Method for improving water quality of aquaculture pond by using nutrient germinator composition and spore hatching method
阅读说明：本技术 用营养萌发剂组合物和芽胞孵化法改善水产养殖池塘水质的方法 (Method for improving water quality of aquaculture pond by using nutrient germinator composition and spore hatching method ) 是由 查尔斯·J·格林沃尔德 加布里埃尔·F·K·埃弗里特 朱迪·普鲁伊特 阿曼达·罗斯马里恩 约 于 2019-01-31 设计创作，主要内容包括：一种通过向池塘水中添加使用营养萌发剂组合物优选从芽胞原位萌发的活性细菌来改善水产养殖应用中池塘水质量的方法,以及一种结合硝化增强剂如碳酸钙或钙化的海藻,和任选的反应表面积改性剂,例如钙化的海藻或塑料或金属颗粒或碎片提高芽胞萌发效率的孵化方法。该营养萌发组合物包含L-氨基酸、D-葡萄糖和/或D-果糖、磷酸盐缓冲液、工业防腐剂,并且可以包括细菌芽胞(优选一种或多种芽孢杆菌属物种)或它们可以单独组合以用于萌发。该孵化方法包括将营养萌发剂组合物和细菌芽胞加热到35℃至60℃的温度范围约2至60分钟,以产生排放到水产养殖应用中的经孵化的细菌溶液。(A method for improving pond water quality in aquaculture applications by adding to the pond water active bacteria that germinate preferentially in situ from spores using a vegetative germination composition, and a hatching method for improving spore germination efficiency in combination with a nitrification enhancer such as calcium carbonate or calcified seaweed, and optionally a reactive surface area modifier such as calcified seaweed or plastic or metal particles or fragments. The nutritional germination compositions comprise L-amino acids, D-glucose and/or D-fructose, phosphate buffer, industrial preservatives, and may include bacterial spores (preferably one or more bacillus species) or they may be combined separately for germination. The hatching method comprises heating the nutrient germinant composition and bacterial spores to a temperature in the range of 35 ℃ to 60 ℃ for about 2 to 60 minutes to produce a hatched bacterial solution for discharge into an aquaculture application.)
1. A method of adding bacteria to water for aquaculture applications, the method comprising:
providing a volume of a nutritional germinant composition and a volume of bacterial spores, which may be pre-mixed together or separated as a nutritional spore composition;
optionally, if separate, mixing a portion of the nutritional germinant composition and a portion of the bacterial spores to form the nutritional spore composition;
heating a portion of the vegetative spore composition to a temperature in the range of about 35 ℃ to 60 ℃ at or near an aquaculture application site;
maintaining the temperature within a hatching period range of about 2 minutes to about 6 hours to form a batch of the germinated bacterial solution;
periodically repeating the heating and maintaining steps during the treatment cycle to form additional batches of the germinating bacteria solution;
dispersing each batch of the germinating bacteria solution into water for said aquaculture application;
providing a nitrification enhancer;
dispersing the nitrification enhancer in water simultaneously with at least one batch of the germinating bacteria solution; and
wherein the bacteria are used to remediate the water by degrading organic waste and inhibiting the growth of pathogenic bacteria, or the bacteria are probiotics of a species used in the aquaculture application.
2. The method of claim 1, wherein the bacteria is selected from the group consisting of Bacillus (Bacillus), bacteroides (Bacteriodes), Bifidobacterium (Bifidobacterium), leuconostoc (luecomotoc), Pediococcus (Pediococcus), Enterococcus (Enterococcus), Lactobacillus (Lactobacillus), Megasphaera (Megasphaera), Pseudomonas (Pseudomonas), and Propionibacterium (Propionibacterium).
3. The method of claim 2, wherein the bacteria is one or more species of Bacillus licheniformis (Bacillus licheniformis) and Bacillus subtilis (Bacillus subtilis).
4. The method according to claim 2, wherein the probiotic bacteria are selected from the group consisting of Bacteroides amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus licheniformis (Bacillus licheniformis), Bacillus pumilus (Bacillus pumilus), Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus coagulans (Bacillus coagulosus), Bacillus megaterium (Bacillus megaterium), Bacillus ruminis (Bacillus ruminicola), Bacteroides (Bacillus adolescentis), Bifidobacterium (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium infantis (Bifidobacterium bifidum), Bifidobacterium thermophilum (Bifidobacterium bifidum), Bifidobacterium lactis (Bifidobacterium), Lactobacillus plantarum (Enterococcus), Bacillus brevis (Enterococcus), Lactobacillus brevis (Lactobacillus), Lactobacillus intermedium), Lactobacillus brevis (Lactobacillus), Lactobacillus (Lactobacillus), Enterococcus), Lactobacillus (Lactobacillus), Lactobacillus (Enterococcus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus, Lactobacillus buchneri (Lactobacillus buchneri), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei), Lactobacillus cellobiosus (Lactobacillus celecoxib), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactobacillus farinosus (Lactobacillus farinosus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus lactis (Lactobacillus lactis), Lactobacillus pentosus plantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus plantarum (Lactobacillus mesenteroides), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus plantarum (Lactobacillus), Lactobacillus reuteri (Lactobacillus reuteri), Streptococcus suis (Streptococcus reuteri), Streptococcus faecalis (Streptococcus faecalis), Propionibacterium (Peucedanii), Propionibacterium strain (Peucedani), and Propionibacterium (Peucedani).
5. The method of claim 1, wherein 1 wherein the nutritional germinant composition comprises:
an L-amino acid;
one or more buffers comprising phosphate buffer, HEPES, Tris base, or a combination thereof;
industrial preservatives;
optionally D-glucose, or optionally D-fructose, or optionally both D-glucose and D-fructose; and
optionally a source of potassium ions.
6. The method of claim 5, wherein the L-amino acid is L-alanine, L-asparagine, L-valine, L-cysteine, a hydrolysate of soy protein, or a combination thereof.
7. The method of claim 5, wherein the nutrient germinant composition comprises a total of from about 17.8g/L to 89g/L of one or more L-amino acids.
8. The method of claim 5, wherein the nutritional germinant composition and spores are pre-mixed and the nutritional spore composition further comprises spores of a Bacillus species and a germination inhibitor.
9. The method of claim 8, wherein the germination inhibitor or preservative comprises sodium chloride, D-alanine, or a combination thereof.
10. A method as claimed in claim 9 wherein the pre-mixed vegetative spore composition comprises about 29 to 117g/L sodium chloride.
11. A method as claimed in claim 9 wherein the pre-mixed vegetative spore composition comprises about 8 to 116g/L D-alanine.
12. The composition of claim 7, wherein the phosphate buffer comprises about 10-36g/L of monobasic sodium phosphate and about 30-90g/L of dibasic sodium phosphate.
13. The method of claim 1, wherein the nutritional germinant composition or if pre-mixed the nutritional spore composition is a concentrate comprising:
about 8.9-133.5g/L of one or more L-amino acids;
about 0.8-3.3g/L in total of one or more industrial preservatives;
about 40-126 total of one or more phosphate buffers, about 15-61g/L Tris base, or about 32.5-97.5g/L HEPES, or combinations thereof;
optionally, about 18-54g/L of D-glucose, D-fructose, or a combination thereof;
optionally KCl at about 7.4-22.2 g/L; and
optionally one or more bacteria in the form of spores.
14. The method of claim 13, further comprising:
adding a diluent to the nutritional germinant composition or if pre-mixed, the vegetative spore composition prior to or during heating; and
mixing the diluted nutritional germinant composition and the bacterial spores or the diluted nutritional spore composition during the incubation period.
15. The method of claim 14, wherein the concentration of the diluted nutritional germinant composition is from about 0.1% to 10%.
16. The method of claim 1, wherein the nitrification enhancer increases the alkalinity of water, provides increased surface area for biofilm growth of nitrifying bacteria, or both.
17. The method of claim 1, wherein the nitrification enhancer is calcium carbonate, calcified seaweed, or both.
18. The method of claim 17, wherein the nitrification enhancer is provided in the form of pellets, prills, or granules.
19. The method of claim 16, further comprising providing and dispersing additional surface area modifier in the water simultaneously with the at least one batch of active bacteria.
20. The method of claim 19, wherein the surface area modifier is selected from the group consisting of particles or chips of plastic or metal.
21. The method of claim 1, wherein the nitrification enhancer comprises an alkalinity enhancer, and wherein the alkalinity enhancer is dispersed in the water simultaneously with one batch of the germinating bacteria solution on a seasonal basis.
22. A method as claimed in claim 1 wherein the bacterial spores are in a separate spore composition comprising:
one or more Bacillus (Bacillus) species in the form of spores;
about 0.002 to 5.0 wt% of a thickener;
about 0.01 to 2.0 wt% of one or more acids or acid salts, based on the total weight; and
optionally from about 0.00005 to about 3.0 wt% of a surfactant;
wherein percentages are by weight of the spore composition.
23. A method as claimed in claim 5 wherein said bacterial spores are in a separate spore composition comprising:
one or more Bacillus (Bacillus) species in the form of spores;
one or more acids or acid salts; and
a thickening agent.
24. The method of claim 23, wherein the bacillus species is one or more wherein the bacterial species includes one or more of: bacillus pumilus (Bacillus pumilus), Bacillus licheniformis (Bacillus licheniformis), Bacillus amylophilus (Bacillus amylophilus), Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus clausii (Bacillus clausii), Bacillus firmus (Bacillus megaterium), Bacillus solani (Bacillus mesentericus), Bacillus natto (Bacillus subtilis var. natto) or Bacillus eastern (Bacillus toyonensis).
25. A method as claimed in claim 23 wherein the pH of the spore composition is from about 4.5 to about 5.5.
26. The method of claim 23, wherein the acid or acid salt is one or more of acetic acid, citric acid, fumaric acid, propionic acid, sodium propionate, calcium propionate, formic acid, sodium formate, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, or calcium sorbate.
27. A method as claimed in claim 23 wherein the spore composition comprises:
about 0.002 to 5.0 wt% of a thickener;
about 0.01 to 2.0 wt% of one or more acids or acid salts, based on the total weight; and
optionally from about 0.00005 to about 3.0 wt% of a surfactant;
wherein percentages are by weight of the spore composition.
28. The method of claim 4, wherein said incubation period is from about 2 minutes to about 5 minutes.
29. The method of claim 28, wherein the aquaculture application is a growing pond comprising fish or eels.
30. The method of claim 1, wherein said incubation period is about 4 to 6 hours.
31. The method of claim 30, wherein the aquaculture application is a growing pond comprising shrimp.
Technical Field
The present invention relates to the treatment of aquaculture pond water with bacteria that germinate in a nutrient germinator composition and employ the use of dot spore hatching to reduce organic waste, ammonia and disease stress in aquatic animal husbandry applications and to provide probiotics to aquaculture species.
Background
Aquaculture refers to the cultivation of aquatic species for use as food sources for humans or animals. This technique applies some type of control to the natural environment of the farmed species to improve overall yield. This may include artificially hatching species to increase commercial harvest of field animals, hatching and rearing species in closed ponds, and hatching and rearing species in tidal drainage closed areas near the shoreline. Problems associated with this process include: the pollution discharged from the farm will deteriorate the surrounding water quality; product loss due to deterioration of water quality in the feedlot; the pathogenic microorganism-associated disease pressure in the breeding facilities increases. Such problems can be determined by testing or monitoring various parameters including pH, conductivity, ammonia, nitrate, phosphate and alkalinity. Conductivity is an indicator of salt content, amounts greater than 1200ppm are no longer considered fresh water; the desired amount is 700ppm and the range is 300 and 1200 ppm. Ammonia levels measure the amount of available oxygen in fish. High levels of ammonia prevent the transfer of oxygen from gills to blood in fish; however, this is also a product of their metabolic waste products. Although the ammonia in fish waste is typically not concentrated enough to be toxic in itself, fish farmers must closely monitor the ammonia levels due to the high concentration of fish in each pond. Nitrifying bacteria in the pond consume oxygen and these bacteria break down toxic ammonia into non-toxic forms. However, the use of oxygen in large quantities reduces the oxygen available for ingestion by the fish. Ammonia levels >1ppm are considered toxic to fish life. In addition, nitrate levels were checked to determine the amount of plant fertilizer in the water. Nitrate can be highly leached from the surrounding soil and can be harmful to children and pregnant women. Nitrate becomes nitrite in the gastrointestinal tract and interacts with the oxygen carrying capacity of the blood. The maximum contamination level of nitrate was 10 ppm. Alkalinity is a measure of the ability of a pond or lake to neutralize acids without changing the pH. Alkalinity decreases over time due to bacterial effects; however, the ideal level is 100ppm, and an acceptable range is 50-200 ppm. The phosphates found in ponds and lakes are mainly derived from human and animal waste. Fertilizer run-off is a major source of phosphate found in golf courses and decorative ponds. The elevated levels result in an increase in the rate of eutrophication, which in turn increases sludge production. Proper amount of phosphate can stimulate plant growth, resulting in increased seaweed yield; levels >0.1ppm indicate an increase in plant growth, considered to be beyond acceptable levels.
Current techniques to address these problems include bioremediation, antibiotics, and chemical additives. Typical bioremediation techniques include the application of supplemental bacteria to the water to enhance microbial activity and thereby improve water quality. It is also known to use nitrifying bacteria to enhance the nitrification process to convert toxic ammonia to non-toxic nitrate salts. The addition of chemical additives improves water quality and aids microbial activity by providing additional nutrients and alkalinity. Antibiotics are added to inhibit the growth of pathogenic microorganisms. Problems associated with the current technology include: high cost, poor water quality improvement due to inactive supplemented bacteria, low nitrification due to the presence of organic waste and lack of nitrifying bacteria growth sites, and biological accumulation of antibiotics in the cultured aquatic species.
According to a preferred method disclosed in U.S. application serial No. 14/720,088, a biomass generator can be used to produce live bacteria on site to grow bacteria from a solid bacterial starter material to a useful population. The viable bacteria may then be discharged from the one or more biomass generators into an aquaculture application. Such biomass generators and methods of using them are disclosed, for example, in U.S. Pat. nos. 6,335,191; 7,081,361, respectively; 7,635,587, respectively; 8,093,040, respectively; and 8,551,762, the contents of which are incorporated by reference into this disclosure. However, when used in aquaculture applications, it would be useful to have an alternative method of producing viable bacteria from spores.
Spore germination is a multistep pathogenic process in which spores are effectively awakened or restored from a dormant state to a vegetative growth state. The first step is the step of activating spores and inducing their germination by an environmental signal called germination. The signal may be a nutrient such as an L-amino acid. The vegetative germinants bind to receptors in the inner membrane of spores to initiate germination. Furthermore, sugars have been shown to increase the binding affinity of L-amino acids to their cognate receptors.
The germination signal then initiates a cascade leading to the release of dipicolinic acid (DPA), which is associated with Ca in the core of the bud2+(CaDPA) was stored in a ratio of 1: 1. The release of CaDPA is a rapid process and is usually completed within 2 minutes>90 percent. CaDPA release represents an importation of spores in which they are dedicated to the germination process. This step is referred to by those skilled in the art as the "sizing" step.
After CaDPA release, the spores were partially hydrated and the core pH increased to about 8.0. The core of the spore then swells and the cortex (consisting mainly of peptidoglycans) is degraded by the core lytic enzyme. Spores absorb water and thus lose their refractive index. This loss of refractive index at the end of the germination process allows monitoring of spore germination by phase contrast microscopy.
The second phase of germination is the step of outgrowth where the spore's metabolic, biosynthetic and DNA replication/repair pathways begin. There are several phases in the anagen phase. The first is called the maturation phase, in which no morphological changes (e.g., cell growth) occur, but the molecular mechanisms of the spore (e.g., transcription factors, translation mechanisms, biosynthesis mechanisms, etc.) are activated. The length of this period may vary depending on the initial resource of spore packaging used during the spore formation process. For example, a preferred carbon source for several species of bacillus (including bacillus subtilis) is malic acid, and bacillus spores typically contain large amounts of malic acid for use during resuscitation. Interestingly, deletion mutants that cannot utilize the malate pool showed an extended maturation period compared to wild-type spores, indicating that the spore malate pool was sufficient to initiate the initial outgrowth process. In addition, spores store small acid soluble proteins that degrade within the first minutes of resuscitation to serve as a direct source of amino acids for protein synthesis. After the outgrowth step, spore resuscitation is complete and the cells are considered to be asexually grown.
It is known that spore germination can be induced by heat activation. Spores of various bacillus species are thermally activated at strain-specific temperatures. For example, spores of Bacillus subtilis (B.subtilis) were heat-activated at 75 ℃ for 30 minutes, whereas spores of Bacillus licheniformis (B.licheniformis) were heat-activated at 65 ℃ for 20 minutes. Thermal activation has been shown to cause transient, reversible unfolding of the spore coat protein. The heat-activated spores may then be germinated for additional time in a germination buffer containing a nutrient germinant (e.g., L-alanine). However, if no vegetative germinant is present, the spores will return to their pre-heated non-germinated state.
It is also known that germination can take place at ambient temperature (close to typical room temperature) without heat activation and a germination buffer containing nutrients, but that this process usually takes longer than heat activation. For example, bacillus licheniformis (b. licheniformis) and bacillus subtilis (b. subtilis) spores will germinate at 35 ℃ or 37 ℃, respectively, but take longer (e.g., 2 hours) in germination buffer with a vegetative germinant. In addition, it is known that non-heat-activated spores of Bacillus subtilis (B.subtilis) have been placed under non-nutrient germinant conditions (e.g., CaCl)2+Na2DPA) for an extended period of time.
It is also known to germinate spores in a laboratory environment by combining the use of heat activation and a nutritional germinant in a two-step process. First, spores are thermally activated by incubation at a temperature of 65-75 ℃ for a period of time (e.g., 30 minutes) (this particular temperature is species dependent). The spores are then transferred to a buffered solution containing a nutrient germinant, such as L-alanine. It is also known to grow bacteria located in a growth chamber near the site of use by feeding granulated nutrients (containing sugars, yeast extract and other nutrients not directly associated with spore germinants), a bacterial starter and water into the growth chamber at a controlled temperature in the range of 16-40 c, more preferably 29-32 c, for approximately 24 hours of growth as disclosed in us patent No. 7,081,361.
There is a need for a rapid spore hatching and activation method that allows for the generation of viable bacteria, such as bacillus species, in a single step at the point of use where the bacteria will be discharged into aquaculture applications. Thus, the present invention describes a simple method for spore germination using a nutrient germinant concentrate in combination with a spore composition, or using a nutrient spore composition, while performing a hot hatch in a single step for use in aquaculture applications.
Disclosure of Invention
The process of the present invention provides an economical and efficient method to deliver active bacteria to pond water (or growing pond) in an aquaculture facility to degrade organic waste and inhibit the growth of pathogenic microorganisms without bioaccumulation. The method of the invention reduces the disease pressure of water livestock and improves the harvest of cultured species in aquaculture operation. As part of this process, the addition of the nitrification enhancer disclosed herein provides a stable source of alkalinity and additional growth sites for nitrifying bacteria to promote nitrification activity and ammonia reduction.
The method of the invention desirably comprises delivering live bacteria, optionally including probiotics, most preferably from bacteria produced from an on-site incubator using a liquid nutrient germinant concentrate and spore form, into aquaculture applications. A nutritional germinant composition according to a preferred embodiment of the present invention comprises one or a combination of a plurality of L-amino acids, optionally D-glucose, which increases the binding affinity of the L-amino acids to their cognate receptors in the spore coat, and a neutral buffer such as a phosphate buffer and an industrial preservative, e.g. the commercially available Kathon/lingatur CG having active ingredients comprising methylchloroisothiazolinone and methylisothiazolinone. A nutritional germinant composition according to another preferred embodiment of the present invention comprises a combination of one or a combination of two or more L-amino acids, optionally D-glucose, which increases the binding affinity of the L-amino acid to its cognate receptor in the spore coat, HEPES sodium salt (a biological buffer providing an appropriate pH for spore germination), and industrial preservatives, such as propyl and methyl parabens or other us federal GRAS (generally recognized as safe) preservatives. According to another preferred embodiment, the composition further comprises a source of potassium ions, such as potassium chloride or potassium dihydrogen phosphate or dipotassium hydrogen phosphate. According to another preferred embodiment, the composition comprises D-glucose and D-fructose.
According to another preferred embodiment, the nutritional germinant composition further comprises spores of one or more bacterial species, preferably a bacillus species, although other bacteria may be used and including germination inhibitors such as NaCl, industrial preservatives or D-alanine, in combination with any of the above spore composition ingredients. The germination inhibitors prevent spores from prematurely germinating in the nutritional germinant composition. Germination inhibitors may include chemicals that prevent spore germination, such as NaCl, industrial preservatives, or D-alanine.
Alternatively, bacterial spores may be provided separately and added to the nutritional germinant composition according to the invention at the point of use and at the time of hatching. When added separately, it is preferred to provide a stable spore suspension spore composition comprising one or more bacterial species, preferably a bacillus species. According to a preferred embodiment, a germ cell composition comprises bacterial spores, about 0.00005 to 3.0 wt% of a surfactant, about 0.002 to 5.0 wt% of a thickening agent and optionally about 0.01 to 2.0 wt% of an acidifying agent, acid or acid salt (including those used as preservatives or stabilizers), the remainder being water. According to another preferred embodiment, a germ composition comprises bacterial spores, about 0.1-5.0 wt% of a thickening agent, about 0.05-0.5 wt% of an acid or acid salt, optionally about 0.1-20 wt% of a water activity reducing agent, and optionally about 0.1-20 wt% of other acidifying agents (acids or acid salts), the remainder being water.
Most preferably, in two preferred spore composition embodiments, the bacterial spores are a dry powder blend combining about 0.1-10% of 40-60% salt (salt) and 60-40% bacterial spores (prior to addition to the spore composition) by weight of the spore composition preferably comprises about 1.0 × 108To about 3.0 × 108Spore composition (spore suspension) cfu/ml providing about 10 in drinking water when diluted with drinking water (for animal watering applications)4To 106cfu/ml of bacterial strain. Most preferably, the thickener in both preferred embodiments is one that also acts as a prebiotic, such as xanthan gum, to provide additional benefits. Although other commercially available sprouts may be usedThe cellular product, but a preferred spore composition for use in the present invention is disclosed in U.S. application serial No. 14/524,858 filed on 27/10/2014, which is incorporated herein by reference.
According to another preferred embodiment, the nutritional germinant composition according to the present invention is in concentrated form and diluted to 0.01% to 10% strength in water or another diluent at the point of use. The use of a concentrated formulation reduces shipping, storage and packaging costs and makes administration of the spore composition at the point of use easier. Most preferably, the concentrated spore composition is in liquid form which is easier and faster to mix with the diluent at the point of use, but solid forms such as pellets or bricks or powders may also be used. The inclusion of a general purpose industrial preservative in the spore composition aids in long term storage and/or germination inhibition, which is particularly useful when the spore composition is in a preferred concentrated form.
In another preferred embodiment, the invention includes a method of using a nutritional germinant composition in combination with a spore composition or germinating bacillus using a nutritional germinant composition at an elevated temperature; preferably in the range of 35-60 c, more preferably in the range of 38-50 c and most preferably in the range of 41-44 c for a period of time (incubation period). The incubation period is preferably 2-60 minutes or more, depending on the application. Most preferably, the nutritional germinant or the vegetative spore composition in a concentrated form according to a preferred embodiment of the present invention is used in the hatching/germination method of the present invention, but other nutritional germinant compositions and spore compositions may also be used. Preferably, the hatching method is carried out at or near the point of use-at or near the aquaculture site where the germinated spores are used or consumed, and further comprising distributing the germinated spores to the point of use/consumption. A preferred method according to the invention can be carried out in any hatching apparatus having a reservoir capable of holding a volume of spores (if added separately), a liquid (typically water as a diluent), a nutritional germinant composition and capable of heating the mixture during hatching. Most preferably, the method is performed in a device that is also capable of mixing these ingredients, automatically switching off the heating at the end of the incubation period, and automatically dispensing the incubated bacterial solution containing the bacteria to the point of use/consumption of the aquaculture. The preferred process can also be carried out as a batch process or as a continuous process. Although it is preferred to use a spore composition according to the invention, any kind of spore form or product, such as a dry powder form, a liquid suspension or a reconstituted aqueous mixture, may be used with the method of the invention.
Preferred embodiments of the present invention allow for the rapid germination of spores of the genus bacillus at the point of use of aquaculture. The active, nutrient bacteria solution discharged from the hatcher can be supplied directly to the growing pond or can be accumulated and diluted with pond water or other similar suitable diluent (e.g., water from a municipal water supply) prior to discharge to the growing pond. Alternatively, the incubator may be configured to heat the vegetative germinant composition and spores, or the vegetative spore composition, between dormant spores and vegetative bacteria for a incubation period and temperature range that produces bacteria in a metastable state. The metastable bacteria solution is then discharged into a growing pond where the bacteria are able to become viable vegetative bacteria. If the incubator is some distance from the rearing tank, dilution may facilitate delivery of treatment solution from the incubator flow to the rearing tank. The active bacteria will degrade the organic waste and inhibit the growth of pathogenic microorganisms in the water in the aquaculture facility without the need to add (or reduce) the amount of chemical treatment and antibiotics used in the grow-out pond.
The present invention also desirably includes simultaneously applying at least one nitrification enhancer to the feedwell. The nitrification enhancer may increase the activity of naturally occurring nitrifying bacteria in the water, thereby reducing the ammonia content. These nitrating agents include alkalinity enhancers which increase the alkalinity of the water, which is necessary for nitrification (7 parts alkalinity to 1 part ammonia) and/or surface area modifiers to increase the surface on which the nitrifying bacteria grow, as the nitrifying bacteria will grow with the biofilm, requiring a surface to which they can attach. Alkalinity enhancing agents may include, for example, calcium carbonate, calcified seaweed, or other similarly effective additives. These agents may be added in amounts above dissolution to provide a continuous source of alkalinity as they slowly dissolve. By providing alkalinity and high surface area, certain nitrating agents (e.g., calcified seaweed) may act as both an alkalinity enhancer and a surface area modifier, providing a supporting surface for the growth of nitrifying bacterial biofilms. Calcified seaweed may also serve as a micronutrient source for bacteria. Other nitrification enhancers simply act as surface area modifiers, such as plastics or metal parts, or other similarly effective materials, to increase the surface area over which favorable reactions and interactions can occur. One or more agents that act solely as surface area modifiers (but not alkalinity enhancers) may also be added to the growth tank, alone or preferably in combination with one or more alkalinity enhancers; however, the agent that merely acts as a surface area enhancer does not degrade in the nutrient bath, nor is it added with each batch of bacterial solution. Such agents, which only act as surface area modifiers, are preferably added to the growth tank only once. The agent acting as an alkalinity enhancing agent may be added to the growing pond at regular intervals, for example seasonally (once per season or once a year in the summer, twice a year, etc.) or as needed, simultaneously with a batch of bacteria. As used herein, the term "simultaneous" is intended to refer to the time "or about" that time when a batch of vegetative bacteria and other ingredients or reagents are added to a growing pond or other growing medium in which aquatic animal species are grown in an aquaculture facility.
Drawings
The system and method of the present invention will be further described and explained with reference to the following drawings
FIG. 1 is a flow diagram of a hatching system and method according to a preferred embodiment of the present invention;
FIG. 2 is a flow diagram of a hatching system and method in accordance with another preferred embodiment of the present invention;
FIG. 3 is a flow diagram of a hatching system and method in accordance with another preferred embodiment of the present invention;
FIG. 4 is a graph of nitrate levels in a laboratory study;
FIG. 5 is a graph of orthophosphate levels in a laboratory study;
FIG. 6 is a graph of turbidity in a laboratory study
FIG. 7 shows a photograph of a bacterial slide using spore compositions and methods according to preferred embodiments of the invention compared to a control slide;
FIG. 8 is a pO showing confirmation of germination levels using spore compositions and methods according to preferred embodiments of the invention compared to control tests2A graph of test data;
FIG. 9 is a pO showing confirmation of germination levels compared to control tests using spore compositions and variation methods according to a preferred embodiment of the invention2A graph of test data; and is
Fig. 10 shows images of three aquaria, each under control (left), treated with calcium carbonate only (middle) or activated vegetative-spore formulation (right).
Detailed Description
Aquaculture treatment method
According to a preferred embodiment, the active bacteria are generated in situ from a vegetative germination composition with a spore composition or a pre-mixed vegetative spore composition, preferably using an incubator system and preferred germination method as described below, and the active bacteria are periodically fed into the growing pond in aquaculture applications. One or more nitrification enhancers are also added to the growing pond simultaneously with the active bacteria.
Satisfactory bacterial growth and delivery devices for use in the methods of the present invention will include on-site incubator systems, such as air incubators, water incubators, or any other chamber or similar device that provides uniform, constant heat over a given temperature range as needed to germinate spores for discharge into aquaculture applications. Referring to fig. 1, preferably, an on-site incubator system 10 includes one or more tanks or containment vessels for containing an initial amount of a nutritional germinant composition 12 and an initial amount of bacterial spore solution 14 (if bacterial spores are not included in the nutritional germinant composition). These spore compositions may also reach the point of use in a container connectable in fluid communication with the incubator, in which case no separate tank or container is required. A water source 16 at or near the aquaculture site is also optional, but preferably is connectable in fluid communication with the incubator system. A well, municipal water supply or aquarium may provide water 16 to the incubator 18. The incubator system 10 also preferably includes a chamber or container 18 configured to receive the nutritional germinant composition and a portion of the spores and to cause them to be heated to germinate the spores; a heater; valves, tubing and pumps (if necessary, if gravity flow is insufficient) to allow the nutrient germinant composition 12, bacterial spore solution 14 and optional water 16 to flow from its storage container/source into the heating chamber or vessel 18 and to drain the incubated bacterial (or activated bacteria)
Alternatively, according to another preferred embodiment as shown in fig. 2, incubator system 110 uses a concentrated nutritional germinant composition 24 and a
Preferably, bacterial spores are germinated in an incubator 18 or other suitable heating device according to the preferred germination methods described herein. According to a preferred embodiment, the nutritional germinant composition and the spore composition (or the nutritional spore composition) are heated in the incubator 18 to a temperature of 35-55 ℃, more preferably 38-50 ℃, and most preferably 41 ℃ to 44 ℃. The incubation period may vary depending on the end use application, but is preferably between about 20 minutes and 60 minutes to produce viable bacteria for aquaculture applications, and most preferably between about 2 minutes and 5 minutes for probiotic applications producing metastable bacteria. In order to provide additional growth time for the vegetative bacteria, the incubation period may be about 4 to 6 hours.
Depending on the desired use of the bacteria in aquaculture applications, such as probiotics for treating water or aquatic animal species, different incubation periods may be used to provide an incubated bacterial solution of bacteria that is predominantly still in the form of spores, predominantly metastable bacteria (where spores are neither dormant nor in vegetative growth stage, also referred to herein as activated state), or predominantly fully vegetative bacteria. In addition, when fully nutritive bacteria are desired, the bacterial solution may be maintained in the incubator 18 or another intermediate container for a period of time after the incubation period to allow the bacteria to multiply and then be discharged into aquaculture applications. Most preferably the bacterial solution is maintained at a temperature of between 30 and 45 ℃, more preferably the nutrient bacterial solution is heated as required to maintain the temperature of the solution in the range of 33 to 42 ℃ and most preferably 36 ℃ to 39 ℃ to promote growth during this post-hatch growth phase. When probiotic applications are required, the bacteria are discharged into aquaculture applications by using a shorter incubation period, mainly remaining in a spore state or metastable state, which gives the bacteria a better chance to survive into the intestinal tract of aquatic animal species where they are most beneficial as probiotics. At the end of the incubation period, the incubated
Each batch of incubated
According to a preferred embodiment for shrimp aquaculture applications, the effective amount of bacteria in the rearing pond can be from about 1 to about 9 × 102According to another preferred embodiment, the effective amount for shrimp aquaculture applications is from about 1 to about 9 × 102To about 108CFU/mL. according to another preferred embodiment, an effective amount of bacteria incubated in the aquarium can be from about 0.001% to about 2% v/v of the total amount of water in the aquarium, and can be any range or value therein As another example, four additions per day to the aquarium comprises about 1 × 109-1×1010cfu/mL of about 500mL of incubated bacterial solution of bacterial species is sufficient to treat a growing pond containing 100,000 gallons of water. Depending on the size of the pond, based on the condition of the pond, the aquatic organism species, the temperature of the pond, and other factors, other volumes of bacterial solution and dosing intervals may be used to treat the growth pond to achieve a desired effective amount of bacteria in the pond, as will be appreciated by one of ordinary skill in the art.
Multiple incubator systems 10, 110, or 210 can be provided to provide a large amount of incubated bacteria solution to the growth pond, achieve a desired effective amount to be added to the pond, to provide different types of bacteria to the growth pond or at different times or rates, and/or to space the discharged incubated bacteria solution around the perimeter of the growth pond to aid in the dispersal of bacteria through the pond. Pumps or other mixing devices may also be added to the growth tank (if not already installed) to help disperse the incubated bacterial solution (and nitrification enhancer or surface area enhancer) throughout the growth tank.
The on-site incubator is preferably configured to incubate batches of incubated bacterial solution from a container of the nutrient germinant composition/spore composition or nutrient spore composition such that batches of bacteria may be drained at periodic intervals for an extended period of time before starting material needs to be replenished. For example, a container of the nutritional germinant composition 12 may initially contain 0.3 to 3 liters of the nutritional germinant composition, which may be fed into the incubator in increments of about 10 to 100 mL/1 to 24 hours. The container of concentrated nutritional starter solution 24 may initially hold 0.2 to 1 liter of solution, diluted with water/diluent from source 26 or 16 to a ratio of concentrate to water/diluent of about 1:50 to about 1:10, and fed to the incubator every 1 to 24 hours in increments of about 0.1 to 200mL of working nutritional starter solution 28. The containers of bacterial spore solution 14 to be fed separately with the nutritional germinant composition may initially hold 0.6 to 6 litres of solution which may be fed to the incubator in increments of about 20 to 200mL every 1 to 24 hours. The container of concentrated
Preferably, the incubated
As described below, various bacillus species are preferably used with the aquaculture treatment method according to the invention, but other bacteria may also be used. For example, bacterial genera believed to be suitable for use in the methods of the present invention include any one or more species of the genera Bacillus (Bacillus), Bacteroides (Bacteroides), Bifidobacterium (Bifidobacterium), Leuconostoc (Lueconosoc), Pediococcus (Pediococcus), Enterococcus (Enterococcus), Lactobacillus (Lactobacillus), Megasphaera (Megasphaera), Pseudomonas (Pseudomonas) and Propionibacterium (Propionibacterium). Probiotics that may be produced in situ include any one or more of the following: bacteroides amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus licheniformis (Bacillus licheniformis), Bacillus pumilus (Bacillus pumilus), Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus coagulans (Bacillus coagulans), Bacillus megaterium (Bacillus megaterium), Bacillus nidulans (Bacillus ruminolyticus), Bacillus suis (Bacillus suis), Bifidobacterium adolescentis (Bifidobacterium adolescentis), Bifidobacterium animalis (Bifidobacterium animalis), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium (Bifidobacterium thermophilum), Lactobacillus (Enterococcus faecalis), Lactobacillus plantarum (Enterococcus), Lactobacillus brevis (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus brevis (Lactobacillus), Lactobacillus plantarum (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus, Lactobacillus casei (Lactobacillus casei), Lactobacillus cellobiosus (Lactobacillus celebiliosus), Lactobacillus curvatus (Lactobacillus curvatus), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactobacillus farinosus (Lactobacillus farinosis), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus lactis (Lactobacillus lactis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), Leuconostoc mesenteroides (Leuconostoc mesenteroides), Streptococcus megaterium (Megasphaerella elsdenii), Pediococcus acidilactici (Pediococcus acidicii), Pediococcus beer (Pediococcus acidicus), Streptococcus faecalis (Pediococcus faecalis), Lactobacillus salivarius (Propionibacterium), Propionibacterium acidiproducens (Propionium), and Propionibacterium acidiproducens (Pezii).
At least one dose (or batch) of the hatching bacterial solution is discharged into the rearing tank, preferably with the simultaneous addition of one or more nitrification enhancers. Alkalinity enhancers including calcium carbonate or calcified seaweed may be added periodically, such as seasonally or as needed, to reduce phosphate rather than with each dose of bacteria. These agents may be added in amounts above dissolution to provide a continuous source of alkalinity as they slowly dissolve. Slowly dissolving alkalinity enhancers (e.g., calcified seaweed) also act as surface area modifiers, providing a supporting surface for the growth of nitrifying bacterial biofilms, and they also aid in nutrient transport. In addition, agents that only act as surface area modifiers (e.g., metal or plastic flakes) may be added to the growth pond as needed to reduce nitrogen or phosphorous, as well as a batch or dose of hatching bacteria solution and one or more alkalinity enhancing agents, but are preferably added only once, and not with each dose of hatching bacteria solution. These surface enhancers similarly provide a supporting surface for biofilm growth by the added bacteria, which contributes to faster development of beneficial bacteria. Most preferably, about 100 pounds of this nitrification enhancer is added per 750 ten thousand gallons of growing pond, and this amount can be scaled to the volume of the other growing ponds. Preferred methods of dispersion for nitrification enhancers may include the use of automated devices or manual application to the water in the culture pond. Automated or manually operated devices that can be used to broadcast or otherwise disperse at least one nitrification enhancer in the form of pellets, prills, or granules are commercially available and well known to those skilled in the art. Additionally, these nitrification enhancers can be dispersed in ponds using a self-dispersing additive system and method that employs effervescent materials and treatments in a water-soluble package, as described in U.S. patent application serial No. 14/689,790 filed 4-17/2015, which is incorporated herein by reference.
Suitable applications for the method of the invention include, for example, but are not limited to, various types of aquaculture applications, such as hatcheries, ponds and tidal aquaculture. Preferably, the combination of germinating or vegetative bacteria grown in situ with at least one nitrification enhancing aid (e.g. calcium carbonate, calcified seaweed) or another material that is similarly effective for use in aquaculture applications to address organic waste, ammonia and pathogenic microorganisms and general water quality issues. It is believed that the effectiveness of the subject methods to achieve these objectives may be further enhanced by the addition of calcified seaweed or other plastic or metal pieces, particles or fragments that increase the available surface area for interaction or reaction to occur.
A laboratory study was conducted to evaluate the benefits of adding beneficial bacteria and nitrification enhancers to pond water. One objective of this study was to evaluate the inhibitory or herbicidal efficacy of added bacteria (a pond blend commercially available from EcoBionics) on seaweed production. Six 2L beakers were used in this study, each beaker containing 1.5L of source water taken from an existing aquarium in which the algae was present. Each beaker also contains a goldfish, a bubble stone and a light source from the fish tank, the light source is alternatively turned on for 12 hours, and thenClose for 12 hours and observe the glass lid to reduce evaporation losses. According to a preferred embodiment of the invention described below, 37g of pond plus particles are used instead of using an incubator and nutrient germinant composition in the BIO-AmpTMA pond blend bacterial solution is produced in the biomass generator. After a 24 hour growth cycle in the biomass generator, an aliquot of the bacterial solution was obtained and diluted to maintain a 3L pond blend ratio: 579024 gallons of pond water, however, the preferred ratio for use in the field is 3 liters of pond blend: 100,000 gallons of pond water. According to this ratio, about 0.4 μ Ι _ of pond blend bacterial solution was added to a special beaker containing 1.5L of aquarium water. Adding calcified seaweed to a specific beaker based on the rate of clarification according to the manufacturer's instructions; this corresponds to 0.045g of calcified seaweed per 1.5L of water. Equal amounts of calcium carbonate were added to some beakers. The additives in each beaker were as follows:
TABLE 1
Beaker 1
Pond blend of only 0.4. mu.L and source water of 1.5
Beaker
2
Only 0.045g of calcified seaweed and 1.5L of source water
Beaker 3
0.4. mu.L of pond blend, 0.045g of calcified seaweed and 1.5L of source water
Beaker 4
Only 0.045g of calcium carbonate and 1.5L of source water
Beaker 5
0.4. mu.L of pond blend, 0.045g of calcium carbonate and 1.5L of source water
Beaker 6
Used as a negative control, only 1.5L of source water was included
Each test beaker is treated according to a preferred dosing regimen to be used in the art. Beakers 1,3 and 5 filled with pond blend were treated (dosed) with an additional 0.4 μ L of pond blend once a week. Other dosing schedules may be used in the field depending on the growing pond conditions. At the beginning of the study, only primary calcified seaweed and calcium carbonate were added, however, other doses may be used in the field.
One pre-treatment sample (before addition of pond blend bacterial solution, calcified seaweed or calcium carbonate) was removed from each beaker and analyzed to obtain a baseline for comparison with the post-treatment results. Chemical analysis was performed weekly using 200-300mL samples from each beaker. These weekly measurements include analyses of pH, conductivity, nitrate, orthophosphate, total alkalinity, and ammonia levels. The turbidity was checked once a month and photographed to assess changes in seaweed growth and overall clarity. Treatment and analysis of the beaker lasted for a total of three months; again reflecting the length of the field study.
Data analysis was performed using Excel 2003, comparing pre-treatment and post-treatment values at a 95% confidence level using a two-tailed t-test for both samples. The two-tailed t-test of the two samples examined the null hypothesis that the mean values before and after treatment were not different, and the alternative hypothesis that the mean values were different.
Before HO is treated as mu and after HO is treated as mu
Before HA ≠ μ treatment
The baseline readings indicated an increase in phosphate levels in all beakers, which was more than 40 times the level indicating increased growth of the algae. All other measurements are within acceptable ranges.
TABLE 2-results of two samples T-tests for nitrate and phosphate
Signifying significant results
The test beakers containing only the bacterial blend pellets did not change statistically significantly over the three month study period. Phosphate levels declined after two weeks but returned to pre-treatment levels within one month. Nitrate levels reflect those of phosphate. In all test beakers, only beaker 1 had similar increases in nitrate and phosphate levels as the negative control. When bacteria are used alone, this means that the main chemical indicators of pond health are of little impact.
The mean values of the two beakers containing calcified seaweed showed statistically significant changes before and after treatment. The nitrate levels in
The turbidity checked throughout the study indicated a sustained decrease in all test beakers. When comparing pre-treatment and post-treatment pictures, the presence of seaweed increased in all beakers, however, by the second month there was evidence of seaweed death in both beakers. Both beaker 4 and beaker 6 containing calcium carbonate (control) were yellow in color, indicating a dying seaweed system. Algae death is a common problem after initial propagation of algae because oxygen in the water has been depleted; despite the presence of the air stones. Nitrate levels increased significantly as the algae died. This is evident from the increase in nitrate levels in this month above pre-treatment, with 14% and 38% increase in beakers 4 and 6, respectively. By the last month, the nitrate level in beaker 4 recovered and dropped, although the system was still dark green, yellow. Nitrate continued to increase to 6 times the level of contamination in beaker 6. Fig. 4-6 are graphs showing the results of this laboratory study.
Field studies focused on improving overall pond health and clarity while reducing sludge have also been conducted in various ponds. Although the study is not aquaculture specific (since the ponds in the study are ornamental or recreational, and not for rearing and harvesting aquatic species), and the use of a biomass generator rather than an incubator and nutrient germinator composition according to the preferred embodiment of the invention, it provides some useful information regarding the addition of bacteria and nitrification enhancer. This study included Irving, Texas and five ponds in its vicinity; ponds (identified as ponds No. 1-5) ranging from 23,400ft3To 720,131ft3. The length of the study was seven months. From once to twice a week, 200-300mL samples of surface water were withdrawn from the bank of each pond. These samples were analyzed for pH, alkalinity,Nitrate, phosphate, ammonia, conductivity, turbidity and escherichia coli (e. Phosphate, ammonia and turbidity analyses were performed using a Hach Dr890 colorimeter. Coli spp was performed using a special medium for coliform growth (3 mptrifilm 6404) incubated at 35 ℃ for 48 hours.
Each pond was sampled once a month to assess sludge depth, clarity and Dissolved Oxygen (DO). These measurements are taken from two to four locations on a boat and marked with GPS coordinates to obtain representative samples. Measuring the depth of the sludge in inches by using a sludge judging instrument; each GPS position is sampled three to four times and averaged. Dissolved oxygen was measured in ppm from the bottom layer using a Hach LDO probe with a hachq 30d meter and again at 18 "from the surface. Clarity was determined in%/ft using Secchi discs, which gave empirical measurements. In addition, photographs were taken once a month at 2 to 4 locations per pond and re-marked with GPS coordinates to give the customer a view of the overall surface condition.
BioAMP was mounted at each siteTM750 climate controllable biomass generator for daily field metering of a specialized pond blend of bacteria. Use of modified FREE-FLOWTMThe preparation is prepared by granulating Bacillus; the granules were fed to a growth vessel where they were grown under optimal conditions for 24 hours and then dispersed directly into the pond. Maintenance of the BioAMP 750 must be modified according to standard protocols, as sodium hypochlorite (bleach) is considered toxic to surface water and is not permitted by the European market. To obtain similar whitening effect as the standard bleach treatment, 155g of sodium bicarbonate (baking soda) was used to remove excess biofilm and clean the growth vessel. In addition to monthly maintenance, the biomass generator is also monitored for any failures and the ability to maintain the programmed temperature despite ambient temperatures exceeding 100 ° F.
Calcified seaweed was also applied to each pond as a partner for bacterial treatment. The amount of calcified seaweed given is dependent on the ratio of 100lbs to 1,000,000ft3The volume of water of (a). Such as U.S. patent application serial No. 14/689,790, the calcified seaweed is metered using a water soluble package containing an effervescent couplant and the calcified seaweed.
Each study pond received the same number of bacteria per day (30 trillion CFU). correlation matrix showed that sludge depth was inversely proportional to dose rate in the two smaller ponds, the greatest reduction in sludge level and clarity was observed, and the highest bacterial dose per day was 2 × 10, respectively7CFU/L and 7 × 106CFU/L. The clarity observed showed a positive effect on all ponds, regardless of size. In the three smallest ponds of this study, the clarity was about 100%. In contrast, by the end of the study, only 20% clarity was achieved for the two largest ponds.
A one-sided 2 sample t test was used to assess whether the treated sludge level was significantly reduced. The P value was 0.006, and a statistically significant average reduction of 31% was found. H0: before or after μ treatment, HA: before mu treatment>Mu after treatment. This average reduction observed equates to an average 3 inch reduction in the sludge blanket. In addition, the average sludge water level per pond was reduced in this study compared to that before treatment (see table 3).
TABLE 3 Pond PO4、NO3And changes in sludge
Pond
％ΔPO4
％ΔNO3
% delta sludge
1
-19
-71
-45
2
-77
-100
-18
3
-40
0
-16
4
-70
-100
-37
5
-48
0
-43
Average value:
-52
-91
-31
the average change observed in E.coli was a 59% reduction. It is important to note that the entire range includes a decrease of 145% up to 100%. Such a wide range coupled with a small sample size makes it difficult to determine the therapeutic effect on E.coli species. Three of the five study ponds had increased coliform concentrations; however, a dramatic decrease was observed for the other two ponds; however, this increase is believed to be a result of runoff of rainwater into the pond.
The overall effect of the treatment on phosphate concentration was examined and the levels before and after treatment were compared. The data is found to beNonparametric, and a unilateral Mann-Whitney test was used to determine if the phosphate concentration decreased significantly after treatment. H0: before or after μ treatment, HA: before mu treatment>Mu after treatment. A P value of 0.0000 was obtained, indicating that a total 52% reduction in phosphate levels after treatment was statistically significant. Detailed examination of changes in phosphate levels in ponds also showed significant decline. The phosphate concentration reduction range for each treated pond was 19% to 77% (see table 3). The reduction, averaging 52%, was similar to the 57% reduction observed in the first phase of the study. This indicates that an increase in the frequency of bacterial administration may not be associated with a decrease in phosphate concentration. Furthermore, a significant decrease in phosphate levels was observed directly after the administration of calcified seaweed. After the phosphate level began to rise at 6 months, the first dose was administered in the spring and the second dose was administered in the summer. The non-chemical, environmentally friendly nature of the powder product provides promising results for controlling phosphate concentration.
Also, nitrate levels were examined using the unilateral Mann-Whitney test to assess whether the nitrate concentration decreased significantly after treatment. H0: before or after μ treatment, HA: before mu treatment>Mu after treatment. At a P value of 0.0000, a 91% reduction in concentration was determined to be statistically significant (see table 3). For this reason, the baseline nitrate concentration is lower than the recommended level and therefore is not expected to decrease, let alone a statistically significant 91% decrease. Furthermore, by month 4, each pool with detectable nitrate was significantly reduced to below the detection limit of 0.01 ppm. This is a great improvement compared to the reduction observed in the first stage (about 69%), indicating that increasing the frequency of bacterial delivery has a direct effect on these concentrations. Overall, this study showed that enhanced treatment with bacteria and calcified seaweed improved pond health through chemical proxy and reduction of sludge levels.
Nutritional germinator composition
A nutritional germinant composition according to a preferred embodiment of the present invention comprises one or more L-amino acids, D-glucose (which increases the binding affinity of the L-amino acid to its cognate receptor in the spore coat and is optional), D-fructose (optional, depending on the bacterial species), a biological buffer providing a suitable pH for spore germination (e.g. HEPES sodium salt, phosphate buffer or Tris buffer), an optional source of potassium ions (e.g. KCl) and industrial preservatives. In another preferred embodiment, the nutritional germinant composition further comprises D-glucose and D-fructose. When D-glucose and D-fructose are used, it is most preferred to include a source of potassium ions, such as KCl. As understood by those of ordinary skill in the art, the use of D-fructose, a combination of D-glucose and D-fructose, and a source of potassium ions depends on the species of bacteria. It is preferred to use a preservative compatible with the pH of the spore composition, which has a relatively neutral pH. According to another preferred embodiment, the vegetative spore composition further comprises spores of one or more bacillus species and preferably one or more germination inhibitors. A vegetative germinant composition comprising spores is referred to herein as a vegetative spore composition, formulation or solution. Alternatively, spores may be added separately to the nutritional germinant composition according to the present invention at the point of use. When added separately, the spores are preferably part of a spore composition or spore formulation as described herein, but other commercially available spore products may also be used. According to another preferred embodiment, the nutritional germinant or nutritional spore composition is in a concentrated form, most preferably as a concentrate, and diluted at the point of use.
Preferred L-amino acids include one or more of L-alanine, L-asparagine, L-valine, or L-cysteine. The L-amino acids may be provided in the form of any suitable source, for example, in their pure form and/or as a hydrolysate of soy protein. In another embodiment of the concentrated nutritional germinant composition, the L-amino acid may be provided in the form of a hydrolysate of soy protein. When in concentrated form, the spore composition preferably comprises a solution of one or more of the aforementioned L-amino acids in a weight range of about 8.9 to about 133.5g/L, more preferably about 13.2 to about 111.25g/L, and most preferably each about 17.8 to about 89 g/L; d-glucose (optional) and/or D-fructose (optional) each in a weight range of from about 18 to about 54g/L, more preferably from about 27 to about 45g/L, and most preferably from about 30 to about 40 g/L; the weight range of KCl (optionally, as a source of potassium ions) is from about 7.4 to about 22.2g/L, more preferably from about 11.1 to about 18.5g/L, and most preferably from about 14 to about 16 g/L; biological buffers, such as sodium phosphate monobasic in the weight range of about 10 to about 36g/L, more preferably about 15 to about 30g/L, and most preferably about 20 to about 24g/L, and/or sodium phosphate dibasic in the weight range of about 30 to about 90g/L, more preferably about 21.3 to about 75g/L, and most preferably about 28.4 to about 60 g/L. The one or more biological buffers help maintain the nutritional germinant composition at a suitable pH for spore germination, about pH 6-8. In addition to or in place of the monobasic/dibasic sodium phosphate buffer, the spore composition may comprise other phosphate buffer(s), Tris base, in a weight range of about 15 to about 61g/L, more preferably about 24 to about 43g/L, and most preferably about 27 to about 33 g/L; or HEPES buffer in a weight range of about 32.5 to about 97.5g/L, more preferably about 48.75 to about 81.25g/L, and most preferably about 60 to about 70 g/L. Optionally, potassium dihydrogen phosphate may also be used as the source of potassium ions, preferably in a weight range of about 13.6 to about 40.8g/L, more preferably about 20.4 to about 34g/L, and most preferably about 26 to about 29 g/L. Optionally, dipotassium hydrogen phosphate may also be used as the source of potassium ions, preferably in a weight range of from about 8.7 to about 26.1g/L, more preferably from about 13 to about 21.75g/L, and most preferably from about 16 to about 19 g/L. According to another preferred embodiment, the amount of KCl, sodium dihydrogen phosphate and/or disodium hydrogen phosphate may be adjusted such that the pH in the nutritional germinant solution and/or the nutritional spore solution may be about 6, about 7 or about 8.
In another preferred embodiment, the nutritional germinant composition further comprises one or more industrial preservatives in a final (total) weight range of from 0.8 to 3.3g/L, more preferably from 1.2 to 2.7g/L, most preferably from 1.6 to 2.2. Preservative(s) may be beneficial for long term storage. Suitable preservatives include NaCl, D-alanine, potassium sorbate and chemical preservatives. The chemical preservative may be a preservative having the active ingredients of methylchloroisothiazolinone (about 1.15% to about 1.18% v/v) and methylisothiazolinone (about 0.35-0.4% v/v); having as active ingredients diazoalkylurea (about 30%), methylparaben (about 11%) and propylparaben (about 3%)A preservative; and a preservative containing only the active ingredient of methylparaben; and other preservatives containing methylparaben, propylparaben, and diazolidinyl urea). Non-limiting examples of chemical preservatives having methylchloroisothiazolinone and methylisothiazolinone as active ingredients are Linguard ICP and KATHONTMCg (active ingredients comprising about 1.15-1.18% methylchloroisothiazolinone and about 0.35-0.4% methylisothiazolinone). Non-limiting examples of chemical preservatives having diazoalkyl ureas, polyparabens, and methylparabens as active ingredients include Germaben II. When the active ingredients of the chemical preservative are methylchloroisothiazolinone and methylisothiazolinone, the chemical preservative may be included in the concentrated nutrient solution in an amount of from about 0.8 to about 3.3g/L, more preferably from about 1.2 to about 2.7g/L and most preferably from about 1.6 to about 2.2 g/L. Where the active ingredient or ingredients of the chemical preservative are diazoalkyl urea, methyl paraben and/or propyl paraben, the chemical preservative may be included in the concentrated nutritional solution from about 0.3% to about 1% (wt/wt). In some aspects, the amount of chemical preservative with diazolidinyl urea, methylparaben, and propylparaben may be included in the nutritional formulation in an amount of about 10 g/L. In the case of methylparaben, the preservative may be included in the concentrated nutritional solution at about 0.27 to about 1.89g/L, more preferably at about 0.81 to about 1.35g/L and most preferably at about 1.0 to about 1.18 g/L. According to another preferred embodiment, where the nutritional formulation may be used to produce a spore nutrient formulation effective for aquaculture applications of prawns or other shellfish, the preservative may comprise an amount of methylparaben and potassium sorbate. According to another preferred embodiment, the nutrient germinant solution may be used to produce a vegetative spore formulation effect effective on plants and/or wastewater, which vegetative spore formulation may include an amount of Linguard ICP or KATHONTMCG。
According to yet another preferred embodiment, the nutritional germinant composition may further optionally comprise an osmoprotectant compound. A preferred embodiment may include a natural osmoprotectant, tetrahydropyrimidine, produced by certain bacteria. The amount of tetrahydropyrimidine (optional) in the concentrated nutritional germinant composition may range from about 0.625 to about 4.375g/L, more preferably 1.875 to 3.125g/L, and most preferably in an amount of about 2 to 3 g/L. According to another preferred embodiment, the nutritional germinant composition may further comprise other standard ingredients including, but not limited to, surfactants to aid in dispersing the active ingredients, additional preservatives to ensure shelf life of the spore composition, buffers, diluents and/or other ingredients typically contained in the nutritional and/or spore formulations.
The amounts of the above components are an important aspect of the present invention, as higher concentrations render some components insoluble and lower concentrations are ineffective for germinating spores.
According to another preferred embodiment, the nutritional starter concentrate composition according to an embodiment of the present invention is in a concentrated form and is diluted to a working solution in water, a spore composition or any other suitable diluent or combination thereof prior to germination at the point of use as described further below according to various preferred embodiments, a working nutritional starter solution may be prepared by diluting the concentrated nutritional starter composition according to a preferred embodiment of the present invention with water or other suitable diluent at a ratio of 0.01% to 50% (v/v) concentrated nutritional starter to diluent, but other amounts may be used6Fold or any range or value therein to produce a working nutritional germinant solution. Most preferably, the dilution is from about 0.1% to about 10% of the concentrate and balance water or other suitable diluent. The amount of the above-mentioned ingredients present in the working nutrient solution (diluted solution from the concentrated formulation) can be calculated based on the above-mentioned dilution factor and the amount of concentration.
The use of a concentrated nutritional germinant composition reduces shipping, storage and packaging costs and makes administration of the spore composition at the point of use easier. Most preferably, the concentrated nutritional starter composition is in liquid form which is easier and faster to mix with the diluent at the point of use, but solid forms such as pellets or bricks or powders may also be used. The inclusion of typical industrial preservatives in the nutritional germinant composition aids long term storage.
Most preferably, all ingredients in the nutritional germinant composition according to or for use with the methods of the present invention meet us federal GRAS standards.
Nutrient spore composition
According to another preferred embodiment, the nutritional spore composition preferably comprises 10% to 90% by weight of one or more bacillus spores or a blend of spores (comprising 40-60% of spore powder with one or more bacillus species and 60-40% of salt) according to another preferred embodiment, the nutritional spore composition comprises about 5% by weight of one or more bacillus spores or a blend of spores according to another preferred embodiment the total concentration of spores in the nutritional spore composition may be at about 1 × 105CFU/mL or spore/g to 1 × 1014CFU/mL or spores/g or any particular concentration or range therein.
The vegetative spore composition preferably further comprises one or more germination inhibitors and/or preservatives. Preferred germination inhibitors or preservatives for concentrated vegetative spore compositions include a relatively high concentration of NaCl ranging from about 29 to about 117g/L, more preferably from about 43 to about 88g/L, and most preferably from about 52 to about 71 g/L; and/or D-alanine in an amount of from about 8 to about 116g/L, more preferably from about 26 to about 89g/L, and most preferably from about 40 to about 50 g/L; and/or potassium sorbate in an amount of from about 1.25 to about 8.75g/L, more preferably from about 3.75 to about 6.25g/L, and most preferably from about 4.5 to about 5.5 g. Other chemical preservatives described above with preferred nutritional germinant compositions may also be used with the vegetative spore compositions according to the present invention. These germination inhibitors or preservatives keep the spores inactive and prevent the spores from prematurely germinating before dilution and activation at their point of use. When a spore composition according to this embodiment is used with the preferred method of the invention, the use of a germination inhibitor is particularly preferred, wherein germination occurs at the point of use. When spores are included, the amount of other ingredients (e.g., L-amino acids, biological buffers, etc.) of the above-described nutritional germinant composition make up for a proportional reduction in the balance of the spore composition to correspond to the above-described preferred ranges. Preferred vegetative spore compositions are also in concentrated form and are diluted into a working solution at the point of use as described above for the vegetative germinant composition and further described below.
Preferred bacillus spores for use in a vegetative spore composition according to a preferred embodiment of the present invention include the following species: bacillus licheniformis (Bacillus licheniformis), Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus polymyxa (Bacillus polymyxa), Bacillus thuringiensis (Bacillus thuringiensis), Bacillus megaterium (Bacillus megaterium), Bacillus coagulans (Bacillus coagulans), Bacillus bradycardia (Bacillus lentus), Bacillus clausii (Bacillus clausii), Bacillus circulans (Bacillus circulans), Bacillus firmus (Bacillus firmus), Lactobacillus plantarum (Bacillus lactis), Bacillus laterosporus (Bacillus laterosporus), Bacillus lactis (Bacillus laevicola), Bacillus polymyxa (Bacillus polymyxa), Bacillus pumilus (Bacillus pumilus), Bacillus simplex (Bacillus pumilus) and Bacillus sphaericus (Bacillus sphaericus). Other bacillus spore species may also be used, as will be appreciated by those of ordinary skill in the art. Preferably, the vegetative spore composition comprises 1 to 20 or more species of bacillus, more preferably 3 to 12 species of bacillus. According to another preferred embodiment, the vegetative Bacillus composition comprises 3 strains of Bacillus bacteria, most preferably 2 strains of Bacillus (Bacillus) bacteria, each of which may be a strain of the species Bacillus licheniformis (Bacillus licheniformis) and the third strain is a species Bacillus subtilis (Bacillus subtilis). According to another preferred embodiment, the spores in the spore blend comprise about 80% bacillus licheniformis (40% of each strain) and 20% bacillus subtilis.
In another preferred embodiment, a vegetative spore composition for use as a probiotic comprises one or more strains of bacillus which are probiotic in nature, as they assist in the breakdown of nutrients in the consumer's digestive tract. The strain preferably produces one or more of the following enzymes: proteases that hydrolyze proteins, amylases that hydrolyze starches and other carbohydrates, lipases that hydrolyze fats, glycosidases that help hydrolyze glycosidic bonds in complex sugars and help degrade cellulose, cellulases that degrade cellulose to glucose, esterases (enzymes similar to lipases), and xylanases that degrade xylans, polysaccharides found in plant cell walls. Bacillus strains that produce these enzymes are well known in the art.
According to another preferred embodiment, the vegetative spore composition is in a concentrated form and is diluted to a working solution in water, or any other suitable diluent or combination thereof prior to germination at a point of use as described further below according to various preferred embodiments, a working vegetative spore solution may be prepared by diluting a concentrated vegetative spore composition according to the preferred embodiments herein with water or other suitable diluent at a ratio of 0.01% to 50% (v/v) concentrated vegetative germination agent to diluent, although other amounts may be used13Fold or any range or value therein to produce a working nutritional germinant solution. Most preferably, the dilution is from about 0.1% to about 10% of the concentrate and balance water or other suitable diluent. The amounts of the above-mentioned ingredients (e.g., L-amino acid and germination inhibitor) present in the working nutrient solution (diluted solution from the concentrated preparation) can be calculated based on the above-mentioned dilution factor and concentration amount.
Most preferably, all of the ingredients in the vegetative spore composition according to or used with the method of the invention meet federal GRAS standards in the united states.
Spore composition
A probiotic spore composition according to a preferred embodiment of the present invention comprises one or more bacterial species, optionally a surfactant, a thickening agent, and optionally one or more acidulants, acids or salts of acids to act as preservatives. According toIn another preferred embodiment, the spore composition further comprises one or more prebiotics, either in the sense that the thickening agent is not a prebiotic, or in addition to any thickening agent that is a prebiotic5CFU/mL or spore/g to 1 × 1014CFU/mL or spores/g or any particular concentration or range therein.
According to a preferred embodiment, a suitable thickening agent is included in the spore composition. The thickener is preferably one that does not separate or degrade at the varying temperatures typically found in non-climate controlled aquaculture environments. The thickening agent helps to stabilise the suspension so that the bacterial mixture remains homogeneous and dispersed in the volume of spore composition and does not settle out of the suspension. When used with the hatching system and aquaculture treatment methods of the preferred embodiments of the invention described herein, this can ensure that the concentration of probiotic material is evenly distributed throughout the container, thereby maintaining a consistent or relatively consistent spore dose delivered to the hatcher (depending on the particular delivery method and control mechanism used throughout the treatment cycle).
The most preferred thickener is xanthan gum, which is a polysaccharide composed of glucose, mannose and glucuronic acid and pentasaccharide repeat units of known prebiotics. Unlike some other gums, xanthan gum is very stable over a wide range of temperatures and pH. Another preferred thickener is gum arabic, which is also known as a prebiotic. Other preferred thickeners include locust bean gum, guar gum and gum arabic, which are also considered prebiotics. In addition to the benefits of prebiotics, these fibers do not bind to minerals and vitamins and therefore do not limit or interfere with their absorption, and may even improve the absorption of certain minerals (e.g., calcium) by aquatic species. Other thickeners not considered prebiotics may also be used.
Preferred embodiments may optionally include one or more prebiotics, preferably if the thickener used is not a prebiotic, but may be used in addition to a prebiotic thickener. Prebiotics are classified as disaccharides, oligosaccharides and polysaccharides and may include inulin, Fructooligosaccharides (FOS), Galactooligosaccharides (GOS), Transgalactooligosaccharides (TOS) and short chain fructooligosaccharides (scFOS), soy fructooligosaccharides (soyFOS), fructooligosaccharides, Glyco-oligosaccharides, lactitol, maltooligosaccharides, xylooligosaccharides, stachyose, lactulose, raffinose. Mannooligosaccharides (MOS) are prebiotics that may not enrich the probiotic bacterial population, but bind to and remove pathogens from the gut and are thought to stimulate the immune system.
Preferred embodiments also preferably include one or more acidulants, acids or salts of acids to act as preservatives or acidified spore compositions. Preferred preservatives are acetic acid, citric acid, fumaric acid, propionic acid, sodium propionate, calcium propionate, formic acid, sodium formate, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate and calcium sorbate. Other known preservatives may also be used, preferably food preservatives that are Generally Recognized As Safe (GRAS). Preferably, the pH of the spore composition is about 4.0 to 7.0. More preferably it is about 4.0 to 5.5, most preferably about 4.5 to prevent premature germination of spores before use or addition to the incubator as described below. Lowering the pH of the spore composition may also have antimicrobial activity against yeast, mold and pathogenic bacteria.
According to any preferred embodiment, one or more water activity reducing agents, such as sodium chloride, potassium chloride or corn syrup (70% solution of corn syrup) is optionally included in the spore composition. The water activity reducing agent helps inhibit the growth of microorganisms so that bacterial spores do not germinate prematurely while being stored prior to the time the spore composition is discharged to the point of consumption by the animal or plant to be treated or prior to discharge to the point of use in the growing pond. They also help inhibit the growth of contaminating microorganisms.
The optional surfactant is preferably one that is safe for ingestion by animals, although other surfactants may be used for other applications, such as delivery to plants. Most preferably, the surfactant is polysorbate 80. Although any GRAS or AAFCO approved surfactant or emulsifier may be used with any of the embodiments, there is a concern that some animals may not be well tolerant of all approved surfactants. Because of the benefit of surfactants in stabilizing the suspension, the bacterial mixture remains homogeneous and does not settle, and can also be achieved by using thickeners, thus eliminating the need for adding surfactants. If a surfactant is used in the spore composition according to this second embodiment, it is preferably used in the same weight percent range as the first embodiment.
Preferred bacteria for use in the spore compositions according to the invention are the same as described above for the preferred vegetative spore compositions. Most preferably, the bacterial species used in the spore composition is one or more species from the genus bacillus. The most preferred species of probiotic bacteria include the following: bacillus pumilus (Bacillus pumilus), Bacillus licheniformis (Bacillus licheniformis), Bacillus amylophilus (Bacillus amylophilus), Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus coagulans (Bacillus coagulosus), Bacillus clausii (Bacillus clausii), Bacillus firmus (Bacillus firmus), Bacillus megaterium (Bacillus megaterium), Bacillus solani (Bacillus mesentericus), Bacillus natto (Bacillus subtilis var. natto), or Bacillus orientalis (Bacillus oyonensis), but any Bacillus species recognized as probiotic in the country of use may also be used. It is preferred that the bacteria are in spore form, as spore forms are more stable to environmental fluctuations such as environmental temperature changes. Most preferably, the spores used in the spore composition according to the invention are a dry powder blend comprising about 40-60% salt (salt) and 60-40% bacterial spores. Preferably, the spores are spray dried from the liquid fermentation concentrate. Pure spray-dried spore powder was diluted with salt to a standard spore count in the final spore powder mixture. During the production fermentation, different bacillus strains will grow at different rates, resulting in a change in the final count of fermentation batch. The fermentation broth was centrifuged to concentrate the spores in the liquid. The concentrate was then spray dried, which resulted in a powder containing only bacillus spores. The addition of salt to the spray dried bacillus spore powder helps to standardize the spore blend count per gram between batches. Other forms of bacterial spores or spore blends may also be used. Most preferably, the dried spore blend is premixed with a portion of the water used in the spore composition, about 3-30% of the total water amount, and the resulting bacterial spore solution is added to the other ingredients, including the remaining water. This helps to disperse bacterial spores throughout the spore composition.
The probiotic spore composition according to the first preferred embodiment of the present invention preferably comprises bacterial spores which provide 108Spore suspension at cfu/ml (most preferably about 1.0 × 10)8To about 3.0 × 108Spore composition of cfu/ml providing about 10 when diluted in a nurturing pool1To 104cfu/ml pond water), 0.00005 to 3.0% of a surfactant and 0.002 to 5.0% of a thickener, and optionally about 0.01 to 2.0% of one or more acids or acid salts as preservatives, all weight percentages of spore composition. A probiotic spore composition according to another preferred embodiment of the present invention comprises bacterial spores which provide 109Spore suspension of cfu/ml (which provides about 10 when diluted in pond water1To 104cfu/ml pond water), from about 0.1 to 5.0% of a thickener (preferably also acting as a thickener for prebiotics), from about 0.05 to 0.5% of one or more preservatives, optionally from about 0.1 to 20% of one or more water activity reducing agents, and optionally from 0.1 to 20% of one or more acidulants, all weight percentages of the composition. In two preferred embodiments, the balance of the spore composition is water and the percentages herein are weight percentages. Deionized or distilled water is preferably used to remove salts or external bacteria, but tap water or other water sources may also be used.
According to another preferred embodiment, a Bacillus composition comprises about 1% to 10% of a bacterial spore blend comprising one or more of Bacillus pumilus (Bacillus pumilus), Bacillus licheniformis (Bacillus licheniformis), Bacillus amylophilus (Bacillus amylophilus), Bacillus subtilis (Bacillus subtilis), Bacillus clausii (Bacillus clausii), Bacillus coagulans (Bacillus coagulans), Bacillus firmus (Bacillus firmus), Bacillus megaterium (Bacillus megaterium), Bacillus solani (Bacillus mesentericus), Bacillus subtilis var natto, or Bacillus toyonensis in salt and spore form; about 0.3% to 1% of the total amount of acid or acid salt; about 0.2% to 0.5% of a thickening agent; about 0.1-0.3% sodium chloride, potassium chloride, or a combination thereof; from about 0.00005% to 3.0% of a surfactant; and 86.2% to 98.4% water. According to another preferred embodiment, a spore composition comprises about 0.01% to 10% of a blend of bacterial spores; about 0.1-0.33% sorbic acid, salts thereof, or combinations thereof; about 0.1-0.34% citric acid, a salt thereof, or a combination thereof; about 0.1-0.33% benzoic acid, a salt thereof, or a combination thereof; about 0.2-0.5% xanthan gum; from about 0.00005% to 3.0% of a surfactant; and about 0.1-0.3% sodium chloride, potassium chloride, or a combination thereof, all weight percentages of the composition. According to yet another preferred embodiment, the spore composition comprises about 5% bacterial spores or spore blend; and about 0.25% of a thickening agent; about 0.3% of the total amount of one or more acids or acid salts; about 0.1% surfactant; about 0.2% sodium chloride, potassium chloride, or a combination thereof (other than any salt in the spore blend); and water. According to another preferred embodiment, the acid or acid salt is one or more of potassium sorbate, sodium benzoate and anhydrous citric acid.
Several examples of probiotic spore compositions according to preferred embodiments of the present invention were prepared and tested for different parameters. These spore compositions are listed in table 4 below.
TABLE 4
The remainder of each spore composition is water (about 1L in these samples). The spore composition uses deionized water in addition to spore composition # 1 using tap water the percentages shown are weight percentages.each formulation target has a pH of 4.0 to 5.5, but the actual pH of certain formulations was found to be much lower than expected.formulation 1 target has a pH of 5.0 to 5.5, but its actual pH is about 2.1-2.3, which is too low and potentially harmful to spores, creates stability problems in packaging, and complies with stricter shipping regulations.formulation 1 also exhibits a weak thickening.formulation 2 is the same as formulation 1 except for the source of water.formulation 2 has an actual pH of about 2.2-2.3 and also exhibits a weak thickening.reducing the amount of acid and acid salt in formulation # 3 to increase the pH and determine if there is an improvement in the thickness of formulation while using the same thickener as in formulation # 1 and 2.3 is about a further improvement in the thickness of formulation # 1 and 2, and the thickening of the concentration of the acid in formulation # 1& 2 is not increased by the target value of the concentration of No. 5, and No. the concentration of no acid in formulation # 1& 2, and No. 5, and No. the concentration of No. 4, which is not increased by the concentration of No. 411cfu/gm, formulation number 8 provides 1 × 1011cfu/gm bacterial spores. In these sample formulations, number 8 is most preferred because it shows sufficient thickening, the actual pH is about 4.5+/-0.2, and less spore blend is used.
Preferably, a spore composition according to an embodiment of the invention uses about 0.01% to about 0.3% of the bacterial spore blend, more preferably about 0.03% to 0.1% of the bacterial spore blend. Reducing the amount of spore blend can greatly reduce the cost of the spore composition. Depending on the end use application, different amounts of spore blends may be used in spore compositions according to the invention. For example, a smaller percentage of spore blend may be used in a spore composition for chicken, while a larger percentage may be used in a spore composition for pig.
A shelf life of spore composition according to formulation number 8 at various temperatures was tested.A sample of formulation number 8 was sealed in a plastic bag, such as the one used in the preferred delivery system described below, and stored at temperatures of about 4-8 ℃ (39-46 ° F), 30 ℃ (86 ° F), and 35 ℃ (95 ° F) for two months to simulate typical temperature ranges in which probiotic spore compositions can be stored and used in an agricultural environment8Bacterial spores of cfu/mL at the end of the one month storage period, approximately 2.09 × 10 was contained in the sample8Bacterial spores of cfu/mL spore suspension (lowest temperature sample), 1.99 × 108cfu/mL (medium temperature sample) and 2.15 × 108cfu/mL (high temperature sample.) bacterial counts varied somewhat in different samples, particularly in thickened samples, however, these were considered comparable counts-each sample was tested again after two months of storage-the samples contained about 2.08 × 108cfu/ml (lowest temperature sample); 2.01 × 108cfu/ml (medium temperature sample), and 2.0 × 108Bacterial spores of cfu/ml (high temperature sample) the target shelf life was about 2 × 108cfu/ml spore suspension, so the sample was within the target shelf life after two months of storage. These test results show that probiotic spore combinations according to a preferred embodiment of the present inventionThe spore blend (40-60% spore powder and 60-40% salt) used in each sample formulation is the same, providing at least about 2 × 1011Spores per gram. The spore species in the blend are a plurality of Bacillus subtilis and Bacillus licheniformis strains. The spore blend powder was premixed with 100mL of water for 30 minutes under stirring and then added to the other ingredients. Pre-mixing with water helps mix the spore blend with the other ingredients and disperse the spores throughout the spore composition.
Although the spore compositions according to the present invention preferably use probiotic spore compositions comprising one or more species of Bacillus, the methods of the present invention may be used with spore compositions comprising other bacterial species and other species. Bacillus (Bacillus), Bacteroides (Bacillus), Bifidobacterium (Bifidobacterium), Pediococcus (Pediococcus), Enterococcus (Enterococcus), Lactobacillus (Lactobacillus), and Propionibacterium (Propionibacterium) (including Bacillus pumilus (Bacillus pumilus), Bacillus licheniformis (Bacillus licheniformis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus clausii (Bacillus clausii), Bacillus coagulans (Bacillus natto), Bacillus firmus (Bacillus firmus), Bacillus megaterium (Bacillus megaterium), Bacillus licheniformis (Bacillus megatericus), Bacillus subtilis (Bacillus megaterium), Bacillus subtilis (Bacillus megaterium), Bacillus megaterium (Bacillus megaterium), Bacillus megaterium, bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium thermophilum (Bifidobacterium thermophilum), Pediococcus acidilactici (Pediococcus cerevisiae), Pediococcus pentosaceus (Pediococcus pentosaceus), Enterococcus kei (Enterococcus cremoris), Enterococcus dibutylosus (Enterococcus diacetylactis), Enterococcus faecium (Enterococcus faecalis), Enterococcus intermedius (Enterococcus intemperatus), Enterococcus lactis (Enterococcus lactis), Enterococcus thermophilus (Enterobacterus thermophilus), Lactobacillus thermophilus (Lactobacillus thermophilus), Lactobacillus lactis (Lactobacillus), Lactobacillus brevis (Lactobacillus), Lactobacillus strain (Lactobacillus), Lactobacillus (Lactobacillus brevis (Lactobacillus), Lactobacillus strain (Lactobacillus), Lactobacillus (Lactobacillus brevis (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus strain (Lactobacillus), Lactobacillus (Lactobacillus), Lactobacillus strain (Lactobacillus), Lactobacillus strain (Lactobacillus), Lactobacillus strain, Lactobacillus casei (Lactobacillus casei), Lactobacillus coli (Lactobacillus brevis), Lactobacillus cellobiosus (Lactobacillus cellulobiosus), Lactobacillus curvatus (Lactobacillus curvatus), Propionibacterium (Propionibacterium acidipronii), Propionibacterium freudenreichii (Propionibacterium freudenreichii), Propionibacterium schermanii (Propionibacterium shermanii) and/or one or more of the following species: leuconostoc mesenteroides (Leuconostoc mesenteroides), and Megasphaeraelsdenii (Megasphaeraelsdenii) may be used with the compositions and methods of the invention.
The spore composition may also be in a concentrated form, using less water, with proportionately greater amounts of the other ingredients as described above. Such concentrated spore compositions may be diluted with a nutritional germinant composition, water, other suitable diluent, or a combination thereof at the time of use prior to germination. Most preferably, all of the ingredients in the spore composition according to the invention or for use with the method of the invention meet federal GRAS standards in the united states.
Germination method
According to a preferred embodiment, a method of germinating spores at a point of use according to the present invention includes providing nutrient and spores (preferably providing a nutrient germinator composition and a spore composition or providing a nutrient spore composition according to the present invention, but other commercially available products containing spores and nutrient may be used simultaneously or separately) and heating it to an elevated temperature or range of elevated temperatures and holding it at that temperature or range for a period of time (hatching period) to allow germination at the point of use location near the point of consumption. The heating during the incubation period is performed in one step, heating the nutrients together with the spores. The method preferably further comprises the step of distributing germinated spores into an aquaculture application as described previously. Preferably, the nutritional germinant composition and the spore composition (or the nutritional spore composition) are heated to a temperature in the range of 35-55 ℃, more preferably in the range of 38-50 ℃, most preferably in the range of 41-44 ℃. The incubation period may vary depending on the end use application. For probiotic applications, where an aquatic species having a digestive system (e.g., fish or eel) is to ingest bacteria, it is preferred that the incubation time last no more than 10 minutes. Most preferably, in probiotic applications, the incubation period is 2-5 minutes. In this way, spores are released into the rearing tank before the spores are fully germinated and there is a greater chance of surviving into the gut of aquatic animal species where they are most beneficial. To treat water in aquaculture applications, such as may be done in shrimp aquaculture applications, the preferred incubation time is at least one hour to allow spores to fully germinate before discharge into the water, more preferably 4 to 6 hours to allow bacteria to become vegetative before discharge into the water. Most preferably, the nutritional germinant composition and the spore composition (or the nutritional spore composition) are added to the incubation period to incubate the spores at the preferred temperature ranges and durations described above to produce a bacterial solution having the bacteria in a vegetative state, preferably according to one embodiment of the invention discussed herein. It may be performed in an air incubation period, a water incubation period, or any other chamber that provides uniform, constant heat over a given temperature range. The bacterial solution is then discharged into aquaculture applications as previously described. If a concentrated nutritional starter composition is used, dilution water is preferably added to the incubator with the nutritional starter composition.
Various nutritional germinant compositions according to preferred embodiments of the present invention were tested according to a preferred method of the present invention. The compositions, methods and results are described below.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：用于冷冻脂肪组织的装置