnamaldehyde were more effective, than alone. L. monocytogenes was reduced to undetectable levels when 195.2 µg/mL carvacrol was used in combination with 50 µg/mL nisin V, where treatment with these compounds alone resulted in final counts of approximately 7 log CFU/mL and 5.5 log CFU/mL, respectively. Combining Nisin A with EO compound was less effective when compared to nisin V [62]. Combinations of eugenol, thymol, cinnamaldehyde or carvacrol paired with nisin at the concentrations of 1/4 MIC for both antimicrobials used in pairs, resulted in lower final bacterial counts than the control, reaching approximately 7 log CFU/mL for pair with eugenol and thymol, 6 log CFU/mL for pair with cinnamaldehyde and 4 log CFU/mL for pair with carvacrol, compared to 9 log CFU/mL in the control and 6 log CFU/mL as an initial bacterial population [57]. Combining nisin with thymol inhibited L. monocytogenes at concentrations 2-fold lower of the two antimicrobials, compared to when used alone. Addition of 1.25 mg/mL of lactobionic acid and creating ternary combination resulted in 4-fold reduction of nisin and thymol [70]. Additionally, binary combinations of eugenol, thymol or carvacrol with nisin or ternary combinations of the three compounds with nisin and diglycerol fatty acid ester, namely diglycerol monolaurate had bactericidal effect on growing L. monocytogenes culture, even at concentrations that were ineffective separately [75]. As mentioned earlier, nisin was also incorporated along with cinnamaldehyde and seed extract grapefruit and showed synergism against L. monocytogenes cocktail on lettuce and pork loin [41]. In other study however, trans-cinnamaldehyde (at concentrations of 0.3 and 0.6 g/kg of curd) showed no synergistic effects with nisin (0.49 g/kg) and caprylic acid (0.36 g/kg) combination in Queso fresco cheese [108]. Experiments with L. innocua showed that combinations of nisin with carvacrol, thymol, eugenol or cinnamic acids resulted in synergistic actions against the bacterium [123].
Carvacrol (30 mM), eugenol (32 mM), thymol (5 mM), thyrosol (0.5 mM) and terpineol (30 mM) paired with enterocin AS-48 (30 µg/g) reduced viable cell counts of L. monocytogenes CECT 4032 inoculated on Russian type salad at 10 • C by 3.57-4.57 log units compared to control after 24 h. In case of thymol, terpineol or thyrosol with enterocin, bacteria were no longer detectable. Reductions obtained in the combined treatments were significantly higher (p < 0.05) than the sum of reductions obtained for each treatment separately, indicating a more than additive effect [109].

Heat Treatments
There are several studies suggesting synergism between natural antimicrobials and mild heat treatments in inhibiting L. monocytogenes. Exponential growth of L. monocytogenes cells cultured at 8 • C showed reductions after 30 min mild heat (45 • C) treatment in the presence of antimicrobials, namely S-carvone, carvacrol, cinnamaldehyde, thymol or decanal, by 1.5 to 2.1 log units, even though reductions achieved by the separate treatments showed no effect [54]. Isothermal heating between 53 • C and 68 • C in the presence of citral, (E)-2-hexenal and carvarcrol at their sublethal concentrations reduced the time required to achieve 5-log reduction of L. monocytogenes counts, e.g., at 55 • C in the control the time was of 145.75 min, whereas in the presence of antimicrobials, the same pathogen reduction was achieved between 40.13 for (E)-2-hexenal and 42.11 min for citral [61]. Thymol or carvacrol (at 0.3 mM for each antimicrobial) combined with 55 • C treatment for 5, 10 and 15 min resulted in significantly higher percent reductions of the pathogen than with the mild heat treatment only. For example, 5 min treatment at 55 • C without additive, with thymol and with carvacrol resulted in 76.27%, 92.76% and 98.19% reduction of the pathogen, respectively [121].
Additionally, a Weibull model of thermal inactivation of L. monocytogenes ATCC 13932 in the presence of cinnamaldehyde in ground pork was created and showed that the supplementation of cinnamaldehyde in ground pork considerably increased the thermal susceptibility of L. monocytogenes. Four levels of temperature (55 • C to 70 • C) and four levels of cinnamaldehyde at concentrations of 0 up to 1% (vol/wt) were used to determine thermal inactivation curves of the pathogen. Time needed to achieve a 5-log pathogen reduction declined from 28.15 min to 17.35 min at the presence of 1% carvacrol compared to the sample without carvacrol. At 70 • C time required declined from 1.95 min to 0.27 min. Created secondary model, based on 5-log lethality was statistically significant (p < 0.0001) [88].
However, phenomena of cross-resistance caused by reaction to sublethal stress exists and due to this phenomena bacteria can gain cross-resistance to EOs or their components after sublethal heat treatment [124]. L. monocytogenes EGD-e was exposed to carvacrol at concentration of 200 µL/L at 4.0 or 7.0 pH buffer. Prior to the carvacrol exposure, bacteria were heat shocked at 45 • C for one hour. When cells were not heat shocked beforehand, they were reduced by more than 5 log CFU (to undetectable levels) at both pH values. Heat shocked cells however were reduced by approximately 2 log CFU at both pH [124].

Antibiotics
Natural plant-derived antimicrobials showed synergistic action with classic antibiotics. Oleanolic acid and ursolic acid used in combination with ampicillin and oxacillin increased susceptibility of L. monocytogenes PCM 2191 to these two antibiotics. Oleanolic acid or ursolic acid (both acids acted identically) at concentration of 1/2 MIC combined with antibiotics resulted in the decrease of the antibiotics MICs values by approximately 2-folds [125]. Additionally, gentamycin combined with erucin, sulforaphane and allylisothiocyanate showed increased antibacterial activity in relation to the action of the phytochemical alone [55]. Citral and carvacrol separately and in combination decreased erythromycin, bacitracin and colistin MIC values against Listeria monocytogenes CECT 4032. MIC values of 0.125, 32 and 96 µg/mL for erythromycin, bacitracin and colistin, respectively, were reduced to 0.002, 0.034 and 0.144, respectively, when 0.100 µg/mL carvacrol and 0.250 µg/mL were present in the culture medium, indicating approximately 62 to almost 1000 times increased sensitivity to antibiotics [126]. Streptomycin with thymol and cinnamaldehyde showed synergism, while eugenol and berberine with streptomycin showed additive effect. Synergy was sufficient to eradicate biofilm formed on polystyrene [53]. Citral at concentration of 0.250 µL/mL increased sensitivity of first and second generation of L. monocytogenes CECT 4032 to 8 antibiotics, out of 9 tested. MIC values of erythromycin, cephalothin, gentamicin, bacitracin, colistin, trimethoprim/sulfamethoxazole, ampicillin and ciprofloxacin were significantly reduced. However, sensitivity to chloramphenicol remained unchanged [127].

Other Factors
Other factors, such as high hydrostatic pressure (HHP), preservatives, condiments or bacteria in combination with NPDA also showed synergy. Combined treatments with carvacrol or thymol and HHP (150, 200, 250 or 300 MPa) at 1 • C, 8 • C or 20 • C on exponentially grown cells of L. monocytogenes were more effective than separate treatments. At 1 • C, more than 5 log reductions in viable counts were achieved by applying pressures of 250 MPa in combination with 2.5 or 3 mmol/L carvacrol or 300 MPa in combination with 2, 2.5 or 3 mmol/L carvacrol, whereas at higher temperatures achieved reductions were less pronounced. Additionally, combined treatments of HHP and thymol were most effective at 1 • C [92]. Combined treatment with carvacrol and HHP was also evaluated in milk [92] and ground chicken meat [98] as a model food, where synergism against L. monocytogenes was confirmed.
Soy sauce with thymol (1 mM) and soy sauce with carvacrol (1 mM), reduced bacteria counts to undetectable levels, i.e., by more than 7 log CFU/mL after 10 min of storage at 22 or 4 • C. Significant reduction have also been achieved by combination of soy sauce with eugenol at 4 • C, where bacteria were reduced by 2 log CFU/mL, whereas soy sauce alone and the compounds in saline alone were not effective in reducing L. monocytogenes [101].
Hydrogen peroxide at concentration of 0.1% resulted in much greater performance of β-resorcylic acid (1.5%), carvacrol (0.75%) and trans-cinnamaldehyde (0.75%) when used in combination as antilisterial agents in frankfurters, leading to complete eradication of the pathogen when dipping treatment was applied at 65 • C [102]. Similarly, hydrogen peroxide at 2% concentration used in combination with carvacrol, thymol, β-resorcylic acid, and caprylic acid in washing solution, resulted in eradication of the pathogen from cantaloupes rind, whereas these substances alone was less effective [110]. Phenolic compounds (thymol, carvacrol, and eugenol) also showed synergism with potassium sorbate against Listeria innocua. Experiments were performed in two levels of water activity (a w ; 0.99 or 0.97) and two levels of pH (5.5 or 4.5). Potassium sorbate alone was inhibitory at 400-600 ppm, whereas phenolic compounds alone were inhibitory at concentrations of 200-250 ppm of eugenol, 100-200 ppm of thymol and 100-150 ppm of carvacrol, depending on the environmental conditions. At a w of 0.99 and pH 5.5 Listeria innocua growth was inhibited by combination of 50 ppm of potassium sorbate combined with 50 ppm of phenolic compound (either thymol, carvacrol or eugenol), proving synergism between the compounds [128].
Carvacrol or gallic acid paired with chitosan also acted synergistically in L. monocytogenes inhibition, as MIC values of the two compounds used together were lower than it would result from an additive effect [103].
Lactic acid bacteria showed synergy with eugenol. Combination of eugenol at 0.04% with either of the lactic acid bacteria tested (Bifidobacterim bifidum, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus plantarum or Lactococcus lactis) reduced invasion of Caco-2 cells to undetectable level, compared to approximately 2-3 log CFU/mL invasion rate when bacteria were used alone or 1.25 log CFU/mL when eugenol was used alone [93].

Alternative Delivery Methods of the Components Used against L. monocytogenes
Alternative ways of delivering antilisterial agents to the pathogen (compared to direct application of NPDA to food products or to laboratory medium) have been developed by many authors. Encapsulation and creating various films with plant derived compounds have been investigated and tested against L. monocytogenes. These systems are developed, because microorganisms proliferate in the aqueous phase of the food system and due to hydrophobic nature of most of the compounds, especially present in violate oils, mass transport of these active compounds to the microorganisms is relatively small [112]. Alternative ways of delivering are proposed to overcome this limitation, but also to prolong efficacies of the compounds, as they are hindered by their rapid depletion in foods [129]. Delivery systems also limit sensory impact of the antimicrobials on the products, as better delivery enable lower concentrations of antimicrobials.
Carvacrol and eugenol were solubilized in nonionic surfactant micelles (Surfynol 465 and 485W) and the activity of the encapsulated compounds depended on the surfactant used as a carrier. For carvacrol lower MICs were achieved with Surfynol 485W and varied from 0.025 to 0.35% (wt/wt), depending on the bacterial strain. For eugenol better results were achieved with Surfynol 465, where MICs varied from 0.1% to 0.3% [112]. Micellar-encapsulated carvacrol and thymol by nonionic surfactants [130,131], as well as carvacrol encapsulated into nanocapsules [80] have been also used as biofilm inhibitors. Citral and linalool alone or in combinations were nanoemulsified with Tween 80 and alternatively with ripening inhibitors, namely medium chain triglycerides, coconut oil, sesame oil or castor oil. Formulation containing citral only (stable throughout storage) showed the lowest MIC value out of formulation tested and reached 0.312%, whereas free citral showed MIC of 0.635%. Free linalool also showed greater MIC (of 1.25%) than emulsified (0.635%). MIC values of emulsions containing both antimicrobials was of 0.635%, whereas ripening inhibitors increased MIC value to 1.25%. Experiments were performed with Listeria monocytogenes ATCC 19111. The anti-biofilm activity of the free citral and citral nanoemulsion (citral at 5% concentration) indicated 81.54% and 83.51% biofilm inhibitions, respectively [132]. Carvacrol encapsulated into nanoliposomes (NLC) by the lipid film hydration technique and into nanocapsules (NCC) by the interfacial deposition technique of the preformed polymer had higher MBC values of 3.31 mg/mL for NCC and 5.30 mg/mL for NLC, compared to 1.77 mg/mL in the free carvacrol solution [80]. However, in the other study MIC value of polylactide nanoparticles with polyethylenimine coating and loaded with carvacrol was of 128 µg/mL, whereas free carvacrol had MIC of 1024 µg/mL against L. monocytogenes. Evaporation rates of the nanoparticles was less than 10% (weight) after 6 days at 37 • C, whereas free carvacrol was reduced to less than 25% [64]. Efficacy of nanoemultions of eugenol and thymol prepared with auric arginate and lecithin (which allowed to stabilize emulsion during storage) was evaluated in 2% reduced fat milk [106], as well as food grade capsules created by spray drying zein with coencapsulated nisin and thymol as antimicrobials, along with glycerin in some formulations were also evaluated in in milk [107] as described earlier in the text and presented in Table 5.
Poly(ethylene-co-vinylacetate) (EVA14) films, containing 3.5 wt% cinnamaldehyde or carvacrol and 7 wt% of carvacrol was not inhibitory against L. monocytogenes, however film containing 7 wt% of cinnamaldehyde resulted in antilisterial activity, with inhibition zone diameter of 15 mm in disc diffusion method. Authors also incubated the films with pathogens at 37 • C for up to 48 h, and relative inhibition of biofilm formed on the polymeric films compared to control was of minimum 48.4 for cinnamaldehyde at 3.5% after 24 h up to 76.3 for 7% carvacrol after 48 h [133]. Poly(lactic acid)-based nanofibres with incorporated 30% gelatin and 12% carvacrol, showed inhibitory effect against L. monocytogenes culture under the film, with average inhibitory zone of 35.9 mm 2 [134].
Nanofibers of fish skin gelatin encapsulating carvacrol (15-30% w/w) were prepared by blow-spinning method. L. monocytogenes 19115 was inhibited by created nanofibers. Nanofiber mats were placed on agar plates and incubated at 35 • C for 24 h. Oppositely to most of the other reports, authors achieved the biggest inhibition zone when fibers had the least carvacrol concentration (15%) and the smallest with 30% of carvacrol [135]. However, the same group prepared also nanofibers containing cinnamaldehyde (5-30%) and measured activity of the fibers using the same method. In this case, inhibition zones were increasing along with increasing cinnamaldehyde concentration [136]. Poly(lactic) acid-based films containing cinnamaldehyde inclusions (CI) at a concentration of at least 10% CI concentration showed 100% inhibition of L. monocytogenes in vitro. What is more, CI at 10% significantly improved tensile strength of the film. Cinnamaldehyde release was the highest at the first day and then smoothly decreased thought 20 days of storage [111]. Carvacrol loaded on electrospun gelatinized soluble potato starch fibers had better thermal stability than the free compound. Growth reduction of L. monocytogenes reached 89.0% [137]. Pea protein isolate-polyvinyl alcohol nanofibers incorporating cinnamaldehyde inhibited L. monocytogenes in diffusion assay, as the diameter of the inhibition zones varied from 10 mm to 15 mm (for concentrations of 1 wt% to 1.5 wt% of carvacrol) [138]. An edible film of Gelidium corneum containing carvacrol was incorporated as ham package against L. monocytogenes [104], films made of apple, carrot and hibiscus purees, incorporated with carvacrol or cinnamaldehyde at 0.5%, 1.5%, and 3.0% concentrations were used to wrap cooked ham and bologna [105], but also cassava starch films containing chitosan along with of gallic acid, as well as films with chitosan and carvacrol were tested on cooked ham [103], as described earlier in the text and presented in Table 5.

Natural Substances against L. monocytogenes Biofilm
As already mentioned, L. monocytogenes is able to form biofilm, where bacteria anchor themselves to surfaces and each other and are covered in extracellular matrix, what makes them especially hard to remove [2,7]. Several experiments showed, that NDPA are able to reduce listerial biofilm and biofilm formation on stainless steel surfaces and on other materials.
On stainless steel, carvacrol at concentration 2 mM removed or inactivated 32% of L. monocytogenes dried biofilm [139], carvacrol-or eugenol-loaded micelles were shown to reduce mature L. monocytogenes films, whereas achieved reduction depended on contact time, bacterial strain and medium on which the biofilm was grown. Eugenol-loaded micelles after 20 min exposure time was able to reduce biofilm to undetectable levels [130]. On the other experiment however nanocapsules containing carvacrol resulted in initial reduction of the pathogen anchored to stainless steel, however within next 5 h of incubation the counts were similar to control untreated sample. At the end of the experiment, after 25 h, the two samples showed no differences, reaching approximately 10 log CFU/cm 2 [80]. Sub-inhibitory concentrations of trans-cinnamaldehyde, carvacrol, thymol and eugenol showed inhibitory effect during biofilm formation on stainless steel and the inhibitory effect was more pronounced at 4 • C growth temperature (compared to 25 and 37 • C). Against pre-formed biofilm, carvacrol, thymol and eugenol resulted in pathogen reduction to undetectable levels after 15 min of incubation at all three temperatures. Trans-cinnamaldehyde resulted in reduction of L. monocytogenes counts, however in all cases the pathogen was still detectable at all temperature levels [79]. Combined action of cinnamaldehyde and eugenol resulted in approximately 90% inhibition of L. monocytogenes on the surface of 96-well microtiter plate [66].
The adherence ability of L. monocytogenes was also significantly (p < 0.05) inhibited by eugenol and citral at the subinhibitory concentration (0.05 mg/mL). Strong reduction (p < 0.05) of the adherence was observer after exposure to the MBC concentration of eugenol or citral during 30 min [68]. Resveratrol at its MIC value of 200 mg/L inhibited approximately 75% of biofilm formed in wells of flat-bottom 96-well polystyrene microtiter plate. Relative biofilm cell metabolic activity of treated strains was reduced to approximately 30% compared to the untreated control [74]. Trans-cinnamaldehyde, terpinene-4-ol or thymol, at their sub-inhibitory concentrations resulted in significant biofilm reduction compared to the control, when they were added to suspensions of 24 h-old L. monocytogenes culture. Trans-cinnamaldehyde performed the best, then followed by (not significantly different between each other) terpinene-4-ol and thymol [67]. Additionally, light controllable micelles incorporating thymol were tested against L. monocytogenes biofilm. Micelles were made of chitosan, toluidine blue O and poly(propylene sulfide) and activated under irradiation, which generates reactive oxygen species (ROS), which result in additional ROS-related bactericidal effects and stimulate thymol release. L. monocytogenes biofilm treated with these micelles after irradiation at 20 mW/cm 2 for 10 min was reduced to approximately 0.3 relative biofilm value compared to the control untreated with micelles, but also irradiated [140].
Experiments focusing on inhibiting L. monocytogenes biofilm are usually based on created homogenous mono-species systems. However, in vivo biofilms are heterogeneous, comprising different microorganisms forming a complex multi-species community [67]. Kerekes et al. [67] created dual-species biofilms where L. monocytogenes was paired with Escherichia coli, Pseudomonas putida and Staphylococcus aureus. Activity of transcinnamaldehyde, terpinene-4-ol and thymol were tested against these biofilms. Inhibition of biofilm formed with E. coli (i.e., eradication of both microorganisms) was achieved by cinnamaldehyde at concentration 0.5 mg/mL, terpinen-4-ol at 8 mg/mL and thymol at 0.2 mg/mL. Biofilm with S. aureus was inactivated by cinnamaldehyde at concentration of 0.8 mg/mL, terpinen-4-ol at 0.5 mg/mL and thymol at 8 mg/mL. Biofilm with P. putida was inactivated by terpinen-4-ol at 0.1 mg/mL and thymol at 8 mg/mL. Cinnamaldehyde at concentration of 1 mg/mL completely inhibited L. monocytogenes, however P. putida in the biofilm still survived at concentration of ca. 5.5 log CFU/mL.

Summary and Conclusions
Many natural chemical compounds show inhibitory or bactericidal activity against L. monocytogenes and bacterial cells are highly influenced by the presence of the compounds in the environment. Primary mechanism of action of eugenol, carvacrol [86], cinnamaldehyde [88], citral and (E)-2-hevenal [89] is cell membrane disruption.
The antimicrobials were used in many food model systems and in some cases complete eradication of the pathogen was achieved, even though food components inhibit action of the antimicrobials [52,63,70,74,92,112]. L. monocytogenes at the end of storage was no longer detectable after treatment with NPDA in raw meat [98,99], teriyaki sauce [99] and in soy sauce [101], in raw fish [100], various meat products [102,105,111], milk [70], salads and vegetable models [65,69,109] and cantaloupes [110]. However, there are no studies where the effect of NPDA at undetectable (in terms of sensory analysis) levels would be measured in food products contaminated with L. monocytogenes and such experiments would be valuable.
Achieving sufficient reductions of the pathogen in food model systems without any sensory impact would probably require using binary or ternary combinations of the natural antimicrobials, as many of them show synergism in combination [41,52,65,76,114,[119][120][121] or combining them with bacteriocins, as bacteriocins also act synergistically with many NPDA [23,41,57,70,75,108,109,122,123]. Other factors, for example mild heat treatments also significantly enhance action of the NPDAs [61,88,92,121]. Alternative methods of delivering the compounds, e.g., microcapsulation, may also reduce sensory impact of the antimicrobials on the food product, as they increase mass transport of active com-pounds to the microorganisms and due to that concentration of the antimicrobial can be decreased [112].
Even though many publications suggest that NPDA could be very beneficial in combating L. monocytogenes, there are also two disturbing finding. Firstly, C. elegans showed loss in life span (p < 0.05) when it was fed in a lawn of L. monocytogenes previously treated carvacrol compared to untreated cells. Additionally, carvacrol treated bacteria reduced statistically significantly the number of laid eggs [97]. Second disturbing finding is that in presence of trans-cinnamaldehyde at concentrations close to MBC, L. innocua cells entered in the VBNC state [87]. This finding raises a question if L. monocytogenes, closely related to L. innocua, in other studies could also survive but was not enumerated, because of methodology limitations. Though some studies indicate that VBNC L. monocytogenes are avirulent [141,142] other study showed that ingestion of VBNC L. monocytogenes by C. elegans resulted in significant reduction in life span, that was not different from life span reductions caused by culturable L. monocytogenes treatments [143].
Areas that require further studies in the field of NPDA as L. monocytogenes inhibitors are especially: ability of the pathogen to enter VBNC state in food products treated with NPDA and also studies focused on creating NPDA food treatments that would be both completely undetectable by consumers and effective against the pathogen.