A glycolipid and its preparation method and application

Abstract

The present invention belongs to the field of microbial fermentation technology, and specifically relates to a glycolipid and a preparation method and application thereof. The present invention provides a method for preparing glycolipid, comprising the following steps: fermenting yeast liquid to obtain glycolipid; and feeding during the fermentation and cultivation process. The method for preparing glycolipid according to the present invention has the advantages of large output, high yield, stable quality, low energy consumption and simple operation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a glycolipid and a preparation method and application thereof. Background Art

[0002] Mannoerythritol lipid (MEL) is an emerging glycolipid with biosurfactant function. It is currently mainly converted by microorganisms into carbon sources such as vegetable oil or glucose. The bacterial species used to convert this substance are mainly some yeasts in basidiomycetes.

[0003] At present, studies have found that this glycolipid has good biological activity in addition to surface activity. For example, some articles show that it has good antibacterial activity, inhibits the growth of melanocytes, and can remove free radicals and reduce skin cell death caused by SDS. This indicates that it may be a potential functional cosmetic raw material. Although there have been studies involving small-scale fermentation of mannose erythritol lipids, such as invention patents 201510170977.4 and 201310540906.8, which disclose two methods for preparing mannose erythritol lipids by shaking flask fermentation, the amount of mannose erythritol lipids produced is only 62.5g / L and 88g / L, respectively, with a low yield, and the total amount of fermentation on this scale is small, which is not enough to provide raw materials for industrial production. Summary of the invention

[0004] The purpose of the present invention is to provide a glycolipid and a preparation method and application thereof. The method of the present invention has the advantages of large output and high yield, and can produce glycolipid in a factory.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing glycolipid, comprising the following steps:

[0007] The yeast liquid is fermented and cultured to obtain glycolipids;

[0008] Feeding is performed during the fermentation culture process.

[0009] Preferably, during the fermentation culture, the fermentation medium used includes the following components in percentage by weight: 10% to 30% vegetable oil, 0.05% to 0.2% yeast extract, 0.02% to 0.06% magnesium sulfate, 0.2% to 0.8% sodium nitrate, 0.02% to 0.08% sodium dihydrogen phosphate, 0.001% to 0.01% defoaming agent and the remainder water.

[0010] Preferably, the feeding method is batch feeding.

[0011] Preferably, the feeding time is after 48 hours of fermentation, and the amount of feeding is 10% to 30% of the yeast solution;

[0012] The batch feeding includes one feeding, two feedings, four feedings or six feedings; the time of the one feeding is preferably the 48th hour of fermentation culture; the time of the two feedings is the 48th hour and the 72nd hour of fermentation culture; the time of the four feedings is the 48th hour, the 72nd hour, the 96th hour and the 120th hour of fermentation culture; the time of the six feedings is the 48th hour, the 72th hour, the 96th hour, the 120th hour, the 144th hour and the 168th hour of fermentation culture; in the case of batch feeding, the amount of each feeding is 5% to 10% of the volume of the yeast liquid;

[0013] Preferably, during the supplementation, the supplemented fermentation feed includes vegetable oil.

[0014] Preferably, the vegetable oil comprises one or more of olive oil, soybean oil, rapeseed oil, sunflower oil, corn oil, castor oil, palm oil, linseed oil, rapeseed oil and wheat germ oil.

[0015] Preferably, the yeast comprises Candida antarctica.

[0016] Preferably, the fermentation culture temperature is 27.5-28.5°C, the time is 5-10 days, the rotation speed is 300-600 rpm, and the ventilation volume is 1-1.5 vvm.

[0017] The present invention also provides a glycolipid, which is obtained by fermentation and culture using yeast liquid; and feed supplement is performed during the fermentation and culture process.

[0018] The present invention also provides the use of the glycolipid described in the above technical solution in the preparation of daily chemical products.

[0019] Beneficial effects:

[0020] The present invention provides a method for preparing glycolipids, comprising the following steps: fermenting and culturing yeast liquid to obtain glycolipids; and feeding during the fermentation and culturing process. The method for preparing glycolipids according to the present invention has the advantages of large output, high yield, stable quality, low energy consumption and simple operation. The results of the embodiments of the present invention show that the output of glycolipids prepared by the preparation method of the present invention reaches 110g / L, which is significantly higher than the output of glycolipids prepared by the preparation method of the prior art in the same time. Therefore, the method of the present invention can be used for the factory production of glycolipids.

[0021] Moreover, the present invention can enrich glycolipids in large quantities by increasing the total amount of substrate, setting the feeding method, time, and amount of feeding, and limiting the condition parameters during the fermentation culture process, thereby expanding the production capacity as much as possible, greatly reducing the production cost of mannitol erythritol lipids, and reducing the difficulty of its industrial production.

[0022] In addition, the glycolipid prepared by the method of the present invention is natural and non-irritating, and can inhibit the damage of the active cell layer in the 3D cell model (keratinocyte model) induced by sodium dodecyl sulfate (SDS), and increase the expression of skin barrier related proteins filaggrin (FLG), keratin transglutaminase (TGM1) and loricrin (LOR). Therefore, the glycolipid has good skin barrier protection, improves the combing and smoothing function of slightly damaged hair bundles, and can be used for the preparation of daily chemical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 The histological observation pictures of skin irritation test in different treatment groups (400×);

[0025] Figure 2 The histological observation pictures of eye irritation test of different treatment groups (400×);

[0026] Figure 3 The staining results of different treatment groups are shown in the figure. The first row shows H&E staining, the second row shows the expression of FLG protein, the third row shows the expression of TGM1 protein, and the fourth row shows the expression of LOR protein. NC is the blank group, MD is the model group, PC is the positive control group, and TA is the glycolipid group.

[0027] Figure 4 It is the change of combing property of slightly damaged hair before and after using shampoo, among which NC is the matrix group and PA is the sample test group.

[0028] Figure 1 and Figure 2 NC1 is negative control repeat 1, NC2 is negative repeat 2, NC3 is negative repeat 3, PC1 is positive control repeat 1, PC2 is positive control repeat 2, PC3 is positive control repeat 3, TA1 is glycolipid repeat 1, TA2 is glycolipid repeat 2, and TA3 is glycolipid repeat 3. DETAILED DESCRIPTION

[0029] Unless otherwise specified, the components, culture media and reagents used in the present invention can be purchased routinely by those skilled in the art.

[0030] The present invention provides a method for preparing glycolipid, comprising the following steps:

[0031] The yeast liquid is fermented and cultured to obtain glycolipids;

[0032] Feeding is performed during the fermentation culture process.

[0033] The present invention ferments and cultures yeast liquid to obtain glycolipid. In the present invention, the yeast preferably includes Candida antarctica, more preferably Candida antarctica numbered JCM 10317 (from: Sugita T. Pseudozyma antarctica genes for ITS1, 5.8S rRNA, ITS2, complete and partial sequence, strain: JCM10317.2002). The present invention has no special limitation on the source of the Candida antarctica, and the yeast can be purchased by a person skilled in the art.

[0034] In the present invention, the yeast liquid preferably includes secondary seed fermentation liquid or tertiary seed fermentation liquid; the preparation method of the yeast liquid preferably includes the following steps: after activating the fermentation bacteria, subculture to obtain a bacterial liquid; subjecting the bacterial liquid to primary fermentation culture to obtain a primary seed fermentation liquid; subjecting the primary seed fermentation liquid to secondary fermentation culture to obtain a secondary seed fermentation liquid; subjecting the secondary seed fermentation liquid to tertiary fermentation culture to obtain a tertiary seed fermentation liquid.

[0035] In the present invention, during the activation, the culture medium used is preferably YM culture medium; during the activation culture, the inoculation amount is preferably 5% of the volume of the YM culture medium; the temperature of the activation culture is preferably 28°C; the rotation speed of the activation culture is preferably 200rpm; and the time of the activation culture is preferably 24h. The present invention has no special requirements for the components of the YM culture medium, and the conventional YM culture medium in the art can be used.

[0036] In the present invention, the culture medium and the condition parameters of the subculture are the same as those of the activation culture described above, so they are not described in detail here.

[0037] In the present invention, the culture medium used in the primary fermentation culture is preferably YM culture medium; the inoculation amount of the primary fermentation culture is preferably 5% of the volume of the YM culture medium; the temperature of the primary fermentation culture is preferably 28°C; the time of the primary fermentation culture is preferably 24h; the pH of the primary fermentation culture is preferably 6.8; the ventilation volume of the primary fermentation culture is preferably 1vvm; the stirring speed of the primary fermentation culture is 225rpm. In the present invention, the culture medium used in the secondary fermentation culture and the condition parameters of the subculture culture are the same as those of the activation culture above, and are not described in detail here.

[0038] In the present invention, during the fermentation culture, the fermentation medium used preferably includes the following components in percentage by mass: 10% to 30% vegetable oil, 0.05% to 0.2% yeast extract, 0.02% to 0.06% magnesium sulfate, 0.2% to 0.8% sodium nitrate, 0.02% to 0.08% sodium dihydrogen phosphate, 0.001% to 0.01% defoaming agent and the remainder water.

[0039] In terms of mass percentage, the fermentation medium preferably includes 10% to 30% of vegetable oil, more preferably 13% to 27%, and more preferably 15% to 23%. In the present invention, the vegetable oil preferably includes one or more of olive oil, soybean oil, rapeseed oil, sunflower oil, corn oil, castor oil, palm oil, linseed oil, rapeseed oil and wheat germ oil, more preferably olive oil or soybean oil. In the present invention, the vegetable oil is a carbon source that can be used by microorganisms.

[0040] In terms of mass percentage, the fermentation medium preferably includes 0.05% to 0.2% yeast extract powder, more preferably 0.08% to 0.18%, and even more preferably 0.1% to 0.15%.

[0041] In terms of mass percentage, the fermentation medium preferably includes 0.02% to 0.06% magnesium sulfate, more preferably 0.03% to 0.05%, and even more preferably 0.035% to 0.045%.

[0042] In terms of mass percentage, the fermentation medium preferably includes 0.2% to 0.8% sodium nitrate, more preferably 0.3% to 0.7%, and even more preferably 0.4% to 0.6%.

[0043] In terms of mass percentage, the fermentation medium preferably includes 0.02% to 0.08% of sodium dihydrogen phosphate, more preferably 0.03% to 0.07%, and even more preferably 0.02% to 0.08%.

[0044] In terms of mass percentage, the fermentation medium preferably includes 0.001% to 0.01% of the defoaming agent, more preferably 0.003% to 0.009%, and even more preferably 0.005% to 0.007%.

[0045] In the present invention, the fermentation culture is preferably carried out in a fermenter; the fermentation culture temperature is preferably 27.5-28.5°C, more preferably 28°C; the fermentation culture time is preferably 5-10 days, more preferably 6-9 days, more preferably 7-8 days; the fermentation culture speed is preferably 300-600 rpm. In the present invention, the fermentation culture is preferably carried out in a ventilated state; the fermentation culture ventilation volume is 1 vvm; the pH of the fermentation culture is preferably controlled to be 6.5±0.5.

[0046] The present invention performs feeding during the fermentation culture process. In the present invention, the feeding method is preferably batch feeding. In the present invention, the feeding time is the 48th hour of fermentation culture; the feeding time is preferably counted from the time when the yeast liquid is placed in the fermentation medium. When feeding, the volume of the supplemented fermentation material is preferably 10% to 30% of the initial fermentation medium volume, more preferably 11 to 28%, and most preferably 13 to 25%. The feeding method described in the present invention can solve the problem of bacterial growth caused by excessive or too fast single feeding and overcome the defects of microbial metabolism inhibition and decreased dissolved oxygen in the fermentation tank caused by too many feedings, thereby achieving the technical effect of increasing production capacity.

[0047] In the present invention, when the feed is supplemented, the supplemented fermentation material preferably includes vegetable oil. In the present invention, the vegetable oil preferably includes one or more of olive oil, soybean oil, rapeseed oil, sunflower oil, corn oil, castor oil, palm oil, linseed oil, rapeseed oil and wheat germ oil, and more preferably includes olive oil or soybean oil. In the present invention, the preparation method of the sterile vegetable oil preferably includes the following steps: subjecting the vegetable oil to high-pressure sterilization treatment. The present invention has no special limitation on the high-pressure sterilization treatment, and the conventional high-pressure sterilization treatment method in the art can be used to achieve the sterilization effect of the present application.

[0048] In the present invention, the batch feeding preferably includes one feeding, two feedings, four feedings or six feedings. When the batch feeding method is one feeding, the feeding time is preferably 48h of fermentation culture; when the batch feeding method is two feedings, the feeding time is preferably 48h and 72h of fermentation culture; the feeding time is preferably counted from the time when the yeast liquid is placed in the fermentation medium. When the batch feeding method is four feedings, the feeding time is preferably 48h, 72h, 96h and 120h of fermentation culture. When the batch feeding method is six feedings, the feeding time is preferably 48h, 72h, 96h, 120h, 144h and 168h of fermentation culture. In the present invention, when feeding in batches, the volume of the fermentation material supplemented each time is preferably 5% to 10% of the volume of the initial fermentation medium, more preferably 5% to 9%, still preferably 5.5% to 8.5%, and more preferably 6% to 8%. The fermentation material has been described in detail in the previous text of the present invention, which will not be repeated here. The feeding method of the present invention can avoid the phenomenon of decreased dissolved oxygen in the tank, excess carbon source, and inability of microorganisms to utilize it caused by premature feeding. This feeding method can increase the total daily product conversion in a single batch fermentation process, and at the same time increase production efficiency.

[0049] In the present invention, in the continuous fermentation process, the fermentation culture is preferably terminated after the feeding is completed by continuing the fermentation for 48 h to 96 h, more preferably 50 h to 90 h, and even more preferably 60 h to 80 h.

[0050] The present invention provides a glycolipid, which is obtained by fermentation and culture using yeast liquid; and feeding is performed during the fermentation and culture process. The preparation method of the glycolipid of the present invention has been discussed in detail in the previous text, so it will not be repeated here. The glycolipid of the present invention is a mixture of various glycolipids and oils and fats containing mannose erythritol lipids. The glycolipid of the present invention is natural and non-irritating, and can inhibit the damage of the active cell layer in the 3D cell model (keratinocyte model) induced by sodium dodecyl sulfate (SDS), and increase the expression of skin barrier-related proteins filaggrin (FLG), keratin transglutaminase (TGM1) and loricrin (LOR) proteins. Therefore, the glycolipid has good skin barrier protection, improves the combing and smoothing function of slightly damaged hair bundles, and can be used for the preparation of daily chemical products.

[0051] The present invention provides the use of the glycolipid described in the above technical solution in preparing daily chemicals, more preferably for preparing daily chemicals with skin barrier protection function and / or hair smoothing effect. In the present invention, the daily chemicals preferably include shampoo, conditioner, cream, cleanser, toner, body lotion, essential oil, facial mask, facial cleanser and soap, but are not limited to the above-mentioned daily chemicals.

[0052] In order to further illustrate the present invention, a glycolipid and its fermentation method and application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] Seed fermentation liquid preparation:

[0055] 1) Taking a low-temperature preserved Candida (JCM 10317) working seed batch, activating it with YM medium (0.3wt.% yeast powder, 0.3wt.% malt powder, 0.5wt.% peptone), expanding it with YM medium to 10mL bacterial liquid, the temperature is 28°C, the shaking speed is 200rpm, the culture time is 24h, and the activated Candida seeds are obtained;

[0056] 2) Inoculate 10 mL of the activated Candida seeds in 1) into a 1L conical flask containing 200 mL YM medium, with an inoculation volume of 5%. Use YM medium to expand to 200 mL of bacterial liquid, at which point the wet bacterial weight is about 160±20 g / L, and the subculture conditions are the same as step 1);

[0057] 3) The bacterial solution in step 2) is aseptically inoculated into a first-level seed fermentation liquid tank containing YM medium, with an inoculation amount of 5% of the volume of the YM medium, and cultured for 24 hours at 28° C., pH=6.8, with a ventilation volume of about 1 vvm and stirring at 225 rpm to obtain a first-level seed fermentation liquid;

[0058] 4) After 24 hours, the first-level seed fermentation liquid is transferred to the second-level seed fermentation liquid tank for cultivation. The culture medium of the second-level seed fermentation liquid tank is YM culture medium. The culture conditions are the same as those of the first-level seed fermentation liquid tank to obtain the second-level seed fermentation liquid;

[0059] 5) After 24 hours, the secondary seed fermentation liquid is transferred to the tertiary seed fermentation liquid tank for cultivation to obtain the tertiary seed fermentation liquid. The culture medium of the tertiary seed fermentation liquid tank is YM culture medium. The culture conditions are the same as those of the primary seed fermentation liquid tank to obtain the tertiary seed fermentation liquid.

[0060] Example 2

[0061] 5T fermentation medium (10% olive oil, 0.05% yeast extract, 0.02% magnesium sulfate, 0.2% sodium nitrate, 0.02% sodium dihydrogen phosphate, 0.001% defoaming agent and balance water) was added to a 10T fermentation tank. When the temperature of the fermentation tank dropped to 28°C after deionization, 250L of the tertiary seed fermentation liquid obtained by culture in Example 1 was transferred into the 10-ton fermentation tank under positive pressure.

[0062] The temperature of the fermentation culture was controlled at 28°C, the pH was controlled at 6.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 8 days.

[0063] During the fermentation process, feeding was carried out in batches. The feeding nodes were 48h, 72h, 96h, and 120h of fermentation, and sterile olive oil (121°C, sterilized for 20min) equivalent to 6%, 9%, 9%, and 6% of the initial fermentation medium volume was added respectively.

[0064] After the last addition of sterile olive oil, the fermentation was continued for 48 hours and then the fermentation ended to obtain the fermentation product;

[0065] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as glycolipid 1.

[0066] Example 3

[0067] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (10% olive oil, 0.05% yeast extract, 0.02% magnesium sulfate, 0.2% sodium nitrate, 0.02% sodium dihydrogen phosphate, 0.001% defoaming agent and the remainder water) for fermentation.

[0068] The temperature of the fermentation culture was controlled at 28°C, the pH was controlled at 6.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 8 days.

[0069] During the fermentation process, feeding was carried out in batches at the 48th, 72nd, 96th and 120th hours of fermentation, with sterile olive oil (121°C, sterilized for 20 minutes) added at 6%, 9%, 9% and 6% of the initial fermentation medium volume, respectively.

[0070] After the last addition of sterile olive oil, the fermentation was continued for 48 hours and then the fermentation ended to obtain the fermentation product;

[0071] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as glycolipid 2.

[0072] Example 4

[0073] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (30% soybean oil, 0.2% yeast extract, 0.06% magnesium sulfate, 0.8% sodium nitrate, 0.08% sodium dihydrogen phosphate, 0.001% defoamer and the remainder water) for fermentation.

[0074] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 8 days.

[0075] At 48 h of fermentation, sterile soybean oil (sterilized at 121°C for 20 min) equivalent to 10% of the initial fermentation medium volume was added to the tank.

[0076] After adding sterile soybean oil, the fermentation was continued for 48 hours and then the fermentation was terminated to obtain a fermentation product;

[0077] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as glycolipid 3.

[0078] Example 5

[0079] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (12% olive oil, 0.1% yeast extract, 0.04% magnesium sulfate, 0.4% sodium nitrate, 0.04% sodium dihydrogen phosphate, 0.001% defoaming agent and the remainder water) for fermentation.

[0080] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 5 days.

[0081] During the fermentation process, sterile olive oil (121°C, sterilized for 20 min) equivalent to 6% and 9% of the volume of the initial fermentation medium was added to the tank at 48h and 72h of fermentation, respectively.

[0082] After the last addition of sterile olive oil, the fermentation was continued for 48 hours and then the fermentation ended to obtain the fermentation product;

[0083] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as glycolipid 4.

[0084] Example 6

[0085] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (10% olive oil, 0.1% yeast extract, 0.04% magnesium sulfate, 0.4% sodium nitrate, 0.04% sodium dihydrogen phosphate, 0.001% defoaming agent and the remainder water) for culture.

[0086] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 10 days.

[0087] During the fermentation process, feeding was carried out in batches at 48h, 72h, 96h, 120h, 144h and 168h of fermentation. Each feeding was sterile olive oil (121°C, sterilized for 20min) equivalent to 5% of the initial fermentation medium volume.

[0088] After the last addition of sterile olive oil, the fermentation was continued for 48 hours and then the fermentation ended to obtain the fermentation product;

[0089] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as glycolipid 5.

[0090] Example 7

[0091] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank filled with 250 mL of fermentation medium (10% olive oil, 0.1% yeast extract, 0.04% magnesium sulfate, 0.4% sodium nitrate, 0.04% sodium dihydrogen phosphate, 0.001% defoamer and the remainder water) for culture.

[0092] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 10 days.

[0093] During the fermentation process, feeding was carried out in batches at the 48th, 72nd, 96th and 120th hours of fermentation, with sterile olive oil (121°C, sterilized for 20 minutes) added at 6%, 9%, 9% and 6% of the initial fermentation medium volume, respectively.

[0094] After the last supplement of sterile olive oil, the fermentation was continued for 96 hours and then the fermentation ended to obtain the fermentation product;

[0095] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as glycolipid 6.

[0096] Comparative Example 1

[0097] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (40% olive oil, 0.2% yeast extract, 0.06% magnesium sulfate, 0.8% sodium nitrate, 0.08% sodium dihydrogen phosphate, 0.001% defoamer and the remainder water) for fermentation.

[0098] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 8 days.

[0099] At 120 h of fermentation, 10% of the volume of the initial fermentation medium was supplemented into the tank with sterile soybean oil (121° C., sterilized for 20 min).

[0100] After adding sterile soybean oil, the fermentation was continued for 48 hours and then the fermentation was terminated to obtain a fermentation product;

[0101] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as control glycolipid 1.

[0102] Comparative Example 2

[0103] 25 L of the secondary seed fermentation liquid obtained by the culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (10% olive oil, 0.1% yeast extract, 0.04% magnesium sulfate, 0.4% sodium nitrate, 0.04% sodium dihydrogen phosphate, 0.001% defoaming agent and the remainder water) for fermentation.

[0104] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 4 days.

[0105] At 48 h of fermentation, sterile olive oil (121°C, sterilized for 20 min) equivalent to 5% of the initial fermentation medium volume was added.

[0106] After adding sterile olive oil, the fermentation was continued for 48 hours and then the fermentation was terminated to obtain a fermentation product;

[0107] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as control glycolipid 2.

[0108] Comparative Example 3

[0109] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (10% olive oil, 0.1% yeast extract, 0.04% magnesium sulfate, 0.4% sodium nitrate, 0.04% sodium dihydrogen phosphate, and 0.001% defoaming agent) for fermentation.

[0110] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 8 days.

[0111] During the fermentation process, feeding was carried out in batches at the 48th, 72nd, 96th and 120th hours of fermentation. The amount of each feeding was sterile olive oil (121°C, sterilized for 20 minutes) equivalent to 10% of the initial fermentation medium volume.

[0112] After the last addition of sterile olive oil, the fermentation was continued for 48 hours and then the fermentation ended to obtain the fermentation product;

[0113] After the fermentation, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain glycolipid, which was recorded as control glycolipid 3.

[0114] Comparative Example 4

[0115] 25 L of the secondary seed fermentation liquid obtained by culture in Example 1 was transferred into a 500 L fermentation tank containing 250 L of fermentation medium (10% olive oil, 0.1% yeast extract, 0.04% magnesium sulfate, 0.4% sodium nitrate, 0.04% sodium dihydrogen phosphate, 0.001% defoaming agent and the remainder water) for culture.

[0116] The temperature of the fermentation culture was controlled at 28±0.5°C, the pH was controlled at 6.5±0.5, the ventilation volume was 1vvm, the rotation speed was 150rpm, and the total fermentation time was 8 days.

[0117] During the fermentation process, feeding was carried out in batches. The feeding points were 72h and 120h of fermentation, and sterile olive oil (121°C, sterilized for 20min) equivalent to 15% of the initial fermentation medium volume was added respectively.

[0118] After the last supplement of sterile olive oil, the fermentation was continued for 12 hours to obtain the fermentation product;

[0119] After the fermentation was completed, the fermentation product was centrifuged at 4000-6000×g for 15 min to obtain control glycolipid 4.

[0120] Example 7

[0121] HPLC Detection of Mannosyl Erythritol Lipids (MELs) in Glycolipids

[0122] (1) Preparation of Mannose Erythritol Lipids (MELs) Standards:

[0123] ① Pretreatment: The fermentation broth was extracted three times with an equal volume of ethyl acetate, the organic phase and the aqueous phase were separated, the organic phases were combined, and the obtained MELs were extracted three times with a solvent system of cyclohexane: methanol: water = 1:6:3, pH 5.5 (adjusted with 3N HCl), the aqueous phase was adjusted to pH 7.0, and rotary evaporated to obtain degreased mixed MELs.

[0124] ②Separation and purification: silica gel column chromatography: dry-mix the sample with silica gel 60-80 mesh (3-4 times the sample weight), load the column with silica gel 300-400 mesh (10-20 times the sample weight), exhaust the air, and pre-balance the column with dichloromethane. Then use dichloromethane: acetone = 5:1 (MEL-A), 3:1 (MEL-B), 2:1 (MEL-C) gradient elution to obtain MEL monomer.

[0125] ③Structural identification: Use HRESIMS and NMR to identify the compound structure and confirm whether it is the target product.

[0126] (2) Standard and sample treatment: Dissolve 2 mg of MELs standard in 1 mL of dichloromethane:methanol (v / v=1:1), filter through a 0.22 μm filter membrane to a concentration of 2.0 mg / mL, dilute to 500 and 250 μg / mL standard solution, filter through a 0.22 μm filter membrane, and store at 4°C for testing.

[0127] 110 mg of the glycolipid prepared in Example 2 was dissolved in 1 mL of chloroform (v / v=1:1), diluted to 500 μg / mL, filtered through a 0.22 μm filter membrane, and stored at 4° C. for testing.

[0128] (3) Instruments: Shimadzu 2030plus high performance liquid chromatograph, equipped with quaternary gradient pump, column oven, UV detector, and evaporative light detector;

[0129] (4) Conditions:

[0130] Chromatographic column: KromasilC18, 4.6×250mm, 5μm; column number: E2717011, mobile phase: A water-B acetonitrile; flow rate: 1mL / min; column temperature: 30℃; injection volume: 10μL; evaporative light detector: gain 6; drift tube temperature: 40℃; pressure: 350kpa; gradient elution: 0-10min, B80%; 10-15min, B90%: 15-23min, B90%; 23-25min, B80%; 25-35min, B80%.

[0131] After testing, the MELs content and yield in the glycolipids obtained in Examples 2 to 7 and Comparative Examples 1 to 4 are shown in Table 1.

[0132] Table 1 Statistics of the content and yield of MELs in different glycolipids

[0133]

[0134] As shown in Table 1, excessive or too fast supplementation of vegetable oil will reduce the content of MELs. At the same time, extending the fermentation time by 48 to 96 hours after the last feeding can increase the content of MELs. This shows that the preferred fermentation parameters of the present invention have excellent daily MELs production and content. While increasing the total output, the microorganisms are given sufficient and reasonable time to transform and metabolize olive oil. The fermentation process improves the quality of MELs and reduces the production cost of a single batch.

[0135] Example 8

[0136] Skin irritation test

[0137] (1) Skin model: 12-well artificial skin model; culture conditions: 37°C, 5% CO2, relative humidity 95%;

[0138] Positive control (PC): 5wt.% SDS (sodium dodecyl sulfate);

[0139] Negative control (NC): ultrapure water;

[0140] Test sample (TA): glycolipid 1 prepared in Example 2;

[0141] Culture medium: special culture medium for skin model (Guangdong Boxi Biotechnology Co., Ltd.).

[0142] (2) Experimental procedures

[0143] 1) Skin model preparation: Add pre-warmed (37°C) maintenance culture medium (Guangdong Boxi Biotechnology Co., Ltd.) to a 12-well culture plate, 2 mL / well, and transfer the skin model to the maintenance culture medium and place it in an incubator overnight.

[0144] 2) Reaction of the sample to be tested with MTT (3-(4,5-dimethylthiazole-2-yl)-2): add 2 mL of 0.3 mg / mL MTT culture medium and 10 μL of the sample to be tested to each well, mix well, and incubate in an incubator at 37° C. for 3 h.

[0145] 3) Sampling: Take 10 μL of the test sample (NC, PC and TA) treated in Example 2, evenly apply it on the surface of the skin model, leave it at room temperature for 15 minutes, rinse the sample thoroughly with DPBS (Dubrich phosphate buffered saline) until there is no residue, and then place it in a well containing fresh maintenance culture medium and incubate at 37° C. for 42 hours. Each treatment is repeated three times.

[0146] 4) The skin model treated in Example 3 was placed in a well containing 2 mL of 0.3 mg / mL MTT solution and placed in an incubator at 37°C for 3 h. After the MTT reaction was completed, the epidermal tissue was removed with a puncher and transferred to a 2 mL EP tube. 500 μL of isopropanol hydrochloride was added to each tube, and after sufficient mixing, it was placed at room temperature and away from light overnight. 200 μL of solution was taken from each tube in a 96-well plate, and the OD value was read at 570 nm. Isopropanol hydrochloride was used as a blank control, NC was recorded as a negative control group, PC was recorded as a positive control group, and TA was recorded as a test group.

[0147] 5) Place the epidermal model in a neutral fixative and fix it at room temperature for at least 24 hours. Embed it as usual, slice it, and observe its morphology under a microscope after HE staining.

[0148] (3) Data analysis

[0149] Isopropyl hydrochloride was used as the blank control, and the activity of the negative control group was 100%. The tissue activity of the test group and the positive control group was calculated using the following formula:

[0150] Relative tissue activity (%) = (OD TA / PC -OD BLANK ) / (OD NC -OD BLANK )×100%; when the average relative tissue activity (%)>50, it is No Category (no classification); when the average relative tissue activity (%)≤50, it is Categoryoey1 / 2.

[0151] (4) Experimental results

[0152] The results of the skin irritation test of different glycolipids are shown in Table 2 and Figure 1 .

[0153] Table 2 Skin irritation test of different glycolipids

[0154] Grouping OD value Average tissue relative viability (%) UN GHS Classification NC 0.6395±0.0176 100.00±2.76 —— PC 0.0281±0.0059 4.40±0.92 Categoey1 / 2 TA 0.4971±0.0495 77.73±7.18 No Category

[0155] From Table 2 and Figure 1 It can be seen from the records that from the histological analysis, the basal layer, spinous layer, granular layer and stratum corneum of the NC and TA groups were basically intact, and the epidermal tissue of the PC group showed coagulation and cavitation, and the cell structure was destroyed. It can be seen that the glycolipid prepared by the preparation method of the present invention has no skin irritation.

[0156] Example 9

[0157] Eye irritation test

[0158] (1) Experimental Materials

[0159] 1) Cow eyes: Cow eyes were obtained from a slaughterhouse in Guangzhou on the morning of the test day and placed in cold HBSS solution (Hank's balanced salt solution, 4-8°C) and transported to the laboratory.

[0160] 2) Culture conditions: 32±1℃.

[0161] 3) Kit control:

[0162] Positive control group (PC): anhydrous ethanol; negative control group (NC): ultrapure water; test group (TA): glycolipid 1 prepared in Example 2.

[0163] MEM medium: MEM medium without phenol red and MEM medium containing phenol red containing 1% newborn calf serum.

[0164] HBSS: HBSS medium containing double antibodies.

[0165] Fluorescein sodium solution: Prepared with DPBS buffer containing calcium ions and magnesium ions, the concentration is 4 mg / mL.

[0166] (2) Experimental steps:

[0167] 1) Corneal preparation: Select an intact bovine cornea and cut it 2-3 mm from the scleral edge. Fix the cornea on a preheated corneal holder with the surface facing up. Fill the anterior and posterior chambers with preheated (32°C) phenol red-free MEM culture medium and equilibrate in a 32°C incubator for 1-2 h.

[0168] 2) Measurement of basal turbidity value: After the equilibrium is completed, the cornea is taken out of the incubator, and the old culture medium in the front and back chambers is replaced with fresh MEM culture medium without phenol red. The basal turbidity of all corneas is measured using a BASF turbidity meter.

[0169] 3) Sample addition: Remove the fluid in the corneal anterior chamber, add 0.75 mL of the test sample (NC, PC and TA) to the corneal epithelial side, and place in a 32°C incubator for incubation for 10 min. After the exposure, wash the test sample in the corneal anterior chamber at least 3 times with MEM medium containing phenol red until there is no residue in the sample. The PC group and the NC group were operated in the same way. Each treatment was repeated 3 times.

[0170] 4) Post-incubation: After washing, fill the anterior and posterior chambers of the cornea with fresh MEM medium without phenol red and incubate at 32°C for 120 min.

[0171] 5) Measure the turbidity value after exposure: replace the old culture medium in the anterior and posterior chambers of the cornea with fresh MEM culture medium without phenol red, and measure the turbidity values ​​of all corneas again using a BASF turbidity meter.

[0172] 6) Permeability test: Remove the culture medium in the anterior chamber and add 1 mL of 4 mg / mL sodium fluorescein solution. Place the holder vertically back into the incubator and incubate for 90 min. Collect the culture medium in the posterior chamber of the cornea and transfer 360 μL / well to a 96-well plate. Measure the absorbance at 490 nm.

[0173] 7) Histological analysis: The corneas were fixed in 4% paraformaldehyde solution for at least 24 h, routinely sectioned in paraffin, stained with HE, and observed under a microscope.

[0174] (3) Data analysis

[0175] Calculate the corresponding turbidity change values ​​and OD values ​​of the test group, positive control group and negative control group, and correct them by the negative control group. In vitro scoring is based on the following formula:

[0176] In vitro score IVS = average corrected turbidity + 15 × average corrected OD value; when IVS ≤ 3, it is No Category; when 3 < IVS ≤ 55, No stand-alone prediction can be made; when IVS ≤ 3, it is Category Oey1.

[0177] (4) Test results

[0178] The results of the eye irritation test of glycolipids are shown in Table 3 and Figure 2 :

[0179] Table 3 Eye irritation test results of different treatments

[0180]

[0181] From Table 3 and Figure 2 It can be seen from the records that from the histological analysis, the corneal epithelium of the NC combination sample TA group was intact, the matrix was neatly arranged, mild edema occurred, and the endothelium was intact. The epithelium of the PC group was coagulated and vacuolated, the matrix was irregularly arranged, the edema was more serious, and the endothelium was intact. It can be seen that the glycolipid prepared by the preparation method of the present invention has no eye irritation.

[0182] Example 10

[0183] Take the cultured keratinocyte model 3D cell model, (Guangdong Boxi Biotechnology Co., Ltd.), set up blank group (NC), model group (MD), positive control group (PC) and experimental group (TA);

[0184] Among them, the keratinocyte model of the blank group was cultured with maintenance culture medium; the model group was cultured with maintenance culture medium with a final concentration of 0.2wt.% SDS; the positive control group was cultured with a final concentration of 0.2wt.% SDS and a final concentration of 50μM WY14643; the experimental group was cultured with a final concentration of 0.2wt.% SDS and a final concentration of 20μg / mL maintenance culture medium, and 100μg / mL of the glycolipid prepared in Example 2 was added.

[0185] The above four groups were placed in a carbon dioxide incubator at 37°C and 5% CO2 by volume for 24 hours.

[0186] After the culture, the keratinocyte model was removed, fixed with 40 g / L paraformaldehyde for 30 min, washed once with PBS, dehydrated with 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% ethanol by volume, transparentized with 50% ethanol-xylene and pure xylene by volume, immersed in paraffin overnight, embedded in paraffin on the second day, and cut into 8 μm thin sections.

[0187] The sections were stained with hematoxylin-eosin, sealed with dry gum after staining, and the skin morphology was observed under a microscope. Another section was taken, dewaxed and placed in water, blocked with a 5% goat serum PBS solution, incubated for 10 minutes, and FLG (ab234406), TGM1 (ab167657), and LOR (ab176322) primary antibodies were added respectively, and incubated on a shaker at 4°C overnight at a speed of 60-70 r / min. The primary antibody was discarded the next day, and the sections were washed three times with TBST for 5 minutes each. Then, the fluorescent dye FITC was added, incubated at 4°C in the dark for 1 hour, and washed three times with TBST for 5 minutes each. DAPI was added for staining for 5 minutes, and then washed three times with TBST for 5 minutes each. The sections were observed under a fluorescence microscope. The results are shown in the figure. Figure 3 : The results showed that, compared with the blank group, the keratinocyte model after the addition of SDS stimulation showed loose and thickened stratum corneum, damaged living cell layer, vacuoles appeared, obvious damage, and decreased FLG, TGM1 and LOR proteins. Compared with the model group, the positive control group had clear boundaries of the four-layer structure, compact arrangement of living cell layer cells, significantly improved loose and thickened stratum corneum, and significantly increased expression of FLG, TGM1 and LOR proteins; the vacuoles in the TA group were significantly reduced, the damage of the living cell layer was significantly improved, and the expression of FLG, TGM1 and LOR proteins was significantly increased.

[0188] Embodiment 11

[0189] Wet hair combing test

[0190] A number of standard hair bundles for testing (each bundle is 250 mm wide, 175-200 mm long, and weighs 3.5-4.0 g including the binding tape) are taken and divided into a matrix group and a sample test group. The matrix group (NC) contains a hair conditioner matrix; the sample test group (PA) contains a hair conditioner matrix containing 0.5 wt.% of the glycolipid prepared in Example 2.

[0191] The test hair tresses were soaked in warm water for 1 minute, and then 1 ml of 10 wt.% SLES (Stepan) was applied to the hair tresses and gently rubbed for 1 minute to clean the hair tresses, and then washed with warm water. The hair tresses were combed 10 times from different sides to remove tangles, and then the hair tresses containing the conditioner base and the conditioner base containing 0.5 wt.% of the glycolipid prepared in Example 2 were applied to different hair tresses, and gently massaged for 1 minute.

[0192] Finally, the hair bundles were fixed on the MTT175 hair tester (Dia-Stron Ltd, Andover, UK) to measure the work required for combing. Figure 4As shown, compared with the matrix group (NC), the sample test group (PA) required significantly less effort when combing the hair bundle, which indicates that the glycolipid of the present invention can soften damaged hair.

[0193] In summary, the yield of glycolipids using the preparation method of the present invention reaches 110g / L, which is 2 to 3 times the yield of glycolipids prepared by the prior art preparation method in the same time. Moreover, the present invention can enrich glycolipids in large quantities by setting the feeding method, time, amount of feeding and the limitation of condition parameters during the fermentation culture process, greatly reducing the production cost of mannitol erythritol lipids. In addition, the glycolipids of the present invention are natural and non-irritating, have good skin barrier protection, improve the combing and smoothing function of slightly damaged hair bundles, and can be used for the preparation of daily chemical products.

[0194] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing glycolipid in an industrial fermentation tank, characterized in that: The following steps are involved: Fermenting the yeast liquid to obtain glycolipid, wherein the glycolipid contains mannose erythritol lipid; Feeding is performed during the fermentation culture process; During the fermentation culture, the fermentation medium used includes the following components in percentage by weight: 10% to 30% vegetable oil, 0.05% to 0.2% yeast extract powder, 0.02% to 0.06% magnesium sulfate, 0.2% to 0.8% sodium nitrate, 0.02% to 0.08% sodium dihydrogen phosphate, 0.001% to 0.01% defoaming agent and the balance water; The feeding method is batch feeding; The time of feeding is after 48 hours of fermentation, and the amount of feeding is 10% to 30% of the yeast liquid; The batch feeding includes one feeding, two feedings, four feedings or six feedings; the time of the one feeding is preferably the 48th hour of fermentation culture; the time of the two feedings is the 48th hour and the 72nd hour of fermentation culture; the time of the four feedings is the 48th hour, the 72nd hour, the 96th hour and the 120th hour of fermentation culture; the time of the six feedings is the 48th hour, the 72th hour, the 96th hour, the 120th hour, the 144th hour and the 168th hour of fermentation culture; in the case of batch feeding, the amount of each feeding is 5% to 10% of the volume of the yeast liquid; The yeast is Candida antarctica numbered JCM 10317; When supplementing the feed, the supplemented fermentation feed includes vegetable oil, and the vegetable oil is olive oil; The fermentation culture temperature is 27.5-28.5°C, the time is 5-10 days, the rotation speed is 300-600 rpm, and the ventilation volume is 1-1.5 vvm.

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