Category: News

 Patent applications by Xianfeng Peng, Guangzhou CN

Inventors:  Xianfeng Peng (Guangzhou, CN)  Zonghua Tan (Guangzhou, CN)
Assignees:  GUANGZHOU INSIGHTER BIOTECHNOLOGY CO., LTD.
IPC8 Class: AA23K116FI
USPC Class: 560130
Class name: Carboxylic acid esters acyclic acid moiety esterified phenolic hydroxy
Publication date: 2013-12-26
Patent application number: 20130345469

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Abstract:

A feed additive includes at least one of p-thymol, a salt derivative and
an ester derivative thereof for animals.

Claims:

1. A feed additive for animals, comprising: at least one of p-thymol, a
salt derivative of said p-thymol and an ester derivative of said
p-thymol.

2. The feed additive for animals of claim 1, wherein said p-thymol is
extracted and purified from a plant or synthesized by a chemical method.

3. The feed additive for animals of claim 1, wherein said salt derivative
is at least one of salts formed by p-thymol and metal ions, p-thymol
ammonium formed by p-thymol and ammonia, and resin salts formed by
p-thymol and negative ion resins.

4. The feed additive for animals of claim 3, wherein said metal is one of
potassium, sodium, calcium, magnesium, copper, iron, manganese, zinc,
cobalt and chromium.

5. The feed additive for animals of claim 1, wherein said ester
derivative is an ester formed by said p-thymol and different carboxylic
acids.

6. The feed additive for animals of claim 5, wherein said carboxylic acid
is one of formic acid, acetic acid, propionic acid and butyric acid.

7. The feed additive for animals of claim 1, wherein said animals are
various bred animals.

8. The feed additive for animals of claim 7, wherein said bred animals
are pigs, chickens, ducks, geese, beef cattle, cows, sheep, fishes,
shrimps, foxes, ermines or raccoon dogs; and the bred animals are those
of all growth stages.

9. The feed additive for animals of claim 1, wherein a quantity of one of
said p-thymol, said salt derivative and said ester derivative thereof
serving as an animal feed growth promoter is 5-500 ppm of a mass of a
complete formula feed.

10. The feed additive for animals of claim 9, wherein said quantity of
one of said p-thymol, said salt derivative and said ester derivative
thereof serving as said animal feed growth promoter is 50-250 ppm of said
mass of said complete formula feed.

 

Description:

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part application of
PCT/CN2011/071533, filed on Mar. 4, 2011, which claims the priority of
China Patent Application Serial No. 201110048452.3, filed on Mar. 2,
2011. The contents of are all hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention belongs to the field of feeds, and particularly
relates to a feed additive of p-thymol, a salt or an ester derivative
thereof for use in a complete formula feed.

[0004] 2. Description of the Prior Art

[0005] The usage of feed antibiotics (AGPs) was regarded as the greatest
biotechnology in animal husbandry production in the 20th century. It was
first reported that streptomycin can stimulate the growth of chicken in
1946, and Food and Drug Administration (FDA) first approved that
antibiotics were used as feed additives in 1950. Hereafter, the
antibiotics are widely used for treating and preventing bacterial
diseases, and are also popularized and applied in a large range as the
feed additives, so the antibiotics play an extremely important role in
controlling infectious diseases of livestock and poultry and promoting
the healthy development of modern breeding industry. However, with more
and more use and more and more abuse of the antibiotics, various
shortcomings of the antibiotics are gradually recognized by people. The
negative effects of the antibiotics serving as the feed additives mainly
comprise the following several aspects: firstly, drug-resistant strains
are generated, and the occurrence of a large quantity of drug-resistant
strains, particularly multiple-resistant strains arouses the worry of
people about bacterial drug resistance transfer, so that concern on the
problem of public health is aroused; secondly, the growth promoting
effect of the antibiotics significantly declines due to the
drug-resistant strains, and the antibiotics overdose often occurs
clinically, so that the drug resistance of bacteria is more serious, and
on the contrary, the toxic and side effects of the antibiotics overdose
inhibit the production performance of animals and affect the normal
physiological functions of the animals; and finally, long-term use and
abuse of the feed antibiotics lead to residue in animal products and the
environment and harm the health of human beings. Based on the
consideration of multiple aspects, the European Union has comprehensively
banned the use of the feed antibiotics as animal growth promoters since
2006, and Korea has also prohibited 8 feed growth promoting antibiotics
including enramycin in feeds since August, 2010. Particularly, after the
“super bacteria” event in 2010, the doubt about the use of the feed
antibiotics and the voice for prohibition has been growing. Therefore,
the research and development of effective antibiotic substitutes become
an important direction in the development of the feed additive industry.

[0006] A lot of work has been done on the research of feed antibiotic
substitutes. In the substitutes such as edible essential oil, organic
acid, enzyme preparation, oligosaccharides and micro-ecological
preparations, the use of essential oil components to substitute the
antibiotic growth promoters is the most requested.

[0007] Majoram, also named as dysentery stop herb, elsholtzia patrini
garcke and lobular mint, is a lamiaceae origanum renascent herb. An
essential oil extract, namely majoram oil, which is extracted from the
natural majoram plant, is light yellow clear oily liquid. At present, the
majoram oil has been approved to be used as a feed additive for improving
the production performance of livestock and poultry, and is greatly used
in European Union countries as a substitute for antibiotic growth
promoters.

[0008] The majoram oil contains more than 30 components, wherein the main
components are carvacrol and thymol which account for 60-70% and 10-20%
of the total amount of the majoram oil respectively. Further research has
proved that the antibacterial activity of the majoram oil and the effect
as the animal growth promoter are mainly from the effects of thymol and
carvacrol. Thymol is also named as `Shexiangcaofen`, of which the
chemical name is 5-methyl-2-isopropyl-phenol, the melting point is
51.5° C., the boiling point is 232° C., and the oral median
lethal dose (LD₅₀) of rats is 980 mg/kg weight. Carvacrol is an
isomeride of thymol, of which the chemical name is
2-methyl-5-isopropyl-phenol, the melting point is about 0° C., the
boiling point is 237-238° C., and the oral median lethal dose
(LD₅₀) of the rats is 810 mg/kg weight.

[0009] Although the production performance of animals can be partially
improved when the majoram oil taking thymol and carvacrol as main
components is used as a feed additive, the defects of the majoram oil
limit the application in the feed industry on three aspects: (1) the
strong pungent smell affects feed processing and also reduces the intake
of the animals; (2) the majoram oil has strong volatility, so that the
majoram oil is easy to volatilize in the feed preservation process and
must be used in a large dose; and (3) thymol and carvacrol have
relatively strong toxicity on the animals (the oral LD₅₀ of the rats
are 980 mg/kg weight and 810 mg/kg weight respectively), narrow safety
range and weak mutability.

SUMMARY OF THE INVENTION

[0010] It is thus an object of the invention to overcome the defects of
majoram oil taking thymol and carvacrol as main components in the use as
an animal growth feed additive, with p-thymol, a salt or an ester
derivative thereof in an animal feed additive provided. P-thymol is an
isomeride of thymol, with the chemical name of
3-methyl-4-isopropyl-phenol.

[0011] The object of the invention is fulfilled through the following
technical scheme: p-thymol, a salt derivative or an ester derivative
thereof as an animal feed additive.

[0012] P-thymol includes: (1) p-thymol extracted from a plant; and (2)
p-thymol synthesized by a chemical method.

[0013] The salt derivatives of p-thymol comprise salts formed by p-thymol
and metal ions, p-thymol ammonium formed by p-thymol and ammonia, resin
salts formed by p-thymol and negative ion resins and the like;

the metal comprises potassium, sodium, calcium, magnesium, copper, iron,
manganese, zinc, cobalt, chromium and the like; the ester derivatives of
p-thymol comprise esters formed by p-thymol and different carboxylic
acids; the carboxylic acids comprise formic acid, acetic acid, propionic
acid, butyric acid and the like; and the animals comprise various
artificially-bred animals such as pigs, chickens, ducks, geese, beef
cattle, cows, sheep, rabbits, fishes, shrimps, foxes, ermines and raccoon
dogs.

[0014] The animal feed of p-thymol and the salt derivatives or the ester
derivatives thereof may be used in all growth, production and propagation
stages of the animals.

[0015] The animal feed of p-thymol and the salt derivatives or the ester
derivatives thereof comprises p-thymol and the salt derivatives or the
ester derivatives thereof in the name of an antibiotic growth promoter
substitute, a growth promoter, a feed flavoring agent, a flavoring agent
and the like in an animal feed.

[0016] When the animal feed is a complete formula feed, the adding
quantity of p-thymol and the salt derivatives or the ester derivatives
thereof serving as a growth promoter is 5-500 ppm of the mass of the
complete formula feed, preferably 50-250 ppm.

[0017] Accordingly, the invention also relates to a formula feed for
animals. The formula feed for animals comprises an animal feed and a feed
additive. The feed additive comprises at least one of p-thymol, a salt
derivative and an ester derivative thereof.

[0018] The invention also relates to a method for manufacturing a formula
feed for animals. First, an animal feed is provided. Second, the animal
feed is mixed/blended with a feed additive to obtain a formula feed for
animals.

[0019] The invention also relates to a method to feed an animal. First, a
formula feed is provided. Second, the formula feed is fed to an animal.

[0020] Compared with the prior art, the invention has the advantages and
beneficial effects that the animal growth promoting effect on p-thymol
and the salt derivatives or the ester derivatives thereof is discovered
for the first time, and p-thymol has the characteristics of low
volatility, low toxicity, low irritation, strong antibacterial activity
and the like compared with thymol and carvacrol, so p-thymol and the salt
derivatives or the ester derivatives thereof are more suitable to be used
as a growth promoting feed additive for animals to substitute feed
antibiotics.

[0021] These and other objectives of the present invention will no doubt
become obvious to those of ordinary skill in the art after reading the
following detailed description of the preferred embodiment that is
illustrated in the various figures and drawings.

DETAILED DESCRIPTION

[0022] The invention is further illustrated in detail in conjunction with
the following embodiments, but the embodiments of the invention are not
limited herein.

Embodiment 1

Synthesis of Salt Derivatives or Ester Derivatives of p-Thymol

[0023] (1) Synthesis of p-thymol sodium: dissolving 75.1 g of p-thymol
into 500 ml of tetrahydrofuran solution, adding 20 g (60% wt) of sodium
hydride while stirring under the condition of ice bath, reacting for 2
hours, and performing vacuum reduced-pressure distillation on the
reaction product to remove the solvent, thus obtaining p-thymol sodium.
(2) Synthesis of p-thymol ammonium: dissolving 75.1 g of p-thymol into
500 ml of tetrahydrofuran solution, continuously introducing ammonia
during continuous stirring (200 rpm), stopping the reaction after 6
hours, and performing vacuum reduced-pressure distillation on the
reaction product to remove the solvent, thus obtaining p-thymol ammonium.
(3) Synthesis of p-thymol resin salt: dissolving 50 g of p-thymol sodium
into 1000 ml of water, adding 200 g of pretreated strongly alkaline
polystyrene anion resin, reacting for 6 hours during continuous stirring
at room temperature, filtering, washing resin granules by using deionized
water, drying to the constant weight, weighing and calculating the
drug-loading rate, wherein the content of p-thymol in the resin salt is
21.5%; and crushing, sieving, and taking the component of less than 100
meshes, thus obtaining the p-thymol resin salt. (4) Synthesis of p-thymol
ethyl ester: dissolving 40 g of p-thymol and 40 ml of acetyl chloride
into 500 ml of tetrahydrofuran solution, dripping 40 ml of triethylamine
during stirring at room temperature, reacting for 4 hours, adding 500 ml
of water, stirring uniformly, then adding 1000 ml of ethyl acetate,
stirring, extracting, and performing vacuum reduced-pressure distillation
on the ethyl acetate phase to remove ethyl acetate, thus obtaining
p-thymol ethyl ester.

Embodiment 2

Volatility Comparison Test of p-Thymol, Thymol and Carvacrol

[0024] Test Materials

p-thymol, thymol and carvacrol: purchased from SIGMA company; vacuum
drying oven and circulating water pump: Shanghai Yiheng Test Equipment
Factory.

[0025] Test Method

1. Volatility Comparison of p-Thymol, Thymol and Carvacrol

[0026] Accurately weighing 10.0 g of p-thymol, 10.0 g of thymol and 10.0 g
of carvacrol, putting into the vacuum drying oven, performing
reduced-pressure drying for 1 hour at the temperatures of 50° C.,
70° C. and 90° C. respectively, then weighing the residual
weight, and comparing the volatility difference of different compounds.

2. Changes of p-Thymol, Thymol and Carvacrol in the Feed Preservation
Process

[0027] Preparing chicken complete formula feeds containing 100 ppm (mass)
of p-thymol, 100 ppm of thymol and 100 ppm of carvacrol respectively,
preserving for a long time at room temperature of 25-30° C.,
sampling after the feeds are preserved for different time (1, 15, 30, 45
and 60 days), extracting with ethanol respectively to recover p-thymol,
thymol and carvacrol in corresponding samples, then measuring the
corresponding content of the components by adopting gas chromatography,
and comparing the change of each component in the feed preservation
process.

[0028] Test Results:

1) The test result of 1. is as follows:

[0029] Under the condition of reduced-pressure drying, thymol and
carvacrol are easy to volatilize; after 1 hour of reduced-pressure drying
at different temperatures, the loss of carvacrol is the highest, and
carvacrol is completely volatilized within 1 hour at the temperatures of
70° C. and over; under the condition of reduced-pressure drying at
the temperature of 70° C., 89.8% of thymol is volatilized within 1
hour; and the volatility of p-thymol is the lowest, and when p-thymol is
dried for 1 hour under reduced pressure at the temperatures of 70°
C. and 90° C., the volatilized parts only account for 11.4% and
36.6% respectively (see Table 1).

TABLE-US-00001
TABLE 1
Loss of P-thymol, Thymol and Carvacrol under the Condition
of Reduced-pressure Drying
Initial
Sample Weight (g) 50° C. 70° C. 90° C.
p-thymol 10.0 9.84 8.86 6.34
thymol 10.0 6.12 1.02 0
carvacrol 10.0 4.84 0 0
Note:
the numerical values in the table represent the weights (unit: g)
remained after reduced-pressure drying

2) The test result of 2. is as follows:

[0030] When p-thymol, thymol and carvacrol are added in the complete
formula feeds, the stability of p-thymol is the best, the loss of
p-thymol is less than 5% after p-thymol is placed for 2 months, and the
loss of carvacrol and thymol exceeds 50% (see Table 2).

TABLE-US-00002
TABLE 2
Loss of P-thymol, Thymol and Carvacrol under the Condition
of Reduced-pressure Drying
Initial
Concentration 15 30 45 60
Sample (ppm) 1 day days days days days
p-thymol 100 99.8 99.2 98.3 97.5 96.4
thymol 100 100.4 90.2 78.9 63.7 49.7
carvacrol 100 99.6 87.7 73.1 58.5 42.6
Note:
the numerical values in the table represent the concentration (unit: ppm)
measured after the extraction from the complete formula feeds.

Experiment 3

Measurement on Safety of p-Thymol and the Like (Rat LD₅₀ Measurement,
and Improved Karber’s Method)

1. Test Materials

[0031] Wistar rats are purchased from Animal Experiment Center of Southern
Medical University, with the weights of 120 to 150 g, and with the number
of males and females respectively accounting for 50%.

[0032] Test samples: p-thymol, thymol and carvacrol.

[0033] Apparatuses: syringes, intragastric administration needles and rat
cages

[0034] Test Methods

[0035] A, Doses for causing animal 0% (Dn) and 100% (Dm) death are
obtained through preliminary experiments, wherein the Dn and the Dm of
p-thymol, thymol and carvacrol are shown in Table 3.

TABLE-US-00003
TABLE 3
Rat LD₅₀ Preliminary Experiment Results (Dn and Dm) of P-thymol,
Thymol and Carvacrol
Compound Dn (mg/kg weight) Dm (mg/kg weight)
p-thymol 4000 8000
thymol 400 1800
carvacrol 400 1800

[0036] B, As required by the experiments, the maximum reaction rate is
100%, the minimum reaction rate is 0%, or at least the reaction rate
approaches to 100% or 0%; and in the dose ratio of the groups, 8 doses
are averagely set between Dn and Dm according to the Dn and Dm values of
each compound, and 10 rats constitute a group. When the adjacent doses
have repeated reaction rates of 100% and 0% in the experiments, the
marginal group is abandoned, so that the big dose group only has a
reaction rate of 100%, and the small dose group only has a reaction rate
of 0%; after grouping, the rats are drenched with different doses of
p-thymol, thymol and carvacrol respectively; and the rats are observed
for 7 days after administration, and the toxic reaction condition of
animals and the distribution of dead animals are recorded day by day
during observation.

[0037] C. Calculation mode: the LD₅₀ and the confidence limit
(P=0.95) of p-thymol, thymol and carvacrol on the rats are calculated
according to the death rate of each formal experiment group and according
to the following formulas:

[0038] When the death rate of the minimum dose group is 0% and the death
rate of the maximum dose group is 100%, the LD₅₀ is calculated
according to the following formula:

LD₅₀=lg^-1[Xm-i(Σp-0.5)]

[0039] B. When the death rate of the minimum dose group is more than 0%
and less than 30% or the death rate of the maximum dose group is less
than 100% and more than 70%, the LD⁵⁰ can be calculated according to
the following correction formula:

LD 50 = lg – 1 [ Xm – i ( p – 3 – Pm – Pn 4 ) ]
##EQU00001##

[0040] The standard error of the LD50 is as follows:

S x 50 = i P – P 2 n – 1 ##EQU00002##

the average confidence limit of the LD₅₀ is as follows:

LD₅₀±4.5 S_x50 LD₅₀ (P=0.95);

and in the above formulas, Xm is the logarithm of the dose of the maximum
dose group, i is the difference (subtracting the small dose group from
the large dose group) of the logarithmic doses of the two adjacent
groups, Pm is the death rate of the maximum dose group, Pn is the death
rate of the minimum dose group, P is the death rate of each group, and n
is the number of each group of animals.

[0041] D. Test Results

[0042] The oral lethal doses 50 (LD₅₀) of the rats on all the test
samples are shown in Table 4, and the results show that the safety of
p-thymol is improved by about 30 folds and 25 folds respectively compared
with that of thymol and carvacrol.

[0043] Compound toxicity judgment standards (compound peroral acute
toxicity grading standards, based on LD₅₀) are as follows: <1,
extreme toxicity; 1-50, violent toxicity; 51-500, medium toxicity;
501-5000, low toxicity; 5001-50000, relative non-toxicity; >50000,
non-toxicity.

TABLE-US-00004
TABLE 4
Research Results on Oral LD50 of P-thymol, Thymol and Carvacrol
on the Rats
Test Samples LD50 (mg/kg weight) Toxicity Judgment
p-thymol 920 low toxicity
thymol 800 low toxicity
carvacrol 6400 relative
non-toxicity

Embodiment 4

In-Vitro Antibacterial Activity Test of p-Thymol and the Salt or Ester
Derivatives Thereof

(1) Test Materials:

1. Culture Medium:

[0044] LB liquid culture medium: for culturing escherichia coli and
salmonella, with the formula shown as follows:

TABLE-US-00005
yeast extract 5 g
tryptone 10 g
sodium chloride 10 g
water added to 1000 ml
pH 7.0-7.2;

[0045] II. TSB liquid culture medium, added with 2 volume percent of
bovine serum in use, used for culturing staphylococcus aureus, with the
formula shown as follows:

TABLE-US-00006
tryptone 17 g
soy peptone 3 g
yeast extract 6 g
NaCl 5 g
K2HPO4•3H2O 2.5 g
glucose 2.5 g
H2O to 1 L
pH 7.0-7.4;

[0046] III. Fluid thioglycollate culture medium (FT culture medium): for
culturing clostridium perfringens, with the formula shown as follows:

TABLE-US-00007
tryptone 15 g
yeast extract powder 5 g
glucose 5 g
sodium thioglycollate 0.5 g
L-cystine 0.5 g
sodium chloride 2.5 g
resazurin 0.001 g
agar 0.75 g
pH value 7.1 ± 0.2;

2. Strains: escherichia coli CAU0159, escherichia coli CAU0195,
escherichia coli CAU0020, escherichia coli CAU0053, escherichia coli
CAU0147, salmonella gallinarum CAU0206, salmonella gallinarum CAU0205,
salmonella gallinarum QAU0399, salmonella bacillus CAU0118, salmonella
choleraesuis CEC19940146, salmonella choleraesuis CEC19940141,
staphylococcus aureus CAU0871, staphylococcus aureus CAU0868,
staphylococcus aureus CAU0869, staphylococcus aureus CAU0866,
staphylococcus aureus CAU0804, staphylococcus aureus CAU0810,
staphylococcus aureus CAU0837, clostridium perfringens CAU0859,
clostridium perfringens CAU0855, clostridium perfringens CAU0795,
clostridium perfringens CAU0591, clostridium perfringens HNAU166 and
clostridium perfringens HNAU16: purchased from China Veterinary Culture
Collection Center.

3. Test Samples:

[0047] p-thymol sodium, p-thymol resin salt, p-thymol ammonium and
p-thymol ethyl ester: prepared in embodiment 1; p-thymol, thymol and
carvacrol: purchased from SIGMA company.

(2) Test Method (Tube Double Dilution Method)

[0048] The test method adopts a tube double dilution method, and the
antibacterial activity test of p-thymol on the staphylococcus aureus is
taken as an example (the methods for testing the antibacterial activities
of different compounds on different bacteria are identical). When the
test samples are used for testing the antibacterial activities for the
escherichia coli and the salmonella, the culture medium adopts the LB
liquid culture medium. When the clostridium perfringens is tested, the
fresh FT culture medium is adopted, and a layer of paraffin oil for
maintaining the anaerobic environment is covered on the surface during
culturing. The specific test method comprises the following steps:

[0049] picking 12 sterile test tubes numbered 1-12;

[0050] B. sterilely adding 9.5 ml of TSB liquid culture medium into the
1st tube, and sterilely adding 5.0 ml of TSB liquid culture medium into
the 2nd to 11th tubes;

[0051] C. adding 0.5 ml of p-thymol (2% by mass/volume) solution into the
1st tube, then mixing the solution in the 1st tube uniformly, adding 5.0
ml of p-thymol solution from the 1st tube to the 2nd tube sequentially
till the 10th tube, and removing 5.0 ml of solution from the 10th tube,
wherein the 11th tube is not added with the test sample and is used as a
positive control;

[0052] D. additionally preparing a tube (12th tube) containing 5.0 ml of
TSB liquid culture medium which is not added with the test sample or
bacteria, serving as a negative control;

[0053] E. adding 50.0 ml of solution of bacteria to be tested (with the
concentration of the bacteria of about 108 cfu/ml, and the age of the
bacteria of 16-18 h) into the 1st to 11th tubes respectively;

[0054] F. performing static culture for 16 h at the temperature of
37° C.; and

[0055] G. judging the results, namely observing whether the bacteria are
grown or not with naked eyes, wherein turbid growth can be seen in the
positive control, and clearness can be seen in the negative control (the
positive and negative controls must be accurate). According to the
numbers of the test tubes, the drug concentration in the last tube
without bacteria growth is the minimum bacteria inhibiting concentration
(μg/mL) of p-thymol on the staphylococcus aureus.

(3) Test Results

[0056] P-thymol and the salt or ester derivatives thereof have
broad-spectrum antibacterial activities and have strong inhibiting
activities for Gram-positive bacteria (staphylococcus aureus and
clostridium perfringens) and Gram-negative bacteria (escherichia coli and
salmonella), the minimum bacteria inhibiting concentration is 125-250
μg/mL, the antibacterial activity of p-thymol and the salt or ester
derivatives thereof is 1-2 times stronger than that of thymol and
carvacrol, and the detailed results are shown in Tables 5-8.

TABLE-US-00008
TABLE 5
Inhibiting Activity (unit: μg/mL) of P-thymol and the
Derivatives Thereof on the Staphylococcus Aureus
P-thymol P-thymol P-thymol
Strain and Number P-thymol sodium resin salt ammonium
s. aureus CAU0871 125 125 125 125
s. aureus CAU0868 125 125 125 125
s. aureus CAU0869 125 125 125 125
s. aureus CAU0866 125 125 125 125
s. aureus CAU0804 250 250 250 250
s. aureus CAU0810 125 125 125 125
s. aureus CAU0837 125 125 125 125
Strain and Number P-thymol ethyl ester Thymol Carvacrol
s. aureus CAU0871 125 250 250
s. aureus CAU0868 125 250 250
s. aureus CAU0869 125 125 250
s. aureus CAU0866 125 250 250
s. aureus CAU0804 250 500 500
s. aureus CAU0810 125 125 250
s. aureus CAU0837 125 250 250

TABLE-US-00009
TABLE 6
Inhibiting Activity (unit: μg/mL) of P-thymol and the
Derivatives Thereof on the Clostridium Perfringens
P-thymol P-thymol P-thymol
Strain and Number P-thymol sodium resin salt ammonium
clostridium 125 125 125 125
perfringens CAU0859
clostridium 125 125 125 125
perfringens CAU0855
clostridium 125 125 125 125
perfringens CAU0795
clostridium 250 250 250 250
perfringens CAU0591
clostridium 125 125 125 125
perfringens HNAU166
clostridium 250 250 250 250
perfringens HNAU160
P-thymol
Strain and Number ethyl ester Thymol Carvacrol
clostridium perfringens CAU0859 125 250 500
clostridium perfringens CAU0855 125 250 500
clostridium perfringens CAU0795 125 250 250
clostridium perfringens CAU0591 250 250 500
clostridium perfringens HNAU166 125 250 250
clostridium perfringens HNAU160 250 500 500

TABLE-US-00010
TABLE 7
Inhibiting Activity (unit: μg/mL) of P-thymol and the
Derivatives Thereof on the Escherichia Coli
p-thymol p-thymol p-thymol
strain and number p-thymol sodium resin salt ammonium
escherichia coli 125 125 125 125
CAU0159
escherichia coli 250 250 250 250
CAU0195
escherichia coli 125 125 125 125
CAU0020
escherichia coli 250 250 250 250
CAU0053
escherichia coli 125 125 125 125
CAU0147
p-thymol
strain and number ethyl ester thymol carvacrol
escherichia coli CAU0159 125 250 250
escherichia coli CAU0195 250 250 500
escherichia coli CAU0020 125 250 250
escherichia coli CAU0053 250 250 500
escherichia coli CAU0147 125 250 250

TABLE-US-00011
TABLE 8
Inhibiting Activity (unit: μg/mL) of P-thymol and the
Derivatives Thereof on the Salmonella
P-thymol P-thymol P-thymol
Strain and Number P-thymol sodium resin salt ammonium
salmonella 125 125 125 125
gallinarum
CAU0206
salmonella 125 125 125 125
gallinarum
CAU0205
salmonella 125 125 125 125
gallinarum
QAU0399
salmonella bacillus 125 125 125 125
CAU0118
salmonella 250 250 250 250
choleraesuis
CEC19940146
salmonella 125 125 125 125
choleraesuis
CEC19940141
P-thymol ethyl
Strain and Number ester Thymol Carvacrol
salmonella gallinarum 125 250 500
CAU0206
salmonella gallinarum 125 500 250
CAU0205
salmonella gallinarum 125 500 500
QAU0399
salmonella bacillus 125 250 250
CAU0118
salmonella choleraesuis 250 250 250
CEC19940146
salmonella choleraesuis 125 250 500
CEC19940141

Embodiment 5

Growth Promoting Effect Test of p-Thymol on Chickens

[0057] Test Materials

1-day-old Lingnan yellow big chicken, provided by the Breeding Chicken
Farm of Institute of Animal Science, Guangdong Academy of Agricultural
Sciences 102-type chicken feed: containing no antibiotics, provided by
the Institute of Animal Science, Guangdong Academy of Agricultural
Sciences p-thymol, thymol and carvacrol: purchased from SIGMA company.

(2) Test Method

[0058] 1. Growth Promoting Effect Test of Different Doses of p-Thymol on
Chickens 400 1-day-old chickens are randomly divided into 8 groups, and
each group comprises 50 chickens. Different drugs (Table 9) are added to
the feed of each group, and the survival rate, the weight gain and the
feed reward of each group of 1 to 21-day-old test chickens are counted.
The test process is cage culture above a net.

TABLE-US-00012
TABLE 9
Growth Promoting Effect Test Groups of Different Doses of
P-thymol on the Chickens
Quantity of Dose
Group Animals Drug (ppm)
control group 50 — 0
without adding
antibiotics
test group 1 50 p-thymol 5
test group 2 50 p-thymol 10
test group 3 50 p-thymol 20
test group 4 50 p-thymol 50
test group 5 50 p-thymol 100
test group 6 50 p-thymol 200
test group 7 50 p-thymol 500

[0059] Comparison of Chicken Growth Promoting Effects of p-Thymol, Thymol
and Carvacrol

[0060] 200 1-day-old chickens are randomly divided into 4 groups, and each
group comprises 50 chickens. Different drugs (Table 10) are added to the
feed of each group, and the survival rate, the weight gain and the feed
reward of each group of 1 to 21-day-old chickens are counted. The test
process is cage culture above the net.

TABLE-US-00013
TABLE 10
Growth Promoting Test Groups of P-thymol, Thymol and
Carvacrol on Chickens
Quantity of Dose
Group Animals Drug (ppm)
control group 50 — —
without drug
test group 1 50 p-thymol 100
test group 2 50 thymol 100
test group 3 50 carvacrol 100

[0061] Test Results

[0062] Growth Promoting Test Results of Different Doses of p-Thymol on
Chickens

[0063] Through the feeding test result analysis of the 1 to 21-day-old
test chickens, different doses of p-thymol have the growth promoting
effect to different degrees. Compared with the control group without
adding the drug, the survival rate of each test group is 100%, but the
weight gain of the test group added with p-thymol is improved by
1.32-7.39% compared with the control group and has certain correlation
with the adding dose. Moreover, the test group added with p-thymol can
improve the feed reward to different degrees, and compared with the
control group without adding the drug, the feed conversion rate is
reduced by 0.003 to 0.108 (Table 11).

TABLE-US-00014
TABLE 11
Chicken Growth Promoting Test Results of Different Doses
of P-thymol
Aver- Relative Total
Sur- age Weight Total Feed Feed
vival Weight Gain Weight Con- Conver-
Dose Rate Gain Rate Gain sumption sion
Group (ppm) (%) (g) (%) (kg) (kg) Rate
control 0 100 326.1 100 16.31 29.02 1.779
group
without
adding
the
drug
test 5 100 330.4 101.32 16.52 29.33 1.776
group 1
test 10 100 338.3 103.74 16.92 29.34 1.734
group 2
test 20 100 341.3 104.66 17.07 29.33 1.718
group 3
test 50 100 343.5 105.33 17.18 29.29 1.705
group 4
test 100 100 348.3 106.81 17.42 29.28 1.681
group 5
test 200 100 347.8 106.65 17.39 29.32 1.686
group 6
test 500 100 350.2 107.39 17.51 29.26 1.671
group 7

2. Comparison Test Results of Chicken Growth Promoting Effects of
p-Thymol, Thymol and Carvacrol

[0064] Through the feeding test result analysis of the 1 to 21-day-old
test chickens, after 100 ppm of p-thymol, thymol or carvacrol is added
into the feed, compared with the control group without adding the drug,
p-thymol has an obvious growth promoting effect, the relative weight gain
rate is improved by 6.82%, and the feed conversion rate is reduced by
0.066. The growth promoting effects of the thymol and carvacrol test
groups are inferior to the growth promoting effect of the p-thymol test
group, the relative weight gain rates are only improved by 3.78% and
3.19% respectively, and the feed conversion rates are reduced by 0.026
and 0.021 respectively (compared with the control group without adding
the drug, as shown in Table 12).

TABLE-US-00015
TABLE 12
Comparison Test Results of Chicken Growth Promoting Effects
of P-thymol, Thymol and Carvacrol
Total
Aver- Relative Feed
Sur- age Weight Total Con- Feed
vival Weight Gain Weight sump- Conver-
Dose Rate Gain Rate Gain tion sion
Group (ppm) (%) (g) (%) (kg) (kg) Rate
control 0 100 322.6 100 16.13 28.74 1.782
group
without
adding
the
drug
p-thymol 100 100 344.6 106.82 17.23 29.57 1.716
thymol 100 100 334.8 103.79 16.74 29.40 1.756
carvacrol 100 100 332.9 103.19 16.65 29.32 1.761

Embodiment 6

Growth Promoting Effect Test of p-Thymol on Pigs

(1) Test Materials

[0065] 120 three-way cross weaned pigs, provided by Guangdong Minfeng
Breeding Pig Farm

[0066] 303-type pig complete formula feed, containing no antibiotics,
provided by the Feed Factory of Guangdong Minfeng Livestock Development
Co., Ltd.

[0067] p-thymol, thymol and carvacrol: purchased from SIGMA company.

(2) Test Method

[0068] 120 weaned pigs are grouped as in Table 13, and each group
comprises 30 pigs. After different growth promoters are added to the feed
of each group, the pigs freely eat the feed, the weight gain and the feed
reward of each group of test pigs after the pigs are weaned for 30 days
are counted, and the difference of the growth promoting effects of
p-thymol, thymol and carvacrol on the pigs is compared.

TABLE-US-00016
TABLE 13
Pig Growth Promoting Test Groups of P-thymol, Thymol and
Carvacrol
Average
Quantity of Initial Growth Concentration
Group Animals Weight (kg) Promoter (ppm)
1 30 8.15 — —
2 30 8.20 p-thymol 100
3 30 8.08 thymol 100
4 30 8.22 carvacrol 100

(3) Test Results

[0069] Through the feeding test result analysis of the weaned pigs, after
p-thymol, thymol and carvacrol are added into the complete formula feed
respectively, compared with the control group without adding the drug,
p-thymol, thymol and carvacrol have the growth promoting effects to
different degrees, the relative weight gain rates are improved by 7.17%,
4.96% and 4.69% respectively, the feed conversion rates are reduced by
0.133, 0.097 and 0.085 respectively, and the growth promoting effect and
the feed reward improvement of p-thymol are superior to those of the
thymol and carvacrol test groups (Table 14).

TABLE-US-00017
TABLE 14
Growth Promoting Test Results of P-thymol, Thymol and
Carvacrol on Pigs
Total
Quan- Aver- Relative Feed
tity Sur- age Weight Total Con- Feed
of vival Weight Gain Weight sump- Conver-
Ani- Rate Gain Rate Gain tion sion
Group mals (%) (g) (%) (kg) (kg) Rate
contro1 30 100 10.88 100 326.4 790.2 2.421
group
p-thymol 30 50 11.66 107.17 349.8 800.2 2.288
group
thymol 30 100 11.42 104.96 342.6 796.3 2.324
group
carvacrol 30 100 11.39 104.69 341.7 798.1 2.336
group

Embodiment 7

Growth Promoting Effect Test of p-Thymol on Ducks

Test Materials

[0070] 1-day-old Chemy Valley ducks, provided by Guangzhou Nongfeng
Breeding Duck Farm

[0071] 807-type complete formula duck feed: containing no antibiotic
growth promoters, provided by Guangzhou Huinong Feed Factory p-thymol,
thymol and carvacrol, purchased from SIGMA company.

(2) Test Method

[0072] 400 1-day-old Chemy Valley ducks are randomly divided into 4
groups, and each group comprises 100 ducks. Different growth promoters
(Table 15) are added to the feed of each group, and the survival rate,
the weight gain and the feed reward of each group of 1 to 46-day-old
ducks are counted. The test process is cage culture above a net.

TABLE-US-00018
TABLE 15
Growth Promoting Test Groups of P-thymol, Thymol and
Carvacrol on Ducks
Quantity of
Group Animals Drug Dose (ppm)
control group without 100 — —
adding the drug
p-thymol group 100 p-thymol 100
thymol group 100 thymol 100
carvacrol group 100 carvacrol 100

(3) Test Results

[0073] As shown by the breeding experimental results, after p-thymol,
thymol and carvacrol are added into the complete formula feed
respectively, compared with the control group without adding the drug,
p-thymol, thymol and carvacrol have obvious growth promoting effects, the
relative weight gain rates are improved by 6.65%, 4.38% and 2.51%
respectively, the feed conversion rates are reduced by 0.112, 0.062 and
0.049 respectively, and the growth promoting effect and the feed reward
improvement of p-thymol are superior to those of the thymol and carvacrol
test groups (Table 16).

TABLE-US-00019
TABLE 16
Comparison Test Results of Duck Growth Promoting Effects
of P-thymol, Thymol and Carvacrol
Total
Aver- Relative Feed
Sur- age Weight Total Con- Feed
vival Weight Gain Weight sump- Conver-
Dose Rate Gain Rate Gain tion sion
Group (ppm) (%) (g) (%) (kg) (kg) Rate
control 0 100 3.306 100 330.6 921.7 2.788
group
without
adding
the drug
p-thymol 100 100 3.526 106.65 352.6 943.6 2.676
group
thymol 100 100 3.451 104.38 345.1 940.7 2.726
group
carvacrol 100 100 3.389 102.51 338.9 928.3 2.739
group

Embodiment 8

Application of p-Thymol in Aquatic Feed

(1) Test Materials

[0074] Test fishes: the test fishes are black carps from annual
fingerlings and are provided by Dafeng Breeding Farm in Huizhou of
Guangdong. The healthy and lively black carps with the consistent size
are bred for 4 weeks in a big net cage (4×2×1.5 m3) and then
are used for formal breeding test, the experimental system comprises
floating small net cages (1.1×1.1×1.1 m3), an inflating head
is arranged in each small net cage, and the small net cages are inflated
all the day. The small net cages and temporary net cages are placed in a
pond of 3500 m2 in a test field, the water depth of the pond is about 1.5
m, and the water in the pond is fully-aerated bottom water. During the
test, 192 black carps which are hungry for 1 day are randomly divided
into 4 groups, each group is repeated for 4 times, 12 black carps are put
into each repeated group, and the whole groups are weighed, then randomly
put into 12 net cages and fed with different test feeds respectively.

[0075] Test feed: the test feed is prepared according to the formula of
Table 17, and different test groups are added with different growth
promoters according to Table 18 respectively. The raw materials for the
feed are super-finely crushed and then prepared into a floating puffed
feed with the grain diameter of 3 mm through a Jiangsu shepherd puffing
unit, the mold stripping temperature is 130° C., the floating
puffed feed is sprayed with 3% of soybean oil through oil spraying
equipment, and the feed is sealed and preserved in the shade for later
use.

TABLE-US-00020
TABLE 17
Formula and Chemical Components (% wt.) of Test Black Carp
Feed
Content Content
Raw Materials (%) Raw Materials (%)
fish meal 9.0 soybean oil 3.0
casing powder 3.0 phospholipid 9.0
rapeseed meal
bean pulp 12.0 wheat gluten 4.0
rapeseed meal 12.0 blood cell meal 2.0
monosodium 3.0 Vc-phosphate 0.1
glutamate
protein
wheat middling 12.6 calcium dihydrogen 1.8
phosphate
flour 17.0 choline chloride 0.2
bentonite 0.70 multivitamins 0.1
rice bran 10.0 micro-mineral 0.5
premix

TABLE-US-00021
TABLE 18
Growth Promoting Test Groups of P-thymol, Thymol and
Carvacrol on Fishes
Dose
Group Drug (ppm)
control group without drug — —
p-thymol group p-thymol 100
thymol group thymol 100
carvacrol group carvacrol 100

[0076] Test Method

[0077] Test management: the test adopts artificial limited feeding, the
feeding quantity is adjusted once a week, the feeding levels (according
to the initial weights) of two groups are completely consistent, and the
fishes are fed twice (7:30 and 15:00) every day. The test period is 8
weeks. The water quality is periodically monitored during the test. In
the whole breeding process, the temperature of water is
26.88±3.08° C., the dissolved oxygen (DO) is more than 5.0 mg
OL-1, the pH is 7.8, the ammonia nitrogen is less than 0.50 mg N L-1, and
the nitrite nitrogen is less than 0.05 mg N L-1.

[0078] Parametric statistics: during the test, each whole net cage is
weighed after feeding is stopped for 1 day, and the weight gain (WG, %),
the feed conversion rate (FCR) and the survival rate (SR, %) are
calculated. The calculation formulas are as follows:

weight gain(WG,%)=100×(average final weight-average initial
weight)/average initial weight;

feed conversion rate(FCR)=feed intake/fish weight gain;

survival rate(SR,%)=100×fish quantity when the test is
completed/fish quantity when the test starts.

[0079] Test Results

[0080] The growth promoting test results of p-thymol, thymol and carvacrol
on the fishes are shown in Table 19. The results show that the test
groups added with p-thymol, thymol and carvacrol are superior to the
control group without the drug on the aspects of weight gain, feed
conversion rate and survival rate, and p-thymol, thymol and carvacrol
have obvious growth promoting effects, wherein the average weight gains
are improved by 7.47%, 4.53% and 3.52% respectively, and the feed
conversion rates are reduced by 5.37%, 3.36% and 2.68%. The results show
that the p-thymol test group is superior to the thymol and carvacrol test
groups on the aspects of growth promotion and feed reward improvement.

TABLE-US-00022
TABLE 19
Application Test Results of P-thymol in Aquatic Feed
Control P-thymol Carvacrol
Parameter group group Thymol group group
initial 251.20 ± 251.60 ± 2.73 251.40 ± 2.35 251.30 ± 2.18
weight (g) 2.43
final 586.20 ± 611.61 ± 26.03 601.58 ± 28.02 598.08 ± 27.11
weight (g) 25.40
weight 133.36 ± 143.09 ± 8.22 139.29 ± 7.37 138.0 ± 7.78
gain (%) 7.97
feed 1.49 ± 1.41 ± 0.09 1.44 ± 0.08 1.45 ± 0.06
conversion 0.11
rate (FCR)
survival 100 100 100 100
rate (SR)
(%)

Embodiment 9

Growth Promoting Effect Research of p-Thymol and Salt or Ester Derivatives
Thereof on Animals and Pigs

Test Materials

[0081] Test animals: 140 50-day-old healthy three-way cross pigs, provided
by Guangdong Minfeng Breeding Pig Farm

[0082] 304-type complete formula pig feed: containing no antibiotics,
provided by the Feed Factory of Guangdong Minfeng Livestock Development
Co., Ltd.

[0083] p-thymol sodium, p-thymol resin salt, p-thymol ammonium and
p-thymol ethyl ester are prepared in embodiment 1, and p-thymol, thymol
and carvacrol are purchased from SIGMA company.

(2) Test Method

[0084] 140 50-day-old pigs are grouped as in Table 20, and each group
comprises 20 pigs. After different test samples are added into the feed,
each group freely eats the feed. The weight gain of test pigs of each
test group and the feed reward on the 30th day after the test starts are
counted, and the influence of different p-thymol derivatives on the
production performance of the test pigs is compared.

TABLE-US-00023
TABLE 20
Test Groups of the Influence of P-thymol Derivatives on the
Production Performance of the Test Pigs
Quantity Average
of Initial Growth Dose
Group Animals Weight (kg) Promoter (ppm)
1 20 15.60 — —
2 20 15.65 thymol 100
3 20 15.42 p-thymol 100
4 20 15.50 p-thymol 100
resin salt
5 20 15.48 p-thymol 100
sodium
6 20 15.52 p-thymol 100
ammonium
7 20 15.55 p-thymol 100
ethyl ester

(3) Test Results

[0085] As shown by animal feeding test results, p-thymol and the salt or
ester derivatives thereof have remarkable improvement on the growth
performance of the test pigs; the average weight gains of the p-thymol
sodium, p-thymol resin salt, p-thymol ammonium and p-thymol ethyl ester
test groups are improved by 9.4%, 9.1%, 8.9% and 10.7% respectively
compared with the control group without adding the growth promoter; and
the feed conversation rates are reduced by 0.199, 0.199, 0.197 and 0.190
respectively. The growth promoting effects of p-thymol and the salt or
ester derivatives thereof are equivalent to the effects of the same dose
of p-thymol test groups but superior to the effect of the thymol test
group (Table 21).

TABLE-US-00024
TABLE 21
Test Results of the Test Influence of P-thymol and Salt or
Ester Derivatives Thereof on the Production Performance of the Pigs
Aver- Relative Total
age Weight Total Feed Feed
Survival Weight Gain Weight Con- Conver-
Rate Gain Rate Gain sumption sion
Group (%) (g) (%) (kg) (kg) Rate
control 100 15.66 100 313.2 694.7 2.218
group
without
drug
thymol 100 16.22 103.6 324.4 680.6 2.098
group
p-thymol 100 17.10 109.2 342.0 693.9 2.029
group
p-thymol 100 17.13 109.4 342.6 693.8 2.025
sodium
group
p-thymol 100 17.08 109.1 341.6 691.7 2.025
resin
salt
group
p-thymol 100 17.05 108.9 341.0 691.6 2.028
ammonium
group
p-thymol 100 17.34 110.7 346.8 701.2 2.022
ethyl
ester
group

[0086] The embodiments are better embodiments of the invention, but the
implementation modes of the invention are not limited by the embodiments,
and other any changes, modifications, replacements, combinations and
simplifications which do not depart from the spirit and principle of the
invention are equivalent replacement modes and shall be encompassed in
the protection scope of the invention.

[0087] The invention discloses p-thymol and salt or ester derivatives
thereof in an animal feed additive. P-thymol is an isomeride of thymol
with the chemical name of 3-methyl-4-isopropyl phenol. The salt
derivatives of p-thymol comprise salts formed by p-thymol and metal ions,
p-thymol ammonium formed by p-thymol and ammonia, resin salts formed by
p-thymol and negative ion resins and the like; and the ester derivatives
of p-thymol comprise esters formed by p-thymol and different carboxylic
acids. The applicants firstly discover that p-thymol and the salt
derivatives or the ester derivatives thereof have stronger antibacterial
activity, lower volatility, higher safety and lower thrill compared with
thymol and carvacrol. When being taken as the additive of the animal
growth promoting feed, p-thymol and the salt derivatives or the ester
derivatives thereof have better palatability and growth promoting effect
compared with thymol or carvacrol.

[0088] Those skilled in the art will readily observe that numerous
modifications and alterations of the device and method may be made while
retaining the teachings of the invention. Accordingly, the above
disclosure should be construed as limited only by the metes and bounds of
the appended claims.

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