KR102689516B1 - Method for producing rumen fluid microbial culture fluid and feed additive containing the same - Google Patents

Abstract

The present invention includes the steps of a) collecting rumen fluid from the rumen of a cow; b) preparing a buffer solution from which dissolved oxygen is removed; c) mixing the rumen fluid and the buffer at a volume ratio of 7:3 to 3:7 in a culture vessel; d) supplying mixed feed for livestock containing concentrated feed and forage in a weight ratio of 7:3 to 3:7 as a substrate for microbial growth to the culture vessel; and e) anaerobically culturing the culture in the culture vessel to obtain a culture solution. A method for producing a rumen fluid microbial culture solution comprising a step and a feed additive containing the rumen fluid microbial culture solution are provided.

Description

Method for producing rumen fluid microbial culture fluid and feed additive containing the same}

The present invention relates to a method for producing a rumen fluid microbial culture medium and a feed additive containing the same.

The word ruminant comes from the Latin word ruminare, meaning to chew several times, and includes herbivores such as cows, sheep, goats, antelopes, and deer. Unlike non-ruminants, the stomach of ruminants is divided into four compartments: rumen, reticulum, omasum, and abomasum. The contents of the stomach freely pass through each other through chewing of the cud, and it has the same fermentation and absorption functions as the first stomach, so the first and second stomachs (rumen-reticulum) are commonly called the rumen. At this time, the rumen provides the environment necessary for the growth and activity of microorganisms, and the rumen fluid microorganisms that inhabit are controlled by the unique ecological balance of the first rumen. The rumen environment is an anaerobic state with an oxygen-reduction potential of about 250-450mV and a very low oxygen concentration. The pH is about 5.5-7.0 and the temperature is about 39-41℃, which is where most enzymes achieve optimal vitality. There are conditions for maintaining it. In addition, the ingested feed and water and saliva secreted through masticatory activities continuously flow into the inside, providing an environment suitable for the growth of microorganisms.

Due to the unique space and environment of the rumen, the rumen contains a very diverse group of microorganisms (bacteria, 10 ^9-10 /ml; protozoa, 10 ^5-6 /ml; fungi, 10 ^3-5 /ml) in a symbiotic relationship with ruminants. This is inhabited. In the process of decomposing ingested feed, ruminal fluid microorganisms synthesize various amino acids through coexistence and competition between microorganisms and produce complex types of metabolites, signaling substances, and unknown growth factors involved in the growth of livestock. Therefore, ruminant fluid microorganisms play a very important role in the digestive physiology of ruminants because they decompose feed and produce various metabolites for use by ruminants.

Most bacteria and fungi living in the rumen have hydrolytic enzymes that break down fiber and convert it into sugar. Therefore, ruminants can absorb and utilize polysaccharides, including fiber, which are most widely distributed in plants and cannot be used by monogastric animals, through the intestinal wall by converting them into volatile fatty acids (VFA _s ) by microorganisms living in the rumen. In addition, among nitrogen compounds, non-protein nitrogen compounds such as urea, biuret, and inorganic ammonium salts, which cannot be used by monolithic animals, can be absorbed and used by microorganisms by synthesizing them into microbial proteins.

These various metabolites and nutrient sources produced by the activities of rumen fluid microorganisms can be said to be materials with very high potential for use as feed additives to improve the productivity of livestock.

However, in order to develop and produce feed additives using rumen fluid, realistically, there is no choice but to collect and utilize the waste rumen fluid generated in large quantities during the slaughter process at the slaughterhouse. However, there is a risk of BSE (Bovine spongiform encephalopathy) occurring in the rumen fluid generated during the slaughter process at the slaughterhouse, as there is a possibility that animal body tissue that has fallen off during the repetitive cutting and separation process may enter. Additionally, it is not easy to safely and quickly collect rumen fluid from spoilage or contamination.

Therefore, it is very difficult to collect rumen fluid in a hygienic and safe manner from the spread of livestock diseases and sources of contamination. Therefore, research into ways to safely utilize this rumen fluid is required.

Korean Patent Publication No. 10-2002-0075804

The present invention was devised to solve the above problems of the prior art,

A rumen fluid culture solution that enables safe industrial use of the useful components of rumen fluid by effectively cultivating rumen fluid under pH, temperature, and anaerobic conditions similar to the rumen environment of ruminants to multiply rumen fluid microorganisms in large quantities. The purpose is to provide a manufacturing method.

In addition, the purpose is to provide a feed additive that improves the health and promotes growth of livestock fed with this feed by including the rumen fluid culture medium.

This invention

a) collecting rumen fluid from the rumen of a cow;

b) preparing a buffer solution from which dissolved oxygen is removed;

c) mixing the rumen fluid and the buffer at a volume ratio of 7:3 to 3:7 in a culture vessel;

d) supplying mixed feed for livestock containing concentrated feed and forage in a weight ratio of 7:3 to 3:7 as a substrate for microbial growth to the culture vessel; and

e) obtaining a culture solution by anaerobic culturing the culture in the culture vessel; providing a method for producing a rumen fluid microbial culture solution including.

also,

Provided is a feed additive manufactured by adsorbing rumen fluid microbial culture fluid to an absorbent excipient.

The method for producing rumen fluid culture fluid is to effectively cultivate rumen fluid under pH, temperature, and anaerobic conditions similar to the environment in the rumen of ruminants to multiply rumen fluid microorganisms in large quantities, thereby safely utilizing the useful components of rumen fluid for industrial purposes. It provides an enabling effect.

In addition, the feed additive of the present invention includes the rumen fluid culture medium, thereby providing the effect of improving the health and promoting growth of livestock fed with this feed.

The method for producing a rumen fluid microbial culture medium of the present invention has the characteristics of comprising the following steps:

a) collecting rumen fluid from the rumen of a cow;

b) preparing a buffer solution from which dissolved oxygen is removed;

c) mixing the rumen fluid and the buffer at a volume ratio of 7:3 to 3:7 in a culture vessel;

d) supplying mixed feed for livestock containing concentrated feed and forage in a weight ratio of 7:3 to 3:7 as a substrate for microbial growth to the culture vessel; and

e) Obtaining a culture medium by culturing the culture in the culture vessel anaerobically.

The rumen fluid of ruminants contains various enzymes and various metabolites produced during the decomposition of feed by numerous microorganisms living in the rumen, as well as unknown growth factors that promote the growth of livestock, providing not only nutritional value but also functional value. Its practical value is very high. However, collecting and utilizing rumen fluid generated in the form of slaughter by-products had many hygienic problems.

In the prior art, feed additive products were produced by collecting rumen fluid discarded in large quantities at slaughterhouses. However, collecting rumen fluid, which is discarded in the form of slaughter by-products, is difficult to manage safely and hygienically from disease spread or contaminants, and because it is classified as waste, export conditions are strict.

The present invention is characterized by effectively solving this problem.

After collecting the rumen fluid in step a), it is filtered, stored in a thermos container at 36 to 42°C filled with CO ₂ without oxygen, and can be used in step c). In other words, in order to increase the efficiency and productivity of rumen mass culture, it is necessary to collect rumen fluid from the rumen so that the vitality of microorganisms is not reduced as much as possible.

In addition, it is important to quickly transport the collected rumen fluid to the culture site while maintaining a temperature of 36 to 42°C, preferably 39°C, under anaerobic conditions. This is because the vitality of collected rumen microorganisms has a significant impact on the growth rate and productivity of rumen fluid microorganisms during mass culture.

In the present invention, rumen fluid is collected from Korean cattle equipped with a canula in the rumen, filtered through a 4-ply gauze mesh, placed in a thermos (39°C) filled with O ₂ free CO _{2 ,} and immediately transported to a culture site for mass culture. It can be used as a seed for . Rumenal fluid can be collected from the rumen of cows equipped with a canula, but it can also be collected directly from the rumen of cows slaughtered at a slaughterhouse. At this time, care must be taken to ensure that the rumen fluid is not mixed with contaminants such as digestive fluid or blood from slaughtered cattle. The method of collecting a small amount of rumen fluid from the rumen of cattle slaughtered at a slaughterhouse and using it as a seed for mass culture is much safer and hygienically free from contaminants than the conventional method of collecting and drying large quantities of rumen fluid discharged to the slaughterhouse. There is a way to do this.

In step b), the buffer solution from which dissolved oxygen has been removed can be used.

During normal fermentation metabolism, the proliferation and activity of rumen fluid microorganisms are greatly affected by pH. Buffers play a very important role in maintaining a constant hydrogen ion concentration and maintaining homeostasis in living organisms. Buffering agents include magnesium oxide, soda ash, sodium bicarbonate, potassium bicarbonate, potassium carbonate, magnesium carbonate, magnesium oxide, and sodium bentonite. sodium bentonite), etc. can be used alone or in mixture form.

In particular, in the present invention, McDougall's buffer solution (Table 1), which is mainly used for research purposes for rapid growth of rumen fluid microorganisms, can be preferably used as a buffer solution.

In the present invention, the buffer solution is preferably used after bubbling with O ₂ free CO ₂ about 30 minutes before culturing rumen microorganisms to remove dissolved oxygen present in the buffer solution. However, if bubbling with O ₂ free CO ₂ for too long a period of time changes the pH to acidic, making it less effective as a buffer solution, so it is necessary to measure the pH and maintain it in the range of 6.8-7.0.

In step c), the rumen fluid and the buffer may be injected into the culture vessel at a volume ratio of 7:3 to 3:7, preferably 1:1.

Step c) can be performed by supplying O ₂ -free CO ₂ to the culture vessel while blocking exposure of the culture to oxygen.

As a substrate for microbial growth in step d), a mixed feed for livestock containing concentrated feed and forage in a weight ratio of 7:3 to 3:7, preferably 6:4 to 4:6, is preferably used. You can. At this time, the mixed feed can be used by pulverizing it to a size of 0.1 mm to 1 mm.

For the above substrates, the ratio of concentrate and roughage is important. If the ratio of concentrated feed with a high proportion of soluble carbohydrates or fast-decomposing starches is too high, starch-decomposing microorganisms rapidly increase, while microorganisms that mainly use fiber decrease, and the pH of the rumen fluid rapidly changes to acidic. It also causes changes in the balance of VFA (Volatile fatty acids), affecting the productivity and quality of rumen fluid produced artificially.

When pulverizing the mixed feed and using it as a substrate, care must be taken because fine feed residues may cause the air supply device inside the incubator to malfunction or feed particles remaining inside the incubator may become a source of contamination.

Additionally, purified diet can be used as a substrate for the growth of the microorganisms. Refined feed is manufactured using pure nutrients rather than the form of natural feed, and varies depending on the type and purpose of the microorganism to be cultivated. Carbohydrate (Cellulose, cellobiose, glucose, maltose, pectin, starch), Tryticase, Yeast extract, Casein, Hemin, VFA mixture(Acetic acid, propionic acid, n-valeric acid, Iso-valeric acid, Iso-butyric acid, DL-α-methylbutyric acid) L-cystein-HCl, Mineral mixture(KH ₂ PO ₄ , NaCl,(NH ₄ ) ₂ SO ₄ , CaCl ₂ , MgSO ₄ , MnSO ₄ ·H ₂ O, ZnSO ₄ ·7H ₂ O, COCl ₂ ·6H ₂ O, Vitamin mixture (Pyridoxine HCl, Riboflavine, Thiamine Pure nutritional sources such as HCl, Nicotinamide, Ca-D-pantothenate p -Amino benzoic acid, etc. can be used.

However, the raw materials of purified diet are highly refined nutritional sources, and most of them are expensive raw materials, which are not only difficult to store and handle, but are also unsuitable for commercial use. Therefore, it is mainly used for the purpose of pure isolation of rumen fluid microorganisms, research on metabolites, and fermentation mechanisms rather than for commercial mass production.

In order to achieve the industrial purpose of the present invention, it is desirable to process and use mixed feed for livestock feeding as a substrate for microbial culture in order to ensure suitability and ease of mass production and sufficient economic efficiency.

Step d) can be performed by supplying O ₂ -free CO ₂ to the culture vessel while blocking exposure of the culture to oxygen.

The culture in step e) may be performed in an incubator maintained at 30 to 45°C, preferably 39°C. For example, a water bath incubator may be used as the incubator.

The culture may be performed as an anaerobic culture for 20 to 30 hours, preferably 24 hours. At this time, fermentation gas generated internally during the growth of microorganisms is discharged to the outside, but a stopper designed to prevent external air from entering the inside must be installed. If the gas generated inside cannot be released to the outside, there is a risk that the culture vessel may burst or the rumen fluid inside may overflow and be lost. Specifically, to explain the structure of the sealing stopper, a hole is drilled through the silicone stopper, a capillary glass tube is installed, and then a rubber tube with sides cut up and down is connected to the capillary glass tube, so that the fermentation gas generated inside is released into the rubber tube. It can be configured to pass between the cut surfaces of the.

In the present invention, the rumen fluid microbial culture medium prepared in steps a) to e) can itself be used in the production of feed additives, etc.

Additionally, steps a) to e) may be used as a pre-culture process for industrial mass culture. In other words, the pre-cultivation may be a step performed to provide adaptation time for rumen microorganisms for mass culture and to increase the capacity of seed required for mass culture. Additionally, the pre-cultivation may be performed to increase the vitality of the spawn and secure the amount of spawn required for main culture.

The present invention involves placing the culture solution obtained in step e) in a large-capacity incubator supplied with a buffer solution and mixed feed for livestock containing concentrated feed and forage as a substrate, and performing anaerobic culture again in step f) (main culture step). may further include.

The main culture in step f) is performed by inoculating an industrial incubator using the rumen fluid microbial culture solution for which the pre-culture has been completed as a seed. In order to use an industrial microbial incubator for mass culturing of rumen fluid microorganisms, a device designed to maintain anaerobic conditions is required in addition to the general sterilization function, temperature control, and pH control of the microbial incubator.

The large-capacity incubator may be equipped with an air sparger that strongly inflows and disperses O ₂ -free CO ₂ from the gas storage tank and an outlet that exhausts the air inside the incubator but blocks the inflow of external air. During cultivation, O ₂ -free CO ₂ is strongly introduced and dispersed through the air sparger to remove oxygen, and then anaerobic culture can be performed.

In other words, in order to create an anaerobic condition, it is necessary to strongly inflow and disperse O ₂ -free CO ₂ from the gas tank through the air sparger at the bottom of the incubator to remove dissolved oxygen.

To describe the main culture method in detail, a pre-prepared buffer is injected into the incubator, and dissolved oxygen is removed by strongly inflowing and dispersing O ₂ -free CO ₂ from the gas tank through an air sparger installed at the bottom of the incubator. At this time, the pH of the buffer solution changes to acidic depending on the injection amount and time of O ₂ -free CO ₂ , so pH adjustment is necessary. The presence or absence of dissolved oxygen in the buffer solution following O ₂ -free CO ₂ injection can be confirmed by injecting Resazuine (0.1%) solution as an indicator. If the indicator reacts with oxygen in the buffer solution and turns red, it can be judged that dissolved oxygen remains. there is. When the removal of dissolved oxygen from the buffer solution is completed, as a step to supply the substrate necessary for the growth of microorganisms, the pulverized mixed feed for livestock described in the pre-culture above is injected into the incubator and O ₂ -free CO ₂ is additionally supplied for about 10 minutes. This removes dissolved oxygen.

Once the supply of buffer and substrate is completed, the pre-cultured rumen fluid microbial culture solution is supplied into the incubator. At this time, by injecting O ₂ -free CO ₂ gas into the incubator and inoculating the rumen fluid microbial culture medium while the internal pressure is slightly high, the inflow of surrounding contaminants and the inflow of oxygen can be reduced.

The main culture can be performed at a temperature of 30 to 45°C, preferably 39°C, pH 6.8-7.0, and stirring at a speed of 38 rpm for 20 to 30 hours, preferably 24 hours. At this time, for the growth of rumen fluid microorganisms, the same amount of substrate can be added for 10 to 14 hours, preferably once every 12 hours. At this time, as described in the preliminary culture above, mixed feed for livestock or purified diet can be used as a substrate for the growth of ruminal fluid microorganisms, but for mass production and economic efficiency for commercial purposes, mixed feed for livestock is used. It is desirable to use

At this time, as the mixed feed, a mixed feed for livestock containing concentrate and forage in a weight ratio of 7:3 to 3:7, preferably 6:4 to 4:6, can be used. When using the above mixed feed, the ratio of concentrated feed and roughage is important. If the ratio of concentrated feed with a high proportion of soluble carbohydrates or fast-decomposing starches is too high, starch-decomposing microorganisms rapidly increase, while microorganisms that mainly use fiber decrease, and the pH of the rumen fluid rapidly changes to acidic. It also causes changes in the balance of VFA (Volatile fatty acids), affecting the productivity and quality of rumen fluid produced artificially.

In addition, the present invention

It relates to a feed additive manufactured by adsorbing the rumen fluid microbial culture prepared by the method described above to an absorbent excipient.

The rumen fluid microbial culture solution from which the pre-culture or main culture has been completed can be used to prepare a feed additive by mixing it with an absorbent excipient and drying it.

The absorbent excipient may be one or more selected from the group consisting of raw materials for concentrated feed (corn, soybean meal, defatted steel, wheat hulls, ethanol bean, etc.), silica, zeolite, bentonite, and vermiculite powder. The adsorbent excipient is a raw material that absorbs and maintains the rumen microbial culture fluid and gives it shape. The grain raw material may be used in the form of a powder with a particle size of approximately 1 to 3 mm, and the inorganic adsorbent excipient such as silica may be used in the form of a powder with a particle size of approximately 200 mesh.

The drying may be performed by hot air or vacuum drying, and may be dried at a temperature of 20 to 200°C, but drying at 40 to 60°C is more preferable. If the drying temperature is lower than 40℃, the drying time takes too long, and if it is higher than 60℃, for example, the carbohydrates contained in the grain raw materials are denatured and the color changes, which has the disadvantage of lowering the sensory preference of the product.

The dried feed additive can be pulverized to produce a feed additive in powder form. The moisture content of the powdered feed additive is preferably up to 11% and at least 9%. If it contains more than 11% moisture, the shelf life will be shortened, and even if it is within the expiration date, there is a high risk of deterioration. In addition, if a formulation is produced with a moisture content of 9% or less, the drying time is too long, the unique odor and appearance characteristics of the dried rumen fluid microorganisms are lost, and only the characteristics of the excipient remain strong.

The present invention will be explained in more detail through the following examples. Since these examples are merely presented as examples to help the present invention be better understood, the scope of the present invention should not be limited by these examples.

Example 1: Preparation for artificial culture

(1) Preparation of buffer solution

McDougall's buffer solution ¹ was prepared as shown in Table 1 below and used as a buffer for culturing rumen fluid microorganisms.

Component Amounts(g) Sodium bicarbonate (NaHCO ₃ )
Disodium phosphate (Na ₂ HPO ₄ )
Potassium Chloride(KCl)
Sodium Chloride (NaCl)
Magnesium Chloride(MgCl ₂ )
Calcium Chloride(CaCl ₂ .2H ₂ O) 29.40
27.90
1.70
1.41
0.18
0.14

¹ Mix the first 6 chemicals and 5L distilled water in volumetric and stir until dissolved (pH=8.0).

(2) Preparation of substrate for culture

As a substrate for culturing ruminal fluid microorganisms, the mixed feed shown in Table 2 below was prepared by grinding it to a size of 1 mm or less using a feed grinder (1093 cyclotec sample mill, FOSS TECATOR, Sweden).

Ingredients % Corn, flaked 39.00 Lupine seeds, flaked 23.00 Italian ryegrass 19.00 Wheat bran 8.00 Corn gluten feed 8.20 Lime stone 1.60 Salt 0.40 Tricalcium phosphate 0.40 Vitamin mineral mix. 0.40 Total 100.0 Nutrients %, as fed basis dry matter 74.68 TDN 64.00 CP 13.80 Fiber 9.62 Fat 3.60 NDF 16.90 Ca 0.69 P 0.41

Example 2: Collection of rumen fluid microorganisms from the rumen of cattle

In this example, about 3 liters of rumen fluid was collected from two castrated Korean cattle equipped with a cannula in the rumen in order to artificially cultivate rumen fluid microorganisms. The collected gastric fluid was filtered through a 4-ply gauze mesh, placed in a thermos (39°C) filled with O ₂ free CO ₂ , and immediately transported to the experimental site.

Example 3: Artificial culture of rumen fluid microorganisms

(1) Pre-culture

Before culturing, O ₂ -free CO ₂ gas was injected into the buffer solution prepared in advance to remove dissolved oxygen, and then injected into a 20L glass culture vessel at a ratio of 1:1 (v/v) with the collected rumen fluid. As a substrate for microbial growth, mixed feed (Table 2) was finely ground and supplied, and anaerobic culture was performed for 2 hours in a water bath incubator at 39°C.

At this time, the inlet of the culture vessel used for pre-culture was equipped with a sealing stopper designed to prevent gases such as carbon dioxide or methane generated during the growth of microorganisms from being discharged to the outside, but to prevent external air from entering the inside.

(2) Main culture

The 500L (working volume 250L) microorganism incubator used in the present invention is equipped with high-temperature and high-pressure sterilization (105℃~130℃), pH control, anti-foam, and automatic temperature control functions, and is externally operated through an air sparger at the bottom. It is designed to maintain anaerobic conditions by flowing and dispersing O ₂ -free CO ₂ from the gas tank into the incubator.

In the mass culture of the present invention, 225L of buffer solution of the same composition used in the pre-culture is injected into the incubator, and O ₂ -free CO ₂ gas is forcibly introduced and dispersed at a rate of 0.5 vvm for about 30 minutes through an air sparger at the bottom of the reactor. to remove dissolved oxygen. To determine the presence of dissolved oxygen, add Resazurine (0.1%) solution (225 ml) and confirm that the red solution has changed to colorless. Then, add 2 kg of pulverized mixed feed (Table 2) as a substrate and O ₂ - for an additional 10 minutes. Preparation for anaerobic culture was completed by introducing and dispersing free CO ₂ gas. Cultivation of rumen fluid microorganisms is performed by inoculating the rumen fluid microorganisms prepared in the pre-cultivation step at a weight ratio of 5% through the inoculum of the incubator, at a temperature of 39°C, pH 6.8-7.0, and stirring at a speed of 38 rpm for 24 hours. During this period, mass culture was performed, and the same amount of substrate for the growth of rumen fluid microorganisms was added once every 12 hours.

Example 4: Drying of rumen fluid microbial culture and production of formulation as feed additive

In this example, a stirrer equipped with a hot air supply device was used. First, 600 kg of wheat hulls, an example of a grain raw material, as an adsorbent excipient, and 300 L of the culture solution with complete cultivation were added to the stirrer, mixed uniformly, and then dried by hot air drying. At this time, the drying conditions were a temperature of 55°C, a stirring speed of 30 rpm, and a drying time of 32 hours. After drying was completed, the powdered feed additive according to the present invention was measured to have a moisture content of 8.3% by weight.

Claims (9)

a) collecting rumen fluid from the rumen of a cow;
b) preparing a buffer solution from which dissolved oxygen is removed;
c) mixing the rumen fluid and the buffer at a volume ratio of 7:3 to 3:7 in a culture vessel;
d) supplying mixed feed for livestock containing concentrated feed and forage in a weight ratio of 7:3 to 3:7 as a substrate for microbial growth to the culture vessel; and
e) obtaining a culture medium by culturing the culture in the culture vessel anaerobically,
Adding the culture solution obtained in step e) to a large-capacity incubator supplied with a buffer solution and mixed feed for livestock containing concentrated feed and forage as a substrate, and performing a large-scale anaerobic culture again;
In step f), anaerobic culture is carried out at pH 6.8-7.0,
Preparation of a feed additive further comprising the step of adsorbing the rumen fluid microbial culture prepared in step f) to one or more adsorptive excipients selected from the group consisting of raw materials for concentrated feed, powders of silica, zeolite, bentonite, and vermiculite. method. According to paragraph 1,
A method for producing a feed additive, comprising collecting the rumen fluid in step a), filtering it, storing it in a thermos container at 36 to 42°C filled with CO ₂ without oxygen, and using it in step c). . According to paragraph 1,
A method for producing a feed additive, characterized in that in step b), the buffer solution is one in which dissolved oxygen has been removed. According to paragraph 1,
A method for producing a feed additive, characterized in that in step d), the mixed feed for livestock is pulverized to a particle size of 0.1 mm to 1 mm. According to paragraph 1,
A method for producing a feed additive, characterized in that each step of steps c) and d) is performed by supplying O ₂ -free CO ₂ to the culture vessel and blocking exposure of the culture to oxygen. delete According to paragraph 1,
The large-capacity incubator is equipped with an air sparger that strongly inflows and disperses O ₂ -free CO ₂ from the gas storage tank and an outlet that exhausts the air inside the incubator but blocks the inflow of external air,
A method for producing a feed additive, characterized in that during cultivation, O ₂ -free CO ₂ is strongly introduced and dispersed through the air sparger to remove oxygen, and then anaerobic culture is performed. delete delete