3 MATERIALS AND METHODS

3.1 SAMPLES OF PAPER

Whatman no. 1 filter paper was used in these experiments because it is a pure cellulose paper that is additive-free and serves as a point of reference in respect to other experimental works. The choice of paper samples was made in accordance with TAPPI standards T400 om-90 and T402 om-93 (TAPPI 1990; TAPPI 1993).

Folding endurance was tested with strips of paper 10 cm long by 1.5 cm wide, in accordance with TAPPI standard T423 om-89 (TAPPI 1989). For the brightness test, octagons of 420 mm2 were used for each sample.

3.2 SIZING OF PAPER AND STERILIZATION

The samples were injected with 2% solutions (w/v) of each of the polymers indicated. The chitosan was prepared in the chemistry laboratory of Karl Augustin Grellman Institute of Wood, Cellulose, and Paper, University of Guadalajara, from deproteinized and decalcified shrimp shells. The shells had been treated with alkali in a two-step process to diminish the presence of N-acetyl groups (percentage of free primary amines) (Wolfrom et al. 1958). The acetylation degree of the sample was 17.3%; the viscosity (measured by a capillary viscometer) of a 1% (w/v) in 1% (v/v) acetic acid solution was 51 cps;and the sample's content in ashes was 0.23%. This chitosan was whiter, purer, and of better quality than commercial chitosan.

Three different 2% (w/v) solutions of chitosan were used, employing in each case 1% glacial acetic acid, butyric acid, and propionic acid to dissolve the chitosan. Therefore, we prepared three products: chitosan acetate, chitosan butyrate, and chitosan propionate. The preparation procedure involved adding 2 g of shredded chitosan to 100 ml of deionized water. The mixture was then stirred continuously while 1 ml of the corresponding acid was slowly added to completely dissolve the chitosan.

To prepare 2% solutions of the cellulose ethers CMC and HPMC, 2 g of powder were added slowly to 100 ml of deionized water, which was stirred continuously to avoid lump formation. In the case of HPMC, cold water was better for the dissolution.

To obtain a homogeneous solution of the third cellulose ether, MC, 100 ml of deionized water was used: 50 ml was placed in a freezer and left to the point of freezing, and the other 50 ml was heated to the boiling point. The hot water was immediately mixed with 2 g of MC, then 50 ml of freezing water was added while the liquid was stirred vigorously. The solution was then immediately placed in a refrigerator for five minutes. A transparent viscous solution was produced.

The viscosity of all 2% solutions was recorded with a Brookfield viscometer through the technique described in TAPPI standard T666 om-91 (TAPPI 1991). The pH at 25�C of all solutions was determined with a benchtop pH-meter.

After the samples were coated with a roll handcoater no. 8, they were left to dry over a nylon screen. The samples were sterilized using ethylene oxide (EtO) in a sterilization chamber. This sterilization method least affected the properties of the paper. The other methods compared in the preliminary tests were sterilization in an autoclave and sterilization with ozone. The former caused significant diminution of the paper's brightness and its resistance properties; the latter was ineffective.

Sterilization of the samples after application of the polymer coatings was necessary to ensure that only the test organism interacted with the paper. It was necessary to evaluate the consequences of the sterilization method on the properties of the paper and coatings.

3.3 PHYSICO-MECHANICAL PROPERTIES

After the sterilization with EtO, the modifications in paper and coating properties were evaluated in terms of folding endurance (TAPPI 1989); zerospan tensile strength (TAPPI 1985; Caulfield and Gunderson 1990); brightness (TAPPI 1992); and pH of the extracts of paper at cold temperatures (TAPPI 1988).

3.3.1 Folding Endurance

Folding endurance is directly related to fiber elasticity and to the polymer elasticity of the fiber coating. This property measures resistance to fatigue, and it has found great acceptance as a means of evaluating paper durability, that is, the degree to which the paper keeps its physical properties in relation to the frequency of use. Folding endurance measures a sheet of paper's capability of keeping its fold line without breaking in the course of repeated folding.

The Schooper tester produced eight measurements for each type of coating, including the control.

3.3.2 Zero-Span Tensile Strength

To test for zero-span tensile strength, the clips of the tester were placed as close as possible to zero separation. The aim is to test the individual fibers caught between the clips in order to show only the resistance of the fiber, not the resistance of the fiber network. (Unlike zero-span tensile strength, folding endurance is related to the intrinsic resistance of the paper and the cohesive force between fibers.)

The zero-span tests were carried out with a modified pendulum-type tensile-strength machine, and strips 1.5 cm wide were utilized in accord with TAPPI T231 cm-85 (TAPPI 1985).

Zero-span tensile strength reflects the intrinsic resistance of the fibers, thus the condition of the fibers. Enzymatic or age deterioration will affect the zero-span tensile strength.

3.3.3 Brightness Test

Brightness tests with an Elrepho Zeiss instrument TAPPI standard T452 om-92 (TAPPI 1992) measured the reflection percentage at 457 nm and noted changes in the color of coated and uncoated paper. In this test, eight replicas of each sample were made, obtaining a mean and standard deviation.

3.3.4 Determination of pH

The final pH measure of the sample aqueous extracts was carried out using TAPPI standard T509 om-88 (TAPPI 1988), a measure that was important because acidity promotes the deterioration of the paper. These pH tests were administered to each sample before and after sterilization with ethylene oxide.