Liquid feed additive containing bacillus and method of preparation

Abstract

The application relates to the field of feed additives, and particularly discloses a liquid feed additive containing bacillus and a preparation method thereof. The liquid feed additive comprises the following raw materials: culture solution, stabilizer, bacillus and acid adjusting solution. The addition amount of the stabilizer is 0.01-0.5 wt% based on the total amount of the culture solution. The stabilizer comprises at least two of xanthan gum, carrageenan, sodium carboxymethyl cellulose and guar gum. The addition amount of the bacillus is 2.5-5 g / L based on the culture solution. The culture solution is prepared through the following steps. Single bacterial colonies of the bacillus are inoculated into culture medium to obtain seed solution, the seed solution is sealed and cultured in a neutral environment, and the supernatant is obtained when the bacillus rate reaches more than 90% to obtain the culture solution. The obtained liquid feed additive is rich in probiotics, has good stability, and does not have phenomena such as stratification, precipitation and peculiar smell after long-time storage. The bacillus has high viable count, and can be directly fed to animals.

Description

Technical Field

[0001] This application relates to the field of feed additives, and more specifically, to a liquid feed additive containing Bacillus subtilis and a method for its preparation. Background Technology

[0002] Liquid feed additives refer to liquid additives used in animal feed. They can be selected and used according to different animal species and growth stages, with the aim of improving the nutritional value, health benefits, or production performance of the feed.

[0003] Probiotics can regulate intestinal flora, maintain intestinal health, promote digestion and absorption, inhibit the colonization of pathogens, promote the growth of anaerobic probiotics, and regulate animal immune responses. They are one of the most effective animal feed additives currently available on the market to replace antibiotics.

[0004] Currently, most liquid feed additives on the market are composed of waste molasses, liquid oils, liquid amino acids, and liquid milk substitutes. These liquid feed additives cannot be directly mixed with probiotics to form a product, mainly because the raw material components have poor compatibility and stability, leading to sedimentation, stratification, and off-odors during storage. This also seriously affects the number of live probiotics. Therefore, probiotic products must be mixed twice before feeding. Summary of the Invention

[0005] This application provides a liquid feed additive containing Bacillus and its preparation method. The resulting liquid feed additive is rich in probiotics, has good product stability, and will not exhibit stratification, sedimentation, or off-odors after long-term storage. It also has a high number of viable Bacillus bacteria and can be directly fed to animals.

[0006] The liquid feed additive containing Bacillus subtilis provided in this application adopts the following technical solution:

[0007] A liquid feed additive containing Bacillus subtilis, comprising the following ingredients: culture medium, stabilizer, Bacillus subtilis, and acidity regulator;

[0008] Based on the total amount of culture medium, the amount of stabilizer added is 0.01-0.5 wt%; the stabilizer includes at least two of xanthan gum, carrageenan, sodium carboxymethyl cellulose, and guar gum;

[0009] Based on the culture medium, the amount of Bacillus added is 2.5-5 g / L;

[0010] The culture medium is prepared by the following steps: a single colony of Bacillus is inoculated into the culture medium to obtain a seed culture, the seed culture is sealed and cultured in a neutral environment, and the supernatant is taken when the spore rate reaches more than 90% to obtain the culture medium.

[0011] Furthermore, the initial bacterial count of Bacillus was greater than 100 million CFU / mL.

[0012] Xanthan gum is a polysaccharide polymer with a secondary structure in which side chains are anti-winding around the main chain backbone, maintaining a rod-like double helix structure through hydrogen bonds. It exhibits unique rheological properties, good water solubility, excellent stability, and compatibility. Carrageenan is a calcium, potassium, sodium, and ammonium salt of a polysaccharide sulfate ester composed of galactose and dehydrated galactose. It is formed by alternating α-1,3 glycosidic bonds and β-1,4 bonds between sulfated or non-sulfated galactose and 3,6-dehydrated galactose. The 1,3-linked D-galactose unit has a sulfate group at C4, which effectively balances the solution system. Sodium carboxymethyl cellulose has good hygroscopicity, easily disperses in water to form a transparent colloidal solution, and also has good emulsifying and thickening effects. Guar gum is a non-ionic galactomannan extracted from the endosperm of the guar bean plant. It has good water solubility, can disperse in solution to form a viscous liquid, and plays a certain role in thickening and stabilizing. At least two of xanthan gum, carrageenan, sodium carboxymethyl cellulose, and guar gum are selected as system stabilizers to work synergistically to ensure good product stability and prevent stratification, sedimentation, or off-odors after long-term storage.

[0013] The culture medium provides nutrients for Bacillus, and the acidic conditioning solution can adjust the pH value of the system, which can play a certain role in inhibiting bacteria, making the system easier for Bacillus to grow and survive, while improving the taste of liquid feed additives.

[0014] Preferably, the step of inoculating a single colony of Bacillus into a culture medium to obtain a seed solution includes: inoculating a single colony of Bacillus into a culture medium for a first culture time of 10-12 hours to obtain a primary seed solution; and inoculating the primary seed solution into a culture medium again for a second culture time of 10-12 hours to obtain a secondary seed solution.

[0015] Preferably, the culture medium comprises, by weight percentage, 20-25% soybean meal, 4-8% glucose, 1.6-2.5% ammonium sulfate, 4-9% corn starch, and the balance being water.

[0016] Preferably, the sealed culture conditions are: cultured at a temperature of 32-40℃ for 30-40 hours.

[0017] The seed culture obtained using the above-mentioned culture medium contains abundant carbon sources, nitrogen sources, trace elements and other nutrients, which can provide nutrients for the growth and survival of Bacillus.

[0018] Preferably, the stabilizer is xanthan gum and carrageenan.

[0019] Preferably, the mass ratio of xanthan gum to carrageenan is (2-3):(0.3-0.4).

[0020] Furthermore, the xanthan gum selected has a relative molecular mass of over 1 million, and the carrageenan has a relative molecular mass of over 200,000.

[0021] Further optimization of the stabilizer component selection helps improve product stability. Experimental research has found that using a combination of xanthan gum and carrageenan as stabilizers results in products with better stability. Even after long-term storage, there will be no stratification, precipitation, or off-odors, and the number of viable Bacillus bacteria is high.

[0022] Preferably, the acid conditioning solution includes a propionic acid solution or an acetic acid solution.

[0023] Furthermore, the acidic conditioning solution is a propionic acid solution.

[0024] Preferably, the mass concentration of the propionic acid solution is 15-25%.

[0025] By adopting the above technical solutions, both propionic acid solution and acetic acid solution can adjust the system to acidity. Experimental studies have found that propionic acid solution is preferred as the acidity adjustment solution. Propionic acid solution can also play a role in inhibiting bacteria, preventing mold and corrosion, and is more conducive to the combination with stabilizers to improve product stability and help Bacillus survive.

[0026] Preferably, the Bacillus includes one of Bacillus licheniformis, Bacillus subtilis, Bacillus belye, Bacillus coagulans, Bacillus cereus, Bacillus tarda, and Bacillus pumilus.

[0027] Secondly, this application provides a method for preparing a liquid feed additive containing Bacillus subtilis, using the following technical solution:

[0028] A method for preparing a liquid feed additive containing Bacillus includes the following steps: mixing an acidic conditioning solution, a culture medium, a stabilizer, and Bacillus evenly.

[0029] Furthermore, after adding stabilizer to the culture medium and stirring to dissolve it, the pH of the system was adjusted to 4.0-4.5 using an acidic adjusting solution, and finally Bacillus was added.

[0030] By adopting the above technical solution, the culture medium and stabilizer are first mixed to form a viscous liquid with no layering and uniform texture. Then, the pH value of the system is adjusted to acidic, and Bacillus is added. This results in a product with good stability. Even after long-term storage, there will be no layering, precipitation, or off-odors. In addition, the number of live Bacillus is high, so there is no need to mix probiotics again, and it can be fed directly.

[0031] In summary, this application has the following beneficial effects: This application uses xanthan gum and carrageenan compound as system stabilizers, combined with acidic conditioning solution, to obtain a product with good stability. It will not exhibit stratification, precipitation, or off-odors after long-term storage. In addition, the number of viable Bacillus bacteria is high, so there is no need to mix probiotics again, and it can be fed directly. Attached Figure Description

[0032] Figure 1 This is a picture of the sample from Example 1 after 60 days.

[0033] Figure 2 This is a picture of the sample from Example 2 after 60 days.

[0034] Figure 3 This is a picture of the sample from Example 3 after 60 days.

[0035] Figure 4 This is a picture of the sample from Example 4 after 60 days.

[0036] Figure 5 This is a picture of the sample from Example 5 after 60 days.

[0037] Figure 6 This is a picture of the sample from Example 6 after 60 days.

[0038] Figure 7 This is a picture of the sample from Example 7 after 60 days.

[0039] Figure 8 This is a picture of the sample from Comparative Example 1 after 60 days.

[0040] Figure 9 This is a picture of the sample from Comparative Example 2 after 60 days.

[0041] Figure 10 This is a picture of the sample from Comparative Example 3 after 60 days.

[0042] Figure 11 This is a picture of the sample from Comparative Example 4 after 60 days.

[0043] Figure 12 This is a picture of the sample from Comparative Example 5 after 60 days.

[0044] Figure 13 This is a diagram showing the mold growth in Example 1.

[0045] Figure 14 This is a diagram showing the mold growth in Comparative Example 2.

[0046] Figure 15 This is a diagram showing the mold growth in Comparative Example 3.

[0047] Figure 16 This is a diagram showing the mold growth in Comparative Example 4.

[0048] Figure 17 This is a diagram showing the mold growth in Comparative Example 5. Detailed Implementation

[0049] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Among them, xanthan gum model number 11138-66-2, carrageenan CAS No. 11114-20-8; sodium carboxymethyl cellulose CAS No. 9004-32-4, and guar gum CAS No. 9000-30-0.

[0050] Culture medium preparation example

[0051] Preparation Example 1

[0052] The culture medium is prepared by the following steps: a single colony of Bacillus licheniformis is picked and inoculated into 75 mL of culture medium and cultured for 12 hours as the primary seed culture. Then, it is inoculated into 750 mL of culture medium and cultured for another 12 hours as the secondary seed culture. The secondary seed culture is inoculated into a 200 L fermenter and cultured at 37 °C for 36 hours in a neutral environment with pH = 7. After the spore rate reaches 90%, the fermenter is centrifuged and the supernatant is taken as the culture medium.

[0053] The culture medium comprises 23.7% soybean meal, 5.8% glucose, 2.1% ammonium sulfate, 7.9% corn starch, and the remainder water.

[0054] Preparation Example 2

[0055] The culture medium is prepared by the following steps: a single colony of Bacillus licheniformis is picked and inoculated into 200 mL of culture medium and cultured for 20 h to obtain seed culture. The seed culture is then inoculated into a 200 L fermenter and cultured at 32 °C for 40 h in a neutral environment with pH = 7. After the spore rate reaches 90%, the fermenter is centrifuged and the supernatant is taken as the culture medium.

[0056] The culture medium components are the same as those in Preparation Example 1.

[0057] Preparation Example 3

[0058] The difference from Preparation Example 1 is that the culture medium consists of 4% corn starch, 25% soybean meal, 8% glucose, 1.6% ammonium sulfate, and the remainder water; the rest are the same as Preparation Example 1.

[0059] Example

[0060] Example 1

[0061] A method for preparing a liquid feed additive containing Bacillus subtilis includes the following steps:

[0062] Take 100 mL of the culture medium prepared in Example 1, add 0.3% xanthan gum, stir to dissolve, and after complete dissolution, add 0.03% carrageenan. After complete dissolution, add 20% propionic acid solution to adjust the pH of the system to the range of 4.0-4.5. Finally, add 0.25 g of Bacillus licheniformis powder, stir evenly, so that the initial bacterial count of Bacillus licheniformis in the culture medium is greater than 100 million CFU / mL, and then dispense into 50 mL sample tubes.

[0063] Example 2

[0064] The difference from Example 1 is that the culture medium of Preparation Example 2 was used; otherwise, it is the same as Example 1.

[0065] Example 3

[0066] The difference from Example 1 is that the culture medium of Preparation Example 3 was used; otherwise, it is the same as Example 1.

[0067] Example 4

[0068] The difference from Example 1 is that 0.3% xanthan gum was added first, stirred and dissolved, and then 0.2% sodium carboxymethyl cellulose was added after complete dissolution. The rest was the same as in Example 1.

[0069] Example 5

[0070] The difference in Example 1 is that 0.2% xanthan gum was added first, stirred and dissolved, and then 0.04% carrageenan was added after it was completely dissolved. The rest was the same as in Example 1.

[0071] Example 6

[0072] The difference from Example 1 is that 0.01% carrageenan was added first and stirred to dissolve, then 0.1% sodium carboxymethyl cellulose was added and stirred to dissolve, and then 0.02% guar gum was added. The rest were the same as in Example 1.

[0073] Example 7

[0074] The difference from Example 1 is that 0.3% sodium carboxymethyl cellulose was added first and stirred to dissolve, and then 0.06% guar gum was added. The rest were the same as in Example 1.

[0075] Comparative Example

[0076] Comparative Example 1

[0077] A method for preparing a liquid feed additive containing Bacillus subtilis includes the following steps:

[0078] Heat 100 mL of culture medium to 90 °C. First, add 0.3% xanthan gum and stir to dissolve. After complete dissolution, add 0.03% carrageenan. After the reagents are completely dissolved, add 0.5 g of Bacillus licheniformis powder (initial viable count greater than 100 billion CFU / mL). Add 20% propionic acid solution to adjust the pH of the system to 4.0-4.5. Dispense into 50 mL sample tubes. The culture medium is prepared by mixing 10.2% corn starch, 17% soybean meal, 12% glucose, 4.8% ammonium sulfate, and the remainder water.

[0079] Comparative Example 2

[0080] The difference from Example 1 is that the stabilizer is sodium carboxymethyl cellulose, and the amount of stabilizer added is 0.2% based on the total amount of culture medium. All other aspects are the same as in Example 1.

[0081] Comparative Example 3

[0082] The difference from Example 1 is that the stabilizer is xanthan gum, and the amount of stabilizer added is 0.1% based on the total amount of culture medium. All other aspects are the same as in Example 1.

[0083] Comparative Example 4

[0084] The difference from Example 1 is that the stabilizer is guar gum, and the amount of stabilizer added is 0.02% based on the total amount of culture medium. All other aspects are the same as in Example 1.

[0085] Comparative Example 5

[0086] The difference from Example 1 is that the stabilizer is carrageenan, and the amount of stabilizer added is 0.03% based on the total amount of culture medium. All other aspects are the same as in Example 1.

[0087] Performance testing

[0088] The samples obtained in Examples 1-7 and Comparative Examples 1-5 were tested as follows:

[0089] pH values ​​were measured using a pH meter. The initial pH value of the samples was determined by adding the appropriate stabilizer to the fermentation broth, dissolving and cooling it, and then measuring the pH value of each group of samples on a pH meter. The results were recorded in Table 1.

[0090] Changes in pH value of samples after 30 days of storage: Samples from each experimental group were taken and measured after 60 days of storage using a pH meter. The results are recorded in Table 1.

[0091] Table 1

[0092] initial pH 60-day pH Example 1 4.1 4.32 Example 2 4.06 4.39 Example 3 4.15 4.43 Example 4 4.08 4.38 Example 5 4.11 4.46 Example 6 4.09 4.4 Example 7 4.12 4.37 Comparative Example 1 4.08 6.88 Comparative Example 2 4.1 6.36 Comparative Example 3 4.15 6.05 Comparative Example 4 4.08 6.74 Comparative Example 5 4.11 6.67

[0093] The viable count was determined according to GB 4789.2-2016 "Total Colony Count Test". The viable count was performed by plating NA medium.

[0094] Initial viable count of samples: Dilute the sample with added bacterial powder to 10⁻⁶, 10⁻⁷, or 10⁻⁸ times, spread the sample, incubate at 37℃ for 24 hours, and count the viable cells. The detailed operating procedure is in accordance with GB 4789.2-2016.

[0095] Changes in viable bacterial counts after 30 and 60 days of sample storage: For each experimental group, samples stored for 30 and 60 days were diluted 10⁻⁵, 10⁻⁶, and 10⁻⁷ times to determine their viable bacterial counts and record the results. The experimental results are expressed in log₁₀ form. The detailed operating procedures were carried out in accordance with GB 4789.2-2016, and the results are recorded in Table 2.

[0096] Table 2

[0097] Initial viable count Day 30 Day 60 Example 1 8.525 8.362 8.439 Example 2 8.447 8.279 8.208 Example 3 9.031 8.781 8.492 Example 4 8.491 8.850 8.308 Example 5 8.787 8.994 8.624 Example 6 8.602 8.336 8.217 Example 7 8.505 8.247 8.176 Comparative Example 1 10.866 7.06 5.57 Comparative Example 2 8.693 5.14 5.15 Comparative Example 3 8.736 6.74 5.38 Comparative Example 4 8.531 6.25 5.11 Comparative Example 5 8.667 6.67 5.52

[0098] According to GB / T-13092-2006 "Determination of Total Mold Count in Feed", mold was determined by taking samples from day 60, spreading them on high-salt Czapek's agar plates, and incubating at 37°C for at least 48 hours. The samples were then diluted in parallel to a concentration of 10⁻⁶. 0 10 -1 10 -2 The coating was doubled, and the growth of mold was observed; the mold growth in Example 1 is shown in [reference needed]. Figure 13 The mold growth in Comparative Examples 2-4 is shown in the figure. Figures 14-17 .

[0099] The appearance of the samples after 60 days of storage was observed with the naked eye: the layering and sedimentation were observed. The presence of any obvious odor was determined by fanning the sample and smelling it. The results are recorded in Table 3.

[0100] Specifically, the sensory characteristics of the samples prepared in Examples 1-7 and Comparative Examples 1-5 after 60 days of storage are shown in the following figures. Figures 1-12 .

[0101] Table 3

[0102]

[0103]

[0104] As can be seen from Examples 1-7 and Tables 1-3, the pH value of each sample showed an increasing trend with prolonged storage time, but remained within the range of 4.0-4.5. The viable bacterial count remained relatively stable, with all counts exceeding 100 million CFU / mL. Figure 1-7 As can be seen, in Example 3, the culture medium used in Preparation Example 3 was used, and the product showed slight stratification. Although Carrageenan, Sodium Carboxymethyl Cellulose, and Guar Gum were used as stabilizers in Example 6, the product also showed slight stratification, with some sediment at the bottom. This indicates that using xanthan gum in combination with carrageenan as stabilizers yields the best results. Furthermore... Figure 13 As can be seen, no mold growth was observed on the surface of the products obtained in Examples 1-6.

[0105] Based on Example 1 and Comparative Example 1, and referring to Tables 1-3, it can be seen that the sample of Comparative Example 1 was very unstable during storage, exhibiting stratification and precipitation. After a period of time, the pH value increased significantly. Although 0.5g of Bacillus licheniformis was added, the viable cell count decreased significantly. This is due to two reasons: firstly, the stabilizer is unstable in the culture medium, and the culture medium cannot provide a favorable environment for the bacteria; secondly, the heating and dissolving process further deteriorated the sample's stability.

[0106] Combining Example 1 and Comparative Examples 2-5 with Tables 1-3, it can be seen that the pH value of the sample groups increased over time during storage. Regarding the viable cell count, the viable cell count of the sample groups gradually decreased over time. Products using sodium carboxymethyl cellulose, carrageenan, or guar gum as stabilizers individually exhibited a relatively clear and transparent upper layer with a large amount of sediment in the lower layer. Samples using xanthan gum as a stabilizer alone showed a semi-solid state. In terms of odor, all samples prepared in Comparative Examples 2-5 showed a decrease in acidity, with Comparative Example 1 exhibiting a strong hydrogen sulfide odor, Comparative Example 2 having a distinct alkaline odor, and Comparative Example 4 also showing a hydrogen sulfide odor. Furthermore, considering… Figure 14-17 As can be seen, the product surface has severe mold growth, and the stability of the products obtained from Comparative Examples 2-5 is poor.

[0107] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A liquid feed additive containing Bacillus subtilis, characterized in that, The formulation includes the following raw materials: culture medium, stabilizer, Bacillus subtilis, and acid-regulating solution; the stabilizer is added at a rate of 0.01-0.5 wt% based on the total amount of culture medium; the stabilizer is xanthan gum and carrageenan, and the mass ratio of xanthan gum to carrageenan is (2-3):(0.3-0.4); the Bacillus subtilis is added at a rate of 2.5-5 g / L based on the culture medium; the culture medium is prepared by the following steps: inoculating a single colony of Bacillus subtilis into the culture medium for a first culture time of 10-12 h to obtain a primary seed culture; inoculating the primary seed culture into the culture medium again for a second culture time of 10-12 h to obtain a secondary seed culture; The secondary seed culture is sealed and cultured in a neutral environment. When the spore rate reaches more than 90%, the supernatant is taken to obtain the culture medium. The culture medium used to prepare the culture medium includes, by weight percentage, 20-25% soybean meal, 4-8% glucose, 1.6-2.5% ammonium sulfate, 4-9% corn starch, and the balance being water.

2. The liquid feed additive containing Bacillus according to claim 1, characterized in that: The sealed culture conditions are: cultured at a temperature of 32-40℃ for 30-40 hours.

3. The liquid feed additive containing Bacillus according to claim 1, characterized in that: The acid conditioning solution includes a propionic acid solution or an acetic acid solution.

4. The liquid feed additive containing Bacillus according to claim 3, characterized in that: The mass concentration of the propionic acid solution is 15-25%.

5. The liquid feed additive containing Bacillus according to claim 1, characterized in that: The Bacillus species include one of Bacillus licheniformis, Bacillus subtilis, Bacillus belye, Bacillus coagulans, Bacillus cereus, Bacillus tarda, and Bacillus pumilus.

6. A method for preparing a liquid feed additive containing Bacillus according to any one of claims 1-5, characterized in that: Includes the following steps: Mix the acid conditioning solution, culture medium, stabilizer and Bacillus evenly.

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