Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
• • 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
  • A23K40/00—Shaping or working-up of animal feeding-stuffs
  • A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
• • 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 probiont- and prebiont-loaded zooplankton for improving health, growth, and survival rates of larval fish, fry fish, and crustaceans, by feeding them probiont- or prebiont-loaded zooplankton.
• microbial flora is a rich ecosystem of enormous complexity containing trillions of bacteria divided into more than 400 species, in addition to yeasts, and other organisms.
• bacteria and yeast are routinely introduced into culture medium as a normal part of food consumption (transient microflora) or as contaminants (accidental microflora).
• the bacteria in the aquatic animal gastrointestinal tract can be divided into several functional groups; important among these are probionts, commensals and pathogens.
• the probionts are beneficial, since they participate in a wide variety of positive and health promoting activities in fish physiology (Gildberg et al., 1997; Hansen, 2000; Irianto et al., 2000).
• Commensals establish a community within the gut and provide continuous interaction with the intestinal contents and the animal.
• the pathogens are harmful, as they disrupt the normal gut flora, or invade the animal to cause disease. Normally the probionts and commensals predominate within the gut, providing normal function.
• the probionts produce many bioactive compounds, such as bacteriocins, hydrogen peroxide, lactocidin, acidophilin, and bulgarican, which have antibiotic properties inhibiting or modifying the growth of a wide spectrum of undesirable organisms, pathogens and potential pathogens.
• the metabolic products of probionts provide many useful enzymes and volatile or non-volatile fatty acids to help digestion and absorption of certain minerals, such as phosphorus, calcium, and iron.
• B3 niacin
• B6 pyridoxine
• folic acid folic acid
• biotin folic acid
• the lactic acid-producing bacteria confer significant protection for the fish through various immune mechanisms, which include interactions between the microbes and the host, as well as interactions among microbes.
• Prebiotics do not colonize the gut of the target animal yet can deliver many of the same benefits derived from probionts.
• Prebionts deliver pre-made enzymes, acids, polysaccharides, secondary metabolites, and other metabolic products that influence the composition of the intestine, as well as the overall health of the target organism.
• Crustaceans, rotifers, nematodes, and caldocerans have been used extensively as live feeds for aquaculture, mostly for larval culture of crustaceans and fmfish (Abu-Rezq et al., 2002; Chu and Ozkizilcik, 1995; Harel et al, 1999; Shields et al., 1999). While many groups are searching for alternatives to live feeds, commercial aquaculture still relies heavily on live feeds for larviculture.
• the present invention relates to both a composition of zooplankton loaded with probiotic or prebiotic organisms, and a method for using these specifically loaded zooplankton for aquaculture, including larviculture, in the production of crustaceans ⁇ e.g., shrimp, lobsters, red claw, and crab), mollusks (oysters and abalone), and fish ⁇ e.g., striped bass, sea bass, sea bream, carp, tilapia, catfish, trout, salmon, halibut, and yellow flounder).
• crustaceans ⁇ e.g., shrimp, lobsters, red claw, and crab
• mollusks oysters and abalone
• fish ⁇ e.g., striped bass, sea bass, sea bream, carp, tilapia, catfish, trout, salmon, halibut, and yellow flounder.
• Probiotic organisms such as Bacillus subtilis or Saccharomyces cerevisiae
• zooplankton live feeds such as Artemia franciscana or Brachionus plicatilis, either as single cells or as microencapsulated cells or cell aggregates.
• the feeding method depends on the maintenance of viability needs for specific probionts in the gut of the zooplankton being fed.
• Types of microencapsulation could be a coating, such as, but not limited to, lipid, chitosan, alginate, or starch.
• Prebiotic organisms are envisioned in this invention, and can be delivered in a manner analogous to that described for probionts, with the limitation on encapsulation being the maintenance of bioactivity provided by the specific prebiont.
• the invention provides for zooplankton comprising at least one microencapsulated, viable, bioactive prebiotic or probiotic.
• the zooplankton can comprise crustaceans, including those of the Artemia species, e.g., Artemia salina.
• the zooplankton can also comprise brine shrimp, fairy shrimp, Daphina sp., Daphina magna, Streptocephalus sp., Streptocephalus dichotomus, Branchinella sp., Artemia salina, Artemia sp., Artemia parthenogenetica, Artemia franciscana, or Triops granaries.
• the zooplankton can comprise rotifers, including those of the Brachionus species, e.g., Brachionus plicatilis, Brachionus rotundiformis, and Brachionus calyciflorus.
• the invention further provides that the zooplankton can comprise Copepods, including Eurytemora velox.
• the invention yet further provides that the zooplankton can comprise nematodes.
• the invention provides that the zooplankton can comprise Panagellus redivivus. [021] The invention also provides that the zooplankton can comprise caldocerans.
• the invention further provides that the zooplankton can comprise a Moina species.
• probiotic and or prebiotic can be selected from fish intestinal aerobic or anaerobic microbial flora, and crustacean intestinal aerobic or anaerobic microbial flora.
• the invention provides that the probiotic and or prebiotic can comprise a Clostridium species.
• the invention also provides that the probiotic and or prebiotic can be selected from Aeromonas sp., Aeromonas media, Bacteriodies sp., Carnobacterium sp., Carnobacterium divergens, Carnobacterium sp.
• strain Kl Clostridium sp., Clostridium difficile, Fusobacterium sp., Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus plantarum, Listonella sp., Listonella anguillarum, Bacillus sp., Bacillus cereus, Bacillus cereus var.
• toyoi Bacillus licheniformis, Bacillus megaterium, Bacillus polymxa, Bacillus subtilis, Streptococcus sp., Streptococcus lactis, Streptococcus thermophilus, Bifidobacterium sp., Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Vibrio sp., Vibrio alginolyticus, Vibrio anguillarum, Vibrio carchariae, Vibrio salmonicida-like, Peptostreptococcus sp., Photobacterium sp., Pseudomonas sp., Pseudomonas corrugata, and Pseudomonas fluorescens .
• the invention also provides that the probiotic and or prebiotic can be selected from Saccharomyces sp., Saccharomyces cerevisiae, Pfaffia sp., Pfaffia rhodomonas, Debaryomyces sp., and Debaryomyces hansenii HF1.
• the invention further provides that the probiotic and or prebiotic can be selected from Aspergillus sp., Aspergillus oryzae, Morteriella sp., and Morteriella alpina.
• the probiotic and or prebiotic can be selected from Tetraselmis sp., Tetraselmis suecica, Myrmecia sp., Myrmecia bissecta, Lyngbya sp., Lyngbya majuscula, Cytospora sp., Scenedesmus sp., Scenedesmus obliquus, Scytonema sp., Scytonema hofmanni, Nostoc sp., Nostoc weissfloggia, Chaetoceros sp., Chaetoceros lauderi, Ecklonia sp., Ecklonia maxima, Duneliella sp., Duneliella salina, Duneliella tertiolecta, Duneliella bardiwal, Pseudoanabaena sp., Anabaena sp., Prorocentrum sp.,
• the invention provides a composition comprising the zooplankton of the invention, further comprising a component selected from alginate, chitosan, starch, cornstarch, and potato starch.
• the invention provides a method for delivery of live, bioactive probionts or prebionts to larval fish, juvenile fish, and crustaceans, by providing the probionts or prebionts; and feeding them to said larval fish, juvenile fish, and crustaceans a feed or feed supplement comprising zooplankton further comprising the probionts or prebionts.
• the invention also provides a method wherein the zooplankton are selected from crustaceans, rotifers, caldocerans, and nematodes.
• the invention further provides a method wherein the crustaceans are selected from Artemia sp., Daphina sp., Daphina magna, Streptocephalus sp., Streptocephalus dichotomus, Branchinella sp., Artemia salina, Artemia sp., Artemia parthenogenetica, Artemia franciscana, and Triops granaries.
• the crustaceans are selected from Artemia sp., Daphina sp., Daphina magna, Streptocephalus sp., Streptocephalus dichotomus, Branchinella sp., Artemia salina, Artemia sp., Artemia parthenogenetica, Artemia franciscana, and Triops granaries.
• the invention yet further provides a method wherein the zooplankton are rotifers.
• the invention provides a method wherein the rotifers are selected from Brachionus sp., Brachionus plicatilis, Brachionus rotundiformis, and Brachionus calyciflorus.
• the invention provides a method wherein the probionts or prebionts are processed in a manner that maintains their viability.
• the invention also provides a method wherein the probionts or prebionts are microencapsulated.
• the invention further provides a method wherein the probionts or prebionts are mixed with alginate, chitosan, starch, cornstarch, or potato starch.
• the invention yet further provides a method wherein the probionts or prebionts are cross-linked for stability. [039] The invention provides a method wherein the probionts or prebionts are in an encysted or resting stage.
• the invention provides a method wherein the microcapsules with the probionts or prebionts are sized to a diameter of from about 2 to about 20 microns.
• the invention also provides a composition wherein the zooplankton are produced by culturing on nutrient culture media comprising probiont- or prebiont-containing microcapsules.
• the invention also provides a method of culturing the zooplankton on nutrient culture media comprising probiont- or prebiont-containing microcapsules.
• a "probiont” is an organism that grows stably or transiently in the intestine of the target animal.
• a “probiotic” is a general term applied to the use of probionts to deliver benefit to the target animal.
• Prebiotic refers to an organism grown in such a method to contain one or several compounds that provide a specific health or nutritional benefit to the target animal.
• a “target animal” as contemplated herein refers to a cultured animal to which a probiotic and/or prebiotic is developed and which provides some health or nutritional benefit.
• a “zooplankton” (plural) or “zooplankter” (singular) as contemplated herein, is an organism or organisms belonging to the animal kingdom, found in the water, including either a fresh water or seawater column,. Examples of zooplankton include, but are not limited to, crustaceans, Artemia, rotifers, caldocerans, nematodes, copepods, and ciliates. Zooplankton can adapt to pelagic (open water), littoral (vegetated), and benthic (bottom) environments, and are widespread in both freshwater and marine environments.
• a "crustacean” is any organism of the class Crustacea.
• a "rotifer” is any of a class of minute, usually microscopic, but many-celled aquatic invertebrate animals having the anterior end modified into a retractile disk bearing circles of strong cilia that often give the appearance of rapidly revolving wheels. Rotifers are used as a source of live feed for aquatic animals during their early larval stage.
• Articleemia include any strain belonging to the phylum Arthropoda in the class Crustacea. They are also referred to as "brine shrimp.” These tiny crustaceans can be found in salt lakes and brine ponds throughout the world, and are used as a source of live feed for aquatic animals.
• a "copepod” is any organism of the subclass Copepoda.
• a "nematode” is any organism of the phylum Nematoda.
• Bioactivity is the ability to affect a living organism.
• An “encysted” stage is a period of time in the course of a process in which an organism is enclosed in a small capsule or sac that encloses it in its dormant or larval stage.
• a “resting stage” is not equivalent to the encysted stage since capsule formation is not included in the cells preparation for lower metabolism, h the resting stage the cell has lowered metabolism compared to actively growing organisms.
• a "caldoceran” is an organism formerly grouped in the branchiopod crustacean group.
• the caldocerans are in the class Branchiopoda, subclass diplostraca, order caldocera and family Daphniidae mainidae. Examples of caldocerans are Daphnina magana, D. galeata, D. pulex and Bosmina logirostins.
• Microencapsulation refers to coating, containing, or incorporating in a matrix an organism or organisms in another compound or mixture of compounds such that a cell or aggregate of cells (homogeneous or a mixture) is sealed within a matrix (liquid, solid or gel) that may be used to conveniently deliver the cell or cell aggregate as described in this invention. Any process that binds up the materials to be provided to the zooplankter for eventual consumption by the target organism is contemplated herein.
• load is used synomously with the term “enrich” for the purposes of this invention.
• zooplankter are fed materials that are taken orally but are not meant for metabolism by the zooplankter, rather to be maintained intact in the gastrointestinal tract and consumed by the target species as a mode of delivery of the material provided to the zooplankter.
• Clostridium difficile spores fed directly to Artemia nauplii [061] Clostridium difficile is cultured using standard anaerobic techniques and allowed to form spores (Holdeman and Moore, 1975). Anaerobic medium for growth of Clostridium is made using the following recipe: 1.75 g K 2 HPO 4 , 0.01 g CaCl 2 '2H 2 O, 0.2 g MgSO 4 » 7H 2 O, 0.01 g FeSO 4 « 7H 2 O, 2.0 g L-arginine HC1, 2.0 g L-lysine HC1, 5.0 g yeast extract, 2.0 g sodium formate, 2.0 g NH C1, 0.002 g methylene blue, and 1 L tap water.
• the spores are mixed with an enrichment supplement for Artemia (such as AquaGrow Enhance ® ) as a dried mix or provided as an oil emulsion (mixed with water/oil/Tween 80 and spores in a Beadbeater ® or sonicator) and fed to Artemia nauplii (Cure et al., 1996).
• an enrichment supplement for Artemia such as AquaGrow Enhance ®
• oil emulsion mixed with water/oil/Tween 80 and spores in a Beadbeater ® or sonicator
• Example 2 Clostridium difficile spores delivered to rotifers in bioencapsulated format.
• Clostridium difficile spores are produced as in Example 1. h a reduced medium, alginate is added at from 1 to 8% and dissolved. Calcium chloride at 2-20% is added to induce cross-linking of the gel and the microbeads are spray dried through a fine nozzle to form microencapsulated beads containing spores.
• Microencapsulated spores can be added to zooplankton cultures as a top dressing to existing feeds or mixed directly into the culture medium for Artemia consumption. Once the medium has been cleared, typically in 4-16 hours, preloaded Artemia can be used to introduce viable C. difficile spores into larval fish and crustaceans by direct feeding using the Artemia of this invention as live feed.
• Example 3 Prebiont delivery to rotifers.
• Clostridium difficile spores are produced as in Example 1. Rotifers are cultured in seawater or artificially made seawater at a salinity of 15-40 psu and
• Example 4 Microencapsulation of Lactobacillus fermentum in alginate.
• Lactobacillus fermentum spores are mixed with 1-3% of an emulsifying agent such as phosphatidyl choline, Tween 80, or Santone, and vortexed vigorously to produce a fine emulsion.
• the emulsion is blended at 10-50% into sodium alginate (1-3%) and cornstarch (1-5%) in deionized water.
• the resultant probiont in gel blend is then atomized through a spray nozzle assembly pressurized with nitrogen gas, into a bath of chilled solution containing 2-20% calcium chloride and 1-5% sodium chloride.
• the resultant microcapsules are sieved through 20 ⁇ m
• Example 5 Microencapsulation of Vibrio alginolyticus in alginate plus additional components.
• Vibrio alginolyticus are produced as in Example 4, microencapsulated and dried into a fine powder.
• AquaGrow ARA and 2-20% of fine powder of Vibrio alginolyticus are formulated and packed under nitrogen for later use as zooplankton enrichment feed.
• Example 6 Feeding probiont-loaded rotifers to shrimp larvae.
• Example 8 Feeding probiont-loaded Artemia to sea bream larvae.

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  • 2003-06-10 AU AU2003239484A patent/AU2003239484A1/en not_active Abandoned
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