Phosphatidylserine: An overview on functionality,processing techniques,patents,and prospects

Jingnn Chen ,Jun Li ,Hoyu Xu,b ,Jixi Li,b ,Yonghong Yun ,Xuebing Xu ,Ynln Bi,＊

a College of Food Science and Engineering,Henan University of Technology,Zhengzhou 450001,China

b Wilmar (Shanghai) Biotechnology Research and Development Center Co.,Ltd.,Shanghai 200137,China

c Enzymecode Biotechnology Co.,Ltd.,Binhai New District,Tianjin 300457,China

Keywords:Phosphatidylserine Functionality Processing techniques Patents evaluation Prospects

ABSTRACT Phosphatidylserine (PS) is the part of cell structure in the body and has many beneficial functions especially in brain-related aging diseases.Although daily foods can provide PS to human body,the amount is very limited due to its poverty in most foods.To overcome the issue,numerous studies based on PS have been reported to develop PS-related supplements.In this review,PS was comprehensively and critically reviewed from the view of resources,functions,processing techniques,patents,and prospects.For resources,animal,plant,and microorganism origins were all covered with their differences in composition profiles.For functions,benefits regarding memory,cognitive enhancement,exercise performance,reducing Alzheimer’s disease,and attention-deficit hyperactivity disorder symptoms were covered as well as the functional differences among animal-,plant-,and microorganism-based PS-related supplements.For processing techniques,traditional extracting methods from animal,plant,and microorganism tissues were comparatively discussed with enzymatic synthesis based on different reaction systems.Finally,patents of PS-related supplements were evaluated as well as their applications.This review could provide scientific and valuable support for PS industry.

1.Introduction

With the advances in living standards and medical standards,human average life expectancy is getting longer and longer[1].The average life of global human expectancy is not more than 40 years before 1900s but reaches 72 years in 2020.However,aging related diseases are increasing with the extension of human lifespan,and the symptoms are becoming prominent increasingly [2].The most common age-dependent neurodegenerative diseases such as dementia and Alzheimer’s disease have become urgent public health problems in many countries around the world [3].Among the studies that attempt to address those problems[4–8],phosphatidylserine(PS)and docosahexaenoic acid(DHA)(Fig.1)are both thought to be important in the structures and functions of human brain and may be relevant to these issues[6,9,10].

Fig.1.Chemical structures of phosphatidylserine and docosahexaenoic acid(R1 and R2 indicate fatty acids which could be same or different).

PS,one aminophospholipid molecular,is widely distributed among animals,plants,and microorganisms.It is a key component of mammalian cell double membranes and plays very important roles in biological processes such as apoptosis (cell death) and cell signaling[11].In the brain,PS comprises up to 15% of the total phospholipid pool.The fatty acid composition of endogenous PS depends on its locale and function in the cell.Approximately 30 g of PS exists in the human body and about half of this amount(13 g)is found in brain tissues[12].Besides,PS can help reduce muscle fatigue and soreness,reduce inflammation,improve memory,reduce depression,etc.[13].Based on its nutrient value and physiologic functions,PS has been granted as the Novel Food Raw Materials in China in October 2010.

DHA,anω-3 unsaturated fatty acid,is widely distributed in animals(e.g.,fish and krill)and microorganisms(e.g.,algae)[14].It enriches in the brain and eye of the human body highly and is essential for the growth and functional development of those brains in infants [15].Although DHA is not the essential fatty acid of human body and can be synthesized from its precursorα-linolenic acid,the synthesis rate is quite low especially in the infants and elderly,which suggests that a continuous supply through external intake is necessary especially for such a group of population.Besides,DHA can help reduce heart disease,improve attention deficit hyperactivity disorder,reduce the risk of early preterm births,reduce inflammation,support muscle recovery after exercise,etc.[16,17].Based on its nutrient value and physiologic functions,DHA has been also granted as the Novel Food Raw Materials in China in March 2010.

In this review,we intended to comprehensively summarize the functions of PS and/or DHA based on their resources such has animalsbased,microorganisms-based,and synthetic.We also reviewed their performance differences in application of physiologic functions.PSand/or DHA-related production techniques were also discussed.In addition,an overall patent evaluation was also covered based on globally related patents granted and issued.

2.Occurrence of natural PS

PS is a structural component of the eukaryotic membrane and accounts for 5%–10% of the total lipids of cells.PS exists naturally in animals,plants,and microbial resources.The most commonly available sources of PS are soy and milk.Soy lecithin contains approximately 0.5%PS [18],whereas milk contains about 5%–10% of the total phospholipids[19].There are trace amounts of PS in vegetable oils such as corn oil,almond oil,and sunflower oil[20,21].PS is commercially available as an oral supplement intended for the cognition-enhancing function.The main sources that have been used to derive PS are brain cortex(BCPS),soybean (S-PS),and egg (E-PS).Many studies were conducted on the physiological functions of BC-PS.Nunzi et al.[22]demonstrated that the administration of BC-PS improved the memory-related behaviors on aged rats.BC-PS intake by elder people also showed a positive impact on the improvement of age-related brain dysfunction including Alzheimer’s disease [23] and depressive symptoms [24].For healthy subjects,the brain performance and stress alleviation were improved after BC-PS intake [25].

S-PS is the preferred supplement for use in humans,however,due to the possibility of the transfer of infectious diseases (e.g.,bovine spongiform encephalopathy) of BC-PS and the low yield of PS from bovine cortex.Acute administration of S-PS to young rodents was suggested to restore the scopolamine-induced memory impairment [26].Clinical studies on S-PS also showed positive effects on improving the agerelated memory impairment [27].In recent years,another source of krill-derived PS has been developed,which is enzymatically made from krill shrimp.Although the fatty acid compositions of krill-derived PS are quite different from that of BC-PS,the effects of krill-derived PS on cognitive function are identical with those of BC-PS.Park et al.[28]demonstrated that krill-derived PS treatment improved the memory in the Morris water maze test and enhanced the expression of brain-derived neurotrophic factor and insulin-like growth factor in the normal young rats.Besides,treatment with the krill-derived PS also significantly enhanced the glucose activity of the hippocampus.These findings are interpreted as being indicative of comparable effectiveness of those PS forms.Blokland et al.[29],however,investigated the cognitionenhancing properties of BC-PS,S-PS,and E-PS in rats and found that S-PS rather than E-PS might have comparable effects on cognition when compared with BC-PS.

Oral supplement has been becoming the choice for administration of humans with exogenous PS.The oral administration of BC-PS 800 mg/day for 10 days was demonstrated to significantly attenuate plasma cortisol concentrations in healthy inactive males [30].Enhancing the higher brain functioning with PS supplement is suggested to employ lower daily doses (<500 mg/day) for longer duration.Cenacchi et al.[23]reported the improvements in behavioral and cognitive functions in a group of geriatric patients with cognitive impairment after BC-PS 300 mg/day for 6 months.Furthermore,S-PS 300 mg/day for 1 month has been shown to be associated with feeling less stressed and having better mood in a sub-group of healthy young males [31].

PS from different sources has a similar molecular structure respecting to glycerol moiety and serine head group,but the fatty acid compositions are origin-dependent and tissue-specific.The fatty acid composition of PS in brain is rich in DHA[32]and in soybean is rich in linoleic acid [33],whereas that from egg yolk contains primarily palmitic acid.S-PS is virtually devoid of arachidonic acid and DHA,whereas BC-PS and E-PS are identically rich with these fatty acids.Moreover,PS in marine organisms is enriched with polyunsaturated fatty acids(PUFA)[34].

3.Nutritional functions of PS

PS,a phospholipid component,is found in mammalian cell membranes.Previous studies indicated that acute and long-term administration of PS dampens cortisol responses to acute exercise and mental stress.In addition,PS has been shown to improve memory,learning,mood,and stress management.Furthermore,the intake of PS has been associated with an improvement of psychiatric disorders,such as bipolar and major depressive disorders and with the prevention of inflammatory neurodegenerative events.

3.1.Effects of PS on human brains

PS is a structural component of endoplasmic reticulum,nuclear envelopes,Golgi apparati,inner(cytosolic)leaflets of plasma membranes,outer mitochondrial membranes,and myelin in human body,which processes 2%–20% of the total phospholipid mass of adult human plasma and intracellular membranes [35,36].Supplemental PS (200 mg/day for 3 months) significantly improved in memory,information processing,and the ability to perform activities of daily living for older patients with severe cognitive impairments secondary to Alzheimer’s disease [37].Moreover,PS significantly improved the attention-deficit hyperactivity disorder (ADHD) symptoms and the short-term auditory memory in children.PS supplement might be a safe and natural nutritional strategy for improving the mental performance in young children suffering from ADHD.However,Jorissen et al.[38] reported that ageassociated memory impairment was allocated at random to one of the three treatment groups:placebo,300 mg S-PS/day,or 600 mg S-PS/day,indicating that a daily supplement of S-PS does not affect memory or other cognitive functions in older individuals with memory complaints.

It is worthy to note that approximately 20%–30%of the PS in human gray matter is in the form of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoserine[39–41].The DHA content of neuronal PS is of functional importance.A study by Cunnane et al.[42] demonstrated that a reduction in the DHA content of PS is associated with the progression of mild cognitive impairment to Alzheimer’s disease.Consequently,the incorporation of PS into human membranes is sensitive to the availability of both PS and DHA [39,40,43].Adequate amounts of DHAenriched PS are required for the fusion of intraneuronal secretory granules with the presynaptic membrane,the release of neurotransmitter molecules and proper postsynaptic neurotransmitter-receptor interactions [41,44].

3.2.Effects of supplement PS on exercising humans

Acute exercises induce active oxygen species in the body and damage cells and even cause cell death.PS has been reported to be an effective supplement for combating exercise-induced stress and preventing the physiological deterioration that accompanies too overtraining.PS has been demonstrated to speed up recovery,prevent muscle soreness,improve well-being,and might possess ergogenic properties in athletes involved in cycling,weight training,and endurance running[45].Fahey and Pearl [46] reported that oral administration of S-PS (800 mg/day)during 2 weeks of intense weight training does not significantly attenuate creatine kinase activity but reduces the subjective ratings of muscle soreness during training.Short-term supplementation with S-PS (750 mg/day) improves exercise capacity during high-intensity cycling and tends to increase performance during intermittent running,indicating that PS might alter neuroendocrine function.

In addition to physical stress,PS supplement benefits subjects suffering from mental stress.PS has been reported to improve the accuracy during tee-off by increasing the golfer’s stress resistance[47]and to reduce stress and increase performance in runners,cyclists,and golfers.Mood improvement was observed in young healthy adults who took 300 mg PS/day supplement for a month [31].Parker et al.[48]demonstrated that PS supplement significantly increased cognitive function prior to exercise in 18 males aged 18–30 years.

Moreover,an excessive cortisol response to exercise-induced stress has been linked to a negative training state,which could lead to overreaching or overtraining.PS supplement has been reported to improve mood in a sub-group of healthy young adults when faced with a stressful mental task[31]and blunted both serum ACTH and cortisol[49].PS has been established as a safe oral supplement capable of attenuating the serum cortisol[50]and creatine kinase[51]responses to acute exercise stress.The study conducted by short-term supplementation with a moderate dose of PS (600 mg/day) promotes a desired hormonal balance for athletes by blunting increases in cortisol levels [52].These findings suggest that PS partly counteracts the stress-induced activation of the hypothalamopituitary-adrenal axis [53].

3.3.Functions of PS formulated with other active ingredients

Acute and long-term administration of PS dampens cortisol responses to acute exercise and mental stress.Phosphatidic acid(PA)is a precursor for other lipids.In combination with PS,PA has shown to lower cortisol levels,improve wellbeing under acute social stress and the premenstrual syndrome symptoms (PMS) of women aged 18–45 years[49,54].The study conducted by Doma et al.[55] demonstrated that a supplement containing whole coffee cherry extract and PS is safe and well tolerated,and significantly improves memory,accuracy,focus and concentration,and learning in a healthy adult population with selfreported memory problems.A recent study demonstrated that the treatment with nutritional formulation containing caprylic and capric acid,PS,and DHA reversed the memory impairment observed in streptozotocin-lesioned rats [56].Supplementing with 400 mg/day PS and 100 mg/day caffeine might be effective for attenuating total mood disturbance and perception of fatigue following an acute exercise stress[57].

Silymarin is a component of milk thistle(Silybummarianum),which has been proved to have a galactagogic effect [58].Zecca et al.[59]investigated the efficacy of a galactagogue containing silymarinphosphatidylserine and galega in mothers of premature infants.The result showed that the mixture increases milk production in mothers without any significant side effects.The underlying mechanism might be that PS acts as a carrier for insoluble molecules,promoting intestinal absorption of Silymarin.Araujo et al.[60]reported that a nutraceutical supplement containing PS,Ginkgobiloba,vitamin E,and pyridoxine could significantly improve the performance accuracy of memory in aged beagles.Regarding the activity of PS on stimulating acetylcholine release [61],modulating acetylcholinesterase activity [62],and inhibiting the age-related loss of muscarinic receptors [63],the antioxidant activity ofGinkgobilobaand vitamin E,and the biological activity of pyridoxine (a cofactor in the synthesis of several neurotransmitters),their combination might be of particular benefit to dogs.

Though the interaction of PS with these nutraceutical supplements,such as vitamin B,long-chainω-3 PUFA and polyphenols was not clearly proved,some underlying mechanisms that can potentially support the function of these nutrients against brain aging are the restoration of the microstructural integrity of the hippocampus,prevention of glutamatergic synapse and astroglia aging,decreased oxidative stress and neuroinflammation,and increased expression of brain neurotrophins.

4.Nutritional functions of DHA and PS-DHA in brain health

The brain has a unique fatty acid composition compared to other organs as it is rich in DHA with the functions of regulates neuroinflammation,cell survival,neuronal homeostasis,and neurotransmission in the brain [64,65].It is widely believed that humans need to supplement DHA in diet for maintaining adequate tissue levels due to the poor synthesis capacity and low partition coefficient of DHA in brain[66,67].DHA is almost exclusively consumed as triacylglycerols in the diet.However,studies [67–69] demonstrated that DHA esterified to phospholipids might be more efficient at targeting the brain compared to DHA esterified to triacylglycerols(TG-DHA).Liu et al.[68]reported that approximately two times more DHA was recovered in the brain of piglets that consumed the labelled DHA esterified at the sn-2 position of PC compared to those esterified at the sn-2 position of triacylglycerol when administered orally.Chizhikov et al.[70]suggested that PS-DHA might support cerebellar development in preterm pigs by enhancing proliferation and increasing the survival of granule cells in the internal granule cell layer.

The most enriched DHA form in the brain phospholipid pools is phosphatidylethanolamine (PE) and PS.The greatest concentration of PS in humans is in the brain,where it comprises about 15%of the total phospholipid pool [71].Except for acting as a structural role in maintaining the integrity of cell membranes,PS is considered critical for the functioning of signal transduction,secretory vesicle release,cell-to-cell communication,and cell growth regulation [72].Compared with DHA attached on triacylglycerol,DHA associated with PS(PS-DHA)possesses a chronic administration and results in a greater DHA accumulation in the cortices of middle-aged rats [73].Chouinard-Watkins et al.[74]found that PC-DHA or PS-DHA were more efficient at targeting the brain than TG-DHA after acute ingestion.Importantly,studies demonstrated that DHA conjugated to PS is included in human milk throughout lactation [75,76],implying the importance of PS as a delivery mechanism.Buddington et al.[77] reported that the supplementing formula with PS-DHA increase the weight of the brain,particularly the cerebellum,and improve the neurodevelopment and survival rate of preterm pigs compared with placebo.These studies indicate that DHA bioavailability is improved in the form of PS-DHA.

5.Processing techniques of PS and DHA

As PS exists in nature especially in animal tissues,an early supply was from the extraction of animal tissues such as cow brains.However,as mad cow disease came as a concern,such extraction was facing a stop.Although such extraction can be sought from other sources,a more economy viable technology appeared using enzymatic synthesis from PC with phospholipase D (PLD) as the biocatalysts.Therefore,more innovation efforts have been given on the enzyme assisted production approach as the technology is viable in terms of resources and sustainability.

5.1.Extraction of PS from animal tissues using organic solvents

The extraction effectiveness of PS is dependent on the selection of raw materials and extracting solvents.Although PS is distributed in animals,plants,and microorganisms widely [72],solvent extraction methods are mostly based on animal tissues due to the low PS content in plants.PS is presented in lipids of tissues.To obtain pure PS,crude phospholipids are firstly concentrated and separated from the tissues,followed by further isolation and purification[78].

Bligh et al.[79] used fish muscle tissue as raw material and chloroform-methanol-water (1:2:1,V/V/V) as solvent for crude phospholipids extraction.This method is simple and has a high sample throughput,but is not suitable for large-scale phospholipids extraction due to the toxicity and high cost of chloroform.Hara and Radin [80]used hexane-isopropanol (3:2,V/V) to extract phospholipids from rat brain.This extracting method is more economical compared to chloroform-methanol-water extracting method and has lower solvent toxicity.Researchers also extract phospholipids from animal brains since phospholipids are insoluble in acetone.A patent by Liu and Yang[81] reported that the fat is removed by washing the animal brain powder with acetone,followed by dissolving the remaining substance in hexane or ethanol and filtering off the supernatant,dissolving the remaining substances in ether,concentrating and adding alkaline aqueous solution,filtering off the precipitate at low temperature and adjusting the pH of the supernatant to 3–6,finally evaporating to obtain crude phospholipids.

After crude phospholipids are extracted using the above method,PS needs to be separated from the phospholipids to obtain a high purity of PS.Common methods include thin-layer chromatography (TLC),preparative high performance liquid chromatography (pre-HPLC).TLC utilizes the differences in the magnitude of the force between each component of phospholipids and the stationary phase to separate the components of phospholipids.PS,phosphatidylcholine (PC),phosphotidylethanolamine (PE),phosphatidylinositol (PI),lysophosphatidylcholine(LPC),diphosphatidylglycerol(DPG)in bovine brain extract can be effectively separated by this method [82].Chen et al.[83] used pre-HPLC with methanol and phosphoric acid(1 mol/L)(90:10,V/V)as the mobile phase to separate PS from bovine brain extractives,and anion-exchange chromatography was used to further purify PS.

Nevertheless,there are still some shortcomings in the above methods: (a) the solvent extracting method is characterized by high consumption of organic solvents and low purity of PS [84],(b) the extracted product is on the edge of being abandoned since the safety of PS derived from animals has been questioned because of mad cow disease [85,86].

5.2.Enzymatic synthesis of PS with phospholipase D

PS has extremely important functions in brain,central nervous system,and cognition.Consequently,PS appears to be an interesting candidate for prevention and treatment of age-associated cognitive diseases.However,the available natural sources of PS are limited.Enzyme-catalyzed synthesis of PS offers a pathway to achieve higher purities of PS.Moreover,these enzymatic methods are generally environmentally-friendly ones,because of the mild conditions and the nontoxic nature of the reaction system (often aqueous).Therefore,enzymatic production of PS (especially PLD) becomes a mainstream of research.PLD is a phospholipase widely distributed in animals,plants,and microorganisms.Currently,industrialized PLDs are mainly extracted from microorganisms.Because of PLD has specific conserved sequenceHxKxxxxD[85],PLD exhibits phosphodiesterase activity towards specific phospholipids and acts on the phosphodiester bond of the substrate phospholipid compounds to catalyze two reactions: (a) the hydrolysis of the substrate PC,which yields choline and PA,and(b)the transphosphatidylation reaction.PS is produced by reacting the substrate lecithin (mainly contains PC) andL-serine [87].The catalytic reaction process of PLD is shown in Fig.2.

Fig.2.The catalytic reaction process of PLD.Abbreviations: PLD-phospholipase D;PC-phosphatidylcholine;PA-phosphatidyl acid;PS-phosphatidylserine.

PA is the main by-product when PC is catalyzed by PLD to produce PS(Fig.2).To minimize PA formation,more diverse reaction systems are now used to produce PS.According to the number of phases and the type of solvents,these reaction systems can be classified into single-phase reaction system,biphasic reaction system,and novel eco-solvent reaction system.

5.2.1.Single-phasereactionsystem

The single-phase reaction system is carried out only in the aqueous phase throughout the process.Due to the amphiphilic nature of lecithin,the reaction system is usually in the form of a suspension.Generally,the substrate lecithin is first broken up by homogenizer and then reacted together withL-serine and PLD.Parameters of single-phase reaction systems used for the preparation of PS are summarized in Table 1[88–92].

Table 1 Parameters of PS production catalyzed by PLD in single-phase reaction system.

Liu et al.[88] catalyzed the transphosphatidylation of soybean lecithin using recombinant PLD in single-phase aqueous system,with 53% conversion of PS.Single-phase aqueous systems with no organic solvent residues are suitable for applications in the pharmaceutical and food industries.However,it is undeniable that the single-phase aqueous system leads to the enhanced hydrolytic activity of PLD.Thus,further optimization of the conditions is necessary to reduce the hydrolysis rate of PC and to increase the generation rate of PS.The main current strategies are: (a) solubilization of PC with surfactant to form mixed micelles,(b) use of TritonX-100 and silica as adsorbents of PC.Pinsolle et al.[89]synthesized PS by adding surfactant sodium deoxycholate or sodium cholate to the reaction system and found that their addition inhibited the hydrolysis reaction of PC.Covalent adsorption of PC in single-phase aqueous system using TritonX-100 and silica is also effective with PS yield of up to 99%[90,91].Although the above methods are effective in avoiding the generation of by-products,there are problems such as the complexity of the process and the toxicity of surfactants.

In addition,immobilized enzymes have been used to produce PS in single-phase aqueous system.Mao et al.[92] immobilized PLD on an epoxy-based carrier and then synthesized PS in single-phase aqueous system with 65% yield.The advantage of the immobilized enzyme technique is the ability to simply strip the enzyme from the reaction system,thus allowing the enzyme to be recycled.However,the high cost of immobilized enzymes still remains an obstacle to industrial applications.

5.2.2.Biphasicreactionsystem

PC and PS are soluble in the organic phase,whereas PLD andL-serine are soluble in the aqueous phase.Therefore,biphasic reaction system is generally a water-organic solvent system.Currently,studies on biphasic reaction systems have focused on the selection of organic phases,the volume ratio of two phases,the mass ratio of PC to serine,the reaction temperature,pH,etc.Parameters of biphasic reaction systems used for the preparation of PS are summarized in Table 2[91,93–97].

Table 2 Parameters of PS production catalyzed by PLD in biphasic reaction systems.

For biphasic reaction system,the enzymatic transphosphatidylation efficiency is highly dependent on the type of organic solvents.Hosokawa et al.[93]compared the effects of different organic solvents(ether,ethyl acetate,benzene,toluene,hexane)on the preparation of PS from squid skin lecithin andL-serine catalyzed by PLD.The results showed that ethyl acetate is the best organic solvent for the reaction.In addition,the use of chloroform as an organic solvent also results in a high transphosphatidylation efficiency.The effect of organic solvents on the reaction efficiency mainly consists of the different arrangements and packing density of PC at the interface between the aqueous and organic phases[98].Moreover,the interfacial pressure between the two phases seems to be an important factor[99].

Higher enzymatic transphosphatidylation efficiency was observed in biphasic reaction system compared to single-phase aqueous system.However,the PS produced by biphasic reaction system will have a certain amount of organic solvent residue,which is toxic to the human body.Therefore,the efficient and safe production technique of PS is the current research hotspot.

5.2.3.Noveleco-solventreactionsystem

The hydrolysis reaction in the single-phase reaction system results in a large amount of by-product PA,and the produced PS by biphasic reaction system is unsuitable for addition to food and pharmaceutical products due to the toxicity of the organic solvents.To overcome these obstacles,several researchers have used novel eco-solvent as reaction systems to produce PS.Novel eco-solvent reaction systems used for production of PS are summarized in Table 3 [100–102].

Table 3 Parameters of PS production catalyzed by PLD in eco-solvent reaction system.

Duan and Hu [100,101] reported the enzymatic transphosphatidylation in 2-methyltetrahydrofuran andγ-valerolactone systems and achieved PS yields of 90%and 95%,respectively.Similarly,Bi et al.[102]reported the enzymatic transphosphatidylation in limonene andp-cymene and achieved PS yields of 88%and 95%,respectively.PS yields in eco-solvent reaction systems are approximately the same as those in the biphasic reaction system.Most importantly,these ecosolvents derive from renewable resources such as 2-methyltetrahydrofuran from corn crops,γ-valerolactone from sucrose,limonene,andpcymene from citrus.Thus,producing PS by novel eco-solvent reaction system is non-pollutant to the environment and the product has a highenzymatic transphosphatidylation efficiency.This seems to be a potential direction for future PS productions.

5.2.4.EnzymaticsynthesisofDHA-PS

As mentioned above,DHA-PS has more robust biological functions than PS with other molecular configurations [103].However,most DHA-PS is naturally occurring in marine organisms,such as krill and fish,and represents only a small fraction of the marine phospholipids.Relying only on DHA-PS extracted from marine phospholipids is far from being able to meet the market demand.Therefore,the production of DHA-PS by PLD-catalyzed marine phospholipids is the most efficient way.Parameters of the production of DHA-PS by PLD-catalyzed marine phospholipids are summarized in Table 4[93,104,105].

Table 4 Parameters of the production of DHA-PS by PLD-catalyzed marine phospholipids.

The yields of DHA-PS produced in the two papers in Table 4 were 60.51%and 43.1%,where DHA accounted for 34.1%and 37.6%of the total fatty acids of PS,respectively.The content of DHA in PS is closely related to the DHA content of the substrate PC.DHA-PC in herring seed phospholipids contains 31.4% DHA,and DHA-PS contains 34.1% DHA after enzymatic transphosphatidylation.Currently there are companies using microreactor for the production of DHA-PS,which contains about 20% DHA in PS.

Moreover,PLD-mediated transphosphatidylation of PC or DHA-PC withL-serine is an effective method for the preparation of PS and DHA-PS.Nevertheless,PC and DHA-PC would be converted to the undesirable byproducts PA and DHA-PA because of the hydrolysis activity of PLD.Mao et al.[105]reported a new PLD cloned fromAcinetobacter radioresistensa2 (PLDa2) and applied it in DHA-PS synthesis with high transphosphatidylation activity and no hydrolysis activity.After reaction of 12 h,both the transphosphatidylation conversion rate and the selectivity of PS and DHA-PS were approximately 100%.The underlying mechanism of PLDa2 was also explained by utilizing homology modeling,two-step docking,and binding energy and conformation analysis.

6.Patent evaluation

This evaluation utilized patent data for PS extracted from the Patsnap database (www.zhihuiya.com),renowned as one of the most reliable and comprehensive patent information providers globally.The search query was set as ‘phosphatidylserine’,with a research domain limitation to encompass medical science,food science,and dairy products.A total of 8394 patent families(14,829 patents documents totally)were retrieved and utilized for further insight analysis.Considering that only scattered works have been reported in the early stage without forming a substantial body,the focus of this study was primarily on the innovation development and market situation in the last two decades(2004–2023).

6.1.Innovation trend and technological lifecycle analysis

The evaluation focused on the annual application and authorization trends of PS-related patents,and a Phase-Average Trend Method model(built by the National Bureau of Economic Research) was used to forecast the total number of applications for the years 2022–2025(Fig.3).In addition,a technological lifecycle assessment (LCA) was conducted for the technology,aimed at understanding its development stage and evaluating its future prospects and investment potentials (Fig.4).

Fig.3.Trends in yearly patent applications,issued patents,issuance rate,and predicted trends (updated to Aug.1,2023).

Fig.4.Lifecycle of PS related technology (updated to Aug.1,2023).

Since 2004,there has been an overall upward trend in the number of patent applications related to PS,and it entered a stable period between 2017 and 2021 (Fig.3).It should be noted that the examination and granting of patents is a complex process that typically takes one to two years or even longer.Therefore,when analyzing innovation trends,it is rational to exclude the most recent two years.This assertion is supported by the data pertaining to the patent legal status,where 22.3% of the patents are in a pending state(data not shown).According to the model predictions,there is a possibility of a slight increase in the application volume during the period from 2022 to 2025.At the same time,the results of the LCA analysis suggest that this technology has experienced a period of consistent and stable growth over the past two decades and is still in a growth phase.The illustration of the analysis of technology development stages based on LCA can be seen in other publication[106].Furthermore,when considering the projected application numbers,it is highly likely that this upward trend will persist in the foreseeable future.Furthermore,it can be observed that between 2004 and 2015,the annual authorization rate of patents related to PS consistently exceeded 60%.This is a positive indicator,suggesting that these patents hold commercial value and may possess high market acceptance and technological competitiveness.However,starting from 2016,the authorization rate has gradually declined.Nonetheless,this does not necessarily imply a decrease in application quality or commercial value.The trend of patent authorization concentration over time is illustrated(Fig.5),aiding in the assessment of competition levels and potential monopolization within the technology field over the specified period.There has been a noticeable decrease in authorization concentration since 2011,indicating an increase in market competition.Additionally,the decline in authorization rate is influenced by policies,application volume,and examination processes.In summary,the market for PS-related technologies is significantly large,and its technological innovation is likely to continue to grow in the foreseeable future.However,the increasing market competitiveness undoubtedly imposes higher demands on future research endeavors.

Fig.5.Annual changes in assignee concentration (updated to Aug.1,2023).

6.2.Geographical distributions

A visual representation demonstrates the countries where the initial patent applications were filed,providing insights into the geographical origins of the technology(Fig.6).This could assist users in evaluating a country’s innovation capacity and might indicate the concentration of organizations or highlight the preferred countries for initial investment by companies.Overall,approximately 80%of technological innovation is generated or applied in the United States (46.11%) and China(29.33%).Following that,there are respective percentages of 6.82%,6.74%,and 10.41% for leading innovation applications in EPO,Japan,and other countries (Fig.6a).In more details,the anticipated adoption of this technology varies across different regions.To illustrate,the United States has emerged as the leading marketplace for this technology over the last twenty years.Simultaneously,China’s aspirations for this technology have been steadily escalating,denoting enhanced competitiveness and the prospect of evolving into the foremost market in the future.Nonetheless,it is essential to exercise caution due to the potential delay in patent data disclosure,necessitating ongoing observation of this trend.Moreover,Japan,Korea,and Europe have collectively witnessed a general upswing in their expectations regarding this technology,although Denmark has observed a marginal descent.The rising proportion of technological innovation signifies the strengthening of research and development capabilities as well as innovative capacities in the region.It also implies that the region’s relevant industries possess greater influence and competitive advantages in the international market.Additionally,it may suggest an increasing dependence of other regions on their technology,necessitating more collaboration and exchange to leverage the innovative achievements of the region.In conclusion,it can be anticipated that China and the United States will continue to be the most active and innovative countries in the foreseeable future.This also implies their potential to become the dominant markets in this field.Moreover,it suggests that expanding the dissemination of this technology to other potential markets is a worthwhile consideration.

Fig.6.The geographical distribution (a) and application trend (b) in main countries of patent applications (updated to Aug.1,2023).

6.3.Research interests and key topics

The patents were classified according to their technological themes,and the top 20 themes in terms of publication number were presented(Fig.7a).It can be seen that these themes hold significant positions in the fields of medicine and biomedical research,due to the important physiological and pathological characteristics of PS discussed earlier.Specifically,the research interests are mainly focused on the treatment and diagnosis of diseases and cancer,drug delivery and targeted therapy,immunotherapy,as well as amino acids and peptide drugs.The top 10 International Patent Classification (IPC) technology classifications with the highest representation in this field of research are summarized in Table 5.This result again underscores the commercial value and market potential of PS in the field of pharmaceutical development.Furthermore,these data also demonstrate that the product form may be a crucial consideration in the application of PS.According to the IPC classification data in the field of food science,the adding of PS as health ingredients to food and utilizing them as components in drugs or pharmaceutical formulations are currently the primary considerations for application.These two aspects account for 42.66%and 44.22%of the total patent count,respectively.

Table 5 Top 10 key technology areas based on IPC classification.

Based on the current situation,a further analysis was conducted on the top 20 technical problems involved in the patents (Fig.7b).The results showed that the most frequently mentioned technical problems in PS-related patents covered clinical application limitations,toxicity and side effects,poor taste,poor efficacy,poor solubility,significant side effects,low activity,instability,and others.These issues have significant implications for the safety,efficacy,taste quality,therapeutic effect,solubility,side effects,activity,cost,stability,and application scope of PS-related technologies.This not only reveals the main technical challenges in current research but also indicates the focus of research interests,providing reference for future development.

Through the correlation analysis of these technical problems with the main technical topics,we found that there are prominent problems in drug and drug carrier development as well as targeted therapy,including low stability,high cost,low bioavailability and encapsulation efficiency,as well as potential toxic side effects.This implies that PSrelated carrier technologies are still in an immature stage,and much research effort has been invested in the development of carriers such as liposomes and nonionic surfactant vesicles.Typically,appropriate carrier technologies can significantly enhance the stability of drugs both in vitro and in vivo,thereby increasing their efficacy.By controlling the composition of the materials,targeted drug release can be achieved to fully exploit their physiological effects.However,developing suitable carriers poses challenges,mainly due to increased costs and material safety considerations.Additionally,during storage and usage,carriers may experience instability issues such as separation,expansion,or aggregation of liposomal components.These can compromise the quality and performance of the carriers,preventing them from achieving the desired outcomes.Furthermore,the size distribution and material characteristics of the carriers also affect the drug release rate,necessitating extensive optimization efforts.In conclusion,the development of stable,safe,and cost-effective drug carriers or appropriate application forms remains a critical issue in this field.

7.Future remarks

As discussed above,PS is part of cell structure in the body.The body can synthesis PS in vivo,but most of what it needs comes from foods.Small amounts are found in most foods.PS is important for many functions in the human body,especially in the brain.It has also be taken as a supplement,which was once made from cow brain.Recently,PS is commonly made from PC through a reaction catalyzed by PLD.

There are a few commonly regarded or claimed health benefits,which are also discussed above.We put them in a conclusive way for easy understanding: (1) Memory improvement,in which the most crucial function as a brain supplement is to provide structural support for brain cells;(2)Aids in cognitive enhancement to prevent age-related decline in creating new brain cells;(3)Boosts exercise performance;(4)Helps Alzheimer’s disease symptoms;(5) Treats ADHD symptoms.

Although there were comments on more evidence,EU has authorized the placing on the market of PS from soya phospholipids as a novel food ingredient under Regulation (EC) No 258/97 of the European Parliament and of the Council.Furthermore,even though FDA did not regulate as nootropics and supplements,PS was recognized as FDA-approved food supplement on the list due to its potent effects.Thus,it is not only accepted by researchers that PS has beneficial effect on human health,but also it is approved by the regulation offices or organizations,including the novel food raw material approval from the Chinese authority as mentioned early.

PS is commercially produced nowadays through the catalysis of PLD from purified PC or egg lecithin.More commonly,soya lecithin as a mixture without oil is widely used.The reaction is not complicated.With proper optimization,good yields can be achieved from the state of the art as shown above.On the other hand,PLD is not a common enzyme which is not widely available.Due to the easy start-ups for the production side,the development of the enzyme becomes part of the mission,meaning commercial PLDs are not readily available as most of other food enzymes which are provided by enzyme producers.Development of microbial enzymes is a starting point for being able to start up a production plant of PS.Good part is the screening of a strain as well as engineering the microorganism for optimal performance getting much easier with the development of modern biotechnology and synthetic biology.Thus,this makes huge interest for the innovation activities in terms of patents especially for enzyme development as well as its applications in PS synthesis.There have been huge number of patents in publications either issued or in pending for such a “small” product in recent years,in which majority of the innovation claims are for the enzyme development.

To put the PS in a stronger application portfolio,the study on functions is still weak or not sufficient.More work is needed to elucidate the performance or more clinical evidence is required in terms of different groups of population or diversified groups of population,PS metabolism mechanisms,delivery and delivery systems,dosage and synergy with other nutrients,etc.as well as negative effects being clarified.

Brain health is getting pandemic especially with increasing appearance of chronic disease.The healthy care of brains is a challenging issue.More understanding of the science behind will definitely help find the solutions.Lipids for the brain nutrition have been gained quite a lot of progress for the last decades including docosahexaenoic acid,medium chain triglycerides,etc.An intensive understanding of PS will contribute to future brain health and care.

CRediT Authorship Contribution Statement

Jingnan Chen:conceptualization,formal analysis,investigation,visualization,writing-original draft.Jun Li:conceptualization,formal analysis,funding acquisition,investigation,visualization,writingoriginal draft.Haoyu Xu:formal analysis,investigation,visualization,writing-original draft.Jiaxi Li:formal analysis,investigation,visualization,writing-original draft.Yonghong Yuan:conceptualization,investigation,writing-original draft.Xuebing Xu:conceptualization;supervision,writing-review &editing.Yanlan Bi:conceptualization,writing-review &editing,funding acquisition.

Declaration of Competing Interest

The author Xuebing Xu is an Associate Editor for Grain&Oil Science and Technology and was not involved in the editorial review or the decision to publish this article.The authors declare that there are no conflicts of interest.

Acknowledgements

This study was financially supported by the Innovative Funds Plan of Henan University of Technology (2020ZKCJ10) and Cultivation Programme for Young Backbone Teachers in Henan University of Technology.