Method for producing astaxanthin through fermentation of phaffia rhodozyma

Abstract

The invention relates to a method for producing astaxanthin through fermentation of phaffia rhodozyma, and belongs to the technical field of microbial fermentation. The invention provides a method for producing astaxanthin by fermenting phaffia rhodozyma, which specifically comprises the following steps: inoculating an activated phaffia rhodozyma single strain into a seed culture medium, culturing to obtain a primary seed solution, inoculating the primary seed solution into the seed culture medium, culturing to obtain a secondary seed solution, inoculating into a fermentation culture medium, and fermenting at 20 DEG C to obtain the astaxanthin. Centrifuging, discarding supernatant, cleaning thalli with normal saline, performing wall breaking to obtain wall-broken liquid, and extracting to obtain astaxanthin. According to the method, seed culture is performed twice to improve the strain activity and enhance the metabolic activity, fermentation is performed on this basis to promote the synthesis and stability of astaxanthin, so that the phaffia rhodozyma thalli with higher astaxanthin content are obtained, and the phaffia rhodozyma thalli are prepared into liquid preparations or dry powder preparations to be applied to aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and relates to a method for producing astaxanthin by fermenting Phaffia rhodozyma. Background Art

[0002] Astaxanthin is a ketocarotenoid and a terpene-based unsaturated compound, typically obtained through algae cultivation and microbial fermentation. Adding astaxanthin to aquaculture feed, such as aquaculture feed, as a feed additive can effectively boost animal immunity and survival, improving the taste and nutritional value of food, and is in high market demand. However, the microorganisms capable of synthesizing astaxanthin in nature are primarily algae, fungi, and bacteria. Most lack industrial application prospects due to low astaxanthin content or difficulty in cultivation. Phaffia rhodozyma is the most studied astaxanthin-producing microorganism, and increasing its astaxanthin production for use in aquaculture remains a major research area. Summary of the Invention

[0003] The object of the present invention is to provide a method for producing astaxanthin by fermenting Phaffia rhodozyma. The method adopts two seed cultures to improve the activity of the strain and enhance the metabolic activity. On this basis, fermentation is carried out to promote the synthesis and stability of astaxanthin, thereby obtaining Phaffia rhodozyma cells with a high astaxanthin content, and preparing liquid preparations or dry powder preparations for use in aquaculture.

[0004] The purpose of the present invention can be achieved through the following technical solutions: A method for producing astaxanthin by fermenting Phaffia rhodozyma, the fermentation method comprising the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2. Inoculate the first-level seed solution with an inoculum size of 4.8-5.2% into the seed culture medium to obtain the second-level seed solution; A3, inoculating the secondary seed solution with an inoculum amount of 10.1-10.5% into the fermentation medium, and fermenting at 20° C. to obtain a fermentation liquid; A4. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

[0005] Furthermore, the seed culture medium in step A1 and step A2 contains 18-22 g glucose, 7.5-8.5 g soybean meal hydrolyzate dry powder, 1.9-2.3 g KH2PO4, 0.8-1.2 g MgSO4·7H2O per liter, and is diluted to 1 L with deionized water, with a pH of 5.4-5.6.

[0006] Furthermore, the culturing in step A1 and step A2 both refers to culturing at 25° C. and shaking at 160-200 rpm for 46-50 hours.

[0007] Furthermore, the fermentation medium in step A3 contains 46-50 g glucose, 10-12 g soybean meal hydrolysate powder, 4.5-5.5 g fish peptone, 1.8-2.2 g (NH4)2SO4, 4.7-5.3 g KH2PO4, 1.9-2.1 g MgSO4·7H2O, 0.08-0.12 g CaCl2, 0.04-0.06 g FeSO4·7H2O, 0.008-0.012 g vitamin B1 per liter, and the volume is adjusted to 1 L with deionized water, and the pH is 5.8-6.2.

[0008] Furthermore, the method for preparing the soybean meal hydrolyzate dry powder comprises the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water in a mass ratio of 1:9.6-10.4. Adjust the pH to 8.4-8.6, and heat at 90° C. for 20-24 minutes to obtain pretreated soybean meal. B2. Mixing the pretreated soybean meal and alkaline protease in a mass ratio of 1:0.02-0.03, and hydrolyzing at 48-52° C. for 3.8-4.2 hours while maintaining the pH at 8.4-8.6 to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and neutral protease in a mass ratio of 1:0.014-0.016, hydrolyzing at 43-47° C. for 2.1-2.5 hours, inactivating the enzyme in a boiling water bath for 10 minutes, and cooling to room temperature to obtain a secondary hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 6000-10000 rpm for 13-17 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 29-31%, and spray dry to obtain the product.

[0009] Furthermore, the fermentation in step A3 refers to setting the fermentation temperature to 20°C, the ventilation volume to 1.5-2.5vvm, and controlling the dissolved oxygen content at 30% by adjusting the stirring speed, and controlling the pH of the fermentation liquid to 5.9-6.1; during the fermentation, when the residual sugar concentration drops to 10g / L, 500g / L glucose solution is added to maintain the residual sugar concentration at 6-10g / L.

[0010] Furthermore, the centrifugation in step A4 refers to centrifugation at 7000-9000 rpm for 10-12 min.

[0011] Furthermore, the cell wall breaking in step A4 refers to suspending the bacteria in 0.1 M phosphate buffer with a pH of 7, adding glass beads with a diameter of 0.5 mm, and shaking at 1800-2200 rpm for 25-35 minutes at 4°C.

[0012] Furthermore, the extraction described in step A4 refers to mixing the cell wall breaking liquid with food-grade 95% ethanol in a volume ratio of 1:3.1-3.3, ultrasonically assisted at 40°C, an ultrasonic frequency of 30-40kHz, and an ultrasonic power of 150-250W for 8-12 minutes, and then centrifuged at 3800-4200rpm for 4-6 minutes, collecting the supernatant, repeating 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0013] Furthermore, the astaxanthin can be prepared into a liquid preparation or a dry powder preparation, and the liquid preparation or the dry powder preparation is used in aquaculture.

[0014] Beneficial effects of the present invention: (1) The present invention uses two seed cultures to improve bacterial activity and enhance metabolic activity, and then ferments to promote the synthesis and stability of astaxanthin, thereby obtaining red Phaffia yeast cells with a high astaxanthin content, and preparing liquid preparations or dry powder preparations for use in aquaculture.

[0015] (2) In the process of fermenting astaxanthin by Phaffia rhodozyma, the present invention uses soybean meal hydrolyzate powder to replace the use of yeast extract in the seed culture medium, and uses soybean meal hydrolyzate powder and fish peptone to replace the use of yeast extract in the fermentation culture medium, thereby reducing the cost of nitrogen sources and providing balanced nutrition at the same time; in the preparation process of soybean meal hydrolyzate powder, alkaline protease is used to effectively degrade the anti-nutritional factors in soybean meal, while promoting the growth and reproduction of beneficial microorganisms, and then neutral protease is used to degrade the macromolecular protein in soybean meal into small molecular peptides and free amino acids, further promoting the growth and metabolism of Phaffia rhodozyma, thereby indirectly contributing to the synthesis of astaxanthin; and fish peptone is rich in proline and glycine, both of which play an important role in the biosynthesis of astaxanthin, that is, they can be converted into precursors of astaxanthin through a series of enzymatic reactions, synergistically promoting the synthesis of astaxanthin. DETAILED DESCRIPTION

[0016] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0017] Alkaline protease in all the examples and comparative examples of the present invention was purchased from Hebei Jiuxing Chemical Products Co., Ltd. with a CAS number of 9014-01-1; neutral protease was purchased from Hebei Jiuxing Chemical Products Co., Ltd. with a CAS number of 9068-59-1; fish peptone was purchased from Beijing Hongrun Baoshun Technology Co., Ltd. with a CAS number of 91079-42-4; The method for obtaining the activated red yeast single strain is to streak the red yeast strain (strain deposit number is CICC 33064) on a YPD solid culture medium containing 2% agar, culture at 25° C. for 48 hours, and pick a single colony.

[0018] Example 1 A method for producing astaxanthin by fermenting Phaffia rhodozyma, the fermentation method comprising the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2, inoculating the first-level seed solution with an inoculum size of 4.8% into the seed culture medium to obtain the second-level seed solution; A3, inoculating the secondary seed solution at an inoculum amount of 10.1% into the fermentation medium, and fermenting at 20°C to obtain a fermentation liquid; A4. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

[0019] The seed culture medium in step A1 and step A2 contains 18 g glucose, 7.5 g soybean meal hydrolyzate powder, 1.9 g KH2PO4, 0.8 g MgSO4·7H2O per liter, and is diluted to 1 L with deionized water, with a pH of 5.4.

[0020] The culturing in step A1 and step A2 refers to culturing at 25° C. and shaking at 160 rpm for 46 h.

[0021] The fermentation medium in step A3 contains 46 g glucose, 10 g soybean meal hydrolysate powder, 4.5 g fish peptone, 1.8 g (NH4)2SO4, 4.7 g KH2PO4, 1.9 g MgSO4·7H2O, 0.08 g CaCl2, 0.04 g FeSO4·7H2O, 0.008 g vitamin B1 per liter, and is diluted to 1 L with deionized water and has a pH of 5.8.

[0022] The preparation method of the soybean meal hydrolyzate dry powder comprises the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:9.6. Adjust the pH to 8.4, and heat at 90° C. for 20 min to obtain pretreated soybean meal. B2. Mixing the pretreated soybean meal and alkaline protease in a mass ratio of 1:0.02, and hydrolyzing the mixture at 48° C. for 3.8 hours while maintaining the pH at 8.4 to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and neutral protease in a mass ratio of 1:0.014, hydrolyzing at 43°C for 2.1 hours, inactivating the enzyme in a boiling water bath for 10 minutes, and cooling to room temperature to obtain a secondarily hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 6000 rpm for 13 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 29%, and spray dry to obtain the product.

[0023] The fermentation in step A3 refers to setting the fermentation temperature to 20°C, the ventilation volume to 1.5 vvm, controlling the dissolved oxygen content at 30% by adjusting the stirring speed, and controlling the pH of the fermentation liquid to 5.9; during the fermentation, when the residual sugar concentration dropped to 10 g / L, 500 g / L glucose solution was added to maintain the residual sugar concentration at 6 g / L.

[0024] The centrifugation in step A4 refers to centrifugation at 7000 rpm for 10 min.

[0025] The cell wall breaking described in step A4 refers to suspending the bacteria in 0.1 M phosphate buffer with a pH of 7, adding glass beads with a diameter of 0.5 mm, and shaking at 1800 rpm for 25 minutes at 4°C.

[0026] The extraction described in step A4 refers to mixing the cell wall breaking liquid with food-grade 95% ethanol in a volume ratio of 1:3.1, ultrasonically assisted at 40°C, an ultrasonic frequency of 30kHz, and an ultrasonic power of 150W for 8 minutes, and then centrifuged at 3800 rpm for 4 minutes, collecting the supernatant, repeating 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0027] The astaxanthin can be prepared into a liquid preparation or a dry powder preparation, and the liquid preparation or the dry powder preparation is used in aquaculture.

[0028] Example 2 A method for producing astaxanthin by fermenting Phaffia rhodozyma, the fermentation method comprising the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2, inoculating the first-level seed solution with an inoculum size of 4.9% into the seed culture medium to obtain the second-level seed solution; A3, inoculating the secondary seed solution with an inoculum amount of 10.2% into the fermentation medium, and fermenting at 20° C. to obtain a fermentation liquid; A4. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

[0029] The seed culture medium in step A1 and step A2 contains 19 g glucose, 7.8 g soybean meal hydrolyzate powder, 2 g KH2PO4, 0.9 g MgSO4·7H2O per liter, and is diluted to 1 L with deionized water, with a pH of 5.4.

[0030] The culturing in step A1 and step A2 refers to culturing at 25° C. and shaking at 170 rpm for 47 h.

[0031] The fermentation medium in step A3 contains 47 g glucose, 10.5 g soybean meal hydrolysate powder, 4.7 g fish peptone, 1.9 g (NH4)2SO4, 4.8 g KH2PO4, 1.96 g MgSO4·7H2O, 0.09 g CaCl2, 0.04 g FeSO4·7H2O, 0.009 g vitamin B1 per liter, and is diluted to 1 L with deionized water and has a pH of 5.9.

[0032] The preparation method of the soybean meal hydrolyzate dry powder comprises the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:9.8. Adjust the pH to 8.4 and heat at 90° C. for 21 minutes to obtain pretreated soybean meal. B2. Mixing the pretreated soybean meal and alkaline protease in a mass ratio of 1:0.022, and hydrolyzing the mixture at 49° C. for 3.9 hours while maintaining the pH at 8.4, to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and neutral protease in a mass ratio of 1:0.014, hydrolyzing at 44° C. for 2.2 h, inactivating the enzyme in a boiling water bath for 10 min, and cooling to room temperature to obtain a secondary hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 7000 rpm for 14 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 29.5%, and spray dry to obtain the product.

[0033] The fermentation in step A3 refers to setting the fermentation temperature to 20°C, the ventilation volume to 1.8 vvm, controlling the dissolved oxygen content at 30% by adjusting the stirring speed, and controlling the pH of the fermentation liquid to 5.9; during the fermentation, when the residual sugar concentration dropped to 10 g / L, 500 g / L glucose solution was added to maintain the residual sugar concentration at 7 g / L.

[0034] The centrifugation in step A4 refers to centrifugation at 7500 rpm for 10 min.

[0035] The cell wall breaking described in step A4 refers to suspending the bacteria in 0.1 M phosphate buffer with a pH of 7, adding glass beads with a diameter of 0.5 mm, and shaking at 1900 rpm for 27 minutes at 4°C.

[0036] The extraction described in step A4 refers to mixing the cell wall breaking liquid with food-grade 95% ethanol in a volume ratio of 1:3.1, ultrasonically assisted at 40°C, an ultrasonic frequency of 30kHz, and an ultrasonic power of 180W for 9 minutes, and then centrifuged at 3900rpm for 4 minutes, collecting the supernatant, repeating 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0037] The astaxanthin can be prepared into a liquid preparation or a dry powder preparation, and the liquid preparation or the dry powder preparation is used in aquaculture.

[0038] Example 3 A method for producing astaxanthin by fermenting Phaffia rhodozyma, the fermentation method comprising the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2, inoculating 5% of the first-level seed solution into the seed culture medium to obtain the second-level seed solution; A3, inoculating the secondary seed solution with an inoculum amount of 10.3% into the fermentation medium, and fermenting at 20° C. to obtain a fermentation liquid; A4. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

[0039] The seed culture medium in step A1 and step A2 contains 20 g glucose, 8 g soybean meal hydrolyzate powder, 2.1 g KH2PO4, 1 g MgSO4·7H2O per liter, and is diluted to 1 L with deionized water, with a pH of 5.5.

[0040] The culturing in step A1 and step A2 refers to culturing at 25° C. and shaking at 180 rpm for 48 h.

[0041] The fermentation medium in step A3 contains 48 g glucose, 11 g soybean meal hydrolysate powder, 5 g fish peptone, 2 g (NH4)2SO4, 5 g KH2PO4, 2 g MgSO4·7H2O, 0.1 g CaCl2, 0.05 g FeSO4·7H2O, 0.01 g vitamin B1 per liter, and is diluted to 1 L with deionized water and has a pH of 6.

[0042] The preparation method of the soybean meal hydrolyzate dry powder comprises the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:10. Adjust the pH to 8.5 and heat at 90° C. for 22 minutes to obtain pretreated soybean meal. B2. Mixing the pretreated soybean meal and alkaline protease in a mass ratio of 1:0.025, and hydrolyzing the mixture at 50° C. for 4 h while maintaining the pH at 8.5 to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and neutral protease in a mass ratio of 1:0.015, hydrolyzing at 45°C for 2.3 hours, inactivating the enzyme in a boiling water bath for 10 minutes, and cooling to room temperature to obtain a secondary hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 30%, and spray dry to obtain the product.

[0043] The fermentation in step A3 refers to setting the fermentation temperature to 20°C, the ventilation volume to 2 vvm, and controlling the dissolved oxygen content at 30% by adjusting the stirring speed, and controlling the pH of the fermentation liquid to 6; during the fermentation, when the residual sugar concentration dropped to 10 g / L, 500 g / L glucose solution was added to maintain the residual sugar concentration at 8 g / L.

[0044] The centrifugation in step A4 refers to centrifugation at 8000 rpm for 11 minutes.

[0045] The cell wall breaking described in step A4 refers to suspending the bacteria in 0.1 M phosphate buffer with a pH of 7, adding glass beads with a diameter of 0.5 mm, and shaking at 2000 rpm for 30 minutes at 4°C to break the cell wall.

[0046] The extraction described in step A4 refers to mixing the cell wall breaking liquid with food-grade 95% ethanol in a volume ratio of 1:3.12, ultrasonically assisted at 40°C, an ultrasonic frequency of 35kHz, and an ultrasonic power of 200W for 10 minutes, and then centrifuged at 4000rpm for 5 minutes, collecting the supernatant, repeating 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 a liquid preparation or a dry powder preparation, and the liquid preparation or the dry powder preparation is used in aquaculture.

[0048] Example 4 A method for producing astaxanthin by fermenting Phaffia rhodozyma, the fermentation method comprising the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2, inoculating 5.1% of the first-level seed solution into the seed culture medium to obtain the second-level seed solution; A3, inoculating the secondary seed solution with an inoculum amount of 10.4% into the fermentation medium, and fermenting at 20°C to obtain a fermentation liquid; A4. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

[0049] The seed culture medium in step A1 and step A2 contains 21 g glucose, 8.2 g soybean meal hydrolyzate powder, 2.2 g KH2PO4, 1.1 g MgSO4·7H2O per liter, and is diluted to 1 L with deionized water, with a pH of 5.6.

[0050] The culturing in step A1 and step A2 refers to culturing at 25° C. and shaking at 190 rpm for 49 h.

[0051] The fermentation medium in step A3 contains 49 g glucose, 11.5 g soybean meal hydrolysate powder, 5.3 g fish peptone, 2.1 g (NH4)2SO4, 5.2 g KH2PO4, 2.04 g MgSO4·7H2O, 0.11 g CaCl2, 0.06 g FeSO4·7H2O, 0.011 g vitamin B1 per liter, and is diluted to 1 L with deionized water and has a pH of 6.1.

[0052] The preparation method of the soybean meal hydrolyzate dry powder comprises the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:10.2. Adjust the pH to 8.6 and heat at 90° C. for 23 minutes to obtain pretreated soybean meal. B2. Mixing the pretreated soybean meal and alkaline protease in a mass ratio of 1:0.027, and hydrolyzing the mixture at 51° C. for 4.1 h while maintaining the pH at 8.6 to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and neutral protease in a mass ratio of 1:0.016, hydrolyzing at 46° C. for 2.4 h, inactivating the enzyme in a boiling water bath for 10 min, and cooling to room temperature to obtain a secondary hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 9000 rpm for 16 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 30.5%, and spray dry to obtain the product.

[0053] The fermentation in step A3 refers to setting the fermentation temperature to 20°C, the ventilation volume to 2.2 vvm, controlling the dissolved oxygen content at 30% by adjusting the stirring speed, and controlling the pH of the fermentation liquid to 6.1; during the fermentation, when the residual sugar concentration dropped to 10 g / L, 500 g / L glucose solution was added to maintain the residual sugar concentration at 9 g / L.

[0054] The centrifugation in step A4 refers to centrifugation at 8500 rpm for 12 minutes.

[0055] The cell wall breaking described in step A4 refers to suspending the bacteria in 0.1 M phosphate buffer with a pH of 7, adding glass beads with a diameter of 0.5 mm, and shaking at 2100 rpm for 33 minutes at 4°C.

[0056] The extraction described in step A4 refers to mixing the cell wall breaking liquid with food-grade 95% ethanol in a volume ratio of 1:3.3, ultrasonically assisted at 40°C, an ultrasonic frequency of 40 kHz, and an ultrasonic power of 220 W for 11 minutes, and then centrifuged at 4100 rpm for 6 minutes, collecting the supernatant, repeating 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0057] The astaxanthin can be prepared into a liquid preparation or a dry powder preparation, and the liquid preparation or the dry powder preparation is used in aquaculture.

[0058] Example 5 A method for producing astaxanthin by fermenting Phaffia rhodozyma, the fermentation method comprising the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2, inoculating 5.2% of the first-level seed solution into the seed culture medium to obtain the second-level seed solution; A3, inoculating the secondary seed solution at an inoculum amount of 10.5% into the fermentation medium, and fermenting at 20° C. to obtain a fermentation liquid; A4. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

[0059] The seed culture medium in step A1 and step A2 contains 22 g glucose, 8.5 g soybean meal hydrolyzate powder, 2.3 g KH2PO4, 1.2 g MgSO4·7H2O per liter, and is diluted to 1 L with deionized water, with a pH of 5.6.

[0060] The culturing in step A1 and step A2 refers to culturing at 25° C. with shaking at 200 rpm for 50 h.

[0061] The fermentation medium in step A3 contains 50 g glucose, 12 g soybean meal hydrolysate powder, 5.5 g fish peptone, 2.2 g (NH4)2SO4, 5.3 g KH2PO4, 2.1 g MgSO4·7H2O, 0.12 g CaCl2, 0.06 g FeSO4·7H2O, 0.012 g vitamin B1 per liter, and is diluted to 1 L with deionized water and has a pH of 6.2.

[0062] The preparation method of the soybean meal hydrolyzate dry powder comprises the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:10.4. Adjust the pH to 8.6 and heat at 90° C. for 24 minutes to obtain pretreated soybean meal. B2. Mixing the pretreated soybean meal and alkaline protease in a mass ratio of 1:0.03, and hydrolyzing the mixture at 2° C. for 4.2 h while maintaining the pH at 8.6 to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and neutral protease in a mass ratio of 1:0.016, hydrolyzing at 47°C for 2.5 hours, inactivating the enzyme in a boiling water bath for 10 minutes, and cooling to room temperature to obtain a secondary hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 10,000 rpm for 17 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 31%, and spray dry to obtain the product.

[0063] The fermentation in step A3 refers to setting the fermentation temperature to 20°C, the ventilation volume to 2.5 vvm, controlling the dissolved oxygen content at 30% by adjusting the stirring speed, and controlling the pH of the fermentation liquid to 6.1; during the fermentation, when the residual sugar concentration dropped to 10 g / L, 500 g / L glucose solution was added to maintain the residual sugar concentration at 10 g / L.

[0064] The centrifugation in step A4 refers to centrifugation at 9000 rpm for 12 minutes.

[0065] The cell wall breaking described in step A4 refers to suspending the bacteria in 0.1 M phosphate buffer with a pH of 7, adding glass beads with a diameter of 0.5 mm, and shaking at 2200 rpm for 35 minutes at 4°C.

[0066] The extraction described in step A4 refers to mixing the cell wall breaking liquid with food-grade 95% ethanol in a volume ratio of 1:3.3, ultrasonically assisted at 40°C, an ultrasonic frequency of 40 kHz, and an ultrasonic power of 250 W for 12 minutes, and then centrifuged at 4200 rpm for 6 minutes, collecting the supernatant, repeating 3 times, combining the supernatants, and concentrating under reduced pressure at 40°C to 1 / 10 of the original volume.

[0067] The astaxanthin can be prepared into a liquid preparation or a dry powder preparation, and the liquid preparation or the dry powder preparation is used in aquaculture.

[0068] Example 6 On the basis of Example 3, the soybean meal hydrolyzate powder in the seed culture medium was removed and replaced with an equal weight of soybean extract purchased from Shandong Xuguang Chemical Co., Ltd. Other conditions remained the same as in Example 3.

[0069] Example 7 Based on Example 3, the components of the seed culture medium per liter were changed to 22 g glucose, 6 g soybean meal hydrolyzate powder, 2.1 g KH2PO4, 1 g MgSO4·7H2O, and the volume was adjusted to 1 L with deionized water, and the pH was 5.5.

[0070] Example 8 On the basis of Example 3, the fish peptone in the fermentation medium was removed and replaced with an equal weight of soybean meal hydrolyzate dry powder. Other conditions remained the same as in Example 3.

[0071] Example 9 Based on Example 3, the components per liter of the fermentation medium were changed to 48 g glucose, 6 g soybean meal hydrolyzate powder, 10 g fish peptone, 2 g (NH4)2SO4, 5 g KH2PO4, 2 g MgSO4·7H2O, 0.1 g CaCl2, 0.05 g FeSO4·7H2O, 0.01 g vitamin B1, and the volume was adjusted to 1 L with deionized water, and the pH was 6.

[0072] Comparative Example 1 On the basis of Example 3, keeping other conditions the same, the method for producing astaxanthin by fermentation of Phaffia rhodozyma was changed to the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2, inoculating the first-level seed liquid with an inoculum amount of 10.3% into the fermentation medium, and fermenting at 20°C to obtain a fermentation liquid; A3. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

[0073] Comparative Example 2 On the basis of Example 3, keeping other conditions the same, the preparation method of soybean meal hydrolyzate powder was changed to the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:10. Adjust the pH to 8.5 and heat at 90° C. for 22 minutes to obtain pretreated soybean meal. B2. The pretreated soybean meal and alkaline protease were mixed in a mass ratio of 1:0.04, and the mixture was hydrolyzed at 50° C. for 6.3 h while maintaining the pH at 8.5. The enzyme was inactivated in a boiling water bath for 10 min, and the mixture was cooled to room temperature to obtain hydrolyzed soybean meal. B3. Centrifuge the hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 30%, and spray dry to obtain the product.

[0074] Comparative Example 3 On the basis of Example 3, keeping other conditions the same, the preparation method of soybean meal hydrolyzate powder was changed to the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:10. Adjust the pH to 8.5 and heat at 90° C. for 22 minutes to obtain pretreated soybean meal. B2. Mix the pretreated soybean meal and neutral protease in a mass ratio of 1:0.015, and hydrolyze at 45° C. for 2.3 h to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and alkaline protease in a mass ratio of 1:0.025, hydrolyzing at 50° C. for 4 h while maintaining the pH at 8.5, inactivating the enzyme in a boiling water bath for 10 min, and cooling to room temperature to obtain a secondary hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 30%, and spray dry to obtain the product.

[0075] Comparative Example 4 On the basis of Example 3, keeping other conditions the same, the preparation method of soybean meal hydrolyzate powder was changed to the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:10. Adjust the pH to 8.5 and heat at 90° C. for 22 minutes to obtain pretreated soybean meal. B2. Mix the pretreated soybean meal, alkaline protease, and neutral protease in a mass ratio of 1:0.025:0.015, hydrolyze at 45° C. for 6.3 h, inactivate the enzymes in a boiling water bath for 10 min, and cool to room temperature to obtain hydrolyzed soybean meal; B3. Centrifuge the hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 30%, and spray dry to obtain the product.

[0076] Comparative Example 5 On the basis of Example 3, keeping other conditions the same, the preparation method of soybean meal hydrolyzate powder was changed to the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water at a mass ratio of 1:10. Adjust the pH to 8.5 and heat at 90° C. for 22 minutes to obtain pretreated soybean meal. B2. Mix the pretreated soybean meal and neutral protease in a mass ratio of 1:0.04, hydrolyze at 45° C. for 6.3 h, inactivate the enzyme in a boiling water bath for 10 min, and cool to room temperature to obtain hydrolyzed soybean meal; B3. Centrifuge the hydrolyzed soybean meal at 8000 rpm for 15 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 30%, and spray dry to obtain the product.

[0077] The astaxanthin extracted from Examples 1-9 and Comparative Examples 1-5 was used as a sample to calculate the amount of astaxanthin. The specific results are recorded in Table 1 below.

[0078] Table 1 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 As shown in Table 1, the method for producing astaxanthin by fermenting Phaffia rhodozyma provided by the present invention can effectively increase the astaxanthin content and has certain application prospects.

[0079] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any indirect modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for producing astaxanthin by fermentation of Phaffia rhodozyma, characterized in that: The fermentation method comprises the following steps: A1. Inoculate the activated Phaffia rhodozyma strain into a seed culture medium to obtain a primary seed solution; A2. Inoculate the first-level seed solution with an inoculum size of 4.8-5.2% into the seed culture medium to obtain the second-level seed solution; A3, inoculating the secondary seed solution with an inoculum amount of 10.1-10.5% into the fermentation medium, and fermenting at 20° C. to obtain a fermentation liquid; A4. Centrifuge the fermentation broth, discard the supernatant, wash the cells with saline, and then break the cell wall to obtain the broken cell liquid, and extract astaxanthin.

2. The method for producing astaxanthin by fermentation of Phaffia rhodozyma according to claim 1, wherein: The seed culture medium in step A1 and step A2 contains 18-22 g glucose, 7.5-8.5 g soybean meal hydrolyzate dry powder, 1.9-2.3 g KH2PO4, 0.8-1.2 g MgSO4·7H2O per liter, and is diluted to 1 L with deionized water, with a pH of 5.4-5.

6.

3. The method for producing astaxanthin by fermentation of Phaffia rhodozyma according to claim 1, wherein: The culturing in step A1 and step A2 refers to culturing at 25° C. with shaking at 160-200 rpm for 46-50 hours.

4. The method for producing astaxanthin by fermentation of Phaffia rhodozyma according to claim 1, wherein: The fermentation medium in step A3 contains 46-50 g glucose, 10-12 g soybean meal hydrolyzate powder, 4.5-5.5 g fish peptone, 1.8-2.2 g (NH4)2SO4, 4.7-5.3 g KH2PO4, 1.9-2.1 g MgSO4·7H2O, 0.08-0.12 g CaCl2, 0.04-0.06 g FeSO4·7H2O, 0.008-0.012 g vitamin B1 per liter, and the volume is adjusted to 1 L with deionized water, and the pH is 5.8-6.

2.

5. The method for producing astaxanthin by fermentation of Phaffia rhodozyma according to claim 2 or 4, characterized in that: The preparation method of the soybean meal hydrolyzate dry powder comprises the following steps: B1. Grind the soybean meal, pass it through a 60-mesh sieve, and mix it evenly with deionized water in a mass ratio of 1:9.6-10.

4. Adjust the pH to 8.4-8.6, and heat at 90° C. for 20-24 minutes to obtain pretreated soybean meal. B2. Mixing the pretreated soybean meal and alkaline protease in a mass ratio of 1:0.02-0.03, and hydrolyzing at 48-52° C. for 3.8-4.2 hours while maintaining the pH at 8.4-8.6 to obtain a preliminarily hydrolyzed soybean meal; B3, mixing the preliminarily hydrolyzed soybean meal and neutral protease in a mass ratio of 1:0.014-0.016, hydrolyzing at 43-47° C. for 2.1-2.5 hours, inactivating the enzyme in a boiling water bath for 10 minutes, and cooling to room temperature to obtain a secondary hydrolyzed soybean meal; B4. Centrifuge the secondary hydrolyzed soybean meal at 6000-10000 rpm for 13-17 min, collect the supernatant, concentrate under reduced pressure at 60°C to a solid content of 29-31%, and spray dry to obtain the product.

6. The method for producing astaxanthin by fermentation of Phaffia rhodozyma according to claim 1, wherein: The fermentation in step A3 refers to aeration of 1.5-2.5 vvm, and the dissolved oxygen content is controlled at 30% by adjusting the stirring speed, and the pH of the fermentation liquid is controlled at 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.

7. The method for producing astaxanthin by fermentation of Phaffia rhodozyma according to claim 1, wherein: The centrifugation in step A4 refers to centrifugation at 7000-9000 rpm for 10-12 minutes.

8. The method for producing astaxanthin by fermentation of Phaffia rhodozyma according to claim 1, characterized in that: The cell wall breaking in step A4 is to suspend the cells in 0.1 M phosphate buffer with a pH of 7, add glass beads with a diameter of 0.5 mm, and shake at 1800-2200 rpm for 25-35 minutes at 4°C.

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

10. A use of astaxanthin obtained by the method for producing astaxanthin by fermentation of Phaffia rhodozyma according to any one of claims 1 to 9, characterized in that: The astaxanthin can be prepared into a liquid preparation or a dry powder preparation, and the liquid preparation or the dry powder preparation is used in aquaculture.