JP7737271B2 - Feed bacteriostatic agent - Google Patents

Description

The present invention relates to a bacteriostatic agent for feed and feed containing the same.

Salmonella infection is known as one of the livestock infectious diseases. In particular, poultry salmonella infection is mainly caused by Salmonella Enteritidis or Salmonella Typhimurium, which enter the intestines through the mouth, enter the bloodstream from the intestinal tract, and circulate through various organs. It often occurs in young chicks within one month of hatching, and many cases result in death within 10 days of hatching.
Feed contaminated with Salmonella is an important source of infection for poultry Salmonella infection, and even small amounts of bacteria can cause infection. For example, even a single bacterium in 15 g of feed can cause infection. For this reason, Salmonella in feed is strictly controlled. Therefore, techniques for inhibiting the growth of Salmonella in feed are being actively studied, and there is a particular need for techniques that can inhibit the growth of Salmonella in oil cake used in animal feed when it becomes wet due to rain or other factors and becomes contaminated with Salmonella.

When examining the prior art, it is known that for the prevention and treatment of Salmonella infection in livestock, feeding feed containing ingredients derived from the fermentation broth of specific lactic acid bacteria is effective in preventing the proliferation of Salmonella in the bodies of livestock (see Patent Document 1).
Furthermore, a technology has been developed that can suppress the growth of Salmonella in oil cake by adding lactic acid bacteria to the oil cake (see Patent Document 2).
However, in these methods, lactic acid bacteria are added to feed in the form of an aqueous solution of a lactic acid bacteria culture solution (fermentation broth). To maintain the hygienic quality of the feed during storage, the water content in the feed must be kept below a certain level, so the water from the lactic acid bacteria culture solution must be dried.

Republished WO00/30661 JP 2011-244704 A

The object of the present invention is to provide a bacteriostatic agent for feed that can stagnate Salmonella in feed or inhibit the establishment of Salmonella in livestock bodies without increasing the moisture content of the feed by adding a lactic acid bacteria culture solution or the like.

After extensive research, the inventors discovered that by using cellulase produced by microorganisms of the genus Acremonium as the fiber-degrading enzyme used in the saccharification of vegetable meal, the resulting high-protein vegetable meal contains components that inhibit the growth of Salmonella and can be used as a bacteriostatic agent for feed, leading to the completion of the present invention.

Specifically, the present invention includes the following aspects.
[1] A bacteriostatic agent for feed containing a high-protein vegetable meal as an active ingredient, characterized in that the high-protein vegetable meal is obtained by a method comprising mixing the vegetable meal with water, subjecting the resulting mixture to saccharification and alcoholic fermentation, adding a fibrous enzyme for the saccharification, adding a microorganism for the alcoholic fermentation, and using cellulase produced by Acremonium cellulolyticus as the fibrous enzyme.
[2] The bacteriostatic agent for feed according to [1], characterized in that the plant meal is rapeseed meal.
[3] A bacteriostatic agent-containing vegetable meal for feed, comprising the bacteriostatic agent for feed according to [1] or [2] and vegetable meal.
[4] A feed containing the bacteriostatic agent for feed according to [1] or [2], or the plant meal containing the bacteriostatic agent for feed according to [3].
[5] A method for raising livestock by feeding the feed according to [4].
[6] A method for inhibiting Salmonella colonization in livestock bodies, using the bacteriostatic agent for feed according to [1] or [2], or a plant meal containing the bacteriostatic agent for feed according to [3].
[7] A method for storing plant meal, comprising adding the bacteriostatic agent for feed according to [1] or [2] to the plant meal.
[8] A method for storing feed, comprising adding the bacteriostatic agent for feed according to [1] or [2], or the plant meal containing the bacteriostatic agent for feed according to [3], to the feed.
[9] A method for preventing Salmonella infection in livestock using the bacteriostatic agent for feed according to [1] or [2], or a plant meal containing the bacteriostatic agent for feed according to [3].

According to the present invention, a bacteriostatic agent for feed can be provided without increasing the moisture content in the feed by adding a lactic acid bacteria culture solution or the like, and by including the bacteriostatic agent in the feed, general viable bacteria and Salmonella present in the feed can be stagnated, and the number of these bacteria can be reduced over time.
Furthermore, according to the present invention, by feeding livestock feed containing a bacteriostatic agent for feed, it is possible to suppress the establishment of Salmonella in the bodies of livestock.
As a result, it contributes to the health of livestock and reduces the decline in livestock numbers due to death.

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

(High protein vegetable meal)
The high-protein vegetable meal of the present invention is a high-protein vegetable meal processed product obtained by subjecting vegetable meal to saccharification and alcohol fermentation, as described below, and contains 44 to 65 mass % of protein, and preferably 47 to 65 mass % of protein, calculated on a dry weight basis.
The protein content referred to here refers to the value obtained by multiplying the total nitrogen determined by the Kjeldahl method by 6.25.
As will be explained in detail below, this high-protein vegetable meal can be used as a bacteriostatic agent for feed as is.

(bacteriostatic agent for feed)
The feed bacteriostatic agent of the present invention is a feed bacteriostatic agent containing high-protein vegetable meal as an active ingredient, and the high-protein vegetable meal is obtained by a method of mixing vegetable meal with water, subjecting the resulting mixture to saccharification and alcoholic fermentation, adding a fibrous decomposing enzyme for the saccharification, adding a microorganism for the alcoholic fermentation, and using cellulase produced by a microorganism of the genus Acremonium as the fibrous decomposing enzyme.
The high-protein vegetable meal of the present invention and its manufacturing method are described in detail in the previously filed Japanese Patent Application No. 2021-028962, and therefore will not be described in detail here. The contents of the above application are incorporated herein by reference.

The bacteriostatic agent for feed of the present invention can be made by using the above-mentioned high-protein vegetable meal as it is as a bacteriostatic agent for feed.
By adding the bacteriostatic agent for feed of the present invention to feed, general viable bacteria and Salmonella present in the feed can be stagnated, and the number of these bacteria can be reduced over time.
Furthermore, by feeding livestock feed containing the bacteriostatic agent for feed of the present invention, it is possible to suppress the colonization of Salmonella in the bodies of livestock.
Here, examples of livestock to which feed containing the bacteriostatic agent for feed of the present invention is to be fed include poultry such as chickens and quails, as well as cattle and pigs, and among these, poultry is preferred.

(Plant 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 that contains the bacteriostatic agent for feed described above.
By adding the bacteriostatic agent for feed of the present invention to a plant-based meal to prepare a plant-based meal containing the bacteriostatic agent for feed, the storability of the plant-based meal can be improved from a hygienic standpoint. The plant-based meal containing the bacteriostatic agent for feed can be used as a raw material for feed.
The content of the bacteriostatic agent for feed in the bacteriostatic agent-containing plant meal for feed of the present invention is preferably 1 to 90% by mass, more preferably 1 to 50% by mass, even more preferably 1 to 25% by mass, even more preferably 1 to 10% by mass, and most preferably 1 to 5% by mass.

Next, the raw materials for the high-protein vegetable meal and the method for producing the same will be briefly explained.
(Plant-based meal)
The vegetable meal used in the present invention is not particularly limited, and any vegetable meal can be used, such as soybean meal, rapeseed meal, flaxseed meal, perilla meal, etc. In particular, it is preferable to use rapeseed meal as the vegetable meal.
Here, soybean meal refers to soybean meal obtained by extracting soybean seeds using an organic solvent such as n-hexane through an oil extraction process, followed by evaporation of the organic solvent, and contains approximately 8 to 15% by mass of water.
Rapeseed meal is a rapeseed meal obtained by extracting rapeseed seeds using a press, then undergoing an oil expression process in which the oil remaining in the press cake is extracted using an organic solvent such as n-hexane, and then evaporating the organic solvent, and contains approximately 8 to 15% by mass of moisture.

(Water and other additives)
The water used in the present invention is not particularly limited and may be, for example, distilled water, pure water, or tap water. When mixing plant-based meal with water before saccharification or alcoholic fermentation, the plant-based meal and water may be mixed in a ratio of 40 to 60 parts by weight of plant-based meal to 60 to 40 parts by weight of water, preferably 45 to 55 parts by weight of plant-based meal to 55 to 45 parts by weight of water, and more preferably 48 to 52 parts by weight of plant-based meal to 52 to 48 parts by weight of water. This water content is preferable because it allows for solid-state fermentation. Here, "solid-state fermentation" refers to a fermentation method in which the water content is set lower than in liquid fermentation, and in which almost no waste liquid is discharged not only during fermentation but also after fermentation is completed.

Additives such as minerals, sugars, amino acids, culture medium components, enzymes, and microorganisms may be added in an amount of 0 to 10 parts by weight per 100 parts by weight of a mixture of plant meal and water before saccharification and alcohol fermentation. Specific examples of minerals include phosphorus, magnesium, sodium, potassium, and iron. Specific examples of sugars include sucrose, glucose, maltose, and oligosaccharides. Specific examples of amino acids include glutamic acid and lysine, and may also be protein-constituting amino acids, non-constituting amino acids such as GABA, and protein hydrolysates. Specific examples of culture medium components include yeast extract, malt extract, and peptone.
Specific examples of enzymes for saccharification include cellulases, hemicellulases, glucosidases, and mixtures thereof. In the saccharification process of the present invention, cellulases produced by microorganisms of the genus Acremonium are used as one type of cellulase (fibrous decomposition enzyme), particularly cellulase F produced by Acremonium cellulolyticus (product name: Meiji Acremonium Cellulase F, manufactured by Meiji Seika Pharma Co., Ltd.) and cellulase KM produced by Acremonium cellulolyticus (product name: Acremonium Cellulase KM, manufactured by Kyowa Kasei Co., Ltd.). In the present invention, it is preferable to use cellulases produced by Acremonium cellulolyticus . As described below, it was discovered for the first time in the present invention that ethanol production ability can be improved by using cellulases produced by these microorganisms of the genus Acremonium.
Specific examples of microorganisms for alcoholic fermentation treatment include filamentous fungi, yeasts such as Saccharomyces cerevisiae, Shizosaccharomyces pombe and Pichia stipitis, Zymomonas mobilis, etc. For the alcoholic fermentation treatment of the present invention, it is preferable to use yeasts such as Saccharomyces cerevisiae, and particularly yeasts that can be used in solid fermentation methods are preferred.
Hereinafter, water or water and these additives will be collectively referred to as "water, etc." Among these additives, water-soluble ones may be solubilized in the water and added.

(mixture)
In the present invention, "mixing of vegetable meal with water, etc.," refers to mixing of vegetable meal with water, etc., manually or by device. A more uniform distribution of water in the solids after mixing is desirable because it improves the efficiency of the saccharification or alcoholic fermentation process, but the purpose of saccharification or alcoholic fermentation can be achieved even if the water distribution in the solids is uneven. It has generally been considered desirable to sterilize a mixture of vegetable meal and water, etc., in an autoclave during saccharification or alcoholic fermentation to prevent the growth of microorganisms other than the desired microorganisms. However, it has been found that the present invention makes it possible to omit the sterilization process prior to saccharification or alcoholic fermentation.

(Saccharification treatment)
The saccharification process in the present invention refers to the decomposition of fiber (e.g., cellulose) contained in vegetable meal into glucose by adding a fibrous enzyme to a mixture of vegetable meal and water at a concentration of 0.01 to 1.0 wt %. The fibrous enzyme used in the saccharification process can be cellulase alone or cellulase in combination with enzymes such as hemicellulase or glucosidase. However, in the present invention, it is essential to use cellulase produced by a microorganism belonging to the genus Acremonium. The cellulase is preferably added in an amount of 0.01 to 1.0 wt % based on the mixture of vegetable meal and water. An addition of 0.05 to 1.0 wt % is more preferred, and an addition of 0.1 to 1.0 wt % is even more preferred. The enzyme used in the saccharification process can be any enzyme capable of decomposing fiber, and may be a commercially available product, a culture medium obtained by culturing a filamentous fungus or a further purified version thereof, or the filamentous fungus itself.

(Alcoholic fermentation treatment)
The alcoholic fermentation treatment in the present invention refers to a process in which a microorganism is inoculated into a mixture of vegetable meal and water, or a saccharified mixture of vegetable meal and water, and the microorganism produces ethanol. As for the microorganisms used, yeasts such as Saccharomyces cerevisiae, Shizosaccharomyces pombe, and Pichia stipitis can be used. Other than yeasts, any microorganism capable of alcoholic fermentation, including genetically modified ones, can be used, such as Zymomonas mobilis, a bacterium capable of alcoholic fermentation. Microorganisms preserved in a slant or frozen state can be used, but when using Saccharomyces cerevisiae, commercially available baker's yeast can also be used. When using microorganisms preserved in a slant or frozen state, it is preferable to pre-culture them in a liquid medium before use and use the pre-culture solution. The liquid medium used for pre-culture may be any medium suitable for culturing yeast or bacteria, such as one containing 1% by mass of yeast extract, 2% by mass of peptone, and 3% by mass of glucose.
In the present invention, yeast such as Saccharomyces cerevisiae is preferably used as the microorganism for alcoholic fermentation, and yeast that can be used in solid-state fermentation is particularly preferred. The amount of yeast used is preferably a yeast suspension prepared so that the absorbance (hereinafter referred to as OD) at 660 nm is 0.2, and added so that the amount of yeast present in the total reaction system (yeast (suspension) + meal + added water) is 0.05 to 0.15. It is more preferred to add the yeast so that the OD is 0.05 to 0.1.

(When done in the same process or in separate processes)
When saccharification and alcoholic fermentation are carried out in the same process (referred to as "parallel multiple fermentation") by adding enzymes and microorganisms to a mixture of vegetable meal and water, the treatment temperature is preferably 25 to 45°C, more preferably 27 to 43°C, and even more preferably 30 to 40°C. When saccharification and fermentation are carried out in separate processes, 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, and even more preferably 25 to 35°C.
The total treatment time is preferably 4 to 336 hours, more preferably 72 to 336 hours, and even more preferably 168 to 336 hours.

(After solid-liquid separation, drying and distillation)
The mixture of vegetable meal and water undergoes saccharification and alcoholic fermentation, followed by solid-liquid separation as needed, and then drying. The resulting product is called high-protein vegetable meal. Drying can be performed by heat drying, vacuum drying, freeze drying, spray drying, or any other method that can evaporate the water and ethanol from the mixture that has been saccharified and/or alcoholic fermented. If ethanol is recovered by steam distillation or the like before drying, the recovered ethanol can be used for industrial purposes or as fuel.
In the present invention, the amount of ethanol produced per 100 g of dry weight of rapeseed meal is preferably 3.5 g or more, more preferably 3.8 g or more, and even more preferably 4.0 g or more.
The saccharification treatment and alcoholic fermentation treatment are preferably carried out by solid fermentation, which is preferable because it does not require the above-mentioned solid-liquid separation and does not produce waste liquid.

(Production of plant meal containing bacteriostatic agent for feed)
Next, a method for producing a plant meal containing a bacteriostatic agent for feed will be described.
A feed containing the plant meal containing the bacteriostatic agent 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 plant meal.
The mixing can be carried out using a mixer such as a ribbon mixer, a V-type mixer, or a W-type mixer.

Next, a feed containing a bacteriostatic agent for feed and a feed containing a plant meal containing a bacteriostatic agent for feed will be described.
(feed, livestock breeding)
The feed of the present invention is a feed containing the above-mentioned bacteriostatic agent for feed, or a feed containing the above-mentioned bacteriostatic agent-containing vegetable meal for feed.
The feed bacteriostatic agent of the present invention or the plant meal containing the feed bacteriostatic agent of the present invention can be used by mixing with feed ingredients or feed additives containing sugars, proteins, amino acids, fiber, minerals, oils, antibacterial components, etc.
Specific examples of feed ingredients and feed additives include corn, sorghum, corn gluten, corn starch, rice bran, soybean meal, ordinary rapeseed meal, bran, oats, milk casein, whey, fish meal, various vitamins, vitamin mixes, mineral mixes, amino acid preparations, calcium carbonate, monocalcium phosphate, vegetable oils and fats, animal oils and fats, and salt.
The content of the bacteriostatic agent for feed in the feed is preferably 1 to 20% by mass, more preferably 1 to 15% by mass, and even more preferably 1 to 10% by mass, when the total amount of the feed is taken as 100% by mass.
The bacteriostatic agent-containing vegetable meal for feed is preferably blended into the feed in an amount such that the content of the bacteriostatic agent in the bacteriostatic agent-containing vegetable meal for feed is the above-mentioned content.
For example, in the case of a feed bacteriostatic-agent-containing vegetable meal containing 25% by mass of the feed bacteriostatic agent, the content of the feed bacteriostatic-agent-containing vegetable meal in the feed is preferably 4 to 80% by mass, more preferably 4 to 60% by mass, and even more preferably 4 to 40% by mass, when the total amount of the feed is 100% by mass.

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 raw materials and feed additives described above.
Furthermore, feed containing the plant-based meal containing the bacteriostatic agent for feed of the present invention can be produced by mixing the plant-based meal containing the bacteriostatic agent for feed with the various feed ingredients and feed additives described above.
These ingredients can be mixed using a mixer such as a ribbon mixer, a V-type mixer, or a W-type mixer.
Feed using the feed bacteriostatic agent produced by the production method of the present invention, or plant meal containing the feed bacteriostatic agent, does not have any adverse effects on the growth or health of chicks and can be used as feed without any problems.
Furthermore, by feeding livestock feed using the bacteriostatic agent for feed of the present invention or plant meal containing the bacteriostatic agent for feed, it is possible to suppress Salmonella colonization in the bodies of livestock and raise the livestock.

(How to store plant-based meals)
By adding the feed bacteriostatic agent of the present invention to plant-based meal, the growth of Salmonella contaminating the plant-based meal can be suppressed, and therefore the feed bacteriostatic agent of the present invention can be used to prevent contamination with Salmonella during storage of plant-based meal.
The content of the bacteriostatic agent for feed of the present invention used to prevent contamination with Salmonella in the vegetable meal is as described above.

(Feed storage method)
The addition of the bacteriostatic agent for feed or the plant-based meal containing the bacteriostatic agent for feed of the present invention to feed can suppress the growth of Salmonella contaminating the feed. Therefore, the bacteriostatic agent for feed or the plant-based meal containing the bacteriostatic agent 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 of the present invention or the plant meal containing the bacteriostatic agent for feed in feed, which is used to prevent contamination with Salmonella, is as described above.

(Methods for preventing livestock Salmonella infection)
By using the bacteriostatic agent for feed or the plant meal containing the bacteriostatic agent for feed of the present invention as a raw material for feed, Salmonella infection in livestock can be prevented.
As will be described in detail in the Examples, by using the bacteriostatic agent for feed of the present invention in plant-based meal or feed, or by using plant-based meal containing the bacteriostatic agent for feed in feed, Salmonella contamination due to feeding to livestock can be prevented, and the onset of Salmonella infection in livestock can be prevented in advance.
Furthermore, by using the feed bacteriostatic agent or plant-based meal containing the feed bacteriostatic agent of the present invention as a raw material for feed and feeding it to chickens, the growth of Salmonella inside the body (in the cecum) can be suppressed, so it can be said that the feed bacteriostatic agent or plant-based meal containing the feed bacteriostatic agent of the present invention can prevent Salmonella infection in livestock.
The content of the bacteriostatic agent for feed or the plant meal containing the bacteriostatic agent for feed of the present invention used to prevent Salmonella infection in livestock is as described above.

The following examples are provided to explain the present invention in more detail, but the present invention is not limited to these examples.

<Feeding test of compound feed containing bacteriostatic agent for feed>
1) Rapeseed meal and high-protein rapeseed meal used The rapeseed meal used was "rapeseed oil cake" (protein content: 44.8% (dry matter equivalent), moisture content: 13.3% by mass) manufactured by Nisshin Oillio Group, Ltd.
The high-protein rapeseed meal was produced as follows.
4000 g of rapeseed meal was added to 4000 g of water to prepare 8000 g of a mixture of rapeseed meal and water. Without autoclave sterilization, a yeast (Saccharomyces cerevisiae A30) suspension was added so that the total reaction system had an OD of 0.1. Furthermore, 0.1% by mass of cellulase F (product name: Meiji Acremonium Cellulase F, manufactured by Meiji Seika Pharma Co., Ltd.) was added, and with the container lid closed, solid fermentation was carried out at 35 ° C for 13 days to produce high-protein rapeseed meal. An 8-liter plastic case was used as the container.
The obtained high-protein rapeseed meal was subjected to a reduced-pressure heating treatment (72°C, -0.1 MPa) for 8 hours. The dried high-protein rapeseed meal was then pulverized to a size suitable for use as a feed ingredient using a pulverizer (Osaka Chemical Co., Ltd., "Absolute mill ABS-W") to produce a dried high-protein rapeseed meal (protein content: 49.4% (dry matter equivalent), moisture content: 9.0% by mass).
The obtained high-protein rapeseed meal was used as it was as a bacteriostatic agent for feed.
Furthermore, the rapeseed meal and the high-protein rapeseed meal (bacteriostatic agent for feed) used were examined for the presence of Salmonella using the enrichment culture method described below, but the results for both were negative/25g.

(Enrichment culture method)
Twenty-five grams of the sample to be analyzed was weighed and placed in buffered peptone water. After shaking, the mixture was incubated at 36±1°C for 18 to 24 hours. Next, 1 ml and 0.1 ml of the pre-enrichment culture were added to Hahna-Tetrathionate and Rappaport-Vassiliadis media, respectively. After shaking, the cultures were subjected to selective enrichment incubation at 42±1°C for 18 to 24 hours. A loopful of each selective enrichment culture was streaked onto DHL agar and BG (Brilliant Green agar) plates, inverted, and incubated at 36±1°C for 18 to 24 hours. Approximately three colonies suspected to be Salmonella were picked from each of the DHL agar and Brilliant Green agar plates and subjected to confirmation testing using LIM and TSI media. If typical characteristics were observed, the presence of Group O sera was confirmed by glass agglutination. If an agglutination reaction was observed, it was judged as positive (positive/25g), and if not, it was judged as negative (negative/25g).

The safety of compound feed containing the obtained feed bacteriostatic agent (high protein rapeseed meal) was confirmed in accordance with the "Chicken Growth Test" in the "Establishment of Feed Safety Evaluation Standards and Evaluation Procedures (Notice No. 20 Shoan-597 dated May 19, 2008, Director-General of the Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries)," and it was confirmed that the feed bacteriostatic agent can be used in feed without any problems.
2) Test Chicks Individuals weighing 42 to 46 g were selected from 8-day-old laying hens (Julia Light) male chicks raised on the basic feed shown in Table 1 (containing 20.00% by mass of rapeseed meal (product name: rapeseed oil cake, manufactured by Nisshin Oillio Group, Ltd.)) at a rate of 10 g per chick for three days at feeding time, and from the fourth day onwards, 3.5 g per chick per day.
The basic feed used was designed so that the contents of metabolic energy, general components such as protein, various minerals, amino acids, etc., would satisfy the nutrient requirements of egg-laying chicks as specified in the Japanese Feeding Standards for Poultry (2011 edition).
3) Setting up of test plots Two test plots were set up, each fed with compound feed 1 (containing 10.00% by mass of rapeseed meal (trade name: rapeseed oil cake, manufactured by Nisshin Oillio Group, Ltd.) and 10.00% by mass of a feed bacteriostatic agent) and compound feed 2 (containing 20.00% by mass of a feed bacteriostatic agent) shown in Table 1.
Formula feeds 1 and 2 were designed so that the contents of metabolic energy, general components such as protein, various minerals, amino acids, etc., would satisfy the nutrient requirements of egg-laying chicks in the Japanese Feeding Standards for Poultry (2011 edition).
The contents of each component in the B vitamin premix used in the compound feed were thiamine nitrate 2.0 g/kg, riboflavin 10.0 g/kg, pyridoxine hydrochloride 2.0 g/kg, nicotinamide 2.0 g/kg, calcium D-pantothenate 4.35 g/kg, choline chloride 138.0 g/kg, folic acid 1.0 g/kg, and cyanocobalamin 10 mg/kg.
The contents of each component in the Vitamin ADE Premix are: Vitamin A oil 10,000 IU/kg, Vitamin _D3 oil 2,000 IU/kg, and dl-α-tocopherol acetate 20 mg/kg.
The contents of each component in the mineral premix were 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 rearing management The test chicks were divided into six groups of six birds each so that the weight distribution was approximately equal, and three replicate groups were allocated to each plot and reared for six days.
The chicks were reared in groups in electrically heated brooders, and were provided with various formulated feeds and drinking water ad libitum. To prevent the effects of environmental conditions, the housing locations of each group were moved daily.
5) Weight gain, feed intake and feed conversion rate of test chicks As reference data, the weight of each test chick was measured at the start and end of the test, and the weight gain (g/bird) during the test period was calculated.
As reference data, the feed intake during the test period was measured for each group, and the feed intake per bird (g/bird) and feed conversion ratio were calculated.
The results of these measurements (mean values ± standard deviations for each of the three groups) are shown in Table 2.

As a result of the feeding test, no abnormalities were observed in the health of any of the individuals in either test group. This confirmed that there is no concern that compound feed containing the feed bacteriostatic agent will have a negative impact on the growth or health of chicks, and that it can be used as feed without any problems.

<Confirmation test of the bacteriostatic effect of rapeseed meal, high-protein rapeseed meal, or a mixture thereof>
1) Rapeseed meal and high-protein rapeseed meal used The rapeseed meal and high-protein rapeseed meal used were the same as those used in the feeding test of the compound feed containing the bacteriostatic agent for feed described above.
This high-protein rapeseed meal was used as a bacteriostatic agent for feed to examine 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 (without the addition of a feed bacteriostatic agent), test samples B to D are meals containing a feed bacteriostatic agent, and test sample E is a feed bacteriostatic agent.

3) Test Microorganisms Salmonella: Salmonella Enteritidis L58 strain The above microorganism was pre-cultured in a nutrient medium and adjusted to a concentration of about 10 ⁹ CFU/mL with sterilized purified water to prepare a test bacterial solution.
4) Setting of plots Test plots A to E shown in Table 4 were prepared using test samples A to E.

5) Test procedure The test to confirm the bacteriostatic effect was carried out with reference to "JIS Z 2801 (antibacterial processed products, antibacterial test methods, bactericidal effect)" and the carbolic acid coefficient method.
The microbiological testing methods and test methods are as follows:
[Microbial testing method (measurement of bacterial count in test solution)]
The test samples were diluted appropriately with sterile saline and cultured on standard agar medium (general viable cell count) and X-Sal selective medium (Salmonella count). The culture was carried out under aerobic conditions at 35°C for 24 to 48 hours, and the number of colonies that grew after the culture was counted to obtain the bacterial count.
[Test method]
20 g of the test sample and control sample were placed in a sterilized wide-mouth glass bottle, and 0.4 mL of the test bacteria solution was added and mixed well. Immediately after mixing and after reacting for a certain period of time at 35°C, the remaining viable bacterial count and Salmonella count were measured according to the microbiological testing method.

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

Regarding the general viable bacterial count, in test plots B, C, and D, which were plant-based meal containing a bacteriostatic agent for feed, and test plot E, which was a bacteriostatic agent for feed, the count began to decrease from the first day, and by the third day it was in the range of 200 to 2700 CFU/g.
On the other hand, in test group A, which was a meal containing no bacteriostatic agent added to the feed, the number of serotonin levels increased from the start of the test until the first day, and then decreased slightly on the third day.
Furthermore, the Salmonella counts in test plots B, C, and D, which were plant-based meal containing a bacteriostatic agent for feed, and in test plot E, which was a bacteriostatic agent for feed, began to decrease from the first day, and were below the detection limit on the first day in test plots D and E, and on the third day in test plots B and C.
On the other hand, in test group A, which was a meal containing no bacteriostatic agent added to the feed, the number of serotonin levels increased from the start of the test until the first day, and then decreased slightly on the third day.
The degree of reduction effect was in the following order: test group D (vegetable meal containing bacteriostatic agent for feed), test group E (bacteriostatic agent for feed) → test group C (vegetable meal containing bacteriostatic agent for feed) → test group B (vegetable meal containing bacteriostatic agent for feed) → test group A (meal without bacteriostatic agent for feed).

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

Regarding the general viable bacterial count, in test group A, which was the meal without feed bacteriostatic agent, the count tended to increase from the start of the test until the first day, and then began to decrease. In test group E, which was the feed bacteriostatic agent, the count decreased from the first day, and was below the detection limit by the 10th day.
Furthermore, in test group A, which was fed meal without feed bacteriostatic agent, the Salmonella count tended to increase from the start of the test until the first day, and then began to decrease. In test group E, which was fed meal with a feed bacteriostatic agent, the count was below the detection limit from the first day.
In test group E, after 10 days, both the general viable cell count and the Salmonella count were below the detection limit.

<Confirmation test for suppression of Salmonella colonization in broiler chickens>
Materials and Methods 1) Rapeseed meal and high-protein rapeseed meal used The rapeseed meal and high-protein rapeseed meal used were the same as those used in the feeding test of the compound feed containing the bacteriostatic agent for feed described above.
This high-protein rapeseed meal was used as a bacteriostatic agent in feed to examine its effect on suppressing Salmonella colonization in broiler chickens.

2) Test chicks Sixty-three one-day-old male broiler chicks (UK Chunky) were introduced and raised to six days of age while being fed ad libitum with the sterilized control feed described below. At the end of rearing, the weight of each chick was measured individually, and 40 chicks with similar weights were selected for use in the test. Furthermore, since no Salmonella was detected (qualitative) in the bedding in the transport box used when the test chicks were introduced, the test chicks were considered to be Salmonella negative.

3) Setting of test groups As shown in Table 8, Group 1 was set up to receive a single forced oral administration of 1 x ^{10 Salmonella} per test chick, and Group 2 was set up to receive a single forced oral administration of 1 x 10 Salmonella per test chick. Within each group, there was set up a control group fed with a control feed containing rapeseed meal, and a test group fed with a test feed containing a feed bacteriostatic agent, for a total of four groups.
At the end of rearing, the test chicks were divided into four groups of 10 chicks each so that the average weight of each group was approximately equal based on their weight at the end of rearing, and one group was assigned to each section.They were fed each feed ad libitum from 7 days to 14 days of age.

4) Feed composition The feed composition is as shown in Table 9. Using the values listed in the Japanese Standard Feed Composition Table (2009 edition), the feed composition was designed to satisfy the feed ingredients for starters (0-10 days old) of Nutritional Composition for Chunky Broilers 2014. Rapeseed meal was used in the control feed, and a feed bacteriostatic agent was used in the test feed. After preparation, both the control feed and the test feed were sterilized by gamma irradiation at 20 kGy.
The contents of each component in the vitamin and mineral premix used in the compound feed were as follows: 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 6,500,000 IU/kg, vitamin D ₃ 2,500,000 IU/kg, dl-α-tocopherol acetate 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, and I 0.5 g/kg.

5) Administration of Salmonella At the start of the test (just before feeding on the day after division), a bacterial solution containing 1 x ¹⁰³ Salmonella Enteritidis (NBRC3313) per ^0.5 mL was administered to each chicken by force into the crop using a stomach tube. The bacterial solution was prepared on the day of administration.

6) Rearing management of test chicks The test chicks were reared in negative isolators throughout the rearing and test periods, and were reared in groups of five chicks in two rooms during the test period. Purified water was available ad libitum throughout the rearing and test periods. The lights were on all day.

- Survey items and methods 1) Body weight and feed intake of test chicks The body weight of each test chick was measured at the time of division, at the start of the test (7 days old), and at the end of the test, and the feed intake from the start to the end of the test was measured for each group.

2) Sample collection [1] Litter Litter was collected from the transport box when the chicks were introduced and analyzed for Salmonella.
[2] Cloaca swabs 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), the cloaca of all test chicks was swabbed with a cotton swab (Seed Swab No. 1, Eiken) and Salmonella was qualitatively analyzed.
[3] Feces and urine mixture The feces trays were cleaned in the afternoon of the day before the end of the test (12 days old), and fresh feces and urine mixture excreted in the morning of the day before the end of the test (13 days old) was randomly collected from each group, 10 samples were mixed, and the Salmonella count was measured (quantified) and the Salmonella was qualitatively analyzed.
[4] Cecal Contents On the final day of the test, all test chicks were bled to death, and the cecal contents were collected from each chick to measure (quantitate) the number of Salmonella bacteria and also to qualitatively analyze Salmonella.

3) Qualitative analysis of Salmonella Qualitative analysis of Salmonella was performed on the litter and cloacal swabs using the following method.
25 g of the bedding was placed in 225 mL of buffered peptone water and pre-enriched (37°C, 24 hours), after which 1 mL was added to 10 mL of Hahna tetrathionate medium to prepare the sample stock solution. 1 g of each cloacal swab specimen was placed in 10 mL of Hahna tetrathionate medium to prepare the sample stock solution.
The sample stock solution was cultured at 41.5°C for 20 hours, and then a loopful of the culture was streaked onto an MLCB agar plate and cultured at 37°C for 24 hours. Typical black colonies grown on the plate were then picked and inoculated onto TSI, SIM, and lysine decarboxylation media, which were cultured at 37°C for 24 hours to confirm their properties. If the colonies were identified as Salmonella, they were confirmed to agglutinate to group O polyvalently using Salmonella immune serum.

4) Quantification and Qualitative Analysis of Salmonella The feces/urine mixture and cecal contents were subjected to the following method for determining the Salmonella count and qualitative analysis. One gram of each sample was diluted 10-fold with Hahna tetrathionate medium and thoroughly mixed to prepare a stock sample solution. This stock sample solution was serially diluted with sterilized 0.1% peptone water at a common ratio of 10 to prepare dilutions up to ^10.5 .
0.5 mL and 0.1 mL of the sample stock solution and each diluted solution were smeared onto two MLCB agar plates, respectively, and cultured at 37°C for 24 hours.
The typical black colonies that appeared on each plate medium were counted, picked up, and inoculated onto TSI and LIM media. They were then cultured at 37°C for 24 hours to confirm their properties. If the colonies were confirmed to be Salmonella, the number of colonies that agglutinated to the Salmonella O group polyvalent antibody using Salmonella immune serum was multiplied by the dilution factor to calculate the number of Salmonella bacteria per 1 g of each sample.
Furthermore, the sample stock solution was subjected to qualitative analysis of Salmonella by the method described in 3) above.

5) Health of the chicks The health of the chicks was observed twice a day, in the morning and in the evening, and the rearing rate was calculated.

Analysis of results The Salmonella counts in the cecal contents were logarithmically transformed and the average value was calculated for each group. This was used as the infection factor (IF value) for each group, and the protection factor (PF value) was calculated by dividing the IF value for the control group by the IF value for the test group. In addition, the Salmonella positivity rate (%) was calculated for each group by dividing the number of chicks that tested positive for Salmonella by the total number of chicks tested.
Analysis of variance was performed for each group using one-way analysis of variance for the weight gain during the test period and the logarithmically transformed Salmonella count, and the significance of the difference between the control and test groups was examined at a risk level of less than 5%.
Furthermore, the significance of the difference in the Salmonella positivity rate between the control and test groups was examined for each group using FISHER's direct probability calculation method at a risk level of less than 5%.

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

2) Salmonella Measurement Results The Salmonella measurement results are shown in Tables 12 and 13.
In both the control group (rapeseed meal feed) and the test group (feed bacteriostatic agent feed) in Groups 1 and 2, no Salmonella was detected in cloacal swabs taken before the start of the test, and the Salmonella positive rate was 0%.
In addition, in Group 1, no Salmonella was detected in the cloacal swabs or feces/urine mixtures taken on the day before the end of the test in either the control group (rapeseed meal mixed feed) or the test group (feed bacteriostatic agent mixed feed).
On the other hand, in Group 2, Salmonella was detected in one bird in the control group (feed containing rapeseed meal) using a cloacal swab taken the day before the end of the test, giving a Salmonella positivity rate of 10%, but no Salmonella was detected in the test group (feed containing bacteriostatic agent).
In Group 1, no Salmonella was detected in the cecal contents of either the control group (rapeseed meal formulated feed) or the test group (bacteriostatic agent formulated feed).
On the other hand, in Group 2, the number of Salmonella bacteria in the test group (feed containing bacteriostatic agent) was significantly (p<0.05) lower than that in the control group (feed containing rapeseed meal), and the Salmonella positive rate also tended to be lower.
Furthermore, the IF value for test group 2 (feed bacteriostatic agent-containing feed) was calculated to be <0.10, and the PF value was calculated to be >7.4. Pivnik et al. reported that feed with a PF value of less than 4.0 is not very effective against Salmonella even when used outdoors, but the results in this study were 4.0 or higher, demonstrating that feeding feed containing a feed bacteriostatic agent was effective in suppressing Salmonella colonization.
For reference, the literature information of Pivnik et al. mentioned above is listed below.
Pivnick, H. ,D. Barnum, S. Stavric, T. Gleeson, and B. 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 for the bacteriostatic effect of the high-protein rapeseed meal, by mixing the bacteriostatic agent for feed of the present invention with plant-based meal (plant-based meal containing bacteriostatic agent for feed), even if the plant-based meal is unexpectedly contaminated with Salmonella, the growth of the contaminated Salmonella can be suppressed, thereby preventing contamination of the plant-based meal itself with Salmonella. Therefore, the bacteriostatic agent for feed of the present invention can be used to prevent contamination of plant-based meal with Salmonella during storage.
Furthermore, by using the bacteriostatic agent for feed or the plant meal containing the bacteriostatic agent for feed of the present invention as a raw material for feed, even if Salmonella is unexpectedly mixed into the feed, the growth of the mixed Salmonella can be suppressed, thereby preventing contamination of the feed itself with Salmonella. Therefore, the bacteriostatic agent for feed of the present invention can be used to prevent contamination with Salmonella during storage of feed.
In this way, by using the feed bacteriostatic agent of the present invention in plant-based meal or feed, or by using plant-based meal containing the feed bacteriostatic agent in feed, Salmonella contamination due to feeding to livestock can be prevented, and the onset of livestock Salmonella infection can be prevented in advance.
Furthermore, as can be seen from the results of the above-mentioned confirmation test for the suppression of Salmonella colonization in broilers, even if Salmonella does invade the chicken body, the growth of Salmonella in the chicken body (in the cecum) can be suppressed by using the feed bacteriostatic agent of the present invention or the plant-based meal containing the feed bacteriostatic agent as an ingredient in feed and feeding it to the chicken. Therefore, it can be said that the feed bacteriostatic agent or the plant-based meal containing the feed bacteriostatic agent of the present invention can prevent livestock Salmonella infection.

Claims (9)

A bacteriostatic agent for feed containing high-protein vegetable meal as an active ingredient, characterized in that the high-protein vegetable meal is obtained by a method of mixing vegetable meal with water, subjecting the resulting mixture to saccharification and alcoholic fermentation, adding a fibrous enzyme for the saccharification, adding a microorganism for the alcoholic fermentation, and using cellulase produced by Acremonium cellulolyticus as the fibrous enzyme . The bacteriostatic agent for feed according to claim 1, characterized in that the plant meal is rapeseed meal. A bacteriostatic agent-containing vegetable meal for feed, comprising the bacteriostatic agent for feed described in claim 1 or 2 and vegetable meal. A feed containing the bacteriostatic agent for feed according to claim 1 or 2, or the plant meal containing the bacteriostatic agent for feed according to claim 3. A method for raising livestock by feeding the feed described in claim 4. A method for inhibiting Salmonella colonization in livestock using the feed bacteriostatic agent described in claim 1 or 2, or a plant meal containing the feed bacteriostatic agent described in claim 3. A method for storing plant-based meal, comprising adding the bacteriostatic agent for feed described in claim 1 or 2 to the plant-based meal. A method for storing feed, comprising adding the feed bacteriostatic agent described in claim 1 or 2, or the plant meal containing the feed bacteriostatic agent described in claim 3, to the feed. A method for preventing Salmonella infection in livestock, comprising using the bacteriostatic agent for feed described in claim 1 or 2, or the plant meal containing the bacteriostatic agent for feed described in claim 3.