Biotechnology Applications in the Pectin Industry

Abstract

Pectin and/or pectin-like polysaccharide (PLP) is one of polysaccharides contained in the plants and algae cell walls, That’s known as a polymer branched from galacturonic acids. Pectins are widely used in industry to remove heavy elements, gel and stabilize materials. Furthermore, its antioxidant properties are considered medically and in healthy eating policies. “Pectin is composed of D-galacturonic acid linked by α-1, 4-glycosidic linkage and in most cases, pectins are classified according to their degree of esterification (DE), which represents the ratio of galacturonic acid groups esterified in the structure of the pectin polysaccharide. The high methyl (HM) ester is a polymer that is methyl esterified in more than 50% of its carboxylate monomers, and conversely, the low methyl (LM) ester is a pectin with a degree of esterification of less than 50%. The bioactive properties of pectin polymers are very wide. For example, pectins, with their antioxidant properties, are anti-cancer and anti-tumor, and help heal patients undergoing chemotherapy. Pectin polymers can help improve diabetes and lower cholesterol. In addition, pectin has received much attention in medicine due to the importance of hydrogels, nanofiber mats and nanoparticles.” The purpose of this chapter is to review and introduce possible applications of biotechnology in pectin industries. We review sections on agricultural production and the enzymatic extraction method, as well as enzymatic-ultrasonic extraction. Finally, some suggestions are made for factory effluents and solid waste.

1. Introduction

“In general, pectin is extracted from agricultural products, especially apples, oranges, carrots, etc” [1, 2, 3, 4, 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 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.

Fruit/fruit by-product	Yield of pectin	Treatment conditions			pH	Galacturonic acid	Degree of esterification
		Temperature (°C)	Time (min)	Acid
Mango peel	5.4%^a	85	30	Nitric acid	2	80.71%	67%
Pomelo peels	3.11%^b	90	90	Hydrochloric acid	2	NS	NS
Cubiu fruits peels	14.2%^c	Temperature not specified (boiling)	120	Nitric acid	1.5	72%	62.0%
Cubiu fruits peels	9.6%^c	100	120	NS	NS	79%	56.9%
Ponkan peels	25.6%^a	Temperature not specified (boiling)	100	Nitric acid	1.6	84.5%	85.7%
Cornelian cherry fruit whole	0.83%^a	Room temperature	60	NS	3	59.1%	84%
Pomegranate peel	8.5%^a	86	80	Nitric acid	1.7	62%	75%
Honey pomelo peels	17.5%^a	85	80	Hydrochloric acid	1.24	749 g/kg	76.6%
Durian rinds	9.1%^a	86	43	Hydrochloric acid	2.8
Fresh watermelon rinds	19.3%	Temperature not specified (boiling)	60	Nitric acid	NS	74.2%	63%
Pomegranate peel	11.34%^c	88	120	Citric acid	2.5	80.95 g/100 g	53.09%
Wolf apple unripe fruit pulp	33.68%^a	80	30	Nitric acid	1	NS	77.15%
Main harvested kiwi fruit	3.27%^a	50	60	Citric acid	2.8	56.08%	82%
Early harvested kiwi fruits	1.43%^a	50	60	Citric acid	2.8	48.80%	88%
Main harvested kiwi fruit	3.27%^a	50	60	Water	NA	51.87	84%
Early harvested kiwi fruits	1.01%^a	50	60	Water	NA	42.88	90%
Citron peel	21.85%^a	90	180	Water	NA	NS	77%

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.

[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

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 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].

5. Quality of pectin molecules from enzymatic extraction

“Studies show that the highest efficiency and quality of pectin extraction is obtained by enzymatic method with simultaneous use of cellulase and xylanase enzymes. For more information, see the following data: All spectra showed the characteristics peaks for pectic rich polysaccharide as follows: stretching vibration of OH groups of carboxylic acid and alcohol at ~3432 cm − 1, stretching vibration ofC-H groups of methyl, methylene groups at ~2900 and 2925 cm − 1, stretching vibration of CO of ester groups at ~1744 cm − 1, symmetric and asymmetric stretching vibration of CO of carboxylate groups at ~1621 cm − 1 and 1433 cm − 1, amide groups of protein linked to pectin at ~1544 cm − 1, stretching vibration of C-N groups (present in protein) and also C-O bending at 1270, 1100 & 1026 cm − 1 for the C-O bonds in glycosidic linkage and alcoholic OH groups of sugars. Quantification the ratio of the intensities of carboxylic group at 1632 cm − 1 or ester group at 1744 cm − 1 to that of methylene C-H group of the backbone at 2925 cm − 1 showed that A1632/A2925 ratios were 1.49, 1.63, and 1.50 for pectin sample extracted using 1:1, 1:1.5, and 1:2 xylanase to cellulase enzymes, respectively, while A1744/A2925 ratios were 0.88, 1.0, and 0.97 for the same samples, respectively.

The results showed noticeably higher ratios of A1632/A2925 and A1744/A2925 in case of using 1:1.5 xylanase to cellulase ratio. The study of sugars in Table 2 shows the highest galacturonic acid content in this sample. It is also shown that the highest extraction performance is obtained at this enzymatic ratio. Ester or carboxylic acid groups are another important factor in the emulsification properties of pectin” [4].

Ratio of xylanase:cellulase enzymes	Sugars content (wt.% based on weight of sample)				Sum of total sugars (wt. %)	Protein content (%)	Ferulic acid content (%)	Degree of esterification (%)	Molecular weight (mol/g)
	GalA	Ara	Xyl + Gal+Rha*	Glc
1:1	55.73 ± 1.30^a	0.95 ± 0.031^c	2.50 ± 0.202^c	10.12 ± 0.11^b	69.30	5.6 ± 0.35^a	0.19 ± 0.013^b	64.5 ± 5.80^a	1.47E+05 ± 2.80E+03^a
1:1.5	58.56 ± 1.23^a	1.58 ± 0.011^b	4.08 ± 0.205^b	16.07 ± 0.84^a	80.29	5.7 ± 0.14^a	0.27 ± 0.016^a	67.7 ± 7.47^a	1.19E+05 ± 2.83E+03^b
1:2	50.51 ± 0.76^b	2.67 ± 0.023^a	5.59 ± 0.48^a	11.03 ± 0.44^b	69.80	6.7 ± 0.26^a	0.23 ± 0.016^ab	65.2 ± 8.35^a	1.21E+05 ± 2.12E+03^b
Acid-extracted pectin sample**	72.56 ± 0.50	3.01 ± 0.19	4.11 ± 0.20	3.52 ± 0.20	83.20	10.5 ± 0.69	0.49 ± 0.016	60.6 ± 4.33	1.15 E+05 ± 1.97E+03

Table 2.

Effect of different doses of cellulase and xylanase enzymes on composition of isolated pectin.

^*

Calculated as galactose. GalA: galacturonic acid, Ara: arabinose, Gal: galactose, Xyl: xylose, Rha: rhamnose, Glc: glucose.

^**

Conditions of extraction were: temperature at 85°C, for 2 h, and at pH 2.

[4], p. 6.

“Table 2 shows galacturonic acid, neutral sugars, protein, ferulic acid contents, degree of esterification, and molecular weight of the extracted pectin samples using different xylanase to cellulase enzymes doses. Examination of the table shows that increasing the ratio of xylanase to cellulase from 1: 1 to 1.5: 1 in the extraction solution does not cause significant changes in the galacturonic acid content of the resulting pectins. While increasing the concentration of cellulase enzyme in a ratio of 2: 1, reduces the amount of galacturonic acid in pectin polymers. Analyzes show that the highest content of neutral sugars is related to glucose and the concentrations of rhamnose, galactose, xylose and arabinose are less than 5%. Enzymatic extraction of pectin with the treatment of xylanase to cellulase ratio of 1.5: 1, releases the highest concentration of neutral sugar. High concentrations of neutral sugar have been reported for the enzymatic-ultrasonic extraction of pectin from sisal lesions as well as for the hydrothermal extraction of pectin from sugar beet. High glucose concentrations are mostly related to pectin-bound hydrolyzed cellulose oligomers. As described, cellulose, pectin and hemicellulose are bound together in the cell wall. In addition, previous studies have shown the concentration of glucose or cellobiose in products obtained from the extraction of pectin by acidic method, also the relationship between the concentration of free sugar and the galacturonic column. In general, the optimal purity of the extracted pectin is related to the concentration of galacturonic acid; However, other sugars are also extracted in the process. In fact, proteins ad esterified carboxylic groups in galactron chains have an important effect on the emulsification properties of pectin. In addition, presence of ferulic acid groups, which are attached to the O-2 position of (1 → 5)-linked arabinose residues in the arabinan side-chains as well as to the O-6 position of galactose residues in (1 → 4)-linked galactans, contributes also to the emulsification efficiency of pectin. Table 2 shows that enzymatic extraction of pectin from SBP results in pectins with a high degree of esterification (>50%). The different samples of pectin extracted by this method do not show much difference in the ester content. The content of galacturonic acid and ester in this table – continuously – are at a very good level. But the protein content in different samples varied from 5.6% to 6.7%. According to FAO standards, the nitrogen content of pectin should not exceed 2.5%, which refers to 15.6% of protein. The concentration of ferulic acid varies in the range of 0.19% to 0.27% between different samples with a maximum solution of xylanase to cellulase 1: 1.5. Increasing the concentration of cellulase enzyme relative to xylanase from 1: 1 to 1: 1.5 reduces the molecular weight of pectin in the extraction process, while increasing the concentration of cellulase in the extraction solution will not further reduce the weight of pectin molecules. The molecular weight loss of pectin in this case may be due to the presence of pectinase in commercial cellulase enzymes, as these enzymes have the ability to hydrolysis pectin.

Comparison of the chemical composition of pectin obtained from acid extraction in the best conditions (temperature 85°C, 2 h, ph 2) with pectin obtained from enzymatic extraction using a combination treatment with cellulase and xylanase shows a higher concentration of galacturonic acid in the acidic method (72.56%). Also lower glucose content (3.52%), lower ester content (60.6%), lower ferulic acid concentration (0.49%), near arabinose (3.01%), galactose + rhamnose + xylose (4.11%) and protein (5.01%) it shows. The molecular weight of pectin in this case was measured as 1.85E + 05, which shows a higher proportion of pectins from enzymatic extraction in the Abou Elseoud study” [4].

6. What efficacy does the use of different concentrations of enzyme have on the quality of emulsification and the stability of pectin emulsions containing oil in water?

“The pectin that isolated using the different xylanase to cellulase ratios gave close EAI and also stability of emulsion after storage in fridge at 4°C for four weeks. The different emulsions prepared with pectin from different percentages of enzyme concentrations didn’t undergo any phase separation. However, after 4 weeks of storage at 4°C, they experienced a decrease in EAI, which was probably the result of the accumulation of emulsion droplets. The yield is almost similar to the emulsification of different samples of enzymatic pectin extracted, probably due to the close protein concentration, and their similar ester content, which plays an essential role in the quality of the emulsion and its stability. In addition, the emulsion extracted from the pectin extracted by the acidic method, EAI showed almost the same as the results obtained by enzymatic extraction. You can compare EAI with other emulsifiers such as soy protein, sodium caseinate and whey protein with EAI of pectin from enzymatic extraction” [4].

7. How does ultrasonic pretreatment affect the performance of pectin from ultrasonic-enzymatic extraction?

“Studies show that the use of ultrasound before treatment with extraction fluid containing enzymes increases the performance of the extracted pectin and also reduces the extraction time. For example, With ultrasonic pretreatment for 15-45 minutes and then treatment with enzymatic extraction fluid for 60 minutes, the yield of pectin shows an increase of about 84-92%. If the enzyme-containing extraction fluid treatment is applied for 120 minutes and the ultrasonic pretreatment is applied for 15-45 minutes, the yield of pectin increases by 67-95%. In addition, if ultrasonic pretreatment is performed for 15-45 minutes and then treated with enzymatic extraction solution for 240 minutes, the pectin yield will not increase significantly (2-16%). The increase in yield of isolated pectin by the ultrasonic treatment is due to the intensification of mass transfer by sonication due to cavitation bubble collapse, which facilitates penetration of the enzymes into the plant tissue because of the increase in porosity and surface area. Ultrasonic applications cause cell swelling and softening of the walls, and by hydrating the pectin in the inner layer, they break down the walls during the process and facilitate the release of pectin” [4].

8. Evaluation of the quality of extracted molecules using ultrasonic-enzymatic technique

“Sugars content was dependent on length of enzymatic treatment time. At the shortest enzymatic time (1 h), increasing ultrasonic treatment from 0 to 45 min resulted in increasing galacturonic acid by about 17%.regarding neutral sugars, the isolated pectin samples after different ultrasonic treatment had close arabinose, rhmanose, galactose, glucose, and xylose contents at the 1-hour enzymatic treatment. At 4-hour enzymatic treatment, ultrasonic treatment time had no effect on galacturonic acid content but arabinose tended to decrease, glucose sugars tended to increase, and no significant effect on rhmanose, galactose, and xxylos was found. There was generally no significant effect for increasing the ultrasonic treatment time on protein content of extracted pectin. Regarding the degree of esterification, pectin extracted using merely enzymatic treatment for 1 h had generally higher degree of esterification than those isolated after 4 h. The effect of ultrasonic treatment time on degree of esterification was depended on enzymatic treatment duration. At 1 h enzymatic treatment, there was no effect for the ultrasonic time on the degree of esterification while in case of 4 h enzymatic treatment the ultrasonic pretreatment resulted in pectin with lower degree of esterification.

Regarding the ferulic acid content, it generally decreased with increasing the ultrasonic pretreatment time in case of the 4-hour enzymatic treatment experiments while no effect was found in case of the 1-Hour enzymatic treatment. This is in accordance with a previous study, which showed that ultrasonic treatment could promote de-esterification of ferulic acid and also methyl and acetyl groups from galacturonic acid units” [4].

9. Evaluation of emulsification quality of pectin extracted by ultrasonic-enzyme method

“Studies show that ultrasonic pretreatment can improve enzyme extraction time. For example, ultrasonic pretreatment for 15-45 minutes and then using enzyme extraction sources for 1 hour, increases the yield of pectin to 92-84%. Also, enzymatic treatment for 2 hours and ultrasonic pretreatment for 15-45 minutes, increases the yield of pectin to 67-95%. However, if the enzyme extraction solution is used for 4 hours and the ultrasonic pretreatment for 15-45 minutes, this number is reduced to 2-16%. Increased yield of pectin extracted by ultrasonic pretreatment and enzymatic treatment, due to increased mass transfer, occurs during ultrasound through the process of cavity bubble collapse, which itself, due to increased porosity and surface area, will increase the penetration of enzymes into plant tissue. On the other hand, ultrasonic pretreatment, by hydrating the pectin in the inner layer, causes more swelling and softening of the cell wall, and ultimately leads to wall destruction during pretreatment” [4].

10. Therefore…

“Today, we can extract pectin with a very good quality by the extraction solution of cellulase and xylanase enzymes, similar to acid extraction under the best possible conditions. Generally, the benefits of enzymatic extraction far outweigh the benefits of acid extraction. Furthermore, many of the disadvantages of traditional extraction don’t exist in green extraction. We must also consider that the enzymatic extraction of pectin in the conditions of using the combined extraction solution of xylanase and cellulase is in its highest quality, which will be much more effective than the method of using single enzymes. In addition, ultrasonic pretreatment in enzymatic extraction method develops and improves pectin processing technology, making extraction time and product quality very desirable. But the use of ultrasonic-enzyme extraction method does not show a significant effect on the emulsification properties of pectins in comparison with enzymatic extraction” [4, 5].

11. Biotechnology ideas for pectin factory solid waste

Since the pectin is extracted from plant products (or pectin-like from algae); Its solid waste can be used to extract or produce valuable materials. One of the ideas for extracting pectin waste is related to substances that can complement your main product. According to studies, if you can extract “cellulose, starch or alginate; You have found a good supplement to increase and improve the effectiveness of your pectin” [11].

Also, if you succeed in commercializing algae to extract pectin-like; You can extract “lipids from its waste to produce biodiesel, carbohydrates to produce bioethanol, protein, or even alginate, and so on” [8, 11, 12, 13].

If none of these extractions are cost-effective for your factory, you may welcome the production of fertilizer (algae waste or plant products) and even biochar. If you own a large pectin plant and produce a lot of waste, it may be cost-effective to produce biochar. You can also collect agricultural waste to mix with your waste.

“Biochar, which is produced from biomass pyrolysis, can be used for water’s heavy element treatment, such as agricultural fertilizer (with the advantage of reducing the amount of water required), or even as fuel” [6, 14].

12. Biotechnology ideas for pectin factory effluent

Plant effluent can be used to grow algae. “These algae may eventually be used as a source for biofuels, fertilizers, and so on” [8, 12, 13]. ”Algae also have the ability to purify water even completely” [15]. “Or you can use these algae to make custom water purification biofilms” [15].

Good luck.