CN112063539A - Culture medium and culture method for producing carotenoid by fermentation of phaffia rhodozyma - Google Patents

Culture medium and culture method for producing carotenoid by fermentation of phaffia rhodozyma Download PDF

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CN112063539A
CN112063539A CN202010973840.1A CN202010973840A CN112063539A CN 112063539 A CN112063539 A CN 112063539A CN 202010973840 A CN202010973840 A CN 202010973840A CN 112063539 A CN112063539 A CN 112063539A
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fermentation
culture medium
extract powder
phaffia rhodozyma
generation
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姜宗然
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Xiamen Canco Biotech Co ltd
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Xiamen Changqing Biotechnology Co ltd
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

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Abstract

The invention relates to a culture medium and a culture method for producing carotenoid by fermentation of phaffia rhodozyma, wherein the seed culture medium comprises: 2g/L of mixed carbon source, 2g/L of yeast extract powder, 2g/L of malt extract powder and 5g/L of peptone; the fermentation medium comprises: 100g/L of mixed carbon source, 7g/L of mixed nitrogen source, 5g/L of yeast extract powder, 5g/L of malt extract powder, 6g/L of peptone, 1.5g/L of magnesium sulfate, 10g/L of tomato powder and 6mL of absolute ethyl alcohol; wherein the mixed carbon source comprises 40% of molasses and 60% of starch sugar; the mixed nitrogen source is 40% of corn steep liquor and 60% of ammonium sulfate. 3 natural carotenoids can be produced by fermentation of this medium: astaxanthin, canthaxanthin and beta-carotene, and the proportion of the three carotenoids is reasonable, so the feed is suitable for being added into feed for producing eggs and prawns, and the best coloring effect is obtained.

Description

Culture medium and culture method for producing carotenoid by fermentation of phaffia rhodozyma
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a fermentation medium for producing carotenoid by fermentation of phaffia rhodozyma and a culture method.
Background
Carotenoids are natural pigments useful as feed additives, food additives, pharmaceuticals, and the like. Carotenoids include: such as astaxanthin, canthaxanthin, zeaxanthin, lycopene, beta-carotene, echinenone, and the like.
The research shows that the addition of the beta-carotene into the daily ration can improve the laying rate of the laying ducks and improve the color of egg yolks. Canthaxanthin can also improve the color of egg yolk. Thus, β -carotene and canthaxanthin are receiving increasing attention for use in animal husbandry.
Phaff and its co-workers found primarily in the exuded mucilage of deciduous trees in high latitudes, Phaff belongs to the genus Zymomonas, which has both aerobic respiration and fermentation metabolic modes and is the fungus commonly used for the production of astaxanthin at present. The media components have a large influence on the growth of Phaffia rhodozyma and the synthesis of astaxanthin.
In the related art, the culture medium of phaffia rhodozyma has been studied more on how to increase the astaxanthin content, and there has been no report on optimizing the culture medium of phaffia rhodozyma to simultaneously produce astaxanthin, canthaxanthin and β -carotene.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a culture medium and a culture method for producing carotenoid by phaffia rhodozyma. The culture medium can make Phaffia rhodozyma simultaneously produce 3 kinds of natural carotenoid, and can be used as feed additive for egg and prawn, thereby obtaining optimum coloring effect.
To this end, according to an embodiment of the present invention, the present invention provides a culture medium for producing carotenoids by fermentation of Phaffia rhodozyma, wherein the seed culture medium comprises: 2g/L of mixed carbon source, 2g/L of yeast extract powder, 2g/L of malt extract powder and 5g/L of peptone; the fermentation medium comprises: 100g/L of mixed carbon source, 7g/L of mixed nitrogen source, 5g/L of yeast extract powder, 5g/L of malt extract powder, 6g/L of peptone, 1.5g/L of magnesium sulfate, 10g/L of tomato powder and 6mL of absolute ethyl alcohol; wherein the mixed carbon source comprises 40% of molasses and 60% of starch sugar; the mixed nitrogen source is 40% of corn steep liquor and 60% of ammonium sulfate.
The Phaffia rhodozyma according to an embodiment of the invention ferments a carotenoid production medium by which 3 natural carotenoids can be produced: astaxanthin, canthaxanthin and beta-carotene, and the proportion of the three carotenoids is reasonable, so the feed is suitable for being added into feed for producing eggs and prawns, and the best coloring effect is obtained.
In addition, the culture medium for producing carotenoids by fermentation of phaffia rhodozyma feed, which is proposed according to the above embodiments of the present invention to increase the astaxanthin content in poultry eggs, may further have the following additional technical features:
optionally, in the fermentation medium, 25g/L of mixed nitrogen source is added at the beginning of fermentation, and 75g/L is fed-batch at the later stage of fermentation; adding 5g/L tomato powder at the beginning of fermentation, and adding 5g/L tomato powder at the later stage of fermentation; 3mL of absolute ethyl alcohol is added at the beginning of fermentation, and 3mL is added at the later stage of fermentation.
According to an embodiment of the present invention, the present invention also provides a method for producing carotenoids using the above-mentioned medium, comprising the steps of:
(1) inoculating Phaffia rhodozyma to the seed culture medium to activate the strain for the third generation;
(2) inoculating the activated strain into the fermentation culture medium, performing fermentation culture, and collecting astaxanthin, beta-carotene and canthaxanthin in fermentation liquor.
In addition, the method for producing carotenoids according to the above embodiment of the present invention may have the following additional technical features:
optionally, in the step (1), phaffia rhodozyma is inoculated into a seed culture medium, and shake cultivation is carried out for 40h at the temperature of 20-22 ℃ and the rotating speed of 150r/min, so as to obtain a first generation of seeds; inoculating the first generation seed in a new seed culture medium at 6% of the inoculum size, and performing shake culture at 30 deg.C and 150r/min for 40h to obtain second generation seed; inoculating the second generation seeds in a new seed culture medium in an inoculation amount of 6%, and performing shake cultivation for 40h at the temperature of 20-22 ℃ and the rotating speed of 150r/min to obtain the third generation seeds.
Optionally, in the step (2), the activated strain is inoculated in the fermentation medium in an inoculation amount of 10%, the pH is adjusted to be 5.0-5.5, the temperature is adjusted to be 20-22 ℃, the weak light culture is performed, and the stirring speed is 250-350 r/min.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Examples
Preparation of seed culture medium: preparing a liquid culture medium, subpackaging the liquid culture medium in 250mL (liquid containing amount of 50-70 mL) and 1000mL (liquid containing amount of 300mL) triangular flasks, adjusting the pH value to 5.5-6.0, and sterilizing at the high temperature of 121 ℃ for 30min for later use; the formula of the seed culture medium comprises 2g/L of mixed carbon source (molasses 40% and starch sugar 60%), 2g/L of yeast extract powder, 2g/L of malt extract powder and 5g/L of peptone.
Seed culture of phaffia rhodozyma: inoculating Phaffia rhodozyma strain (from China center for type culture Collection, with the preservation number: CCTCC M2019083) into a seed culture medium (250mL triangular flask) from a biological safety cabinet, and performing shake culture for 40h at the temperature of 20-22 ℃ and the rotating speed of 150r/min to obtain a first-generation seed; then continuously inoculating the first generation seeds into a seed culture medium (250mL triangular flask), wherein the inoculation amount is 6%, and performing shake culture for 40h at 30 ℃ and the rotation speed of 150r/min to obtain second generation seeds; inoculating the second generation seeds into a seed culture medium (1000mL triangular flask), wherein the inoculation amount is 6%, and performing shake culture for 40h at the temperature of 20-22 ℃ and the rotating speed of 150r/min to obtain the third generation seeds.
Fermentation culture: inoculating the cultured third-generation seeds into a 5L fermentation tank, wherein the inoculation amount is 10%, the liquid loading amount is 3L, the pH is 5.0-5.5, the temperature is 20-22 ℃, culturing is carried out for 24h under low light, the stirring speed is 250-350 r/min, and fermentation liquid is collected. Wherein the fermentation medium is: 100g/L of mixed carbon source (40% of molasses and 60% of starch sugar) (wherein 25g/L is added at the beginning of fermentation and 75g/L is added at the beginning of 18h of fermentation), 7g/L of mixed nitrogen source (40% of corn steep liquor and 60% of ammonium sulfate), 5g/L of yeast extract powder, 5g/L of malt extract powder, 6g/L of peptone, 1.5g/L of magnesium sulfate, 10g/L of tomato powder (5 g/L is added at the beginning of fermentation and 5g/L is added at 18h of fermentation), and 6mL of absolute ethyl alcohol (3 mL is added at the beginning of fermentation and 3mL is added at 18h of fermentation).
5ml of fermentation broth is taken and subjected to RCF: centrifuging at 3000r/min for 10min, washing with deionized water twice, adding 55 deg.C and 2ml dimethyl sulfoxide into centrifuged yeast, shaking, stirring, adding 2ml acetone extraction reagent, stirring, centrifuging (3000r/min) for 5min to obtain extractive solution. Collecting supernatant, and detecting by liquid chromatography. The results of the three carotenoids were 906ug/g astaxanthin, 302ug/g canthaxanthin and 302ug/g beta-carotene.
Test examples
Selecting 28-31 week-old Luhua chickens with healthy constitutions, similar body weights and body sizes and similar laying rates, randomly dividing the Luhua chickens into 3 groups (a control group, a test 1 and a test 2), wherein each group has 5 repetitions, and each repetition has 30 chickens. The control group was fed a basal diet (commercially available, new wisdom), and test 1 added 1g of the fermentation product obtained in example 1 (astaxanthin: canthaxanthin: β -carotene ═ 3:1:1) per kg of basal diet, and test 2 added 1g of astaxanthin per kg of basal diet. Feeding test chickens in a single cage in the whole process, freely taking food and drinking water, carrying out conventional feeding management, feeding once every 08:00 and 14:00 days, picking up eggs and cleaning manure twice respectively, disinfecting 1 time every week, and carrying out conventional immunization on the test chickens. The test is divided into a pre-test period and a test period, wherein the pre-test period is 1 week, and the test period is 8 weeks. On days 0, 3, 7, 10, 21, 35 and 56 of the experiment, 50 eggs of consistent egg weight were collected from each group and assayed.
The whole yolk was separated by yolk separator (H-Y2033 model, beth-honest hardware and plastic works in Yangjiang city, etc.), the color grade of the yolk was measured by a roche colorimetric fan (Nanjing Minao instruments and equipments Co., Ltd.), the measured value was represented by the RYCF yellow value, and the yolk yellow value b was measured by a colorimeter (CR-10 model, Shanghai Dingyun International trade Co., Ltd.).
Table 1: yellowness index of egg yolk
Group of Test group 1 Test group 2 Control group
Day 0 8.62±0.42 8.60±0.43 8.68±0.41
Day 3 9.77±0.45 8.77±0.45 8.57±0.45
Day 7 11.58±0.52 9.58±0.52 8.77±0.43
Day 10 11.66±0.47 9.66±0.47 8.81±0.45
Day 21 11.75±0.56 10.75±0.56 8.97±0.43
Day 35 11.87±0.58 10.87±0.58 9.14±0.44
Day 56 12.08±0.51 10.88±0.51 9.16±0.49
As can be seen from the above table, 3 natural carotenoids can be produced by fermentation of this medium: astaxanthin, canthaxanthin and beta-carotene, and the proportion of the three carotenoids is reasonable, so the feed is suitable for being added into feed for producing eggs and prawns, and the best coloring effect is obtained.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (5)

1. A culture medium for producing carotenoid by fermentation of phaffia rhodozyma is characterized in that a seed culture medium comprises: 2g/L of mixed carbon source, 2g/L of yeast extract powder, 2g/L of malt extract powder and 5g/L of peptone; the fermentation medium comprises: 100g/L of mixed carbon source, 7g/L of mixed nitrogen source, 5g/L of yeast extract powder, 5g/L of malt extract powder, 6g/L of peptone, 1.5g/L of magnesium sulfate, 10g/L of tomato powder and 6mL of absolute ethyl alcohol; wherein the mixed carbon source comprises 40% of molasses and 60% of starch sugar; the mixed nitrogen source is 40% of corn steep liquor and 60% of ammonium sulfate.
2. The culture medium for producing carotenoids by fermentation of Phaffia rhodozyma according to claim 1, wherein the mixed nitrogen source is added at 25g/L at the beginning of fermentation and is fed at 75g/L at the later stage of fermentation; adding 5g/L tomato powder at the beginning of fermentation, and adding 5g/L tomato powder at the later stage of fermentation; 0.1 percent of absolute ethyl alcohol is added at the beginning of fermentation, and 0.1 percent is added at the later stage of fermentation.
3. A method for producing carotenoids using the medium of claim 1 or 2, comprising the steps of:
(1) inoculating Phaffia rhodozyma to the seed culture medium to activate the strain for the third generation;
(2) inoculating the activated strain into the fermentation culture medium, performing fermentation culture, and collecting astaxanthin, beta-carotene and canthaxanthin in fermentation liquor.
4. The method of claim 3, wherein in the step (1), Phaffia rhodozyma is inoculated into a seed culture medium and subjected to shake cultivation for 40 hours at the temperature of 20-22 ℃ and the rotating speed of 150r/min to obtain a first generation of seeds; inoculating the first generation seed in a new seed culture medium at 6% of the inoculum size, and performing shake culture at 30 deg.C and 150r/min for 40h to obtain second generation seed; inoculating the second generation seeds in a new seed culture medium in an inoculation amount of 6%, and performing shake cultivation for 40h at the temperature of 20-22 ℃ and the rotating speed of 150r/min to obtain the third generation seeds.
5. The method as claimed in claim 3, wherein in the step (2), the activated strain is inoculated into the fermentation medium in an inoculation amount of 10%, the pH is adjusted to 5.0-5.5, the temperature is adjusted to 20-22 ℃, the culture is performed under the weak light, and the stirring speed is 250-350 r/min.
CN202010973840.1A 2020-09-16 2020-09-16 Culture medium and culture method for producing carotenoid by fermentation of phaffia rhodozyma Pending CN112063539A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114045226A (en) * 2021-11-19 2022-02-15 山东省食品发酵工业研究设计院 Low-cost culture medium for culturing phaffia rhodozyma producing astaxanthin and preparation method and application thereof

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CN1544647A (en) * 2003-11-25 2004-11-10 华南理工大学 Course feeding fermentation method for preparing astaxanthin with molasses or starch sugar as materials
CN103649328A (en) * 2011-06-30 2014-03-19 株式会社钟化 Method for producing carotenoid composition
CN108893517A (en) * 2018-07-19 2018-11-27 威海利达生物科技有限公司 A kind of fermentation medium and method of red phaffia rhodozyma fermenting and producing astaxanthin

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Publication number Priority date Publication date Assignee Title
WO2004029261A2 (en) * 2002-09-27 2004-04-08 Dsm Ip Assets B.V. Production of canthaxanthin by phaffia
CN1544647A (en) * 2003-11-25 2004-11-10 华南理工大学 Course feeding fermentation method for preparing astaxanthin with molasses or starch sugar as materials
CN103649328A (en) * 2011-06-30 2014-03-19 株式会社钟化 Method for producing carotenoid composition
CN108893517A (en) * 2018-07-19 2018-11-27 威海利达生物科技有限公司 A kind of fermentation medium and method of red phaffia rhodozyma fermenting and producing astaxanthin

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114045226A (en) * 2021-11-19 2022-02-15 山东省食品发酵工业研究设计院 Low-cost culture medium for culturing phaffia rhodozyma producing astaxanthin and preparation method and application thereof

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