Optimization of Enzyme-Assisted Isolation of Bioactive Polysaccharides and its Characterization from Pumpkin (Cucurbita Moschata)

Pumpkin is a seasonal vegetable crop that belongs to the Family Cucurbitaceae and genus Cucurbita [1]. This plant family is considered as one of the largest families in the plant Kingdom with a large number of edible plants with 118 genera and 825 species [2,3]. In India, large number of pumpkin varieties varying in shape, size and colour of flesh are available. Largely found common varieties are CM-14, Pusa vishwas, Arka chandan, Arka suryamukhi, CM-350 and NDPK-24 [4]. The main growing season for cucurbits in India is the summer and rainy months. The winter pumpkins are grown in some parts of southern and western India. Pumpkin is utilised for its leaves, marrow, fruit pulp and seeds and are all appropriate resources for multifarious food. Unripe, still small and green pumpkin is eaten in the same way as zucchini. Whereas pumpkin ripe can be boiled, baked, steamed or roasted and can be frozen or canned. The people of the rural areas of India have been growing it on the roofs of the house or in the backyard of the house using their traditional knowledge. In Himachal Pradesh, the ripe pumpkin is utilized in preparation of traditional dish (sweet or savoury) during Himachali weddings and festivals. Whereas, in Kerala, they used in preparation of ellisserry and olan, a curry prepared using red cowpea and ripe pumpkin [5]. 

Over the past countless decades, researchers have engrossed their work on the scientific assessment of nutritional components of pumpkin and squashes, which has been frequently led to its utilization as a functional food or exploitation in medicines in several epidemiological studies [6]. Pumpkin is a valuable source of functional components mainly carotenoids, lutein, zeaxanthin, vitamin E, ascorbic acid, phytosterols, selenium and lenoleic acid, which act as antioxidants in human nutrition [7]. Pumpkin are also known to contain bioactive materials such as polysaccharide, protein-bound polysaccharides, carotenoids, minerals, amino acids and active proteins. The polysaccharides present in pumpkin are cellulose, hemicellulose and pectin [8]. Protein bound polysaccharides from the pumpkin pulp could help to regulate the serum insulin level, reduce blood glucose levels and improve glucose tolerance [9]. Pumpkin is rich in pectin which when consumed is supposed to control glycaemic levels and reduce the need for insulin [10]. Pumpkin has no cholesterol, low in fat and sodium. They are recommended by dieticians in cholesterol controlling and weight reduction programs. Danilchenko et al. [11], has suggested pumpkin for atherosclerosis as it helps to reduce cholesterol in people suffering from obesity.

Pectin is a multifunctional polymer occurring naturally in cell wall of all non-woody plants. It is found in higher concentration in middle lamella, with a gradual decrease as one passes through the primary wall towards the plasma membrane. It is very difficult to determine the structure of pectin because they change with variation in source and location of source, storage and processing of raw material, condition of isolation and extraction and other environmental factors [12]. Fruits like apples, guavas, quince, plums and gooseberries also possess high levels of pectin while soft fruits like cherries, grapes and strawberries contain lower amounts [13]. Pectin is getting more and more application in food, pharmaceutical and biotechnology industry. It is being successfully used in food and beverage industry as thickener, emulsifier, texturizer, stabilizer and gelling agent. It is also being used as a fat substitute in spreads, ice-cream and salad dressings. Pectin also have been widely applied in feed or pharmaceutical industries for their functionality. Pectin and their derivatives have been observed anticancer, immunostimulation, anti-inflammatory, gastroprotection, antibacterial, antiadhesive, and complement fixing activities. The immunostimulating, antibacterial, anti-inflammatory, and prebiotic activities of pectin synergistically contribute to the anticancer activity. Moreover, some pectins could improve regeneration of bone and connective tissue, wound healing, and bio-accessibility of encapsulated ingredients with good biological compatibility, and greater heat resistance and electrolyte tolerance. 

There are diverse alternatives for the extraction of pectic polysaccharides for industrial applications such as chemical, physical and biotechnological. Chemical extraction processes are the most used and consist of using strong acids or bases. Physical extraction involves the use of microwaves, autoclaving, extrusion while biotechnological processes involve the use of enzymes for the pectin extraction process. There is an increasing interest in using environmentally friendly technologies for pectin extraction such as enzymes, ultrasonic and microwave technologies. Enzymes are widely used in several industries as more environmentally friendly technology than use of chemicals, while the use of biotechnological methods are still confined to laboratories. The use of enzymes for pectin extraction brings two major advantages associated to selectivity and efficiency, especially in terms of the recovery. Two main approaches in enzyme-assisted extraction is (i) the use of enzymes that degrade and isolate specific pectin fragments and (ii) the use of enzymes capable of deconstructing the plant cell wall and isolating the whole pectin molecule [14,15]. In the process of enzyme-assisted isolation of pectin, various enzymes can be employed to break down the plant cell wall and release the pectin. For extraction of pectin, celluclast is commonly used enzyme to promote the release of pectin substances. This enzyme acts as catalyst to breakdown cellulose in cell wall and turns them into glucose, cellobiose and higher glucose polymers [15]. Other enzymes intended for extraction of pectin are protease, α-amylase, cellulase, viscozyme, hemicellulase etc. These enzymes are sometime known to have the ability to degrade and modify the physico-chemical properties of the pectin [16]. 

Therefore, in this study pectin extraction from pumpkin was investigated by the use of cellulase enzymes from two different source that degrades the plant cell wall components. The extraction of pectin was conducted on a laboratory scale and the influence of the enzyme dose, extraction temperature and time was studied.

Procurement of Raw Materials 

The ripe pumpkins were procured from the local fruit and vegetable market of Solan, Himachal Pradesh, India. The chemicals used were of Loba Chemie and purchased from International Scientifics and Surgicals, Solan, HP, India. The experimentation was carried out in the Fruit Processing Research Laboratory of Department of Food Science and Technology, UHF, Nauni, Solan (HP) Himachal Pradesh. 

Preparation of Raw Material 

Pumpkin, with its hard texture, typically needs water for pulp conversion, subsequently affecting pulp-water ratio and phase separation during experimentation. To ease preparation, pumpkins were shredded instead of grating. For this, ripe pumpkins were halved, deseeded, sliced, peeled, and chopped. The pieces were then juiced in a juicer, yielding pumpkin pomace and juice separately. Pomace and juice were then blended in a mixer for 3-4 minutes to ensure homogeneity. This method reduced water usage and delayed phase separation, enhancing stability. 

Enzyme-Assisted Extraction of Pectin 

Pectin was extracted from ripe pumpkin by employing enzymes as per the method followed by Ptichkina et al. [17]. The parameters like enzyme concentration, extraction time and temperature (Table 1) were modified to obtain the desirable combination. Two different enzyme preparations viz. Trichoderma viride cellulase and commercial cellulase were used to isolate the pumpkin pectin by this method. 

Table 1: Optimization of parameters for pumpkin pectin isolation with enzyme extraction. 

Pumpkin pulp and water were taken in 1000 mL conical flask in a ratio of 1:4. The mixture was subjected to pre-heating treatment by autoclaving at 121°C for 20min and was then allowed to cool down. The flask was allowed to cool and the pH of the mixture was adjusted to 5.5 by adding citrate buffer. The enzyme was added and mixed properly. Mouth of flask was covered with aluminium foil and was incubated in water bath at specified time temperature combination. The sample was agitated with continuous stirring at constant intervals of 15min. Once the desired time was attained, the temperature of the mixture was raised to 60°C and immediately cooled to approximately 3-4ºC in a bath of crushed ice to stop further enzymatic reactions. The mixture was filtered through double layer cheese cloth to separate residue and extract was collected. The extract was centrifuged at 3000rpm for 20min and the supernatant was siphoned off and collected in beaker (Figure 1).  

 Figure 1: Unit operations for preparation of pectin extract using enzyme extraction.        

The filtrate obtained above was concentrated two fold by using buchy type vacuum evaporator and precipitated with 95% ethanol twice the volume of filtrate. The solution was stirred continuously for 10min and allowed to stand for 4 h at 4ºC. The pectin was separated from the alcohol using a double layer cheese cloth. Samples were washed thrice with 70% alcohol and once with undiluted alcohol to remove any impurities. The resulting pectin was transferred to petri-dish covered with aluminium foil and kept in hot air oven at a temperature of 50ºC. The sample was dried up to the extent (approximately 2h) when it was easily removable from the foil. The samples were allowed to cool at room temperature, weighed and ground to obtain fine powder. The samples were stored in polyethylene pouches (Figure 2).

 Figure 2: Unit operations for precipitation of pectin obtained by enzyme extraction. 

Yield and Characterization of Extracted Pectin 

Yield of Pectin     

The yield of pectin obtained was calculated by the formula given by Ptichkina et al. [17], and is as under:

Moisture Content     

Moisture content was determined as per the method described by Owen et al. [18]. One-gram sample was weighed and transferred into tarred metal dish (5cm in diameter with cover). The sample was dried for 4h at 100ºC. The moisture content was expressed as per cent and calculated as given below:

Ash Content

Ash content of pectin was determined by the procedure detailed by Owen et al. [18]. One to two gram of pectin was taken into a tarred crucible. It was slowly ignited on heater and the heated in muffle furnace (Relitech) for 3 to 4 h at 600ºC. Sample was then cooled at room temperature in desiccators and weighed.

Equivalent Weight 

A known weight (0.5g) of ammonia and ash free pectin was weighed into a 250ml conical flask and was moisten with 5ml ethanol. One-gram sodium chloride was added to sharpen the end point. To the flask, 100ml carbon dioxide free distilled water and 6 drops of Hinton’s indicator was added. Precaution was taken to prevent lump formation of pectic substances on the sides of flask so as to ensure complete dissolving. Sample was then titrated with 0.1N NaOH until the colour of the indicator changed (pH 7.5) to magenta and persisted for at least 30 sec Owen et al. [18].

The neutralised solution was further used for determination of methoxyl content.

Methoxyl Content 

To the neutral solution titrated for equivalent weight, containing 0.5g pectin, 25ml of 0.25N sodium hydroxide was added, shaken thoroughly, and was allowed to stand for 30 min at room temperature in a stoppered flask. To this 25ml 0.25N HCl was added and titrated with 0.1N NaOH to the end point as mentioned above in equivalent weight Owen et al. [18].

Anhydrogalacturonic Acid (AGA) Content 

Making use of equivalent weight and methoxyl content, anhydrogalacturonic acid content was calculated from the expression given below [19]:

When molecular weight of AGA (1 unit) = 176g

Where,

z = ml (titre) of NaOH from equivalent weight determination.

y = ml (titre) of NaOH from methoxyl content determination.

w = weight of sample.

Degree of esterification 

Degree of esterification of pectin was measured as the ratio of methoxyl content and anhydrogalacturonic acid content [18], and was calculated by following formula:

Acetyl Value 

In a 250ml conical flask, 0.5g pectin was weighed and 25ml 0.1N NaOH was added. Flask was stoppered and the content was stirred till all the pectin got dissolved. It was allowed to stand overnight, after which, it was diluted to 50ml with water. Twenty millilitres of sample were pipette to distillation flask along with 20ml magnesium sulphate-sulphuric acid solution. It was then steam distilled and 100ml distillate was collected in lower flask. Distillate was now titrated against 0.05N NaOH using phenol red as indicator [18]. Acetyl value was calculated as:

Jelly Grade 

For determining the jelly grade, test jellies were made with the test sample and were compared with the jelly made under similar condition with a standard 110-grade pectin sample [20]. 

Cold distilled water (320ml) was added to cooking vessel. Sugar was weighed 500g. If the assumed grade of pectin is 150, 3.3g of pectin was added to sugar (5 times the weight of pectin) and mixed. To the water in kettle, 0.5ml citric acid solution and 1ml sodium citrate was added. Then the pectin-sugar mixture was added to water and stirred to ensure dispersion. The mixture was heated rapidly to boiling with constant stirring to prevent lumping or sticking to the sides of the kettle. The mixture was boiled for 30 seconds and was removed from flame, it was stirred until the pectin was completely dissolved in the solution. And then the remaining sugar was added. Solution was heated again and brought to boiling point with continuous stirring till the weight of 770g was achieved. Heat was turned off and the mixture was allowed to cool for 30sec. 

Hot jelly was poured into jelly glasses with 2ml citric acid solution and 0.5ml sodium citrate and was mixed with glass rod. Time was noted immediately after the hot jelly was poured into the glass. Jelly was allowed at 26ºC for 18h. The jelly was then transferred from glass to flat surface. Overall firmness and degree of resilience of the test jelly with a standard jelly prepared under similar condition was compared.

Statistical Analysis 

The data of recovered pectin extracted from pumpkin using Trichoderma viride cellulase and commercial cellulase enzyme at varying concentration, temperature and time were analysed by using Completely Randomized Design (CRD) as given by Cochran and Cox [21]. However, the results on quality characterization of pectin were demonstrated with mean and standard error (dry wt. basis) of triplicate observations.

Influence of Enzyme Concentration, Temperature and Time on Pumpkin Pectin Extracted with Trichoderma Viride Cellulase 

The results pertaining to yield of extracted pectin using Trichoderma viride cellulase at varying concentrations along with different temperature and time combination are given in table 2. Among different extraction time, 9h of extraction gave maximum yield (13.25%) followed by12h (12.48%) and 6h (10.19%) of extraction time (Figure 3). Out of different concentrations of enzyme and extraction temperature, 4mL/mg at 45°C gave maximum release of pectin irrespective of the extraction time applied (Figure 4). The interaction of enzyme concentration, extraction time and temperature revealed the maximum yield of 14.93 per cent pectin by using 4ml/mg of Trichoderma viride cellulase at 45°C for 9h. An interaction between extraction time and temperature was found to be significant whereas the rest of the interactions were non-significant. The lower yield of pectin at higher enzyme concentration, extraction temperature and longer extraction time might be due to hydrolysis of pectin. 

Table 2: Effect of extraction parameters on yield (%) of pumpkin pectin isolated with Trichoderma viride cellulase. 

Figure 3: Effect of extraction hour on yield (%) of pumpkin pectin isolated with Trichoderma viride cellulase.

Figure 4: Effect of temperature and concentration of enzyme on yield (%) of pumpkin pectin at extraction of 6 (a), 9 (b) and 12 (c) hour with Trichoderma viride cellulase.

Influence of Enzyme Concentration, Temperature and Time on Pumpkin Pectin Extracted with Commercial Cellulase 

Table 3 reveals the effect of using commercial cellulase enzyme at varying concentration with different combination of time and temperature on the yield of pectin. A mean maximum yield of 13.19% was recovered at an incubation time of 9h whereas, minimum (10.75 %) was seen in 6h (Figure 5). An interaction of extraction time, temperature and concentration of enzyme reflected the maximum recovery of pectin at 45°C by using 6mL/mg of cellulose enzyme for 9h (Figure 6). However, an interaction between extraction temperature and concentration of enzyme revealed that the mean maximum (12.39%) yield of pectin was at 45°C while minimum (11.42%) was at 25°C. The overall interaction between extraction time, temperature and concentration of enzyme was non-significant while the interaction between extraction time and temperature was significant. 

Table 3: Effect of extraction parameters on yield (%) of pumpkin pectin isolated with commercial cellulase. 

Figure 5: Effect of extraction time on yield (%) of pumpkin pectin isolated with commercial cellulase.

Figure 6: Effect of temperature and concentration of enzyme on yield (%) of pumpkin pectin at extraction of 6 (a), 9 (b) and 12 (c) hour with commercial cellulase. 

The lower yield of pectin at higher enzyme concentration, temperature and longer time might be due to hydrolysis of pectin. Whereas, the minimum yield at lower enzyme concentration, extraction temperature and small extraction time can be attributed to insufficient enzyme concentration, contact time and heat penetration to cell wall of raw material to hydrolyse insoluble pectic substances into soluble pectin. Lower yield of pectin at higher and low concentration of enzyme was also observed by Yuliarti et al. [22], in gold kiwifruit.

Effect of Trichoderma Viride Cellulase and Commercial Cellulase Enzyme on Quality Characteristics of Extracted Pectin               

The chemical characteristic of pectin isolated from pumpkin using is Trichoderma viride cellulase and commercial cellulase enzyme was presented in Table 4. It was found that the type of enzyme had significant effect on the yield and chemical characteristic of pectin. The yield of pectin using Trichoderma viride cellulase and commercial cellulase enzyme was 14.93 and 13.45 per cent, respectively using an enzyme concentration of 4 mg/mL at 45°C for 9h. The pectin obtained in recent work showed a promising yield when compared with commercialized raw materials. Therefore, the use of cellulase enzymes for extraction of pectin can be an effective way of solving effluent problem caused by traditional method. The quality characteristics of pectin extracted from both the enzymes are presented in Table 4.

Table 4: Effect of Trichoderma viride cellulase and commercial cellulase enzyme on quality characterization of recovered pectin.

The highest moisture content (8.45 %) was observed in pectin recovered by commercial pectin but it was under the acceptable limit as prescribed by FDA (up to 12 %). There was significant effect on the ash content of the extracted pectin. The increased ash content of commercial enzyme extracted pectin might be due leaching of indigenous minerals during the action of enzymes on cell wall of pumpkin which gets precipitate with pectin during alcohol precipitation. Trichoderma viride cellulase extracted pectin had higher equivalent weight (820.00) which might be due to higher rate of depolymerisation and de-esterification reaction of pectin which led to the increased formation of pectic acid. Higher anhydrogalacturonic acid content is one of the indicators of good quality pectin. There was non-significant effect of type of enzyme on anhydrogalacturonic acid. The variation in anhydrogalacturonic acid content may due to hydrolysis reaction of protopectin to D-anhydrogalacturonic acid. The pectin obtained contained the methoxyl content and degree of exterification above 8 and 50 per cent, respectively, therefore, can be classified under high methoxyl pectin. The difference in degree of esterification and methoxyl content might be due to degradation of methyl ester [23]. The difference in the acetyl value of both the pectin may be owed to hydrolysis of acetic acid group from galacturonic acid [24,25]. The pectin with higher jelly grade was obtained by Trichoderma viride cellulase extraction. 

The variation in quality characteristic of pectin may be attributed to different ability of the extracting enzymes to penetrate into the cell structure of the tissue and come in contact with the pectic acid substances present on or in between the cell walls and convert the insoluble pectic substances into soluble pectin.

In this study, the applicability of pumpkin for extraction of pectin using Trichoderma viride cellulase and commercial cellulase enzyme was studied with variation in pH, extraction time and temperature. All parameters presented a significant effect on the yield of pectin signally and in combination. The highest yield (14.93%) was obtained with Trichoderma viride cellulase (4mL/mg, 45°C, 9h). The properties of the extracted pectin were comparable to that of commercial pectin. The extracted pectin had methoxyl content and degree of esterification above 8 and 50 percent therefore, is categorised as HM pectin. Henceforth, it is concluded that pumpkin is a source of good quality pectin having the property of gelling ability, emulsifying and storage stability and hence, can be successfully utilized for production of different value-added products. It can be a substantial raw material for isolation of pectin due to its higher availability and lower cost. As the requirement of this food additive is steadily increasing in the country, this approach can consequently solve the problem of new sources required for extraction of pectin to meet the growing demand in food industry as well as other industries. The use of enzyme for extraction will serve the purpose of getting rid of effluent treatment at industrial level and reducing the impact caused by traditional extraction techniques on environment.

The support by the Department of Science and Technology (DST), New Delhi, India, through their Project “Development of low-cost value-added processed products from ripe pumpkin (Curcurbita moschata) and dissemination of technology to the farm women of Himachal Pradesh” and Department of Food Science and Technology, Dr YS Parmar University of Horticulture and Forestry, Himachal Pradesh are gratefully acknowledged in this work.

All authors declare no conflict of interest.

1. Dhiman AK, Sharma KD, Attri S (2009) Functional constituents and processing of pumpkin: A review. Journal of food science and technology 46: 411-417.
2. Dar HA, Sofi SA, Rafiq S (2017) Pumpkin, the functional and therapeutic ingredient: A review. International journal of food science and nutrition 10: 168-173.
3. Kiramana JK, Khasungu ID (2017) First detailed morphological characterisation of qualitative traits of extensive naturalized pumpkin germplasm in Kenya. International journal of development and sustainability 6: 500-525.
4. Kalloo G, Rai M, Kumar R, Prasana HI, Singh M, et al. (2006) New vegetable varieties from IIVR Varanasi. Indian Horticulture 51: 16-22.
5. Ramachandran P, Dhiman AK, Attri S, Vikram A, Rai S, et al. (2022) Comparative study on physical characteristics and nutritional composition of pumpkin (Cucurbita moschata) at different stages of maturity. Indian Journal of Traditional Knowledge 21: 856-864.
6. Ang-Lee MK, Moss J, Yuan CS (2001) Herbal medicines and perioperative care. JAMA 286: 208-216.
7. Ramachandran P, Dhiman AK, Attri S, Kaur T (2020) Influence of various factors on yield and quality of pumpkin pectin extracted with water. Chemical Science Review and Letters 9: 388-397.
8. Serocynska A, Antezak A, Korytowska M, Kaminska K, Radomski A, et al. (2014) Evaluation of the selected forms of winter squash (Cucurbita maxima ) for the content of free sugar and polysaccharides. Polish Journal of Agriculture 16: 69-73.
9. Li QH, Fu CL, Rui YK, Hu GH, Cai TY (2005) Effects of protein-bound polysaccharide isolated from pumpkin on insulin in diabetic rats. Plant Foods for Human Nutrition 60: 13-16.
10. Guillon F, Champ M (2000) Structural and physical properties of dietary fibres, and consequences of processing on human physiology. Food Research International 33: 233-245.
11. Danilchenko H, Paulauskiene A, Dris R, Niskanen R (2003) Biochemical composition and processability of pumpkin cultivars. Acta Horticulture 510: 493-497.
12. Goycoolea FM, Cardenas A (2003) Pectin from Opuntia spp.: A short review. Journal of PACD 1: 17-29.
13. Srivastava P, Malviya R (2011) Sources of pectin, extraction and its applications in pharmaceutical industry: An overview. Indian Journal of Natural Products and Resources 2: 10-18.
14. Adetunji LR, Adekunle A, Orsat V, Raghavan V (2017) Advances in the pectin production process using novel extraction techniques: A review. Food Hydrocolloids 62: 239-250.
15. Panouillé M, Thibault JF, Bonnin E (2006) Cellulase and protease preparations can extract pectins from various plant byproducts. Journal of Agriculture and Food Chemistry 54: 8926-8935.
16. Sandarani MDJC (2017) A review: Different extraction techniques of pectin. Journal of Pharmacognosy and natural products 3: 143-148.
17. Ptichkina NM, Markina OA, Rumyantseva GN (2008) Pectin extraction from pumpkin with the aid of microbial enzymes. Food Hydrocolloid 22: 192-195.
18. Owens HS, McCready RM, Shepard AD, Schultz TH, Pippen EL, et al. (1952) Methods used at Western Regional Research Laboratory for extraction of pectic materials. USDA Bur Agric Ind Chem Albany, California, USA.
19. Mohamed S, Hasan Z (1995) Extraction and characterization of pectin from various tropical agrowaste. ASEAN Food Journal 2: 43-50.
20. Ranganna S (2009) Handbook of analysis and quality control for fruit and vegetable products. Tata McGraw Hill, New Delhi, India.
21. Cochran WG, Cox CM (1965) Experimental Design. John Wiley and Sons, New York, USA.
22. Yuliarti O, Lara M, Goh KT, Mawson J, Brennan C (2012) Characterisation of gold kiwifruit pectin isolated by enzymatic treatment. International Journal of Food Science and Technology 47: 633-639.
23. Ramli N, Asmawati (2011) Effect of ammonium oxalate and acetic acid at several extraction time and pH on some physicochemical properties of pectin from cocoa husk (Theobroma cacao). African journal of Food Science 5: 790-798.
24. Garna H, Mabon N, Robert C, Cornet C, Nott K, et al. (2007) Effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol. Journal of Food Science 72: 1-9.
25. Sundar A, Rubila S, Jayabalan R, Ranganathan TV (2012) A Review on Pectin: Chemistry due to General Properties of Pectin and its Pharmaceutical Uses. Sci Rep1: 1-4.