**1. Introduction**

"In general, pectin is extracted from agricultural products, especially apples, oranges, carrots, etc" [1–5]. But some environmental controls in plant breeding, as well as genetic engineering may increase the production of pectin in the plant. You may also be able to specialize more economically organisms to produce pectin.

For example, "Ajaya K Biswal, et al reported in 2015 that "Mutation of GAUT12 in *Populus deltoides* by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a plant biofuel feedstock". The family name GAUT was coined after the discovery of Arabidopsis galacturonosyltransferase 1 (GAUT1). GAUT1 is a pectin biosynthetic homogalacturonan (HG): α-1,4 galacturonosyltransferase (GalAT) that functions in an HG:GalAT protein complex with GAUT7. The highest amount of Arabidopsis GAUT12 / irx8 was measured in cell tissues containing secondary wall that Its molecular structure is about 61% similar to that of GAUT1 monomers" [1].

"GAUT12 is thought to be a type II membrane protein and a Golgi target. If irx8 / GAUT12 mutates, GX decreases. In the Biswal study, microsomes from irx8 mutant stems didn't show any reduction in xylan XylT activity or xylan GlcAT (glucuronosyltransferase) activity compared to microsomes from wild type (WT)" [1].

Therefore, it's possible to increase pectin production in plants by UP-regulation or GAUT cloning. For any case, it is important to do a thorough study of pectin production control genes first. You may be able to DNA editing and programming for cells with greater, and more cost-effective reproduction.

Also, the environmental conditions' control of the plant, such as light, moisture, soil elements, fertilizers, temperature and etc., may affect of pectin production. Since these plants are primarily edible, this works can hurt food and agricultural policies. For this reason, in the pectin industries are primarily used, agricultural wastes.

"1.3 billion tons of human's food produced consumption is lost or wasted each year. In the meantime, 45% of food wasted are vegetative and herbal." "Food industries based on fruits and plants produce significant amounts of solid waste that can be used for animal feed, fertilizer, biochar production [6] or biogas [7]". However, the bulk of this waste is transported to landfills. While waste includes "avoidable" and "unavoidable" items; Waste of food, such as discarding edible parts of fruits that aren't suitable for the food industry, as well as other untreated/unrecycled waste, causes the spread of greenhouse gases, global warming and "carbon footprints". For this reason, the potential use of agricultural by-products and solid waste of the food industry creates a comprehensive opportunity for the development of functional ingredients industries [3].

"Pectin methyl esterases (PMEs) are important for the regeneration of pectins during plant growth. These enzymes have been reported in both plants and plant pathogens (such as bacteria and fungi). Plant and microbial PMEs aren't the same in enzyme substrate properties and pH tolerance. Furthermore, differences have also been reported between plant PMEs. Different types of PMEs act differently in response to different chemical compounds. For example, the inhibitory composition of plant PMEs, proposed by Melanie L'Enfant Jean et al, had completely no effect on the PMEs of pathogenic fungi. However, this study could open the way to control the properties of pectin methyl esterases" [2].

#### **2. Pectin-like carbohydrates in algae**

Today, algae is considered as one of the best and most cost-effective sources for many organic products. The ability to produce a product per unit time for algae can be much higher than plants. They do not need agricultural land and consume less water and resources. On the other hand, the harvest of algae in a photobioreactor can be about 24 times per year. Studies show that "the ability to produce lipids in algae is higher than plants. The percentage of oil and the quality of essential fatty acids - such as omega-3 and omega-6 - in algae is even higher than in fish oil. For these reasons, the consumption of algae - as human food - has attracted the attention of many in the international community, especially vegetarians. Also, the special properties of algae have made them the main candidates for providing new biofuel production resources" [8]. The algae ability of produce is not limited to lipids, some types of algae produce the high of proteins, carbohydrates, etc. Also with the use genetic engineering or even controls of the culture medium, they can easily be made more specialized.

The use of algae, to provide sources of pectin extraction is very attractive; Because the use of algae as a source, does not require agricultural land, abundant

#### *Biotechnology Applications in the Pectin Industry DOI: http://dx.doi.org/10.5772/intechopen.100470*

water and abundant and expensive resources, also its harvest per unit area is faster and more. On the other hand, increasing the percentage of pectin production in plants can be contrary to global food supply and biofuels policies.

"Magdalena Eder & Ursula Lütz-Meindl, identified pectin-like carbohydrate molecules in the green alga *Netrium digitus*. Pectins known to be involved in cell-wall expansion and representing major components of mucilage were the main objectives of this study. In more evolved plants, low methyl esterified pectins occur at cell corners, in middle lamellae and around air spaces, It's thought that the placement of pectins in these areas was done with the aim of creating resistance to environmental stresses. By forming a stable gel using calciumBridges, low methyl-esterified pectins prevent separation of cells as frequently induced by stress factors" [9].

"Lee, Kyung-Ah et al also reported that they were able to extract pectin-like polysaccharides from marine algae. They extracted Pectin-like from 5 kinds of microalgae and 9 kinds of macroalgae with different extraction methodologies. High yield of PLP was extracted in distilled water (DW) as 21.06±3.5% from *A. maxima*. In general, pectin-like extraction from macroalgae was more satisfactory than microalgae. In acidic condition (AC), PLP from Undaria pinnatifida was not precipitated. However, the yields of galacturonic acid was higher in Hizikia fusiformis (80.28±4.58%) and Laminaria japonica (65.85±0.61%), respectively. In biological activity tests, Fe2+ chelating activity was 26% higher than pectin from citrus peel as control. ABTS scavenging assay showed 100% antioxidative activity based on DW extraction from Ecklonia cava, whereas not detected from other macroalgae. The results of this study show new hopes for the use of gelling and stabilizing properties of PLP in various industries" [10].

"The total amount of carbohydrates in macroalgae is about 40 to 65% and in microalgae is about 10 to 50%. Kyoung-Ah Lee et al reported that they investigated the composition of PT and the anti-oxidant activities of 5 species of micro-algae (Spirulina maxima, Leptolyngbya sp, Tetraselmis sp, Dunaliella sp, and Chlorella sp) and 9 species of macro-algae (Saccharina japonica, Sargassum fulvellum, Undaria pinnatifida, Ecklonia cava, *Gracilaria verrucosa*, *Gelidium amansii*, Sargassum fusiforme, *Ulva pertusa*, and Sargassum horneri). Furthermore, Edirisinghe, S.L. et al and Chandrarathna, H.P.S.U. et al observed immunologic values of Spirulina maxima PT that have the potential to modulate gut microbial population, en- hance the expression of immune related genes, and boost gut morphology in zebrafish larvae" [10, 11].

"Rajapaksha, Dinusha C. et al and Edirisinghe, S.L. et al reported that the performance of pectin-like extracted from Spirulina.maxima was significant for wound healing. Also, D.S. Domozych et al wrote that the green alga Penium margitaraceum shows pectin metabolism in its cell wall. In addition, EDER et al extracted Pectin-like polymers from the cell wall and mucosa of the green alga *Netrium digitus*. In fact, many researchers have been able to extract pectin-like molecules from algae and have studied the properties of these molecules; But we still need more evidence on the effectiveness and industrial value of pectin-like extraction" [11].

"Kyoung-Ah Lee, et al reported, that they were able to extract pectin-like biomolecules from 5 species of microalgae and 9 species of macroalgae. MP extraction yields were higher in the distilled water (DW) extracts of Spirulina maxima and *Ulva pertusa* (yield extractions of 21.90±1.12% and 18.80± 0.97%, respectively). These results confirm that a large amount of MP was extracted by DW from marine algae, and MP is unlike the pectin derived from land plants. The MP extraction conditions were established using different solvents for each marine algae, and optimum extraction conditions exist for each species. Regarding biological activity, The MPs from Ecklonia cava and Sargassum horneri showed 99% ABTS radical scavenging activity, and the Fe2+Chelating activity of the MP from Dunaliella sp. Was confirmed to be higher than those of other MPs. The results of this study potentially indicate the potential for industrialization of pectin-like extraction from algae. This article also shows that the development of the use of DW materials in industry can help reduce environmental pollution" [10].

### **3. Pectin extraction and biotechnology**

"Approximately 30% of the primary cell wall in apples and citrus fruits contains PT pectin. There are several methods for extracting PT; Such as extraction with acid, hot water, enzyme, microwave, ultrasonic as well as combined methods. Acid and hot water extraction are the oldest methods in the pectin industry. The biggest drawback of traditional methods is the degradation of pectin polysaccharides and the long extraction time. In contrast, enzymatic extraction has many advantages such as no degradation of pectin polymers, low extraction temperature, shorter extraction time, reduction of environmental pollution, requires very low acidity and so on. For this reason, various green extraction techniques have received much attention" [11].

• biotechnology methods in pectin extraction include: enzymatic extraction and enzymatic-ultrasonic extraction (and etc). Which we examine.

#### **4. Enzymatic extraction of pectin**

"The analysis in **Table 1** shows that, the polysaccharides xylan and xyloglucan form the crosslinker between pectin, cellulose and hemicellulose. For this reason, the use of cellulase and xylanase can break the bond between these polysaccharides and separate pectin molecules from the cell wall. Based on studies and laboratory evidence, the highest enzymatic extraction efficiency of pectin is in the simultaneous use of cellulase and xylanase. Because using a mixture of cellulase and xylanase, it destroys the bond between xylan, xyloglucan and cellulose and causes the separation of pectin. While enzymatic treatment with xylanase alone reduces the extraction efficiency. The reason for this reduction in efficiency is probably the strong properties of the bonds between hemicellulose xyloses, not the bonds between hemicellulose and pectin or cellulose. The use of xylanase can improve cellulase function by degrading xylan / xyloglucan from the SBP matrix and further release pectin. However, the use of a higher cellulase ratio (2:1) reduced the pectin extraction efficiency in the study by Abou Elseoud et al. However, this may be due to pectinase activity remaining in the cellulase enzyme derived from Trichoderma Iongibrachiatum – reported by him. Because pectinase enzymes cause the hydrolysis of pectin polymers to soluble sugars, thereby reducing the efficiency of pectin extraction.

Comparison of pectin extracted in the best conditions of traditional extraction with sulfuric acid (temperature 85°C, pH = 1, time: 2 h) against pectin extracted by combined treatment of xylanase and cellulase enzymes (8.28%) shows that the extraction efficiency is similarity with the acid extraction method (5.26%). In addition, the maximum enzymatic extraction efficiency of pectin in the Abou Elseoud study is equal to or greater than the acidic extraction of beet pulp" [4].

In enzymatic Pectin extraction GalA efficiency from passion fruit peel were between 17.0 and 25.8 g/100 g of dry peel, which were similar to those obtained for the more commonly used citrus peel substrates with PPase-SE. Efficiencies were also comparable with those obtained from lemon pomace using a different polygalacturonase from Aspergillus niger and from pumpkin using a cellulase from Trichoderma viride and a multi-enzyme crude extract from *Bacillus polymyxa*. GalA efficiency from acid extraction was 15.9 ± 0.1 g/100 g dry peel, Which shows a much lower result


*[3], p. 6.*

*a Considered on dry weight of example [dry weight of extracted pectin [g] ÷dry weight of example [g] ×100] b Considered on dry weight of oil free example [dry weight of extracted pectin [g] ÷dry weight of oil free example [g] ×100]*

*c Considered on dry weight of alcohol insoluble residue of example [dry weight of extracted pectin [g] ÷dry weight alcohol insoluble residue [g] ×100];*

*NA: not applicable / NS: not specified*

#### **Table 1.**

*Repercussion of classical extraction method on the efficiency, quality, degree of esterification and galacturonic acid amount of extracted pectin from tropical and sub tropical fruit /by products.*

than other extraction conditions (p < 0.05). This was probably due to the decomposition of a percentage of soluble GalAs due to very low pH and high temperatures. Therefore, due to the lower extraction temperature as well as the milder pH required for enzymatic extraction; The performance and quality of pectin molecules obtained by enzymatic extraction method have been reported to be significantly higher than the chemical method. A significant number of reports show similar results in studies of pectin from passion fruit. However, most published reports are about the extraction of pectin from the dried peel. Juliana Vasco-Correa and Arley D. Zapata-Zapata [5], used the fresh peel with a relative high particle size, which it need to less energy intensive since high energy would be needed for drying and milling the peel. In Liew et al. study and Kulkarni & Vijayanand obtained maximum efficiency of 14.6 g/100 g of peel and 14.8 g/100 g of peel, respectively, by extracting pectin from passion fruit peel citric acid at pH 2 was used, which are similar to the conditions and results of the chemical extraction performed in the Vasco-Correa's studys. Canteri et al. obtained a slightly higher yield of 20.3 g/100 g of rid flour from passion fruit, using nitric acid for the extraction. Contreras234 Esquivel et al. achieved a yield of 25 g/100 g of dry passion fruit fiber using citric acid and autoclaving for 20 min. Kliemann et al. (2009) Enzyme loading had a significant effect on GalA yield (p < 0.05) (**Table 1**) [5].

The results do not show a significant difference between the performance of GalA prepared at 30 and 40 U / mL. But these two groups recorded higher solubility than 20 U / mL. Therefore, increasing the enzyme charge to 30 U / mL can improve the solubility of GalA, but increasing the enzyme load can not produce more positive results. According to the results, the maximum adsorption of PPase on protopectin (the pure substrate) obtained at 30 U/mL of PGase. So, 30 U/mL can considered as optimum enzyme loading. So, excessive increase in enzyme concentration at 40 U/ml not only increases project costs, but can also degrade soluble pectins, as PPase has significant endopolygalacturonase activity and can degrade internal bonds between GalAs. Although agitation speed has had a significant impact on other PPase-SE processes, in this case, it has had very little effect on GalA performance (p > 0.05). This is due to the excellent permeability and solubility of the enzyme anywhere, at 120–180 rpm. It's also possible that in the Vasco-Correa report, vortex flasks led to better mass transfer. Other reports indicate that agitation speeds do not have a significant effect on enzymatic heterogeneous processes in low solids loading, which in the study was about 2.5 g/100 ml. In addition, temperature and pH variables affect the rate of GalA dissolution (p > 0.05). Maximum yield was measured at 37° C and pH 3.0. This temperature was also the optimum value found for lemon peel pectin extraction using PPase-SE. Minimum yields were obtained at 44°C, perhaps because the enzyme wasn't stable at this heat temperature. While, changing the pH in the range of 4.0–5.0 does not cause much change; GalA performance increased significantly at pH 3.0 (p < 0.05). However, these results contradict the results of the study of pectin extraction with PPase-SE from lemon peel, under similar conditions and higher pH. Therefore, it is necessary to determine the best pH for each system, based on the study of the specific enzyme and substrate of the project. In the acidic extraction method of pectin, the more acidic degree of pH will be in favor of optimizing the extraction process, while the same can cause the destruction of pectin molecules. Acid also kills microbial pathogens.

The report of the periodic study of enzymatic extraction in flasks shows the maximum extraction efficiency of 21.9 g / 100 g of dry peel in 120 minutes. The longer the extraction time, the lower the efficiency and quality of the product, because there is a possibility of unwanted enzymatic hydrolysis due to the endopolygalacturonase activity of the enzyme. While some studies have used enzymatic therapy for longer than 12 to 20 hours, no significant increase in efficiency, quality, or optimization that benefits the pectin extraction industry has been reported.

Generally, yield is normally requested in scale-up of biocatalytic processes, and as the size of the stirred-tank increases, an increment of the mixing time is expected. Agitation speed in the range studied in the bioreactor had a great effect on GalA. The lower the energy consumption of a biochemical process, the greater the potential for industry expansion. This is one of the most important things to consider when planning your business. However, calculating the actual energy consumption for a floating particle heterogeneous system can be difficult without direct measurements" [5].
