Method for high-density culture of rhodotorula mucilaginosa and production of carotenoid by using Xinhui orange juice and application

Abstract

The invention discloses a method for high-density culture of rhodotorula mucilaginosa and production of carotenoid by using Xinhui orange juice and application, and relates to the technical field of biological fermentation application. Rhodotorula mucilaginosa can be rapidly bred in a Xinhui orange juice liquid culture medium at high density, can reach 35-45g / L of dry weight under the optimal shake flask condition, is improved by 5-6 times compared with a common culture medium, can effectively improve the biomass and carotenoid yield, can be directly used as a feed additive to form a carotenoid and single-cell protein double-product system, and has the advantages of high yield, high yield and the like. The resource utilization rate is increased, antioxidant active substances are provided for aquaculture animals, and the immunity of the animals is promoted; the palatability of the feed additive is enhanced, the feed intake of animals is increased, and the palatability of the liquid feed additive meets the requirements of feed industry. The method solves the ecological environment problem of treatment of citrus flesh in the Xinhui district, also can meet the requirements of various aquaculture products, improves the added value, and has significant economic benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation applications, and specifically provides a method and application for high-density cultivation of Rhodotorula mucilaginosa and production of carotenoids using Xinhui orange juice, and particularly relates to a feed additive rich in carotenoids and high-density single-cell protein obtained by fermentation of Xinhui orange juice and a preparation method thereof. Background Art

[0002] Using microorganisms to ferment natural plants is a green and ecological preparation method. Fermenting natural plants can decompose macromolecular substances in natural plants and convert them into small-molecular substances that can be directly utilized by animals, thereby enhancing their usage effects. Currently, fermented natural plants are widely used as feed additives in animal production.

[0003] Carotenoids belong to lipophilic isoprenoid metabolites and are the most common and stable natural pigments in nature. Due to their antioxidant properties, they can effectively inhibit the generation of free radicals, delay the aging process of the body, enhance immune function, resist radiation, and have important effects such as anti-tumor. However, since animal carotenoids rely on food intake, carotenoids have good prospects in the feed additive industry.

[0004] Marine Rhodotorula is a single-celled organism widely present in the ocean. Studies have shown that using Rhodotorula mucilaginosa as a feed additive in the aquaculture production of aquatic animals not only improves the survival rate of aquatic animals but also promotes growth performance, disease resistance, and improvement of the intestinal flora. In feed additives, Rhodotorula mucilaginosa can utilize waste resources for proliferation and fermentation and produce microbial protein rich in protein, nucleotides, carotenoids, oils, growth factors, and multivitamins. And containing a high protein content and a certain amount of unsaturated fatty acids can help improve the antioxidant capacity in the bodies of aquatic animals, making them have characteristics such as strong disease resistance, high survival rate, bright skin and muscle color, and good taste.

[0005] Citrus is a fruit with high nutritional value, rich in nutrients and active ingredients such as dietary fiber, vitamin C, minerals, polyphenols, and carotenoids. And the polyphenols and flavonoids in citrus pulp have functions such as antioxidant, prevention of cardiovascular diseases, and blood pressure reduction.

[0006] Specifically, Xinhui tangerine peel is a national geographical indication product, mainly produced in Xinhui area of Jiangmen City, Guangdong Province. However, because of its sour taste, it is difficult to be fully eaten, with high preservation difficulty and high processing cost, so the pulp is discarded. When the tangerine fruits are harvested every year, after peeling to obtain the peel, a large amount of pulp is peeled off and discarded at will, becoming waste that no one cares about. According to media reports, the amount of pulp discarded in Xinhui District every year is as high as 80,000 tons. Therefore, developing a method for highly efficient production of carotenoid-rich feed additives by Rhodotorula mucilaginosa fermenting Xinhui tangerine juice, solving the problem of environmental pollution caused by the waste of Xinhui tangerine pulp, and at the same time producing carotenoid-rich probiotic feed and feed additives with higher added value has great economic and social benefits. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a method and application for high-density cultivation of Rhodotorula mucilaginosa and production of carotenoids using Xinhui tangerine juice.

[0008] The present invention adopts a preparation process for highly efficient production of carotenoid-rich feed additives by Rhodotorula mucilaginosa fermenting Xinhui tangerine peel juice, including optimization of the Xinhui tangerine peel juice culture medium and process conditions, expansion culture, preparation of feed additives, and application of feed additives.

[0009] The purpose of the present invention is achieved by the following technical solutions:

[0010] The present invention provides a Xinhui tangerine juice liquid culture medium for high-density growth of Rhodotorula mucilaginosa, which is prepared by mixing the main material of Xinhui tangerine juice with auxiliary materials evenly and sterilizing; wherein, the auxiliary materials include 0-5% w / v glucose and 0-6% w / v ammonium sulfate.

[0011] Preferably, the total sugar in the Xinhui tangerine juice liquid culture medium is 95 g / L - 100 g / L, the total nitrogen content is 1 - 6 g / L, and the pH is 5.8 - 6.2.

[0012] More preferably, the total sugar in the Xinhui tangerine juice liquid culture medium is 95 g / L - 100 g / L, the total nitrogen content is 2 - 5 g / L, and the pH is 5.8 - 6.2.

[0013] Even more preferably, the total sugar in the Xinhui tangerine juice liquid culture medium is 95 g / L - 100 g / L, the total nitrogen content is 3 - 4 g / L, and the pH is 5.8 - 6.2.

[0014] The preparation method of the Xinhui tangerine juice includes the following steps: peeling the fresh Xinhui tangerine fruits and then juicing, filtering, centrifuging, and taking the supernatant to obtain Xinhui tangerine juice.

[0015] The filtering is carried out with a filter paper of 300 - 500 meshes; further preferably, it is carried out with a 400-mesh filter paper.

[0016] The centrifugation conditions are centrifuging at 3000 - 5000 rpm for 5 - 15 min; further preferably, centrifuging at 4000 rpm for 10 min.

[0017] The application of the above-mentioned Xinhui tangerine juice liquid medium in the high-density culture of Rhodotorula mucilaginosa and the production of carotenoids.

[0018] A method for high-density culturing Rhodotorula mucilaginosa and producing carotenoids using Xinhui tangerine juice, comprising the following steps:

[0019] Inoculate the seed liquid of Rhodotorula mucilaginosa into the above-mentioned Xinhui tangerine juice liquid medium, ferment to obtain the Rhodotorula mucilaginosa fermentation broth, which is the liquid feed additive; achieving the purpose of high-density culturing Rhodotorula mucilaginosa and efficiently producing carotenoids.

[0020] The inoculation amount of the Rhodotorula mucilaginosa seed liquid is 2 - 6% (v / v); further preferably, it is 4 - 6% (v / v).

[0021] The fermentation conditions are 26°C - 30°C, 150 - 300 rpm for 72 - 120 h; further preferably, 28°C, 240 - 250 rpm for 96 h.

[0022] The preparation method of the Rhodotorula mucilaginosa seed liquid comprises the following steps: inoculate the activated Rhodotorula mucilaginosa into the seed medium for cultivation to obtain the Rhodotorula mucilaginosa seed liquid.

[0023] The cultivation conditions are 26 - 30°C, 150 - 300 r / min for 24 - 48 h; further preferably, 28°C, 180 rpm for 36 h.

[0024] The seed medium includes PDB liquid medium.

[0025] The Rhodotorula mucilaginosa includes Rhodotorula mucilaginosa GDMCC NO.2.30.

[0026] A liquid feed additive is prepared by the above method.

[0027] A powder feed additive is obtained by spray drying after adding 10 - 15% w / v soluble corn starch as a carrier to the above liquid feed additive.

[0028] The spray drying conditions are inlet air temperature 160 - 200°C, outlet temperature 80 - 90°C, feed rate 10 - 12 L / min, and atomization head speed 18000 - 25000 r / min.

[0029] Preferably, the viable count of Rhodotorula mucilaginosa in the powder feed additive is 1 - 5×10 9CFU / g.

[0030] Use of the above liquid feed additive or powder feed additive in the preparation of feed; further, use in the preparation of aquaculture animal feed.

[0031] The addition amount of the liquid feed additive or powder feed additive in the feed is 5-10% w / w; further 7.5%-10% w / w. The growth test results show that: adding this additive to the Litopenaeus vannamei feed increases the survival rate, weight gain rate and specific growth rate of the shrimp compared with the control group. At the same time, the feed conversion ratio decreases.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] (1) Fresh citrus fruits are not easy to store and transport due to their high water content. The feed additive produced by fermenting citrus juice with Rhodotorula mucilaginosa has antibacterial properties and acidity, can be stored for a long time, and can also be in the form of powder by spray drying technology, which is easier to transport and store;

[0034] (2) The Xinhui tangerine peel citrus juice has a high sugar content and is rich in natural carotenoids, which enables Rhodotorula mucilaginosa to grow and reproduce in large numbers. While increasing the viable count of Rhodotorula mucilaginosa, it can also significantly increase the content of carotenoids produced by the bacteria. And Rhodotorula mucilaginosa contains rich microbial protein, and the protein content accounts for about 50% of the dry weight, rich in B vitamins and unsaturated fatty acids. After the citrus juice is fermented, various substances with aromatic flavors such as alcohols, aldehydes, ketones, acids and esters are produced, which enhances the palatability of the feed additive, has the effect of increasing the feed intake of animals, and the palatability of the liquid feed additive meets the requirements of the feed industry. While solving the ecological environment problem of the disposal of citrus pulp in Xinhui District, it can also meet the needs of various aquaculture products, improve its added value, and the economic benefits are significant.

[0035] (3) Safe, pollution-free, simple process and convenient to use. Xinhui tangerine peel citrus is safe to eat, and Rhodotorula is also widely selected as live bait for aquatic animals such as fish and shrimp. While obtaining a feed additive rich in carotenoids, the Xinhui citrus fruits are processed. It achieves the purpose of low cost, pollution-free and turning waste into treasure.

[0036] (4) Rhodotorula mucilaginosa can reproduce rapidly and at high density in the Xinhui citrus juice liquid medium, and reaches a cell dry weight of 35-45 g / L under the optimal conditions of the shake flask, which is 5-6 times higher than that of the ordinary medium. Directly used as a feed additive, it forms a "carotenoid + single cell protein" dual product system, improving the resource utilization rate.

[0037] (5) The discarded Xinhui tangerine fruits are utilized to prepare a feed additive rich in sugars, organic acids, flavonoids and carotenoids. The optimal fermentation condition parameters are determined in shake flask culture as follows: rotation speed: 240 r / min, inoculum size: 4% (v / v), temperature: 28 °C. The total carotenoid yield is 13.06 mg / L, and its biomass reaches 44.62 g / L. Compared with the ordinary medium, Rhodotorula mucilaginosa cultured with Xinhui tangerine juice can effectively improve the biomass and carotenoid yield, providing antioxidant active substances for aquaculture animals and promoting the immunity of the animals themselves. Description of the Drawings

[0038] Figure 1 It is a schematic diagram of the wet mycelium after centrifugation and water washing of 5 mL of fermentation broth of Rhodotorula mucilaginosa cultured with the optimized Xinhui tangerine juice liquid medium and the ordinary medium; among them, a is obtained by culturing with the ordinary medium, and b is obtained by culturing with the optimized Xinhui tangerine juice liquid medium. Detailed Embodiments

[0039] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0040] For the test methods without specifying specific experimental conditions in the following embodiments, they are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0041] The strain used in the present invention is: Rhodotorula mucilaginosa, which is preserved in the Guangdong Provincial Culture Collection Center, and the preservation number is GDMCC NO.2.30.

[0042] Example 1

[0043] The specific steps for the present invention to efficiently produce a carotenoid-rich feed additive by fermenting Xinhui tangerine peel juice with Rhodotorula mucilaginosa are as follows:

[0044] 1) Preparation of the seed liquid: Take Rhodotorula mucilaginosa and inoculate it into the prepared sterilized PDB liquid medium, and ferment at 150 - 250 rpm on a shaker at 26 - 30 °C for 36 h to obtain the Rhodotorula mucilaginosa seed liquid.

[0045] 2) Pretreatment of Xinhui tangerine peel juice: The Xinhui tangerines in the present invention are Xinhui tangerines grown in Xinhui District, Jiangmen City, Guangdong Province, China. The pretreatment method of the Xinhui tangerine juice is as follows: After peeling the fresh Xinhui tangerine fruits, put them into a juicer to make pulp, filter, put them into a centrifuge and centrifuge at 4000 rpm for 10 min, and then take the supernatant for sterilization treatment, that is, the unoptimized Xinhui tangerine juice, as the unoptimized Xinhui tangerine juice liquid medium.

[0046] 3) Fermentation: Inoculate the prepared Rhodotorula mucilaginosa seed liquid in step 1) into a 250 mL Erlenmeyer flask of the well-mixed and filled Xinhui tangerine juice liquid medium in step 2), with an inoculation amount of 2 - 6%, and ferment at 26 - 30 °C on a shaker at 150 - 250 rpm for 3 - 5 days. After the fermentation is completed, the liquid feed additive of Rhodotorula mucilaginosa fermentation broth is obtained.

[0047] Example 2

[0048] A method for efficiently producing a carotenoid-rich feed additive by fermenting Xinhui tangerine peel tangerine juice with Rhodotorula mucilaginosa, comprising the following steps:

[0049] Step 1. Select materials: The Xinhui tangerine pulp after peeling from October to December, and the tangerine pulp should have bright color, no pests and diseases, and no mechanical damage;

[0050] Step 2. Pulp treatment: Put the tangerine pulp into a juicer to extract juice;

[0051] Step 3. Filtration and centrifugation: Filter the Xinhui tangerine juice with a 400-mesh filter paper, and then put it into a centrifuge for centrifugation (centrifuge at 4000 rpm for 10 min) to obtain the supernatant;

[0052] Step 4. Adjust the medium components. Dilute or add glucose according to different batches of tangerines pretreated so that the total sugar is 95 g / L - 100 g / L; supplement ammonium sulfate according to the batch of tangerines so that the total nitrogen content is 1 - 6 g / L for the nitrogen source.

[0053] Step 5. Adjust the pH value to 6.

[0054] Step 6. Sterilization: Adopt the high-temperature and high-pressure sterilization method, with the conditions of 115 °C for 15 min. After sterilization, immediately cool to room temperature, that is, the optimized Xinhui tangerine juice, as the optimized Xinhui tangerine juice liquid medium.

[0055] Step 7. Inoculation: Inoculate the sterilized tangerine juice liquid medium with the Rhodotorula mucilaginosa seed liquid at an inoculation amount of 2% - 6%.

[0056] Step 8. Fermentation: Ferment at 26 - 30 °C on a shaker at 150 - 250 rpm for 3 - 5 days. After the fermentation is completed, the liquid feed additive of Rhodotorula mucilaginosa fermentation broth is obtained.

[0057] Strain activation: After streaking the strain on the slant medium, activate and culture it in an aerobic incubator at 28 °C for 48 h; until single colonies grow;

[0058] Flask fermentation culture: Inoculate the activated seed liquid into a triangular flask, with an inoculation amount of 5% (v / v), a shaker speed of 180 r / min, ferment and culture at 28 °C, and the pH is 6;

[0059] Transfer the slant culture of Rhodotorula glutinis to the seed medium and activate it at 26 - 30°C and 150 - 250 r / min for 36 h to obtain the activated seed liquid. The activated seed liquid is inoculated into the optimized liquid medium of Xinhui orange juice at an inoculation amount of 2 - 6% (v / v). The liquid loading is 20 ml / 250 mL. After culturing at 180 r / min for 96 h, it is diluted and spread on the PDA medium, and the viable count is calculated by the number of single colonies.

[0060] Determination of total carotenoids: Yeast cells were lysed using dimethyl sulfoxide. Take 5 mL of cell suspension, centrifuge it, wash it twice with distilled water, centrifuge at 4000 rpm for 10 min, add 5 mL of 99% anhydrous ethanol and wash once, centrifuge at 4000 rpm for 10 min, discard the supernatant, add 3 mL of dimethyl sulfoxide, resuspend the cell pellet and stir evenly, then incubate in a water bath at 65°C for 50 min. If the lysis is not complete, continue the water bath. After complete lysis, add 6 mL of acetone and extract by shaking for 10 min, shake well, centrifuge at 4000 rpm for 10 min, take the supernatant and measure the absorbance value A at 480 nm on a spectrophotometer; if the extraction is incomplete, add acetone for extraction until the cell pellet becomes colorless. Calculate the total carotenoid yield using the following formula:

[0061] Total carotenoid yield (mg / L) = A 480 ×D×V / (0.16×W)

[0062] Where: D ------- Dilution factor

[0063] V ------- Volume of organic solvent used for extraction (mL)

[0064] W ------- Volume of fermentation broth used for extraction (mL)

[0065] 0.16 ----- Extinction coefficient of organic solvent

[0066] Dry weight of bacteria in fermentation medium: Dry the tin foil dish to a constant weight W0. Take 5 mL of cell suspension and put it into a centrifuge tube for centrifugation, wash it twice with distilled water, discard the supernatant, dry the precipitate in an oven at 105°C, and finally take it out and weigh it to a constant weight W1.

[0067] W = W1 - W0

[0068] The formula of the fermentation medium is as follows: For the carbon source, according to different batches of oranges, after pretreatment, dilute or add glucose to make the total sugar 95 g / L - 100 g / L; for the total nitrogen source, according to different batches of oranges, supplement ammonium sulfate to make the total nitrogen 1 - 6 g / L.

[0069] Example 3, Optimization of orange juice fermentation medium

[0070] Using the total carbon source (95 g / L - 100 g / L), total nitrogen source (1 - 6 g / L), and initial pH (pH = 4 - 7) as experimental factors, single-factor experiments were conducted separately according to the following culture conditions. Pick the Rhodotorula mucilaginosa slant culture and inoculate it into the potato dextrose medium, and culture it at 28 °C and 180 r / min for 36 h to obtain the seed liquid. The fermentation conditions were an initial pH of the medium of 6, the unoptimized Xinhui tangerine juice of Example 1, 28 °C, 180 r / min, an inoculation amount of 5% (v / v), and a culture time of 96 h. Optimization was carried out with the total carotenoid yield as the index.

[0071] The results of medium optimization were as follows: Glucose was supplemented to 95 g / L - 100 g / L according to the total sugar of different batches of tangerines; when the total nitrogen source was supplemented with ammonium sulfate to a total nitrogen of 3 g / L and the initial pH = 6, the total carotenoid yield reached an optimal 11.27 mg / L, and the biomass reached 43.61 g / L.

[0072] Example 4. Optimization of fermentation process conditions by response surface methodology

[0073] On the basis of the optimal fermentation medium in Example 3, the best fermentation process conditions were preliminarily explored, and three levels of three factors, namely rotation speed (200 / 250 / 300 r / min), inoculation amount (2 / 4 / 6%), and temperature (26 / 28 / 30 °C), were studied using Design-Expert 13.0.

[0074] The optimal fermentation conditions were obtained, that is, the rotation speed was 240 rpm, the temperature was 28 °C, and the inoculation amount was 4% (v / v). Under these fermentation process conditions, the average carotenoid production of this strain was 13.06 mg / L, and the biomass reached 44.62 g / L.

[0075] Example 5. Comparative test between ordinary medium and Xinhui tangerine juice liquid medium

[0076] On the basis of the optimal fermentation medium in Example 3, with the best culture conditions of a rotation speed of 240 r / min, an inoculation amount of 4%, and a temperature of 28 °C, fermentation was carried out for 96 h under these conditions, and it was repeated 3 times; the results of the verification test are shown in Table 1 and Figure 1 as follows. The dry cell weight of Rhodotorula mucilaginosa cultured with Xinhui tangerine juice was more than 5 times higher than that of the ordinary medium. And it effectively increased the carotenoid yield. Among them, the composition of the ordinary medium was as follows: ammonium sulfate 3 g / L, glucose 95 g / L, magnesium sulfate heptahydrate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, ferrous sulfate 0.01 g / L, potassium chloride 0.5 g / L, thiamine 0.4 μg / L, made up to volume with distilled water, pH 6.

[0077] Table 1. Results of verification test and comparison between optimized medium and ordinary medium

[0078]

[0079] Example 6 Preparation of High-Density Fermentation Broth of Rhodotorula mucilaginosa

[0080] (1) Strain activation: After the strain is streaked on the slant medium, it is activated and cultured in an aerobic incubator at 28 °C for 48 h until single colonies grow;

[0081] (2) Culture of seed liquid: The activated strain is transferred to the seed medium (PDB liquid medium), and the shaker speed is 180 rpm. It is cultured at 28 °C for 36 h as the seed liquid;

[0082] (3) Preparation of Rhodotorula mucilaginosa fermentation broth: The seed liquid prepared in step (2) is inoculated into a 7 L fermenter containing 5 L of fermentation medium at an inoculation amount of 4%. The temperature is 28 °C, the rotation speed is 400 r / min, the tank pressure is 0.05 Mpa, and the ventilation ratio is 1:0.5. It is cultured for 96 h to obtain a Rhodotorula mucilaginosa fermentation broth with a viable cell count of 5×10 10 cfu / mL, that is, the liquid feed additive is obtained;

[0083] In the fermentation medium, the carbon source is diluted after pretreatment of different batches of oranges or glucose is added until the total sugar is 95 g / L - 100 g / L; the nitrogen source is supplemented with ammonium sulfate according to the batch of oranges until the total nitrogen content is 3 - 4 g / L, and the initial pH = 6.

[0084] Example 7 Preparation of Powdered Feed Additive

[0085] The preparation method of the powdered feed additive is as follows: 10 - 15% w / v soluble corn starch is added as a carrier to the Rhodotorula mucilaginosa fermentation broth after the fermentation in Example 6, and spray drying is carried out. The drying conditions are: the inlet air temperature is 160 - 200 °C, the outlet temperature is 80 - 90 °C. The fermentation broth is put into a spray dryer for drying, the feeding speed is 10 - 12 L / min, and the atomization head speed is 18000 - 25000 r / min, to prepare a powdered feed additive containing 1 - 5×10 9 CFU / g.

[0086] Example 8 Feeding Experiment on Litopenaeus vannamei

[0087] The feed additive of Rhodotorula mucilaginosa fermented Xinhui orange juice provided by the present invention is applied to the feeding of Litopenaeus vannamei:

[0088] The feed with a fish meal content of 28% w / w is used as the basal feed and serves as the control group; a single-factor experimental design is adopted. The powdered feed additive in Example 7 is mixed into the basal feed at a ratio of 5 - 10% w / w, so that the content of carotenoids in the feed is 40, 60, 80 mg / kg.

[0089] The experiment was divided into 4 groups, including 1 control group (G0) and 3 experimental groups (G40, G60, G80). There were 3 replicates in each group, and each replicate had 10 Litopenaeus vannamei. The control group was fed with basal diet, and the experimental groups were fed with the basal diet supplemented with the above feed additives respectively. The shrimp were fed twice a day with the same feeding amount each time. Litopenaeus vannamei with an initial weight of 0.26 g were cultured for 21 days, and Litopenaeus vannamei with an initial weight of 0.11 g were cultured for 41 days to study the effects of adding the feed additive to the formulated feed of Litopenaeus vannamei on its growth, survival rate, feed utilization and body composition, etc. The experimental results are shown in Tables 2 - 3.

[0090] The survival rate, weight gain rate and specific growth rate of the experimental Litopenaeus vannamei were calculated using the following formulas respectively:

[0091] Weight gain rate (WGR, %) = 100×(final average weight - initial average weight) / initial average weight;

[0092] Specific growth rate (SGR, % / d) = 100×(ln final average weight - ln initial average weight) / number of days of culture;

[0093] Survival rate (SR, %) = (number of shrimp at the end of the experiment / number of shrimp at the beginning of the experiment)×100;

[0094] Feed conversion ratio (FCR) = total amount of feed fed / (final weight + weight of dead shrimp - initial weight);

[0095] Table 2 Growth performance of Litopenaeus vannamei with an initial body weight of 0.26 g cultured for 21 days

[0096]

[0097] Note: *p < 0.05, vs G0.

[0098] Table 3 Growth performance of Litopenaeus vannamei with an initial body weight of 0.11 g cultured for 41 days

[0099]

[0100] Note: *p < 0.05, vs G0.

[0101] The growth experiment results showed that adding the feed additive to the feed of Litopenaeus vannamei had no adverse effects on the growth, survival rate and feed utilization rate of the shrimp (P > 0.05). When the feed contained 60 - 80 mg / kg carotenoid, the survival rate, weight gain rate and specific growth rate of the shrimp were all increased compared with the control group. At the same time, the feed conversion ratio decreased, and the decrease range was 4.00% - 6.52%.

[0102] When the Rhodotorula glutinis of the present invention is fermented, the raw materials used are all raw materials adopted by feed animals. After fermenting the above raw materials with the Rhodotorula glutinis, a large amount of live cells of Rhodotorula glutinis rich in carotenoids can be produced. The results of the examples show that after fermenting the Rhodotorula glutinis, the obtained fermentation culture can be directly fed to Litopenaeus vannamei, and it can promote the growth of the shrimp and enhance its immunity.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A liquid medium of Xinhui tangerine juice for high-density growth of Rhodotorula glutinis, characterized in that The Xinhui tangerine juice liquid medium is prepared by taking Xinhui tangerine juice as the main raw material, mixing it evenly with auxiliary materials, and then sterilizing; wherein, the auxiliary materials include 0-5% w / v glucose and 0-6% w / v ammonium sulfate.

2. The Xinhui tangerine juice liquid medium according to claim 1, characterized in that, In the Xinhui tangerine juice liquid medium, the total sugar is 95 g / L - 100 g / L, the total nitrogen content is 1 - 6 g / L, and the pH is 5.8 - 6.2; further, the total sugar is 95 g / L - 100 g / L, the total nitrogen content is 2 - 5 g / L, and the pH is 5.8 - 6.2; still further, the total sugar is 95 g / L - 100 g / L, the total nitrogen content is 3 - 4 g / L, and the pH is 5.8 - 6.

2.

3. The Xinhui tangerine juice liquid medium according to claim 1 or 2, characterized in that The preparation method of the Xinhui tangerine juice includes the following steps: peeling the fresh Xinhui tangerine fruits, juicing, filtering, centrifuging, and taking the supernatant to obtain Xinhui tangerine juice.

4. Application of the Xinhui tangerine juice liquid medium according to any one of claims 1 - 3 in high-density culture of Rhodotorula mucilaginosa and production of carotenoids.

5. A method for high-density cultivation of Rhodotorula glutinis and production of carotenoids using Xinhui tangerine juice, characterized in that, Including the following steps: Inoculating the seed liquid of Rhodotorula mucilaginosa into the Xinhui tangerine juice liquid medium according to any one of claims 1 - 3, fermenting to obtain the Rhodotorula mucilaginosa fermentation broth, which is the liquid feed additive.

6. According to the method of claim 5, characterized in that: The inoculation amount of the Rhodotorula mucilaginosa seed liquid is 2 - 6% v / v; The fermentation conditions are fermentation at 26°C - 30°C and 150 - 300 rpm for 72 - 120 h; The Rhodotorula mucilaginosa includes Rhodotorula mucilaginosa GDMCC NO.2.

30.

7. A liquid feed additive, characterized in that, Prepared by the method according to claim 5 or 6.

8. A powdery feed additive, characterized in that, Adding 10 - 15% w / v soluble corn starch as a carrier to the liquid feed additive according to claim 7, and performing spray drying to obtain it.

9. Application of the liquid feed additive according to claim 7 or the powdered feed additive according to claim 8 in the preparation of feed.

10. According to the application of claim 9, characterized in that: The addition amount of the liquid feed additive or the powdered feed additive in the feed is 5 - 10% w / w.

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