Aims: Chitosan has gained wide applications in the food industry and biomedical field owing to its biodegradability, biocompatibility, nontoxicity and its antimicrobial activity against a wide spectrum of micro-organisms. However, the methods used to investigate antimicrobial effects of chitosan vary considerably among studies, making comparisons difficult. Methods and Results: One diffusion (disc diffusion) and two dilution (agar dilution and broth microdilution) methods commonly used in clinical laboratories to assess microbial susceptibility/resistance to antimicrobial agents were comparatively used to determine the antimicrobial activity of two watersoluble chitosan derivatives (molecular weights of 43 and 67 kDa) against 31 representative foodborne pathogens. When tested at 1Á6% for the 43-kDa chitosan and 3Á2% for the 67-kDa chitosan, by disc diffusion, approximately 10-to 11-mm-diameter inhibition zones were observed for all of the bacterial groups, except for Salmonella tested for the 67-kDa chitosan where no inhibition zone was observed. By agar dilution and broth microdilution, the minimal inhibitory concentration (MIC) values varied largely dependent upon the molecular weight of chitosan, bacterial genus/species and the testing method. The agreement between MIC values obtained by the two methods was poor, with broth microdilution generally having lower MIC values than agar dilution. Regardless of the testing method, Salmonella strains were the least susceptible among Gram-negative strains for both chitosans, followed by Escherichia coli and Vibrio. Conclusions: Besides chitosan's molecular weight and bacterial genus/species, the antimicrobial activity of chitosan was also influenced largely by the susceptibility testing method used. Significance and Impact of the Study: This is the first study that comparatively evaluated these diffusion and dilution methods, particularly two quantitative methods (agar dilution and broth microdilution), to assess the antimicrobial activity of two water-soluble chitosans against a large number of foodborne pathogens. The study highlights the need for standardized methods to be used in evaluating chitosan's antimicrobial properties in future studies.

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In the present study, a 2-stage solid-state fermentation process using followed by was performed with the purpose of improving the nutritional quality of corn and soybean meal (SBM) mixed feed (MF). The matrix of native MF and fermented MF (FMF) was analyzed using scanning electron microscopy to determine external structures after solid-state fermentation. Results of the present study indicated that the FMF had a greater concentration of CP and total P compared with the unfermented feed, whereas the contents of β-conglycinin, glycinin, phytate P, raffinose, stachyose, and NDF in FMF declined by 77.6, 86.7, 41.2, 47.2, 36.6, and 38.9%, respectively. The content of trichloroacetic acid-soluble protein, particularly those of small peptides and free AA, increased more than 3 times after fermentation. Scanning electron microscopy revealed that irregular shapes and a lot of porous structures were observed in the SBM of FMF, the cell wall of corn in FMF was destroyed, and the number of starch granules was decreased after 2-stage fermentation. Two animal experiments were conducted to evaluate the digestibility of CP and P in FMF when fed to pigs. In Exp. 1, 6 barrows (15.20 ± 1.27 kg initial BW) fitted with a T-cannula in the distal ileum were allotted to a replicated 3 × 3 Latin square with 3 diets and 3 periods per square. Two diets containing unfermented MF or FMF as the only source of protein and a N-free diet were formulated. Results indicated that the apparent ileal digestibility (AID) of total AA and Lys in FMF (74.98 and 75.34%) were greater ( < 0.05) than in untreated MF (69.66 and 68.56%). The standard ileal digestibility (SID) of Lys in FMF (82.17%) was also greater ( < 0.05) than in MF (75.91%). In Exp. 2, 16 barrows (17.57 ± 1.08 kg initial BW) were allotted to 2 diets formulated to contain MF or FMF as the sole source of P to determine the digestibility of P. The apparent total tract digestibility and standardized total tract digestibility of P in FMF were 58.14 and 64.72%, respectively. These values were greater ( < 0.01) than in untreated MF (37.11 and 44.89%, respectively). Our results indicate that a solid-state fermentation process using followed by offers an effective approach to improving the quality of corn and SBM MF.

Supplemental Figure S1. Toxicity of diquat in mice. Thirty mice (five-week-old) were chosen to establish the mouse model of intestinal oxidative stress. After one week of adaption, these mice were randomly allocated into six groups (n=5) and treated as follows: (a) PBS (control group), (b) 12 mg/kg diquat group, (c) 25 mg/kg diquat group, (d) 50 mg/kg diquat group, (e) 75 mg/kg diquat group, (f) 100 mg/kg diquat group. Equal volumes of diquat or PBS was injected intraperitoneally. Twelve hours after treatment, the mice that survived were counted and samples of their blood, liver, duodenum, jejunum, ileum and colon were collected.

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