MODERN APPLICATIONS OF CHITOSAN IN FOOD SCIENCES
6.4 MODERN APPLICATIONS OF CHITOSAN IN FOOD SCIENCES
The data so far available clearly show that chitosan may be either bactericidal or bacteriostatic, or even be a growth promoter depending on the bacterial strain. For each strain, the characteristics of chitosan may be more or less significant. While general statements are therefore to be avoided, it appears that chitosans are certainly active against most human pathogens and food spoiling microbes.
Functional Food Carbohydrates
Chitosan is not an antimicrobial per se, but its performances can occasionally
be superior to biocides. For example, the antimicrobial efficacy of 0.5, 1.0, and 2.0% chitosan and a commercial biocide based on hydrogen peroxide was determined at
20˚C against Listeria monocytogenes, Salmonella enterica serovar, Typhimurium, Staphylococcus aureus , and Saccharomyces cerevisiae adhered to stainless steel. Dried films of S. aureus were most sensitive to chitosan but relatively resistant to the biocide. By contrast, yeast films were least sensitive to chitosan. 37
6.4.1 A NTIBACTERIAL A CTIVITY
The antibacterial activity of chitosan was originally documented by Muzzarelli et al., 38 who published electron micrographs showing the alterations produced in the bacterial cell wall and organelles. Those results were brilliantly confirmed more than a decade later by Helander et al., 39 who studied the mode of antimicrobial action of chitosan on Gram-negative bacteria, with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane. Chitosan (250 ppm) at pH 5.3 induced significant uptake of the hydrophobic probe 1-N-phenyl- naphthylamine in Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium . Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction of chitosan with E. coli and the salmonellae involved no release of lipopolysaccharide or other membrane lipids. Highly cationic mutants of S. typhimurium were more resistant to chitosan than the parent strains. Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the outer membrane with vesicular structures. Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function. This property makes chitosan useful for food protection. It was also found that the antibacterial activity of quaternized chitosan against E. coli is stronger than that of chitosan. 40
New interest has emerged in partially hydrolyzed chitosan and chitosan oligosac- charides. Enzymatic preparation methods captured interest due to safe and nontoxic conditions, and production has been developed into a continuous process. Many of the biological activities reported for chitosan oligosaccharides, such as antimicrobial, anticancer, antioxidant, and immunostimulant effects, depend on their physicochemical
properties. In a review, Kim and Rajapakse 41 have summarized different enzymatic preparation methods of chitosan oligosaccharides and their biological activities. Chitosan activity is synergistically enhanced by traditional preservatives such as benzoic acid, acetic acid, and sulfite. For fresh pork sausages, two pilot-scale trials showed that 0.6% chitosan combined with low sulfite (170 ppm) retarded the growth of spoilage organisms more effectively (3 to 4 log cfu/g) than high
levels (340 ppm) of sulfite alone at 4˚C for up to 24 days. 42 Trials in real foods showed that dipping of standard and skinless pork sausages in chitosan solutions
(1.0%) reduced the native microflora (total viable counts, yeasts and molds, and lactic acid bacteria) by approximately 13 log cfu g –1 for 18 days at 7˚C. Chitosan
treatment increased the shelf life. 43–45 The combined use of chitosan and sulfite permitted the slowing down of deterioration of chilled pork sausages. 42 In the case
Chitosan as a Dietary Supplement
of the preservation of herring and Atlantic cod, chitosan as an edible invisible film enhanced the quality of seafood during storage. 46
Recent investigations point out that chitosan in emulsions might be particularly more effective than in aqueous systems; for example, Jumaa et al. 47 found that lipid emulsions containing 0.5% chitosan conformed to the requirements of the preservation efficacy test for topical formulations according to the European Pharmacopoeia.
6.4.2 A NTIFUNGAL A CTIVITY
The use of chitosan to control postharvest fungal decay has attracted much attention due to problems associated with chemical agents, consumer reluctance against fun- gicide-treated produce, and an increasing number of fungicide-tolerant postharvest pathogens. Chitosan reduces the in vitro growth of numerous fungi with the exception of Zygomycetes, i.e., the fungi containing chitosan as a major cell wall component.
Tripathi and Dubey 48 reviewed the exploitation of some natural products, such as flavor compounds, acetic acid, jasmonates, glucosinolates, propolis, fusapyrone
and deoxyfusapyrone, chitosan, essential oils, and plant extracts, for the management of fungal rotting of fruits and vegetables, capable of prolonging shelf life.
The antifungal effect of chitosan on in vitro growth of common postharvest fungal pathogens in strawberry fruits consists of the marked reduction of the radial growth of Botrytis cinerea and Rhizopus stolonifer, with a greater effect at higher concentrations. Signs of infection in chitosan-coated fruits appeared after 5 days of storage at 13˚C compared with 1 day for the control treatment. After 14 days of storage, chitosan coating at 15 mg/ml reduced decay of strawberries caused by the same fungi by more than 60%, and coated fruits ripened normally and did not show any apparent sign of phytotoxicity. Similarly, the preservative effect of chitosan was observed on low-sugar candied kumquat. The growth of Aspergillus niger was inhibited by the addition of chitosan (0.1 to 5 mg/ml) to the medium (pH 5.4). Cuero
et al. 49 observed that N-carboxymethylchitosan reduced aflatoxin production in Aspergillus flavus and Aspergillus parasiticus by more than 90%, while fungal growth was reduced to less than one half.
Table grape treated with 1% chitosan showed increased phenylalanine ammonia- lyase activity, besides a direct activity against Botrytis cinerea. 50 Chitosan coatings reduced the incidence of molds occurring on apples over 12 weeks. The combination of hypobaric and chitosan treatments was found to be a valid strategy for decreasing
the decay of sweet cherries. 51 The effect of glycol chitosan applied as a coating to act as a biocontrol treatment of postharvest diseases of apple and citrus fruits was evaluated under simulated commercial packinghouse conditions by El-Ghaouth et al. 52
Devlieghere et al. 53 studied the antimicrobial effects of chitosan coatings on decay of minimally processed strawberries and lettuce. Several bacteria and yeasts were exposed to chitosan concentrations varying from 40 to 750 mg/l. Generally, Gram-negative bacteria seemed to be very sensitive to chitosan minimum inhibitory concentration (MIC 0.006% (w/v)), while the sensitivity of Gram-positive bacteria was variable and that of yeast was 0.01% (w/v). A chitosan coating was formed by dipping the products in a chitosan–lactic acid/Na–lactate solution; the pH was adjusted to the pH of the products. These products were equilibrium modified
Functional Food Carbohydrates
atmosphere packaged, stored at 7˚C, and during storage sensorially and microbio- logically evaluated. The microbiological load on the chitosan-treated samples was
lowered for both products. The antimicrobial effect of chitosan on lettuce disap- peared after 4 days of storage, while on the strawberries, it lasted 12 days. The treatment of mandarins and oranges with a chitosan coat produced excellent results in terms of percentage of weight loss and visual appearance. 54
A study carried out on chitosan coating for the inhibition of Sclerotinia sclero- tiorum rot of carrot showed that the incidence of rotting was reduced from 88 to 28% by coating carrot roots with 2% chitosan. 55–57 Carrot slices coated with a starch and chitosan mixture showed reductions in mesophilic aerobes, mold and yeast, and psychrotrophic amounting to 1.34, 2.50, and 1.30 log cycles, respectively. The presence of 1.5% chitosan in the coatings inhibited the growth of total coliforms and lactic acid bacteria throughout the storage period (10˚C for 15 days). The use of edible antimicrobial yam starch and chitosan coating was deemed to be a viable alternative for controlling microbial growth in minimally processed carrot.
Coating fruits and vegetables with chitosan or its derivatives has positive advantages for long-term storage of these foods, particularly fruits of exotic origin such as mango. 58 The preliminary treatment of the plants provided further advan- tages: Pseudomonas fluorescens, Bacillus subtilis, and Saccharomyces cerevisiae were evaluated for their potential to attack the mango (Mangifera indica L.) anthracnose pathogen Colletotrichum gloeosporioides Penz. under endemic con- ditions. The plant growth-promoting rhizobacteria P. fluorescens amended with chitin sprayed at fortnightly intervals gave the maximum induction of flowering,
a yield attribute in the preharvest stage; consequently, reduced latent symptoms were recorded at the postharvest stage. An enormous induction of the defense- mediating lytic enzymes chitinase and beta-1,3-glucanase was recorded. 59
Mangosteen, an economically important fruit of Thailand, has a short shelf life. Kungsuwan et al. 60 showed that the most effective chitosan concentration for aerial spraying was 2%, which could extend the shelf life of mangosteens to more than
23 days. Extension of the storage life and better control of decay of peaches, pears, and kiwi fruits by application of chitosan film have been documented. 61 Cucumbers, bell peppers, strawberries, and tomatoes could be stored for long periods after coating with chitosan. These results may be attributed to decreased respiration rates, inhi- bition of fungi development, and delayed ripening due to the reduction of ethylene and carbon dioxide evolution.
A series of O-acyl chitosans with a degree of substitution between 0.02 and 0.28 were synthesized by reaction of alkanoic acid derivatives with chitosan in the presence of H 2 SO 4 as a catalyst. O-Decanoyl chitosan (mole ratio of 1:2 chitosan to decanoic acid) was the most active compound against Botrytis cinerea and O- hexanoyl chitosan displayed the highest activity against Pyricularia grisea. Some derivatives also repressed spore formation at rather high concentrations (1.0, 2.0, and 5.0 g l –1 ). 62
When administered to a plant, chitosan has a dual function, i.e., direct interfer- ence of fungal growth and activation of several defense processes that include accumulation of chitinases, which degrades fungal cell walls’ synthesis of proteinase
Chitosan as a Dietary Supplement
inhibitors, lignification, and induction of callous synthesis. The microbial transport systems seem to be highly affected by the presence of chitosan. 63,64 Chitosan induced
the accumulation of the antifungal phytoalexin pisatin in pea pods. 65,66 Saprolegnia parasitica is responsible for infection of fish and eggs in aquaculture facilities and grows on injured, stressed, or infected fish. Electron microscopy obser- vation provided evidence of ultrastructural alteration, damaged fungal structure, hyphal distortion, and retraction. The antifungal action of chitosan could be modulated by proper chemical modification and put to use in protecting aquacultured fish. 67
The quantitative determination of chitin, a constituent of the fungal cell walls, offers the advantage that it reflects the total amount of mycelium. Bishop et al. 68
used chitin to further evaluate the detection of mold in tomato products, ketchup, paste, and puree. Variations were observed in chitin content among different fungal species, depending upon age and growth conditions. Insect contamination did not change the glucosamine level significantly except in cases of extremely high contamination.
In addition to its direct antimicrobial activity, chitosan induces a series of defense reactions correlated with enzymatic activities. Chitosan increases the production of glucanohydrolases, phenolic compounds, and synthesis of specific phytoalexins with antifungal activity, and reduces macerating enzymes such as polygalacturonases and pectin metil esterase. For some horticultural and ornamental commodities, chitosan increased the harvested yield. Due to its ability to form a semipermeable coating, chitosan extends the shelf life of treated fruit and vegetables by minimizing the rate of respiration and reducing water loss. As a nontoxic biodegradable material, as well as an elicitor, chitosan has the potential to become a new class of plant protectant, thus assisting toward the goal of sustainable agriculture. 69
6.4.3 E DIBLE F ILMS AND T EXTURAL A GENTS
Edible films can provide supplementary and sometimes essential means of control- ling physiological, morphological, and physicochemical changes in food products.
High-density polyethylene film, a common packaging material used to protect foods, has disadvantages like fermentation due to the depletion of oxygen and condensation
of water, which promotes fungal growth. Due to their filmogenicity, chitin and chitosan are satisfactorily used as food
wraps. Semipermeable chitosan films modify the internal atmosphere, decrease the transpiration, and delay the ripening of fruits. 70,71 For the preparation of chitosan/pec-
tin-laminated films and chitosan/methylcellulose films, several approaches have been used, including simple coacervation. Chitosan films are tough, flexible, and tear resistant; moreover, they have favorable permeation characteristics for gases and water vapor.
Chitosan is also suitable as a texturizing agent for perishable foods: for instance, high-viscosity chitosan solutions were used to prepare tofu, a widely consumed Oriental food, for which the organoleptic properties did not vary appreciably, while shelf life was extended. 72–74
Functional Food Carbohydrates
6.4.4 C ONTROL OF E NZYMATIC B ROWNING IN F RUITS
Mechanical injury during postharvest handling and processing causes browning of fruits and vegetables with loss of quality and value. Polyphenol oxidase is respon- sible for this phenomenon that affects color, taste, and nutritional value of fruits and
vegetables. 75 Dark-colored pigments are generated from o-quinones, under the effect of polyphenol oxidase activity. Concern over the adverse health effects of sulfite,
the most effective browning inhibitor, has stimulated a search for surrogate anti- browning compounds. The effect of a chitosan film on the enzymatic browning of
litchi fruit (Litchi chinensis Sonn.) was studied by Zhang and Quantick, 76 who reported that chitosan film coating delayed changes of contents of anthocyanins,
flavonoids, and total phenolics. It also delayed the increase in polyphenol oxidase activity and partially inhibited the increase in peroxidase activity.
Manually peeled litchi fruits were treated with aqueous solutions of 1, 2, or 3% of chitosan, placed into trays overwrapped with plastic film, and then stored at –1˚C.
Application of chitosan coating retarded weight loss and the decline in sensory quality, with higher contents of total soluble solids, titratable acid, and ascorbic acid, and suppressed the increase of polyphenol oxidase and peroxidase. Application of
a chitosan coating effectively maintained quality attributes and extended shelf life of the peeled fruit. 77
6.4.5 C LARIFICATION AND D E - ACIDIFICATION OF F RUIT J UICES
Processing of clarified fruit juices commonly involves the use of clarifying agents, including gelatin, bentonite, tannins, potassium caseinate, and polyvinyl pyrrolidone.
Chitosan is a de-hazing agent used to control acidity in fruit juices, besides being
a good clarifying agent for grapefruit juice, with or without pectinase treatment, and apple, lemon, and orange juices, as well as a fining agent for apple juice, which can
afford zero turbidity products with as little as 0.8 kg/m 3 of chitosan. No impact on the biochemical parameters of the juices was found. 78 Apple juice can be protected from fungal spoilage with the aid of modest additions of chitosan glutamate. 79 Spagna et al. 80 observed that chitosan has a good affinity for polyphenolic com- pounds, such as catechins, proanthocyanidins, cinnamic acid, and their derivatives, which can change the color of white wines due to their oxidative products. By adding chitosan to grapefruit juice (15 g/l), the total acid content (citric, tartaric, malic, oxalic, and ascorbic acid) was sharply reduced.
6.4.6 R ECOVERY OF S OLIDS FROM F OOD P ROCESSING W ASTES
Complying with water quality regulations is one of the major endeavors of the food industry. Effluents from food processing plants are characterized by high chemical
oxygen demand, biochemical oxygen demand, and total suspended solids. Recovery of suspended solids by coagulation and decanting may also be convenient in view of their utilization. 81
Chitosan as a coagulating agent for waste treatment systems is particularly effective in removing proteins from butchery and fishery wastes: the coagulated by-
products serve as animal feed, chitosan being digestible by most animals. Similarly,
Chitosan as a Dietary Supplement
plant proteins can be recovered from waters used to prepare vegetables for canning. In the cheese manufacture, for example, Fernandez and Fox 82 reported the use of chitosan to remove proteins and peptides from whey, while Ausar et al. 83 precipitated casein with chitosan. Altieri et al. used chitosan to prolong mozzarella cheese shelf life by taking advantage of the growth inhibition of spoilage microorganisms such as coliforms; in this context chitosan was found to stimulate lactic acid bacteria. 84
In fact, the presence of chitosan together with growth of milk fermentative bacteria was found useful in cheese making. In nutrient broth, all chitosans showed
a dose-dependent inhibition of Streptococcus thermophilus and Lactobacillus del- brueckii ssp. bulgaricus growth. Chitosan of high and low molecular weight, but not chitosan oligosaccharides, showed a dose-dependent inhibition of Propionibac-
terium freudenreichii . The effect of chitosan on milk fermentative processes depended not only on its molecular weight and concentration, but also on the presence of casein micelles or milk fat, which could prevent the inhibitory activity of these biopolymers on bacterial growth. 85