Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.     

Preparation and characterisation of selected physicochemical and functional properties of β‐chitosans from squid pen

Squid pen β‐chitosans prepared under various deacetylation conditions (30%, 35%, 40% and/or 45% NaOH for 15, 30 and/or 60 min) were characterised. β‐Chitosans (deacetylated with 35–45% NaOH for 15–60 min) had 87.1–96.2% degree of deacetylation (DD), 93.5–96.7% solubility and 120.5–654.9 mPa s viscosity. Treatment with 30% NaOH for 15–60 min yielded inadequately deacetylated β‐chitosans (DD = 51.9–80.2%). Two chitosans prepared under 35% NaOH for 15 min and 45% NaOH for 30 min (designated as 35%–15 and 45%–30, respectively) were further compared. Drying (sun‐drying vs. oven‐drying) methods did not affect DD. 35%–15 chitosan exhibited lower nitrogen, DD and bulk density, but higher viscosity compared with 45%–30 chitosan. Higher water‐ and fat‐binding capacity but lower DPPH radical scavenging activity were observed for 35%–15 chitosan compared with 45%–30 chitosan. Compared with 45%–30 chitosan, 35%–15 chitosan exhibited higher antibacterial activity against Salmonella Enteritidis and Listeria monocytogenes, but lower antibacterial activity against Escherichia coli.     

Low molecular weight chitosan prepared with the aid of cellulase,lysozyme and chitinase: Characterisation and antibacterial activity

A continuous set of low molecular weight chitosan (LMWC) products was successfully made for this study by coordinating three enzymes (chitinase, lysozyme and cellulase) and two different deacetylated chitosan substrates (80% and 92%). It was observed that the intrinsic viscosity molecular weight (M_V) and SEC-HPLC-determined M_W distribution of LMWC were directed by both the used enzyme and the degree of chitosan substrate acetylation. LMWC prepared using lysozyme and 92%-deacetylated chitosan had larger M_W and, therefore, possessed higher antibacterial activity, as compared to other combinations. LMWC enzyme-directed properties suggest chitinase is more predictable and flexible to produce the specified M_V of LMWC. LMWC’s solubility and antibacterial activity, determined as minimum inhibitory concentration (MIC), against Escherichia coli exhibited a negative linear relationship with log M_V. E. coli strains showed much higher susceptibility to LMWCs than Staphylococcus aureus strains did. Both species also showed intra-species sensitivity diversity toward LMWC.     

Effect of molecular weight of chitosans on their antioxidative activities in apple juice

This work studies how chitosans with low molecular weight (LMWC, M_W = 12 kDa), medium molecular weight (MMWC, M_W = 95 kDa) and high molecular weight (HMWC, M_W = 318 kDa) affect antioxidant activity in an aqueous system and in apple juice. Antioxidant activity was determined, including that of DPPH radicals, hydrogen peroxide and superoxide anion radicals, as well as metal ion chelating capacity, ABTS radicals of chitosans with different molecular weights (DMWCs) in apple juice.     

Enzymatic preparation of chitooligosaccharides by commercial lipase

The effect of a commercial lipase on chitosan degradation was investigated. When four chitosans with various degrees of deacetylation were used as substrates, the lipase showed higher optimal pH toward chitosan with higher DD (degree of deacetylation). The optimal temperature of the lipase was 55 °C for all chitosans. The enzyme exhibited higher activity to chitosans which were 82.8% and 73.2% deacetylated. Kinetics experiments show that chitosans with DD of 82.8% and 73.2% which resulted in lower K_m values had stronger affinity for the lipase. The chitosan hydrolysis carried out at 37 °C produced larger quantity of COS (chitooligosaccharides) than that at 55 °C when the reaction time was longer than 6 h, and COS yield of 24 h hydrolysis at 37 °C was 93.8%. Products analysis results demonstrate that the enzyme produced glucosamine and chitooligosaccharides with DP (degree of polymerization) of 2–6 and above, and it acted on chitosan in both exo- and endo-hydrolytic manner.     

Molecular characteristics of partially hydrolyzed fucoidans from sporophyll of Undaria Pinnatifida and their in vitro anticancer activity

The effects of molecular characteristics on the anticancer activity of fucoidans were investigated after hydrolysis by copper acetate and then fractionation with 30 and 5 kDa membranes, which produced three fucoidan fractions: F_>30 K, F_5–30 K and F_<5 K. The F_>30 K and F_5–30 K consisted of mostly carbohydrate (58.2–61.3%) and sulphate (31.7–35.5%) with small amounts of proteins (1.2–6.4%). However, the major constituents of F_<5 K were sulphate (31.8%) and ash (37.5%) with smaller amounts of carbohydrate (15.5%) and protein (1.2%). The molecular weights (M_w) of F_>30 K, F_5–30 K and F_<5 K, obtained by a light scattering technique, were 262, 5.6 and 1.6 kDa, respectively. The observed anticancer activities were 18.0–28.5% for F_>30 K, 19.2–57.5% for F_5–30 K and 26.5–36.5% for F_<5 K, respectively, in the concentration range of 0.2–0.8 mg/mL. The results suggest that the anticancer activity of fucoidans could be considerably improved by lowering their M_w and by improving the binding properties of sulphate groups possibly through changing the molecular conformation.     

Determination of physicochemical properties of sulphated fucans from sporophyll of Undaria pinnatifida using light scattering technique

The weight average molecular weight (M_w) of the intact sulphated fucans extracted with water from the sporophyll of Undaria pinnatifida without acid treatment was determined using the high performance size exclusion chromatography coupled to multiangle laser light scattering and refractive index detection (HPSEC-MALLS-RI) system. The effects of different heating conditions on the determination of M_w were investigated. The extracted intact fucoidans mostly consisted of carbohydrates (54.9%) and sulphate (41.5%) with monosaccharide composition of 78.8% fucose and 21.2% galactose. The M_w of the intact fucoidans was reduced from 23,600 to 5200 kDa when heated in boiling water for 1–15 min. Microwave heating for 30 s decreased the M_w of fucoidans to 2400 kDa despite no significant polymer degradation. The results indicate that the 30 s-microwave heating yielded a more accurate M_w value of the intact fucoidans than any other heat treatments used in this study.     

Physicochemical and functional properties of chitosans affected by sun drying time during decoloration

The typical production of chitosan from crustacean shell consists of demineralization, deproteinization, decoloration, and deacetylation. Selected physicochemical and functional properties of chitosans as affected by various decoloration times (4, 5, 6, 7 or 8 h) using sun drying were evaluated. Moisture content (6.67-6.89 g/100 g), degree of deacetylation (81.91-82.73%), and color L^∗ value (78.32-79.43) of chitosans were not affected by sun drying time. However, color a^∗ and b^∗ values decreased when sun drying time was over 4 h. The viscosity of chitosan solution (0.5 g/100 mL acetic acid) decreased gradually with increasing sun drying time, with a more pronounced effect observed at 8 h of sun drying. There was no change in water-binding capacity (WBC) of chitosans decolorized by sun drying between 4 and 6 h; however, further increase in sun drying time from 6 to 7 or 8 h increased WBC of chitosans. DPPH radical scavenging activity of chitosans increased with increasing sun drying time. This study demonstrated that white-colored chitosans could be produced by an alternative decoloration step using sun drying for 4 h following the deacetylation step. However, increasing sun drying time to 7 h produced chitosan with increased WBC and DPPH radical scavenging activity.     

几种壳聚糖的抑菌性能

通过壳聚糖的抑菌实验和几种壳聚糖的最低抑菌浓度的测定 ,比较了相同脱乙酰度不同分子量 ,以及分子量相近脱乙酰度不同的壳聚糖对金黄色葡萄球菌、枯草杆菌、大肠杆菌和假单胞菌的抑菌作用。结果表明 ,实验中用到的壳聚糖都对金黄色葡萄球菌有很强的抑菌作用 (抑菌率接近 10 0 % ,最低抑菌浓度为 0 0 3% ) ;对于其他 3种细菌 ,脱乙酰度相同 (为 75 3%或 93 7% ) ,粘均分子量不同 (在 4 0～ 80万之间 )的壳聚糖 ,抑菌作用随分子量的升高而增强 ;而分子量相近脱乙酰度不同的壳聚糖对上述 4种细菌的抑菌作用差别不大 ;在 pH 5 5左右至 pH 6 0左右壳聚糖能够发挥最强的抑菌作用 ;总体看来 ,壳聚糖对金黄色葡萄球菌的抑菌作用最强 ,其次是对假单胞菌和枯草杆菌 ,壳聚糖对大肠杆菌的抑菌作用相对弱一些 ;实验条件下的壳聚糖对上述 4种细菌的抑菌作用普遍比苯甲酸钠强。     

Efficacy of various plant hydrosols as natural food sanitizers in reducing Escherichia coli O157:H7 and Salmonella Typhimurium on fresh cut carrots and apples

In the present study, inhibitory effects of the hydrosols of thyme, black cumin, sage, rosemary and bay leaf were investigated against Salmonella Typhimurium and Escherichia coli O157:H7 inoculated to apple and carrots (at the ratio of 5.81 and 5.81 log cfu/g for S. Typhimurium, and 5.90 and 5.70 log cfu/g for E. coli O157:H7 on to apple and carrot, respectively). After the inoculation of S. Typhimurium or E. coli O157:H7, shredded apple and carrot samples were washed with the hydrosols and sterile tap water (as control) for 0, 20, 40 and 60 min. While the sterile tap water was ineffective in reducing (P > 0.05) S. Typhimurium and E. coli O157:H7, 20 min hydrosol treatment caused a significant (P < 0.05) reduction compared to the control group. On the other hand, thyme and rosemary hydrosol treatments for 20 min produced a reduction of 1.42 and 1.33 log cfu/g respectively in the E. coli O157:H7 population on apples. Additional reductions were not always observed with increasing treatment time. Moreover, thyme hydrosol showed the highest antibacterial effect on both S. Typhimurium and E. coli O157:H7 counts. Inhibitory effect of thyme hydrosol on S. Typhimurium was higher than that for E. coli O157:H7. Bay leaf hydrosol treatments for 60 min reduced significantly (P < 0.05) E. coli O157:H7 population on apple and carrot samples. In conclusion, it was shown that plant hydrosols, especially thyme hydrosol, could be used as a convenient sanitizing agent during the washing of fresh-cut fruits and vegetables.     

Changes in vitamin content of powder enteral formulas as a consequence of storage

The thiamine, vitamin E (α-, γ- and δ-tocopherol) and vitamin A (all-trans and 13-cis-retinol) contents of four commercial powder enteral formulas (A, B, C and D) have been determined. The vitamin intake provided by the studied formulas was always above the US daily recommendations. Powder enteral formulas A and D were stored at 30 °C for up to 6 months with a water activity of 0.44 (A_w = 0.44), and formula A was also stored under atmospheric conditions for 3, 4 and 6 months. Formulas A and D kept at 30 °C and A_w = 0.44 suffered a gradual loss in vitamin content (from 3% to 4% after 1 month to 58–60% after 6 months). Formula A, stored at 30 °C under atmospheric conditions, underwent a slight reduction in vitamin content after 3 months, similar to that found after 1 month with A_w = 0.44, and from that time onward, this decreased steadily (to 30% after 6 months). The RDA of thiamine, vitamin E and vitamin A for women and men were met only when the powder enteral formulas were stored at 30 °C with A_w = 0.44 up to 1 month and without A_w up to 3 months. These results show that A_w and storage period have a marked effect on the stability of thiamine, vitamin E and A during the storage of powder enteral formulas and should be taken into consideration for the shelf-life of the product.     

Effects on Salmonella shell contamination and trans-shell penetration of coating hens' eggs with chitosan

Chitosan is a biopolymer with antimicrobial activity and film-forming properties. In this study, the effects on Salmonella shell contamination and trans-shell penetration of coating hens' eggs with chitosan was evaluated. A chitosan was selected from eight types (four non-commercial and four commercial) based on its antimicrobial activity against Salmonella enterica serovar Enteritidis (S. Enteritidis). For this purpose, a contact plate method was developed and chitosans were applied at a concentration of 0.25% (w/v). A commercial type with a molecular weight of 310-375 kDa and a deacetylation degree of 75% that reduced S. Enteritidis by 0.71 log₁₀ colony forming units compared to the control (without chitosan) was selected for further studies. The chitosan was shown to have antimicrobial activity against other egg borne bacteria, i.e., Acinetobacter baumannii, Alcaligenes sp., Carnobacterium sp., Pseudomonas sp., Serratia marcescens and Staphylococcus warneri, and against S. enterica serovar Typhimurium, Escherichia coli and Listeria monocytogenes. The effects of various concentrations of the selected chitosan (0.25%, 1% and 2%) on Salmonella shell contamination and trans-shell penetration were assessed using the agar molding technique. Effective reduction of eggshell contamination could not be demonstrated, but trans-shell penetration was significantly reduced in the presence of a 2% chitosan eggshell coating, with only 6.1% of the eggs being penetrated compared to 24.5% of the uncoated eggs. It was concluded that the 2% chitosan coating has the potential to reduce contamination of egg contents resulting from trans-shell penetration by S. Enteritidis.     

Rhizosphere effect on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure-amended soil during cabbage (Brassica oleracea) cultivation under tropical field conditions in Sub-Saharan Africa

The effect of cabbage (Brassica oleracea) rhizosphere on survival of Escherichia coli O157:H7 and Salmonella Typhimurium in manure-amended soils under tropical field conditions was investigated in the Central Agro-Ecological Zone of Uganda. Three-week old cabbage seedlings were transplanted and cultivated for 120 days on manure-amended soil inoculated with 4 or 7 log CFU/g non-virulent E. coli O157:H7 and S. Typhimurium. Cabbage rhizosphere did not affect survival of the 4 log CFU/g inocula in manure-amended soil and the two enteric bacteria were not detected on/in cabbage leaves at harvest. The 7 log CFU/g E. coli O157:H7 and S. Typhimurium survived in bulk soil for a maximum of 80 and 96 days, respectively, but the organisms remained culturable in cabbage rhizosphere up to the time of harvest. At 7 log CFU/g inoculum, E. coli O157:H7 and S. Typhimurium contamination on cabbage leaves occurred throughout the cultivation period. Leaf surface sterilisation with 1% AgNO₃ indicated that the organisms were present superficially and in protected locations on the leaves. These results demonstrate that under tropical field conditions, cabbage rhizosphere enhances the persistence of E. coli O157:H7 and S. Typhimurium in manure-amended soil at high inoculum density and is associated with long-term contamination of the leaves.     

Molecular mass distribution of dextran in Brazilian sugar and insoluble deposits of cachaça

The dextran molecular mass distribution profile in 77 sugar samples from Brazil and twelve insoluble deposits (alcoholic flocks) samples from sugared cachaças (Brazilian sugar cane spirit) is described in terms of number-average molecular mass M_n, weight-average molecular mass M_w, Z-average molecular mass M_z, and polydispersity. The analyses were performed by size-exclusion chromatography, using a refractive index detector. In most of the sugar samples, it was possible to identify two major groups of dextrans with M_w averages of 5 × 10⁶ and 5 × 10⁴ Da. Based on the evaluated parameters, the dextran distribution profile is about the same in samples analyzed over five seasons, and, therefore, it is likely that the Brazilian product pattern will not change very much over the years. In insoluble deposits from sugared cachaças, dextrans with M_w values in the order of the 10⁵ Da were the most frequent ones, being present in 58% of the samples.     

Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate

The efficacy of commercially available plant extracts and essential oils used extensively as flavour ingredients in confectionery products were used as antimicrobials in laboratory media against the following microorganisms: Escherichia coli O157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. Using the disc diffusion method, inhibition zones in diameter >20 mm were observed by adding 10 μl of each antimicrobial substance on the following microorganisms: lemon flavour applied on E. coli O157:H7, lemongrass essences against S. aureus, plum using a B. cereus strain and strawberry flavour using a L. monocytogenes strain. E. coli O157:H7 strains were the most susceptible microorganisms inhibited by 18 extracts, followed by S. Typhimurium and S. aureus which were inhibited by 17 extracts. Lemon flavour, lemongrass essences, pineapple and strawberry flavour inhibited the foodborne pathogens at the lowest concentration (5 ml/100 ml). Plant extracts and essential oils with potent antimicrobial activities were tested in chocolate held at different temperatures (7 and 20 °C) in dry or humidified environment, which resulted in different a_w values of the product (i.e. 0.340, 0.450, and 0.822), in order to determine their efficacy on the fate of the inoculated pathogens. The most inhibitory action was observed by lemon flavour applied on chocolate inoculated with E. coli cocktail culture after storage at 20 °C for 9 days. Plant extracts tested on chocolate show an enhanced inhibitory effect during storage at 20 °C indicating that their application may provide protection in case of storage at the above temperature or even higher.     

Antioxidative activity of chitosans with varying molecular weights

Antioxidant activity of chitosans of different molecular weights (30, 90 and 120 kDa chitosan) in salmon (Salmo salar) was investigated. The progress of oxidation was monitored by employing the 2-thiobarbituric acid-reactive substances (TBARS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assays. In general, all chitosans exhibited antioxidative activities in salmon. The addition of chitosans to salmon reduced lipid oxidation for seven days of storage. The TBARS values of salmon containing chitosan were significantly lower than those of the control (p < 0.01). At 0.2% (w/v) and 0.5% (w/v) concentrations, the TBARS with chitosan addition was decreased by 75% and 45%, respectively, over 15 days. At 1% concentration, the TBARS value with native chitosan addition was decreased by 32% after 15 days of storage. 90 kDa chitosan showed an increased DPPH free radical-scavenging activity with increasing concentration in the range of 0.2–1% (w/v). The free radical-scavenging activity of the 0.2 mM DPPH solution was saturated by 30 kDa chitosan at a concentration of ?0.7% (w/v), resulting in a strong antioxidant activity of approximately 85%. This was comparable to the DPPH free radical-scavenging activity of BHT.     

Inhibitory effects of 4-chlorosalicylic acid on mushroom tyrosinase and its antimicrobial activities

The inhibitory effects of 4-chlorosalicylic acid on the activity of mushroom tyrosinase have been investigated. The results showed that 4-chlorosalicylic acid could strongly inhibit both monophenolase activity and diphenolase activity. The IC₅₀ values were estimated as 1.89 mM and 1.10 mM for monophenolase and diphenolase activities, respectively. For the monophenolase activity, 4-chlorosalicylic acid could not only lengthen the lag time, but also decrease the steady-state rate. For the diphenolase activity, kinetic analyses showed that the inhibition by 4-chlorosalicylic acid was reversible and its mechanism was mixed-II type, which is different from salicylic acid. The inhibition constants (K_I and K_IS) were determined to be 1.51 mM and 0.82 mM, respectively. Furthermore, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Aspergillus niger and Candida boidinii were investigated. The results showed that 4-chlorosalicylic acid was the most effective against E. coli with the MIC of 250 μg/ml and with the MBC of 500 μg/ml.     

Synergistic effect of chitooligosaccharides and lysozyme for meat preservation

The objective of this study was to enhance the antibacterial spectrum of lysozyme with the use of chitooligosaccharides (COS) produced by radiation treatment of chitosan. Exposure of chitosan solution to γ radiation led to formation of oligosaccharides of different molecular weights. COS with molecular weight of 8.3 kDa were found to exhibit highest antioxidant potential in free radical scavenging assay but antibacterial activity decreased with decrease in molecular weight. Combination of COS and lysozyme was more effective against Gram-negative bacteria than when used alone. This clearly indicated the synergistic effect of the two antibacterial agents added together. When tested in meat model system, the combination treatment resulted in complete elimination of Escherichia coli, Pseudomonas fluorescens and Bacillus cereus and reduced the load of Staphylococcus aureus cells in packed inoculum and storage studies. The shelf life of minced meat containing COS-lysozyme mixture was extended up to 15 days at chilled temperatures.     

Use of chitosan for the removal of metal ion contaminants and proteins from water

Naturally abundant biosorbants such as chitin and chitosan are recognized as excellent metal ligands, forming stable complexes with many metal ions, and serving as effective protein coagulating agents. Chitin isolated from crab processing discards was deacetylated over different time periods in order to obtain three types of chitosans [Type 1 (20 h), Type 2 (10 h) and Type3 (4 h)]. Chitosans so prepared were evaluated for their capacity to chelate metal ions in samples of water obtained from a zinc mining site (Buchans, Newfoundland). Metal chelation capacity of chitosan for wastewater was determined by inductively coupled plasma-mass spectrometry (ICP-MS) at three different pH (5, 6 and 7). Chitosan served as an effective coagulating agent in removing proteins from wastewater as well as for the removal of metal ions [Hg(II), Fe(II), Ni(II), Pb(II), Cu(II) and Zn(II)] from industrial wastewater, especially at pH 7, as measured by ICP-MS. Mercury was best chelated by all three types of chitosan under all pH conditions tested. In the protein flocculation study, Type 1 chitosan showed the best flocculation ability followed by Type 2 and Type 3 chitosans.     

Synergistic effect of tannic acid and modified atmospheric packaging on the prevention of lipid oxidation and quality losses of refrigerated striped catfish slices

Chemical, microbiological and sensorial changes of striped catfish (Pangasius hypophthalmus) slices treated without and with tannic acid (100 and 200 mg/kg) were determined during 15 days of storage at 4 °C in air and under modified atmospheric packaging (MAP, 60% N₂/35% CO₂/5% O₂). The slices consisted of 9.2 g lipid/100 g and the lipid contained 64.55% unsaturated fatty acids and 33.87% saturated fatty acids. During the storage, the sample treated with 200 mg/kg tannic acid and stored under MAP (M₂) had the lowest peroxide value (PV) and thiobarbituric acid-reactive substances (TBARS) with the coincidental lowest non-haem iron content, indicating the retarded lipid oxidation. Fourier transform infrared (FTIR) spectra indicated the formation of primary oxidation products and free fatty acids in M₂ sample after 15 days. Conversely, these compounds were found at lower contents in the control samples kept in air without tannic acid treatment (A₀), suggesting that the deterioration was more advanced. Myosin heavy chain of A₀ was degraded by 17.85% after 15 days of storage, whereas no change was noticeable in M_2, compared with the fresh sample (F). Based on microbiological acceptability limit (10⁷ cfu/g), the shelf-life of A₀ and M₂ was estimated to be 3 and 15 days, respectively. M₂ had the acceptable scores for all sensory attributes up to 15 days, while A₀ was acceptable when stored for 9 days. Therefore, tannic acid exhibited a synergistic effect with MAP on retarding lipid oxidation and microbial growth, thereby increasing the shelf-life of striped catfish slices during refrigerated storage.