A Simple Method for Measuring Agar Gel Strength

Abstract

Seaweeds are the only source for phycocolloids. Commercial applications of phycocolloids depend on their chemical/physical properties, including their gel forming ability. Thus, gel strength values are important for seaweed growers and scientists. Gel strength measurements include the use of texture analyzers or rheometers, which are not always available for seaweed growers and scientists. Here, we describe a home-made apparatus for assessing gel strength through the weight of a water column required for breaking a gel surface. The system worked well at gel concentrations between 0.5 and 1.5%, giving values of 82–535 g cm⁻² and 163–754 g cm⁻² for agar and agarose gels, respectively. The lowest variations were obtained for gel sample volumes between 25 and 30 mL. The system was manually operated but no significant variations were observed between measurements conducted by the five different users. The readings were independent of the water column fill rate. The variations in gel strength values were similar to reports using other gel strength measuring devices. We propose the use of our apparatus as a flexible, affordable tool for the assessment of gel quality, which is suitable for research groups or seaweed farmers without access to expensive equipment, and with a need to quickly assess their seaweed of interest at a relevant time scale for cultivation or harvest.

1. Introduction

Seaweeds are the only available source for commercially important phycocolloids, such as agar, agarose, carrageenan, and alginate [1,2,3]. Phycocolloids are usually used as additives that give products their desired thickness and texture, or serve as emulsifying agents, thus the chemical properties of phycocolloids often determine their end use [1,2]. Phycocolloids can often form hydrogels, with each phycocolloid type having different gel properties, such as melting and settling (gelling) temperatures and gel strength. [4]. Gel strength is a measure of the force required for breaking the gel’s surface, with rigid gels having higher values. It is dependent on the concentration of the gelling agent in the gel solution and on the chemical structure of the phycocolloid carbohydrate backbone [5], which is determined by the genetic information of the seaweed [1,2,4,5]. Different seaweed species synthesize different kinds of phycocolloids, each with their unique gel properties [4,6,7]. In agar or carrageenan gels—produced by several red seaweed species—chemical residues covalently bound to the carbohydrate backbone may determine their gelling properties as interruptions to the gel matrix can occur either from steric interruptions or from electrostatic interferences [5]. In general, phycocolloid abundance and quality can be influenced by the seaweed’s cultivation conditions [1,7,8,9,10,11,12,13] or manipulated by the extraction method [4,5,14], or through the addition of salts to the gel solution [5]. For example, agar extraction methods usually include boiling seaweeds in an aqueous solution, either by using heating elements, autoclaving, or through microwave assistance, followed by filtration, freezing and thawing, and finally dehydration. Sometimes a pre-treatment in basic solution is performed. The resulting dry agar can be further bleached for a clearer hue, or washed to remove contaminants [[4] and references within].

Since gel strength is an important phycocolloid trait, it is beneficial for seaweed growers and scientists to measure it. Several methods have been reported for measuring gel strength. Most of them consist of calculating the amount of force required for a rod or disc shaped probe—placed on or in the gel—to penetrate the gel surface. Such methods include the use of weights [15], a mercury load [15,16], or the use of a metal rod plunger [6,8,13]. Sometimes, only a vague description of the gel strength measurement is reported [4,8,10]. Special equipment is also commercially available, including gelometers or texture analyzers [4,7,12,17]. Home-made devices usually require either technical knowledge or workshop labor to build, or require the use of toxic materials like mercury. Due to their usually high prices, texture analyzers may not be available for many seaweed farmers and researchers. The use of service laboratories can also be costly and requires a time lag between sample preparation and analysis, due to shipment times, service laboratory availability, and more. Given that gelling properties depend on the usually fluctuating growth conditions of the seaweeds—at least in outdoor growth facilities—the real-time assessment of the gel strength, even at a qualitative level, of crude gels can be crucial for seaweed farmers and scientists.

In this work, we describe the setup and some parameters of a simple, flexible, and cost-efficient apparatus for the qualitative assessment of gel strength using readily available laboratory equipment. It is based on water flow, rather than mercury, which simplifies its operation in terms of toxicity and liquid handling. This paper outlines a specific system built in our lab, yet it can be easily modified to fit the needs and available resources of seaweed growers and researchers alike.

2. Materials and Methods

2.1. Gel Preparation

The gels in this work were prepared using commercial agarose bought from HyLabs LTD (Rehovot, Israel). The agarose had a reported gel strength of 1200 gr cm⁻² according to the information on the container. Agar gels were made using BD^® BACTO^TM Agar (Becton, Dickinson and Company, Sparks, MD, USA). The gels were prepared by mixing the agar or agarose powder with distilled water at concentrations of 0.5, 0.75, 1, and 1.5%. The mixture was then boiled using an electric pressure cooker (Ninja^® Foodi^®, SharkNinja Europe Ltd, Leeds, UK) set on HI for 10 min. The hot solution was poured into plastic measuring cylinders and distilled water was added to the desired final volume when required. The hot solution was then transferred using pipettes, at the desired volume (20 mL, except when noted differently), to glass crystallizing-dishes (50 mm D, 30 mm H). The solutions were allowed to cool and gel at room temperature and then covered and transferred to a refrigerator where they were kept at 4 °C until use. It is important to note that gel strength may vary as a result of water loss during storage. A tight covering for the gels is recommended. For this reason, it is also preferable that all tested gel samples be prepared and stored together. In addition, crude agar was prepared from sun-dried Gracilaria cornea (Rhodophyta). In brief, the seaweed material was coarsely ground and boiled for two hours in water (50:1 V:W ratio). The hot fluid was filtered through a 25 μm mesh and the filtrate was cooled and frozen overnight. The frozen sample was then thawed and allowed to drip, and the resulting gel was oven-dried at 70 °C overnight. The dry agar was ground to a coarse powder and agar gels (1%) were prepared as described above.

2.2. Gel Strength Apparatus Setup

A chromatography glass column was held loosely in place by two or three metal rings (funnel holders) connected to a laboratory stand holder. The size of the column was determined by the concentration range of the gel (bigger, heavier columns for stronger gels and vice versa). A home-made flat probe was inserted into the glass tip. The probe was built from a series of 2 × 2 round Lego^® bricks and plates with axle holes (part numbers 6143 and 4032, respectively) covered with a 2 × 2 round tile (part number 14769). A Lego^® axle (such as part number 3706) connected the bricks to the column tip. The probe had a surface area of 2 cm². An electronic scale was placed under the glass column. The column was free to move vertically above the scales, guided by the metal rings. A peristaltic pump connected to a water reservoir was placed next to the stand holder and a silicone tube was fitted into the pump, with one end attached to the water reservoir, and the other end placed in the bottom of the glass column. A simple peristaltic pump, which is easily available for purchase online, is sufficient. The full setup is illustrated in Figure 1.

2.3. Gel Strength Apparatus Operation

The general manner of operation was similar in all the experiments, with some variations between them that are described, for clarity, in the Results section. The apparatus was manually operated by two people, although one should suffice. For each measurement, a single gel was taken out of refrigerated storage and placed on the electronic scale for taring. The probe tip was then placed on the center of the gel and water pumping into the gel was immediately started. As the water volume inside the column increased, so did the total weight pressed upon the gel by the tip, as observed through the electronic scales. At the point when the tip broke the gel surface and penetrated the gel, the weight display on the scales was noted, and the pump was immediately stopped by the operator. Full gel penetration once the surface broke took less than a second. The gel break point was determined as the weight at which the probe completely broke the gel surface divided by the surface area of the probe (2 cm²). The broken gel was then removed, and the tip probe cleaned. Once all the water was removed from the column—either by reverse pumping or by decanting—the process could be repeated for the next gel sample. Each measurement took between 30 and 90 s to complete. The factors investigated in this report include the ratio between gel break point and gel concentration (0.5, 0.75, 1, and 1.5%), effect of the gel volume (10, 15, 20, 25, and 30 mL) on the gel break readings, effect of the water fill rate (270, 350, and 450 mL min⁻¹) on the gel break readings, user effect on the readings, and the variation in the readings, for different batches, from a single user. Each data point was measured using at least 6 separate gel samples. Statistics were calculated using the Excel^® data analysis package. ANOVA (single factor) was used to check for variation between samples. All samples had just one variable (ANOVA single factor) in the Excel data analysis package. To compare two specific sample means to each other, we used a two-tailed t-test (paired two samples for means) in the Excel data analysis package. A minimum of 6 independent repeats were made for each sample point.

3. Results

3.1. Gel Break Point to Gel Concentration Curve

Our system was able to distinguish between the quality and concentrations of different gelling agents. Commercial agar and agarose gels were prepared and their gel breakpoints were determined (Figure 2). As expected, agar had lower gel breakpoint values than agarose at all gel concentrations. The standard error values ranged between 15.1 and 35.2 g cm⁻² for agar gels and from 12.5 to 26 g cm⁻² for agarose gels. The differences in the gel breakpoints at the different concentrations were much higher than the error values. This allows for a clear distinction between the different concentrations, at least in 0.25 % intervals. At the measured gel concentrations, both the agar and the agarose measurements could be fitted to a linear curve with R² > 0.99. This implies that there was no noticeable effect when using a lighter column for the lowest gel concentration measurement. In addition, to test our system on a crude “home-made” gel, we extracted agar from sun-dried Gracilaria cornea and prepared 1% agar gel samples, which gave a gel breakpoint value of 307.8 ± 5 g cm⁻² (n = 3). This value is similar to the value obtained for 1% BD^® BACTO™ Agar (Figure 2). Once the ability of our system to qualitatively distinguish between gels was established, we set up further measurements to characterize other system variables, including the effects of the gel properties, column operation, and user. Hereafter, only agarose gels were used.

3.2. Characterization of the System

We tested the effect of the gel volume on the gel breakpoint measurements. We expected that thicker gels would have more mechanical durability. Figure 3 shows the ratio between the gel volume (or thickness) and the weight required to achieve gel breakage. We indeed found that thicker gels have more apparent durability in our system in a stepwise manner. The 10 mL gels showed the lowest gel breakpoint, with a high variability. Gels of 15–20 mL volumes gave roughly similar results, and gels of 25–30 mL volumes showed the highest gel breakpoint values with the lowest variability.

Next, we tested the effect of the water fill rate on the measured gel breakpoint. The peristaltic water pump was set at either 50, 75, or 100% capacity, which gave flow rates of 270, 350, and 450 mL min⁻¹, respectively. No statistical difference was found in the measured gel breakpoints between replicate samples of 1% agarose gels (Figure 4).

The system relies on the operator’s skill to note the exact point when the gel surface is broken and simultaneously stop the pump and note the weight reading. In order to check for the variability of the system in terms of user identity, we selected four random volunteers from our institution, none of them familiar with our system, and after a short demonstration, we asked them to measure the gel breakpoint of 20 mL 1% agarose gels with six replicates (Figure 5a, users A–D). Their results were plotted on a box chart and compared to our measurements (Figure 5a, user E). Although there was some variability between the users, no statistically significant differences were found.

Finally, we collected our accumulated data from all our measurements and plotted them on a box chart to compare the measurements done by the research team on different dates with the same gel conditions (20 mL of 1% agarose gel, Figure 5b). In this case, we did find some limited variability between the measurements, which can be attributed to either different gel storage times, or to small variations in the preparation of the gel solutions at the different times (e.g., small variations in weight and volume measurements).

4. Discussion

Gel strength is an important parameter for defining phycocolloid quality [1,5,9]. Gel strength is related to the chemical nature of the different portions of the phycocolloids, either that of the main carbohydrate chain or of the chemical residues covalently bound to them [5]. These variations can occur either due to the genetic information of the seaweed species, or as an acclimation to their growth conditions [4,5,6,8,9,10,11,12,13]. Thus, the assessment of gel strength is important for seaweed growers, as well as researchers, for identifying seaweed species with commercially desired phycocolloid qualities [1,2], or for optimizing growth conditions for improved gel strength. Texture analyzers, usually used today for measuring gel strength, are generally expensive and may not be readily available for growers or scientists. This is especially true if the only use for a texture analyzer in the laboratory will be for gel strength measurements, when it takes up valuable bench space when not in use. Using service laboratories is a good option, but often the service can be expensive and the lag time between the samples being sent and analyzed may slow down research or be out of sync with the seaweed growth season.

In this work we demonstrated a simple, flexible, and affordable way to assess agar gel strength. The system is easily set up or stored, and it uses commonly available laboratory equipment (Figure 1), which can be disassembled after the measurements for other laboratory usages. The apparatus can be easily adjusted for the required application; different size columns can be used for varying gel strengths, with smaller volumes and lower weight columns used for weaker gels, and larger columns for stronger or more concentrated gels. To further simplify and reduce costs, we suggest that water be added directly from the tap or added in pre-measured quotas, thus eliminating the need for a peristaltic pump. For very weak gels, the water can be replaced by plastic or metal beads, which can be added manually. Further system simplification and reductions in cost can be achieved by not using an electronic scale at all, but rather by predetermining the weight of the empty column, probe, and tube apparatus, and then measuring the water volume required for gel breakage. Simply converting the volume to grams (1 mL water = 1 g) and adding the apparatus weight will give the gel breakpoint value. The Lego^® probe can be replaced by any other means, such as a silicone stopper, cut to the desired dimensions. Instead of using a flat tip, the end of the tip can be replaced by a concave part (e.g., 2654).

Our system does not give information on gel resistance parameters while the weight increases, and thus we named the measurement “gel breakpoint” rather than “gel strength”, with units of gram cm⁻². The system can sometimes have a variability up to tens of grams. These values are similar to those reported in another study using a home-made device [6]. The variation values of our system are also comparable to those reported while using a commercial texture analyzer [18], and the references within]. Surprisingly, in several other reports, gel strength variation values were not available [12,13,14,15,16,17]. We believe that the accuracy of our measurement system is good enough for differentiating between different gels through the assessment of gel breakpoints, as it is repetitive across different sampling trials and different users, and we suggest it for use as a simple, available, and easy-to-use tool.

The system sufficiently differentiated between agar and agarose gels in the range of 0.5 to 1.5%, with a linear correlation found between gel concentrations and gel breakpoints in the measured gel concentrations (Figure 2). These are gel concentrations that are commonly used in biological applications, such as Petri dish microbial culturing and DNA separation. Our apparatus was also able to measure home-made, crude agar gels prepared from dry G. cornea and gave comparable results to those of commercial agar. This implies that seaweed farmers or researchers can prepare crude seaweed extracts at their facility and easily assess the gel breaking point using a similar system.

The lowest variability between measurements was found for gel volumes of 25–30 mL (Figure 3). The reason for this is not clear. It is possibly due to the physical force applied by the glass plate sides on the gels, countering the force pressed down on the gel by the probe tip. The recordings can of course be affected by the diameter of the glass dishes, yet we chose a dish size that was easily available on the market. The system gave good results for lower volumes, which can be critical for such studies where the available biological material is limited. The researchers can then decide on the best gel volumes to use. Alternatively, they could use smaller dishes for the lower volumes. We did not see any effect from the water fill rate (Figure 4) or any user effect (Figure 5A), but it is preferable that all samples to be compared are made together and measured at the same time (Figure 5B). Another key issue for conducting the measurements is keeping the column vertically positioned above the gel. The iron rings make this easy, and untrained people were able to conduct these measurements without significant variations in the results (Figure 5A).

It is important to note that the values for the 1% agarose gels measured in our system were about one-third less than the stated gel strengths as printed on the agarose packaging. The reason for this is probably due to the use of different measurement techniques, noting that there were no details on the protocol used to determine the gel strength by the manufacturer. In home-made devices, it is expected to find some variations across measurements done in different laboratories [6]. To address this, we suggest that for each measurement of an unknown gel sample, another measurement, or a gel concentration–gel breakpoint calibration curve, should be conducted as a control, using a commercial agar gel, such as BD^® BACTO™ Agar, or another well-known brand. Such a practice was also suggested by Levy et al. [6]. Also, since the correlation between the gel breakpoint and the gel concentration was linear in the tested range, it should be possible to prepare a calibration curve using a commercial phycocolloid with a known gel strength, at a concentration range of 0.5–1.5%, for the gel strength assessment of an unknown sample.

The main time and effort requirement for laboratories or farms using this apparatus is the preparation of gel samples from their raw seaweed material to conduct the measurements. Our system was able to clearly distinguish between agar qualities—agar and agarose (Figure 2)—and worked well with a home-made agar gel. For more standardized methods, select samples can be sent to expert service laboratories.

In conclusion, the system described here can be used for an affordable, fast, in-house determination of the gel strength properties of different gel samples. Using such a flexible system allows a seaweed farmer or researcher to establish a gel strength measurement system with tailor-made parameters for each batch of seaweed or experiment, and to gain qualitative and timely information on their products or research subjects.