Methods and applications for producing natural astaxanthin in poultry compound feed using Pharbitis rubescens solid-state fermentation

Abstract

This invention discloses a method for producing natural astaxanthin in poultry compound feed using Pharfovia rubescens solid-state fermentation. The method mainly involves the following steps: (1) slant culture; (2) seed culture; (3) solid-state fermentation culture, where the cultured seed solution is inoculated into the fermentation substrate and cultured at 20-26°C for 6-12 days. This invention features a short fermentation cycle and a simple process. The resulting solid-state fermented feed directly contains natural astaxanthin. On the one hand, it avoids the extraction and additional addition of astaxanthin, reducing production costs; on the other hand, it solves the problem of antibiotic restriction in feed, improving the conversion and absorption of astaxanthin, and enhancing feed quality and palatability.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, specifically to a method for producing natural astaxanthin in poultry compound feed using Pharbitis rubescens solid-state fermentation and its application. Background Technology

[0002] Astaxanthin belongs to the carotenoid family and can be used as a natural colorant and feed additive. The US FDA approved astaxanthin as a pigment additive for animal and fish feed in April 2009, and the European Commission has also approved astaxanthin as a food coloring agent for use in the food industry.

[0003] Currently, astaxanthin is mainly used in feed by adding it to poultry feed to improve poultry farming efficiency. This method has the following drawbacks:

[0004] First, natural astaxanthin is difficult to obtain, the extraction process is complicated, the production cost is high, and there may be problems such as pollutant residue and concentration during the extraction and processing.

[0005] The production of natural astaxanthin in my country is currently in its early stages, with much of the work concentrated in laboratory research. Purchasing natural astaxanthin powder in China costs approximately 7900 yuan / kg, making it relatively expensive. Microbial preparation is more complex. Taking *Phaeodactylum rubrum* as an example, liquid fermentation of the yeast is generally required before astaxanthin extraction. Examples include Chinese patents CN103820520A ("A Fermentation Method for High-Yield Natural Astaxanthin"), CN102864087A ("A *Phaeodactylum rubrum* Strain with High-Yield Natural Astaxanthin and its Breeding Method and Application"), and CN 106701880 A ("A Method for Improving Astaxanthin Production by *Phaeodactylum rubrum* Strain"). Their processes all include *Phaeodactylum rubrum* cell activation, inoculation, fermentation, centrifugation to collect cells, drying, cell wall disruption, extraction, concentration, and analytical extraction of astaxanthin.

[0006] Secondly, astaxanthin is artificially added, and the entire process is not only complex, but it also leads to poor feed taste, which affects feed intake and astaxanthin intake, resulting in low astaxanthin content in poultry and produced eggs.

[0007] For example, Chinese patent CN109258966A discloses "A method for increasing the natural astaxanthin content in eggs," in which the feed consists of a basic feed and a premix containing natural astaxanthin. The feed preparation process involves manually or mechanically mixing the basic feed and the premix until homogeneous. This simple mixing method makes astaxanthin easily decomposed, ultimately preventing its effective intake. Chinese patent CN115606707A discloses "A poultry feed rich in natural astaxanthin and its preparation method," which improves the feed preparation method, increasing the shelf life of astaxanthin in the feed and promoting the intake of astaxanthin by poultry. However, the entire process is relatively complex, and palatability enhancers need to be added to improve palatability.

[0008] Therefore, it is necessary to provide a new method for producing astaxanthin feed. Summary of the Invention

[0009] The purpose of this invention is to solve the above-mentioned problems by providing a method for producing natural astaxanthin in poultry compound feed using solid-state fermentation of Pharbitis rubescens yeast.

[0010] The present invention achieves the above objectives through the following technical solutions:

[0011] A method for producing natural astaxanthin in poultry compound feed using Pharbitis rubrum yeast solid-state fermentation, characterized by comprising the following steps:

[0012] (1) Slant culture: The red Pharf yeast strain was inoculated onto a slant culture medium and cultured.

[0013] (2) Seed culture: The slant culture obtained in step (1) is inoculated into seed culture medium for culture;

[0014] (3) Solid-state fermentation culture: The cultured seed liquid is fermented at 1-15×10 8 Inoculate the fermentation substrate with an inoculum of CFU / kg and culture for 6-12 days at an initial pH of 5.5-7.0 and a temperature of 20-26℃.

[0015] Furthermore, the slant culture medium is PDA culture medium, which contains the following components per 1L: 6g potato extract powder and 20g glucose.

[0016] Furthermore, the slant culture is carried out at 22-26℃ for 3-4 days.

[0017] Furthermore, the seed culture medium is YM medium, which contains the following components per 1L: 10 g glucose, 5 g peptone, 3 g yeast extract, and 3 g malt extract.

[0018] Furthermore, the seed culture conditions are as follows: cultured in a constant temperature shaker at 180-200 r / min and 22-26℃ for 3-4 days.

[0019] Furthermore, the fermentation substrate is composed of the following components by mass percentage: 60% corn flour, 25% soybean meal, and 15% wheat bran; wherein the corn flour, soybean meal, and wheat bran all contain water, and the water content is 35%-45% of the total mass of the fermentation substrate.

[0020] Preferably, the red phage seed culture is prepared at a concentration of 1.5 × 10⁻⁶. 9 The inoculum was inoculated into solid fermentation medium at a rate of CFU / kg and cultured at pH 6.5 and 24°C for 12 days.

[0021] Furthermore, in the above method, the *Phaeodactylum rubrum* is *Phaeodactylum rubrum* P406. The *Phaeodactylum rubrum* ( Phaffia rhodozyma P406, with accession number GDMCC No.63339, was deposited on April 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0022] This invention also provides applications of poultry compound feed produced according to the above method, wherein fermented feed can be mixed with ordinary feed to obtain biological feed. When the biological feed is used for laying hens, the fermented feed accounts for 10%-30% of the biological feed, preferably 30%, and is fed twice daily.

[0023] Preferably, the common feed ingredients are: 60% corn, 22% soybean meal, 8% wheat bran, 8% limestone powder, 1% dicalcium phosphate, and 1% premix (providing inorganic salts and vitamins).

[0024] The beneficial effects of this invention are as follows:

[0025] This invention provides a method for producing natural astaxanthin in poultry compound feed using Pharbitis rubescens solid-state fermentation. This method has a short fermentation cycle and a simple process. The obtained solid-state fermented feed directly contains natural astaxanthin. On the one hand, it avoids the extraction and additional addition of astaxanthin, reducing production costs, and the single-cell protein of the bacteria can also be used as feed. On the other hand, it solves the problem of "antibiotic prohibition" in feed, improves the conversion and absorption of astaxanthin, and improves feed quality and palatability. Attached Figure Description

[0026] Figure 1 The images show the comparison between egg yolk color and a color chart. The left image shows the egg yolk color of the control group, and the right image shows the egg yolk color of the group fed 30% fermented feed. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] Red Pfaff yeast ( Phaffia rhodozyma P406 was isolated from water samples collected in Baiyangdian in 2020. It can utilize a variety of carbon sources, has simple growth conditions, and can produce astaxanthin.

[0029] The specific separation method is as follows:

[0030] Take 10 mL of water sample and add it to 90 mL of yeast extract peptone dextrose medium (YPD): 10.0 g yeast extract, 20.0 g peptone, 20.0 g glucose, and 1 L water for enrichment culture. Then, through serial dilution, spread it on YPD fermentation substrate with 0.1 mg / mL chloramphenicol and separate it using conventional separation methods.

[0031] Preparation of culture medium:

[0032] Slant culture medium (PDA medium): 6g potato extract powder, 20g glucose, 1000mL water, pH 6.5, sterilized at 121℃ for 20 minutes.

[0033] Seed culture medium (YM medium): 10g glucose, 5g peptone, 3g yeast extract, 3g malt extract, 1000mL water, pH 6.0-6.4, sterilized at 121℃ for 20 minutes.

[0034] Fermentation substrate composition: 60% corn flour, 25% soybean meal, 15% wheat bran, with a moisture content of 40%, and no sterilization is performed.

[0035] Example 1

[0036] (1) Slant culture: P406 of Pharfogel's red yeast strain was inoculated onto slant culture medium and cultured at 22°C for 3 days;

[0037] (2) Seed culture: The slant culture obtained in step (1) is inoculated into seed culture medium and cultured in a constant temperature shaker at 200 r / min and 22℃ for 3 days;

[0038] (3) Solid-state fermentation culture: The cultured seed liquid was fermented at 1.5×10 9The inoculum was inoculated into a solid fermentation medium at a rate of CFU / kg and cultured for 12 days at pH 6.5 and 24°C to obtain fermented feed.

[0039] Comparative Example

[0040] (1) Slant culture: The Rhodopseudomonas rubrum strain CGMCC 2.1557 purchased from the strain preservation center was inoculated onto slant culture medium and cultured at 22℃ for 3 days;

[0041] (2) Seed culture: The slant culture obtained in step (1) is inoculated into seed culture medium and cultured in a constant temperature shaker at 200 r / min and 22℃ for 3 days;

[0042] (3) Solid fermentation culture: The cultured seed liquid was inoculated into the solid fermentation medium at an inoculation amount of 1.5×109 CFU / kg and cultured for 12 days at pH 6.5 and 24℃ to obtain fermented feed.

[0043] The astaxanthin content in the fermented feed of Rhodopseudomonas aeruginosa P406 (Example 1) and the fermented feed of CGMCC 2.1557 (Comparative Example) was determined. It was found that the astaxanthin content in the fermented feed of Rhodopseudomonas aeruginosa P406 reached 17.50±0.31 mg / kg, while the astaxanthin content in the fermented feed of CGMCC 2.1557 was only 0.97±0.34 mg / kg.

[0044] Example 2: Determination of relevant indicators in fermented feed

[0045] Further analysis was conducted on the fermented feed and fermentation substrate of Example 1 to determine relevant indicators. The relevant indicators of the fermented feed and fermentation substrate are shown in Table 1. Compared with the fermentation substrate, after fermentation, the peptide content (increased by 52.81%), soluble polysaccharide content (increased by 24.64%), and organic acid content in the fermented feed were all significantly increased. The fermented feed also showed a significant increase in soybean globulin and... β- Most of the soybean globulin was degraded, with degradation rates reaching 38.07% and 37.61% respectively compared to the fermentation substrate. The trypsin inhibitor content was 19.71% lower than that of the fermentation substrate, showing a highly significant difference. P <0.01). Although aflatoxin B1 was not detected in either the fermentation substrate or the fermented feed, vomitoxin was detected in the fermentation substrate. Compared to the fermentation substrate, the zearalenone content in the fermented feed was reduced by 29.21%.

[0046] Table 1 Measurement of relevant indicators

[0047]

[0048] Note: Different lowercase letters in the same row's shoulder inscription indicate significant differences. P<0.05), different capital letters on the shoulder insignia indicate extremely significant differences ( P <0.01); "-" indicates a negative test result, and "+" indicates a positive test result.

[0049] Example 3: Application of Pharbitis purpureus solid-state fermentation poultry compound feed in laying hen feeding.

[0050] Two hundred healthy, 40-week-old laying hens with similar body condition, weight, and egg production rate were randomly divided into four groups, with five replicates per group and ten hens per replicate. The four groups underwent the following treatments:

[0051] Control group (A): fed with basal diet; Experimental group (B): fed with basal diet + 10% fermented feed from Example 1; Experimental group (C): fed with basal diet + 20% fermented feed from Example 1; Experimental group (D): fed with basal diet + 30% fermented feed from Example 1.

[0052] The basal diet consisted of: 60% corn, 22% soybean meal, 8% wheat bran, 8% limestone, 1% dicalcium phosphate, and 1% premix (providing minerals and vitamins). A one-week pre-trial period was conducted, during which the basal diet was fed. A six-week formal trial period followed. The laying hens underwent performance and egg quality testing.

[0053] Egg production performance testing: Egg production rate, average egg weight, average daily feed intake, and feed conversion ratio are calculated on a group basis.

[0054] Egg quality testing: At the beginning of the experiment, the middle of the experiment (3 weeks), and the end of the experiment (6 weeks), three eggs with a weight close to the average weight were selected from each replicate to determine the eggshell thickness, yolk color, albumen height, and Haugh units.

[0055] At the end of the experiment, blood was collected from the wing vein, 5 mL from each chicken. The blood collection tubes were then left to stand until the serum separated naturally. The tubes were labeled, and serum antioxidant capacity, including glutathione peroxidase (GSH-Pxg), superoxide dismutase (SOD), catalase (CAT), total serum antioxidant capacity (T-AOC), and malondialdehyde (MDA) content, was measured using kits.

[0056] As shown in Table 2, initially, the egg production rates of the four groups of hens were basically the same. Generally, egg production rates decrease with increasing age. However, after 6 weeks of feeding, the egg production rate of the experimental groups showed almost no decrease compared to the initial rate, and group D even showed a slight increase. The egg production rate of the control group, however, decreased significantly and was lower than that of the three experimental groups, with a statistically significant difference. P <0.05).

[0057] The average egg weight was lowest in the control group, significantly lower than that of the three experimental groups. P<0.05 (Table 3). There was no significant difference in average daily feed intake between the control and experimental groups. However, the feed conversion ratio (FCR) was significantly higher in the control group than in the experimental group. Specifically, the FCR in the group supplemented with 20% fermented feed was 6.45% lower than that in the control group. There were no significant differences among the three experimental groups. P >0.05 (Table 4).

[0058] Table 2. Effects of astaxanthin-added fermented feed on egg production rate of laying hens (%)

[0059]

[0060] Table 3. Effect of astaxanthin addition on average egg weight (g) in fermented feed

[0061]

[0062] Table 4. Effects of adding astaxanthin to fermented feed on average daily feed intake and feed conversion ratio.

[0063]

[0064] As shown in Table 5, after 3 and 6 weeks of feeding, the yolk color of the eggs in all three experimental groups was significantly higher than that in the control group. P <0.01), the egg yolk color value of the group with 30% fermented feed added could be increased by up to 46.30% compared with the control group ( Figure 1 ).

[0065] Table 5. Effects of astaxanthin-added fermented feed on egg yolk color value

[0066]

[0067] After feeding, there was no significant difference in eggshell thickness and yolk ratio between the control group and the experimental group. P >0.05), but for Haugh units, the control group was significantly lower than the experimental group, and the Haugh units in the group with 30% fermented feed were 7.04% higher than the control group ( P <0.01 (Table 6).

[0068] Haugh units, also known as Haugh units, are an indicator of egg freshness and protein quality. The higher the albumen density, the fresher the egg, and the larger the Haugh units.

[0069] Table 6. Effects of Astaxanthin Addition on Egg Quality in Fermented Feed

[0070]

[0071] After feeding laying hens a certain proportion of fermented feed, the antioxidant levels of the experimental group of laying hens were improved to varying degrees compared with the control group (Table 7). After adding 30% fermented feed, the contents of CAT, SOD and GSH-PX in the serum of laying hens increased by 44.60%, 34.32% and 29.87% respectively, while the MDA content decreased by 28.94%.

[0072] Table 7 Serum antioxidant capacity

[0073]

[0074] The above experiments show that fermented compound feed produces a sour aroma, increasing palatability and feed intake in animals. Fermentation produces organic acids such as lactic acid, acetic acid, and hexanoic acid. When fermented feed rich in organic acids enters the intestines, it lowers the pH of the digestive tract, inhibits the growth of harmful pathogens, and prevents disease. Simultaneously, the content of acid-soluble proteins increases significantly after fermentation, and various digestive enzymes are produced, breaking down large molecules that the animal body cannot or does not easily absorb into smaller molecules, such as peptides and soluble polysaccharides, making the feed easier to absorb and utilize. Most anti-nutritional factors in feed ingredients can be eliminated under microbial fermentation conditions. Therefore, the natural astaxanthin poultry compound feed described in this invention not only promotes the absorption of nutrients in the intestinal tract of laying hens but also improves the utilization rate of nutrients in bio-fermented feed.

[0075] After feeding laying hens, it was shown that adding a certain proportion of astaxanthin-fermented feed to the basal diet effectively improved the laying rate and average egg weight, reduced the feed conversion ratio, and significantly increased egg yolk color, eggshell thickness, and Haugh unit. It also improved the antioxidant capacity of laying hens. Under the conditions of this experiment, adding 30% astaxanthin-fermented feed to the basal diet showed the best effect in improving the laying performance and egg quality of laying hens.

[0076] Example 4: Detection of digestion and absorption of astaxanthin from different sources with the same content in laying hens.

[0077] One hundred and fifty healthy 40-week-old laying hens with similar body condition, weight, and egg production rate were randomly divided into three groups, with five replicates per group and ten hens per replicate. The three groups were then treated as follows:

[0078] Control group (I): fed with basal diet;

[0079] Experimental group (II): Astaxanthin additive (astaxanthin additive extracted by liquid fermentation and purchased from Jinan Dewen Chemical Co., Ltd.) was added to the basal diet until the astaxanthin content in the diet was the same as that in 30% of the fermented feed in Example 1.

[0080] Experimental group (III): fed with basal diet + 30% of fermented feed from Example 1.

[0081] After 4 weeks of feeding, 20 eggs were randomly selected from each group, and the astaxanthin content in the yolks was measured (Table 8).

[0082] Table 8 Astaxanthin Content

[0083]

[0084] It can be seen that the digestibility and absorption rate of solid-state fermented feed by red yeast in laying hens is higher than that of astaxanthin additives with the same astaxanthin content.

Claims

1. A method for producing natural astaxanthin in poultry compound feed using Pharfovia rubescens solid-state fermentation, characterized in that, Includes the following steps: (1) Slant culture: Red Pavlova yeast ( Phaffia rhodozyma P406 strain was inoculated onto slant culture medium and cultured; the red Paffozyme ( Phaffia rhodozyma P406, accession number GDMCCNo.63339; (2) Seed culture: The slant culture obtained in step (1) is inoculated into seed culture medium for culture; (3) Solid-state fermentation culture: The cultured seed liquid is fermented at 1-15×10 8 Inoculate the fermentation substrate at an inoculum rate of CFU / kg and culture for 6-12 days at an initial pH of 5.5-7.0 and a temperature of 20-26℃. The fermentation substrate consists of the following components by weight percentage: 60% corn flour, 25% soybean meal, 15% wheat bran, with a moisture content of 40%, and is not sterilized.

2. The method for producing natural astaxanthin in poultry compound feed using Pharrellis rubrum fermentation as described in claim 1, characterized in that, The slant culture medium is PDA medium, which includes the following components per 1L: 6g potato extract powder and 20g glucose.

3. The method for producing natural astaxanthin in poultry compound feed using Pharbitis rubrum yeast solid-state fermentation as described in claim 1, characterized in that, The culture conditions for the slant culture are to culture at 22-26℃ for 3-4 days.

4. A method for producing natural astaxanthin in poultry compound feed using Pharrellis rubrum fermentation as described in claim 1, characterized in that, The seed culture medium is YM medium, which contains the following components per 1L: 10 g glucose, 5 g peptone, 3 g yeast extract, and 3 g malt extract.

5. A method for producing natural astaxanthin in poultry compound feed using Pharrellis rubrum fermentation as described in claim 1, characterized in that, The seed culture conditions are as follows: cultured in a constant temperature shaker at 180-220 r / min and 22-26℃ for 3-4 days.

6. A method for producing natural astaxanthin in poultry compound feed using Pharrellis rubrum fermentation as described in claim 1, characterized in that, The red phaf yeast seed culture was prepared at 1.5 × 10⁻⁶. 9 The inoculum was inoculated into the fermentation substrate at a rate of CFU / kg and cultured for 12 days at pH 6.5 and 24°C.

7. The application of the natural astaxanthin-containing poultry compound feed prepared according to any one of claims 1-6 in the preparation of biological feed, characterized in that, The compound feed accounts for 10%-30% of the biological feed.

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