KR20000010060A - Phytase-secreting lactic acid bacterium - Google Patents

Abstract

PURPOSE: A phytase-secreting lactic acid bacterium is provided which can catalyze the release of inorganic phosphorous from phytic acid, an anti-nutritional component to livestock such as chickens or pigs. CONSTITUTION: Lactobacillus paracasei subsp. paracasei (KFCC-11042) showing high phytase activity is isolated from livestock intestinal tract and produced on a large scale, which can be useful as direct-fed-microbes or strains for producing animal fermentation feed.

Description

Phytate secretion lactic acid bacteria

The present invention relates to lactic acid bacteria excellent in the decomposition and lactic acid production of phytic acid, and more particularly, the production of acid, including lactic acid separated from the intestinal tract of livestock, and anti-nutritional factors of unit livestock feed such as chicken and pig The present invention relates to a lactic acid bacterium showing the resolution of inphytic acid.

Phytic acid is a substance in which six molecules of Inositol is combined with six molecules of Phosphorus of anion. It contains 0.14 ~ 7.50% by weight depending on the kinds of grains, seed oil, legumes, and protein by-products of livestock feed. According to the anionic nature of phosphorus, it exists in the state of being combined with cationic groups such as proteins, fats, and starches as well as minerals of cations important for livestock such as zinc, iron and magnesium. In addition, about 30 to 70% by weight of phosphorus contained in such feedstock is present in the form of phytic acid.

In the case of unit livestock such as chicken and pig, the secretion of phytate, an enzyme that breaks down the phytic acid in the intestine, is very low, and the availability of livestock of phosphorus bound to phytic acid is very low. At the same time, the availability of major minerals and proteins is low.

Phytase is known to be present in or secreted from plants and microorganisms, and industrially, phytase is used. Pytease-producing microorganisms currently used are mainly produced using Aspergillus ssp. Fungi and Bacillus ssp. Bacteria such as Aspergillus ficuum.

However, as with other enzyme production, many facilities and manpower are required to obtain phytize from microorganisms, so there is a problem in terms of cost to use it as an animal feed additive.

Therefore, the researchers have isolated the lactic acid bacteria that show the lactic acid production and phytic acid decomposition ability to solve the above problems from the intestinal tract of the livestock, and can feed the livestock as a strain for producing fermented feed and livestock probiotic strains. Its purpose is to improve economic efficiency by improving efficiency.

1 is a lactic acid by Lactobacillus paracasei subsp. Paracasei (LPP), the lactic acid bacterium of the present invention in broccoli (BCP) and phytase search medium. Photograph showing production (A) and phytic acid degradation (B).

Figure 2 is a figure showing the phytase activity of the Lactobacillus paracasei subspis paracaze of lactic acid bacteria of the present invention

Hereinafter, the decomposition of phytic acid and lactic acid production according to the present invention will be described in detail with respect to the novel lactic acid bacteria.

The present invention relates to a lactobacillus paracase, which is excellent in phytic acid degradation and lactic acid production, and to subspis paracasei.

The strain of Lactobacillus paracasein subspis paracasei lactic acid bacterium of the present invention is collected from the young livestock fecal and grafted in physiological saline (0.85% by weight NaCl), and then isolates strains that turned yellow using a BC medium first Then, it was plated again in a phytease screening medium and incubated at 37 ° C. for 24 hours. Secondly, 5 strains showing growth and phytase production were selected secondly, and the whey medium (lactose, 109 / L; whey powder, 10g / L; Sodium acetate, 5g / L; Sodium phytate, 0.6g / L) was incubated for 48 hours at 37 ℃, 150rpm and measured to select the final 1 strain with the best pH change, growth and phytic acid degradation You get

The strain of the present invention has been deposited with the Accession No. KFCC-11042 by the Korean spawn association on July 13, 1998.

In addition, the following experiment was carried out to determine the properties of the lactobacillus paracase, the subspec Paracasei, the lactic acid bacterium of the present invention.

① Measurement of phytase activity of LLP:

In a shaker bath at 40 ° C, lactic acid bacteria culture medium was added to a solution containing 0.1 M sodium acetate buffer adjusted to pH 5.5 and phytic acid to a final concentration of 1 g / L. The reaction was performed by adding hydrochloric acid to a final concentration of 1 M, followed by centrifugation at 1,800 × g for 10 minutes to determine free phosphate activity of the supernatant. In this experiment it was shown that the amount of free phosphorus increases as the incubation time elapsed (FIG. 2).

② Resistance to acid and bile:

Acid and bile resistance tests were performed according to the methods of Lee and No (1997).

1% (w / v) of pepsin (1: 10,000) was added to MRS liquid medium adjusted to pH 2.5 using HCl, which was used as artificial gastric juice. After incubation for 24 hours at 37 ℃, 170rpm and centrifuged at 3,000 × g for 10 minutes, the supernatant was discarded, the cells were recovered and the pH 2.5 solution maintained at 37 ℃ was added in the same amount as the supernatant and incubated for 3 hours at 37 ℃, After incubation, the number of bacteria was measured by smearing on BCP medium, and the number of bacteria before culture was compared.

Artificial bile solution is sterilized by adding 1% (w / v) pancreatin to MLS liquid medium, and then adding sterile 10% (w / v) oxagall solution to 1% (w / v) of the medium. PH was adjusted to 6.8 and used. Resistant lactic acid bacteria against artificial bile were incubated for 3 hours in artificial gastric juice, centrifuged at 3,000 × g for 10 minutes, discarded supernatant, and only cells were recovered. Bacterial counts were measured by smearing on a medium of B. medium, and in order to compare acid resistance and bile resistance under these experimental conditions, Lactobacillus GG. Isolated from commercial yogurt known to be excellent in acid resistance and bile resistance was used as a reference. Compared.

Compared with the strains known to be excellent in acid and bile resistance in this experiment, the bacterium according to the present invention showed a high bacterial count even under conditions with low pH and bile acids. 4).

③Pure culture using Lactobacillus paracasei subspis paracasei:

5 g of ground soybean meal, rice bran and wheat bran and 45 ml of distilled water were mixed in a 250 ml Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes, followed by incubation for 24 hours with Lactobacillus paracasei subspis Paracazei 11% ( v / v) and then incubated at 37 rpm for 24 hours at 150rpm to analyze the pH, phytic acid content of the raw material feed, phosphorus content released into the supernatant and water-soluble protein content of the supernatant.

As a result of this experiment, except for phytic acid content of rice bran, it was slightly different for each feed, but with decreasing pH, phytic acid content and mineral phosphorus and water soluble protein contents of supernatant decreased.

④ Piglet Specification Test:

Pigs were fed to the hog feed with Lactobacillus paracasei subspis paracaze at a level of 0.4% (w / w). As shown in Table 7, the results of this test did not affect the weight, but the improvement of feed demand (feed intake / weight) was found and 0.4% (w / w) zeolite was used as an excipient of lactic acid bacteria. Compared with the reference group, the weight gain and feed rate were not different from the control group, and the feed rate improvement effect of the 0.4% LPP treatment group was due to the lactobacillus paracasei subspis paracasei, a lactic acid bacterium of the present invention. It is considered to be.

Hereinafter, the present invention will be described in more detail with reference to Examples.

<Example 1>

○ Isolation and Identification of Strains

This strain was prepared by diluting fecals from several young livestock with sterilized saline solution (0.85 wt.% NaCl) and using Bissippi medium, which is a medium for measuring the number of lactic acid bacteria. The strains excellent in lactic acid production were first isolated, and then spread on a phytate screening medium (Table 1) at pH 5.5 to incubate at 37 ° C. for 24 hours at 5 ° C. to show growth and phytase production (transparent ring). Secondarily, the cells were incubated at 37 ° C. and 150 rpm for 48 hours using whey medium (lactose, 10 g / L; whey powder, 10 g / L; sodium acetate, 5 g / L; sodium phytate, 0.6 g / L). The final 1 strain was then selected by measuring pH, growth and phytic acid degradation.

Identification of the selected strains was carried out in accordance with the Burgy's Mannual of Systematic Bacteriology (Kandler and Weiss, 1986) by examining the morphological, physiological and biochemical properties of the strains. The medium was stored in the culture medium, and the liquid medium was cultured as the basic culture medium.

The growth of microorganisms was determined by measuring absorbance (O.D.) at 535 nm using a spectrophotometer (Kontron, Model No. 922).

Table 1 Composition of the Pietease Screening Medium

The phytic acid content was analyzed by the method of Hug and Ranch (Lantzch) (1983), and the phosphorus (P) content was analyzed by the same method as the phosphorus content analysis in the enzyme analysis according to the method of Shimizu (1992). It was.

After selection of 4 strains showing growth and phytic acid clear zone in the calcium-phytate-containing medium using lactic acid production and phytase screening medium primarily 6-5 strains with the best pH change, growth and phytic acid resolution in whey medium were selected as the final strain (Figure 1) (Table 2).

Shiral et al. (1994) found that the decrease in the content of phytic acid in the medium by lactic acid bacteria was affected not only by the metabolism of the strain but also by the pH lowered as a result of the lactic acid bacteria metabolism. The lower the pH value and the better the growth, the higher the phytic acid degradation rate.

Finally, the selected strains were examined using microscopy, similar production on BAC medium, Gram staining, catalase test and API 50 CHL kit (bio Merieux Vitek, Inc.) as shown in Table 2. As a result of the identification through 50 kinds of sugar availability tests, it was identified as Lactobacillus paracasei subspecs paracasei, the lactic acid bacterium of the present invention.

Table 2 After 48 hours of incubation in whey media of primary selection strains

Table 3 Biochemical Properties of Selected Strains

<Example 2>

○ Measurement of phytase activity of LPP

The activity was measured in a shaker bath at 40 ° C. The reaction was carried out in a solution containing lactic acid bacteria in a solution containing 0.1 M sodium acetate buffer adjusted to pH 5.5 and phytic acid added to a final concentration of 1 g / L. The reaction was terminated by adding 6M hydrochloric acid at a final concentration of 1M at 15 minute intervals, followed by centrifugation at 1,800 × g for 10 minutes, and the free phosphorus content of the supernatant was measured to become phytate activity. .

The result is shown in FIG.

<Example 3>

○ Acid and bile resistance test

Acid and bile resistance tests were performed according to the method of Lee and No (1997). Artificial gastric juice was used by adding 1% (w / v) of pepsin (Sigma, 1: 10,000) to the liquid liquid medium adjusted to pH 2.5 using hydrochloric acid (HCl). Incubate twice at 37 ℃ for 24 hours at 170rpm, centrifuged at 3,000 × g for 10 minutes, discard the supernatant, recover the cells, and add pH 2.5 solution maintained at 37 ℃ in the same amount as the supernatant. After incubation, the cultures were plated on BC medium and the number of bacteria was measured.

Artificial bile solution was sterilized by adding 1% (w / v) pancreatin to MLS liquid medium, and then sterile 10% (w / v) solution of oxagall (manufactured by Difco) in 1% (w / v) v) was added to adjust the pH to 6.8. Resistant lactic acid bacteria against artificial bile were incubated for 3 hours in artificial gastric juice, centrifuged at 3,000 × g for 10 minutes, discarded supernatant, and only cells were recovered. The number of bacteria was measured by smearing on a medium medium, and lactic acid bacteria isolated from commercial yogurt known to be excellent in acid and bile resistance were compared for reference in order to compare acid resistance and bile resistance under these experimental conditions.

As a result of acid and bile resistance test of the final selected Lactobacillus paracasei subspis paracaze, compared with strains known to be excellent in acid and bile resistance under these test conditions, as shown in Table 4 It has been shown to have excellent bile resistance, which may be effective in the intestines of livestock.

According to Lee and Noh (1997), the lactic acid bacteria found in the pH range of 3.0-4.0 during kimchi fermentation are generally resistant to acids. When fermentation was carried out at 37 ° C. at 170 rpm for 24 hours on MRS broth, the pH was 3.76 and 3.80, respectively, and the number of bacteria was 5.1 × 109 and 1.2 × 10 ¹⁰ CFU / mL, respectively. Able to know. Therefore, when primary strains were selected for excellent acid resistance, it was determined that selecting a strain reaching a pH range of 3.0 to 4.0 after incubation for a predetermined time (24 hours).

Table 4 Lactobacillus in artificial gastric juice (pH 2.5) and artificial bile (pH 6.8)

^{a, b} Different superscript means that the mean value between lines is significantly different

¹ Standard error of the mean (*, P <0.05; ***, P <0.001)

² LPP-Lactobacillus paracasei subspecosis paracasei

³ Reference Sphere-Lactobacillus GG. Isolated from Commercial Yogurt

<Example 4>

○ Pure culture using Lactobacillus paracasei subspis Paracasei

In order to investigate the effect of pure culture using Lactobacillus paracasei subspis paracaze on phytic acid content and phosphorus decomposition of raw feed, the 250ml Erlenmeyer flask was crushed in a 1 mm sieve using a Wiley mill. 5 g of soybean meal, rice bran and bran, respectively, and 45 ml of distilled water were mixed and sterilized at 121 ° C. for 15 minutes, followed by inoculation of 11% (v / v) of Lactobacillus paracasei subspis Paracaze with starter for 24 hours. Incubation at 150 rpm for 24 hours at 37 ℃ to analyze the pH, phytic acid content of the raw material feed, phosphorus content released into the supernatant and water soluble protein content.

The phosphinic acid content of the feed was dried and pulverized until the content was reached at 60 ° C, and analyzed by the method of Haug and Lantzch (1983). The phosphorus content released into the supernatant was 8,000 in culture. Phosphorus content of the supernatant obtained by centrifugation at × g for 5 minutes was analyzed by the method of Shimizu (Shimizu. 1992), and the water-soluble protein content of the supernatant was analyzed according to the Bradford method (1976).

Inoculated with sterile soybean meal, rice bran and bran inoculated with Lactobacillus paracasei subspis paracazei and cultured for 24 hours. And the content of inorganic phosphorus and water soluble proteins in the supernatant (Table 5).

According to Khetarpaul and Chauhan (1989), fermentation of pearl millet using lactic acid bacteria resulted in a decrease in phytic acid content and inorganic phosphorus content of pearl millet. The increase is said to be the result of the degradation of phytic acid. The results showed that the inorganic phosphorus content was decreased by the fermentation of lactic acid bacteria because the inorganic phosphorus content of the culture supernatant was used in large part by the lactic acid bacteria, and the decrease in the water-soluble protein content was also due to the decomposition and use by the lactic acid bacteria. It became.

Reduction of phytic acid content by fermentation depends on the type of raw materials besides the treatment method. According to de Boland et al. (1975), the rate of reduction of phytic acid in various grains according to sterilization time was determined. Wheat decreased only 17% and 77% of phytic acid, respectively, but only 5% of rice, which is estimated to be due to differences in protein and cationic environment in raw materials.

In this study, phytic acid reduction rates of soybean meal, rice bran and bran were 27.07, 0 and 12.18%, respectively. Judging from the location of the distribution and the structural differences in the feed, it is necessary to take into account the difference of phytic acid decomposition in the production of fermented feed using lactic acid bacteria.

In addition, in order to accurately confirm the improvement of utilization efficiency by fermentation, pH, protein and starch digestibility in vitro and in vivo experiments should be performed. In consideration of the improvement of mineral availability and protein and starch digestibility, it is considered a preferable method to use phytic acid content change as an indirect index with pH measurement.

Table 5 Effects of Lactobacillus paracasei subspecs paracasease on phytic acid content and phosphate release to supernatant and soluble protein content of soybean meal, rice bran and bran fermentation (mean ± standard deviation)

<Example 5>

○ Piglet Specification Test

Strains incubated on MS medium were inoculated in MS liquid medium and shaken for 18 hours at 37 ° C. and 150 rpm as a starter. % (v / v) inoculation and were prepared by drying 24 hours incubation and then mixed with a zeolite as an excipient 12 hours blowing at 45 ℃, wherein lactic acid bacteria can have non-see, the results measured using a medium 2.0 × 10 ⁷ CFU / g Contained.

In this experiment, a total of 30 heads of four-month-old boars at 4 months of age were treated by the control group (control: commercial hog feed, treatment: hog feed + zeolite and LPP 0.4% (w / w), and reference: hog feed + zeolite). 0.4 (w / w)] and 10 heads were tested for a total of 45 days. At this time, the formula and chemical composition of the salary feed are shown in Table 5, and the salary feed was twice a day (07:00, 18:00 hours). Unlimited salary.

The obtained results were analyzed by variance analysis using the GLM procedure (SAS, 1985), and the significance test of the mean value for each treatment was Duncan's multiple range test (Duncan's multiple range test; Duncan, 1955).

Table 6 Formulation and Chemical Composition of Test Feed (Wet Basis)

¹ ㎏ sugar content: vitamin A, 4,400 IU; Vitamin E, 44 IU; Pantothenic acid, 40 mg; Niacin, 40 mg; Riboflavin, 40 mg; Vitamin B ₁₂ , 0.04 mg; Biotin, 2.8 mg; Pyridoxine, 4.0 mg; Hydrochloric acid, 0.9 mg; Thiamine, 3.9 mg; Choline, 600 mg, 600 mg, selenium, 0.5 mg; Manganese, 0.08 g; zinc. 0.2 g; iron. 0.2 g; Copper, 0.02 g; Potassium, 0.43 g; Iodine, 0.2mg

² calculations

As a result of feeding the lactobacillus paracasei subthress paracasei, the lactobacillus of the present invention at 0.4% (w / w) level in the feed for pigs, did not affect the weight gain as shown in Table 7, In the case of (feed intake / weight gain), the improvement effect was shown and 0.4% (w / w) zeolite was used as an excipient of lactic acid bacterium, so it was compared with reference. Feed-through rate improvement effect of 0.4% LPP treatment is believed to be due to the lactobacillus paracasei subspis paracasei, the lactic acid bacteria of the present invention contained.

However, due to the nature of the farm, it is difficult to identify individual feed intake and fecal phosphorus excretion due to the characteristics of the farm, and compared only the productivity of each treatment group. It is obvious that the treatment effect is recognized. After all, the improvement of feed demand means that the utilization of nutrients is increased. This is because the utilization efficiency of phosphorus, minerals, protein and starch has increased due to the increase of phytic acid degradation rate. In addition, it is believed that the digestibility is increased by increasing the activity of the digestive enzymes and the enzymes produced by the intestinal microorganisms due to the decrease in intestinal pH.

According to Nahashon et al. (1994), the rate of phosphorus accumulation was improved in the diets fed low-level (0.25%) phosphorus-containing diets by feeding lactobacillus as an excipient for laying hens. This was attributed to the increased absorption of phosphate by the phytic acid activity of molasses and lactic acid bacteria and the decrease in pH of the vesicles and small intestine contents.

In general, the effect of increasing lactic acid bacteria and improving feed efficiency is known to be due to the promotion of digestive enzyme activity and the maintenance of normal flora of the intestinal pH, and the phytate activity of lactic acid bacteria reported so far is very low. Et al. (1994) showed that both molasses and mixtures of molasses and lactic acid bacteria exhibited phytase activity, and that the mixture of molasses and lactic acid bacteria had higher phytase activity. Molasses is slightly acidic (pH 5.0) and varies depending on the strain in the case of lactic acid bacteria, but in the case of the culture medium, pH 3.0-4.5 is considered, and the phytase activity is considerably lower in pH (3.0-5.0) and pH lowering. According to Shiral et al. (1994), the pH was lowered during fermentation of lactic acid bacteria in whey medium containing phytic acid. Given the fact that the reduction of phytic acid contained in a whey medium by lowering the pH with an acid Sikkim effect appears phytic acid the decomposition effect of the lactic acid bacteria was that the effect of significantly lowering pH.

In conclusion, the effects of the fermentation of raw material feed and improvement of feed demand by lactic acid bacteria are considered to be due to the effect of lowering of pH due to the fermentation of lactic acid bacteria of the present invention. It can be said that the use of Lactobacillus paracasei subspis paracaze, the lactic acid bacterium of the present invention, which has excellent bile resistance, suggests the possibility of simultaneously obtaining probiotic and phytase addition effect. The availability of phosphorus varies depending on age and feed, but averages about 34% of phytic acid-phosphorus are available (Anon, 1984; Fisher, 1983). In view of the fact that a large part of phytic acid-phosphorus is broken down by intestinal phytase and endogenous phytase in feed, The use of phytic acid-phosphorus by adding lactic acid bacteria of the present invention to pig feed may be improved due to the degradation of phytic acid-phosphorus due to pH decrease in the intestinal tract due to the addition of lactic acid bacteria and the promotion of endogenous phytate of feed.

In the case of chickens, the availability of phytic acid-phosphorus is generally lower than that of pigs, which is considered to be due to relatively short digestion time and low intestinal phytase activity. Since the lowering effect can be obtained, it promotes the degradation of phytic acid and the activity of endogenous phytase in the feed according to the pH decrease, thereby increasing the phytic acid-phosphorus decomposition rate, thereby inhibiting the activity of phytic acid by phytic acid amylase and intestinal digestion It is thought that the efficiency of feed can be increased by increasing the activity of enzyme.

In conclusion, it is necessary to verify the effectiveness of feed efficiency and phosphorus excretion into feces compared with phytize treatment, but the acid resistance and bile resistance obtained from livestock feces obtained in this study were excellent and the pH drop rate during fermentation was excellent. In the case of Lactobacillus paracasei subspis paracasei, the lactic acid bacterium of the present invention, it is considered that probiotic and phytase addition effects can be simultaneously obtained.

Table 7 Effect of Lactobacillis paracasease subspace paracasease on feed conversion and mean weight gain in finishing pigs

Lactobacillus paracasei subsp. Paracasei of the present invention is a lactic acid bacterium excellent in phytic acid degradation and lactic acid production. It increases the utilization efficiency of starch, and together with the digestion efficiency according to the intestinal pH decrease and the activity of the enzymes produced by the intestinal microorganism to increase the digestion rate. The effects of improving feed efficiency and controlling disease can be enhanced.

Claims (1)

Lactobacillus paracasei subsp. Paracasei (Accession No. KFCC-11042) Lactobacillus, characterized by excellent resistance to acidic pH and bile acids, phytic acid degradation and lactic acid production.