Potential of chitosan from Mucor rouxxi UCP064 as alternative natural compound to inhibit Listeria monocytogenes.

Listeria monocytogenes is widely distributed in nature and the infection listeriosis is recognized as a potential threat for human health because of its mortality rate. The objective of this study was to evaluate the growth profile and chitosan production by Mucor rouxxi UCP 064 grown in yam bean (Pachyrhizus erosus L. Urban) medium. It was also to assess the anti-L. monocytogenes efficacy of the obtained chitosan. Higher values of biomass of M. rouxxi (16.9 g.L(-1)) and best yield of chitosan (62 mg.g(-1)) were found after 48 h of cultivation. Residual glucose and nitrogen in the growth media were 4.1 and 0.02 g.L(-1) after 96 h, respectively. Obtained chitosan presented 85 % of degree of deacetylation and 2.60 x 10(4) g.mol(-1) of viscosimetric molecular weight. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of chitosan against L. monocytogenes ATCC 7644 were, respectively, 2.5 and 5.0 mg.mL(-1). At 2.5 and 5.0 mg.mL(-1) chitosan caused cidal effect in a maximum time of 4 h. Bacterial count below 2 log cfu.mL(-1) were found from 2 h onwards and no recovery in bacterial growth was noted in the remainder period. These results show the biotechnological potential of yam bean medium for chitosan production by Mucor rouxxi and support the possible rational use of chitosan from fungi as natural antimicrobial to control L. monocytogenes.

INTRODUCTION

Listeria monocytogenes is widely distributed in nature and has been frequently isolated from a range of sources such as soil, decaying vegetables, vegetal matter, silage, sewage, animal feed, fresh and processed meats, dairy products, slaughter-house waste and food processing plants (3, 22). Listeriosis is recognized as a potential threat for human health because of its high mortality rate, particularly in immune-suppressed patients, neonates, children, pregnant woman and elderly (16).

Since the majority of human listeriosis is caused by the consumption of contaminated food, alternative anti-L. monocytogenes strategies to control this pathogen in foods are of particular interest. Particularly, the increased demand for safe and natural foods has provoked researchers to investigate the antimicrobial efficacy of many natural compounds against some food related pathogen microorganisms (13). Concern over the negative consumer perception to chemical preservatives prompts an increased interest in the antimicrobial properties of chitosan to be applied in food conservation (4, 8).

Chitosan is a cationic amino polysaccharide, common constituent of fungal cell walls, particularly the class of Zygomycetes, essentially composed of β- 1,4 D-glucosamine linked to N-acetyl-D-glucosamine residues. This polymer is traditionally obtained by chemically deacetylation of crustacean chitin (5, 15). Antimicrobial efficacy of chitosan depends on its molecular weight and methods used to convert chitin to chitosan because it could affect the characteristics of deacetylation and distribution of acetyl groups, chain length, and the structure conformation of chitosan molecule (4).

In order to obtain chitosan of greater quality, filamentous fungi have been known as attractive source for industrial applications (1, 2) since it can be carried out by a simple process of extraction resulting in the obtainment of chitosan with no amount of proteins involved in human allergic reactions to shellfish (24). This study aimed to evaluate the chitosan production by Mucor rouxxi UCP 064 growing in the alternative yam bean (Pachyrhizus erosus L. Urban) media, and also to verify the efficacy of the obtained chitosan as anti-L. monocytogenes compound.

MATERIAL AND METHODS

Microorganism and culture conditions

Mucor rouxxi UCP 064 (Microorganism Culture Collection, Catholic University of Pernambuco, Recife, Brazil) isolated from mangrove sediment (Rio Formoso, Pernambuco, Brazil) was assayed for chitosan production. The strain was kept at Potato Dextrose Agar slants at 4 °C. For chitosan production, spores were harvest of 7 days old cultures grown on Potato Dextrose agar Petri dishes by adding sterile NaCl (0.85 g.100 mL-1) on the medium growth followed for gentle shaking during 30 s. Spores suspension was adjusted with sterile NaCl (0.85 g.100 mL-1) to have approximately 108 spores.mL-1 using a hemocytometer.

Listeria monocytogenes ATCC 7644 (Laboratory of Food Microbiology, Department of Nutrition, Federal University of Pernambuco, Recife, Brazil) was assayed as revealed strain. Stock culture was kept on Muller Hinton agar slants at 4 °C. Inocula used in antimicrobial assays were obtained from overnight cultures grown on BHI broth at 37 °C. After incubation, bacterial cells were separated from the growth medium by centrifugation (10000 rpm, 10 min), washed three times in buffered KCl (0.05 M KCl, 1 mM KH2PO4, 1 Mm CaCl2, 0.1 Mm MgCl2, pH 6.0) and resuspended in buffered KCl. Cell suspension was adjusted for a optical density at 600 nm of 1.5 providing a bacterial inocula of approximately 1.0 x 108 cfu.mL-1.

Chitosan production

The production of chitosan was carried out by submerse cultivation in yam bean (Pachyrhizus erosus L. Urban) medium (protein 8.72; starch 40.9; glucose 11.4 g.L-1; pH 7.0) prepared according to Stamford et al. (24). For this, 10 mL of M. rouxxi UCP 064 spores suspension (ca 108 spores.mL-1) was inoculated in Erlenmeyer flasks of 1000 mL containing 290 mL of growth medium, followed for incubation at 28 °C in an orbital shaker (150 rpm) for 96 h. Mycelia were harvested, washed twice in distilled water and submitted to lyophilization. Afterward, the obtained mass was maintained in a vacuum dissecator until constant weight.

During the cultivation aliquots were collected every 24 h for determination of biomass, chitosan production, pH, glucose and total nitrogen. Measure of biomass (g) was performed by a gravimetric procedure; glucose consumption (g.L-1) was determined by an enzymatic colorimetric method (Labtest® Kit - Glucose oxidase); nitrogen consumption (g.L-1) was found by a colorimetric method (Labtest® Kit for protein); pH was measured using a potentiometer (Digital Pontentiometer Quimis Mod. 400 A). All assays were performed twice.

Chitosan extraction

The chitosan extraction involved deproteination with 2% (w/v) sodium hydroxide solution (30:1 v/w, 90°C, 2 h), separation of alkali-insoluble fraction (AIF) by centrifugation (4000 rpm, 15 min), extraction of chitosan from AIF under reflux (10% v/v acetic acid 40:1 v/w, 60°C, 6 h), separation of crude chitin by centrifugation (4000 rpm, 15 min) and precipitation of chitosan from the extract at pH 9.0 (adjusted with a 4 M NaOH solution). Crude chitin and chitosan were washed with distilled water, ethanol and acetone and air-dried at 20 °C (10).

Chitosan was assessed in a range of 160–0.06 mg.mL-1 for anti-L. monocytogenes effect. Chitosan solutions were prepared in acetic acid (1 %) and adjusted to pH 5.8 using NaOH or HCl (21).

Determination of deacetylation degree (DD) and molecular weight (MWv) of chitosan

DD of chitosan was determined using infrared spectroscopy, and an absorbance ratio (A %) was calculated as follow: (9A1655/A3450) x 100/1.3. For this, two milligrams of chitosan overnight dried at 60 °C were mixed with 100 mg of KBr to provide 0.5 mm thick disks. The disks were dried for 24 h at 110 °C under reduced pressure. Infrared spectroscopy was recorded using a Bruker 66 Spectrometer and 100 mg KBr disks for reference. Intensity of maximum absorbance bands were found by the baseline method (19).

MWv of chitosan was found by viscosity (20). The viscosity of chitosan was determined using an AVS-350 viscosimeter (Schott Geräte), type/capillary: Cannon-Fenske dinside= 1.01 mm, at 25°C. After getting the intrinsic viscosity from tables K and a, were obtained for HAc/NaAc. K = 0.076, a = 0.76. The flow time was determined in seconds. Mark-Houwinks equation was used to find the average viscosimetric molecular weight in g.mol-1.

Determination of Minimum Inhibitory Concentration – MIC and Minimum Bactericidal Concentration – MBC

MIC and MFC of chitosan was found by macrodilution in broth. 5 mL of double strength BHI broth was inoculated with 1 mL of the bacterial inocula (ca 108 cfu.mL-1). After that, 4 mL of the chitosan solution at different concentrations (160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.06, 0.03 mg.mL-1) was added and followed by shaking for 30 s. The system was statically incubated for 24 h at 35 °C. MIC was defined as the lowest concentration required to completely preventing visible bacterial growth. An aliquot (100 μL) of the tubes with no visible bacterial growth was subcultured on sterile Muller-Hinton agar at 35 °C for 48 h to determine if the inhibition was reversible or permanent. MBC was defined as the lowest concentration which no growth was noted on Muller-Hinton agar. Control flasks without chitosan were tested in the same way.

Time-kill assay

For analyzing 96-h time-kill curves chitosan was assayed at 5.0 and 2.5 mg.mL-1. For this, 5 mL of double strength BHI broth was inoculated with 1 mL of the bacterium suspension (c.a. 106 cfu.mL-1). After that, 4 mL of chitosan was added to obtain the final proper concentrations, and the culture statically incubated at 37 °C. Aliquots (100 μL) were taken at 0, 1, 2, 4, 8, 12, 24, 48, 72 and 96 h, serially diluted in sterile peptone water (0.1 % w/v) and spread plated onto sterile Muller-Hinton agar. After 48 h of incubation at 37 °C, colonies were counted and the results were expressed in log of count forming unit per mL (cfu.mL-1). Control flasks without chitosan were tested in the same way.

Statistical analysis

The data of kill times were analyzed for significance (p<0.01) by the Tukey test using STATISTICA program version 6.0 of Statsolt Inc., USA. All experiments were carried out in triplicate and the results are expressed as mean of the parallel assays.

RESULTS AND DISCUSSION

Results of biomass, chitosan production, glucose and nitrogen consumption of Mucor rouxxi UCP 064 growing in yam bean medium at 28 °C are showed in Figure 1. Biomass production increased rapidly within 48 h, and reached the highest value (16.9 g.L-1) after 72 h of cultivation. Synowiecki and Al Khatteb (25) found highest amount of biomass (4 g.L-1) of Mucor rouxii growing in yeast extract and glucose 2 % medium after 48 h. Residual glucose and nitrogen in the growth medium were 4.1 and 0.02 g.L-1, respectively. Similar results were reported by Franco et al. (10) and Stamford et al. (24).

Figure 1

Curve of growth, chitosan production, pH, glucose and nitrogen consumption of Mucor rouxxi UCP 064 grown in yam bean medium at 28°C, 150 rpm, during 96 h of cultivation.

pH of the medium dropped to 5-4 in the first 48 h during the growth of M. rouxxi, and stayed near to 4 for the remainder period. pH values decreased during the exponential growth phase, probably, because the high amount of glucose and starch in yam bean medium resulting in production of pyruvic acid. Best yield of chitosan (mg per gram of dry mycelia biomass) was obtained with 48 hours of culture (62 mg.g-1). This result is in agreement with Stamford et al. (24) investigating the production of chitosan from C. elegans in yam bean medium, where noted best yield of chitosan (66 mg.g-1) with 48 hours of cultivation.

Chitosan obtained from M. rouxxi grown on yam bean medium presented 85 % DD and 2.60 x 104 g.mol-1 of MWv. In general, DD of chitosan from fungi is in a range of 80 to 90 % (5, 11, 17). 2.60 x 104 g.mol-1 of MWv characterizes the tested chitosan as having low molecular weight. Chitosan of low molecular weight presents reduced tensile strength resulting in elongation of its molecule and increased permeability in for bacterial membrane (17).

MIC and MBC values of chitosan from M. rouxxi UCP 064 grown in yam bean medium against L. monocytogenes ATCC 7644 were, respectively, 2.5 and 5.0 mg.mL-1. Previous studies reported on the antimicrobial property of a variety of chitosan toward some food related microorganisms, including the bacteria Staphylococcus aureus, Escherichia coli (26), Salmonella tiphymurium (6), and the fungi Fusarium and Alternaria (7).

Kanatt et al. (12) states that most studies has noted MIC values of chitosan in a range of 0.1 - 10 mg.mL-1 against food-related microorganisms. Even the MIC and MBC values of chitosan are regarded high in comparison to other alternative antimicrobial compounds (e.g. essential oils, phenolics, bacteriocins), broad spectrum of antimicrobial activity and low toxicity to mammalian cells makes it a promising antimicrobial compound to be applied in foods (18).

Kill-curves of L. monocytogenes ATCC 7644 when treated with chitosan from M. rouxxi UCP 064 along 96 h is showed in Figure 2. At 2.5 mg.mL-1 chitosan caused cidal effect (≥3 log10 reduction in viable cells, i.e., ≥99.9 % kill rate) after 4 h, compared to 2 h for concentration 5.0 mg.mL-1. Values smaller than 2 log cfu.mL-1 were found at the remainder periods with no recovery in bacterial growth. Chitosan at 2.5 and 5 mg.mL-1 caused significant decrease (p<0.01) of the bacterial count in comparison to the control assay. Results from the kill-curves demonstrated a clear concentration and time-dependent inhibitory effect of the assayed chitosan.

Figure 2

Survivors curves for L. monocytogenes ATCC 7644 in Brain Heart Infusion Broth at 35 °C during 96 h as a function of chitosan concentration: (▲): control (0 UL.mL-1); (♦): chitosan CIM (2.5 Ug.mL-1); (■): chitosan CBM (5.0 Ug.mL-1). The detection limit for viable cells was 2 log cfu.mL-1. Where no cells were recovered, the detection limit is indicated.

Although some researchers have reported antimicrobial property of commercial chitosan there has been few data about the antimicrobial potential of chitosan from fungi. Fai (9) found MIC values of 1.25 mg.mL-1 for chitosan from Mucor circinelloides UCP 050 (MWv 2.7 x 104 g.mol-1, DD 83 %) against L. monocytogenes, Staphylococcus aureus, E. coli, Salmonella enterica and Yersinia enterocolitica, while MBC were two or four-fold higher. Stamford (23) reported MIC values of chitosan from Cunninghamela elegans UCP 542 (MWv 4.02 x104 g.mol-1, DD 85 %) of 1.25 mg.mL-1 against Streptococcus mutans, S. sanguis, S. mitis and S. oralis.

Antimicrobial activity of chitosan is believed to originate from its polycationic nature, being hypothesized to be mediated by the electrostatic forces between the protonated amino group (NH2) in chitosan and the negative residues at cell surfaces (4). The cidal effect of chitosan toward L. monocytogenes is probably caused by the electrostatic interaction between NH3+ groups of chitosan and the negatively charged phosphoryl groups of phospholipid components of the cell membrane (14).

The results obtained in this study show the yam been medium as suitable media for production of chitosan by Mucor rouxxi. Moreover, the chitosan studied here presented interesting anti-L.monocytogenes property in synthetic media. Chitosan from fungi could become a promising compound for controlling L. monocytogenes in human and/or veterinary medicine and in foods. Particularly, the results of this study encourage further researches in our laboratory about the antimicrobial effect of the assayed chitosan toward other food-related pathogens in synthetic media and food matrix.