Method for separating and removing microbial cells from fermentation broth

Abstract

The invention provides a method for separating and removing bacillus subtilis cells from bacillus subtilis fermentation liquor and a composition for removing the bacillus subtilis cells from the bacillus subtilis fermentation liquor.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a method of isolating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth, and a composition for removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth. BACKGROUND

[0002] Generally, fermentation processes using microorganisms are mainly used for mass production of target products such as enzymes, amino acids, etc. However, a problem with the production of target products through a microbial fermentation process is that a large amount of purification cost is required because various by-products are present as impurities in the fermentation broth, including microbial cells and organic acids accompanying the target products.

[0003] Meanwhile, it is known that Bacillus subtilis cells are difficult to isolate and remove due to their small size and high viscosity. Therefore, in order to remove the microbial cells, methods such as dilution or heating of the fermentation broth, use of a large amount of filter aid, etc. are used. However, there is a problem in that these methods can inactivate the target substance, protein (enzyme), or can leave it in the process, thereby reducing the efficiency of the process of isolating and removing the microbial cells and the subsequent process. For example, the retention of unremoved microbial cells can reduce the flux and membrane life in the MF / UF membrane process, thereby generating shear stress during the process. Ultimately, there is a problem in that the recovery rate of the target protein (enzyme) is reduced.

[0004] In this regard, KR 10-2013-0022059A discloses, as a method of isolating microbial cells from a fermentation broth, centrifugation, filter press, pressure filter, diatomaceous earth filter, rotary vacuum filter, membrane separator, and flocculation and flotation methods, etc. However, this prior art does not mention a method of isolating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth using calcium salt and phosphate salt.

[0005] Therefore, there is still a need to research a method of isolating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth using a simpler and more economical method. SUMMARY

[0006] TECHNICAL PROBLEM

[0007] An object of the present disclosure is to provide a method of isolating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth, the method comprising the steps of: preparing a Bacillus subtilis fermentation broth; adding calcium salt and phosphate salt to the Bacillus subtilis fermentation broth; and removing the Bacillus subtilis cells from the fermentation broth to which the calcium salt and the phosphate salt have been added.

[0008] Another object of the present disclosure is to provide a composition for removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth, the composition comprising calcium salt and phosphate salt.

[0009] Technical Solution

[0010] Provided is a method of separating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth, the method including the steps of: preparing a Bacillus subtilis fermentation broth; adding a calcium salt and a phosphate salt to the Bacillus subtilis fermentation broth; and removing the Bacillus subtilis cells from the fermentation broth to which the calcium salt and the phosphate salt have been added.

[0011] Also provided is a composition for removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth, the composition including a calcium salt and a phosphate salt.

[0012] Advantageous Effects

[0013] The method of the present disclosure for separating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth has the excellent effect of increasing process yield and economic feasibility, as calcium chloride and sodium phosphate dihydride are used to effectively remove microbial cells and impurities produced in the fermentation broth as a fermentation byproduct in a fermentation process using Bacillus subtilis without including a step of diluting the Bacillus subtilis fermentation broth, and thus, the method can be effectively applied to industrial production of a target product. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a flowchart illustrating a method of separating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth according to one embodiment of the present disclosure. DETAILED DESCRIPTION

[0015] The present disclosure will be described in detail hereafter. Meanwhile, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below.

[0016] In addition, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. In addition, such equivalents are intended to be within the scope of the present disclosure.

[0017] As used in the specification and the appended claims of the present disclosure, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Singular terms will include the plural unless the context clearly dictates otherwise. As used in the specification and the appended claims of the present disclosure, the use of “or” can be used to mean “and / or” unless otherwise indicated.

[0018] As used herein, the term "about" can precede a particular numerical value. The term "about," as used herein, does not just include the precise listed value but also a range around that value. Whether a number is around or approximate to a particular number can be determined in view of the context in which the number is presented. In one example, the term "about" can refer to a range of -10% to +10% of an index value. In another example, the term "about" can refer to a range of -5% to +5% of a given numerical value, but is not limited thereto.

[0019] As used herein, descriptions such as the terms "first, second, third," "i), ii), iii)," or "(a), (b), (c), (d)" are used to distinguish between like elements and are not meant to be sequential or ordered. For example, when the terms are used to refer to steps of a method, use, or assay, there can be no time interval between the steps, or they can be performed simultaneously, or they can be performed apart by seconds, minutes, hours, days, or months.

[0020] As used herein, the term "comprising" means the presence of the recited features, steps, or components, and does not exclude the presence or addition of one or more features, steps, or components. Components or features recited after the term "comprising" herein can be essential or mandatory. However, in some embodiments, the term can also include any other or non-essential components or features.

[0021] One aspect of the present disclosure provides a method of separating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth, the method comprising the steps of: preparing a Bacillus subtilis fermentation broth; adding a calcium salt and a phosphate salt to the Bacillus subtilis fermentation broth; and removing the Bacillus subtilis cells from the fermentation broth to which the calcium salt and the phosphate salt have been added.

[0022] In a specific embodiment, the calcium salt can be calcium chloride (CaCl2).

[0023] In a specific embodiment, the phosphate salt can be selected from the group consisting of sodium phosphate monobasic (NaH2PO4), phosphoric acid (H3PO4), sodium phosphate dibasic (Na2HPO4), potassium phosphate monobasic (KH2PO4), potassium phosphate dibasic (K2HPO4), ammonium phosphate monobasic (NH4H2PO4), ammonium phosphate dibasic ((NH4)2HPO4), and combinations thereof, specifically, sodium phosphate monobasic (NaH2PO4), but is not limited thereto.

[0024] In a specific embodiment, the method for separating and removing Bacillus subtilis cells from a Bacillus subtilis fermentation broth disclosed herein may include the following steps: preparing a Bacillus subtilis fermentation broth; adding calcium chloride (CaCl2) and sodium dihydrogen phosphate (NaH2PO4) to the Bacillus subtilis fermentation broth; and removing Bacillus subtilis cells from the fermentation broth containing the added calcium chloride and sodium dihydrogen phosphate, comprising 0.5 to 0.75 parts by weight of calcium chloride, based on a total composition of 100 parts by weight, comprising 0.9 to 1.5 parts by weight of sodium dihydrogen phosphate, based on a total composition of 100 parts by weight, and comprising calcium chloride and sodium dihydrogen phosphate in a mixed ratio of 1:1.6 to 1:2.

[0025] Typically, microbial fermentation methods are primarily used for the large-scale production of target products such as enzymes and amino acids. However, a problem with producing target products through microbial fermentation is the high purification costs required, as various byproducts, including microbial cells and organic acids accompanying the target product, exist in the fermentation broth as impurities.

[0026] In particular, during fermentation using Bacillus subtilis, Bacillus subtilis cells are known to be difficult to separate and remove due to their small size and high viscosity. Therefore, methods such as diluting or heating the fermentation broth and using large amounts of filter aids are employed to remove microbial cells. However, the problem is that these methods may inactivate the target substance, proteins (enzymes), or may leave them in the process, thereby reducing the efficiency of the separation and removal of microbial cells and subsequent processes. For example, retaining unremoved microbial cells can reduce flux and membrane lifetime in MF / UF membrane processes, thereby generating shear stress during the process. Ultimately, the problem is a reduced recovery rate of the target protein (enzyme).

[0027] The method disclosed herein for isolating and removing Bacillus subtilis cells from Bacillus subtilis fermentation broth has the excellent effect of increasing process yield and economic feasibility, because calcium chloride and sodium dihydrogen phosphate are used to effectively remove microbial cells and impurities generated in the fermentation broth as fermentation byproducts during the fermentation process using Bacillus subtilis through a simple process.

[0028] As used in this article, the term "fermentation" refers to the process by which bacteria use their own enzymes to break down organic matter, excluding putrefaction. Fermentation and putrefaction occur through similar processes. However, when the result of decomposition is the production of useful substances, it is called fermentation; when it produces unpleasant odors or harmful substances, it is called putrefaction.

[0029] In this disclosure, there are no particular limitations on the method for obtaining Bacillus subtilis fermentation broth, and the fermentation product can be obtained according to methods commonly used in the art or similar fields.

[0030] As used herein, the term "fermentation broth" includes not only the fermentation material itself, but also all kinds of substances, including fermentation products produced by strains, such as strain culture media in which strains and cultures coexist, fermentation products obtained by filtering strains from a culture medium, fermentation products obtained by sterilizing strains from a culture medium and filtering them, extracts obtained by extracting fermentation products or culture media containing fermentation products, dilutes obtained by diluting fermentation products or their extracts, concentrates obtained by drying fermentation products or their extracts, lysates obtained by collecting and breaking microbial cells of strains, etc.

[0031] In specific embodiments, the composition may contain 0.1 to 2 parts by weight, 0.2 to 2.0 parts by weight, 0.3 to 2 parts by weight, 0.4 to 2 parts by weight, 0.5 to 2.0 parts by weight, 0.5 to 1.5 parts by weight, or 0.5 to 0.75 parts by weight, based on a total composition of 100 parts by weight, but is not limited thereto.

[0032] It may contain 0.5 to 4 parts by weight, 0.6 to 4 parts by weight, 0.7 to 4 parts by weight, 0.8 to 4 parts by weight, 0.8 to 3 parts by weight, or 0.9 to 1.5 parts by weight of sodium dihydrogen phosphate, based on a total composition of 100 parts by weight, but is not limited thereto.

[0033] In specific implementation schemes, calcium chloride and sodium dihydrogen phosphate may be mixed in ratios of 1:1 to 1:3, 1:1.1 to 1:2.9, 1:1.2 to 1:2.8, 1:1.3 to 1:2.7, 1:1.4 to 1:2.6, 1:1.5 to 1:2.5, or 1:1.6 to 1:2, but are not limited thereto.

[0034] By using calcium chloride and sodium dihydrogen phosphate in the range and / or mixing ratios described above, microbial cells and impurities generated as fermentation byproducts in the fermentation broth during the fermentation of Bacillus subtilis can be effectively removed through a simple process without the need for diluting the Bacillus subtilis fermentation broth. This results in significantly increased process yield and economic feasibility. Specifically, because the step of diluting the Bacillus subtilis fermentation broth is omitted, the amount of calcium chloride and sodium dihydrogen phosphate added is minimized while achieving the optimal amount that exhibits the greatest effect in separating and removing microbial cells. This is more efficient in terms of process cost and time compared to existing methods for separating and removing microbial cells that include a dilution step.

[0035] Regarding the method for isolating and removing Bacillus subtilis cells from Bacillus subtilis fermentation broth disclosed herein, the fermentation broth with removed Bacillus subtilis cells can have a Bacillus subtilis cell removal rate of 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0036] The method disclosed herein for isolating and removing Bacillus subtilis cells from Bacillus subtilis fermentation broth may exclude the step of diluting the Bacillus subtilis fermentation broth.

[0037] Typically, when using additives for the microbial cells contained in the flocculation fermentation broth, the fermentation broth is diluted 2 to 10 times before use, which results in an increase in the amount of solution to be treated, an increase in the amount of flocculant used, and an increase in process time.

[0038] The method for separating and removing Bacillus subtilis cells from Bacillus subtilis fermentation broth disclosed herein does not include the step of diluting the Bacillus subtilis fermentation broth, thereby shortening the process time and reducing losses in subsequent processes. Specifically, because the method does not include a dilution step, the amount of solution to be processed is reduced by 2 to 10 times, thereby reducing the amount of flocculant used and the amount of auxiliary materials such as filter aids, filters, and electricity used for the separation and removal of microbial cells and impurities after flocculation, and shortening the filtration and concentration process time. Therefore, it has the excellent effect of significantly reducing manufacturing costs.

[0039] In a specific implementation plan, Bacillus subtilis fermentation broth can be prepared by culturing Bacillus subtilis in a culture medium, but is not limited to this.

[0040] As used herein, the term "culture" refers to the growth of the strains of this disclosure under appropriately regulated environmental conditions. The culture procedures of this disclosure can be performed according to suitable culture media or culture conditions known in the art. Such culture procedures can be readily adapted for use by those skilled in the art based on the selected strain. Specifically, the culture can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0041] As used herein, the term "culture medium" refers to a mixture containing, as a major component, the nutrients required for culturing the microorganisms of this disclosure, wherein the culture medium provides nutrients including water, growth factors, etc., necessary for survival and growth. Specifically, for the culture medium and other culture conditions used to culture the strains of this disclosure, any culture medium commonly used for culturing microorganisms can be used without particular limitation. However, the microorganisms of this disclosure can be cultured under aerobic conditions in a general culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0042] In this disclosure, carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and various other carbon sources may be used without limitation in appropriate amounts. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0043] As for nitrogen sources, inorganic nitrogen sources can be used, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids, such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources, such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean meal or its decomposition products, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0044] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. As for inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used, and amino acids, vitamins, and / or suitable precursors may also be included. These components or precursors may be added to the culture medium in batches or continuously. However, this disclosure is not limited thereto.

[0045] Furthermore, during the cultivation of the microorganisms disclosed herein, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid in an appropriate manner. Additionally, antifoaming agents, such as polyethylene glycol fatty acids, can be used to inhibit foam formation during cultivation. Furthermore, oxygen or oxygen-containing gases can be injected into the culture medium to maintain an aerobic state, or no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gases may be injected to maintain an anaerobic or non-anaerobic state, but these are not limited to these methods.

[0046] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 27°C and 37°C, specifically between 30°C and 33°C, and the cultivation can be carried out for approximately 20 hours to 120 hours, but is not limited thereto.

[0047] As used herein, the term "culture" refers to a culture medium, concentrated culture medium, dried culture medium, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing specific microorganisms in a culture medium, and a culture medium refers to those containing specific microorganisms, while a culture filtrate refers to those substantially not containing specific microorganisms (meaning that microorganisms to be separated by filtration, etc., are substantially excluded, but not necessarily that microorganisms are completely excluded from the filtrate). The dosage form of the culture is not limited, but may be, for example, a liquid, emulsion, or solid. Specifically, for the purposes of this disclosure, the culture may include the target product.

[0048] Regarding the methods of this disclosure, the microorganisms can be cultured using any culture conditions and methods known in the art. Such culture procedures can be readily adapted for use by those skilled in the art based on the selected strain.

[0049] In a specific implementation, prior to the step of removing Bacillus subtilis cells from the fermentation broth that has already been supplemented with calcium chloride and sodium dihydrogen phosphate, the method may further include the step of adjusting the pH of the Bacillus subtilis fermentation broth to 6.5 to 7.5, 6.6 to 7.4, 6.7 to 7.3, 6.8 to 7.2, 6.9 to 7.1, or 6.95 to 7.05.

[0050] Another aspect of this disclosure provides a composition for removing Bacillus subtilis cells from Bacillus subtilis fermentation broth, the composition comprising calcium chloride and sodium dihydrogen phosphate.

[0051] The calcium chloride, sodium dihydrogen phosphate, and Bacillus subtilis fermentation broth are as described above.

[0052] In a specific embodiment, the composition is a composition for removing Bacillus subtilis cells from Bacillus subtilis fermentation broth. The composition comprises calcium chloride and sodium dihydrogen phosphate, comprising 0.5 to 0.75 parts by weight of calcium chloride based on a total of 100 parts by weight, 0.9 to 1.5 parts by weight of sodium dihydrogen phosphate based on a total of 100 parts by weight, and comprising calcium chloride and sodium dihydrogen phosphate in a mixed ratio of 1:1.6 to 1:2.

[0053] In specific embodiments, the composition may contain 0.1 to 2 parts by weight, 0.2 to 2.0 parts by weight, 0.3 to 2 parts by weight, 0.4 to 2 parts by weight, 0.5 to 2.0 parts by weight, 0.5 to 1.5 parts by weight, or 0.5 to 0.75 parts by weight, based on a total composition of 100 parts by weight, but is not limited thereto.

[0054] It may contain 0.5 to 4 parts by weight, 0.6 to 4 parts by weight, 0.7 to 4 parts by weight, 0.8 to 4 parts by weight, 0.8 to 3 parts by weight, or 0.9 to 1.5 parts by weight of sodium dihydrogen phosphate, based on a total composition of 100 parts by weight, but is not limited thereto.

[0055] In specific implementation schemes, calcium chloride and sodium dihydrogen phosphate may be mixed in ratios of 1:1 to 1:3, 1:1.1 to 1:2.9, 1:1.2 to 1:2.8, 1:1.3 to 1:2.7, 1:1.4 to 1:2.6, 1:1.5 to 1:2.5, or 1:1.6 to 1:2, but are not limited thereto.

[0056] By using calcium chloride and sodium dihydrogen phosphate in the range and / or mixing ratios described above, microbial cells and impurities generated as fermentation byproducts in the fermentation broth during the fermentation of Bacillus subtilis can be effectively removed through a simple process without the need for diluting the Bacillus subtilis fermentation broth. This results in significantly increased process yield and economic feasibility. Specifically, because the step of diluting the Bacillus subtilis fermentation broth is omitted, the amount of calcium chloride and sodium dihydrogen phosphate added is minimized while achieving the optimal amount that exhibits the greatest effect in separating and removing microbial cells. This is more efficient in terms of process cost and time compared to existing methods for separating and removing microbial cells that include a dilution step.

[0057] In specific embodiments, the pH of the composition disclosed herein for removing Bacillus subtilis cells from Bacillus subtilis fermentation broth may be 6.5 to 7.5, 6.6 to 7.4, 6.7 to 7.3, 6.8 to 7.2, 6.9 to 7.1, or 6.95 to 7.05.

[0058] Embodiments of the present invention

[0059] The present disclosure will now be described in more detail by way of exemplary embodiments. However, the following exemplary embodiments are merely preferred embodiments for illustrating the present disclosure and are not intended to limit the scope of the disclosure thereto. Furthermore, those skilled in the art to which this disclosure pertains or similar art will fully understand and readily implement technical matters not described in this specification.

[0060] Example: Evaluation of the microbial cell removal rates of NaH2PO4 and CaCl2

[0061] The aim was to evaluate the microbial cell removal rates of NaH2PO4 and CaCl2.

[0062] <Experimental Methods>

[0063] CaCl2 (Jiangsu Kolod Food Ingredients Co., Ltd.) and NaH2PO4 (Jiangsu Runpu Food Technology Co., Ltd.) were used. The amount of CaCl2 added was 0.5% to 0.75% of the fermentation broth weight, and the amount of NaH2PO4 added was 0.9% to 1.5% of the fermentation broth weight. Furthermore, the CaCl2:NaH2PO4 ratio ranged from 1:1.6 to 1:2.0. A centrifuge (Shanghai Anting Science Instrument Factory, TDL-60B) used to confirm the flocculation effect and analyze physical properties was run at 3,000 to 4,000 rpm for 5 to 15 minutes.

[0064] Fermentation of Bacillus subtilis strains (KCTC 18039P, KR 100411238 B1) was carried out using fed-batch culture. Dissolved oxygen (DO) was used as an indicator; the fed-batch culture medium was increased as DO increased. The main fermentation medium consisted of lactose, corn steep liquor, calcium chloride, sodium dihydrogen phosphate, ammonium sulfate, and magnesium sulfate. The fed-batch culture medium consisted of lactose, manganese sulfate, ferric sulfate, and zinc sulfate. Meanwhile, regarding fermentation conditions, the culture temperature was maintained at 32°C, and the stirring speed was adjusted to avoid DO limitation.

[0065] Specifically, without a dilution step, the flocculant was added to the stock solution of Bacillus subtilis fermentation broth at a ratio of 1:1.6, 1:1.8, or 1:2.0. After gentle stirring for approximately 1 hour and settling, microbial cells were removed using centrifugation or filtration. The microbial cell removal rate was calculated by analyzing the physical properties of the solution before and after microbial cell removal. The weight of microbial cells (DCW, stem cell weight) was calculated by measuring total solids (TS) and total dissolved solids (TDS). The methods and formulas for measuring the physical properties of the liquid are as follows:

[0066] TS: Accurately weigh 2 g to 3 g of sample into a weighing bottle with an accuracy of 0.001 g, place it in an oven and dry at 105°C for 3 hours. Then, place the sample in a desiccator, cool to room temperature (30 minutes), and dry. The calculation formula is as follows:

[0067] A: Weight of weighing bottle (g), B: Weight of sample and weighing bottle (g), C: Weight of weighing bottle and dry weight (g)

[0068] TS = 1 - (BC) / (BA) * 100%

[0069] TDS: Place the sample in a centrifuge and centrifuge at 4000 rpm for 5 minutes (if centrifugation is not possible, filter with 0.45 μm). Then, accurately weigh 2 g to 3 g of the centrifuged supernatant into a weighing bottle with an accuracy of 0.001 g. Place the bottle in an oven and dry at 105°C for 3 hours. Then, place the bottle in a desiccator to cool to room temperature (30 minutes) and dry. The calculation formula is as follows.

[0070] A: Weight of weighing bottle (g), B: Weight of sample and weighing bottle (g), C: Weight of weighing bottle and dry weight (g)

[0071] TDS = 1 - (BC) / (BA) * 100%

[0072] PCV: Place the sample in a centrifuge tube and centrifuge at 3,000 rpm for 15 minutes. The calculation formula is as follows:

[0073] A: Volume of the sample before centrifugation; B: Volume of the supernatant after centrifugation.

[0074] PCV = (AB) / A*100%

[0075] DCW: The weight of microbial cells is calculated using measured TS and TDS. The calculation formula is as follows:

[0076] DCW=TS-(TDS*((100-TS) / (100-TDS)))

[0077] <Comparative Example>

[0078] For the evaluation of the control group, no NaH2PO4 and CaCl2 were added to the Bacillus subtilis fermentation broth, and the pH was not adjusted. The removal rate of microbial cells was assessed by centrifugation or filtration. The results are shown in Table 1 below.

[0079] [Table 1]

[0080]

[0081] As shown in Table 1 above, it was confirmed that the microbial cell removal rate was 52.7% and the turbidity was 1000 NTU or higher when NaH2PO4 and CaCl2 were not added.

[0082] <Example 1>

[0083] The microbial cell removal rates of NaH2PO4 and CaCl2 were assessed by adding 0.5% CaCl2 and 0.8% to 1.0% NaH2PO4 based on the weight of the Bacillus subtilis fermentation broth, and adjusting the pH of the fermentation broth from 6.7 to 7.0 or higher using a 10% NaOH solution. The results are shown in Table 2 below.

[0084] [Table 2]

[0085]

[0086] As shown in Table 2 above, the microbial cell removal rate was confirmed to be 90.3% to 99.6% when NaH2PO4 and CaCl2 were added. It was also confirmed that the microbial cell removal efficiency was improved by 37.7% to 46.9% compared to the control group without the addition of NaH2PO4 and CaCl2.

[0087] Example 2

[0088] The microbial cell removal rates of NaH2PO4 and CaCl2 were assessed by adding 0.75% CaCl2 and 1.2% to 1.5% NaH2PO4 based on the weight of the Bacillus subtilis fermentation broth, and adjusting the pH to 7.0. The results are shown in Table 3 below.

[0089] [Table 3]

[0090]

[0091] As shown in Table 3 above, the microbial cell removal rate was confirmed to be 99.0% to 99.3% when NaH2PO4 and CaCl2 were added. It was also confirmed that the microbial cell removal efficiency was improved by 46.3% to 46.6% compared to the control group without the addition of NaH2PO4 and CaCl2.

[0092] <Example 3>

[0093] The microbial cell removal rates of NaH2PO4 and CaCl2 were assessed by adding 1.0% CaCl2 and 1.6% to 2.0% NaH2PO4 based on the weight of the Bacillus subtilis fermentation broth, and adjusting the pH to 7.0. The results are shown in Table 4 below.

[0094] [Table 4]

[0095]

[0096] As shown in Table 4 above, the microbial cell removal rate was confirmed to be 92.2% to 97.9% when NaH2PO4 and CaCl2 were added. It was also confirmed that the microbial cell removal efficiency was improved by 39.5% to 45.2% compared to the control group without the addition of NaH2PO4 and CaCl2.

[0097] <Example 4>

[0098] The microbial cell removal rates of NaH2PO4 and CaCl2 were assessed by adding 1.5% CaCl2 and 2.4% to 3.0% NaH2PO4 based on the weight of the Bacillus subtilis fermentation broth, and adjusting the pH to 7.0. The results are shown in Table 5 below.

[0099] [Table 5]

[0100]

[0101] As shown in Table 5 above, the microbial cell removal rate was confirmed to be 81.5% to 81.9% when NaH2PO4 and CaCl2 were added. It was also confirmed that the microbial cell removal efficiency was improved by 28.8% to 29.2% compared to the control group without the addition of NaH2PO4 and CaCl2.

[0102] <Example 5>

[0103] The microbial cell removal rates of NaH2PO4 and CaCl2 were assessed by adding 2.0% CaCl2 and 3.2% to 4.0% NaH2PO4 based on the weight of the Bacillus subtilis fermentation broth, and adjusting the pH to 7.0. The results are shown in Table 6 below.

[0104] [Table 6]

[0105]

[0106] As shown in Table 6 above, the microbial cell removal rate was confirmed to be 69.0% to 69.6% when NaH2PO4 and CaCl2 were added. It was also confirmed that the microbial cell removal efficiency was improved by 16.3% to 16.9% compared to the control group without the addition of NaH2PO4 and CaCl2.

[0107] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit or essential features. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims rather than by their preceding description; therefore, all changes and modifications, or equivalent substitutions falling within the boundaries and scope of the claims, are covered by the claims.

Claims

1. A method for isolating and removing Bacillus subtilis cells from Bacillus subtilis fermentation broth, the method comprising the following steps: Preparation of Bacillus subtilis fermentation broth; Calcium chloride (CaCl2) and sodium dihydrogen phosphate (NaH2PO4) were added to the Bacillus subtilis fermentation broth; and Remove Bacillus subtilis cells from fermentation broth that has been treated with calcium chloride and sodium dihydrogen phosphate. It contains 0.5 to 0.75 parts by weight of calcium chloride, based on a total composition of 100 parts by weight. Containing 0.9 to 1.5 parts by weight of sodium dihydrogen phosphate, based on a total composition of 100 parts by weight, and Contains calcium chloride and sodium dihydrogen phosphate in a mixed ratio of 1:1.6 to 1:

2.

2. The method according to claim 1, excluding the step of diluting the Bacillus subtilis fermentation broth.

3. The method according to claim 1, wherein the Bacillus subtilis fermentation broth is prepared by culturing Bacillus subtilis in a culture medium.

4. The method according to claim 1, wherein the fermentation broth from which Bacillus subtilis cells have been removed has a Bacillus subtilis cell removal rate of 99% or more.

5. The method of claim 1, further comprising adjusting the pH of the Bacillus subtilis fermentation broth to 6.5 to 7.5 prior to the step of removing Bacillus subtilis cells from the fermentation broth to which calcium chloride and sodium dihydrogen phosphate have been added.

6. A composition for removing Bacillus subtilis cells from Bacillus subtilis fermentation broth, said composition comprising calcium chloride and sodium dihydrogen phosphate. It contains 0.5 to 0.75 parts by weight of calcium chloride, based on a total composition of 100 parts by weight. Containing 0.9 to 1.5 parts by weight of sodium dihydrogen phosphate, based on a total composition of 100 parts by weight, and Contains calcium chloride and sodium dihydrogen phosphate in a mixed ratio of 1:1.6 to 1:

2.

7. The composition according to claim 6, wherein the pH of the composition is 6.5 to 7.5.

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