The shrimp shells were washed under

The shrimp shells were washed under running warm tap water to

remove soluble organics, adherent proteins and other impurities. The

shells were then collected and boiled in water for 1 h to remove the

tissue, followed by drying in an oven (Prolabo, model Volca MC18,

French) at 160 C for 2 h to make themmore brittle and to break down

the crystalline structure of chitin (Mukherjee, 2002). At the end, the

dried shells were ground into a fine powder using a standard grinder

(Model KU-2, PredomMesko, SkarzyskoKam., Poland).

2.2.1. Demineralization

Calcium carbonate constitutes the main inorganic component of

the shells. To remove the calcium carbonate, only dilute hydro-
chloric acid was used to prevent hydrolysis of chitin (No et al.,

1995). The hydrochloric acid concentrations ranged from 0.25 to

2 M and the reaction time was varied from 10 to 120 min. The ratio

of dried shells to acid solution used during the extraction of chitin

ranged from 1/10 to 1/30 (w/v). The experiments were carried out

at room temperature under constant stirring of 150 rpm. The

decalcified shells were collected on a 250 mm sieve, washed to

neutrality with tap water, rinsed with deionised water, and then

oven-dried at 80 C overnight. The rate of demineralization was

evaluated by determining ash contents in the solid.

2.2.2. Deproteinization

Similar experimental conditions were applied for the deminer-
alization of dried shells. The sodium hydroxide concentration was

varied from 0.5 to 5 M, the reaction time ranged from 10 to 400 min

and the temperature was varied from 20 to 100 C. At the end of this

process the material was filtrated, washed and dried, as previously

described in the demineralization process. In order to evaluate the

extent of deproteinization, the protein concentration in the

supernatant was determined according to Biuret’s method (Fine,

1935); It remains the most widely used method for protein

determination.

50 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012) 48e56

chitosan is hydrolyzed by 50 ml HCl 6.27 N at 56 C during 3 h

(Chang, Lee, & Fu, 2000).

2.5. Antibacterial tests

Minimum inhibitory concentrations are important to confirm

resistance of microorganisms to an antimicrobial agent and also to

monitor the activity of new antimicrobial agents. The minimal

inhibitory concentration (MIC) is generally regarded as the most

basic laboratory measurement of the activity of an antimicrobial

agent against an organism. Antibacterial activities of chitin, chito-
san, N-acetyl chito-oligosaccharides (NAc-COS) and chito-
oligosaccharides (COS) were examined as the inhibitory effects

against the growth of four Gram-positive bacteria: Staphylococcus

aureus ATCC 25923, S. aureus ATCC 43300, Bacillus subtilis and

Bacillus cereus and seven Gram-negative bacteria: Escherichia coli

ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella

typhimurium, Vibrio cholerae, Shigella dysenteriae, Prevotella mela-
ninogenica and Bacteroides fragilis. All bacteria were supplied by

Algeria Pasteur Institute. 0.1 g of sterile chitin, chitosan, N-acetyl

chito-oligosaccharides (NAc-COS) or chito-oligosaccharides (COS)

was added in 100 ml of cultured bacteria suspension in a flask and

incubated with shaking at 37 C. The inhibitory effect was esti-
mated periodically by measuring turbidity of the cultured medium

at 640 nm using a spectrophotometer UVeVisible mini 1240 SHI-
MADZU. The minimum inhibitory concentration (MIC) was defined

as the lowest concentration of chitin, chitosan, NAc-COS or COS

required to completely inhibit bacterial growth after incubation at

37 C for 72 h (No, Park, Lee, & Meyers, 2002).

The anaerobic bacteria (P. melaninogenica and B. fragilis) were

cultured in anaerobic atmosphere (Jeon & Kim, 2000). For deter-
mination of the minimum inhibitory concentration (MIC) of chitin,

chitosan, N-acetyl chito-oligosaccharides (NAc-COS) and chito-
oligosaccharides (COS), solutions (1% (w/w) in 1% (w/w) acid) of

each substances were added to Muller Hinton agar (supplemented

with blood for anaerobic bacteria) for final chitin, chitosan, NAc-
COS or COS concentrations of 0.1%, 0.08%, 0.05%, 0.03%, 0.01%,

0.006% and 0.003% (w/v).

Plots were made of the optical density (OD) (i.e. Absorbance at

640 nm) versus the culture time for each of the four Gram-positive

bacteria and the seven Gram-negative bacteria tested by the

shaking flask method.

Inhibitory effects against growth due to antibacterial activities

of chitin, chitosan, N-acetyl chito-oligosaccharides (NAc-COS) and

chito-oligosaccharides (COS) would be indicated by a levelling off of

the slopes of the curves.

3. Results and discussion

ผลลัพธ์ (อังกฤษ) 2:

The shrimp shells were washed under running Warm Tap Water to Remove soluble Organics, Adherent Proteins and Other impurities. The shells were then Collected and boiled in Water for 1 H to Remove the tissue, followed by Drying in an oven (Prolabo, Model Volca MC18, French) at 160 C for 2 H to Make Themmore Brittle and to Break down the crystalline structure of. chitin (Mukherjee, 2002). At the End, the Ground Dried shells were using a standard Into a Fine Powder Grinder (Model KU-2, PredomMesko, SkarzyskoKam., Poland). 2.2.1. Demineralization Calcium carbonate inorganic Constitutes the Main Component of the shells. Remove the calcium carbonate to, only dilute Hydro Chloric acid was used to Prevent hydrolysis of chitin (No et al., 1 995). The hydrochloric acid concentrations ranged from 00:25 to 2 M and the Reaction time was varied from 10 to 120 min. The ratio of acid to Dried shells used during the extraction of chitin Solution ranged from 1/10 to 1/30 (W / v). The experiments were carried out at Room Temperature under Constant stirring of 150 RPM. The decalcified shells were Collected on a 250 mm Sieve, washed to neutrality with Tap Water, rinsed with deionised Water, and then oven-Dried at 80 C Overnight. The rate of demineralization was evaluated by determining the ash contents in Solid. 2.2.2. Deproteinization Similar Applied Experimental conditions were for the Deminer- Alization of Dried shells. Sodium hydroxide the concentration was varied from 0.5 to 5 M, the Reaction time ranged from 10 to 400 min and the Temperature was varied from 20 to 100 C. At the End of this Process was the Material Filtrated, washed and Dried, as previously described. in the demineralization process. In Order to evaluate the extent of Deproteinization, the protein concentration in the supernatant was determined according to Biuret's method (Fine, 1,935th); It remains widely used method for the Most protein determination. 50 MS et al Benhabiles. / Food Hydrocolloids 29 (in 2012) 48e56 Chitosan is Hydrolyzed by 50 ml 27.06 N HCl at 56 C during 3 H (Chang, Lee, & Fu, two thousand). 2.5. Antibacterial tests Minimum inhibitory concentrations are important to Confirm resistance of microorganisms to an antimicrobial Agent and also to the Activity Monitor of New antimicrobial agents. The Minimal inhibitory concentration (MIC) is generally regarded as the Most Basic Laboratory measurement of the Activity of an antimicrobial Agent against an organism. Antibacterial activities of chitin, Chito- San, N-acetyl Chito-oligosaccharides (Nac-COS) and Chito- oligosaccharides (COS) were examined as the inhibitory effects against Gram-positive bacteria the growth of Four: Staphylococcus aureus ATCC 25923, S. aureus ATCC the 43300th, Bacillus subtilis and Bacillus cereus and Gram-negative bacteria Seven: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium, Vibrio cholerae, Shigella dysenteriae, Mela- Prevotella Bacteroides fragilis and Ninogenica. All bacteria were supplied by Algeria Pasteur Institute. 0.1 G of sterile chitin, Chitosan, N-acetyl Chito-oligosaccharides (Nac-COS) or Chito-oligosaccharides (COS) was added in 100 ml of cultured bacteria suspension in a Flask and incubated with shaking at 37 C. The inhibitory Effect was. Esti- mated periodically by measuring Turbidity of the Medium cultured using a spectrophotometer at 640 NM UVeVisible Mini Shi-one thousand two hundred and forty MADZU. Minimum inhibitory concentration the (MIC) was defined as the lowest concentration of chitin, Chitosan, Nac-COS or COS required to completely inhibit bacterial growth after Incubation at 37 C for 72 H (No, Park, Lee, & Meyers, 2002nd). the anaerobic bacteria (P. melaninogenica and B. fragilis) were cultured in anaerobic atmosphere (Jeon & Kim, 2000). For Deter- mination of the Minimum inhibitory concentration (MIC) of chitin, Chitosan, N-acetyl Chito-oligosaccharides (Nac-COS) and Chito- oligosaccharides (COS), Solutions (1% (W / W) in 1% (W. / W) acid) of each Substances were added to Muller Hinton agar (supplemented with anaerobic bacteria for Blood) for Final chitin, Chitosan, NAc- COS or COS concentrations of 0.1%, twelve eight%, .05%, twelve three%, twelve one a.m.%,. 0.006% and 0.003% (W / v). Plots were Made of the Optical density (OD) (IE Absorbance at 640 NM) Versus the Culture time for each of the Four Gram-positive bacteria and the Seven Gram-negative bacteria tested by. the Flask shaking method. Inhibitory effects Due to Antibacterial activities against growth of chitin, Chitosan, N-acetyl Chito-oligosaccharides (Nac-COS) and Chito-oligosaccharides (COS) would be indicated by a Levelling off of the slopes of the curves. 3. Results and discussion

ผลลัพธ์ (อังกฤษ) 3:

The shrimp shells were washed under running warm tap water to.Remove soluble organics adherent proteins, and other impurities. The.Shells were then collected and boiled in water for 1 h to remove the.Tissue followed by, drying in an oven (Prolabo model Volca MC18,,French) at 160 C for 2 h to make themmore brittle and to break down.The crystalline structure of, chitin (Mukherjee 2002). At, the end the.Dried shells were ground into a fine powder using a standard grinder.(Model KU-2 PredomMesko SkarzyskoKam,,, Poland).2.2.1. Demineralization.Calcium carbonate constitutes the main inorganic component of.The shells. To remove the calcium carbonate only dilute, hydro -.Chloric acid was used to prevent hydrolysis of chitin (No et, al.1995). The hydrochloric acid concentrations ranged from 0.25 to.2 M and the reaction time was varied from 10 to 120 min. The ratio.Of dried shells to acid solution used during the extraction of chitin.Ranged from 1 / 10 to 1 / 30 (w / V). The experiments were carried out.At room temperature under constant stirring of 150 rpm. The.Decalcified shells were collected on a 250 mm sieve washed to,,Neutrality with tap water rinsed with, deionised water and then,,Oven-dried at 80 C overnight. The rate of demineralization was.Evaluated by determining ash contents in the solid.2.2.2. Deproteinization.Similar experimental conditions were applied for the deminer -.Alization of dried shells. The sodium hydroxide concentration was.Varied from 0.5 to 5 M the reaction, time ranged from 10 to 400 min.And the temperature was varied from 20 to 100 C. At the end of this.Process the material was filtrated washed and dried as previously,,,Described in the demineralization process. In order to evaluate the.Extent of deproteinization the protein, concentration in the.Supernatant was determined according to Biuret ', s method (Fine1935); It remains the most widely used method for protein.Determination.50 M.S. Benhabiles et al. / Food Hydrocolloids 29 (2012 48e56.)Chitosan is hydrolyzed by 50 ml HCl 6.27 N at 56 C during 3 h.,, & (Chang Lee, Fu 2000).2.5. Antibacterial tests.Minimum inhibitory concentrations are important to confirm.Resistance of microorganisms to an antimicrobial agent and also to.Monitor the activity of new antimicrobial agents. The minimal.Inhibitory concentration (MIC) is generally regarded as the most.Basic laboratory measurement of the activity of an antimicrobial.Agent against an organism. Antibacterial activities, of chitin Chito -.San N-acetyl, chito-oligosaccharides (NAc-COS) and Chito -.Oligosaccharides (COS) were examined as the inhibitory effects.Against the growth of four Gram-positive bacteria: Staphylococcus.Aureus, ATCC 25923 S. Aureus ATCC 43300 Bacillus subtilis, and.Bacillus cereus and seven Gram-negative bacteria: Escherichia coli.ATCC 25922 Pseudomonas aeruginosa ATCC 27853 Salmonella,,,Typhimurium Vibrio, dysenteriae, cholerae Shigella, Mela Prevotella.Ninogenica and Bacteroides fragilis. All bacteria were supplied by.Algeria Pasteur Institute. 0.1 g of sterile chitin chitosan N-acetyl,,,Chito-oligosaccharides (NAc-COS) or chito-oligosaccharides (COS).Was added in 100 ml of cultured bacteria suspension in a flask and.Incubated with shaking at 37 C. The inhibitory effect was esti -.Mated periodically by measuring turbidity of the cultured medium.At 640 nm using a spectrophotometer UVeVisible Mini 1240 SHI -.MADZU. The minimum inhibitory concentration (MIC) was defined.As the lowest concentration of chitin chitosan NAc-COS or COS,,,Required to completely inhibit bacterial growth after incubation at.37 C for 72 h (No Park Lee,,,, & Meyers 2002).The anaerobic bacteria (P. Melaninogenica and B. Fragilis were.)Cultured in anaerobic atmosphere (Jeon, & Kim 2000). For deter -.Mination of the minimum inhibitory concentration (MIC), of chitinChitosan N-acetyl, chito-oligosaccharides (NAc-COS) and Chito -.Oligosaccharides (COS), solutions (1% (w / W) in 1% (w / W) acid of.)Each substances were added to Muller Hinton agar (supplemented.With blood for anaerobic bacteria) for final chitin chitosan NAc -,,,COS or COS concentrations of 0.1% 0.08% 0.05%,,,,, 0.03% 0.01%0.006% and 0.003% (w / V).Plots were made of the optical density (OD) (i.e. Absorbance at.640 nm) versus the culture time for each of the four Gram-positive.Bacteria and the seven Gram-negative bacteria tested by the.Shaking flask method.Inhibitory effects against growth due to antibacterial activities.Of chitin chitosan N-acetyl chito-oligosaccharides (,,), NAc-COS andChito-oligosaccharides (COS) would be indicated by a levelling off of.The slopes of the curves.3. Results and discussion.