USE OF UNCONVENTIONAL FEEDSTOCKS AND CO-PRODUCTS OF FOOD PROCESSING TO PRODUCE PROTEIN FOR ANIMAL FEED AND OTHER VALUABLE COMPOUNDS VIA FERMENTATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013943
• Project Start Date: Oct 3, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Food Science

Non Technical Summary
As we try to move from a petroleum-based economy to a more sustainable biobased-economy, we confront a number of challenges that need to be addressed. The National Renewable Energy Laboratory established a list of twelve building block chemicals that can be produced chemically or via fermentation of biomass and eventually be transformed into other compounds by chemical routes. However, in most cases, the methods to produce these compounds economically remain to be developed. Other compounds that are not in this list, such as lactic acid and ethanol, are in high demand and the procedure to produce them are well established. However, the substrates used to produce these compounds via fermentation come from the food supply, which promotes the food vs. fuel debate. Therefore, there is a need for new sources of carbohydrates to fuel these fermentations.

Animal Health Component

60%

Research Effort Categories

Basic

0%

Applied

60%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	1599	2000	100%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
1599 - Grain crops, general/other;

Field Of Science
2000 - Chemistry;

Keywords

food byproduct

food co-product

lactic acid

ethanol

sorghum

fermentation

protein

Goals / Objectives
Develop pre-treatments to transform unconventional feedstocks and co-products of food processing into fermentable sugars using chemical, enzymatic, or microbiological methods (saccharifying microorganisms).Transform the pre-treated substrates into protein and valuable compounds (for instance lactic acid) via fermentation with simple cultures or mixed cultures of bacteria, molds, and yeast.

Project Methods
Objective 1: Develop pre-treatments to transform unconventional feedstocks and co-products of food processing into fermentable sugars using chemical, enzymatic, or microbiological methods (saccharifying microorganisms).Materials that will be used for this research are byproducts and co-products of food production as well as grains (sorghum grain and soybean) that are not already widely used in the fermentation industry. Treatments will be developed to transform structural carbohydrates into fermentable sugars using chemical, enzymatic, or microbiological methods. Chemical methods consist of treatments with dilute acid or hot water. Enzymatic methods may include treatments with α-amylase, glucoamylase, and cellulases. Microbiological methods consist of using bacteria or molds capable of transforming structural carbohydrates into fermentable sugars. Then, these sugars can be fermented into lactic acid, ethanol, or other compounds in a two-sequence fermentation or using simultaneous saccharification and fermentation.The efficacy of the treatments will be evaluated by quantifying the amounts of fermentable sugars using the National Renewable Energy Laboratory (NREL) method. This method uses an HPLC with a refractive index detector and a Biorad Aminex HPX-87P column with 0.005M sulfuric acid as the mobile phase (Sluiter et al 2006).For starch-rich byproducts, the effectiveness of the treatment will be followed also by analyzing residual starch using the Megazyme total starch assay (AOAC method 996.11).Objective 2: Transform the pre-treated substrates into protein and valuable compounds (for instance lactic acid) via fermentation with simple cultures or mixed cultures of bacteria, molds, and yeast.Microbial CulturesPotential microorganisms that will be used in this proposal are the following: Bacillus stearothermophilus, Bacillus subtilis, Bacillus licheniformis, Lactobacillus plantarum, Aspergillus orizae, Aspergillus niger, Candida utilis, Rhizopus oligosporus, Saccharomyces cerevisiae, and Endomycopsis fibuligera. Cultures will be obtained from the USDA Agricultural Research Service and the American Type Culture Collection. Once received, microorganisms will be reanimated according to specific procedures for each of them, multiplied, and saved as frozen cultures for the preparation of inoculum for the fermentations.Preparation of inoculumMold spores will be prepared as reported by Feng et al. 2005, by inoculating three to five grains of spore-containing silica onto a 100-mL MEA agar (Oxoid) slant in a 200-mL bottle with a loosely tightened lid (for air access), and incubating at 30 oC for 4 to 5 days. Spores will be harvested and washed several times with NaCl solution (0.9%) to remove residual nutrients from the medium. After counting the spore concentration with a hemocytometer, the spore suspension will be stored and at 2oC until used.will be cultured in 5 mL of YPD broth (10 g yeast extract, 20 g peptone, 20 g glucose L-1) for 24 h. Hundred microliters of this culture will be transferred into a new 5-mL YPD broth and incubated for 24 h. Yeast cells will be washed twice with NaCl solution, and re-suspended in 1 mL of NaCl solution. Concentration of cell suspension need to be approx 108 cells/mL as estimated with a hemocytometer.Lactic acid bacterial inoculum will be prepared in MRS sterilized medium inoculated with two mL of frozen cells (107 cells/mL) and incubated at 37°C for 24 h in a benchtop orbital shaker incubator. Next, the cells will be transferred to new sterilized media (100 mL) to continue incubation up to three generations before inoculation. Samples will be inoculated with 10 mL of bacteria suspension containing 108 cells/mL. Bacillus subtilis will be cultivated on a 5 % nutrient agar slant and the slants will be then incubated at 37 °C for 2 days. All suspensions and slants will be kept in vials at −20 °C until used.Fermentation ProcessFermentations will be conducted using two techniques: submerged liquid culture and solid substrate.Submerged Culture:All substrates for submerged liquid fermentation will be prepared as follows: A slurry will be prepared by adding 90 g of DI water to 30 g of the sorghum flour contained in an 250 mL Erlenmeyer flask. The required nutrients will be added to the medium according to the combination of microorganisms used. The fermentation medium will be sterilized for 20 minutes at 121°C and the pH adjusted (with NH4OH or HCl) before inoculation. The incubation process will be monitored at regular intervals to adjust and take samples. For non-amylolytic strains, liquefaction and saccharification of sorghum flours will be done with thermostable α-amylase from Bacillus licheniforms (≥500U/mg) and amyloglucosidase from Aspergillus niger (≥300 U/mL) from Sigma-Aldrich Co. (St. Louis, MO, USA).Solid-substrate fermentationSolid-state fermentation will be performed in 250 mL Erlenmeyer flasks containing 30 g of dry substrates with different amounts of distilled water. The inoculums containing 10% spore and/or cells suspension (107~108 spores or cells) will be used to inoculate the substrates. All the substrates will be sterilized at 121 °C for 20 min before inoculation. Incubation temperature, initial moisture content, and fermentation duration are three factors that will be optimized.Evaluation of fermentation processChemical analysisTotal phenols content and total starch in sorghum flours before and after fermentation, will be determined using Folin Ciocalteu method (Singleton and Rossi 1965) and the Megazyme total starch assay (AOAC method 996.11) respectively. Sugars and lactic acid concentrations will be estimated by HPLC. Crude protein content and protein digestibility will be determined via total nitrogen combustion with Elementar Variomax, and a multi-enzyme pH-drop method.Microbial developmentSince fungal growth is difficult to quantify in mixed species fermentations, microbial growth will be evaluated by real-time PCR using specific primers for each of the microorganisms according to the method reported by Feng et al, 2007.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems:The main problem has been the SARS-CoV-2 pandemic that limited laboratory access for extended periods. Fermentations require continuity of work, and disruptions caused by the pandemic took a toll on the progress. These delays have not altered the project's goals but just delayed them. We expect during 2021, we will be able to get plans back on track. What opportunities for training and professional development has the project provided?The next step will be testing different microorganisms and conditions to maximize protein production and minimize antinutritional factors on grain sorghum or other grains. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? With the limited time we had this period before the Covid-19 pandemic shut down our laboratory, we could reconfirm results obtained in the previous period on protein gain and antinutritional factors reductions. By conducting solid-state fermentations of grain sorghum with Aspergillus oryzae and Bacillus subtilis, we can report protein increments of up to 10 percentage points and a phytic acid reduction of 97 percent in reference to the unfermented grain.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Lactic Fermentation of Grain Sorghum: Effect of Variety and Pretreatment on the Production of Lactic Acid and Biomass, Journal of Food Processing and Preservation

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The scientific comunity,the food industry, and the animal feed industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Part of this research has been presented atICEF13 - 13th International Congress on Engineering and Food. 23-26 September 2019, Melbourne, Australia. The title of the paper(Peer Reviewed) wasEnhancement of The Nutritional Value and Functionality of Grain Sorghum via Solid State Fermentation with Co-culture of Aspergillus oryzae and Bacillus subtilis What do you plan to do during the next reporting period to accomplish the goals?The next step will be to assess the nutritional value and functionality including protein digestibility, amino acid profile.

Impacts
What was accomplished under these goals? Our lab has been focused on exploring the suitability of unconventional feedstock and co-products of food processing as substrates to produce protein for animal feed and other valuable compounds via fermentation. During the past year, we used solid-state fermentation withAspergillus oryzaeandBacillus subtilis, alone or in combination of the two on several varieties of grain sorghum, with the primary objective of increasing the protein content, quality, and digestibility while decreasing anti-nutritional factors, such as phytic acid and trypsin inhibitors. Results indicated an increment in crude protein content from the naturally contained 10 percent to 16 percent withAspergillus oryzaealone and 18 percent with bothAspergillus oryzaeandBacillus subtilis. At the same time, the phytic acid content in the grain was reduced by 88 percent withAspergillus oryzaeand 96 percent with both,Aspergillus oryzaeandBacillus subtilis.

Publications

• Type: Book Chapters Status: Published Year Published: 2019 Citation: Morawicki RO. 2019. Cooking Technologies. Annual Review on Heat Transfer. Chapter 7, pp 245-265 10.1615/AnnualRevHeatTransfer.2019027380
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Diaz Gonzalez D, Morawicki R, Mauromoustakos A. 2019.Effect of nixtamalization treatment of three varieties of grain sorghum on the reduction of total phenolics and their subsequent enzymatic hydrolysis. J Food Process Preserv. https://doi.org/10.1111/jfpp.14067

Progress 10/03/17 to 09/30/18

Outputs
Target Audience:Corn and grain sorghum growers and researchers in the field of aquaculture Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We theorized that the protein content would increase as a result of biomass production due to the development of microorganisms, and the preliminary results are in agreement with these predictions. In addition, we also hypothesize that the co-culture fermentation process will improve the amino acid profile of the existing protein while reducing some antinutritional factors present in grain sorghum, thus producing protein-rich biomass with less anti-nutritional factors as a nutrient source for animal feed. Consequently, during the next year, our plan is to continue the optimization of the fermentation process using different conditions to improve even further the protein content of sorghum. Once the solid-state co-culture fermentation process has been optimized, the fermented samples will be tested not only for protein content, but also for protein quality and digestibility. A second objective for next year is to asses the solid-state fermentation with co-culture of Aspergillus oryzae and Bacillus subtilis using corn as a substrate.

Impacts
What was accomplished under these goals? During 2018, we focused on the development of new processes to enhance the nutritional value and functionality of grain sorghum via fermentation. The main goal was to improve the protein content, quality, and digestibility of grain sorghum via solid state fermentation with a co-culture of Aspergillus oryzae and Bacillus subtilis. Results show that the protein content of grain sorghum can be increased significantly by fermentation, from 10% to 17%, which could have an impact on the potential applications of grain sorghum as feed. Aspergillus oryzae and Bacillus subtilis were obtained from the United States Department of Agriculture's Agricultural Research Service (USDA ARS) Culture Collection. Microbial cultures arrived as lyophilized pellets in glass vials that were activated in liquid medium following specific instructions for each microbe. For inoculum preparation, Aspergillus oryzae was streaked to a sterilized PDA (potato dextrose agar) petri dish and incubated at a stationary state at 30 ?C for seven days. Fungal spores grown for seven days in PDA were harvested by gently washing them with 0.1% Tween 80 polyethylene sorbitol ester, and then filtered with a miracloth to remove all fragments of mycelium. Cells were counted with a hemocytometer to choose the right volume of inoculum needed for fermentation (107 spores/mL). Spore suspension was stored at 4°C until further use. After reanimation, Bacillus subtilis was propagated using Difco Sporulation Media (DSM), cultured at 200 rpm and 37 ?C, for 48 h. The composition (g/L) of the DSM was: Nutrient broth (Difco) 8.0 g, Potassium Chloride (KCl) 1.0 g, 1 mL of Magnesium Sulfate 1M solution, 1 mL of Manganese Chloride 10 mM solution (MnCl2), 0.5 mL Calcium Chloride (CaCl2) 1M solution, and 1 ml of Iron II Sulfate (FeSO4) 1mM solution, completed to 1 litter with deionized water. Upon 48 hours incubation, cells were counted, and stock pre-culture solutions (2 mL) were prepared and frozen with 30% v/v of a sterile 87% glycerol solution for long-term storage at low temperature. 48 hours before inoculation of the substrate with Bacillus subtilis, the inoculum was prepared by transferring a stock culture (2 mL) onto a 125 mL flask containing 50 mL of sterile DSM broth and was let to incubated at 37°C and 200 rpm for 24 hours in a MaxQ 4450 orbital shaker (ThermoScientific, Waltham, MA, USA). At least two pre-cultures were cultivated before inoculation. Next, the inoculum was transferred to centrifuge tubes and centrifuged in and Allegra X-22R (Beckman Coulter, Brea, CA, USA) at 1450 g for 10 minutes. The supernatant was discarded, and the cell pellets were resuspended in D.I. water (5 mL) to be used as the inoculum. For the fermentation process, grain sorghum coarsely ground (20g) was mixed with water (2:1 ratio) and placed in 250 mL Erlenmeyer flasks, sterilized at 121 °C for 20 minutes. The sterilized samples were let to cool down before inoculation with 3 ml of Aspergillus oryzae (107 spores/mL), and incubated at a stationary state at 30 ?C for a week. After fermenting for 7 days at 30 °C, the samples were inoculated with Bacillus subtilis (10% ratio) and let to incubate for another 48 hours at 37 °C, with 200 round per minute (rpm). Upon fermentation the samples were oven dried at 60 ?C, re-ground and storage at 4 ?C for further analysis. Nitrogen content in the samples was determined using total nitrogen by combustion with an Elementar Rapid N III (Elementar Analysensysteme GmbH, Langenselbold, Germany). The preliminary results from the co-culture fermentation with Aspergillus oryzae and Bacillus subtilis were very encouraging. The results showed an increase of protein content from 10% to 17% without any supplementation of the media culture. These results were also confirmed by colorimetric assay Lowry as described by Peterson, 1977.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Akoetey W, Morawicki R.The effect of adaptation of Lactobacillus amylovorus to increasing concentrations of sweet potato starch on the production of lactic acid for its potential use in the treatment of cannery waste. J Environ Sci Health B. 2018 Sep 10:1-8. doi: 10.1080/03601234.2018.1505076.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Christina N. Day and Ruben O. Morawicki, Effects of Fermentation by Yeast and Amylolytic Lactic Acid Bacteria on Grain Sorghum Protein Content and Digestibility, Journal of Food Quality, vol. 2018, Article ID 3964392, 8 pages, 2018. https://doi.org/10.1155/2018/3964392.
• Type: Book Chapters Status: Submitted Year Published: 2018 Citation: Cooking Technologies. Annual Review of Heat Transfer. Begell house, inc. publishers