A qualitative method for gel strength assessment of phycocolloid-based gels

doi:10.21203/rs.3.rs-3748600/v1

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A qualitative method for gel strength assessment of phycocolloid-based gels

https://doi.org/10.21203/rs.3.rs-3748600/v1

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Seaweeds are the natural, sustainable, source for phycocolloids. Phycocolloids such as agar and agarose, carrageenan and alginate have many usages in a variety of industries including health, science, food, and cosmetics. The commercial application of each phycocolloid, and its commercial value, depend on its chemical/physical properties. One of the unique characteristics of certain phycocolloids is their ability to form gels. Gel properties such as gel strength, gel setting and melting temperatures vary between different seaweed species and among a single species, can vary depending on their growth conditions. Thus, measuring gels properties including gel strength is important for seaweed growers and scientist. State of the art gel strength measurements mainly includes the use of texture analyzers. However, such instruments are not always available for growers and scientists, and alternative methods for obtaining gels strength are desired. In this work we describe a home-made apparatus for assessing gel strength, or gel break point, of phycocolloid gels. The system measures the weight of water in a column required for breakage and penetration of a flat tip, pressed against gels at a concentration range of 0.5–1.5%. We found that the system gave repeatable results, both across different sampling trials, as well as across different users. The variation in the measurement was similar to that reported for other gel strength measuring devices. We propose the use of our system as a simple, flexible, and affordable method for quickly assessing gel quality of phycocolloid samples.

Gel strength

phycocolloids

agar

agarose

macroalgae

seaweed

Seaweeds are the only available source for commercially important phycocolloids such as agar and agarose, carrageenan, and alginate (Pangestuti and Kim 2015; Padam and Chye 2020; Lomartire and Gonçalves 2023). One of the important characteristics of phycocolloids is their ability to form gels. The gelling properties of the different phycocolloids often determines their end use in various industries (Pangestuti and Kim 2015; Lomartire and Gonçalves 2023). Gel strength is an important parameter of phycocolloids (Lee et al. 2017). The strength of a gel is dependent on the concentration of the gelling agent in the gel solution and by the chemical structure of the carbohydrate backbone (Lahaye 2001). Phycocolloid backbone chemical structure is determined by the seaweed genetic information (Lahaye 2001; Pangestuti and Kim 2015; Lee et al. 2017; Lomartire and Gonçalves 2023). Different species synthesize different kinds of phycocolloids, each with their unique gel properties (Levy et al. 1990; Lee et al. 2017; Vuai 2022). In agar or carrageenan gels chemical residues covalently bound to the carbohydrate backbone also determine their gelling properties as interruption to gel matrix can occur either from steric interruption or from electrostatic interferences (Lahaye 2001). Generally, phycocolloid abundance and quality can be influenced by growth conditions of the seaweeds(Bird et al. 1981; Christiaen et al. 1987; Friedlander et al. 1987; Lahaye and Yaphe 1988; Correa and McLachlan 1992; Pangestuti and Kim 2015; Firdaus et al. 2021; Vuai 2022) or manipulated by the extraction method (Lahaye 2001; Lee et al. 2017; Fateha et al. 2021a), or addition of salts (Lahaye 2001).

Since gel strength is an important phycocolloid trait, it is very beneficial both for seaweed growers and for scientist to be able to measure it. Several methods have been reported for measuring gels 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 (Czapke 1979), mercury load (Hamer 1947; Czapke 1979)or the use of a metal rod plunger (Christiaen et al. 1987; Friedlander et al. 1987; Levy et al. 1990). Sometimes only a vague description of the gel strength measurement is reported (Friedlander et al. 1987; Lee et al. 2017; Firdaus et al. 2021). Special equipment is also commercially available, including gelometers or texture analyzers (Guerin and Bird 1987; Lahaye and Yaphe 1988; Lee et al. 2017; Fateha et al. 2021b; Vuai 2022). Home-made devices usually require either technical knowledge and workshop labor to build, or the use of toxic materials like mercury. Due to their high prices texture analyzers may not be available for many seaweed farmers and researchers. Use of service lab can also be costly and requires a time lag between sample preparation and analysis, due to shipment time, service lab availability and more. Given that the gelling properties depend on the usually fluctuating growth conditions of the seaweeds—at least in outdoor growth facilities, —real time assessment of gel strength, even at a qualitative level 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 approach for qualitative assessment of gel strength using readily available laboratory equipment. It is based on water flow, rather than mercury, which simplify things in means of toxicity and liquid handling. This paper gives the outline of a specific system built in our lab, yet it can be easily modified to fit needs and available resources of seaweed growers and researchers alike.

Gel preparation

The gels in this work were prepared using agarose bought from HyLabs LTD (Rehovot Israel). The agarose had a reported gel strength of 1,200 gr cm^− 2 according to the information on the container. Agar gels were made using BD® BACTO™ Agar (Becton, Dickinson and Company, Sparks, MD, USA) The gels were prepared by mixing the agar or agarose powder with distilled water at appropriate ratios for each desired concentration. The mixture was then boiled using an electric pressure cook (Ninja® Foodi®, SharkNinja Europe Ltd, Leeds, UK) set up on HI for ten minutes. The hot solution was poured into plastic measuring cylinders and distilled water were added to the correct volume, when needed. 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 in room temperature, and then covered and transferred to a refrigerator where they were kept at 4°C until use.

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 homemade flat probe was inserted into the glass tip. The probe was built from a series of 2X2 round Lego® bricks and plates with axle holes (part number 6143 and 4032, respectively) covered with a 2X2 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 are of 2 cm². An electronic scale was place 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 place in the bottom of the glass column. A simple peristaltic pump, easily available for purchase online is sufficient. The full setup is illustrated in Fig. 1.

Gel strength apparatus operation

The apparatus was manually operated by two people, although one should suffice. For each measurement, a single gel was taken out of the 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 by the electronic scales. When the tip broke the gel surface and penetrated the gels, the pump was immediately stopped by the observant operator and the weight display was recorded. The gel break point was determined as the weight in which the probe completely broke the gel, and the tip touched the bottom of the crystalizing dish, 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 were 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 seconds to complete. Each data point was measured using at least 6 separate gel samples. Statistics were calculated using the Excell® data analysis package.

Gel break point to gel concentration curve

Our system was able to distinguish between different gelling agent quality and concentrations. Agar and agarose gels at concentrations of 0.5, 0.75, 1 and 1.5% were prepared and their gel break point determined (Fig. 2). As expected, agar had lower gel break point values than agarose, at all gel concentrations. Standard error values ranged between 15.1 to 35.2 grams cm^− 2 for agar gels and from 12.5 to 26 grams cm^− 2 for agarose gels. The differences in gel break point at the different concentration was much higher than the error values. This allows for a clear distinction between the different concentrations, at least in 0.25% intervals. 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 of using a lighter column for the lowest gel concentration measurement. Now that the ability of our system to qualitatively distinguish between gels was established, we set up further measurements to characterize other system variables, including effect of the gel properties, column operation and user effect. Hereafter only agarose gels were used.

Characterization of the system

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

Next, we tested the effect of the water fill rate on the measured gel break point. The peristaltic water pump was set up at either 50, 75 or 100% capacity, which gave flow rates of 270, 350 and 450 ml min^− 1, respectively. No statistically difference was found in the measured gel break points between replicate samples of 1% agarose gels (Fig. 4).

In order to see if the variability of the system in terms of the 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 break point of 20 ml 1% agarose gels, in six replicates (Fig. 5A, users A-D), their results were plotted on a box chart and compared to our measurements (Fig. 5A, user E). Although there was some variability between 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 measurements done by us on different dates, in the same gel conditions (20 ml 1% agarose gel, Fig. 5B). In this case we did find some small 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).

Gel strength is an important parameter for defining phycocolloid quality (Bird et al. 1981; Lahaye 2001; Pangestuti and Kim 2015). Gel strength is related to the chemical nature of the different of the phycocolloids, either those of the main carbohydrate chain or of chemical residues covalently bound to them (Lahaye 2001). These variations can occur either due to the genetic information of the seaweed species or as an acclimation to their growth conditions (Bird et al. 1981; Christiaen et al. 1987; Friedlander et al. 1987; Lahaye and Yaphe 1988; Levy et al. 1990; Correa and McLachlan 1992; Lahaye 2001; Lee et al. 2017; Firdaus et al. 2021). Thus, assessment of gel strength is important for seaweed growers as well as researchers, either for identifying seaweed species with commercially desired phycocolloid quality (Pangestuti and Kim 2015; Lomartire and Gonçalves 2023), or for optimizing growth condition for improved gel strength. Texture analyzers, usually used today for measuring gel strength are generally expensive and not readily available for growers or scientists. Using service labs is a good option but often the service can be expensive and the lag time between 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 gel strength. The system is easily set up or stored (Fig. 1). It can be easily adjusted for the required application; different size columns can be used for varying gels strengths, with smaller volume and lower weight columns used for weaker gels, and larger columns for stronger or more concentrated gels. To further simplify and reduce cost, we suggest that the water can be added directly from the tap or added in pre-measured quotas, thus eliminating the need for a peristaltic pump. Further system simplification, and reduction in costs can be achieved by not using an electronic scale at all, but rather by predetermining the weight of the empty column, probe and tubes apparatus, and then measuring the water volume required for gel breakage. Simply converting the volume to grams (1 ml water = 1 gram) and adding the apparatus weight will give the gel break point value. The Lego® probe can be replaced by any other means, such as a silicone stopper, cut to the desired dimensions.

Our system does not give information on gel resistance parameters while the weight increases, and thus we named the measurement as "gel break point" 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 (Levy et al. 1990). Surprisingly, in several other reports gel strength variation values were not available (Guerin and Bird 1987; Christiaen et al. 1987; Lahaye and Yaphe 1988). We believe that the accuracy of our measurement system is good enough for qualitatively differentiating between different gels through assessment of gel break point, 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 gel concentrations in the range of 0.5 to 1.5%, with a linear correlation found between gel concentration and gel break point (Fig. 2). These are gel concentrations commonly used in biological applications such as petri dish microbial culturing and DNA separation.

The lowest variability between measurements was found for gel volumes of 25–30 ml (Fig. 3). Although the system still gave good results for lower volumes. This can be critical for such studies where the available biological material is limited. The researchers can then decide on the best gels volumes to use. Alternatively, they could use smaller dishes for the lower volumes. We did not see any effect of the water fill rate (Fig. 4) or any user effect (Fig. 5A) but it is preferable that all samples to be compared will be made together and measured at the same time (Fig. 5B).

It is important to note that the values for 1% agarose gels, measured in our system were about one third than the stated gel strength as printed on the agarose packaging. The reason for this is probably due to different measurement technique, noting that there were no details on the protocol used to determine the gel strength by the manufacturer. In such home-made devices it is expected to find some variations between measurements done in different labs (Levy et al. 1990). To overcome this, we suggest that for each measurement of an unknown gel sample, another measurement, or a gels concentration-to-gel-break-point calibration curve will be conducted as a control, using a commercial agar gel, such as BD® BACTO™ Agar, or another well-known brand. This was also suggested by Levy et al. (Levy et al. 1990). For more standardized methods, select samples can be sent to expert service labs.

In conclusion, the system described here can be used for an affordable, fast, in-house determination of 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 seaweeds or experiment, and to gain qualitative and timely, information on their products or research subjects.

Funding – This work was supported by the Israel Ministry of Agriculture & Rural Development, grant number 30-01-0003.

Competing interests – The authors declare no competing interests.

Availability of data and material – The data of this study are available upon request from the authors.

Code availability - not applicable.

Authors' contributions – ES conceptualized and designed the study. Material preparation, data collection and analysis were performed by LS and ES. The first draft of the manuscript was written by ES. LS and ES read and approved the final manuscript.

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No competing interests reported.

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A qualitative method for gel strength assessment of phycocolloid-based gels

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