CN118028388A

CN118028388A - Synthesis process for preparing glyceride rich in palmitoleic acid

Info

Publication number: CN118028388A
Application number: CN202410146407.9A
Authority: CN
Inventors: 曾春来; 屠越兴; 沈清
Original assignee: Zhejiang Provincial Tongde Hospital Zhejiang Institute Of Mental Health; Zhejiang Gongshang University; Lishui City Peoples Hospital
Current assignee: Zhejiang Provincial Tongde Hospital Zhejiang Institute Of Mental Health; Zhejiang Gongshang University; Lishui City Peoples Hospital
Priority date: 2024-02-02
Filing date: 2024-02-02
Publication date: 2024-05-14

Abstract

The invention discloses a synthesis process for preparing glyceride rich in palmitoleic acid, which comprises the following steps: extracting and separating lipid, refining fish oil and enriching by enzyme-catalyzed transesterification. According to the invention, TAG type crude fish oil extracted from marine fish raw materials is refined, the existing enriched POA-EE is used as an acyl donor, and the enzymatic synthesis reaction parameters of the POA-TAG are optimized by adopting a single-factor optimization method, so that glyceride rich in palmitoleic acid, which is high in content, relatively low in preparation cost and good in economic benefit, is effectively prepared; the physical and chemical indexes of the fish oil are effectively improved through refining; the catalytic enzymes used for the reaction and the optimal reaction conditions are determined so that high levels of POA-TGA can be produced at relatively low cost. The invention utilizes the viscera of fish which are not effectively utilized originally to prepare glyceride rich in palmitoleic acid by a scientific method, and has great economic benefit.

Description

Synthesis process for preparing glyceride rich in palmitoleic acid

Technical Field

The invention relates to the technical field of glyceride preparation, in particular to a synthesis process for preparing glyceride rich in palmitoleic acid.

Background

Palmitoleic acid (palmitoleic acid, POA) is a monounsaturated fatty acid (monounsaturated FATTY ACIDS, MUFA) consisting of 16 carbon atoms and 1 double bond, the unsaturated double bond being located on the 7 th carbon atom of the methyl end (16:1n-7). POA exists in two isomeric forms, including the cis (16:1c9) and trans (16:1t9) isomers. The form of cis-isomer, which is the same as the form of POA endogenously synthesized in humans, is mainly present in nature and has been receiving attention in recent years.

In nature, the POA content of most vegetable sources is relatively low, and the oil crop (calculated as total fatty acid content/%) contents are respectively: 0.25% of camellia, 0.26% of corn, 0.27% of quinoa, 0.15% of buckwheat, 0.08% of linseed, 0.07% of pea, 0.15% of peanut, 0.63% of almond, 0.23% of walnut, 0.29% of hazelnut and 0.23% of chickpea, wherein the contents of the chickpea in the macadamia nut oil and the sea buckthorn fruit oil are more, and 15-22.0% and 12.1-39.0% respectively. The oleum Hippophae is oil extracted from fructus Hippophae pulp, and POA content in oleum Hippophae with high oil content is only 0.64-1.03%. In addition, the POA content varies depending on the place of production and the variety.

In animal sources, POA is common in deep sea fish, such as by analysis of fatty acid composition of 22 important commercial fish in the mouth of the pearl river, the POA content was found to be 6.06-15.5%. The lipid components of different fish products are distinguished from fatty acid structures by natural and genetic characteristics and external factors such as fishing season, geographical source, habitat, diet and the like. For characterization of the fatty acid composition of 34 marine fishes in Mediterranean sea, the POA content is found to be 1.48-19.61%. In addition to deep sea fish, POA is also present in beef and pork, where POA represents 4.7% and 7.4% in the form of triglycerides (triacylglycerol, TAG) and 2.2% and 2.3% in the form of phospholipids (phospholipid, PL), respectively. The POA content in poultry eggs is 2.62-6.54%.

Besides animals and plants, algae are also an important source of POA, and researches show that the POA in chlorella oil, trichlella febrile, phaeodactylum tricornutum and Sclerotinia aurita respectively account for 30.4%, 18.34% and 16.53% of total fatty acids.

POA has the functions of regulating obesity, improving type II diabetes, preventing cardiovascular diseases, preventing nonalcoholic fatty liver diseases and the like, and even has reported that POA also has the functional activities of treating xerophthalmia, preventing osteomalacia, improving skin conditions, resisting inflammation and the like.

Functional fatty acids represented by deep sea fish oil and the like have become important types of dietary supplements, and POA has various physiological functions of regulating obesity, reducing blood sugar, preventing cardiovascular diseases and the like, and has wide development prospect.

In nature, the POA content is low, nuts and seabuckthorn in plants are the best sources of the POA, but the POA is limited by the reserve and the high price of raw material resources, and the development and the utilization of the POA from the plants are not already in the process. The grease rich in the viscera of the fish has rich nutritive value, and is one of important sources of UFA in nature. In recent years, people have generated great interest in comprehensive utilization of lipids in marine byproducts, and the POA in the marine byproducts is effectively screened by combining with a lipidomic technology, so that the method not only responds to a green development strategy, but also has important significance in low exploitation and high utilization of circulating marine resources, accelerating development of comprehensive utilization technology of the marine product processing byproducts, maintaining marine ecological balance and promoting development of circulating economy of the marine product resources.

The natural oil exists in the TAG form in the nature, and has higher bioavailability and bioactivity, and the enrichment method of POA-TAG is rarely studied at present.

Currently, many biopharmaceutical and nutritional supplement enterprises are actively developing POA-based health products and pharmaceutical formulations, some of which have been successfully marketed. POA generally exists in 4 forms: TAG, PL, EE and FFA. The majority of the currently commercial POA-rich products exist in EE and TAG forms. Studies have shown that different administration forms and lipid classes have significant differences in bioavailability, absorption, metabolism and biological activity in vivo. Because the human lipase can catalyze and hydrolyze TAG, the human lipase can provide energy for organisms, promote absorption and metabolism of human bodies and the like, compared with EE type TAG, the human lipase has higher oxidation stability and bioavailability, and can be more effectively digested and absorbed by human bodies. FFA is easily oxidized and PL is low in total lipid and is generally not used for commercial production.

At present, the main methods for enriching POA include urea inclusion method, low-temperature crystallization method, molecular distillation method, microbial bioconversion, chemical method, enzymatic method and the like. Physical or chemical methods are mainly used to enrich POA in EE, ME or FFA form and are not suitable for preparing high levels of POA-TAG. The specific fatty acid TAG can be synthesized under mild condition by using the biological enzyme preparation, and has the advantages of small environmental pollution, high yield and the like. Several methods have been developed to date including esterification, transesterification and hydrolysis to produce specific structured TAGs by lipase-catalyzed reactions. However, in recent years, enzyme-catalyzed enrichment studies have been mainly focused on TAG synthesis rich in n-3 PUFAs, and studies on the synthesis of POA-TAG by an enzyme method have been rarely reported.

The available parts of the edible fishes only account for 50-70% of the weight of the edible fishes, and during the processing, the processing byproducts of viscera, bones, heads and the like of the fishes cannot be well utilized, so that the resource waste is caused. Whether the glyceride rich in the palmitoleic acid can be prepared stably and efficiently by using processing byproducts such as fish viscera and the like through a feasible method has great significance.

Disclosure of Invention

The invention aims to provide a synthesis process for preparing glyceride rich in palmitoleic acid. The invention utilizes the viscera of fish which are not effectively utilized originally to prepare glyceride rich in palmitoleic acid by a scientific method, and has great economic benefit.

The technical scheme of the invention is as follows: the synthesis process for preparing the glyceride rich in palmitoleic acid comprises the following steps:

A. extracting and separating lipid: homogenizing marine fish raw material with ethanol, extracting and separating glyceride, and refrigerating;

B. Refining fish oil: refining the extracted glyceride;

C. enrichment of enzyme-catalyzed transesterification reactions: mixing refined fish oil, POA-EE and immobilized lipase for transesterification.

In the aforementioned synthesis process for preparing glycerol ester rich in palmitoleic acid, the lipid extraction and separation step a comprises the following specific contents:

a1, cleaning marine fish raw materials and carrying out homogenization treatment;

A2, weighing a proper amount of homogenized sample, adding absolute ethyl alcohol according to a feed liquid ratio of 1:10g.mL ^–1, performing ultrasonic treatment for 10min, wherein the ultrasonic specification is that the frequency is 20kHz and the power is 100W;

a3, collecting filtrate, and re-extracting the solid residues with an equal volume of absolute ethyl alcohol for two times;

A4, combining the organic phases, and removing the organic phases by using a rotary evaporator under the vacuum condition of 40 ℃ to obtain crude fat with constant weight;

A5, settling the extracted crude fat for 30min at the temperature of-4 ℃ by using cold acetone, centrifuging to obtain supernatant, and repeating the operation until the supernatant is clear and colorless;

a6, merging the supernatant, removing the organic phase by rotary evaporation to obtain glyceride, and refrigerating at the temperature of-4 ℃ for later use.

In the aforementioned synthesis process for preparing glycerol ester rich in palmitoleic acid, the refined fish oil in step B comprises the following specific contents:

B1, degumming: taking a certain amount of glyceride extracted from marine fish raw materials, adding phosphoric acid with concentration of 60% and adding amount of 1%, continuously stirring at 65 ℃ for 10min, centrifuging for 10min at 5000 r.min ^–1 while hot, and taking the upper layer to obtain degummed fish oil;

B2, deacidification: adding 0.9% NaOH solution with concentration of 10% into degummed fish oil, heating in 70deg.C water bath, stirring for 10min, centrifuging at 5000 r.min ^–1 for 15min, and collecting upper oil phase; adding ultrapure water accounting for 10% of the volume of the fish oil, washing for multiple times, centrifuging for 10min at 5000 r.min ^–1, and separating an oil phase;

B3, decoloring and deodorizing: heating deacidified fish oil in water bath to 60deg.C, adding 5% of the compound agent, stirring at 60deg.C for 30min, centrifuging at 5000 r.min ^–1 min for 15min, collecting upper oil phase to obtain refined fish oil, sealing with nitrogen, and refrigerating at-4deg.C; the compound agent is prepared from active carbon and attapulgite in a ratio of 1.25:1.

In the aforementioned synthesis process for preparing glycerol ester rich in palmitoleic acid, the enzyme-catalyzed transesterification reaction in step C is enriched, and the specific contents thereof are as follows:

Adding POA-EE, refined fish oil and water according to a certain proportion, and stirring and uniformly mixing by using a mechanical stirrer at 200 r.min ^–1; the mass ratio of the refined fish oil to the POA-EE substrate is 1:1-1:5; the addition amount of the water is 0.5% -2.5%; the POA-EE is palmitoleic acid of ester type;

Adding immobilized lipase at 50-65deg.C, filling N2, sealing, placing in a constant temperature water bath shaker with rotation speed of 200r.min ^–1, timing reaction for 20 hr, filtering to remove enzyme, and collecting reaction solution; the immobilized lipase is Novozym 435, lipozyme RM IM or Lipozyme TL IM; the addition amount of the immobilized lipase is 5% -25%.

In the synthesis process for preparing the glyceride rich in palmitoleic acid, the immobilized lipase is Lipozyme RM IM, and the enrichment condition of the enzyme catalysis transesterification reaction is that the mass ratio of the substrate is 1:3, the enzyme addition amount is 15%, and the water addition amount is 2.5%.

In the synthesis process for preparing the glyceride rich in palmitoleic acid, the immobilized lipase is Novozym 435, and the enrichment condition of the enzyme catalysis transesterification reaction is that the mass ratio of the substrate is 1:3, the enzyme addition amount is 15%, and the water addition amount is 0.5%.

In the aforementioned synthesis process for preparing glycerol ester rich in palmitoleic acid, the reaction temperature at which the immobilized lipase is added is 60 ℃.

In the aforementioned synthetic process for preparing glycerol ester rich in palmitoleic acid, the marine fish raw material at least comprises tuna, pomfret, engraulis japonicus Temminck et Schlegel, anchovy and sardine; the marine fish raw material comprises at least viscera, fish heads and fish flesh.

In the synthesis process for preparing the glyceride rich in palmitoleic acid, the marine fish raw material adopts tuna, and the part adopts viscera.

Compared with the prior art, the TAG type crude fish oil extracted from marine fish raw materials is subjected to refining treatment, is used as an enzyme catalytic transesterification reaction substrate, is matched with the existing enriched POA-EE as an acyl donor, optimizes the enzymatic synthesis reaction parameters of the POA-TAG by adopting a single-factor optimization method, and can effectively prepare glyceride rich in palmitoleic acid, high in content, relatively low in preparation cost and good in economic benefit;

According to the invention, physical and chemical indexes of the fish oil are effectively improved through refining, peroxide value, acid value and unsaponifiable matter index are reduced, iodine value is increased, and lipid quality reaches the first-grade refined fish oil industry standard;

the catalytic enzymes used for the reaction and the optimal reaction conditions are determined so that high levels of POA-TGA can be produced at relatively low cost.

Therefore, the invention utilizes the viscera of the fish which are not effectively utilized originally to prepare glyceride rich in palmitoleic acid by a scientific method, and has great economic benefit.

Drawings

FIG. 1 is the effect of different lipases on POA-TAG synthesis in examples of the invention;

FIG. 2 is the effect of substrate mass ratio on POA-TAG transesterification in the examples of the invention;

FIG. 3 is the effect of enzyme addition on POA-TAG transesterification in the examples of the present invention;

FIG. 4 is the effect of the amount of moisture added on POA-TAG transesterification in an embodiment of the invention;

FIG. 5 is a raw material and product POA-TAG content analysis in examples of the present invention;

FIG. 6 is a graph of PCA score of a multivariate statistical analysis of POA-TAG molecular species in the raw material and lipase-catalyzed product of an embodiment of the invention;

FIG. 7 is a graph of PCA load of a multivariate statistical analysis of POA-TAG molecular species in the raw materials and lipase-catalyzed products in an example of the present invention;

FIG. 8 is a graph showing the multivariate statistical analysis of the molecular species POA-TAG in the raw materials and lipase-catalyzed products in the examples of the present invention;

FIG. 9 is a graph showing the multivariate statistical analysis of the molecular species POA-TAG in the raw materials and lipase-catalyzed products in the examples of the present invention;

FIG. 10 is a cluster map of POA-TAG in the raw materials and lipase-catalyzed products in an embodiment of the invention.

Detailed Description

The invention is further illustrated by the following figures and examples, which are not intended to be limiting.

Example 1. The synthesis process for preparing the glyceride rich in palmitoleic acid comprises the following steps:

B. Refining fish oil: refining the extracted glyceride;

The materials and reagents used are:

the used instruments and equipment are as follows:

The common solvents for extracting lipid by solvent method are chloroform-methanol, petroleum ether, isopropanol, n-hexane, ethanol, etc., but the extraction process can pollute the sample and the environment. The ethanol has lower toxicity, the extraction process is greener and safer, and the content of the extracted crude fat is higher, so the invention selects the ethanol as the extraction solvent.

The lipid is extracted and separated in the step A, and the specific content is as follows:

The refined fish oil in the step B comprises the following specific contents:

B3, decoloring and deodorizing: heating deacidified fish oil in water bath to 60deg.C, adding 5% of the compound agent, stirring at 60deg.C for 30min, centrifuging at 5000 r.min ^–1 min for 15min, collecting upper oil phase to obtain refined fish oil, sealing with nitrogen, and refrigerating at-4deg.C; the compound agent is prepared from active carbon and attapulgite in a ratio of 1.25:1

The enrichment of the enzyme-catalyzed transesterification reaction in the step C comprises the following specific contents:

Adding POA-EE, refined fish oil and water according to a certain proportion, and stirring and uniformly mixing by using a mechanical stirrer at 200 r.min ^–1; the mass ratio of the refined fish oil to the POA-EE substrate is 1:1-1:5; the addition amount of the water is 0.5% -2.5%;

The marine fish raw materials at least comprise tuna, pomfret, anchovy and sardine; the marine fish raw material comprises at least viscera, fish heads and fish flesh.

Example 2. The synthesis process for preparing glycerol ester rich in palmitoleic acid was otherwise the same as in example 1, except that: the marine fish raw material adopts tuna viscera.

Example 3. The synthesis process for preparing glycerol ester rich in palmitoleic acid was otherwise identical to example 2, except that:

the immobilized lipase is Lipozyme RM IM, and the enrichment condition of the enzyme catalysis transesterification reaction is that the mass ratio of the substrate is 1:3, the enzyme addition amount is 15%, and the water addition amount is 2.5%.

The reaction temperature at which the addition of the immobilized lipase was carried out was 60 ℃.

Example 4. The synthesis process for preparing glycerol ester rich in palmitoleic acid was otherwise identical to example 2, except that:

the immobilized lipase is Novozym 435, and the enrichment condition of the enzyme catalyzed transesterification reaction is that the mass ratio of the substrate is 1:3, the enzyme addition amount is 15%, and the water addition amount is 0.5%.

Product verification

Measuring physical and chemical indicators

Glyceride extraction rate: m _{Glyceride esters} is the mass (g) of extracted glycerides; m _{Crude fat}: crude fat mass (g) extracted with absolute ethanol;

Recovery rate of refined fish oil: m1 is the mass (g) of the refined fish oil; m2 is the mass (g) of fish oil before refining;

Measurement of peroxide value: the determination is carried out by referring to GB 5009.227-2016 "determination of acid value in food";

determination of acid value: the determination is carried out by referring to GB 5009.229-2016 "determination of acid value in food";

determination of iodine value: the determination is carried out by referring to GB/T5532-2008 animal and plant grease iodine value determination;

determination of unsaponifiable matter: the measurement was carried out with reference to GB/T5535.1-2008 "measurement of unsaponifiable matter of animal and vegetable fats & oils".

Raw material selection

The invention screens viscera POA lipid of six marine fishes such as large-eye tuna, white pomfret, yellow croaker, anchovy and sardine by adopting UHPLC-QE/MS and GC/FID technologies, and analyzes the composition of total fatty acid and FFA in a sample and the molecular species composition and content difference of POA-PL and POA-TAG.

(1) Among the total fat, POA is the main MUFA in the viscera byproducts of 6 fishes, the POA in sardine has the highest relative content of total fatty acid, and then Engraulis japonicus Temminck et Schlegel, tuna, anchovy, yellow croaker and white pomfret have the lowest content; in FFA, the content of POA in viscera by-products of yellow croaker is highest (10.28%), and the content of tuna and white pomfret is lowest, accounting for only 1.55% and 0.63%.

(2) Within the m/z 600-1000, 9 POA-PL, 40 POA-TAG were detected. The results show that the POA-PL content difference in the samples is obvious, and the POA-PL content of white pomfret, tuna and anchovy is highest; the content of POA-TAG in the anchovy is highest, and the tuna and yellow croaker are the next.

(3) The multivariate statistical analysis results show that: the molecular compositions and the contents of POA-PL and POA-TAG of byproducts of different fish species are obviously different: PCA score graph shows that POA-PL and POA-TAG in visceral fish oil of different fish species are well distributed in space; the correlation coefficient heat map constructed based on the pearson coefficients shows the correlation coefficients of the key POA-PL and POA-TAG molecules and reveals highly correlated ions.

In conclusion, the POA-TAG in the viscera byproducts of the tuna is found to have rich composition and higher total content. And the tuna is popular with consumers because of high nutritive value and delicious meat quality, and is always an important biological resource in the ocean fishing industry in China as one of the fishes with the highest economic value. Therefore, the invention adopts TAG type fish oil extracted from the viscera byproducts of tuna as enzyme-catalyzed transesterification substrates.

Results and discussion

Determination result and analysis of physical and chemical indexes of fish oil

Lipid extracted from viscera of tuna has poor fluidity, contains impurities such as protein, phospholipid, pigment, carbohydrate, FFA, and the like, and is not beneficial to research and development and preparation of functional food. Therefore, the extracted glyceride was subjected to a refining treatment before the enrichment of POA-TAG by enzyme-catalyzed transesterification, and the physicochemical index results of the refined sample are shown in Table 1.

TABLE 1 physical and chemical index of fish oil

The results show that: the yield of glyceride extracted from the viscera by-product of tuna is 77.48%, the recovery rate of glyceride is higher and reaches 90.84% after degumming, deacidification, decoloration, deodorization and deodorization operations, the quality of lipid is obviously improved, and the index of peroxide value, acid value and unsaponifiable matters in the refined fish oil is reduced. For example, the unsaponifiable matter content in the crude fish oil reaches the standard of the secondary refined fish oil (less than or equal to 3.0 percent), the unsaponifiable matter content in the fish oil is reduced again after refining, and the unsaponifiable matter content reaches the standard of the primary refined fish oil (less than or equal to 1.0 percent) and is 0.72 percent. In addition, the iodine value increased to eventually reach 163.85g/100g.

Enzymatic transesterification enrichment of POA-TAG Condition optimization

According to the invention, the relative content of POA-TAG in the total TAG in the transesterification product is used as an index, and three common immobilized lipases of Novozym 435, lipozyme RM IM and Lipozyme TL IM are experimentally selected as transesterification catalysts. Since EE and glycerides have good miscibility, they are carried out in the absence of solvents in order to be suitable for functional food or nutraceutical applications. The stirring speed is kept 200 r.min ^–1 in the reaction process, the effective collision between the substrate oil and the enzyme particles is increased, the reaction rate is accelerated, and the mechanical damage to the immobilized lipase is avoided.

The optimal range of the enzymatic synthesis transesterification reaction temperature is 50-65 ℃. The increase of the temperature can accelerate the mass transfer effect and increase the enzyme catalytic reaction speed, but the excessive temperature can cause partial enzyme inactivation, and simultaneously, the fish oil is easy to oxidize and degrade, so that the product quality is influenced. In comprehensive consideration, the fixed reaction temperature is 60 ℃, the reaction time is 20 hours, and the influence of the mass ratio of the substrate, the addition amount of the enzyme (based on the total mass of the reaction substrate) and different water contents (based on the total mass of the reaction substrate) on the efficiency of the catalytic synthesis of POA-TAG by the enzyme is studied.

1. Effect of different lipases on POA-TAG Synthesis

The sources of lipases are different, as are the catalytic capabilities of TAGs. The effect of Novozym 435, lipozyme RM IM and Lipozyme TL IM lipase on POA-TAG synthesis was examined as an acyl donor under the conditions of no solvent system, substrate (refined fish oil) mass 1:5, enzyme loading 10%, water content 1.0%, N ₂ filling and reaction time 20h, and the results are shown in FIG. 1.

The difference of the results of the transesterification synthesis of POA-TAG by different lipases is remarkable, and compared with the POA-TAG content (26.97%) of the refined fish oil, the POA-TAG content in the catalytic product of the Lipozyme RM IM enzyme is remarkably increased to 48.85%, but the catalytic effect is inferior to that of Novozyme 435 (61.62%) and Lipozyme TL IM (60.26%).

Lipozyme RM IM and Lipozyme TL IM enzymes can specifically hydrolyze TAG sn-1,3 fatty acid acyl chains, and are commonly used for acyl exchange between short chain, medium chain or long chain unsaturated fatty acids and sn-1,3 saturated acyl groups to synthesize functional lipids. Palm oil and oleyl alcohol are catalyzed by Lipozyme TL IM enzyme to synthesize palm oil ester, and the conversion rate reaches 79.54%. The transesterification reaction of coconut oil and high oleic rapeseed oil is catalyzed by Lipozyme RM IM enzyme to promote the acyl transfer, and 45.65% novel lipid rich in 1, 3-oleic acid-2-medium chain structure TAG is synthesized. Unlike Lipozyme RM IM and Lipozyme TL IM and enzymes, the structural lipids sn-2 and sn-1,3 synthesized by the Novozyme 435 enzyme under the catalysis of nonpolar conditions are randomly distributed, have no position selectivity, but the enriched POA-TAG has better effect.

In addition, although the acyl transfer positions of the Lipozyme RM IM and the Lipozyme TL IM are the same, the acyl transfer capability of the Lipozyme TL IM is higher and the price is relatively low. Thus, lipozyme TL IM and Novozym 435 enzymes were selected for subsequent preparation.

2. Effect of substrate Mass ratio on POA-TAG Synthesis

The refined fish oil and POA-EE are respectively mixed according to substrate mass ratio of 1:1, 1:2, 1:3, 1:4 and 1:5, and under the conditions that Lipozyme TL IM and Novozyme 435 enzyme loading amount is 15% and water content is 1.0%, the result of synthesizing POA-TAG is shown in figure 2.

In the enzyme catalytic reaction process, the substrate mass ratio is obvious in the content difference of the synthetic POA-TAG. The binding rate of the catalytic synthesis of POA-TAG by Novozym 435 and Lipozyme TL IM enzyme is in a trend of rising and then falling along with the increase of the POA-EE content in the substrate mass ratio. Since the viscosity of glyceride is higher than that of POA-EE, excessive glyceride may form a film on the surface of lipase during the reaction process, which hinders the contact between the substrate and the enzyme active site, thus inhibiting the enzyme-catalyzed transesterification reaction.

The catalytic effects of Lipozyme TL IM and Novozyme 435 were the worst with substrate mass ratios of 1:1, and the relative POA-TAG contents were 47.41% and 46.42%, respectively. As the reaction proceeds, the fatty acid on the glycerol backbone is replaced, resulting in a decrease in the POA content in the reaction system and a decrease in the reaction rate, and thus, an increase in the POA content contributes to the progress of the reaction. However, too high a substrate mass ratio, an excessive saturation of the substrate concentration may prevent adequate contact of the lipase with the substrate, resulting in a reduction of the final target lipid content. The preparation of DHA-rich medium-long chain TAG by lipase-catalyzed acidolysis of schizochytrium microbial oil and caprylic acid shows that a higher substrate molar ratio (caprylic acid/microbial oil) accelerates the initial reaction rate, but excessive substrate reduces the reaction sites, thereby resulting in reduced content of synthesized target products.

The invention discovers that the catalytic effect of Lipozyme TL IM and Novozyme 435 enzyme is best when the substrate mass ratio is 1:4, and the relative content of POA-TAG reaches 61.82 percent and 63.30 percent respectively. However, excessive reaction substrates can cause great difficulty in separation and purification of POA-TAG, and the production cost is high, so that the preparation is carried out by selecting the substrate mass ratio of 1:3.

3. Effect of enzyme addition on POA-TAG Synthesis

The large enzyme load can shorten the reaction time, quicken the reaction speed and weaken the influence of acyl migration to promote the synthesis of structural lipid. The immobilized lipase participates in the reaction but is not consumed, and thus can be recycled. However, in the actual reaction process, the structure of the lipase may be irreversibly changed due to high temperature or physical stirring, the enzyme activity may be reduced in the presence of an organic solvent, and the recovery of the immobilized enzyme may be destroyed due to elution. Therefore, in the preparation process, the balance of reaction efficiency and economic benefit is required, and the use amount of lipase is reduced as much as possible so as to further reduce the production cost.

The invention determines the effect of Lipozyme TL IM or Novozyme 435 enzyme loading on the catalytic effect when the substrate mass ratio is 1:3 and the water content is 1.0%, and the result of synthesizing POA-TAG is shown in figure 3.

As the catalytic effect trend of Lipozyme TL IM and Novozyme 435 is the same with the increase of the enzyme addition amount (5-15%), the binding rate of POA-TAG is on the whole on the rising trend, probably because the enzyme catalyzed transesterification reaction rate is improved with the increase of the enzyme content, more reaction sites are provided at the same time, and the binding rate of POA-TAG starts to rise slowly with the continuous increase of the enzyme content (15-25%) and then falls. At 20% enzyme addition, lipozyme TL IM and Novozyme 435 enzymes had the best catalytic effects, and POA-TAG contents were 62.59% and 64.17%, respectively; the difference in POA-TAG content was not significant at enzyme addition levels of 15% -25% (p < 0.05). Excessive enzyme load can cause lipase aggregation, increase mass transfer resistance of the reaction and is unfavorable for the reaction. In comprehensive consideration, the preparation is carried out by selecting the addition amount of enzyme to be 15%.

4. Effect of moisture addition on POA-TAG Synthesis

In immobilized lipase-catalyzed reactions, water can maintain the structure and flexibility of the enzyme and form acyl-enzyme complexes, and can also enhance enzyme activity by providing a good protective layer around the enzyme and lowering the dielectric constant of the reaction mixture.

The optimal water content in the reaction is dependent on the type of lipase, the immobilization technique used, the solvent system, etc.

To investigate the effect of moisture addition on the enzyme-catalyzed synthesis of POA-TAG, 0.5-2.5% water was added to the reaction mixture and the results of the synthesis of POA-TAG at a substrate mass ratio of 1:3, lipozyme TL IM or Novozyme 435 enzyme loading of 15% were shown in FIG. 4.

The relative amount of POA-TAG in the Lipozyme TL IM enzyme catalyzed product slowly increased with increasing moisture addition, with the POA-TAG content being highest when the moisture content was 2.5% (67.50%). However, the catalysis effect of Novozym 435 enzyme is opposite, the increase of moisture content inhibits the synthesis of POA-TAG, for example, the catalysis effect of Novozym 435 enzyme is optimal when the minimum water content is 0.5%, and the binding rate of POA is up to 67.45%; whereas when the water content was increased to 2.5%, the POA binding rate was only 52.85%. Water may participate in the transesterification reaction, thereby affecting the reaction equilibrium. But when the water content reaches a certain level, fatty acid acyl transfer to the active site is prevented, hydrolysis of TAG is promoted, and the conversion rate of target lipid is reduced. In conclusion, high moisture content may lead to aggregation of immobilized Novozym 435 enzyme, so catalytic reaction is more prone to be carried out under low moisture content (0.5%). Whereas Lipozyme TL IM enzyme-catalyzed transesterification reactions are more prone to proceed at higher moisture levels, with an optimum moisture content of 2.5%.

Structure characterization of refined fish oil and catalytic product

From the analysis, the substrate mass ratio (1:3) and enzyme loading (15%) were the same in the optimal conditions for Lipozyme TL IM and Novozyme 435 enzyme-catalyzed transesterification to POA-TAG, but there was a significant difference in moisture content. Thus, the catalytic products POA-TAG molecules were characterized for two enzymes under different moisture levels (0.5% and 2.5%), respectively, with the aim of exploring the mechanism of influence of moisture levels on the catalytic reactions of the different enzymes.

1. Analysis of the composition of the starting Material and catalytic product POA-TAG

41 POA-TAG molecules were detected in total from the purified fish oil and the products, and the relative content of the identified POA-TAG molecules was determined by normalization, and the results are shown in Table 2.

A total of 30 TAG molecules containing single strand POA and 10 TAG molecules containing double strand POA were detected. The TAG skeleton mainly comprises C12:0, C14:0, C15:0, C16:0, C17:0, C17:1, C18:0, C18:1, C18:2, C18:3, C19:0, C20:1, C20:4, C20:5, C22:1, C22:5 and C22:6 except for the POA acyl chain. The relative content of TAG 16:1/20:5/22:6、TAG 16:1/18:1/18:1、TAG 16:1/16:1/20:4、TAG 16:1/16:1/18:1、TAG 16:1/16:1/16:1、TAG 16:0/16:1/22:6 and TAG 16:0/16:1/18:1 in the raw materials is higher (> 1.0%), wherein the content of TAG 16:1/16:1/20:4 is the highest and reaches 5.56%. The relative amounts of TAG 16:1/16:1/18:1, TAG 16:1/16:1/16:1, TAG 16:0/16:1/18:1 in the enzyme catalytic products were higher (> 3.0%) at different moisture levels of Lipozyme TL IM and Novozyme 435. Wherein, the highest content of POA-TAG molecules is TAG 16:1/16:1/16:1, the water addition amount is respectively 15.92% and 28.55% when 0.5%, and the water addition amount is 30.55% and 27.10% when 2.5%

TABLE 2 analysis of the composition of the raw materials and products POA-TAG

Note that: ¹: at 0.5% moisture, lipozyme tlim and Novozym 435 enzymes catalyze transesterification reactions; ²: at a water content of 2.5%, lipozymeTL IM and Novozym 435 enzymes catalyze transesterification reactions.

The relative content of TAG 16:1/18:2/19:0、TAG 16:1/18:1/22:1、TAG 16:1/18:1/19:0、TAG 16:1/16:1/22:5、TAG 16:1/16:1/19:0、TAG 16:1/16:1/18:1、TAG 16:1/16:1/17:0、TAG 16:1/16:1/16:1、TAG 16:0/16:1/18:2、TAG 15:0/16:1/16:1 in the product is obviously increased (p < 0.05). In the above products, other fatty acid chains than POA chains on the glycerol backbone are mostly SFA or MUFA (chain length. Ltoreq.20), and small amounts are PUFAs, indicating that the reaction is inert to the melting of SFA or MUFA due to lipase selectivity, or the reaction is near equilibrium but does not reach equilibrium point [118]. The relative content of POA-TAG products containing long-chain (chain length is more than or equal to 20) PUFAs on the TAG glycerol skeleton in the products is obviously reduced (p < 0.05), such as TAG 16:1/20:5/22:6, TAG 16:1/16:1/20:4 and TAG 16:0/16:1/22:6, and the TAG content of PUFAs in the system is reduced probably because the Lipozyme TL IM and Novozyme 435 enzymes have stronger activity on the PUFAs, so that the POA and the PUFAs in the substrate are effectively replaced.

2. The moisture content influences the search of the enzyme catalysis mechanism

The difference in POA composition and content on TAG backbone in the catalytic products of Lipozyme TL IM and Novozyme 435 enzyme is shown in FIG. 5. The content of TAG molecules (P-TAG or P-P-TAG) containing single-stranded or double-stranded POA varies smoothly, but the difference of TAG molecules (P-P-P) containing three-stranded POA is significant. The content of P-P-P in refined fish oil is only 1.74%, and when the water content is 2.5%, the content of P-P-P generated by the catalysis of Lipozyme TL IM enzyme is the highest, namely 30.55%.

In FIG. 5, P-TAG: one chain of the TAG framework is POA; P-P-TAG: the two chains on the TAG framework are POA; P-P-P: the three chains on the TAG framework are POA; total POA-TAG: the TAG carbon skeleton contains POA.

The effect of moisture content on the catalysis of the Novozym 435 enzyme to produce P-P-P was insignificant, but at low moisture content, P-TAG and P-P-TAG were more readily produced. The moisture content can stabilize the overall structure of the protein via hydrogen bonding, helping to stabilize the active site of the enzyme so that more substrate is bound to the active site. And a water molecule with a lone pair of electrons can form a nucleophilic attack on the carbon atom of the secondary carbonyl group of the DAG-enzyme complex, resulting in an accelerated acyl migration rate [125]. In the study, the catalytic activity of the Lipozyme TL IM enzyme is stronger when the moisture content is 2.5%, and the sn-2 position sn-2/3 or sn-1/2 position in the raw material possibly contains rich POA fatty acid acyl chains, which is more favorable for the selective catalysis of the sn-1/3 position by the Lipozyme TL IM enzyme to generate P-P-P. However, with the increase of the water activity in the reaction system, the polarity of the reaction medium is increased, so that the activation energy of the reaction is finally influenced, and the acyl migration rate is further influenced, and the Novozym 435 enzyme is more suitable for catalysis under the condition of 0.5% of the water addition amount. Different immobilization supports or different physicochemical properties may also lead to different parameters of the enzyme-catalyzed reaction, such as immobilization of Novozym 435 with macroporous acrylic resin and immobilization of LipozymeTL IM with silica gel.

POA-TAG molecular species change characterization

As a result of the above analysis, the difference between the species and the content of POA-TAG molecules was found. Thus, based on chemometrics, multiplex statistical analyses were performed on POA-TAG products produced by transesterification of Lipozyme TL IM and Novozyme 435 enzymes at different moisture levels, and the results are shown in FIGS. 6-9.

In FIGS. 6-9, A: refining fish oil; b: lipozyme TL IM catalytic reaction at 0.5% moisture; c: novozym 435 enzyme catalyzed reaction at 0.5% moisture; d: lipozyme TL IM enzyme catalyzed reaction at 2.5% moisture; e: novozym 435 enzyme catalyzed reaction at 2.5% moisture content.

From the PCA score plot (FIG. 6), the raw material and the catalytic product were divided into two clusters and dispersed in different regions, indicating that the difference in POA-TAG molecular species and content of the raw material and the enzymatic catalytic product was significant. In addition to the Lipozyme TL IM enzyme catalytic product at a water content of 0.5%, the catalytic products under the other three conditions were found to aggregate in the figure, indicating that the composition and content of POA-TAG in the product were relatively similar compared to the starting material. Because the difference between the raw materials and the products is large, the raw materials are removed for dimension reduction treatment, and the 4 enzyme catalysis samples are found to be well distributed in space.

The load diagram shows the characteristic molecular species of the differences in the feedstock and catalytic products: TAG 16:1/16:1/18:1, TAG16:1/16:1/16:1 and TAG 16:0/16:1/22:6, the content differences are shown in FIG. 7.

The variable significance map (variable importance, VIP) analysis is a weighted sum of squares of partial least squares (PARTIAL LEAST squares, PLS) loadings, and the results of fold-of-variance obtained in combination with univariate analysis are used to screen for significant differential markers, typically setting a threshold VIP >1, describing the contribution of a single variable to the sample population.

From the VIP graph, TAG16:1/16:1, TAG 16:1/20:4, TAG 16:0/16:1/22:6 and TAG 16:0/16:1/18:1 are main characteristic molecular species of enzyme-catalyzed transesterification products.

The cluster heat map is subjected to visual analysis through color intensity and similarity degree, and further reflects the similarity and difference of POA-TAG molecular species and content in the raw materials and enzyme catalysis products (red represents relatively higher content of corresponding ions and blue represents opposite). As can be seen from FIG. 10, the refined fish oil feedstock and the enzyme-catalyzed products fall into two main categories. Among the enzyme-catalyzed reaction groups, the Novozym 435 enzyme-catalyzed reaction group at a water content of 2.5% had a significant difference in content from the other three enzyme-catalyzed reaction groups, while the Novozym 435 enzyme-catalyzed reaction group at a water content of 0.5% was classified into a subclass with the Lipozyme TL IM enzyme-catalyzed reaction group at a water content of 2.5%, indicating that the two enzymes had a high similarity in the molecular species and content of POA-TAG produced, although the catalytic conditions were different.

In fig. 10, a: refining fish oil; b: lipozyme TL IM catalytic reaction at 0.5% moisture; c: novozym 435 enzyme catalyzed reaction at 0.5% moisture; d: lipozyme TL IM enzyme catalyzed reaction at 2.5% moisture; e: novozym 435 enzyme catalyzed reaction at 2.5% moisture content.

Claims

1. The synthesis process for preparing the glyceride rich in palmitoleic acid is characterized by comprising the following steps of:

B. Refining fish oil: refining the extracted glyceride;

2. The synthetic process for preparing palmitoleic acid-rich glycerides according to claim 1, wherein the lipid extraction and separation in step a is as follows:

3. The synthetic process for preparing glycerol ester rich in palmitoleic acid according to claim 1, wherein the refined fish oil in step B comprises the following specific contents:

4. The synthetic process for preparing glycerol ester enriched in palmitoleic acid according to claim 1, wherein the enzyme-catalyzed transesterification enrichment of step C is as follows:

Adding immobilized lipase at 50-65deg.C, filling N ₂, sealing, placing in a constant temperature water bath shaker with rotation speed of 200r.min ^–1, timing reaction for 20 hr, filtering to remove enzyme, and collecting reaction solution; the immobilized lipase is Novozym 435, lipozyme RM IM or Lipozyme TL IM; the addition amount of the immobilized lipase is 5% -25%.

5. The synthetic process for preparing palmitoleic acid-rich glycerides according to claim 4, wherein:

6. The synthetic process for preparing palmitoleic acid-rich glycerides according to claim 4, wherein:

7. The synthetic process for preparing palmitoleic acid-rich glycerides according to claim 4, wherein: the reaction temperature at which the addition of the immobilized lipase was carried out was 60 ℃.

8. The synthetic process for preparing palmitoleic acid-rich glycerides according to claim 1, wherein: the marine fish raw materials at least comprise tuna, pomfret, anchovy and sardine; the marine fish raw material comprises at least viscera, fish heads and fish flesh.

9. The synthetic process for preparing palmitoleic acid-rich glycerides according to claim 8, wherein: the marine fish is prepared from tuna, and viscera are used at the position.