Research progress on the genesis and removal methods of non-hydratable phospholipids from vegetable oils

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INTRODUCTION
Vegetable oils, such as soybean oil, sunflower oil, olive oil, or peanut oil, can replace animal fat as a source of energy.Moderate intake of vegetable oils has many beneficial effects on health, including reducing the risk of cardiovascular disease and improving inflammatory diseases and the immune system (Hashempour et al., 2022).Vegetable oil mainly comes from all kinds of oil-bearing crops, and the crude oil is obtained by pressing or extracting, and then processed by degumming, alkaline refining, decolorization, deodorization and other refining processes to finally obtain finished, edible oil.Plant phospholipids mainly exist in the seeds of oil-bearing crops, an important class of lipid concomitants.Most of them exist in the colloid phase, binding with proteins, sugars, fatty acids, sterols, vitamins and other substances.In the process of oil production, phospholipids are extracted with the oil, and the effect of phospholipid removal directly determines the quality index of the refined oil.
Phospholipids are an amphiphilic molecule composed of a water-soluble phosphate group and two hydrophobic fatty acid groups.Phosphate groups usually bind to substances such as choline, ethanolamine, or glycerol to form different types of phospholipids, as shown in Figure 1.The phospholipids in vegetable oils can be divided into hydratable and non-hydratable phospholipids.Hydratable phospholipids are hydrophilic and can be removed by hydration, but in general, some phosphorus in oil exists in the form of non-hydratable phospholipids.A hydratable phospholipid has obvious hydrophobicity, which cannot be completely removed even after washing 16 times.acts as a substituent to connect with glycerophospholipid.Lipid head group (Z) acts as a substituent to connect with glycerophospholipid.
In the refining process of vegetable oils, the content and nature of non-hydratable phospholipids af-fect the quality and stability of vegetable oils.Since non-hydratable phospholipids are difficult to remove, their presence increases the cost of oil treatment and leads to problems such as deepening the color of the oil and reducing storage stability (Jiang et al., 2015).Vegetable oils with a high content of non-hydratable phospholipids are often accompanied by more metal ions, which will not only lead to vegetable oil acid and bad flavor, but also affect the post-sequence refining process (Zhao et al., 2020).Therefore, the removal of non-hydratable phospholipids is the main research goal of vegetable oil degumming.
Furthermore, owing to the coexistence of hydrophobic and hydrophilic elements within phospholipids, the phosphate groups of phosphatides undergo esterification through the hydroxyl groups of diverse alcohols (e.g., ethanolamine, choline, inositol, and glycerol).Such diversity poses challenges in their isolation and analysis using conventional methodologies.Conventional phospholipid detection relies on chemical analysis techniques like thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and mass spectrometry (MS) (Antonelli et al., 2020).These approaches often entail intricate sample pretreatment and specialized equipment, making them time-consuming and costly.However, recent technological advancements have introduced novel methods for detecting phospholipids in vegetable oils.Spectroscopic techniques, such as near-infrared spectroscopy, offer non-destructive, real-time, and highly efficient means for rapid detection and quantitative analysis (Meng et al., 2014).To delve deeper into understanding and studying non-hydratable phospholipids, the following section systematically elaborates on previous research in this domain.
storage time, high water content and excessive phospholipase D activity all contribute to an increase in non-hydratable phospholipids in oil, which often leads to the hydrolysis of hydratable phospholipids into phosphatidic acid (Zhao et al., 2020).Non-hydratable phospholipids form when they bind to divalent metal ions such as calcium and magnesium.The main formation process of non-hydratable phospholipids is shown in Figure 2.

Major morphology of non-hydratable phospholipids in vegetable oils
In vegetable oils, most NHP is mainly in the form of calcium-magnesium salts of phosphatidic acid and phosphatidic acid.Non-hydratable phospholipids (NHP) mainly include phosphatidic acid (PA), lysophosphatidic acid (LPA), lysophosphatidyl serine (PS), lysophosphatidyl ethanolamine (LPE) and calcium and magnesium salts of lysophosphatidic acid, etc.It was confirmed that phosphatidic acid is generated by phosphatidylcholine and phospholithyl ethanolamine subjected to phospholipase D, and can react with calcium to form non-hydratable phospholipids.The percentage of its composition obtained after detection and analysis is shown in Table 1 (Dijkstra, 2017).The common features are that they contain fewer polar groups, have obvious hydrophobicity, and are more difficult to bind with water in hydration degumming .Hydratable phospholipids and non-hydratable phospholipids are mainly distinctive in the different functional groups linked to the hydroxyl group of the acid.The former contains more polar groups, such as choline, ethanolamine, serine, and inositol.Hydratable Phospholipids (HP) are mainly Phosphatidyl Choline (PC) and Phosphatidyl Ethanolamine (PE), so they are highly hydrophilic.They can precipitate in contact with water, but their hydration rate differs.

The structure of non-hydratable phospholipids
NHPs are a class of compounds with a phosphate ester structure in a nonaqueous environment, and their structure consists of three main parts: glycerol backbone, phosphate group and fatty acid chain.The hydroxyl clusters on the second and third carbon atoms of the glycerol backbone are esterified to phosphorylated functional groups, which can further esterify the hydroxyl groups of various alcohol groups including ethanolamine, inositol, choline and serine.The fatty acid tails of phospholipids are nonpolar and are mainly composed of two fatty acid chains linked to the glycerol backbone via ester bonds.The general formula of the non-hydratable phospholipid structure is shown in Figure 3.

COMPOSITION OF NHP IN DIFFERENT KINDS OF OILS
All kinds of oil processed as crude oil will contain a certain amount of NHP.This is especially relevant in the case of soybean, rapeseed and other major oils which are processed as crude oil.The composition of phospholipids in different kinds of vegetable oils is shown in Table 2.
As can be seen from Table 2, the proportion of HP and NHP in some vegetable oils is different, and is related to the longer storage time of crude oil and the conversion of some HP.Oil should be refined immediately after pressing, and soaked before steam heating or microwave treatment to inactivate phospholipase, prevent HP conversion, and lessen the burden of refining.
Stable and accurate PL quantification methods are needed to detect and analyze the process of oil degumming.Currently, techniques such as liquid chromatography, mass spectrometry, and spectrophotometry have been applied to the quantitative analysis of PL in edible oils.The Table 3 shows the  Not suitable for the determination of oils with low phospholipids.
detection limit for these methods, along with their advantages and disadvantages.Due to the high sensitivity and selectivity of detecting phospholipids, more and more researchers have established qualitative and relative quantitative methods of non-hydratable phospholipids in vegetable oils, which are applied to vegetable oil categories of different characterizations.These methods to reveal the changes in composition can lead to the removal of non-hydratable phospholipids and the quality improvement of refined oil products.

REMOVAL TECHNIQUES FOR NON-HY-DRATABLE PHOSPHOLIPIDS
Degumming is an important step in refining, where PLs are removed from crude vegetable oils.NHP is a world-class problem in the oil industry.Water, acid, enzyme and membrane degumming methods have been used in vegetable oil degumming in recent years.

Traditional degumming method
The traditional methods are mainly hydration and the simple acid method.

Hydration degumming
In the industry, the method of removing PL is mainly hydration.That is, a certain amount of hot water is added to crude oil so that the phospholipids in crude oil expand and are then separated after condensation to be removed, The process is generally carried out at 40-45 ºC, which is relatively energy-efficient, has a short production cycle, and reduces the acid value.The crude oil phospholipid content and temperature determine the amount of water added during degumming.For example, at high temperatures, the amount of water added is about 3-5 times that of the crude oil PL content (Dijkstra, 2017).In addition, metal ions in tap water may combine with HP in oils to form NHP, which affects the degumming effect.
The degumming process parameters and the final degumming results differ for different oil samples.The results show that the phospholipid removal rates for soybean oil, rapeseed oil, rice bran oil, sunflower oil and olive oil are 98.3, 67.1, 89.8, 80.6 and 48% respectively.The phospholipid removal rate of mixed algal oil was only 19.4% (Supansa et al., 2017).Hydration degumming can only remove hydratable phospholipids with strong hydrophilic properties, while NHP with weak polarity and hydrophobicity is difficult to remove.After degumming, the phospholipid content is still 80-200 mg/kg, which cannot guarantee low phosphorus content (Aleksandra et al., 2003).Moreover, due to the high content of NHP, it is not always the best for all kinds of oil.

Acid degumming
The acid degumming method entails adding a certain amount of phosphoric acid or citric acid in order to dissociate NHP by adjusting the pH, and then cool the phospholipids to form crystals.The phosphorus content can be reduced to less than 30 mg/kg.
Phosphoric acid was used to refine palm oil to remove phospholipids, and the optimal dose of phosphoric acid was determined to be 0.05 wt% by adopting the wet degumming process (Harrison et al., 2022).The researchers used a 1:1 mixture of citric acid and phosphoric acid to degum sunflower oil, and the degumming effect was better and the amount of phospholipid remaining was 0.05 mg/kg (Pan et al., 2000).
Some studies have shown that phosphoric acid is used for degumming vegetable oils with high phospholipid contents, such as soybean and algal oil.As a result, the phosphorus content of crude algal oil was reduced from 530 ppm to 93 ppm in the optimal acid degumming process, with a phospholipid removal rate of 82.5%, which is much higher than that of 19.4% for water degumming (Supansa et al., 2017).
At present, acid degumming has been widely used in oil processing.The technology is simple, but closely correlated with the crude oil quality, and is suitable for low phosphorus oil such as palm oil.Some vegetable oils with high non-hydratable phosphorus contents are still not up to the requirements.

New degumming method
The main disadvantages of the above conventional degumming processes are considerable oil loss, significant wastewater, and high energy consumption, which cannot guarantee the low phosphorus levels required for physical refining.These techniques are not suitable for oils containing high levels of non-hydratable phospholipids.

Enzymatic degumming
In recent years, research on the application of phospholipase in vegetable oil degumming has become more and more in-depth.Enzymatic degumming uses the biological enzymatic method to convert NHP into HP, and then the hydration method to maximize the phospholipids' removal and recovery.There are four types of phospholipases used for vegetable oil degumming.Phospholipase A 1 (PLA 1 ) and phospholipase A 2 (PLA 2 ) remove fatty acids from positions 1 and 2, respectively, and phospholipids are hydrolyzed to free fatty acids and soluble phospholipids.Phospholipase B (PLB) eliminates fatty acids from glyceryl.Phospholipase C (PLC) hydrolyzes the bond between acylglycerol and the phosphate group to release Diacylglycerol (DAG) (Jiang et al., 2015).The site of action of phospholipase D is after the phosphate group.The action sites of four different phospholipases are shown in Figure 4. Applying PLA 1 and PLC to eight crude oils, the researchers found that the PLA 1 degumming process significantly reduced residual phosphorus to less than 10 mg/kg and that PLC degumming improved the oil yield of most oil samples (Jiang et al., 2015).The enzyme mixture Purifine® 3G was applied to crude soybean oil to reduce the residual phosphorus content to 8.9 mg/kg (dos Passos et al., 2022).To improve the stability and reusability of phospholipase, magnetic immobilized PLA was used for degumming soybean oil.The results showed that even after 5 cycles of degumming, magnetic immobilized PLA 2 retained more than 80% of its initial activity (Qu et al., 2016).Immobilized phospholipases show excellent potential for enzymatic degumming.
Enzymatic degumming has many advantages over traditional methods, including higher degumming efficiency, lower chemical requirements, higher product yield, and less environmental pollution.Despite the obvious advantages of this approach, it has drawbacks, particularly the cost of the enzyme.Moreover, the quality of different crude oils is different, so a specific enzymatic degumming process should be selected (Jiang et al., 2015).For example, for oil samples with low NHP content and high initial phosphorus content, PLC degumming with acid pretreatment can meet the requirements of physical refining and improve the yield simultaneously.For oil samples with high NHP content and low initial phosphorus content, acid-pretreated PLA 1 degumming is the best choice.

Membrane filtration degumming process
Membrane degumming is a non-thermal process that selectively separates phospholipids based on the size or properties of the membrane and is an emerging method for efficient degumming at low energy costs.Different types of membranes are applied to the crude vegetable oil membrane degumming process, such as polyethersulfone (PES) membranes, polysulfone (PSf) membranes, polyvinylidene fluoride (PVDF) membranes and ceramic membranes have shown good permeation flow with good degumming effect.
The PVDF membrane is more stable to hexane reagent than PES and PSf membranes (de Souza Araki et al., 2010).The filtration of rice bran gross oil (CRBO) using 50 kDa PVDF membranes mixed with hexane solvent achieved 95.5% phospholipid removal (Sehn et al., 2016).It was shown that membrane degumming combined with hydration degumming and acid degumming could improve the phospholipid removal rate of crude oil.By combining conventional acid degumming with PES membrane separation, the phospholipid content of jatropha oil can be reduced to less than 20 ppm (Liu et al., 2012).
In addition to the above polymeric membranes, ceramic membranes have a highly repulsive reaction to phosphorus.In a recent study using ceramic membranes to degum CRBO, phosphorus was reduced by 95%, along with a 42-62% reduction in color.The average phospholipid retention was 95±2% using 5 kDa ceramic membranes (Abdellah et al., 2020).It has been shown that a three-stage hybrid membrane process of ultrafiltration, nanofiltration and permeation vaporization treats crude canola oil with almost complete rejection of phospholipids (> 99.9%) (Abdorrezaee et al., 2021).
The membrane filtration de-adhesive process replaces the traditional energy-intensive process.It has the advantages of low energy consumption, safety, no chemicals, good retention of nutrients, reduced wastewater production, simple operation and so on.Despite these advantages, the poor stability and low osmotic flux of some organic membranes during membrane filtration and degumming lead to problems such as scaling and clogging, requiring high operation and maintenance costs, making the process economically unfeasible and unlikely to be applied on a large scale.

Other degumming processes
In addition to the above degumming processes, some novel degumming technologies are less widely used, such as Ultrasonic Assisted Enzymatic Degumming (UAED), saturated steam degumming, cavitation reactor-assisted ethanolamine degumming and electrolyte degumming.The comparison of new degumming processes for different kinds of vegetable oils is shown in Table 4 below.
The ultrasonic treatment of crude vegetable oil can separate the phospholipids completely, which has the enhanced benefit of reducing the reaction time and processing cost.When water, acid and enzyme degumming are combined with ultrasonic degumming, phosphorus content significantly reduces, which can eliminate alkali refining neutralization, improve degumming efficiency, and reduce the use of chemical degumming reagents, temperature and processing time.Cavitation reactor-assisted degumming is used to purify crude soybean oil.The degumming treatment based on cavitation can produce high-quality oil, and reduce reaction time and energy consumption, but this process has high requirements for equipment.Compared to other methods, saturated steam degumming does not add any chemical reagent and has no residual problem, which can provide a theoretical basis for industrial production.The ethanolamine degumming can be carried out at an ambient temperature, and does not need additional energy, uses simple technology, has low production cost, and avoids energy and equipment-intensive problems.The degumming agent is cheap, highly efficient and easy to obtain.By contrast, the electrolyte degumming uses potassium chloride and sodium chloride aqueous solution to improve the degumming performance, with water, total phenol, phytosterol content and taste well retained.The recycled gelatin can be used in commercial applications, which has good potential.

Model of dynamics
To meet the phosphorus content specifications of the vegetable oil refining process and better optimize the process parameters, many researchers have proposed kinetic models.These models enable the estimation of crucial parameters, such as time, tem-perature, and acid concentration, based on the initial phospholipid content, thereby optimizing the efficiency of the degumming process.For the refining process already in operation, the data on the degumming process can be dynamically monitored by using the kinetic model to optimize the parameters in real time, so as to improve the effect of the degumming and reduce the production cost.Hydrodynamic models, especially computational fluid dynamic (CFD) models, play an important role in predicting the concentration and polarization thickness of corn oil in the drying process using tubular ceramic films (Wibisono et al., 2015).By simulating the flow of fluid in the tubular ceramic film, this model can accurately calculate the concentration distribution of corn oil in the pipeline, thus predicting the concentration polarization thickness of corn oil throughout the treatment.To solve the problem of longer computation time, a method combined with an artificial neural network is proposed to enable the model to predict the corn oil concentration polarization thickness more quickly and optimize the production process.The study showed that the kinetic models developed for the de- gumming and bleaching stages determined the doses of 0.015 wt% and 0.047 wt% for citric acid and bleaching clay, respectively.The phosphorus content in degummed palm oil was reduced from 35 ppm to product specifications and the reversible first order kinetic model was optimal (Serrano-Bermúdez et al., 2021).Furtherdelving into kinetic studies concerning the refining stages, notably degumming and bleaching in vegetable oil, facilitates a comprehensive grasp of the reaction mechanisms.These studies not only enhance our predictive capabilities regarding these processes but also enable a profound comprehension of the underlying principles governing non-hydratable phospholipid (NHP) removal from vegetable oil.Moreover, they furnish a robust theoretical framework, pivotal in augmenting the efficacy of dephosphorylation techniques.

CONCLUSIONS
Non-hydratable phospholipids (NHP) play a crucial role in both the taste and revitalization of oils, making their elimination a primary focus in the realm of vegetable oil refinement.Comprised of a mixture of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and other components, NHP remain a crucial target for purification.In the quest for purifying these non-hydratable phospholipids, delving into the specific impact of distinct phospholipid components on oil quality warrants further investigation.Attention to the variations in species, origin, and processing techniques of plant oil, coupled with a meticulous exploration of pertinent factors, serves as the bedrock for understanding the nuances within this domain.
By scrutinizing the origins, physical attributes, and chemical properties of non-hydratable phospholipids, a more precise segregation of these compounds from other impurities becomes achievable.This in-depth comprehension paves the way for designing the NHP removal process in vegetable oil more effectively.In summary, the systematic investigation and isolation of non-hydratable phospholipids in vegetable oils hold promise for significant advancements in the oil industry.Through a comprehensive understanding of NHP components and their effects on oil quality, the refinement process can be optimized, producing oils that meet the rigorous benchmarks of safety and health, thereby fortifying their integration into industrial production.

Figure 1 .
Figure 1.Schematic representation of lipid head group (Z) that acts as a substituent to connect with glycerophospholipid.Lipid head group (Z) acts as a substituent to connect with glycerophospholipid.

Table 3 .
Advantages and disadvantages of each detection method for NHP