WO2020060034A1 - A double-coating and non-drying preparation method of whole cottonseed with improved storage stability - Google Patents

Abstract

The present invention relates to a preparation method of the double-coated and non-dried whole cottonseed for animal feed using by (a) the storage stability enhancer; (b) the 1^st coating agent; and (c) the 2^nd coating agent. The inventive novel industrialized method for preparing the coated whole cottonseed for animal feed can provide with more favorable advantages such as stronger coating ability, prolonged storage stability, efficient economic cost due to simple preparation steps by dint of non-drying step etc comparing with the prior preparation methods already known in the art, especially, a method for preparing a high-strength, coated, whole cottonseed for stock feed disclosed in PCT/WO2015186935 (A1), in which the germination function of the whole cottonseed is removed by using a high temperature and high pressure steam, and high-viscosity gelatinized starch.

Description

A DOUBLE-COATING AND NON-DRYING PREPARATION METHOD OF WHOLE COTTONSEED WITH IMPROVED STORAGE STABILITY

The present invention relates to a double-coating and non-drying preparation method of whole cottonseed with improved storage stability.

A coated whole cottonseed has been reported to be palatable to a livestock with high energy (2.3Mcal NEI/kg) and to comprise about 15-17% lipid, 15-21% protein, 85-90% TDN, about 24% crude cellulose (26-31% ADF) etc, which is rare feed with such various nutritional property (Pires, A. V. et al., 2007. Effects of Heat Treatment and Physical Processing of Cottonseed on Nutritional Digestibility and Production Performance by Lactating Cows, Journal of Dairy Science, (8): pp1685-1695).

The high cellulose content of coated whole cottonseed provides a high producing cow requiring the raring with concentrated feedstuff with normal maintenance. The whole cottonseed has been frequently used during late fattening phase on the growth performance and the used amount has been rapidly increased till now.

Generally, the cottonseed reproduced in cotton industry has been used in high grade clothes or bedding and the seed is easily separated through the processing process but a little cottonseed attached to the fruit husk could not be completely eliminated. Accordingly, it is common that the cottonseed for use as feed in livestock industry is surrounded with a little amount of cotton and fruit with rigid husk (Bernard, J. K. 1999, Performance of Lactating Dairy Cows Fed Whole Cottonseed Coated with Gelatinized Cornstarch. Journal of Dairy Science. 82:1305-1309).

The sticky cotton tangled with each other and get together gives rise to several problems in the processing, for example, the difficulty in mixing with feed raw material causing to the separation, dust occurrence and the deterioration of work environment etc (Moreira, V. R eta l., 2004, Comparison of Conventional Linted Cottonseed and Mechanically Delinted Cottonseed in Diets for Dairy Cows, Journal of Dairy Science, 87:131-138).

The present inventors has developed the preparation method of coated whole cottonseed using by appropriate mixture of binding agent such as the mixture of pre-gelatinized starch, lignosulfate etc., specifically, it comprises several step of (a) mixing a whole cottonseed with water using by sprayer to prepare the mixture of whole cottonseed and water at the 1st step; (b) stirring the mixture of whole cottonseed and water at the 2^nd step; (c) pouring a binding agent into the stirred mixture and mixing together to prepare the bound substance with binding agent at 3^rd step; and (d) drying the bound substance with binding agent at high temperature at 4^th step to prepare a coated whole cottonseed which provide with more favorable advantages with keeping its nutritional advantage comparing with the prior preparation methods already known in the art, for example, preventing effect from tangling and lumping phenomenon as well as to provide inhibitory effect from the dust problem in work environment and improving effect on the convenience of several works such as measuring work or mixing work in the preparation of livestock feedstuffs. (Korea Patent Registration No. KR 10-1337883 B1 and PCT/WO2014/104651 A1).

However, the pre-gelatinized starch used as a bing agent in the above invention, could not throughly be mixed with whole cottonseeds due to sticky hole cottonseeds resulted from the sticky gelatinized starch mixed with water, which causes to several problems, for example, unfavourable work environment such as the entangled and clustered cottonseed etc; un-economic problems such as increased production cost caused by an expensive binding agent, for example, expensive pre-gelatinized starch comparing with conventional gelatin; the limited selection range on the coating agent and the like. Furthermore, the method requires drying step caused by the abundant use of distilled water (5 - 30% w/w of cottonseed); the use of certain amount of starch to improve the coating strength of cottonseed resulting in the increased use of starch and the relatively increased use of distilled water, which causes the cost of drying process among the total preparation process to be increased (Laird W., T.C., Wedegaertner and T.D. Valoco, 1997, Coating cottonseed for improved handling characteristics, Proc. Belt Wide Cotten Conf. New Orleans, LA 1599-1602).

Additionally, the present inventors also has developed a method for preparing a high-strength, coated, whole cottonseed for stock feed, in which the germination function of the whole cottonseed is removed by using a high temperature and high pressure steam, and high-viscosity gelatinized starch, which is obtained by gelatinizing starch in a preparation process, is used. Specifically, it comprises (1) a first step of removing lint from whole cottonseed; (2) a second step of preparing a mixture by adding a coating agent and purified water to whole cottonseed, followed by mixing; (3) a third step of disfunctioning a sprouting function of whole cottonseed while agitating the mixture in order to obtain coating agent-bound whole cottonseed by gelatinizing the mixed coating agent by spraying high-temperature and high-pressure steam into the mixture; and (4) a fourth step of preparing coated whole cottonseed as a final product by cooling the coating agent-bound whole cottonseed without a drying process. (Korea Patent Registration No. KR 10-1551333 B1; PCT/WO2015186935 (A1)/ US 2017196241 (A1)/AU 2015269119 (B2)).

Additionally, as a simple method, the present inventors also has developed a method for preparing a whole cottonseed for stock feed, in which the coating step on the surface of whole cottonseed is omitted among whole process disclosed in PCT/WO2015186935 (A1). Specifically, it comprises (1) a first step of removing lint from whole cottonseed; (2) a second step of preparing a humidified whole cottonseed by adding a purified water to whole cottonseed; (3) a third step of disfunctioning a sprouting function of whole cottonseed by spraying high-temperature and high-pressure steam into the humidified whole cottonseed; and (4) a fourth step of preparing un-coated whole cottonseed as a final product by drying and cooling the whole cottonseed. (Korea Patent Registration No. KR 10-1824133 B1).

Additionally, as an alternative simple method, the present inventors also has developed a method for preparing a whole cottonseed for stock feed, in which the coating step on the surface of whole cottonseed as well as delinting step from whole cottonseed are omitted among whole process disclosed in PCT/WO2015186935 (A1). Specifically, it comprises (1) a first step of preparing a humidified whole cottonseed by adding a purified water to whole cottonseed; (2) a second step of disfunctioning a sprouting function of whole cottonseed by spraying high-temperature and high-pressure steam into the humidified whole cottonseed; and (3) a third step of preparing un-coated and linter containing cottonseed as a final product by drying and cooling the whole cottonseed. (Korea Patent Registration No. KR 10-1824132 B1).

However, the above-mentioned methods removing the drying step consuming high energy among the previous methods, have also several problems in respect of economic cost, for example, I. e., additional cost for using high-temperature and high-pressure steam.

However, there has been not reported or disclosed about the double-coating and non-drying preparation method of whole cottonseed with improved storage stability in the above cited literatures, the disclosures of which are incorporated herein by reference.

Accordingly, the present inventors have been focused to solve the previous severe problems in use of the whole cottonseed as a feed material and to provide more cost-effective preparation method of whole cottonseed with improved storage stability and lower cost.

Finally, the present inventors have found the novel double-coating and non-drying preparation method of whole cottonseed with improved storage stability which comprises double-coating step on the coated whole cottonseed prepared at 4^th step (See claim 2 of PCT/WO2014/104651 A1) rapidly to reduce the certain amount of free water contained in the 1^st coated whole cottonseed and then the inventive method provide several advantages, for example, (1) reduced production cost and simple production process due to the omitted drying step requiring high energy among previous process and (2) prevention of several problems caused by long term distribution channel by dint of it’s improved storage stability.

The present invention provides a double-coating and non-drying preparation method of whole cottonseed with improved storage stability.

Accordingly, it is an object of the present invention provides a double-coating and non-drying preparation method of whole cottonseed with improved storage stability.

Specifically, the present invention provides a preparation method of the a double-coated and non-dried whole cottonseed for animal feed using by (a) the storage stability enhancer selected from the group consisting of sodium chloride (NaCl), Magnesium chloride (MgCl₂), Potassium chloride (KCl) and Calcium chloride (CaCl₂); (b) the 1^st coating agent selected from the group consisting of pre-gelatinized starch, lignosulfate, gelatin, molasses, corn starch, tapioka starch, wheat starch, rice starch, potato starch, saccharides, CMS (Condensed Molasses Fermentation Solubles), corn flour, tapioka flour, wheat flour, rice flour, and cellulose; and (c) the 2^nd coating agent selected from at least one group consisting of (1) vegetable oil selected from mineral oil, essential oil, soybean oil, corn oil, canola oil, olive oil, sunflower seed oil, coconut oil, palm oil, or cottonseed oil; and/or (2) clay minerals selected from bentonite, monmorillo- nite, zeolite, illite, or silica gel.

More specifically, the present invention provides a preparation method of the double-coated and non-dried whole cottonseed for animal feed comprising the steps of; (1) removing linter from whole cottonseed at the 1^st step; (2) adding the mixture of a storage stability enhancer and water to the de-linted cottonseed and stirring together to prepare the 1^st cottonseed mixture at 2^nd step; (3) adding the 1^st coating agent to the 1^st cottonseed mixture and stirring together to prepare the 1^st coated cottonseed at 3^rd step; (4) adding the 2^nd coating agent to the 1^st coated cottonseed and stirring together to prepare the 2^nd coated cottonseed at 4^th step; and then (5) cooling the 2^nd coated cottonseed without drying step.

At the 1^st step in the above-described method, the term “whole cottonseed” disclosed herein is characterized by being manufactured from Australia, United State of America, China, India, Pakistan, Brazil, Turkey, Uzbekistan, Greece, Vietnam, Korea etc.

At the 1^st step in the above-described method, the lint is finally removed to an amount of 1 to 10%(w/w), preferably, 1 to 5%(w/w), by weight based on a weight of the whole cottonseed.

At the 2^nd step in the above-described method, the term “storage stability enhancer” disclosed herein is selected from the group consisting of sodium chloride (NaCl), Magnesium chloride (MgCl₂), Potassium chloride (KCl) and Calcium chloride (CaCl₂).

At the 2^nd step in the above-described method, the water used with the storage stability enhancer for increasing the coating strength of the de-linted cottonseed disclosed herein is characterized by being mixed the whole cottonseed with water in the amount of ranging from 1 to 20% (v/w), preferably, 3 to 10% (v/w) based on the weight of the whole cottonseed.

At the 2^nd step in the above-described method, the water is used to separate the entangled and lumped linter from the cottonseed easily by way of being smeared to linter and being stirred for ceratin period.

In case that the amount of water exceeds the range, it gives rise to longer drying period resulting in un-economic problem, whereas in case that the amount of water is below the range, it is difficult to separate the linter from the cottonseed and the coating strength of de-linted cottonseed has reduced.

At the 2^nd step in the above-described method, the above storage stability enhancer is characterized by being mixed with water in the amount of ranging from 1 to 15% (v/w), preferably, 3 to 10% (v/w) based on the weight of water. The amount of storage stability enhancer is finally poured to an amount of 1 to 10%(w/w), preferably, 3 to 8%(w/w), by weight based on a weight of water considering the adding amount of water is variable according to the water content of whole cottonseed.

At the 2^nd step in the above-described method, the mixture of a storage stability enhancer and water is characterized by being stirred for the period ranging from 1 hr to 6 hrs, preferably, 2 to 4 hrs to provide water with enough time to be penetrated into the center of cottonseed.

At the 3^rd step in the above-described method, the “1^st coating agent” disclosed herein is characterized by being used as a sole or combinations of coating agent selected from the group consisting of pre-gelatinized starch, lignosulfate, gelatin, molasses, corn starch, tapioka starch, wheat starch, rice starch, potato starch, saccharides, CMS (Condensed Molasses Fermentation Solubles), corn flour, tapioka flour, wheat flour, rice flour, and cellulose.

At the 3^rd step in the above-described method, it is preferable to use the 1^st coating agent with the combination of pre-gelatinized starch, and lignosulfate with the relative mixed ratio of 0.5-10: 1(w/w), more preferably, 1-8: 1(w/w), in case of using the combination of binding agents.

At the 3^rd step in the above-described method, it is preferable to use the 1^st coating agent in the amount of ranging from 0.01 to 30% (w/w), preferably, 0.1 to 10% (w/w) based on the weight of the whole cottonseed. The pouring amount of the 1^st coating agent into the stirred mixture may be variable depending on the kinds of the 1^st coating agent, for example, 2-5%(w/w) in case of pre-gelatinized starch, 1-3% (w/w) lignosulfate, 0.5-2% (w/w) gelatin based on the weight of the whole cottonseed, which may be variable in case that the combination of binding agent is used.

At the 3^rd step in the above-described method, it is preferable to consecutively pour the 1^st coating agents into the stirred mixture by dividing into several times, preferably, 5 to 100 times, more preferably, 10 to 50 times, for example, the 1^st coating agent can be dividedly poured in an amount of about 1/100 to 1/5 (w/w), preferably, about 1/50 to 1/10 (w/w), at every time, based on the total weight of the 1^st coating agent.

In case that the 1^st coating agent is evenly distributed on the humidified cottonseed, it can provide with the concrete coating of the linter attached to cottonseed and the efficient economic effect by reducing the amount of coating agent.

It is very important in the present invention to pour the 1^st coating agent dividedly in order to distributing the 1^st coating agent on the wet whole cotton with water evenly. In case that the 1^st coating agent is evenly distributed on the wet whole cotton with water, it allows the strong binding effect of the 1^st coating agent and reduced use of the 1^st coating agent to provide with economic advantage.

In a while, in case that the 1^st coating agent is unevenly distributed on the wet whole cotton with water, it cause to irregular binding of the 1^st coating agent to the wet whole cotton with water.

At the 4^th step in the above-described method, “the 2^nd coating agent to coat again to the 1^st cottonseed mixture” disclosed herein is characterized by being used as a sole or combinations of coating agent selected from the group consisting of (1) vegetable oil selected from mineral oil, essential oil, soybean oil, corn oil, canola oil, olive oil, sunflower seed oil, coconut oil, palm oil, or cottonseed oil; and/or (2) clay minerals selected from bentonite, monmorillo- nite, zeolite, illite, or silica gel.

At the 4^th step in the above-described method, it is preferable to use a mineral oil or bentonite as the 2^nd coating agent, for example, in the amount of 0.1 - 2%(w/w), preferably, 0.2 - 0.6 % (w/w) based on the weight of the whole cottonseed in case of mineral oil which is used to coat the oily surface of cottonseed and improve storage stability; and 0.1 - 1%(w/w), preferably, 0.2 - 0.6 % (w/w) based on the weight of the whole cottonseed in case of bentonite which is used to rapidly reduce the amount of free water, which may be variable in case that the combination of 2^nd coating agent is used.

It is preferable to consecutively pour the 2^nd coating agent into the stirred mixture by dividing into several times, preferably, 2 to 100 times, more preferably, 3 to 50 times, for example, the 2^nd coating agent can be dividedly poured in an amount of about 1/100 to 1/2 (w/w), preferably, about 1/50 to 1/3 (w/w), at every time, based on the total weight of the 2^nd coating agent.

At the 5^th step in the above-described method, the term “cooling the 2^nd coated cottonseed without drying step” disclosed herein is characterized by performing cooling step with variable condition according to the amount of poured water at the 2^nd step and the purposed controlled final water content of final coating product, preferably, 10 - 18%(w/w), more preferably, 11 - 15%(w/w) of final water content of final coating product. Accordingly, it is preferable to perform the cooling step in the air for the period ranging from about 1 mins to 60 mins, preferably, 10 mins to 30 mins.

Most specifically, the present invention also provides a preparation method of the double-coated and non-dried whole cottonseed for animal feed comprising the steps of; (1) removing linter from whole cottonseed to the extent that the amount of linter based on the weight of whole cottonseed reaches to 1 -10 % (w/w) at the 1^st step; (2) preparing the storage stability enhancer solution in water, preferably, sodium chloride in water, more preferably, 1 - 24% (w/w) sodium chloride in water ; adding 1 - 15 % (w/w) the storage stability enhancer solution to the de-linted cottonseed and stirring together to prepare the 1^st cottonseed mixture at 2^nd step; (3) adding 0.1 - 30% (w/w) 1^st coating agent based on the weight of whole cottonseed, preferably, sole or combinations of 1^st coating agent selected from the group consisting of pre-gelatinized starch, lignosulfate, gelatin, molasses, corn starch, tapioka starch, wheat starch, rice starch, potato starch, saccharides, CMS (Condensed Molasses Fermentation Solubles), corn flour, tapioka flour, wheat flour, rice flour, and cellulose to the 1^st cottonseed mixture and stirring together to prepare the 1^st coated cottonseed at 3^rd step; (4) adding the 2^nd coating agent, preferably, sole or the combinations of 1 - 2% (w/w) mineral oil and/or 0.1 - 2% (w/w) bentonite based on the weight of whole cottonseed to the 1^st coated cottonseed and stirring together to prepare the 2^nd coated cottonseed at 4^th step; and then (5) cooling the 2^nd coated cottonseed without drying step to the extent that the water content of final produce ranges from 10 - 17% (w/w), preferably, 11 - 15% (w/w).

The term “free water” disclosed herein denotes the water which can be freely accessible by microbes and it is known to the person skilled in the art that the form in feedstuff is transformed into the form of “solid water” where free water is impregnated into the cell of feedstuff.

The present invention also provides a double-coated and non-dried whole cottonseed for animal feed prepared by the above-described preparation method.

The double-coated and non-dried whole cottonseed prepared by the above-described method can provide with more favorable advantages such as stronger coating ability, prolonged storage stability, efficient economic cost due to simple preparation step by dint of non-drying step etc comparing with the prior preparation methods already known in the art through various experiments, for example, reduced generated CO₂ gas in Experimental Example 1 (Determination of the content of CO₂ gas in cottonseed; prolonged occurrence of fungal growth on condition with various sodium chloride concentration in Experimental Example 2 (Determination of the occurring date of fungal growth in 1^st coated cottonseed); prolonged occurrence of fungal growth on condition with 4% sodium chloride in Experimental Example 3 (Determination of the occurring date of fungal growth in 2^nd coated cottonseed); rapidly reduced water activity in Experimental Example 4 (Determination of the water activity of cottonseed); prolonged aerobic stability and improved stability in Experimental Example 5 (Determination of the aerobic stability of 2^nd coated cottonseed); improved quality having improved passage ratio in Experimental Example 6 (Determination of the passage ratio of 2^nd coated cottonseed); improved aerobic stability and prolonged occurrence of microbial growth in Experimental Example 7 (Determination of the storage stability of 2^nd coated cottonseed); improved palatibility to cows in Experimental Example 8 (Determination of the palatibility of 2^nd coated cottonseed) etc.

As described in the present invention, inventive novel industrialized method for preparing the a double-coated and non-dried whole cottonseed for animal feed can provide with more favorable advantages such as stronger coating ability, prolonged storage stability, efficient economic cost due to simple preparation steps by dint of non-drying step etc comparing with the prior preparation methods already known in the art, especially, a method for preparing a high-strength, coated, whole cottonseed for stock feed disclosed in PCT/WO2015186935 (A1), in which the germination function of the whole cottonseed is removed by using a high temperature and high pressure steam, and high-viscosity gelatinized starch.

The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

EXAMPLES

The following Comparative Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Comparative Example 1. Preparation of binding agent treated whole cottonseed (See Example 1 in Korea Patent Registration No. KR 10-1337883 B1 and PCT/WO2014/104651 A1)

200kg of whole cottonseed imported from Australia (Cargill Australia Limited, Australia) was poured to a stirrer (5m³, CML-200, Korea) and 10L of distilled water was added thereto to stir at the speed of 30rpm for 10mins. After checking the separation of cotton, 4kg of pre-gelatinized starch (suprex corn, FFA Co. Ltd., Switzerland) as a binding agent was consecutively poured thereto with stirring by dividing into 200g per every time at the interval of every five mins. At the end of addition of the binding agent, the bound substance with binding agent was dried at 100℃ for 30 mins in hot-wind dryer (Bio-celtec Co, ltd, Korea). Through the drying step, the humidity of coated whole cottonseed for animal feed was determined to about 10.5%, and the binding affinity of its surface was confirmed to be very favorable apparently. The final coated whole cottonseed for animal feed is used as a comparative example 1 to compare with the inventive invention.(designated as “ce1-cotton” hereinafter).

Comparative Example 2. Preparation of high-strength, coated, whole cottonseed for stock feed, in which the germination function of the whole cottonseed is removed by using a high temperature and high pressure steam, and high-viscosity gelatinized starch (See Examples in Korea Patent Registration No. KR 10-1551333 B1; PCT/WO2015186935 (A1))

2-1. Preparation of Whole Cottonseed from which Lint is Removed

To prepare whole cottonseed from which lint is removed, the weight ratio of lint to whole cottonseed was first measured. Whole cottonseed used in the preparation was American (MOTU0796660, FEEDWILL, Korea) and Australian (UACU8445073, FEEDWILL, Korea) whole cottonseed.

Whole cottonseed was used in experiments after being dried in a drying oven (JSON-050, JCR, Korea) at 105℃, for 24 hours. Completely dried whole cottonseed was treated with 95% H₂SO₄ (DAEJUNG chemicals & metals Co. LTD, Korea) to melt lint. Here, 100ml of H₂SO₄ per 1 kg of completely dried whole cottonseed was used, followed by washing with water and drying at 105℃, for 24 hours. Thereafter, the weight ratio of lint to whole cottonseed was obtained by calculating weight ratio (Khan, N. U., K. B. Marwat, G. Hassan, Farhatullah, S. Batool, K. Makhdoom, W. Ahmad and H. U. Khan., 2010. Genetic variation and heritability for cottonseed, fiber and oil traits in GOSSYPIUM HIRSUTUM L. Pak. J. Bot., 42 (1):615-625).

In the case of American whole cottonseed, the weight ratio of lint to whole cottonseed was about 12.8±0.4 (w/w), and in the case of Australian whole cottonseed, the weight ratio of lint to whole cottonseed was about 7.5±0.3 (w/w). The weight ratio of lint in American whole cottonseed was about 5.3% higher than in Australian whole cottonseed.

A lint remover (MR114D, SDMT, China), by which the desired amount of lint can be removed, was used in preparing whole cottonseed, from which lint is removed. When the weight ratio of lint to whole cottonseed was taken as 100, American whole cottonseed having various weight ratios of removed lint was prepared while increasing the ratio of removed lint in units of 10%. When the weight ratio of the removed lint was 70% or more, the back region of the inside of whole cottonseed was exposed to the outside, and thus the whole cottonseed showed a tendency to have a black color. On the other hand, whole cottonseed, wherein the weight ratio of the removed lint was about 50%, retained the original color thereof. On the basis of the results, it was identified that the proper ratio of removed lint is about 50% by weight.

2-2. Preparation of Whole Cottonseed, the Germination Ability of which is Removed

To prepare whole cottonseed, the germination ability of which is removed, the following experiments were performed. Steam was applied to American whole cottonseed by means of a steam generating device (STE2600, SANG ROK Electronics, Korea). Particularly, the steam application was performed at 120℃, under a pressure of 6kg/cm². Steam was sprayed for up to a maximum of 10 minutes while increasing a spraying time in steps of 1 minute. To measure the germination rate of each group consisting of 50 seeds, wherein each group had been treated with steam for each spraying time, as described in above experimental condition, each steam-treated group was sowed in a germination bed (120 mm×115 mm), and then cultured in an incubator (JSGI-100T, JS Research, Inc., Korea), a culture condition of which was set up as follows: 25℃, under a dark condition. Seed germination was observed and recorded every 24 hours for two weeks. In the case of a non-treated group, which had not been treated with steam, seeds began to germinate at three days after culture, and 87% of seeds germinated within two weeks. In the case of a treated group, which had been treated with steam for 1 minute, 13% of seeds germinated within two weeks. In the treated groups, which had been treated with steam for 2 or more minutes, none of the seeds germinated.

2-3. Preparation of Gelatinized Starch having Viscosity Improved by Steam Treatment

Generally, when starch is heated with water, starch is gelatinized and forms colloid solution with very high viscosity, which is transparent or has an ivory white color (Booyong Lee, Chulkyun Mok, Chulho Lee. 1993. Comparison of differential scanning calorimetry with enzymatic method for the determination of gelatinization degree of cornstarch. Korean J. FOOD SCI. TECHNOL. Vol. 25:400-403).

Cornstarch (C0022, SAMYANG Co., Korea) was used as a coating agent. To fully gelatinize cornstarch, moisture content of 65% or more is necessary (Wang, etc., 1991, Experimental analysis and computer simulation of starch-water interaction during phase transition, J. Food Sci., 56 121).

To measure gelatinization degree according to steam treatment, suspension composed of cornstarch and distilled water was prepared, wherein the weight ratio of cornstarch to purified water was 35:55 and the moisture content of the suspension was about 65%.

100ml of a resultant starch suspension was added to a 500ml Erlenmeyer flask having a branch at the bottom thereof, and then 120℃, steam was sprayed into the Erlenmeyer flask through the branch of the Erlenmeyer flask using a steam generating device (STE2600, SANG ROK Electronics, Korea). When spraying steam, the suspension was stirred using a magnetic stirrer inserted into the flask. Varying a steam spraying time from 1 to up to 10 minutes, steam treatment was respectively carried out by a total of 10 steam treatment methods. As a result, variously gelatinized starch suspensions were prepared.

To measure the gelatinization degree of the variously gelatinized starch suspension samples, the starch suspension samples were subjected to rapid freezing (-70℃), and then freeze-drying (operating conditions: -50℃, 1.33 Pa) was carried out using a freeze-drying apparatus (Ilshin Co., Korea). After powderizing each freeze-dried sample, each sample was passed through a standard sieve (100 mesh; 150μm), to obtain solid content of a certain particle size, and then the solid content was used as a sample for measurement.

20mg of each of the powdered samples was added into a 50ml centrifuge tube, 5ml of distilled water was added thereinto, and then the powdered samples were dispersed. Thereafter, 25ml of α-amylase solution (A3306, SIGMA, USA) (20.1 units/g solid, acetate buffer, pH 4.5) was added to each of the tubes, and then incubated at 40℃, for 1 hour, and subsequently 2ml of 25% trichloroacetic acid (204-02405, WAKO, Japan) was added to stop the reaction. The reaction product was subjected to centrifugation (16,000×g, 5 min) using a centrifugal separator (UNION32R, HANIL, Korea) to separate a supernatant, followed by measurement of gelatinization degree.

0.5ml of supernatant was diluted with a proper amount of distilled water to achieve an absorbance at 550nm of 0.1 to 0.9, and the diluted supernatant was added in a test tube and mixed with 1.5ml of a DNS reagent. The mixture was incubated in boiling water for 5 minutes and subsequently placed in ice water to cool to room temperature, and then subjected to absorbance measurement at 550nm using a spectrometer (UV-Vis spectrophotometer, S-100, SCINCO, Korea). Based on a calibration curve calculated using pure glucose (glucose, G0350500, SIGMA, USA), reducing sugar of each sample was calculated from the measured absorbance.

The ratio of reducing sugar of sample for each time period to reducing sugar of fully gelatinized cornstarch, which was prepared by the same method except for a spraying time of 30 minutes, was used to calculate specific gelatinization degree. When whole cottonseed was not treated with steam, gelatinization degree was about 26%. On the other hand, when whole cottonseed was treated with steam for 2 or more minutes under conditions of 120℃, and 6kg/cm², gelatinization degree increased up to about 73%, and colloid was formed, which indicates that the method is suitable to improve the viscosity of gelatinized starch.

2-4. Preparation of Coated Whole Cottonseed, the Germination Ability of which is Removed

To prepare coated whole cottonseed, the germination ability of which is removed, as in Comparative Example 2-1, Australian whole cottonseed (UACU8445073, FEEDWILL, Korea) was delinted using a lint remover (MR114D, SDMT, China) to prepare delinted whole cottonseed, from which 2% by weight of lint was removed. With the same method as described in Comparative Example 2-2, the delinted whole cottonseed was treated with steam for 2 minutes under conditions of 120℃, and 6kg/cm², using a steam generating device (STE2600, SANG ROK Electronics, Korea), thereby removing the germination ability of the whole cottonseed.

Using the same method as described in Comparative Example 2-3, a starch suspension having a moisture content of about 65% was prepared using cornstarch (C0022, SAMYANG Co., Korea), and then, using a steam generating device (STE2600, SANG ROK Electronics, Korea), the starch suspension was treated with steam under conditions of 120℃, and 6kg/cm², for 2 minutes to prepare gelatinized starch with high viscosity. 100 kg of whole cottonseed, the germination ability of which was removed, was added to an agitator (DDK-801M, DAEDONG, Korea), and then 5kg of purified water was added to the agitator, followed by agitation for 5minutes. Thereafter, 5.7kg of the gelatinized starch was added, and then the mixture of the whole cottonseed and the gelatinized starch was agitated for 5 minutes to prepare coated whole cottonseed, the germination ability of which had been removed.

The final coated whole cottonseed for animal feed is used as a comparative example 2 to compare with the inventive invention.(designated as “ce2-cotton” hereinafter).

Example 1. Preparation of de-linted whole cottonseed

The water content of American whole cottonseed was determined to prepare the de-linted whole cottonseed of the present invention as follows:

The water content of American whole cottonseed was determined by using drying oven (JSON-050, JCH, Korea) at 130℃, for 3 hrs according to AOCS Aa3-38 analysis (American Oil Chemists’s Society) and found to 9.8% (w/w).

The amount of lint adhered to whole cottonseed was determined as follows: Completely dried whole cottonseed was treated with 95% H₂SO₄ (DAEJUNG chemicals & metals Co. LTD, Korea) to melt lint. Here, 100ml of H₂SO₄ per 1kg of completely dried whole cottonseed was used, followed by washing with water and drying at 105℃, for 24 hours. Thereafter, the weight ratio of lint to whole cottonseed was obtained by calculating weight ratio (Khan, N. U., K. B. Marwat, G. Hassan, Farhatullah, S. Batool, K. Makhdoom, W. Ahmad and H. U. Khan., 2010. Genetic variation and heritability for cottonseed, fiber and oil traits in GOSSYPIUM HIRSUTUM L. Pak. J. Bot., 42 (1):615-625).

In the case of American whole cottonseed, the weight ratio of lint to whole cottonseed was about 11.4±0.3 (w/w). The removed lint in whole cottonseed was made to about 50% by weight using by a lint remover (MR114D, SDMT, China).

Example 2. Preparation of de-linted whole cottonseed treated with storage stability enhancer and 1^st coating agent

The de-linted whole cottonseed prepared in Example 1, was treated with storage enhancer for increase it’s storage stability and 1^st coating agent to prepare the 1^st coated cottonseed as follows:

Various concentrations Sodium chloride (NaCl, storage stability enhancer) in 5.7kg of purified water (0, 2, 4, 6, 8% w/w) was poured to 100kg of the de-linted whole cottonseed showing water content of 9.8% (w/w) in Example 1 to prepare the final product showing water content of 14.5% (w/w). 2.0kg of starch (Suprexcorn, FFA, 1^st coating agent) was added to the 1^st cottonseed mixture and stirred together using by stirrer (DKM-250Food, DAEDONG, Korea) to prepare the 1^st coated cottonseed.

Example 3. Preparation of a double-coated and non-dried whole cottonseed treated with 2^nd coating agent

The 1^st coated whole cottonseed prepared in Example 2, was treated with 2^nd coating agent to prepare the 2^nd coated cottonseed as follows:

200kg of 1^st coated whole cottonseed was prepared by the identical procedure with those in Example 2 using by 4% Sodium chloride (NaCl, storage stability enhancer) in purified water and starch (Suprexcorn, FFA, 1^st coating agent). 0.4% mineral oil (PCF-S, CALS NBT Co. Ltd., Hwaseong-si, Gyeonggi-do, Korea) and 0.4% bentonite (FFA) used as the 2^nd coating agent were poured to 1^st coated whole cottonseed and mixed together using by stirrer (DKM-250Food, DAEDONG, Korea) for 5 mins. The mineral oil was dividedly sprayed several times and bentonite was dividedly poured three times in an amount of about 1/3 (w/w) at every time, based on the total weight of bentonite. The 2^nd coated cottonseed was cooled in the air for 20 mins to prepare the double-coated and non-dried whole cottonseed of the present invention (designated as “dc-cotton” hereinafter).

Experimental Example 1. Determination of the content of CO₂ gas in cottonseed

In order to confirm the storage stability of the double-coated and non-dried whole cottonseed of the present invention, various generated contents of CO₂ gas in cottonseed with various concentration of sodium chloride (storage stability enhancer), were determined according the method disclosed in the previous literature (Korea Patent Publication No. KR 10-2016-0011967 A).

The generated amount of CO₂ gas is regarded as indirect criteria for determining the growth and division of fungi since it generates CO₂ gas in the presence of oxygen.

Various broths (Potato dextrose broth, 254920, Difco) adjusted to the concentration of 0, 2, 4, 6, 8% sodium chloride in 1L of pyrex bottle (BT100-1000, NewKukjekwahak Co. Ltd, Korea) were sterilized with autoclave (HK-AC200, HanKuk Scientific Instrument Company, Korea) at 135℃, for 20 mins and the 500mL of sterilized broth was mixed with 20g of whole cottonseed to be sealed up.

The broth in the pyrex bottle of which upper side had been equipped with CO₂ detector (CO₂, 2H, Gas tech) was incubated at 35℃, in an incubator (JSGI-100T, JS Research Inc., Korea) and the generated amount of CO₂ gas was determined.

At the result, it has been confirmed that the negative control group (whole cottonseed without NaCl) consistently generate CO₂ gas (more than 10%) 48 hrs after the incubation whereas the test group treated with 2 - 6 % sodium chloride generate CO₂ gas (0.5%) 12 hrs after the incubation. The test group treated with more than 4 % sodium chloride generate CO₂ gas (2 - 2.2%) 36 hrs before the incubation and did not generate CO₂ gas 48 hrs after the incubation. The test group treated with 2% sodium chloride showed reduced generated CO₂ gas by 50 - 75% comparing with negative control and the test group treated with more than 4% sodium chloride showed reduced generated CO₂ gas by about 80 - 90% comparing with negative control. It has been confirmed that the optimum concentration of sodium chloride is 4% considering the above experimental results (See Table 1).

The generated amount of CO₂ gas according to various sodium chloride

Incubation time(hr)	NaCl 0%*	NaCl 2%	NaCl 4%	NaCl 6%	NaCl 8%
	- - % (generated amount of CO2 gas)
0	-	-	-	-	-
12	8	0.5	0.5	0.5	0.5
24	10.0	2.5	1.0	1.0	1.0
36	10.0	5.5	2.2	2.0	2.0
48	10.0	4.2	-	-	-
*:negative control

Experimental Example 2. Determination of the occurring date of fungal growth in 1^st coated cottonseed

In order to confirm the storage stability of the double-coated and non-dried whole cottonseed of the present invention, the occurring date of fungal growth in the 1^st coated cottonseed with various concentration of sodium chloride (storage stability enhancer), were determined according the method disclosed in the previous literature (Korea Patent Publication No. KR 10-2016-0011967 A).

To determine the occurring date of fungal growth in the 1^st coated cottonseed with various concentration of sodium chloride, the water content of the 1^st coated cottonseed was adjusted to 18% for shortening the determination period of the occurring date of fungal growth.

10 test groups consisting of 10g of each 1^st coated cottonseed with various concentration of sodium chloride, were placed on 10 petri dishes and incubated at 35℃ in an incubator (JSGI-100T, JS Research Inc., Korea) to determine the occurring date of fungal growth. 1L of distilled water in beaker was also placed on the incubator to maintain the relative humidity to be routine high level and the occurring date of fungal growth was regarded where more than 5 coated cottonseeds were found to occur fungal growth.

At the result, it has been confirmed that the negative control group (whole cottonseed without NaCl) has been found to occur fungal growth at 12^th day of incubation whereas the test group treated with 4 % sodium chloride has been found to occur fungal growth at 17^th day of incubation. It has been confirmed that the occurring date of fungal growth in the 1^st coated cottonseed with the higher concentration of sodium chloride has been found to prolong the occurrence of fungal growth considering the above experimental results (See Table 2).

The occurring date of fungal growth according to various sodium chloride concentration

	NaCl 0%*	NaCl 2%	NaCl 4%	NaCl 6%	NaCl 8%
occurring date of fungal growth (day)	12th day	13rd day	17th day	19th day	21st day
*:negative control

Experimental Example 3. Determination of the occurring date of fungal growth in 2^nd coated cottonseed

In order to confirm the storage stability of the double-coated and non-dried whole cottonseed of the present invention, the occurring date of fungal growth in the 2^nd coated cottonseed with various concentration of sodium chloride (storage stability enhancer), were determined according the method disclosed in the previous literature (Korea Patent Publication No. KR 10-2016-0011967 A).

To determine the occurring date of fungal growth in the 2^nd coated cottonseed with various concentration of sodium chloride, the water content of the 2^nd coated cottonseed using by 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite was adjusted to 18% for shortening the determination period of the occurring date of fungal growth.

As a negative control group, the 1^st coated cottonseed was treated with purified water without sodium chloride and was not treated with 2^nd coating agent to be used as a negative control.

The determination of the occurring date of fungal growth was performed according to the method disclosed in Experimental Example 2.

At the result, it has been confirmed that the negative control group has been found to occur fungal growth at 14^th day of incubation whereas the test group treated with 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite as the 2^nd coating agent as well as 4 % sodium chloride as a storage stability enhancer has been found to occur fungal growth at 22^nd day of incubation. It has been confirmed that the occurring date of fungal growth in the 2^nd coated cottonseed with 4 % sodium chloride as a storage stability enhancer has been found to prolong the occurrence of fungal growth to about 8 days comparing with negative control group (See Table 3).

The occurring date of fungal growth using by 4% sodium chloride

	negative control*	ce1-cotton	ce2-cotton	dc-cotton**
occurring date of fungal growth (day)	14th day	15th day	16th day	22nd day
*: negative control ; cottonseed treated with 2% starch and 10% purified water,**: dc-cotton ; double coated cottonseed treated with 2% starch; 10% purified water(4% NaCl) and 2nd coating agent (0.4% (w/w) mineral oil and 0.4% (w/w) bentonite).

Experimental Example 4. Determination of the water activity of cottonseed

In order to confirm the storage stability of the double-coated and non-dried whole cottonseed of the present invention, the water activity of the 2^nd coated cottonseed, were determined according the method disclosed in the previous literature (Effect of Packaging Methods on the Quality Properties of Stick Type Restructured Jerky, Korean Journal for Food Science of Animal Resources, 27(3), pp290-298, 2007).

To determine the water activity of the 2^nd coated cottonseed, the test group was prepared by the treatment with 4% sodium chloride and then mixture of 0.4% mineral oil and bentonite and as a negative control group, the 1^st coated cottonseed was treated with purified water without sodium chloride and was not treated with 2^nd coating agent to be used as a negative control.

The determination of the water activity of the 2^nd coated cottonseed was performed after completing the preparation of 2^nd coated cottonseed and checked at the interval of one hour after then using by available water analyzer (HP-23-AW-A, ROTRONIC Switzerland).

The purified water (free water) added in the 1^st coating step, mainly exists on the outer surface of seed after the completion of 2^nd coated cottonseed and then evaporates little by little or smeared into the center of seed, resulting in the water equilibrium between the center part and outer husk of the seed.

The “free water”, the water which can be freely accessible by microbes, reduces the storage stability and the reduced water activity inhibits from the microbial grow.

At the result, it has been confirmed that the negative control group has been found to be rapidly reduced water activity within 6 - 7 hrs after the completion and to be slowly reduced to 0.82 whereas the test group treated with 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite as the 2^nd coating agent as well as 4 % sodium chloride as a storage stability enhancer has been found to be rapidly reduced water activity. It has been confirmed that the 0.4% (w/w) mineral oil formed an oil membrane and bentonite used as the 2^nd coating agent absorbed the free water existing on the surface of coated cottonseed, which resulted in rapidly reduced free water accessible by various microbes and proved the storage stability of the inventive double-coated and non-dried whole cottonseed of the present invention (See Table 4).

The change of water activity according to storage stability

Incubation time(hr)	negative control*	dc-cotton**
0	1.00	1.00
1	0.98	0.95
2	0.97	0.92
3	0.95	0.90
4	0.93	0.89
5	0.91	0.89
6	0.89	0.88
7	0.88	0.88
8	0.88	0.88
9	0.87	0.87
18	0.87	0.87
19	0.86	0.85
24	0.84	0.85
48	0.83	0.82
*: negative control ; cottonseed treated with 2% starch and 10% purified water,**: dc-cotton ; double coated cottonseed treated with 2% starch; 10% purified water(4% NaCl) and 2nd coating agent (0.4% (w/w) mineral oil and 0.4% (w/w) bentonite).

Experimental Example 5. Determination of the aerobic stability of 2^nd coated cottonseed

In order to confirm the storage stability of the double-coated and non-dried whole cottonseed of the present invention, the aerobic stability of the 2^nd coated cottonseed were determined using by aerobic stability analyzer.

To determine the aerobic stability of the 2^nd coated cottonseed, the water content of the 2^nd coated cottonseed using by 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite was adjusted to 18% for shortening the determination period of the aerobic stability. 2.5kg of the 2^nd coated cottonseed was placed on plastic vessel (20L, round neck) equipped with thermometer sensor to determine the temperature of the vessel at the interval of every one hour.

The temperature was determined using by aerobic stability analyzer (EG-1, Ubikorea Co. Ltd) automatically recording the temperature of sensor as well as that of environment. The degree of aerobic stability was determined by the base of the number of day which showed more than 2℃ of discrepancy between the temperature of sensor as well as that of environment.

As a negative control group, the 1^st coated cottonseed with 2% starch was treated with purified water without sodium chloride and then was not treated with 2^nd coating agent to be used as a negative control.

As a comparative test group (3), the 1^st coated cottonseed with 2% starch was treated with purified water containing 0.3% propionate instead of sodium chloride and was not treated with 2^nd coating agent to be used as a comparative group (3)(designated as "T1" hereinafter).

As a comparative test group (4), the 1^st coated cottonseed with 2% starch was treated with purified water containing 4% sodium chloride and was not treated with 2^nd coating agent to be used as a comparative group (4)(designated as "T2" hereinafter).

As an inventive test group, the 1^st coated cottonseed with 2% starch was treated with purified water containing 4% sodium chloride and was treated with 2^nd coating agent to be used as an inventive test group (designated as "T3" hereinafter).

At the result, it has been confirmed that the negative control group has been found to showed the aerobic stability of 9.1 days and the comparative test group (3) treated with 0.3% propionate instead of sodium chloride showed the aerobic stability of 13.7 days whereas comparative test group (4) containing 4% sodium chloride showed the aerobic stability of 15.4 days and inventive test group treated with the 2^nd coating agent such as 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite showed the aerobic stability of 46.2 days. It has been confirmed that the aerobic stability of the 2^nd coated cottonseed with 4 % sodium chloride as well as the 2^nd coating agent such as 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite, has been found to prolong the aerobic stability and to have more improved stability without drying process comparing with control group or comparative groups (See Table 5).

The comparison of the aerobic stability between an inventive test group and comparative groups

	negative control*	ce1-cotton	ce2-cotton	T1**	T2***	T3(dc-cotton)****
aerobic stability (day)	9.1	9.4	9.6	13.7	15.4	46.2
*: negative control ; cottonseed treated with 2% starch and 10% purified water (0% NaCl),**: T1; cottonseed treated with 2% starch and 10% purified water (0% NaCl) containing 0.3% propionate.***: T2 cottonseed treated with 2% starch and 10% purified water (4% NaCl). ****: T3(dc-cotton) ; double coated cottonseed treated with 2% starch; 10% purified water(4% NaCl) and 2nd coating agent (0.4% (w/w) mineral oil and 0.4% (w/w) bentonite).

Experimental Example 6. Determination of the passage ratio of 2^nd coated cottonseed

In order to confirm the improved quality of the double-coated and non-dried whole cottonseed of the present invention, the passage ratio of the 2^nd coated cottonseed through the sieve with the diameter of 8mm were determined.

The coating method and the sort of coating agent takes an effect on the surface of coated seed by changing the physical property of whole cottonseed, resulting in the change of its passage ration. The improved quality of passage ratio is confirmed to be good coating quality of coated cottonseed.

To determine the passage ratio of the 2^nd coated cottonseed, 150g of the 2^nd coated cottonseed using by 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite was placed on the sieve with the diameter of 8mm prepared by the present inventors and was let to pass the sieve with stirring five times at every direction and rotating at the degree of 90°to determine the mean passaged weight ratio of the 2^nd coated cottonseed from the performed test results of four times.

As a negative control group, the 1^st coated cottonseed with 2% starch was treated with purified water without sodium chloride and then was not treated with 2^nd coating agent to be used as a negative control.

At the result, it has been confirmed that the negative control group has been found to show the passage ratio of 81.2% whereas the inventive test group treated with the 2^nd coating agent such as 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite showed the improved quality having the passage ratio of 84.3% (See Table 6).

The comparison of the passage ratio between an inventive test group and negative control group

	negative control*	ce1-cotton	ce2-cotton	dc-cotton**
passage ratio (%)	81.2±3.1	79.3±2.3	80.2±1.6	84.3±2.8
*: negative control ; cottonseed treated with 2% starch and 10% purified water (0% NaCl),**: dc-cotton ; double coated cottonseed treated with 2% starch; 10% purified water(4% NaCl) and 2nd coating agent (0.4% (w/w) mineral oil and 0.4% (w/w) bentonite).

Experimental Example 7. Determination of the storage stability of 2^nd coated cottonseed

In order to confirm the improved quality of the double-coated and non-dried whole cottonseed of the present invention by comparing with the comparative example 2, the storage stability of the 2^nd coated cottonseed was determined according to the previous known literature (Asian Australas, J. Anim. Sci. Vol. 28(6), pp816-826, 2015).

7-1. Determined content

Aerobic stability and the occurring day of microbial growth.

7-2. Treatment of test samples

The test samples consisting of five group of the comparative example 2 and five group of inventive 2^nd coated cottonseed, were humidified to the extent that final water content was reached to 14% and all the test samples were poured to 20kg of glass bottle of which upper side had been open, and the bottle was placed on an incubator (JSGI-100T, JS Research Inc., Korea) which maintains 30℃ of temperature and 70% of relative humidity to determine the storage stability fo thest samples.

7-3. Test procedure

(1) Determination of the occurring day of microbial growth: the occurring day of microbial growth in the groups of comparative example 2 and inventive 2^nd coated cottonseed were determined by optical observation.

(2) Determination of the aerobic stability: the aerobic stability in the groups of comparative example 2 and inventive 2^nd coated cottonseed were determined by using aerobic stability analyzer (EG-1, Ubikorea Co. Ltd) equipped in the center of 20kg of glass bottle and automatically recording the temperature of sensor as well as that of environment.

7-4. Test result

At the result, it has been confirmed that the comparative group has been found to show the mean occurring day of microbial growth of 13^th day whereas the inventive test group treated with the 2^nd coating agent such as 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite showed the mean occurring day of microbial growth of 22^nd day (See Table 7).

As to the aerobic stability, it has been confirmed that the comparative group has been found to show the mean aerobic stability of 9^th day whereas the inventive test group treated with the 2^nd coating agent such as 0.4% (w/w) mineral oil and 0.4% (w/w) bentonite showed the mean aerobic stability of 49^th day (See Table 7).

It has been confirmed that inventive test group treated with the 2^nd coating agent has been found to prolong the occurrence of microbial growth to 23 days comparing with comparative group.

The comparison of storage stability between an inventive test group and comparative group

SAMPLE**	ce1-cotton	ce2-cotton	dc-cotton
mean occurring day of microbial growth (day)	13th day	10th day	22nd day
mean aerobic stability(day)*	9th day	8th day	49th day
*: aerobic stability denotes the occurring day of which temperature is higher than environmental temperature; **: ce1-cotton denotes comparative cottonseed prepared in Comparative example 1; : ce2-cotton denotes comparative cottonseed prepared in Comparative example 2; dc-cotton ; double coated cottonseed treated with 2% starch; 10% purified water(4% NaCl) and 2nd coating agent (0.4% (w/w) mineral oil and 0.4% (w/w) bentonite).

Experimental Example 8. Determination of the palatibility of 2^nd coated cottonseed

In order to confirm the improved palatibility of the double-coated and non-dried whole cottonseed of the present invention by comparing with the comparative cottonseed prepared in Comparative example 2, the palatibility of the 2^nd coated cottonseed was determined according to the previous known literature (Han I. K., 1991, Feed & Nutrition, Korea Society of Animal Nutrition and Feedstuffs, Sunjin Culture Press) ).

The inventive double-coated and non-dried whole cottonseed of the present invention and the comparative cottonseed prepared in Comparative example 2 were performed to palatibility test for 10 head of Halstein dairy cattle having fed with formula feed and three hours after the feeding, the cattle were fed with the coated whole cottonseed according to Top Dressing method to test their palatibility.

At the result, it has been confirmed that the inventive double-coated and non-dried whole cottonseed is more palatable to the cattle, i.e., immediate intake-6 head; intake within 5 mins-2 head; feed refusal- 2 head comparing with the comparative cottonseed prepared in Comparative example 2. (See Table 8).

The comparison of palatibility between an inventive test group and comparative group

TEST SAMPLE*	ce1-cotton	ce2-cotton	dc-cotton
immediate intake (head)	4	4	6
intake within 5 mins (head)	3	2	2
feed refusal (head)	3	4	2
sum	10	10	10
*: ce1-cotton denotes comparative cottonseed prepared in Comparative example 1; : ce2-cotton denotes comparative cottonseed prepared in Comparative example 2; : dc-cotton ; double coated cottonseed treated with 2% starch; 10% purified water(4% NaCl) and 2nd coating agent (0.4% (w/w) mineral oil and 0.4% (w/w) bentonite).

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

As described in the present invention, inventive novel industrialized method for preparing the a double-coated and non-dried whole cottonseed for animal feed can provide with more favorable advantages such as stronger coating ability, prolonged storage stability, efficient economic cost due to simple preparation steps by dint of non-drying step etc comparing with the prior preparation methods already known in the art, especially, a method for preparing a high-strength, coated, whole cottonseed for stock feed disclosed in PCT/WO2015186935 (A1), in which the germination function of the whole cottonseed is removed by using a high temperature and high pressure steam, and high-viscosity gelatinized starch.

Claims (16)

1. A preparation method of the double-coated and non-dried whole cottonseed for animal feed using by (a) the storage stability enhancer selected from the group consisting of sodium chloride (NaCl), Magnesium chloride (MgCl₂), Potassium chloride (KCl) and Calcium chloride (CaCl₂); (b) the 1^st coating agent selected from the group consisting of pre-gelatinized starch, lignosulfate, gelatin, molasses, corn starch, tapioka starch, wheat starch, rice starch, potato starch, saccharides, CMS (Condensed Molasses Fermentation Solubles), corn flour, tapioka flour, wheat flour, rice flour, and cellulose; and (c) the 2^nd coating agent selected from at least one group consisting of (1) vegetable oil selected from mineral oil, essential oil, soybean oil, corn oil, canola oil, olive oil, sunflower seed oil, coconut oil, palm oil, or cottonseed oil; and/or (2) clay minerals selected from bentonite, monmorillo- nite, zeolite, illite, or silica gel.
2. The method according to claim 1, said method comprises the steps of; (1) removing linter from whole cottonseed at the 1^st step; (2) adding the mixture of a storage stability enhancer and water to the de-linted cottonseed and stirring together to prepare the 1^st cottonseed mixture at 2^nd step; (3) adding the 1^st coating agent to the 1^st cottonseed mixture and stirring together to prepare the 1^st coated cottonseed at 3^rd step; (4) adding the 2^nd coating agent to the 1^st coated cottonseed and stirring together to prepare the 2^nd coated cottonseed at 4^th step; and then (5) cooling the 2^nd coated cottonseed without drying step.
3. The method according to claim 2, said whole cottonseed at the 1^st step is characterized by being manufactured from Australia, United State of America, China, India, Pakistan, Brazil, Turkey, Uzbekistan, Greece, Vietnam, or Korea.
4. The method according to claim 2, the lint at the 1^st step is characterized by being finally removed to an amount of 1 to 10%(w/w) by weight based on a weight of the whole cottonseed.
5. The method according to claim 2, the storage stability enhancer at the 2^nd step is characterized by being selected from the group consisting of sodium chloride (NaCl), Magnesium chloride (MgCl₂), Potassium chloride (KCl) and Calcium chloride (CaCl₂).
6. The method according to claim 2, the storage stability enhancer at the 2^nd step is characterized by being mixed with water in the amount of ranging from 1 to 15% (v/w) based on the weight of water.
7. The method according to claim 2, the storage stability enhancer at the 2^nd step is characterized by being stirred for the period ranging from 1 hr to 6 hrs to provide water with enough time to be penetrated into the center of cottonseed.
8. The method according to claim 2, the 1^st coating agent at the 3^rd step is characterized by being used as a sole or combinations of coating agent selected from the group consisting of pre-gelatinized starch, lignosulfate, gelatin, molasses, corn starch, tapioka starch, wheat starch, rice starch, potato starch, saccharides, CMS (Condensed Molasses Fermentation Solubles), corn flour, tapioka flour, wheat flour, rice flour, and cellulose.
9. The method according to claim 2, the 1^st coating agent at the 3^rd step is characterized by being used in the amount of ranging from 0.01 to 30% (w/w) based on the weight of the whole cottonseed.
10. The method according to claim 2, the pouring amount of the 1^st coating agent into the stirred mixture at the 3^rd step is characterized by being dividedly poured in an amount of about 1/100 to 1/5 (w/w) with consecutively pouring into the stirred mixture by dividing into 5 to 100 times, based on the total weight of the 1^st coating agent.
11. The method according to claim 2, the 2^nd coating agent at the 4^th step is characterized by being used as a sole or combinations of coating agent selected from the group consisting of (1) vegetable oil selected from mineral oil, essential oil, soybean oil, corn oil, canola oil, olive oil, sunflower seed oil, coconut oil, palm oil, or cottonseed oil; and/or (2) clay minerals selected from bentonite, monmorillo- nite, zeolite, illite, or silica gel.
12. The method according to claim 11, said mineral oil or bentonite as the 2^nd coating agent at the 4^th step is characterized by being used in the amount of 0.1 - 2%(w/w), based on the weight of the whole cottonseed in case of mineral oil; and 0.1 - 1%(w/w), based on the weight of the whole cottonseed in case of bentonite.
13. The method according to claim 2, said cooling step of “the 2^nd coated cottonseed without drying step”at the 5^th step is characterized by performing the cooling step to the extent to the final water content of final coating product reach to 10 - 18% (w/w) of final water content of final coating product.
14. The method according to claim 2, said cooling step of “the 2^nd coated cottonseed without drying step” at the 5^th step is characterized by performing in the air for the period ranging from about 1 mins to 60 mins.
15. The method according to claim 2, said method comprises the steps of; (1) removing linter from whole cottonseed to the extent that the amount of linter based on the weight of whole cottonseed reaches to 1 -10 % (w/w) at the 1^st step; (2) preparing the 1 - 24% (w/w) sodium chloride in water as a storage stability enhancer solution ; adding 1 - 15 % (w/w) the storage stability enhancer solution to the de-linted cottonseed and stirring together to prepare the 1^st cottonseed mixture at 2^nd step; (3) adding sole or combinations of 0.1 - 30% (w/w) 1^st coating agent based on the weight of whole cottonseed, wherein said 1^st coating agent selected from the group consisting of pre-gelatinized starch, lignosulfate, gelatin, molasses, corn starch, tapioka starch, wheat starch, rice starch, potato starch, saccharides, CMS (Condensed Molasses Fermentation Solubles), corn flour, tapioka flour, wheat flour, rice flour, and cellulose to the 1^st cottonseed mixture and stirring together to prepare the 1^st coated cottonseed at 3^rd step; (4) adding sole or the combinations of 1 - 2% (w/w) mineral oil and/or 0.1 - 2% (w/w) bentonite based on the weight of whole cottonseed as the 2^nd coating agent to the 1^st coated cottonseed and stirring together to prepare the 2^nd coated cottonseed at 4^th step; and then (5) cooling the 2^nd coated cottonseed without drying step to the extent that the water content of final produce ranges from 10 - 17% (w/w).
16. The double-coated and non-dried whole cottonseed for animal feed prepared by the preparation method of claim 1.