AU2021102064A4

AU2021102064A4 - Fermented astragalus propinquus (a. propinquus) and preparation method thereof, feed additive and preparation method thereof, and beef cattle feed

Info

Publication number: AU2021102064A4
Application number: AU2021102064A
Authority: AU
Inventors: Yueping GAO; Pengfei Li; Jiayu Liu; Liuliu LIU; Qiang Liu
Original assignee: Shanxi Fanshi County Tianhemuye Co Ltd; Shanxi Agricultural University
Current assignee: Shanxi Fanshi County Tianhemuye Co Ltd; Shanxi Agricultural University
Priority date: 2020-06-19
Filing date: 2021-04-20
Publication date: 2021-06-03
Anticipated expiration: 2029-04-20
Also published as: CN111789186B; CN111789186A

Abstract

The present disclosure belongs to the technical field of fermented Astragalus propinquus (A. propinquus), and specifically relates to fermented A. propinquus and a preparation method thereof, a feed additive and a preparation method thereof, and a beef cattle feed. The fermented A. 5 propinquus is prepared from the following raw materials in parts by mass: 90,000 to 110,000 parts of A. propinquus, 1,800 to 2,200 parts of sugar, 18 to 22 parts of laccase, 36 to 44 parts of acid cellulase, 36 to 44 parts of acid xylanase, 23 to 27 parts of lactobacilli, 45 to 55 parts of cellulose-decomposing bacteria, and 60,000 to 80,000 parts of water. The lactobacilli have a viable count of 9 x 10" CFU/g to 11 x 10 1CFU/g and the cellulose-decomposing bacteria have 10 a viable count of 1.5 x 109 CFU/g to 1.7 x 109 CFU/g. In the present disclosure, cellulose and xylan are decomposed through the synergistic effect of laccase, cellulose-decomposing bacteria, lactobacilli, acid cellulase, and acid xylanase to finally improve a release rate of active ingredients in A. propinquus, thereby increasing the utilization of A. propinquus.

Description

FERMENTED ASTRAGALUSPROPINQUUS (A. PROPINQUUS) AND PREPARATION METHOD THEREOF, FEED ADDITIVE AND PREPARATION METHOD THEREOF, AND BEEF CATTLE FEED TECHNICAL FIELD

The present disclosure belongs to the technical field of fermented Astragaluspropinquus (A.

propinquus), and specifically relates to fermented A. propinquus and a preparation method

thereof, a feed additive and a preparation method thereof, and a beef cattle feed.

BACKGROUND

Astragalus polysaccharides (APSs), saponins, flavonoids, and other compounds in A.

propinquus have strong biological activity. Astragalus saponins play an important role in

promoting the proliferation and differentiation of lymphocytes and the regulation of

proto-oncogene transcription, which can also influence macrophages to enhance the cellular

immunity. Astragalus saponins can lower the blood pressure by dilating blood vessels, and

protect cardiac functions by improving the myocardial contraction and relaxation and increasing

the coronary flow. Modern medical research shows that the active ingredients in A. propinquus

have the effects of enhancing immunologic functions in an organism, promoting cellular

metabolism, anti-oxidation, regulating the synthesis of DNA, RNA, and protein, and reducing

the disease attack, so A. propinquus is an excellent health and preventive medicine. In order to make full use of the resource endowment of the local medicinal material of A. propinquus in

China, A. propinquus beef cattle are raised, so that people can enjoy the delicacy of beef and the

health function at the same time; and dominant beef cattle breeds with independent

characteristics, an A. propinquus beef cattle brand, and functional beef products with a unique

flavor are produced to lead the development of the beef cattle industry and realize the industrial

goal of "technical support, farming and animal husbandry cycle, industrial efficiency increase,

and farmers' income increase".

However, the method of directly mixing A. propinquus into a beef cattle feed commonly

used at a current stage cannot achieve the effective release of active ingredients wrapped by

lignocellulose in A. propinquus, which reduces the utilization of A. propinquus.

SUMMARY

The present disclosure is intended to overcome the shortcomings existing in the prior art and

provide fermented A. propinquus and a preparation method thereof, a feed additive and a

preparation method thereof, and a beef cattle feed. The fermented A. propinquus of the present

disclosure improves a release rate of active ingredients in A. propinquus, thus increasing the

utilization of A. propinquus.

To achieve the above objective, the present disclosure provides the following technical

solutions:

The present disclosure provides fermented A. propinquus prepared from the following raw

materials in parts by mass:

90,000 to 110,000 parts of A. propinquus, 1,800 to 2,200 parts of sugar, 18 to 22 parts of

laccase, 36 to 44 parts of acid cellulase, 36 to 44 parts of acid xylanase, 23 to 27 parts of

lactobacilli, 45 to 55 parts of cellulose-decomposing bacteria, and 60,000 to 80,000 parts of

water.

The lactobacilli have a viable count of 9 x 10" CFU/g to 11 x 1011 CFU/g and the

cellulose-decomposing bacteria have a viable count of 1.5 x 109 CFU/g to 1.7 x 109 CFU/g.

Preferably, the A. propinquus may be selected according to the following criteria: moisture

content: not higher than 13%; ash content: not higher than 5%; astragaloside IV content: not

lower than 0.04%; and calycosin content: not less than 0.05%.

Preferably, the cellulose-decomposing bacteria may include Bacillus subtilis (B. subtilis).

The present disclosure provides a method for preparing the fermented A. propinquus

according to the above solution, including the following steps:

crushing the A. propinquus, mixing crushed A. propinquus with the water, lactobacilli, and

sugar, and conducting sealed fermentation for 2 d to 3 d to obtain a first fermented product;

mixing the first fermented product with the laccase, cellulose-decomposing bacteria, acid

cellulase, and acid xylanase, and conducting sealed fermentation for 5 d to 8 d to obtain a second fermented product; and air-drying and crushing the second fermented product to obtain the fermented A. propinquus; where, the sealed fermentation to obtain the first fermented product and the sealed fermentation to obtain the second fermented product are both conducted at 30°C to 45°C.

Preferably, a preparation method of the second fermented product may include: mixing the

first fermented product with the laccase, and conducting sealed fermentation for 2 d to 3 d; and

mixing a resulting product with the cellulose-decomposing bacteria, acid cellulase, and acid

xylanase, and conducting sealed fermentation for 3 d to 5 d to obtain the second fermented

product.

The present disclosure provides a feed additive including the fermented A. propinquus

according to the above solution and a coating. The fermented A. propinquusand the coating may

have a mass ratio of 100:(12-20); and the coating may include grease with a melting point of

37°C to 40°C.

Preferably, the coating may further include calcium fatty acid, and the grease may have a

mass ratio of 3:2 with the calcium fatty acid.

The present disclosure provides a method for preparing the feed additive according to the

above solution, including: spray-mixing the grease with fermented A. propinquus to obtain the

feed additive.

The present disclosure provides a beef cattle feed, and based on dry matters in parts by mass,

the beef cattle feed may include the following components: 99.25 to 99.75 parts of basal feed

and 0.25 to 0.75 parts of the feed additive according to claim 6 or 7.

Preferably, based on dry matters in parts by mass, the basal feed may include the following

components: 32 to 36 parts of corn silage, 6 to 10 parts of alfalfa hay, 7 to 9 parts of

Arrhenatherum elatius, 28.2 to 30.2 parts of corn, 3.6 to 4.5 parts of bran, 5.4 to 6.6 parts of

soybean meal, 2.3 to 2.7 parts of rapeseed meal, 5.4 to 6.6 parts of cotton meal, 0.5 part of

calcium carbonate, 0.5 part of salt, 0.3 part of dicalcium phosphate (DCP), and 0.5 part of

mineral-vitamin premix.

Beneficial effects:

materials in parts by mass: 90,000 to 110,000 parts of A. propinquus, 1,800 to 2,200 parts of

sugar, 18 to 22 parts of laccase, 36 to 44 parts of acid cellulase, 36 to 44 parts of acid xylanase,

23 to 27 parts of lactobacilli, 45 to 55 parts of cellulose-decomposing bacteria, and 60,000 to

80,000 parts of water. The lactobacilli have a viable count of 9 x 1011 CFU/g to 11 x 1011 CFU/g

and the cellulose-decomposing bacteria have a viable count of 1.5 x 109 CFU/g to 1.7 x 109

CFU/g. In the present disclosure, lignin is oxidated by laccase to break a link between cell wall

lignin and hemicellulose, and then cellulose and xylan are decomposed through the synergistic effect of cellulose-decomposing bacteria, lactobacilli, acid cellulase, and acid xylanase to finally improve a release rate of active ingredients in A. propinquus, thereby increasing the utilization of

A. propinquus.

Furthermore, in the feed additive provided by the present disclosure, the surface of the

fermented A. propinquusis evenly coated with a layer of film through high-melting-point grease,

which reduces the degradation of rumen microorganisms to active ingredients in the fermented A.

propinquus and thus increases the content of the active ingredients of A. propinquus in beef,

thereby further improving the economic benefits of beef.

Furthermore, the beef cattle feed provided in the present disclosure is added with the feed

additive to increase the dry matter intake (DMI) and average daily weight increase of beef cattle

and improve the digestibility of beef cattle and the deposition of active ingredients of A.

propinquus in beef, thereby increasing the weight-gaining performance and functional

characteristics of beef cattle to improve the economic benefits.

DETAILED DESCRIPTION

CFU/g.

Unless otherwise specified, the present disclosure has no special limitations on sources of

the components in the fermented A. propinquus, and commercially-available products well

known to those skilled in the art may be adopted.

In parts by mass, the fermented A. propinquus provided in the present disclosure may

include 90,000 to 110,000 parts, more preferably 95,000 to 105,000 parts, and most preferably

100,000 parts of A. propinquus. In the present disclosure, the A. propinquus may preferably be

selected according to the following criteria: moisture content: not higher than 13%; ash content:

not higher than 5%; astragaloside IV content: not lower than 0.04%; and calycosin content: not

less than 0.05%. In the present disclosure, the moisture content not higher than 13% in A.

propinquus can meet storage conditions and prevent mildew; and the ash content not higher than

5%, astragaloside IV content not lower than 0.04%, and calycosin content not lower than 0.05%

can ensure that obtained A. propinquus beef has a calycosin glycoside content not less than 0.15

pg/g and an astragaloside IV content not less than 75 pg/g.

Based on the parts of the A. propinquus, the fermented A. propinquusprovided in the present

disclosure includes 1,800 to 2,200 parts, more preferably 1,900 to 2,100 parts, and most preferably 2,000 parts of sugar. The sugar of the present disclosure can provide a carbon source required for fermentation and can be decomposed into lactic acid by lactobacilli to achieve acidification of the fermentation environment, which is more conducive to the subsequent fermentation of A. propinquus. In the present disclosure, the sugar may preferably include glucose.

disclosure may include 18 to 22 parts, more preferably 19 to 21 parts, and most preferably 20

parts of laccase. The laccase of the present disclosure oxidizes the lignin in A. propinquus to

break a link between cell wall lignin and hemicellulose, which is more conducive to the release

of subsequent active ingredients in A. propinquus by the combination of enzymes and bacteria.

In the present disclosure, the laccase may have an enzymatic activity preferably of 90,000 U/g to

110,000 U/g. Based on the parts of the A. propinquus, the fermented A. propinquusprovided in

the present disclosure may include 36 to 44 parts, more preferably 38 to 42 parts, and most

preferably 40 parts of acid cellulase. The acid cellulase of the present disclosure can decompose

the cellulose in A. propinquus to release the active ingredients in A. propinquus. In addition, the

active ingredients of A. propinquus are released in an acidic environment, which is more

conducive to the adaptation to the rumen environment and thus reduces the degradation of the

active ingredients of A. propinquus by rumen. In the present disclosure, the acid cellulase may

have an enzymatic activity preferably of 90,000 U/g to 110,000 U/g.

disclosure may include 36 to 44 parts, more preferably 38 to 42 parts, and most preferably 40

parts of acid xylanase. The acid xylanase of the present disclosure can decompose the xylan in A.

propinquus to release the active ingredients in A. propinquus. In addition, the active ingredients

of A. propinquus are released in an acidic environment, which is more conducive to the

adaptation to the rumen environment and thus reduces the degradation of the active ingredients

of A. propinquusby rumen. In the present disclosure, the acid xylanase may have an enzymatic

activity preferably of 45,000 U/g to 55,000 U/g.

disclosure may include 23 to 27 parts, more preferably 24 to 26 parts, and most preferably 25

parts of lactobacilli. In the present disclosure, the lactobacilli may have a viable count of 9 x 10"

CFU/g to 11 x 10" CFU/g, more preferably of 9.5 x 10" CFU/g to 10.5 x 10" CFU/g, and most

preferably of 10 x 1011 CFU/g. The lactobacilli of the present disclosure can decompose the

sugar into lactic acid to achieve the acidification of the fermentation environment, which is more

conducive to the subsequent fermentation of A. propinquus. Moreover, the lactobacilli can also

synergize with the acid cellulase and acid xylanase to decompose cellulose and xylan, thereby

improving a release rate of active ingredients in A. propinquus. In the present disclosure, the

lactobacilli may preferably be Lactobacillus acidophilus (L. acidophilus).

Based on the parts of the A. propinquus, the fermented A. propinquusprovided in the present disclosure may include 45 to 55 parts, more preferably 47 to 53 parts, and most preferably 50 parts of cellulose-decomposing bacteria. In the present disclosure, the cellulose-decomposing bacteria may have a viable count of 1.5 x 109 CFU/g to 1.7 x 109 CFU/g, more preferably of

1.55 x 109 CFU/g to 1.65 x 109 CFU/g, and most preferably of 1.6 x 109 CFU/g. The

cellulose-decomposing bacteria of the present disclosure can not only decompose the cellulose in

A. propinquus to release the active ingredients in A. propinquus, but also can synergize with the

acid xylanase and lactobacilli to decompose xylan, thereby improving a release rate of active

ingredients in A. propinquus. In the present disclosure, the cellulose-decomposing bacteria may

preferably include B. subtilis.

The present disclosure also provides a method for preparing the fermented A. propinquus

according to the above solution, including the following steps:

cellulase, and acid xylanase, and conducting sealed fermentation for 5 d to 8 d to obtain a second

fermented product; and

air-drying and crushing the second fermented product to obtain the fermented A. propinquus;

where, the sealed fermentation to obtain the first fermented product and the sealed fermentation to obtain the second fermented product are both conducted at 30°C to 45°C.

In the present disclosure, A. propinquus is crushed and mixed with water, lactobacilli, and

sugar and then sealed fermentation is conducted for 2 d to 3 d to obtain the first fermented

product, which makes sugar decomposed into lactic acid by lactobacilli to achieve the

acidification of the fermentation environment, thus facilitating the subsequent fermentation of A.

propinquus. In addition, the active ingredients in A. propinquus are released in an acidic

environment, which is more conducive to the adaption to the rumen environment and reduces the

degradation of the active ingredients in A. propinquusby rumen.

In the present disclosure, a method for preparing the second fermented product may

preferably include: mixing the first fermented product with the laccase, and conducting sealed

fermentation for 2 d to 3 d; and mixing a resulting product with the cellulose-decomposing

bacteria, acid cellulase, and acid xylanase, and conducting sealed fermentation for 3 d to 5 d to

obtain the second fermented product. In the present disclosure, the laccase is used to ferment the

first fermented product, where, the laccase oxidizes the lignin in A. propinquus to break a link

between cell wall lignin and hemicellulose, which is more conducive to the subsequent release of

active ingredients in A. propinquusby the combination of enzymes and bacteria.

The present disclosure also provides a feed additive including the fermented A. propinquus

according to the above solution and a coating. The fermented A. propinquusand the coating may have a mass ratio of 100:(12-20); and the coating may include grease with a melting point of

37°C to 40°C. In the present disclosure, the fermented A. propinquusand the coating may a mass

ratio more preferably of 100:(12-15) and most preferably of 100:12. The coating may preferably

include grease with a melting point of 37°C, and the grease may preferably include palm oil.

In the present disclosure, the coating may preferably further include calcium fatty acid; and

the grease and the calcium fatty acid may have a mass ratio of 3:2.

The present disclosure also provides a method for preparing the feed additive according to

the above solution, including: spray-mixing the grease with the fermented A. propinquus to

obtain the feed additive.

In the present disclosure, when the coating also preferably includes calcium fatty acid, the

preparation method of the feed additive may preferably include: spray-mixing 5/6 of the total

mass of grease with the fermented A. propinquus to obtain coated A. propinquus; and mixing the

coated A. propinquuswith the calcium fatty acid, and spray-mixing a resulting mixture with 1/6

of the total mass of grease to obtain the feed additive.

In the present disclosure, a spray-mixing method may preferably include: heating the grease

to 60°C to 70°C, and spray-mixing in a mixer (GHJ-10) for 2 min to 3 min.

The present disclosure also provides a beef cattle feed, including the feed additive according

to the above solution and a basal feed. Based on dry matters in parts by mass, the beef cattle feed may include the following components: 99.25 to 99.75 parts of basal feed and 0.25 to 0.75 parts of the feed additive according to the above solution.

In the present disclosure, based on dry matters in parts by mass, the basal feed may

preferably include the following components: 32 to 36 parts of corn silage, 6 to 10 parts of

alfalfa hay, 7 to 9 parts of Arrhenatherum elatius, 28.2 to 30.2 parts of corn, 3.6 to 4.5 parts of

bran, 5.4 to 6.6 parts of soybean meal, 2.3 to 2.7 parts of rapeseed meal, 5.4 to 6.6 parts of cotton

meal, 0.5 part of calcium carbonate, 0.5 part of salt, 0.3 part of DCP, and 0.5 part of

mineral-vitamin premix.

In the present disclosure, the above mineral-vitamin premix may preferably include the

following components in parts by mass: 3.66 parts of ferrous sulfate monohydrate, 3.85 parts of

manganese sulfate monohydrate, 1.3 parts of copper sulfate pentahydrate, 4.27 parts of zinc

sulfate monohydrate, 0.03 part of potassium iodide, 0.03 part of sodium selenite, 0.01 part of

cobalt chloride, 10 parts of zeolite, 40.48 parts of bentonite, 2 parts of edible oil, 0.32 part of

vitamin A, 0.05 part of vitamin D3, 4 parts of vitamin E, and 30 parts of wheat middling.

The present disclosure preferably provides a method for preparing the mineral-vitamin

premix according to the above solution, including the following steps: starting a mixer, and

adding 80% of the bentonite and the zeolite to the mixer; then adding the trace elements in the

descending order of the proportion, adding the remaining 20% of the bentonite, and thoroughly mixture; and adding the vitamins and the wheat middling, and thoroughly mixing a resulting mixture to obtain the mineral-vitamin premix.

To further illustrate the present disclosure, the fermented A. propinquus and the preparation

method thereof, the feed additive and the preparation method thereof, and the beef cattle feed

provided in the present disclosure are described in detail below with reference to examples, but

these examples should not be interpreted as a limitation to the protection scope of the present

disclosure.

Example 1

Fermented A. propinquus was prepared from the following raw materials: 90 kg of A.

propinquus, 1.8 kg of sugar, 18 g of laccase, 36 g of acid cellulase, 36 g of acid xylanase, 23 g of

L. acidophilus (10 x 1011CFU/g), 45 g of B. subtilis (1.6 x 109 CFU/g), and 60 kg of water.

Preparation of the fermented A. propinquus:

A. propinquus was crushed (sieved through a 2 mm sieve) and mixed with water, L.

acidophilus, and sugar, and a resulting mixture was subjected to sealed fermentation at 30°C for

2 d to obtain a first fermented product;

Laccase was added to the first fermented product, and a resulting mixture was subjected to

sealed fermentation at 30°C for 2 d; and then cellulose-decomposing bacteria, acid cellulase, and

acid xylanase were added, and a resulting mixture was subjected to sealed fermentation at 30°C for 3 d to obtain a second fermented product; and the second fermented product was air-dried (a moisture content not higher than 13%) and crushed (sieved through a 2 mm sieve) to obtain the fermented A. propinquus.

Example 2

Fermented A. propinquus was prepared from the following raw materials: 100 kg of A.

propinquus, 2 kg of sugar, 20 g of laccase, 40 g of acid cellulase, 40 g of acid xylanase, 25 g of L.

acidophilus (10 x 1011 CFU/g), 50 g of B. subtilis (1.6 x 109 CFU/g), and 70 kg of water.

A preparation method of the fermented A. propinquuswas the same as in Example 1.

Example 3

Fermented A. propinquus was prepared from the following raw materials: 110 kg of A.

propinquus, 2.2 kg of sugar, 22 g of laccase, 44 g of acid cellulase, 44 g of acid xylanase, 27 g of

L. acidophilus (10 x 1011 CFU/g), 55 g of B. subtilis (1.6 x 109 CFU/g), and 80 kg of water.

Example 4

A feed additive was prepared from the following components: 100 kg of the fermented A.

propinquusprepared in Example 2 and 12 kg of palm oil with a melting point of 37C.

Preparation of the feed additive: the palm oil was heated to 60°C, then spray-mixing was conducted in a mixer (GHJ-10) for 2 min, and a resulting mixture was cooled to obtain the feed additive.

Example 5

A feed additive included the following components: 100 kg of the fermented A. propinquus

prepared in Example 2, 12 kg of palm oil with a melting point of 37°C, and 8 kg of calcium fatty

acid.

Preparation of the feed additive: 10 kg of palm oil was heated to 60°C, then spray-mixing

was conducted in a mixer (GHJ-10) for 2 min, and a resulting mixture was cooled to obtain

coated A. propinquus; then the remaining 2 kg of palm oil was heated to 60°C, and the calcium

fatty acid and coated A. propinquus were added; and spray-mixing was conducted in a mixer

(GHJ-10) for 1 min, and a resulting mixture was cooled to obtain the feed additive.

Example 6

prepared in Example 2, 9 kg of palm oil with a melting point of 37°C, and 6 kg of calcium fatty

acid.

Preparation of the feed additive: 7.5 kg of palm oil was heated to 60°C, then spray-mixing

coated A. propinquus;then the remaining 1.5 kg of palm oil was heated to 60°C, and the calcium fatty acid and coated A. propinquus were added; and spray-mixing was conducted in a mixer

Example 7

A beef cattle feed was prepared from the following components: 34 kg of corn silage, 8 kg

of alfalfa hay, 8 kg of Arrhenatherum elatius, 29.2 kg of corn, 4.25 kg of bran, 6.0 kg of soybean

meal, 2.5 kg of rapeseed meal, 6.0 kg of cotton meal, 0.25 kg of feed additive (Example 4), 0.5

kg of calcium carbonate, 0.5 kg of salt, 0.3 kg of DCP, and 0.5 kg of mineral-vitamin premix.

The above mineral-vitamin premix was prepared from the following raw materials: 3.66 kg

of ferrous sulfate monohydrate, 3.85 kg of manganese sulfate monohydrate, 1.3 kg of copper

sulfate pentahydrate, 4.27 kg of zinc sulfate monohydrate, 30 g of potassium iodide, 30 g of

sodium selenite, 10 g of cobalt chloride, 10 kg of zeolite, 40.48 kg of bentonite, 2 kg of edible oil,

320 g of vitamin A, 50 g of vitamin D3, 4 kg of vitamin E, and 30 kg of wheat middling.

Preparation of the mineral-vitamin premix:

A mixer was started, and 80% of the bentonite and the zeolite were first added to the mixer;

then the trace elements were added in the descending order of the proportion, the remaining 20%

of the bentonite was added, and a resulting mixture was thoroughly mixed; then 2% of the edible

oil was sprayed, and a resulting mixture was thoroughly mixed; and the vitamins and the wheat

middling were finally added, and a resulting mixture was thoroughly mixed to obtain the mineral-vitamin premix.

The above components were directly mixed thoroughly to obtain the beef cattle feed.

Example 8

of alfalfa hay, 8 kg of Arrhenatherum elatius, 29.2 kg of corn, 4 kg of bran, 6.0 kg of soybean

meal, 2.5 kg of rapeseed meal, 6.0 kg of cotton meal, 0.5 kg of feed additive (Example 4), 0.5 kg

of calcium carbonate, 0.5 kg of salt, 0.3 kg of DCP, and 0.5 kg of mineral-vitamin premix.

The mineral-vitamin premix was the same as in Example 7.

Example 9

of alfalfa hay, 8 kg of Arrhenatherum elatius, 29.2 kg of corn, 3.75 kg of bran, 6.0 kg of soybean

meal, 2.5 kg of rapeseed meal, 6.0 kg of cotton meal, 0.75 kg of feed additive (Example 4), 0.5

The mineral-vitamin premix was the same as in Example 7.

Comparative Example 1

of alfalfa hay, 8 kg of Arrhenatherum elatius, 29.2 kg of corn, 4.5 kg of bran, 6.0 kg of soybean

meal, 2.5 kg of rapeseed meal, 6.0 kg of cotton meal, 0.5 kg of calcium carbonate, 0.5 kg of salt,

0.3 kg of DCP, and 0.5 kg of mineral-vitamin premix.

The mineral-vitamin premix was the same as in Example 7.

Application Example 1

Comparative test for the release rate of active ingredients in Examples 1 to 3 and

unfermented A. propinquus

200 g of each of unfermented A. propinquus and the fermented A. propinquusin Examples 1

to 3 was taken. Then the following methods were used to determine the contents of astragaloside

IV and calycosin glycoside. Determination results were shown in Table 1.

Astragaloside IV (C41H68014) was determined in accordance with the high-performance

liquid chromatography (HPLC) (General Principle 0512) described on page 302 of the Chinese

Pharmacopoeia (version 2015). The difference lies in the preparation of test solutions. In the

present disclosure, 4 g of each of the unfermented A. propinquus and fermented A. propinquusin

Examples 1 to 3 was weighed accurately and added to a Soxhlet extractor, with 3 replicates for

each sample.

Calycosin glycoside (C22H22010) was determined in accordance with the HPLC (General

Principle 0512) described on page 302 of the Chinese Pharmacopoeia (version 2015). The

difference lies in the preparation of test solutions. In the present disclosure, 1 g of each of the

unfermented A. propinquus and fermented A. propinquus in Examples 1 to 3 was weighed

accurately and added to a round-bottomed flask, with 3 replicates for each sample.

Release rate of active ingredients = A. propinquus active ingredient content/A. propinquus

mass x 100%

Table 1 Release rate of active ingredients in unfermented A. propinquus and fermented A.

propinquus

Item Unfermented A. propinquus Example 1 Example 2 Example 3

Astragaloside IV (%) 0.041 0.054 0.055 0.054

Calycosin glycoside(%) 0.046 0.061 0.063 0.062

It can be seen from Table 1 that the release rate of Astragaloside IV in the fermented A.

propinquus of Examples 1 to 3 is increased by 32.5% compared with that in the unfermented A.

propinquus, and the release rate of calycosin glycoside in the fermented A. propinquus of

Examples 1 to 3 is increased by 36.4% compared with that in the unfermented A. propinquus.

Astragaloside IV and calycosin glycoside are distributed in A. propinquus cells and intercellular

matrix, mainly in cells, and cells have a diameter of 30 pm 80 pm. A crushing granularity in general production cannot allow the effective release of active ingredients in A. propinquus. The solvent extraction alone requires the active ingredients to pass through various cell walls, which is difficult to achieve the effective release. Therefore, the fermentation method of the present disclosure can effectively destroy cell walls to allow the effective release of both astragaloside

IV and calycosin glycoside.

Application Example 2

Comparative test for the rumen degradation rate and the small intestinal release rate of the A.

propinquus active ingredients in the uncoated fermented A. propinquus of Example 2 and the

coated fermented A. propinquusof Examples 4 to 6

In this experiment, 12 dairy bulls (with a weight of 557 9.6 kg) with permanent rumen

fistulas and duodenal fistulas were selected and randomly divided into 4 groups, with 3 bulls in

each group. In order to eliminate the differences among different animals, this experiment

adopted the 4 x 4 Latin square design and was conducted in 4 phases. For test at each phase, a

pre-feeding period was 10 days and a trial period was 5 days.

The basal diet adopted 0.5 kg of the daily ration in Example 6 without the feed additive

(Example 4). The test bulls were fed twice a day (7:00 and 19:00) in a single trough, with 15 kg

of total mixed ration (TMR) each time, and had free access to water.

In each phase of test, 5 g of each of the uncoated fermented A. propinquusin Example 2 and the coated fermented A. propinquus in Examples 4 to 6 was accurately weighed and put into a nylon bag (5 cm x 8 cm) with a known mass, the mouth of the bag was tightened with nylon thread, and then the bags were put at position at 50 cm in the ventral sac of the rumen after the morning feeding on the first day of the trial period, with 8 bags for each bull. 4 of the bags were taken out at 12 h and 24 h. 2 bags taken out from the rumen of each bull were immediately rinsed with water until obtained water was completely clear, and then oven-dried at 65°C to a constant weight. The method in Application Example 1 was used to determine the astragaloside IV and calycosin glycoside contents before and after degradation. The other two bags were put into the small intestine of a bull from the duodenal fistula. Then the nylon bags were collected from the cow dung, immediately rinsed with water until obtained water was completely clear, and then oven-dried at 65°C to a constant weight. The method in Application Example 1 was used to determine the astragaloside IV and calycosin glycoside contents in the residue. The rumen degradation rate and the small intestinal release rate were calculated, separately. Calculation results were shown in Table 2.

Rumen degradation rate = (A. propinquus mass before degradation x A. propinquus active

ingredient content before degradation - A. propinquus mass after degradation x A. propinquus

active ingredient content after degradation)/(A. propinquus mass before degradation x A.

propinquus active ingredient content before degradation) x 100%

Small intestinal release rate = (A. propinquus mass before degradation x A. propinquus active ingredient content before degradation - A. propinquus residue mass in nylon bags collected from dung x active ingredient content in residue)/(A. propinquus mass before degradation x A. propinquus active ingredient content before degradation) x 100%

Table 2 Rumen degradation rate and small intestinal release rate of fermented A. propinquus

Item Example 2 Example 4 Example 5 Example 6

Astragaloside Calycosin Astragaloside Calycosin Astragaloside Calycosin Astragaloside Calycosin

IV glycoside IV glycoside IV glycoside IV glycoside

Rumen 39.6 41.3 9.8 10.3 9.9 10.5 10.1 10.6

degradation

rate at 12 h

(0%)

Rumen 65.4 68.5 18.2 19.9 18.5 20.1 18.7 20.4

degradation

rate at 24 h

(%o)

Small 25.4 23.6 65.7 66.3 65.5 66.1 65.2 65.9

intestinal

release rate

(0%)

It can be seen from Table 2 that, compared with the uncoated fermented A. propinquus in

Example 2, in the coated fermented A. propinquus in Examples 4 to 6, a degradation rate of

astragaloside IV is decreased by 74.5% to 75.3% at 12 h and decreased by 71.4% to 72.2% at 24 h in the rumen; and a degradation rate of calycosin glycoside is decreased by 74.3% to 75.1% at

12 h and decreased by 70.2% to 70.9% at 24 h in the rumen. It indicates that the coated

fermented A. propinquusin Examples 4 to 6 can effectively pass through the rumen and enter the

small intestine compared with the uncoated fermented A. propinquus in Example 2.

Example 2, in the coated fermented A. propinquus in Examples 4 to 6, a small intestinal release

rate of astragaloside IV is increased by 156.7% to 158.7%, and a small intestinal release rate of

calycosin glycoside is increased by 179.2% to 180.9%. It indicates that, compared with the

uncoated fermented A. propinquus in Example 2, in the coated fermented A. propinquus in

Examples 4 to 6, the release of A. propinquus active ingredients is increased in the small

intestine, which can increase the contents of A. propinquus active ingredients in beef.

Application Example 3

32 Angus fattening cattle were divided into 4 groups using a single-factor randomized block

design (according to body weight, age, and fat condition), with 8 cattle in each group. The beef

cattle feeds prepared in Examples 7 to 9 and Comparative Example 1 were fed, separately.

This feeding test was conducted for 70 d in total, including a 10-day pre-feeding period and

a 60-day trial period.

The test cattle were fed three times a day (6:00, 12:00, and 18:00) in a single trough, with a manner of self-feeding. The drinking water should be sufficient, clean, and readily available to cattle. Before the experiment was started, a cattle barn was disinfected, and all the test cattle were dewormed. During the experiment, the feed intake, water drinking, dung, behavior, and mental status were observed every day for the test cattle.

During the experiment, corn silage and TMR were collected every 15 days, a fresh weight

was recorded, and then the corn silage and TMR were oven-dried, weighed, and stored for later

use. An initial moisture content was recorded. In the three days before the trial period ended,

dung was collected from the rectum in the morning, at noon, and in the evening every day, and a

weight of the dung was recorded. A 10% tartaric acid solution was added to the dung sample at

an amount 25% of the weight of the dung sample, and a resulting mixture was thoroughly stirred

and stored at -20°C for later use.

During the trial period, the feeding amount and the remaining feed amount were recorded

for each cattle to calculate DMI. The body weight was measured at the beginning, day 30, and

day 60 of the trial period, which was recorded for 2 consecutive days each time. Measurement

results were shown in Table 3.

The feed samples and dung samples were oven-dried at 65°C for 72 h, then dampened for 24

h, and weighed, and an obtained weight was recorded. Then the feed samples and dung samples

were oven-dried at 65°C for 24 h and then dampened for 24 h, and an obtained weight was recorded. The feed samples and dung samples, when at a constant weight, were crushed and sieved through a 1 mm sieve. The contents of DM, OM, CP, EE, and crude fiber were determined according to routine laboratory analysis methods. The contents of NDF and ADF were determined according to the method of Van Soest. The digestibility was calculated for nutrients according to the determination results, and calculation results were shown in Table 4.

After the feeding test was completed, the cattle were transported to the Hulan Food Co., Ltd.

in Wenshui County for slaughter, and a carcass weight and a net meat weight were measured.

500 g of a longissimus sample was collected to determine the A. propinquus active ingredients.

The muscle samples were sent to Shanghai Duma Biological Technology Co., Ltd. for the

determination of calycosin and astragaloside IV, and determination results were shown in Table

5.

The above data were subjected to analysis of variance (ANOVA) with one-way-anova of the

statistical analysis software SAS 9.0 and to LSD multiple comparison, and linear and Quadratic

analysis were also conducted.

Table 3 Effect of the feed additive added to a beef cattle feed on the DMI, average daily

weight gain, and feed conversion ratio (FCR) of Angus fattening cattle

Experimental treatment P-value

Comparative Among Item Example 7 Example 8 Example 9 SEM L Q Example 1 groups

DMI (kg/d)

0 d to 30 d 12.70 12.81 12.86 13.04 0.073 0.445 0.126 0.927

31 d to 60 d 13.11 13.25 13.26 13.45 0.071 0.458 0.143 0.906

Whole phase 12.91 13.03 13.06 13.25 0.052 0.155 0.049 0.896

Body weight (kg)

0d 684.9 682.9 680.4 681.2 10.91 0.999 0.897 0.952

30 d 721.6 722.7 723.6 723.7 10.52 0.997 0.942 0.982

60 d 756.3 761.2 763.1 763.2 10.55 0.996 0.821 0.914

Average daily weight gain (kg/d)

0 d to 30 d 1.22 1.33 1.44 1.42 0.056 0.523 0.181 0.574

31 d to 60 d 1.16 1.29 1.32 1.32 0.057 0.732 0.332 0.588

Whole phase 1.19 1.31 1.38 1.37 0.041 0.342 0.092 0.420

FCR (kg/kg)

0 d to 30 d 10.57 10.03 9.70 9.32 0.360 0.681 0.232 0.919

31 d to 60 d 12.34 10.72 10.61 10.57 0.542 0.626 0.288 0.483

Whole phase 11.45 10.38 10.16 9.95 0.334 0.399 0.117 0.778

Note: The P values for DMI and average daily weight gain at different time points are 0.001

and 0.327, respectively. There is no significant difference among interactions of all index time

with treatment (P > 0.05).

It can be seen from Table 3 that there was no interaction between the addition of the feed additive to a beef cattle feed and a feeding time (P > 0.05). With the extension of the growth period, the DMI increased significantly (P < 0.05), the DMI increased linearly with the increase of the feed additive at day 0 to day 60 (P < 0.05), and the average daily weight gain showed a linear upward trend with the increase of the feed additive at day 0 to day 60 (0.05 < P < 0.1). It can be seen that the beef cattle feed provided in the present disclosure improved the weight-gaining performance of beef cattle, thereby improving economic benefits.

Table 4 Effect of the feed additive added to a beef cattle feed on the digestibility of Angus

fattening cattle for nutrients.

Experimental treatment P-value

Comparative Among Item Example 7 Example 8 Example 9 SEM L Q example 1 groups

Dry matter 0.762c 0780c 0.827a 0.803b 0.006 0.001 0.001 0.001

Organic matter 0.736b 0.752b 0.799a 0.779a 0.006 0.001 0.001 0.002

Crude protein 0.682b 0.688b 0.753a 0.714ab 0.008 0.002 0.006 0.071

Crude fat 0.643c 0.673b 0.727a 0.663bc 0.008 0.001 0.002 0.001

Neutral detergent fiber 0.615b 0.646b 0.702a 0.685a 0.009 0.001 0.001 0.018 (NDF)

Acid detergent fiber (ADF) 0.575c 0.601bc 0.645a 0.639ab 0.008 0.001 0.001 0.131

The different letters of a, b, and c in the same line indicate significant differences (P < 0.05).

It can be seen from Table 4 that the digestibility of DM, OM, EE, and NDF increased

linearly and quadraticly with the increase in the amount of fermented A. propinquus(P < 0.05). It

can be seen that the beef cattle feed provided in the present disclosure improved the digestibility of beef cattle, thereby improving the weight-gaining performance of beef cattle and increasing economic benefits.

Table 5 Effect of the feed additive added to a beef cattle feed on the deposition of A.

propinquus active ingredients in Angus fattening cattle.

Experimental treatment P-value

Comparative Among Item Example 7 Example 8 Example 9 SEM L Q Example 1 groups

Calycosin glycoside (pg/g) 0.00c 0.08b 0.15a 0.18a 0.002 0.001 0.001 0.506

Astragaloside IV (pg/g) 0.00c 37.3lb 75.43a 80.36a 0.753 0.001 0.001 0.438

The different letters of a, b, and c in the same line indicate significant differences (P< 0.05).

It can be seen from Table 5 that the beef cattle feeds of Examples 7 to 9 can make the

deposition of calycosin glycoside in beef reach 0.08 pg/g to 0.18 pg/g and the deposition of

astragaloside IV in beef reach 37.31 ptg/g to 80.6 pg/g, which improves the functional

characteristics ofbeef.

Although the present disclosure has been described in detail through the above examples, the

examples are only a part rather than all of the examples of the present disclosure. All other

examples obtained by a person based on these examples without creative efforts shall fall within

a protection scope of the present disclosure.

Claims

1. Fermented Astragalus propinquus (A. propinquus), prepared from the following raw

materials in parts by mass:

water;

wherein, the lactobacilli have a viable count of 9 x 10" CFU/g to 11 x 10" CFU/g and

the cellulose-decomposing bacteria have a viable count of 1.5 x 109 CFU/g to 1.7 x 109 CFU/g;

wherein, the A. propinquus is selected according to the following criteria: moisture

lower than 0.04%; and calycosin content: not less than 0.05%;

wherein, the cellulose-decomposing bacteria comprise Bacillus subtilis (B. subtilis).

2. A method for preparing the fermented A. propinquus according to claim 1, comprising the

following steps:

crushing the A. propinquus, mixing crushed A. propinquus with the water, lactobacilli,

and sugar, and conducting sealed fermentation for 2 d to 3 d to obtain a first fermented product;

mixing the first fermented product with the laccase, cellulose-decomposing bacteria, acid cellulase, and acid xylanase, and conducting sealed fermentation for 5 d to 8 d to obtain a second fermented product; and air-drying and crushing the second fermented product to obtain the fermented A.

propinquus;

wherein, the sealed fermentation to obtain the first fermented product and the sealed

fermentation to obtain the second fermented product are both conducted at 30°C to 45°C;

wherein, a preparation method of the second fermented product comprises: mixing the

product.

3. A feed additive, comprising the fermented A. propinquus according to claim 1 and a

coating, wherein, the fermented A. propinquus and the coating have a mass ratio of 100:(12-20);

and the coating comprises grease with a melting point of 37°C to 40°C;

wherein, the coating further comprises calcium fatty acid, and the grease has a mass ratio

of 3:2 with the calcium fatty acid.

4. A method for preparing the feed additive according to claim 3, comprising: spray-mixing

the grease with fermented A. propinquusto obtain the feed additive.

5. A beef cattle feed, wherein, based on dry matters in parts by mass, the beef cattle feed

comprises the following components: 99.25 to 99.75 parts of basal feed and 0.25 to 0.75 parts of

the feed additive according to claim 3;

wherein, based on dry matters in parts by mass, the basal feed comprises the following

mineral-vitamin premix.