A food-grade composite FAS inhibitor for increasing the DHA production of Schizochytrium Sn-2, method and application thereof

Abstract

The present invention belongs to the technical field of biochemical engineering, and discloses a food-grade composite FAS inhibitor for increasing the production of Sn-2DHA of Schizochytrium, wherein the inhibitor is composed of clove extract, resveratrol and epigallocatechin gallate (EGCG). The present invention uses Schizochytrium as a production strain. A food-grade composite FAS inhibitor is added during the fermentation process to reduce the content of saturated fatty acids and increase the content of Sn-2DHA. The present invention first determines the optimal addition amounts of clove extract, resveratrol, and epigallocatechin gallate (EGCG) respectively through a single factor experiment; and further determines the optimal ratio of the composite FAS inhibitor through an orthogonal experiment. In addition, the present invention also conducts a synergistic effect test to determine that the three FAS inhibitors have a synergistic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular to a food-grade composite FAS inhibitor for improving the DHA yield of Schizochytrium Sn-2, a method and an application thereof. Background Art

[0002] Unsaturated fatty acids, especially Sn-2 DHA, can not only promote the development of infant brains, but also play an important role in anti-inflammation, anti-oxidation, and immune regulation. It is an essential ω-3 PUFA for the human body and one of the raw materials for infant formula and various health products. Saturated fatty acids are usually the most direct source of increased cholesterol. Therefore, increasing unsaturated fatty acids and reducing saturated fatty acid intake has become a consensus in the public diet. In addition, due to the current pollution of the marine environment, blindly catching deep-sea fish oil to obtain unsaturated fatty acids is not sustainable. Therefore, biomanufacturing is considered to be a green and highly promising path.

[0003] Schiochytrium sp. is a marine heterotrophic eukaryotic microorganism with advantages such as fast growth rate and high robustness. It has been widely used in scientific research and industrial production. Among them, the biggest advantage of Schiochytrium sp. is that it has an efficient polyketide synthase pathway (PKS), which can efficiently synthesize polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (Sn-2DHA). Some wild-type Schiochytrium sp., without fermentation condition optimization or metabolic engineering, have a total lipid content of more than 55% of cell dry weight (DCW), and Sn-2 DHA accounts for 40-50%. However, when the fatty acid profile of the fermentation product was analyzed, it was found that there were still high levels of saturated fatty acids (e.g., C14:0, C16:0). The possible reason is that fatty acid synthase (FAS) exists in Schiochytrium sp., and this hypothesis has also been confirmed in the sequencing results of the Schiochytrium sp. genome. Therefore, the present invention hopes to reduce the content of saturated fatty acids by utilizing FAS inhibitors, so that more carbon metabolic flow will flow to the production of unsaturated fatty acids, especially Sn-2 DHA.

[0004] Although related FAS inhibitors are currently used in Schizochytrium fermentation culture to increase the production of polyunsaturated fatty acids. For example, Sun Haoxin et al. (CN202310422205.8) increased EPA (eicosapentaenoic acid) production by adding iodoacetamide (a FAS inhibitor). However, iodoacetamide has a certain irritation and a high production cost (120 yuan / g), so this FAS inhibitor is not suitable for industrial production. Based on this, the three food-grade FAS inhibitors selected by the present invention are not only safe, but also inexpensive (<5 yuan / g). Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a food-grade composite FAS inhibitor, method and application for increasing the yield of Schizochytrium SN-2DHA.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] A food-grade composite FAS inhibitor for increasing the DHA production of Schizochytrium Sn-2. The inhibitor consists of clove extract, resveratrol and epigallocatechin gallate (EGCG).

[0008] Furthermore, the final concentration of the clove extract added is 120 mg / L, the final concentration of resveratrol added is 60 mg / L, and the final concentration of epigallocatechin gallate EGCG added is 160 mg / L.

[0009] The fermentation method for increasing the DHA content of Schizochytrium Sn-2 by using the food-grade composite FAS inhibitor as described above comprises the following steps:

[0010] (1) Take a tube of Schizochytrium preservation tube culture medium from a -80°C refrigerator, add 50 mL of Schizochytrium seed liquid culture medium, culture at 28°C, 170 rpm, and add the corresponding FAS inhibitor;

[0011] (2) Every 24 hours, 1 mL of Schizochytrium seed liquid was transferred to fresh seed liquid culture medium, and the transfer was repeated twice to obtain the third generation seed liquid;

[0012] (3) 10 mL of the third generation seed solution was added to 100 mL of Schizochytrium fermentation medium, and the glucose content was measured every 12 h. The glucose concentration was supplemented to 80 g / L, and the culture conditions were the same as above, and the culture was continued for 5 days to obtain Sn-2DHA by fermentation;

[0013] Each time the cells are transferred to fresh culture medium, corresponding FAS inhibitors need to be added.

[0014] Furthermore, the Schizochytrium is selected from the wild type Schizochytrium HX-308, and the deposit number is CCTCC No. M209059.

[0015] The use of the food-grade composite FAS inhibitor as described above in increasing the DHA content of Schizochytrium Sn-2.

[0016] The advantages and positive effects achieved by the present invention are:

[0017] 1. In the process of Schizochytrium fermentation, the present invention additionally adds a composite FAS inhibitor to inhibit the content of saturated fatty acids, increase the content of Sn-2 DHA, improve the nutritional value of Schizochytrium algae oil, and expand its market application.

[0018] 2. The FAS inhibitors added in the present invention are all food-grade and cheap, which not only avoids the cost of removing the inhibitors, but also does not significantly increase the production cost by adding the inhibitors. At the same time, the present invention is simple to operate and has universal applicability.

[0019] 3. The present invention obtains a food-grade composite lipase (FAS) inhibitor through experiments, the saturated fatty acid content is reduced by 6.47%, and Sn-2 DHA is increased by 17.39%, providing a wider market prospect for Schizochytrium algae oil.

[0020] 4. The present invention uses single factor test, orthogonal test and other optimization tests to determine the optimal formula of the optimal composite FAS inhibitor: the addition amount of clove extract is 120 mg / L, the addition amount of resveratrol is 60 mg / L, and the addition amount of EGCG is 160 mg / L). The fatty acid profile data at the end of fermentation showed that the saturated fatty acid content decreased by 6.47% and Sn-2 DHA increased by 17.39%. It provides a wider market prospect for Schizochytrium algae oil. In addition, the present invention is simple and easy to operate and has strong universal adaptability.

[0021] 5. The present invention uses Schizochytrium as a production strain. A food-grade composite FAS inhibitor is added during the fermentation process to reduce the content of saturated fatty acids and increase the content of Sn-2 DHA. The present invention first determines the optimal addition amounts of clove extract, resveratrol, and epigallocatechin gallate (EGCG) through single-factor experiments; and further determines the optimal ratio of the composite FAS inhibitor through orthogonal experiments. In addition, the present invention also conducts a synergistic effect test and determines that the three FAS inhibitors have a synergistic effect.

[0022] 6. According to the search of existing patents, although Sun Haoxin et al. (CN202310422205.8) increased EPA production by adding iodoacetamide, the three food-grade FAS inhibitors used in the present invention have certain advantages in terms of product safety and low production cost. At the same time, according to experimental results, the three FAS inhibitors have certain synergistic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of different addition amounts of clove extract on the content of C14:0+C16:0 in the fermentation product of the present invention;

[0024] Figure 2This is a graph showing the effect of different addition amounts of clove extract on the content of DHA in the fermentation product Sn-2;

[0025] Figure 3 This is a graph showing the effect of different amounts of resveratrol added on the content of C14:0+C16:0 in the fermentation product of the present invention;

[0026] Figure 4 This is a graph showing the effect of different amounts of resveratrol added on the content of DHA in the fermentation product Sn-2 in the present invention;

[0027] Figure 5 This is a graph showing the effect of different addition amounts of EGCG on the content of C14:0+C16:0 in the fermentation product of the present invention;

[0028] Figure 6 This is a graph showing the effect of different addition amounts of EGCG on the DHA content of the fermentation product Sn-2 in the present invention;

[0029] Figure 7 This is a graph showing the effects of different composite FAS inhibitors on the content of C14:0+C16:0 in the fermentation product of the present invention;

[0030] Figure 8 This is a graph showing the effect of different composite FAS inhibitors on the DHA content of the fermentation product Sn-2 in the present invention;

[0031] Fig. 9 This is a diagram for verifying whether there is a synergistic effect between the three FAS inhibitors using the C14:0+C16:0 content as an indicator in the present invention;

[0032] Fig.10 This is a diagram for verifying whether there is a synergistic effect between the three FAS inhibitors in the present invention using the Sn-2 DHA content as an indicator. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0034] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0035] A food-grade composite FAS inhibitor for increasing the DHA production of Schizochytrium Sn-2. The inhibitor consists of clove extract, resveratrol and epigallocatechin gallate (EGCG).

[0036] Preferably, the final concentration of the clove extract is 120 mg / L, the final concentration of resveratrol is 60 mg / L, and the final concentration of epigallocatechin gallate EGCG is 160 mg / L.

[0037] The fermentation method for increasing the DHA content of Schizochytrium Sn-2 by using the food-grade composite FAS inhibitor as described above comprises the following steps:

[0038] (1) Take a tube of Schizochytrium preservation tube culture medium from a -80°C refrigerator, add 50 mL of Schizochytrium seed liquid culture medium, culture at 28°C, 170 rpm, and add the corresponding FAS inhibitor;

[0039] (2) Every 24 hours, 1 mL of Schizochytrium seed liquid was transferred to fresh seed liquid culture medium, and the transfer was repeated twice to obtain the third generation seed liquid;

[0040] (3) 10 mL of the third generation seed solution was added to 100 mL of Schizochytrium fermentation medium, and the glucose content was measured every 12 h. The glucose concentration was supplemented to 80 g / L, and the culture conditions were the same as above, and the culture was continued for 5 days to obtain Sn-2DHA by fermentation;

[0041] Each time the cells are transferred to fresh culture medium, corresponding FAS inhibitors need to be added.

[0042] Preferably, the Schizochytrium is wild-type Schizochytrium HX-308, with the deposit number being CCTCC No.M209059.

[0043] The use of the food-grade composite FAS inhibitor as described above in increasing the DHA content of Schizochytrium Sn-2.

[0044] Specifically, the relevant preparation and detection are as follows:

[0045] The culture medium used in the following examples is as follows:

[0046] Seed liquid culture medium: glucose: 50g / L, potassium dihydrogen phosphate: 4g / L, yeast extract: 4g / L, sodium sulfate: 20g / L, magnesium sulfate heptahydrate: 4.6g / L, ammonium sulfate: 4.8g / L, sodium chloride: 1.25g / L, potassium chloride: 1g / L, monosodium glutamate: 20g / L, 0.1% trace elements, including: vitamin B10.5 mg / L, vitamin B60.5 mg / L, vitamin B 12 0.25μg / L.

[0047] Fermentation medium: glucose: 10g / L, potassium dihydrogen phosphate: 4g / L, yeast extract: 4g / L, sodium sulfate: 12g / L, magnesium sulfate heptahydrate: 2g / L, ammonium sulfate: 4g / L, sodium chloride: 0.5g / L, potassium sulfate: 0.7g / L, calcium chloride: 0.15g / L, monosodium glutamate: 20g / L, 0.1% trace elements, including: vitamin B10.5 mg / L, vitamin B60.5 mg / L, vitamin B 12 0.25μg / L.

[0048] During the fermentation process of Schizochytrium, the corresponding concentration of FAS inhibitor needs to be added to the fresh culture medium each time.

[0049] The present invention designs and implements a single factor experiment. First, different concentrations of FAS inhibitor solutions are prepared, the required reagent addition amount in a certain volume is calculated, and all are dissolved in ddH2O, and then sterilized by filtering with a 0.22 μm filter membrane. During the extraction process of Schizochytrium fermentation, the control experiment is to add no FAS inhibitor, and the test group 1 is to add clove extract to the culture medium, adding 30 mg / L, 60 mg / L, 90 mg / L, 120 mg / L, 150 mg / L, 180 mg / L respectively; the test group 2 is to add resveratrol to the culture medium, adding 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L respectively; the test group 3 is to add epigallocatechin gallate (EGCG) to the culture medium, adding 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L, 160 mg / L respectively. Then, fermentation experiments were carried out and the fatty acid content at the end point of fermentation was determined.

[0050] The present invention designs and implements an orthogonal experiment. According to the results of the single factor experiment, three factors and three levels of the orthogonal experiment are determined (the amount of clove extract added is 90 mg / L, 120 mg / L, 150 mg / L; the amount of resveratrol added is 60 mg / L, 80 mg / L, 100 mg / L; the amount of EGCG added is 120 mg / L, 140 mg / L, 160 mg / L). Subsequently, a fermentation experiment is performed, and the fatty acid content at the fermentation end point is determined.

[0051] The present invention designs and implements a synergistic effect test. According to the orthogonal test results, the optimal FAS inhibitor complex ratio for improving Sn-2 DHA is determined. The synergistic effect test is further used to verify whether the three groups of components have a synergistic effect. Example 1 is to add the optimal composite surfactant during the fermentation process of Schizochytrium; Comparative Example 1 is to add only clove extract and resveratrol; Comparative Example 2 is to add only clove extract and EGCG; Comparative Example 3 is to add only resveratrol and EGCG. Subsequently, a fermentation experiment was carried out, and the fatty acid content at the end of the fermentation was determined.

[0052] Example 1

[0053] Single factor experiment was used to determine the optimal addition amount of clove extract in three groups.

[0054] (1) Take a tube of HX-308 bacterial culture medium from a -80°C refrigerator, add 50 mL of Schizochytrium seed culture medium, and culture at 28°C and 170 rpm;

[0055] (2) Every 24 hours, 1 mL of Schizochytrium seed liquid was transferred to fresh seed liquid culture medium. This was repeated twice to obtain the third generation of seeds.

[0056] (3) Take 10 mL of the third generation seed solution and add it to 100 mL of Schizochytrium fermentation medium. Measure the glucose content every 12 hours and add the glucose concentration to 80 g / L. Culture for 5 consecutive days under the same culture conditions as above. Measure the remaining glucose in the fermentation medium every 12 hours and add the glucose concentration to 80 g / L. Each time the culture medium is transferred to fresh medium, different concentrations of clove extract (30 mg / L, 60 mg / L, 90 mg / L, 120 mg / L, 150 mg / L, 180 mg / L) need to be added to the medium at the same time.

[0057] (4) Treatment of bacterial samples. After the fermentation is completed, take 1 mL of bacterial solution into a 2 mL centrifuge tube and vacuum freeze-dry it (vacuum degree: ≤1Pa, temperature: ≤-50℃) for 48 hours. Then, the sample is methylated. The specific operation is as follows: add 600uLNaOH / methanol solution (NaOH concentration: 1M) to the 2 mL centrifuge tube containing the sample, shake overnight, add 30μL sulfuric acid and 1mL n-hexane, and continue shaking for 2 hours to complete the methylation reaction. Centrifuge the sample at 13,000g for 1 minute, aspirate the supernatant, and filter it through a 0.22μm filter membrane into a gas phase vial.

[0058] (5) Sample gas phase treatment and data analysis. The gas chromatography system is GC-7890A produced by Agilent. The GC system is equipped with a capillary column (HP-88, 60m×0.25mm×0.2μm) and a hydrogen flame ionization detector (FID). The injection temperature is 220℃ and the detector temperature is 250℃. Program temperature rise: initial temperature is 150℃, lasting for 5min; 150-170℃, heating rate is 3℃ / min, maintained for 5min; 170-210℃, heating rate is 3℃ / min, maintained for 5min. Carrier gas: helium; split ratio is 10:1; injection volume: 1μL. The results are as follows: Figure 1 , Figure 2 As shown, when the addition amount of clove extract was 90 mg / L, 120 mg / L, and 150 mg / L, the C14:0+C16:0 content in the fermentation broth at the end of fermentation was lower, while the Sn-2 DHA content was higher.

[0059] Example 2

[0060] Single factor experiment was used to determine the optimal amount of resveratrol added to the three groups.

[0061] During the fermentation of Schizochytrium, different concentrations of clove extract were replaced with different concentrations of resveratrol. Other fermentation conditions and sample treatment of Schizochytrium were the same as those in Example 1. Figure 3 , Figure 4 As shown, when the resveratrol addition amount was 60 mg / L, 80 mg / L, and 100 mg / L, the C14:0+C16:0 content in the fermentation broth at the end of fermentation was lower, while the Sn-2 DHA content was higher.

[0062] Example 3

[0063] Single factor experiment was used to determine the optimal amount of EGCG added to the three groups.

[0064] During the fermentation of Schizochytrium, different concentrations of clove extract were replaced with different concentrations of resveratrol. Other fermentation conditions and sample treatment of Schizochytrium were the same as those in Example 1. Figure 5 , Figure 6 When the EGCG addition amount was 120mg / L, 140mg / L, and 160mg / L, the C14:0+C16:0 content in the fermentation broth at the end of fermentation was lower, while the Sn-2 DHA content was higher.

[0065] Example 4

[0066] Design and implement orthogonal experiment. According to the single factor experiment, three factors and three levels were determined, as shown in Table 1.

[0067] level A Clove extract (mg / L) B Resveratrol (mg / L) CEGCG (mg / L) 190 60 120 2120 80 140 3150 100 160

[0068] Design an orthogonal experiment as shown in Table 2.

[0069]

[0070]

[0071] The control group of the orthogonal test was not added with any inhibitor. According to the experimental steps of Schizochytrium fermentation and sample treatment in Implementation 1, the fatty acid profile of the key fermentation broth was determined. Figure 7 , Figure 8 When the addition amount of clove extract was 120 mg / L, resveratrol was 60 mg / L, and EGCG was 160 mg / L, the saturated fatty acid content in the fermentation broth at the end of fermentation decreased by 6.47%, and Sn-2 DHA increased by 17.39%.

[0072] Example 5

[0073] In order to verify whether there is a synergistic effect among the three components, a synergistic test is performed as follows:

[0074] In addition to adding the optimal composite FAS inhibitor screened by the present invention (clove extract: 120 mg / L, resveratrol: 60 mg / L, EGCG: 160 mg / L) to the culture medium during the fermentation of Schizochytrium, other fermentation conditions and sample treatment of Schizochytrium were the same as those in Example 1. After 5 days of fermentation, the fatty acid profile of the endpoint sample was determined.

[0075] Comparative Example 1

[0076] The fermentation conditions and sample treatment steps of Schizochytrium were the same as those of Example 1. In addition to adding two components of the optimal composite FAS inhibitor obtained by screening in the present invention to the culture medium during the fermentation of Schizochytrium: clove extract (120 mg / L) and resveratrol (60 mg / L), the other fermentation conditions and sample treatment of Schizochytrium were the same as those of Example 1. After 5 days of fermentation, the fatty acid profile of the endpoint sample was determined.

[0077] Comparative Example 2

[0078] The fermentation conditions and sample treatment steps of Schizochytrium were the same as those in Example 1. In addition to adding two components of the optimal composite FAS inhibitor obtained by screening of the present invention: clove extract (120 mg / L) and EGCG (160 mg / L) to the culture medium during the fermentation of Schizochytrium, the other fermentation conditions and sample treatment of Schizochytrium were the same as those in Example 1. After 5 days of fermentation, the fatty acid profile of the endpoint sample was determined.

[0079] Comparative Example 3

[0080] The fermentation conditions and sample treatment steps of Schizochytrium were the same as those in Example 1. In addition to adding two components of the optimal composite FAS inhibitor obtained by screening of the present invention, resveratrol (60 mg / L) and EGCG (160 mg / L), to the culture medium during the fermentation of Schizochytrium, the other fermentation conditions and sample treatment of Schizochytrium were the same as those in Example 1. After 5 days of fermentation, the fatty acid profile of the endpoint sample was determined.

[0081] The results are as follows Fig. 9 , Fig.10 As shown. When the three components were added at the same time during the fermentation of Schizochytrium, the saturated fatty acids (C14:0 and C16:0) decreased by 5.92% compared with the group without FAS inhibitor, and the Sn-2 DHA content increased by 17.79%. At the same time, the significance analysis of the experimental results was carried out. For the saturated fatty acid content, Comparative Examples 1 and 3 were significantly different from Example 5 (P < 0.05), and Comparative Example 2 was extremely significantly different from Example 5 (P < 0.001) ( Fig. 9 ). As for the Sn-2 DHA content, Comparative Examples 1 and 2 have extremely significant differences compared with Example 5 (P < 0.01), and Comparative Example 3 has significant differences compared with Example 5 (P < 0.05) ( Fig.10 ). Therefore, in summary, the three components have a synergistic effect and can synergistically increase the yield of Sn-2 DHA fermented by Schizochytrium.

[0082] The present invention screens and obtains a formula of a food-grade composite FAS inhibitor by combining a single factor test with an orthogonal test. The fatty acid profile of the end-point sample of Schizochytrium fermentation is measured, and the saturated fatty acid is reduced by 5.92%, while the Sn-2DHA content is increased by 17.79%. The formula of the optimal composite FAS inhibitor is: clove extract (120 mg / L), resveratrol (60 mg / L), and EGCG (160 mL / L).

[0083] The present invention optimizes the fermentation process of Schizochytrium, and additionally adds a food-grade FAS inhibitor during the fermentation process, thereby further increasing the content of saturated fatty acids in Schizochytrium lipids and the content of Sn-2 DHA, further improving the nutritional value of Schizochytrium algae oil and expanding its market application.

[0084] According to the search of existing patents, although Sun Haoxin et al. (CN202310422205.8) increased EPA production by adding iodoacetamide, the three food-grade FAS inhibitors used in the present invention have certain advantages in terms of product safety and low production cost. At the same time, according to experimental results, the three FAS inhibitors have certain synergistic effects.

[0085] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. Using food-grade composite FAS inhibitors to improve the growth of Schizochytrium Sn-2 The fermentation method for increasing the DHA content is characterized by: The steps include: (1) Take a tube of Schizochytrium preservation tube culture medium from the -80℃ refrigerator, add 50 mL of Schizochytrium seed culture medium, culture at 28℃, 170 rpm, and add the corresponding FAS inhibitor; (2) Every 24 hours, transfer 1 mL of Schizochytrium seed liquid to fresh seed liquid culture medium, and transfer twice in succession to obtain the third generation seed liquid; (3) Take 10 mL of the third generation seed solution and add it to 100 mL of Schizochytrium fermentation medium. Measure the glucose content every 12 h, add glucose concentration to 80 g / L, and continue culturing for 5 days under the same culture conditions as step (1). Sn-2 DHA; Each time the cells are transferred to fresh culture medium, the corresponding FAS inhibitor needs to be added; The Schizochytrium is selected from the wild type Schizochytrium HX-308, with the deposit number of CCTCC No.M209059; The food-grade composite FAS inhibitor consists of clove extract, resveratrol and epigallocatechin gallate (EGCG); The final concentration of the clove extract is 120 mg / L, the final concentration of resveratrol is 60 mg / L, and the final concentration of epigallocatechin gallate EGCG is 160 mg / L; Saturated fatty acids decreased by 5.92%. Sn-2 The DHA content increased by 17.79%.

2. The food-grade composite FAS inhibitor as claimed in claim 1 is effective in improving the activity of Schizochytrium Sn-2 Application of DHA content.

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