JP2023039375A - Bacteriostatic agent for feed - Google Patents

Abstract

To provide a bacteriostatic agent for feed that can perform bacteriostasis on Salmonella in feed without increasing the water content of the feed by adding a lactic acid bacteria culture solution or the like, or can suppress the establishment of Salmonella in livestock.SOLUTION: Provided is a bacteriostatic agent for feed containing high-protein vegetable meal as an active ingredient, the bacteriostatic agent being characterized in that the high-protein vegetable meal is obtained by a method involving mixing a vegetable meal and water, performing saccharification treatment and alcoholic fermentation treatment on the resulting mixture, adding a fiber-degrading enzyme for the saccharification treatment, adding a microorganism for the alcohol fermentation treatment, and using cellulase produced by a microorganism of the genus Acremonium as the fiber-degrading enzyme.SELECTED DRAWING: Figure 2

Description

The present invention relates to feed bacteriostats and feeds containing the same.

Salmonella infection is known as one of livestock infectious diseases. In particular, poultry Salmonella infections are mainly caused by Salmonella Enteritidis and Salmonella Typhimurium entering through the mouth, going to the intestine, entering the blood from the intestinal tract, and circulating in various organs. develop. It often develops during the juvenile stage within one month after hatching, and in many cases it dies within 10 days after hatching.
Salmonella-contaminated feed is an important source of Salmonella infection in poultry, and infection can occur even with small amounts of bacteria. For example, even one fungus in 15g of feed can cause infection. Therefore, Salmonella in feed is strictly controlled. Therefore, techniques for suppressing the proliferation of Salmonella in feed are being actively investigated. In particular, when the oil cake used for animal feed gets wet due to rain, etc., and is further contaminated with Salmonella, the proliferation of Salmonella in the oil cake is inhibited. A technology capable of suppressing the noise is required.

Examining the prior art, for the prevention and treatment of salmonella infection in livestock, administration of feed containing ingredients derived from fermentation broth of specific lactic acid bacteria is effective in preventing the growth of salmonella in the body of livestock. is known (see Patent Document 1).
Also, a technique has been developed in which proliferation of Salmonella in oil cake can be suppressed by adding lactic acid bacteria to oil cake (see Patent Document 2).
However, the addition of lactic acid bacteria to feed in these methods involves adding an aqueous solution of lactic acid bacteria culture solution (fermentation broth). In order to maintain hygiene during feed storage, it is necessary to keep the water content in the feed below a certain amount, so it was necessary to dry the water derived from the lactic acid bacteria culture solution.

Republished WO 00/30661 JP 2011-244704 A

An object of the present invention is to make it possible to bacteriostasize Salmonella in feed without increasing the water content in the feed by adding a lactic acid bacteria culture solution or the like, or to suppress the colonization of Salmonella in livestock. It is to provide a feed bacteriostatic agent.

As a result of extensive studies, the present inventors have found that a high-protein plant obtained by using cellulase produced by Acremonium genus microorganisms as a fiber-degrading enzyme used for saccharification of plant meal The present invention was completed based on the discovery that toxic meal has a component that inhibits the growth of Salmonella and can be used as a bacteriostatic agent for feed.

Specifically, the present invention includes the following aspects.
[1] A feed bacteriostatic agent containing a high-protein vegetable meal as an active ingredient, wherein the high-protein vegetable meal is mixed with a vegetable meal and water, and the resulting mixture is subjected to saccharification treatment and alcoholic fermentation treatment. and adding a fibril-degrading enzyme for the saccharification treatment, adding a microorganism for the alcohol fermentation treatment, and using cellulase produced by Acremonium genus microorganisms as the fibrous-degrading enzyme. A bacteriostatic agent for feed, characterized in that it is obtained.
[2] The bacteriostatic agent for feed according to [1], wherein the vegetable meal is rapeseed meal.
[3] A feed bacteriostat-containing plant meal containing the feed bacteriostat described in [1] or [2] and a plant meal.
[4] A feed containing the bacteriostatic agent for feed according to [1] or [2], or the bacteriostatic agent-containing vegetable meal for feed according to [3].
[5] A method of raising livestock by feeding the feed according to [4].
[6] A method for inhibiting Salmonella colonization in livestock, using the feed bacteriostat described in [1] or [2] or the feed bacteriostat-containing vegetable meal described in [3].
[7] A method for storing plant meal, which comprises adding the feed bacteriostat described in [1] or [2] to the plant meal.
[8] Feed storage characterized by adding the feed bacteriostat described in [1] or [2] or the feed bacteriostat-containing vegetable meal described in [3] to the feed. Method.
[9] A method for preventing livestock Salmonella infection using the feed bacteriostat described in [1] or [2] or the feed bacteriostat-containing vegetable meal described in [3].

According to the present invention, it is possible to provide a bacteriostatic agent for feed without increasing the water content of the feed by adding a lactic acid bacteria culture solution or the like, and by including the bacteriostatic agent for feed in the feed, It is possible to bacteriostasize common viable bacteria and Salmonella, and reduce the number of those bacteria over time.
In addition, according to the present invention, salmonella colonization in livestock can be suppressed by feeding livestock with feed containing a bacteriostatic agent for feed.
As a result, it contributes to the health of livestock and suppresses the decrease in the number of livestock due to death.

FIG. 1 is a graph showing the general viable cell count (CFU/g) on the test start date, the 1st day, and the 3rd day of the test plots A to E. FIG. 2 is a graph showing the number of Salmonella bacteria (CFU/g) on the test start date, 1st day, and 3rd day in test plots A to E. FIG. 3 is a graph showing the general viable cell count (CFU/g) on the test start date, 1st day, 2nd day, 3rd day, 5th day, and 10th day of Test Groups A and E. be. FIG. 4 is a graph showing the salmonella counts (CFU/g) on the test start date, 1st day, 2nd day, 3rd day, 5th day, and 10th day of Test Groups A and E.

(high protein vegetable meal)
The high-protein vegetable meal in the present invention is a processed high-protein vegetable meal obtained by subjecting vegetable meal to saccharification treatment and alcoholic fermentation, as described later. It contains protein, preferably 47 to 65% by weight.
The protein content referred to here refers to a value obtained by multiplying the total nitrogen obtained by the Kjeldahl method by 6.25.
As described in detail below, this high-protein vegetable meal can be used directly as a feed bacteriostat.

(Feed bacteriostatic agent)
The bacteriostatic agent for feed of the present invention is a bacteriostatic agent for feed containing a high-protein vegetable meal as an active ingredient, wherein the high-protein vegetable meal is a mixture obtained by mixing vegetable meal and water. A saccharification treatment and an alcohol fermentation treatment are performed, a fiber degrading enzyme is added for the saccharification treatment, a microorganism is added for the alcohol fermentation treatment, and an Acremonium genus microorganism is used as the fiber degrading enzyme. It is obtained by a method using the produced cellulase.
The high-protein vegetable meal of the present invention and the method for producing the same are described in detail in Japanese Patent Application No. 2021-028962 filed earlier, so the details are omitted here. Also, the content of said application is incorporated herein.

For the feed bacteriostat of the present invention, the above-mentioned high-protein vegetable meal can be used as it is as a feed bacteriostat.
By adding the feed bacteriostatic agent of the present invention to the feed, it is possible to bacteriostatic common viable bacteria and Salmonella present in the feed and reduce the number of these bacteria over time.
In addition, colonization of Salmonella in livestock can be suppressed by feeding livestock with feed containing the bacteriostatic agent for feed of the present invention.
Livestock to which the feed containing the bacteriostatic agent for feed of the present invention is fed includes poultry such as chickens and quails, cattle and pigs, among which poultry is preferred.

(Vegetable meal containing bacteriostatic agent for feed)
The bacteriostatic agent-containing vegetable meal for feed of the present invention is a bacteriostatic agent-containing vegetable meal for feed containing the above-described bacteriostatic agent for feed.
By adding the bacteriostatic agent for feed of the present invention to the vegetable meal to obtain a bacteriostatic agent-containing vegetable meal for feed, the sanitation of the vegetable meal can be improved. The bacteriostatic agent-containing vegetable meal for feed can be used as a raw material for feed.
The content of the bacteriostatic agent for feed in the plant meal containing the bacteriostatic agent for feed of the present invention is preferably 1 to 90% by mass, more preferably 1 to 50% by mass, and 1 to 25% by mass. %, even more preferably 1 to 10% by mass, most preferably 1 to 5% by mass.

The following is a summary of the ingredients for the high protein vegetable meal and the method of making it.
(vegetable meal)
The vegetable meal used in the present invention is not particularly limited, but general vegetable meals such as soybean meal, rapeseed meal, linseed meal, and perilla meal can be used. In particular, it is preferable to use rapeseed meal as the vegetable meal.
Here, soybean meal refers to soybean meal obtained by evaporating the organic solvent after passing through an oil extraction process in which soybean seeds are extracted using an organic solvent such as n-hexane, and has a water content of about 8 to 15%. % by mass.
In addition, rapeseed meal is obtained by extracting rapeseed seeds with a press, followed by an oil extraction process in which the oil remaining in the pressed lees is extracted using an organic solvent such as n-hexane, and then the organic solvent is evaporated. It is a rapeseed meal made from rice and contains about 8 to 15% by mass of water.

(water, other additives)
Water in the present invention is not particularly limited, and may be, for example, distilled water, pure water, or tap water. When vegetable meal and water are mixed before saccharification treatment or alcoholic fermentation treatment, 40 to 60 parts by weight of vegetable meal and 60 to 40 parts by weight of water, preferably 45 to 55 parts by weight of vegetable meal and 55 to 55 parts by weight of water 45 parts by weight, more preferably 48 to 52 parts by weight of vegetable meal and 52 to 48 parts by weight of water may be mixed. Such a water content is preferable because it becomes a solid fermentation method. Here, the term "solid fermentation method" refers to a fermentation method in which the water content is set lower than that of the liquid fermentation method, and little waste liquid is discharged not only during the fermentation period but also after the fermentation is completed.

0 to 10 parts by weight of additives such as minerals, sugars, amino acids, medium components, enzymes and microorganisms may be added to 100 parts by weight of the mixture of vegetable meal and water before saccharification treatment and alcoholic fermentation treatment. Specific examples of minerals include phosphorus, magnesium, sodium, potassium, iron and the like. Specific examples of sugars include sucrose, glucose, maltose, oligosaccharides and the like. Specific examples of amino acids include glutamic acid and lysine, and may be protein-constituting amino acids, non-constituting amino acids such as GABA, or protein hydrolysates. Specific examples of medium components include yeast extract, malt extract, peptone, and the like.
Specific examples of enzymes for saccharification include cellulase, hemicellulase, glucosidase, or mixtures thereof. In the saccharification treatment of the present invention, cellulase produced by Acremonium genus microorganisms, in particular, cellulase F produced by Acremonium cellulotycus (product Name: Meiji Acremonium Cellulase F, manufactured by Meiji Seika Pharma Co., Ltd.) and Cellulase KM produced by Acremonium cellulotycus (product name: Acremonium Cellulase KM, manufactured by Kyowa Kasei Co., Ltd.) are used. In the present invention, it is preferred to use cellulase produced by Acremonium cellulotycus. As will be described later, it was found for the first time in the present invention that ethanol production ability is improved by using cellulases produced by these Acremonium genus microorganisms.
Specific examples of microorganisms for alcoholic fermentation include filamentous fungi, yeasts such as Saccharomyces cerevisiae, Shizosaccharomyces pombe, and Pichia stipitis, and Zymomonas mobilis. Yeast such as Saccharomyces cerevisiae is preferably used for the alcoholic fermentation treatment of the present invention, and yeast that can be used for solid-state fermentation is particularly preferred.
Hereinafter, water or water and these additives are collectively referred to as "water or the like". Among these additives, water-soluble ones may be added after being solubilized in the water.

(mixture)
The mixing of vegetable meal and water or the like in the present invention means mixing the vegetable meal and water or the like manually or with a device. The more uniform the distribution of water in the solid content after mixing, the better the efficiency of the saccharification treatment or the alcoholic fermentation treatment. be. In general, it has been thought that it is desirable to sterilize a mixture of vegetable meal and water etc. in an autoclave in order to prevent the growth of microorganisms other than the desired microorganisms during saccharification or alcoholic fermentation. It was found that the present invention can omit the sterilization treatment before the saccharification treatment or the alcoholic fermentation treatment.

(saccharification treatment)
The saccharification treatment in the present invention is the addition of 0.01 to 1.0% by weight of a fiber-degrading enzyme to the fiber (cellulose etc.) contained in the vegetable meal to the mixture of the vegetable meal and water etc. It refers to the decomposition of the fiber contained in rapeseed meal into glucose. As the fiber-degrading enzyme used for saccharification, cellulase alone or cellulase in combination with enzymes such as hemicellulase and glucosidase can be used. It is mandatory to use The amount of cellulase used is preferably 0.01 to 1.0% by weight based on the mixture of vegetable meal and water. Further, it is more preferable to add 0.05 to 1.0% by weight, more preferably 0.1 to 1.0% by weight. Other than that, the enzyme used for the saccharification treatment may be any enzyme that can decompose the fiber, and even if it is a commercial product, a culture solution in which filamentous fungi are cultured, or a further refined product, the filamentous fungus itself can be used. It can be.

(alcohol fermentation treatment)
The alcoholic fermentation treatment in the present invention refers to a process of inoculating a mixture of vegetable meal and water or the like or a saccharified mixture of vegetable meal and water or the like with microorganisms to produce ethanol by the microorganisms. Say. Regarding the microorganisms to be used, yeasts such as Saccharomyces cerevisiae, Shizosaccharomyces pombe, and Pichia stipitis can be used. Anything can be used, including genetically modified ones. Microorganisms that have been preserved by slant or freezing may be used, but when using Saccharomyces cerevisiae, commercially available baker's yeast may be used. When using slant- or frozen-preserved strains, it is desirable to pre-culture them in a liquid medium before use, and use the pre-culture solution or the like. The liquid medium used for the pre-culture may be one suitable for culturing yeast or bacteria, such as 1% by mass yeast extract, 2% by mass peptone, and 3% by mass glucose.
In the present invention, yeast such as Saccharomyces cerevisiae is preferably used as the microorganism used for alcoholic fermentation, and yeast that can be used for solid fermentation is particularly preferred. In addition, the amount of the yeast used is a yeast suspension prepared so that the absorbance (hereinafter referred to as OD) when measured at 660 nm is 0.2, and the total reaction system (yeast (suspension) + It is preferable to add so that the amount of yeast present in the meal + added water is OD = 0.05 to 0.15. Further, it is more preferable to add so that OD=0.05 to 0.1.

(When performed in the same process or in separate processes)
In the saccharification treatment and alcohol fermentation treatment, enzymes and microorganisms are added to a mixture of vegetable meal and water, etc., and when saccharification and fermentation are performed in the same process (referred to as "multiple parallel fermentations"), the treatment temperature is 25. It is preferably carried out at ~45°C, more preferably at 27-43°C, and even more preferably at 30-40°C. When saccharification and fermentation are performed in separate steps, the saccharification temperature is preferably 40 to 60°C, more preferably 42 to 58°C, and even more preferably 45 to 55°C. On the other hand, the fermentation temperature is preferably 20 to 40°C, more preferably 22 to 38°C, even more preferably 25 to 35°C.
The total treatment time is preferably 4 hours to 336 hours, more preferably 72 to 336 hours, even more preferably 168 to 336 hours.

(After solid-liquid separation, drying and distillation)
A mixture of vegetable meal and water, subjected to saccharification treatment and alcoholic fermentation treatment, is subjected to solid-liquid separation and dried as necessary. The result is called a high protein vegetable meal. Drying may be dry heat drying, vacuum drying, freeze drying, spray drying, or the like, as long as the moisture and ethanol of the mixture obtained by saccharification treatment and/or alcohol fermentation treatment can be evaporated. If ethanol is recovered by steam distillation or the like before drying, the recovered ethanol can be used for industrial purposes or fuel.
The ethanol production amount in the present invention is preferably 3.5 g or more, more preferably 3.8 g or more, and more preferably 4.0 g or more per 100 g dry weight of rapeseed meal. More preferred.
It is preferable that the saccharification treatment and the alcoholic fermentation treatment are performed by a solid-state fermentation method. The use of the solid-state fermentation method eliminates the need for the above-described solid-liquid separation and is preferable because no waste liquid is produced.

(Production of vegetable meal containing bacteriostat for feed)
Next, a method for producing a bacteriostatic agent-containing vegetable meal for feed will be described.
The feed containing the bacteriostatic agent-containing plant meal for feed of the present invention can be produced by mixing the bacteriostatic agent for feed produced by the production method of the present invention with the plant meal.
Mixing can be performed using a mixer such as a ribbon mixer, a V-type mixer, or a W-type mixer.

Next, the feed containing the bacteriostatic agent for feed and the feed containing the bacteriostatic agent-containing vegetable meal for feed will be described.
(fodder, livestock rearing)
The feed of the present invention is a feed containing the bacteriostatic agent for feed described above, or a feed containing the bacteriostatic agent-containing vegetable meal for feed described above.
The bacteriostatic agent for feed of the present invention or the vegetable meal containing the bacteriostatic agent for feed of the present invention is combined with feed raw materials and feed additives containing sugar, protein, amino acid, fiber, minerals, oil, antibacterial components, etc. Can be mixed and used.
Specific examples of feed ingredients and feed additives include corn, sorghum, corn gluten field, corn starch, rice bran, soybean meal, regular rapeseed meal, bran, oats, milk casein, whey, fishmeal, various vitamins, vitamin mixes, and minerals. Mixes, amino acid preparations, calcium carbonate, monobasic calcium phosphate, vegetable oils and fats, animal oils and fats, salt and the like.
The content of the feed bacteriostat in the feed is preferably 1 to 20% by mass, more preferably 1 to 15% by mass, and 1 to 10% by mass when the total amount of the feed is 100% by mass. is more preferable.
In addition, the bacteriostatic agent-containing vegetable meal for feed is preferably added to the feed in such an amount that the content of the bacteriostatic agent for feed in the bacteriostatic agent-containing vegetable meal for feed becomes the content described above.
For example, in the case of a feed bacteriostat-containing vegetable meal containing 25% by mass of a feed bacteriostat, the content of the feed bacteriostat-containing plant meal in the feed is 100% by mass of the total amount of the feed. , preferably 4 to 80% by mass, more preferably 4 to 60% by mass, even more preferably 4 to 40% by mass.

A feed containing the bacteriostatic agent for feed of the present invention can be produced by mixing the bacteriostatic agent for feed with the various feed ingredients and feed additives described above.
Further, the feed containing the bacteriostatic agent-containing plant meal for feed of the present invention can be produced by mixing the bacteriostatic agent-containing plant meal for feed with the above-described various feed raw materials and feed additives. can.
These can be mixed using a mixer such as a ribbon mixer, a V-type mixer, and a W-type mixer.
Feeds using the bacteriostatic agent for feed produced by the production method of the present invention or the vegetable meal containing the bacteriostatic agent for feed have no concerns of adversely affecting the growth and health of chicks, and are satisfactory as feed. can be used.
In addition, by feeding livestock feed using the bacteriostatic agent for feed or the vegetable meal containing the bacteriostatic agent for feed of the present invention, it is possible to suppress colonization of salmonella in the livestock and raise the livestock. .

(Method for storing vegetable meal)
By adding the bacteriostatic agent for feed of the present invention to vegetable meal, it is possible to suppress the growth of Salmonella that is mixed in the vegetable meal. It can be used to prevent contamination with Salmonella when storing meals.
The content of the feed bacteriostat of the present invention used to prevent contamination with Salmonella in the vegetable meal is as described above.

(Method of storing feed)
By adding the bacteriostat for feed or the plant meal containing the bacteriostat for feed of the present invention to the feed, it is possible to suppress the proliferation of Salmonella contaminating the feed. Therefore, the bacteriostat for feed or the plant meal containing the bacteriostat for feed of the present invention can be used to prevent contamination with Salmonella during storage of feed.
The content of the bacteriostatic agent for feed or the vegetable meal containing the bacteriostatic agent for feed of the present invention used to prevent contamination with Salmonella in the feed is as described above.

(Method for preventing livestock salmonella infection)
Livestock Salmonella infection can be prevented by using the bacteriostatic agent for feed or the vegetable meal containing the bacteriostatic agent for feed of the present invention as a raw material for feed.
As will be described in detail in Examples, by using the bacteriostatic agent for feed of the present invention in plant meal or feed, or by using the bacteriostatic agent-containing plant meal for feed in feed, Since salmonella contamination can be prevented, the onset of livestock salmonella infection can be prevented in advance.
In addition, the bacteriostatic agent for feed of the present invention or the vegetable meal containing the bacteriostatic agent for feed is used as a raw material for feed, and by feeding it, it is possible to suppress the growth of Salmonella in the chicken body (inside the cecum). Therefore, it can be said that the bacteriostatic agent for feed or the vegetable meal containing the bacteriostatic agent for feed of the present invention can prevent livestock salmonella infection.
The content of the bacteriostatic agent for feed or the vegetable meal containing the bacteriostatic agent for feed of the present invention used for preventing salmonella infection in domestic animals is as described above.

EXAMPLES In order to describe the present invention in more detail, examples are shown below, but the present invention is not limited to these.

<Feeding test of compound feed containing bacteriostatic agent for feed>
1) Rapeseed meal and high-protein rapeseed meal used The rapeseed meal was a product “rapeseed cake” manufactured by Nisshin Oillio Group Co., Ltd. (protein content: 44.8% (on a dry matter basis), moisture content: 13.3% by mass. )It was used.
Also, a high-protein rapeseed meal was produced as follows.
4000 g of water was added to 4000 g of rapeseed meal to prepare 8000 g of a mixture of rapeseed meal and water. A suspension of yeast (Saccharomyces cerevisiae A30) was added to bring the total reaction to OD=0.1 without autoclaving. In addition, 0.1% by mass of Cellulase F (product name: Meiji Acremonium Cellulase F, manufactured by Meiji Seika Pharma Co., Ltd.) was added, and solid fermentation was performed at 35 ° C. for 13 days with the lid of the container closed. A high protein rapeseed meal was produced. As the container, an 8-liter resin case was used.
The resulting high-protein rapeseed meal was heat-treated under reduced pressure (72° C., −0.1 MPa) for 8 hours. After that, the dried high-protein rapeseed meal was pulverized with a grinder (manufactured by Osaka Chemical Co., Ltd., device "Absolute mill ABS-W") to a size suitable for feed ingredients, and dried high-protein rapeseed meal (protein content: 49 .4% (converted to dry matter), water content 9.0% by mass).
The obtained high-protein rapeseed meal was used as it was as a feed bacteriostat.
The rapeseed meal and the high-protein rapeseed meal (feed bacteriostat) used were examined for the presence of Salmonella by the following enrichment culture method, and the results were negative/25 g for both.

(Enrichment culture method)
A 25 g assay sample was weighed into buffered peptone water, shaken and incubated at 36±1° C. for 18-24 hours. Next, 1 ml and 0.1 ml of the pre-enrichment cultures were added to the Hannah Tetrathionate medium and the Rappaport-Vassiliadis medium respectively, shaken, and subjected to selective enrichment culture at 42±1° C. for 18-24 hours. A loopful of each selective enrichment culture was taken, streaked on DHL agar medium and BG (brilliant green agar plate medium), inverted, and streaked and cultured at 36±1° C. for 18 to 24 hours. About 3 colonies suspected to be Salmonella on the surfaces of the DHL agar medium and the brilliant green agar medium were picked from each of them, and confirmation tests were conducted using LIM medium and TSI medium. Group O sera were confirmed by glass agglutination when they exhibited typical characteristics. It was judged as positive (positive/25 g) when an agglutination reaction was observed, and as negative (negative/25 g) when no agglutination reaction was observed.

Regarding the compounded feed containing the obtained bacteriostatic agent for feed (high protein rapeseed meal), "Establishment of safety evaluation standards and evaluation procedures for feed (20 Shoan No. 597 dated May 19, 2008, By confirming the safety according to the "chicken chick growth test" by the Ministry of Agriculture, Forestry and Fisheries Consumer Affairs and Safety Bureau Notification), it was confirmed that the feed bacteriostat can be used in feed without any problems.
2) Test chicks The basic feed shown in Table 1 (rapeseed meal (trade name: rapeseed meal, manufactured by Nisshin OilliO Group Co., Ltd.) containing 20.00% by mass) was fed at 10 g per chick for 3 days. From the 4th day onwards, 8-day-old egg-laying hens (Julia Light) male chicks raised by feeding 3.5 g per day were selected and tested. bottom.
In addition, the basic feed used should meet the nutritional requirements for egg-laying young chicks in the Japanese Feeding Standards for Poultry (2011 edition) in terms of metabolic energy, general ingredients such as protein, various minerals, amino acids, etc. designed to
3) Setting test plots Formulated feed 1 shown in Table 1 (rapeseed meal (trade name: rapeseed meal, manufactured by Nisshin OilliO Group Co., Ltd.) 10.00% by mass and 10.00% by mass of bacteriostatic agent for feed ) and compound feed 2 (containing 20.00% by mass of bacteriostat for feed) were set.
The contents of metabolic energy, general components such as protein, various minerals, amino acids, etc. in compound feeds 1 and 2 satisfy the nutrient requirements for egg-laying young chicks in the Japanese Feeding Standards for Poultry (2011 edition). It was designed to
The content of each component in the vitamin B group premix used for the mixed feed was thiamine nitrate 2.0 g/kg, riboflavin 10.0 g/kg, pyridoxine hydrochloride 2.0 g/kg, and nicotinamide 2.0 g/kg. Calcium D-pantothenate 4.35 g/kg, Choline chloride 138.0 g/kg, Folic acid 1.0 g/kg, Cyanocobalamin 10 mg/kg.
The contents of each component in the vitamin ADE premix are 10000 IU/kg of vitamin A oil, 2000 IU/kg of vitamin _D3 oil and 20 mg/kg of dl-α-tocopherol acetate.
The contents of each component in the mineral premix are Mn 80 g/kg, Zn 50 g/kg, Fe 6 g/kg, I 1 g/kg and Cu 0.6 g/kg.

4) Establishment of Test Plots and Feeding Management The test chicks were divided into 6 groups with 6 chicks each so that the weight distribution was almost even.
The test chicks were reared in groups in electric heating type brooders, and fed with various mixed feeds and drinking water without interruption. In addition, the housing position of each group was changed every day to prevent the effects of environmental conditions.
5) Body weight gain, feed intake, and feed conversion rate of test chicks As reference data, the body weight of each test chick was measured at the start and end of the test, and the body weight gain during the test period (g/chicken ) was calculated.
In addition, as reference data, the feed intake was measured for each group during the test period, and the feed intake per bird (g/bird) and the feed conversion rate were calculated.
Table 2 shows the results of these measurements (average ± standard deviation for each of the three groups).

As a result of the feeding test, no abnormal health condition was observed in any of the individuals in both test groups. From this, it was confirmed that the compounded feed containing the bacteriostatic agent for feed can be used as a feed without any concern about adverse effects on the growth and health of the chicks.

<Confirmation test of bacteriostatic effect of rapeseed meal, high-protein rapeseed meal, or mixture thereof>
1) Rapeseed meal and high-protein rapeseed meal used Rapeseed meal and high-protein rapeseed meal are the same as the rapeseed meal and high-protein rapeseed meal used in the feeding test of the feed bacteriostatic agent-containing compound feed described above. It was used.
This high-protein rapeseed meal was used as a feed bacteriostatic agent to investigate its bacteriostatic effect on feed.
2) Test Samples Test samples A to E shown in Table 3 were prepared using rapeseed meal and high-protein rapeseed meal.
Here, test sample A is rapeseed meal (no feed bacteriostat added), test samples B to D are meals containing feed bacteriostat, and test sample E is feed bacteriostat. be.

3) Test microorganism Salmonella: Salmonella enteritidis L58 strain The above microorganism was pre-cultured in a nutrient medium, and the concentration was adjusted to about 10 ⁹ CFU/mL with sterilized purified water. liquid.
4) Setting of Sections Using test samples A to E, test sections A to E shown in Table 4 were prepared.

5) Test procedure The confirmation test of the bacteriostatic effect this time was carried out with reference to "JIS Z 2801 (Antibacterial processed product/Antibacterial test method/Bactericidal effect)" and the carbolic acid coefficient method.
Microbial inspection methods and test methods are shown below.
[Microorganism test method (measurement of bacterial count in test solution)]
The test sample was appropriately diluted with sterilized physiological saline and cultured on a standard agar medium (general viable cell count) and X-Sal selective media (Salmonella count). Cultivation was carried out under aerobic conditions at 35° C. for 24 to 48 hours, and colonies that developed after cultivation were counted to obtain the number of bacteria.
[Test method]
20 g of the test sample and control sample were placed in a sterile wide mouth glass bottle, 0.4 mL of the test bacteria solution was added and mixed well. Immediately after mixing and after reacting at 35° C. for a certain period of time according to the test setting, the number of remaining viable bacteria and the number of Salmonella bacteria were measured according to the microbiological test method.

6) Test results Table 5 shows the test results for the general viable cell count on the test start date, the 1st day, and the 3rd day of the test plots A to E, and Table 6 shows the test results for the Salmonella count.

Regarding the number of general viable bacteria, in test groups B, C, and D, which are vegetable meals containing bacteriostatic agents for feed, and test group E, which is bacteriostatic agents for feed, they tend to decrease from the first day, and on the third day ranged from 200 to 2700 CFU/g.
On the other hand, in the test group A, which is the meal with no added bacteriostatic agent for feed, it increased from the test start day to the 1st day, and decreased slightly on the 3rd day.
In addition, regarding the number of Salmonella bacteria, in test plots B, C, and D, which are vegetable meals containing bacteriostatic agents for feed, and test plot E, which is a bacteriostatic agent for feed, decreased from the first day, and test plots D, It became below the detection limit on the 1st day in E, and on the 3rd day in test plots B and C.
On the other hand, in the test group A, which is the meal with no added bacteriostatic agent for feed, it increased from the test start day to the 1st day, and decreased slightly on the 3rd day.
The degree of the reduction effect is as follows: test area D (feed bacteriostatic agent-containing vegetable meal), test area E (feed bacteriostatic agent) → test area C (feed bacteriostatic agent-containing vegetable meal) → test area B The order was (vegetable meal containing bacteriostatic agent for feed) → test group A (meal without bacteriostatic agent for feed).

Next, the test results for the general viable cell count and the Salmonella count on the test start date, the 1st day, the 2nd day, the 3rd day, the 5th day, and the 10th day of the test groups A and E are shown. 7.
The values on the test start date, the 1st day, and the 3rd day are the same as the values shown in Tables 5 and 6.

Regarding the number of general viable bacteria, in the test group A, which is the feed bacteriostatic agent-free meal, there was an increasing trend from the start of the test to the first day, and then it tended to decrease. A decrease was observed from the first day in test group E, which was a bacteriostatic agent for feed, and became below the detection limit on the 10th day.
In addition, regarding the number of Salmonella bacteria, in the test group A, which is the meal with no added bacteriostatic agent for feed, it tended to increase from the test start day to the first day, and then decreased. In test group E, which is a bacteriostatic agent for feed, it became below the detection limit from the first day.
In test group E, both the general viable cell count and Salmonella were below the detection limit after 10 days.

<Confirmation test of suppression of Salmonella colonization in broilers>
・Materials and methods 1) Rapeseed meal and high-protein rapeseed meal used I used the same as the meal.
This high-protein rapeseed meal was used as a feed bacteriostat to investigate the inhibition of Salmonella colonization in broilers.

2) Test Chicks Sixty-three 1-day-old broiler-only breed (UK chunky) male chicks were introduced, and sterilized control diets described later were fed ad libitum to raise them until they were 6 days old. At the end of rearing, the weight of all chicks was measured individually, and 40 individuals with similar weights were selected and used for the test. In addition, since no Salmonella was detected in the litter in the transport boxes used to introduce the chicks (qualitative), the chicks were considered negative for Salmonella.

3) Setting test plots As shown in Table 8, Group 1 and Group 1 where 1 x 10 ³ Salmonella per chick were administered by single gavage, and Group 1 where 1 x 10 ⁶ Salmonella per chick were administered by single gavage. 2, and within each group, a total of four groups were set: a control group fed with a control feed containing rapeseed meal and a test group fed with a test feed containing a feed bacteriostat.
At the end of rearing, the test chicks were divided into 4 groups of 10 chicks based on the body weight at the end of rearing so that the average body weight of each group was approximately equal. Each feed was fed ad libitum until day-old.

4) Mixing ratio of feed The mixing ratio of feed is as shown in Table 9, using the values listed in the Japanese Standard Feed Composition Table (2009 edition), chunky broiler nutritional ingredients 2014 starter (0 to 10 days old) Rapeseed meal was used for the control diet, and bacteriostat for feed was used for the test diet. Moreover, both the control feed and the test feed were sterilized by 20 kGy γ-ray irradiation after preparation.
The content of each component in the vitamin/mineral premix used in the mixed feed was thiamine nitrate 2.0 g/kg, riboflavin 4.5 g/kg, pyridoxine hydrochloride 2.0 g/kg, cyanocobalamin 10.0 mg/kg, Nicotinic acid 30.0 g/kg, D-calcium pantothenate 7.5 g/kg, d-biotin 75.0 mg/kg, folic acid 1.0 g/kg, vitamin A 6500000 IU/kg, vitamin D ₃ 2500000 IU/kg, acetic acid dl- α-tocopherol 40 mg/kg, vitamin K ₃ 3.836 g/kg, Mn 50 g/kg, Zn 50 g/kg, Fe 20 g/kg, Cu 7.5 g/kg, I 0.5 g/kg.

5) Administration of Salmonella At the start of the test (immediately before feed feeding on the day after sorting), in Group 1, a bacterial solution containing 1 × 10 ³ cells/0.5 mL of Salmonella Enteritidis (NBRC3313) was administered to the group. In 2, 0.5 mL of the bacterial solution containing 1×10 ⁶ cells/0.5 mL of the same was forcibly orally administered into the sac of each bird using a gastric tube. The bacterial solution was prepared on the day of administration.

6) Breeding management of test chicks The test chicks were reared in a negative isolator throughout the growing period and the test period, and were kept in groups of five chicks each using two rooms during the test period. As for drinking water, purified water was given ad libitum throughout the growing period and the test period. The lights were on all day.

・Survey items and methods 1) Body weight and feed intake of test chicks At the time of sorting, at the start of the test (7 days old), and at the end of the test, the weight of each individual test chick was measured, and at the start of the test. Feed intake at the end of the period was measured for each plot.

2) Collection of specimens [1] Bedding The bedding was collected from the shipping boxes at the time of chick introduction, and salmonella was qualitatively determined.
[2] Cloaca swabs Cloaca swabs of all test chicks were swabbed with cotton swabs (Seed Swab No. 1, Eiken) at the time of sorting before administration of Salmonella (6 days old, at the end of rearing) and the day before the end of the test (13 days old). A swab was performed and a Salmonella qualitative analysis was performed.
[3] Manure mixture The feces pan was cleaned in the afternoon two days before the end of the test (12 days old), and the fresh manure mixture excreted in the morning the day before the end of the test (13 days old) was randomly distributed to each plot. 10 points each were sampled and mixed, and the number of Salmonella bacteria was measured (quantified), and Salmonella was qualitatively determined.
[4] Cecal Contents On the test completion day, all the chicks tested were bled and sacrificed, and the cecal contents were collected from each individual, and the number of Salmonella bacteria was measured (quantitatively) and Salmonella was qualitatively determined.

3) Qualification of Salmonella In the litter and cloac swab, Salmonella was qualitatively determined by the following method.
25 g of the bedding was added to 225 mL of buffered peptone water and subjected to pre-enrichment culture (37° C., 24 hours). For cloaca swabs, 1 g of each sample was placed in 10 mL of Hana tetrathionate medium to prepare a sample stock solution.
After the sample stock solution was subjected to enrichment culture at 41.5°C for 20 hours, one platinum loop of the enrichment culture solution was streaked on an MLCB agar plate medium and cultured at 37°C for 24 hours. Next, the typical black colonies grown on the plate medium were picked, inoculated into TSI, SIM, and lysine decarboxylation media, cultured at 37°C for 24 hours, and the properties were confirmed. When it was obtained, it was confirmed that the Salmonella immune serum was used to agglutinate group O polyvalently.

4) Quantification and Qualification of Salmonella For feces and urine mixtures and cecal contents, the number of Salmonella was measured and qualitative by the following methods. After 1 g of each sample was diluted 10-fold with Hana tetrathionate medium, the mixture was thoroughly mixed to obtain a sample stock solution. Sterilized 0.1% peptone water was added to this sample undiluted solution to dilute stepwise by a common ratio of 10 to prepare diluted solutions up to 10 ⁵ times.
0.5 mL and 0.1 mL of each of the undiluted sample solution and each diluted solution was smeared on two MLCB agar plates, and cultured at 37° C. for 24 hours.
A typical black colony that appeared on each plate medium was measured, picked, inoculated into TSI and LIM media, cultured at 37°C for 24 hours, and the properties were confirmed. When this colony was recognized as Salmonella 2, the number of Salmonella group O polyvalently aggregated colonies was multiplied by the dilution factor to calculate the number of Salmonella bacteria per 1 g of each sample.
In addition, the sample undiluted solution was qualitatively tested for Salmonella by the method described in 3) above.

5) Health status of chicks The health status of the chicks was observed twice daily, in the morning and in the evening, and the breeding rate was calculated.

・Analysis of results The Salmonella count in the cecal contents was logarithmically transformed, then the average value was calculated for each section, and this was used as the infection coefficient (IF value) for each section. Then, the infection protection factor (PF value) was calculated. In addition, the salmonella positive rate (%) was calculated by dividing the number of chicks that were positive for salmonella by the total number of chicks tested.
The weight gain during the test period and the Salmonella count after logarithmic transformation were analyzed for each group by one-way analysis of variance, and the significance of the difference between the control group and the test group was examined at a risk rate of less than 5%.
In addition, the significance of the difference between the control group and the test group with a risk rate of less than 5% was examined by FISHER's direct probability calculation method for each group regarding the salmonella positive rate.

Test results 1) Breeding results Body weight, weight gain, feed intake, feed conversion rate, and rearing rate are shown in Tables 10 and 11, and both groups were the weight gain of the test group and the control No significant difference was observed between the weight gain of the group and no difference was observed between the control group and the test group in each of the other items. In addition, no abnormality was observed in the health condition of any individual in each group.

2) Salmonella Measurement Results Tables 12 and 13 show the measurement results of Salmonella.
In both the control group (rapeseed meal-containing feed) and the test group (feed bacteriostatic agent-containing feed) of Groups 1 and 2, Salmonella was not detected in the cloaca swabs before the start of the test, and the Salmonella positive rate was 0%. .
Moreover, in Group 1, Salmonella was not detected from the cloaca swab and the manure mixture on the day before the end of the test in both the control group (rapeseed meal-containing feed) and the test group (feed bacteriostatic agent-containing feed).
On the other hand, in Group 2, in the control group (rapeseed meal-containing feed), Salmonella was detected from one chicken in the cloac swab on the day before the end of the test, and the salmonella positive rate was 10%. ), no Salmonella was detected.
Salmonella was not detected in the contents of the cecum in Group 1 in both the control group (rapeseed meal-containing feed) and the test group (feed containing bacteriostatic agent for feed).
On the other hand, in Group 2, the number of Salmonella bacteria in the test group (feed containing bacteriostat for feed) was significantly (p<0.05) lower than that in the control group (rapeseed meal containing feed), and the positive rate of Salmonella tended to be low. was taken.
In addition, the IF value of test group 2 (feed containing bacteriostatic agent for feed) was calculated to be <0.10, and the PF value was calculated to be >7.4. Pivnik et al. reported that those with a PF value of less than 4.0 have little effect on Salmonella even when used outdoors. It was found that there was an effect of suppressing colonization of Salmonella when the mixed feed was given.
For reference, the literature information of Pivnik et al. mentioned above is described below.
Pivnick, H.; , D. Barnum, S.; Stavric, T.; Gleeson, andB. Blanchfield, 1985.
Investigations on the use of competitive exclusion to control Salmonella in poultry. Pages 80-87 in: Proceedings of the International Symposium on Salmonella. G. H. Snoeyenbos, ed. American Association of Avian Pathology, University of Pennsylvania, Philadelphia, PA.

As can be seen from the results of the confirmation test of the bacteriostatic effect of the high-protein rapeseed meal, the bacteriostatic agent for feed of the present invention can be mixed with vegetable meal (bacteriostatic agent-containing vegetable meal for feed). Even if the vegetable meal is unexpectedly contaminated with Salmonella, the proliferation of the contaminating Salmonella can be suppressed, thereby preventing contamination of the vegetable meal itself with Salmonella. Therefore, the feed bacteriostat of the present invention can be used to prevent contamination with Salmonella during storage of plant meals.
In addition, by using the bacteriostatic agent for feed or the vegetable meal containing the bacteriostatic agent for feed of the present invention as a raw material for feed, even if the feed is unexpectedly contaminated with Salmonella, the contaminated Salmonella can be prevented. As a result, the feed itself can be prevented from being contaminated with Salmonella. Therefore, the feed bacteriostat of the present invention can be used to prevent contamination with Salmonella during storage of feed.
Thus, by using the bacteriostatic agent for feed of the present invention in vegetable meal or feed, or by using the bacteriostatic agent-containing vegetable meal for feed in feed, salmonella contamination due to feed feeding to livestock can be prevented. Therefore, the onset of livestock Salmonella infection can be prevented in advance.
In addition, as can be seen from the results of the confirmation test for suppressing colonization of Salmonella in broiler bodies, even if Salmonella invades chicken bodies, the bacteriostatic agent for feed of the present invention or the bacteriostat for feed of the present invention can be used. Since the growth of Salmonella in the chicken body (cecum) can be suppressed by using the plant meal containing the agent as a raw material for feed, the bacteriostatic agent for feed or the static feed for feed of the present invention can be used. It can be said that the fungicide-containing vegetable meal can prevent livestock Salmonella infections.

Claims (9)

A bacteriostatic agent for feed containing a high-protein vegetable meal as an active ingredient, wherein the high-protein vegetable meal is mixed with a vegetable meal and water, and the resulting mixture is subjected to saccharification treatment and alcoholic fermentation treatment, Obtained by adding a fiber-degrading enzyme for the saccharification treatment, adding a microorganism for the alcoholic fermentation treatment, and using cellulase produced by a microorganism of the genus Acremonium as the fiber-degrading enzyme. A feed bacteriostatic agent characterized by: 2. The feed bacteriostat according to claim 1, wherein said vegetable meal is rapeseed meal. A plant meal containing a bacteriostat for feed, comprising the bacteriostat for feed according to claim 1 or 2 and a plant meal. A feed comprising the bacteriostat for feed according to claim 1 or 2 or the bacteriostat-containing plant meal for feed according to claim 3. A method of raising livestock by feeding the feed according to claim 4. A method for inhibiting colonization of Salmonella in livestock, using the feed bacteriostat according to claim 1 or 2 or the feed bacteriostat-containing vegetable meal according to claim 3. A method for storing plant meal, which comprises adding the feed bacteriostat according to claim 1 or 2 to the plant meal. A method for storing feed, which comprises adding the bacteriostat for feed according to claim 1 or 2 or the plant meal containing the bacteriostat for feed according to claim 3 to feed. A method for preventing livestock Salmonella infection, which comprises using the bacteriostatic agent for feed according to claim 1 or 2 or the bacteriostatic agent-containing vegetable meal for feed according to claim 3.

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