WO2019040266A1 - Agents microbiens alimentés directement pour améliorer l'état général et la santé des poissons - Google Patents

Agents microbiens alimentés directement pour améliorer l'état général et la santé des poissons Download PDF

Info

Publication number
WO2019040266A1
WO2019040266A1 PCT/US2018/045368 US2018045368W WO2019040266A1 WO 2019040266 A1 WO2019040266 A1 WO 2019040266A1 US 2018045368 W US2018045368 W US 2018045368W WO 2019040266 A1 WO2019040266 A1 WO 2019040266A1
Authority
WO
WIPO (PCT)
Prior art keywords
fish
bacillus subtilis
strain
subtilis strain
vibrio
Prior art date
Application number
PCT/US2018/045368
Other languages
English (en)
Inventor
David Drahos
Original Assignee
Novozymes A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to MX2020001687A priority Critical patent/MX2020001687A/es
Priority to CN201880050775.6A priority patent/CN111801019A/zh
Priority to BR112020003799-0A priority patent/BR112020003799A2/pt
Publication of WO2019040266A1 publication Critical patent/WO2019040266A1/fr
Priority to PH12020500167A priority patent/PH12020500167A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs

Definitions

  • the present invention relates to a bacterial strain for improving the well-being, general condition, health and/or yields of fish such as Nile Tilapia fish.
  • Aquaculture is the cultivation of freshwater and saltwater populations under controlled conditions and has been found to be beneficial as they reduce the impacts and pressures of commercial fishing on wild fisheries as well as reduce the human ingestion of toxins (e.g., heavy metals such as mercury) which are often found in wild caught fish such as tuna.
  • toxins e.g., heavy metals such as mercury
  • challenges remain in the aquaculture industry. In particular, maintaining a healthy gut in the aquatic animal can improve aquatic animal weight and/or aquatic animal yields.
  • the invention provides bacterial strains for improving the well-being, general condition, health and/or yields of fish such as Nile Tilapia fish, improving, e.g., the weight of Nile Tilapia fish, the overall gut health of Nile Tilapia fish, and/or the immune response in Nile tilapia.
  • the invention provides an isolated Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof for use in a method for improving the well-being, general condition, health and/or yields of fish such as Nile Tilapia fish.
  • the invention provides an isolated Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof for use in a method for increasing the weight of Nile Tilapia fish, improving the overall gut health of Nile Tilapia fish, and/or improving the immune response of fish such as Nile Tilapia fish.
  • the invention provides an isolated Bacillus subtilis strain which has a 16S rDNA sequence having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at
  • the invention further provides a composition comprising an isolated Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B- 67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof and a carrier, wherein the composition is a fish feed or a fish feed additive.
  • Bacillus subtilis strain 014VRQ having deposit accession number NRRL B- 67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof and a carrier, wherein the composition is a fish feed or a fish feed additive.
  • SEQ ID NO: 1 is 16S rDNA of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221.
  • SEQ ID NO: 2 is the forward primer for strain NZ86.
  • SEQ ID NO: 3 is the reverse primer for strain NZ86.
  • SEQ ID NO: 4 is the probe for strain NZ86.
  • SEQ ID NO: 5 is the forward primer for strain 014VRQ.
  • SEQ ID NO 6 is the reverse primer for strain 014VRQ.
  • SEQ ID NO 7 is the probe for strain 014VRQ.
  • SEQ ID NO 8 is the forward primer for Oreochromis niloticus gene TNF-a.
  • SEQ ID NO 9 is the reverse primer for Oreochromis niloticus gene TNF-a.
  • SEQ ID NO 10 is the probe for Oreochromis niloticus gene TNF-a.
  • SEQ ID NO 11 is the forward primer for Oreochromis niloticus gene IL-1 ⁇ .
  • SEQ ID NO 12 is the reverse primer for Oreochromis niloticus gene IL- ⁇ ⁇ .
  • SEQ ID NO 13 is the probe for Oreochromis niloticus gene I L- 1 .
  • SEQ ID NO: 14 is the forward primer for Oreochromis niloticus gene act .
  • SEQ ID NO: 15 is the reverse primer for Oreochromis niloticus gene act .
  • SEQ ID NO: 16 is the probe for Oreochromis niloticus gene actp.
  • Fig. 1 shows the concentration of peripheral blood neutrophils on day 28 (upper) and lysozyme in the blood (middle and lower) on days 14 and 51 , respectively, of Nile tilapia after feeding the fish B. subtilis 014VRQ compared to the concentration of peripheral blood neutrophils and lysozyme in the blood of a control group of Nile tilapia which had not been fed the strain (CTL).
  • Fig. 2 shows expression of pro-inflammatory cytokine mRNA from the mid intestine of
  • Nile tilapia ⁇ Oreochromis niloticus after probiotic supplementation for 51 days. TNFa in the intestine (A), and I L- ⁇ in the intestine (B).
  • the term "about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • aquaculture As used herein, the terms “aquaculture”, “aquaculturing”, “aquafarm”, and “aquafarming” can be used interchangeably and refer to the cultivation, breeding, raising, production, propagation and/or harvesting of an aquatic or marine animal such as fish, generally in an artificial environment such as a tank (e.g., an aquarium), a pond, a pool, a paddy, a lake, etc., or in an enclosed or fenced off portion of the animals natural habitat, such as a pond, a pool, a paddy, a lake, an estuary, an ocean, a marsh (e.g., a tidal marsh), a lagoon (e.g., a tidal lagoon), etc.
  • a tank e.g., an aquarium
  • a pond e.g., a pond, a pool, a paddy, a lake, etc.
  • an estuary an ocean
  • a marsh
  • fish refers to all gill-bearing aquatic craniate animals that lack limbs with digits.
  • Non-limiting examples include, e.g., osteichthyes (including, but not limited to catfish, tilapia, trout, salmon, perch, bass, tuna, wahoo, tuna, swordfish, marlin, grouper, sturgeon, snapper, eel and walleye) and chondrichthyes (including, but not limited to sharks, rays, and skates.
  • the fish is Nile tilapia.
  • Neile tilapia refers to a fish of the family Cichlidae, Oreochromis niloticus, which is native to Africa. Other commercially known names of the fish include mango fish, nilotica, and boulti.
  • supply As used herein, the terms “supply”, “supplied”, “supplying”, “administer”, “administered”, or “administering”, are used interchangeably, and are intended to mean bringing a fish, e.g. , Nile tilapia fish, into contact with a Bacillus strain or a composition as described herein.
  • a preferred form of supplying or administration is oral administration such as administration via water or via fish feed.
  • control or “controlling” as in, e.g., the phrase: the "control” of pathogenic microorganisms, or “controlling” pathogenic microorganisms, or as in the phrase: “controlling” species of pathogenic microorganisms, refers to any means for preventing infection by pathogenic microorganisms, reducing the number of pathogenic microorganisms, killing the pathogenic microorganisms, or elimination the pathogenic microorganisms as defined herein. Indeed, "control” or “controlling” as used herein refers to any indicia of success in prevention, killing, elimination, reduction or amelioration of one or more pathogenic bacteria.
  • spore and “endospore” are interchangeable and have their normal meaning which is well known and understood by those of skill in the art.
  • spore refers to a microorganism in its dormant, protected state.
  • isolated means that the bacterial strain described herein are in a form or environment which does not occur in nature, that is, the bacterial strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
  • the term “blend” means more than one of the bacterial strains described herein.
  • the term “pathogenic microorganism” means any microorganism that can adversely affect the health, yield, or environment of one or more fish.
  • Health refers to the state or condition of an organism or one of its parts.
  • the terms "effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the health or yield of a fish such as Nile tilapia.
  • the actual effective dosage in absolute numbers depends on factors including: the state of health of the fish in question; whether the aim is prevention or reduction of a pathogenic organism, to improve overall health, gut health, etc.; other ingredients present, and also the surface or aqueous environment in question.
  • an effective dosage of bacteria e.g., of the one or more of the Bacillus subtilis strains disclosed, would be in the range from 1x10 2 to 1x10 12 CFU/g of the composition, preferably 1x10 4 to 1x10 9 CFU/g of the composition, more preferably 1x10 5 to 1x10 8 CFU/g of the composition, and even more preferably 1x10 6 to 5x10 8 CFU/g of the composition.
  • the ratio between the bacteria strain or blends concerned herein and the undesired microorganism(s) in question may be between 1 : 100,000 and 100,000: 1 (strain/blend: undesired microorganism), preferably 1 : 10,000 to 10,000: 1 , more preferably 1 : 1 ,000 to 1 ,000: 1 , more preferably 1 : 100 to 100: 1 , even more preferably 1 : 10 to 10: 1.
  • the "effective amount", “effective concentration”, or “effective dosage” of the bacterial strains may be determined by routine assays known to those readily skilled in the art.
  • fish feed or fish feed ingredient refers to any compound, preparation, or mixture suitable for, or intended for intake by fish such as Nile tilapia fish.
  • the term "vegetable protein(s)” refers to any compound, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives.
  • pellets and/or “pelleting” refer to solid rounded, spherical and/or cylindrical tablets or pellets and the processes for forming such solid shapes, particularly feed pellets and solid extruded fish feed.
  • extrusion or “extruding” are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure.
  • composition refers to a composition comprising a carrier and at least one bacterial strain as described herein.
  • the compositions described herein may be mixed with a fish feed(s) and referred to as a "mash feed.”
  • the composition is, e.g., sinking feed pellets.
  • bacterial strains for improving the well-being, general condition, health and/or yields of fish such as Nile Tilapia fish, improving, e.g., the weight of Nile Tilapia fish, the overall gut health of Nile Tilapia fish, and/or the immune response in Nile tilapia.
  • a strain as disclosed herein is supplied to fish such as Nile tilapia for decreasing lymphocytes, increasing neutrophils and monocytes in circulating blood, decreasing plasma lysozyme and/or increasing the proportion of rhodamine (RHO)-positive cells.
  • RHO rhodamine
  • the bacterial strains provided herein will improve the health of Nile Tilapia fish and/or survival rate of the Nile Tilapia fish. It is a further object of the embodiments disclosed herein, that the bacterial strains will improve fish prophylaxis, reduce the need for antibiotics, lower production costs and/or improve environmental conditions in fish culture.
  • the bacterial strain improves the health of the fish by enhancing the immune system and/or immune response of the fish.
  • the Bacillus subtilis strain has activity against pathogens such as one or more Vibrio strains and/or Photobacterium strains.
  • the Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, or any combination thereof.
  • the Vibrio strain is Vibrio parahaemolyticus.
  • the Bacillus subtilis strain is isolated. In another preferred embodiment, the Bacillus subtilis strain is on spore form.
  • a method for improving the well-being, general condition or health of a fish such as Nile tilapia comprises supplying such as administering to a fish such as Nile tilapia the bacterial strain disclosed herein in an effective amount.
  • the method comprises contacting the gut of a fish with the bacterial strain disclosed herein.
  • the bacterial strains can control and/or inhibit one or more Vibrio strains such as Vibrio parahaemolyticus.
  • the invention relates to a method for improving the well-being, general condition or health of a fish such as Nile tilapia
  • the method comprises the step of supplying such as administering one or more of the isolated Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof to fish such as Nile tilapia.
  • the invention further relates to a method for improving the well-being, general condition or health of a fish such as Nile tilapia comprising the step of supplying such as administering one or more of isolated Bacillus subtilis strains having a 16S rDNA sequence which has at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least
  • a method for improving the immune response of a fish such as Nile tilapia comprises supplying such as administering to a fish such as Nile tilapia a bacterial strain in an effective amount.
  • the method comprises contacting the gut of a fish with a bacterial strain disclosed herein.
  • the bacterial strains can control and/or inhibit lymphocytes, neutrophils and monocytes, plasma lysozyme, and/or the proportion of rhodamine (RHO)-positive cells.
  • the invention relates to a method for improving the immune response of a fish such as Nile tilapia comprising the step of supplying such as administering one or more of the isolated Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof to fish such as Nile tilapia.
  • the invention yet further relates to a method for improving immune response of a fish such as Nile tilapia comprising the step of supplying such as administering one or more of isolated Bacillus subtilis strains having a 16S rDNA sequence which has at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at
  • a method for obtaining a decrease in lymphocytes, an increase in neutrophils and monocytes, increase in plasma lysozyme, and/or an increase in the proportion of rhodamine (RHO)-positive cells in a fish such as Nile tilapia comprises supplying such as administering to a fish such as Nile tilapia a bacterial strain in an effective amount.
  • the method comprises contacting the gut of a fish with a bacterial strain disclosed herein.
  • the bacterial strains can control and/or inhibit pathogens such as one or more Vibrio strains and/or Photobacterium strains.
  • the Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, or any combination thereof.
  • the Vibrio strain is Vibrio parahaemolyticus.
  • the invention relates to a method for obtaining a decrease in lymphocytes, an increase in neutrophils and monocytes and/or an increase in plasma lysozyme in a fish such as Nile tilapia comprising the step of supplying such as administering one or more of the isolated Bacillus subtilis strain 014VRQ having deposit accession number NRRL B- 67221 or a strain having the identifying characteristics of the Bacillus subtilis strain NRRL B- 67221 or a mutant thereof to fish such as Nile tilapia.
  • the invention further relates to a method for obtaining a decrease in lymphocytes, an increase of neutrophils and monocytes, and/or an increase in plasma lysozyme in a fish such as Nile tilapia comprising the step of supplying such as administering one or more of isolated Bacillus subtilis strains having a 16S rDNA sequence which has at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%
  • the method preferably comprises a daily supplying such as administration for at least 2 days such as 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 days (coherent or non-coherent days).
  • the daily supplying such as administration is for up to 100 days, such as up to 90 days, 80 days, 70 days, 60 days or 51 days.
  • the daily supplying such as administration is for 1 to 100 days, such as for 2 to 80 days, 10 to 70 days, 20 to 60 days, or 28 to 51 days.
  • the method preferably comprises a daily dose of 1.5x10 6 CFU of the Bacillus subtilis strain according to the invention per fish such as per Nile tilapia.
  • the method comprises a daily dose of from 0.9x10 s to 6.0x10 6 CFU of the Bacillus subtilis strain according to the invention per fish such as per Nile tilapia.
  • the method comprises a daily dose of from 0.1x10 s to 1.0x10 7 CFU of the Bacillus subtilis strain according to the invention per fish such as per Nile tilapia such as a dose selected from the group consisting of from 0.1x10 6 to 0.5x10 6 , from 0.5x10 6 to 1.0x10 6 , from 1.0x10 s to 2.0x10 s , from 2.0x10 s to 3.0x10 6 , from 3.0x10 s to 4.0x10 6 , from 4.0x10 s to 5.0x10 6 , from 5.0x10 s to 6.0x10 6 , from 6.0x10 s to 7.0x10 6 , from 7.0x10 s to 8.0x10 s , from 8.0x10 s to 9.0x10 s , from 9.0x10 s to 1.0x10 7 CFU of the Bacillus subtilis strain according to the invention per fish such as per Nile tilapia, or any combination
  • the treatment results in survival of at least 20% more of the Nile tilapia compared to the control (no supplying such as administration of the isolated Bacillus subtilis strains according to the invention) such as at least 30% more, such as at least 40% more, such as at least 50% more or such as at least 60% more survival.
  • the invention also relates to the isolated Bacillus subtilis strain 01 VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof for use in a method for improving the well-being, general condition or health of a fish such as Nile tilapia.
  • the invention also relates to an isolated Bacillus subtilis strain having a 16S rDNA which has at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%
  • the bacterial strain for use in of the method are present in the form of a stable spore.
  • the stable spore will germinate in the gut of the fish.
  • the method comprises supplying such as administering to a fish such as Nile tilapia a bacterial strain described herein, wherein the bacterial count of the bacterial strain is between 1x10 2 and 1x10 12 CFU/g of the composition, particularly 1x10 4 and 1x10 9 CFU/g of the composition, and more particularly 1x10 5 and 5x10 8 CFU/g of the composition.
  • the bacterial count of the bacterial strain described herein is between 1x10 6 and 1x10 8 CFU/g of the composition.
  • the method comprises the step of contacting the gut of a fish such as Nile tilapia with Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or one or more strains having the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof.
  • the method comprises the step of contacting the gut of a fish such as Nile tilapia with one or more of the bacterial strains described herein wherein the bacterial strain improves the health of the fish.
  • parameters suitable for measuring improved health include but are not limited to body weight gain, improved body condition score, increased food intake, improved antioxidant status, decrease in markers of oxidative stress, optimal serum protein levels, optimal serum mineral levels, improved immune system function, improved health and diversity of gut microflora, decrease in fecal bacteria, etc.
  • suitable performance parameters include weight gain, feed:gain ratio, nutrient digestibility, feed conversion ratio, meat grade, meat yield, meat protein to fat ratio, mortality, mortality rate and similar performance parameters.
  • the method comprises the step of contacting the gut of a fish such as Nile tilapia with one or more of the bacterial strains described herein wherein the bacterial strain improves the health of the fish by increasing the weight of the fish.
  • the bacterial strain improves the health of the fish by enhancing the immune system and/or immune response of the fish.
  • the bacterial strain improves the health of the fish by improving the overall health of the gut of the fish.
  • Non- limiting examples of improved gut health include reducing gut inflammation, increasing the length and/or surface area of intestinal villi, increasing intestinal crypt depth, improving nutrient absorption by the gut, maintaining and/or improving healthy gut microflora, enhancing peripheral blood neutrophils, lymphocytes, and monocytes or combinations thereof.
  • the bacterial strain improves the health of the fish by controlling pathogenic microorganisms.
  • the bacterial strain improves the health of the fish by controlling pathogenic microorganisms in the gut of the fish.
  • the fish may be selected from the group consisting of osteichthyes and chondrichthyes.
  • the first is selected from the group consisting of catfish, tilapia, trout, salmon, perch, bass, tuna, wahoo, tuna, swordfish, marlin, grouper, sturgeon, snapper, eel, walleye, sharks, rays, and skates.
  • the fish is an osteichthye, such as a tilapia.
  • the fish is Nile tilapia.
  • the invention also relates to use of the bacterial strains according to the invention in aqua culturing.
  • a composition for use in the method of the invention comprises a fish feed ingredient such as a Nile tilapia fish feed ingredient and a bacterial strain according to the invention.
  • the bacterial strain is a stable strain when the strain is subjected to an extrusion process having a pressure of 1 bar to 35 bar; the strain is subjected to an extrusion process wherein the extrusion process temperatures are temperatures from 80°C to 120°C; and the strains that control one or more pathogenic microorganisms.
  • the bacterial strain is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof.
  • the composition includes Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof; and a carrier.
  • the carrier can, e.g., be calcium carbonate, other carriers mentioned elsewhere herein or other carriers described in the prior art.
  • the composition comprises a Bacillus subtilis strain having a 16S rDNA sequence having at least 98.00%, e.g. , at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least
  • the one or more of the isolated Bacillus subtilis strains in the composition are on spore form.
  • the composition is preferably a fish feed or a fish feed additive such as a Nile tilapia fish feed or a Nile tilapia fish feed additive.
  • the fish feed is feed pellets such as sinking feed pellets.
  • the feed pellets are surface coated with the Bacillus subtilis strain for use according to the invention.
  • the feed pellets are surface coated onto standard fish feed with the Bacillus subtilis strain in the form of spores.
  • the composition comprises from 10 4 to 10 8 CFU/g feed such as from 10 5 to 10 7 CFU/g feed or such as from 5 x 10 6 to 9 x 10 6 CFU/g feed of the Bacillus subtilis strains according to the invention.
  • the compositions described herein can be of any form so long as the carrier is able to support the bacterial strain, regardless of form (e.g. , a vegetative state or a dormant state), and the composition is suitable for intake by a fish such as Nile tilapia fish.
  • the composition may be in the form of a liquid, a slurry, a solid, or a powder (wettable powder or dry powder).
  • the composition disclosed herein, regardless of form, e.g. , a liquid, slurry, or powder (e.g. , wettable powder or dry powder), is suitable for use as an ingredient in a fish feed.
  • compositions described herein are suitable for use as an ingredient in a pelleted fish feed. In still a more particular embodiment, the compositions described herein are suitable for use as an ingredient in a fish feed produced via an extrusion process.
  • Carriers in the composition:
  • the carriers described herein will allow the bacterial strain described herein to remain efficacious (e.g. , capable of improving the well-being, general condition, health or survival rate of fish such as Nile tilapia fish) and viable once formulated.
  • Carriers described herein include liquids, slurries, or solids (including wettable powders or dry powders).
  • the carrier is a liquid carrier.
  • liquids useful as carriers for the compositions disclosed herein include water, aqueous solutions, or nonaqueous solutions.
  • the carrier is water.
  • the carrier is an aqueous solution, such as sugar water.
  • the carrier is a non- aqueous solution.
  • the liquid (e.g., water) carrier may further include growth media to culture the bacterial strain.
  • suitable growth media for the deposited bacterial strains include arabinose-gluconate (AG), yeast extract mannitol (YEM), G16 media, or any media known to those skilled in the art to be compatible with, and/or provide growth nutrients to the bacterial strain.
  • the carrier is a slurry.
  • the carrier is a solid.
  • the solid is a powder.
  • the powder is a wettable powder.
  • the powder is a dry powder.
  • the solid is a granule.
  • Non-limiting examples of solids useful as carriers for the compositions disclosed herein include calcium carbonate, sodium bicarbonate, sodium chloride, peat, wheat, wheat chaff, ground wheat straw, bran, vermiculite, cellulose, starch, soil (pasteurized or unpasteurized), gypsum, talc, clays ⁇ e.g. , kaolin, bentonite, montmorillonite), and silica gels.
  • the carrier is calcium carbonate.
  • the carrier is sodium bicarbonate.
  • composition for use in the method of the invention comprises a bacterial strain according to the invention.
  • the bacterial strain is stable when the strain is subjected to a feed manufacturing process.
  • the bacterial strain is stable when the strain is subjected to an extrusion process for pelleting.
  • the bacterial strain is stable when subjected to pressures achieved during an extrusion process for pelleting.
  • the bacterial strain is stable at pressures ranging from 1 bar to 40 bar, particularly 10 bar to 40 bar, more particularly 15 bar to 40 bar, even more particularly 20 bar to 40 bar, still even more particularly 35 bar to 37 bar, even still more particularly 36 bar.
  • the bacterial strain is stable at high temperatures.
  • the bacterial strain is stable when they are subjected to temperatures achieved during an extrusion process for pelleting.
  • the bacterial strain is stable at temperatures format about 80°C.
  • the extrusion stability of the bacterial strain(s) is determined by extrusion at 100°C or 1 10°C, exhibiting 50% or more survival at 100°C or 25% or more survival at 1 10°C.
  • the bacterial strain is stable when the strain is subjected to an extrusion process wherein the extruder has a die diameter of 0.5 mm to 5.0 mm.
  • the bacterial strain control one or more pathogenic microorganisms.
  • the bacterial strain is stable strains when the strain is subjected to an extrusion process having a pressure of 1 bar to 40 bar; the strain is subjected to an extrusion process wherein the extrusion process temperatures are temperatures from
  • strains control one or more pathogenic microorganisms.
  • the bacterial strain will be present in a quantity between
  • the bacterial strain will be present in a quantity between 1x10 6 and 1x10 12 CFU/g of the composition, particularly 1x10 4 and 1x10 9 CFU/g of the composition, and more particularly 1x10 5 and 5x10 8 CFU/g of the composition.
  • the bacterial strain will be present in a quantity between 1x10 6 and
  • the fermentation of the bacterial strain may be conducted using conventional fermentation processes, such as, aerobic liquid-culture techniques, shake flask cultivation, and small-scale or large-scale fermentation (e.g., continuous, batch, fed-batch, solid state fermentation, etc.) in laboratory or industrial fermentors, and such processes are well known in the art. Notwithstanding the production process used to produce the bacterial strain, the bacterial strain may be used directly from the culture medium or subject to purification and/or further processing steps (e.g., a drying process).
  • conventional fermentation processes such as, aerobic liquid-culture techniques, shake flask cultivation, and small-scale or large-scale fermentation (e.g., continuous, batch, fed-batch, solid state fermentation, etc.) in laboratory or industrial fermentors, and such processes are well known in the art. Notwithstanding the production process used to produce the bacterial strain, the bacterial strain may be used directly from the culture medium or subject to purification and/or further processing steps (e.g., a drying process).
  • the bacterial strain may be recovered using conventional techniques (e.g. , by filtration, centrifugation, etc.).
  • the bacterial strain may alternatively be dried
  • a suitable temperature e.g. , room temperature
  • the one or more bacteria disclosed herein are stable and retain a sufficient effective amount of activity when used. Methods for producing stabilized microorganisms are known in the art. In one embodiment, the one or more bacteria disclosed herein are present in the composition in the form of a stable spore.
  • compositions for use in the method of the invention may further comprise one or more optional ingredients that are suitable for consumption by an aquatic fish such as Nile tilapia fish.
  • optional ingredients include enzymes. Such ingredients are known to those skilled in the art. Enzymes
  • compositions for use in the method of the invention optionally include one or more enzymes as described herein.
  • the one or more enzymes may be any enzyme or combination of different enzymes that are suitable to be given to a fish, meaning that it in one way or the other will be good for the fish nutritionally to eat the enzyme. Accordingly, when reference is made to "an enzyme” this will in general be understood to include one or more feed enzymes. In a particular embodiment, it is not construed as including enzymes which has a therapeutic function in medical sense.
  • the feed enzymes should be feed/food grade, thus meaning that they may not be harmful to fish such as Nile tilapia fish and be a feed/food grade meaning that it should comply with recommended purity specifications for food grade enzymes.
  • JECFA Joint FAO/WHO Expert Committee on Food Additives
  • FCC Food Chemical Codex
  • the enzyme comprises less than 30 coliform bacteria per gram and comprise a viable count of less than 50,000/g.
  • compositions described herein optionally include one or more enzymes.
  • Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: expasy.ch/enzyme/.
  • ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch, 2000, The ENZYME database, Nucleic Acids Res. 28: 304-305).
  • This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.
  • glycoside hydrolase enzymes such as endoglucanase, xylanase, galactanase, mannanase, dextranase, lysozyme and galactosidase is described in Henrissat et al., 2014, "The carbohydrate-active enzymes database (CAZy) in 2013", Nucl. Acids Res. (D1): D490-D495; see also malariay.org.
  • composition of the invention may also comprise at least one other enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); arabinofuranosidase (EC 3.2.1.55); beta-xylosidase (EC 3.2.1.37); acetyl xylan esterase (EC 3.1.1.72); feruloyl ester este
  • composition of the invention comprises a phytase (EC
  • phytases examples include Bio-FeedTM Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), NatuphosTM (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma) Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont).
  • Other preferred phytases include those described in, e.g., WO 98/28408, WO 00/43503, and WO 03/066847.
  • the composition of the invention comprises a xylanase (EC 3.2.1.8).
  • xylanases include Ronozyme® WX and Ronozyme® G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma) and Axtra® XB (Xylanase/beta-glucanase, DuPont).
  • composition of the invention comprises a protease (EC
  • proteases examples include Ronozyme® ProAct (DSM Nutritional Products).
  • the compositions for use in the method of the invention are suitable for use in fish feed(s) such as Nile tilapia fish feed(s).
  • fish feed(s) such as Nile tilapia fish feed(s).
  • the characteristics of the compositions described herein allow its use as a component which is well suited for inclusion with a fish feed.
  • the compositions described herein are mixed with a fish feed ingredient and/or fish feed(s) and referred to as a mash feed. In certain embodiments, the mash feed is subsequently pelletized.
  • the fish feed may comprise any ingredient suitable for intake by fish (such as Nile tilapia fish), e.g., comprising sources of protein, lipids, carbohydrates, salts, minerals and vitamins.
  • the fish feed ingredients may be selected, and mixed in any proportions, suitable to meet the nutritional needs of the fish to be fed with the feed and/or to keep the raw material cost of the feed within desired limits and/or to achieve other desired properties of the feed.
  • Non- limiting examples of fish feed ingredients may include one or more of the following materials: plant derived products, such as seeds, grains, leaves, roots, tubers, flowers, pods, husks, oil, soybean meal, soy protein isolate, potato protein powder, wheat, barley, corn, soybean oil, and corn gluten meal; animal derived products, such as fish meal, fish oil, milk powder, skim milk powder, bone extract, meat extract, blood extract, and the like; additives, such as minerals, vitamins, aroma compounds, and feed enhancing enzymes.
  • plant derived products such as seeds, grains, leaves, roots, tubers, flowers, pods, husks, oil, soybean meal, soy protein isolate, potato protein powder, wheat, barley, corn, soybean oil, and corn gluten meal
  • animal derived products such as fish meal, fish oil, milk powder, skim milk powder, bone extract, meat extract, blood extract, and the like
  • additives such as minerals, vitamins, aroma compounds, and feed enhancing enzymes.
  • the fish feed may comprise 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0-20% whey.
  • the fish feed may comprise vegetable proteins.
  • the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w).
  • Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.
  • the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean.
  • the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g., beet, sugar beet, spinach or quinoa.
  • Other examples of vegetable protein sources are rapeseed, and cabbage.
  • soybean is a preferred vegetable protein source.
  • Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.
  • the fish feed may optionally comprise one or more suitable fish feed additives.
  • suitable fish feed additives include enzyme inhibitors, fat-soluble vitamins, water soluble vitamins, trace minerals, macro minerals, and combinations thereof.
  • the fish feed may further optionally comprise one or more feed- additive ingredients.
  • feed-additive ingredients include coloring agents, aroma compounds, stabilizers, anti-microbial peptides (non-limiting examples of anti-microbial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1 , Thanatin, Defensin, Ovispirin such as Novispirin (Robert Lehrer, 2000), and variants, or fragments thereof which retain antimicrobial activity), anti-fungal polypeptides (AFP's) (non-limiting examples include the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and PCT/DK02/00289), and/or at least one other enzyme selected from amongst phytases EC 3.1.3.8 or 3.1.3.26; xy
  • the fish feed may still further optionally include one or more fat- and water soluble vitamins, trace minerals and macro minerals.
  • fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
  • Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.
  • Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. , Ca-D- panthothenate.
  • Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, etc.
  • Non-limiting examples of macro minerals include calcium, magnesium, potassium, sodium, etc.
  • methods described herein comprise administering to a fish such as a Nile Tilapia fish and/or contacting the gut of a fish such as a Nile Tilapia fish with a composition described herein.
  • the composition is an ingredient in a fish feed (i.e., a mash feed) as described herein.
  • bacterial spores according to the invention are blended into a Calcium carbonate carrier (such as #1491 Vicron 45-3FG), to form the product (target is preferably 1x10 8 CFU/g to 1x10 10 CFU/g such as, e.g., 1x10 9 CFU/g) from an initial spray-dried spore concentrate (usually at 5-9x10 9 to 5-9x10 11 CFU/g such as, e.g., CFU/g 5-9x10 10 CFU/g).
  • This formulation is typically stable under dry conditions for at least 2 years at RT (23°C).
  • the product is typically suspended 1 :5 g/g to 1 :20 g/g, such as 1 : 10 g/g in, e.g., water or vegetable oil to provide a suspension of, e.g., 1x10 8 CFU/ml.
  • This material is preferably sprayed evenly on the pre-formed feed pellets, e.g., at a 5% to 20% v/g rate such as a 10% v/g rate, and dried, e.g., overnight at 21-30°C.
  • the resultant recoverable level of spores according to the invention on the pellets is preferably 6-8 x 10 5 CFU/g feed to 6-8 x 10 8 CFU/g feed, such as, e.g., about 6-8 x 10 6 CFU/g feed.
  • the bacterial concentration on the feed pellets is from 1 x 10 5 CFU/g feed to 8 x 10 8 CFU/g feed such as from 1 x 10 5 CFU/g feed to 8 x 10 5 CFU/g feed, such as from 8 x 10 5 CFU/g feed to 1 x 10 6 CFU/g feed, such as from 1 x 10 6 CFU/g feed to 1 x 10 7 CFU/g feed, such as from 1 x 10 7 CFU/g feed to 1 x 10 8 CFU/g feed, or any combination of these intervals.
  • the feed mash is pelleted. Pelleting processes are known in the art.
  • the pellets are manufactured through a pelleting process.
  • the pellets are manufactured through an extrusion process.
  • the feed mixture (mash feed) may be prepared by mixing the composition comprising the bacterial strain with desired feed components.
  • the mash feed may be conditioned or unconditioned.
  • the mash feed is conditioned prior to the pelleting process.
  • the mash feed is led to a conditioner, e.g., a cascade mixer with steam injection.
  • the feed is in the conditioner heated up to a specified temperature such as, e.g., 60- 120°C, e.g., 60°C, 70°C, 80°C, 90°C, 100°C, 105°C, 110°C, 115°C, or 120°C by injecting steam, measured at the outlet of the conditioner.
  • the residence time can be variable from seconds to minutes and even hours.
  • the process temperature during steam treatment is at least 60°C. In a more particular embodiment, the process temperature during steam treatment is at least 70°C. In an even more particular embodiment, the process temperature during steam treatment is at least 80°C. In a most particular embodiment, the process temperature during steam treatment is at least 90°C.
  • the pelleting process is an extrusion process.
  • extrusion processes for manufacturing feed pellets are known to those skilled in the art. Extrusion or pelletized products, wherein the feed mixture (mash feed) is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the mash feed. (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker).
  • the mash feed is led to an extruder to form pellets of variable length from the extrudate.
  • the extrusion apparatus may be any screw-type extruder known in the art.
  • the extruder is a double screwed extruder, e.g., a Werner & Pfleiderer Type continua 37" extruder.
  • Extrusion parameters ⁇ e.g., capacity, screw speed, die diameter, drying temperatures, drying time, etc.) are dependent upon the particular extrusion process and/or extrusion apparatuses employed.
  • the screw speed of the extruder is 1-1 ,000 RPM. In a more particular embodiment, the screw speed of the extruder is 100 RPM. In an even more particular embodiment, the screw speed of the extruder is 150 RPM. In yet an even more particular embodiment, the screw speed of the extruder is 200 RPM. In still an even more particular embodiment, the screw speed of the extruder is 250 RPM. In still yet an even more particular embodiment, the screw speed of the extruder is 300 RPM.
  • the die diameter is 0.5-5.0 mm. In a more particular embodiment, the die diameter is 0.5 mm. In an even more particular embodiment, the die diameter is 1.0 mm. In yet an even more particular embodiment, the die diameter is 1.5 mm. In a most particular embodiment, the die diameter is 2.0 mm.
  • pellets are placed then dried for a specified time, e.g., at least 15 minutes, preferably 20 minutes, at temperatures of 60-100°C, preferably 90-100°C, more preferably 90°C, even more preferably 95°C, even still more preferably 100°C.
  • a specified time e.g., at least 15 minutes, preferably 20 minutes, at temperatures of 60-100°C, preferably 90-100°C, more preferably 90°C, even more preferably 95°C, even still more preferably 100°C.
  • Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of
  • Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of
  • An isolated Bacillus subtilis strain which has a 16S rDNA sequence having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.
  • An isolated Bacillus subtilis strain which has a 16S rDNA sequence having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.55%, at least 99.
  • Vibrio strain for use according to aspect 5, wherein the Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis,
  • Vibrio parahaemolyticus Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, or any combination thereof.
  • Bacillus subtilis strain for use according to any of aspects 1 to 7, wherein the Bacillus subtilis strain is supplied to said fish at least one time, at least two times, at least three times, at least four times or at least five times.
  • Bacillus subtilis strain for use according to any of aspects 1 to 8, wherein the Bacillus subtilis strain is supplied to said fish at least 6 times, at least 8 times, at least 10 times, at least 15 times or at least 20 times. 10. The Bacillus subtilis strain for use according to any of aspects 1 to 9, wherein the Bacillus subtilis strain when supplied to fish cultures results in improved fish prophylaxis, reduced need for antibiotics, lower production costs and/or improved environmental conditions in fish cultures compared to fish cultures which have not received said Bacillus subtilis strain. 11.
  • Bacillus subtilis strain for use according to any of aspects 1 to 10, wherein the Bacillus subtilis strain when supplied to fish cultures results in a decrease in lymphocytes, an increase in neutrophils and monocytes in circulating blood and/or an increase in plasma lysozyme in said fish in comparison to fish which have not received said Bacillus subtilis strain. 12.
  • the Bacillus subtilis strain for use according to any of aspects 1 to 11 wherein the Bacillus subtilis strain results in a fish survival of at least 15% after 104 hours, such as a fish survival of at least 20%, at least 25% or at least 30% after 104 hours.
  • Bacillus subtilis strain for use according to any of aspects 1 to 15, wherein said Bacillus subtilis strain is supplied such as administered in a daily dose of from 0.9x10 6 to
  • Bacillus subtilis strain for use according to any of aspects 1 to 15, wherein said Bacillus subtilis strain is supplied such as administered in a daily dose of from 0.1x10 6 to 1.0x10 7 CFU of the Bacillus subtilis strain per fish such as per Nile tilapia.
  • Bacillus subtilis strain for use according to any of aspects 1 to 15, wherein said Bacillus subtilis strain is supplied such as administered in from 0.1x10 6 to 0.5x10 6 , from 0.5x10 6 to 1.0x10 6 , from 1.0x10 6 to 2.0x10 s , from 2.0x10 6 to 3.0x10 5 , from 3.0x10 6 to 4.0x10 6 , from 4.0x10 6 to 5.0x10 6 , from 5.0x10 6 to 6.0x10 6 , from 6.0x10 6 to 7.0x10 6 , from 7.0x10 6 to 8.0x10 6 , from 8.0x10 6 to 9.0x10 s , or from 9.0x10 6 to 1.0x10 7 CFU of the Bacillus subtilis strain per fish such as per Nile tilapia.
  • Bacillus subtilis strain for use according to any of aspects 1 to 18, wherein the use results in survival of at least 10% more of the fish compared to a control group of fish such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, or such as at least 70% higher survival, wherein the control group is not receiving said isolated Bacillus subtilis strain or a composition comprising said isolated Bacillus subtilis strain. 20.
  • Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of the Bacillus subtilis strain NRRL B-67221 or a mutant thereof in a method for improving the well- being, general condition, health and/or yields of fish such as Nile Tilapia fish. 21 .
  • Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof in a method for increasing the weight of Nile Tilapia fish, improving the overall gut health of Nile Tilapia fish, and/or improving the immune response of fish such as Nile Tilapia fish.
  • the Bacillus subtilis strain has activity against pathogens such as one or more Vibrio strains and/or Photobacterium strains.
  • the Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, or any combination thereof.
  • Bacillus subtilis strain results in a fish survival of at least 15% after 104 hours such as a fish survival of at least 20%, at least 25% or at least 30% after 104 hours.
  • said Bacillus subtilis strain is supplied such as administered daily for at least 2 days such as 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 days.
  • Bacillus subtilis strain is supplied such as administered daily for up to 100 days, such as up to 90 days, 80 days, 70 days, 60 days or 51 days.
  • Bacillus subtilis strain is supplied such as administered daily for 1 to 100 days, such as for 2 to 80 days, 10 to 70 days, 20 to 60 days, or 28 to 51 days.
  • Bacillus subtilis strain is supplied such as administered in from 0.1x10 6 to 0.5x10 6 , from 0.5x10 s to 1.0x10 6 , from 1.0x10 6 to 2.0x10 6 , from 2.0x10 6 to 3.0x10 s , from 3.0x10 s to 4.0x10 s , from 4.0x10 s to 5.0x10 s , from 5.0x10 s to 6.0x10 s , from 6.0x10 s to 7.0x10 6 , from 7.0x10 s to 8.0x10 6 , from 8.0x10 s to 9.0x10 6 , or from 9.0x10 s to 1.0x10 7 CFU of the Bacillus subtilis strain per fish such as per Nile tilapia.
  • any of aspects 20 to 37 wherein the use results in survival of at least 10% more of the fish compared to a control group of fish such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, or such as at least 70% higher survival, wherein the control group is not receiving said isolated Bacillus subtilis strain or a composition comprising said isolated Bacillus subtilis strain.
  • a method for improving the well-being, general condition, health and/or yields of fish comprising the step of supplying such as administering an isolated
  • Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof to said fish.
  • a method for increasing the weight of Nile Tilapia fish, improving the overall gut health of Nile Tilapia fish, and/or improving the immune response of fish such as Nile Tilapia fish comprising the step of supplying such as administering an isolated Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof for use in a method to said fish.
  • a method for improving the well-being, general condition, health and/or yields of fish such as Nile Tilapia fish
  • the step of supplying such as administering an isolated Bacillus subtilis strain which has a 16S rDNA sequence having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least
  • a method for increasing the weight of Nile Tilapia fish, improving the overall gut health of Nile Tilapia fish, and/or improving the immune response of fish such as Nile Tilapia fish comprising the step of supplying such as administering an isolated Bacillus subtilis strain which has a 16S rDNA sequence having at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.1
  • Bacillus subtilis strain has activity against pathogens such as one or more Vibrio strains and/or Photobacterium strains.
  • Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, or any combination thereof.
  • Vibrio strain is Vibrio parahaemolyticus.
  • Bacillus subtilis strain is supplied to said fish at least one time, at least two times, at least three times, at least four times or at least five times.
  • the Bacillus subtilis strain results in a fish survival of at least 15% after 104 hours such as a fish survival of at least 20%, at least 25% or at least 30% after 104 hours.
  • the supplying such as administering comprises a daily supplying such as administration for at least 2 days such as 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 days.
  • Bacillus subtilis strain is supplied such as administered daily for up to 100 days, such as up to 90 days, 80 days, 70 days, 60 days or 51 days.
  • Bacillus subtilis strain is supplied such as administered daily for 1 to 100 days, such as for 2 to 80 days, 10 to 70 days, 20 to 60 days, or 28 to 51 days.
  • Bacillus subtilis strain is supplied such as administered in from 0.1x10 6 to 0.5x10 6 , from 0.5x10 6 to 1.0x10 6 , from 1.0x10 6 to 2.0x10 6 , from 2.0x10 6 to 3.0x10 s , from 3.0x10 6 to 4.0x10 s , from 4.0x10 s to 5.0x10 s , from 5.0x10 s to 6.0x10 s , from 6.0x10 s to 7.0x10 6 , from 7.0x10 s to 8.0x10 6 , from 8.0x10 s to 9.0x10 6 , or from 9.0x10 s to 1.0x10 7 CFU of the Bacillus subtilis strain per fish such as per Nile tilapia.
  • a composition comprising an isolated Bacillus subtilis strain which is Bacillus subtilis strain 014VRQ having deposit accession number NRRL B-67221 or a strain having the identifying characteristics of Bacillus subtilis strain NRRL B-67221 or a mutant thereof and a carrier, wherein the composition is a fish feed or a fish feed additive.
  • a composition comprising an isolated Bacillus subtilis strain having a 16S rDNA sequence which has at least 98.00%, e.g., at least 98.05%, at least 98.10%, at least 98.15%, at least 98.20%, at least 98.25%, at least 98.30%, at least 98.35%, at least 98.40%, at least 98.45%, at least 98.50%, at least 98.55%, at least 98.60%, at least 98.65%, at least 98.70%, at least 98.75%, at least 98.80%, at least 98.85%, at least 98.90%, at least 98.95%, at least 99.00%, at least 99.05%, at least 99.10%, at least 99.15%, at least 99.20%, at least 99.25%, at least 99.30%, at least 99.35%, at least 99.40%, at least 99.45%, at least 99.50%, at least 99.5
  • composition according to aspect 60 wherein the Vibrio strain is selected from the group consisting of Vibrio fischeri, Vibrio vulnificus, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio cholera, Vibrio harveyi, or any combination thereof.
  • composition according to aspect 61 wherein the Vibrio strain is Vibrio parahaemolyticus.
  • the carrier is a solid which is selected from the group consisting of: a powder, a wettable powder, a dry powder, a granule, calcium carbonate, sodium bicarbonate, sodium chloride, peat, wheat, wheat chaff, ground wheat straw, bran, vermiculite, cellulose, starch, soil (pasteurized or unpasteurized), gypsum, talc, clay (e.g., kaolin, bentonite, montmorillonite) and silica gel.
  • composition according to any of aspects 58 to 67, wherein the fish feed is fish feed pellets such as sinking feed pellets or extruded pellets such as floating pellets.
  • Bacillus subtilis strain 014VRQ was isolated from a soil sample from Virginia, USA on May 5, 2006 by Michael Frodyma from Novozymes North America Inc.
  • the strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto.
  • the deposit represents a substantially pure culture of the deposited strain.
  • the deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
  • Nile tilapia fish were fed standard amounts of commercial fish feed pellets which had been treated with Bacillus subtilis strain NRRL B-67221 (014VRQ) or NRRL B-50136 by surface spray directly on the feed pellet 24 hours prior to feeding or which had been fed non-treated standard amounts of commercial fish feed pellets.
  • a spore-suspension in water of the treatment bacterium was prepared at a preferable concentration of 1.0 x 10 7 CFU/g-feed.
  • the suspension was sprayed evenly onto dry feed pellets (sinking) at a preferable rate of 1.0 ml spore suspension to 10.0 g of feed pellets, and allowed to dry overnight at 23°C.
  • the resulting recoverable bacterial concentration on the pellets was 0.9-1.2 x 10 7 CFU/g-feed CFU/g-feed.
  • the treated pellets were added to each specified Tank at a rate of 3% (w/w) of Nile tilapia body weight per day.
  • the target inoculation was an average of 4.8 g- feed/fish/day, or 4.8 x 10 7 CFU/fish per day. At test initiation (Time 0 hrs.), all fish (100%) were healthy and similar in size.
  • Table 3 shows the percentage of peripheral blood leukocyte levels of Nile tilapia fed with Bacillus subtilis strain NRRL B-67221 (014VRQ) or NRRL B-50136 (NZ86) on days 14 and 28 compared to a control.
  • Lymphocytes % 82.8 86.0 80.8
  • Lymphocytes % 88.9 a 92.5 a 81.7 b
  • the percentage of peripheral blood neutrophils is significantly increased by the feeding of B. subtilis 014VRQ at day 14 and even more significantly by day 28, compared with B. subtilis NRRL B-50136 and the control. In addition, by day 28 the Monocytes are also significantly enhanced by 014VRQ.
  • Nile tilapia were treated according to example 2. Kidney and spleen blood leukocyte profile was measured for the treated Nile tilapia as a measure for the immune defense mechanism.
  • RHO rhodamine
  • MFI median fluorescence intensity
  • Nile tilapia were treated according to example 3 and the proportion of neutrophil levels in the blood cells in Tilapia fish was measured on day 28. The proportion of lysozyme levels was measured on days 14 and 51 , respectively.
  • Figure 1 illustrates the proportion of neutrophil and lysozyme levels in the blood cells of
  • Nile tilapia fish were fed B. subtilis 014VRQ for 51 days, at which time the mRNA expression of the pro-inflammatory cytokines IL-1 B, TNFa, and actB were quantitatively assessed by RT-qPCR using primers and probes specific to these cytokines as shown in Table 5.
  • Omni TH homogenizer Omni TH homogenizer
  • NCBI primer-BLAST tool was used to design primers and probes of interleukin- 1 beta (IL- ⁇ ), tumor necrosis factor alpha (TNF-a) and beta-actin (actp), with sequences spanning exon-exon regions (Table 5).
  • Gene expression analysis was carried out through RT- qPCR in a total sample volume of 10 ⁇ _, containing 300 nM both forward and reverse primers (Integrated DNA Technologies, Coralville, IA), 150 nM 6-carboxyfluorescein (FAM) probe (Integrated DNA Technologies), and 2x Primetime Gene Expression Master Mix and 12.5 ng of cDNA. All samples were run in triplicate on a CFX Connect Real Time System (Bio-Rad Laboratories, Hercules, CA, USA).
  • FIG. 2 shows expression of pro-inflammatory cytokines TNFa and I L- 1 ⁇ from the mid intestine of Nile tilapia ⁇ Oreochromis niloticus) after probiotic supplementation for 51 days.
  • the present work showed that the functionality of the tilapia gut was influenced immunologically following administration of the B. subtilis strains 014VRQ and NZ86. Such supplementation resulted in elevated levels of the pro-inflammatory cytokines I L- 1 ⁇ and TNF-a, where the heightened effect was significant (p ⁇ 0.05) with diet 014VRQ.
  • These cytokines are biological markers that promote inflammatory responses during the event of invasion or colonization of the gut.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physiology (AREA)
  • Insects & Arthropods (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Molecular Biology (AREA)
  • Birds (AREA)
  • Inorganic Chemistry (AREA)
  • Fodder In General (AREA)
  • Feed For Specific Animals (AREA)
  • Fish Paste Products (AREA)
  • Meat, Egg Or Seafood Products (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une souche bactérienne pour améliorer le bien-être, l'état général, la santé des poissons et/ou les rendements piscicoles.
PCT/US2018/045368 2017-08-23 2018-08-06 Agents microbiens alimentés directement pour améliorer l'état général et la santé des poissons WO2019040266A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
MX2020001687A MX2020001687A (es) 2017-08-23 2018-08-06 Agentes microbianos para alimentacion directa para mejorar el estado general y salud de los peces.
CN201880050775.6A CN111801019A (zh) 2017-08-23 2018-08-06 直接饲喂微生物用于改善鱼的一般状况和健康
BR112020003799-0A BR112020003799A2 (pt) 2017-08-23 2018-08-06 microrganismo de alimentação direta para melhorar a condição geral e saúde de peixes
PH12020500167A PH12020500167A1 (en) 2017-08-23 2020-01-23 Direct fed microbials for improving the general condition and health of fish

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201762549030P 2017-08-23 2017-08-23
US62/549,030 2017-08-23
US201862664644P 2018-04-30 2018-04-30
US62/664,644 2018-04-30

Publications (1)

Publication Number Publication Date
WO2019040266A1 true WO2019040266A1 (fr) 2019-02-28

Family

ID=63364181

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/045368 WO2019040266A1 (fr) 2017-08-23 2018-08-06 Agents microbiens alimentés directement pour améliorer l'état général et la santé des poissons

Country Status (6)

Country Link
CN (1) CN111801019A (fr)
BR (1) BR112020003799A2 (fr)
EC (1) ECSP20019404A (fr)
MX (1) MX2020001687A (fr)
PH (1) PH12020500167A1 (fr)
WO (1) WO2019040266A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116948890B (zh) * 2023-07-13 2024-03-22 江苏三仪生物工程有限公司 一种抑制水产弧菌的复合微生态菌剂及其制备方法

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001459A1 (fr) 1992-07-10 1994-01-20 Novo Nordisk A/S Compose ayant une activite fongicide
WO1998028408A1 (fr) 1996-12-20 1998-07-02 Novo Nordisk A/S Phytase induite par peniophora
WO2000043503A1 (fr) 1999-01-22 2000-07-27 Novozymes A/S Phytases ameliorees
WO2003066847A2 (fr) 2002-02-08 2003-08-14 Novozymes A/S Variants de phytase
WO2013029013A1 (fr) * 2011-08-24 2013-02-28 Dupont Nutrition Biosciences Aps Souches de bacillus produisant une enzyme
WO2017044953A1 (fr) * 2015-09-10 2017-03-16 Locus Solutions, Llc Production microbienne améliorée de biosurfactants et d'autres produits, et leurs utilisations
WO2017081105A1 (fr) * 2015-11-09 2017-05-18 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Souches de bacillus et agents ayant des propriétés bénéfiques
WO2017147130A2 (fr) * 2016-02-23 2017-08-31 Novozymes A/S Produits microbiens administrés directement pour la prévention de la maladie des crevettes
US20170340683A1 (en) * 2016-05-31 2017-11-30 Evonik Degussa Gmbh Bacillus subtilis strain with probiotic activity
WO2017207372A1 (fr) * 2016-05-31 2017-12-07 Evonik Degussa Gmbh Souche debacillus subtilis ayant une activité probiotique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150216915A1 (en) * 2012-04-12 2015-08-06 Dupont Nutrition Biosciences Aps Microbial Strains and Their Use in Animals
WO2014169046A1 (fr) * 2013-04-09 2014-10-16 Novozymes A/S Compositions et procédés pour améliorer la santé d'animaux aquatiques

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001459A1 (fr) 1992-07-10 1994-01-20 Novo Nordisk A/S Compose ayant une activite fongicide
WO1998028408A1 (fr) 1996-12-20 1998-07-02 Novo Nordisk A/S Phytase induite par peniophora
WO2000043503A1 (fr) 1999-01-22 2000-07-27 Novozymes A/S Phytases ameliorees
WO2003066847A2 (fr) 2002-02-08 2003-08-14 Novozymes A/S Variants de phytase
WO2013029013A1 (fr) * 2011-08-24 2013-02-28 Dupont Nutrition Biosciences Aps Souches de bacillus produisant une enzyme
WO2017044953A1 (fr) * 2015-09-10 2017-03-16 Locus Solutions, Llc Production microbienne améliorée de biosurfactants et d'autres produits, et leurs utilisations
WO2017081105A1 (fr) * 2015-11-09 2017-05-18 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Souches de bacillus et agents ayant des propriétés bénéfiques
WO2017147130A2 (fr) * 2016-02-23 2017-08-31 Novozymes A/S Produits microbiens administrés directement pour la prévention de la maladie des crevettes
US20170340683A1 (en) * 2016-05-31 2017-11-30 Evonik Degussa Gmbh Bacillus subtilis strain with probiotic activity
WO2017207372A1 (fr) * 2016-05-31 2017-12-07 Evonik Degussa Gmbh Souche debacillus subtilis ayant une activité probiotique

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB", 1992, ACADEMIC PRESS, INC.
BAIROCH: "The ENZYME database", NUCLEIC ACIDS RES., vol. 28, 2000, pages 304 - 305
HADI ZOKAEIFAR ET AL: "Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei", FISH AND SHELLFISH IMMUNOLOGY, vol. 33, no. 4, 30 March 2012 (2012-03-30), GB, pages 683 - 689, XP055367147, ISSN: 1050-4648, DOI: 10.1016/j.fsi.2012.05.027 *
HENRISSAT ET AL.: "The carbohydrate-active enzymes database (CAZy) in 2013", NUCL. ACIDS RES., vol. D1, 2014, pages D490 - D495
JOSÉ LUIS BALCÁZAR ET AL: "Inhibitory Activity of Probiotic Bacillus subtilis UTM 126 Against Vibrio Species Confers Protection Against Vibriosis in Juvenile Shrimp (Litopenaeus vannamei)", CURRENT MICROBIOLOGY, SPRINGER-VERLAG, NE, vol. 55, no. 5, 7 August 2007 (2007-08-07), pages 409 - 412, XP019561489, ISSN: 1432-0991, DOI: 10.1007/S00284-007-9000-0 *
MICHAEL S. SHOWELL: "Powdered detergents", vol. 71, 1998, MARCEL DEKKER, article "Surfactant Science Series", pages: 140 - 142

Also Published As

Publication number Publication date
CN111801019A (zh) 2020-10-20
ECSP20019404A (es) 2020-04-22
MX2020001687A (es) 2020-07-13
PH12020500167A1 (en) 2020-10-12
BR112020003799A2 (pt) 2020-09-08

Similar Documents

Publication Publication Date Title
US20240108666A1 (en) Compositions and methods for improving the health of aquatic animals
Menaga et al. Effect of in-situ and ex-situ biofloc on immune response of Genetically Improved Farmed Tilapia
Zhou et al. Enhancement of growth and intestinal flora in grass carp: the effect of exogenous cellulase
Abdel-Latif et al. Evaluation of two phytobiotics, Spirulina platensis and Origanum vulgare extract on growth, serum antioxidant activities and resistance of Nile tilapia (Oreochromis niloticus) to pathogenic Vibrio alginolyticus
Yu et al. Monitoring of growth, digestive enzyme activity, immune response and water quality parameters of Golden crucian carp (Carassius auratus) in zero-water exchange tanks of biofloc systems
WO2017147130A2 (fr) Produits microbiens administrés directement pour la prévention de la maladie des crevettes
US20160128337A1 (en) Antibacterial compositions and methods of use
CN109563472B (zh) 芽孢杆菌属组合物及用于反刍动物的方法
Gómez et al. Influence of probiotics on the growth and digestive enzyme activity of white Pacific shrimp (Litopenaeus vannamei)
WO2016118840A1 (fr) Souches de bacillus utiles pour améliorer la santé et les performances d'animaux de rente
JP2015142566A (ja) 養魚用飼料
Saad et al. Growth response of the freshwater prawn, Macrobrachium rosenbergii (De Man), to diets having different levels of Biogen®
JP2020528414A (ja) ソホロリピッドの飼料添加物としての使用
Xu et al. Dietary Azomite, a natural trace mineral complex, improved the growth, immunity response, intestine health and resistance against bacterial infection in largemouth bass (Micropterus salmoides)
Chama et al. Methanotroph (Methylococcus capsulatus, Bath) as an alternative protein source for genetically improved farmed tilapia (GIFT: Oreochromis niloticus) and its effect on antioxidants and immune response
El Basuini et al. Microbial, immune and antioxidant responses of Nile tilapia with dietary nano-curcumin supplements under chronic low temperatures
KR100887334B1 (ko) 아라자임을 유효성분으로 함유하는 어류 또는 갑각류양식용 사료첨가제
Ding et al. Effects of tributyrin on the growth performance, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei)
Liu et al. Rhodopseudomonas palustris in effluent enhances the disease resistance, TOR and NF‐κB signalling pathway, intestinal microbiota and aquaculture water quality of Pelteobagrus vachelli
Anuar et al. Effect of commercial probiotics on the survival and growth performance of goldfish Carassius auratus.
KR100857771B1 (ko) 바실러스 폴리퍼멘티쿠스, 바실러스 리케니포르미스 및사카로마이세스 세르비지에를 포함하는 어류사료첨가용조성물
WO2019040266A1 (fr) Agents microbiens alimentés directement pour améliorer l'état général et la santé des poissons
Hassanien et al. Physiological and haematological responses of the Nile tilapia (Oreochromis niloticus) fed on diets supplemented with probiotics
Ghosh et al. Feed utilization efficiency and growth performance in rohu, Labeo rohita (Hamilton, 1822), fingerlings fed yeast extract powder supplemented diets
KR102174589B1 (ko) 뱀장어 사료내 항생제 대체를 위한 신바이오틱스 개발

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18759482

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112020003799

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112020003799

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20200221

122 Ep: pct application non-entry in european phase

Ref document number: 18759482

Country of ref document: EP

Kind code of ref document: A1