TW202100743A - Method for preparing fermented composition with improved odor using yeast - Google Patents

Abstract

The present disclosure relates to a method for preparing a fermented composition, and more specifically, to a method for preparing a fermented composition with improved odor, which comprises preparing grain flour; performing primary fermentation of the grain flour using yeast; performing secondary fermentation of the primary fermented product using a strain of the genus Bacillus; and obtaining the secondary fermented product. The fermented composition of the present disclosure has a high content of a peptide with a low molecular weight, and thus enables the increase of digestibility and absorption rate of proteins during ingestion while also improving the peculiar odor of a fermented product to enhance its palatability.

Description

Method of using yeast to prepare fermented composition with improved smell

The present invention relates to a method for preparing a fermentation composition, and more specifically, to a method for preparing a fermentation composition having an improved odor, which includes preparing grain flour and performing the fermentation using yeast The primary fermentation of cereal flour; the secondary fermentation of the product of primary fermentation using strains of Bacillus ( Bacillus ), and the product of post-fermentation; related to improving the smell of the fermentation product of Bacillus, Yeast that produces α-galactosidase, protease and phytase; related to the composition used to ferment grains and containing the yeast; related to the fermentation composition prepared by the above method ; And related to feed composition containing yeast or fermentation composition.

Based on high feed efficiency due to high energy content and good digestibility due to low crude fiber content, grains have been widely used as feed for livestock. However, grain feed has a low content of protein and amino acids, so in order to balance nutrition, supplementary supplements are necessary. For protein sources, animal protein sources (such as fish meal, powdered skim milk, meat meal, blood, etc.) and plant protein sources (such as soybeans, seeds, flax, etc.) are used. Corn gluten (corn gluten), which is a plant protein source, is a by-product of corn starch preparations. It has a high protein content (about 3% of general plant protein sources). The fish meal of times) is similar and low in price. Therefore, it has been widely used as a protein source for feed. In addition, soybean meal, which is a by-product remaining after soybean oil is manufactured from soybeans, is used as the main protein source in feed due to its high protein content.

However, indigestible oligosaccharides and insoluble proteins, such as indigestible oligosaccharides and insoluble proteins contained in soybean meal or zein produced during the manufacturing process, will reduce the digestibility, so it is problematic when used as feed. Therefore, there is a need to develop a novel processing method that can improve the digestibility of the protein portion so that soybean meal or corn gluten can be used as high-quality protein feed.

In addition, in this respect, various complex microbial agents that can be used in feed compositions have recently been developed, but these complex microbial agents have problems with physiological effects and interactions between microorganisms that have not been considered.

In this situation, the inventor of the present invention has made efforts to address the above-mentioned problems. As a result, they have found that it is possible to increase the water-soluble sugar content of the raw materials through the enzyme treatment of cereal flour, and to concentrate the protein contained in it through yeast fermentation and Bacillus subtilis fermentation. Therefore, it is possible to increase the protein content in the cereal flour and The number of viable bacteria improves the functionality of the composition, thereby completing the present invention.

One aspect of the present invention is to provide a method for preparing a fermented composition with an improved odor, which includes preparing cereal flour; initial fermentation of the cereal flour with yeast; post-fermentation of the product of the initial fermentation using a strain of Bacillus subtilis ; And obtain the product of post-fermentation.

Another aspect of the present invention is to provide yeast for improving the odor of fermentation products of Bacillus subtilis and producing α-galactosidase, protease and phytase.

Yet another aspect of the present invention is to provide a composition for grain fermentation, which contains the yeast.

Yet another aspect of the present invention is to provide a fermentation composition prepared by the above method.

Yet another aspect of the present invention is to provide a feed composition containing the yeast or fermentation composition.

The present invention will be described in detail as follows. Meanwhile, the descriptions and embodiments disclosed in the present invention can be applied to other descriptions and embodiments, respectively. That is, all the combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention is not intended to be limited by the following specific description.

In order to achieve the above object, one aspect of the present invention provides a method for preparing a fermentation composition with an improved smell, which includes preparing cereal flour; using yeast for primary fermentation of the cereal flour; using a strain of Bacillus subtilis for primary fermentation Post-fermentation of the product; and obtaining the product of post-fermentation.

In the preparation of cereal flour by the above preparation method, the cereal flour may include, but is not limited to, any raw materials commonly used in the method of preparing feed. Specifically, cereal flour may include soybeans, soybean meal, corn or corn gluten, etc., more specifically, soybean meal or corn gluten, and even more specifically, both soybean meal and corn gluten, but cereal flour does not limited Here.

Cereal flour can be the one that has been adjusted for moisture content and heat treatment.

It is known that microorganisms need at least a certain degree of moisture to grow, and they are difficult to grow under moisture with a concentration of 5% to 12%, which is the concentration of moisture contained in the raw material itself. In addition, it is known that most enzymes (for example, glucoamylase, protease, etc.) undergo decomposition reactions based on hydrolysis, and therefore in order to enable smooth enzyme reactions, at least a certain degree of water content is necessary.

Specifically, the adjusted water content in the cereal flour may be in the range of 30% to 60%, more specifically in the range of 35% to 55%, and more specifically in the range of 40% to 50%. Within the range, but the adjusted water content is not limited to this. The advantage of a composition having an adjusted water content within the above range is that it can prevent the decrease in fermentation rate (fermentation rate) caused by low water content and improve the high temperature incurred during raw material transfer and post-fermentation drying procedures. Cost is a problem, and in addition, the composition is beneficial from the viewpoint of heat efficiency.

In particular, the water content can affect the degree of increase in the protein content during the fermentation of the composition. Specifically, as the low water content becomes lower, it becomes more unfavorable for the growth of microorganisms, and the degree of increase in the protein content of the composition obtained by fermentation may be reduced. In addition, when the water content is excessively high, the drying cost to remove the contained moisture at the end of the fermentation increases, and therefore, the manufacturing cost may increase and the competitiveness of the product may decrease.

At the same time, the heat treatment process can sterilize harmful microorganisms contained in the raw material itself, and reduce the digestibility of materials such as anti-nutritional factors (anti-nutritional factors). factors, for example, trypsin inhibitors found in soybean meal or zein). In addition, since zein has low hygroscopicity, it can help ensure that sufficient moisture is contained in the zein through the heat treatment process after hydrolysis.

The heat treatment can be performed using various methods known in this technical field, for example, using steam or superheated steam.

Specifically, the heat treatment may be performed at 90°C to 110°C for 20 to 40 minutes, more specifically, 95°C to 105°C for 25 to 35 minutes, and more specifically, 100°C for 30 minutes, But the heat treatment is not limited to this.

When the heat treatment temperature is low or the treatment time is short, the bactericidal effect of various bacteria may be reduced, and the subsequent fermentation process may not proceed smoothly. When the heat treatment temperature is high or the treatment time is long, the composition The digestibility of the protein may be reduced due to the denaturation of the protein, and therefore the quality of the final product may be degraded.

In the preparation method of the present invention, the initial fermentation uses yeast to ferment cereal flour.

As used here, the term "yeast" is a general term for single-celled organisms, which is a group of fungi or mushrooms, but does not have hyphae, nor photosynthesis or photosynthesis. The function of motility. Yeast itself is used as a cheap source of fat/protein and can also be used for the fermentation of food or feed. Specifically, the yeast used for the yeast fermentation of the fermentation composition may be Saccharomyces cerevisiae , but the yeast is not limited thereto.

Since yeast secretes enzymes that can decompose oligosaccharides, it can decompose oligosaccharides in plant materials used in feed. Furthermore, the cell wall of yeast can be used as a beneficial component for animals, and because Therefore, the method of preparing fermentation composition using yeast can improve the composition and functionality of plant raw materials.

The yeast of the present invention may be a yeast that produces α-galactosidase, protease, and phytase, and more specifically, a brewer's yeast registered with the access number of KCCM12123P or the access number of KCCM12124P. Generally speaking, not all yeasts can produce α-galactosidase, protease and phytase, but the yeast of the present invention can produce these enzymes, and therefore, compared with other yeast species, it has an improved fermentation effect by Bacillus subtilis The fermented fermented product and composition have excellent odor.

The amount of yeast inoculated into the fermentation composition is an important factor affecting fermentation. The amount of inoculated yeast can be such that the amount of yeast immediately after inoculation is in the range of 10 ⁵ CFU/g to 10 ⁹ CFU/g, and more specifically, in the range of 10 ⁶ CFU/g to 10 ⁸ CFU/g However, the amount of yeast inoculated is not limited to this.

When the amount of inoculation is too small, the amount of the fermentation liquid of the consumed seed bacteria is small, but it takes a longer time to ferment the composition. As a result, the fermentation time required to produce the product becomes longer, which increases the chance of contamination by various bacteria. At the same time, when the amount of inoculation is too large, the fermentation time can be significantly shortened, but it has the disadvantage of needing to provide inoculum for inoculation. In particular, since the efficiency of fermentation is mainly determined by the growth characteristics of the fermentation strains to be used and the type of fermentation equipment, those skilled in the art will be able to consider the characteristics of these strains in the production stage and appropriately select the inoculation. the amount.

The initial fermentation of the cereal flour in the present invention may further include treating the cereal flour with an enzyme. More specifically, this step may include adding alpha-amylase or glucoamylase.

The preparation method of the present invention can increase the water of the raw material through the enzyme treatment of the cereal flour The content of soluble sugars can be concentrated by yeast fermentation and Bacillus subtilis fermentation to concentrate the protein contained therein, and therefore the functionality of the composition can be improved by increasing the protein content ratio in the cereal flour.

The enzymatic treatment of cereal flour corresponds to the decomposition of structural carbohydrates.

As used herein, the term "structural carbohydrates" represents carbohydrates with low utilization (for example, cellulose, hemicellulose, pectin, etc.), which constitute the cell wall of plant cells . Structural carbohydrates can inhibit the utilization of fermenting microorganisms for the raw materials used in feedstuffs (such as soybean meal, corn protein, etc.), and also reduce the absorption rate and digestibility of livestock. Therefore, enzyme treatment can be performed to improve the rate of substrate utilization by fermenting microorganisms and the absorption rate and digestibility of livestock.

Specifically, examples of enzymes that can decompose structural carbohydrates include α-amylase, glucoamylase, cellulase, pectinase, etc., and more specifically, α-amylase or glucoamylase, but the enzyme Not limited to this.

Depending on the type of enzyme, the enzyme treatment can be carried out simultaneously or sequentially with the yeast inoculation process to the grain flour to be fermented.

Specifically, whether the enzyme treatment is performed simultaneously or sequentially may vary depending on the type of enzyme.

Since the activity conditions are different depending on the enzyme, the enzyme treatment step and the yeast fermentation step can be carried out considering the enzyme activity conditions and the yeast fermentation conditions. For example, when using thermophilic alpha-amylase, high temperature conditions are required for enzyme activity. Therefore, heat treatment The procedure and the enzyme treatment step can be simultaneously performed by adding enzymes before the heat treatment of the cereal flour and after the moisture treatment. That is, the yeast fermentation step may be performed after the heat treatment process and the enzyme treatment step.

At the same time, when using glucoamylase or mesophilic α-amylase, high temperature conditions are not required for enzyme activity. Therefore, an enzyme treatment step can be performed after the moisture treatment and heat treatment are completed. In this case, the enzyme reaction and yeast fermentation can be performed separately or simultaneously. For example, the enzyme reaction can be carried out by adding glucoamylase or mesophilic α-amylase to cereal flour at 50°C to 70°C for 30 minutes to 1 hour 30 minutes, and then yeast fermentation can be carried out by inoculation with yeast. Or, in order to optimize the process, the enzyme reaction step and the yeast fermentation step can be performed simultaneously by adding enzyme and yeast simultaneously.

The initial fermentation of cereal flour may further include adding glucoamylase to the cereal flour in an amount of 0.1% by weight (wt%) to 1.0% by weight, 0.3% to 0.7% by weight, or 0.5% by weight; adding yeast culture Inoculate in an amount of 1 wt% to 30 wt%, 1 wt% to 20 wt%, 5 wt% to 15 wt%, 8 wt% to 12 wt%, or 10 wt%; or at 10°C to 50°C, Anaerobic fermentation of grain flour inoculated with yeast culture at 20°C to 40°C, 25°C to 35°C or 30°C for 1 to 10 hours, 2 to 10 hours, 4 to 8 hours, 5 to 7 Hours, or 6 hours.

In the preparation method of the present invention, the post-fermentation step corresponds to a step in which the product of the primary fermentation is further fermented using a Bacillus subtilis strain, and the post-fermentation is performed in sequence following the primary fermentation.

Bacillus, which is a genus belonging to the family of subtilis, and the bacteria of the genus Bacillus are rod-shaped Gram-positive bacteria (Gram-positive). Specifically, the strain of Bacillus subtilis may be selected from Bacillus subtilis, Bacillus licheniformis, Bacillus toyoi, Bacillus coagulans, and Bacillus polyenzymes. At least one strain of the group consisting of Bacillus polyfermenticus and Bacillus amyloliquefaciens, more specifically, Bacillus subtilis, and even more specifically, the strain of Bacillus subtilis may be a KCCM11471P Take the number of Bacillus subtilis registered, but the strains of Bacillus subtilis are not limited to this.

The amount of Bacillus subtilis strains to be inoculated into the fermentation composition is the same as the amount inoculated against yeast as described above.

By producing ammonia (ammonia) during fermentation, Bacillus subtilis exhibits an acrid taste, and this acrid taste may affect the preference for the fermentation composition. However, by improving the off-flavor of the fermentation product of Bacillus subtilis by using yeast and Bacillus subtilis in sequence, the preparation method of the present invention can improve the preference for the fermentation product of Bacillus subtilis.

In addition, by sequentially performing yeast fermentation and fermentation by Bacillus subtilis strains, the viable cell count in the fermentation composition can be significantly increased, and thus the viable cell count is increased It can improve the effect of probiotics in the fermentation composition.

In the preparation method of the fermentation composition of the present invention, the fermentation composition can be produced in a situation where the characteristics and advantages of each of yeast and Bacillus subtilis are combined. In general, it is known that Bacillus subtilis cannot produce α-galactosidase and therefore cannot completely decompose polysaccharides having glycosidic bonds. However, some yeast strains can produce α-galactosidase, and therefore the carbohydrate component of soybean meal or corn gluten-it is difficult to separate from Bacillus subtilis strains alone. Decomposition can be decomposed by yeast, and therefore the carbohydrate component of soybean meal or corn protein can be effectively used as a substrate for the growth and metabolism of fermentation strains.

Specifically, the functionality of the fermentation composition can be improved by using the oligosaccharide decomposing enzyme expressed in the yeast to decompose the oligosaccharides present in the flour and increasing the number of viable yeasts themselves, thereby increasing the yeast The content of functional components. In addition, the digestibility and absorption rate of the feed composition can be improved by using the protease expressed in Bacillus subtilis to decompose the protein in the flour.

In the method for preparing the fermentation composition of the present invention, the fermentation may be solid-state fermentation or liquid-state fermentation, and specifically, the fermentation may be solid-state fermentation.

As used herein, the term "solid fermentation" refers to a method of fermented production by microorganisms using solid raw materials containing a certain amount of water.

After fermentation of the present invention, after 2 to 10 hours, 4 to 8 hours, 5 to 7 hours, or 6 hours after yeast inoculation, the method may further include adding the culture of the Bacillus subtilis strain to 1% to 30% by weight, 1 to 20% by weight, 5% to 15% by weight, 8% to 12% by weight, or 10% by weight for inoculation, and at 35°C to 40°C, or 37°C, at 80% to Anaerobic fermentation is carried out at 100%, 90% to 100%, 93% to 97%, or 95% humidity.

The fermented product after performing the post-fermentation step may be a product in which the content of low molecular weight peptide is 30% to 100%, and more specifically, 40% to 100%, 50% to 100%, 60% to 100%, 40% % To 90%, 50% to 90%, 40% to 80%, 50% to 80%, 40% to 70%, or 50% to 70%.

"Low molecular weight peptide" means 30 A peptide with a molecular weight of kDa or lower, and more specifically, a peptide having a molecular weight of 0.1 kDa to 30 kDa, 1 kDa to 30 kDa, 0.1 kDa to 20 kDa, 1 kDa to 20 kDa, 0.1 kDa to 10 kDa, 1 kDa to 10 kDa, or 10 kDa or lower The molecular weight of the peptide.

Yet another aspect of the present invention provides yeast for improving the odor of fermentation products of Bacillus subtilis, which produces α-galactosidase, protease and phytase.

Yet another aspect of the present invention provides a composition for grain fermentation containing the above-mentioned yeast, and a feed composition containing the above-mentioned yeast.

Yet another aspect of the present invention provides a fermentation composition prepared by the above method.

Yet another aspect of the present invention provides a feed composition including the above-mentioned fermentation composition.

Alpha-galactosidase, protease, phytase, fermentation products of Bacillus subtilis and yeast are as described above.

Yeast may be a producer of α-galactosidase, protease, and phytase, and more specifically, it is a brewer's yeast registered with the access number of KCCM12123P or the access number of KCCM12124P.

As used herein, the term "feed composition" represents materials that supply organic or inorganic nutrients that are necessary for maintaining the life of the subject and nurturing the subject. The feed composition can contain nutrients (for example, energy, protein, lipid, vitamins, minerals, etc.) required by the test target of the consuming feed, and can be used as a plant feed (for example, grains, root meal). ), by-products of food processing, seaweed, fiber, fat and oil, starch (starch), gourd, grain by-products, etc.) or animal feed (for example, protein, inorganic matters) , Fats and oils, Minerals, single-cell protein, zooplanktons, fish meal, etc.), but the use of the feed composition is not particularly limited thereto. In the present invention, the feed composition is a concept that includes all materials added to the feed (ie, feed additives), raw materials used in the feed, or the feed itself supplied to the test target.

The subject is representative of the cultivated person, and may include, but is not limited to, organisms that can ingest the feed of the present invention. In this way, the feed composition of the present invention can be applied to a variety of diets (feeds) for animals including mammals, poultry, fish, and crustaceans. It can be used for commercially important mammals (for example, pigs, cattle, goats, etc.), zoo animals (for example, elephants, camels, etc.), or domestic animals (for example, dogs, cats, etc.). Commercially important poultry may include chickens, ducks, geese, etc., and commercially-raised fish and crustaceans (for example, trout and shrimp) may also be included.

The content of soybean meal or corn protein in the feed composition according to the present invention can be appropriately adjusted to, for example, 1% by weight according to the type and age of the animal to be applied, the application form, the desired effect, etc. To 99% by weight, specifically, 10% to 90% by weight, and more specifically 20% to 80% by weight, but the content of soybean meal or zein is not limited thereto.

For administration, in addition to soybean meal or zein, the feed composition of the present invention may further include the following mixture: at least one organic acid (such as citric acid, fumaric acid, adipic acid, lactic acid, etc.) ; Phosphate (such as potassium phosphate, sodium phosphate, polyphosphate, etc.); natural antioxidants (such as polyphenols, catechins, tocopherols, vitamin C, green tea extract, chitin, tannic acid, etc.). If necessary, other typical additives (such as anti-influenza agents, buffers, bacteriostatic agents, etc.) can be added. In addition, diluent, dispersant, surface Active agents, binders or lubricants may be additionally added to formulate the composition into injectable preparations (such as aqueous solutions, suspensions, emulsions, etc.), capsules, granules or lozenges.

What's more, in addition to the main ingredients-including vegetable protein feed (for example, powdered or chopped wheat, barley, corn, etc.), animal protein feed (for example, blood meal, meat meal, fish meal, etc.), animal fat and vegetable oil- , The feed composition of the present invention can be combined with various auxiliary components (such as amino acids, inorganic salts, vitamins, antioxidants, antifungal agents, antibacterial agents, etc.) as well as nutritional supplements, growth accelerators, digestion/ Absorption accelerators are used together with prophylactic agents.

When the feed composition of the present invention is used as a feed additive, the feed composition may be added alone or used together with other components, and may be appropriately used according to a conventional method. The feed composition can be prepared into an immediate-release formulation or a sustained-release formulation in a dosage form, and combined with a non-toxic pharmaceutically acceptable carrier. The edible carrier can be corn starch, lactose, sucrose or propylene glycol. The solid carrier can be in the form of tablets, powders, troches, etc., and the liquid carrier can be in the form of syrups, liquid suspensions, emulsions, and solutions. In addition, the administration agent may include preservatives, lubricants, solution accelerators, or stabilizers, and may also include other auxiliary agents for improving inflammatory diseases and substances effective for preventing viruses.

The feed composition according to the present invention can be mixed in an amount of about 10 grams (g) to 500 grams per kilogram (kg), preferably 10 grams to 100 grams, based on the dry weight of the livestock feed, and is completely mixed Later, the feed composition can be provided as mash, or be further subjected to pelletizing, extensification or extrusion processes, but it is not limited to this.

According to the method of the present invention, the content of water-soluble sugars in the raw material can be increased by the enzyme treatment of the cereal flour, and the protein contained in the raw material can be concentrated by the yeast fermentation and the fermentation of Bacillus subtilis, and thus the cereal flour can be increased by The ratio of protein content and the number of viable bacteria in the composition improve the functionality, digestibility and absorption rate of the composition.

In addition, as described above, the fermentation product of Bacillus subtilis produces peculiar smell due to ammonia and the like during the fermentation process, which may cause problems in using the corresponding fermentation product as feed. The yeast of the present invention is used in the complex fermentation related to the fermentation of Bacillus subtilis, and therefore can significantly reduce the odor of the product fermented by the fermentation of Bacillus subtilis. In addition, the yeast of the present invention contains the yeast, and thus can provide a composition for grain fermentation and a feed composition having an improved smell.

The method for preparing the fermentation composition of the present invention can increase the water-soluble sugar content in the raw material by enzymatic treatment of the cereal flour, and condense the protein contained in the raw material through the yeast fermentation, and thus can increase the cereal flour The ratio of the protein content and the number of viable bacteria improve the functionality of the composition. In particular, since yeast secretes an enzyme capable of decomposing oligosaccharides, it can decompose oligosaccharides in plant materials used for feed. Furthermore, the cell wall of yeast can be used as a component beneficial to animals, and therefore, yeast is used to prepare fermentation The composition method can improve the composition and functionality of plant raw materials. In addition, the method for preparing the fermentation composition of the present invention may further include Bacillus subtilis fermentation after yeast fermentation. Since Bacillus subtilis produces protease, the protein in the fermentation composition can be peptidized. In this way, the feed Protein digestibility and absorption rate can be improved. Furthermore, the method for preparing a fermentation composition of the present invention can improve the off-flavor caused by the fermentation of Bacillus subtilis through sequential yeast fermentation and Bacillus subtilis fermentation, thereby enhancing the preference of the composition.

Figure 1 shows an image showing a group of grain raw materials, a group of mixed grains that are individually fermented with Bacillus subtilis, and a group of mixed grains that are individually fermented with yeast Group, and the measurement results of the sugar components in the group of mixed grains fermented by a combination of yeast and Bacillus subtilis, where (1) represents the standard material (from bottom to top): stachyose , Raffinose, sucrose and glucose; (2) represents the raw material of soybean meal; (3) represents the raw material of corn gluten; (4) represents the mixed grain (large Soybean meal + corn gluten); (5) represents a mixed grain (soybean meal + corn gluten) that is fermented by yeast (CJN1697) alone; (6) represents a mixed grain that has been fermented with yeast (CJN1697) and Bacillus subtilis Soybean meal + corn protein); (7) represents the combined yeast (CJN2343) and the mixed grain (soybean meal + corn protein) fermented by Bacillus subtilis; and (8) represents the combined yeast (Angest ^® ) and Mixed grains (soybean meal + corn gluten) for fermentation of Bacillus subtilis.

Figure 2 shows an image of the analysis results of the protein components of each of the following groups: the group of cereal raw materials (soybean meal, corn gluten, and mixed cereal raw materials); the individual cereal raw materials fermented by Bacillus subtilis Group; a group of grain raw materials subjected to yeast fermentation alone; and a group of grain raw materials subjected to combined yeast and Bacillus subtilis fermentation.

Figure 3 shows a graph showing the phylogenetic analysis of strain CJN1697 the result of.

Figure 4 shows a graph showing the results of the lineage analysis of the CJN2343 strain.

Hereinafter, the present invention will be described in detail through exemplary embodiments. However, these exemplary embodiments are for illustrative purposes only, and are not intended to limit the scope of the present invention.

Example 1: Screening of yeast strains

Among yeast strains, select those strains with excellent ability to produce α-galactosidase, protease and phytase. In order to confirm the ability to produce α-galactosidase, X-gal agar (NaCl (0.5%), peptone (1%), raffinose (1%), agar (1.5%), and X -gal (0.5%)) is prepared. In addition, in order to confirm the ability to produce protease, YM agar culture medium (powdered skim milk (2%), yeast extract (0.3%), malt extract (0.3%), egg whites (1%) and agar ( 1.5%)) was prepared, and in order to measure the phytase activity, the medium was prepared by adding phytin to the above medium.

The yeast strains were cultured in the YPD medium (glucose (2%), yeast extract (0.8%), and soy protein poultry (0.2%)) at 30°C for 12 hours, whereby the beer yeast was prepared. The prepared yeast culture solution (5μL) was added dropwise to each agar-agar culture medium and incubated at 30°C for about 24 hours. The ability to produce α-galactosidase, protease and phytase was determined by The clear zone produced by each drop of yeast culture solution is measured.

The existence of α-galactosidase was confirmed by X-gal agar culture medium, and the ability to produce protease and phytase was measured and compared with each colony and the net produced around the colony. The size of the area (clear area size/colony size) was tested (Table 1). When the production of clean areas of strains is not observed due to lack of protease activity, these strains are indicated as "0". As a result, among about 100 strains of S. cerevisiae, 14 strains showed α-galactosidase activity, and among the 14 strains, two strains (namely CJN1697 and CJN2343) finally did not exist for cereals by eliminating Strains with protease activity necessary for fermentation were selected.

Example 2: Preparation of fermentation composition

2-1 Moisture treatment and heat treatment method of cereal flour

Soybean meal flour and corn gluten flour are prepared separately. Water was added to the soybean meal to adjust the moisture content of the soybean meal to about 45% based on the weight of the soybean meal, and the mixture was subjected to heat treatment at 100° C. for 30 minutes. For the corn gluten meal, phosphoric acid in an amount of 1.5% by weight based on the weight of the corn gluten was added, and then water was added to adjust the water content of the corn gluten to about 43%, and the mixture was subjected to heat treatment at 100° C. for 30 minutes. Sodium hydroxide (NaOH) was added to the heat-treated zein in an amount of 2.4% by weight based on the weight of the zein, and then water was added to adjust the water content of the zein to about 45%.

2-2. Enzyme treatment method and preparation of yeast fermentation group

The soybean meal and corn gluten meal prepared by the method of Example 2-1 were mixed in the same weight ratio to prepare mixed grain flour. Next, glucoamylase (0.5% by weight) was added to the mixed cereal flour and each with 10% by weight of the culture of three kinds of beer yeast (CJN1697, CJN2343, and commercial baker's yeast (available from Angel Yeast Co., Ltd.)) Inoculated and mixed, and this was allowed to carry out anaerobic fermentation at 30°C for 6 hours, and thereby it was prepared as a group of yeast fermentation.

2-3. Preparation method of group fermented by yeast and Bacillus subtilis

The yeast fermentation group prepared by the method of Example 2-2 was further subjected to fermentation by Bacillus subtilis. More specifically, as described in Example 2-2, each fermentation group, in which the fermentation is performed using three types of brewer’s yeast (CJN1697, CJN2343, and commercial baker’s yeast), and 6 hours after yeast inoculation, the system A culture of Bacillus amyloliquefaciens (KCCM11471P) was inoculated in an amount of 10% by weight, and then aerobic fermentation was performed in a thermo-hygrostat (thermo-hygrostat, temperature: 37°C, humidity: 95%) 24 hour.

2-4. Measurement of components in each experimental group

For each experimental group prepared by the methods of Examples 2-2 and 2-3, the water content, the number of viable bacteria of Bacillus subtilis, the number of viable yeasts, and the protein content were measured according to time. The protein content is provided by the Kjeldahl device, after drying and the fermentation product is powdered (pulverized) after measurement. The measurement results are shown in Table 2 below.

As a result, the amount of surviving yeast increased from 10 ⁷ CFU/g to 10 ⁸ CFU/g in all experimental groups where yeast fermentation was performed. In addition, the amount of surviving Bacillus subtilis increased from 10 ⁹ CFU/g to 10 ¹⁰ CFU/g in all experimental groups where fermentation of Bacillus subtilis was performed. In addition, it has been confirmed that the amount of protein in the composition is increased by yeast fermentation, and it has been confirmed that adding Bacillus subtilis fermentation after yeast fermentation is more effective for increasing the amount of protein.

Therefore, that is, when the fermentation of Bacillus subtilis is performed on the grain material after the yeast fermentation, the protein content of the grain material can be further increased by Bacillus subtilis without inhibiting the growth of the yeast.

Example 3: Confirmation of the degree of decomposition of oligosaccharides by fermentation

A group of two kinds of grain raw materials (a group of soybean meal raw materials and a group of corn gluten raw materials), a group of mixed grains (soybean meal + corn gluten) that are separately fermented by Bacillus subtilis, and a mixed grain that is separately fermented by yeast ( The group of soybean meal + corn gluten) and the group of mixed grains (soybean meal + corn gluten) fermented by combined yeast and Bacillus subtilis were prepared, and the carbohydrate component was analyzed for each group.

The group of soybean meal raw materials (Figure 1 (2)) and the group of corn gluten raw materials (Figure 1 (3))-which have not been pre-treated-are prepared. The carbohydrate components of each group are passed through thin layer chromatography (Thin layer chromatography, TLC) is quantitatively analyzed. 25 milliliters (mL) of distilled water was added to 1 gram of each sample, and the mixture was heated in boiling water for 15 to 20 minutes and extracted by shaking at 37°C for 2 hours. The extract was centrifuged and the supernatant was recovered and used as a TLC sample. 2 microliters (μL) of the supernatant solution was spotted on the silica gel TLC plate, dried to a constant level, and developed in the developing solution 3 hours. After the development is completed, the spots of oligosaccharides and monosaccharides are confirmed by color development and drying procedures.

The group of mixed grains (Fig. 1(4)) separately subjected to the fermentation of Bacillus subtilis is made by mixing the soybean meal and corn gluten meal prepared by the method of Example 2-1 in the same weight ratio, with a ratio of 10% by weight A culture of Bacillus subtilis was inoculated to the mixture, and then aerobic fermentation was performed in a thermostat (temperature: 37°C, humidity: 95%) for 24 hours to prepare.

The group of mixed grains (Fig. 1(5)) subjected to yeast fermentation alone was prepared by the method of Example 2-2. For the group of mixed grains subjected to combined yeast and Bacillus subtilis fermentation, there are 3 groups (Figure 1(6) (CJN1697+Bacillus subtilis), Figure 1(7) (CJN2343+Bacillus subtilis) and Figure 1(8) (Bakery yeast + Bacillus subtilis)) is prepared using three kinds of yeast.

The sugar component of each fermentation group is measured in the same way as the sugar component of the soybean meal raw material group and the corn protein raw material group. For each fermentation group, 25 ml of distilled water was added to 1 gram of each sample, and the mixture was heated in boiling water for 15 to 20 minutes and extracted by shaking at 37°C for 2 hours. The extract is centrifuged and the supernatant is recovered and used Make TLC samples.

In Figure 1, the first column (lane(1)) represents the carbohydrate component markers of stachyose, raffinose, sucrose and glucose from bottom to top; the second column represents the soybean meal raw materials Group; the third column represents the group of zein raw materials; the fourth column represents the group of mixed grains (soybean meal + corn gluten) that undergoes yeast fermentation alone; the fifth column represents the group of yeast (CJN1697) fermentation alone The group of mixed grains (soybean meal + corn gluten); columns 6 to 8 each represent the group of mixed grains (soybean meal + corn gluten) undergoing combined yeast and Bacillus subtilis fermentation ((6) CJN1697+ Bacillus subtilis , (7) CJN2343 + Bacillus subtilis, and (8) Baker's yeast + Bacillus subtilis).

Referring to Figure 1, in the group that uses Bacillus subtilis for single fermentation (Figure 1(4)), it has been confirmed that the sugar components contained in soybean meal have not been completely decomposed. This is because the microorganisms are in The carbohydrate component cannot be fully utilized during the fermentation process, and therefore oligosaccharides are included in the fermentation product. At the same time, in the group that uses yeast for fermentation (Figure 1(5)), it has been confirmed that oligosaccharides are decomposed by yeast and the microorganisms have fully utilized the decomposed sugar components, thus resulting in low oligosaccharide content .

In addition, in the fermentation groups (columns 6 and 7) where CJN1697 and CJN2343 yeast strains are used, no specific oligosaccharide spots (spots) were observed in the fermentation products. This is due to the enzyme's α-galactosidase In the fermentation group (column 8) where commercial baker’s yeast (Angest ^® ) is used, the α-galactosidase activity of baker’s yeast is low and therefore there are specific oligosaccharide spots observed.

Therefore, the fermentation composition prepared using yeast is characterized in that oligosaccharides are decomposed in the composition, and therefore, when ingested by animals through feed, the fermentation composition does not require any individual components for the decomposition of the oligosaccharides. Digestive enzymes, thus making the digestion of feed easier. In particular, it has been confirmed that yeast strains using CJN1697 or CJN2343 are more effective.

Example 4: Confirmation of the degree of protein degradation by fermentation

The group of grain raw materials (soybean meal, corn gluten, and mixed grain raw materials), the group of grain raw materials that are individually fermented by Bacillus subtilis, the group of grain raw materials that are individually fermented by yeast, and the combined yeast and The groups of grain raw materials fermented by Bacillus subtilis were prepared in the same manner as in Example 3, and an attempt was made to analyze the protein components in each group.

Just like the group of grain raw materials, there are three groups in total (namely, a group of soybean meal raw materials (group 2), a group of corn gluten raw materials (group 3), and a group of mixed grains-the soybean meal and corn gluten are the same Weight ratio mixing (group 4))-is prepared. The molecular weight pattern of the protein of each raw material was confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 100 milligrams (mg) of each sample was mixed with 5 ml of 8M urea solution, and the mixture was sonicated for extraction and centrifugation, and the supernatant was recovered. In each supernatant solution, the protein content was quantified using bicinchoninic acid and confirmed by SDS-PAGE by loading a certain amount of each protein sample.

The group of grain raw materials (groups 5 to 8) that are separately subjected to the fermentation of Bacillus subtilis is obtained by inoculating a flour mixture with a culture of Bacillus subtilis in an amount of 10% by weight-among which, from Example 2-1 The soybean meal and corn gluten meal prepared by the method are mixed in the same weight ratio-and then aerobic fermentation is carried out in a constant temperature humidifier (temperature: 37°C, humidity: 95%) for 24 hours. The fermentation time (0 hour, 16 hours, 20 hours and 24 hours) of each group is shown in Table 3 below.

The group of grain raw materials (groups 9 to 12) that are individually fermented with yeast is prepared by the method of Example 2-1, and the group of grain raw materials (groups 13 to 12) that undergo combined yeast and Bacillus subtilis fermentation 24) is prepared by the method of Example 2-3 using three kinds of yeast. The strains used for the fermentation of each group and the fermentation time of each group are shown in Table 3 below. In the group of combined fermentations in which Bacillus subtilis fermentation is performed after yeast fermentation, Bacillus subtilis fermentation is performed for 6 hours after yeast fermentation.

The degree of protein degradation of each fermentation group was confirmed by SDS-PAGE. The samples were pretreated in the same way as to confirm the degree of molecular weight distribution of the protein in the group of cereal raw materials. The protein molecular weight map of each raw material was confirmed by SDS-PAGE. 100 mg of each sample was mixed with an 8M urea solution, and the mixture was ultrasonically decomposed for extraction and centrifuged, and the supernatant was recovered. The protein content was quantified by using Dicinchonic Acid and confirmed by SDS-PAGE by filling a certain amount of each protein sample. The results are shown in Figure 2.

In Table 3 above, the time of the group of sequential fermentation by yeast and Bacillus subtilis refers to the total fermentation time, which is equal to the sum of the fermentation time by Bacillus subtilis and the yeast fermentation time ( That is, 6 hours).

Figure 2 shows images illustrating the SDS-PAGE results of the supernatant protein in groups 1-24.

Referring to Table 3 and Figure 2, Bacillus subtilis can produce proteases, and therefore can use proteases during the fermentation process to break down proteins into low molecular weight peptides. Therefore, in the combined fermentation group of yeast and Bacillus subtilis, it has been confirmed that the proteins of soybean meal and zein are decomposed. At the same time, because yeast cannot produce protease at all, in the group that performs yeast fermentation alone, protein cannot be decomposed at all, and therefore the protein profile of the raw material is indicated as its original state. That is, since the composition that undergoes fermentation by Bacillus subtilis after yeast fermentation contains low molecular weight peptides, the composition can improve the protein absorption rate of feed.

In order to measure the content of low-weight peptides in the fermentation product in more detail, the molecular weight distribution of low-molecular-weight peptides is measured by gel permeation chromatography (GPC).

GPC is a method for confirming retention time (RT) by analyzing standard proteins with different molecular weights, and using a standard curve of molecular weight and RT to measure the protein distribution of analytes based on molecular weight. In order to confirm the degree of protein degradation of the raw materials due to fermentation, the protein distribution in the fermentation product was analyzed by the GPC method.

GPC analytes are pretreated in the same way as the SDS-PAGE method. 100 mg of each sample was suspended in 5 ml of 8M urea solvent, and the mixture was ultrasonically decomposed for extraction and centrifuged, and the recovered supernatant was filtered with a syringe filter and used as a Analytes analyzed by GPC. As analytes, groups from group 4 (mixed raw material: soybean meal + corn starch), group 8 (Bacillus subtilis fermentation alone), group 12 (yeast fermentation alone), and groups 16, 20 and 24 (combined fermentation by Bacillus subtilis + yeast) of each fermentation product. The results of the GPC analysis are shown in Table 4 below.

Referring to Table 4, the raw material contains more than 82% of 30kDa or larger polymer peptides. In the case of Bacillus subtilis fermentation alone, the content of low molecular weight peptides of 30 kDa or less is about 71%, and in the case of combined fermentation by Bacillus subtilis and yeast, low molecular weight peptides of 30 kDa or less are better than The peptide content is 83%, so it shows a significant low molecular weight peptide content Is increasing. In addition, when the fermentation product is obtained from Bacillus subtilis and yeast, the content of low molecular weight peptides of 10kDa or less in the fermentation product is in the range of about 66% to about 69%, so it is different from subtilis alone. The content of low-molecular-weight peptides of 10kDa or lower in the fermented product of bacillus fermentation increased by about 40%. That is, in the case of fermentation by Bacillus subtilis and yeast, the protein decomposition efficiency increases, and the content of low molecular weight peptides in the fermentation product also increases. Therefore, it can be seen that the digestion and absorption rate can be significantly improved when the products fermented by Bacillus subtilis and yeast are used as raw materials for food or feed.

Example 5: Comparison of simultaneous or sequential fermentation of yeast and Bacillus subtilis

Between the case where the yeast fermentation and the Bacillus subtilis fermentation are performed simultaneously and the case where the yeast fermentation and the Bacillus subtilis fermentation are performed sequentially, the increase in the number of viable bacteria and the protein quality is measured and compared.

The group for performing yeast fermentation was prepared by the method of Example 2-2, and the group for performing sequential fermentation with yeast and Bacillus subtilis was prepared by the method of Example 2-3. The group for simultaneous fermentation with yeast and Bacillus subtilis is by mixing the soybean meal and corn gluten meal prepared by the method of Example 2-1 in the same weight ratio, and adding glucoamylase (0.5%) to it , And simultaneously inoculated with yeast and Bacillus subtilis, followed by aerobic fermentation. The group of fermentation with yeast and Bacillus subtilis in sequence is prepared by fermentation with Bacillus subtilis for 6 hours after yeast fermentation. The water content, the number of live bacteria of Bacillus subtilis, the number of live bacteria of yeast, and the amount of protein in each group were measured according to time, and the results are shown in Table 5 below.

In Table 5 above, the time of the group of sequential fermentation by yeast and Bacillus subtilis represents the total fermentation time, which is equal to the fermentation time by Bacillus subtilis and the sum of the fermentation time by yeast (ie, 6 hours ).

Referring to Table 5, when the fermentation of Bacillus subtilis is carried out after the fermentation of yeast, both the number of viable bacteria of Bacillus subtilis and the number of viable yeasts increase in proportion to their fermentation time. However, it has been confirmed that when yeast and Bacillus subtilis are simultaneously inoculated and fermented together, the viable cell count of Bacillus subtilis increases in proportion to the fermentation time, but the viable cell count of yeast remains at 10 ⁷ CFU/g degree. That is, when yeast and Bacillus subtilis are simultaneously inoculated and fermented together, yeast does not affect the growth of Bacillus subtilis, and Bacillus subtilis inhibits the growth of yeast.

Therefore, it is presumed that the protease of Bacillus subtilis reduces the population of yeast (which is proliferation through budding), and it has been confirmed that the order of microbial inoculation has an effect on two microorganisms (ie yeast and Bacillus subtilis) in solid state fermentation. The growth in all has a significant impact. In particular, it has been confirmed that both Bacillus subtilis and yeast can make better use of water-soluble sugar components. This is because the oligosaccharides in soybean meal are first subjected to 6 hours of yeast fermentation during yeast fermentation. Decomposed.

Example 6: The problem of smell improvement

The fermentation product of Bacillus subtilis produces odor due to ammonia and the like during the fermentation process, and this may be one of the limiting factors in the use of feed. Therefore, in this example, it has been confirmed whether the complex fermentation performed by yeast and Bacillus subtilis can reduce the odor of Bacillus subtilis fermentation products. Odor test for the mixed raw materials of soybean meal and corn gluten, the product fermented with Bacillus subtilis (KCCM11471P) alone, and the product obtained by the compound fermentation of yeast (baker's yeast; CJN1697 or CJN2343) and Bacillus subtilis (KCCM11471P) (50 subjects) proceed. The score is based on a point scale from 0 to 5, where the higher the score, the higher the intensity of the odor of Bacillus subtilis (Table 6).

As a result, it was confirmed that the product fermented by Bacillus subtilis fermentation alone (using Bacillus subtilis (KCCM11471P)) showed the highest score. In addition, it has been confirmed that when CJN1697 or CJN2343 yeast is used for sequential fermentation of Bacillus subtilis, the odor is significantly reduced compared with the use of commercially used yeast.

Example 7: Analysis of the nucleotide sequence and phylogeny of the 18S rRNA gene of the beer yeast strain of the present invention

To analyze the strain isolated in Example 1, 18S ribosomal DNA sequencing was performed in the following manner. The chromosomes of CJN1697 and CJN2343 strains were isolated using Wizard genomic DNA purification kit (Promega, USA), and then received using NS1 (5 ' -GTAGTCATATGCTTGTCTC-3 ' ) and NS8 (5 ' - TCCGCAGGTTCACCTACGGA-3 ' ) primer-which is a universal primer used for 18S rRNA sequencing-PCR amplification (PCR amplification). The amplified PCR product was purified using Wizard SV gel and PCR purification system (clean-up system, Promega, USA). As a result, the purified amplified PCR product was compared with the ribosomal DNA sequence of GENEBANK using the BLASTN program, and sequence homology was compared and analyzed using the Clustal X and Mega 2 programs.

The results of the pedigree analysis showed that the two strains of the present invention (ie, CJN1697 and CJN2343) showed 99% homology with S. cerevisiae-reference strain (Figures 3 and 4). The strains of the present invention (ie CJN1697 and CJN2343) were named Saccharomyces cerevisiae CJN1697 and Saccharomyces cerevisiae CJN2343, respectively, and were deposited at the Korean Culture Center of Microorganisms (KCCM) on October 11, 2017 according to the Budapest Agreement. , The access numbers are KCCM12123P and KCCM12124P.

Based on the foregoing, those with ordinary knowledge in the technical field of the present invention can understand that the present invention can be implemented in other specific forms without modifying the technical concept or essential features of the present invention. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the present invention. In contrast, the present invention is intended to cover not only exemplary embodiments, More as defined by the scope of the attached patent application, various alternatives, modifications, equivalent content, and other embodiments may also be included in the spirit and scope of the present invention.

【Biological Material Deposit】

Domestic deposit information [please note in order of deposit institution, date and number]

1. Food Industry Development Research Institute, June 27, 2019, BCRC 910907.

2. Food Industry Development Research Institute, July 15, 2019, BCRC 920115.

3. Food Industry Development Research Institute, July 15, 2019, BCRC 920116.

Foreign hosting information [please note in the order of hosting country, institution, date and number]

1. South Korea, Korea Microbial Culture Center (KCCM), November 07, 2013, KCCM11471P.

2. South Korea, Korea Microbial Culture Center (KCCM), October 11, 2017, KCCM12123P.

3. South Korea, Korea Microbial Culture Center (KCCM), October 11, 2017, KCCM12124P.

Claims (16)

A method for preparing a fermentation composition with an improved odor, comprising: preparing cereal flour; using yeast to perform primary fermentation of the cereal flour; using a strain of Bacillus subtilis to perform post-fermentation of the product of the primary fermentation; and obtaining the The product of post-fermentation. The method according to claim 1, wherein the yeast produces α-galactosidase, protease and phytase. The method according to claim 1, wherein the yeast is a brewer's yeast registered with the access number of KCCM12123P or the access number of KCCM12124P. The method according to claim 1, wherein a peptide having a molecular weight of 30 kDa or less is contained in an amount of 40% to 100% in the product of the post-fermentation. The method according to claim 1, wherein the cereal flour comprises soybean meal or corn gluten. The method according to claim 1, wherein performing the initial fermentation of the cereal flour comprises adding α-amylase or glucoamylase. The method according to claim 1, wherein the cereal flour is adjusted for moisture content and then subjected to heat treatment. The method according to claim 7, wherein the adjusted water content is in the range of 30% to 60%. The method according to claim 1, wherein the strain of Bacillus subtilis is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus toyoi, Bacillus coagulans, Bacillus polyenzymes, and Bacillus subtilis At least one strain. The method according to claim 1, wherein the strain of Bacillus subtilis is Bacillus subtilis registered with KCCM11471P access number. A yeast used to improve the odor of fermentation products of Bacillus subtilis, which produces α-galactosidase, protease or phytase. The yeast according to claim 11, wherein the yeast is a beer yeast registered with the access number of KCCM12123P or the access number of KCCM12124P. A composition for grain fermentation, which comprises the yeast described in claim 12. A feed composition comprising the yeast according to claim 12. A fermentation composition prepared by the method according to any one of claims 1 to 10. A feed composition comprising the fermentation composition according to claim 15.

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