Method for synergistic fermentation of poultry compound feed by phaffia rhodozyma and bacillus subtilis and application thereof

Abstract

The application discloses a method for synergistically fermenting poultry compound feed by phaffia rhodozyma and bacillus subtilis and application thereof, and is mainly realized through the following steps: (1) phaffia rhodozyma slant culture; (2) phaffia rhodozyma seed culture; (3) bacillus subtilis slant culture; (4) bacillus subtilis seed culture; and (5) phaffia rhodozyma and bacillus subtilis synergistic solid-state fermentation culture. The application fully gives play to the product and metabolic advantages of the two strains, directly contains natural astaxanthin in the obtained poultry compound feed, improves the nutritional value of the fermented material, produces more active substances, enhances the disease resistance of animals, realizes the whole-course antibiotic-free breeding of laying hens, and improves the egg quality.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, specifically to a method and application of co-fermentation of poultry compound feed by Pharfovia rubescens and Bacillus subtilis. Background Technology

[0002] In July 2019, the Ministry of Agriculture and Rural Affairs issued Announcement No. 194, stipulating that all growth-promoting drug feed additives would be phased out after July 1, 2020. The trend of "antibiotic ban" in feed, "reduced and limited antibiotics" in animal husbandry, and "antibiotic-free" products has become an inevitable trend in the development of the feed and livestock industries. Astaxanthin, used as a feed additive for laying hens and livestock in the poultry industry, is a safe bacterial strain permitted for use in my country's "List of Feed Additive Varieties (2013)".

[0003] However, existing astaxanthin feeds primarily improve poultry farming efficiency by adding astaxanthin to the feed. This method has the following drawbacks:

[0004] Firstly, poultry's enzyme system is incomplete, resulting in insufficient enzyme levels. Furthermore, current astaxanthin-containing compound feeds typically consist mainly of indigestible plant-based materials such as corn, bran, oilseed cake, and crop straw, leading to low utilization rates of crude protein and energy in existing diets. Additionally, pathogenic bacteria such as Salmonella and Escherichia coli can affect the intestinal microenvironment of poultry, disrupting intestinal structural integrity and reducing their production performance and product quality.

[0005] Secondly, 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.

[0006] 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 typically required before astaxanthin extraction. Examples include Chinese patents CN103820520A ("A Fermentation Method for High-Yield Natural Astaxanthin"), CN102864087 A ("A *Phaeodactylum rubrum* Strain with High-Yield Natural Astaxanthin and its Breeding Method and Application"), and CN106701880 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.

[0007] Third, 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.

[0008] 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. Summary of the Invention

[0009] The purpose of this invention is to solve the above-mentioned problems by providing a method and application for the co-fermentation of poultry compound feed by Pharfovia rubescens and Bacillus subtilis.

[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 of Pharrellis redis: Pharrellis redis strain was inoculated onto slant culture medium and cultured.

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

[0014] (3) Bacillus subtilis slant culture: Bacillus subtilis strains were inoculated onto slant culture medium and cultured.

[0015] (4) Seed culture of Bacillus subtilis: The slant culture obtained in step (3) is inoculated into seed culture medium for culture;

[0016] (5) Co-fermentation culture: The seed liquid cultured in steps (2) and (4) is inoculated into the fermentation substrate at a ratio of 1:(1-10) and cultured for 6-12 days at an initial pH of 6.2-6.6 and a temperature of 20-26℃.

[0017] Furthermore, the red Pharfovia slant culture medium is a PDA medium, containing the following components per 1L: 6g potato extract powder, 20g glucose, and 20g agar.

[0018] Furthermore, the culture conditions for the red phaf yeast slant culture are as follows: culture at 22-26℃ for 3-4 days.

[0019] Furthermore, the Bacillus subtilis slant culture medium is an NA medium, containing the following components per 1L: 3.0g beef extract, 10.0g peptone, 5.0g NaCl, 20.0g agar, pH 7.2-7.4.

[0020] Furthermore, the Bacillus subtilis slant culture conditions are as follows: culture at 28-30℃ for 2-3 days.

[0021] Furthermore, the Pharbitis rubescens seed culture medium is YM medium, which contains the following components per 1L: 10.0 g glucose, 5.0 g peptone, 3.0 g yeast extract, and 3.0 g malt extract.

[0022] Furthermore, the culture conditions for the Rhodopseudomonas erythrosporum seed culture are as follows: cultured in a constant temperature shaker at 180-200 r / min and 22-26℃ for 3-4 days.

[0023] Furthermore, the Bacillus subtilis seed culture medium is NB medium, which contains the following components per 1L: 3.0g beef extract, 10.0g peptone, 5.0g NaCl, pH 7.2-7.4.

[0024] Furthermore, the conditions for culturing the Bacillus subtilis seeds are as follows: cultured in a constant temperature shaker at 180-200 r / min and 28-30℃ for 2-3 days.

[0025] 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.

[0026] Preferably, the Rhodopseudomonas erythrosporum seed culture and Bacillus subtilis seed culture are inoculated into a fermentation substrate with a water content of 40% at a ratio of 1:5, and cultured for 12 days at pH 6.5 and 24°C.

[0027] Furthermore, in the above method, the red phaf yeast is red phaf yeast (… Phaffia rhodozyma P406. The aforementioned Pharfovia p406, with accession number GDMCC No. 63339, was deposited in April 2023 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0028] Furthermore, in the above method, the Bacillus subtilis is Bacillus subtilis (… Bacillus subtilis B24. The Bacillus subtilis B24, with accession number GDMCC No. 63457, was deposited in May 2023 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0029] 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.

[0030] Preferably, the common feed composition is: 60% corn, 22% soybean meal, 8% wheat bran, 8% limestone powder, 1% dicalcium phosphate, and 1% premix (providing inorganic salts and vitamins).

[0031] The beneficial effects of this invention are as follows:

[0032] This invention provides a method for co-fermenting poultry compound feed with Pharfovia rubescens and Bacillus subtilis. The method uses Pharfovia rubescens, an astaxanthin-producing strain, and Bacillus subtilis functional strain as fermentation strains to carry out solid-state fermentation of the poultry compound feed base. The two strains work synergistically and enhance each other's effects, giving full play to the product and metabolic advantages of the two strains.

[0033] The compound feed fermented with red phloxera directly contains natural astaxanthin. Bacillus subtilis can produce more digestive enzymes, organic acids, antimicrobial proteins, lipopeptides, and other substances to inhibit pathogenic microorganisms in the intestines, keeping the body in a healthy, non-inflammatory state. This allows the astaxanthin in the feed to have a greater effect on the body, improving the body's antioxidant capacity, egg freshness, and yolk color. Furthermore, the two work synergistically, resulting in fermented feed with higher nutritional value of astaxanthin and acid-soluble proteins, a uniform light red color, a sour aroma, and good palatability for animals. It can produce more active substances and beneficial bacteria, improving feed digestibility and absorption, degrading anti-nutritional factors, antagonizing the growth of harmful intestinal pathogens, enhancing the animal's disease resistance, achieving antibiotic-free farming of laying hens throughout their lives, and improving egg quality. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] Bacillus subtilis ( Bacillus subtilis B24 was isolated from soil samples collected in Qingyuan District, Baoding in 2020. It can produce high levels of antibacterial active substances, proteases, and amylases.

[0037] The specific separation method is as follows:

[0038] Isolation of Pharfogel's red yeast: 10 mL of water sample was added 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 were used for enrichment culture. Then, the sample was serially diluted and spread onto YPD solid medium supplemented with 0.1 mg / mL chloramphenicol. The sample was then separated using conventional isolation methods.

[0039] Isolation of Bacillus subtilis: Take 1g of soil sample, add it to 99mL of sterile water, shake on a shaker for 20-30min, serially dilute, incubate in a water bath at 85℃ for 20-30min, take 100μL of the diluted sample, spread it on NA medium plates, and isolate single colonies.

[0040] Preparation of culture medium:

[0041] Pharvestia redissima slant culture medium (PDA medium): 6g potato extract powder, 20g glucose, 1000mL water, pH 6.5, sterilized at 121℃ for 20 minutes.

[0042] Bacillus subtilis slant culture medium (NA medium): 3.0g beef extract, 10.0g peptone, 5.0g NaCl, 20.0g agar, 1000 mL water, pH 7.2-7.4, sterilized at 121℃ for 30 minutes.

[0043] Red Pharbitis yeast seed culture medium (YM medium): 10.0 g glucose, 5.0 g peptone, 3.0 g yeast extract, 3.0 g malt extract, 1000 mL water, pH 6.0-6.4, sterilized at 121℃ for 20 minutes.

[0044] Bacillus subtilis seed culture medium (NB medium): 3.0g beef extract, 10.0g peptone, 5.0g NaCl, 1000mL water, pH 7.2-7.4, sterilized at 121℃ for 30 minutes.

[0045] Fermentation substrate composition: 60% corn flour, 25% soybean meal, 15% wheat bran, with a moisture content of 40%, and no sterilization is performed.

[0046] Example 1

[0047] (1) Red Pharf yeast slant culture: Red Pharf yeast strain P406 was inoculated onto slant culture medium and cultured at 22℃ for 3 days;

[0048] (2) Seed culture of Pharfogel's red yeast: The slant culture obtained in step (1) was inoculated into seed culture medium and cultured in a constant temperature shaker at 200 r / min and 22℃ for 3 days;

[0049] (3) Bacillus subtilis slant culture: Bacillus subtilis strain B24 was inoculated onto slant culture medium and cultured at 28°C for 2 days;

[0050] (4) Seed culture of Bacillus subtilis: The slant culture obtained in step (3) was inoculated into seed culture medium and cultured in a constant temperature shaker at 200 r / min and 28℃ for 2 days;

[0051] (5) Co-solid fermentation culture: The seed liquid cultured in steps (2) and (4) was inoculated into the fermentation substrate at a ratio of 1:5 and cultured for 12 days at an initial pH of 6.5 and 24°C.

[0052] Comparative Example 1

[0053] (1) Red Pharbitis yeast slant culture: Red Pharbitis yeast strain CGMCC2.1557 purchased from the strain preservation center was inoculated onto slant medium and cultured at 22℃ for 3 days;

[0054] (2) Seed culture of Pharfogel's red yeast: The slant cultures obtained in step (1) were inoculated into seed culture medium and cultured in a constant temperature shaker at 200 r / min and 22℃ for 3 days;

[0055] (3) Bacillus subtilis slant culture: Bacillus subtilis B24 was inoculated onto slant culture medium and cultured at 28℃ for 2 days;

[0056] (4) Seed culture of Bacillus subtilis: The slant culture obtained in step (3) was inoculated into seed culture medium and cultured in a constant temperature shaker at 200 r / min and 28℃ for 2 days;

[0057] (5) Co-solid fermentation culture: The seed liquid cultured in steps (2) and (4) is inoculated into the fermentation substrate at a ratio of 1:5, and fermented feed is obtained after 12 days at an initial pH of 6.5 and 24℃.

[0058] Comparative Example 2

[0059] (1) Slant culture: P406 of Pharfogel's red yeast strain was inoculated onto slant culture medium and cultured at 22°C for 3 days;

[0060] (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;

[0061] (3) Solid-state fermentation culture: The cultured seed liquid was fermented at 1.5×10 9 The 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.

[0062] After fermentation, the astaxanthin content in the fermented feed samples of Example 1, Comparative Examples 1 and 2 was determined. The astaxanthin content in the co-fermented feed of Pharrellis erythropoietin strain P406 and Bacillus subtilis B24 in Example 1 was similar to that in the solid-state fermented feed of Pharrellis erythropoietin strain P406 alone in Comparative Example 2, which were 17.08 mg / kg and 17.19 mg / kg, respectively. However, the astaxanthin content in the co-fermented feed of Pharrellis erythropoietin strain CGMCC2.1557 and Bacillus subtilis B24 in Comparative Example 1 was only 0.78 ± 0.29 mg / kg.

[0063] Example 2

[0064] Further tests were conducted on the digestion and absorption of the fermented feed described in Example 1 and Comparative Example 2, as well as the common astaxanthin additive, by laying hens.

[0065] One hundred and fifty 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 were then treated as follows:

[0066] Control group (I): fed with basal diet;

[0067] 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.

[0068] Experimental group (III): fed with basal diet + 30% of fermented feed from comparison ratio 2;

[0069] Experimental group (Ⅳ): fed with basal diet + 30% of fermented feed from Example 1.

[0070] After 4 weeks of feeding, 20 eggs were randomly selected from each group, and the astaxanthin content in the yolks was measured (Table 1).

[0071] Table 1. Astaxanthin content in egg yolks

[0072]

[0073] It can be seen that even though the astaxanthin content in the co-fermented feed of Pharrellis rubrum strain P406 and Bacillus subtilis B24 in Example 1 is similar to that in the solid-state fermented feed of Pharrellis rubrum strain P406 alone in Comparative Example 2, the digestion and absorption of astaxanthin in the fermented feed of Example 1 by laying hens is higher than that in the fermented feed of Comparative Example 2.

[0074] Under the same astaxanthin content ingested by laying hens, the final digestion and absorption of astaxanthin from the fermented feed in Example 1 by the laying hens was also higher than that of astaxanthin additives.

[0075] Example 3

[0076] Further analysis of relevant indicators in solid-state fermented feed of *Phaeopsyrum rubrum* strain P406 and co-fermented feed of *Phaeopsyrum rubrum* strain P406 and *Bacillus subtilis* B24 was conducted (Table 2). Compared with the *Phaeopsyrum rubrum* fermented feed, the co-fermented feed showed a significantly higher acid-soluble protein / crude protein content (P<0.01); peptide and soluble polysaccharide contents were also significantly increased; and the contents of acetic acid, hexanoic acid, and lactic acid in the organic acids were all significantly increased. Most of the glycinin and β-conglycinin in the co-fermented feed were degraded, showing a significant difference compared to the *Phaeopsyrum rubrum* fermented feed (P<0.05); the zearalenone content was somewhat reduced.

[0077] Table 2 Results of relevant indicator measurements

[0078]

[0079] 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.

[0080] Example 4: Feeding Trial for Laying Hens

[0081] One hundred healthy, 40-week-old laying hens with similar weight and egg production rate were randomly divided into two groups, with five replicates per group and ten hens per replicate. The two groups were treated as follows: a basal diet plus 30% red phaffeine fermented feed group and a basal diet plus 30% co-fermented feed group. The pre-trial period was one week, during which the basal diet was used. The formal trial period was six weeks. The laying hens' production performance and egg quality were tested.

[0082] Egg production performance testing: Egg production rate, average daily feed intake, and feed conversion ratio are calculated on a group basis.

[0083] Egg quality testing: At 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 yolk color and Haugh unit.

[0084] 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 then the serum antioxidant capacity, including glutathione peroxidase (GSH-Pxg), superoxide dismutase (SOD), catalase (CAT), total serum antioxidant capacity (T-AOC), and malondialdehyde (MDA) levels, was measured using kits.

[0085] As shown in Table 3, after 6 weeks of feeding, the egg production rate of the basal diet + 30% co-fermented feed group was higher than that of the basal diet + 30% red Paffia yeast fermented feed group; the average daily feed intake and feed conversion ratio were not significantly different; while the Haugh unit and egg yolk color value were both improved.

[0086] Table 3. Egg production performance and egg quality of laying hens

[0087]

[0088] Compared with the basal diet + 30% red Paffia yeast fermented feed group, the antioxidant levels of laying hens fed the basal diet + 30% co-fermented feed group were improved to varying degrees (Table 4). The contents of T-AOC, CAT, SOD and GSH-PX in the serum of laying hens increased by 12.07%, 12.05%, 10.79% and 7.28% respectively, while the MDA content decreased by 6.27%.

[0089] Table 4 Serum antioxidant capacity

[0090]

[0091] The above experiments show that feed co-fermented with Bacillus subtilis and Pharrellis rubrum exhibits superior effects in both feed quality and laying hen feeding. Adding a certain proportion of co-fermented feed to the basal diet, compared to adding Pharrellis rubrum-fermented feed, can further improve the egg production rate of laying hens, increase egg yolk color and Haugh units. Simultaneously, it can also enhance the antioxidant capacity of laying hens.

Claims

1. A method for the synergistic fermentation of a poultry compound feed with Phaffia rhodozyma and Bacillus subtilis, characterized in that, Includes the following steps: (1) Red Pharf yeast slant culture: Red Pharf yeast strains were inoculated onto slant culture medium and cultured; (2) Seed culture of Pharfogel's red yeast: The slant culture obtained in step (1) is inoculated into seed culture medium for culture; (3) Bacillus subtilis slant culture: Bacillus subtilis strains were inoculated onto slant culture medium and cultured. (4) Seed culture of Bacillus subtilis: The slant culture obtained in step (3) is inoculated into seed culture medium for culture; (5) Co-fermentation culture: The seed culture prepared in steps (2) and (4) is inoculated into the fermentation substrate at a ratio of 1:(1-10) and cultured for 6-12 days at an initial pH of 6.2-6.6 and a temperature of 20-26℃. The said Rhodotorula is Rhodotorula mucilaginosa (R. mucilaginosa) Phaffia rhodozyma ) P406, with the preservation number of GDMCC No. 63339; The Bacillus subtilis is Bacillus subtilis (B24), with the preservation number of GDMCC No. 63457. Bacillus subtilis ) B24, with the preservation number of GDMCC No. 63457. The fermentation substrate consists of 60% corn flour, 25% soybean meal, and 15% wheat bran, with a moisture content of 40%, and is not sterilized.

2. The method for co-fermenting poultry compound feed with *Phaeophyte vulgaris* and *Bacillus subtilis* as described in claim 1, characterized in that... The red phafungi slant culture medium is PDA medium, which contains the following components per 1L: 6g potato extract powder, 20g glucose, and 20g agar; the red phafungi slant culture conditions are 22-26℃ for 3-4 days.

3. A method of co-fermenting a poultry compound feed with Rhodotorula mucilaginosa and Bacillus subtilis according to claim 1, characterized in that, The Bacillus subtilis slant culture medium is a nano medium, containing the following components per 1L: 3.0g beef extract, 10.0g peptone, 5.0g NaCl, 20.0g agar, pH 7.2-7.4; the Bacillus subtilis slant culture conditions are 28-30℃ for 2-3 days.

4. A method of co-fermenting poultry compound feed with Rhodotorula mucilaginosa and Bacillus subtilis according to claim 1, characterized in that, The Phaefuros rhamnosus seed culture medium is YM medium, which contains the following components per 1L: 10.0 g glucose, 5.0 g peptone, 3.0 g yeast extract, 3.0 g malt extract; the conditions for culturing the red Pharf yeast seed were 22-26℃ and 180-220 r / min for 3-4 days.

5. The method for co-fermenting poultry compound feed with *Pharbitis erythropoietin* and *Bacillus subtilis* as described in claim 1, characterized in that... The Bacillus subtilis seed culture medium is NB medium, which contains the following components per 1L: 3.0g beef extract, 10.0g peptone, 5.0g NaCl, pH 7.2-7.4; the conditions for Bacillus subtilis seed culture are 28-30℃ and 180-200 r / min for 2-3 days.

6. The method for co-fermenting poultry compound feed with *Pharbitis erythropoietin* and *Bacillus subtilis* as described in claim 1, characterized in that... The Rhodopseudomonas erythrosporum seed culture and Bacillus subtilis seed culture were inoculated into the fermentation substrate at a ratio of 1:5 and cultured for 12 days at pH 6.5 and 24°C.

7. Use of the fermented feed prepared according to any one of claims 1 to 6 for the preparation of a bio-feed, characterized in that, Fermented feed accounts for 10%-30% of biological feed.

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