KR100943891B1 - Process for preparing ruminally protected soybean peptide and the use thereof - Google Patents

Abstract

The present invention relates to a method for preparing ruminally protected soybean peptides and their use. According to the present invention, the soybean meal is converted into soybean peptide by enzymatic treatment by injecting 2.4 L of bacteria-derived protease Alcalase into soybean meal soaked at low temperature, and the soybean peptide is converted to formaldehyde (formaldehyde). By spreading treatment, soybean peptides are not degraded by microorganisms in the rumen, so that they can be stably moved to the lower digestive tract and absorbed, thereby increasing the nutrient utilization efficiency of ruminants and increasing livestock productivity.

Proteolytic enzymes, formaldehyde, ruminally protected soybean peptides

Description

Process for preparing ruminally protected soybean peptide and the use

The present invention relates to a method for producing ruminally protected soybean peptides and its use, and more particularly, Bacillus subtilis GR-101 (KCCM 10673P) having excellent protease-producing ability. Enzymatic treatment by inoculating and fermenting or injecting 2.4 liters of Alcalase, a bacteria-derived protease into cold soaked soybean meal, converts the protein of soybean meal into soybean peptide, and converts the soybean peptide into formaldehyde ( Formaldehyde) ruminant soybean peptide prepared by spraying treatment to be protected from microorganisms in the rumen of ruminant, rumen protective soybean peptide prepared by the method and used as feed additives and rumen protective soybean peptide as an active ingredient It relates to a feed for livestock containing.

According to the Association of American Feed Control, 'ruminal protection' means that nutrients are protected from rumen microorganisms in the rumen and are absorbed in the small intestine of the livestock. Therefore, ruminally protected protein or ruminally protected peptide refers to a protein or peptide that has been processed and processed so as not to be degraded by microorganisms or proteolytic enzymes present in the rumen. Say.

The importance of rumen protective protein feed in ruminants has been known through many studies to date. In ruminant protein metabolism, ruminant protein (RDP) overfeeding decreases protein synthesis of ruminant microorganisms and increases urea content in ruminant blood due to increased ammonia concentrations in rumen animals. In addition, during the detoxification process, not only does the ruminant's liver function in large numbers, but also the glucose metabolism is disturbed, resulting in decreased reproduction and milk production. Therefore, in order to solve these problems related to protein metabolism, the use of rumen protective protein feed with proper energy supply is essential.

However, these rumen protective proteins are not completely available in the small intestine, whether in natural or processed form. The optimal way to solve the problem of incomplete utilization of expensive rumen protective proteins is to decompose the proteins into peptides in advance. And then protect them. In the present invention, the peptide is formed of a combination of various amino acids and generally refers to 20 kDa or less.

In addition, in recent years, many studies have reported that the availability of peptides to ruminants is superior to that of amino acids. Some dipeptides are absorbed faster or in quantity than the individual amino acids that make up the peptide. In particular, comparing the absorption capacity of peptides and amino acids in the small intestine allows the animal to feed more amino acids than if the peptide absorption rate was given in the form of free amino acids. In fact, 79% of the amino acids in the calf's blood are in the form of peptides, and more than 50% of the amino acids absorbed, regardless of essential or non-essential amino acids, are in peptide form.

Conventional technology for preparing low molecular soybean peptides is described in International Patent WO 97/01966, which has been subjected to proteolytic enzymes or acid degradation using whey isolate protein powder (crude protein content of 90% or more) as a raw material, followed by microfiltration or ultrafiltration. A method for preparing a low molecular peptide having a crude protein content of 80% or more through filtration, reverse osmosis, and electrodialysis has been proposed.

However, the above manufacturing method is essential to prepare soy protein separated from skim soybean meal. The manufacturing process is acid precipitation at pH 4.5 of isoelectric point using hydrochloric acid and neutralization of pH 7.0 using caustic soda and ion purification. Desalination process should be carried out.

Such a manufacturing process is difficult to remove salts generated by using hydrochloric acid and caustic soda, a large amount of wastewater, and a high manufacturing cost due to a complicated process.

In order to solve this problem, Korean Patent Application No. 2003-959777 (Method for preparing low molecular soybean peptides) heat-processes soybean meal with steam at 100-120 ° C to remove protein solubles, remove soluble components such as water, and remove soybean peptides through protease. It was prepared and separated and purified.

However, such a manufacturing process heat treatment of soybean meal at high temperature may negatively affect the protein diffusion index (PDI) and KOH solubility, thereby reducing the protein utilization efficiency in ruminants. When manufacturing costs are expensive.

Therefore, the present invention has been made in order to solve the above problems, the soybean meal protein soybean peptide by enzymatic treatment of inoculation, fermentation or infusion of soybean meal or microorganism or protease with excellent proteinase production ability It is an object of the present invention to provide a method for producing a rumen protective soybean peptide, which is converted to and sprayed with formaldehyde on the soybean peptide, thereby protecting it from rumen microorganisms in the rumen and enhancing the absorption efficiency in the small intestine.

Another object of the present invention is to provide a rumen protective soybean peptide prepared by the above method and used as a feed additive.

Another object of the present invention is to provide a livestock feed containing the rumen protective soybean peptide prepared by the above method as an active ingredient.

The present invention provides a method for preparing soybean peptides through solid phase fermentation of Bacillus subtilis GR-101 (KCCM 10673P), which has excellent protease-producing ability, and confirming a conversion rate of soy protein to soybean peptides; Preparing soybean peptides by treatment with bacterial degrading enzyme alkalase 2.4L and confirming the conversion rate from soy protein to soybean peptides; Spraying formaldehyde on the soybean peptide obtained from the above; In order to evaluate the soybean peptide treated with the formaldehyde In Identifying the rate of building loss in the rumen by the situ method.

In particular, the present invention is inoculated and cultured soybean meal adjusted to the initial water content of 40 ~ 50wt% with Bacillus subtilis GR-101 (KCCM 10673P) having a protease production capacity to prepare soybean peptide In addition, the soybean peptide provides a method of producing a rumen protective soybean peptide, characterized in that by maturing by spraying with formaldehyde 2 ~ 4% (wt / v).

At this time, the Bacillus subtilis ( Bacillus subtilis ) GR-101 (KCCM 10673P) is inoculated and cultured for 70 to 80 hours at a culture temperature of 30 to 40 ° C. and a culture humidity of 70 to 80%, and the ripening of the soybean peptide sprayed with formaldehyde is aged at 70 ° C. It is characterized in that it is carried out for 24 to 48 hours at ~ 80 ℃.

In addition, the present invention is to prepare a soybean peptide by the enzyme treatment to inject 2.4L of bacteria-derived protease Alcalase 2.4 soybean meal to adjust the initial moisture content to 40 ~ 50wt%, to form a formaldehyde to the soybean peptide It provides a method for producing a rumen protective soybean peptide, characterized in that the aging by spraying at 1 ~ 4% (wt / v).

At this time, the enzyme treatment of injecting 2.4L of bacteria-derived protease, alkalase, prepares soybean peptides of 20 kDa or less, which is performed for 1 to 2 hours at a culture temperature of 60 to 70 ° C., and the soybean treated with formaldehyde is sprayed. The maturation of the peptide is characterized in that it is carried out for 24 to 48 hours at the aging temperature 70 ~ 80 ℃.

In addition, the present invention is characterized in that it provides a rumen protective soybean peptide prepared according to the production method used as a feed additive.

In addition, the present invention is characterized by providing a feed for livestock containing the rumen protective soybean peptide prepared according to the production method as an active ingredient.

As described above, the present invention provides excellent protein resolution Bacillus subtilis ( Bacillus subtilis ) Soybean meal is converted to soybean peptide using GR-101 (KCCM 10673P) or Alkalase 2.4L, a bacteria-derived protease, and it is an efficient and inexpensive rumen-protected soybean peptide that is sprayed with formaldehyde and matured. By the manufacturing method of the soybean peptide is not decomposed by the microorganisms in the rumen to be stably moved to the lower digestive system to be absorbed to increase the nutrient utilization efficiency of ruminants to increase productivity.

In addition, the present invention can produce a feed for livestock containing the rumen protective soybean peptide as an active ingredient has an excellent effect of improving the productivity of the livestock. Therefore, the present invention is a very useful invention in the feed industry.

Hereinafter, the specific configuration of the present invention will be described in detail with reference to Examples and drawings, but the present invention is not limited to the following Examples.

EXAMPLE

Example  1: protease Production capacity  Excellent Bacillus  Subtilis ( Bacillus  subtilis ) GR -101 ( KCCM  Soybean by 10673P) Peptide  Conversion and Rumen Protection of Soy Peptides Loss rate  evaluation

Peptide of soybean meal protein was performed using a solid fermentation technique. Three treatment groups were inoculated and cultured with soybean meal protein, Aspergillus oryzae , and fermented product for 72 hours at 37 ° C and 80% humidity. Bacillus subtilis subtilis ) Fermented product inoculated and incubated with GR-101 (KCCM 10673P) for 72 hours at 37 ° C. and 80% humidity, and Aspergillus oryzae and Bacillus subtilis subtilis ) GR-101 (KCCM 10673P) was inoculated and cultured 1: 1, and the peptide conversion rate of the fermentation product fermented at a culture temperature of 37 ° C. and a culture humidity of 80% for 72 hours was investigated.

Generally, the peptide may be formed by a combination of various amino acids, but since the utilization efficiency of the peptide of 20 kDa or less is high in ruminants, the peptide in the present invention refers to 20 kDa or less.

In order to investigate the conversion rate of the soybean peptide, a tricin SDS-PAGE method and Experion 206 were used, and the results are shown in FIG. 1. In FIG. 1, SBM represents stripped soybean meal, AO represents Aspergillus duck, and BA represents Bacillus subtilis GR-101 (KCCM 10673P).

As can be seen in Figure 1, Bacillus subtilis ( Bacillus subtilis ) GR-101 (KCCM 10673P) of the present invention The treatment group formed the most peptides of 20 kDa or less, and the results of the quantification using Experion 260 are shown in Table 1.

 Peptide Conversion Rate of Soybean Meal by Fermentation of Microorganisms with Protease Production Capacity (%) 20kDa or less 20 ~ 60kDa 60kDa or more Stripping Soybean Meal 1.2 82.2 16.6 Aspergillus Duck 1.9 97.9 0.3 Bacillus subtilis GR-101 45.6 54.2 0.2 Aspergillus duck + Bacillus subtilus GR-101 3.8 96 0.2

Subsequently, the Bacillus subtilis ( Bacillus) subtilis)-GR 101 (KCCM 10673P After aging in) the soybean peptide 80 ℃ treated spraying formaldehyde or glutaraldehyde at different concentrations for 24 hours in the production by the In The loss rate of situ buildings was investigated. That is, the chemical treatment of the soybean peptide using the formaldehyde or glutaraldehyde, Bacillus subtilis ( Bacillus subtilis ) 1% (wt / v), 2% (wt / v), 4% (wt / v) of formaldehyde or glutaraldehyde in fermented products fermented by inoculation and culture of GR-101 (KCCM 10673P). It means sparging and aging at a temperature of 80 ° C. for 24 hours. Table 2 shows the results of this chemical treatment and In situ dryness loss.

In The method of measuring situ building loss rate is as follows. 2 g of each sample was placed in a nylon bag, the inlet was sealed, and placed in the rumen of the ruminant immediately before feeding the morning food, followed by incubation for 72 hours. The incubated nylon bag was removed from the rumen fistula, immersed in iced water and washed, and dried in a 60 ° C. dry oven for 48 hours.

The dried samples were stored at 4 ° C. for component analysis. In After completion of the situ incubation, dry matter loss rate is calculated using a simple exponential model according to the method of Ørskov and McDonald (1979). The equation is

P = a + b (1-e ^{- kdt} )

P: loss of nutrients at time t

a: Nutrients that are easily dissolved (dissolved amount at 0 hours,%)

b: Digestible insoluble nutrients (degraded amount from 0 hours before reaching the lag phase,%)

t: incubation time

k _d : dissipation rate constant (hourly dissipation rate of b, h-1)

The digestibility of each sample was measured while staying in the rumen by the following equation using the k _d value calculated by regression analysis of the above result using SAS statistical analysis. And this database construction was calculated by applying the rumen pass rate of 5% (0.05 h-1) per hour feed.

ED = a + b × [k _d / (k _d + k _p )]

ED: rumen nutrient digestibility

a: Nutrients that are easy to dissolve (, dissolved amount at 0 hours,%)

b: Digestible insoluble nutrients (degraded amount from 0 hours before reaching the lag phase,%)

k _d : dissipation rate constant (hourly dissipation rate of b, h-1)

k _p : Rumen pass rate constant

Bacillus subtilis in subtilis)-GR 101 (KCCM 10673P) after fermentation and chemical treatment protected soybean peptide In situ building loss rate (%) Treatment method Treatment Concentration (% (w / t)) a b k _d ED (0.05) Bacillus subtilis GR-101 fermented soybean meal (without chemical treatment) 39.5 55.4 0.140 80.3 Formaldehyde Treatment after Fermentation of Bacillus Subtilis GR-101 One 27.8 71.7 0.131 79.8 2 24.1 73.8 0.103 73.7 4 23.6 74.8 0.062 65.2 Glutaraldehyde Treatment after Bacillus subtilis GR-101 Fermentation One 33.3 62.8 0.202 83.6 2 33.8 64.2 0.175 83.7 4 20.9 70.4 0.175 75.7

As the treatment concentration of formaldehyde increased, the values of a, k _d (loss rate constant) and ED (ruminant nutrient digestibility) decreased. In particular, only Bacillus subtilis, without chemical treatment with formaldehyde subtilis (Bacillus subtilis ) soybean meal fermented by GR-101 (KCCM 10673P) has a high ED (rumen nutrient digestibility) value of 80.3, whereas the fermentation is sprayed with formaldehyde at 2-4% (wt / v). In the case of ripening, the ED value was significantly reduced from 73.7% to 65.2%.

That is, Bacillus subtilis ( Bacillus subtilis ) soybean meal fermented by GR-101 (KCCM 10673P) was sprayed with formaldehyde 2-4% (wt / v) and aged at 80 ° C. for 24 hours, soybean peptides compared to the other cases. It can be seen that is much more protected from microorganisms in the rumen.

Example  2: Protected Soybean by Protease Peptide  Transition and Protection Soybeans Peptide Loss rate  evaluation

Enzymatic treatment was performed by injecting 2.4L of bacteria-derived alkalase 2.4L into 0.01% (wt / wt), 0.1% (wt / wt) and 1% (wt / wt) into the peeled soybean meal. After the temperature was corrected to 60 ° C., the reaction rate was investigated for 1 hour, and then the conversion rate of the soybean peptide was examined. In order to investigate the conversion rate of soybean protein to soybean peptide by Alkalase 2.4L, Tricin SDS-PAGE method and Experion 206 were used as in Example 1, and the results are shown in FIG. 2. In FIG. 2, SBM represents peeled soybean meal and Ala represents alkalize.

In addition, the results of FIG. 2 are shown in Table 3 numerically using the Experion 260. As can be seen in Table 3 below, the peeled soybean meal that had not been treated with Alkalase 2.4L, which is a bacterial proteinase, had only 1.2% conversion to a peptide of 20 kDa or less, whereas the alkaline soybean meal contained in the peeled soybean meal. When 0.1L (0.1% (wt / wt)) of Izu was treated, the conversion rate to peptide of 20kDa or less was 47.6%, and 20LD or less when Alkalase 2.4L was treated at 1% (wt / wt) concentration in peeled soybean meal. Conversion to the peptide increased significantly to 66.6%.

Peptide Conversion Rate of Peeled Soybean Meal Treated with Alkalase 2.4L Treatment Concentration (% (wt / wt)) 20kDa or less 20 ~ 60kDa 60kDa or more Stripped Soybean Meal (SBM) 1.2 82.2 16.6  Alcalase 2.4L 0.01 10.2 80.0 9.7 0.1 47.6 52.3 0.1 One 66.6 33.3 0.1

According to Table 3, when the enzyme was treated by injecting 2.4L of alcalase in a concentration of 1% (wt / wt) into the peeled soybean meal, the conversion to soybean peptide was the highest. In practice, however, the best peptide conversion to price was achieved by treating Alcalase 2.4L at a concentration of 0.1% (wt / wt).

Therefore, soybean peptide prepared by enzymatic treatment by injecting 2.4L of bacteria-derived protease Alcalase 2.4L into 0.1% (wt / wt) concentration in peeled soybean meal, formaldehyde or glue as in the method described in Example 1 above. after the spraying process the tar aldehyde concentration by aging at 80 ℃ for 24 hours to investigate the disappearance in situ building. Table 4 shows the results.

Loss rate of in situ dryness of protective peptides chemically treated with Alkalase 2.4L (0.1%), a bacteria-derived protease Treatment method Treatment Concentration (% (wt / v)) a b k _d ED (0.05) Alcalase 2.4L (0.1%) Fermented Soybean Meal (No Chemical Treatment) 16.9 80.5 0.244 83.7 Formaldehyde treatment after fermenting Alcalase 2.4L (0.1%) One 12.2 54.8 0.136 52.3 2 12.1 51.9 0.102 46.9 4 10.6 35.8 0.114 35.5 Glutaraldehyde Treatment after Alcalase 2.4L (0.1%) Fermentation One 13.4 72.1 0.126 65.0 2 11.3 69.6 0.084 54.9 4 11.4 48.9 0.082 41.7

As the concentration of formaldehyde increased, the values of a, k _d (loss rate constant) and ED (ruminant nutrient digestibility) decreased. In particular, fermented soybean meal fermented with Alcalase 2.4L at a concentration of 0.1% (wt / wt) without chemical treatment with formaldehyde has a high ED (ruminant nutrient digestibility) value of 83.7, whereas the fermentation Treatment of formaldehyde with water at 1-4% (wt / v) significantly reduced ED (ruminant nutrient digestibility) values from 52.3% to 35.5%.

That is, the fermented product of soybean meal fermented with Alcalase 2.4L, a bacteria-derived protease, was sparged with formaldehyde 1-4% (wt / v) and aged at 80 ° C. for 24 hours. It can be seen that soybean peptides are much more protected from the microorganisms in the rumen.

1 is a trisine SDS-PAGE photograph showing the peptide conversion rate of soybean meal according to the fermentation treatment of microorganisms having protease production capacity, and

2 is a tricine SDS-PAGE photograph showing the peptide conversion rate of soybean meal according to the concentration of alkalase treatment.

Claims (6)

delete delete Enzymatic treatment of injecting 2.4 liters of alkalase, a bacteria-derived proteolytic enzyme, into soybean meal with an initial moisture content of 40-50 wt% was carried out at a temperature of 60-70 ° C. for 1-2 hours. Preparing soybean peptide to increase the content of soybean peptide of 20 KDa or less in the soybean peptide, Formaldehyde treatment of soybean peptide with 1 to 4% (wt / v) and aging for 70 to 80 hours at a aging temperature of 70 to 80 ℃ for the preparation method of the rumen protective soybean peptides. delete Rumen protective soybean peptide prepared according to claim 3 and used as feed additive. Animal feed containing the rumen protective soybean peptide prepared according to claim 3 as an active ingredient.

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