M US 20210137135A1 INI ( 19 ) United States ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2021/0137135 A1 Hastard et al . ( 43 ) Pub . Date : May 13 , 2021 ( 54 ) MICROBIAL - BASED PROCESS FOR Publication Classification IMPROVED QUALITY PROTEIN ( 51 ) Int. Ci . CONCENTRATE A23 ) 1/14 ( 2006.01 ) A23 ) 3/16 ( 2006.01 ) ( 71 ) Applicant: Prairie AquaTech LLC , Brookings, SD A23 ) 3/30 ( 2006.01 ) ( US ) A23K 10/12 ( 2006.01 ) A23K 10/18 ( 2006.01 ) ( 72 ) Inventors: Dennis Hastard , Brookings, SD (US ); A23K 20/147 ( 2006.01 ) Sergio F. Nates, Haymarket, VA ( US ) A23K 50/80 ( 2006.01 ) A23L 33/185 ( 2006.01 ) ( 73 ) Assignee: Prairie Aqua Tech LLC , Brookings , SD A23 ) 3/22 ( 2006.01 ) ( US ) ( 52 ) U.S. CI . CPC A23J 1/148 ( 2013.01 ) ; A23J 3/16 ( 2013.01 ) ; A23J 3/30 ( 2013.01 ) ; A23K 10/12 ( 21 ) Appl. No .: 17 / 093,557 ( 2016.05 ) ; A23V 2002/00 ( 2013.01 ) ; A23K 20/147 ( 2016.05 ) ; A23K 50/80 ( 2016.05 ) ; ( 22 ) Filed : Nov. 9 , 2020 A23L 33/185 ( 2016.08 ) ; A23J 3/227 ( 2013.01 ) ; A23K 10/18 ( 2016.05 ) Related U.S. Application Data ( 57 ) ABSTRACT ( 60 ) Provisional application No. 63 / 052,745 , filed on Jul . The present invention describes a bio - based process to 16 , 2020 , provisional application No. 63 / 039,694 , produce high quality protein concentrate ( HQPC ) by con filed on Jun . 16 , 2020 , provisional application No. verting plant derived celluloses and carbohydrates into bio 63 / 036,274 , filed on Jun . 8 , 2020 , provisional appli available protein via aerobic incubation , including the use of cation No. 63 / 035,797 , filed on Jun . 7 , 2020 , provi such HQPC so produced as a nutrient, including use as a fish sional application No. 62 / 932,684 , filed on Nov. 8 , meal replacement in aquaculture diets . 2019 . Specification includes a Sequence Listing. Fresh Water cooking White Flakes extrusion pH 5 30 min incubation sarcharification { noculum 32 pH 3 50CPH 5 2-4 days 3-5 h precipitation centrifugation 10 min Ethanol Supernatant Condensate RO evaporation distillation Condersed Solids final drying Patent Application Publication May 13 , 2021 Sheet 1 of 17 US 2021/0137135 A1 Fresh Water Acid cooking extrusion incubation Saccharification 32 ° C PH3 centrifugation Supernatant Condensate H20 evaporation Condensed Solids HO - SPC FIG . 1 Patent Application Publication May 13 , 2021 Sheet 2 of 17 US 2021/0137135 A1 Hydrolysis and Aurobasidium : FIG . 2 Patent Application Publication May 13 , 2021 Sheet 3 of 17 US 2021/0137135 A1 80 40 FIG . 3 Patent Application Publication May 13 , 2021 Sheet 4 of 17 US 2021/0137135 A1 FIG . 4 Patent Application Publication May 13 , 2021 Sheet 5 of 17 US 2021/0137135 A1 9 ? W FIG. 5 Patent Application Publication May 13 , 2021 Sheet 6 of 17 US 2021/0137135 A1 103(d) Boves 106 Sep4 105 104 SeedTrain 102(d) 103(0) Mix4 Centrate4 3Sep 103(b) 102(c) FIG.6 sep Mix3 100 Centrate3 ProductionProcess 102(b) Condensate Mix2 1Sep 1 Centrate 103(a) Centrate2 Mix1 Mill 107 101 108 SOV 102(a) colonies Patent Application Publication May 13 , 2021 Sheet 7 of 17 US 2021/0137135 A1 102(b) 103(a) Mix2 Sep1 1 Centrate FIG.7 Centrate1 TOT Mix1 107 ??? 102(a) EVAP SON Subles 108 100a)( Patent Application Publication May 13 , 2021 Sheet 8 of 17 US 2021/0137135 A1 103(d) 104 4Sep 102(d) 4Centrate 103(c) Fermentation FIG.8 Mix4 Centrate4 3Sep evan, 108 100(b) Patent Application Publication May 13 , 2021 Sheet 9 of 17 US 2021/0137135 A1 103(d) water 106 SeparationDiscStackiplids 105 4,Sep SeedTrain 109 104 Uzobroteill'sUFProteins 103(c) Mix4 102(d) Centrate4 Sep3 103(b) 100(c) Mix3 102(c) FIG.9 Sep Centrate3 102(b) Condensate Mix2 Sept 1 Centrate Oilsludge+DeProteins-UF,112 103(a) Centrate2 Mix1 109 Mill 107 1 1 } 1 { 3 ? 1 } ca NF-Sugars 101 102(a) 108 Patent Application Publication May 13 , 2021 Sheet 10 of 17 US 2021/0137135 A1 5 WAVEHUMBER OLON Patent Application Publication May 13 , 2021 Sheet 11 of 17 US 2021/0137135 A1 NAVEHUMBER FIG.11 ? 958 ) Patent Application Publication May 13 , 2021 Sheet 12 of 17 US 2021/0137135 A1 800 0 36 FIG . 12 Patent Application Publication May 13 , 2021 Sheet 13 of 17 US 2021/0137135 A1 FIG . 13a Patent Application Publication May 13 , 2021 Sheet 14 of 17 US 2021/0137135 A1 Negative control FIG . 13b Patent Application Publication May 13 , 2021 Sheet 15 of 17 US 2021/0137135 A1 59,0 53.0 Survival(%) FIG . 14 Patent Application Publication May 13 , 2021 Sheet 16 of 17 US 2021/0137135 A1 FIG . 15 Patent Application Publication May 13 , 2021 Sheet 17 of 17 US 2021/0137135 A1 FIG . 16 US 2021/0137135 Al May 13 , 2021 1 MICROBIAL - BASED PROCESS FOR [ 0005 ] Animal protein producers face a range of sustain IMPROVED QUALITY PROTEIN ability risks . By diversifying into sustainable proteins, these CONCENTRATE businesses can hedge these risks and enter a fast - growing market. CROSS REFERENCE TO RELATED accelerating[ 0006 ] At theand same is creating time , innovationprotein production in food technologyopportunities is APPLICATION with the potential to disrupt the incumbent industry . For [ 0001 ] This application claims benefit under 35 U.S.C. intensive animal protein producers, a failure to engage with 119 ( e ) to U.S. Provisional Application No. 63 / 052,745 , filed this innovation is a risk . Diversification into producing on Jul. 16 , 2020 ; 63 / 039,694 , filed on Jun . 16 , 2020 ; alternative ( i.e. , non - animal) proteins is therefore key for 63 / 036,275 , filed on Jun . 8 , 2020 ; 63 / 035,797 , filed on Jun . both managing the risks of resource - constrained supply 7 , 2020 , and 62 / 932,684 , filed Nov. 8 , 2019 , which are chains and for seizing opportunities for market grow incorporated by reference in their entireties . [ 0007 ] Meat alternatives and broader protein alternatives that can act as substitutes for traditional animal -based food are attracting considerable financial investment. BACKGROUND OF THE INVENTION [ 0008 ] Therefore , a plant - derived protein source which is cost - effective and sustainable , and that is of a high - enough Field of the Invention quality to fully or substantially replace more of the animal protein in an animal diet is needed , including the use of such [ 0002 ] The invention generally relates to incubation pro plant -derived source in meat alternatives in general ( e.g. , for cesses , and specifically microbial - based incubation pro human consumption ) . cesses to produce high quality protein concentrates , includ ing products made therefrom and use of such products in the SUMMARY OF THE INVENTION formulation of nutrient feeds . [ 0009 ] The present disclosure relates to an organic, micro bially - based system to convert plant material into a highly Background Information digestible , concentrated protein source that also contains a microbial gum ( exopolysaccharide) binder , including such a [ 0003 ] In 2008 , approximately 28 % of the world's wild , concentrated source which is suitable for use as a feed for marine fish stocks were overexploited and 52 % were fully animals and a foodstuff for human consumption. exploited , even as the demand for per capita consumption of [ 0010 ] In embodiments, a composition is disclosed includ fish and shellfish products have increased with the increas ing a non - animal based protein concentrate , wherein the ing human population . With dwindling wild fish stocks , in concentrate contains a fermented plant product containing an effort to meet this increased demand, commercial aqua low -pullulan yielding A. pullulans and from at least about culture production has increased dramatically. However, one 65 % to about 75 % protein content ( dry matter basis ) , where of the primary constituents of dietary formulations for the protein concentrate exhibits one or more of the proper aquaculture , fish meal protein , is also derived from wild ties including a degree of hydrolysis ( DH ) of at least about capture fisheries. It was estimated that at least 6.7 mmt of 2 % , an ash content of up to about 4 % , or a potassium and fish meal will be required to support commercial aquaculture magnesium content of less than about 0.1 ppm . production by 2012. This is clearly unsustainable based on [ 0011 ] In one aspect , the A. pullulans produces less than present trends . about 3.0 g / L pullulan when grown in a medium comprising [ 0004 ] Lower cost , more sustainable plant - derived sources between 0.35 and 0.5 g / L yeast extract. of protein have been used to partially replace fish meal in [ 0012 ] In another aspect , the non - animal based protein aquaculture diets . Defatted soybean meal ( SBM , 42-48 % concentrate is isolated from plant material including soy protein ) has commonly been used to replace up to 20 % of beans, sorghum , peanuts , pulses, Rapeseeds, oats , barley, total protein in grower diets for several species , while soy rye, lupins , fava beans, canola , peas , sesame seeds , cotton protein concentrate ( SPC , 65 % protein ) has been tested seeds , palm kernels , barley, grape seeds , olives , safflowers, successfully at higher total protein replacement levels , sunflowers, copra , corn , coconuts , linseed , hazelnuts, wheat, largely governed by the trophic status of the species . These rice , potatoes, cassavas, legumes, camelina seeds , pen soybean products provide high protein and relatively good nycress seeds , mustard seeds , wheat germ meal , corn gluten amino acid profiles, but are still deficient in some critical meal , corn gluten feed , distillery / brewery by - products , and amino acids ( e.g. , taurine ) required by carnivorous marine combinations thereof. fishes . SPC can be used at higher levels than soybean meal , [ 0013 ] In a related aspect , the plant material is from primarily because the solvent extraction process used to soybeans in the form of soy flakes or soy meal . produce SPC removes anti - nutritional factors ( e.g.