**4. Agar**

The development of solid culture media was fundamental not only for bacteriology but also for biotechnology. Robert Koch and his assistant Walter Hesse are known as the inventors of solid media, in 1882 they replaced animal gelatin with agar gelatin, however, this technique already had precursors. Some authors mentioned that agar was discovered in Japan in 1658 by Tarazaemon Minoya, agar was used at the time as a gelling agent for food [25]. Agar is extracted from the cell membrane wall of red algae in a fibrous crystallized form and is mainly composed of sulfated galactan [26].

Around the world, there are different genera of algae such as Gelidium, Gracilaria, Gelidiella, Pterocladia, Gracilariopsis, Ahnfeltia, and their respective species. The characteristics of each of these provide the extracted agar with different properties. However, the best quality agar is derived from Gelidium, algae that are only found in the wild. It is important to mention that the composition of the agar is not affected by seasonality, but the yield is [27].

Agar is classified as a non-branched, high molecular weight, reserve natural polysaccharide with mineral content including Copper (Cu), Zinc (Zn), Manganese (Mn), Calcium (Ca), and Magnesium (Mg). Agar is composed of agaropectin and agarose, the latter is the main constituent (70%). Both components have the same basic structure, formed by alternating units of D-galactose and 3,6-anhydro-Lgalactopyranose linked by α-(1–3) and β-(1–4) bonds [28].

Agarose has the following formula [C 12H14 O5 (OH) 4], and is classified as a neutral polysaccharide. The α and β galactoses present in their molecular structure are responsible for the mechanical property of gelation which is linked to the formation of gels, caused by the interaction of helicoids. Agarose and agaropectin are differentiated by the presence of sulfate and pyruvate residues. Agaropectin is classified as a charged polymer, where D-galacturonic acid and pyruvic acid provide it with viscosity property [26, 29]. The aforementioned composition is similar to that of the natural polymer starch [30].

#### **4.1 General structure and properties of agar**

The FTIR spectrum of agar (Gelidium) has vibrations in the absorption bands at 3291–3390 cm−1 associated with O-H stretching, followed by vibration at 2932– 2922 cm−1 for CH2 stretching, and the absence of a band at 2845 cm−1 corresponding to the O-CH3 groups indicates a degree of low methoxylation. A vibration in the

**Figure 1.** *Chemical structure of agar [33].*

1642 cm−1 band is associated with the stretching vibration of the conjugated peptide bond formed by amine (NH) and acetone (CO) groups. Vibrations for CH2 groups are found at 1413–1370 cm−1, and a vibration that is identified for bridging a sulfated ester at 1179 cm −1. In addition, the most represented vibration band in the agars is found at 930 cm −1 and attributed to the bridging of 3,6-anhydro-galactose [30–32]. A schematic of the agar structure is shown in **Figure 1**.

Agar is a hydrophilic hydrocolloid that has different properties, including the ability to form colorless, thermo-reversible gels that do not lose their original characteristics with changes in temperature; the gel forms at 30°C and dissolves at temperatures between 75°C and 90°C. The gels are not digested by plant enzymes. Important properties of these gels, such as yield and gel strength, may vary depending on the genus and location of the algae (**Table 2**).

For the extraction of agar there are different methodologies that can alter the above-mentioned properties (yield and gel strength). The most used methodologies are direct extraction in the water bath, the Freeze–Thaw method, syneresis method, and the alkaline treatment. Recent extraction processes are photobleaching, microwave-assisted extraction, and enzymatic method assisted by hydrogen peroxide [40, 41].

On the other hand, agar has important uses in different industries in addition to the elaboration of solid culture media for plant growth and micropropagation. It was the first hydrocolloid used as a gelling and stabilizing agent in the food industry. Agar is also used in the dental industry for the manufacture of the mold, in the medical industry for pharmaceutical formulations, in other industries for biodegradable films as packaging film; and its use in the cosmetic industry is well known [32].

The industry of agar production continues growing, Asia and the Pacific dominate the manufacture of this product, Indonesia and China play an important role, but Chile has prospered in that sense. The main suppliers of Gelidium are Spain, Portugal,


#### **Table 2.**

*Yield and strength properties of gel in different species of algae***.**

#### *A Potential Alternative for Agar in In Vitro Culture Media Based on Hydrocolloids Present… DOI: http://dx.doi.org/10.5772/intechopen.101745*

Morocco, Japan, Republic of Korea, China, Chile, and South Africa [42]. Annually, 55,000 tons of seaweed are extracted, which produce 7500 tons of agar with a value of US\$ 132 million [43]. The growth of this industry has caused ecological problems that have hindered agar temporalities and supply; therefore, scientists have been inclined to find total or partial substitutes for agar for different industries. The following section explores some commercial gelling agents and natural agar substitutes with successful results.

#### **4.2 Agar substitutes for gelling agents in culture media**

Currently, research is seeking to reduce the ecological effects of agar extraction, while reducing the costs of this biotechnology [6]. Therefore, in 1986, the carrageenan was used as a substitute for the gelling agent agar in plant tissue cultures; according to the results obtained in the research, the tissues grew better on this gel than when the medium was solidified with agar [44].

In Cuba, the Center for Research and Development of Medicines (CIDEM) investigated the use of Aloe vera (Aloe barbadensis miller) and sago flour (Maranta arundinacea) as solid support in culture media for medicinal plants. The research demonstrated that partial or total substitution of agar by A. vera gel or sago flour is possible. In addition, this culture medium has been used for the in vitro propagation of agraz (Vaccinium meridionale Swartz) [45, 46].

Starches from cassava (Manihot esculenta), corn (Zea mays), and rice (Oryza sativa) have been investigated for their high availability in local markets and low cost. The use of starches in the partial replacement of Phytagel® in the modified MS medium for sweet potato (Ipomoea batatas) and cassava (M. esculenta) crops was investigated in Honduras at the Plant Tissue Culture Laboratory. As a result, it can replace up to 72% of the Phytagel® dose in sweet potato (I. batatas) and cassava (M. esculenta) crops.

Isabgol, which is the seed of Plantago psyllium, a herbaceous species from Spain and Morocco, used commercially for the production of mucilage for dietary fiber, in conjunction with commercial sugar was used as an alternative agar in in vitro culture media for plantain. The results showed that not only can isabgol be a solidifying agent in culture media, but also a preservation medium for germplasm banks [5].

In 2012, a study conducted in Ethiopia investigated the efficiency of ensete (bulla) starch as a gelling agent, significantly improving the number of sprouts and saving about 72.5% in costs [47]. In another study, the partial substitution of agar by the starch of the Diacol Capiro variety in the micropropagation of lulo Solanum quitoense Lam. was carried out, with a positive result [48].

Future perspectives in the development of this biotechnology are directed towards the partial or total substitution of agar as a gelling agent by solidifying agents that can be used more efficiently, easily extracted, locally acquired, and not temporary. In addition to improving plant production in reduced spaces and the resources derived to face future challenges with micropropagation without the risk of crop contamination.

#### **5. Nopal hydrocolloids: potential substitutes of agar**

Opuntia, better known as Nopal cactus is native to the American continent and belongs to the subfamily Opuntioideae (Cactaceae) that consists of 181 known species that are present throughout the American territory in the wild and are characterized by their easy reproduction and their ability to adapt to different climatic conditions [49]. Nopal cactus is produced worldwide and has been used by different industries in research related to natural medicine and human body benefits because it has essential nutrients for human beings such as dietary fiber, vitamin C and A, calcium, phosphorus, potassium, magnesium, chlorophyll, and antioxidants. The content of these nutrients is related to variables such as the age of the plant, the place where it grows, and the climate to which it has been exposed [50, 51].

However, it has been determined that the amino acid content of nopal cactus consists of aspartic acid, glutamic acid, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and tryptophan [52]. Within its mineral composition, there is manganese, iron, zinc, magnesium, and sodium.

Dietary fiber is the major component of the nopal cactus, which can range from 11.0 g to 23.33 g depending on the age of the stalk and has been related to health benefits in different research studies due to its high content of bioactive compounds such as phenols and carotenoids [53]. Dietary fiber is composed of insoluble fiber and soluble fiber.

Soluble dietary fiber is formed by hydrocolloids (pectin and mucilage), which are named like that for their great capacity to capture and retain water [54]. These hydrocolloids are classified as natural polymers that have recently been studied for their importance and technological advantages. They are composed of arabinose, galactose, rhamnose, xylose, and galacturonic acid residues [55]. The mucilages of the nopal cactus have a similar composition to the exudates of Sterculia trees (Sterculia and Khaya gum), which are used as stabilizers [56].

However, due to the molecular composition and characteristics of the hydrocolloids present in the nopal cactus, this work revises definitions and characteristics of mucilage and pectin from nopal cactus stalks, the extraction methods, and mechanical properties.

#### **5.1 Structure and general properties of mucilage**

Mucilage is a complex neutral polymeric substance that is part of the carbohydrates and has a yield of 1.0% to 1.2% in fresh weight and about 17.9% in dry weight [57]. It is present in the Golgi apparatus and functions as a chelator capable of binding calcium and controlling the amount of free soluble calcium [58]. Mucilage has a branched chemical structure with approximately 55 sugars where L-arabinose, D-galactose, L-rhamnose, D-xylose, and D-galacturonic acid are present in different proportions [59–61].

Sáenz et al., [62] mention that McGarvie and Parolis (1981) presented the first suggested structure for O. ficus-indica mucilage, where they describe the molecule as a linear repeating central chain of α-D-linked (1–4) and β-L-linked (1–2) rhamnose with side chains of (1–6)-β-D-galacturonic acid linked to O-4 rhamnose residues. The galactose residues substituents at the O-3 positions, or double substituents at O-3 and O-4 (**Figure 2**) [63, 64].

Sepúlveda [57] mentions that the structure of mucilage is proposed as two distinct water-soluble fractions, where one is identified as pectin with gelling properties with Ca2 +and the other is a mucilage without gelling properties that swells when dissolved in water and shows characteristics of high viscosity [61, 65].

The property of viscosity, a physical characteristic of fluids, has a relationship to ionic strength, pH, and slightly to temperature in the Opuntia spp. As pH increases from acidic to alkaline conditions, viscosity increases. In addition, viscosity decreases *A Potential Alternative for Agar in In Vitro Culture Media Based on Hydrocolloids Present… DOI: http://dx.doi.org/10.5772/intechopen.101745*

as temperature increases just as it does in Xanthan gum. It is also mentioned that Opuntia mucilage has high elastic properties, the higher the concentration of mucilage, the lower the normal stresses [60]. The concentration of mucilage is important in the characterization of certain properties.

The extraction of mucilage can be carried out by different methods, and its yield depends on this. First, there is the extraction by water bath and the use of ethanol (95%) or isopropyl alcohol (95%), with a yield of 1.58% fresh weight [57]. The microwave-assisted extraction, the mechanical pressing system, and its lyophilization are other extraction methods.

The FTIR analysis of the mucilage of O. ficus indica and O. robusta performed in this study, compared with that performed in other species such as O. jonostle, O. streptacantha, O. tomentosa, O. atropes, and O. hyptiacantha shows that the functional groups present in all cactus mucilages are: absorption bands at 3500–3200 cm−1 that represent the carboxylic acid -OH groups involved in the intermolecular bond as mentioned by [66] Contreras-Padilla (2016). At 2975–2919 cm −1, a band may appear which is related to the stretching of the -CH groups belonging to the pyranose groups, then a softer absorption band at 2850 cm−1which is related to the stretching of -CH2 groups of the carboxylic group [67]. It is shown the lack of waveband at 1749 cm −1 is linked to the low degree of esterification, as mentioned by [68] Rodriguez-Gonzalez et al., (2014) which indicates that the carboxyl groups are free and available to interact with water molecules and this results in their high capacity to absorb water; as well as if the free carboxyls are mixed with Ca 2+ in the presence of water, they can form viscous structural networks. However, for O. robusta and O. atropes a slight vibration can be identified at 1730 cm −1related to C=O stretching. In addition, it was found two bands at 1593 and 1388 cm −1 related to symmetric and asymmetric COOstretching, which confirms the low degree of mucilage esterification. Finally, it was found a band at 1030 cm −1, due to the vibration of C-O molecules attributed to the

stretching of secondary cyclic alcohols [67]. The bands below 1000 cm −1 correspond to β-D-glucose and below 800 cm −1 are attributed to vibrations of N-H and O-H groups (**Figure 3**) [69].

### **5.2 Structure and general properties of pectin**

Pectin is part of the structural tissues of plants and vegetables, it is present in the skin of certain fruits such as apples or in the pulp of other vegetables such as citrus fruits, strawberries, quince, and carrots. Pectin contains mainly galacturonic acid (GalA) with residues partially esterified with methanol and is the main component of the middle lamella in plants and primary cell wall, it has the function of providing cohesion and stability to tissues [70]. Pectin is formed by long chains of α-D- (1 → 4) linked to galacturonic acid interspersed by the insertion of residues (1 → 2) linked to L rhamnose residues with side chains of neutral sugars, the linear segments are predominantly composed of homogalacturonan [71]. Within its structure homogalacturonan, rhamnogalacturonan I and II, and xylogalacturonan are identified. **Figure 4** shows the basic molecular structure of pectin, where it can be seen that each ring has a carboxyl group that can be esterified with methanol-producing methyl esters [72].

The importance of pectin lies in its ability to form gels in the presence of Ca+2 ions or in solute at low pH, hence its importance and multiple applications as a thickening agent, gelling agent, binder, and stabilizer in industries such as pharmaceuticals for gastrointestinal treatments, in the food industry for the production of jams and frozen foods, and recently, innovations in its use for edible coatings and foams. However, for the formation of gels, the most important characteristic is the quality of the extracted pectin, which is classified into two types: pectin with a high degree of methylation and pectin with a low degree of methylation.

**Figure 3.** *ATM-FTIR mucilage extracted from Opuntia ficus-indica (OFI) and Opuntia robusta (OR) by alkaline hydrolysis.*

*A Potential Alternative for Agar in In Vitro Culture Media Based on Hydrocolloids Present… DOI: http://dx.doi.org/10.5772/intechopen.101745*

**Figure 4.** *Basic molecular structure of pectin [72].*

The degree of methylation is the indicator of galacturonic acid residues esterified or methoxylated by the methyl group and are classified into low (<50%) and high methoxyl (>50%) pectins [73]. For low methoxyl pectin "gelation results from ionic bonding through calcium bridges between two carboxyl groups that belong to two different chains in close contact with each other. In high methoxyl pectin, cross-linking of pectin molecules involves a combination of hydrogen bonding and hydrophobic interactions between the molecules" mentions [74] Thakur et al., (2009). The yield of pectin can be from 4.42 to 10.39%, depending on conditions such as time, temperature, pH, and dry weight extraction method [75]. With the microwave-assisted extraction method, yields of 12.56% dry weight were obtained [70]. Recently, the enzymatic method with xylanase and cellulase was used for the extraction of pectin from O. ficus indica where yields of 17.91% in dry weight were obtained [76].

The conditions and method of pectin extraction directly affect the GalA content and therefore its gelation capacity; in the case of requiring pectin as a functional additive, hot acid extraction is recommended; for the use of de-esterified pectin, with

**Figure 5.** *ATM-FTIR of pectin extracted from Opuntia ficus-indica (OFI) and Opuntia robusta (OR) by alkaline hydrolysis.*

high GalA content and gelation capacity with calcium ions, an alkaline soluble extraction is recommended [77].

According to the alkaline, extraction carried out in this study and compared to that performed by [61] Goycoolea & Cárdenas (2003), [77] Cárdenas et al., (2008) and [76] Bayar et al., (2017), the FITR analysis shows the following vibrations. A strong vibration at 3283 cm −1 related to the stretching vibrations of the -OH groups of alcohol and carboxylic acid involved in inter-and intramolecular hydrogen bonds of the galacturonic acid polymer [78]. Subsequently at 2968 cm−1 a pronounced band corresponding to the absorption of the O-CH3 extension bonds the methyl ester of galacturonic acid [79]. A 1720 cm−1 vibration caused by stretching vibration C∙O methyl esterified carboxyl groups, at 1624 cm−1 vibration is related to the stretching of carboxylate ions and the relative ester band, which is more intense in pectins of a high degree of esterification. The bands found between 1600 to 1400 cm−1 correspond to the antisymmetric and COO - symmetric stretching characteristic of carboxylic acid salts. Some of the carboxyl group signals may also originate from phenolic compounds as indicated by the presence of peaks at 1530 cm−1 for aromatic ring vibrations [80]. It has been shown that the relative intensity of the last two peaks is related to the degree of methoxylation. The bands found between 1250 to 1140 cm−1 correspond to the C-O-C ether stretching [81]. The last strong bands found between 1140 to 1100 cm−1 are due to C-O-stretching of secondary alcohols and C-O- stretching of H in cyclic alcohols respectively [76] (**Figure 5**).

### **6. Conclusion**

According to the analysis of the hydrocolloids extracted by acid hydrolysis contained in O. ficus-indica and O. robusta, analyzed by FTIR and compared with other research, it is concluded that the functional groups found in the mucilage are characteristic of proteins and polysaccharides, that have mechanical properties of viscosity, which can be used in industries such as food, pharmaceutical, construction, cosmetics, and biotechnology. However, when separating the solid residue from the mucilage extraction and performing acid hydrolysis to it, we obtain (according to the FTIR analysis) pectin with a low degree of methoxylation, because a small absorption peak is observed at 1732 cm−1, which is attributed to the C = O stretching vibration of the carboxyl groups esterified with methyl. Due to this, the use of this pectin in combination with the indicated substances and the selected culture medium is suggested as a potential partial or total substitute for agar as a gelling agent in In vitro culture media for the development of plant cultures under laboratory conditions.

#### **Acknowledgements**

I thank the Universidad Autónoma de Querétaro and the bromatology laboratory staff of this university. To Dr. Liliana España Sánchez, research professor at the Centro de Investigación y Desarrollo Tecnológico en Electroquímica (CIDETEQ ) and Dr. Reyna Araceli Mauricio Sánchez (CINVESTAV) for their assistance in the FTIR measurement and characterization performed. To my thesis advisors. Also, to the Consejo Nacional de Ciencia y Tecnología (CONACyT) for the scholarship granted to carry out this research and Call Fund for the Promotion of Entrepreneurial Culture 2021, School of Engineering, Universidad Autónoma de Querétaro.

*A Potential Alternative for Agar in In Vitro Culture Media Based on Hydrocolloids Present… DOI: http://dx.doi.org/10.5772/intechopen.101745*
