U.S. patent application number 15/745087 was filed with the patent office on 2019-01-17 for feed additive comprising bacillus subtilis and bacillus licheniformis, a feed composition comprising the feed additive and a method for producing the feed additive.
This patent application is currently assigned to CJ CHEILJEDANG CORPORATION. The applicant listed for this patent is CJ CHEILJEDANG CORPORATION. Invention is credited to Yu Jin KIM, Eun Seon OH, Min Ah PARK, Seo Hyung WOO.
Application Number | 20190014795 15/745087 |
Document ID | / |
Family ID | 65002439 |
Filed Date | 2019-01-17 |
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United States Patent
Application |
20190014795 |
Kind Code |
A1 |
OH; Eun Seon ; et
al. |
January 17, 2019 |
FEED ADDITIVE COMPRISING BACILLUS SUBTILIS AND BACILLUS
LICHENIFORMIS, A FEED COMPOSITION COMPRISING THE FEED ADDITIVE AND
A METHOD FOR PRODUCING THE FEED ADDITIVE
Abstract
The present invention relates to a feed additive comprising a
Bacillus subtilis strain and a Bacillus licheniformis strain, a
feed composition comprising the feed additive, and a method for
producing the feed additive.
Inventors: |
OH; Eun Seon; (Seongnam-si,
KR) ; KIM; Yu Jin; (Suwon-si, KR) ; PARK; Min
Ah; (Suwon-si, KR) ; WOO; Seo Hyung;
(Suwon-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CJ CHEILJEDANG CORPORATION |
Seoul |
|
KR |
|
|
Assignee: |
CJ CHEILJEDANG CORPORATION
Seoul
KR
|
Family ID: |
65002439 |
Appl. No.: |
15/745087 |
Filed: |
August 4, 2017 |
PCT Filed: |
August 4, 2017 |
PCT NO: |
PCT/KR2017/008446 |
371 Date: |
January 15, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12R 1/10 20130101; A23K
50/10 20160501; A23K 10/16 20160501; A23K 10/18 20160501; C12R
1/125 20130101 |
International
Class: |
A23K 10/18 20060101
A23K010/18; C12R 1/125 20060101 C12R001/125; C12R 1/10 20060101
C12R001/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2017 |
KR |
10-2017-0088646 |
Claims
1. A feed additive for enhancing milk fat and milk yield comprising
a Bacillus subtilis strain and a Bacillus licheniformis strain.
2. The feed additive according to claim 1, wherein the Bacillus
subtilis strain is Bacillus subtilis CJBS62 (KCCM12039P).
3. The feed additive according to claim 1, wherein the Bacillus
licheniformis strain is selected from the group consisting of
Bacillus licheniformis CJBL215 (KCCM12040P) and Bacillus
licheniformis CJBL219 (KCCM12041P).
4. The feed additive according to claim 1, wherein the Bacillus
subtilis strain and the Bacillus licheniformis strain are each
independently present at a concentration of at least
1.times.10.sup.7 cfu per gram of the feed additive.
5. The feed additive according to claim 1, wherein the Bacillus
licheniformis strain produces acetate when cultured in a
lactate-containing medium for 9 to 48 hours and converts 30% to 70%
of the initial amount of lactate to acetate.
6. The feed additive according to claim 1, wherein the Bacillus
subtilis strain and the Bacillus licheniformis strain are blended
in a weight ratio of 1:9 to 9:1.
7. A feed composition comprising the feed additive according to
claim 1.
8. A feed composition comprising the feed additive according to
claim 2.
9. A feed composition comprising the feed additive according to
claim 3.
10. A feed composition comprising the feed additive according to
claim 4.
Description
TECHNICAL FIELD
[0001] The present invention relates to a feed additive comprising
a Bacillus subtilis strain and a Bacillus licheniformis strain, a
feed composition comprising the feed additive, and a method for
producing the feed additive.
BACKGROUND ART
[0002] Forage for ruminants, such as cattle, accounts for about 20%
of feeds for beef cattle and about 60% of feeds for dairy cattle.
After ingestion, forage is degraded by microorganisms and essential
nutrients and energy are absorbed in the rumen, also known as the
first stomach. From a nutritional/physiological point of view,
ruminants ingest considerable amounts of protein, energy, fatty
acids, minerals, and vitamins, which are essential to
microorganisms inhabiting the rumen and the tissues of the animals,
from forage. Forage takes the form of fresh grass, dry grass or
silage. Dry forage contains .gtoreq.20-30% of crude fiber. Forage
is composed of hemicellulose, lignin, and cellulose that are
relatively difficult to degrade. A great deal of research has been
conducted on methods for increasing the digestibility of forage to
enhance the value of feeds. Representative examples of such methods
include physical treatment methods (immersion, pulverization,
pressure steam treatment, expansion, gamma-ray irradiation, and
pelletizing), chemical treatment methods (sodium hydroxide, urea,
ammonia, lime, calcium hydroxide, potassium hydroxide, sodium
carbonate, chlorine, ozone, hydrogen peroxide, etc.), and
biological treatment methods (fermentation, enzymatic modification,
and silage, etc.). However, the physical treatment methods involve
high processing costs. The chemical treatment methods incur
increased processing costs and pose danger during handling. The
chemical treatment methods also cause soil contamination or affect
the normal physiological functioning of animals. For these reasons,
use of the physical and chemical treatment methods is avoided.
Thus, biological treatment methods based on the use of cellulolytic
enzymes or the addition of microorganisms producing large amounts
of such enzymes are mainly used. Particularly, much research has
focused on increasing the degradation rate of cellulose.
[0003] Forage is fed in combination with concentrated feeds to
dairy cattle and beef cattle to replenish a necessary amount of
energy and to improve the productivity of milk and beef.
Concentrated feeds are small in volume and high in energy content.
Concentrated feeds are easily digestible compared to forage.
However, feeding of large amounts of concentrated feeds increases
the concentration of lactate in the course of starch degradation,
leading to a significant decrease in the rumen pH. The decreased
rumen pH adversely affects the growth of beneficial microorganisms
and leads to acidosis, causing reduced feed intake, maldigestion,
severe hypohydration, and diarrhea.
[0004] There is thus a need to develop a feed additive highly
capable of degrading cellulose and lactate.
PRIOR ART DOCUMENTS
Patent Documents
[0005] Korean Patent No. 10-1721900
DISCLOSURE
Technical Problem
[0006] It is one object of the present invention to provide a feed
additive comprising a Bacillus subtilis strain and a Bacillus
licheniformis strain that is helpful in improving milk fat and is
useful in enhancing milk yield.
[0007] It is another object of the present invention to provide a
feed composition comprising the feed additive comprising a Bacillus
subtilis strain and a Bacillus licheniformis strain.
Technical Solution
[0008] Hereinafter, embodiments of the present invention will be
described in detail. It should be noted that descriptions of
details apparent to those skilled in the art will be omitted for
clarity.
[0009] One embodiment of the present invention provides a feed
additive for enhancing milk fat and milk yield comprising a
Bacillus subtilis strain and a Bacillus licheniformis strain.
[0010] Any strain belonging to Bacillus subtilis capable of
producing digestive enzymes, for example, cellulase and/or mannase,
to degrade cellulose may be used. Bacillus subtilis is a
gram-positive, aerobic bacterium that commonly inhabits the soil,
fermented soybean paste or red pepper paste. The Bacillus subtilis
strain may be, for example, Bacillus subtilis CJBS62 or Bacillus
subtilis CJBS16. Particularly, Bacillus subtilis CJBS62 can be
used. Bacillus subtilis CJBS62 was deposited with the Korean
Culture Center of Microorganisms (KCCM) (Yurim Building 45,
Hongjenae-2ga-Gil, Seodaemun-Ku, Seoul, Korea) on Jun. 22, 2017 and
received deposit number KCCM12039P. The sequence (5'.fwdarw.3') of
16s ribosomal DNA of the Bacillus subtilis CJBS62 is set forth in
SEQ ID NO. 3.
[0011] Any strain belonging to Bacillus licheniformis capable of
degrading lactate to acetate may be used. When cultured in a
lactate-containing medium for 9 to 48 hours, the Bacillus
licheniformis strain can convert 30% to 70%, specifically 50% to
65%, of the initial amount of lactate to acetate or can convert at
least 50%, at least 60%, at least 70% or at least 80% of the
consumed amount of lactate to acetate. The conversion rate of the
initial amount of lactate to acetate and the conversion rate of the
consumed amount of lactate to acetate can be calculated by Formulas
1 and 2, respectively:
Conversion rate of initial amount of lactate to acetate (%)=(Amount
of acetate produced/Initial amount of lactate)*100 [Formula 1]
Conversion rate of consumed amount of lactate to acetate
(%)=(Amount of acetate produced/Consumed amount of lactate)*100
[Formula 2]
[0012] The Bacillus licheniformis strain can additionally produce
cellulase and/or mannase.
[0013] Bacillus licheniformis is a gram-positive, aerobic bacterium
that commonly inhabits fermented soybean paste or red pepper paste.
The Bacillus licheniformis strain may be, for example, Bacillus
licheniformis CJBL215 or Bacillus licheniformis CJBL219.
Particularly, Bacillus licheniformis CJBL219 can be used. Bacillus
licheniformis CJBL215 or Bacillus licheniformis CJBL219 were
deposited with the Korean Culture Center of Microorganisms (KCCM)
(Yurim Building 45, Hongjenae-2ga-Gil, Seodaemun-Ku, Seoul, Korea)
on Jun. 22, 2017 and received deposit numbers KCCM12040P and
KCCM12041P, respectively. The sequences (5'.fwdarw.3') of 16s
ribosomal DNA of Bacillus licheniformis CJBL215 and Bacillus
licheniformis CJBL219 are set forth in SEQ ID NOS. 1 and 2,
respectively.
[0014] The feed additive comprising the Bacillus subtilis strain
and the Bacillus licheniformis strain can stabilize the rumen pH of
ruminants and can increase feed digestibility of ruminants. The
feed additive is effective in improving milk fat production in
milking cows and can efficiently increase the amount of milk
produced.
[0015] The Bacillus subtilis strain and the Bacillus licheniformis
strain may be each independently present at a concentration of at
least 1.times.10.sup.7 cfu, specifically at least 1.times.10.sup.8
cfu, more specifically 1.times.10.sup.9 cfu, per gram of the feed
additive. The presence of the strains at the concentrations defined
above makes the feed additive effective in degrading cellulose and
increasing milk fat.
[0016] The feed additive may be used in an amount of 0.1 g to 1 kg,
specifically 5 g to 900 g, more specifically 10 g to 800 g per head
of animal per day.
[0017] The weight ratio of the Bacillus subtilis strain to the
Bacillus licheniformis strain may range from 1:9 to 9:1,
specifically from 2:8 to 8:2, more specifically from 3:7 to 7:3,
most specifically from 1:2 to 2:1.
[0018] The feed additive may be a liquid or solid. When the feed
additive is a liquid, the Bacillus subtilis strain and the Bacillus
licheniformis strain may exist in the form of their biomass,
culture broth or concentrate. The solid feed additive may include a
biomass, culture broth or concentrate of the Bacillus subtilis
strain and the Bacillus licheniformis strain. For example, the
solid feed additive may take the form of a powder, tablet, pellet,
granule or coating that is prepared by heat- or freeze-drying.
[0019] Another embodiment of the present invention provides a feed
composition comprising the feed additive. The feed composition may
include 0.1% to 50% by weight, specifically 0.1% to 40% by weight,
more specifically 1% to 20% by weight of the feed additive based on
the weight of the composition.
[0020] The feed composition may further include at least one
ingredient selected from animal growth promotors, nutrients,
nutritional supplements, storage stabilizers, and coating agents.
The feed composition may include at least one ingredient selected
from: other probiotics; enzymes, such as amylase and lipase;
vitamins, such as L-ascorbic acid, choline chloride, and inositol;
minerals, such as potassium chloride, iron citrate, magnesium
oxide, and phosphates; amino acids, such as lysine, alanine, and
methionine; organic acids, such as fumaric acid, butyric acid, and
lactic acid, and salts thereof; antioxidants, such as vitamin C and
vitamin E; antifungal agents, such as calcium propionate;
emulsifying agents, such as lecithin and glycerin fatty acid
esters; and colorants.
[0021] The feed may be an animal feed, specifically a ruminant
feed. Examples of such ruminants include, but are not limited to,
cows, water buffalo, mountain goats, sheep, goats, and deer. Intake
of the feed can be appropriately determined depending on the kind,
weight, age, sex, and general health of the animal, the ingredients
of the feed, and other factors.
[0022] A further embodiment of the present invention provides a
method for producing the feed additive. The method comprises
culturing a Bacillus subtilis strain and a Bacillus licheniformis
strain and drying a cultured biomass, culture broth or concentrate
of the strains. Specifically, the culturing may comprise
inoculating with the strains and culturing the strains at 30 to
50.degree. C. for 2 to 60 hours, for example, at 32 to 40.degree.
C. for 5 to 50 hours. More specifically, the culturing may comprise
inoculating with the strains, primarily culturing the strains at 30
to 50.degree. C. for 2 to 60 hours, for example, at 32 to
40.degree. C. for 5 to 50 hours until the level of the strains
reaches at least 1.times.10.sup.7 cfu per gram of culture broth,
adding one or more raw materials selected from corn, wheat gluten,
soybean meal, and sugar syrup to the primary culture, and
secondarily culturing the strains at 30 to 50.degree. C. for 2 to
60 hours, for example, at 32 to 40.degree. C. for 5 to 50 hours.
The cultured biomass, culture broth or concentrate may be dried at
40 to 70.degree. C. for 5 to 120 hours, for example, at 40 to
60.degree. C. for 5 to 60 hours. In one embodiment, the method may
further comprise pulverizing the dried cultured biomass, culture
broth or concentrate.
[0023] Yet another embodiment of the present invention provides a
method for increasing milk fat and milk yield of an animal,
comprising administering the feed additive comprising the Bacillus
subtilis strain and the Bacillus licheniformis strain to the
animal. A detailed description of this embodiment is the same as
that of other embodiments described herein.
[0024] Yet another embodiment of the present invention provides a
method for stabilizing the rumen pH of an animal, particularly a
ruminant, comprising administering the feed additive comprising the
Bacillus subtilis strain and the Bacillus licheniformis strain to
the animal. A detailed description of this embodiment is the same
as that of other embodiments described herein.
[0025] Yet another embodiment of the present invention provides a
method for enhancing the digestibility of an animal, particularly a
ruminant, comprising administering the feed additive comprising the
Bacillus subtilis strain and the Bacillus licheniformis strain to
the animal. A detailed description of this embodiment is the same
as that of other embodiments described herein.
Advantageous Effects
[0026] The feed additive according to the present invention is
helpful in improving milk fat of a ruminant and can keep milk fat
from decreasing when exposed to high-temperature stress in summer.
In addition, the feed additive according to the present invention
is effective in degrading lactate in the rumen. Therefore, the feed
additive according to the present invention can prevent the rumen
pH from decreasing by lactate and thus is useful in preventing
rumen acidosis.
DESCRIPTION OF DRAWINGS
[0027] FIG. 1 shows electron microscopy images of Bacillus subtilis
CJBS62 and Bacillus licheniformis CJBL215 and CJBL219 isolated in
one example of the present invention.
[0028] FIG. 2 shows color change of a medium used for screening a
lactate-consuming strain in one example of the present invention.
When the color of the medium turned from yellow (left) to purple
(right), a strain in the medium was judged to consume lactate.
[0029] FIG. 3 is a histogram showing the conversion rates to
acetate by strains as measured in one example of the present
invention.
[0030] FIG. 4 shows the amounts of lactate consumed and acetate
produced by Bacillus licheniformis CJBL215 and CJBL219 (left, y
axis) and the conversion rates to acetate (right, x-axis) upon 9 h
and 48 h culture in one example of the present invention.
[0031] FIG. 5 is an image confirming whether Bacillus subtilis
CJBS62 and Bacillus licheniformis CJBL215 and CJBL219 were
hemolyzed in one example of the present invention.
[0032] FIG. 6 shows culturing time-dependent changes in lactate
concentration when a feed additive according to one example of the
present invention was added to a concentrated feed.
[0033] FIG. 7 shows culturing time-dependent changes in acetate
concentration when a feed additive according to one example of the
present invention was added to a concentrated feed.
[0034] FIG. 8 compares the effect of a mixture of a feed additive
according to one example of the present invention and a
concentrated feed on the stabilization of pH, compared to that of a
control group.
[0035] FIG. 9 is a histogram showing the effect of a mixture of a
feed additive according to one example of the present invention and
a concentrated feed on the improvement in digestibility.
[0036] FIG. 10 is a histogram showing the effect of a mixture of a
feed additive according to one example of the present invention and
a TMR feed on the improvement in digestibility.
MODE FOR INVENTION
[0037] Next, the present invention will be described in more detail
with reference to examples. However, it should be noted that these
examples are provided for illustration only and should not be
construed in any way as limiting the invention.
EXAMPLES
Example 1: Strain Isolation and Screening
[0038] (1) Sampling and Strain Isolation
[0039] Samples were collected from traditionally fermented soybean
paste and pepper paste. The samples were diluted stepwise, plated
on brain heart infusion (BHI) (Difco) solid media, and cultured at
37.degree. C. for 24 h. The cultures were transferred to and
cultured in fresh media. Pure strains were isolated, placed in
media supplemented with 20 wt % glycerol with respect to the total
weight, and stored at .ltoreq.-70.degree. C. The strains were
divided into strains with good cellulase activity and strains
capable of degrading lactate to acetate by below described
method.
[0040] (2) Investigation of Morphological and Biochemical
Properties
[0041] First, morphological and biochemical properties of the
isolated strains were investigated to identify the strains. As a
result of gram staining for morphological investigation, all of the
isolated strains were found to be gram positive. Electron
microscopy revealed that the strains were bacillus sp. (FIG.
1).
[0042] The biochemical properties of the isolated strains were
analyzed. To this end, the sugar fermentation patterns of the
strains were analyzed using an API 50 CHB system (biomerieux Vitek,
Inc., France). The results are shown in Table 1.
TABLE-US-00001 TABLE 1 Results Sugar CJBL215 CJBL219 CJBS62 Control
- - - Glycerol + + + Erythritol - - - D-Arabinose - - - L-Arabinose
+ + + Ribose + + + D-Xylose + - - L-Xylose - - - Adonitol - - -
.beta. Methyl-xyloside - - - Galactose - + - D-Glucose + + +
D-Fructose + + + D-Mannose + + - L-sorbose - - - Rhamnose - + -
Dulcitol - - - Inositol + - + Mannitol + + + Sorbitol - + + .alpha.
Methyl-D-mannoside - - - .alpha. Methyl-D-glucoside + + - N Acetyl
glucosamine - - - Amygdaline + + - Arbutin + + - Esculine + + +
Salicine + + - Cellobiose + + - Maltose + + + Lactose - + -
Melibiose - - - Saccharose + + + Trehalose + + + Inulin - - +
Melezitose - - - D-Raffinose + - + Amidon + + + Glycogen + + -
Xylitol - - - .beta. Gentiobiose - - - D-Turanose + + - D-Lyxose -
- - D-Tagatose + + - D-Fucose - - - L-Fucose - - - D-Arabitol - - -
L-Arabitol - - - Gluconate - - - 2-keto-gluconate - - -
5-keto-gluconate - - - +: Positive, -: negative
[0043] As a result of analyzing the sugar fermentation patterns of
the strains, CJBL215 was found to belong to Bacillus licheniformis
(reliability 99.7%) and CJBL219 was found to belong to Bacillus
licheniformis (reliability 99.9%). CJBS62 was found to belong to
Bacillus subtilis/amyloliquefaciens (reliability 99.6%).
[0044] (3) Strain Identification
[0045] For more accurate identification of the strains, molecular
systematics based on DNA sequencing was carried out. For DNA
sequencing, 16s rDNA gene amplification was performed using PCR
premix (Bioneer, Korea) and universal primers 27F (5'
AGAGTTTGATCMTGGCTCAG 3') and 1492R (5' GGTTACCTTGTTACGACTT 3') The
entire reaction solution was adjusted to 20 .mu.l and gene
amplification was repeated a total of 30 times at 94.degree. C. for
1 min, at 56.degree. C. for 1 min, and at 72.degree. C. for 1 min.
The amplified DNA sequences were analyzed. The sequences of 16s
rDNA of the isolated strains are set forth in SEQ ID NOS. 1 to 3.
As a result of the analysis, each of the sequences of CJBL215 and
CJBL219 had a homology of 99% with that of Bacillus licheniformis
and the sequence of CJBS62 had a homology of 99% with that of
Bacillus subtilis. The isolated stains were named "Bacillus
licheniformis CJBL215", "Bacillus licheniformis CJBL219", and
"Bacillus subtilis CJBS62". The newly identified microorganisms
Bacillus licheniformis CJBL215, Bacillus licheniformis CJBL219, and
Bacillus subtilis CJBS62 were deposited with the Korean Culture
Center of Microorganisms (KCCM) on Jun. 22, 2017 and received
deposit numbers KCCM12040P, KCCM12041P, and KCCM12039P,
respectively.
Example 2: Digestive Enzyme Activities of the Isolated Strains
[0046] (1) Digestive Enzyme Activities of the Strains
[0047] The complex digestive enzyme activities of the isolated
bacteria derived from pastes were evaluated for mannase and
cellulase as digestive enzymes. The digestive enzyme activities of
the strains were measured depending on whether clear zones were
formed in media containing substrate for the enzymes.
[0048] The isolated strains were cultured in brain heart infusion
(BHI) (Difco) liquid media for 24 h. The resulting culture broths
were collected and used as crude enzyme solution for enzyme
activity analysis. The degrees of degradation of the substrate in
the media were determined as follows.
[0049] 1) Measurement of Mannase Activity
[0050] Matrix media (Yeast extract 3 g/L, Peptone 5 g/L,
KH.sub.2PO.sub.4 1 g/L, Agar 20 g/L, pH 5) supplemented with 1%
mannan (locust bean gum, sigma, USA) were prepared. The crude
enzyme solution (1.5 .mu.l each) were dropped into the matrix
media. After the reactions were allowed to proceed at 37.degree. C.
for 15-18 h, the activities of the enzymes were measured depending
on whether clear zones were formed. The results are shown in Table
2.
[0051] 2) Measurement of Cellulase Activity
[0052] YM media supplemented with 1% carboxymethylcellulose (CMC)
substrate were prepared. The crude enzyme solution (1.5 .mu.l each)
were dropped into the substrate media. Reaction was allowed to
proceed at 37.degree. C. for 15-18 h. The reaction solutions were
stained with a 0.2% Congo red solution for 30 min and bleached with
a 1 M aqueous NaCl solution to measure clear zones. The activities
of the enzymes were measured depending on whether clear zones were
formed as a result of degradation of the substrate around the
strains. The results are shown in Table 2.
[0053] Based on the results of evaluations, Bacillus subtilis
CJBS16 and CJBS62 were found to have the best mannase activities
and the best cellulase activities.
TABLE-US-00002 TABLE 2 Digestive enzyme activities of the screened
strains (mm) CJBS16 CJBL215 CJBL219 CJBS62 Mannase 5 0 2.5 3.5
Cellulase 2 3 2 3.5
[0054] (2) Cellulolytic Activities of Culture Supernatants
[0055] The cellulolytic abilities of culture supernatants of the
screened strains were confirmed. First, each of the strains was
cultured for 24 h and centrifuged at 10,000 rpm for 5 min. The
resulting supernatant was filtered through a 0.2 .mu.m syringe
filter. 20 .mu.l of the supernatant was dropped into a medium
supplemented with cellulose (carboxymethylcellulose sodium salt 10
g/L, Bacto agar 15 g/L). Reaction was allowed to proceed at
37.degree. C. for 1 day. The reaction solution was stained with a
0.4% Congo red solution for 20 min and bleached with a 1 M NaCl
solution. The size of clear zones was measured to confirm the
cellulolytic ability of the culture supernatant. The results are
shown in Table 3. The culture supernatant of the Bacillus subtilis
CJBS62 was found to have the best cellulolytic activity.
TABLE-US-00003 TABLE 3 Cellulolytic activities of the culture
supernatants of the screened strains (mm) CJBS16 CJBL215 CJBL219
CJBS62 Cellulolytic activity 16.5 16.5 14 24
Example 3: Screening of Lactate-Consuming Strains and
Acetate-Producing Strains
[0056] Lactate-consuming strains were qualitatively screened by the
chromogenic method using BCP after culture in media supplemented
with lactate. First, media supplemented with 15 mM lactate, yeast
extract 10 g/L, peptone 20 g/L, NaCl 10 g/L, and bromophenol blue
(BCP) 0.0004 g/L were prepared. The media (1.5 ml each) were plated
on microcentrifuge tubes and the tubes were inoculated with strains
(50 .mu.l each) pre-cultured in brain heart infusion (BHI) (Difco)
media. The strains were stationary cultured in the tubes at
37.degree. C. for 4-5 days. When the color of the medium turned
from yellow to purple, the strain was preliminarily judged to
consume lactate in the medium. 117 strains isolated from the
samples were cultured and a total of 4 strains CJBS16, CJBL215,
CJBL219, and CJBS62 were screened (FIG. 2).
[0057] Brain heart infusion (BHI) (Difco) media were inoculated
with the screened CJBS16, CJBL215, CJBL219, and CJBS62 and cultured
at 37.degree. C. and 200 rpm for 16 h for activation thereof. A
medium supplemented with 15 mM lactate, 10 g/L of yeast extract, 20
g/L of peptone, and 10 g/L of NaCl was inoculated with each strain
(5%) and cultured at 37.degree. C. and 200 rpm for 48 h. After
completion of culture, 10% BCP was added to the culture broth to
reconfirm whether lactate was consumed. As a result, the four
strains were confirmed to consume lactate (the colors of the
culture broths turned from yellow to purple).
Example 4: Measurement of Amounts of Lactate Consumed and Amounts
of Acetate Produced
[0058] (1) Measurement Amounts of Acetate Produced
[0059] Brain heart infusion (BHI) (Difco) media were inoculated
with the screened CJBS16, CJBL215, CJBL219, and CJBS62 and cultured
at 37.degree. C. and 200 rpm for 16 h for activation thereof. A
medium supplemented with 15 mM lactate, 10 g/L of yeast extract, 20
g/L of peptone, and 10 g/L of NaCl was inoculated with each strain
(5%) and cultured at 37.degree. C. and 200 rpm for 48 h.
[0060] The culture broth was centrifuged, 0.2 ml of 25%
metaphosphoric acid was added to 1 ml of the collected supernatant,
followed by centrifugation at 10,000 rpm for 5 min. The collected
supernatant was filtered through a 0.2 .mu.m filter and acetate
content thereof was analyzed by GC (Agilent Technologies
7890A).
[0061] The four lactate-consuming strains consumed lactate to
produce acetate. The acetate contents were measured. The conversion
rates were represented by Formula 1:
Conversion rate of initial amount of lactate to acetate (%)=(Amount
of acetate produced/Initial amount of lactate)*100 [Formula 1]
[0062] As a result, the lactate-to-acetate conversion rate of
CJBL219 was highest (59.4%) and that of CJBL215 was 44.9% (Table 4,
FIG. 3).
TABLE-US-00004 TABLE 4 Amounts of acetate produced after
consumption of lactate in media and conversion rates Acetate
Conversion rate calculated Strain No. content (mM) by Formula 1 (%)
CJBL219 8.91 59.4 CJBL215 6.74 44.9 CJBS16 3.74 24.9 CJBS62 1.99
13.3
[0063] (2) Measurements of Amounts of Lactate Consumed and Amounts
of Acetate Produced by the Screened Strains
[0064] The amounts of lactate consumed and acetate produced by the
screened strains in media were quantified.
[0065] Brain heart infusion (BHI) (Difco) media were inoculated
with the screened CJBS16, CJBL215, CJBL219, and CJBS62 and cultured
at 37.degree. C. and 200 rpm for 16 h for activation thereof.
[0066] A medium supplemented with 15 mM lactate, 10 g/L of yeast
extract, 20 g/L of peptone, and 10 g/L of NaCl was inoculated with
each strain (5%) and cultured at 37.degree. C. and 200 rpm for 48
h. At 9 h and 48 h after initiation of culture, samples were
collected.
[0067] 1) Quantification of Lactate
[0068] Each culture broth was centrifuged and the collected
supernatant was filtered through a 0.2 .mu.m filter. After
filtration, the culture supernatant was placed in a microcentrifuge
tube and lactate content thereof was measured using a lactate bio.
test kit for Cedex Bio Analyzer (ROCHE). The results are shown in
Table 5.
[0069] 2) Quantification of Acetate
[0070] Each culture broth was centrifuged, 0.2 ml of 25%
metaphosphoric acid was added to 1 ml of the collected supernatant,
followed by centrifugation at 10,000 rpm for 5 min. The collected
supernatant was filtered through a 0.2 .mu.m filter and acetate
content thereof was analyzed by GC (Agilent Technologies 7890A).
The results are shown in Table 5 and FIGS. 4 and 5.
Conversion rate of consumed amount of lactate to acetate
(%)=(Amount of acetate produced/Consumed amount of lactate)*100
[Formula 2]
[0071] Referring to table 5, CJBL215 consumed 49.8% of the initial
amount of lactate during culture for 48 h and converted about 91.1%
of the consumed lactate to acetate. CJBL219 consumed 48.6% of the
initial amount of lactate during culture for 48 h and converted all
consumed lactate to acetate (FIG. 4).
TABLE-US-00005 TABLE 5 Contents of lactate and acetate in media at
different time points during culture and conversion rates 0 h 9 h
48 h CJBL215 Lactate amount (mM) 13.12 8.35 6.59 Acetate amount
(mM) 0 2.50 5.94 Conversion rate calculated 52.4 91.1 by Formula 2
(%) CJBL 219 Lactate amount (mM) 13.12 8.52 6.74 Acetate amount
(mM) 0 2.87 6.49 Conversion rate calculated 62.4 101.8 by Formula 2
(%)
Example 5: Stability of the Strains
[0072] (1) Confirmation of Hemolysis of the Strains
[0073] .beta.-Hemolysis refers to a phenomenon in which
phospholipids supplied by erythrocytes are hydrolyzed by
phospholipase produced from harmful bacteria, resulting in
hemolysis of erythrocytes. Hemolysis of the isolated strains was
investigated using blood agar plate media (sheep blood 5%, Hanil
Komed Co. Ltd., Korea). The strains were streaked onto the blood
agar plate media and cultured at 37.degree. C. for 24 h. An
observation was made as to whether hemolysis occurred. No hemolysis
was observed, as shown in FIG. 5.
[0074] (2) Confirmation of Susceptibilities of the Strains to
Antibiotics
[0075] The susceptibilities of the screened strains to antibiotics
were confirmed by the following procedure. First, brain heart
infusion (BHI) (Difco) media were inoculated with the screened
strains and cultured at 37.degree. C. and 200 rpm for 16 h. Sterile
cotton swabs soaked with the cultured strains were used to plate
the strains on Mueller Hinton II Agar plates (Difco). Antibiotic
discs were placed on the plate media, followed by culture at
37.degree. C. for 15-18 h. Ampicillin, clindamycin, gentamicin,
kanamycin, tetracycline, vancomycin, erythromycin,
ampicillin/sulbactam, chloramphenicol, and streptomycin discs
(OXOID) were prepared for antibiotic testing. The susceptibilities
of the strains to the antibiotics were confirmed depending on the
formation of clear zones around the antibiotic discs after culture.
As a result of the antibiotic susceptibility tests, the screened
strains were found to be less resistant to the antibiotics (Table
6).
TABLE-US-00006 TABLE 6 Degrees of growth inhibition of the strains
by antibiotics Radii of clear zones around antibiotics (mm)
Antibiotics CJBL215 CJBL219 CJBS62 Amp 10 (Ampicillin) 10 8 10 C30
(Clindamycin) 14 4 9 CN120 (Gentamicin) 18 12 12 K30 (Kanamycin) 13
9 10 TE30 (Tetracycline) 5 8 12 VA30 (Vancomycin) 9 6 7 E15
(Erythromycin) 13 13 11 SAM20 15 12 13 (Ampicillin/Sulbactam) S10
(Chloramphenicol) 3 3 6 DA2 (Streptomycin) 6 7 9
Example 6: Preparation of Feed Additive Including the Strains
[0076] 9 L of tryptic soy broth was inoculated with each of the
bacillus strains CJBL215, CJBL219, and CJBL62 (two species of
Bacillus licheniformis and one species of Bacillus subtilis) and
cultured at 36.degree. C. for 36 h.
[0077] The strain was plated on a tryptic soy agar medium and the
number of colonies was measured. At that time, the strain was
cultured until the number of colonies reached
.gtoreq.1.times.10.sup.9 cfu per gram of strain. A mixture of 20 kg
of corn, 30 kg of wheat gluten, 45 kg of soybean meal, and 5 kg of
sugar syrup was prepared as a raw material for solid state
fermentation. The culture broths of the three species of bacillus
strains (9 L each) were mixed together. The mixture of the culture
broths (total 27 L) was added to 100 kg of the raw material for
solid state fermentation. The strains were homogenously fermented
with stirring in the raw material for solid state fermentation at a
temperature of 34.degree. C. for 48 h. After the fermentation was
finished, the mixture was dried at a temperature of 50.degree. C.
for 48 h and pulverized to produce a feed additive.
Example 7: Effect of the Feed Additive on the Fermentation Behavior
in the Rumen Depending on Matrices
[0078] Tests were conducted to investigate the effect of the feed
additive on the improvement of dry matter digestibility and the
enhancement of acetate using a stationary culture system in a rumen
model.
[0079] Treatment group: 50 mg of the feed additive produced in
Example 6 and 0.5 g of a matrix were fed into a 200 ml serum
bottle. 37.5 ml of a buffer reduced by carbon dioxide gas and 12.5
ml of a rumen fluid were maintained in an anaerobic state using
carbon dioxide gas. After the serum bottle was filled with carbon
dioxide gas for .about.30 sec, the inlet of the serum bottle was
closed with a septum, the serum bottle was sealed with an aluminum
cap, followed by culture in a stationary incubator at 39.degree. C.
for 24 h. At that time, a concentrated feed or total mixed ration
(TMR) was used as the matrix. The feed additive was added in an
amount of 10 wt % per 0.5 g of the matrix. The buffer was prepared
by mixing 9.3 g/L sodium phosphate monobasic
(NaH.sub.2PO.sub.4.2H.sub.2O), 9.8 g/L sodium bicarbonate
(NaHCO.sub.3), 0.47 g/L sodium chloride (NaCl), 0.57 g/L potassium
chloride (KCl), 0.256 g/L magnesium chloride (MgCl.sub.2), 0.106
g/L calcium chloride (CaCl.sub.2), 2.5 g/L casein (N-Z-Amine), and
1.25 ml/L resazurin solution.
[0080] * Control group: The strains were cultured under the same
conditions as in the treatment group, except that the feed additive
was not added.
[0081] Each of the treatment group and the control group was
cultured in a stationary incubator at 39.degree. C. and lactate
content thereof, acetate content, pH, and dry matter digestibility
were measured by the following methods:
[0082] Each of the culture broths was centrifuged and the collected
supernatant was filtered through a 0.2 .mu.m filter. After
filtration, the culture supernatant was placed in a microcentrifuge
tube and lactate content thereof was measured using a lactate bio.
test kit for Cedex Bio Analyzer (ROCHE). Each culture broth was
centrifuged, 0.2 ml of 25% metaphosphoric acid was added to 1 ml of
the collected supernatant, followed by centrifugation at 10,000 rpm
for 5 min. The collected supernatant was filtered through a 0.2
.mu.m filter and acetate content thereof was analyzed by GC
(Agilent Technologies 7890A). Dry matter digestibility was
calculated by Formula 3:
Dry matter digestibility (%)=(Amount of matrix before
culture-Amount of matrix after culture)/Amount of matrix before
culture*100 [Formula 3]
[0083] The amount of the matrix before culture and the amount of
the matrix after culture were measured after filtration of the
culture broth through filter paper using a vacuum pump and drying
at 60.degree. C. overnight.
[0084] Changes in the concentration of lactate and acetate in the
treatment group and the control group were measured at different
time points during culture. The results are shown in FIGS. 6 and
7.
[0085] Referring to FIG. 6, the minimum concentration of lactate in
the treatment group was .about.10% lower than in the control group.
Referring to FIG. 7, the maximum concentration of acetate in the
treatment group was .about.2.5 times that in the control group.
[0086] The pH values and the dry matter digestibility values (%) of
the treatment group and the control group are shown in FIGS. 8, 9,
and 10 (respectively).
[0087] Referring to FIG. 8, the pH of the control group was 0.5
lower than its initial value while the pH of the treatment group
was 0.42 lower than its initial value. These results indicate that
the feed additive according to the present invention is effective
in stabilizing the rumen.
[0088] Referring to FIGS. 9 and 10, the dry matter digestibility
values of the treatment group using the concentrated feed and the
TMR matrices were measured to be higher by 4% and 1.3% than those
of the control group, respectively. In conclusion, the feed
additive according to the present invention can improve the
digestibility in the rumen and can increase the production of
acetate, indicating positive influence on increase of milk fat.
Example 8: Effect of the Feed Additive for Enhancing Milk Fat on
Milk Productivity of Milking Cows
[0089] The feed additive according to the present invention was
produced by the method mentioned in Example 6 and was used for feed
testing on milking cows. The influence of the feed additive on the
milk productivity of milking cows was evaluated through a known
dairy feed test method. First, 72 milking cows were divided into a
control group and a treatment group, 36 cows per group. A
conventional feed was administered to the control group for a test
period of 4 weeks and a mixture of the feed additive according to
the present invention and the conventional feed in the form of a
top-dressing was fed to the treatment group (each 20 g per head of
animal per day). Before initiation of feeding and after feeding,
the amounts of milk fat and milk protein and milk yields were
measured. The results are shown in Tables 7, 8, and 9.
[0090] As a result of continuous feeding of the feed additive to
the milking cows (each 20 g per head of animal per day), milk fat
was increased by 0.3% p, as shown in Table 7. As can be seen from
the results in Table 8, the control group showed no change in the
level of milk protein but the treatment group showed a significant
improvement in the level of milk protein (0.1% p). No significant
change in milk yield was observed in the control group but a
significant increase in milk yield (0.4 kg) was observed in the
treatment group (Table 9).
[0091] The above results conclude that the feed additive according
to the present invention has a positive influence on the
improvement of milk fat, milk protein, and milk yield, achieving
high productivity and quality of milk from milking cows.
TABLE-US-00007 TABLE 7 Effect of the feed additive on improvement
in milk fat Control Treatment group group Before initiation
(average for 3 weeks) 4.7% 4.7% After feeding (average for 4 weeks)
4.7% 5.0% Increment in milk fat (% p) 0.0 0.3
TABLE-US-00008 TABLE 8 Effect of the feed additive on improvement
in milk protein Control Treatment group group Before initiation
(average for 3 weeks) 3.4% 3.2% After feeding (average for 4 weeks)
3.4% 3.3% Increment in milk protein (% p) 0.0 0.1
TABLE-US-00009 TABLE 9 Effect of the feed additive on improvement
in milk yield Control Treatment group group Before initiation
(average for 3 weeks) 27.9 kg 31.9 kg After feeding (average for 4
weeks) 28.0 kg 32.3 kg Increment in milk yield (kg) 0.1 0.4
Example 9: Effect of the Feed Additive for Enhancing Milk Fat on
the Productivity of Milking Cows when Exposed to High-Temperature
Stress in Summer
[0092] The feed additive according to the present invention was
produced by the method mentioned in Example 6 and was used for feed
testing on milking cows. The influence of the feed additive on the
milk productivity of milking cows was evaluated through a known
dairy feed test method. First, a feed including 0.2 wt % of the
feed additive according to the present invention was fed to 150
milking cows over 4 weeks (from May 26 to June 23), which is a
common period for which dairy cattle are exposed to a
high-temperature stress environment. The productivities of milk
from the milking cows before and after feeding were compared.
[0093] For a control group, a feed without the feed additive was
fed to the milking cows over 4 weeks in an environment without
high-temperature stress. For a treatment group, a feed including
0.2 wt % of the feed additive was fed to the milking cows in a
high-temperature stress environment over 4 weeks. The basic
composition of the feed fed to the treatment group was the same as
that of the feed fed to the control group.
TABLE-US-00010 TABLE 10 Effect of the feed additive on the
productivity of milk in summer Milk fat Milk yield Control group
3.7% 36.2 kg Treatment group 3.9% 36.7 kg Increment .sup. 0.2% p
0.5 kg
[0094] Referring to Table 10, the milk fat and milk yield measured
in the treatment group were found to be higher by 0.2% p and 0.5 kg
than those measured in the control group, respectively.
[0095] It is generally known that exposure of milking cows to a
high-temperature stress environment decreases milk yield and milk
fat produced from milking cows. However, feeding of the feed
additive according to the present invention to milking cows can
improve milk fat and milk yield even when exposed to a
high-temperature stress environment.
Sequence CWU 1
1
311506DNAArtificial SequenceCJBL215 16s rDNA 1tcaccttttt cgggtttccc
ctttacgact tcaccccaat catctgtccc accttcggcg 60gctggctcca aaaggttacc
tcaccgactt cgggtgttac aaactctcgt ggtgtgacgg 120gcggtgtgta
caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc
180gattccagct tcacgcagtc gagttgcaga ctgcgatccg aactgagaac
agatttgtgg 240gattggctta gcctcgcggc ttcgctgccc tttgttctgc
ccattgtagc acgtgtgtag 300cccaggtcat aaggggcatg atgatttgac
gtcatcccca ccttcctccg gtttgtcacc 360ggcagtcacc ttagagtgcc
caactgaatg ctggcaacta agatcaaggg ttgcgctcgt 420tgcgggactt
aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca
480ctctgccccc gaaggggaag ccctatctct agggttgtca gaggatgtca
agacctggta 540aggttcttcg cgttgcttcg aattaaacca catgctccac
cgcttgtgcg ggcccccgtc 600aattcctttg agtttcagtc ttgcgaccgt
actccccagg cggagtgctt aatgcgtttg 660ctgcagcact aaagggcgga
aaccctctaa cacttagcac tcatcgttta cggcgtggac 720taccagggta
tctaatcctg ttcgctcccc acgctttcgc gcctcagcgt cagttacaga
780ccagagagtc gccttcgcca ctggtgttcc tccacatctc tacgcatttc
accgctacac 840gtggaattcc actctcctct tctgcactca agttccccag
tttccaatga ccctccccgg 900ttgagccggg ggctttcaca tcagacttaa
gaaaccgcct gcgcgcgctt tacgcccaat 960aattccggac aacgcttgcc
acctacgtat taccgcggct gctggcacgt agttagccgt 1020ggctttctgg
tcaggtaccg tcaaggtacc gccctattcg aacggtactt gttcttccct
1080aacaacagag ttttacgatc cgaaaacctt catcactcac gcggcgttgc
tccgtcagac 1140tttcgtccat tgcggaagat tccctactgc tgcctcccgt
aggagtctgg gccgtgtctc 1200agtcccagtg tggccgatca ccctctcagg
tcggctacgc atcgttgcct tggtgagccg 1260ttacctcacc aactagctaa
tgcgccgcgg gtccatctgt aagtggtagc taaaagccac 1320cttttatgtt
tgaaccatgc ggttcaaaca agcatccggt attagccccg gtttcccgga
1380gttatcccag tcttacaggc aggttaccca cgtgttactc acccgtccgc
cgctaacatc 1440agggagcaag ctcccatctg tccgctcgac ttgcatgtat
taggcacgcc gccagcgttc 1500gtctga 150621504DNAArtificial
SequenceCJBL219 16s rDNA 2ttttttccgg tttccccttt acgacttcac
cccaatcatc tgtcccacct tcggcggctg 60gctccaaaag gttacctcac cgacttcggg
tgttacaaac tctcgtggtg tgacgggcgg 120tgtgtacaag gcccgggaac
gtattcaccg cggcatgctg atccgcgatt actagcgatt 180ccagcttcac
gcagtcgagt tgcagactgc gatccgaact gagagcagat ttgtgggatt
240ggcttagcct cgcggcttcg ctgccctttg ttctgcccat tgtagcacgt
gtgtagccca 300ggtcataagg ggcatgatga tttgacgtca tccccacctt
cctccggttt gtcaccggca 360gtcaccttag agtgcccaac tgaatgctgg
caactaagat caagggttgc gctcgttgcg 420ggacttaacc caacatctca
cgacacgagc tgacgacaac catgcaccac ctgtcactct 480gcccccgaag
gggaagccct atctctaggg gtgtcagagg atgtcaagac ctggtaaggt
540tcttcgcgtt gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc
cccgtcaatt 600cctttgagtt tcagtcttgc gaccgtactc cccaggcgga
gtgcttaatg cgtttgctgc 660agcactaaag ggcggaaacc ctctaacact
tagcactcat cgtttacggc gtggactacc 720agggtatcta atcctgttcg
ctccccacgc tttcgcgcct cagcgtcagt tacagaccag 780agagtcgcct
tcgccactgg tgttcctcca catctctacg catttcaccg ctacacgtgg
840aattccactc tcctcttctg cactcaagtt ccccagtttc caatgaccct
ccccggttga 900gccgggggct ttcacatcag acttaagaaa ccgcctgcgc
gcgctttacg cccaataatt 960ccggacaacg cttgccacct acgtattacc
gcggctgctg gcacgtagtt agccgtggct 1020ttctggttag gtaccgtcaa
ggtaccgccc tgttcgaacg gtacttgttc ttccctaaca 1080acagagtttt
acgatccgaa aaccttcatc actcacgcgg cgttgctccg tcagactttc
1140gtccattgcg gaagattccc tactgctgcc tcccgtagga gtctgggccg
tgtctcagtc 1200ccagtgtggc cgatcaccct ctcaggtcgg ctacgcatcg
tcgccttggt gagccgttac 1260ctcaccaact agctaatgcg ccgcgggtcc
atctgtaagt ggtagctaaa agccaccttt 1320tataattgaa ccatgcggtt
caatcaagca tccggtatta gccccggttt cccggagtta 1380tcccagtctt
acaggcaggt tacccacgtg ttactcaccc gtccgccgct aacctaaggg
1440agcaagctcc cgtcggttcg ctcgacttgc atgtattagg cacgccgcca
gcgttcgtcc 1500tgac 150431490DNAArtificial SequenceCJBS62 16s rDNA
3ccccttacga cttcacccca atcatctgtc ccaccttcgg cggctggctc ctaaaaggtt
60acctcaccga cttcgggtgt tacaaactct cgtggtgtga cgggcggtgt gtacaaggcc
120cgggaacgta ttcaccgcgg catgctgatc cgcgattact agcgattcca
gcttcacgca 180gtcgagttgc agactgcgat ccgaactgag aacagatttg
tgggattggc ttaacctcgc 240ggtttcgctg ccctttgttc tgtccattgt
agcacgtgtg tagcccaggt cataaggggc 300atgatgattt gacgtcatcc
ccaccttcct ccggtttgtc accggcagtc accttagagt 360gcccaactga
atgctggcaa ctaagatcaa gggttgcgct cgttgcggga cttaacccaa
420catctcacga cacgagctga cgacaaccat gcaccacctg tcactctgcc
cccgaagggg 480acgtcctatc tctaggattg tcagaggatg tcaagacctg
gtaaggttct tcgcgttgct 540tcgaattaaa ccacatgctc caccgcttgt
gcgggccccc gtcaattcct ttgagtttca 600gtcttgcgac cgtactcccc
aggcggagtg cttaatgcgt tagctgcagc actaaggggc 660ggaaaccccc
taacacttag cactcatcgt ttacggcgtg gactaccagg gtatctaatc
720ctgttcgctc cccacgcttt cgctcctcag cgtcagttac agaccagaga
gtcgccttcg 780ccactggtgt tcctccacat ctctacgcat ttcaccgcta
cacgtggaat tccactctcc 840tcttctgcac tcaagttccc cagtttccaa
tgaccctccc cggttgagcc gggggctttc 900acatcagact taagaaaccg
cctgcgagcc ctttacgccc aataattccg gacaacgctt 960gccacctacg
tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttaggta
1020ccgtcaaggt accgccctat tcgaacggta cttgttcttc cctaacaaca
gagctttacg 1080atccgaaaac cttcatcact cacgcggcgt tgctccgtca
gactttcgtc cattgcggaa 1140gattccctac tgctgcctcc cgtaggagtc
tgggccgtgt ctcagtccca gtgtggccga 1200tcaccctctc aggtcggcta
cgcatcgtcg ccttggtgag ccgttacctc accaactagc 1260taatgcgccg
cgggtccatc tgtaagtggt agccgaagcc accttttatg tttgaaccat
1320gcggttcaaa caaccatccg gtattagccc cggtttcccg gagttatccc
agtcttacag 1380gcaggttacc cacgtgttac tcacccgtcc gccgctaaca
tcagggagca agctcccatc 1440tgtccgctcg acttgcatgt attaggcacg
ccgccagcgt tcgtctgacg 1490
* * * * *