KR101197532B1 - Feed additive using citrus peel and method for manufacturing thereof - Google Patents

Abstract

The present invention relates to a feed additive using a citrus gourd and a method for manufacturing the same, and more particularly, to an antimicrobial feed additive containing a culture solution inoculated with the bacteria of Lactobacillus in a citrus gourd or citrus gourd extract and a method for producing the same. It is about. The feed additive of the present invention has a superior antimicrobial activity compared to the conventional medium, exhibits a formal effect of inhibiting the growth of harmful bacteria in the intestines of livestock, and is a probiotic substitute for antibiotics, and also produces GABA, a bioactive substance. And can be used as a high value added probiotic feed.

Description

Feed additive composition using citrus gourd and its manufacturing method {Feed additive using citrus peel and method for manufacturing

The present invention relates to a composition for feed addition using a citrus gourd, and a method for preparing the same, and more particularly, a composition for feed addition containing a culture solution inoculated with a strain of the genus Lactobacillus in a citrus gourd or a citrus gourd extract and its preparation It is about a method.

Recently, with the development of agricultural technology, the production of fruits such as citrus fruits is also increasing. This increase in production has led to industrial necessity of increasing the availability of citrus fruits due to price imbalances and supply and demand imbalances, thereby increasing the production of processed products such as citrus juice and citrus liquor. However, with the increase in production, the generation of citrus peels and debris as by-products or wastes has also increased, and the remaining citrus gourds after these citrus juices are not decayed, which is a serious environmental factor.

On the other hand, citrus gourd is known to contain pectin, flavonoids, alkaloids, hesperidin and vitamins. They are important because they show detoxification, lower blood cholesterol, antibacterial, anti-inflammatory and immunity. Therefore, in recent years, methods for effectively treating citrus gourds have been studied in various ways, and methods for making citrus gourds by pulverizing and drying them to feed, fertilizers, or compost, and citrus gourd fermented feed production apparatuses have been studied. In the case of fermenting citrus gourd, the pH of the citrus gourd may be lowered due to the fermentation of the citrus gourd, and thus an antimicrobial may be used (eg, Jeju Shaft Hyup Fiber Feed).

As a solution to this problem, many studies are being conducted to produce high value added probiotic feed through bacterial fermentation. Existing feed markets have benefited from the widespread use of antibiotics for the purpose of promoting growth in the livestock industry, increasing feed conversion efficiency, and preventing intestinal infections. Resulted in the appearance of strains. Recently, the European Commission has banned the use of antibiotics added to feeds as animal growth promoters, and in particular, poultry farms strongly recommend antimicrobial preparations to prevent pathogens. As a solution to this problem, probiotics that produce antimicrobial substances have emerged as substitutes for antibiotics.

Probiotics (probiotics) refers to the culture of microorganisms containing the cells, including probiotics, these microorganisms can be said to be a 'microbial agent' that is supplied to livestock in the state of live or dead bacteria to produce a useful effect. Such feed probiotics prevent harmful bacteria from adhering to the intestinal wall and produce organic acids, antimicrobial substances, enzymes, etc. to enhance immunity. Currently used probiotic strains are mainly Gram-positive bacteria Lactobacillus, Streptococcus, Bacillus, Clostridium genus and mold, yeast and the like.

Among them, Lactobacillus is anaerobic or some aerobic bacteria, Bacillus Sp . Lactobacillus, as a major beneficial bacterium of intestinal bacteria, is advantageous for forming a large intestinal flora. In addition, Lactobacillus bacteria to produce lactic acid (Lactic acid) to maintain the intestinal environment at a low pH to hinder the growth of harmful or pathogenic bacteria.

The lactobacillus is known to produce lactic acid and antibiotics as a main probiotic, as well as the recent research on the production of GABA (γ-Aminobutyric acid) using lactobacillus. GABA is a nonprotein amino acid and is well known as the major inhibitory neurotransmitter of the central nervous system in animals. GABA is known to be involved in many physiological mechanisms to stimulate the blood flow of the brain in animals and to increase the oxygen supply to promote the metabolism of brain cells.It is also involved in the regulation of prolactin and growth hormone secretion. It is known to be effective in lowering and pain relief, so it is a pharmacologically very high substance. GABA, which is involved in these various physiological activities, is also used in animal feed because it has a sedative and stress-inhibiting effect.

However, MRS medium, which is a culture medium of Lactobacillus, a major probiotic, is an expensive medium with a price of 20,000-30,000 won / L. In the livestock industry, which must be mass-produced, the price of feed additives is important. As a solution to this problem, the aim was to produce high value added probiotic feed through the fermentation of Lactobacillus strain by adding nitrogen source and trace element to citrus gourd or citrus gourd extract using inexpensive waste citrus gourd.

Therefore, the present inventors prepare a feed additive containing a culture medium cultured by inoculating the bacterium of the genus Lactobacillus to the citrus gourd or citrus gourd extract, and the culture solution has superior antimicrobial activity than the conventional medium depending on the nitrogen source and the trace element. The present invention was completed by preparing a high-value probiotic feed containing a bioactive substance GABA as well as a substitute for antibiotics.

It is an object of the present invention to provide a composition for feed addition comprising a high antibacterial activity from a citrus gourd or citrus gourd extract and containing GABA, and a method for preparing the same.

In order to achieve the above object, the present invention provides a feed additive for antimicrobial containing a culture solution inoculated with the genus Lactobacillus bacteria in citrus gourd or citrus gourd extract.

In addition, the present invention comprises the steps of 1) obtaining a citrus gourd after the tangerine juice;

2) mixing the citrus fruit foil and calcium hydroxide (Ca (OH) ₂ ) of step 1) and dehydrating using an extruder to separate the solid portion and the liquid portion to obtain a citrus fruit extract from the liquid portion; And

3) The method of preparing a feed additive comprising the step of obtaining a culture after adding the culture medium of the strain of Lactobacillus bacteria to the citrus gourd of step 1) or the citrus gourd of step 2).

The present invention also provides an antimicrobial feed additive containing a supernatant obtained by storing citrus gourd in a silo.

In the present invention, by adding a nitrogen source and a trace element to the culture medium containing citrus gourd or citrus gourd extract, Lactobacillus cultivation exhibits excellent antimicrobial activity and can replace antibiotics and at the same time produce GABA, a bioactive substance. The antimicrobial feed additive of the present invention can be used as an economical and high value probiotic feed. In addition, the present invention can be used as a probiotic feed additive antimicrobial feed additive containing a supernatant obtained by storing citrus gourd in silos.

1 is a process chart showing the waste citrus fruit processed waste feed process.
Figure 2 is a graph of GABA production capacity according to the medium composition of L. brevis IFO 12005 fermented in lung citrus gourd medium.

Hereinafter, the present invention will be described in detail.

The present invention provides a feed additive containing a culture solution cultured by inoculating Lactobacillus bacteria in a culture medium containing a citrus gourd or citrus gourd extract.

Citrus gourd in the present invention refers to the remaining residue after citrus juice, in the present invention can be usefully recycled not only the liquid extract of citrus gourd but also citrus gourd.

In this case, the culture medium may include a nitrogen source of peptone, beef extract or yeast extract, or the nitrogen source and trace element (see Table 1 ).

At this time, the bacteria of the genus Lactobacillus used for inoculation may be applied to all kinds of bacteria of the Lactobacillus, preferably Lactobacillus brevis ( Lactobacillus) brevis), Lactobacillus Planta Rum (L. plantarum), Lactobacillus liquid filler's attempt (L. acidophilus), Lactobacillus casei (L. casei), Lactobacillus momentum spread (L. fermentum), Lactobacillus Caucasus ramno ( L. rhamnosus ) and L. reuteri are preferably selected from the group consisting of (see Table 2 ). The bacterium Lactobacillus is a strain that can be used as probiotics (effective probiotic). More preferably, it is preferably selected from the group consisting of L. fermentum , L. reuteri and L. plantarum according to the medium composition (see Table 2 ). ).

The antimicrobial activity of the feed additive including the Lactobacillus culture medium according to the culture medium using the citrus gourd was confirmed by the agar disk diffusion test method for various pathogens. Compared with the size of the inhibitory ring of ampicillin 1000 mg / ml, which is a positive control group, it showed significant antibacterial activity against pathogens. Although the antibacterial activity of the liquid obtained by storing citrus gourd in silos was confirmed (see Table 3 ), the antimicrobial activity of the feed additive including Lactobacillus culture was better (see Tables 4 to 6 ). In particular, feed additives prepared from cultures comprising L. casei , L. fermentum , L. fermentum and L. reuteri have superior antimicrobial activity compared to ampicillin antibiotics. Activity is shown (see Table 3 ).

The feed additive exhibiting the antimicrobial activity may contain a supernatant obtained by storing citrus gourd in a silo, and the lactobacillus culture solution is preferably included in the citrus gourd or citrus gourd extract.

In the culture medium, a nitrogen source is preferably added to the citrus gourd or citrus gourd extract, and more preferably, peptone, beef extract or yeast extract is added as the nitrogen source.

The Lactobacillus is preferably selected from the group consisting of Lactobacillus casei ( L. casei ), Lactobacillus fermentum ( L. fermentum ) and Lactobacillus luteri ( L. reuteri ) (see Table 3 ).

The feed additives collectively refer to substances added in trace amounts to the feed for nutritional or specific purposes. Preferably the feed additive is added to the feed for livestock, the range of livestock includes mammals, birds, fish, insects, more preferably for feed for cattle, beef, cows, pigs, poultry, dogs and cats do.

In addition, whether the Lactobacillus strain produced GABA according to the culture medium of the present invention was confirmed by the thin film chromatography (TLC) method of the culture supernatant. It was confirmed that Lactobacillus strains produce GABA using MSG as a negative control and GABA as a positive control (see FIG. 2 ).

In the culture medium, it is preferable to add a nitrogen source and a trace element to the citrus gourd or citrus gourd extract, a peptone, a beef extract or a yeast extract as a nitrogen source, and a MgSO as a trace element. _4? it is more preferred to add _{H 2 O, MnSO 4? H} 2 O or K ₂ PO ₄ (see Table 7).

The Lactobacillus strain is preferably Lactobacillus brevis ( L. brevis ).

In addition,

The present invention is 1) obtaining a citrus gourd juice after the tangerine;

2) mixing the citrus fruit foil and calcium hydroxide (Ca (OH) ₂ ) of step 1) and dehydrating using an extruder to separate the solid portion and the liquid portion to obtain a citrus fruit extract from the liquid portion; And

3) The method of preparing a feed additive comprising the step of obtaining a culture after adding the culture medium of the strain of Lactobacillus bacteria to the citrus gourd of step 1) or the citrus gourd of step 2).

Step 1) of the present invention is a step of obtaining a citrus gourd extract from the citrus gourd. In a preferred embodiment of the present invention, the citrus gourd was mixed with calcium hydroxide and then dehydrated to obtain a citrus gourd extract.

Step 2) of the present invention is a step of obtaining a culture cultured by inoculating the citrus gourd or the citrus gourd extract obtained in step 1 with bacteria of the genus Lactobacillus. Lactobacillus strains used for inoculation may be applied to all kinds of Lactobacillus bacteria, but preferably Lactobacillus brevis ( Lactobacillus) brevis), Lactobacillus Planta Rum (L. plantarum), Lactobacillus liquid filler's attempt (L. acidophilus), Lactobacillus casei (L. casei), Lactobacillus momentum spread (L. fermentum), Lactobacillus Caucasus ramno ( L. rhamnosus ) and L. reuteri are preferably selected from the group consisting of (see Table 2 ). The bacterium of the genus Lactobacillus grows in the citrus gourd or citrus gourd extract because it decomposes pectin in the citrus gourd or citrus gourd extract using the protopectinase produced by the bacterium. can do.

In addition, it may further comprise the step of preparing a powder by drying the culture solution obtained after step 2) of the present invention.

In addition, the present invention confirmed the antimicrobial activity of the supernatant obtained by storing citrus gourd in silos (see Table 3 ). The period of storage of citrus gourds in silos is preferably between one and three years.

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

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> Citrus gourd  Preparation of Feed Additives

The strain used in the present invention is Lactobacillus brevis ( Lactobacillus) brevis ) IFO12005, L. plantarum ATCC 8014, L. acidophilus ATCC 4356, L. casei ATCC 393, Lactobacillus fermentum ( L. fermentum ) ATCC 9338, L. rhamnosus ATCC 10863 and L. reuteri ATCC 23272 were distributed at the Korea Microbial Conservation Center.

Citrus gourd corresponds to the residue left after the tangerine juice, and the citrus gourd medium used the processed wastewater that went through the extrusion drying process using the citrus juice processing waste.

In order to manufacture citrus fruit medium, waste citrus fruit (Jeju Island, citrus processing complex; 90% water content), which is a processed waste from the citrus juice factory in Jeju, is subjected to the first two stages of grinding and extrusion process and calcium hydroxide (Ca ( OH) ₂ ) was separated into a solid phase part of 27% (water content 55% or less) and the liquid phase part 73% (water content 95% or more). The solid phase produced CPP (citrus pulp pellet) with water content of 5% or less through hot air drying and pellet molding, and the liquid phase (citrus waste water) was used as a feed additive as a culture medium for bacteria ( 1 ).

< Experimental Example  1> Citrus gourd  On the badge Lactobacillus  Check the growth rate of the strain

Lactobacillus strains, except L. brevis , were precultured at 37 ° C. for 24 hours using MRS medium and 1% (v / v) of the medium in which the nitrogen source (Table 1) was added to the citrus fruit medium. The rate of inoculation was 37 ℃. Lactobacillus brevis (L. brevis ) was incubated at 30 ℃, and sampled (sampling) at 0, 24, 48 hours to determine the growth was measured the number of viable cells.

As a result, when the viable cell count was measured after 48 hours of cultivation, most of the strains were grown more than 100 times the initial inoculation concentration in MRS medium, almost no growth in citrus gourd medium (CJM). However, when the nitrogen source was added to the citrus fruit medium (CJM), L. fermentum , L. reuteri , L. brevis increased more than 100-fold over the initial inoculation concentration ( Table 2 ).

Composition of the badge One 2 3 4 5 6 7 8
Lactobacillus
Brevis
Except
Lactobacillus strains
MRS
CJM
CJM +
1% peptone
CJM
+
1% BE
CJM
+
0.5%
YE
CJM
+
0.5% peptone
+
0.5% BE
+
0.25%
YE
Lactobacillus
Brevis
IFO 12005
MRS
+
2% MSG
CJM
+
2% MSG
CJM +
1% peptone
+
2% MSG
CJM
+
1% BE
+
2% MSG
CJM
+
0.5%
YE
+
2% MSG
CJM
+
0.5%
peptone
+
0.5% BE +
0.25% YE +
2% MSG
CJM
+
0.5%
peptone
+
0.5% BE
+
0.25% YE
+
0.1% MgSO4? H2O
+
2%
MSG

CJM
+
0.5%
peptone
+
0.5% BE
+
0.25% YE
+
0.01% MgSO ₄ ? H ₂ O
+
0.005% MnSO ₄ ? H ₂ O
+
0.2% K ₂ PO ₄
+
2% MSG

CJM: 5 brix citrus fruit medium

B.E: Beef extract

Y.E. : Yeast extract

MSG: Sodium L (+)-Glutamate Monohydrate

Growth Rate of Lactobacillus Strains According to Medium Composition
L.
Axidophilus
L.
Rhamnosus
L.
Permanent
L.
Kazei
L.
Lutheri
L.
Plantarum
L.
Brevis
(2% MSG
adding) MRS + ++ +++ ++ +++ +++ +++ CJM - - +++ - + - +++ CJM
+
1% peptone - - + - +++ - +++ CJM
+
1% BE - - +++ - +++ - +++ CJM
+
0.5% YE - - +++ - +++ - +++ CJM
+
0.5% peptone
+
0.5% BE
+
0.25% YE - - ++ + +++ - +++

+++: 1000x increase

++: 100 to 1000 times increase

+: 10 to 100 times increase

-: 10 times or less of initial concentration

< Experimental Example  2> Lactobacillus  Determination of antimicrobial activity by strain

The supernatant of the solution in which citrus gourds were stored in silos without adding Lactobacillus strains and the supernatant incubated for 48 hours in MRS medium and citrus gourd medium were filtered by 0.2 μm centrifuged at 8000 rpm for 20 minutes. Samples were prepared. Pathogen Salmonella Entebbe utility disk (Salmonella enteritidis) and Escherichia coli (E. coli) O-157, Escherichia coli (E. coli) K88, Salmonella aureus (aureus Salmonella) as, the ampicillin (ampicillin positive control to measure the antimicrobial activity ) 1000ppm (1mg / ml) was compared with the size of the inhibitory ring.

Lactobacillus supernatant obtained without adding the strain Bacillus storage foil on the citrus silo is E. coli O-157 with Salmonella Entebbe utility disk (Salmonella enteritidis ) was confirmed to have antimicrobial activity (Table 3).

Antimicrobial activity by Lactobacillus strains showed antimicrobial activity in cultured strains in most MRS medium, but L. casei , in Escherichia coli O-157, In case of L. reuteri , L. reuteri showed more antimicrobial activity when added nitrogen source to citrus gourd medium than MRS culture, and L. reuteri showed antibacterial activity even on E. coli K88 (Table 4). 6).

Determination of Antimicrobial Activity of the Supernatant from Citrus Fruits in Silos Escherichia coli O-157 Salmonella Enteritidis Salmonella Aureus 1 year storage in silos - + - 3 years storage in silos ++ + -

++++: 75 ~ 100% of Ampicillin 1000 ppm

+++: 50 ~ 75% of Ampicillin 1000 ppm

++: 25-50% of Ampicillin 1000 ppm

+: 25% less than 1000 ppm Ampicillin

-: No activity

Determination of Antimicrobial Activity against Salmonella enteritidis of Lactobacillus Strains According to Medium Composition
Salmonella Enteritidis
L.
Axidophilus
L.
Rhamnosus
L.
Permanent
L.
Kazei
L.
Lutheri
L.
Plantarum MRS ++ ++ +++ +++ - ++++ CJM - - ++ - - - CJM
+
1% peptone
-
-
++
-
-
- CJM
+
1% BE
-
-
++
-
-
-
CJM
+
0.5% YE
-
-
++
-
-
- CJM
+
0.5% peptone
+
0.5% BE
+
0.25% YE
-
-
++
-
-
-

++++: 75 ~ 100% of Ampicillin 1000 ppm

+++: 50 ~ 75% of Ampicillin 1000 ppm

++: 25-50% of Ampicillin 1000 ppm

+: 25% less than 1000 ppm Ampicillin

-: No activity

Determination of Antimicrobial Activity against E. coli O-157 of Lactobacillus Strains According to Medium Composition
Escherichia coli O-157
L.
Axidophilus
L.
Rhamnosus
L.
Permanent
L.
Kazei
L.
Lutheri
L.
Plantarum MRS +++ ++ - ++ - - CJM - - - ++ - - CJM
+
1% peptone
-
-
-
++
-
- CJM
+
1% BE
-
-
-
++
++++
-
CJM
+
0.5% YE
-
-
-
-
++++
- CJM
+
0.5% peptone
+
0.5% BE
+
0.25% YE
-
-
-
++++
++++
-

++++: 75 ~ 100% of Ampicillin 1000 ppm

+++: 50 ~ 75% of Ampicillin 1000 ppm

++: 25-50% of Ampicillin 1000 ppm

+: 25% less than 1000 ppm Ampicillin

-: No activity

Determination of Antimicrobial Activity against E. coli K88 of Lactobacillus Strains According to Medium Composition
Escherichia coli K88
L.
Axidophilus
L.
Rhamnosus
L.
Permanent
L.
Kazei
L.
Lutheri
L.
Plantarum MRS ++ ++ - - - ++ CJM - - - - - - CJM
+
1% peptone
-
-
-
-
-
- CJM
+
1% BE
-
-
-
-
-
-
CJM
+
0.5% YE
-
-
-
-
-
- CJM
+
0.5% peptone
+
0.5% BE
+
0.25% YE
-
-
-
-

+++
-

++++: 75 ~ 100% of Ampicillin 1000 ppm

+++: 50 ~ 75% of Ampicillin 1000 ppm

++: 25-50% of Ampicillin 1000 ppm

+: 25% less than 1000 ppm Ampicillin

-: No activity

< Experimental Example  3> Lactobacillus  By strain GABA Generation  Measure

GABA production capacity is Lactobacillus brevis (L. brevis) IFO 12005 was inoculated 1% in a medium containing a nitrogen source in MRS medium and citrus gourd medium (CJM), and then cultured at 30 ° C. for 7 days (Table 7). The culture supernatant was filtered by 0.2um centrifuged at 8000 rpm for 20 minutes to prepare a sample. Incubate supernatant samples with TLC silica gel 60 F to confirm the production of GABA.₂₅₄After loading by 3μl each was confirmed by separating with a solvent. The solvent composition used was n-butanol: acetic acid: water in a 5: 2: 2 ratio, and MSG and GABA were used as controls. The Rf value of GABA was 0.54 and the Rf value of MSG was 0.42. GABA is the source of nitrogen and MgSO in MRS and citrus gourd medium.₄ ? H₂It was produced when a trace element such as O was added (Fig. 2).

Medium composition for GABA production Badge number Badge composition One MRS + 2% MSG 2 CJM + 0.5% Peptone + 0.5% Beef Extract + 0.25% Yeast Extract + 0.1% MgSO ₄ ? H ₂ O + 2% MSG 3 CJM + 0.5% Peptone + 0.5% Beef Extract + 0.25% Yeast Extract + 0.01% MgSO ₄ ? H ₂ O + 0.005% MnSO ₄ ? H ₂ O + 0.2% K ₂ PO ₄ + 2% MSG 4 MRS

Claims (10)

Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus casei ( L. casei ), L. fermentum ( L. fermentum ), Lactobacillus L. rhamnosus , Lactobacillus L. reuteri ( L. reuteri ) containing a culture medium inoculated by inoculating any one of the bacteria of the genus Lactobacillus, the culture medium contains GABA (-Aminobutyric acid) produced by Lactobacillus bacteria, Lactobacillus Antimicrobial feed additive having antibacterial activity of the genus bacteria.
delete The antimicrobial feed additive of claim 1, wherein the culture medium further comprises a nitrogen source, or a nitrogen source and a trace element.
delete According to claim 1, wherein the antimicrobial activity is antimicrobial feed additive, characterized in that the antimicrobial activity against the pathogen Salmonella enteritidis , E. coli O-157 or E. coli K88 .
delete 1) after the citrus juice to obtain a citrus gourd;
2) mixing the citrus fruit foil and calcium hydroxide (Ca (OH) ₂ ) of step 1) and dehydrating using an extruder to separate the solid portion and the liquid portion to obtain a citrus fruit extract from the liquid portion; And
3) The method of preparing an antimicrobial feed additive according to claim 1, comprising the step of culturing by adding the culture medium of the Lactobacillus bacterial strain to the citrus gourd of step 1) or the citrus gourd extract of step 2).
The method of claim 7, further comprising the step of preparing a powder by drying the culture solution obtained after step 3).
delete delete

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