TW202239332A - Feeding stuff for poultry with high surfactin content, fermentation product with high surfactin content, and its manufacturing method and use accelerating the growth of the poultry and lowering the manufacturing cost - Google Patents

Abstract

Disclosed are a fermentation product with high surfactin content and its manufacturing method and use. Bacillus subtilis is utilized to perform solid state fermentation on soybean meal as a fermentation substrate, and then the resulting fermentation product is applied to a feed for the poultry after being dried and crushed, which can accelerate the growth of the poultry, improve the height of intestinal villi in the duodenum and the jejunum and the ratio of villi height to glandular fossa depth, and lower the manufacturing cost.

Description

Poultry feed with high supersurin content, fermented product with high supersurin content, production method and use thereof

The invention is related to poultry feed, especially a fermented product with high surfin content for promoting poultry growth, its production method and application.

Surfactin is a secondary metabolite unique to Bacillus strains. It has a hydrophilic and hydrophobic structure and can reduce surface tension or dissolve hydrophobic substances in water (Soberón-Chávez, 2010). Stable under extreme temperature, pH and salinity conditions, and can use renewable raw materials as its fermentation production substrate (Yeh et al., 2005; Cameotra et al., 2010), has anti-bacteria, virus, fungi and mycoplasma and other properties (Meena et al., 2016). Due to the high production cost and low yield of surfin, its large-scale industrial application is limited (Makkar and Cameotra, 2002; Chtioui et al., 2010). Compared with liquid fermentation, solid-state fermentation technology can reduce production costs, shorten the process and increase secondary metabolites (Beltran-Gracia et al., 2017). Many studies have successfully used solid-state fermentation technology to use low-cost agricultural and industrial by-products as fermentation substrates for the production of lipopeptides, which can greatly reduce production costs (Das and Mukherjee, 2007; Mizumoto and Shoda, 2007). Zhu et al. (2013a) used liquid and solid-state fermentation of soybean flour to produce surfin, respectively, and the yield of surfin was significantly higher in solid-state fermentation than in liquid-state fermentation (11.61 VS 4.97 mg/gds).

In recent years, due to the overuse of antibiotics, some European countries and South Korea have banned the addition of antibiotic growth promoters (antibiotic growth promoters) to animal diets (Castanon, 2007; Alloui et al., 2013). Surfactin has a special bactericidal mechanism, broad antibacterial activity and is not easy to produce drug resistance, so it has great potential to replace antibiotics (Zhai et al., 2015). In recent years, studies have shown that the addition of Bacillus subtilis (B. subtilis), which can produce surfin, to the diet can improve the negative impact of Clostridium perfringens (Clostridium perfringens) on necrotic enteritis (NE) in white broiler chickens. And it has the effect of replacing antibiotics to promote the growth of chickens. Adding 0.05% B. subtilis PB6 to the diet can improve the feed conversion rate of white meat chickens, the height of intestinal villi and reduce the bacterial count of C. perfringens in the intestine compared with the NE group (Jayaraman et al., 2013), and adding 0.05% B. subtilis PB6 improved the growth traits and intestinal villi of white broiler chickens compared with avilamycin (Jayaraman et al., 2017). Adding 0.3% B. subtilis NIU068 fermented product for four days to the diet of white-broiled chickens can improve the body weight and feed intake of the chickens compared with the NE group, and can improve the feed conversion rate compared with the antibiotic group, and slow down the intestinal tract caused by C. perfringens Damage and improve intestinal villi height and crypt depth (Cheng et al., 2018). However, the current literature still lacks the effect of dietary surface protein content on poultry growth.

The main purpose of the present invention is to provide a fermented product with a high content of surfin and its production method and application. It utilizes Bacillus subtilis, uses soybean meal as a fermentation substrate, and performs solid-state fermentation, and then dries and pulverizes the fermented product obtained Applied to poultry feed, it can promote the growth of poultry, improve the villi height of poultry duodenum and jejunum, and the ratio of villi height to gland depth, and reduce manufacturing costs.

In order to achieve the aforementioned purpose, the present invention provides a method for producing a fermented product with high surfin content that promotes the growth of poultry, which includes: A, Bacillus subtilis is inoculated in soybean meal solid-state fermentation substrate; B. Carry out solid-state aerobic fermentation, the conditions are temperature 30°C to 40°C, moisture 55%, 62.5% or 70% (v/w), and fermentation time 2 days; C, the fermentation product is dried and pulverized; D. Using a high-resolution orbitrap mass spectrometer to analyze the fermented product, the fermented product yield was 10.79 mg/g when the water content was 55%; when the water content was 62.5%, the fermentin yield was 14.62 mg /g; when the water content was 70%, the yield of surfin was 18.40 mg/g.

The inoculation amount of the Bacillus subtilis in the above step A is 5% to 10% (v/w) of the solid fermentation substrate, and the concentration of the bacteria solution is 8.74 Log CFU/ml.

The analysis conditions of above-mentioned step D are: a. Full mass scan in positive charge mode; b. The scanning range is 988 to 1200 mass-to-charge ratio, the analytical column is Mightysil RP-18 GP (250 × 4.6 mm; 5 μm), the flow rate is 1.0 mL/min, and the injected sample volume is 5 μL; c. The mobile phases are 0.1% formic acid aqueous solution and 0.1% formic acid in acetonitrile; d. The analysis time is 25 minutes; e. Take the mass-to-charge ratio of the superficial standard product as the qualitative basis, and carry out quantification according to the calibration line of the standard product.

Preferably, the poultry is chicken.

The present invention further provides a poultry diet with high supersurin content produced according to the above-mentioned manufacturing method. The fermented product is added to the diet at a weight ratio of 2.5%. The content is 270 mg/kg; when the water content is 62.5%, the content of surfin is 365 mg/kg; when the water is 70%, the content of surfin is 460 mg/kg.

The present invention also provides an application of poultry feed with high surfin content produced by the above-mentioned production method, which is used to promote the growth of chickens, improve the height of villi in duodenum and jejunum of chickens, and the ratio of villi height to gland depth.

The above objects and advantages of the present invention can be easily understood from the following detailed descriptions and accompanying drawings of selected embodiments.

The present invention uses Bacillus subtilis LYS and uses soybean meal as a solid-state fermentation substrate, adjusts the water content to 55, 62.5 or 70% (v/w) respectively, and performs high-pressure sterilization (121°C, 1.2 kg/cm ² ) After cooling for 30 minutes, inoculate 5-10% (v/w) bacterial solution (the number of bacteria is more than 8.74 Log CFU/ml) and ferment aerobically at 30-40°C for 2 days. Dried to a moisture content below 12% (v/w), dried and pulverized to prepare a fermented product.

The fermentation product was mixed with LC grade methanol (methanol, Merk, Germany) at a ratio of 1:100 (W/V), shaken by ultrasonic waves (TP690, Elma Schmidbauer GmbH, Germany) at room temperature for one hour, and then heated at 4°C at 10,000 Centrifuge at g for 10 minutes to remove insoluble matter, and then filter the supernatant with a 0.22 μm syringe filter (13mm - PVDF syringe Filter - 0.22 μm, PureTech™, Taiwan) to extract surfin from the product.

The extracted samples were analyzed with a high-resolution orbitrap mass spectrometer (LC-ESI-HRMS) (Thermo Fisher Scientific, Germany). LC-ESI-HRMS analysis conditions: full mass scan (full scan) in positive charge mode, scan The range is 988-1200 m/z, the analytical column is Mightysil RP-18 GP (250 × 4.6 mm; 5 μm, Kanto Kagaku, Japan), the flow rate is 1.0 mL/min, the injected sample volume is 5 μL, and 0.1% formic acid (formic acid, Merk, Germany) aqueous solution and 0.1% formic acid in acetonitrile (acetonitrile, Anaqua™ Chemicals Supply, USA) were used as the mobile phase (mobile phase), the analysis time was 25 minutes, and surfactin standard (surfactin, ≥ 98.0%, Sigma-Aldrich®, USA) mass-to-charge ratio ( m/z ) was used as the qualitative basis, and the calibration line of the Surfactin standard was used for quantification, and different concentrations of Surfactin were prepared by serial dilution method, each concentration 3 repetitions, using Xcablibur (Thermo Fisher Scientific, Germany) for automatic integration, and substituting the integrated area of the sample into the curve formula of surfin to quantify the concentration of surfin in the sample.

The analysis method of the present invention proves that C _{12-17 surfin} peaks can be detected in the standard product and fermentation products with different moisture content, and it is found that C ₁₅ surfin contains linear and ring structures. The mass-to-charge ratios are 994.64, 1008.66, 1022.67, 1036.69, 1054.70, 1050.71, and 1064.72. The analysis results are shown in Figure 1 and Figure 2.

Figure 3 shows the quasisurin quantitative results of B. subtilis LYS fermentation products with different moisture content. The results showed that the yield of surfin increased significantly with the increase of fermentation water (P < 0.05). ) and 55% water fermentation group (10.79 mg/g). Zhu et al. (2013b) used Bacillus amyloliquefaciens XZ-173 and reaction surface method to produce surfin from soybean flour. The optimal solid-state fermentation conditions were 15.96% inoculum, 62.8% moisture and 26.9℃ temperature. After 48 hours of fermentation, the yield of surfin could reach 15.03 mg/gds. This is similar to the results of the present invention. When B. subtilis LYS fermented soybean meal with 70% moisture, the yield of surfin could reach 18.40 mg/g, surpassing the highest yield of surfin (15.03 mg/gds) known in the current literature.

Animal experiments on B. subtilis LYS fermentation products with different water content. The experiments were divided into 5 groups. 1. Control group: fed with normal diet; 2. Fishmeal group: 2.5% Danish whitefish meal (crude protein 70%, TripleNine Group) was added to the diet ;3. Moisture 55% fermentation group: add 2.5% fermentation product to the diet (the dietary surface protein content is 270 mg/kg); 4. Water 62.5% fermentation group: add 2.5% fermentation product to the diet (the dietary surface protein content is 360 mg/kg); mg/kg); 5. Fermentation group with 70% moisture: add 2.5% fermentation product to the diet (surfactin content of the diet is 460 mg/kg). Each treatment group has 4 repetitions, and each repetition has 10 white meat chickens. The experiment lasted for 35 days. The body weight, feed intake and survival rate were recorded weekly to calculate body weight and production efficiency factors. At the end of the experiment, the chickens were sacrificed, and the duodenum and jejunum were collected to analyze the intestinal tissue type. The effects of B. subtilis LYS fermentation products with different water content on body weight gain and production efficiency factors of white-broiled chickens are shown in Figure 4 and Figure 5, respectively. The results showed that at 0-3 weeks, the fermented group with a water content of 55% had the best performance on the growth of chickens, and it could improve the body weight of white-broiled chickens and the intestinal tissue type of white-broiled chickens fermented with different moisture B. subtilis LYS compared with the control group The effects of the states are shown in Figures 6 and 7, respectively. The results showed that the 55% water fermentation group had the best performance on the intestinal tissue type of the 5-week-old chickens. Compared with the duodenum and jejunum of the control group, the height of villi and the ratio of villi height/glandular crypt depth were respectively improved (P < 0.05), and There was no significant difference with the fish meal group (P > 0.05).

However, the disclosure of the above embodiments is only used to illustrate the present invention, not to limit the present invention, and any replacement of equivalent elements should still fall within the scope of the present invention.

To sum up, those skilled in the art can understand that the present invention can clearly achieve the above-mentioned purpose, and it actually complies with the provisions of the Patent Law, so they should file an application according to the law.

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Figure 1 is a chromatogram of the Surfactin Standard of the present invention containing different variants; Figure 2 is a chromatogram of surfin in the fermentation product of Bacillus subtilis with different water content of the present invention; Fig. 3 is a figure of surficin yield of different moisture Bacillus subtilis fermentation products of the present invention; Figure 4 is a schematic diagram of the influence of different water contents of Bacillus subtilis fermentation products on body weight gain of white broiler chickens according to the present invention; Figure 5 is a schematic diagram of the influence of different moisture content of Bacillus subtilis fermentation products of the present invention on the production efficiency factor of white broiler chickens; Figure 6 is a schematic diagram of the influence of different water contents of Bacillus subtilis fermentation products of the present invention on the height of intestinal villi of white-broiled chickens; Fig. 7 is a schematic diagram showing the influence of different water contents of Bacillus subtilis fermentation products of the present invention on the ratio of villus height/glandular depth in white broiler chicken.

Claims (6)

A method for producing a fermented product with high surfin content to promote poultry growth, comprising: A, Bacillus subtilis is inoculated in soybean meal solid-state fermentation substrate; B. Carry out solid-state aerobic fermentation, the conditions are temperature 30°C to 40°C, moisture 55%, 62.5% or 70% (v/w), and fermentation time 2 days; C, the fermentation product is dried and pulverized; D. Using a high-resolution orbitrap mass spectrometer to analyze the fermented product, the fermented product yield was 10.79 mg/g when the water content was 55%; when the water content was 62.5%, the fermentin yield was 14.62 mg /g; when the water content was 70%, the yield of surfin was 18.40 mg/g. The manufacturing method as described in Claim 1, wherein the inoculation amount of Bacillus subtilis in step A is 5% to 10% (v/w) of the solid fermentation substrate, and the concentration of the bacterial solution is 8.74 Log CFU/ml. The manufacturing method as described in claim 1, wherein the analysis conditions of step D are: a. Full mass scan in positive charge mode; b. The scanning range is 988 to 1200 mass-to-charge ratio, the analytical column is Mightysil RP-18 GP (250 × 4.6 mm; 5 μm), the flow rate is 1.0 mL/min, and the injected sample volume is 5 μL; c. The mobile phases are 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile; d. The analysis time is 25 minutes; e. Take the mass-to-charge ratio of the superficial standard product as the qualitative basis, and carry out quantification according to the calibration line of the standard product. The manufacturing method according to claim 1, wherein the poultry is chicken. A poultry feed with high surface protein content produced according to the production method described in claim 1, which is to add the fermented product to the feed in a proportion of 2.5% by weight, and when the water content is 55%, the surface Surfactin content is 270 mg/kg; in the case of 62.5% water, the content of surfin is 365 mg/kg; in the case of 70% water, the content of surfin is 460 mg/kg. A use of poultry feed with high surface protein content as described in Claim 5, which is used to promote the growth of chickens, improve the height of villi in duodenum and jejunum of chickens, and the ratio of villi height to gland depth.

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