A method for producing astaxanthin by phaffia rhodozyma fermentation

Abstract

The present application relates to a method for producing astaxanthin by fermenting phaffia rhodozyma, and belongs to the technical field of microbial fermentation. The present application provides a method for producing astaxanthin by fermenting phaffia rhodozyma, specifically, the activated phaffia rhodozyma is inoculated into seed culture medium, and first-stage seed liquid is obtained by culture; the first-stage seed liquid is inoculated into seed culture medium, and second-stage seed liquid is obtained by culture, and then the second-stage seed liquid is inoculated into fermentation culture medium, and fermentation is carried out at 20 DEG C to obtain fermentation liquid; the fermentation liquid is centrifuged, the supernatant is discarded, the bacterial body is washed with physiological saline, and then the broken wall liquid is obtained by breaking the wall, and astaxanthin is extracted. The present application uses twice seed culture to improve the activity of the bacterial strain and enhance the metabolic activity, and on this basis, fermentation is carried out to promote the synthesis and stability of astaxanthin, so that the phaffia rhodozyma bacterial body with high astaxanthin content is obtained, and liquid preparation or dry powder preparation is prepared and applied in aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and relates to a method for producing astaxanthin by fermentation of Pharfovia rubescens. Background Technology

[0002] Astaxanthin is a ketocarotenoid and a terpene unsaturated compound, typically obtained through algal cultivation and microbial fermentation. Adding astaxanthin to aquaculture feed, such as livestock feed, can effectively improve animal immunity and survival, as well as enhance palatability and nutritional value, resulting in high market demand. However, the microorganisms capable of synthesizing astaxanthin in nature are mainly algae, fungi, and bacteria. Most of these species lack industrial application prospects due to low astaxanthin content or difficulties in cultivation. Among them, *Phaffia rhodozyma* is the most studied astaxanthin-producing microorganism, and increasing its astaxanthin production and its application in aquaculture remains a major research direction. Summary of the Invention

[0003] The purpose of this invention is to provide a method for producing astaxanthin by fermentation of Pharfogel's red yeast. This invention uses two seed cultures to improve the activity of the strain and enhance its metabolic activity. On this basis, fermentation is carried out to promote the synthesis and stability of astaxanthin, thereby obtaining Pharfogel's red yeast cells with high astaxanthin content, which can be used to make liquid or dry powder preparations for aquaculture.

[0004] The objective of this invention can be achieved through the following technical solutions:

[0005] A method for producing astaxanthin by fermentation of Pharfovia rubescens, the fermentation method comprising the following steps:

[0006] A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution;

[0007] A2. Inoculate 4.8-5.2% of the primary seed culture into the seed culture medium and culture to obtain the secondary seed culture;

[0008] A3. Inoculate 10.1-10.5% of the secondary seed culture into the fermentation medium and ferment at 20°C to obtain the fermentation broth;

[0009] A4. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, and then break the cell wall to obtain the cell wall-broken liquid. Extract astaxanthin from the broken liquid.

[0010] Further, the seed culture medium described in steps A1 and A2 contains 18-22g glucose, 7.5-8.5g soybean meal hydrolysate powder, 1.9-2.3g KH2PO4, 0.8-1.2g MgSO4·7H2O, and deionized water to a final volume of 1L, with a pH of 5.4-5.6.

[0011] Furthermore, the cultivation described in steps A1 and A2 both refer to cultivation at 25°C with shaking at 160-200 rpm for 46-50 hours.

[0012] Further, the fermentation medium described in step A3 contains per liter the following components: 46-50g glucose, 10-12g soybean meal hydrolysate powder, 4.5-5.5g fish peptone, 1.8-2.2g (NH4)2SO4, 4.7-5.3g KH2PO4, 1.9-2.1g MgSO4·7H2O, 0.08-0.12g CaCl2, 0.04-0.06g FeSO4·7H2O, 0.008-0.012g vitamin B1, and deionized water to a final volume of 1L, with a pH of 5.8-6.2.

[0013] Furthermore, the method for preparing the soybean meal hydrolysate dry powder includes the following steps:

[0014] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:9.6-10.4, adjust the pH to 8.4-8.6, and heat at 90℃ for 20-24 minutes to obtain pretreated soybean meal.

[0015] B2. After mixing the pretreated soybean meal and alkaline protease at a mass ratio of 1:0.02-0.03, the mixture is hydrolyzed at 48-52℃ for 3.8-4.2 hours, while maintaining the pH at 8.4-8.6, to obtain pre-hydrolyzed soybean meal.

[0016] B3. Mix the pre-hydrolyzed soybean meal and neutral protease at a mass ratio of 1:0.014-0.016, hydrolyze at 43-47℃ for 2.1-2.5h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature to obtain the second-hydrolyzed soybean meal.

[0017] B4. Centrifuge the soybean meal after secondary hydrolysis at 6000-10000 rpm for 13-17 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 29-31%, and spray dry to obtain the final product.

[0018] Further, the fermentation mentioned in step A3 refers to setting the fermentation temperature to 20℃, the aeration rate to 1.5-2.5 vvm, and controlling the dissolved oxygen content to 30% by adjusting the stirring speed, and controlling the pH of the fermentation broth to 5.9-6.1; during the fermentation, when the residual sugar concentration drops to 10 g / L, 500 g / L glucose solution is added to maintain the residual sugar concentration at 6-10 g / L.

[0019] Furthermore, the centrifugation mentioned in step A4 refers to centrifugation at 7000-9000 rpm for 10-12 minutes.

[0020] Further, the cell disruption mentioned in step A4 refers to suspending the bacterial cells in 0.1M phosphate buffer at pH 7, adding glass beads with a diameter of 0.5mm, and shaking at 1800-2200rpm for 25-35 minutes at 4°C.

[0021] Further, the extraction described in step A4 refers to mixing the cell wall disruption liquid with food-grade 95% ethanol at a volume ratio of 1:3.1-3.3, then ultrasonically assisted at 40°C, ultrasonic frequency of 30-40kHz, and ultrasonic power of 150-250W for 8-12 minutes, followed by centrifugation at 3800-4200rpm for 4-6 minutes, collecting the supernatant, repeating the process 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0022] Furthermore, the astaxanthin can be prepared into liquid or dry powder formulations, which are then used in aquaculture.

[0023] The beneficial effects of this invention are:

[0024] (1) This invention uses two seed culture to improve the activity of the strain and enhance its metabolic activity. On this basis, fermentation is carried out to promote the synthesis and stability of astaxanthin, thereby obtaining red Paffia yeast cells with high astaxanthin content, and making liquid or dry powder preparations for use in aquaculture.

[0025] (2) In the process of producing astaxanthin by fermentation of Pharrellis rubrum, this invention uses soybean meal hydrolysate powder instead of yeast extract in seed culture medium, and uses soybean meal hydrolysate powder and fish peptone instead of yeast extract in fermentation culture medium to reduce nitrogen source cost and provide balanced nutrition. In the preparation of soybean meal hydrolysate powder, alkaline protease is used to effectively degrade anti-nutritional factors in soybean meal and promote the growth and reproduction of beneficial microorganisms. Then, neutral protease is used to degrade the macromolecular proteins in soybean meal into small molecule peptides and free amino acids, further promoting the growth and metabolism of Pharrellis rubrum, thereby indirectly contributing to the synthesis of astaxanthin. Fish peptone is rich in proline and glycine, which play an important role in the biosynthesis of astaxanthin. They can be converted into precursors of astaxanthin through a series of enzymatic reactions, thus synergistically promoting the synthesis of astaxanthin. Detailed Implementation

[0026] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0027] The alkaline protease used in all embodiments and comparative examples of this invention was purchased from Hebei Jiuxing Chemical Products Co., Ltd., CAS No. 9014-01-1; the neutral protease was purchased from Hebei Jiuxing Chemical Products Co., Ltd., CAS No. 9068-59-1; and the fish peptone was purchased from Beijing Hongrun Baoshun Technology Co., Ltd., CAS No. 91079-42-4.

[0028] The method for obtaining the activated Pharfovia rubescens single strain is as follows: streak the Pharfovia rubescens strain (strain preservation number CICC 33064) onto YPD solid medium containing 2% agar, incubate at 25°C for 48 hours, and pick a single colony to obtain the strain.

[0029] Example 1

[0030] A method for producing astaxanthin by fermentation of Pharfovia rubescens, the fermentation method comprising the following steps:

[0031] A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution;

[0032] A2. Inoculate the primary seed culture with an inoculum of 4.8% into the seed culture medium and culture to obtain the secondary seed culture;

[0033] A3. Inoculate the secondary seed culture at an inoculation rate of 10.1% into the fermentation medium and ferment at 20°C to obtain the fermentation broth;

[0034] A4. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, and then break the cell wall to obtain the cell wall-broken liquid. Extract astaxanthin from the broken liquid.

[0035] The seed culture medium described in steps A1 and A2 contains 18g glucose, 7.5g soybean meal hydrolysate powder, 1.9g KH2PO4, 0.8g MgSO4·7H2O, and deionized water to a final volume of 1L, with a pH of 5.4.

[0036] The cultivation described in steps A1 and A2 both refer to cultivation at 25°C with shaking at 160 rpm for 46 hours.

[0037] The fermentation medium described in step A3 contains the following components per liter: 46g glucose, 10g soybean meal hydrolysate powder, 4.5g fish peptone, 1.8g (NH4)2SO4, 4.7g KH2PO4, 1.9g MgSO4·7H2O, 0.08g CaCl2, 0.04g FeSO4·7H2O, 0.008g vitamin B1, and deionized water to a final volume of 1L, with a pH of 5.8.

[0038] The method for preparing the soybean meal hydrolysate dry powder includes the following steps:

[0039] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:9.6, adjust the pH to 8.4, and heat it at 90℃ for 20 minutes to obtain the pretreated soybean meal.

[0040] B2. The pretreated soybean meal and alkaline protease were mixed at a mass ratio of 1:0.02 and hydrolyzed at 48℃ for 3.8h, while maintaining the pH at 8.4, to obtain pre-hydrolyzed soybean meal.

[0041] B3. Mix the pre-hydrolyzed soybean meal and neutral protease at a mass ratio of 1:0.014, hydrolyze at 43℃ for 2.1h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature to obtain the second-hydrolyzed soybean meal.

[0042] B4. Centrifuge the soybean meal after secondary hydrolysis at 6000 rpm for 13 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 29%, and spray dry to obtain the final product.

[0043] The fermentation described in step A3 refers to setting the fermentation temperature to 20℃, the aeration rate to 1.5 vvm, and controlling the dissolved oxygen content to 30% by adjusting the stirring speed, and controlling the pH of the fermentation broth to 5.9; during the fermentation, when the residual sugar concentration drops to 10 g / L, 500 g / L glucose solution is added to maintain the residual sugar concentration at 6 g / L.

[0044] The centrifugation mentioned in step A4 refers to centrifugation at 7000 rpm for 10 minutes.

[0045] The cell disruption mentioned in step A4 refers to suspending the bacterial cells in 0.1M phosphate buffer at pH 7, adding glass beads with a diameter of 0.5mm, and shaking at 1800rpm for 25min at 4℃.

[0046] The extraction described in step A4 refers to mixing the cell wall disruption liquid with food-grade 95% ethanol at a volume ratio of 1:3.1, then ultrasonically assisted for 8 minutes at 40°C, ultrasonic frequency of 30kHz, and ultrasonic power of 150W, followed by centrifugation at 3800rpm for 4 minutes, collecting the supernatant, repeating the process 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0047] The astaxanthin can be prepared into liquid or dry powder formulations, which are then used in aquaculture.

[0048] Example 2

[0049] A method for producing astaxanthin by fermentation of Pharfovia rubescens, the fermentation method comprising the following steps:

[0050] A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution;

[0051] A2. Inoculate the primary seed culture with an inoculum of 4.9% into the seed culture medium and culture to obtain the secondary seed culture;

[0052] A3. Inoculate the secondary seed culture at an inoculation rate of 10.2% into the fermentation medium and ferment at 20°C to obtain the fermentation broth;

[0053] A4. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, and then break the cell wall to obtain the cell wall-broken liquid. Extract astaxanthin from the broken liquid.

[0054] The seed culture medium described in steps A1 and A2 each contains 19g glucose, 7.8g soybean meal hydrolysate powder, 2g KH2PO4, 0.9g MgSO4·7H2O, and deionized water to a final volume of 1L, with a pH of 5.4.

[0055] The cultivation described in steps A1 and A2 both refer to cultivation at 25°C with shaking at 170 rpm for 47 h.

[0056] The fermentation medium described in step A3 contains the following components per liter: 47g glucose, 10.5g soybean meal hydrolysate powder, 4.7g fish peptone, 1.9g (NH4)2SO4, 4.8g KH2PO4, 1.96g MgSO4·7H2O, 0.09g CaCl2, 0.04g FeSO4·7H2O, 0.009g vitamin B1, and deionized water to a final volume of 1L, with a pH of 5.9.

[0057] The method for preparing the soybean meal hydrolysate dry powder includes the following steps:

[0058] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:9.8, adjust the pH to 8.4, and heat it at 90℃ for 21 minutes to obtain the pretreated soybean meal.

[0059] B2. The pretreated soybean meal and alkaline protease were mixed at a mass ratio of 1:0.022 and hydrolyzed at 49℃ for 3.9h, while maintaining the pH at 8.4, to obtain pre-hydrolyzed soybean meal.

[0060] B3. Mix the pre-hydrolyzed soybean meal and neutral protease at a mass ratio of 1:0.014, hydrolyze at 44℃ for 2.2h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature to obtain the second-hydrolyzed soybean meal.

[0061] B4. Centrifuge the soybean meal after secondary hydrolysis at 7000 rpm for 14 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 29.5%, and spray dry to obtain the final product.

[0062] The fermentation described in step A3 refers to setting the fermentation temperature to 20℃, the aeration rate to 1.8 vvm, and controlling the dissolved oxygen content to 30% by adjusting the stirring speed, and controlling the pH of the fermentation broth to 5.9; during the fermentation, when the residual sugar concentration drops to 10 g / L, 500 g / L glucose solution is added to maintain the residual sugar concentration at 7 g / L.

[0063] The centrifugation mentioned in step A4 refers to centrifugation at 7500 rpm for 10 minutes.

[0064] The cell disruption mentioned in step A4 refers to suspending the bacterial cells in 0.1M phosphate buffer at pH 7, adding glass beads with a diameter of 0.5 mm, and shaking at 1900 rpm for 27 min at 4°C.

[0065] The extraction described in step A4 refers to mixing the cell wall disruption liquid with food-grade 95% ethanol at a volume ratio of 1:3.1, then ultrasonically assisted for 9 minutes at 40°C, ultrasonic frequency of 30kHz, and ultrasonic power of 180W, followed by centrifugation at 3900rpm for 4 minutes, collecting the supernatant, repeating the process 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0066] The astaxanthin can be prepared into liquid or dry powder formulations, which are then used in aquaculture.

[0067] Example 3

[0068] A method for producing astaxanthin by fermentation of Pharfovia rubescens, the fermentation method comprising the following steps:

[0069] A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution;

[0070] A2. Inoculate 5% of the primary seed culture into the seed culture medium and culture to obtain the secondary seed culture;

[0071] A3. Inoculate the secondary seed culture at an inoculation rate of 10.3% into the fermentation medium and ferment at 20°C to obtain the fermentation broth;

[0072] A4. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, and then break the cell wall to obtain the cell wall-broken liquid. Extract astaxanthin from the broken liquid.

[0073] The seed culture medium described in steps A1 and A2 each contains 20g glucose, 8g soybean meal hydrolysate powder, 2.1g KH2PO4, 1g MgSO4·7H2O, and deionized water to a final volume of 1L, with a pH of 5.5.

[0074] The cultivation described in steps A1 and A2 both refer to cultivation at 25°C with shaking at 180 rpm for 48 hours.

[0075] The fermentation medium described in step A3 contains the following components per liter: 48g glucose, 11g soybean meal hydrolysate powder, 5g fish peptone, 2g (NH4)2SO4, 5g KH2PO4, 2g MgSO4·7H2O, 0.1g CaCl2, 0.05g FeSO4·7H2O, 0.01g vitamin B1, and deionized water to a final volume of 1L, with a pH of 6.

[0076] The method for preparing the soybean meal hydrolysate dry powder includes the following steps:

[0077] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:10, adjust the pH to 8.5, and heat it at 90℃ for 22 minutes to obtain the pretreated soybean meal.

[0078] B2. The pretreated soybean meal and alkaline protease were mixed at a mass ratio of 1:0.025 and hydrolyzed at 50°C for 4 hours, while maintaining the pH at 8.5, to obtain pre-hydrolyzed soybean meal.

[0079] B3. Mix the pre-hydrolyzed soybean meal and neutral protease at a mass ratio of 1:0.015, hydrolyze at 45℃ for 2.3h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature to obtain the second-hydrolyzed soybean meal.

[0080] B4. Centrifuge the soybean meal after secondary hydrolysis at 8000 rpm for 15 min, take the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 30%, and spray dry to obtain the final product.

[0081] The fermentation described in step A3 refers to setting the fermentation temperature to 20°C, the aeration rate to 2 vvm, and controlling the dissolved oxygen content to 30% by adjusting the stirring speed, and controlling the pH of the fermentation broth to 6; during the fermentation, when the residual sugar concentration drops to 10 g / L, a 500 g / L glucose solution is added to maintain the residual sugar concentration at 8 g / L.

[0082] The centrifugation mentioned in step A4 refers to centrifugation at 8000 rpm for 11 minutes.

[0083] The cell disruption mentioned in step A4 refers to suspending the bacterial cells in 0.1M phosphate buffer at pH 7, adding glass beads with a diameter of 0.5mm, and shaking at 2000rpm for 30min at 4℃.

[0084] The extraction described in step A4 refers to mixing the cell wall disruption liquid with food-grade 95% ethanol at a volume ratio of 1:3.12, then ultrasonically assisted for 10 minutes at 40°C, ultrasonic frequency of 35kHz, and ultrasonic power of 200W, followed by centrifugation at 4000rpm for 5 minutes, collecting the supernatant, repeating the process 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0085] The astaxanthin can be prepared into liquid or dry powder formulations, which are then used in aquaculture.

[0086] Example 4

[0087] A method for producing astaxanthin by fermentation of Pharfovia rubescens, the fermentation method comprising the following steps:

[0088] A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution;

[0089] A2. Inoculate 5.1% of the primary seed culture into the seed culture medium and culture to obtain the secondary seed culture;

[0090] A3. Inoculate the secondary seed culture at an inoculation rate of 10.4% into the fermentation medium and ferment at 20°C to obtain the fermentation broth;

[0091] A4. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, and then break the cell wall to obtain the cell wall-broken liquid. Extract astaxanthin from the broken liquid.

[0092] The seed culture medium described in steps A1 and A2 each contains 21g glucose, 8.2g soybean meal hydrolysate powder, 2.2g KH2PO4, 1.1g MgSO4·7H2O, and deionized water to a final volume of 1L, with a pH of 5.6.

[0093] The cultivation described in steps A1 and A2 both refer to cultivation at 25°C with shaking at 190 rpm for 49 hours.

[0094] The fermentation medium described in step A3 contains the following components per liter: 49g glucose, 11.5g soybean meal hydrolysate powder, 5.3g fish peptone, 2.1g (NH4)2SO4, 5.2g KH2PO4, 2.04g MgSO4·7H2O, 0.11g CaCl2, 0.06g FeSO4·7H2O, 0.011g vitamin B1, and deionized water to a final volume of 1L. The pH is 6.1.

[0095] The method for preparing the soybean meal hydrolysate dry powder includes the following steps:

[0096] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:10.2, adjust the pH to 8.6, and heat it at 90℃ for 23 minutes to obtain the pretreated soybean meal.

[0097] B2. The pretreated soybean meal and alkaline protease were mixed at a mass ratio of 1:0.027 and hydrolyzed at 51℃ for 4.1h, while maintaining the pH at 8.6, to obtain pre-hydrolyzed soybean meal.

[0098] B3. Mix the pre-hydrolyzed soybean meal and neutral protease at a mass ratio of 1:0.016, hydrolyze at 46℃ for 2.4h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature to obtain the second-hydrolyzed soybean meal.

[0099] B4. Centrifuge the soybean meal after secondary hydrolysis at 9000 rpm for 16 min, take the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 30.5%, and spray dry to obtain the final product.

[0100] The fermentation described in step A3 refers to setting the fermentation temperature to 20℃, the aeration rate to 2.2 vvm, and controlling the dissolved oxygen content to 30% by adjusting the stirring speed, and controlling the pH of the fermentation broth to 6.1; during the fermentation, when the residual sugar concentration drops to 10 g / L, 500 g / L glucose solution is added to maintain the residual sugar concentration at 9 g / L.

[0101] The centrifugation mentioned in step A4 refers to centrifugation at 8500 rpm for 12 minutes.

[0102] The cell disruption mentioned in step A4 refers to suspending the bacterial cells in 0.1M phosphate buffer at pH 7, adding glass beads with a diameter of 0.5mm, and shaking at 2100rpm for 33min at 4℃.

[0103] The extraction described in step A4 refers to mixing the cell wall disruption liquid with food-grade 95% ethanol at a volume ratio of 1:3.3, then ultrasonically assisted for 11 minutes at 40°C, ultrasonic frequency of 40kHz, and ultrasonic power of 220W, followed by centrifugation at 4100rpm for 6 minutes, collecting the supernatant, repeating the process 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0104] The astaxanthin can be prepared into liquid or dry powder formulations, which are then used in aquaculture.

[0105] Example 5

[0106] A method for producing astaxanthin by fermentation of Pharfovia rubescens, the fermentation method comprising the following steps:

[0107] A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution;

[0108] A2. Inoculate the primary seed culture with an inoculum of 5.2% into the seed culture medium and culture to obtain the secondary seed culture;

[0109] A3. Inoculate the secondary seed culture at an inoculation rate of 10.5% into the fermentation medium and ferment at 20°C to obtain the fermentation broth;

[0110] A4. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, and then break the cell wall to obtain the cell wall-broken liquid. Extract astaxanthin from the broken liquid.

[0111] The seed culture medium described in steps A1 and A2 each contains 22g glucose, 8.5g soybean meal hydrolysate powder, 2.3g KH2PO4, 1.2g MgSO4·7H2O, and deionized water to a final volume of 1L, with a pH of 5.6.

[0112] The cultivation described in steps A1 and A2 both refer to cultivation at 25°C with shaking at 200 rpm for 50 h.

[0113] The fermentation medium described in step A3 contains the following components per liter: 50g glucose, 12g soybean meal hydrolysate powder, 5.5g fish peptone, 2.2g (NH4)2SO4, 5.3g KH2PO4, 2.1g MgSO4·7H2O, 0.12g CaCl2, 0.06g FeSO4·7H2O, 0.012g vitamin B1, and deionized water to a final volume of 1L, with a pH of 6.2.

[0114] The method for preparing the soybean meal hydrolysate dry powder includes the following steps:

[0115] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:10.4, adjust the pH to 8.6, and heat it at 90℃ for 24 minutes to obtain the pretreated soybean meal.

[0116] B2. The pretreated soybean meal and alkaline protease were mixed at a mass ratio of 1:0.03 and hydrolyzed at 2°C for 4.2 hours, while maintaining the pH at 8.6, to obtain pre-hydrolyzed soybean meal.

[0117] B3. Mix the pre-hydrolyzed soybean meal and neutral protease at a mass ratio of 1:0.016, hydrolyze at 47℃ for 2.5h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature to obtain the second-hydrolyzed soybean meal.

[0118] B4. Centrifuge the soybean meal after secondary hydrolysis at 10,000 rpm for 17 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 31%, and spray dry to obtain the final product.

[0119] The fermentation described in step A3 refers to setting the fermentation temperature to 20℃, the aeration rate to 2.5 vvm, and controlling the dissolved oxygen content to 30% by adjusting the stirring speed, and controlling the pH of the fermentation broth to 6.1; during the fermentation, when the residual sugar concentration drops to 10 g / L, 500 g / L glucose solution is added to maintain the residual sugar concentration at 10 g / L.

[0120] The centrifugation mentioned in step A4 refers to centrifugation at 9000 rpm for 12 minutes.

[0121] The cell disruption mentioned in step A4 refers to suspending the bacterial cells in 0.1M phosphate buffer at pH 7, adding glass beads with a diameter of 0.5mm, and shaking at 2200rpm for 35min at 4℃.

[0122] The extraction described in step A4 refers to mixing the cell wall disruption liquid with food-grade 95% ethanol at a volume ratio of 1:3.3, then ultrasonically assisted for 12 minutes at 40°C, ultrasonic frequency of 40kHz, and ultrasonic power of 250W, followed by centrifugation at 4200rpm for 6 minutes, collecting the supernatant, repeating the process 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0123] The astaxanthin can be prepared into liquid or dry powder formulations, which are then used in aquaculture.

[0124] Example 6

[0125] Based on Example 3, the soybean meal hydrolysate powder in the seed culture medium was removed and replaced with an equal weight of soybean extract, which was purchased from Shandong Xuguang Chemical Co., Ltd., and other conditions remained the same as in Example 3.

[0126] Example 7

[0127] Based on Example 3, the components per liter of seed culture medium were changed to 22g glucose, 6g soybean meal hydrolysate powder, 2.1g KH2PO4, 1g MgSO4·7H2O, and deionized water was brought to a final volume of 1L, with a pH of 5.5.

[0128] Example 8

[0129] Based on Example 3, the fish peptone in the fermentation medium was removed and replaced with an equal weight of soybean meal hydrolysate powder, while other conditions remained the same as in Example 3.

[0130] Example 9

[0131] Based on Example 3, the components per liter of the fermentation medium were changed to 48g glucose, 6g soybean meal hydrolysate powder, 10g fish peptone, 2g (NH4)2SO4, 5g KH2PO4, 2g MgSO4·7H2O, 0.1g CaCl2, 0.05g FeSO4·7H2O, 0.01g vitamin B1, and deionized water was brought to a final volume of 1L, with a pH of 6.

[0132] Comparative Example 1

[0133] Based on Example 3, while keeping other conditions consistent, the method for producing astaxanthin by fermentation of Pharfia redis yeast was modified to the following steps:

[0134] A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution;

[0135] A2. Inoculate 10.3% of the primary seed culture into the fermentation medium and ferment at 20°C to obtain the fermentation broth;

[0136] A3. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, and then break the cell wall to obtain the cell wall-broken liquid, from which astaxanthin is extracted.

[0137] Comparative Example 2

[0138] Based on Example 3, while keeping other conditions consistent, the preparation method of soybean meal hydrolysate dry powder was changed to the following steps:

[0139] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:10, adjust the pH to 8.5, and heat it at 90℃ for 22 minutes to obtain the pretreated soybean meal.

[0140] B2. The pretreated soybean meal and alkaline protease were mixed at a mass ratio of 1:0.04 and hydrolyzed at 50℃ for 6.3h, during which the pH was maintained at 8.5. The enzyme was inactivated by boiling in a water bath for 10min and then cooled to room temperature to obtain the hydrolyzed soybean meal.

[0141] B3. Centrifuge the hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 30%, and spray dry to obtain the final product.

[0142] Comparative Example 3

[0143] Based on Example 3, while keeping other conditions consistent, the preparation method of soybean meal hydrolysate dry powder was changed to the following steps:

[0144] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:10, adjust the pH to 8.5, and heat it at 90℃ for 22 minutes to obtain the pretreated soybean meal.

[0145] B2. The pretreated soybean meal and neutral protease were mixed at a mass ratio of 1:0.015 and hydrolyzed at 45℃ for 2.3 hours to obtain pre-hydrolyzed soybean meal.

[0146] B3. Mix the pre-hydrolyzed soybean meal and alkaline protease at a mass ratio of 1:0.025, hydrolyze at 50℃ for 4 hours, maintain pH at 8.5 during the process, inactivate the enzyme by boiling in a water bath for 10 minutes, and cool to room temperature to obtain the second-hydrolyzed soybean meal.

[0147] B4. Centrifuge the soybean meal after secondary hydrolysis at 8000 rpm for 15 min, take the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 30%, and spray dry to obtain the final product.

[0148] Comparative Example 4

[0149] Based on Example 3, while keeping other conditions consistent, the preparation method of soybean meal hydrolysate dry powder was changed to the following steps:

[0150] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:10, adjust the pH to 8.5, and heat it at 90℃ for 22 minutes to obtain the pretreated soybean meal.

[0151] B2. The pretreated soybean meal, alkaline protease and neutral protease were mixed at a mass ratio of 1:0.025:0.015, hydrolyzed at 45℃ for 6.3h, inactivated by boiling in a water bath for 10min, and cooled to room temperature to obtain hydrolyzed soybean meal.

[0152] B3. Centrifuge the hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 30%, and spray dry to obtain the final product.

[0153] Comparative Example 5

[0154] Based on Example 3, while keeping other conditions consistent, the preparation method of soybean meal hydrolysate dry powder was changed to the following steps:

[0155] B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:10, adjust the pH to 8.5, and heat it at 90℃ for 22 minutes to obtain the pretreated soybean meal.

[0156] B2. The pretreated soybean meal and neutral protease were mixed at a mass ratio of 1:0.04, hydrolyzed at 45℃ for 6.3h, inactivated by boiling in a water bath for 10min, and cooled to room temperature to obtain hydrolyzed soybean meal.

[0157] B3. Centrifuge the hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 30%, and spray dry to obtain the final product.

[0158] Astaxanthin extracted in Examples 1-9 and Comparative Examples 1-5 were used as samples, and the yield of astaxanthin was calculated. The specific results are recorded in Table 1 below.

[0159] Table 1

[0160] Astaxanthin yield (mg / L) Example 1 92.9 Example 2 93.8 Example 3 94.5 Example 4 94.2 Example 5 93.5 Example 6 80.3 Example 7 86.1 Example 8 88.6 Example 9 89.7 Comparative Example 1 76.4 Comparative Example 2 83.8 Comparative Example 3 78.3 Comparative Example 4 85.4 Comparative Example 5 73.9

[0161] As shown in Table 1, the method for producing astaxanthin by fermentation of Pharfogel's red yeast provided by this invention can effectively increase the astaxanthin content and has certain application prospects.

[0162] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any indirect modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for the production of astaxanthin by fermentation of Phaffia rhodozyma, characterized in that: The fermentation method includes the following steps: A1. Inoculate the activated Pharfovia rubescens single strain into the seed culture medium and culture to obtain the primary seed solution; A2. Inoculate the primary seed culture solution with an inoculation volume of 4.8-5.2% into the seed culture medium and culture to obtain the secondary seed culture solution; A3. Inoculate 10.1-10.5% of the secondary seed culture into the fermentation medium and ferment at 20℃ to obtain the fermentation broth; A4. Centrifuge the fermentation broth, discard the supernatant, wash the bacterial cells with physiological saline, then break the cell wall to obtain the cell wall-broken liquid, and extract astaxanthin. The seed culture medium described in steps A1 and A2 contains 18-22g glucose, 7.5-8.5g soybean meal hydrolysate powder, 1.9-2.3g KH2PO4, 0.8-1.2g MgSO4·7H2O, and deionized water to a final volume of 1L, with a pH of 5.4-5.

6. The fermentation medium described in step A3 contains the following components per liter: 46-50g glucose, 10-12g soybean meal hydrolysate powder, 4.5-5.5g fish peptone, 1.8-2.2g (NH4)2SO4, 4.7-5.3g KH2PO4, 1.9-2.1g MgSO4·7H2O, 0.08-0.12g CaCl2, 0.04-0.06g FeSO4·7H2O, 0.008-0.012g vitamin B1, and deionized water to a final volume of 1L, with a pH of 5.8-6.

2. The method for preparing the soybean meal hydrolysate dry powder includes the following steps: B1. After crushing the soybean meal and passing it through a 60-mesh sieve, mix it evenly with deionized water at a mass ratio of 1:9.6-10.4, adjust the pH to 8.4-8.6, and heat at 90℃ for 20-24 minutes to obtain pretreated soybean meal. B2. After mixing the pretreated soybean meal and alkaline protease at a mass ratio of 1:0.02-0.03, the mixture is hydrolyzed at 48-52℃ for 3.8-4.2 hours, while maintaining the pH at 8.4-8.6, to obtain pre-hydrolyzed soybean meal. B3. Mix the pre-hydrolyzed soybean meal and neutral protease at a mass ratio of 1:0.014-0.016, hydrolyze at 43-47℃ for 2.1-2.5h, inactivate the enzyme in a boiling water bath for 10min, and cool to room temperature to obtain the second-hydrolyzed soybean meal. B4. Centrifuge the soybean meal after secondary hydrolysis at 6000-10000 rpm for 13-17 min, collect the supernatant, concentrate it under reduced pressure at 60℃ until the solid content is 29-31%, and spray dry to obtain the final product.

2. The method for producing astaxanthin by fermentation of Phaefuer's red yeast according to claim 1, characterized in that: The cultivation described in steps A1 and A2 refers to cultivation at 25°C with shaking at 160-200 rpm for 46-50 hours.

3. The method for producing astaxanthin by fermentation of Pharfovia rubescens according to claim 1, characterized in that: The fermentation described in step A3 refers to an aeration rate of 1.5-2.5 vvm, with dissolved oxygen controlled at 30% by adjusting the stirring speed, and the pH of the fermentation broth controlled at 5.9-6.

1. During the fermentation, when the residual sugar concentration drops to 10 g / L, a 500 g / L glucose solution is added to maintain the residual sugar concentration at 6-10 g / L.

4. The method for producing astaxanthin by fermentation of Phaffia rubra according to claim 1, characterized in that: The centrifugation mentioned in step A4 refers to centrifugation at 7000-9000 rpm for 10-12 minutes.

5. The method for producing astaxanthin by fermentation of Pharfovia rubescens according to claim 1, characterized in that: The cell disruption mentioned in step A4 refers to suspending the bacterial cells in 0.1M phosphate buffer at pH 7, adding glass beads with a diameter of 0.5mm, and shaking at 1800-2200rpm for 25-35 minutes at 4°C.

6. The method for producing astaxanthin by fermentation of Phaffia rubra according to claim 1, characterized in that: The extraction described in step A4 refers to mixing the cell wall disruption liquid with food-grade 95% ethanol at a volume ratio of 1:3.1-3.3, then ultrasonically assisted at 40℃, ultrasonic frequency of 30-40kHz, and ultrasonic power of 150-250W for 8-12 minutes, followed by centrifugation at 3800-4200rpm for 4-6 minutes, collecting the supernatant, repeating the process 3 times, combining the supernatants, and concentrating under reduced pressure at 40℃ to 1 / 10 of the original volume.