Antibacterial activity of crude extract of Tabernaemontana catharinensis latex (A. DC) against Alicyclobacillus spp

Alicyclobacillus spp. is composed of Gram-positive, aerobic, thermoacidophilic, endospore-forming bacteria that cause food and beverage spoilage. The presence of Alicyclobacillus spp. may result in the production of guaiacol, which leads to sensory changes in the odour and taste of citrus juices and acidic foods. Tabernaemontana catharinensis (A. DC) is a plant belonging to the family Apocynaceae that produces milky latex with several biological activities described as antioxidant, antiviral, antimicrobial, trypanocidal and anti-leishmanicidal. Therefore, this study aims to evaluate the antimicrobial activity of the crude latex of T. catharinensis (A. DC) against microorganisms of the genus Alicyclobacillus spp. The minimum inhibitory concentration of latex was 7.81 μg/ml for the five Alicyclobacillus species analysed. The minimum bactericidal concentration for the species Alicyclobacillus acidoterrestris 0244T, A. hesperidum 0298T, A. acidiphilus 0247T and A. cycloheptanicus 0297T was 250 μg/ml. Cytotoxicity analysis demonstrated that latex was toxic to Vero cells at concentrations greater than 84.67 μg/ml. Scanning electron microscopy revealed changes in the cell wall of A. acidoterrestris 0244T present in orange juice when treated with crude latex. The results obtained suggest that the crude latex of T. catharinensis (A. DC) displays an antimicrobial effect against Alicyclobacillus, with potential for application in the food industry.


Introduction
The juice deterioration process caused by Alicyclobacillus acidoterrestris was first described in 1984 in apple juice, with changes in taste and odour resulting from the production of 2-methoxyphenol (guaiacol) and 2,6-dibromophenol by the microorganism (Chang and Kang, 2005;Yamazaki et al., 1996).
The cell wall of species of the genus Alicyclobacillus is composed of cyclic fatty acids that confer resistance at temperatures of 25 to 70 ºC and low pH (2.0 to 6.5), which together with the potential to form spores, causes problems in the citrus juice and acidic foods industry, with consequent economic losses (Cai et al., 2015;Chang and Kang, 2005).
Brazil is responsible for 34% of the production of oranges and more than half of the production of concentrated orange juice (66°Brix) worldwide, and, consequently, juice exports contribute significantly to the country's trade balance (Neves and Trombin, 2017). Therefore, ensuring the quality of concentrated juice, increasing its shelf life and finding natural substances to prevent the development of spoilage species of this product is critical to the industry (Neves and Trombin, 2017).
Tabernaemontana catharinensis (A. DC) is a lactescent tree belonging to the family Apocynaceae, found in Brazil, Argentina, Paraguay and Uruguay, used in folk medicine as an antidote for snake bites, toothache and vermifuge relief. It presents biological action, such as antioxidant, antiviral, antimicrobial, trypanocidal and antileishmanial (Janning et al., 2011;da Silva Brum et al., 2016;Soares et al., 2007).
This plant has lactiferous cells that secrete latex, an aqueous emulsion with various chemical substances and varied bioactive compounds, such as polyphenols, flavonoids, sugars, amino acids, n-alkanes, alkaloids and proteins that are secreted in response to tissue damage in the plant (Boligon et al., 2015;Dussourd, 2017;Lewinsohn, 1991;da Silva Menecucci et al., 2019).
The chemical composition of T. catharinensis latex used in the current study presents 52.5% protein, 21.2% free amino acids, and a strong proteolytic activity in addition to other properties as described by Menecucci et al., 2019. This research aimed to evaluate the antimicrobial activity of the crude latex of T. catharinensis against microbial species of the genus Alicyclobacillus.

Bacterial strains and culture media
The bacterial reference strains used in this study (A. acidoterrestris 0244 T , A. hesperidum 0298 T , A. acidiphilus 0247 T , A. cycloheptanicus 0297 T and A. acidocaldarius 0299 T ) originated from the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI), located at the Centre for Chemical, Biological and Agricultural Research (CPQBA/UNICAMP). The strains were stored in cryotubes containing Bacillus acidoterrestris BAT medium (Deinhard et al., 1987) with 30% glycerol (Thermo Fisher Scientific, Waltham, MA, USA) at -20 °C at the Laboratory of Microbiology of Water, Environment and Food of the State University of Maringá (Maringá, Brazil).
The culture medium used in the assays was the BAT with final pH adjusted to 4.0 with 1 M NaOH or 1 M HCl solutions. Concentrated orange juice (66°Brix, pH 4.0) used in the assays was provided by a citrus juice industry in the northwestern state of Paraná, Brazil and reconstituted to 11°Brix in sterile distilled water under aseptic conditions and free of Alicyclobacillus spp., as analysed in the laboratory. Latex was obtained through superficial incisions in the stem of T. catharinensis (A. DC), collected in an equal volume of water. Samples were centrifuged (5,000g) at 10 °C for 25 min. The precipitate was discarded, and the resulting supernatant was lyophilised and maintained at -20 °C until assays were performed. The plot comprising almost all compounds of watersoluble latex was named latex of T. catharinensis (A. DC) (Mousinho et al., 2011;da Silva Menecucci et al., 2019).

Chemical characterisation of the crude latex of Tabernaemontana catharinensis
The biochemical composition of the crude latex of T. catharinensis presented 52.5% protein, 21.2% free amino acids, and potent proteolytic activity, as described by da Silva Menecucci et al. (2019). The analysis also revealed that crude latex contains 26.3% of total sugars.
Latex was also chemically analysed to determine the presence of bioactive secondary metabolites (da Silva Menecucci et al., 2019), which demonstrated that T. catharinensis latex is also a renewable non-wood source of biologically active monoterpenoid indole alkaloids.

Determination of minimum inhibitory concentration and minimum bactericidal concentration
The minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) for crude latex were determined by the 96-well plate microdilution technique (TPP Techno Plastic Products AG, Trasadingen, Switzerland), according to the Clinical and Laboratory Standards Institute (CLSI, 2018) guidelines, using BAT medium for culture (Deinhard et al., 1987). The crude latex was serially diluted in the wells, obtaining concentrations ranging from 0.49 to 1000 μg/ml. Next, 5 μl of vegetative cells of the microorganism previously standardised to 0.5 McFarland (10 8 CFU/ml) was added and re-diluted 1:10 (v/v) to obtain the standard concentration used (10 4 CFU/ml). Each well contained 100 μl of the culture.
The assays were performed individually for each species. The 96-well plate was incubated at 45 °C for 24 h, and then the turbidity of the well was evaluated visually. The MIC was considered the lowest concentration resulting in inhibition of growth, as visually assessed. The MBC was determined by sub-culturing 20 μl of each negative well to the surface of a BAT agar plate that was incubated at 45 °C for 24 h. The assays were performed in triplicate, according to the methodology described by (Anjos et al. 2016).

Cytotoxicity analysis
The cytotoxic activity of T. catharinensis latex crude (A. DC) was evaluated by the colorimetric method using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as described by Mosmann (1983). Accordingly, 96-well plates were prepared with a monolayer of Vero cells and incubated until confluence. For the cytotoxicity assays, serial dilutions (31.25, 62.50, 125, 250, 500 and 1000 μg/ml) of the crude latex were prepared, and 200 μl/well of each concentration was added, followed by incubation of the plates at 37 °C and 5% CO2 for 72 h. After incubation, the old medium was removed, the wells were washed with 100 μl of phosphate-buffered saline (PBS), and then 50 μl/well of MTT (2 mg/ml) was added, and the plates incubated in an oven at 37 °C for 4 h under light. The reagent was then removed, 150 μl/well of dimethyl sulfoxide was added, and the reading was carried out at 570 nm. The cytotoxicity was determined according to Eq. (1): Cellular destruction (%) = 1 -(ODt/ODcc) where ODt is the optical density of the treated sample and ODcc is the optical density of the control cells.
The selectivity index (SI) was obtained through the formula: SI = LC50/MIC (Makhafola et al., 2014) where LC50 denotes the lethal concentration at which 50% of the cells are killed. The experiment was performed in triplicate, and the result expressed Research, Society and Development, v. 10, n. 9, e16310917907, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org /10.33448/rsd-v10i9.17907 5 as the average of three independent experiments. Positive and negative controls of the test were analysed simultaneously as the samples.  (Haddad et al., 2007). All the assays were performed in triplicate.

Minimum inhibitory concentration and minimum bactericidal concentration
The MIC and MBC of the crude latex are shown in Table 1. For all strains examined, the MIC was 7.81 μg/ml, and the most effective MBC result of 250 μg/ml occurred for strains A. acidoterrestris 0244 T , A. hesperidum 0298 T , A. acidophilus 0247 T and A. cycloheptanicus 0297 T . For the strain A. acidocaldarius 0299 T , the MBC was 500 μg/ml. According to the study by Holetz et al. (2002), which evaluated the antimicrobial activity of several plants against various bacteria, a MIC below 100 μg/ml is classified as good antimicrobial activity, 100-500 μg/ml is described as weak activity, and a MIC above 1000 μg/ml is considered inactive. In this context, the MIC result of <100 μg/ml obtained in the current study, demonstrated that the crude latex of T. catharinensis could be considered to exert good activity against the Alicyclobacillus species evaluated.  Research, Society and Development, v. 10, n. 9, e16310917907, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org /10.33448/rsd-v10i9.17907 6 of energy metabolism. In the present study, the crude latex was used, and therefore, it is likely to contain the same compounds plus others not extracted by n-butanol.
When investigating the antibacterial activity of Piperaceae extracts and nisin against A. acidoterrestris 0244 T , Ruiz et al. (2013) reported Piper aduncum as having the lowest MIC of 15.6 μg/ml although nisin also exhibited strong antibacterial activity. Nisin is an antibacterial polypeptide produced by Lactococcus lactis sp., used as a food preservative, which has a high production cost. Therefore, when compared with the current results, the crude latex of T. catharinensis shows a great potential application in the inhibition of Alicyclobacillus spp.
Previously, dos Anjos et al. (2016) suggested the antimicrobial activity of papain and bromelain against Alicyclobacillus spp. could be associated with the high proteolytic capacity of these enzymes. According to da Silva Menecucci et al. (2019), the crude latex of T. catharinensis has a strong proteolytic activity, and this might explain its good antibacterial action against Alicyclobacillus spp.

Cytotoxicity
The in vitro cytotoxic effect of the latex of T. catharinensis (A. DC) was evaluated by the MTT assay in Vero cells.
The LC50 value found for the crude latex was 84.67 ± 8.14 μg/ml. At this concentration, the crude latex was able to reduce 50% of the viable Vero cells.
The SI indicates the relative safety of the plant extract according to its toxicity-activity, which presented a result equivalent to 10.84. High SI values (>1.0) indicate a large safety margin between the crude latex concentration capable of eliminating the bacterium and the toxic concentration for mammalian cells (Makhafola et al., 2014). SI values lower than 1.0 are considered to be less selective for microorganisms, indicating that the sample tested is more toxic (Santos et al., 2008).
An investigation into the cytotoxic activity of the crude extract of leaves and stem bark of T. catharinensis (A. DC) conducted by Boligon et al. (2015), determined LC50 values of 59.53 ± 4.56 and 62.82 ± 4.75 μg/ml, respectively. In comparison, the crude latex of the present study presented a higher LC50, allowing the use up to nine times the MIC, remaining within the limits of cytotoxicity.  Similar characteristics regarding the morphological alterations were reported by Molva and Baysal (2015) when examining Alicyclobacillus acidoterrestris bacterial and spore cells in apple juice treated with pomegranate fruit extract and grape seed, Likewise, de Pascoli et al. (2018) noted morphological changes to A. acidoterrestris 0244 T cells exposed to Piperaceae extracts that also corroborated our observations.

Conclusion
The results of the present study showed that T. catharinensis latex displays good antimicrobial activity against A. acidoterrestris bacteria. The images captured by scanning electron microscopy allowed the visualisation of the morphological changes caused to the cells by the crude latex. Therefore, the crude latex of T. catharinensis is a promising alternative for the concentrated orange juice industry. The cytotoxicity test presented satisfactory results, allowing the use of crude latex at up to nine times the MIC without reaching the limits of cytotoxicity, although further studies of in vivo toxicity for the application of this compound in food are still necessary. This study is the first to evaluate the antimicrobial activity of T. catharinensis against Alicyclobacillus spp. and the use of this compound as a food preservative is a sustainable alternative.
The proteolytic enzymes isolated from the latex of Tabernaemontana catharinensis are of great interest mainly because they are non-wood products that avoid destructive harvesting of plant sources. The appropriate processing of the crude latex and isolating of protein and/or peptides is very important to identify the major component responsible for antibacterial activity. We suggest as future studies the verification of antimicrobial activity of latex in other food matrices.