Enzymatic Degumming of Soybean Oil for Raw Material Preparation in BioFuel Production

Abstract

The paper investigates the process of degumming substandard soybean oil using an enzyme complex of phospholipases to prepare it as a feedstock for biodiesel production. Dehumidification is an important refining step aimed at reducing the phosphorus content, which exceeds the permissible limits according to ASTM, EN, and ISO standards, by re-moving phospholipids. The enzyme complex of phospholipases includes phospholipase C, which specifically targets phosphatidylinositol, and phospholipase A2, which catalyzes the hydrolysis of phospholipids into water-soluble phosphates and lysophospholipids. This process contributes to the efficient removal of phospholipids, increased neutral oil yield, and reduced residual oil in the humic phase. The use of an enzyme complex of phospholipases provides an innovative, cost-effective, and environmentally friendly method of oil purification. The results of the study demonstrate the high efficiency of using the phospholipase enzyme complex in the processing of substandard soybean oil, which allows reducing the content of total phosphorus to 0.001% by weight, turning it into a high-quality raw material for biodiesel production. The proposed approach contributes to increasing the profitability of agricultural raw materials and the introduction of environmentally friendly technologies in the field of renewable energy.

1. Introduction

Degumming of vegetable oil is a key stage in its refining, aimed at reducing the con-tent of phospholipids and free fatty acids (FFA) to ensure the quality of the final product. Phospholipids, which are the main carriers of phosphorus in crude soybean oil, negative-ly affect the stability and storage of the oil and the quality of the finished product. High phosphorus levels limit the further use of the oil as a raw material for the production of biofuels. Phospholipids are divided into hydrated (HPL) and non-hydrated (NHP). HPL is removed by water, while NHP is removed by acid degumming [1,2,3,4,5,6].

Traditional oil refining methods, such as chemical treatment or physical processes, are often characterized by high oil losses and negative environmental impacts due to the formation of large amounts of wastewater and emulsions, which complicates their dis-posal [7,8,9]. As a biotechnological alternative to traditional methods of degumming, enzymatic degumming has been actively implemented in recent decades, which provides deeper cleaning with lower losses and environmental burden. In particular, dos Passos et al. [5] demonstrated that enzymatic degumming provides a reduction in wastewater volumes and oil losses, as well as a reduction in the use of reagents (phosphoric acid, soda), which ultimately reduces operating costs. Kahmies et al. [10] also indicated environmental benefits, in particular a reduction in the phosphate load on treatment systems and a simplification of filtration stages.

A commonly used chemical treatment involves conditioning the oil with phosphoric acid, followed by neutralization with caustic soda and multi-stage separation. The pro-cess provides effective removal of phospholipids but is accompanied by high oil losses (up to 3%) due to emulsification of soap and oil. In addition, large volumes of wastewater with a high phosphate content are generated, which require complex treatment [10,11,12,13]. A hybrid modern degumming technology has also been developed, which includes acid treatment of the oil with the addition of 85% phosphoric acid, which provides a pH reduc-tion of 3.8–4.2 for Quara LowP and 4.5–5.5 for Lecitase Ultra, and a subsequent enzymatic reaction with the addition of water (5%) and the enzymes Quara LowP or Lecitase Ultra [10]. The use of enzymes such as phospholipase-A1, commercially known as Quara LowP and Lecitase Ultra, allows the conversion of non-hydrated phospholipids into hydrated forms. This process is selective, which ensures a reduction in oil loss [14,15,16]. This method is effective in achieving phosphorus levels of <10 ppm but requires the use of acid to sta-bilize the pH level, high energy, and reagent consumption. In addition, the process lasts 4 h, which lengthens the overall production cycle.

Of particular interest is the immobilization of phospholipases on solid supports (chitosan, calcium alginate, polymer gels), which provides multiple uses of the enzyme, improves its stability to temperature and pH, and also simplifies the purification of the final product from protein residues [17,18,19]. This opens up new prospects for scaling up enzymatic degumming in industrial conditions with high requirements for environmental friendliness and economic efficiency.

This work is aimed at assessing the effectiveness of the application of the enzyme complex Purifine^® 3G (manufactured by DSM Food Specialties (Royal DSM), Netherlands, Heerlen; distributor in Ukraine Tempo LLC, Horodok, Lviv region, Ukraine) to crude soybean oil obtained from soybean seeds grown in Ukraine. The content and qualitative composition of phospholipids significantly depend on agroclimatic conditions, soil-climatic factors, soybean variety, and technological parameters of its primary processing, which, in turn, directly affect the efficiency of the degumming process [10]. Therefore, even when using the same enzyme complex, the optimal degumming conditions can vary significantly and require local adaptation—this is exactly what became the subject of our study.

In addition, this work focuses on the study of enzymatic degumming without prior treatment with phosphoric acid, which is typical for hybrid enzyme technologies. This approach minimizes the formation of phosphate-containing effluents and simplifies the integration of the degumming process into first-generation biofuel (FAME) production lines.

Thus, the applied goal of our research is to verify experimentally the possibility of environmentally safe and technologically efficient enzymatic preparation of Ukrainian plant raw materials for further use in the production of biofuels.

The scientific novelty of the work lies in the comprehensive study of the adaptation of the process of enzymatic degumming of crude soybean oil of Ukrainian origin using Purifine^® 3G without prior acid activation. The establishment of optimal process parameters that ensure a reduction in the residual phosphorus content to a level of <10 ppm—which is a critical indicator for the use of oil in the production of FAME without additional purification.

2. Materials and Methods

2.1. Materials

Soybeans were grown in the Lviv region, Ukraine, on the acreage of the «Agro Center» farm. Soybean vegetable oil was obtained by extraction using hexane as a solvent at the production facilities of D-MIX LLC (Zolochiv, Lviv region, Ukraine). The physical and chemical properties of soybean oil are given in

Table 1. Physical and chemical properties of crude soybean oil.

Properties	Units of Measurement	Value	Methods
Density at 15 °C	kg/m3	922	[20]
Kinematic viscosity at 40 °C	mm2/s	30.7	[21]
Flash point	°C	345	[22]
Sulphur content	ppm	15	[23]
Phosphorus content	ppm	421	[24]
Water content	ppm	200	[25]
Copper strip test (3 h at 50 °C)	-	withstands class 1	[26]
Acid value	mg KOH/g	0.49	[27]
Iodine value	g I2/100 g	39.0	[28]

.

The phospholipase enzyme complex under the brand name Purifine^® 3G, manufactured by DSM Food Specialties (Royal DSM), belongs to the third-generation enzymes. It is a combination of three separate phospholipases obtained by three independent degumming processes. Phospholipase C (PL-C), which catalyzes the hydrolysis of phospholipids to diglycerides and water-soluble phosphate residues, is cultivated using a strain of Pichia pastoris. Phospholipase C specific for phosphatidylinositol (PI), which hydrolyzes phosphatidylinositol to water-soluble residues, is produced by a strain of Pseudomonas fluorescence. Phospholipase A2 (PL-A2), which catalyzes the hydrolysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), forming lysophospholipids, is produced by the Aspergillus niger strain. Enzymatic processing of soybean oil using the Purifine^® 3G enzyme complex is carried out under strictly regulated conditions that ensure optimal process efficiency. The temperature regime is set within 50–60 °C, which is optimal for achieving maximum enzyme activity. The pH value of the reaction medium is maintained in the range of 6.5–7.5, which ensures the highest efficiency of enzymatic hydrolysis of phospholipids. The reaction duration depends on the type and quality of the oil and varies within 1–3 h. The enzyme dosage is determined by the concentration of phospholipids in the raw material and is in the range of 50–200 ppm. Compliance with these parameters is critical to ensure effective degumming, reduce the content of phospholipids, and obtain high-quality raw materials for further use in biofuel synthesis.

2.2. Methods

2.2.1. Enzymatic Reaction

The optimal laboratory method of enzymatic degumming was developed based on the general recommendations for temperature, pH of the medium and enzyme concentration, set out in the technical sheet for the enzyme complex Purifine^® 3G, produced by DSM Food Specialties (Royal DSM). The final laboratory protocol was adapted and refined in the process of conducting our own research, taking into account the specifics of Ukrainian raw materials and target process parameters for enzymatic treatment of 1 kg of crude soybean oil using Purifine^® 3G. The obtained experimental results, which confirm the effectiveness of the proposed approach, are described in detail in Section 3. For the enzymatic treatment of 1 kg of crude soybean oil, 0.1 g of Purifine^® 3G enzyme was used, which corresponded to a concentration of 100 ppm per ton of oil. The enzyme complex was pre-dissolved in 20 mL of water, which was 2.5% of the oil mass, pH in the range of 6.5–7.5. The prepared enzyme solution was added to 1 kg of crude soybean oil preheated to 57 °C under continuous stirring conditions. The components were mixed using a laboratory mixer at a speed of 500 rpm. A biocatalytic reaction was carried out at a temperature of 57 °C, which is optimal for enzyme activity, at a neutral pH of the medium. The duration of the enzymatic process was 2 h. During this time, constant stirring ensured uniform distribution of the enzyme and effective contact with the phospholipids. After the enzymatic treatment was completed, the oil was heated to 80 °C and kept at this temperature for 30 min to inactivate the enzyme. Then, stirring was stopped, and the treated oil was transferred to a separatory funnel for settling. During the settling process, the humic precipitate containing hydrophilic hydrolysis products settled in the lower part of the funnel, while the purified oil accumulated in the upper layer. After the separation was completed, the purified oil was carefully collected for further use in biofuel synthesis.

2.2.2. Physical and Chemical Properties of Raw Materials/Products

The determination of physical and chemical properties of raw materials/products was carried out according to the methods presented in Table 1 and using the methods described in [20,21,22,23,24,25,26,27,28,29,30,31].

2.2.3. GC-MS Analysis

Gas Chromatography Mass Spectrometry (GC-MS) to confirm the target products was conducted using the Agilent 8890 gas chromatograph (Agilent Technologies, Inc. Headquarters, Santa Clara, CA 95051, USA) equipped with a DB-5ms capillary column (30 m × 0.32 mm) and Agilent 5977C mass detector, at mass scan range 45.00–450.00 m/z, using methanol as a solvent.

2.2.4. FTIR Spectra Record

The Fourier-transform infrared (FTIR) spectra of samples were recorded on a Spectrum Two spectrometer PerkinElmer, using a diamond UATR single reflection accessory. PerkinElmer Spectrum 10 software was used to draw the spectra. The spectra (16 scans per spectrum) of the samples were collected in the mid-infrared wavenumbers range from 4000 to 400 cm⁻¹, with a spectral resolution of 0.5 cm⁻¹.

3. Results

To achieve maximum efficiency of the process, experiments were conducted to determine the optimal parameters: enzyme dose, temperature, pH value, reaction time, and amount of added water. Enzymatic treatment was carried out on samples of crude soybean oil obtained after pre-pressing the seeds. The initial parameters of crude soybean oil were determined and were (phosphorus level 421 ppm and free fatty acids (acid number 0.49 mg KOH/g of oil) Table 1.

To achieve maximum results, the following general enzymatic degumming conditions were observed:

• Temperature: 50–70 °C, optimal for enzyme activity.
• pH value: 5.5–8.0, which ensures stable enzyme operation.
• Reaction duration: 1–5 h, depending on the quality of the starting material.
• Enzyme dose: 50–250 ppm, determined according to the concentration of phospholipids in the raw material.

The degumming process was accompanied by regular monitoring of the level of residual phosphorus and the acid number, which are important indicators when planning the technology for obtaining biofuels.

3.1. Effect of Enzyme Dose on Degumming Efficiency

A study was conducted to determine the effect of the dosage of the enzyme complex Purifine^® 3G on the efficiency of degumming of 1 kg of crude soybean oil. The dosage of the enzyme is an important factor that determines the efficiency of degumming and eco-nomic feasibility. Within the framework of the experiments, concentrations of 50, 100, 150, 200, and 250 ppm of the enzyme were tested. The level of phosphorus and free fatty acids in the final product was determined; the results are given in

Table 2. Effect of enzyme dose on degumming efficiency.

Dosage of Purifine® 3G Enzyme Complex (ppm)	Phosphorous Content (ppm)	FFA (Acid Value, mg KOH/g)
50	12.0	0.67
100	9.5	0.85
150	8.7	1.17
200	8.3	1.59
250	7.8	2.15

. Degumming was carried out for 3 h at fixed values of pH = 7.0; T = 60 °C; 2% H₂O by weight of the oil.

The results showed that at a dose of 100 ppm, the phosphorus level decreased from 421 to 9.5 ppm, and the FFA content acid value was 0.85 mg KOH/g oil. Increasing the dose to 150, 200, or 250 ppm had a slight effect on the phosphorus level (7.8–8.7 ppm), but was accompanied by an increase in the cost of the enzyme preparation and an increase in the level of free fatty acids. Reducing the enzyme dose to 50 ppm does not provide a phosphorus level of <10 ppm. Therefore, 100 ppm is the optimal dose that allows obtaining a phosphorus content of <10 ppm, which meets industrial standards and provides a balance between efficiency and costs.

3.2. The Effect of Temperature on the Enzymatic Degumming Process

The effect of enzymatic treatment temperature using the Purifine^® 3G enzyme complex on the efficiency of degumming 1 kg of crude soybean oil was studied. Temperature is a critical factor for ensuring enzyme activity. The temperature range from 50 °C to 70 °C was studied. Degumming was carried out for 3 h at fixed pH values of 7.0; Purifine^® 3G enzyme amount of 100 ppm; 2% H₂O by weight of oil. The level of phosphorus and free fatty acids in the final product was determined; the results are given in

Table 3. The effect of temperature on the enzymatic degumming process with Purifine^® 3G enzyme complex.

Enzymatic Degumming Temperature (°C)	Phosphorous Content (ppm)	FFA (Acid Value, mg KOH/g)
50–52	35.0	0.61
53–55	15.0	0.79
56–58	10.0	0.86
59–61	10.2	0.82
62–64	18.1	0.80
65–67	52.3	0.80
68–70	128.4	0.79

.

Purifine^® 3G enzyme efficiency was maintained in the temperature range of 53–65 °C. The best results were observed at a temperature range of 56–58 °C: phosphorus level—10.0 ppm, FFA, acid value—0.86 mg KOH/g oil. Lowering the temperature below 53 °C led to an increase in phosphorus levels in soybean oil. Raising the temperature above 65 °C led to an increase in phosphorus levels and increased FFA.

3.3. Determining the Optimal pH Value

The influence of the pH level of the reaction medium on the enzymatic degumming process was investigated using the enzyme complex Purifine^® 3G, when degumming 1 kg of crude soybean oil. The enzymatic activity of Purifine^® 3G is most pronounced at pH 6.5–7.5. Degumming was carried out for 3 h at fixed values of T = 57 °C; the amount of enzyme Purifine^® 3G 100 ppm; 2% H₂O by weight of oil. The level of phosphorus and free fatty acids in the final product was determined; the results are given in

Table 4. Determining the optimal pH value of enzymatic degumming with Purifine^® 3G enzyme complex.

pH Value	Phosphorous Content (ppm)	FFA (Acid Value, mg KOH/g)
6.3–6.5	26.2	0.59
6.6–6.8	12.1	0.81
6.9–7.1	9.5	0.88
7.2–7.4	19.2	0.85
7.5–7.7	37.5	0.71

.

Experiments showed that at pH = 6.9–7.1 the phosphorus level was 9.5 ppm, which is optimal for the degumming process. Deviation from this range reduced the enzyme activity due to changes in its structure.

3.4. Effect of Water Dosage on Degumming Efficiency

A study on the effect of water quantity on the efficiency of degumming of 1 kg of crude soybean oil was conducted. Water dosage is an important factor that determines the efficiency of degumming and economic feasibility. Within the framework of the experiments, concentrations of 0.50–5.00% water to the amount of oil being processed were tested. The level of phosphorus and free fatty acids in the final product was determined, and the results are given in

Table 5. Effect of water dosage on degumming efficiency.

Dosage of Water (%)	Phosphorous Content (ppm)	FFA (Acid Value, mg KOH/g)
0.50	254.2	0.59
1.00	171.9	0.67
1.50	56.2	1.07
2.00	10.0	0.93
2.50	9.1	0.87
3.00	9.0	0.85
3.50	8.8	0.81
4.00	8.8	0.78
4.50	8.7	0.74
5.00	8.6	0.73

. Degumming was carried out for 3 h at fixed values of pH = 6.9–7.1; T = 57 °C; amount of enzyme Purifine^® 3G 100 ppm.

The amount of water determines the formation of the humic phase and its ability to remove hydrolysis products. The best values were obtained when using 5% water to the mass of oil. However, the optimal ratio was 2.5% water to the mass of oil, which ensured a sufficient level of phosphorus 9.1 ppm and minimized VFA (acid value 0.87 mg KOH/g oil) and reduced the amount of wastewater.

3.5. Study of Enzymatic Degumming Duration on Degumming Efficiency

A study to determine the effect of enzymatic degumming duration on the efficiency of degumming of 1 kg of crude soybean oil was conducted. The reaction time determines how complete the hydrolysis of phospholipids is. The experiments investigated the reaction from 30 min to 5 h. The level of phosphorus and free fatty acids in the final product was determined, the results are given in

Table 6. Influence of the enzymatic degumming time on degumming efficiency.

Enzymatic Degumming Time (min)	Phosphorous Content (ppm)	FFA (Acid Value, mg KOH/g)
30	178.2	0.58
60	59.2	0.65
90	25.3	0.73
120	9.2	0.84
150	9.0	0.95
180	8.8	1.18
210	8.5	1.47
240	8.4	1.95
270	8.1	2.49
300	8.0	3.18

. Degumming was carried out at fixed values of pH = 6.9–7.1; T = 57 °C; amount of enzyme Purifine^® 3G 100 ppm; 2.5% water by weight of oil.

The optimal enzymatic degumming time was 2 h, during which the phosphorus level decreased to 9.2 ppm, and the fatty acids remained stable (acid value 0.84 mg KOH/g oil). Increasing the duration to 3 h or more slightly improved the efficiency of dephosphorylation but was accompanied by an increase in fatty acids and increased energy costs.

3.6. Analysis of the Transesterification Product by GC-MS

To confirm the composition of the transesterification products of soybean oil, gas chromatography-mass spectrometry (GC-MS) analysis was performed. The study was performed on an Agilent 8890 gas chromatograph equipped with a DB-5ms capillary column (30 m × 0.32 mm) coupled to an Agilent 5977C mass detector. The mass scan range was 45.00–450.00 m/z, and methanol was used as the solvent. The main objective was to determine the content of fatty acid methyl esters (FAME) in the 240–320 °C fraction of the biofuel. The results of the study showed that the biofuel contains the following main fractions, the main ones of which were observed at retention times of about 8.193; 8.675; 8.814; 8.882; 8.973, and 9.196 min.

Analysis of GC-MS results confirmed the presence of fatty acid methyl esters as the main components in the studied biofuel fraction (

Table 7. GC-MS analysis of the fatty acid methyl ester composition of the transesterification product of degummed soybean oil.

FAME	R.T. (min)	Contents (% of Total)	Characteristic Fragments with Masses m/z
Palmitic acid, methyl ester (C17H34O2)	8.193	9.808	74; 87; 43; 55; 41; 143; 75; 57; 69; 227
Linolenic acid, methyl ester (C19H32O2)	8.675	4.448	79; 67; 95; 81; 41; 55; 108; 93; 80; 94
Linoleic acid, methyl ester (C19H34O2)	8.725	2.808	67; 81; 41; 55; 95; 43; 82; 79; 96; 68
Oleic acid, methyl ester (C19H36O2)	8.814	35.651	55; 69; 74; 83; 97; 96; 84; 41; 98; 87
Stearic acid, methyl ester (C19H38O2)	8.882	10.075	74; 87; 298; 143; 255; 75; 43; 55; 199; 57
Isolinoleic acid, methyl ester (C19H34O2)	8.973	10.686	67; 81; 95; 82; 55; 41; 96; 68; 79; 294
Isolinoleic acid, methyl ester (C19H34O2)	9.136	8.207	67; 81; 95; 82; 55; 41; 96; 68; 79; 294

).

3.7. FTIR Spectral Analysis of Crude Soybean Oil, Enzymatically Degummed Soybean Oil, and the Resulting Biofuel

The Fourier-transform infrared (FTIR) spectra of samples were recorded on a Spectrum Two spectrometer PerkinElmer. The FTIR spectra of crude soybean oil and degummed soybean oil were nearly similar, and there were no significant differences, as shown in

Table 8. Results of crude soybean oil, degummed soybean oil and biofuel research using FTIR.

Observation	Frequency, cm−1
	Crude Soybean Oil	Degummed Soybean Oil	BioFuel
–OH	3475.00	3475.46	3464.00
–C=C–H	3008.93	3008.93	3009.00
–CH2	2922.00	2922.81	2923.49
–CH2	2853.00	2853.27	2853.70
C=O	1741.30	1741.35	1741.38
–CH2	1463.96	1463.97	1459.29
C(O)–O–CH3 asσ	-	-	1435.62
C(O)–O–CH3 sσ	-	-	1361.78
C(O)–O–	-	-	1241.02
P=O	1237.08	1237.15	-
C–O	-	-	1195.43
C–O	1159.97	1160.00	1169.58
P=O	1144.24	1144.24	-
P=O	1118.77	1119.22	-
P–O	1099.38	1099.92	-
P–O	1032.31	1032.89	-
-CH2	719.93	720.93	722.59

. The main differences were observed in the spectrum of biofuel, which is explained by the transesterification reaction. The identification of functional groups and bands corresponding to different stretching and bending vibrations clearly indicates the corresponding characteristic bands of the expected functional groups.

3.8. Pilot Industrial Implementation

The pilot industrial implementation was carried out in two stages:

(1)

enzymatic degumming of crude soybean oil;

(2)

production of BioFuel from degumming soybean oil (using a cavitation unit BIOTRON-R 1000 (CT Systems Corporation, 2910-567 Setúbal, Portugal)).

Enzymatic degumming improves soybean oil quality for further processing. Using Purifine^® 3G, 20,000 kg of oil was degummed at the “Agro Center” farm in Ukraine. The process involved adding 2 kg of enzyme (100 ppm per ton) to 500 L of water (2.5% of oil mass) with pH control (6.9–7.1). The reaction occurred in a heated, stirred reactor at 57 °C for 2 h. After enzymatic treatment, the oil was heated to 80 °C for 30 min to inactivate the enzyme, followed by settling to separate impurities. The purified oil was used for biofuel production. Quality control assessed phospholipid content and acid value, ensuring compliance with processing standards.

The hydraulic schematic diagram is shown in Figure 1. The operating principle is based on alternate filling of tanks E1 and E2 with source components, precise dosing and preliminary mixing, with subsequent alternate draining of the mixtures into the cavitation reactor R. Feed pumps D1, D2, and D3 are intended to supply source products into the system. Regulating valves V1, V2 and V3 are intended to control tank-filling speed with corresponding products. Pumps E1 and E2 are intended for product mixing and subsequent supply to the cavitation reactors. Regulating valve V4 is intended to control the necessary flow level (efficiency) of the preliminary processing system. Dosing of products 1, 2 and 3 is conducted via weighing with strain sensors W1 and W2.

The conditions of the soybean oil enzymatic degumming process and the production of BioFuel are given in

Table 9. Process conditions.

Process Parameter	Units of Measurement	Value
1. The stage of enzymatic degumming
Enzyme	-	Purifine® 3G enzyme complex
Enzyme content	Ppm	100
Temperature	°C	56–58
pH value	-	6.9–7.1
Dosage of water	wt. %	2.5
Time	Min	120
2. The stage of biodiesel production by cavitation
Catalyst	-	32% solution of potassium methoxide (CH3OK) in methanol
Content catalyst	wt. %	3.9
Degumming Soybean Oil	wt. %	86.6
CH3OH (Methanol)	wt. %	9.5
Cavitation temperature	°C	80

. Characteristics of crude soybean oil, degummed soybean oil, and BioFuel are given in

Table 10. Physical and chemical properties of raw materials/products.

Properties	Units of Measurement	Value			Methods	Requirements for Biofuels According to Ukrainian Regulations [30]
		Crude Soybean Oil	Degummed Soybean Oil	BioFuel
Content of fatty acids methyl esters	wt. %	-	-	97.2	[29]	≥96.5
Density at 15 °C	kg/m3	922	917	865	[20]	860–900
Kinematic viscosity at 40 °C	mm2/s	30.7	24.1	4.1	[21]	3.5–5.0
Flash point	°C	345	326	156	[22]	≥120
Sulphur content	ppm	15	12	7	[23]	≤10
Phosphorus content	ppm	421	9.2	3.9	[24]	≤10
Water content	ppm	200	500	430	[25]	≤500
Copper strip test (3 h at 50 °C)	-	withstands class 1			[26]	withstands class 1
Acid value	mg KOH/g	0.49	0.83	0.46	[27]	≤0.5
Iodine value	g I2/100 g	39.0	53.4	80.7	[28]	≤120
Linoleic acid methyl ester content	wt. %	-	-	2.808	[29]	≤12
Methanol content	wt. %	-	-	0.05	[30]	≤0.2
Monoglycerides content	wt. %	-	-	0.4	[31]	≤0.8
Diglycerides content	wt. %	-	-	0.08	[31]	≤0.2
Triglycerides content	wt. %	-	-	0.18	[31]	≤0.2
Free glycerol content	wt. %	-	-	0.008	[31]	≤0.02

.

4. Discussion

In this work, we focused on evaluating the effectiveness of the Purifine^® 3G enzyme complex in the degumming of crude soybean oil, as well as determining the optimal process conditions, taking into account the enzyme dose, temperature, pH, reaction time, and the amount of water. The use of the Purifine^® 3G enzyme complex, which includes phospholipases C and A2, provides a modern approach to the purification of soybean oil. This method allows reducing the phosphorus content to acceptable limits while maintaining a high yield of neutral oil. Purifine^® 3G hydrolyzes all four phospholipids in crude oil, namely PC, PE, PI, and phosphatidic acid (Figure 2).

Enzymatic degumming of soybean oil using the Purifine^® 3G enzyme complex represents a modern approach that effectively reduces phospholipid content through the synergistic action of two key enzymes: PL-C and PL-A2. These enzymes perform specific functions aimed at the hydrolysis of phospholipids, which allows reducing the phosphorus content to the level required for biofuel production.

PL-C is the first component in the Purifine^® 3G pathway. It catalyzes the cleavage of the phosphodiester bond between the glycerol and phosphate groups in phospholipid molecules (Figure 2). This process leads to the conversion of phospholipids into diglycerides (DAG) and water-soluble phosphate residues (Figure 3a). The main substrates for PL-C are PI and PC, which constitute a significant proportion of phospholipids in crude soybean oil. During the reaction, the water-soluble phosphate residues migrate into the humic phase along with water, while the lipophilic diglycerides remain in the oil phase, increasing its yield (Figure 3a). This step results in a significant reduction in phosphorus, which is critical for further use of the oil.

The second step is the action of PL-A2, which cleaves the ester bond of glycerophospholipid molecules (Figure 2). This process results in the formation of lysophospholipids and FFA (Figure 3b). The main substrates for PL-A2 are PC and PE, which are also important components of the phospholipid composition of crude oil. The formed lysophospholipids have high surface activity and remain in the humic phase, facilitating the removal of residual phospholipids (Figure 3b). Free fatty acids are also removed during subsequent processing steps.

The combined action of PL-C and PL-A2 allows for the effective hydrolyzation of phospholipids to water-soluble and lipophilic components. This provides a significant reduction in phosphorus content to <10 ppm, which meets international standards for biofuel feedstocks. It contributes to an increase in the yield of neutral oil due to the formation of diglycerides that remain in the product. It ensures minimization of oil losses due to the reduction of the formation of emulsions and humic phase and the environmental friendliness of the process due to the reduction of wastewater volumes and the reduction of the use of chemical reagents.

The results of the study confirmed the high efficiency of using the Purifine^® 3G enzyme complex for degumming soybean oil, which allows for significantly reducing the phosphorus content and ensuring the content of FFA to levels that meet industrial standards in the preparation of raw materials for biofuel production. Optimization of process parameters ensures cost-effectiveness and environmental friendliness of the technology, making it competitive in comparison with traditional degumming methods.

The results of the experiments showed that the optimal dose of the enzyme is 100 ppm. At the same time, the phosphorus level decreased to 9.5 ppm, and FFA was 0.85 mg KOH/g oil. Increasing the dose to 150–250 ppm was accompanied by only a slight decrease in the phosphorus level (to 7.8–8.7 ppm), but the economic feasibility of such an increase is questionable due to the increased costs of the enzyme and the increase in the level of FFA. On the other hand, reducing the dose to 50 ppm did not ensure a decrease in phosphorus to an acceptable level, which is explained by the insufficient number of active enzymes for complete hydrolysis of phospholipids. Thus, 100 ppm provides a balance between efficiency and economic feasibility.

The temperature range of 53–65 °C was found to be the most favourable for the enzyme complex to work. The best results were achieved at a temperature of 56–58 °C, when the obtained phosphorus level was 10.0 ppm, and FFA remained at the level of acid value 0.86 mg KOH/g oil. Lowering the temperature to 50 °C reduced the enzyme activity, and increasing it above 65 °C caused thermal destruction of the enzyme and led to an increase in FFA (Figure 3).

The pH level is an important parameter that determines the conformation of the enzyme and, consequently, its activity. The best results were obtained at pH 6.9–7.1, when the resulting phosphorus level was 9.5 ppm, and FFA remained at the level of acid value 0.88 mg KOH/g oil. Deviation from this pH range led to a decrease in the efficiency of degumming, which confirms the importance of maintaining stable process conditions (Figure 4).

The amount of added water affects the formation of the humic phase and the efficiency of removal of hydrolysis products. At a water concentration of 2.5% of the oil mass, the obtained phosphorus level was 9.1 ppm, and FFA remained stable (acid value 0.87 mg KOH/g oil). Although increasing the water content to 5% further reduced the phosphorus level, it led to an increase in wastewater volumes, which requires additional costs for treatment systems, making this option less environmentally friendly.

The optimal duration of enzymatic degumming was 120 min. During this period, the enzymatic complex ensured complete hydrolysis of phospholipids, reducing phosphorus to 9.2 ppm and changing the FFA content to an acid value of 0.84 mg KOH/g oil. Extending the time to 180 min and more slightly improved the results but was accompanied by an increase in FFA due to secondary reactions of triglyceride cleavage. Considering the increase in production costs with an increase in enzymatic degumming time, the optimal reaction period is exactly two hours.

The Purifine^® 3G enzyme action diagram (Figure 3) demonstrates how the synergy of two enzymes creates optimal conditions for highly efficient degumming. This process not only improves the quality of the final product but also ensures economic feasibility and environmental responsibility in industrial production.

A comparison of the degumming efficiency using Purifine^® 3G and methods described in the literature, in particular using the Quara LowP and Lecitase Ultra enzymes, demonstrates a number of fundamental differences in mechanisms of action and technological requirements.

Thus, the Quara LowP and Lecitase Ultra enzymes are phospholipases A₁, the action of which is based on the conversion of non-hydrated phospholipids (in particular, phosphatidylcholine) into hydrated forms, which can subsequently be removed. However, an important condition for their effective action is the preliminary acidification of the oil with phosphoric acid (85%) to a pH level of 3.8–5.5, which requires additional technological stages—acidity regulation, neutralization, introduction of buffers, and significant consumption of reagents. The total duration of the process can reach 4 h and is also accompanied by increased energy consumption and the formation of a large volume of wastewater with a high phosphate content, which complicates cleaning [10].

In contrast, the proposed work uses the enzyme complex Purifine^® 3G, which combines the activity of phospholipase C (PL-C) and phospholipase A2 (PL-A2). This complex allows degumming without prior acidification, since its optimal activity is achieved at neutral or near-neutral pH (6.9–7.1). This approach eliminates the need for additional acidity control steps, the use of acids and alkalis, and also reduces the risk of emulsions and by-product formation.

In addition, an important technological advantage of Purifine^® 3G is the significantly lower water requirement for the reaction. As shown in Section 3, effective phosphorus reduction to <10 ppm is achieved with as little as 2.5% added water by weight of oil, while Quara LowP and Lecitase Ultra enzymes require at least 5% water, which increases waste and the workload of the separation stage.

The degumming reaction with Purifine^® 3G lasts only 2 h and allows for the reduction of the phosphorus content to a level of <10 ppm, which fully meets the industry standards for the further use of the oil as a raw material in the production of biofuels. In addition, the action of phospholipase C leads to the formation of diglycerides, which remain in the oil phase, increasing the yield of neutral oil, in contrast to the formation of soaps and product losses characteristic of traditional methods.

Enzymatic processes can reduce total energy consumption by 20–30% compared to classical methods [5]. Moreover, a number of technological parameters established in this study indicate the increased energy efficiency of the proposed approach. Enzymatic degumming using the Purifine^® 3G complex is carried out at a temperature of 50–55 °C, which is lower than that used in traditional acid or hydration methods, where temperatures reach 60–85 °C. In addition, this technology does not require multi-stage heating, neutralization, or washing, and consumes a minimal amount of water (2–5%), which significantly reduces thermal energy costs and the load on wastewater treatment systems.

Thus, compared to other enzymatic approaches (Quara LowP, Lecitase Ultra), the use of Purifine^® 3G has obvious advantages—simplification of the process, reduction of reagent and water consumption, reduction of processing time, increased oil yield, and environmental friendliness.

It is important to emphasize that one of the key technological advantages of the Purifine^® 3G enzyme complex is the lack of necessity for preliminary treatment of the oil with phosphoric acid or pH adjustment using acids or alkalis to achieve optimal reaction conditions. This neutral pH range (6.9–7.1) not only simplifies the process by reducing the need for reagents, but also creates favorable conditions for the potential utilization of the resulting sludge (rubber phase). Unlike acid-enzymatic methods, the sludge formed after treatment with Purifine^® 3G does not contain residual acids or inorganic salts, which significantly expands its potential for further utilization.

Although a comprehensive quantitative analysis of the rubber phase composition was not conducted within this study, its chemical nature allows for several substantiated assumptions. Given the specificity of enzymatic hydrolysis of soybean oil, the primary substrates for enzymatic action are phosphatidylinositol and phosphatidylcholine [1,32,33]. As a result of their transformation during the degumming process, a complex multicomponent water–lipid emulsion is formed, containing significant concentrations of hydrolysis products such as inositols, glycerophosphates, lysophospholipids, diglycerides, and amphiphilic residues [1,32,33]. Additionally, the phase includes denatured proteins, non-emulsified free fatty acids, and soluble phosphorus-containing compounds.

A preliminary qualitative analysis of the rubber phase was carried out using infrared (IR) spectroscopy, which confirmed the presence of key functional groups: amine groups (1540 cm⁻¹), phosphate residues (1240–1260 cm⁻¹), carbonyl groups (1740–1760 cm⁻¹), and alkyl chains of fatty acids (3008–3010 cm⁻¹; 2850–2920 cm⁻¹).

Based on the obtained data, the gum phase can be preliminarily assessed as a potentially suitable medium for cultivating microorganisms, particularly those requiring sources of phosphorus, nitrogen, and carbon. However, due to the absence of completed quantitative elemental analyses and microbiological tests, these statements are currently regarded as prospective hypotheses requiring further confirmation in subsequent stages of the research.

At the current stage, samples of the obtained sludge have been transferred for a separate study aimed at evaluating its potential as a nutrient medium for the cultivation of biosurfactant-producing microorganisms. This approach opens prospects for the circular utilization of the by-product, which in the future may increase the economic efficiency of enzymatic degumming and reduce its environmental impact.

The results of the study showed that the biofuel contains such fatty acid methyl esters as Palmitic acid, methyl ester (C₁₇H₃₄O₂), Linolenic acid, methyl ester (C₁₉H₃₂O₂), Linoleic acid, methyl ester (C₁₉H₃₄O₂), Oleic acid, methyl ester (C₁₉H₃₆O₂), Stearic acid, methyl ester (C₁₉H₃₈O₂) and Isolinoleic acid, methyl ester (C₁₉H₃₄O₂). Palmitic acid, methyl ester and Stearic acid, methyl ester, which belong to saturated fatty acids, were identified on the chromatogram with retention times of 8.193 min and 8.882 min, respectively. Oleic acid, methyl ester, which is a monounsaturated fatty acid, exhibited the highest peak intensity at 8.814 min, while polyunsaturated fatty acids such as Linolenic acid, methyl ester, Linoleic acid, methyl ester, and Isolinoleic acid, methyl ester, appeared at 8.675 min, 8.725 min, and 9.136 min, respectively (Figure 5 and Table 7).

Mass spectrum analysis of the fraction with a retention time of 8.814 min revealed characteristic fragments with masses m/z 55; 69; 74; 83; 97; 96; 84; 41; 98; 87, confirming the identification of Oleic acid methyl ester (Figure 6). Similar spectra were recorded for all components of the biofuel, and FAME was confirmed and identified in the composition of the biofuel. The total content of fatty acid esters was 97.2%. Characteristic fragments with masses m/z of the main products of the transesterification reaction are listed in Table 7.

The overall composition of the fraction is characterized by a balanced ratio of saturated fatty acids (19.88%), monounsaturated (35.65%) and polyunsaturated (26.14%). In addition, 18.33% are methyl esters of fatty acids with a content below 2.00%. A high proportion of methyl oleate indicates the stability of the fraction, while the presence of saturated acids, such as Palmitic acid, methyl ester and Stearic acid, methyl ester, contributes to increasing energy efficiency. However, a significant content of esters of polyunsaturated acids, in particular Linolenic acid, methyl ester, reduces the resistance of biofuel to oxidation during long-term storage, which may require additional stabilization measures.

The absence of by-products and impurities in the fraction indicates a high selectivity of the transesterification process. The components of biofuel meet the necessary characteristics for use in diesel engines. The high proportion of FAME provides good compatibility with internal combustion engines, improves the combustion process, and contributes to the reduction of emissions of harmful substances. Thus, the obtained biofuel fraction is a promising source of environmentally friendly energy, characterized by high operational properties.

The FTIR spectra of crude soybean oil, degummed soybean oil, and biofuel were nearly similar, and no significant difference were observed as shown in Figure 7 and Figure 8. The identification of functional groups and bands corresponding to different stretching and bending vibrations clearly indicates the corresponding characteristic bands of the expected functional groups.

According to the FTIR spectrum, the band at 3475 cm⁻¹ represents the O–H stretching vibrations in crude soybean oil and degummed soybean oil. The absorption band shifted at 3464 cm⁻¹ in biofuels is attributed to O–H stretching vibrations, which confirm the lower polarity in the absence of phospholipid groups.

In all three spectra, the band at 3009 cm⁻¹ represents the C=C–H stretching vibrations of the cis double bond of unsaturation, while the bands at 2922 and 2853 cm⁻¹ are characteristics of the asymmetric and symmetric vibrations of the hydrocarbon chain –CH₂ in the aliphatic fatty acid.

Asymmetric stretching vibrations of the –CH₃ group at 1435 and 1361 cm⁻¹ increased in the biofuel spectrum, indicating a change in the phospholipid group to methyl.

In oils, the ester carbonyl (C=O) functional group shows a characteristic stretching band of triglyceride at 1743 cm⁻¹. In biofuel, this band shifted to 1741 cm⁻¹, indicating the absence of a polar phospholipid group. In all spectra, bending vibrations of –CH₂ scissoring aliphatic groups were observed at 1463–1455 cm⁻¹.

The strongest evidence of transesterification is the appearance of new signals at 1435 and 1361 cm⁻¹, which are certainly asymmetric and symmetric bending vibrations of the CH₃ group methyl ester. Another visible transformation is in the ester control signal area at approximately 1241cm⁻¹. The broad and strong signal at 1160 cm⁻¹ in oils will separate into two specific signals at 1195 and 1169 cm⁻¹ in biofuel. The averaging of the energy over the triple ester group of the triglycerides is gone. The broad signal in all spectra at 719 cm⁻¹ is due to overlapping the CH₂ rocking vibration and the out-of-plane vibration of cis-disubstituted olefins (Figure 8).

The variability of the vibrational frequency values of the same functional groups listed in Table 8 (in particular –CH₂, C–O, P=O, P–O) is due to changes in their chemical environment resulting from enzymatic degumming and further transesterification. The change in the structure of the samples affects the electron density around the corresponding atoms, the polarity of chemical bonds, and the strength of hydrogen bonding and dipole interactions, which, in turn, leads to a slight shift of the absorption bands in the IR spectra. Thus, the presence of different frequencies for the same types of vibrations is an expected consequence of the chemical modification of the system during the processing and serves as additional confirmation of structural changes in the samples. It has been established that during the pilot industrial implementation using the cavitation method (Figure 1) and pre-fermented crude soybean oil, it was possible to obtain BioFuel that meets the operational requirements for biofuels according to Ukrainian regulations [34], particularly with an exceptionally low phosphorus content of 3.9 ppm (Table 10).

Despite the numerous advantages of using the Purifine^® 3G enzyme complex-such as its high selectivity, the ability to operate under mild technological conditions, reduced energy consumption, and minimized environmental impact-several critical factors must be considered when evaluating its potential for industrial-scale implementation. These factors require further investigation and techno-economic assessment.

One of the key limitations is the cost of the enzymatic preparation, which may be a determining factor for enterprises operating with low profit margins. According to publicly available sources, the average market price of enzyme formulations like Purifine^® 3G ranges from approximately €25–50/kg, depending on the region of distribution, supply terms, and purchase volumes [35]. Based on the experimentally established optimal dose of 100 ppm, enzyme consumption per ton of crude oil does not exceed 10 g, which corresponds to €0.25–0.50 per ton of processed product. Compared to traditional degumming approaches involving phosphoric acid, caustic soda, and large volumes of hot water, this figure is economically competitive.

However, full-scale industrial implementation of the technology may face potential economic risks. In particular, for low value-added production chains, the enzyme cost can significantly affect the final product cost. Therefore, future research should focus on enhancing the efficiency of enzyme utilization, especially through dosage optimization based on feedstock type, reuse of the enzyme suspension, and enzyme immobilization for prolonged activity in reactor systems. Implementation of these strategies may reduce unit costs, improve process reproducibility, and broaden the potential for integrating this technology into industrial lines in the food, biochemical, and fuel industries, making enzymatic degumming not only an effective but also an economically viable solution for wide-scale application.

Although the developed enzymatic degumming method using the Purifine^® 3G complex demonstrated high efficiency in the case of soybean oil, its universal applicability to other types of vegetable oils requires separate evaluation. Soybean oil was selected for initial testing due to its industrial prevalence and well-characterized phospholipid composition. However, each oil type features a unique phospholipid profile and a range of minor components that may influence the enzymatic process. Different oils-such as castor, sunflower, and rapeseed-possess distinct phospholipid compositions and accompanying compounds (e.g., ricinoleic acid, phosphatidylethanolamine, glucosinolates), which can affect enzyme activity and necessitate adjustment of technological parameters [36]. Thus, although the preliminary results obtained with soybean oil are promising, further research should focus on evaluating the efficiency of enzymatic degumming for other types of vegetable feedstocks. This will allow for the adaptation of the methodology to broader industrial applications, taking into account the specific composition of each oil type.

5. Conclusions

Enzymatic degumming using Purifine^® 3G demonstrates high efficiency in removing phospholipids from crude soybean oil, providing phosphorus levels below 10 ppm, which provides high-quality raw materials for further use in biofuel synthesis, meeting ASTM, EN, and ISO standards. The optimal process conditions were found to be an enzyme dose of 100 ppm, a temperature of 57 °C, a pH of 7.0, a reaction time of 2 h, and 2.5% water by weight of the oil. Compared to chemical refining methods and using Quara LowP or Lecitase Ultra enzymes, the proposed technology eliminates the use of phosphoric acid, which indicates the economic and environmental benefits of using Purifine^® 3G, including providing phosphorus levels below 10 ppm, reducing wastewater volumes, and reducing energy costs. This approach is promising for widespread implementation in the production of edible oil and biofuels, contributing to the sustainable development of the industry.