Changes in pH affects Bioactivity of Chitosans from Callinectes sapidus

: The effect of pH on the antimicrobial activity of chitosans was evaluated with five different molecular weight chitosans (DMPAC: M W 152; DMPCA: M W 338; DPMCA (2) : M W 418; DMCPA: M W 550 and DCMPA,M W 558) at 500µg/ml concentration on different food-borne bacteria and fungi. Studies on pH was carried out at pH 3.0, 3.5, 4.0, 5.0 and 5.8 with 500µg/ml chitosan using an overnight broth of bacteria (0.05ml) sub-cultured in nutrient broth or MRS broth (for lactic acid bacteria). Fungi were incubated at 28+ 2 0 C for 72h and enumerated on sabouraud dextrose agar. The viable cell count of Staphylococcus aureus at pH 3.0 for all chitosans ranged between 1.23-1.76Log 10 CFU/ml while at pH 5.8 viable cell numbers was 2.38-5.26log 10 CFU/ml compared to the initial inoculum number of 7.06Log 10 CFU/ml. The growth of Listeria monocytogenes was totally suppressed by 500µg/ml chitosan at or below pH 5.0. Bacillus subtilis was susceptible to inhibition at low pH and had no detectable viable cell counts at pH 3.0-4.0. The viable cell numbers of Escherichia coli 0157:pH7 were reduced by approximately 2 log 10 cycles at pH 5.8 and by more than 5 logs at pH3.0 with DMPAC chitosan. Rhizopus Stolonifer was reduced to non-detectable levels by DMPAC chitosan at pH 3-3.5.This mould was more sensitive to chitosan (500µg/ml) at all pH compared to Penicillium expansum and Aspergillu sniger. Saccharomyces cerevisiae and Pichia fermentans were similarly affected by low pH. The results of the present study show that application of chitosan to acidic foods such as fruit juices will enhance its effectiveness as a natural preservative.

Chitosan is a natural polysaccharide comprising copolymer of glucosamine and N-acetylglucosamine. It can be obtained by the deacetylation of chitin from crustacean shells. (No and Meyers, 1989). Chitin and chitosan have very similar chemical structures. Chitin exhibits structural similarity to cellulose and differs from it with the replacement of C-2 hydroxyl residues by acetamide groups (Kurita, 1998). Chitin can be transformed into chitosan that has free amino groups by removing acetyl groups (CH3-CO) from chitin molecules. Thus chitosan is the deacetylated form of chitin, meaning that the acetamide groups (CH3CO-NH) in chitin are substituted into amino groups (-NH2) in chitosan (Sakthivel et al., 2015). Hsu et al. (2002) reported that chitosan is insoluble in water, alkali and organic solvents, but soluble in most diluted acids with pH less than 6. When chitosan is dissolved in an acid solution, it becomes a cationic polymer due to the protonation of free amino groups on the C-2 position of the pyranose ring. The cationic properties of chitosan in acidic solutions give it the ability to interact readily with negatively charged molecules such as lipids and cholesterol. In this respect, chitin and chitosan have attained increasing commercial interest as suitable resource materials due to their excellent properties including biocompatibility, biodegradability, absorption, ability to form films and to chelate metal ions (Li et al., 1992).Chitosan have been reported to have antimicrobial activity (Omogbai and Ikenebomeh,2013;Sakthivel et al., 2015).In the light of the above, this paper examines the effect of changes in acidic pH on the antimicrobial properties of chitosan.  Gram-stained (Roberts et al., 1995). Phenotypic profiling of both Gram-positive and Gram-negative bacteria was undertaken using API 50CHB and API 20E strips (BioMerieux, Marsielle, France) respectively. Fungi (Saccharomyce scerevisiae, Pichia fermentans, Penicillium expansum, Aspergillus niger and Rhizopus stolonifer) employed in the studies were isolated from tropical fruits wastes of pineapple and watermelon.

Source of chitosan: This was obtained from
Callinectes sapidus by unconventional methods outlined by Omogbai and Ikenebomeh (2016).
Effect of pH on the antimicrobial activity of chitosan: modified method of Youssuf and Munir (2007) was employed. The effect of pH on the antimicrobial activity of chitosans was evaluated with five different molecular weight chitosans (DMPAC,MW 152; DMPCA, MW 338;DPMCA(2),MW 418; DMCPA MW, 550;DCMPA,MW 558) at a 0.05% (500µg/ml) concentration on different food-borne bacteria and fungi. The upper pH value studied was limited to 5.8 because chitosan is soluble in most organic acid solutions with less than pH 6 (Muzzarelli, 1973). Studies on pH were carried out at pH 3.0, 3.5, 4.0, 5.0 and 5.8. The solutions were adjusted to these pH values with 1N HCL and 1N NaOH. Then 0.05ml of overnight broth of each microorganism subcultured in nutrient broth or MRS broth (for lactic acid bacteria) were inoculated into 10ml of nutrient broth or MRS broth containing 0.05% chitosan and incubated at 37 0 C for 24h. Viable cells were enumerated on nutrient agar or MRS agar by pour plating 1ml after serial dilutions of the chitosan solutions followed by incubation at 37 0 C for 24h.Fungi were enumerated on sabouraud dextrose agar and incubated at 28+ 2 0 C for 72h.

RESULTS AND DISCUSSION
The effects of pH 3.0-5.8 on antimicrobial activity of crab chitosans are shown in Tables 1-3. The effect of pH on the antibacterial activity of chitosans was evaluated with five different molecular weight chitosans at 0.05% (500µg/ml) concentration on five Gram-positive bacteria ( Table 1).As shown in Table  1, the antibacterial activity of chitosan was affected by pH, with greater activity at lower pH values. The viable cell count of Staphylococcus aureusat pH 3.0 for all chitosans ranged between 1.23-1.76Log10CFU/ml while at pH 5.8 viable cell numbers was 2.38-5.26log10CFU/ml compared to the initial inoculum number of 7.06Log10CFU/ml. Among the chitosans, DMPAC had the lowest viable cell count of 1.23Log10CFU/ml at pH3.0. Chitosan DCMPA had the highest viable cell count (5.26Log10CFU/ml) for this organism at pH 5.8. The growth of Listeria monocytogenes was totally suppressed by 500µg/ml chitosan at or below pH 5.0. Bacillus subtilis was susceptible to inhibition at low pH and had no detectable viable cell counts at pH 3.0-4.0 using 500µg/ml with chitosans DMPAC, DMPCA, DPMCA (2) and DMCPA. The chitosan DCMPA although suppressed the growth of this organism had viable cell numbers of 1.18Log10CFU/ml at pH 3.0 and 4.06Log10CFU/ml at pH 5.8.
Lactobacillus casei and Lactobacillus plantarum also had their cell numbers reduced at low pH than at higher pH.At pH 3.0 the viable cell numbers of Lactobacillus casei ranged between 1.08-2.03Log10CFU/ml, 1.21 -2.16Log10CFU/ml (at pH 4.0) and 2.60-4.03Log10CFU/ml (at pH 5.8). The antibacterial activity of all chitosans was stronger with decrease in pH against Lactobacillus plantarum. At pH 3.0 and 3.5 no viable cells were detected for DMPAC chitosan, 2.05 Log10CFU/ml at pH 4.0, and 2.52Log10CFU/ml at pH 5.8. Thus for all grampositive bacteria tested, the lower the pH, the greater the antimicrobial activity of all chitosans used.
The effect of pH on the antibacterial activity of chitosans on Gram-negative bacteria is illustrated in Table 2. The most antimicrobial activity was found at low pH values. The viable cell numbers of Escherichia coli 0157:H7 were reduced by approximately 2 log10 cycles pH 5.8 and by more than 5 logs at pH3.0 with DMPAC chitosan. The range of viable cell reduction by chitosans at pH 3.0 for Escherichia coli 0157:H7 was 2.05-2.42Log10CFU/ml, 3.30-3.62Log10CFU/ml (pH 3.5), 4.65-4.92Log10CFU/ml (pH 4.0), 5.32-5.83Log10CFU/ml (pH5.0) and 5.68-5.95Log10CFU/ml (pH 5.8) respectively (Table 2). The viable cell numbers of Salmonella typhimuriumin a control experiment at pH 3.0 increased from 7.57 -9.23Log10CFU/ml. The addition of chitosans to the medium caused a reduction in the viable cell count in the range 2.08-2.61Log10CFU/ml. At pH 3.5, the viable cell count also reduced to 3.41 -4.82Log10CFU/ml. With DMPAC chitosan, Salmonella typhimurium was reduced by approximately 5.5 log cycle at pH 3.0 compared to 2 log cycle at pH 5.8.The growth of Pseudomonas fluorescens and Vibrio parahaemolyticus were similarly affected by low acidic pH values. With DMPCA chitosan addition at pH 3.0 the viable cell count of these bacteria was reduced to non-detectable levels but at pH 5.8 the viable cell counts were reduced to 1.94 and 3.22Log10CFU/ml respectively ( Table 2).
The effect of pH on the antifungal activity of chitosan on yeast and moulds is shown in Table 3. Although yeasts and moulds can survive in acidic pH, their numbers were decimated considerably with the addition of 500µ g/ml of chitosan to the growth medium. Saccharomyces cerevisiae for example at pH 3.0 was reduced to 1.35 -1.59Log10CFU/ml compared to 4.25-4.46Log10CFU/ml at pH 5.8. Saccharomyces cerevisiae at pH 3.5 was reduced by 3log cycles by DCMPA (Mw, 558 KDA) compared to less than 1 log cycle reduction at pH 5.8. The yeast Pichia fermentans was similarly affected by pH. At pH3.0 the log reduction was in the range 1.48-2.20Log10CFU/ml, 2.31-2.50Log10CFU/ml at pH 3.5, 2.62-2.72Log10CFU/ml at pH 4.0, 2.70-2.85Log10CFU/ml at pH 5.0 and 3.17-3.41Log10CFU/ml, at pH 5.8. The log reduction in counts of this organism decreased with increasing pH ( Table 3).
The antimicrobial activity of chitosan was found to increase with decreasing pH. Decrease in pH increased solubility by forming polycationic polymer. This is due to the fact that amino groups of chitosan become ionized at pH below 6 and carry a positive charge. (Muzzarelli, 1973).
The greater the positive charge the more active this polymer becomes. It is worthwhile to note that growth of Listeria monocytogenes and Bacillus subtilis were completely deactivated or suppressed by 500µg/ml chitosan at or below pH 5.5. The presence of Listeria monocytogenes in foods has become a concern in recent years.  Confirmed outbreaks of human Listeriosis have been associated with consumption of contaminated foods from plant and animal sources. The ability of Listeria monocytogenes to proliferate at refrigeration temperatures and cause serious illness have been reported (Ahamad and Marth, 1989), Thus, a significant health hazard could result by consumption of foods contaminated with this organism but possibly could be reduced or prevented by proper chitosan treatment. The pH values of tropical fruit juices are usually acidic and range between 3.54 and 5.56. At low pH, both Escherichia coli 0157:H7 and Salmonella typhimurium survive for several days, especially when stored at refrigeration temperatures as reported by McClure and Hall (2000); Youssuf and Munir (2007). Thus the acidic nature of unpasteurized fruit juices does not ensure its safety as some pathogens may survive for extended periods of time and cause disease (CDC, 1997). While some pathogens gradually died off with chitosan addition at low pH, others were killed rapidly showing the potential efficacy for use of chitosan for fruit juice preservation.

Conclusion:
The bioactivity of chitosan was affected by acidic pH showing the inhibition and total killing of food-borne bacteria and fungi which are either pathogenic or spoilage organisms. Thus the results of the present study clearly show that application of chitosan to acidic foods such as fruit juices will enhance its effectiveness as a natural preservative.