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Homogenization of Lemon Grass via Bead Beating

By Lindsay Gibbons, OPS Diagnostics, LLC

Tall perennial true grasses of the Andropogoneae tribe include many of the most economically important crops.  Sorghum, corn, and sugarcane are three of the more important members of this group.  Considerable attention has been given to the processing of leaves and seeds of these grasses for both genetic and biochemical testing.  However, less information is available on processing of woody stem regions of these true grasses.  Stems are characterized by a high concentration of vascular tissue as well as a higher overall thickness of the samples.  This fibrous element of the plant can make sample processing, and in particular generating a homogenate, relatively difficult as compared to leaves and seeds.  Although seeds often have hard coatings, the endosperm is generally easy to process.  Vascular tissue presents a greater challenge in that xylem and phloem are predominately cellulose and resistant to shearing and grinding.

This short study examines different options for homogenizing fibrous stems from the tall perennial true grass, lemongrass.  In order to ascertain the degree of homogenization, the liberation of peroxidase will be used as a marker of the efficiency of processing.

Materials and Methods

Plant Material: Lemongrass (Cymbopogon citratus) was obtained commercially, stored at 4°C and used within five days of purchase.

Homogenization: Lemongrass was homogenized and lysates were assayed for the hydrogen peroxide- scavenging enzyme in plants. A known mass, 60 mg cross-section of lemongrass stem, was placed in a microfuge tube or polycarbonate vial with 200 µl of 0.1 M sodium citrate buffer, pH 6.0, and various grinding media, as described in Table 1. The samples were processed by bead beating with the HT Mini™, HT 24™, GenoGrinder 2010™ or HT Homogenizer™ II bead beaters. A summary of each method is found in Table 1.

Table 1: Methods Used to Homogenize Lemongrass Stem Samples.

HT Mini™

The sample was homogenized in a 2 ml unskirted disruption tube with 200 µl of 0.1 M sodium citrate buffer, pH 6.0, and one 5/16” stainless steel grinding ball (GBSS 312-1000-08) or one 6 mm ceria based zirconium oxide satellite and 220 mg Garnet. The tube was placed into the HT Mini™ and homogenized at 4000 rpm for 2 minutes.

HT 24™

The sample was homogenized in a 2 ml skirted disruption tube with 200 µl of 0.1 M sodium citrate buffer, pH 6.0, and one 5/16” stainless steel grinding ball (GBSS 312-1000-08) or one 6 mm ceria based zirconium oxide satellite and 220 mg Garnet. The tube was placed into the HT 24™ and homogenized at 4800 rpm for 2 minutes.

GenoGrinder 2010™

The sample was homogenized in a 2 ml skirted disruption tube with 200 µl of 0.1 M citrate buffer, pH 6.0, and one 6 mm ceria based zirconium oxide satellite and 220 mg (PFMM 500-100-25). The sample was also homogenized in a 4 ml polycarbonate vial with 200 µl of 0.1 M sodium citrate buffer, pH 6.0, and one 3/8” Stainless Steel Grinding Ball. The tube/vial was placed into the GenoGrinder 2010™ and homogenized at 1500 rpm for 2 minutes.

HT Homogenizer™ II

The sample was homogenized in a 2 ml skirted disruption tube with 200 µl of 0.1 M citrate buffer, pH 6.0, and one 6 mm ceria based zirconium oxide satellite and 220 mg (PFMM 500-100-25). The sample was also homogenized in a 4 ml polycarbonate vial with 200 µl of 0.1 M sodium citrate buffer, pH 6.0, and one 3/8” stainless steel grinding ball. The tube/vial was placed into the HT Homogenizer™ and homogenized at 1500 rpm for 2 minutes.

Peroxidase Assay: Homogenates were transferred to glass culture tubes and brought up to a final concentration of 1 mg lemongrass/10 µl citrate buffer. The lemongrass homogenates were then cleared by centrifugation prior to enzyme analysis to remove peroxidases bound to debris and tissue particles that were not thoroughly processed. Peroxidase activity was measured using a TMB substrate solution. This was prepared by diluting a 10 mg/ml TMB in DMSO solution 1:100 into 0.1 M sodium citrate buffer, pH 6.0. The TMB solution was added to citrate buffer drop wise while swirling. A 3% hydrogen peroxide solution was then added in a ration of 30 µl/ 10 ml TMB/citrate buffer. The assay for peroxidase was performed by dispensing 100 µl of substrate buffer prepared above to a well of a microplate followed by the addition of 1 µl of cleared homogenate. The enzyme reaction is placed in a kinetic plate reader and optical densities at 650 nm are measured every 30 seconds for 10 minutes. Peroxidase activity is calculated as the maximum slope of the reaction.

Results

The yield of peroxidase liberated from lemongrass samples was influenced by a combination of the homogenizer and grinding formats. Homogenizers, vials, tubes, balls, satellites, and garnet all seemed to impact results.

Generally, peroxidase was most effectively liberated when the lemongrass stem samples were beat with stainless steel grinding balls as compared to a zirconium oxide grinding satellite and garnet (Figure 1). Garnet in combination with a grinding satellite was less effective consistently than homogenization with grinding balls, though the satellite/garnet format was disproportionately better with the GenoGrinder 2010™ than the other homogenizers. Lemongrass homogenized in the HT Homogenizer™ with 3/8” stainless steel balls in 4 ml polycarbonate tube liberated the most peroxidase from samples (Figure 1). Stainless steel grinding balls also worked well with the 2 ml disruption tubes and the HT Mini™. The GenoGrinder 2010™ and HT 24™ also yielded comparable results when stainless steel grinding balls were employed.

Peroxidase liberated from Lemongrass by Bead Beating.

Figure 1. Relative activity of liberated peroxidase from disrupted lemongrass samples with various homogenizers.

Discussion

Fibrous vascular stem tissues are resistant to damage and are challenging to effectively homogenize. However, effective processing of stem tissue was possible using large grinding balls and the linear high throughput homogenizers. Though all methods used did liberate peroxidase, the fibrous nature of the sample required much greater force to more thoroughly homogenize the tissue. This can be associated with stainless steel balls by which the size and density creates more force upon impact.

Bead beating instruments vary in the number of samples that can be processed at one time, physical motion and speed at which the samples are agitated. Three of the four homogenizers tested move the tubes/vials in a linear or near linear motion. The HT Mini™ shakes one to three 2 ml screw cap unskirted microfuge tubes in a slightly arching motion. The tubes are oscillated with a small shaking arm between 2800 and 4000 rpm. The GenoGrinder 2010™ and HT Homogenizer™ both have platforms that oscillate linearly. The GenoGrinder 2010™ has a vertical motion while the HT Homogenizer™ moves horizontally. All three of these machines generated comparable results with grinding balls. The HT 24™, which holds up to 24 microfuge tubes, uses high-speed (2400-4200 rpm) figure-8 multidirectional motion disrupt up to 24 samples. For hard tissue samples, the “figure 8” motion appears less effective than the linear motion of the other homogenizers.

In all cases, homogenization with a large stainless steel ball was more effective at releasing peroxidase in lemongrass in comparison to homogenization with a zirconium oxide satellite and garnet (Figure 1). Although homogenization with stainless steel balls in the HT Mini™ and HT Homogenizer™ liberated similar amounts of peroxidase, the number of samples that can be processed daily is vastly different. Contrasting the HT Mini™ and HT 24™ which can only homogenize samples in microfuge tubes, high throughput homogenizers such as the GenoGrinder 2010™ and HT Homogenizer™ can process samples in microfuge tubes, deep well plates and vials. These high throughput homogenizers are not only efficient at processing hundreds to thousands of samples daily, but they can also accommodate large vials to process samples that cannot be processed in bead beaters designed for microfuge tubes.

Mixed mode matrices using media such as garnet and grinding satellites have proved very effective for homogenizing many difficult to tissues, such as skin and sclera. However, the effectiveness of this media on Lemongrass was much lower than the grinding balls. This illustrates how samples must be matched to a particular grinding media to ensure efficient homogenization.