Microbial microparticle formulation

Abstract

The present invention relates to formulations of spore-producing bacteria. According to a preferred embodiment, the spore-producing bacteria belong to the genus Bacillus (Bacillus), for example Bacillus subtilis (Bacillus subtilis). The invention also relates to a method for producing the spore-producing bacteria.

Description

Technical Field

[0001] The present invention generally relates to methods for producing microbial particulate formulations, and in particular to microbial particulate formulations for agricultural applications. Background Art

[0002] Wettable powders (WPs) are a common product form for chemical pesticides and microbial agricultural formulations, where the active ingredient is formulated and dry-blended with adjuvants (excipients). However, when it comes to end-use, WPs have some inherent drawbacks. Due to the mixing of dry powders with various particle sizes and densities, the smaller particles settle at the bottom while the larger particles remain at the top, creating non-uniformity over time. When the formulation is vibrated during transportation, powder separation becomes more severe. This non-uniformity leads to dose variations during application. Additionally, farmers often complain about the dust of WPs. For WPs, fine powder is almost inevitable, and inhaling the dust from excipients or active ingredients in the air poses a health hazard to end-users. This problem is more severe for microbial formulations because spray-dried spores quickly spread in the air and pose an inhalation risk. Therefore, new product forms are needed to overcome these challenges and methods for manufacturing these products.

[0003] Overview

[0004] Improved formulations of spore-forming bacteria and methods for producing such formulations are provided herein. The particular formulation is referred to as a water-dispersible microgranule (WDMG), and is the result of a production method according to which adjuvants are mixed with a wet Bacillus spore concentrate and then spray-dried.

[0005] Accordingly, in a first aspect, there is provided a method for producing spore-forming bacteria, comprising the steps of: a) adding a bacterial cell starter culture comprising at least one spore-forming bacterial strain to a growth medium; b) propagating the cells by fermenting the medium for a period of time until the cells form spores; c) centrifuging the medium to obtain a spore concentrate; d) mixing the spore concentrate with a co-formulant; e) drying the mixture to obtain a dried product; and f) optionally packaging the dried product, wherein the spore concentrate and the co-formulant are liquids, and the mixing step d) is carried out before the drying step e).

[0006] In one embodiment, mixing is carried out using a high-shear mixer.

[0007] In one embodiment, the co-formulant comprises a disintegrant, a dispersant or an antifoaming agent.

[0008] In a preferred embodiment, the disintegrant is sodium carboxymethyl starch.

[0009] In one embodiment, the drying step e) is carried out using a spray dryer.

[0010] In one embodiment, the spore-forming bacterium belongs to the genus Bacillus.

[0011] In a preferred embodiment, the spore-forming bacterium is Bacillus subtilis.

[0012] In a particularly preferred embodiment, the spore-forming bacterium is Bacillus subtilis, and the excipient comprises the disintegrant sodium carboxymethyl starch.

[0013] According to a second aspect, there is provided a product obtainable by the method according to the first aspect.

[0014] According to a third aspect, there is provided a preparation of a spore-forming bacterium, which comprises a spore-forming bacterium and an excipient.

[0015] In one embodiment, the spore-forming bacterium belongs to the genus Bacillus.

[0016] In a preferred embodiment, the spore-forming bacterium is Bacillus subtilis.

[0017] In one embodiment, the excipient comprises a disintegrant, a dispersant or an antifoaming agent.

[0018] In a preferred embodiment, the disintegrant is sodium carboxymethyl starch.

[0019] In a particularly preferred embodiment, the spore-forming bacterium is Bacillus subtilis, and the excipient comprises the disintegrant sodium carboxymethyl starch.

[0020] According to a fourth aspect, there is provided a product comprising the preparation according to the third aspect.

[0021] According to a fifth aspect, there is provided the use of the product according to the second aspect or the fourth aspect in agriculture.

[0022] In one embodiment, the use in agriculture is as a biopesticide.

[0023] Brief Description of the Drawings

[0024] Figure 1 is a schematic overview of a process line according to a reference embodiment.

[0025] Figure 2 is a schematic overview of a process line according to an embodiment.

[0026] Figure 3 is a graph showing the particle size distribution according to different embodiments.

[0027] Figure 4It is a chart comparing the particle sizes of different embodiments.

[0028] Figure 5 It is a photograph showing the test results according to the embodiments.

[0029] Figures 6 to 11 It is a microscopic image showing multiple embodiments.

[0030] Detailed description

[0031] The present invention relates to the preparation of spore-forming bacteria. According to a preferred embodiment, the spore-forming bacteria belong to the genus Bacillus, such as Bacillus subtilis. The present invention also relates to a method for producing spore-forming bacteria.

[0032] According to the standard protocols known to those skilled in the art, spore-forming bacteria (such as Bacillus subtilis) are fermented in a fermentation vessel based on a starter culture ( Figure 1 a). Spores are formed from vegetative cells. Next, the fermentation medium (including the cells) is transferred to a centrifuge ( Figure 1 b), where the spores are concentrated into a spore concentrate and separated from the fermentation medium supernatant. The spore concentrate is spray-dried in a spray dryer using protocols well-known to those skilled in the art ( Figure 1 c). Subsequently, the resulting dry spore powder is transferred to a powder mixer ( Figure 1 d), where it is mixed with an excipient powder to form a final wettable powder product, which is packaged ( Figure 1 e) and transported.

[0033] A method for manufacturing spore-forming bacteria (such as Bacillus subtilis) is disclosed herein, which has several advantages. According to the standard protocols known to those skilled in the art, spore-forming bacteria (such as Bacillus subtilis) are fermented in a fermentation vessel based on a starter culture ( Figure 2 a). When the growth cycle enters the starvation phase, spores are formed from vegetative cells. Next, the fermentation medium (including the spores) is transferred to a centrifuge ( Figure 2 b), where the spores are concentrated to form a spore concentrate and separated from the fermentation medium supernatant. The spore concentrate is transferred to a mixing tank ( Figure 2 c) and mixed with an excipient. Mixing is preferably carried out using a high-shear homogenizer. The resulting mixture is spray-dried in a spray dryer using protocols well-known to those skilled in the art ( Figure 2 d). Subsequently, the resulting water-dispersible microparticulate agent (WDMG) is packaged ( Figure 2 e) and transported.

[0034] This method is advantageous, especially because it eliminates the problem of powder separation during transportation and storage, reduces dust, and ensures consistent and uniform dosing.

[0035] According to a preferred embodiment, the excipient is sodium carboxymethyl starch. By using sodium carboxymethyl starch as the excipient, microparticles with more uniform size and potency are obtained. In a particularly preferred embodiment, the sodium carboxymethyl starch excipient is LV. Surprisingly, it has been found that LV can very effectively capture spores and release them after rehydration of the dry powder. In addition, the powder formulated with LV has the strongest free-flowability and the lowest content of fine particles.

[0036] The disclosed method is safer than the currently used methods for manufacturing spore-forming bacteria because the mixing of spores and excipient is carried out in the liquid state and thus no dust is generated. By using sodium carboxymethyl starch as the excipient, such pre-drying mixing becomes possible and also improves the properties of the final product.

[0037] Preservation and expert solutions

[0038] The applicant requests that, in accordance with the current regulations of the industrial property offices of the Contracting States to the Budapest Treaty, samples of the following deposited microorganisms can only be provided to experts until the date of patent grant.

[0039] Table 1: Deposits made at the Leibniz DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D - 38124 Braunschweig, Germany, a depository institution having the status of an international depository authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0040] Strain Accession number Deposit date Bacillus subtilis DSM32324 June 8, 2016

[0041] Formulation components

[0042] The composition of the present invention may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, colorants (pigments), excipients, clays, silica, aluminosilicates, fibers, moisture scavengers, or mixtures thereof. The composition may be in frozen or lyophilized form. The composition preferably comprises one or more formulation ingredients, antioxidants, and / or nutrients. The use of standard formulation ingredients is known to those skilled in the art. Suitable formulation ingredients may include monosaccharides, disaccharides, trisaccharides, and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch, and gum arabic, etc.), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol, etc.), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptone, gelatin, yeast extract), and inorganic compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and its salts, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B group, vitamin C). The composition may optionally comprise additional substances, including fillers (such as lactose, maltodextrin) and / or flavors. Examples

[0043] Example 1

[0044] Materials and methods

[0045] The active ingredient in the form of a spore concentrate of Bacillus subtilis (DSM32324) was used. The final dry matter (DM) content of the concentrate was 13% (w / w). The concentration of the active ingredient in the final spray-dried powder was 50% (w / w).

[0046] Table 2 summarizes the ingredients used in this example, the composition of the test formulations (by dry weight), the suppliers of the ingredients, and their functions. Three formulations were used, named Formulation 1, Formulation 2, and Formulation 3. In addition, a control sample using only the spore concentrate without excipients was used.

[0047] Table 2. Name of ingredients, composition of formulations, suppliers of ingredients, and functions of excipients

[0048]

[0049] Formulations 1, 2, and 3 have 50% (w / w) spores and the same amount (3%, w / w) of sodium salt of naphthalene sulfonate condensate ( D425) as a dispersant. In all three formulations, the antifoaming agent ( The amount of [[SP 150]] also remained constant at 0.1% (w / w). In addition to these ingredients, two carrier materials were used, such as maltodextrin (DE 12) and LV (sodium carboxymethyl starch). LV is a modified cross-linked corn starch and is called a disintegrant in tablet formulations. In formulation 2, the ratio of maltodextrin and LV is 1:1. Formulation 1 does not contain LV, and formulation 3 does not contain maltodextrin.

[0050] Slurry preparation

[0051] In a manner well known to those skilled in the art, the excipients were mixed with water at 10,000 RPM for at least 5 minutes and, if necessary, up to 30 minutes using an Ultra- (Ultra- T50, max 10,000 rpm, Janke & Klunkel IKA Labortechnik) high-shear mixer to prepare the slurries of formulation 1, formulation 2, and formulation 3. Formulation 2 and formulation 3 require more time to mix because of the presence of LV which absorbs water and then swells. Therefore, additional water was added to formulation 3 to obtain a viscosity that could be pumped by the pump of the spray dryer. After obtaining a homogenized solution, the concentrate containing the active ingredient was added under high shear to obtain the final slurry. After adding the concentrate, high shear was continued for more than 10 minutes.

[0052] Spray drying of the slurry

[0053] For spray drying, a pilot spray dryer SD / FSD-4.0 (GEA Niro, Germany) was used, and its parameters are as follows:

[0054] · Chamber feed flow rate: 425 kg / h

[0055] · Inlet temperature of the spray dryer: 190 °C

[0056] · Outlet temperature: 90 °C

[0057] · Spray drying time: 13 min, 17 min, 18 min, and 36 min for the control, formulation 1, formulation 2, and formulation 3, respectively.

[0058] · Flow rate: 15.37 kg / h, 17.65 kg / h, 16.67 kg / h, and 14.00 kg / h for the control, formulation 1, formulation 2, and formulation 3, respectively.

[0059] CFU measurement

[0060] Colony-forming unit (CFU) measurement is a quantitative method, and the results are reported as CFU / g. 10 g of the powder is homogenized by paddle homogenization with 190 g of diluent (Maximum Recovery Diluent (MRD)), heated at 80 °C for 10 minutes, cooled and serially diluted in MRD. Then, appropriate dilutions are plated on the surface of tryptic soy agar (TSA) plates. After aerobic incubation at 37 °C for 16 - 24 hours, the colonies are counted.

[0061] The results are summarized in Table 3.

[0062] Bulk density

[0063] Bulk density is calculated from the mass and the corresponding volume of the powder sample. The compressibility is determined using a tapped density apparatus (JEL STAVII, J.Engelsmann AG), where 250 strokes ensure powder compaction.

[0064]

[0065] The results are summarized in Table 3.

[0066] Carr's index

[0067] Carr's index is the ratio of the tapped bulk density of the powder to the free fall bulk density of the powder.

[0068]

[0069] The results are summarized in Table 3.

[0070] Flowability

[0071] The flowability parameter describes the time required for 100 g of the powder to flow through a funnel. After 10 s, the tapped density apparatus starts to tap the sample. After 180 s, the measurement is stopped even if there is still some powder in the funnel.

[0072] The results are summarized in Table 3.

[0073] Water content

[0074] The determination of moisture or loss on drying is analyzed in a dry matter balance / oven (HX204 Moisture Analyzer, Metter Toledo) at 105 °C until constant weight. Moisture is calculated based on the weight difference.

[0075] The results are summarized in Table 3.

[0076] Table 3. Bulk properties of the spray-dried powders of the control sample and Formulations 1, 2, and 3.

[0077] Component Control Formulation 1 Formulation 2 Formulation 3 CFU / g (Bacillus subtilis) <![CDATA[9.7x10 11 > <![CDATA[4.2x10 11 > <![CDATA[4.0x10 11 > <![CDATA[3.7x10 11 > <![CDATA[Bulk density (kg / m 3 )]]> 436 488 521 534 Carr index (%) 18.2 23.3 24.6 13.6 Flowability (s) >180 >180 150 32 Water content (%) 5.1 5.3 5.5 4.6

[0078] Particle size distribution

[0079] The particle size distribution of the sample in both dry and wet states was measured by static light scattering (SLS) using a Sympatec Helos BR laser diffraction system (Sympatec Inc., Clausthal, Germany), with Rodos (for powders) and Quixel (for liquids) attachments.

[0080] Dry particle size

[0081] The particle size distribution of the dry powder was measured as usual.

[0082] Wet particle size

[0083] Slurries of different formulations were prepared by adding 1% (w / w) SD powder to water, and then the particle size was measured.

[0084] Wettability

[0085] Wettability was measured by adding 5 ± 0.1 g of the powder to a beaker containing 100 ± 1 ml of standard water D (342 ppm hardness (Ca 2+ :Mg 2+ = 4:1), pH 6.0 - 7.0), and using a stopwatch to record the time to complete wetting.

[0086] Results and discussion

[0087] Table 3 gives the survival rates of the control spores and the formulated spores. The CFU of the control SD powder was the highest, at 9.7×10 11 . For all the WDMGs, the CFU counts were approximately 4x10 11 (±0.3x10 11 ), which was as expected due to the dilution of the formulation components.

[0088] The bulk density of formulation 3 was the highest among all the formulations, while the Carr's index was the lowest. This must be due to the presence of LV. The Carr's index is an indicator of compressibility and, in turn, of the powder flow behavior, and is obtained from the bulk density and tapped density of the powder. If the Carr's index of a powder is between 5 and 15, the powder has excellent flowability; if between 16 and 18, it has good flowability; between 19 and 21, it has average flowability; and between 22 and 35, it has poor flowability. As shown in Table 3, the flowability was as follows: formulation 3 > spores only > formulation 1 > formulation 2. The flowability was also measured directly, and it also indicated that the best candidate was formulation 3. This means that the presence of the disintegrant acted as a flow modifier, i.e., an adjuvant in the formulation. The water content of formulation 3 was also the lowest among the four.

[0089] The dry particle sizes of the four SD powders were as Figure 3as shown

[0090] The relationship between the density distribution (%) and the size clearly shows that the distribution changes when we move from only spores to formulation 3. The particle size distribution (PSD) of formulation 3 is unimodal, contrary to the bimodal distribution of formulation 2. In addition, formulations 1 and 2 show a wider distribution compared to formulation 3. This indicates that formulation 3 has fewer fine particles and a more uniform particle size among the four. This has a great impact on the dry formulation for field applications. Moreover, the absence of fine particles or smaller particles means that the internal flow of the powder can also be reduced, and thus the separated mixture can be reduced. Therefore, the smaller particles at the bottom and the larger particles at the top do not separate in the package.

[0091] To further describe the results, the d 10 、d 50 and d 90 values are plotted in Figure 4 . It can be clearly seen that for the control (only spores) and formulation 1, d 10 and d 50 are within 1 μm and just greater than 1 μm respectively. But for formulations 2 and 3, d 10 and d 50 shift towards 100 μm, and d 50 and d 90 are close to each other. This indicates that the particle size distributions of formulations 2 and 3 are uniform.

[0092] The wettability of the microparticles is also worthy of attention, and the wetting behavior of the formulations was tracked according to the method described in CIPAC MT 53.3.1. Figure 5 Snapshots of the beaker were captured, where the wettability tests were carried out at the start of the test (0 s), after 30 s, and after 60 s. From the turbidity of the water, we can clearly see that formulation 3 makes the water the most turbid compared to the other three. This is due to the presence of the disintegrant LV (sodium carboxymethyl starch). The turbidities of formulations 2 and 3 are close because both of these formulations contain LV. Once the formulated microparticles come into contact with water, the disintegrant absorbs a large amount of water and swells, and the microparticles lose their cohesion, and the presence of D425 disperses the spores in water. For formulations 2 and 3, the combined action of both the disintegrant and D425 was observed. However, since formulation 2 is a mixture of maltodextrin and the disintegrant, its effect is not as obvious as that of formulation 3, which does not contain maltodextrin.

[0093] Optical micrographs of WDMG dispersed in water at a concentration of 0.1% (w / w) can be observed from Figures 6 to 9 . Figure 6is a microscopic image of the control (diluted 0.1% in water). Figure 7 is a microscopic image of formulation 1 (diluted 0.1% in water). Figure 8 is a microscopic image of formulation 2 (diluted 0.1% (w / w) in water). Figure 9 is a microscopic image of formulation 3 (0.1% (w / w) aqueous solution). The purple dots are individual spores, and the black entities are residues from the fermentation leftovers. The black residue is a problem, and it is desired to reduce the residue by adding a dispersant to the formulation.

[0094] In Figure 7 it can be clearly seen that the residue in formulation 2 is less than that in formulation 1.

[0095] Interestingly, in formulation 2, well - defined regions can be seen, which, according to the non - limiting theory of the present inventors, are the result of two starches, maltodextrin and LV. These regions are clearer in formulation 3, and it can be seen that these particles absorb spores. Interestingly enough, the residue in formulation 3 is much less. In addition, in formulation 3, very round starch particles embedding spores can be seen( Figure 10 , which is a microscopic image of formulation 3 (0.1% (w / w) aqueous solution)).

[0096] In Figure 11 (which is also a microscopic image of formulation 3 (0.1% (w / w) aqueous solution)), but at a higher magnification, it can be seen that the spores drill out of the particles through the pores on the particle surface.

Claims

1. A method for producing spore-forming bacteria, comprising the following steps: a) adding a bacterial cell starter culture comprising at least one spore-forming bacterial strain to a growth medium; b) propagating the cells by fermenting the medium for a period of time until the cells form spores; c) centrifuging the medium to obtain a spore concentrate; d) mixing the spore concentrate with excipients; e) drying the mixture to obtain a dry product; and f) optionally packaging the dry product, wherein the spore concentrate and the excipients are liquids, and the mixing step d) is carried out before the drying step e).

2. The method according to claim 1, wherein the mixing is carried out using a high-shear mixer.

3. The method according to any one of the preceding claims, wherein the excipients comprise a disintegrant, a dispersant or an antifoaming agent.

4. The method according to any one of the preceding claims, wherein the disintegrant is sodium carboxymethyl starch.

5. The method according to any one of the preceding claims, wherein the drying step e) is carried out using a spray dryer.

6. The method according to any one of the preceding claims, wherein the spore-forming bacteria belong to the genus Bacillus.

7. The method according to claim 6, wherein the spore-forming bacteria are Bacillus subtilis (DSM32324).

8. A product obtainable by the method according to any one of claims 1 to 7.

9. A preparation of spore-forming bacteria, comprising spore-forming bacteria and excipients.

10. The preparation according to claim 9, wherein the spore-forming bacteria belong to the genus Bacillus.

11. The preparation according to claim 10, wherein the spore-forming bacteria are Bacillus subtilis (DSM32324).

12. The preparation according to any one of claims 9-11, wherein the excipients comprise a disintegrant, a dispersant or an antifoaming agent.

13. The preparation according to claim 12, wherein the disintegrant is sodium carboxymethyl starch.

14. A product comprising the preparation according to any one of claims 9 to 13.

15. Use of the product according to claim 8 or claim 14 in agriculture.

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