Bacillus safensis and applications thereof

Abstract

This invention relates to the field of biotechnology, and particularly to a strain of Bacillus salafulae subsp. salafulae and its applications. This invention uses a strain of Bacillus salafulae subsp. salafulae as the core functional bacterium to construct a solid / semi-solid fermentation process window that can operate stably at high temperatures of 30-55℃. Through "high-temperature steady-state enzyme production and hydrolysis + process temperature and humidity control and impurity suppression + optional compatibility for synergistic nitrogen locking / adsorption," low ammonia odor and nutrient release are achieved, resulting in fermented feed products with higher nutrient availability, milder odor, and better batch consistency.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Bacillus salaf subsp. salaf and its applications. Background Technology

[0002] In tobacco agricultural production, topping and pruning during the field management stage of tobacco leaves generate a large number of tobacco buds and forks, which have a crude protein content of 15% to 25% and a starch content of 10% to 15%, making them high-quality high-protein feed ingredients. Meanwhile, the lower-grade tobacco leaves (leftover leaves, diseased leaves, and unripe leaves) removed during the harvesting and initial curing stages are also rich in protein and carbohydrates. However, these tobacco wastes have traditionally been mostly disposed of in the field through piling or simple silage, resulting in long processing cycles, high nicotine residues, incomplete protein degradation leading to severe ammonia odor, and susceptibility to contamination and spoilage by miscellaneous bacteria, resulting in extremely low resource utilization rates. In recent years, solid-state fermentation technology has been introduced into the field of tobacco waste treatment. Through microbial transformation, it reduces nicotine toxicity, decomposes large protein molecules into easily absorbed small peptides, and improves palatability, providing a new approach for the feed utilization of by-products from tobacco-growing areas.

[0003] However, the superposition of substrate heat release and ambient temperature in large-scale solid-state fermentation can easily lead to an increase in pile temperature (especially in summer, in closed containers, and when ventilation is insufficient). This problem is particularly prominent in the fermentation of tobacco waste: on the one hand, the respiration of young tissues such as tobacco buds and branches is vigorous after cell breakage, resulting in concentrated metabolic heat production; on the other hand, tobacco planting areas are mostly located in subtropical / temperate zones, with high summer temperatures. The tobacco waste harvesting period (June to August) coincides with the high-temperature season, and the ambient temperature often reaches above 35°C, making it very easy for the fermentation pile temperature to exceed 45°C or even 50°C. Traditional processes often rely on acidification and spoilage inhibition by lactic acid bacteria / yeast. However, published research suggests that the activity and survival of lactic acid bacteria inoculants significantly decrease under heat stress conditions. For example, the survival rate can decrease by nearly 2 logs after 24 hours at 45°C (related to strain / formula). Furthermore, the high protein content of tobacco waste fermentation (superior to conventional straw-based raw materials) increases the risk of abnormal protein degradation and ammonia production. Insufficient acidification due to heat stress can quickly trigger a vicious cycle—a rise in pH promotes the proliferation of harmful bacteria (such as Bacillus putrefactive bacteria and Escherichia coli), further exacerbating protein deammoniation and hindering nicotine degradation, resulting in NH4+. + Significant increases in -N, TVB-N, and ammonia odor lead to decreased palatability and safety of fermentation products, as well as poor batch stability, severely hindering the industrial application of tobacco waste fermentation feed technology. Therefore, there is an urgent need to develop functional microorganisms and supporting processes capable of withstanding the heat stress of tobacco waste fermentation and maintaining efficient protein degradation and detoxification capabilities, in order to achieve stable, low-ammonia conversion of high-protein waste from tobacco-growing areas into feed.

[0004] Current fermented feed processes mostly employ solid-state fermentation using one or more of lactic acid bacteria, yeast, or Bacillus, supplemented with sugar sources, acidifiers, or adsorbents to control pH and inhibit spoilage. Some schemes use Bacillus to produce enzymes to promote hydrolysis, but these often emphasize mesophilic fermentation (25-40℃) or lack a reproducible temperature control window for the high-temperature exothermic phase. Under exothermic conditions, problems such as an imbalance between enzyme production and spoilage inhibition, and unstable ammonia odor control may still occur. Specifically:

[0005] (1) Temperature sensitivity leads to fermentation instability: Exothermic and high ambient temperature can cause the pile temperature to rise to above 45°C, the activity of some commonly used strains (especially lactic acid bacteria / yeast) decreases, acidification is insufficient, putrefactive bacteria are easy to grow, and ammonia and other odors increase.

[0006] (2) Insufficient co-hydrolysis of protein and starch: Insufficient efficiency of simultaneous hydrolysis and saccharification of high protein / high starch substrates, limiting the release of small peptides and available sugars, thereby limiting the improvement of palatability.

[0007] (3) Over-reliance on external inhibitors for ammonia odor control: Relying solely on acidifiers or adsorbents may lead to changes in taste, loss of nutrients, or increased costs; and if there is a lack of evidence of synergy with core bacteria, the subordinate scheme is not stable enough. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a high-yield cellulase strain of Bacillus safensis subsp. safensis, and to provide a method for preparing low-ammonia fermented feed using this strain, which can stably produce enzymes and rapidly hydrolyze proteins at higher temperatures. At the same time, by using reasonable moisture content, aeration and optional compatibility measures, ammonia generation and ammonia odor intensity are reduced, thereby obtaining fermented feed products with higher nutritional availability, milder odor and better batch consistency.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides Bacillus safensis subsp. safensis, with accession number CGMCC No. 36429, which has the ability to stably produce protease and promote the directed hydrolysis of proteins to generate soluble proteins and small peptides under fermentation conditions of 30~55℃, thereby reducing the accumulation of ammonia nitrogen and ammonia odor in the fermentation system.

[0011] The present invention also provides a bacterial agent comprising the above-mentioned Bacillus salaf subsp. salaf.

[0012] In some specific embodiments of the present invention, the above-mentioned bacterial agent is a bacterial solution of Bacillus salsa subsp. salsa, which may contain excipients or adjuvants.

[0013] The present invention also provides the use of the above-mentioned Bacillus salaf subsp. salaf or the above-mentioned bacterial agent in any of the following:

[0014] (i) Preparing feed;

[0015] (ii) Reduce feed NH4 + -N content, TVB-N content, ammonia concentration and / or ammonia odor intensity.

[0016] In some specific embodiments of the present invention, the above-mentioned application uses tobacco waste as the main raw material to prepare feed.

[0017] In some specific embodiments of the present invention, the feed described above is a low-ammonia fermented feed;

[0018] The NH4 in the low-ammonia fermented feed + Lower N-N content, TVB-N content, ammonia concentration, and / or ammonia odor intensity.

[0019] In some specific embodiments of the present invention, the above preparation method includes preparation based on any of the following:

[0020] (i) The above-mentioned *Bacillus safusiforme* subsp. *safusiforme*;

[0021] (ii) The above-mentioned microbial agents.

[0022] In some specific embodiments of the present invention, the above preparation method includes the following steps:

[0023] Adjust the moisture content of the substrate to 35%~60%;

[0024] The *Bacillus salaf* subsp. *salaf* or its inoculum was inoculated onto the substrate to achieve a viable count of 102. 6 ~10 9 CFU / g;

[0025] Fermentation is carried out in a microaerophilic or sealed manner at a temperature of 30~55℃. After fermentation, the product is dried or packaged to obtain feed.

[0026] In some specific embodiments of the present invention, the above preparation method includes the following steps:

[0027] (1) Preparation of bacterial culture: The *Bacillus sabolicus* subsp. *sabolicus* was activated and cultured to obtain bacterial culture;

[0028] (2) Substrate preparation: Select feed ingredients containing starch and protein, crush and mix them, and adjust the moisture content to 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%;

[0029] (3) Inoculation: The bacterial solution is inoculated onto the substrate to make the initial viable count of the substrate 102. 6 CFU / g, 10 7 CFU / g, 10 8 CFU / g or 10 9 CFU / g;

[0030] (4) Fermentation: Solid or semi-solid fermentation is carried out at 30~55℃ (which may fluctuate within this range, or may be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃ or a combination of at least two of the above temperatures) for 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, 60 h, 66 h or 72 h. During the fermentation process, the microaerophilic or sealed anaerobic state is maintained and the mixture is turned over as needed.

[0031] (5) Post-processing: After fermentation, the feed is dried, granulated or sealed and stored to obtain low-ammonia fermented feed.

[0032] In some specific embodiments of the present invention, the above preparation method includes:

[0033] The substrate includes at least one of the following: green tobacco leaves, low-grade tobacco leaves, tobacco buds, soybean meal, distiller's grains, wheat bran, corn flour, and mineral premix; and / or

[0034] The C / N of the substrate is adjusted to 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; and / or

[0035] The substrate comprises 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% (w / w) of a fermentable sugar source; and / or

[0036] The substrate comprises 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, or 2.0% (w / w) of organic acid or buffer salt; and / or

[0037] The substrate comprises 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5.0% (w / w) of ammonia adsorbent material, wherein the ammonia adsorbent material comprises at least one of zeolite, biochar, and bentonite; and / or

[0038] The amount of the bacterial agent added is 0.1%, 0.5%, 1%, 1.5% or 2.0% (w / w) on a wet basis.

[0039] In some specific embodiments of the present invention, the sugar source in the above preparation method is molasses, glucose, maltodextrin, or a combination thereof; and compared to the treatment group that was only inoculated with the bacterial agent, the sugar source makes the fermentation endpoint (i.e., at the time of "completion of fermentation") NH4 + -N further decreases by ≥20% and / or ammonia odor score further decreases by ≥1 point.

[0040] In some specific embodiments of the present invention, the organic acid in the above preparation method is lactic acid, citric acid, or a combination thereof; and compared to the treatment group inoculated only with the bacterial agent, the organic acid or buffer salt causes the pH to decrease by more than 0.2 and / or NH4+ at the fermentation endpoint (i.e., at the "completion of fermentation" mentioned above). + -N is further reduced by ≥10%.

[0041] In some specific embodiments of the present invention, the ammonia adsorbent material in the above preparation method is zeolite, biochar, or bentonite; and compared to the treatment group that is only inoculated with the bacterial agent, the ammonia adsorbent material further reduces the ammonia odor score by ≥0.5 points and / or NH4. + -N is further reduced by ≥10%.

[0042] In some specific embodiments of the present invention, the fermentation of the above preparation method is solid-state fermentation or semi-solid-state fermentation, including (i) a growth and enzyme production initiation stage, (ii) a high-temperature steady-state hydrolysis stage and (iii) a stabilization stage;

[0043] The temperature for the growth and enzyme production initiation phase is 35-40℃ (which may fluctuate within this range, or may be at least one or two of the following temperatures: 35℃, 36℃, 37℃, 38℃, 39℃, and 40℃), and the time is 6-18 h.

[0044] The temperature of the high-temperature steady-state stage is 45~55℃ (it can fluctuate within this range, or it can be at least one or two of the following temperatures: 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, and 55℃), and the time is 18~30 h.

[0045] The stabilization phase is carried out at a temperature of 35-45°C (which may fluctuate within this range, or may be a range consisting of at least one or two of the following temperatures: 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, and 45°C), and for a duration of 6-18 hours.

[0046] In some specific embodiments of the present invention, the criterion for the completion of fermentation in the above preparation method is at least one of the following:

[0047] (i) Ammonia odor score ≤ 1, wherein the ammonia odor score is a scale of 0 to 5;

[0048] (ii) NH4 + -N content decreased by ≥20% compared to the control group that was not inoculated with the described Bacillus salaf subsp. salaf or the bacterial agent;

[0049] (iii) TVB-N decreased by ≥15% compared to the control group that was not inoculated with the aforementioned Bacillus salaf subsp. safoetida or bacterial agent.

[0050] The beneficial effects of this invention are as follows: Compared with traditional fermentation routes that are sensitive to temperature or highly dependent on strong anaerobic acidification, this invention maintains the activity and enzyme production capacity of dominant bacteria under exothermic conditions through segmented temperature control, enabling faster hydrolysis of proteins / starches and the release of small peptides and available sugars; at the same time, it reduces NH4 by inhibiting putrefactive deammoniation pathways and using an optional "assimilation and nitrogen locking / adsorption of free ammonia" strategy. + -N and TVB-N significantly reduce ammonia odor, thereby improving palatability, reducing irritating off-flavors, and enhancing batch stability.

[0051] Biological Preservation Instructions

[0052] Biological material: BSA1, taxonomically named Bacillus safensis subsp. safensis, was deposited on October 30, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; accession number: CGMCC No. 36429. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0054] Figure 1 The flowchart of the method for preparing low-ammonia fermented feed of the present invention is shown, including raw material pretreatment, moisture content control / optional C:N, inoculation with microbial agent, segmented temperature-controlled solid / semi-solid fermentation, and post-treatment;

[0055] Figure 2The diagram shows the control of fermentation temperature and moisture content. In actual production, the temperature and moisture content can be adjusted within the window defined in the claims according to the type and scale of raw materials. However, it is preferred to set a heating stage and a high-temperature steady-state stage to balance rapid enzyme production and hydrolysis with low ammonia odor control.

[0056] Figure 3 This diagram shows the clear zone on a protease screening plate of Bacillus safensis subsp. safensis (CGMCC No. 36429), used to demonstrate that the strain has the ability to secrete extracellular proteases. Detailed Implementation

[0057] This invention discloses a high-yield cellulase strain of *Bacillus safusiforme* subsp. *safusiforme*. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0058] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0059] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0060] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0061] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0062] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0063] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0064] The technical terms and their explanations are as follows:

[0065] Fermented feed: refers to feed products obtained by fermenting plant or animal feed ingredients with microorganisms. They usually have better palatability, stability and nutritional availability.

[0066] Low ammonia odor: refers to a state in which ammonia (NH3) and its odor intensity are reduced during fermentation and in finished feed. This can be assessed through ammonia odor scoring, ammonia concentration, or ammonia nitrogen (NH4) levels. + Indicators such as -N) characterize:

[0067] Solid-state / semi-solid-state fermentation: refers to fermentation methods where the substrate moisture content is typically in the range of 35% to 60%, and the substrate is in a moist granular or aggregated state.

[0068] Microaerophilia refers to a substrate environment where the interior is predominantly low-oxygen, but a small amount of oxygen exchange exists on the surface or in the pores, which is conducive to the growth and enzyme production of Bacillus.

[0069] Degree of protein hydrolysis: This indicates the extent to which proteins are broken down into small peptides / free amino acids, and can be characterized by soluble protein, TCA soluble nitrogen, and small peptide content.

[0070] TVB-N: Total volatile basic nitrogen, often used to characterize protein putrefaction / amine accumulation levels, and can be used as one of the objective chemical indicators of low ammonia odor / low putrefaction.

[0071] The *Bacillus safensis* subsp. *safensis* (CGMCC No. 36429) of this invention possesses the ability to rapidly hydrolyze proteins at 55°C and is beneficial for reducing ammonia odor. The protease plate screening and judgment process is as follows, wherein the screening uses skim milk powder as a substrate, and the extracellular protease secretion capacity is evaluated by observing the clear zone. Specifically:

[0072] (1) Protease screening medium formulation: 10 g / L tryptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L skim milk powder, 18 g / L agar powder, and deionized water to 1 L; autoclave at 121℃ for 15 min, cool to about 50~55℃ and pour into plates (about 20 mL per plate), and let stand at room temperature until solidified;

[0073] (2) Inoculation and culture: After the strain is recovered from slant or glycerol tube, single colonies are obtained. Single colonies are picked and inoculated into LB liquid seed medium and cultured at 30-37℃ and 150-200 r / min for 12-18 h with shaking. The culture is then serially diluted 10-fold with sterile physiological saline. -1 ~10 -6 Invert the plates and incubate at 30-37℃ for 24 h;

[0074] (3) Determination and recording of clear zone: After the culture is completed, observe the clear zone (or the area of ​​reduced turbidity) formed by the hydrolysis of skim milk powder around the colony. The ratio of the diameter of the clear zone D to the diameter of the colony d (D / d) is used as the screening index. Use vernier calipers to measure D and d and take pictures for record. The larger the D / d ratio, the stronger the extracellular protease secretion ability.

[0075] (4) Screening results: The strain formed a clear zone on the screening plate (see Figure 3 This indicates that it possesses a strong extracellular protease secretion capacity, making it suitable for rapidly hydrolyzing proteins and reducing the accumulation of ammonia precursors during feed fermentation. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36429.

[0076] In one implementation of the present invention, Bacillus safensis subsp. safensis (CGMCC No. 36429) is selected as the core functional bacterium for the preparation of low-ammonia fermented feed. By utilizing its ability to maintain growth and enzyme production at high temperatures, it enables rapid hydrolysis of high-protein substrates, and significantly reduces ammonia odor through process window control and optional compatibility measures.

[0077] In a specific implementation, an example of the preparation of the low-ammonia fermented feed can be found here. Figure 1 The flowchart specifically includes the following steps:

[0078] S1 Raw material pretreatment: Crush, sieve and mix the substrate such as soybean meal, distiller's grains, low-grade tobacco leaves, tobacco buds and shoots produced by topping tobacco, and green tobacco leaves removed during harvest. If necessary, add loosening material (such as straw powder and rice husk powder) to improve aeration.

[0079] S2 Moisture and Nutrient Window: Adjust the moisture content to 35%~60% (preferably 40%~55%), and optionally adjust the C / N ratio to 18~30;

[0080] S3 Inoculation: Add the bacterial agent at 0.1%~2.0% (w / w) on wet substrate to achieve an initial viable count of 10⁻⁶ on the substrate. 6 ~10 9 CFU / g;

[0081] S4 Fermentation: Solid / semi-solid fermentation is carried out in the range of 30~55℃ for 12~48 h, using microaerophilic or sealed methods, and stirring is necessary to ensure uniform temperature and humidity.

[0082] S5 Post-processing: After fermentation, dry (≤60℃ hot air or low temperature vacuum), granulate or seal and store.

[0083] In practical implementation, segmented temperature control and moisture content control can be referred to... Figure 2 An example of a three-stage control strategy is as follows: 0~12 h (35~40℃) is used for cell growth and enzyme production initiation; 12~36 h (45~55℃) is used for high-temperature steady-state hydrolysis and inhibition of contaminating bacteria; 36~48 h (40~45℃) is used for system stabilization and flavor convergence.

[0084] In practice, the moisture content is generally maintained at 40% to 55%, and decreases slightly as fermentation releases heat and ventilation increases. Water can be added or ventilation adjusted as needed.

[0085] In practice, the strain stably produces enzymes at high temperatures, and the enzymes promote the targeted hydrolysis of proteins: CGMCC No. 36429 can still secrete extracellular proteases / amylases at higher temperatures, rapidly hydrolyzing large protein molecules into small peptides and amino acids, and simultaneously promoting starch saccharification to release fermentable sugars.

[0086] In practice, the above methods reduce putrefactive deamination and amine accumulation: Under traditional unstable conditions, putrefactive bacteria and strong deamination microorganisms readily deamination amino acids to generate NH3 / NH4. + Accompanying amines and off-odors; through "rapid hydrolysis + process window impurity suppression," the substrate is occupied by dominant bacteria within the critical window, reducing putrefactive metabolic flux and thus reducing NH4. + -N and TVB-N.

[0087] In one example, the above method can also add fermentable sugar sources to promote the production of organic acids (acidification), producing a synergistic effect: adding 0.5%~5% sugar sources can provide carbon sources for dominant bacteria, promote the assimilation and fixation of nitrogen sources ("nitrogen lock-in" in the form of biomass), and promote the production of organic acids to lower the pH of the system, thereby inhibiting putrefactive bacteria from the source.

[0088] In one example, an ammonia adsorbent material can be added to produce a synergistic effect: the ammonia adsorbent material includes, but is not limited to, zeolite, biochar, and bentonite, which can adsorb NH4. + Ion exchange or adsorption occurs, further reducing the free ammonia odor.

[0089] In practice, the bacterial agent can be in the form of bacterial liquid, spore powder, or carrier adsorbed particles.

[0090] In practice, the fermentation method can be sealed anaerobic, microaerobic, or intermittent ventilation.

[0091] In practice, the temperature program can be adjusted from two stages to three stages.

[0092] In practice, post-processing can be low-temperature vacuum drying, hot air drying, direct silage sealing, or coupled with granulation.

[0093] In practice, Bacillus safensis subsp. safensis (CGMCC No. 36429) is used as the core functional bacterium to construct a solid / semi-solid fermentation process window that can still operate stably in the high-temperature range of 30~55℃. Low ammonia odor and nutrient release are achieved through "high-temperature steady-state enzyme production and hydrolysis + process temperature and humidity control and impurity suppression + optional compatibility synergistic nitrogen locking / adsorption".

[0094] In this embodiment, the detection indicators and their determination methods are as follows:

[0095] (1) Experimental design: 3 biological replicates, with an uninoculated control group;

[0096] (2) Sampling and physicochemical analysis: Samples were taken at 0 h, 12 h, 24 h, and 48 h; moisture content, pH, and NH4 were measured. + -N, TVB-N, soluble proteins, small peptides (TCA soluble nitrogen conversion), reducing sugars, etc.

[0097] (3) Microorganisms: Measure the total number of bacteria, the count of Bacillus, and the count of Enterobacteriaceae / putrefactive bacteria indicator bacteria to support the logic of "suppression of contaminants and stability";

[0098] (4) Statistics: Use t-test for comparison of two groups, and one-way ANOVA for comparison of multiple groups; give the p-value in the table;

[0099] (5) Sensory evaluation: The ammonia odor score is based on a scale of 0 to 5 (0 = none, 5 = strong pungent odor), and is evaluated blindly by a training group of ≥5 people. The mean score is ±SD.

[0100] The present invention will be further illustrated below with reference to the embodiments.

[0101] Example 1: Preparation of low-ammonia fermented feed using soybean meal

[0102] (1) Raw materials and formulation: Take 40% soybean meal, 40% wheat bran, 18% corn flour and 2% (w / w) mineral premix as substrate; crush and pass through a 40-mesh sieve and mix thoroughly. Add water to adjust the moisture content to 45%±1%.

[0103] (2) Preparation of bacterial agent: Bacillus safensis subsp. safensis (CGMCC No. 36429) was cultured in LB liquid medium at 30-37℃ with shaking for 12-18 h to prepare seed culture; 2% (v / v) was inoculated into expansion medium and cultured to the end-log / stationary phase, and the bacterial concentration was adjusted to 10. 9 CFU / mL.

[0104] (3) Inoculation and loading: Take 1000 g of raw material and spray the bacterial solution at 0.5% (w / w) on a wet basis and mix thoroughly so that the initial viable count in the substrate is about 1×10⁻⁶. 8 CFU / g. The mixed substrate was placed in the fermentation chamber, with a thickness controlled at 10-20 cm; venting holes were left to create a microaerophilic environment. A control group without inoculated strains was set up.

[0105] (4) Fermentation conditions: 0~12 h, control the temperature at 35~40℃ (growth and enzyme production initiation); 12~36 h, control the temperature at 45~55℃ (high-temperature steady-state hydrolysis); 36~48 h, cool down to 40~45℃ (stabilization). During this period, stir once every 12 h to ensure uniform temperature and humidity and prevent local spoilage. The total fermentation time is 48 h.

[0106] (5) Post-processing: After fermentation, the sample was divided into two parts: one part was dried with hot air at ≤60℃ until the moisture content was ≤12% and then granulated; the other part was directly sealed and packaged and stored at 4~25℃ for stability observation.

[0107] (6) Detection method:

[0108] ① Sampling: Take 3 parallel samples at 0 h and 48 h (each sample not less than 20 g), mix well and then dispense into smaller containers;

[0109] ②pH: Add distilled water at a ratio of 1:10 (w / v), shake for 30 min, and take the supernatant to measure the pH;

[0110] ③NH4 + -N: Nessler's reagent colorimetric method was used;

[0111] ④TVB-N: Distillation titration method is used;

[0112] ⑤ Soluble proteins: Coomassie brilliant blue method;

[0113] ⑥ Small peptides / soluble nitrogen: After precipitation with 10% TCA, the soluble nitrogen content of TCA was measured and converted.

[0114] ⑦ Reducing sugars: DNS method;

[0115] ⑧ Ammonia odor rating: Blind evaluation by ≥5 people, on a scale of 0 to 5, with the mean of the reports ± SD.

[0116] (7) Results

[0117] See Table 1.

[0118] Table 1

[0119]

[0120] Conclusion: Compared with the uninoculated control, inoculation with Bacillus safensis subsp. Safensis (CGMCC No. 36429) resulted in lower and more stable pH levels and lower NH4+. + -N and TVB-N decreased significantly, while the contents of soluble protein, small peptides and reducing sugar increased, indicating enhanced protein / starch hydrolysis and nutrient release; the sensory score of ammonia flavor decreased significantly, reflecting low ammonia flavor and improved palatability.

[0121] Example 2: Preparation and Effect Evaluation of Low-Ammonia Fermented Feed Compared with Uninoculated, Lactic Acid Bacteria Inoculated, and Common Bacillus Inoculated Feeds

[0122] Based on Example 1, the following comparative treatment group was added:

[0123] A = Unvaccinated (blank control);

[0124] B = Inoculation with lactic acid bacteria (LAB, control bacteria);

[0125] C = Inoculation with common Bacillus subtilis (control bacteria);

[0126] D = Inoculation with Bacillus safensis subsp. safensis (the strain of this invention, CGMCC No. 36429).

[0127] Except for the inoculum, all groups maintained the same process conditions, including raw material formulation, moisture content, packing density, sealing method, fermentation temperature, and time. The NH4 endpoint at 48 h was compared. + -N and ammonia odor score.

[0128] The results are shown in Table 2.

[0129] Table 2

[0130]

[0131] Conclusion: Compared with the uninoculated control, inoculation with both lactic acid bacteria and common Bacillus species reduced NH4 levels. + -N and ammonia odor score, but strain CGMCC No. 36429 of the present invention can further reduce NH4 under the same fermentation conditions. + -N significantly reduces the sensory ammonia odor, resulting in a lower ammonia odor and a milder smell, demonstrating its advantage in the preparation of low-ammonia fermented feed.

[0132] Example 3: Preparation of low-ammonia fermented feed using tobacco leaves

[0133] (1) Raw materials and formula: Take 35% green tobacco leaves, 35% wheat bran, 20% corn flour, 8% soybean meal, and 2% (w / w) mineral premix as substrate; crush the tobacco buds through a 20-mesh sieve, and mix the other raw materials thoroughly after passing them through a 40-mesh sieve. Add water to adjust the moisture content to 50%±1%.

[0134] (2) Preparation of bacterial agent: Bacillus safensis subsp. safensis (CGMCC No. 36429) was cultured in LB liquid medium at 30-37℃ with shaking for 12-18 h to prepare seed culture; 2% (v / v) was inoculated into expansion medium and cultured to the end-log / stationary phase, and the bacterial concentration was adjusted to 10. 9 CFU / mL.

[0135] (3) Inoculation and loading: Take 1000 g of raw material and spray the bacterial solution at 0.6% (w / w) on a wet basis and mix thoroughly so that the initial viable count in the substrate is about 1.2 × 10⁻⁶. 8 CFU / g. The mixed substrate was placed in the fermentation chamber, with a thickness controlled at 15-25 cm; venting holes were left to create a microaerophilic environment. A control group without inoculated strains was set up.

[0136] (4) Fermentation conditions: 0-12 h: temperature controlled at 35-40℃ (initiation of growth and enzyme production, promoting protease secretion based on the high protein content of tobacco buds); 12-36 h: temperature controlled at 45-50℃ (medium-high temperature steady-state hydrolysis, inhibiting excessive ammonia production); 36-48 h: temperature reduced to 35-40℃ (stabilization). During this period, the mixture should be turned over every 12 h to ensure uniform temperature and humidity and prevent localized spoilage. Total fermentation time: 48 h.

[0137] (5) Post-processing: After fermentation, the sample was divided into two parts: one part was dried with hot air at ≤60℃ until the moisture content was ≤12% and then crushed and granulated; the other part was directly sealed and packaged and stored at 4~25℃ for stability observation.

[0138] (6) Detection method:

[0139] ① Sampling: Take 3 parallel samples at 0 h and 48 h (each sample not less than 20 g), mix well and then dispense into smaller containers;

[0140] ②pH: Add distilled water at a ratio of 1:10 (w / v), shake for 30 min, and take the supernatant to measure the pH;

[0141] ③NH4 + -N: Nessler's reagent colorimetric method was used;

[0142] ④TVB-N: Distillation titration method is used;

[0143] ⑤ Soluble proteins: Coomassie brilliant blue method;

[0144] ⑥ Small peptides / soluble nitrogen: After precipitation with 10% TCA, the soluble nitrogen content of TCA was measured and converted.

[0145] ⑦ Reducing sugars: DNS method;

[0146] ⑧ Nicotine residue: Ultraviolet spectrophotometry;

[0147] ⑨ Ammonia odor rating: ≥5 people blind review, 0~5 points, report mean ±SD.

[0148] (7) Results

[0149] See Table 3.

[0150] Table 3

[0151]

[0152] Conclusion: Compared with the uninoculated control, inoculation with Bacillus safensis subsp. safensis CGMCC No. 36429 resulted in more stable pH control and significantly lower NH4 levels, taking advantage of the high protein and nicotine content of tobacco buds. + -N and TVB-N decreased by 53.6% and 54.7% respectively, and the ammonia odor score was significantly reduced. At the same time, the content of soluble protein, small peptides and reducing sugar increased significantly, and the nicotine degradation rate increased by 3 times. This indicates that the strain can effectively degrade the protein and starch in tobacco buds, promote nutrient release, and achieve the preparation of fermented feed with low ammonia odor and low nicotine.

[0153] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bacillus safensis subsp. safensis, characterized by, The accession number is CGMCC No. 36429.

2. A microbial agent, characterized in that, Includes *Bacillus salafus* subsp. *salafus* as described in claim 1.

3. The use of *Bacillus sabophilus* subsp. *sabophilus* as described in claim 1 or the bacterial agent as described in claim 2 in any of the following: (i) Preparing feed; (ii) Reduce feed NH4 + -N content, TVB-N content, ammonia concentration and / or ammonia odor intensity.

4. The application as described in claim 3, characterized in that, include: Feed is prepared using tobacco waste as the main raw material.

5. The application as described in claim 3, characterized in that, The feed is a low-ammonia fermented feed. The NH4 in the low-ammonia fermented feed + Lower N-N content, TVB-N content, ammonia concentration, and / or ammonia odor intensity.

6. A method for preparing feed, characterized in that, Including preparation based on any of the following: (i) The *Bacillus salaf* subsp. *salaf* as described in claim 1; (ii) The microbial agent according to claim 2.

7. The preparation method according to claim 6, characterized in that, Includes the following steps: Adjust the moisture content of the substrate to 35%~60%; The *Bacillus salaf* subsp. *salaf* or the bacterial agent was inoculated onto the substrate to achieve a viable count of 102. 6 ~10 9 CFU / g; Fermentation is carried out in a microaerophilic or sealed manner at a temperature of 30~55℃. After fermentation, the product is dried or packaged to obtain feed.

8. The preparation method according to claim 7, characterized in that, include: The substrate includes at least one of the following: green tobacco leaves, low-grade tobacco leaves, tobacco buds, soybean meal, distiller's grains, wheat bran, corn flour, and mineral premix; and / or The C / N ratio of the substrate is adjusted to 18-30; and / or The substrate comprises 0.5%–5% w / w fermentable sugar source; and / or The substrate comprises 0.2% to 2.0% w / w organic acid or buffer salt; and / or The substrate comprises 0.5% to 5.0% w / w ammonia adsorbent, wherein the ammonia adsorbent comprises at least one of zeolite, biochar, and bentonite; and / or The amount of the bacterial agent added is 0.1%~2.0% w / w on a wet basis.

9. The preparation method according to claim 7, characterized in that, The fermentation is a solid-state fermentation or a semi-solid-state fermentation, including (i) a growth and enzyme production initiation stage, (ii) a high-temperature steady-state hydrolysis stage and (iii) a stabilization stage; The temperature for the growth and enzyme production initiation phase is 35-40℃, and the time is 6-18 h. The temperature of the high-temperature steady-state stage is 45~55℃, and the time is 18~30 h; The stabilization phase is carried out at a temperature of 35-45°C for 6-18 hours.

10. The preparation method according to claim 7, characterized in that, The criterion for determining the completion of fermentation is at least one of the following: (i) Ammonia odor score ≤ 1, wherein the ammonia odor score is a scale of 0 to 5; (ii) NH4 + -N content decreased by ≥20% compared to the control group that was not inoculated with the described Bacillus salaf subsp. salaf or the bacterial agent; (iii) TVB-N decreased by ≥15% compared to the control group that was not inoculated with the aforementioned Bacillus salaf subsp. safoetida or bacterial agent.

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